ISOT2016 17th International Symposium on Olfaction and Taste

Transcription

th
17
International Symposium on
Olfaction and Taste
JASTS 50th Annual Meeting
Date
Venue
June 5 - 9, 2016
PACIFICO Yokohama, JAPAN
Abstract
Welcome Letter
Dear Colleagues,
We are delighted to welcome you to the 17th International Symposium on Olfaction and Taste
(ISOT2016) in Yokohama, Japan. This is a particular honor for us since this ISOT is meeting in
conjunction with the 50th anniversary of the Japanese Association for the Study of Taste and Smell
(JASTS).
The purpose of ISOT is to bring together researchers from around the world to present and discuss
their latest research on chemoreception broadly defined, including consciously perceived taste, smell,
and chemestheses as well as interoceptive systems mediated by homologous sensory detection
mechanisms. ISOT began in 1962 with its inaugural meeting in Stockholm, Sweden. Since then it has
convened every 3-4 years as a meeting venue that rotated among the United States, Europe and
Japan. This reflects the close cooperation of the three largest regional organizations: the Association
for Chemoreception Sciences (AChemS), the European Chemoreception Research Organization
(ECRO), and JASTS. This ISOT2016 is co-hosted by JASTS in conjunction with the Australasian
Association for ChemoSensory Science (AACSS) and the Korean Society of Chemoreception and
Ingestive behavior (KoSCI).
We are grateful that the program committee of ISOT2016, consisting of members of JASTS, AChemS,
ECRO, AACSS and KoSCI, has created an engaging and scientifically interesting program that will be
of keen interest to all participants. The program consists of 2 plenary lectures, a Presidential
Symposium, 18 separate symposia, and over 400 poster presentations. The talks and posters address
diverse areas of research ranging from the molecular analysis of chemoreception to central
processing of chemosensory input in the brain to behavior. Both basic and clinical approaches are
represented.
The meeting venue, Yokohama, is Japan’s second largest city with a population of 3.7 million. It is an
attractive tourist city, full of traditional culture, a variety of cuisines, diverse entertainment and a
beautiful cityscape that provides a fusion of lush nature with modern urbanism. The beautiful presence
of Mt. Fuji overlooks it from afar.
We are confident that all participants will enjoy and learn from the meeting. Welcome!
Yours Sincerely,
Yuzo Ninomiya, ISOT2016 President
Takenori Miyamoto, Local Committee Chair
Kazushige Touhara, Program Chair
Organizer / Host
Organizing Committee of the 17th International Symposium on Olfaction and Taste (ISOT2016)
Co-host
The Japanese Association for the Study of Taste and Smell (JASTS)
Association for Chemoreception Sciences (AChemS)
European Chemoreception Research Organization (ECRO)
Australasian Association for ChemoSensory Sciences (AACSS)
Korean Society for Chemical Senses and Ingestive Behavior (KoSCI)
In Cooperation with
・The Japan Neuroscience Society
・The Biophysical Society of Japan
・The Japanese Society for Comparative Physiology and Biochemistry
・The Japanese Association for Oral Biology
・Japanese Society for Sensory Evaluation
・Physiological Society of Japan
・The Zoological Society of Japan
・Japan Society for Bioscience, Biotechnology, and Agrochemistry
・The Pharmaceutical Society of Japan
・The Japanese Biochemical Society
・Japan Society for the Study of Obesity
・The Society for Promotion of International Oto-Rhino-Laryngology
・Japan Society of Nutrition and Food Science
・Japanese Society of Aromatherapy
・The Japanese Rhinologic Society
・Japan Society of Stoma-pharyngology
Organizer / Host, Co-host, In Cooperation with　｜　3
ISOT2016 Organizing Committee
President
Yuzo Ninomiya
Distinguished Professor, Section of Oral Neuroscience,
Graduate School of Dental Sciences, Kyushu University
Division of Sensory Physiology, Research and Development Center for Taste and Odor Sensing,
Kyushu University, Japan
Takenori Miyamoto (Chair, Local Committee)
Professor, Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s
University, Japan
Kazushige Touhara (Chair, Program Committee)
Professor, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, Japan
Takaki Miwa (Chair, Finance Committee)
Professor, Department of Otorhinolaryngology, Kanazawa Medical University, Japan
Ken Iwatsuki (Acting Chair, Public Relations Committee)
Tokyo University of Agriculture, Japan
Thomas E. Finger
Professor, Cell and Developmental Biology, University of Colorado, USA (AChemS)
Charles A. Greer
Professor, Biological & Biomedical Sciences, Yale University, USA (AChemS)
Leslie B. Vosshall
Professor and HHMI Investigator, The Rockefeller University, USA (AChemS)
Wolfgang Meyerhof
Professor, German Institute of Human Nutrition, Germany (ECRO)
Heinz Breer
Professor, Institute of Physiology, University of Hohenheim, Germany (ECRO)
Eugeni Roura
Senior Research Fellow, Centre for Nutrition and Food Sciences, Queensland Alliance for
Agriculture and Food Innovation, The University of Queensland, Australia (AACSS)
Kyung-Nyun Kim
Professor, Gangneung-Wonju National University, Korea (KoSCI)
Minmin Luo
Investigator, National Institute of Biological Sciences, Professor, Tsinghua University, China
Kensaku Mori
Professor, Department of Physiology, Cellular and Molecular Physiology, Graduate School of
Medicine, The University of Tokyo, Japan
Hideto Kaba
Professor, Department of Physiology, Kochi Medical School, Kochi University, Japan
4　｜　ISOT/JASTS Program 2016
Kumiko Sugimoto
Professor, Basic Oral Health Science, Course for Oral Health Engineering, School of Oral Health
Care Sciences, Faculty of Dentistry, Tokyo Medical and Dental University, Japan
Kiyoshi Toko
Distinguished Professor, Graduate School of Information Science and Electrical Engineering,
Research and Development Center for Taste and Odor Sensing, Kyushu University, Japan
Tohru Fushiki
Professor, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto
University, Japan
Satoshi Wakisaka
Professor, Department of Oral Anatomy and Developmental Biology, Graduate School of Dentistry,
Osaka University, Japan
Makoto Kashiwayanagi
Professor, Department of Sensory Physiology, Asahikawa Medical University, Japan
Mamiko Ozaki
Professor, Department of Biology, Graduate School of Science, Kobe University, Japan
Michio Komai
Professor, Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University,
Japan
Hisao Nishijo
Professor, System Emotional Science, Graduate School of Medicine and Pharmaceutical Sciences,
University of Toyama, Japan
Noritaka Sako
Professor, Asahi University School of Dentistry, Japan
Noriatsu Shigemura
Associate Professor, Kyushu University, Japan
Takeshi Kimura
Member of the Board & Corporate Vice President
Management of R&D; Quality Assurance & External Scientific Affairs;
Intellectual Property; Wellness Business, AJINOMOTO CO., INC., Japan
Masanori Kohmura
Associate General Manager, External Scientific Affairs, Quality Assurance & External Scientific
Affairs Department, AJINOMOTO CO., INC., Japan
Makoto Emura
Director, Fragrance & Aroma Chemical Business Planning, Aroma Chemical Global Business
Headquarters, TAKASAGO International Corporation, Japan
Advisors:
Takashi Yamamoto
Professor, Graduate School of Faculty of Health Science, Kio University, Japan
Keiko Abe
Professor, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan
Committee　｜　5
ISOT2016 Program Committee
Chair
Kazushige Touhara
The University of Tokyo, Japan
Co-chairs
Leslie B. Vosshall
Rockefeller University, USA
Wolfgang Meyerhof
German Institute of Human Nutrition Potsdam-Rehbruecke, Germany
Members
Yuichi Iino
Ryohei Kanzaki
Makoto Kashiwayanagi
Asahikawa Medical College, Japan
Teiichi Tanimura
Makoto Tominaga
National Institutes of Natural Sciences, Japan
Yoshihiro Yoshihara
RIKEN Brain Science Institute, Japan
Satoshi Wakisaka
Richard D. Newcomb
University of Auckland, New Zealand
Minmin Luo
National Institute of Biological Sciences,
China
Jeong Won Jahng
Seoul National University, Korea
Nirupa Chaudhari
University of Miami, USA
Robert F. Margolskee
Monell Chemical Senses Center, USA
Steve D. Munger
University of Florida, USA
Dana M. Small
Yale University, USA
Alan C. Spector
The Florida State University, USA
Donald A. Wilson
New York University, USA
Thomas Hummel
University of Dresden, Germany
Peter Mombaerts
Max Planck Institute of Biophysics, Germany
Frederic Marion-Poll
CNRS, France
Marc Spehr
RWTH Aachen University, Germany
Frank Zufall
University of Saarland, Germany
Noam Sobel
Weizmann Institute of Science, Israel
Local Committee
Finance Committee
Chair
Takenori Miyamoto
Japan Women’s University, Japan
Chair
Takaki Miwa
Kanazawa Medical University, Japan
Co-chairs
Noritaka Sako
Asahi University School of Dentistry, Japan
Noriatsu Shigemura
Members
Kiyoshi Toko
Masanori Kohmura
AJINOMOTO CO., INC., Japan
Makoto Emura
TAKASAGO International Corporation, Japan
Toshio Miyazawa
Ogawa & Co., Ltd., Japan
Members
Aki Ejima
Yukako Hayashi
Kyoto University, Japan
Masashi Inoue
Tokyo University of Pharmacy and Life
Sciences, Japan
Takayuki Kawai
Sensory and Cognitive Food Science
Laboratory, Japan
Hiroya Kawasaki
Yuko Kusakabe
Sensory and Cognitive Food Science
Laboratory, Japan
Yutaka Maruyama
Kazunari Miyamichi
Yoshihito Niimura
Masako Okamoto
Fumino Okutani
Kochi Medical School, Japan
Tsuyoshi Shimura
Makoto Sugita
Hiroshima University, Japan
Advisors
Tohru Fushiki
Ryukoku University, Japan
Keiko Abe
Public Relations Committee
Acting Chair
Ken Iwatsuki
Tokyo University of Agriculture, Japan
Ryohei Kanzaki
Saho Ayabe-Kanamura
University of Tsukuba, Japan
Jun Inouchi
National Institute of Agrobiological Sciences,
Japan
Takamichi Nakamoto
Tokyo Institute of Technology, Japan
Masayoshi Kobayashi
Mie University Graduate School of Medicine,
Japan
Masashi Inoue
Tokyo University of Pharmacy and Life
Sciences, Japan
Tadashi Inui
Committee　｜　7
Special Thanks
We would like to send special thanks and appreciation to the following companies and organizations
for their generous support of the 2016 ISOT/50th JASTS Annual Meeting.
Diamond Sponsors
Platinum Sponsor
Gold Sponsors
Kikkoman Corporation
Kirin Company, Limited
Sensonics International
T.HASEGAWA CO.,LTD.
Luncheon Sponsors
Ajinomoto Co., Inc.
Intelligent Sensor Technology, Inc.
Kao Corporation
Suntory Global Innovation Center Limited
ZENSHO HOLDINGS CO., LTD.
Exhibitors
Agilent Technologies
Alpha M.O.S. S.A.
Burghart Messtechnik GmbH
DAIICHI YAKUHIN SANGYO CO., LTD.
Entrex. Inc
Fragrance Journal Ltd.
GL Sciences Inc.
Hitachi High-Technologies Corporation
JAPAN TABACCO INC.
SHIMADZU CORPORATION
Taste and Aroma Strategic Research Institute Co., Ltd
Sponsors
ADEKA CORPORATION
Alfresa Pharma Corporation
ASAHI KASEI PHARMA CORPORATION
ASKA Pharmaceutical. Co., Ltd.
Astellas Pharma Inc.
AstraZeneca K.K.
Bayer Yakuhin, Ltd.
BMS K.K
CHUGAI PHARMACEUTICAL CO., LTD.
DAIICHI SANKYO COMPANY, LIMITED
Eiken Chemical Co., Ltd.
Eisai Co., Ltd.
ELMED EISAI Co., Ltd.
Fuso Pharmaceutical Industries, Ltd.
GlaxoSmithKline K.K.
J.MORITA CORPORATION
JAPAN TOBACCO INC.
KAKEN PHARMACEUTICAL CO.,LTD.
Kissei Pharmaceutical Co., Ltd.
Kowa Company, Ltd.,
Kracie Pharma, Ltd.
KYORIN Pharmaceutical Co.,Ltd.
Kyowa Hakko Kirin Company, Limited
Lotte Co., Ltd.
Maruho Co.,Ltd.
Maruishi Pharmaceutical Co.Ltd
Meiji Seika Pharma Co., Ltd.
Minophagen Pharmaceutical Co., LTD.
Mitsubishi Tanabe Pharma Corporation
MOCHIDA PHARMACEUTICAL CO.,LTD.
MSD K.K.
NIHON PHARMACEUTICAL CO., LTD.
Nippon Boehringer Ingelheim Co., Ltd.
Nippon Chemiphar Company, Limited
Nippon Kayaku Co.,Ltd.
Nippon Shinyaku Co.,Ltd.
Nippon Zoki Pharmaceutical Co.,ltd.
NIPRO PHARMA CORPORATION
Novartis Pharma K.K.
Ogawa & Co., Ltd
Ono Pharmaceutical Co.,Ltd.
Otsuka Pharmaceutical Co., Ltd.
Otsuka Pharmaceutical Factory, Inc.
Pfizer Japan Inc.
ROHTO Pharmaceutical Co., Ltd.
Sanofi K.K.
SANTEN PHARMACEUTICAL CO., LTD.
SANWA KAGAKU KENKYUSHO CO.,LTD.
SATO PHARMACEUTICAL CO., LTD.
Sawai Pharmaceutical Co., Ltd.
SHIONOGI & CO., LTD.
SSP Co., Ltd.
Sumitomo Dainippon Pharma Co., Ltd.
Tablemark Co.,Ltd.
TAIHO Phamaceutical Co.,Ltd.
Taisho Pharmaceutical Co., Ltd.
Takeda Pharmaceutical Company Limited.
Taste and Food Preference Laboratory
TEIJIN PHARMA LIMITED.
TERUMO CORPORATION
The Japan Food Chemical Research Foundation
The Tojuro Iijima Foundation for Food Science
and Technology
The Tokyo Biochemical Research Foundation
The Uehara Memorial Foundation
TOA EIYO LTD.
Torii Pharmaceutical Co., Ltd.
Towa Pharmaceutical Co., Ltd.
TOYAMA CHEMICAL CO., LTD.
Tsumura & Co.
Umami Manufacturers Association of Japan
(UMAJ)
Wakamoto Pharmaceutical Co., Ltd.
Yakult Honsha Company, Limited
YAMASA CORPORATION
Zeria Pharmaceutical Co., Ltd.
(In alphabetical order/As of 10th May, 2016)
Special Thanks　｜　9
XVII ISOT 2016 The Young Investigator Awards
The below participants have been awarded the XIV ISOT 2016 Young Investigator Award
Toshihide Hige (USA)
Zhenbo Huang (USA)
Takaaki Miyazaki (Japan)
Sebastien Kessler (UK)
Nicolas Thiebaud (USA)
Florence Marianne Kermen (Norway)
Brian C. Lewandowski (USA)
Chryssanthi Tsitoura (Germany)
Vladimiros Thoma (Japan)
Kentaro Ishii (Japan)
Sayaka Katsunuma (Japan)
Claire A. de March (USA)
Tomohiro Tanaka (USA)
Ayumi Nagashima (Japan)
Nami Suzuki-Hashido (Japan)
Ruchira Sharma (USA)
Casey Trimmer (USA)
Julia Yu Qing Low (Australia)
Jean-Baptiste Cheron (France)
Norihiro Fujimoto (Japan)
Smiljana Mutic (Germany)
Artin Arshamian (Sweden)
Mai Tsunoda (Japan)
Emiko Nishi (Japan)
Nao Horio (Japan)
Yuko Nakamura (USA)
Raena Bianca Joy Mina (USA)
Sachiko Haga-Yamanaka (USA)
Notes and Errata
P.12-17

Poster session room 304 has changed to 304 AND 316, 317, 318.

Room 316 (Secretariat) has changed to Room 313

Room 317(Cloak) has changed to Room 312

Room 318(Accompanying person’s program) has changed to Room 314
P.14-15
Room has changed from 301 to 303 for the session below;
09:00-11:30 [PA-04] Parallel Symposium 4
Digitizing olfaction
Chairs: Joel Mainland, Noam Sobel
Room has changed from 303 to 301 for the session below;
09:00-11:30 [PA-06] Parallel Symposium 6
No! Aversion and avoidance of chemosensory stimuli
Chairs: Steven Munger, Frank Zufall
P.18
312: Cloak
313: Secretariat
P. 48
314: Accompanying Person’s Program
Poster Sessions
Poster Sessions
There have been changes in speakers of Parallel Symposium V “Umami and amino acid
tastes: Ligands, receptors, appetites and perceptions”.
ISOT/JASTS Program 2016
Program at a Glance
Sunday, June 5
Location
/Time
3rd Fl. Foyer
Main Hall
301
302
17:00
Registration
18:00
19:00
18:30-20:30
Welcome Reception
PACIFICO Yokohama [301+302]
20:00
21:00
Program at a Glance　｜　11
Monday, June 6
Location
/Time
3rd Fl. Foyer
Main Hall
301
302
8:00
9:00
9:30-11:30
[PL]
Plenary lectures
10:00
Chairs: Kazushige
‌
Touhara,
Takashi Yamamoto
[PL-01] David Anderson
[PL-02] Kenji Kangawa
11:00
12:00
Luncheon Seminar Tickets will be distributed at
the foyer of the 3rd floor in the morning of each
seminar day, on a first-come first-serve basis.
11:45-12:45
Luncheon Seminar 2
Registration
P. 20
Sponsor: Kao Corporation
13:00
14:00
15:00
13:00-16:00
[PS]
Presidential symposium
Chairs: Yuzo
‌
Ninomiya,
Kazushige Touhara
[PS-01]Linda Buck
[PS-02]Robert Margolskee
[PS-03]Leslie Vosshall
[PS-04]Wolfgang Meyerhof
[PS-05]Yuichi Iino
[PS-06]Kensaku Mori
16:00
17:00
16:15-18:45
[PA-01]
Parallel Symposium 1
25 years of odorant receptors
Chairs: ‌Peter Mombaerts,
Stuart Firestein
18:00
19:00
20:00
21:00
16:15-18:45
[PA-02]
Recognizing taste: coding along
the neural axis in mammals
Chairs: ‌Nirupa Chaudhari,
Kathrin Ohla
303
304
304 Foyer
316
318
Location
/Time
8:00
9:00
10:00
Luncheon Seminar Tickets will be distributed at
the foyer of the 3rd floor in the morning of each
seminar day, on a first-come first-serve basis.
11:45-12:45
Luncheon Seminar 1
11:00
P. 20
11:45-12:45
JASTS Executive
Committee
Sponsor: ZENSHO HOLDINGS CO., LTD.
12:00
13:00
14:00
9:00-21:00
Exhibition
12:00-19:00
Poster Exhibition
16:15-18:45
[PA-03]
Insect Taste : Behavioral
Plasticity and Evolution
15:00
15:00-17:00
Accompanying
Person’s Program
16:00
Origami
(Art of folding
paper)
*Online reservation
required
17:00
Chairs: Teiichi
‌
Tanimura,
Frédéric Marion-Poll
18:00
19:00
19:00-21:00
Poster Session with
drinks
20:00
21:00
Tuesday, June 7
Location
/Time
3rd Fl. Foyer
Main Hall
301
302
8:00
9:00
09:00-11:30
[PA-04]
Digitizing olfaction
10:00
Chairs: ‌Joel Mainland,
Noam Sobel
09:00-11:30
[PA-05]
Umami and amino acid tastes:
Ligands, receptors, appetites
and perceptions
Chairs: ‌Paul Breslin,
Masanori Komura
11:00
12:00
Registration
Luncheon Seminar Tickets will
be distributed at the foyer of
the 3rd floor in the morning of
each seminar day, on a firstcome first-serve basis.
13:00
14:00
P. 21
11:45-12:45
Luncheon Seminar 3
Sponsor:
Intelligent Sensor Technology, Inc.
13:00-15:30
[PA-07]
Mechanisms of background
segregation and source
localization of odors
Chairs: ‌Brian H Smith,
Takeshi Sakurai
11:45-12:45
Luncheon Seminar 4
Sponsor: Ajinomoto Co., Inc.
13:00-15:30
[PA-08]
Structure-function relationships
of olfactory and taste receptors
Chairs: ‌Wolfgang Meyerhof,
Simone Weyand
15:00
16:00
17:00
18:00
19:00
20:00
15:30-18:00
[PA-10]
Neuromodulation in
chemosensory pathways
Chairs: ‌Jeremy McIntyre,
Markus Rothermel
P. 21
15:30-18:00
[PA-11]
Chemosensory regulation of
energy homeostasis and
metabolism
Chairs: ‌Robert F. Margolskee,
Ivan E de Araujo
303
304
304 Foyer
316
318
Location
/Time
8:00
9:00
09:00-11:30
[PA-06]
No! Aversion and avoidance of
chemosensory stimuli
10:00
Chairs: Steven
‌
Munger,
Frank Zufall
11:00
11:45-12:45
ICOT meeting
13:00-15:30
[PA-09]
Future strategies for the assessment and treatment of smell
and taste disorders
9:00-20:00
Exhibition
12:00
13:00
9:00-18:00
Poster Exhibition
14:00
Chairs: Masayoshi
‌
Kobayashi,
Richard Costanzo
*Online reservation
required
15:30-18:00
[PA-12]
Advances in human chemosensory neuroimaging
Chairs: Masako
‌
Okamoto,
Dana Small
Kodo (Art of
Incense burning)
Accompanying
Person’s Program
*Online reservation
17:00-18:00
required
18:00-20:00
Poster Session with
drinks
15:00
15:00-17:00
Accompanying
Person’s Program
16:00
Shodo
(Japanese
Calligraphy)
17:00
18:00
Open Program
18:00-19:00
19:00
20:00
Wednesday, June 8
Location
/Time
3rd Fl. Foyer
Main Hall
301
302
8:00
9:00
10:00
09:00-11:30
[PA-13]
Emerging topics in olfactory
sensorimotor behavior
09:00-11:30
[PA-14]
Neurotransmission from taste
buds to nerves
Chairs: ‌Yun Zhang,
Yuichi Iino
Chairs: ‌Sue Kinnamon,
Thomas Finger
11:00
Registration
11:45-12:45
Oxford Press Editorial Board
Meeting
12:00
13:00
14:00
13:00-15:30
[PA-16]
Neural circuits processing
pheromones and odor valence
in mice
13:00-15:30
[PA-17]
Respiratory taste receptors:
new arms of respiratory innate
defense
Chairs: ‌Kazunari Miyamichi,
Stephen Liberles
Chairs: ‌Noam Cohen,
Yehuda Ben-Shahar
15:00
15:30-16:30
JASTS General Meeting
16:00
17:00
18:00
19:00
Reservation available
until 6th Jun.
20:00
19:00-21:00
GALA Dinner
The Yokohama Bay Hotel Tokyu [Queen’s Grand Ballroom]
21:00
303
304
304 Foyer
316
318
Location
/Time
8:00
9:00
09:00-11:30
[PA-15]
What drives cultural difference
in chemosensory perception
10:00
Chairs: Tatsu
‌
Kobayakawa,
Han-Seok Seo
Luncheon Seminar Tickets will be
distributed at the foyer of the 3rd
floor in the morning of each seminar
day, on a first-come first-serve basis.
11:00
P. 22
11:45-12:45
Luncheon Seminar 5
Sponsor: Suntory Global Innovation
Center Limited
11:45-12:45
JASTS Editorial
Board Meeting
9:00-16:30
Poster Exhibition
13:00-15:30
[PA-18]
Stem cells and the self-renewal
of chemosensory epithelia:
mechanisms of regeneration in
taste buds versus the olfactory
epithelium
12:00
13:00
9:00-18:30
Exhibition
14:00
*Online reservation
required
Chairs: Bradley
‌
Goldstein,
Linda Barlow
14:00-17:00
15:00
Accompanying
Person’s Program
Yukata
(Japanese traditional clothes)
16:00
17:00
16:30-18:30
Poster Session with
drinks
18:00
19:00
Reservation available
until 6th Jun.
19:00-21:00
GALA Dinner
20:00
The Yokohama Bay Hotel Tokyu [Queen’s Grand Ballroom]
21:00
会場周辺図
Access Guide
Floor Map
パシフィコ横浜
Paciﬁco Yokohama
PACIFICO Yokohama
Location Map
1st. Fl.
PACIFICO Yokohama
Entrance from Inter Continental YOKOHAMA GRAND
ConferenceCenter
Entrance
Plenary Lectures,
Presidential Symposium
The Yokohama
Bay Hotel Tokyu
（Main Hall）
横浜美術館
Yokohama Museum of Art
桜木町駅へ
To Sakuragicho Sta.
3rd. Fl.
⬆
Room 311～315
Foyer:
Registration Desk
Welcome
Reception
Parallel
Session
Poster Session,
Exhibition
Parallel
Session
Parallel
Session
（302）
（301+302）
（301）
316
Secretariat
Foyer: Poster Session,
Exhibition
（304）
（303）
317
Cloak
318
Accompanying
Person’s Program
The Yokohama Bay Hotel Tokyu
B2nd. Fl.
EV
Escalator
GALA Dinner
（Queen’s Grand Ballroom）
Posters/Exhibit Hall Floor Plan
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②
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⑤ ⑥ ⑦ ⑧
⑨ ⑩
⑪ ⑫ ⑬ ⑭
⑯ ⑰ ⑱
⑮
017　019　021　023　025
018　020　022　024
103　105
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123　125　127
122　124　126　128
130 132 134
129　131　133　135
Exhibitors’ List
1
Kikkoman Corporation
①
Entrex Inc.
②
Oxford University Press
③④
TAKASAGO INTERNATIONAL CORPORATION
⑤⑥
AJINOMOTO CO.,INC.
⑦
DAIICHI YAKUHIN SANGYO CO., LTD.
⑧
Alpha M.O.S. S.A.
⑨
GL Sciences Inc.
⑩
⑪
Hitachi High-Technologies Corporation
⑫
Mitsubishi Corporation Life Sciences Limited
⑬
Agilent Technologies
⑭
Burghart Messtechnik GmbH
⑮
Taste and Aroma Strategic Research Institute Co., Ltd
⑯
JAPAN TABACCO INC.
⑰⑱
302 Foyer
Sensonics International
Fragrance Journal Ltd.
Posters/Exhibit Hall Floor Plan　｜　19
EDAIN WING 4.0 星野
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Luncheon Seminar
Luncheon Seminar Tickets will be distributed at the foyer of the 3rd floor
in the morning of each seminar day, on a first-come first-serve basis.
Monday, June 6
Luncheon Seminar 1
11:45-12:45
Sponsored by: ZENSHO HOLDINGS CO., LTD
Room 303
Cross-cultural study for olfactory and taste sensations:
Application for product developments of food and beverage
Chair:
Gary K. Beauchamp. Monell Chemical Senses Center
LS1-1
Cultural differences in flavor perception and preference:
Nature and nurture
Gary K. Beauchamp. Monell Chemical Senses Center
LS1-2
Cross-culture study for foods and beverages in Asian
countries
Nguyen Ba Thanh. Industrial University of Ho Chi Minh City
LS1-3
Application for product development: Sensory studies for
Matcha green tea
Hajime Nagai. Zensho Holdings Co., Ltd.
Luncheon Seminar 2
Sponsored by: Kao Corporation
Room 302
Analysis of olfactory receptors for industrial application
Chair:
Yoshihiro Yoshihara. RIKEN Brain Science Institute
LS2-1
Identification of musk odor receptors and their ligand
specificities
Mika Shirasu. Department of Applied Biological Chemistry Graduate School of Agricultural and
Life Sciences The University of Tokyo
LS2-2
An approach to blockade of malodors by focusing on
olfactory receptors
Keiichi Yoshikawa. Kansei Science Research, Kao Corporation
20 ｜ ISOT/JASTS Program 2016
2016.05.27 09.35.00 Page 3(1)
Tuesday, June 7
Luncheon Seminar 3
11:45-12:45
Sponsored by: Intelligent Sensor Technology, Inc.
Room 301
Taste Sensing System and Its Application
Chair:
Yusuke Tahara. Graduate School of Information Science and Electrical Engineering
LS3-1
Taste Sensing System - Basic Concept and Its Application Masaaki Habara. Solution Service Dept. Intelligent Sensor Technology, Inc.
LS3-2
Application Examples of Taste Information
Nozomi Yajima. Research and Development Dept. Taste and Aroma Strategic Research Institute
Co., Ltd.
Luncheon Seminar 4
Sponsored by: Ajinomoto Co., Inc.
Room 302
Significance of taste and olfaction in healthy life
Chair:
Thomas Hummel. Department of Otorhinolaryngology, Smell and Taste Clinic, University of
Dresden Medical School.
LS4-1
The key role of umami taste in oral and overall health
Takashi Sasano. Department of Oral Diagnosis, Tohoku University Graduate School of Dentistry,
Japan
LS4-2
Taste and smell sensitivity and health Status
Aytug Altundag. Otorhinolaryngology, Head&Neck Surgery Department, Istanbul Surgery Hospital,
Turkey
Luncheon Seminar ｜ 21
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Wednesday, June 8
Luncheon Seminar 5
11:45-12:45
Sponsored by: Suntory Global Innovation Center Limited
Room 303
FUNCTIONALITY OF PALATABLE BEVERAGE
Chair:
Masaaki Uchida. Suntory Global Innovation Center Ltd.
LS5-1
The marriage of Tea and Food: Tea makes fatty meals taste
better
Emi Mura. Suntory Global Innovation Center Ltd.
LS5-2
Anti-stress effect of whisky aroma
Hirofumi Koda. Suntory Global Innovation Center Ltd.
22 ｜ ISOT/JASTS Program 2016
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ABSTRACTS
17th International Symposium on Olfaction and Taste (ISOT2016)
PACIFICO Yokohama, Yokohama, Japan, June 5 - 9, 2016
President: Yuzo Nimoniya
Plenary Lecture
PL1 Social behavior circuits in flies and mice
David J. Anderson
Department of Biology and Biological Engineering, California Institute of Technology, Howard Hughes Medical Institute, USA
Animals often have to make rapid decisions between different, competing behaviors, such as fighting, mating, or
freezing. These decisions are controlled by sensory cues, the animal’s internal state and its previous history. In
humans, these innate behaviors are associated with emotion states such as fear, anger and love. We are
studying the control of aggression vs. mating, in both mice and fruit flies, as a model for understanding how
internal states, such as arousal or other so-called “emotion primitives,” influence decisions between innate
behaviors. This talk will focus on how aggression circuits are organized in the brain, and their relationship to
circuits that control mating behavior. Our studies have revealed that mice and flies contain “modules” (relatively
small groups of neurons) that control both aggression and mating, suggesting that this is an evolutionarily
ancient circuit “motif.” The role of these modules, and their relationship to decision-making and internal brain
states, will be discussed. The long-term objective of these studies is to provide insights into the brain
mechanisms that link emotion and decision-making, and their evolutionary origins.
PL2 Challenge to novel peptide hormones
Kenji Kangawa
National Cerebral and Cardiovascular Center, Japan
A complex network of cell-cell communication system by peptide hormones works for maintaining the
mammalian homeostatic balance. To further clarify the intricate mechanisms of the regulation, we have been
searching for still unidentified bioactive peptides by using our own methods. In the course of these studies, we
discovered novel bioactive peptides such as three natriuretic peptides, ANP (1984), BNP (1988) and CNP
(1990), in mammalian heart and brain, and adrenomedullin (1993) in human pheochromocytoma. The
identification of ANP from the human heart elucidated a new regulatory mechanism of cardiovascular system by
the peptide hormone from the heart. It showed that the heart is not only a pump, but also has a simultaneous
function as an endocrine organ involved in the cardiovascular regulation. These discoveries showed the
existence of new regulatory mechanisms of cardiovascular system, and also brought their therapeutic potentials
on cardiovascular diseases.
Moreover in 1999, we discovered ghrelin from the stomach as an endogenous ligand for the growth hormone
(GH) secretagogues receptor (GHS-R), an orphan GPCR. Beside the potent stimulatory effect of GH release,
ghrelin is also involved in the stimulation of feeding and the regulation of energy metabolism. Recently we have
demonstrated that ghrelin has protective effects in the cardiovascular system. Thus, ghrelin has multifaceted
roles in the metabolic and cardiovascular systems, and also has therapeutic potentials in various diseases by
GH-dependent and independent mechanisms.
Our most recent topic is “atrial natriuretic peptide prevents cancer metastasis through inhibition of cancer cell
adhesion to vascular endothelial cells”. Why cancer rarely metastasizes to the heart? This question is a
beginning of our most recent challenge. I will show you the answer of the question.
In this lecture, I will briefly summarize my challenge to novel peptide hormones over 40 years; their
discoveries, physiological significance and therapeutic potentials.
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Presidential Symposium
PS1 Mechanisms of odor perception and instinctive odor responses in mammals
Linda B. Buck
Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, USA
We are interested in the mechanisms underlying the sense of smell and instinctive odor responses in mammals.
Our previous studies showed that odor perception in the mouse nose is mediated by ~1000 different odorant
receptors (ORs), that ORs are used combinatorially to encode odor identities, that each olfactory sensory neuron
(OSN) is likely to express a single OR gene, and that OSNs with the same OR are scattered in one nasal zone,
but synapse in a few specific glomeruli in the olfactory bulb. We recently used single cell RNA sequencing
(RNA-Seq) to analyze OR gene expression during OSN development. In a prevailing model of OR gene choice,
the OSN selects a single OR gene and feedback from the encoded OR protein prevents the expression of other
OR genes. However, our studies indicate that immature OSNs can initially express low levels of up to 12
different OR genes and then progress over development to express a high level of only one OR gene in the
mature state. OR genes coexpressed in the same OSN have related expression patterns in adults, suggesting
regional biases that restrict OR gene activation to a subset of OR genes. However, a single OSN can coexpress
genes located on multiple chromosomes, arguing against the possibility that coexpression of multiple OR genes
results from chromatin changes at a single locus containing those genes. In other studies, we have developed
viral tracers to explore how volatile predator odors induce fear-associated increases in stress hormones, which
are controlled by hypothalamic corticotropin releasing hormone (CRH) neurons. We found that the amygdalopiriform transition area (AmPir), a minor area of the olfactory cortex, plays a key role in this response and that the
hormonal and behavioral components of the fear response are likely to involve different olfactory cortical areas.
PS2 Taste cells of the gut and endocrine cells of the tongue
Robert F. Margolskee
We have found that many of the receptors and downstream signalling elements involved in taste transduction
are expressed also in intestinal hormone producing (endocrine) cells where they underlie key chemosensory
functions of the gut. In one example of gastrointestinal chemosensation it is known that glucose given orally, but
not systemically, induces secretion of the “incretin” hormone GLP-1 (glucagon like peptide-1), which in turn
regulates insulin secretion and glucose homeostasis. We have found that intestinal endocrine cells express
sweet taste receptors, gustducin, and several other taste transduction elements. Knockout mice lacking
gustducin or the sweet taste receptor subunit T1R3 have deficiencies in secretion of GLP-1 and in the regulation
of plasma levels of insulin and glucose. In another example of gastrointestinal chemosensation we have found
that tuft cells of the gut express multiple taste proteins that are involved in the host’s innate immune responses to
parasites. We have also found that intestinal-type glucose transporters and pancreatic-type ATP-gated
K+ channels (K-ATP metabolic sensors) are present in taste cells and likely function in the detection of the sweet
taste of sugars. Most recently, we have found that the intestinal brush border disaccharidase enzymes maltase
and sucrase are expressed in T1R3+ taste cells. In both wildtype and T1R3 knockout mice disaccharidase
inhibitors significantly reduced gustatory nerve responses to the disaccharides sucrose and maltose, but not to
the monosaccharides glucose and fructose or the noncaloric sweeteners. It appears that these orally expressed
enzymes act in concert with salivary amylase to generate free glucose from sucrose, maltose and starch that
can activate the T1R3-independent sugar detection pathway. In sum our studies point out similarities in
gustation and gut chemosensation and indicate the importance of “taste cells of the gut” and “endocrine cells of
the tongue” in coordinating the body’s hormone responses to regulate glucose homeostasis.
PS3 Building a mosquito sensory system to hunt humans
Leslie B. Vosshall
Rockefeller University, USA
My group is interested in the molecular neurobiology of mosquito host-seeking behavior. Female mosquitoes
require a blood meal to complete egg development. In carrying out this innate behavior, mosquitoes spread
dangerous infectious diseases such as malaria, dengue, Zika, Chikungunya, and yellow fever. Humans attract
mosquitoes via multiple sensory cues including emitted body odor, heat, and carbon dioxide in the breath. The
2016.05.27 16.58.58 Page 3(1)
mosquito perceives differences in these cues, both between and within species, to determine which animal or
human to target for blood-feeding. We have developed CRISPR/Cas9 genome-editing in the Aedes aegypti
mosquito with the goal of understand how sensory cues are integrated by the female mosquito to lead to
host-seeking behavior. Some of the questions we are currently addressing are: Why are some people more
attractive to mosquitoes than others? How do insect repellents work? How are multiple sensory cues integrated
in the mosquito brain to elicit innate behaviors? How do female mosquitoes select a suitable body of water to lay
their eggs?
PS4 The complex molecular and cellular basis of bitterness perception
Wolfgang Meyerhof
Department Molecular Genetics, German Institute of Human Nutrition, Germany
Bitterness in food is brought about by numerous and structurally diverse chemicals of natural origin or produced
during food processing. It is commonly avoided, yet in many cases we accept and enjoy bitter foods and
beverages. This variability suggests a complex organization of the bitter sensing system. Vertebrates recognize
bitter compounds by means of taste 2 G protein-coupled receptors, termed Tas2rs, that extensively differ in
tuning breadth. Their combined agonist profiles and gene repertoire sizes determine the bitter perceptual range
of the various species. In mice and men distinct subsets of their Tas2r repertoires are expressed by the oral
taste bud cells resulting in overlapping but distinct molecular receptive ranges. Accordingly, genetic ablation of
the bitter receptor cell population characterized by the expression of one Tas2r, TAS2r131, causes partial but
not complete impairment of bitter recognition. The functional diversity of the peripheral bitter sensing cells is
propagated by peripheral ganglionic neurons to 1st order central gustatory neurons in the brain stem. In these
structures as well as in other brain areas gustatory neurons are found that express bitter taste receptors.
Blocking synaptic transmission by genetic manipulation in Tas2r131 expressing neurons results in partly
impaired behavioral bitter responses whereas responses to other taste qualities are not affected. However, the
extent to which the bitter sensing system is affected is smaller than that seen in the animals with the ablated Tas
2r131 bitter cell population. The data propose that bitter recognition is mediated by many functionally distinct yet
overlapping peripheral gustatory pathways that partially converge in the central nervous system raising the
question if and to which extend discrimination of bitter compounds is possible and how the complex cellular
organization of the bitter sensing system affects ingestion of food differing in the content of bitter substances.
PS5 Decoding chemical gradient by a simple neural circuit
Yuichi Iino
Department of Biological Sciences, Graduate School of Science, the University of Tokyo, Japan
In the wild, animals navigate by using chemical cues to find food or mates. The value of each chemical as a
navigation cue is determined adaptively through experience. How do the sensory neurons and downstream
neural circuits decode the chemical information and generate appropriate navigation behaviors? To understand
the basic operation of the neural circuit, we study the nematode C. elegans as one of the simplest animal
models, with only 302 neurons in the whole body. C. elegans shows chemotaxis to various odorants and
tastants, and associate the chemicals with food availability leading to changes in the subsequent behaviors. At
least two mechanisms have been found for chemotaxis, the pirouette mechanism (biased random walk,
klinokinesis) and the weathervane mechanism (klinotaxis), which define response to chemical gradient in two
directions, respectively, relative to head direction. In this talk I will show our results on the neural mechanism of
chemotaxis to the salt NaCl. Our analyses suggest the following: 1) both of these behaviors can be generated by
inputs to a single sensory neuron, 2) chemotaxis is generated by biasing the probability of component behaviors,
3) there is redundancy and functional segregation between interneuron classes, 4) behavioral stochasticity is
generated at the level of interneurons, 5) experience-dependent changes in chemotaxis are mainly caused by
changes in the synaptic transmission between the sensory neuron and primary interneurons.
PS6 Respiration phases and olfaction
Kensaku Mori
Olfaction depends critically on respiration rhythms. The olfactory system in the brain receives external odor
information during the inhalation phase of the respiration cycle, whereas it is isolated from the external odor
2016.05.27 16.58.58 Page 4(1)
world during the exhalation phase. Tufted cells and mitral cells are principal neurons in the olfactory bulb
receiving synaptic inputs from olfactory sensory neurons and projecting axons to a variety of areas in the
olfactory cortex. Recent studies show that tufted cells and mitral cells have distinct pattern of axonal projection to
the olfactory cortex and play distinct functional roles in processing of olfactory sensory inputs. Most importantly,
tufted cells and mitral cells convey their sensory signals to the olfactory cortex at different time windows during
the inhalation-exhalation cycle. In coordination with the inhalation-phased inputs from the olfactory bulb,
olfactory cortex areas actively perform inhalation/exhalation-phased switching of operation modes.
Parallel Symposium I: 25 years of odorant receptors
PA01-1 The Many Receptor Problem
Stuart Firestein
Department of Biological Sciences, Columbia University, USA
The seminal discovery 25 years ago by Buck and Axel of the largest gene family of receptors in the vertebrate
genome seem to hold the promise of a molecular understanding of olfaction, all the way from the peripheral
detection to perception. While that has been harder to realize than we had optimistically expected, it has opened
up new sets of questions that are in many ways yet more interesting. The earliest ideas were understandably
that olfaction is based on a combinatorial code of relative simplicity since it there were so many receptors. There
would be no need for primarys or fundamentals as in other systems. The explanation would be molecular rather
than psychological. However over the past few years it has become clearer that while a combinatorial
mechanism is at work for discriminating the large and diverse odor environment, understanding the formation of
an odor perception or appreciating how odors are processed at higher levels cannot be explained entirely on a
the basis of combinations of activated odor receptors. Even with such large numbers. Thus there is a renewed
interest in discovering possible primary odors or, more likely, primary chemical structures or characteristics. One
method for doing this is by applying medicinal chemistry approaches to organizing odors by biological response
properties than by chemical classification schemes. The focus remains on the receptors, but as biologically
specialized chemical detectors. The incomparably large number of odor receptors and ligands provides a unique
perspective for understanding the dynamics of chemical coding in a way that the pharmacology of single
receptors with limited ligands cannot.
PA01-2 How the identification of odorant receptors changed our concept of olfactory glomeruli
Charles Greer
Departments of Neurosurgery and Neuroscience, Yale University, School of Medicine, New Haven, USA
Monro, in a 1783 address to the Royal College of Physicians of Edinburgh, described the approach of the
olfactory nerve to a “cineritous bulb, [situated] within the cranium, and which is evidently of the [same] texture
with the cortical matter of the brain.” However, a first description of olfactory glomeruli awaited the application of
microscopy. Sanders, in an 1882 issue of the Philosophical Transactions of the Royal Society, credits Leydig
with the first description and naming of glomeruli in his 1852 monograph on “Mikroskopischen Anatomie.” The
current anatomy of the glomerulus was presented by Golgi in 1874 followed by Cajal’s confirmation in 1894.
Beginning with the electrophysiological studies of Adrian in the ‘50s, the idea of odor-specific functional
localization in the olfactory bulb began to emerge. The notion was later supported in the late ‘70’s with the first
application of the then new 2-deoxyglucose technique showing that different odors induced spatially distinct
puncta of 2DG accumulation that corresponded to glomeruli. Within 3 years of the identification of the large
family of odor receptors (ORs) by Buck and Axel (1991) we saw the first molecular confirmation that glomeruli
were specific for OR expression based on in situ analyses of OR mRNA (Ressler et al., 1994; Vassar et al.,
1994). In the 25 years since the ’91 report interest has focused on the characterization of individual glomeruli for
odor responses using increasingly sensitive measures of functional/synaptic activity and on understanding the
molecular mechanisms that mediate the convergence of axons expressing the same OR into glomerular-specific
fascicles. I will review some of the most essential steps in the evolution of our conceptual model of glomeruli,
with apologies to those whose work I am unable to review due to time limitations, and propose some of the
questions that will frame our future studies.
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PA01-3 Deciphering ligand-receptor pairs in the olfactory and vomeronasal systems
Kazushige Touhara
Department of Applied Biological Chemistry, The University of Tokyo, Japan
After the discovery of the olfactory receptor gene family by Buck and Axel in 1991, the functional proof had not
been obtained for several years. In 1998-1999, various ligand-receptor pairs began to be reported by using
either homologous or heterologous expression system. The odorant-binding site was then revealed by computer
model-based mutagenesis studies, providing direct evidence for the stereochemical receptor theory. Unlike
vertebrate olfactory receptors that are G protein-coupled, insect odorant or pheromone receptors turned out to
be a ligand-gated heterodimeric channel, which has raised interesting evolutional questions. Another
chemosensory pathway called the vomeronasal system exists in mice to detect various substances utilized for
intra- or inter-species communication. Mainly, two types of G protein-coupled receptors are expressed; the
rhodopsin-type V1R family that recognize small semiochemicals, and the Class-C type V2R family that
recognize peptide or protein cues such as ESP1. In this talk, I will overview our work in the last two decades on
olfactory and vomeronasal receptor biochemistry including our recent progresses, and discuss what we do not
know about chemosensory receptors.
PA01-4 Retargeting olfaction
Peter Mombaerts
Max Planck Research Unit for Neurogenetics, Germany
It has been known for 20 years that odorant receptors (ORs) are mechanistically involved in determining the
positions of the sites of coalescence of axons of the OR-expressing olfactory sensory neurons (OSNs), the
3,600 glomeruli in the olfactory bulb. A model has been proposed whereby OR-derived cAMP signals, rather
than direct action of OR molecules, determine the target destinations (glomeruli) of OSNs in the bulb.
Specifically, the model proposes that anterior-posterior patterning of glomeruli is mediated by OR-derived cAMP
signals that determine the expression levels of neuropilin-1 (Nrp1) in OSN axon termini, forming a gradient of
Nrp1 from anterior-low to posterior-high. We have tested this Nrp1 model for anterior-posterior patterning of
glomeruli, based on the well-characterized mouse OR M71 and with publicly available gene-targeted strains. M
71 glomeruli reside posteriorly in the bulb and are strongly Nrp1+. Our results do not support the generality of the
model of Nrp1 levels patterning glomerular positions in an anterior-posterior axis. We suggest that the validity of
this model is, thus far, restricted to a microdomain of the bulb, wherein a gradient-like pattern of Nrp1 levels can
be discerned along a selected anterior-posterior axis over a row of 20 glomeruli. Combined with our analyses of
Nrp1 levels in 3D reconstructed bulbs by serial two-photon tomography and a survey of the literature on Npr1
patterns in the bulb, we conclude that it is unlikely that Nrp1 levels determine glomerular positioning along an
anterior-posterior axis for all 3,600 glomeruli of the bulb. In any case, OR-specific glomeruli show considerable
positional variability.
PA01-5 Odorant receptor expression is perturbed in mice following recovery from genetically-mediated
lesion
Jessica Brann1, Xinmin Zhang2, Eleonora Spinazzi3, Claire Franklin1, Lis Paulina1, Natasha Kharas3, Clara Altomare3,
Stuart Firestein3
1
Department of Biology, Loyola University Chicago, USA, 2BioInfoRx, USA, 3Department of Biological Sciences, Columbia
University, USA
A repository of basal neural stem cells in the olfactory epithelium (OE) generates excitatory projection neurons
that extend long axons from the OE to the olfactory bulb. Each sensory neuron expresses a single odorant
receptor (OR), conferring an identity required for odorant detection and appropriate axon targeting to the OB. It
has long been known that these stem cells generate sensory neurons, but their ability to faithfully recapitulate
the OE after lesion is relatively unexplored. In particular, aging may alter OR gene choice in newborn neurons.
Here we probe the ability of the stem cell to generate a diverse array of sensory neurons expressing the
appropriate repertoire of odorant receptors in aged animals. To this end we generated a line of mice, iDTR+
OMPCre/+, whereby a Cre-mediated excision of a STOP cassette renders mature neurons sensitive to
diphtheria toxin (DT) via activation of the DT receptor. This method permits a specific and reversible ablation of
mature (OMP-expressing) neurons upon DT administration. We administered either DT or saline to male mice of
several age groups (2-18 months) for six days. RNAs were harvested 30 days following ablation, to allow for
2016.05.27 16.58.58 Page 6(1)
recovery of the OE. Results reveal that age does not affect the cohort of OR genes expressed following recovery
from lesion in DT injected mice. In addition we observe that age does not affect OR expression in saline injected
mice. However, the OR repertoire does significantly change following ablation and these effects are observed at
all ages tested. These results provide evidence that the regenerative potential of the neuronal stem cell is not
altered by age per se, as a wide array of sensory neurons are generated. However, lesion induced ablation of a
large number of sensory neurons disrupts the typical OR expression patterns observed in intact mice.
PA01-6 Transcriptomic deconstruction of mammalian olfaction: From human to mouse.
Luis R. Saraiva1,2,3, Ximena Ibarra-Soria2, Fernando R.M. Aguilera2, Mona Khan4, Laura Van Gerven5, Mark Jorissen5,
Peter Mombaerts4, John C. Marioni2,3, Darren W. Logan2,6
1
Department of Experimental Genetics, Sidra Medical & Research Center, Qatar Foundation, Qatar, 2Wellcome Trust Sanger
Institute, Wellcome Genome Campus, UK, 3European Bioinformatics Institute (EMBL-EBI), European Molecular Biology Laboratory,
Wellcome Genome Campus, UK, 4Max Planck Research Unit for Neurogenetics, Germany,
5
Department of ENT-HNS, Belgium, 6Monell Chemical Senses Center, USA
The importance of sensing the molecular environment is reflected in the genetic investment in encoding olfactory
receptors (ORs), which constitute the largest mammalian gene family. The OR repertoire is largely
species-specific, and shaped by the nature and necessity of chemosensory information for survival in each
species’ niche. In addition to differences in the ORs, the morphology, size, neural projections and organization of
chemosensory epithelia vary remarkably across mammals, suggesting differences in wider gene expression
networks. By combining RNA-seq with FACS in a hierarchical fashion from whole olfactory mucosa (WOM) to
single olfactory sensory neurons (OSNs), we have identified the complete transcriptional profile of mouse OSNs,
and their heterogeneity at the single cell level. But 25 years after the discovery of the ORs, the interspecific
molecular heterogeneity of the olfactory system still remains largely unknown. To study the evolutionary
dynamics of gene expression in the olfactory system among species with different chemosensory niches, we
performed RNA-seq of the WOM of six species of rodents, carnivores and primates (including humans). Our
comparative transcriptome-wide analysis reveals a high degree of molecular conservation across 95 million
years of mammalian evolution. We found that ORs are expressed across a large dynamic range in these six
species. RNA abundances correlate well with the number of OSNs expressing an OR. Combining RNA-seq with
a phylogeny-based method that classifies ORs into orthologous gene groups (OGGs), we found that
phylogenetic conservation does not imply conservation in OR gene expression: we find numerous examples of
highly-expressed ORs specific for a single species or order. Of the 73 highly conserved one-to-one OGGs, one
only case was the OR orthologue highly expressed in all species analysed. This experimental strategy has
identified OR genes that may have been selected for different niches, contributing to a better understanding of
the evolution of olfaction.
Parallel Symposium II: Recognizing taste: Coding along the neural axis in mammals
PA02-1 Gustatory responses of taste receptor cells expressing fluorescent proteins in transgenic mice
Ryusuke Yoshida1,2, Keiko Yasumatsu-Nakano3, Keisuke Sanematsu1, Noriatsu Shigemura1, Yuzo Ninomiya1,3,4
1
Section of Oral Neuroscience, Graduate School of Dental Sciences, Kyushu University, Japan, 2OBT Research Center, Faculty of
Dental Science, Kyushu University, Fukuoka, Japan, 3Division of Sensory Physiology, R&D Center for Taste and Odor Sensing,
Kyushu University, Fukuoka, Japan, 4Monell Chemical Senses Center, PA, USA
Taste compounds are detected by taste receptor cells in the oral cavity. When taste receptor cells are activated
by tastants, they elicit action potentials and release transmitters to gustatory afferent axons. During this process,
taste sensitivities of taste receptor cells and connections between taste receptor cells and gustatory nerve fibers
may be key factors to understand coding of taste signals. To know taste sensitivities of taste receptor cells, we
recorded electrical responses to apically restricted taste stimuli from single taste cells expressing fluorescent
proteins in isolated taste buds of transgenic mice. In fungiform papillae, gustducin-GFP taste cells (Type II cells)
responded best to sweet, bitter or umami stimuli. Bitter best taste cells showed specific responses to bitter
compounds whereas a subset of sweet best taste cells also responded to umami and/or salt stimuli.
GAD67-GFP taste cells (Type III cells) responded to sour stimuli and a subset of them showed responses to
multiple electrolytes. These results suggest that the major discrimination of taste qualities could be done among
taste bud cells. Regarding umami taste, T1R1-expressing taste cells may not contribute to discrimination of
umami taste because most of T1R1-mCherry taste cells responded to not only umami but also sweet stimuli. To
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test whether taste receptor cells respond to multiple taste compounds which represent the same taste, we
examined responses of gustducin-GFP taste cells to multiple bitter compounds (quinine, denatonium,
cyclohexamide, caffeine, sucrose octaacetate, tetraethylammonium, phenylthiourea, L-phenylalanine, MgSO4,
and high concentration of saccharine) in fungiform and circumvallate papillae. In both papillae, bitter sensitive
taste cells were classified into several groups, suggesting heterogeneous population of bitter taste cells. Such
heterogeneity may contribute to detection of subtle difference among bitter compounds.
PA02-2 Coding in the taste periphery: To label or not to label?
Stephen D. Roper, Nirupa Chaudhari
Department of Physiology & Biophysics, Miller School of Medicine, University of Miami, USA
A major question in taste research is how gustatory signals are encoded in the periphery and in the CNS. We
now know that there are abundant opportunities for signal processing in sensory end organs, taste buds. Several
transmitters, including ATP, GABA, serotonin, acetylcholine, and norepinephrine are found in taste buds.
Release of these transmitters from taste cells has been measured and many of their postsynaptic targets in taste
buds have been identified. All indications are that there are local feed-forward and feedback circuits in taste
buds, including both excitation and inhibition. Yet, how is the output from taste buds encoded into the trains of
impulses that are transmitted to the hindbrain? One simple concept is “labeled lines” where each gustatory
sensory afferent fiber is dedicated solely to one of the basic tastes, such as a “sweet” fiber or a “bitter” fiber. This
is an extension and narrowing of a concept originating with Rene Descartes in 1680 and later elaborated by
Johannes Müller and Sir Charles Bell in the early 19th century. These early scientists attempted to explain how
sensory signals reached the brain and evoked perceptions. More recent research in sensory neurobiology, and
in taste in particular, indicates that coding is more complex than simple labeled lines. Instead of information
being carried in single afferent axons, combinatorial activation of several neurons seems to be involved to code
taste. Additionally, in certain regions of the nervous system the temporal pattern of action potentials seems to
code taste. My presentation is designed to review and explore these questions and the research that has been
directed to their answers.
PA02-3 Encoding taste and food in the hindbrain through cooperative activity in a variety of cell types
Patricia M. Di Lorenzo1, Josua D. Sammons1, Jonathan D. Victor2
1
Department of Psychology, Binghamton University, USA, 2Departmetn of Neurology, Weill Cornell Medical College, NY, USA
Information about food processed by the mouth arrives in the nucleus of the solitary tract (NTS) and, in rodents,
is relayed to the parabrachial nucleus of the pons. Many decades of analyses of recordings from anesthetized
rodents have produced evidence for both rate and temporal coding of taste information. More recent recordings
from awake, freely licking rats has uncovered a rich variety of cell types in addition to those that respond to the
sensation of taste. These include cells with firing pattern closely related to the lick pattern. Moreover, many of
those cells that appear to be specialized to convey taste information also respond to or are modified by olfactory
stimuli, suggesting that complex stimuli, such as actual foods, are more effective than pure chemicals at driving
cells in these brainstem nuclei. In all, the patterns of firing in the NTS and PbN point to the integration of the
sensory and motor components of taste. Layered onto this ongoing sensorimotor integration is the influence of
centrifugal input, which may refine the neural code and modify performance in taste-related tasks. In all, we
suggest that the variety of responses types and influences contained in brainstem taste-related nuclei position
them as pivotal nodes in the neural circuit regulating ingestion.
PA02-4 Spatial organization of complex taste responses in gustatory cortex
John Boughter, Max Fletcher
Department of Anatomy & Neurobiology, University of Tennessee Health Science Center, USA
Previous approaches towards understanding the topography of taste responses in gustatory cortex (GC) have
yielded often-disparate conclusions, with evidence for both clusters of homogenous, narrowly-tuned taste cells,
as well as regions where the representation of primary taste qualities overlap. To shed additional light on this
issue, we conducted two-photon (2P) imaging of cortical neurons in anesthetized mice in response to basic taste
stimuli, and asked the following questions: 1. Is the representation of taste quality heterogeneous within an
anatomically defined area of GC. 2. Does the taste quality representation stay constant throughout various
depths of GC. GC was defined by injecting an anterograde tracer into the taste-responsive region of the
2016.05.27 16.58.58 Page 8(1)
thalamus (VPMpc). For 2P imaging of taste responses, we injected AAV1.GCaMP6 into the GC. Focusing on an
area of cortical surface just posterior to the middle cerebral artery, we recorded taste responses from over 740
neurons in 7 mice. Over 70% of cells responded significantly to more than one basic tastant (NaCl, sucrose,
citric acid and quinine). However, a non-biased, hierarchical cluster analysis revealed the existence of multiple
cell types, including those possessing robust responses to single taste qualities and only smaller sideband
responses (narrowly tuned cells), whereas other cells responded strongly to multiple tastants (broadly tuned
cells). In terms of topography, cells responding best to particular primary tastes were found interspersed in all
areas, regardless of depth within layer 2/3 or position relative to cortical surface. This study suggests the
presence of a fairly large area of GC where primary taste representation overlaps, a finding consistent with some
earlier physiological and intrinsic imaging studies.
PA02-5 Cortical population coding of taste decisions and orofacial behavior
Donald B. Katz
Volen Center for Complex Systems, Brandeis University, Waltham, MA, USA
It has long been known that CNS neural responses provide information about the identity of tastes on the
tongue. Our lab has revealed, however, that taste identity is only the first aspect of a more complex temporal
code, documenting that in awake rats information reliably related to taste palatability emerges in gustatory
cortical (GC) responses about 500 msec after identity-related information. These results suggest that GC neural
populations, far from simply coding what the taste IS, may process that information to output an action (e.g.,
accept or reject) code. Here we present evidence that this is in fact the case, showing that: 1) population coding
analysis allows us to identify both WHETHER the rat will gape in response to taste stimulation and precisely
WHEN (r=0.75) that gape will occur, in single trials; 2) using cuing to manipulate the latency of gaping has an
almost identical impact on neural coding; and 3) optogenetic silencing of GC neurons changes the likelihood of
gaping. We conclude that GC is a part of a distributed system responsible for transforming an incoming identity
code into a taste decision, and discuss how our results relate to classic findings in the taste field.
Parallel Symposium III: Insect taste: Behavioral plasticity and evolution
PA03-1 Starvation-induced depotentiation of bitter taste in Drosophila
Emily LeDue, Kevin Mann, Ellen Koch, Bonnie Chu, Roslyn Dakin, Michael Gordon
University of British Columbia, Canada
Animals display dramatically different responses to food depending on their satiety state-a food that is normally
rejected may be accepted following prolonged nutrient deprivation. In flies, this behavioral change depends on
reciprocal sensitization and desensitization of sweet and bitter taste, respectively. However, the mechanisms for
bitter taste modulation remain unclear. Here, we identify a pair of octopaminergic/tyraminergic neurons, named
OA-VLs, that directly modulate bitter sensory neuron output in response to starvation. OA-VLs contact bitter
sensory neuron terminals and potentiate their taste-evoked calcium response. Starvation reduces OA-VL
activity, thereby diminishing bitter sensitivity under nutrient-poor conditions. Moreover, artificial silencing OA-VL
activity in fed flies phenocopies the sensory and behavioral effects of starvation, suggesting that OA-VLs
underlie a critical step in bitter taste modulation. We propose that OA-VLs are the missing link between
previously identified peptidergic regulators of bitter sensitivity and the observed physiological changes in taste
sensory neurons.
PA03-2 Sugar-aversion: Polymorphism of the peripheral gustatory system drives adaptive foraging
behavior in the German cockroach
Ayako Katsumata, Jules Silverman, Cby Schal
Department of Entomology and W.M. Keck Center for Behavioral Biology, North Carolina State University, USA
In response to strong selection with insecticidal baits containing glucose as a phagostimulant, populations of the
German cockroach have rapidly evolved a novel behavior, aversion of glucose, which is highly adaptive because
cockroaches avoid toxic baits. Previously we reported that the glucose-averse (GA) trait is heritable and
electrophysiologically demonstrated that bitter-responding gustatory receptor neurons (GRNs) in the mouthparts
of GA cockroaches acquired a new function―they detect glucose as a bitter compound, while the GRNs of wild-
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type (WT) cockroaches process glucose as a phagostimulant. We hypothesized that polymorphism of the
peripheral gustatory system may be responsible for the evolution of other adaptive feeding behaviors. We
screened field-collected cockroaches for aversive responses to other sugars and demonstrated
electrophysiological changes in their gustatory function in fructose-averse cockroaches. Survivorship of
sugar-averse cockroaches was significantly higher than WT roaches under the strong selective pressure of
sugar-containing toxic baits, demonstrating the fitness value of sugar-aversion. Finally we compared GA and WT
cockroaches in olfactory associative learning experiments using general food odors. Although chocolate is
innately preferred over vanilla by both WT and GA cockroaches, GA roaches rapidly learned to associate the
odor of chocolate with glucose (as a deterrent) and avoided chocolate in foraging preferences. When GA
roaches received two types of conditioning―vanilla odor paired with fructose (reinforcement) and chocolate odor
paired with glucose (punishment)―avoidance of the chocolate odor was enhanced. The avoidance response
was observed for three days after one-hour of self-training. On the other hand, food odors paired with glucose
positively reinforced the food preferences of WT roaches. The results demonstrate that the sugar-aversion traits
impact not only ad-hoc food choices, but also olfactory learning and foraging behaviors. We suggest that local
selection pressures drive polymorphisms in peripheral gustatory function that contribute to the evolution of
locally adaptive foraging behaviors.
PA03-3 Internal nutritional requirement modulates feeding behavior in Drosophila
Teiichi Tanimura, Naoko Toshima
Department of Biology, Kyushu University, Japan
Animals need to intake appropriate nutrients to maintain their life. Recent studies demonstrate that insects can
be used as an excellent model to explore neuronal and molecular mechanisms involved in feeding regulation of
homeostasis. Amino acids are important nutrients for all animals. Our work showed that the adult fruit fly,
Drosophila melanogaster, prefers to ingest amino acids when they were deprived of them (Toshima & Tanimura,
2012). How can animals detect and regulate amino acid intake? Not only vertebrates, but also insects, have a
decision-making process that determines the intake of appropriate nutrients to maintain health. To identify the
gustatory receptor neurons required for amino acid feeding responses, we performed two-choice preference
tests using several mutants and transgenic flies. Our findings suggest that several different parts sets of
gustatory neurons are involved in amino acid sensing and feeding response depending on the internal nutritional
state.
PA03-4 The sweet tooth of the bumblebee: The temporal pattern of firing of sugar-sensing neurons
encodes concentration
Geraldine Wright, Ashwin Miriyala, Sebastien Kessler
Institute of Neuroscience, Newcastle University, UK
Bees and other insect pollinators have mouthparts are specialized consuming sugary solutions such as floral
nectar. Bees prefer to drink solutions high in sugar concentrations but the composition of nectar varies in
concentration over a range from 8-70% (wt/vol). We predict, therefore, that the gustatory neurons in the sensilla
of bees should be sensitive to changes in sugar concentration and capable of detecting sugars over a large
range. Here, we report electrophysiological recordings of the population of neurons housed in the galeal sensilla
of the buff-tailed bumblebee, Bombus terrestris. The gustatory neurons of B. terrestris begin responding to 2.5-5
mM solutions of sucrose. The detection threshold for fructose was between 5-10mM and for glucose was
between 10-25mM. Surprisingly, stimulation with sucrose solutions of greater than 50mM produced a distinctive
bursting pattern of firing in two neurons in each sensillum, in which a single action potential fired by one neuron
silences the rapid firing of the other for a 20 ms period of inhibition in a cycles of 40 Hz. This pattern of bursting
activity was also observed when galeal sensilla were stimulated with >50mM fructose solutions, maltose and
melezitose, but only rarely with glucose or other sugars. Interestingly, this response was completely ablated by
stimulation with a sucrose solution containing 1 mM quinine. Thus we show that the identity and concentration of
sugars and the presence of toxins is represented by a temporal pattern of firing of the gustatory neurons from the
mouthparts and propose that the brain uses this information to guide feeding behaviour.
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PA03-5 Interactions in Drosophila taste decoding
Young Taeg Jeong, Seok Jun Moon
Department of Oral Biology, Yonsei University College of Dentistry, South Korea
Feeding is one of the most important animal behaviors. Taste is a major player in the regulation of feeding
behavior, which allows animals to discriminate nutritious foods from toxic substances in their natural
environment. In actual life, food contains multiple compounds rather than pure chemical. Thus, taste receptor
cells for different taste modalities are activated at the same time. In addition, a variety of sensory information
such as olfaction, texture, and temperature is also involved in the identification and evaluation of the food. Over
the last few decades, there has been much progress in our understanding how animals can taste single taste
modality in peripheral and central levels. However, relatively little is known about how taste perception changes
when multiple taste stimuli are present together or when taste is combined with other sensory modalities
Drosophila taste a diversity of molecules which mediate stereotypical behaviors by activating taste receptor
cells. Combination with the molecular, genetic, calcium imaging and electrophysiological approaches available
in Drosophila offers an ideal opportunity to study these interactions in taste. I will present two examples showing
how taste-taste interactions and taste-mechanosensory interactions change animal feeding behavior.
Parallel Symposium IV: Digitizing olfaction
PA04-1 Using an effective algorithm for predicting odor similarity from odor structure in order to
explore the boundaries of olfactory stimulus space
Noam Sobel
Department of Neurobiology, Weizmann Institute of Science, Israel
Several studies have linked physicochemical properties to discreet perceptual properties of monomolecules.
The real olfactory world, however, is made not of monomolecules, but rather of molecular mixtures. To predict
how such complex odor mixtures might smell based on their structure alone, in three separate experiments we
asked 139 subjects to rate the pairwise perceptual similarity of 64 odorant-mixtures ranging in size from 4 to 43
monomolecular components. We then tested alternative models to link odorant-mixture structure to odorantmixture perceptual similarity. Whereas a model that considered each monomolecular component of a mixture
separately provided a poor prediction of mixture similarity, a model that represented the mixture as a single
structural vector provided consistent correlations between predicted and actual perceptual similarity (r>0.49,
p<0.001). An optimized version of this model yielded a correlation of r=0.85 (p<0.001) between predicted and
actual mixture similarity. In other words, we developed an algorithm that can look at the molecular structure of
two novel odorant-mixtures, and predict their ensuing perceptual similarity. This published algorithm was
developed and tested under conditions of equated perceptual intensity of all mixture components. Here I will first
present an extension of this effort, adding a factor that allows prediction on mixtures with components of varying
intensities, with marginal reductions in performance (r=0.67, p<0.001). I will then demonstrate how this algorithm
provides for a prediction on the total number of odorants humans can discriminate, a question that has recently
captured the attention of olfaction research. Finally, I will point to the major gaps still remaining in our
understanding of olfactory stimulus space.
PA04-2 Human olfactory interfaces
Takamichi Nakamoto
Precision and Intelligence Laboratory, Tokyo Institute of Technology, Japan
Man-machine interface related to olfaction is called human olfactory interface. It mainly consists of odor sensing
system as machine input and olfactory display as machine output. An odor sensing is typically composed of an
array of sensors with partially overlapping specificities. Here an odor sensing system robust against
environmental change is explained. Moreover, a palm-top odor sensing module is presented. An olfactory
display is relatively new technology. Since an olfactometer is often used to investigate human behavior under
olfactory stimuli, it is huge and expensive. Olfactory display is a cheap and tiny gadget easy to use for everyone.
It can blend multiple ingredients at arbitrary ratio. Two types of olfactory displays such as those based upon
solenoid valves and SAW (Surface Acoustic Wave) device are introduced. Recently we have made a wearable
olfactory display utilizing a latter type of olfactory displays for virtual reality applications. Another topic is to
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explore a set of odor components to cover wide area of scents. The method using mass-spectrum database and
nonnegative matrix factorization method is described. Then, the experiment on odor approximation using odor
components was performed. Moreover, the distance to evaluate the data difference in multidimensional data
space was investigated. The sensory test revealed that we could approximate aromas of essential oils to some
extent if the thirty odor components were used. The final topic is teleolfaction when the olfactory display
generates scents classified by an odor sensing system remotely located from the olfactory display in real time. A
user can search for the odor source located at remote site by sniffing its smell as if it were located in front of the
user. Since human olfactory interface is not matured in comparison with human interfaces with other senses, a
variety of new technologies related to digitizing olfaction might evolve soon.
PA04-3 Sensing odor mixtures
Terry E. Acree, Geraldine Prevost, Charlotte Maxa, Manon Gros
Food Science, Cornell University, USA
The perception of odor mixtures is shaped by two established ideas. First the number of identifiable component
stimuli is small (1-4) especially during brief encounters. This limitation has been observed many times during the
last 30 years with various olfactory stimuli including cross modal studies with tastants, indicating a top-down
process that is not unique to olfaction (Laing1986, Wilson 2006). Secondly, the number of sensible or key
odorants reported in any given food is less than 230. Although, hundreds of chemicals with odor in the pure state
have been found in foods (Nijssen 1963-2011), less than 1000 have been detected in all foods (Arn 1997,
Dunkle 2014) and less than 10 of these are key odorants in any particular food. Using GCO and a new
psychophysical technology, Sniff Olfactometry (Wyckoff 2016), we have studied the behavior of binary mixtures
of key odorants in 70 ms encounters. Results confirm that odorant mixture sensations are not synthetic but
analytic and that the range of odorant ratios, that are analyzable, is less than 10 to 1. The impact of these results
on ideas of odor image formation will be discussed.
PA04-4 Biosensors based on odorant binding proteins and major urinary proteins for security
applications
Krishna C. Persaud1, Khasim Cali1, Mara Bernabei1, Jimena Ricatti2, Carla Mucignat2
1
School of Chemical Engineering and Analytical Science, The University of Manchester, UK, 2Department of Molecular Medicine,
The University of Padova, Italy
Odorant Binding Proteins and Major Urinary Proteins are small soluble proteins that contain binding sites that
are selective to a range of small molecules. They have high conformational stability, making them of interest as
biorecognition elements for new biosensors. Using in silico mutagenesis and docking screening techniques, the
binding pockets of major urinary proteins from the mouse and wild type mosquito Anopheles Gambiae Odorant
Binding Protein (OBP1) were modified to produce theoretical variants with enhanced sensitivity to selected
explosives. A subset of mutants and wild type proteins were expressed and their binding properties to target
analytes were assessed using a fluorescent competitive binding assay in solution. Some of these proteins do
indeed show enhanced sensitivity compared to wild type proteins. When immobilised on to quartz crystal
microbalances (QCM) it was found that they remain stable for many months and are able to sensitively detect
analytes in the vapour phase. Selected proteins were incorporated into an array of sensors which were tested
against traces of target explosives. During field trials at an airport, the device positively detected traces of TNT,
PETN and NH4NO3. This opens an new approach for designing biosensors that target specific analytes in the
vapour phase.
PA04-5 Mixing molecules
Christophe Laudamiel
DreamAir, NY, USA
Creating a perfume is akin to playing a chord on a piano with 1500 moving keys (the typical size of their palette)
using 100 fingers (the typical number of compounds in a formula). Here, I will discuss how professional
perfumers organize their palettes and will outline several case studies where I attempt to copy a particular odor,
mask a malodor, or replace an ingredient.
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PA04-6 Receptor representations of odorants
Joel D. Mainland
In the current consensus model of the olfactory code, the perception of an odorant molecule depends on which
receptors are activated and to what extent. Currently, however, there is no method for determining olfactory
perception based on receptor activation patterns. We have taken two major approaches to cracking this code.
First, we took advantage of the natural genetic variation in human olfactory receptors to identify the role of a
single receptor in olfactory perception. We identified sixteen different olfactory receptors in nine gene clusters
that are associated with alterations in odorant perception. Second, we used in vitro screening to measure the
receptor activation patterns for odors where pairwise perceptual similarity had previously been measured. A
model that incorporates both molecular structure and receptor activation patterns to predict the perceptual
similarity of odors outperformed the angle-distance model in predicting the similarity of monomolecular odor
pairs (angle-distance model, r=0.32, our model r=0.77). Understanding how receptor responses translate into
perception is a crucial step toward digitizing odors.
Parallel Symposium V: Umami and amino acid tastes: Ligands, receptors, appetites and
perceptions
PA05-1 Taste interaction between umami and bitter or sweet occurring at taste receptors
Mee-Ra Rhyu
Korea Food Research Institute, Korea
Among the five basic tastes, sweet, bitter and umami taste are recognized via the respective G-protein coupled
receptors, T1R2/T1R3, T2Rs and T1R1/T1R3 in taste cells. Unlike the primary mechanisms for individual sweet,
bitter and umami, interactions between these tastes each other remain unexplored in taste receptors. Although
umami peptides are widely regarded as bitter taste suppressants in food consumption, it has not been
demonstrated that bitter taste receptors, T2Rs, interact with umami peptide, which alters binding of bitter
substances. We recently demonstrated the interaction between umami and bitter taste by presenting the effect
of umami peptides on a bitter substance, salicin, in Ca2+-flux signaling cascade using hT2R16-expressing cells.
Five representative umami peptides (Glu-Asp, Glu-Glu, Glu-Ser, Asp-Glu-Ser, and Glu-Gly-Ser) markedly
attenuated the salicin-induced intracellular calcium influx in a time-dependent and a noncompetitive manner,
while Gly-Gly, a tasteless peptide, did not. These results may provide provisional evidence that the umami
peptides affect binding of bitter substances to T2Rs. Interaction between umami and sweet was also observed in
hT1R2/hT1R3-expressing cells. Umami peptides (Glu-Glu and Glu-Asp) significantly attenuated the stimulation
of hT1R2/hT1R3 by sucrose and Ace K binding to the extracellular domain (ECD) of T1R2. However, the
stimulation of hT1R2/hT1R3 by cyclamate interacting with T1R3 transmembrane domain (TMD) was not
inhibited by the umami peptides, while the inhibition was observed in the T1R3 TMD mutant receptor. Taken
together, these suggest that umami peptides prevent binding of sweet ligands to the T1R2 ECD in an allosteric
manner, not to the T1R3. This presentation summarizes our recent studies in this field, which was supported by
the Korea Food Research Institute. Flp-In 293 cells stably expressing hT1R2/hT1R3 and plasmid of hT2R16
were a gift from Dr. Takumi Misaka (The University of Tokyo, Tokyo, Japan).
PA05-2 Umami and the control of appetite
Martin R. Yeomans
School of Psychology, University of Sussex, UK
This talk contrasts two opposing effects of umami on human appetite, evidenced by recent laboratory-based
studies with human volunteers. The immediate impact of umami on appetite is consistent with an appetizer effect
generated by the flavour-enhancing effects of compounds like monosodium glutamate (MSG): hunger ratings
increase after tasting foods made palatable by manipulation of umami taste. Not only does the experience of
MSG in a savoury context enhance immediate liking, it can also enhance liking for flavour components that were
experienced alongside MSG, suggesting that the appetizing effects of umami can be conditioned to associated
flavours. However, the experience of umami during ingestion also has implications for satiety. A key suggestion
2016.05.27 16.58.58 Page 13(1)
is that we have evolved the ability to taste umami in order to sense the presence of free amino acids as an
indication of the likely presence of protein. Accordingly, experience of umami may potentiate the degree of
satiety experienced after consuming protein-enriched foods. A series of studies tested this, with clear evidence
that the addition of MSG both retarded the recovery of hunger after ingesting a protein-enriched low-MSG soup
and increased the accuracy of compensation for the added protein at a subsequent meal. A further study using a
stronger umami taste by combining MSG and IMP found evidence that umami could enhance satiety even after
a low-energy soup, and further confirmed the enhancement of protein-induced satiety. Together these data
provide clear evidence of a dual-effect of umami in control of appetite: both enhanced enjoyment during eating
but, at least where foods are protein-rich, enhanced satiety after ingestion.
PA05-3 Oral and post-oral determinants of umami preference in rodents
Anthony Sclafani
Department of Psychology, Brooklyn College, USA
The preference for umami compounds such as monosodium glutamate (MSG) has been extensively studied in
rodents. Although MSG preference is typically attributed to umami taste, mouse studies reveal an important
post-oral influence on glutamate preference. Naive mice (C57BL/6, B6) show little or no preference for MSG, but
prior MSG consumption enhances glutamate intake and preference. Intake of other nutrients (sugar,
maltodextin, fat) also enhances glutamate preference whereas non-nutritive sweeteners are ineffective. Direct
evidence for post-oral glutamate effects is provided by intragastric flavor conditioning studies. Mice and rats
trained to drink a flavored solution paired with IG infusions of MSG learn to prefer that flavor over a different
flavor paired with IG water infusions. B6 mice also learn to prefer a flavor (e.g., grape) added to an orally
consumed MSG solution. B6 mice missing glutamate taste signaling components (T1r3, Trpm5) are similar to
control mice in acquiring a MSG preference after exposure to post-oral glutamate effects. Thus, these taste
components, which are found in the mouth and gut, do not mediate post-oral glutamate conditioning. In contrast
to B6 mice, other inbred mouse strains (129, FVB) fail to acquire a MSG preference indicating genetic
differences in post-oral glutamate conditioning. Details of the post-oral glutamate detection process and the
nature of the signal involved in learned preferences are uncertain but probably involve gastric and/or intestinal
sensors and vagal transmission.
PA05-4 Amino acids, food processing, and protein appetite
Paul A.S. Breslin
Rutgers University, Monell Center, USA
Foraging omnivores, such as humans and fruit flies, tend to prefer processed foods during critical stages of life.
Processing here refers to cooking, desiccating, and fermenting. In the case of flies, fermentation is the principal
form of food processing encountered, and it is the oldest. These three food processes all share the common trait
of breaking down food macronutrients into component units: fats in fatty acids, starches and sugars into
malto-oligosaccharides and mono-saccharides, proteins into amino acids, and DNA and RNA into
ribonucleotides. These processes are critical for us because large macromolecules tend not to elicit tastes. The
taste intensities of these individual components are indicators of their levels in foods and mark the nutritionally
“essential” building elements for reproduction, development, and growth. Thus, both pregnant and developing
omnivores in need of essential nutrients, such as amino acids, should seek out processed foods high in umami
taste. This talk will connect ’need states’ to the tastes of requisite essential nutrients that are released from their
macromolecules during food processing.
Supported by NIH DC014286.
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PA05-5 Genetics of amino acid taste and appetite
Alexander A. Bachmanov1, Natalia P. Bosak1, John I. Glendinning2, Masashi Inoue1,3, Xia Li1,6, Satoshi Manita1,3,7,
Stuart A. McCaughey1,8, Yuko Murata1,4, Danielle R. Reed1, Michael G. Tordoff1, Gary K. Beauchamp1,5
1
Monell Chemical Senses Center, Philadelphia, PA, USA, 2Department of Biology, Barnard College, Columbia University, New York,
USA,
3
Laboratory of Cellular Neurobiology, Department School of Life Sciences, Tokyo University of Pharmacy and Life Science, Tokyo,
Japan, 4National Research Institute of Fisheries Science, Yokohama, Japan, 5Department of Psychology and School of Veterinary
Medicine, University of Pennsylvania, Philadelphia, PA, USA, 6Cincinnati Childrens Hospital, Cincinnati, OH, USA, 7Department of
Physiology, Division of Medicine, University of Yamanashi, Yamanashi, Japan,
8
Center for Medical Education, IUSM-Muncie at Ball State University, IN, USA
Our studies have shown that inbred mouse strains differ in voluntary amino acid consumption, and these
differences depend on sensory and nutritive properties of amino acids. Like humans, mice perceive some amino
acids as having sweet (sucrose-like) taste and others as having umami (glutamate-like) taste. Mouse strain
differences in the consumption of some sweet-tasting amino acids (D-phenylalanine, D-tryptophan and
L-proline) are associated with polymorphisms of a taste receptor gene, Tas1r3, and involve differential
peripheral taste responsiveness. Strain differences in the consumption of some other sweet-tasting amino acids
(glycine, L-alanine, L-glutamine and L-threonine) do not depend on Tas1r3 polymorphisms and so must be due
to allelic variation of other, as yet unknown, genes involved in sweet taste. Strain differences in the consumption
of L-glutamate may depend on postingestive rather than taste mechanisms. Thus, genes and physiological
mechanisms responsible for strain differences in consumption of each amino acid depend on the nature of its
taste and post-ingestive properties. Overall, mouse strain differences in amino acid taste and appetite have
complex genetic architecture：In addition to the Tas1r3 gene, these differences depend on other genes likely
involved in determining the taste and postingestive effects of amino acids. Identification of these genes may lead
to discovery of novel mechanisms that regulate amino acid taste and appetite.
PA05-6 An examination of the motivational taste properties of L-glutamate in wild-type and T1R1+3
double knock-out mice
Ginger Blonde, Alan Spector
Program in Neuroscience & Department of Psychology, Florida State University, USA
We recently demonstrated that mice lacking either subunit of the L-amino acid receptor T1R1+3 can detect high
concentrations of monosodium glutamate (MSG) in the presence of inosine 5’-monophosphate (IMP, 2.5mM),
with the sodium component reduced by amiloride (A, 100μM). To investigate the hedonic characteristics of this
stimulus complex (M+A+IMP), we tested knock-out (KO) mice missing both T1R1+3 subunits and wild-type (WT,
both n=13) mice in a battery of behavioral tests. Food-deprived mice were first trained and tested in gustometers
with a concentration series of Maltrin-580, a maltodextrin, in a brief-access (10-s trials) test that minimizes
postingestive feedback. Similar tests followed using M+A+IMP and M+A. The brief-access series was repeated
following both short-term (five 30-min sessions) and long-term (48h) exposures to 0.2M M+A+IMP. Additional
brief-access tests were conducted with NaGluconate replacing MSG. Finally, progressive ratio tests (ratio= 3),
which assess appetitive behavior while minimizing satiation, were conducted using 8% Maltrin-580 and 0.4M M+
A+IMP. Although KO mice took significantly fewer licks than WT mice, Maltrin-580 elicited robust concentrationdependent responding in both groups in brief-access tests. In contrast, M+A+IMP elicited very weak licking
responses across the concentration range in WT mice, and KO mice were virtually unresponsive to this stimulus.
Both groups initially showed no concentration-dependent licking to M+A, implicating IMP as an important
component for consummatory responses to M+A+IMP. WT and KO mice initiated a similar number of trials
during MSG tests, and initiated significantly fewer for NaGluconate, suggesting that appetitive (approach)
behavior was maintained by the glutamate anion. In the progressive ratio task, both groups completed a similar
number of ratios for M+A+IMP. Overall, these results suggest that the T1R1+3 heterodimer is necessary for M+A
+IMP to elicit relatively weak taste-guided consummatory licking in brief-access tests, but is unnecessary for
appetitive responses to be expressed to this “umami” stimulus complex in mice.
2016.05.27 16.58.58 Page 15(1)
Parallel Symposium VI: No! Aversion and avoidance of chemosensory stimuli
PA06-1 Ionotropic Receptors mediate sour and fatty acid taste in Drosophila
Hubert Amrein, Yan Chen, Ji-Eun Ahn
Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M University, USA
Adult Drosophila melanogaster exhibit strong appetitive behavior to sugars (their main dietary nutrient) and
low concentrations of NaCl, but they avoid bitter chemicals and highly concentrated NaCl solutions. Drosophila
taste is mediated by Gustatory Receptor Neurons (GRNs) of taste sensilla (corresponding to taste buds), which
are distributed across the labial palps and legs. Each of the four GRNs in a sensillum is thought to be tuned to
one of several different taste modalities: sweet, bitter/high salt, low salt, water. Genetic, physiological and
behavioral studies have shown that sweet taste is mediated by eight Gustatory receptor (Gr) proteins, while
bitter taste is mediated by another, larger group of Gr proteins. Moreover, axons of sweet and bitter GRNs
project to distinct locations in the subesophagal zone in the brain (labeled lines).
Here, we identify two Ionotropic receptors (Ir) genes required for fatty acid and carboxylic acid taste. Ir25a and
Ir76b are co-expressed in up to three GRNs of most sensilla, two of which correspond to the sweet and bitter
GRNs, respectively. Ca2+ imaging experiments show that the third Ir25a/Ir76b expressing GRN, which does not
express any Gr gene, responds to carboxylic acids, responses that are impaired in Ir25a or Ir76b mutant flies.
Moreover, we confirm previous studies showing that sweet GRNs not only respond to sugars, but also to fatty
acids. Intriguingly, these responses are also dependent on Ir25a and Ir76b. Taste behavior and oviposition site
preference assays confirm that both Ir genes are essential in relevant behavioral paradigms. We propose that Ir
25a and Ir76b are common subunits of multimeric, multimodal taste receptors necessary for the perception of
both appetitive and repulsive taste cues. Our findings necessitate a reevaluation of the labeled line theory for
Drosophila taste perception.
PA06-2 Non-gustatory functions of aversive taste receptors: TAS2R-dependent modulation of thyroid
function
Steven D. Munger1,2
1
Center for Smell and Taste, University of Florida, USA, 2Department of Pharmacology and Therapeutics, University of Florida,
USA
Chemosensory receptors couple stimulus detection to cellular responses. But while the stimulus selectivity of a
response is largely dictated by the ligand selectivity of the receptor(s) expressed on that cell, the “meaning” of
the stimulus depends on the identity of the cell or tissue in which it resides. In the gustatory system, type 2 taste
receptors (TAS2Rs) are employed to recognize myriad chemicals that are both typically aversive and which elicit
a bitter taste. However, these G protein-coupled receptors have also been found in gastrointestinal,
cardiovascular, reproductive and endocrine organs, where their non-gustatory functions remain poorly
understood. We will discuss the TAS2Rs, their contribution to thyroid function, and their potential as novel
therapeutic targets for thyroid disease. Supported by the National Institute on Deafness and Other
Communication Disorders and the University of Florida.
PA06-3 Parallel, but convergent, olfactory subsystems that mediate innate avoidance
Frank Zufall
Department of Physiology, University of Saarland, Germany
This lecture will present neural strategies for the detection and avoidance of chemostimuli that signal potential
danger through the mouse olfactory system. Specifically, we will focus on recent evidence supporting a novel
“multi-subsystem-parallel-processing” model underlying the recognition and avoidance of innately aversive odor
cues. During the past decade, we have made significant progress in elucidating the molecular signatures,
physiological mechanisms and behavioral roles of several olfactory subsystems. However, little is still known
about how olfactory information encoded by these olfactory subsystems is integrated in the brain to evoke a
coherent behavioral response. Here, we will discuss the evidence that olfactory information driving innately
aversive behaviors is conveyed by multiple olfactory subsystems that function in parallel and that this information
is integrated, at least in part, by specific neural circuits in the brain.
2016.05.27 16.58.58 Page 16(1)
PA06-4 Htr2a-expressing cells in the central amygdala control the hierarchy between innate and
learned fear
Reiko Kobayakawa, Ko Kobayakawa
Kansai Medical University, Japan
Fear is powerful behavior-determining emotion induced by innate and learned mechanisms. Both these fear
mechanisms involve separate pathways; however, the mechanism of potential integration of these pathways is
poorly understood. In this study, we aimed to decipher the underlying molecular and cellular mechanisms of this
integration process using an olfactory-mediated innate fear versus learned fear paradigm. Notably, prior
presentation of innate fear stimuli inhibited learned freezing response, but not vice versa. Whole-brain mapping
and pharmacological screening indicated that serotonin-2A receptor (Htr2a)-expressing cells in the central
amygdala (CeA) control both innate and learned freezing, but in opposing directions. In vivo fiber photometry
analyses in freely moving mice indicated that innate but not learned fear stimuli suppressed the activity of
Htr2a-expressing CeA cells. Artificial inactivation of these cells upregulated innate freezing response and
downregulated learned freezing response. Thus, Htr2a-expressing CeA cells serve as a hierarchy generator,
prioritizing innate fear over learned fear.
PA06-5 Identification of a specific area of olfactory cortex involved in stress hormone responses to
predator odors
Kunio Kondoh1, Zhonghua Lu1, Xiaolan Ye1, David P. Olson2, Bradford B. Lowell2, Linda B. Buck1
1
Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, USA, 2Beth Israel Deaconess Medical Center and
Harvard Medical School, Boston, USA
Animals sense environmental dangers that challenge their survival and respond with behavioral and
physiological changes to eliminate the challenge. The olfactory system detects chemical cues emitted from
predators that stimulate an instinctive fear response. In addition to characteristic behaviors, this response
includes increases in blood stress hormones that mobilize multiple body systems to escape impending danger.
These are controlled by corticotropin-releasing hormone (CRH) neurons in the hypothalamus of the brain. To
explore the neural circuits that convey predator odor signals from the nose to CRH neurons, we made
neurotropic viruses that allowed visualization of neurons either one or multiple synapses upstream of CRH
neurons. We discovered that neurons presynaptic to CRH neurons are present in multiple areas of the olfactory
cortex (OC), which receives signals from the nose, as well as in vomeronasal amygdala (VA) areas that receive
olfactory information from the vomeronasal organ. However, only one minor area in the OC, the amygdalopiriform transition area (AmPir), contained neurons upstream of CRH neurons that were activated by volatile
predator odors. Chemogenetic stimulation of AmPir activated CRH neurons and induced an increase in blood
stress hormone, mimicking an instinctive fear response. Moreover, chemogenetic silencing of AmPir markedly
reduced the stress hormone response to predator odors without affecting a fear behavior. These findings
suggest that AmPir plays a key role in generating hormonal fear responses to volatile predator cues that signal
danger.
PA06-6 Pharyngeal taste function in mediating feeding behavior in Drosophila
Yu-Chieh Chen, Anupama Dahanukar
Interdepartmental Neuroscience Graduate Program, University of California, Riverside, USA
In Drosophila, sweet and bitter tastes are mediated mainly by gustatory receptors (Grs) expressed in gustatory
receptor neurons (GRNs). GRNs are housed within taste sensilla that are distributed in various body parts,
including the labellum, pharynx, distal segments of the legs (tarsi), wing margins, and ovipositor. Although
extensive studies have described taste coding in the labellum and tarsi, little is known about the molecular and
cellular basis of pharyngeal taste. Given that the pharynx lies in an anatomical position where it may serve as the
last checkpoint for food ingestion, exploration of pharyngeal taste is imperative for a comprehensive
understanding of how gustatory inputs from different peripheral taste organs are integrated and translated into
appropriate feeding behaviors. To specifically study the function of pharyngeal GRNs in feeding behaviors, we
took advantage of pox-neuro (poxn) mutants, in which the external taste bristles are transformed into
mechanosensory bristles but internal pharyngeal taste organs are retained. Using binary choice feeding assays,
we found that poxn mutants are capable of making similar feeding choices as wild-type flies, suggesting that
pharyngeal input is sufficient for driving appropriate behavioral responses. Moreover, poxn mutants show
2016.05.27 16.58.58 Page 17(1)
behavioral responses to a wide range of tastants representing sweet, bitter, salt, amino acid, and acid
categories, suggesting that the pharynx has broad taste receptivity. Notably, the degree of aversiveness of bitter
compounds is correlated with pharyngeal taste sensitivity to these compounds rather than their toxicity.
Together, our results highlight the key role of this organ in food evaluation. Next, we constructed a
Gr-to-pharyngeal GRN map with Gr-Gal4 lines, which revealed a distinct molecular organization as compared to
that observed in external taste organs. By selectively manipulating subsets of pharyngeal neurons, our initial
results reveal several classes of pharyngeal GRNs in mediating attractive and aversive feeding behaviors.
Parallel Symposium VII: Mechanisms of Background Segregation and Source Localization
of Odors
PA07-1 Odor segregation based on temporal stimulus cues in insects
Paul Szyszka1, Alpha Renner1, Christoph J. Kleineidam1, Brian H. Smith2, C. Giovanni Giovanni1
1
University of Konstanz, Germany, 2Arizona State University, USA
Insects primarily rely on olfaction when locating resources such as food or mating partners. Tracking down the
source of an odor poses a particular challenge: air turbulences break odor plumes into intermittent filaments
which intermingle with filaments of background odors. This creates temporally and spatially complex patterns of
different odor concentrations and mixtures. How do insects detect, recognize and find odor sources in such a
complicated odor environment?
Odorants from the same source fluctuate synchronously, whereas odorants from spatially separated sources
fluctuate asynchronously. I will show that the insect olfactory system is capable of resolving fast odor plume
dynamics, which would allow it to exploit this (a)synchrony of odor fluctuations to detect whether odorants
originate from the same or separate sources. Electrophysiological recordings from insect olfactory receptor
neurons show that they (1) can respond to odorants within less than 2 ms (they are fast), and (2) follow repetitive
odorant pulses above 100 Hz (they have high temporal resolution). Calcium imaging in the honey bee antennal
lobe shows that projection neurons can resolve few millisecond asynchrony in the arrival of two odorants; and
behavioral experiments in honeybees show that the asynchrony between odorants facilitates perceptual odor
segregation.
PA07-2 Processing a dynamic odor in a noisy chemical environment
Jeff Riffell
University of Washington, Department of Biology, USA
Insects use their sense of smell to locate hosts, mates and food resources from long distances, but little is known
about how they are able to discriminate their target scent from a melange of other volatiles. Here, we measured
the plume from Datura wrightii flowers, a nectar resource for Manduca sexta moths, and show that the scent was
dynamic and rapidly embedded among background volatiles. The moths ability to track the odor was dependent
on the background and scent frequency. By influencing the balance of excitation and inhibition in the antennal
lobe, background volatile altered the neuronal representation of the target scent and the ability of the moth to
track the plume. These results show that the mix of odors present in the environment influence the pollinator’s
olfactory ability.
PA07-3 Odor mixture decomposition by mice and machines
Venkatesh N. Murthy, Dan Rokni, Alexander Mathis, Vikrant Kapoor, Matthias Bethge
Harvard University, USA
In a cluttered sensory environment, animals must segregate objects of interest from ever varying backgroundsfor example, picking out a single speaker’s voice at a noisy cocktail party. We have been investigating the
algorithmic and neural bases of such figure-background separation in the olfactory system in mice. We have
found that mice can be trained to recognize target odorants embedded in unpredictable and variable background
mixtures with high degree of success (>95%). Performance drops with increasing number of background odors,
and was well predicted by the extent of overlap between the neural representations of the target and the
2016.05.27 16.58.58 Page 18(1)
background odors. Using empirical measurements of glomerular responses to odors, we built a model of mixture
responses that incorporated nonlinear interactions and trial-to-trial variability, and explored potential decoding
mechanisms that can mimic mouse performance when given glomerular responses as input. We found that
linear classifiers with sparse weight vectors could match mouse performance using just a small subset of the
glomeruli (~15). Thus, despite nonlinear interactions and variability in the representations of odor mixtures by
olfactory receptors, a simple multi-glomerular, feedforward approach is sufficient to explain the performance of
mice in a seemingly difficult odor segregation task.
Support: NIH, Marie Curie Fellowship, DFG
PA07-4 Concentration invariant odor identity coding
Dmitry Rinberg
Neuroscience Institute, New York University, USA
Invariant object recognition is one of the basic properties of all sensory systems, including olfaction. Humans can
identify smells independent of their concentrations and presence of background odors. Here we propose that
concentration invariant odor identification can be achieved if one assumes that only a small population of first
activated receptor neurons fully define an odor identity. To test this novel computational principle, which we call
‘primacy coding’ we performed optogenetic masking experiments, and demonstrated the relevance of short
temporal interval at the beginning of the sniff cycle for odor identification. We propose a simple model how a
neural code responsible for primacy coding can be formed in the nose and and can be read by the cortex. I will
discuss relevance of the primacy coding hypothesis coding for known olfactory psychophysical phenomena,
including odor background separation problem.
PA07-5 A specific subtype of olfactory bulb interneurons is necessary for odor detection and odorbackground segregation
Hiroo Takahashi1, Sei-ichi Yoshihara1, Yoichi Ogawa2, Ryo Asahina1, Tatsuro Kitano1, Akio Tsuboi1
1
Laboratory for Molecular Biology of Neural System, Nara Medical University, Japan, 2Department of Physiology I, Nara Medical
University, Japan
Neural circuits that undergo reorganization by newborn interneurons in the olfactory bulb (OB) are necessary for
odor detection, odor-background segregation, olfactory memory and innate olfactory responses, including
predator-avoidance, food-finding and sexual behaviors. The OB possesses a battery of interneurons, including
various types of granule cells (GCs); however, the contribution that each type of interneuron makes to the control
of olfactory behavior remains unknown. Here, we investigated the in vivo functional role of oncofetal trophoblast
glycoprotein 5T4, a regulator for the dendritic arborization of 5T4 -expressing GCs (5T4 GCs), which is reduced
in its knockout OB (Yoshihara et al , J Neurosci, 32;2217, 2012). Electrophysiological recordings with acute OB
slices indicated that external tufted cells (ETCs) can be divided into two types, bursting and non-bursting.
Optogenetic stimulation of 5T4 GCs revealed their connection to both bursting and non-bursting ETCs, as well
as to MCs. Interestingly, in 5T4 knockout mice, non-bursting ETCs received fewer inhibitory inputs from 5T4 GC
dendrites than in the wild type, while bursting ETCs and MCs received the similar inputs from 5T4 GCs,
respectively. We are in the process of recording the excitatory inputs into 5T4 GCs in 5T4 knockout OB,
compared with the wild type. Notably, in olfactory behavior tests, 5T4 knockout mice had higher odor-detection
thresholds than the wild type, and were impaired in odor-discrimination learning, such as segregating the food
odor from the irrelevant background odor for finding food. Taken together, our findings suggest that loss of 5T4
attenuates inhibitory inputs from 5T4 GCs to non-bursting ETCs, thus probably leading to superfluous firing of
the ETCs and consequent disturbances in olfactory behaviors. Hence, we demonstrated for the first time that
among various types of OB interneurons, the 5T4 GC subtype is required for odor-detection and discrimination
behaviors (Takahashi et al , revised).
2016.05.27 16.58.58 Page 19(1)
PA07-6 Automatic orientation of spatial visual attention from lateralized olfactory-trigeminal
stimulations
Matthieu Jeremiah Ischer1,2, Sylvain Delplanque1,2, Myriam Esselier1,2, Christelle Porcherot3, Nadine Gaudreau3,
Christophe Mermoud4, Isabelle Cayeux3, David Sander1,2
Swiss Center for Affective Sciences (CISA), University of Geneva, Switzerland, 2E3Lab, Department of Psychology, University of
Geneva, Switzerland, 3Firmenich, S.A., Switzerland, 4Department of Medicine, University of Geneva, Switzerland
1
Almost all perceived volatile compounds stimulate both the main olfactory system and the trigeminal system.
Through the trigeminal system, it is possible to intentionally pay attention to a scent and spatially localize it, but
whether a scent is capable of automatically orienting spatial attention intermodally remains unknown. Yet, i)
there is a common pool of attentional resources shared across sensory modalities, ii) the trigeminal system
enables humans to differentiate a stimulation delivered to the left or the right nostril, and iii) strong trigeminal
stimulations create alert responses and can thus be considered relevant stimuli, which are particularly prone to
capturing an individual s attention. Based on these findings, we predicted that lateralized olfactory trigeminal
information would automatically guide visual spatial attention. In four studies, we used CO2-a gas considered to
be a pure trigeminal stimulant-as lateralized cues in a variant of the visual spatial cueing paradigm. Participants
were required to detect as fast and as accurately as possible the orientation of a visual target following an
olfactory trigeminal cue. In valid trials, cues and targets were presented on the same side (e.g., cue in left nostril
and target in the left visual field), whereas in invalid trials, they were presented on opposite sides (e.g., cue in the
left nostril and target in the right visual field). We also varied the delay between the cue and the target in the 4
different studies. As predicted, an intermodal effect was observed: reaction times in valid trials were faster than
invalid trials. We also found that this effect varied as a function of the delay between the olfactory cue and the
visual target. This series of studies demonstrates, for the first time to our knowledge, the automatic orientation of
visual spatial attention from olfactory-trigeminal stimulation.
Parallel Symposium VIII: Structure-function relationships of olfactory and taste receptors
PA08-1 Towards a structural understanding of taste receptors
Simone Nicole Weyand
University of Cambridge, UK
The biology and molecular mechanism of taste receptors has been extensively studied during recent years,
however their molecular structures still remain unknown at the atomic level. In particular the taste recognition of
many more different bitter or sweet substances with only a much smaller, limited number of receptors is still a
conundrum. A high resolution structure could solve these mysteries and shed light into the structure function
relationship of taste receptors and the code which enables us to recognize the countless bitter taste compounds.
Several pre-settings like low affinity ligands have caused major bottlenecks in addressing X-ray crystallographic
studies. In a multidisciplinary collaborative effort we try to overcome these bottlenecks. We aim to tackle the high
resolution structure of bitter taste receptors and initial work with different complementary and multidisciplinary
approaches towards their structure determination has started. Techniques involving computational and
mutational analysis, biochemical and biophysical characterization will allow a holistic understanding of the
structure-function relations of these receptors but also it will facilitate to tackle their structure.
PA08-2 Mammalian odorant receptors: Heterologous expression and deorphanization
Hiro Matsunami
Duke University, USA
Impressive progress in membrane biology has revealed important relationships in G-protein coupled receptor
(GPCR) structure and function, including dynamic interactions with agonists and antagonists at the atomic level.
Despite this progress, understanding of the largest family of GPCRs, the odorant receptors (ORs), remains
elusive. To date, there is no crystal structure of any ORs and many remain orphans without known ligands. Both
of these challenges must be overcome in order to understand the molecular and biophysical mechanisms of
olfaction. Poor expression of ORs in heterologous cells represents a major challenge in expressing sufficient
quantities of functional OR proteins for purification and structure determination. We have identified the RTP
family of accessory proteins that facilitate OR cell surface expression in heterologous cells. However, the
2016.05.27 16.58.58 Page 20(1)
expression levels of ORs are generally lower than canonical GPCRs. A transcriptomic analysis of RTP1/2
knockout mice has revealed that the majority of ORs are downregulated, whereas a subset of ORs are
upregulated, suggesting differential dependence on the RTPs. When tested in heterologous systems, this small
subset of ORs demonstrate robust expression at the cell surface similar to canonical GPCRs. Therefore, we
propose that these ORs serve as good candidates for large scale production. Similarly, we have developed a
new strategy that enables comprehensive screening of ORs in freely behaving animals by odor stimulation. We
combine phosphorylated ribosomal protein S6 immunoprecipitation with next generation sequencing to profile
OR expression in active olfactory sensory neurons. Our method is capable of not only identifying a repertoire of
odorant-OR pairs, but also reveals the most robust and sensitive ORs. This deorphanization is key to
understanding structure-function relationships of odorant-OR interactions. Furthermore, identification of high
affinity ORs for odorants can serve as model systems for detailed biophysical analysis as well as forming a basis
for future development of OR-based chemical sensors.
PA08-3 Bitter taste receptors, nature’s flexible sensors for changing chemical environments
Maik Behrens1, Kristina Lossow1, Stefanie Nowak1, Antonella Di Pizio2, Anat Levit2, Masha Y. Niv2, Wolfgang Meyerhof1
1
Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Germany, 2Institute of Biochemistry, Food
Science, and Nutrition, Robert H. Smith Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem, Israel
Recognition of potentially dangerous bitter substances is mediated by a set of G protein-coupled taste receptors
belonging to the taste 2 receptor family (TAS2R). Interestingly, expression of these receptor genes is not
restricted to the oral cavity, but extends to numerous tissues including respiratory and gastrointestinal mucosae,
brain, and heart to name just a few. In order to match the large number and chemical diversity of bitter
compounds vertebrates possess in general several, sometimes even dozens of bitter taste receptor genes, of
which some facilitate the detection an enormous variety of chemicals. In humans there are 3 of such broadly
tuned receptors that disproportionally contribute to the chemical space recognized as bitter. Similar experiments
performed with receptors of other vertebrates revealed that such generalist receptors also exist in other species.
By a combination of functional assays in mammalian cell lines, point-mutagenesis, and in silico modeling we
have characterized the binding pockets of the 3 most broadly tuned bitter taste receptors, the TAS2R10, TAS2R
14, and TAS2R46. These experiments revealed the existence of single orthosteric agonist binding pockets,
which may be combined with transient vestibular binding sites, as well as a balanced relationship between
tuning breadth and receptor sensitivity. The focus of this presentation will be on the architecture of the binding
pockets of the broadly tuned receptors and the consequences for agonist selectivity, receptor activation, and
receptor evolution.
PA08-4 Interaction of the human T1R2 taste receptor ligand-binding domain with sweeteners and
sweet-tasting proteins
Anni Laffitte1, Fabrice Neiers1, Anne Brockhoff2, Wolfgang Meyerhof2, Loic Briand1
1
Centre des Sciences du Gout et de l’Alimentation, Universite de Bourgogne, France, 2Department of Molecular Genetics, German
Institute of Human Nutrition Potsdam-Rehbruecke, Germany
Humans perceive sweet-taste through a unique heterodimeric receptor composed of two class C G-protein
coupled receptors (GPCR), T1R2 and T1R3. Both of these subunits possess a large extra-cellular domain,
composed of the N-terminal domain (NTD) followed by the cysteine-rich domain that links the NTD to the
heptahelical transmembrane domain. Cellular assays, molecular docking and site-directed mutagenesis studies
have revealed that the NTD of T1R2 (T1R2-NTD) contain the primary binding site for most of the sweet ligands
including natural sugars and, artificial and natural sweeteners including sweet-tasting proteins. To elucidate the
contribution of T1R2-NTD to sweet tastant binding, we recombinantly expressed human T1R2-NTD in
Escherichia coli. Then, we tested T1R2-NTD for its ability to interact with known sweet tasting ligands using
intrinsic tryptophan fluorescence. To validate the method, the same experiment was performed with two T1R2NTD mutants (D278A and E382A) known to abolish the response of the full-length receptor. We then measured
the interaction of T1R2-NTD with the two recombinant sweet-tasting proteins, brazzein and monellin, using BioLayer Interferometry (BLI). This optical technique analyzes the signal variations in the interference pattern
generated from visible light reflected from an optical layer and a biolayer containing the immobilized protein of
interest. This method is powerful for studying protein-protein interactions and measuring both affinity constants
and kinetic parameters. BLI experiments demonstrated that T1R2-NTD binds these two sweet-tasting proteins
with affinities values in agreement with the physiological range. This strategy will be useful to decipher the
2016.05.27 16.58.58 Page 21(1)
essential role of T1R2-NTD in the detection of sweet tastants including sweet-tasting proteins.
PA08-5 Taste substance binding to the ligand-binding domains of T1r taste receptor heterodimer
Atsuko Yamashita1, Eriko Nango2, Shuji Akiyama3, Saori Maki-Yonekura2, Yuji Ashikawa2, Yuko Kusakabe4,
Elena Krayukhina5, Takahiro Maruno5, Susumu Uchiyama5, Nipawan Nuemket1, Koji Yonekura2
1
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Japan, 2RIKEN SPring-8 Center,
Japan, 3Research Center of Integrative Molecular System, Institute for Molecular Science, Japan, 4National Food Research
Institute, Japan, 5Graduate School of Engineering, Osaka University, Japan
Sweet and umami taste substances are perceived by T1r taste receptors expressed in taste buds. In human, the
heterodimer of T1r2 and T1r3 recognizes sugars, while the heterodimer of T1r1 and T1r3 recognizes
L-glutamate. T1r proteins possess a large ligand-binding domain (LBD) at the extracellular side. The T1r LBDs
are response for ligand recognition, binding, and resultant receptor activation, and thus play crucial roles for
taste receptor functions. So far, preparation of the purified sample of T1r LBDs have been hampered by
difficulties in heterologous expression, and thus the detailed analyses of structure and protein-ligand interactions
have been impractical. To overcome the bottlenecks, we extensively screened the expression conditions for T1r
LBDs, and found a combination of specific T1r genes and an expression host, amenable to large-scale
expression and sample preparation. The purified T1r LBD proteins are found to form a stable heterodimer,
analyzed by analytical ultracentrifugation and electron microscopy. We confirmed that the sample retains the
ability of ligand binding, by thermodynamic analysis. The Förster resonance energy transfer analysis indicated
the conformational change of T1r LBD heterodimer upon taste substance binding. Solution X-ray scattering
analysis of T1r LBD heterodimer in the presence and absence of a taste substance revealed two different
conformational state, a compact one and a spread one, which may correspond to distinct T1r receptor functional
states.
PA08-6 Molecular Dynamics simulations reveal the ligand-dependent activation of hOR7D4
Claire A. de March1, Elise Bruguera1, Jeremie Jacques Topin2, Hiroaki Matsunami1, Jerome Golebiowski2
1
Duke University Medical Center, USA, 2Institut de Chimie de Nice, UMR CNRS, Universite de Nice Sophia Antipolis, France
The complexity of the odor chemical space and the large number of odorant receptors (ORs) associated to their
combinatorial activation make understanding odor coding an enormous challenge. More specifically, being able
to predict the behavior of an OR in front of an agonist, an antagonist or a non-agonist remains to be done.[1]
Using a joint approach combining molecular modeling and experimental data on several ORs[2], we have built a
model that can capture the active or inactive state of these proteins when bound to ligands with different
potencies.[3] The methodology is illustrated on a challenging case. By the aim of computational tools combined
with site directed mutagenesis, we predict the activation of human OR7D4 by its strong agonists, androstenone
and androstadienone, and its inactivated form by a non-agonist the (Z)-2-decenal.[4] These models allow us to
investigate the activation mechanism of other mammalian olfactory receptors from the binding cavity to the G
protein binding site, revealing the network of conserved amino acids involved in ORs activation. Such powerful
approaches will help unravel odor-coding in the nervous system and facilitate the understanding of general rules
of neuronal activation induced by an odor. [1] C. A. de March, S. Ryu, G. Sicard, C. Moon and J. Golebiowski,
Flavour and Fragrance Journal 2015, 30, 331-410. [2] C. A. de March, S.-K. Kim, S. Antonczak, W. A. Goddard
and J. Golebiowski, Protein Science 2015, 24, 1543-1548. [3] C. A. de March, Y. Yu, M. J. Ni, K. A. Adipietro, H.
Matsunami, M. Ma and J. Golebiowski, Journal of the American Chemical Society 2015, 137, 8611-8616. [4] A.
Keller, H. Y. Zhuang, Q. Y. Chi, L. B. Vosshall and H. Matsunami, Nature 2007, 449, 468-U466.
Parallel Symposium IX: Future strategies for the assessment and treatment of smell and
taste disorders
PA09-1 Clinical value of olfactory molecular imaging
Hideaki Shiga, Takaki Miwa
Otorhinolaryngology, Kanazawa Medical University, Japan
Currently, there are no accepted prognostic markers that can guide the selection and treatment of patients with
olfactory dysfunction, including those with idiopathic dysosmia. We show the prognostic value of migration of
2016.05.27 16.58.58 Page 22(1)
nasally administered thallium-201 to the olfactory bulb in patients with olfactory disorders treated with the
traditional Japanese herbal (Kampo) medicine (tokishakuyakusan). Log-rank tests were performed to assess the
relationship between percentage migration of nasally administered thallium-201 to the offactory bulb and the
duration until recovery of odor recognition. The duration until recovery of odor recognition was significantly
shorter in the patients with high nasal thallium-201 migration to the olfactory bulb than in those with low nasal
thallium-201 migration to the olfactory bulb (P=0.008). Nasal thallium-201 migration to the olfactory bulb, a
measure of olfactory nerve damage, could be used to predict the prognosis of olfactory-impaired patients in the
treatment of the tokishakuyakusan.
PA09-2 Quantifying taste deficits and recovery, a new tool
Annick Faurion1, Patrick Mac Leod2, Pierre Antoine Bastian3
1
NEURO-PSI NBS, France, 2Institut du Gout, France, 3Myrobotics, France
Electrogustometry (EGM) allows iontophoretic application of the cations of the subject s saliva onto small tongue
loci. It checks the taste sensory pathway, allows distinguishing taste and somatosensory modalities, explores
independently the territories of taste nerves. Reversing the polarity of electrodes allows perceiving a sweet taste
when a sweet anion is introduced at the electrode-tongue interface: the response is due to chemical stimulation,
not to electrical stimulation. Both cathodal and anodal EGM elicit chorda tympani (CT) responses in rodents.
With EGM, we demonstrated taste deficits due to chemotherapy, or to manipulating the CT nerve during
stapedectomy in the middle ear, and to follow recovery over time. We showed dental desafferentations increase
detection thresholds in tongue loci which depend on the location of the desafferented teeth and found a
CT-lingual convergence on NTS second order neurons. Deficits measured in tobacco smokers recovered after
quitting, with a significantly higher percent of success for patients participating in the experiment. We have
recently developed an easy to use new electrogustometer. Digital, pocket size, bluetooth connected, telephone
or tablet controlled (android software), it includes the automatic monitoring of the stimulation, data acquisition
and calculation of the result. Three different protocols of stimulation are available: the usual standard single shot
trial; an automatic staircase procedure (Dixon, 1948, 1965) applied to a monadic stimulation leading to a
detection threshold calculated at P 0,05; the same staircase protocol applied to a blind and randomized paired
comparison between the appropriate stimulating current and a null current. Data are easily transferred to a
computer. This tool can be used by Medical Doctors, Dentists, ENTs, Oncology services, etc. in order to check
for deficits so as not to let patients in denial and confirm eventually a future recovery. It is also used in quitting
tobacco and in prevention programs.
PA09-3 Electrophysiological recordings from the tongue for the objective evaluation of individual
variations of 6-n-propylthiouracil (PROP) sensitivity
Melania Melis1, Giorgia Sollai1, Danilo Pani2, Piero Cosseddu2, Roberto Crnjar1, Annalisa Bonfiglio2,
Iole Tomassini Barbarossa1
1
Department of Biomedical Sciences, University of Cagliari, Italy, 2Department of Electrical and Electronic Engineering, University of
Cagliari, Italy
Several studies have focused on identifying the factors that lead to divergent conclusions about the role of the
PROP phenotype in taste perception, food preferences, and nutrition. One of the major issues is the difficulty in
obtaining an objective measure of the taste phenotype. Here we present direct measures of the gustatory
system activation, by electrophysiological recordings from the tongue, in thirty subjects classified by PROP
taster status and genotyped for TAS2R38. The potentials generated by PROP stimulation were recorded by
means of differential electrophysiological derivations between two silver electrodes, one in contact with the
ventral surface of the tongue and one in perfect adhesion with the dorsal surface. The signals were recorded by
a polygraph for human use and visualized in real time on PC. The electrotastegrams (ETGs) were analyzed by
Clampfit 10.0 software and validated by comparing the voltage amplitude of signals with the perceived PROP
bitterness intensity, PROP taster status and TAS2R38 genotypes. The ETGs showed that PROP stimulation
evoked negative monophasic potentials characterized by a fast initial rise followed by a slow decline. These
potentials represent the measure of the summated voltage change resulting from the response of stimulated
taste cells. The amplitude of potentials was highly correlated with the perceived PROP bitterness intensity
(p<0.0001), with voltage reaching as high as 120 mV in super-taster who were homozygous for the tasting
variant (PAV) of TAS2R38. The depolarization amplitude, as well as depolarization rate, were associated with
PROP taster status and TAS2R38 diplotypes. The direct relationship between the potentials recorded and the
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parameters defining PROP phenotype show that ETG is a simple and reliable technology for the quantitative
measure of the peripheral taste function, which may find application as an objective investigation tool in studies
aimed at evaluating eating behavior and taste function impairment.
PA09-4 Control of local inflammation ameliorates olfactory system recovery following head injury
Mohammed Omar Al Salihi1, Masayoshi Kobayashi1, Kengo Tamari1, Tomotaka Miyamura1, Kazuhiko Takeuchi1,
Richard M. Costanzo2
1
Department of Otorhinolaryngology-Head and Neck Surgery, Japan, 2Department of Physiology and Biophysics, Virginia
Commonwealth University School of Medicine, USA
The olfactory system has a remarkable capacity for neural regeneration and recovery following injury. Clinically,
however, the prognosis of olfactory dysfunction by head injury is reported to be poor. We investigated factors
that influence the degree of recovery. Using transgenic (OMP-tau-lacZ) mice, we studied mild and severe
olfactory nerve transection (NTx) injury models.. Histological assessment was made for regenerating olfactory
nerves (ON), astrocytes and macrophages. With mild injury we observed less injury-associated tissue and more
ON regeneration. At 42 days recovery, more astrocytes and macrophages were observed in the severe injury
model. Dexamethazone sodium phosphate (DXM)-injected mice, anti-interleukin-6 receptor antibody-injected
mice and tumor necrosis factor-alpha blocker-injected mice with severe injury all showed less injury-associated
tissue, better ON recovery and fewer astrocytes and macrophages. These results indicate that ON recovery
depends on the severity of injury and that treatment with anti-inflammatory drug is effective in improving recovery
outcome during the acute phase of head injury. Patients with head injury usually notice their olfactory
dysfunction several weeks or months after the injury, which may be a factor in poor recovery outcomes.
Therefore, we examined the effect of anti-inflammatory treatment at different time points. In the severe injury
model, DXM injection was started at 7, 14, 28 and 42 days after NTx injury. DXM treatment was effective when
initiated at 7 days but ineffective 14 days or longer after head injury. These findings suggest that different
therapeutic strategies for inhibition of inflammation are needed for traumatic olfactory dysfunction in the chronic
phase of recovery. Finally, we confirmed clinical application of these results by administering steroids to patients
with olfactory dysfunction due to head trauma in the acute phase, resulting in good outcomes.
PA09-5 Trans-nasal electrical stimulation of the olfactory bulb
Eric H. Holbrook
Department of Otolaryngology, Massachsetts Eye & Ear Infirmary, Harvard Medical School, USA
Our ability to provide prognostic information and therapeutic intervention for human olfactory disorders outside of
inflammatory causes is severely limited. Scientific advancement has added greatly to our understanding of the
physiology of the sense of smell from stimulus coding to the regeneration of the olfactory epithelium. However,
the basic pathophysiology underlying the most common forms of smell loss experienced by humans is unknown,
contributing to our inability to develop effective intervention strategies. As science advances, there is an
expectation that medical therapies will eventually follow. Taking the lead from strategies developed in restoration
of hearing, the olfactory system may also benefit from artificial electrical stimulation and restoration of smell
caused by a variety of etiologies. This presentation will review the history of artificial stimulation of the olfactory
system in humans and will describe a pilot study exploring the potential for future implant therapy. In addition,
the stimulation technique described may be useful in identifying central (olfactory bulb and above) vs. peripheral
(epithelial) damage that could further classify olfactory disorders and direct useful intervention.
PA09-6 A novel method to restore the sense of smell: The olfactory implant system
Daniel H. Coelho1,2, Richard M. Costanzo1,2
1
Department of Otolaryngology - Head & Neck Surgery, Virginia Commonwealth University School of Medicine, USA,
Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, USA
2
The projection of odorant receptors onto glomeruli in the olfactory bulb provides a framework for the spatial
mapping of odors in the olfactory bulb. This odor mapping is essential for normal olfactory function and the
perception different of different odors. Using a rat model we recorded the responses from an array of 32
electrodes positioned at different locations within the olfactory bulb. We measured different spatial patterns of
neural activity for different odors (odor maps). Direct stimulation of the olfactory bulb with electrical current
pulses from electrodes positioned at different locations were also effective in generating spatial patterns of
neural activity. These findings provide the foundation for the development of a new device (Olfactory Implant
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System) that could restore the sense of smell following olfactory nerve injury. By programming an array of
stimulating electrodes, similar to programming a cochlear implant, it should be possible to selectively activate
different regions of the olfactory bulb and generate unique patterns of neural activity that lead to the restoration
of smell perception. Although in the early prototype stages, the ongoing development of the Olfactory Implant
System (OIS) may ultimately do for acquired anosmia what the cochlear implant has done for sensorineural
hearing loss.
Parallel Symposium X: Neuromodulation in chemosensory pathways
PA10-1 Cortical and neuromodulatory inputs to the mouse olfactory bulb
Markus Rothermel1, Matt Wachowiak2
1
Department of Chemosensation, AG Neuromodulation, Institute for Biology II, RWTH Aachen University, Germany, 2Brain Institute
and Department of Neurobiology and Anatomy, University of Utah, USA
The olfactory bulb (OB) receives centrifugal input from two major types of modulatory systems whose role in
shaping early olfactory processing remains unclear: classical neuromodulatory inputs and cortical inputs. These
systems are thought to differ substantially, with respect to their general mode of action, and the behavioral
paradigms under which they are engaged.
We found that neuromodulatory cholinergic projections from the horizontal limb of the diagonal band of broca
(HDB) regulate OB output by increasing the spiking frequency of mitral/tufted cells, the principal OB output
neurons. This modulation was rapid and transient. Cholinergic enhancement of mitral/tufted cell odorant
responses was robust and occurred independent of the strength or even polarity of the odorant-evoked
response, indicating that cholinergic modulation adds an excitatory bias to mitral/tufted cell responses as
opposed to increasing response gain or sharpening response spectra. These results are consistent with a role
for the HDB in dynamically regulating the sensitivity to or salience of odors during active sensing.
Additionally we investigated projections to the OB from the anterior olfactory nucleus (AON), a major source of
cortical feedback to the OB. We expressed GCaMP selectively in AON projection neurons and imaged
fluorescence signals from their axon terminals in the OB. Surprisingly, odorants evoked large signals that were
transient and coupled to odorant inhalation both in the anesthetized and awake mouse, suggesting that
feedback from AON to the OB is rapid and robust across different brain states. The strength of AON feedback
signals increased during wakefulness, suggesting a state-dependent modulation of cortical feedback. Finally, we
demonstrated that AON feedback projections were also activated when stimulating other neuromodulatory
centers-for example, the HDB. Our results point to the AON as a multifunctional cortical area that provides
ongoing feedback to the OB and also serves as a descending relay for other neuromodulatory systems.
Supported by DFG (MR) and NIDCD (MW).
PA10-2 Neuromodulation of olfactory bulb activity driven by neuroendocrine hormones and dietinduced obesity
Debra Ann Fadool1,2,3, Genevieve Ann Bell1,2, Kassandra Lynn Ferguson1,2, Kristal R. Tucker1,2, Nicolas Thiebaud1,2
1
Department of Biological Science, The Florida State University, USA, 2Program in Neuroscience, The Florida State University,
USA, 3Institute of Molecular Biophysics, The Florida State University, USA
Energy homeostasis is achieved through a coordinated regulation between the peripheral organs and the brain.
Mice challenged with long-term high-fat diets exhibit a loss of olfactory sensory neurons, a reduction in
glomerular projections, an associated reduction in electro-olfactogram amplitude, and olfactory dysfunction in
odor discrimination and odor reversal learning assessed by olfactometry. We currently hypothesize that the
olfactory system is designed to encode external and internal chemical information, the latter being energy
important molecules that modulate mitral cell firing frequency to give information about the state of nutrient
availability. In this symposium, we will present electrophysiological evidence for the modulation of the primary
output neurons of the olfactory bulb by insulin, glucose, and glucagon-like peptide-1 (GLP-1) by targeting the
voltage-dependent potassium channel, Kv1.3. Using an acute, ex vivo slice preparation of the olfactory bulb
(OB), we will present the distribution and regulation of excitability by insulin and glucose via insulin receptor
kinase and glucose transporter 4 signaling pathways. Using a transgenic mouse line with the preproglucagon
promoter (PPG) upstream of a YFP reporter, we characterized a unique class of deep short axon cells that may
be the source of GLP-1 peptide in the OB. GLP-1 and its synthetic agonist Exendin-4 increase firing frequency of
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mitral cells in a dose- and Kv1.3-dependent manner. Immunocytochemical, RT-PCR, binding assays, and
tracking of fluorescently-conjugated exendin approaches provide parallel lines of evidence for the expression of
the GLP-1R on mitral cells. Using an optogenetic activation of the ChR gene in PPG+ neurons contained in
progeny where recombination was successful based upon Cre/lox technology, we discovered a novel
microcircuit involving the deep short axon cells, granule cells, and mitral cells. Uniquely, the circuit uses
glutamatergic synaptic transmission, rather than inhibitory interneurons to regulate the excitability of mitral cells.
PA10-3 Surprise and expectation modulate early olfactory processing in mice
John P. McGann, Lindsey A. Czarnecki, Cynthia D. Fast, Marley D. Kass, Michelle C. Rosenthal
Behavioral & Systems Neuroscience, Psychology Department, Rutgers University, USA
Olfactory signaling in the brain is shaped not only by the external odor stimulus but also by the organism’s prior
knowledge of the sensory world. This knowledge can include the relative frequency that odors are encountered
in the environment, the significance of olfactory stimuli, and the relationships between odors and other sensory
stimuli. By using this knowledge, the olfactory system can potentially adapt to different behavioral or sensory
circumstances by incorporating expectations into its analysis of ongoing sensory input. Information on odor
frequency is available locally within the olfactory system and appears to be directly encoded by the olfactory
sensory neurons (OSNs) themselves. However, incorporating information about the relationships between odors
and behavioral outcomes or stimuli in other sensory modalities requires descending connections from other
brain regions, including neuromodulatory input. This talk will present neurophysiological evidence that these
types of information are also incorporated into early olfactory processing, including changes in OSN and
periglomerular cell activity. For instance, odor-cued fear learning causes OSN synaptic terminals in the olfactory
bulb to become selectively hyper-responsive to the threat-predictive odorant. Similarly, establishing and violating
expectations about stimulus sequence can strongly impact the early olfactory system’s response to odors. Using
optical neurophysiological techniques in gene-targeted mice, we have found that certain expectation violations
induce a burst of activity in GABAergic interneurons that suppresses the synaptic output of OSNs via GABAB
receptor-mediated presynaptic inhibition. This information can occur even in the absence of peripheral sensory
input and reflects a change in neuromodulatory state. The incorporation of stimulus contingency information into
peripheral sensory processing is unexpected and may help to relate sensory processing to higher-level cognitive
functions like attention and memory retrieval.
PA10-4 Balancing the robustness and efficiency of olfactory representations during learning
Takaki Komiyama, Monica W. Chu, Wankun L. Li
University of California San Diego, USA
Sensory representations are dynamically shaped with experience and learning. Using longitudinal imaging of
mitral cell ensemble activity in mice learning an odor discrimination task over a week, we asked how odor
representations in the olfactory bulb are modified by discrimination learning. When mice were trained with two
very dissimilar odors, they mastered the task within tens of trials on the first day and maintained a high level of
performance throughout the week of experiments. During this period, mitral cell ensemble responses to the two
odors gradually sparsened and became less distinct. In contrast, when mice were trained with two very similar
odors, the performance of the mice gradually improved over the course of 3-4 days. In this version of the task,
mitral cell ensembles initially responded to the two odors in a highly overlapping fashion but these responses
gradually decorrelated with learning. These results suggest that odor representations are flexibly adjusted to
balance the robustness of odor identification and coding efficiency depending on the behavioral requirements.
PA10-5 Trigeminal modulation of olfactory signal processing
Federica Genovese, Hanke Gwendolyn Bauersachs, Ines Graesser, Janina Kupke, Marion Thews, Frank Moehrlen,
Stephan Frings
Department of Molecular Physiology, University of Heidelberg, Heidelberg, Germany
Olfactory perception can be described as the integration of olfactory and trigeminal systems, as most odors can
act as irritants, and almost every irritant has an odor. Peptidergic trigeminal fibers have been found in close
association with the olfactory system. In the main olfactory epithelium, fibers that contain calcitonin gene-related
peptide (CGRP) are found among olfactory sensory neurons (OSNs) and between epithelial supporting cells.
CGRP inhibits the odor response of OSNs, an effect that may cause the reduced odor perception during
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trigeminal co-stimulation. In the olfactory bulb, the function of CGRP-positive fibers is not well understood. We
performed a study of trigeminal innervation of the olfactory system and traced the trajectory of the ethmoid
branch of the trigeminal nerve from the trigeminal ganglia through the main olfactory bulb to the nasal cavity. A
detailed examination of the trigeminal innervation of the glomerular layer revealed a close proximity of
CGRP-positive fibers with juxtaglomerular interneurons that expressed calbindin, calretinin or tyrosine
hydroxylase, but not those expressing parvalbumin. Serial section of the mouse olfactory bulb showed that half
of all glomeruli showed this pattern of innervation and may be considered possible targets for trigeminal
neuromodulation. CGRP release from bulbar fibers was induced by agonists of TRPA1 and TRPV1. Results of
functional studies suggested that CGRP influenced signal processing in the bulb through an inhibitory effect on
periglomerular microcircuits. This became evident through a CGRP-induced reduction of c-Fos immunosignals
in interneurons of the glomerular layer. Furthermore, the spontaneous activity pattern of mitral cells, the output
neurons of the olfactory bulb, was altered upon application of CGRP to acute slices. Our data point to a
modulatory role of trigeminal neuropeptides in the first steps of olfactory signal processing.
PA10-6 Differential serotonergic modulation across the main and accessory olfactory bulb
Zhenbo Huang1, Nicolas Thiebaud1, Debra Ann Fadool1,2
1
Program in Neuroscience, Department of Biological Science, The Florida State University, USA, 2Institute of Molecular Biophysics,
The Florida State University, Florida, USA
Because mitral cells of the main olfactory bulb (MOB) typically project one apical dendritic tuft into a single
glomerulus, whereas those of the accessory olfactory bulb (AOB) extend several apical dendrites to multiple
glomeruli, differential anatomical features across the two systems may lead to functionally distinct information
coding. Recent studies have demonstrated that MOB vs. AOB mitral cells have distinct intrinsic membrane
properties but differential neuromodulation has not been well explored. Herein, we investigated a
widely-distributed CNS modulator, serotonin (5-HT), for its ability to modulate the biophysical properties of mitral
cells, using an in vitro , brain slice approach in postnatal 15-30 day mice. Using a whole-cell configuration in
current-clamp mode, bath application of 20 μm 5-HT elicited one of three responses for MOB mitral cells,
namely, cells were either excited (73%, n=115), inhibited (9%, n=15), or exhibited a mixed response-first
inhibition followed by excitation (12%, n=19). For the prevailing excitatory responses, 5-HT elicited a 4.6±3.1
(Mean±SD) fold increase in firing frequency, which could be blocked by 5-HT2A receptor antagonist spiperone.
In contrast, mitral cells of the AOB were predominantly inhibited by 5-HT (83%, n=95). For these inhibitory
responses, 83% appeared to be indirectly inhibited through 5-HT2 receptors on GABAergic interneurons based
upon gabazine sensitivity-a GABAA receptor antagonist-and 17% were insensitive to gabazine, suggesting
involvement of 5-HT1 receptors via direct inhibition. These data suggest that serotonergic pathways may be
largely excitatory for MOB mitral cells (membrane capacitance, 117.1±24.2 pF; input resistance, 80.6±21.7
MΩ) but predominantly inhibitory for AOB mitral cells (membrane capacitance, 84.3±29.9 pF; input resistance,
162.4±67.2 MΩ).
Parallel Symposium XI: Chemosensory regulation of energy homeostasis
PA11-1 Circuit logic for sugar sensing
Ivan E. De Araujo
The J.B. Pierce Laboratory, Yale University, USA
The potent reinforcing properties of sugars arise from the complex interplay between gustatory and nutritive
signals. I will address the unique organization of the neuronal circuitry that mediates the relative influences of
taste and metabolism in sugar reinforcement. Up-to-date evidence will be reviewed to support the existence of a
general circuit model where separate populations of dopaminergic neurons encode the gustatory and nutritional
values of sugar. Intriguingly, the same general circuit organization appears to hold for both vertebrate and
invertebrate organisms. This arrangement allows animals to prioritize energy seeking over taste quality, and
implies that specialized subpopulations of dopamine-containing neurons form a class of evolutionary conserved
chemo- and nutrient-sensors.
2016.05.27 16.58.58 Page 27(1)
PA11-2 The sense of smell impacts metabolic health and obesity
Celine E. Riera1,2,3,4, Patricia Follett2,3, Jonathan Halloran1,4, Kevin Tharp5, Courtney M. Anderson5, Andreas Stahl5,
Andrew Dillin1,2,3,4
1
Molecular and Cell Biology, University of California, Berkeley, USA, 2Howard Hughes Medical Institute, 3the Salk Institute for
Biological Studies, USA, 4Glenn Center for Research on Aging, University of California at Berkeley, USA, 5Nutritional Sciences and
Toxicology, University of California, Berkeley, USA
In order to adapt quickly to variations in environmental conditions and maintain global body homeostasis,
mammalian systems have developed neuronal circuits that integrate intrinsic and external signals into adaptive
physiological responses to adjust food intake, energy expenditure and fuel fluxes across different organs.
Particularly, increasingly well characterized neurocircuits within the hypothalamus translate intrinsic information
into autonomic neuronal responses to adapt whole-body energy homeostasis. Surprisingly, less is known about
the effect of upstream sensory inputs on regulatory mechanisms of energy metabolism. Because olfactory
signals can dynamically influence decisions about food preference and feeding behavior, we investigated the
output of manipulating olfactory acuity on energy metabolism. We conditionally ablated mature olfactory sensory
neurons using diphtheria toxin receptor-mediated cell death. Remarkably, mice with decreased olfactory acuity
are resistant to diet-induced obesity accompanied by increased thermogenesis in brown and inguinal fat depots.
Furthermore, acute loss of smell perception after obesity onset not only retards further weight gain, but strikingly
reduces obesity and fat mass. We find that reduction of olfactory input engages central and parasympathetic
nerve activity, resulting in activation of beta-adrenergic receptors on white and brown adipocytes to promote
lipolysis. Taken together, these findings highlight the existence of an active communication from the olfactory
sensory system to the central nervous system, adjusting energy homeostasis to nutrient sensing signals.
PA11-3 Interoceptive sugar sensing by the brain
Monica Dus1,2, Jason Lai1, Holly Mills1, Yangkyun Oh1, Greg S. B. Suh1
1
Skirball Institute, Department of Cell Biology, NYU School of Medicine, USA, 2Department of Molecular, Cellular, and
Developmental Biology, University of Michigan, USA
Sugar in the natural environment can be detected through taste-independent and taste-dependent modalities.
Taste-independent modalities consist mainly of peripheral chemosensory neurons such as sweet taste
receptors, which primarily detect the orosensory value of sugar (i.e. sweetness). My laboratory and others have
shown that there exist taste-independent, internal sensors that detect the nutritional value of sugar in Drosophila
and mammals. However, the identity of the post-ingestive sugar sensor has not been clearly understood. We
recently found that six neurons in the fly brain that produce diuretic hormone (Dh44), a homologue of the
mammalian corticotropin-releasing hormone (CRH), directly detect the nutritional value of sugar. We showed
that DH44 neurons are both necessary and sufficient for selecting nutritive sugars in the two-choice assay and
are activated by nutritive D-glucose, but not by nonnutritive L-glucose in calcium imaging experiment.
Furthermore, we made a surprising observation that artificial activation of DH44 pathway resulted in rapid
extensions of the mouthpart, and frequent episodes of excretion. These actions would facilitate the ingestion and
digestion of nutritive foods. We propose that activation of DH44 pathway leads to a rapid increase of ingestion
and digestion through a positive feedback loop to continue consumption of nutritive foods. Identification and
characterization of the taste-independent sugar sensor in Drosophila would provide a framework to understand
how appetite is regulated by energy needs in normal and eating disorder patients. Given its strong sequence
homology, CRH and its neurons in the hypothalamus may offer similar functions in mammals.
PA11-4 Glucagon like peptide-1, sweet taste and metabolic modulation of peripheral taste information
Shingo Takai1, Noriatsu Shigemura1, Keiko Yasumatsu-Nakano2, Mayuko Inoue2, Shusuke Iwata1,3, Ryusuke Yoshida1,
Robert F. Margolskee3, Yuzo Ninomiya2,3
1
Section of Oral Neuroscience, Graduate School of Dental Sciences, Kyushu University, Japan, 2Division of Sensory Physiology,
Research and Development Center for Taste and Odor Sensing, Kyushu University, Japan, 3Monell Chemical Senses Center, USA
Recent progress in exploring oral taste perception and gut nutrient sensing have revealed that there are a lot of
common molecules are expressed in both taste bud cells and enteroendocrine cells, such as several taste
receptors or glucose transporters, and some gut peptides, including GLP-1 (glucagon like peptide-1), NPY
(neuropeptide Y), and glucagon. It was reported that some of these gut peptides are expressed in specific
clusters of taste cells. However the function of these peptides in peripheral taste system is still largely unclear. In
this study, we report that an insulinotropic gut peptide GLP-1 is expressed in taste cells which possess sweet
2016.05.27 16.58.58 Page 28(1)
taste receptor subunit T1R3, and GLP-1R (GLP-1 receptor) is expressed in gustatory nerve neurons in wild type
mice. Mice genetically lacking of GLP-1R showed reduced neural and behavioral sweet taste responses. GLP-1
is secreted from a subset of sweet responsive cells by apical sweet taste stimulation in a concentration
dependent manner. Moreover i.v. injection of GLP-1 activated a subset of sweet specific single nerve fibers
without affecting those of other taste fibers. This activation by injected GLP-1 was observed also in CT nerve of
ATP receptor knockout (P2X2/3 KO) mice. Finally we found that two single nucleotide polymorphisms of the
human GLP-1R associate specifically with altered sweet taste sensitivity in humans. Altogether, in both mice and
human, GLP-1 may be contributed to sweet taste quality coding and the regulation of feeding behavior.
PA11-5 Transduction of a sense in the gut
Melanie M. Kaelberer1, Diego V. Bohorquez1,2,3
1
Department of Medicine, Duke University, USA, 2Department of Neurobiology, Duke University, USA, 3Duke Institute for Brain
Sciences, USA
Gastrointestinal chemosensation has been studied so far from an endocrine perspective. The reason is that
enteroendocrine cells, the sensory epithelial cells of the gut, were thought to communicate with nerves indirectly,
only through hormones, such as Peptide YY (PYY). However, enteroendocrine cells have the striking features of
epithelial cell transducers: they are electrically excitable, fire action potentials, possess voltage-gated channels,
express synaptic proteins; and recently, we reported that they are innervated by nerves in the small intestine and
colon. We have explored the components and functional connectivity of this novel gut neuroepithelial circuit.
First, to define if peripheral nerves connect with enteroendocrine cells, we used the monosynaptic rabies virus B
19G SADdG-GFP, and developed a complementary transgenic mouse model, PyyCRE_tdTomato_rabG, to
enable cell-specific spread of the virus. We found that when delivered in the lumen of the colon, there was visible
GFP in mucosal and vagal nerves PyyCRE_tdTomato_rabG mice. These data show that colonic
enteroendocrine cells are innervated by vagal nerve fibers. Second, to test neurotransmission in this
neuroepithelial circuit, we developed an in vitro co-culture system using purified enteroendocrine cells and
dissociated vagal nodose neurons. The two cell types connect in 12-36 hours and often remain viable for at least
5 days, showing that this neuroepithelial circuit can be isolated and recapitulated in vitro. Third, we then tested
the possibility of afferent gut-to-brain transduction using whole cell electrophysiology. We discovered that a
stimulus of 10mM of glucose applied to the enteroendocrine cell induces excitatory post-synaptic potentials and
action potentials in the connected neuron. The same stimulus does not activate a nodose neuron by itself. These
findings unveil a gut-brain neuroepithelial circuit with the ability to transduce a chemical sense.
Parallel Symposium XII: Advances in human chemosensory neuroimaging
PA12-1 Using brain-state triggering of taste stimulation to explore gustatory perception in humans
Dana M. Small, Maria G. Veldhuizen
The John B Pierce Laboratory and Yale University, USA
Recent increases in computing power have made it possible to measure, model and display brain response in
real time. We have recently developed a brain-state triggering protocol in which spontaneous intrinsic
fluctuations (SIFs) of activity in taste-responsive regions of the amygdala are measured in real time while
participants are at rest. SIFs have a frequency of less than 0.1 Hz, are of equal magnitude to task or stimulusrelated responses and have reliable spatial correlates across the brain (Fox et al., 2007). Based on amygdala
SIF characteristics we defined low and high state activity levels and used this predefined threshold to trigger
delivery of a taste stimulus to test predictions about the role of the amygdala in taste perception. Specifically, we
propose that the amygdala exerts an inhibitory influence on insular sensitivity to afferent taste signals to create a
gain mechanism to modulate sensitivity to taste. Variations in this central gain mechanism are then predicted to
account for individual differences in taste intensity perception. In support of the model we found that the same
salty stimulus is rated as more intense during low compared to high spontaneous activity and that the difference
in spontaneous activity correlates positively with the ratings. In contrast, although activity in the insular gustatory
cortex co-varied with amygdala response, it was not associated with intensity ratings. Dynamic causal modeling
then revealed that activity in the amygdala causes changes in activity in insula. These results demonstrate a
new method by which fMRI can be used to study taste perception and they provide preliminary evidence for a
central gain mechanism in which the amygdala exerts a tonic inhibitory influence on the primary gustatory cortex
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to modulate overall sensitivity to taste. Supported by R01DC006706.
PA12-2 Dissecting the natural landscape of odors in the human brain
Jay A. Gottfried
Department of Neurology, Northwestern University, USA
This presentation will consider the natural form of odor stimuli in the real-world environment, and how functional
MRI techniques and psychophysical approaches can be used to gain insights about olfactory coding and
categorization in the human brain. Odors naturally lend themselves to the study of naturalistic stimuli, and in
experiments described here, I will illustrate the roles of piriform cortex, amygdala, and orbitofrontal cortex in
extracting olfactory meaning from complex odor stimuli. Our findings demonstrate how discrete network
interactions between these brain areas help sculpt learning-related value and perceptual changes, and together
support a map of olfactory state space.
PA12-3 Dynamic coding of taste categories in the human brain
Kathrin Ohla1, Niko A. Busch2, Sebastien M. Crouzet3
1
Psychophysiology, German Institute of Human Nutrition, Germany, 2Institute of Psychology, University of Muenster, Germany,
Centre de Recherche Cerveau et Cognition, CNRS, University Paul Sabatier, France
3
The repertoire of taste percepts that humans can reliably discriminate consists of five basic qualities: salty,
sweet, sour, bitter, and umami. Specific receptors on the tongue are activated by chemicals signifying a taste
quality before the signal is conveyed to the brain. How this peripheral signal is used by the central nervous
system to encode taste quality is largely unknown. We investigated when and where taste quality
representations are established in the cortex and whether these representations are used for perceptual
decisions. Time-resolved multivariate pattern analyses of head-surface electrophysiological brain responses
evoked while human participants assessed salty, sweet, sour, and bitter tasting solutions revealed that global
neuronal response patterns allow to decode which of tastant participants tasted on a given trial. The onset of this
prediction coincided with the earliest taste evoked response (at 175 ms) originating from the insula and
opercular cortices indicating that quality is among the first attributes of a taste represented in the central
gustatory system. These response patterns correlated with perceptual decisions of taste quality: tastes that
participants discriminated less accurately (i.e. salty versus sour) also evoked less discriminated brain response
patterns. The results therefore provide the first evidence for a link between taste-related decision-making and
the predictive value of these brain response patterns.
PA12-4 Electrophysiological recordings from the olfactory epithelium, activation in the olfactory bulb,
analysis of chemosensory induced EEG activity in the frequency domain, and source-localization of
chemosensory event-related potentials
Thomas Hummel
Smell and Taste Clinic, Department of Otorhinolaryngology, Germany
Human olfactory function can be assessed at various levels. In the periphery the electro-olfactogram can be
recorded which is thought to be the summated generator potential of olfactory receptor neurons; although this
elegant technique has been around for centuries, few labs have made advantage of it, probably because it
requires professional skills with nasal endoscopy. Among others it allows to investigate the hypothesis whether
[olfactory training] induces the expression of olfactory receptors/the growth of olfactory receptor neurons. More
recently, apart from the morphometric assessment, functional activation has been recorded from the next level of
olfactory processing, the olfactory bulb exploiting the FMRI approach. Using specific head coils reproducible
activation can be seen in response ipsilateral olfactory stimulation. At the level of the EEG the [reinvention] of
time-frequency-analyses provides a very practical, [objective]approach for example to the presence or absence
of olfactory function. And finally, source-localization of chemosensory event-related potentials provides a new
approach to the segmentation of brain responses with high temporal resolution.
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PA12-5 Olfactory attention modulates gamma band activity in healthy humans
Archana K. Singh, Masako Okamoto, Kazushige Touhara
Aims: Attention is a cognitive process that requires self-control to focus on the stimuli of our interest. However,
the role of attention is still not clearly established for olfaction. Specifically, there is a lack of information on how
the attention to an odor modulates gamma band activity, i.e., the spectral power in 30-80 Hz frequency band,
which has been linked with the cognitive processing of a stimulus (e.g., in vision). In this EEG study, we
examined the changes in the gamma-band activity during two olfactory tasks with different levels of attention.
Methods: Eighteen healthy subjects participated. The odor of 2-phenylethyl alcohol (a rose-like smell) was
presented to the nose during inspiration using an olfactometer and a breathing sensor. We computed the
gamma band activity from the wavelet-decomposed EEG signals time-locked to the odor stimulus. A bootstrap
test was used for the statistical comparison of spectral-power between attended and ignored odor tasks.
Results: Several instances of significantly enhanced gamma-band synchronization (p<0.05, fdr corrected) were
observed in the early as well as late stages of olfactory neural processing, consistent with the findings from
visual attention studies*. Conclusion: These results lend support to the commonly held notion that attention to a
stimulus enhances its neural processing. As the experimental reports of spectral-power associated with human
olfactory attention are scarce, this study may provide a useful direction for future research on the topic.
Acknowledgement: ERATO Touhara Chemosensory Signal Project from JST, Japan Ref: *Tallon-Baudry,C.,et
al. (2005). Attention modulates gamma-band oscillations differently in the human lateral occipital cortex andf
usiform gyrus. Cereb. Cortex
PA12-6 Defining a human olfactory network based on resting-state functional connectivity
Thomas Campbell Arnold1, Ivan de Araujo2, Wen Li1
1
Psychology, Florida State University, Tallahassee, USA, 2John Pierce Laboratory, Yale University, New Haven, USA
Recent application of resting-state functional magnetic resonance imaging (rs-fMRI) has unveiled several key
inherent networks in the human brain.
While networks for the physical senses (sensorimotor, auditory, and visual) have been defined with this new
technique, networks for chemical senses of olfaction and gustation are still unknown. Here, we explored the
olfactory network by analyzing inter-regional correlation between rs-fMRI timeseries.
Rs-fMRI data (to date, N=229, each containing 4,800 scans) were obtained from a large data set provided by the
Human Connectome Project. We examined regions known to be involved in or related to olfaction, including
orbital frontal cortex (OFC, including anterior, posterior and olfactory OFC), anterior and posterior piriform cortex
(APC/PPC), amygdala, anterior and posterior hippocampus (AH/PH), insula (divided into eight subdivisions to
roughly equate the size to other regions), and thalamus (divided into four subdivisions). Three modules (defined
by strong internal correlations and weak external correlations) emerged from the analysis-a thalamus module,
an insula module and importantly, an olfactory module (encompassing olfactory OFC, piriform cortex, amygdala
and hippocampus). Representing the olfactory network, the olfactory module was characterized by these
specific connections: the APC connection to olfactory OFC and PPC; PPC connection to APC and amygdala;
and amygdala connection to PPC and both AH and PH (p’s=.001 -.05; the other inter-region connections did not
survive t-tests of significance). Besides these intra-module connections, the olfactory module was linked to the
insula via APC and olfactory OFC, which connected with dorsal anterior insula (a region implicated in human
olfaction). Lastly, connection between the olfactory network and the thalamus appeared indirect and was
mediated by the dorsal anterior insula that linked the dorsal medial thalamus with APC and olfactory OFC. Our
findings thus provided insights into the olfactory network while validating current knowledge of olfactory
functional neuroanatomy.
Parallel Symposium XIII: Emerging topics in olfactory sensorimotor behavior
PA13-1 Olfactory neuromodulation of visual behavior in Drosophila
Mark A. Frye
Department of Integrative Biology and Physiology, USA
Like any animal with an image forming eye, flying Drosophila engages powerful behavioral reflexes during flight
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to stabilize its gaze against perturbations in the field of view. This optomotor response is strengthened by the
paired presentation of a food odor, which presumably helps the animal maintain course in an invisible odor
plume. Motion vision is computed by specialized neurons in the optic lobe that detect movement of the visual
panorama, which presumably feed into the optomotor response. One identified motion detecting neuron shows
larger responses when visual stimuli are paired with a food odor. The optic lobe is innervated by octopaminergic
neurosecretory cells. GFP Reconstitution Across Synaptic Partners labels cell membrane contacts between the
motion detecting neuron and octopaminergic cells. Mutating the vesicular monoamine transporter for
octopamine compromises flies ability to track food odor. Flies distinguish salient visual features from the visual
panorama. In flight, they robustly track a single vertical bar, presumably reflecting desirable natural landscape
features such as plant stalks. However, they actively avoid small contrasting objects, presumably reflecting an
approaching threat. Whereas odor does not influence bar tracking, it reverses the aversion to a small object. We
are testing the hypothesis that odor activates neuromodulatory systems within the optic lobe to enhance the
perceptual salience or valence of visual objects. However, optogenetically depolarizing octopaminergic cells has
no influence over bar tracking or object aversion.
PA13-2 Drosophila neuroecology
Marcus Carl Stensmyr
Lund University, Sweden
The sense of smell is not only of pivotal importance to most animals-it also serves as a significant model system
in biological research, in areas ranging from neuronal information processing to developmental biology. In the
past 15 years, great strides in our understanding of how the olfactory system is organized and operates have
been made. Instrumental in these efforts has been work performed in the vinegar fly Drosophila melanogaster.
In fact, for no other complex organism do we have a similar level of in-depth understanding of the olfactory
system’s physiological, molecular and anatomical organization. However, in spite of the wealth of information
gathered, we still do not know the functional significance of the observed organization. How the fly’s olfactory
system, or in fact any other olfactory system, is used to actually decode the chemical environment remains
unknown. My lab combines neurophysiological, molecular, phylogenetic, behavioral, ecological and chemical
approaches in order to pinpoint specific neural circuits and molecular mechanisms underlying olfactory-guided
behaviors triggered by ecologically relevant odors. I will here outline recent work from our lab aiming at
unraveling the ecological significance of discrete olfactory pathways in the fly.
PA13-3 Olfactory alarm reaction in zebrafish
Yoshihiro Yoshihara
RIKEN Brain Science Institute, Japan
Many olfactory cues pervade the aquatic environment of fish and induce various behaviors important for their
individual survival and species preservation, such as searching foods, escaping from danger, finding potential
mates, and memorizing physiologically important contexts. The zebrafish has become one of the most useful
model organisms in neurobiology. In addition to its general advantageous properties (external fertilization, rapid
development, transparency of embryos, etc.), the zebrafish is amenable to various genetic engineering
technologies including transgenesis, mutagenesis, gene knockout, and transposon-mediated gene transfer. The
zebrafish genome harbors approximately 300 olfactory receptor genes consisting of about 150 ORs, 6 V1Rs, 50
V2Rs, and 100 Taars. One neuron-one receptor rule and glomerular convergence of like axons are essentially
preserved in the zebrafish. A functional odor map is developed on the glomerular array of the olfactory bulb,
based on chemical structures of odorants and pheromones: e.g. amino acids in the lateral glomerular cluster,
bile acids in the dorsal glomerular cluster, and prostaglandin F2alpha in the ventromedial glomeruli. The mitral
cells in the olfactory bulb project axons in different patterns to the four major target areas in the forebrain (the
posterior zone of dorsal telencephalic area, the ventral nucleus of ventral telencephalic area, the right habenula,
and the posterior tuberculum) and the odor information is processed differentially in these four higher olfactory
centers. Thus, the overall organization of the olfactory circuitry and the information processing logic in fish are
remarkably similar to those in mammals. In this talk, I will introduce our recent findings on neural circuit
mechanism underlying the olfactory alarm reaction, which is induced by putative alarm pheromones
(Schreckstoff) released from injured skin of conspecifics. The result indicates a unique coincidence detection
mechanism of two pheromone components activating two distinct glomeruli for eliciting the alarm reaction in
zebrafish.
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PA13-4 Sensory-motor integration regulates olfactory learning
Yun Zhang
Dept of Organismic & Evolutionary Biology, Center for Brain Science, Harvard University, USA
During sensory-guided locomotion, such as chemotactic movement in response to odors, the position and
locomotory pattern of an animal are constantly changing. Therefore, sensory-motor integration is critical for the
animal to steer the movement towards the goal. However, the underlying mechanisms remain largely unknown.
Here, we show that the complex activity in a C. elegans interneuron, RIA, integrates the information of odor and
locomotory pattern to mediate olfactory chemotaxis. Previously, we have shown that the activity in RIA neurite
compartmentalizes in the axonal domains that are localized on the dorsal or ventral side of the animal and
represents the dorsal or ventral head bending, respectively, during the sinusoidal body waves on the
dorsal-ventral plane. The motor-encoding activity of RIA restricts head bending. Now, we further show that the
RIA axonal activity not only encodes the direction of head bending, but also the amplitude. Olfactory stimuli
evoke global axonal activity and regulate the encoding of motor information. These results indicate that distinct
activity patterns representing sensory versus motor information integrate within RIA to mediate sensory-directed
movement. Previously, we have also shown that RIA plays a critical role in aversive olfactory learning through
which C. elegans learns to avoid the smell of pathogenic bacteria. Here, we show that the aversive training
modulates the dynamics of RIA activity, revealing a mechanism whereby sensory-motor integration regulates
learning.
PA13-5 Bioluminescence imaging reveals dynamic presentation and reception of pheromonal cues
that mediate attraction in Drosophila melanogaster
Damien Mercier1,2, Yoshiko Tsuchimoto1, Hokto Kazama1
1
Brain Science Institute, RIKEN, Japan, 2graduate school of science and engineering, Saitama University, Japan
The fruit fly Drosophila melanogaster wears odors with complex profiles believed to mediate pheromone
communication. The chemical composition of the fly cuticle is heavily studied and some odorants, such as
cis-vaccenyl acetate (cVA), are shown to influence fly’s behavior. In particular, cVA, is known for playing a major
role in social behaviors including courtship and aggression. However, when and how a fly is presenting or
receiving these chemical cues under ethological settings remains elusive.
To study the dynamics of pheromone presentation and reception by animals, we developed a method to monitor
responses of identified neurons to natural stimuli in freely behaving adult Drosophila. Neural activity was probed
by detecting the bioluminescence emitted from a genetically encoded calcium indicator, while the fly’s behavior
was recorded by a camera.
Using the Gal4-UAS system, we expressed the calcium indicator in Or67d-positive olfactory receptor neurons
that have been shown to respond exclusively to cVA. Strikingly, we found that these pheromone-sensing
neurons remained unresponsive to other male’s cuticle, on which cVA is reported to be present, and rather
responded strongly to markings placed by males in the landscape. These markings were locally and transiently
attractive for the flies. Genetic mutation and targeted expression of tetanus-toxin demonstrated that this
attraction is largely mediated by Or67d-positive neurons.
These results suggest that this actively placed marking may enhance social behaviors by both creating a local
attraction landmark that increases the chance of interaction and by activating the pheromone-sensing neurons
that trigger courtship and aggression.
PA13-6 Flexible olfactory coding by mushroom body output neurons of Drosophila
Toshihide Hige1,2, Yoshinori Aso1, Mehrab N. Modi2, Gerald M. Rubin1, Glenn C. Turner1,2
1
HHMI, Janelia Research Campus, USA, 2Cold Spring Harbor Laboratory, USA
One of the most important higher-order functions of sensory systems is to flexibly interpret stimulus information
to select appropriate behaviors depending on the animal’s experience and states. In Drosophila olfactory
system, we recently demonstrated such flexible sensory processing at the mushroom bodies, suggesting their
important roles in adaptive sensory-motor transition. However, it has not been clear what kind of synaptic
modulations underlie the flexible odor coding. Here we provide the first demonstration of a long-term synaptic
plasticity at the output synapses of the Drosophila mushroom body, in the context of associative learning. Using
in vivo whole-cell recordings, we show that brief pairing of odor delivery and optogenetic activation of specific
dopamine neurons, which mimics aversive reinforcing stimuli, is sufficient both to induce synaptic plasticity and
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to induce memory formation in a behavioral assay. The induction of plasticity strictly depends on the temporal
order of the two stimuli with sub-second precision, replicating the logical requirement for associative learning.
Furthermore, by precisely manipulating individual circuit elements, we reveal that dopamine action is confined to
and distinct across different anatomical compartments of the mushroom body lobes. Finally, we find that overlap
between sparse representations of different odors determines both how stimulus-specific the plasticity is and
how generalizable the odor memories are. Thus, the plasticity we find here not only manifests important features
of associative learning that are apparent at the behavioral level but also provides general insights into how a
sparse sensory code is read out at the stage of sensory-motor transition.
Parallel Symposium XIV: Neurotransmission from taste buds to nerves
PA14-1 Introduction
Sue Kinnamon
Department of Otolaryngology, University of Colorado, Anschutz Medical Cumpus, USA
PA14-2 Purinergic neurotransmission of taste by CALHM channel
Akiyuki Taruno1, Yoshinori Marunaka1,2
1
Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Japan, 2Department of Bio-Ionomics, Kyoto
Prefectural University of Medicine, Kyoto, Japan
In taste buds, adenosine triphosphate (ATP) serves as the primary neurotransmitter, which conveys information
on sweet, bitter, and umami tastes from taste cells lacking synapses to gustatory nerves. Recently, calcium
homeostasis modulator 1 (CALHM1) was identified as an essential component of the ATP release machinery in
taste cells. CALHM1 monomers homo-multimerize to form a voltage-gated ion channel. ATP can travel across
the plasma membrane through the wide pore of the channel (diameter approx. 1.4 nm). Mice lacking Calhm1
are severely deficient in recognition of sweetness, bitterness, and umami due to impared taste-evoked ATP
release from taste buds. These discoveries brought up two questions. The first question is: What is the full
picture of the CALHM1 channel complex in taste cells? Involvement of unknown regulatory subunit(s) was
suggested because heterologous CALHM1 currents show slower activation kinetics compared to
CALHM1-dependent currents in taste cells. Here we found that CALHM1 and its homologous subunits
expressed in taste buds are assembled to form heteromeric channels with functional properties distinct from
those of homomeric CALHM1 channel. When co-expressed, CALHM1 was co-immunoprecipitated with its
homologs and vice versa. Co-expression of CALHM1 and its homologs gave rise to a novel ATP-permeable
membrane conductance with a more negative V50 value and faster gating kinetics than CALHM1 channel.
Collectively, our data raise the possibility that heteromeric CALHM channels are bona fide mediators of
neurotransmission of tastes. The second question is: Where are CALHM channels localized in a taste cell?
CALHM activity within a taste cell is likely confined to specific regions to serve its function and minimize its
potential cytotoxicity. To approach this problem, we have generated polyclonal antibodies against CALHM1 and
a knock-in mouse model in which endogenous CALHM1 is carboxy-terminally tagged with an epitope tag. Our
recent results will be discussed.
PA14-3 A chemical synapse without veicles: ATP release from Type II taste cells
Thomas E. Finger
Rocky Mountain Taste & Smell Center, University of Colorado School of Medicine, USA
Type II cells of taste buds, the receptor cells that respond to sweet, umami, or bitter, utilize ATP as a
neurotransmitter to activate the gustatory nerve fibers which express P2X purinergic receptors. The mechanisms
for release of ATP is, however, unconventional involving voltage-gated CALHM1 release channels rather than
on vesicular fusion with the plasma membrane. We utilized light microscopic immunocytochemistry and serial
blockface scanning electron microscopy to re-examine the functional relationships and ultrastructure of points of
contact between the Type II taste cells and the sensory nerve fibers in taste buds in the circumvallate papilla of
mice. Immunohistochemistry shows that CALHM1 release channels lie at apparent points of contact between
Type II cells and nerve fibers. These points of contact also are characterized by noncanonical (“atypical”), large
mitochondria with tubular, rather than parallel, cristae. While the surface area of the cristae of atypical
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mitochondria is similar to that of conventional mitochondria, the tubular configuration of the cristae results in a
higher relative volume of the intermembrane space, i.e. the region where ATP accumulates after synthesis.
Atypical mitochondria occur only in Type II cells and only at points of contact with nerve fibers, suggesting that
these are part of a synaptic specialization. These mitochondria lie at a fixed, spacing (about 40nm) from the
external plasma membrane indicative of a scaffolding that maintains this fixed spacing. Similarly, adjacent to the
mitochondria, the plasma membrane of the taste cell maintains an even 20nm spacing from the membrane of
the nerve fiber reminiscent of the spacing of the synaptic cleft of conventional synapses. The assembly of
CALHM1 channels, specialized mitochondria and the postsynaptic nerve fiber strongly suggest that these
represent the structural correlate of a chemical synapse releasing ATP.
PA14-4 What biophysics can tell us about afferent output in taste cell of the type I
Stanislav S. Kolesnikov1, Roman A. Romanov2, Olga A. Rogachevskaya1, Marina F. Bystrova1
1
Institute of Cell Biophysics, Russian Academy of Sciences, Russia, 2Center for Brain Research, Medical University of Vienna,
Austria
A variety of different cells employ ion channels for slow and continuous release of ATP into extracellular media,
where it is involved in paracrine and autocrine regulations. In contrast, type II cells release ATP to relay sensory
information, and therefore ATP efflux should be well-quantified, depending on taste stimulation. This
necessitates discontinuous ATP release governed by tastant-elicited action potentials (APs). The advantage of
this mechanism is that by initiating short and synchronous openings of multiple ATP-permeable channels, each
releasing a small portion of the neurotransmitter, AP makes channel-mediated ATP secretion in fact quantal, as
is the case with exocytotic secretion. At high depolarization elicited by AP, ATP efflux is driven by a small
electrochemical force that rises essentially at post-AP cell re-polarization. Thus, ATP-permeable channels
deactivating slowly allow for more countable ATP release on AP, compared to channels deactivating instantly.
Consistently, recent evidence validated the slowly deactivating CALHM1 as an ATP-release channel, although
instantly deactivating Panx1 hemichannels were considered for a long time to serve this function. Being strongly
dependent on the metabolic status of a cell and activity of ATP-consuming enzymes, the bulk ATP cannot serve
as a releasable pull to reliably encode taste information since channel-mediated ATP efflux is proportional to
ATP concentration. It thus appears that releasable ATP should be compartmentalized to be maintained at a
constant level irrespective of bulk ATP. Despite that the CALHM1 pore of nearly 1.4 nm is Ca2+ permeable, ATP
release is not associated with marked Ca2+ signals in type II cells. Based on electrophysiological and Ca2+
imaging data, pharmacology of ATP release, and dye loading, we suggest the existence of a special intracellular
compartment for ATP release to account for available inconsistencies.
PA14-5 Multiple neurotransmitters working in concert within the taste bud imply peripheral processing
of taste signals
Robin Dando
Department of Food Science, Cornell University, USA
The taste bud utilizes ATP to pass signals to afferent taste nerves, and on to the brain. Ablation of ionotropic
purine receptors leave mice largely taste blind, again emphasizing the importance of ATP in taste. Nonetheless,
cursory screens for neurotransmitter receptors in taste reveal receptors for multiple other neurotransmitters, as
well as the molecular machinery necessary for the synthesis, packaging and release of such signaling molecules
in taste. There exist reports of a number of paracrine and autocrine interactions within the taste bud, involving
several such transmitters. Type III taste cells, which display conventional synaptic connections to the afferent
taste nerve, release serotonin in response to direct acid (sour) stimulation, electrochemical depolarization, or
indirect stimulation of neighboring type II cells, activated by sweet, bitter or umami, and often co-released with
norepinephrine. Thus, paracrine transmission in taste may contribute to an underlying taste code. In addition to
serotonin, evidence exists from biosensor measurements for acetylcholine acting as an autocrine amplifier of
type II taste signals, boosting sweet, bitter and umami taste seemingly not specific to modality. Adenosine is
released from type II cells, selectively enhancing sweet signals within the taste bud. GABA is involved in the
inhibition of taste signaling, likely from type III cells, excited by sour stimuli. This suite of interactions within the
taste bud, along with the many others recorded, suggest a rudimentary form of signal coding occurring within the
taste bud, which may act to process signals from taste buds before report to the CNS.
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PA14-6 Analysis of peptidergic signaling-related gene expression in RNA-seq data of individual type II
and III mouse taste cells
Brian C. Lewandowski, Sunil K. Sukumaran, Robert F. Margolskee, Alexander A. Bachmanov
Monell Chemical Senses Center, Philadelphia, PA, USA
Information from taste cells is sent to gustatory brain regions that underlie taste perception and help drive
feeding related behaviors. The body’s metabolic state is also a key driver of feeding related behaviors. There is
growing evidence that signaling pathways related to metabolic state and energy homeostasis, including
peptidergic hormones, can influence the activity of taste cells. Taste cells are known to produce and/or detect a
number of peptide hormones and neuropeptides, including glucagon-like peptide-1, cholecystokinin and
neuropeptide Y. However, a global survey of the genes and receptors for peptidergic signaling in individual taste
cells has not been conducted. In this study, we used next generation RNA sequencing (RNA-seq) of single taste
cells isolated from the circumvallate papillae of C57BL/6 wild-type and transgenic mice to examine and compare
the expression of peptidergic signaling-related genes in several subpopulations of taste cells: Tas1r3-expressing
type II taste cells, gustducin-expressing type II taste cells, and sour-responsive type III taste cells. Our single-cell
RNA-seq data met high quality control standards and accurately detected differential expression of known
marker genes for type II (e.g., Trpm5, PlcB2, Tas1r’s) and type III (e.g., Ncam1, Pkd2l1, Gad1) taste cells.
Analysis of peptidergic signaling-related genes revealed expression of both known and previously unreported
peptides and their receptors in taste cells. For example, we detected transcripts for adiponectin (Adipor1, Adipor
2) and thrombin (F2r, F11r) receptors in both type II and type III taste cells, for somatostatin receptor 3 (Sstr3) in
type II cells, for apelin (Apln) in gustducin-expressing and sour-responsive taste cells, and for apelin receptor
(Aplnr) in Tas1r3-expressing type II cells. Our analysis of peptidergic signaling-related gene expression should
greatly benefit the effort to understand the crosstalk between the peripheral sensory mechanisms of taste and
the internal signaling pathways associated with energy homeostasis and metabolic state.
Parallel Symposium XV: What drives cultural difference in chemosensory perception
PA15-1 Food noticeability is altered by familiarity and retronasal aroma: A cross-cultural comparison
between Japanese and German consumers
Tatsu Kobayakawa1, Wolfgang Skrandies2, Takefumi Kobayashi3, Naomi Gotow1
1
Human Informatics Research Institute, National Institute of Advanced Industrial Science and Technology, Japan, 2Institute of
Physiology, Justus Liebig University Giessen, Germany, 3The Faculty of Human Studies, Bunkyo Gakuin University, Japan
Common foods consist of several taste qualities. Consumers perceive intensity of a particular taste quality after
noticing it among other taste qualities when they eat common foods. We supposed that while one is eating the
facility for noticing a taste quality present in a common food will differ among taste qualities which compose the
common food. We, therefore, proposed a new measurement scale for food perception named ‘noticeability’. In
our previous study (Gotow, Kobayashi, Kobayakawa, 2013), Japanese consumers evaluated noticeability and
perceived intensity of five fundamental taste qualities (sweetness, saltiness, sourness, bitterness, and umami)
under open- and closed-nostril conditions, using a popular traditional Japanese confection, yokan . The
correlation between noticeability and perceived intensity of sweetness was significantly reduced under
open-nostril conditions. Therefore, we hypothesized that both the retronasal aroma of a common food and a high
degree of familiarity with the food might be necessary to decrease the correlation decrease between these two
scales. In order to verify this hypothesis, we subjected Japanese and German consumers to psychological
evaluations using yokan , which is familiar to Japanese people but unfamiliar to Germans, and marshmallow,
which is familiar to both nationalities. Participants ate each food under open- and closed-nostril conditions, and
evaluated the noticeability and perceived intensity of the five fundamental taste qualities. Only for sweetness of a
highly familiar food (yokan for Japanese), the correlation between noticeability and perceived intensity was
significantly reduced under open-nostril conditions. These results support our hypothesis that high familiarity
with a common food might be necessary in order to decrease the correlation between the noticeability and
perceived intensity of certain taste qualities. And this means information of retronasal aroma in food, will not be
able to be accessed effectively without eating experience.
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PA15-2 Chemosensory experiences during childhood: Foundation for cultural learning
Julie A. Mennella, Loran L. Daniels, Ashley R. Reiter
Breastfeeding is fundamentally a biological process in which the mother continues to nourish her infant after
birth. Like other human behaviors, breastfeeding is influenced by a variety of cultural forces including the types
of foods eaten by the mothers. The flavors of the maternal diet change the flavor profile of human milk, providing
infants with chemosensory signals about their food environment. To experimentally investigate postnatal flavor
learning, we randomized lactating women to one of four diets that differed in the amount and timing of vegetable
consumption during the first four months of life. When their infants were weaned to solid foods, we tested their
acceptance of a variety of vegetables. This body of research demonstrated that maternal diet provides human
infants with chemosensory experiences that bias their response to the foods that are part of the mothers’ diets.
These early experiences not only serve to alter later responsiveness to flavors but, in a sense, educate and
civilize children to appreciate the flavors typical of the culture into which they were born. This research was
supported by Award Number R01HD37119 from the Eunice Kennedy Shriver National Institute of Child Health
and Human Development.
PA15-3 Cross-cultural difference in chemosensory perception: A multisensory perspective
Charles Spence
Crossmodal Research Laboratory, The University of Oxford, UK
In this talk, I want to review the laboratory, online, and real-world (i.e., marketing) research concerning
cross-cultural differences in chemosensory perception. In particular, I will take a multisensory perspective to
flavour perception. I will argue that while we all use the same rules to integrate the inputs from the different
senses that give rise to flavour expectations and flavour perception, differences occur in which combinations of
stimuli are considered as congruent and which are treated as incongruent, or unrelated. I will focus on
cross-cultural differences in the integration of olfactory and gustatory cues. I will then go on to consider whether
the oral-somatosensory and/or auditory attributes of flavour perception also exhibit any meaningful cross-cultural
variation. From there, I will go on to look at the differing meaning of colour in both food and drink, and food and
beverage packaging, in different parts of the world. I will show how the meaning that the consumer attaches to
colour (be it the colour of the food/beverage, or the colour of the packaging) can influence the taste and flavour
of that which is being consumed. I will also address the question of where these cross-cultural differences come
from, and how quickly they change (or are updated).
PA15-4 Effects of ethnicity, primary language and acculturation on odor identification
Claire Murphy, Nobuko Kemmotsu, Yurika Enobi
San Diego State University, USA
Elegant studies have investigated odor identification in Japanese participants with odors chosen for use in
Japan. Informed by the work of Kobayakawa and colleagues, here we focused on cultural factors that drive
differences in performance on an American odor identification test, with a particular interest in
Japanese-American participants. First we studied 71 Japanese-American participants and 71 CaucasianAmerican participants, aged 45 to 91 yrs, who completed the San Diego Odor Identification Test. Age and
gender were related to performance in both ethnic groups. Japanese-American participants tended to score
higher on odor identification at younger ages and lower at older ages. In a second study, we investigated
performance on the same measure in 100 bilingual Japanese-Americans. For 50 the primary language was
Japanese and for 50 the primary language was English. Hierarchical regression analyses examined the effects
of predictor variables: 1) primary language spoken, 2) acculturation level as measured on the Suinn-Lew Asian
Self-Identity Acculturation Scale (SL-ASIA test), 3) years of residence in the US and 4) years of education in
English. Age and gender were entered into the model at step 1, predictor variables at step 2. Interestingly, in
those whose primary language was Japanese, there were no gender differences; whereas in those whose
primary language was English, females performed at a higher level than males. Primary language spoken and
acculturation were more predictive than years of residence in the US or years of education in English.
Acculturation was not a significant factor in the group whose primary language was Japanese. In those whose
primary language was English the SL-ASIA scores predicted odor identification. A deeper understanding of what
drives cultural differences in chemosensory perception is becoming increasingly important as we consider
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olfactory function as a contributing biomarker for detection of neurodegenerative disease. Supported by NIH
grant AG004085-26.
PA15-5 Cross-cultural influences of eating behavior and meal pattern on chemosensory perception of
food
Han-Seok Seo
Department of Food Science, University of Arkansas, USA
There are cultural variations in meal pattern, eating behavior, and eating environment. For example, North
American people are used to drinking iced water/beverages with their meal, while European or Asian people
prefer to drink room temperature or hot water/tea, respectively. In addition, some cultures are used to eating
soup directly from a soup bowl, while other cultures are used to eating soup using a spoon. As another example,
some cultures, as a whole, prefer to eat in a quiet manner, while other cultures show a preference to eat their
meal with dynamic interpersonal communication. Based on the observations in everyday life, there was the
question raised whether such cross-cultural variations in meal pattern and eating behavior can affect
chemosensory perception of food and beverages. This talk was designed to answer the question. In other
words, this talk will provide better understanding of the cross-cultural impacts of eating behavior and
environment as well as meal pattern on chemosensory perception of food and beverages.
Parallel Symposium XVI: Neural circuits processing pheromones and odor valence in mice
PA16-1 A Novel Mechanism and Neural Logic for Mammalian Olfaction
Sandeep Robert Datta
Harvard Medical School Department of Neurobiology, USA
The Datta lab studies how information from the outside world is detected, encoded in the brain, and transformed
into meaningful behavioral outputs. We address this fundamental problem by characterizing the olfactory
system, the sensory system used by most animals to interact with their environment. Here we discuss recent
results relevant to understanding sensorimotor coupling, the process of linking sensation to action, in the
olfactory system. In particular, we describe a novel molecular mechanism and neural logic that underlies odor
perception, one that may be specialized for the detecting and processing of odors with innate meaning. This
mechanism defines a new mode of sensory encoding in mammals, and may be relevant to odor perception
across deuterostomal lineages, including humans.
PA16-2 Olfactory inhibition of dominance rank
Lisa Stowers, Tsung-Han Kuo, Tomo Tanaka
Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, USA
Leveraging the olfactory system provides a robust means to study the coding logic of behavior. We are currently
investigating how internal state changes alter an individual’s behavioral response to the olfactory environment.
Male mice in social groups establish dominance hierarchies which are thought to result in unknown internal state
changes. Dominant males respond to other male olfactory signals with aggression while subordinate males
respond passively. How state changes that result in the dominance hierarchy alters olfactory behavior is largely
unknown. We are using behavioral analysis, genetics, and neural activity reporters to identify differences in
olfactory coding between dominant and subordinate males. Such information is expected to reveal general
coding mechanisms of the olfactory system.
PA16-3 Dissecting neural circuits processing a sex pheromone in mice
Kazunari Miyamichi1,2, Kentaro Ishii1,2, Kazushige Touhara1,2
1
Department of Applied Biological Chemistry, Graduate school of Agricultural and Life Sciences, The University of Tokyo, Japan,
ERATO Touhara Chemosensory Signal Project, JST, The University of Tokyo, Japan
2
Semiochemical compounds and their peripheral receptors underlying various olfactory-mediated innate
behaviors have begun to be identified in mice. What remain mostly elusive are the neuronal circuits in the brain
that process distinct semiochemical signals to evoke appropriate behavioral responses. We here functionally
2016.05.27 16.58.58 Page 38(1)
dissect neuronal circuits for a mouse sex pheromone ESP1 as a model, by using viral and genetic toolkits. ESP1
is produced by male mice, secreted into their tears, received by a single vomeronasal receptor V2Rp5, and
robustly enhances the female sexual receptive behavior, lordosis, upon male mounting. We first identified a
specific type of projection neurons in the medial amygdala that convey ESP1 signals selectively to a
downstream target in the hypothalamus. We next functionally identified a specific hypothalamus-midbrain
pathway necessary for the enhanced lordosis by ESP1. Although natural predator signals that induce defensive
behaviors in mice activated seemingly similar brain regions as ESP1, neuronal representations of ESP1 and
snake exuvia, which contains predator cues, were mostly non-overlapping throughout the
amygdala-hypothalamus axis. Indeed, forced activation of ESP1-responding neurons, but not snake
exuvia-responding neurons, in the hypothalamic region markedly enhanced lordosis even in the absence of ESP
1, demonstrating the presence of heterogeneous neuronal types with distinct behavioral impacts. Collectively,
these data reveal at cellular resolution the neuronal circuits responsible for ESP1-mediated control of sexual
behaviors in female mice. Our study also highlight parallel dedicated pathways consist of distinct neuronal
ensembles for mediating sex versus predator semiochemical cues in the amygdala-hypothalamus of mice.
PA16-4 Ventral striatum systems for assigning odor valence and motivating goal-directed behaviors
Daniel W. Wesson, Marie A. Gadziola, Kate A. White
Case Western Reserve University, USA
Sensory information acquires meaning to adaptively guide behaviors. Despite odors mediating a number of vital
behaviors, the components of the olfactory system responsible for assigning meaning to odors and therefore
guiding odor-directed behaviors remain unclear. The olfactory tubercle (OT) receives monosynaptic input from
the olfactory bulb and is uniquely positioned within the ventral striatum to transform odor information into
behaviorally-relevant neural codes. We have uncovered that the firing rate of mouse OT neurons robustly and
flexibly encode the valence of conditioned odors over identity, with rewarded odors evoking greater firing rates.
This coding of rewarded odors occurs prior to behaviors indicating decisions and represents subsequent
behavioral responses. In support of the view that the OT is critical for motivationally-relevant information
processing, we also find that OT neurons encode goal-directed actions and their outcomes, in manners
dependent upon the motivational state of the animal. Together, this work illustrates the integral relationship of
ventral striatum systems in assigning odor valence and the direct translation of these coding schemes into goaldirected responses.
PA16-5 Oxytocin mediates entrainment of sensory stimuli to social cues of opposing valence
Gloria B. Choi, Han K. Choe, Michael D. Reed
McGovern Institute, Brain and Cognitive Sciences, Massachusetts Institute of Technology, USA
Meaningful social interactions modify behavioral responses to sensory stimuli. The neural mechanism
underlying the entrainment of neutral sensory stimuli to salient social cues to produce social learning remains
unknown. We used odordriven behavioral paradigms to ask if oxytocin, a neuropeptide implicated in various
social behaviors, plays a crucial role in the formation of learned associations between odor and socially
significant cues. Through genetic, optogenetic and pharmacological manipulations, we show that oxytocin
receptor signaling is crucial for entrainment of odor to social cues. Furthermore, we demonstrate that oxytocin
directly impacts the piriform, the olfactory sensory cortex, to mediate social learning. Lastly, we provide evidence
that oxytocin plays a role both in appetitive and aversive social learning. Taken together, these results suggest
that oxytocin conveys saliency of social stimuli to sensory representations in the piriform cortex during
odordriven social learning.
2016.05.27 16.58.58 Page 39(1)
Parallel Symposium XVII: Respiratory taste receptors: New arms of respiratory innate
defense
PA17-1 Nasal trigeminal chemoreception: From free nerve endings to epithelial chemosensors
Wayne L. Silver1, Cecil J. Saunders2
1
Department of Biology, Wake Forest University, USA, 2Department of Otorhinolaryngology, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, PA, USA
In 1912, G.H. Parker coined the term common chemical sense to describe a chemical sense separate from taste
and smell with receptors on exposed mucosal surfaces. The receptors of the common chemical sense were
identified as free nerve endings (FNEs). Years later, research demonstrated that these FNEs were distinct from
the olfactory and gustatory systems, and actually part of the somatosensory system. Since then, the term
chemesthesis has been used to describe the chemical stimulation of somatosensory nerves. Many chemical
irritants activate a class of somatosensory FNEs referred to as peptidergic nociceptors containing substance P
(SP) and calcitonin gene-related peptide (CGRP). In vertebrates, chemesthesis in the mouth, nose and eyes is
largely subserved by peptidergic nociceptors in the trigeminal nerve. Nasal trigeminal nerve fibers respond to a
wide variety of chemicals but typically the more lipid soluble a chemical the more effective stimulus it is, in terms
of threshold and latency. This is largely explained by FNEs terminating just below a line of tight junctions in the
nasal epithelium, necessitating that most chemical irritants pass through a membrane before stimulating
trigeminal nerves. In contrast to direct stimulation of FNEs, M. Whitear, in 1965, described solitary
chemoreceptor cells (SCCs) in fish skin innervated by somatosensory nerves. In the early 2000s cells with
similar morphology and innervation were observed in mammalian nasal epithelia. SCCs possess components of
taste transduction pathways, including receptors (TAS1; TAS2), G-protein (gustducin), phospholipase C, and
TRPM5 cation channels and respond to bitter compounds. At their apical end, SCCs possess microvilli
extending into the nasal lumen. At their basal end, they are innervated by SP and CGRP containing TNs. Thus,
these cells are positioned to detect chemicals (possibly water soluble) in the nasal cavity and relay information to
chemesthetic trigeminal nerve fibers.
PA17-2 Pou2f3/Skn-1a is required for the functional differentiation of Trpm5-expressing cells in
multiple tissues
Junpei Yamashita1, Makoto Ohmoto2, Tatsuya Yamaguchi1, Ichiro Matsumoto2, Junji Hirota1,3
1
Department of Bioengineering, Tokyo Institute of Technology, Yokohama, Japan, 2Monell Chemical Senses Center, USA, 3Center
for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama, Japan
Skn-1a (also known as Pou2f3), a POU (Pit-Oct-Unc) transcription factor, is expressed in Trpm5 (transient
receptor potential channel M5) -expressing cells：sweet, umami and bitter taste cells in taste buds of the
oropharyngeal epithelium, solitary chemosensory cells (SCCs) in the nasal respiratory epithelium, and
microvillous cells (MVCs) in the main olfactory epithelium. Loss of Skn-1a function resulted in defective
generation and/or functional differentiation of these taste cells, SCCs and MVCs, indicating that Skn-1a
functions as a determinant for their generation and functional differentiation.
Recent studies have shown that Trpm5, an indispensable molecule of taste signaling, is expressed not only in
sweet, umami and bitter taste cells, SCCs and MVCs, but also in the tracheal epithelium, thymic medulla, and
urethral epithelium, etc. These Trpm5-expressing chemosensory cells have an apical process with microvilli that
reaches epithelial surface of these tissues. In this study, we examined the expression of Skn-1a in these tissues,
and conducted functional analyses of Skn-1a using Skn-1a-deficient mice. In the absence of Skn-1a, marker
genes for Trpm5-expressing cells were not detected, suggesting that Skn-1a is a master regulator for the
functional differentiation of the Trpm5-expressing cells in multiple tissues.
PA17-3 Solitary Chemosensory Cells: Mechanisms of avoidance behavior and meningeal neurogenic
inflammation in response to airborne Irritants
Marco Tizzano
The nasal epithelium houses a population of solitary chemosensory cells (SCCs) that express T2R (bitter) taste
receptors and other taste transduction cascade elements (α-gustducin and TrpM5). These cells are innervated
by trigeminal polymodal nociceptive nerve fibers. Nasal SCCs respond to bitter compounds including
2016.05.27 16.58.58 Page 40(1)
bacterially-produced molecules, to evoke protective respiratory reflexes and neurogenic inflammation. Here we
show whether activation of the SCCs by inhaled irritants triggers avoidance responses and long-range
inflammation of the meninges consistent with migraine and other types of debilitating headache. To do so, we
developed novel protocols and a custom-built dual-chamber forced-choice device for bitter irritant and bacterial
molecule exposure in genetically modified mice, as well as methods to quantify meningeal neurogenic
inflammation in vivo. When denatonium benzoate or cycloheximide are nebulized in one of the two chambers of
the forced-choice device, wildtype mice avoid the chamber with the irritant, whereas TrpM5- or gustducin-KO
(bitter signaling cascade disrupted) and Skn-1a deficient mice (taste receptor cells and SCCs missing) show no
aversion for the irritant compound. The taste-blind P2X2/P2X3 double knockout mice avoid denatonium and
cycloheximide, showing that mice use SCCs but not the taste system to avoid the irritant chamber. In the
meningeal inflammation experiments, inhaled denatonium triggers neurogenic inflammation (plasma leakage) in
the dura mater of the meninges, which does not occur in genetically engineered (transgenic) mice that lack
either of two critical elements (TrpM5 or gustducin) of the SCC signaling cascade. Using retrograde labeling
staining, we determine that exists a transmission pathway linking activation of the nasal sensors to the
meningeal inflammation. Our results suggest that activation of the SCCs by irritants can lead to rapid avoidance
responses and can trigger meningeal neurovascular inflammation (migraine). Understanding the role for SCCs
in driving both protective and pathological responses will help identify potential treatment targets.
PA17-4 Role of chemosensation in innate immunity of the lower airways
Gabriela Krasteva-Christ1,6, Peter Koenig2,6, Wolfgang Kummer3,6, Emmanuel Saliba4, Peter Reeh6, Rajender Nandigama1
1
Institute of Anatomy and Cell Biology, Julius-Maximilians-University Wuerzburg, Germany, 2Institute for Anatomie, University of
Luebeck, Germany,
3
Institute of Anatomy and Cell Biology, Justus-Liebig-University Giessen, Germany, 4Research Center for Infectious Diseases,
Wuerzburg, Germany,
5
Institute of Physiology and Pathophysiology, University of Erlangen-Nuremberg, Germany, 6German Center for Lung Research,
Germany
Recognition of invading pathogens is a prerequisite for the initiation of immune responses and their subsequent
elimination. The generally accepted view is that the host innate immune system recognizes invading pathogens
using classical pattern recognition receptors. However, recent studies have shown that bacterial products such
as quorum sensing molecules (QSM) act as agonists for non-immune receptors like canonical bitter taste
receptors (T2R). In the last years, several groups including our own have identified T2Rs to be components of
the canonical taste transduction cascade in a specialized epithelial cell type (termed solitary chemosensory cells
or brush cells) in a variety of locations beyond the tongue, from the airway and gastrointestinal epithelia to the
urethra.
An appealing concept has emerged that brush cells in the lower airways serve as sentinels to protect against the
further ingression of potentially harmful bacterial substances into the lung. We have developed animal models to
study tracheal evoked reflexes in vivo. Application of bitter substances and bacterial products (among them
QSM from P. aeruginosa), to the tracheal mucosa resulted in the release of acetylcholine (ACh) from brush cells
followed by activation of sensory nerve fibers and depression of respiration. In addition, ACh influenced in
paracrine manner the mucoliary clearance. Recently, we have been exploring the hypothesis that ACh mediates
a subsequent release of neuropeptides from nerve fibers innervating the trachea leading to neurogenic
inflammation in the airways. Inhalative tracheal challenge of anesthetized spontaneously breathing mice with
bitter/bacterial substances evoked CGRP release, extravasation of Evans blue from subepithelial blood vessels
and recruitment of neutrophils. Interestingly, the number of brush cells was increased 12 hour after infection with
P. aeruginosa and this correlated with bacterial clearance.
In conclusion, chemosensation is responsible for triggering local inflammation and initiating immune responses
to the presence of bacterial metabolites in the lower airways.
PA17-5 Clinical implications of bitter taste receptor genetics in human respiratory diseases
Noam A. Cohen
University of Pennsylvania Dept of Otorhinolaryngology-Head and Neck Surgery, USA
Taste receptors were first identified on the tongue, where they initiate a signaling pathway that communicates
information to the brain about the nutrient content or potential toxicity of ingested foods. However, recent
research has shown that taste receptors are also expressed in a myriad of other tissues, from the airway and
gastrointestinal epithelia to the pancreas and brain. The functions of many of these extra-oral taste receptors
2016.05.27 16.58.58 Page 41(1)
remain unknown, but emerging evidence suggests that taste receptors in the airway are important sentinels of
innate immunity. We will discuss our recent findings demonstrating that a subset of bitter taste receptors
expressed in human ciliated cells regulate rapid nitric oxide production in response to microbial factors. We have
also demonstrated that bitter and sweet taste receptors expressed in human sinonasal solitary chemosensory
cells regulate a complimentary arm of innate immunity, the release of antimicrobial peptides. Lastly, common
genetic polymorphisms of the taste receptor genes that render these receptors non-functional appear to result in
a weakened respiratory innate defense contributing to therapeutically recalcitrant upper respiratory infections.
Parallel Symposium XVIII: Stem cells and the self-renewal of chemosensory epithelia:
Mechanisms of regeneration in taste buds versus the olfactory epithelium
PA18-1 Taste papilla and taste bud maintenance, function and renewal are dependent on epithelial
hedgehog signaling
Charlotte M. Mistretta
School of Dentistry, The University of Michigan, USA
Taste organ cells reside in tissue domains and stem/progenitor cell niches of the tongue epithelium and
connective tissue stroma. The particular domains and niches provide local cues for interactions with signaling
pathways and functional homeostasis to maintain various stem and progenitor cells. In adult tongue we located
Hedgehog (HH) signaling domains, and probed HH signaling roles in fungiform and circumvallate papilla and
taste bud differentiation, maintenance, renewal, regeneration, and function. We repressed the HH pathway in
mouse genetic models with conditional Gli2 transgene expression or Gli2 deletion in tongue epithelium; inhibited
the pathway with conditional Smoothened deletion in epithelium or epithelium and stroma cells; and, blocked
Smoothened systemically by drug administration (LDE225). Across approaches: effects of canonical HH
suppression are rapid, profound, and increase with time, eliminating taste buds and altering papilla integrity; cell
proliferation is reduced in epithelial compartments/niches known to contribute to taste bud cells; robust
innervation is maintained in the papilla and into the epithelium even as taste buds are reduced; stromal
fibroblasts remain in contact with papilla basal lamina. Therefore, epithelial HH signaling is essential for
differentiation of taste bud cells, and a robust innervation and stroma cells are not sufficient to maintain taste
buds in the context of compromised HH signaling. After withdrawing treatments, to determine if HH suppression
effects were reversible, the papilla epithelium, taste buds and taste nerve responses recovered progressively
over time. Thus taste cell progenitors apparently were spared and retained; and, were poised to regenerate taste
buds after HH signaling was restored, with the sustained innervation and papilla stromal core elements already
in place. Overall, comparing epithelium-specific and broader inhibition, we conclude that taste papilla epithelial
cells, which include taste bud progenitors, represent the key targets for physiologic HH signaling that is required
for taste organ homeostasis. (NIDCD DC014428)
PA18-2 Molecular regulation of taste bud cell renewal.
Linda A. Barlow
Dept of Cell and Developmental Biology, University of Colorado School of Medicine, USA
Taste is a fundamental sense and is crucial for human health. Like our other primary senses, we consider our
ability to appreciate sweet, sour, salty, bitter and umami tastes to be relatively constant, even though taste bud
cells that transduce these stimuli are renewed rapidly and regularly. The importance of the sense of taste is
particularly evident for cancer patients receiving a range of radiation and chemotherapies, as these individuals
often experience significant taste loss or dysfunction, and as a result, a significantly diminished quality of life. In
the past decade, understanding of cellular and molecular mechanisms governing taste bud renewal has
expanded significantly. In numerous renewing epithelia, the Hedgehog (HH) and Wnt/beta-catenin signaling
pathways are key regulators of homeostasis. Using an arsenal of multi-allelic mouse models, we have found that
HH and Wnt signaling also control taste bud cell renewal. Specifically, Wnt signaling is required to maintain taste
cell renewal and is key for continued proliferation of taste progenitor cells, while the level of Wnt/beta-catenin
signal impacts the cell fate of new cells generated from the progenitor population. Our working model of HH
pathway function is that it promotes taste bud cell differentiation from progenitors, but does not regulate
progenitor proliferation. Going forward, it will be important to understand how these pathways function together,
2016.05.27 16.58.58 Page 42(1)
as well as how these may be impacted by cancer therapies.
Supported by NIH/NIDCD DC012383 and DC012375 to L.A.B.
PA18-3 In vivo and ex vivo characterization of taste stem/progenitor cells
Peihua Jiang
Taste cells undergo constant turnover throughout life. Until recently, the identity of the adult taste stem cells
responsible for taste tissue regeneration was unknown. We and others reported that the Leucine-rich
repeat-containing G protein-coupled receptor 5 (Lgr5) marks adult taste stem/progenitor cells in posterior
tongue, and Lgr6 may mark stem/progenitor cells in both anterior and posterior tongue. Recently, we have
established a three-dimensional culture system to expand and differentiate single Lgr5-expressing (Lgr5+) or
Lgr6-expressing (Lgr6+) cells isolated from taste tissue into taste bud like “organoids”. Many cells within these
organoids cycle; they are positive for proliferative cell markers (e.g., cytokeratin K5, Sox2), and they incorporate
5-bromo-2’-deoxyuridine. As expected, taste organoids include mature taste receptor cells that express
gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate
diphosphohydrolase-2, or cytokeratin K8. Functional assays demonstrate that cells grown out from taste
organoids respond to tastants in a dose-dependent manner. Taste organoids derived from individual Lgr5+ or
Lgr6+ cells can be passaged multiple times in culture. Transcriptome analyses of organoids at different
developmental stages indicate that multiple pathways are involved in the generation of mature taste cells.
Moreover, we can now grow taste organoids from fresh dissociated lingual epithelial cells culled from transgenic
mouse models without isolating stem/progenitor cells. Together, our data demonstrate that single taste stem/
progenitor cells can generate functional taste cells in vivo and ex vivo. Our taste organoid culture system is a
valuable tool for studying the molecular mechanisms underlying taste cell regeneration and physiology.
PA18-4 Dynamic regulation and maintenance of two olfactory cell populations
Tanu Sharma1, Mengfei Chen3, Andrew Lane3, Randall Reed1,2,3
1
Department of Neuroscience Johns Hopkins Universtity Baltimore, USA, 2Department of Molecular Biology and Genetics Johns
Hopkins University, Baltimore, MD, USA, 3Department of Otolaryngology Johns Hopkins University Baltimore, MD, USA
The turnover of olfactory sensory neurons (OSNs) in normal olfactory epithelium (OE) is well documented.
Recent studies suggest that the robust replacement of OSNs after injury, toxins and pathogens derive from
distinct populations dependent on the nature of the lesion. The mechanisms that control the proliferation of these
precursors and their differentiation are poorly understood. Using a variety of mouse genetic models, we have
examined the ability of globose basal cells (GBCs) and horizontal basal cells (HBCs) to contribute to these
processes and the mechanisms regulating neuroregeneration. Importantly, specific lesion-associated signaling
pathways may suppress proliferation of basal cell populations under some conditions. A second major cell
population in the nose, olfactory ensheathing cells (OECs) are specialized glial cells found exclusively in the
olfactory system. The regenerative capacity of OSNs makes this tissue an excellent model for studying neuronglial interactions. We focused on the close interaction between OSNs and the OECs that ensheath the axons
projecting from the OE to the bulb. The main objective was to elucidate the molecular and cellular characteristics
of OECs in adult mice during acute OSN degeneration and regeneration. We ascertained OEC morphology,
proliferation and gene expression using RNA-seq in normal mucosa and at several points during neuron
regeneration and axon extension. OECs were sparsely-labeled using cre-mediated recombination and imaged in
whole mucosa by confocal microscopy. Our analysis revealed a remarkable stability in morphological
parameters post-neuronal injury. Finally, methyl bromide-induced lesions did not induce significant turnover of
OECs. Overall, our results indicate that OECs remain largely unaffected by the loss and regeneration of OSN
and we conclude that the role for OECs in adult mice is to maintain an overall stable environment for continuous
regeneration of olfactory neurons. Currently, we are examining the effects of OEC ablation on surrounding OSN
and OEC dynamics.
PA18-5 Taking poietic license: Stem and progenitor cells of the olfactory epithelium
James E. Schwob
Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, USA
The capacity of the olfactory epithelium (OE) to maintain a full complement of olfactory sensory neurons (OSNs)
2016.05.27 16.58.58 Page 43(1)
during piecemeal turnover and to reconstitute itself completely following injury depends upon the persistence of
neurocompetent stem cells. Horizontal basal cells (HBCs) and some among the molecularly heterogeneous
population of globose basal cells (GBCs) are both broadly multipotent (able to generate all epithelial cell types)
and self-renewing, thereby fulfilling the usual criteria for stemness. Among the GBCs are both proliferating and
mitotically quiescent cells that are directed toward neuro- vs. multipotency depending on epithelial status and the
surrounding signaling environment. However, HBCs are normally dormant and held in reserve; epithelial injury
triggers them into active multipotency. The transcription factor (TF) p63 is the master regulator of HBC
dormancy, and a decrease in p63 expression is both necessary and sufficient to accomplish activation. The cues
that control p63 levels are complex. Sustentacular cell injury but not selective neuronal death will shift HBCs out
of dormancy. Sus cells express the Notch ligand Jagged1 while HBCs express both Notch1 and Notch2
receptors. Notch signaling increases p63 levels, while HBC-specific knockout of Notch1 but not Notch2
decreases p63 and enhances spontaneous activation of the affected HBCs. Thus, activating HBCs by reducing
Notch signaling might potentially restore GBCs and OSNs to the aged OE. However, HBCs make respiratory
epithelial cells following very severe OE injury suggesting that complex manipulation of the molecular
environment may be required to accomplish recovery. GBC potency is also regulated by epithelial status, and
the category of GBCs that generate only OSNs in normal OE or following transplantation are reprogrammed to
multipotency by prior epithelial lesion either by bulbectomy or methimazole intoxication. The plasticity of
progenitor cell potency presents opportunities for therapeutic intervention designed to ameliorate olfactory
dysfunction.
Support: R01 DC002167, R01 DC014217.
2016.05.27 11.25.45 Page 1(1)
ＫｙｏｒｉｎＷＰＳ／３５０３３−５２２２／ｉｓｏｔｐ１７−１３−２＿／ｋｙ８１３９２３５２２２１０００２９９８
Poster Session
P1-001 Supersensitive detection and discrimination of enantiomers by dorsal olfactory receptors:
Evidence for hierarchical odor coding
Takaaki Sato1, Reiko Kobayakawa2, Ko Kobayakawa2, Makoto Emura3, Shigeyoshi Itohara4, Miwako Kizumi1,
Hiroshi Hamana5, Akio Tsuboi6, Junzo Hirono1
1
Biomedical Research Institute/National Institute of Advanced Industrial Science and Technology, Ikeda, Japan, 2Institute
Biomedical Science, Kansai Medical University, Hirakata, Japan, 3Takasago Int. Corp., Hiratsuka, Japan, 4Brain Science Institute/
RIKEN, Wako, Japan, 5Graduate School of Medicine Pharmachology Science, University of Toyama, Toyama, Japan, 6Research
Institute of Frontier Medicine, Nara Medical University, Kashihara, Japan
Pairs of enantiomeric odor ligands are difficult to resolve by instrumental analyses because compounds with
mirror-image molecular structures have almost identical physicochemical properties. The olfactory system,
however, discriminates (-)-forms of enantiomers from their (+)-forms within seconds. To investigate key olfactory
receptors (ORs) for enantiomer discrimination, we compared behavioral detection and discrimination thresholds
of wild-type (WT) mice with those of ∆D mice that lack all dorsal ORs. Surprisingly, WT mice displayed a
“supersensitivity” to enantiomeric pairs of wine lactones and carvones in both detection and discrimination tasks
using odor plume-like flows in a Y-maze. In contrast, ∆D mice showed selective major loss of sensitivity to the
(+)-enantiomers. The resulting 108-fold differential sensitivity of ∆D mice to (-)- vs. (+)-wine lactone matched that
observed in humans. This suggests that humans lack highly sensitive orthologous dorsal receptors for the (+)enantiomer, similarly to ∆D mice. Moreover, ∆D mice showed >1010-fold reductions in enantiomer discrimination
sensitivity compared to WT mice. ∆D mice detected one or both of the (-)- and (+)-enantiomers over a wide
concentration range, but were unable to discriminate them. This “enantiomer odor discrimination paradox”
indicates that the most sensitive dorsal ORs play a critical role in hierarchical odor coding for enantiomer
identification. Using Ca2+ imaging to profile odorant responses of olfactory sensory neurons of WT mice, we
found that OR coding was three-fold sparser for wine lactones than carvones. Using the 15 identified carvone
ORs, our model explained how a deletion of just one most sensitive dorsal OR (mORcar-c5 ) could significantly
alter early OR signaling for (R )-(-)-carvone to those of (R )-(-)-/(S )-(+)-carvone-overlapping ORs that would code
common elemental odors as the principal elemental odors of (R )-(-)-carvone in ∆D mice. [Reference; Sci. Rep.,
5:14073(2015).]
P1-002 Expression of synaptosome-associated protein 25 kDa (SNAP25) in the salmon brain
Takashi Abe1, Yui Minowa1, Masaki Kobayashi1, Hideaki Kudo2
1
Laboratory of Humans and the Ocean, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Japan, 2Laboratory
of Humans and the Ocean, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan
Synaptosome-associated protein 25 kDa (SNAP25) constitutes the soluble N-ethylmaleimide-sensitive factor
attachment protein receptor (SNARE) complex that mediates pre-synaptic vesicle exocytosis, regulates synaptic
transmission and neuronal plasticity. It is generally accepted that anadromous Pacific salmon (Oncorhynchus
spp.) imprint some odorants of their natal streams at the seaward migration, and use their olfaction for
discriminating those streams during spawning migration. Despite the importance of the synaptic plasticity for the
olfactory imprinting, the expression of SNARE complex is not well understood in salmon brain. In this study,
SNAP25 was detected in the chum salmon (O. keta ) brain as one of SNARE complex by molecular biological
techniques. Expressions of snap25a and snap25b mRNA were detected in the olfactory center (olfactory bulb
and telencephalon) by reverse transcriptase polymerase chain reaction. By quantitative-PCR of each brain
region in mature salmon, snap25a were mainly detected in rostral regions of brain (i.e., forebrain), and its
expression especially in the olfactory bulb was the highest. On the other hand, expressions of snap25b were
detected in all regions of brain. In juvenile salmon at the imprinting period, expressions of both snap25s in the
olfactory centers were the highest seawater life stage compared with the freshwater life stage at natal stream
during the seaward migration. In comparison of both snap25s , the expression level of the snap25b was high.
Our results provide the first detection of snap25 gene expressions in the salmon brain, and indicate that snap25
s are involved in the synaptic plasticity for the olfactory imprinting and/or olfactory memory retrieval in Pacific
salmon.
2016.05.27 11.25.45 Page 2(1)
P1-003 Olfactory network function is modulated by flight motor pattern generating centers: Evidence
for the first corollary discharge circuit to an olfactory pathway
Kevin C. Daly, Samual P. Bradley, Phillip D. Chapman, Ariel B. Thomas
West Virginia University, USA
Active odor sampling behaviors shape spatiotemporal olfactory input, processing and perception. These
behaviors also drive rhythmic reafferent signals driving olfactory responses in the absence of odor. Other
sensory domains receiving reafferent signals also typically receive a corollary discharge signal from motor
pathways driving reafference, yet little is known about the presence and function of these circuits in olfactory
systems. We characterize a novel corollary discharge circuit the moth Manduca sexta. Using anatomical
techniques we characterized two histamine (HA) immunoreactive cells projecting from flight motor centers to
antennal lobe (AL), where <20 local interneurons express the HA-B receptor and co-express GABA, and one or
two also expressing Allatotropin or fmrfamide. We show that these HA cells are the only source of AL HA and the
circuit is only complete in adults. Using paired intra- and extracellular techniques we confirm that the HA cells
spiking activity is positively correlated to motor neuron output. Multiunit recordings of AL responses to temporally
structured stimuli meant to simulate wing beating revealed that populations of AL neurons track and clarify
periodic antennal input resulting in enhanced AL representations as measured by power spectral and population
analyses. Pharmacological manipulation of the HA pathway reveals that HA modulates the ability of AL neurons
to entrain to temporally structured stimuli. These findings are further supported by psychophysical results
demonstrating lowered detection and discrimination thresholds to rhythmical stimuli in a HA pathway-dependent
manner. Overall these results establish that this simple and elegant motor-to-sensory circuit provides a corollary
discharge that modulates AL function, presumably enhancing odor representations during odor-guided flight.
This is the first known corollary discharge circuit to an olfactory pathway and the first higher order corollary
discharge in arthropods, however preliminary comparative immunolabeling suggests this circuit is present in
other moths but not butterflies.
P1-004 A functional atlas of serotonin receptor expression in the Antennal Lobe
Andrew M. Dacks, Ayad Auda, Kaylynn E. Coates, Tyler M. Sizemore
Department of Biology, West Virginia University, Morgantown, WV, USA
The behavioral relevance of an odor changes from one moment to the next depending on the physiological state
of an animal and, as a result, sensory networks often use neuromodulators to optimize how they encode
information to meet these ongoing demands. Neuromodulators alter the biophysical properties and synaptic
efficacy of individual neurons within a network resulting in changes in both the direct output of a network and the
lateral interactions within a network. For instance, within the antennal lobe (AL) of Drosophila melanogaster
serotonin (5-HT) enhances the odor-evoked responses of projection neurons (PNs), while also enhancing the
degree of pre-synaptic inhibition exerted by local interneurons (LNs) on olfactory receptor neurons (ORNs).
However, without knowing the functional identity of those neurons that express each neuromodulatory receptor,
it is difficult to determine if specific changes in olfactory processing are due to direct changes in biophysical
properties or the synaptic input that a particular neuron receives. To this end, we used Drosophila to generate a
functional atlas of AL neurons that express each of the five insect 5-HT receptor subtypes using a reporter of
endogenous receptor translation and functionally identified each population of neurons based on their
transmitter content and morphological characteristics. Each 5-HT receptor was expressed by distinct functional
populations of neurons and, overall, subpopulations from each of the 3 major neuron classes (ORNs, LNs and
PNs) expressed a 5-HT receptor. For instance, the 5-HT1A receptor was expressed by a specific population of
peptidergic LNs and the GABAergic PNs, while the 5-HT2A receptor was expressed only by cholinergic PNs.
Our results suggest that the expression of different 5-HT receptors by each neuronal class allows 5-HT to have
distinct effects on individual features of olfactory coding.
P1-005 Feedforward excitation entrains oscillatory microcircuits in the mouse accessory olfactory bulb
Chryssanthi Tsitoura, Kira Gerhold, Monika Gorin, Julia Mohrhardt, Katja Watznauer, Marc Spehr
Department of Chemosensation, RWTH Aachen University, Aachen, Germany
The accessory olfactory system is a key component in rodent conspecific chemical communication. In the
accessory olfactory bulb (AOB), the first stage of information processing in the mouse vomeronasal pathway,
mitral cells (MCs) receive sensory input from peripheral vomeronasal neurons and relay this information to the
2016.05.27 11.25.45 Page 3(1)
vomeronasal amygdala and the hypothalamus. Despite its fundamental function, however, sensory coding in the
AOB is poorly understood. Recently, we demonstrated that a subpopulation of MCs is intrinsically rhythmogenic
and exhibits slow stereotypical oscillatory discharge triggered by cyclic activation of three interdependent ionic
conductances: subthreshold persistent Na+ current, R-type Ca2+ current, and Ca2+-activated big conductance K+
current. Here, we identify an excitatory circuit within the AOB network that entrains oscillatory activity in a second
MC subpopulation. Using a battery of physiological techniques in acute AOB tissue slices, we investigate the
mechanisms underlying oscillatory entrainment and synchronization. Entrained MCs display periodically
increased excitatory synaptic input that correlates with their respective rhythmic discharge patterns. Several
such MCs are often organized into synchronized microcircuits. Block of fast glutamatergic synaptic transmission
reveals that entrainment depends on an intact glutamatergic network. Ongoing experiments aim to identify the
detailed mechanisms of MC entrainment and the role of slow rhythmic activity in AOB information processing.
This work was supported by the Deutsche Forschungsgemeinschaft (SP724/9-1; SPP 1392: “Integrative
Analysis of Olfaction”).
P1-006 Neural circuits controlling pup-directed behaviors in male mice
Kenichi Tokita1, Yousuke Tsuneoka2, Taiju Amano3, Manami Sato4, Kumi Ozeki Kuroda1
1
Research Team for Affiliative Social Behavior, RIKEN Brain Science Institute, Saitama, Japan, 2Department of Anatomy, Toho
University School of Medicine, Tokyo, Japan, 3Department of Pharmacoloy, Graduate School of Pharmaceutical Sciences,
Hokkaido University, Hokkaido, Japan, 4Department of Animal Sciences, Teikyo University of Science, Tokyo, Japan
Parental behavior, provided by mother and father, is essential for the survival of pups in mammalian species.
Therefore, it is plausible to assume that the basic central neural mechanisms in mammals including humans are
highly conserved. Understanding the central mechanisms of parental behavior should significantly benefit our
society by promoting ideal parental care and preventing its malfunction such as child neglect and infanticide. In
the present study, we investigated both paternal behavior in mice by combining variety of techniques. We
demonstrated that the c-Fos expression pattern in the four nuclei of the preoptic-bed nuclei of stria terminalis
(BST) region could robustly discriminate five kinds of previous social behavior of male mice. Specifically,
neuronal activation in the central part of the medial preoptic area (cMPOA) and rhomboid nucleus of the BST
(BSTrh) retroactively detected paternal and infanticidal motivation with more than 95% accuracy. Moreover,
cMPOA lesions switched behavior in fathers from paternal to infanticidal, while BSTrh lesions inhibited
infanticide in virgin males. Optogenetic or pharmacogenetic activation of cMPOA attenuated infanticide in virgin
males. We also found that excitotoxic lesions centered in the central nucleus of the amygdala attenuated
infanticide in virgin males. These results identified the preoptic-BST nuclei underlying social motivations in male
mice, and amygdala’s involvement in this circuit is also suggested. Afferent and efferent connections of mouse
cMPOA are also described and discussed.
P1-007 Investigation of morphological differences between mitral cell subpopulations in the accessory
olfactory bulb of mice
Katja Watznauer, Monika Gorin, Chryssanthi Tsitoura, Marc Spehr
Department of Chemosensation, RWTH Aachen University, Aachen, Germany
The accessory olfactory system is crucial for controlling both inter- and intraspecific communication as well as
sexual behavior in rodents. Astonishingly, its sensory coding and morphology is still poorly understood
compared to the main olfactory system. The first stages of the vomeronasal pathway comprise the vomeronasal
organ and the accessory olfactory bulb (AOB). In the AOB, vomeronasal sensory information is processed by
mitral cells (MCs), the AOB’s sole excitatory projection neurons, which relay information to higher brain centers
like the amygdala and the hypothalamus. Recently, we demonstrated that at least two subpopulations of MCs
can be distinguished based on different spontaneous discharge patterns. While members of one population fire
action potentials in a seemingly random fashion, others are intrinsically rhythmogenic and exhibit slow oscillatory
discharge. In order to analyze potential morphological differences between both MC populations, we performed
patch-clamp experiments in acute mouse AOB slices. MCs were diffusion-loaded with biocytin for post-hoc
staining and 3-dimensional morphological reconstruction. In-depth comparative investigation revealed that many
morphological properties are remarkably similar between rhythmogenic and non-oscillating MCs. However, our
results indicate that glomerular size is a notable exception. We observed that primary dendrites of intrinsically
oscillating MCs form glomerular tufts that appear significantly larger than observed in randomly firing MCs. On-
2016.05.27 11.25.45 Page 4(1)
going experiments aim to characterize and quantify these distinct features that will likely affect information
coding in the AOB.
FUNDING ACKNOWLEDGMENTS: This work was supported by the Deutsche Forschungsgemeinschaft SPP
1392: “Integrative Analysis of Olfaction” and the Volkswagen Foundation (83533).
FCOI DECLARATIONS: none
P1-008 Scent of ATP: Olfactory circuit mediating attraction to nucleotides in Zebrafish
Noriko Wakisaka, Nobuhiko Miyasaka, Tetsuya Koide, Miwa Masuda, Yoshihiro Yoshihara
Brain Science Institute, RIKEN, Saitama, Japan
Many olfactory cues pervade the aquatic environment of fish and elicit various behavioral and endocrine
responses essential for their survival and species preservation. Amino acids and nucleotides have been
reported to act as feeding cues that attract fish through the olfactory system. Here we report molecular, cellular
and neural circuit mechanisms underlying the attractive response of zebrafish to adenosine triphosphate (ATP)
and related nucleotides. Immunohistochemical staining with antibody against phosphorylated Erk, a marker for
neuronal excitation, revealed that ATP activates a small population of olfactory sensory neurons located in the
apical portion of the olfactory epithelium. Double fluorescence in situ hybridization analysis with cRNA probes
for c-Fos and various seven-transmembrane-type receptors identified a unique, fish-specific receptor expressed
in the ATP-responsive olfactory sensory neurons. In the olfactory bulb, ATP specifically activates only a single
identifiable glomerulus in the latero-posterior region, which is different from the amino acid-activated lateral
glomeruli. The information about ATP is then conveyed to higher olfactory centers including two areas in the
telencephalon and two nuclei in the hypothalamus. One of these hypothalamic nuclei is activated specifically by
ATP but not by amino acids., A behavioral analysis revealed that zebrafish show more sensitive attraction to
ATP than to amino acids. These findings will provide a fundamental basis for understanding neural circuit
mechanism linking food-derived attractive cues to foraging behavior that is critical and common to all animal
species.
P1-009 P-mix, pyrazine analogues contained in wolf urine, induced innate fear in immature and mature
rats
Makoto Kashiwayanagi1, Sadaharu Miyazono1, Kazumi Osada2
1
Department of Sensory Physiology, Asahikawa Medical University, Asahikawa, Japan, 2Department of Oral Biology, School of
Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan
Urine excreted from common grey wolf (Canis lupus) has been used as a repellent for various kinds of
mammals, because their odors act as a kiromone, which induce fear-related behaviors in various kinds of
mammalian. Various fear-inducing substances activate neurons at the main and/or accessory olfactory bulb
(AOB), medial and central amygdala, and ventromedial hypothalamus (VMHdm). Previous our study showed
that pyrazine analogues (P-mix) contained in wolf urine induced avoidance and fear-related behaviours in
laboratory mice and domestic deer in Japan. Exposure to wolf urine and P-mix induces Fos-expression, a
marker of excitation in neurons, in the AOB of mice. In the present study, we explored effects of P-mix on
fear-related behaviours and Fos-expression in the immature and mature rats. Exposure to P-mix induced
avoidance and immobilization in mature rats, while that to the mixture of i-amyl acetate, linalool and d-carvone
(O-mix), which have flower and fruit flavour, induced avoidance but not immobilization. P-mix but not O-mix
increased Fos-immunoreactivity at the AOB, several regions of the amygdala and VMHdm of mature rats.
Exposure to P-mix also induced avoidance, immobilization and Fos-expression at the AOB, amygdala and
VMHdm in immature rats. The present results suggest that P-mix odour induces innate fear-related behaviours
in rats.
P1-010 The vigilance behaviors provoked by various kinds of alkylpyrazine analogues to mice
Kazumi Osada1, Sadaharu Miyazono2, Makoto Kashiwayanagi2
1
Department of Oral Biology, Health Scienses University of Hokkaido, Japan, 2Department of Sensory Physiology, Asahikawa
Medical University, Asahikawa, Japan
Our previous studies indicated that pyrazine analogues, identified in wolf urine, induced a series of vigilance
behaviors in mammals. To know the structure-activity correlation between alkyl pyrazine analogues, we
2016.05.27 11.25.45 Page 5(1)
determined vigilance behaviors of female C57BL/6J mice confronting with each one of sixteen pyrazine
analogues. 2, 3-Diethylpyrazine(DEP)out of tested pyrazine analogues, was the chemical that induced the
longest freezing behavior, the lowest locomotor activities and the shortest investigation time. In addition, 3-ethyl2, 5-dimethyl pyrazine (3EDMP), 5-ethyl-2, 3-dimethylpyrazine (5EDMP) and 2, 3, 5-trimethylpyrazine (TMP)
also significantly induced freezing behaviors than control. The locomotor activity were significantly decreased by
DEP, 3EDMP, 5EDMP and marginally decreased by TMP. Investigation time was significantly decreased by
thirteen out of all pyrazine analogues. There are negative correlations between the freezing duration and both of
the total locomotor activities and the investigation duration. These results suggest that a couple of pyrazines
which induced freezing behavior can also induce the decrease of the locomotor activity and investigation time.
Finally, avoidance behaviors were tested in a Y-maze system to some of pyrazine analogues which were found
to release the other vigilance behaviors significantly. Taken together, the active pyrazine analogues which
release the all four types of vigilance behaviors possess a couple of methyl or ethyl group(s), but not other longer
carbonic chain groups. In addition, these alkyl side chains were constructed with four carbon atoms in each of
the powerful analogues. This study is the first experimental demonstration of the structure-activity correlation of
alkyl pyrazine analogues in mice vigilance behaviors.
P1-011 Sampling mode- and concentration-invariant temporal odor coding by airflow-driven neuronal
oscillations
Ryo Iwata1,2, Takeshi Imai1,3
1
Lab for Sensory Circuit Formation, RIKEN CDB, Kobe, Japan, 2JSPS, Japan, 3JST PRESTO, Japan
Sensory systems have an ability to recognize specific features from noisy and fluctuating background signals in
the external world. A central question in sensory systems is how they can quickly and reliably extract specific
sensory features from such dynamic stimuli. In recent years, it is getting realized that neuronal representation is
achieved not only by the changes of neuronal firing rate (rate coding), but also by altering the temporal pattern of
firing (temporal coding), such as spike latency, oscillation phase, and temporal trajectories. In the rodent
olfactory bulb, odors produce rich temporal patterns of activity in mitral/tufted cells; however, it remains unclear
what kind of features of an odor is encoded in the fine temporal patterns and how it is beneficial for olfaction.
Here we show that the temporal coding distinguishes two sensory modalities detected by input sensory neurons:
odor and airflow-driven mechanical signals. Odor stimuli, but not changes in airflow speed, produced phase
shifts (both delay and advance) in sniff-coupled oscillatory activities. The phase shift was glomerulus-specific as
well as odor-specific. We also found that the odor-evoked phase shifts are invariant across a wide range of odor
concentrations and sniff cycles, whereas the rate code pattern was dynamically reorganized at different odor
concentrations and across sniff cycles. The loss of airflow-driven oscillations impaired stability of the temporal
coding, demonstrating a role of mechanosensation in olfaction. We propose that the phase coding is a robust
encoding strategy for an odor identity under fluctuating sampling and environmental conditions.
P1-012 Olfactory receptor for prostaglandin F2α mediates courtship behavior of male zebrafish
Yoichi Yabuki1,2, Tetsuya Koide1, Nobuhiko Miyasaka1, Noriko Wakisaka1, Miwa Masuda1,3, Masamichi Ohkura4,
Junichi Nakai4, Kyoshiro Tsuge5, Soken Tsuchiya5, Yukihiko Sugimoto5, Yoshihiro Yoshihara1,2,3
1
RIKEN Brain Science Institution, Saitama, Japan, 2Department of Bioengineering, Nagaoka University of Technology, Niigata,
Japan, 3ERATO Touhara Chemosensory Signal Project, JST, Tokyo, Japan, 4Brain Science Institution, Saitama University Saitama,
Japan, 5Department of Pharmaceutical Biochemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University,
Kumamoto, Japan
Pheromones play vital roles in various organisms for survival and reproduction. In many fishes, prostaglandin F2α
acts not only as a female reproductive hormone facilitating ovulation and spawning, but also as a sex
pheromone inducing male reproductive behaviors. Here we unravel the molecular and neural circuit
mechanisms underlying the pheromonal action of prostaglandin F2α in zebrafish. Prostaglandin F2α specifically
activates two OR-type olfactory receptors with different sensitivities, which are expressed in distinct populations
of ciliated olfactory sensory neurons. The information is then transmitted to two glomeruli within the ventromedial
glomerular group (vmG) in the olfactory bulb and further to at least four brain regions: the ventral nucleus of
ventral telencephalon (Vv), anterior part of parvocellular preoptic nucleus (PPa), lateral hypothalamus (LH), and
caudal zone of periventricular hypothalamus (Hc). Mutant male zebrafish deficient in the high-affinity olfactory
receptor exhibit loss of attractive response to prostaglandin F2α. Furthermore, the mutant male fish spent
significantly less time in courtship toward female fish, including chase, touch, and encircle behaviors, than did
2016.05.27 11.25.45 Page 6(1)
wild-type male fish. These findings demonstrate functional significance of the sex pheromone prostaglandin F2α
and its cognate olfactory receptor activating selective neural circuitry in fish reproductive behavior.
P1-013 The main olfactory and vomeronasal systems modulate fear of predator wolf odor, pyrazine
analogs
Sadaharu Miyazono1, Kazumi Osada2, Hitoshi Sasajima1, Tomohiro Noguchi1, Makoto Kashiwayanagi1
1
Department of Sensory Physiology, Asahikawa Medical University, Japan, 2School of Dentistry, Health Sciences University of
Hokkaido, Japan
When prey animals detect predator odor, a spectrum of fear-related behavioral, autonomic and endocrine
responses innately occur to promote survival. How olfactory systems process predator odor to induce the
responses is not clearly understood. Our previous study identified a couple of pyrazine analogs from wolf urine
as a novel fear-eliciting odor. The pyrazine-analogs mixture (P-mix) induces the fear responses in laboratory
mice which possess two major olfactory systems, the main olfactory system (MOS) and the vomeronasal system
(VNS). Here, we investigate how the two olfactory systems contribute to induction of the fear responses. For this
purpose, we interfered with the murine two olfactory systems either independently or simultaneously. Mice were
treated intranasally with ZnSO4 (Zn mice) to block the MOS, while leaving the VNS intact. The vomeronasal
organs of mice were surgically removed (VNX mice) to block the VNS, while leaving the MOS intact. We
measured behavioral, autonomic and endocrine responses in Zn, VNX and Zn/VNX mice when exposed to the
P-mix. Among our measured responses, stimulus contact and grooming behaviors were reduced in VNX and
control mice, but not in Zn mice. Temperature decline of body surface was observed on Zn mice as well as
control mice, but not in VNX mice. Moreover, although risk assessment and rearing behaviors were observed in
control mice, they were observed in neither Zn nor VNX mice. We are further quantifying neuronal activation in
brain regions implicated in individual fear responses, using c-Fos immunohistochemistry. The results suggest
that the MOS and VNS could process the odor of a predator either in collaboration or independently to induce a
set of the fear-related responses.
P1-014 Dissecting the neural circuit responsible for sex pheromone-mediated behavior
Kentaro Ishii1,2, Kazunari Miyamichi1,2, Kazushige Touhara1,2
1
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan, 2
JST ERATO Touhara Chemosensory Signal Project, Japan
Pheromones are chemical compounds secreted by an animal and influence various behaviors of conspecific
individuals. Exocrine gland-Secreting Peptide 1 (ESP1), a peptide isolated from the tear gland of male mice, has
been shown to enhance female sexual receptive behavior, called lordosis reflection (Haga et al, Nature 466,
118, 2010). This pheromone is detected by a single pheromone receptor, V2Rp5, expressed in the vomeronasal
organ. However, the neural circuit that conveys the sensory information to the higher brain regions has remained
to be explored.
We first focused on the Medial amygdala (MeA), the second order center of the pheromone circuit. By using
neuronal tracers and genetic tools, here we show that MeA contains a heterogeneous population in regard of
their axonal projections to various brain areas. Some MeA neurons projected mainly to the Ventromedial
hypothalamus (→VMH neurons), while some exhibit denser projections to the medial preoptic area (→MPA
neurons). Interestingly, ESP1 preferentially induced expression of cFos , a histochemical marker of neural
activity, in the →VMH neurons rather than in the →MPA neurons. We next investigated genetic markers of VMH
neurons activated by ESP1. We found a specific neuron subtype in the VMH that highly responded to ESP1. Cell
type specific loss-of-function experiments revealed that these neurons in the VMH were dispensable for the
lordosis behavior in itself, and necessary for ESP1-mediated enhancement of sexual receptiveness.
Furthermore, by using projection selective synaptic silencing techniques, we revealed the Periaqueductal gray
as a necessary downstream area of the VMH neurons that mediate ESP1-mediated behavior.
Taken together, our results suggest that ESP1 information is sequentially conveyed from the MeA to the VMH,
and further to the PAG to modulate sexual behavior in female mice.
2016.05.27 11.25.45 Page 7(1)
P1-015 The emotional and electrophysiological effects of odor masking
Maki Shigyo1, Nao Tsuzuki1, Masayuki Hamakawa1, Kaori Tamura1, Yuki Yamada2, Hiroya Ishikawa3, Ayumi Kimura4,
Mai Morinaga4, Norihiko Matsumune4, Tsuyoshi Okamoto1,2
1
Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, Japan, 2Faculty of Arts and Science, Kyushu University,
Fukuoka, Japan, 3The International College of Arts and Sciences, Fukuoka Women’s University, Fukuoka, Japan, 4Kobayashi
Pharmaceutical Co., Ltd., Osaka, Japan
Odors can produce or modulate human’s emotion. Many commercial fragrances have been developed to give
out pleasant odors or mask unpleasant odors. Although several previous studies have investigated the effects of
such fragrances themselves, there are not many studies about the emotional effects of odor masking such as a
male body odor with floral fragrance. In order to prove whether such effects could suppress negative emotion
caused by an unpleasant odor, we investigated psychological and neurophysiological activities.
Forty healthy young adult females participated in our experiment. They were divided into four groups depending
on the odorant conditions: 1) no odor (control), 2) male body odor, 3) a unisex fragrance and 4) male body odor
masked by the unisex fragrance. Under each condition, electroencephalogram (EEG) was measured during the
following task: participants were asked to classify gender from several face images displayed on a CRT monitor.
The participants’ mood and emotion were measured by the second edition of the Profile of Mood States (POMS
2) before and after the experiment. Emotional and intensity ratings of the presented faces and odors were also
measured after the experiment.
The acquired EEG data was analyzed via Fourier transform, and the amplitudes of alpha band were
computed. As a result, alpha band (8-13 Hz) amplitudes were significantly larger in 4) male body odor masked
by the light fragrance group than 1) control and 2) male body odor groups. Moreover, the emotional and intensity
ratings of the presented odors showed that participants felt negative emotion in 2) male body odor group,
whereas in 4) fragrance masking male body odor group felt positive emotion.
The above results are evidence suggesting that the masking by the light fragrance improves the negative
emotion caused by unpleasant odor.
P1-016 Transcriptional profiling of olfactory imprinting in the olfactory center of Pacific salmon
Yui Minowa1, Seishi Hagihara2, Takashi Abe1, Shigeho Ijiri2, Shinji Adachi2, Hideaki Kudo3
1
Laboratory of Humans and the Ocean, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Japan, 2Laboratory
of Aquaculture Biology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan, 3Laboratory of Humans and the
Ocean, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan
It is generally accepted that information about some of the odorants in the natal streams of anadromous Pacific
salmon (Oncorhynchus spp.) is imprinted during seaward migration, and that anadromous Pacific salmon use
olfaction to identify their natal streams during spawning migration. However, little is known about the molecules
that are important for olfactory imprinting and memory retrieval in the salmon brain. In this study, a search for the
genes involved in olfactory imprinting and memory retrieval was performed in the chum salmon (O. keta )
olfactory center (the olfactory bulb and telencephalon). The olfactory centers of seaward-migrating (the olfactory
imprinting period) juveniles and homeward-migrating (the olfactory memory retrieval period) adult salmon were
subjected to transcriptome (RNA sequencing) analyses using a next-generation sequencer. A mean of
45,898,780 (4,636 Mb) and 45,409,999 (4,584 Mb) quality controlled reads were obtained in the
seaward-migrated juveniles and homeward-migrated adults, respectively, and these reads were assembled into
253,872 and 226,232 contigs, respectively. tblastx analyses identified 57 and 60 transcripts that be involved in
olfactory imprinting and memory retrieval, respectively. In each period; i.e., during seaward and homeward
migration, the selected genes exhibited minimal expression during the silent stage and >10-fold higher
expression compared with the silent stage during the active stage. Different groups of genes were selected in
the seaward- and homeward-migrated fish. Genes encoding molecules involved in pre-synaptic vesicle
exocytosis, cell-growth/differentiation, etc., were included in the olfactory imprinting-related candidate genes. On
the other hand, the genes for certain neurotransmitter receptors were included in the olfactory memory retrievalrelated candidate genes. This study is the first to attempt to identify transcripts that contribute to olfactory
imprinting and memory retrieval in the olfactory center, and suggests that different gene groups are responsible
for imprinting and memory retrieval in Pacific salmon.
2016.05.27 11.25.45 Page 8(1)
P1-017 Predatory odor induced stress responses can be allayed by habitat odor
Mutsumi Matsukawa1, Masato Imada1, Shin Aizawa1, Takaaki Sato2
1
Division of Anatomical Science, Department of Functional Morphology, Nihon University School of Medicine, Tokyo, Japan,
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Osaka, Japan
2
Odors from predatory animals, such as 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), are known to induce innate
fear and stress responses in prey animals. Exposure to a predator or predatory odor is used to create animal
models of some neuropsychological disorders including anxiety, phobia and post-traumatic stress disorder
(PTSD). We have previously shown that mice have an innate classification system for deciding how to respond
to odors. According to this classification, odors can be divided into at least three types in nature: (1) odors that
can innately induce stress-like behavior, such as predatory odors like TMT; (2) odors that can innately alleviate
predatory odor-induced stress-related activities, such as rose and hinokitiol odor and (3) odors that have no
effect, such as caraway odor. Hence, we attempted to verify whether the postnatal experiences may have a
modifying effect on the innate classification criteria, leading to changes that may cause individual differences in
odor response in adulthood. In this study, we demonstrated that the innate classification system was changed to
counteract predatory odor induced stress responses following a postnatal experience. Moreover, we suggest
that inhibitory circuits are involved in stress-related neuronal networks and the concentrations of norepinephrine
in the hippocampus may be crucial in alleviating stress induced by the predatory odor. We confirm that early life
environment can have a modifying effect not only on innate classification system for odor responses but also in
the development of individual responses to odors in adulthood. Take account of the fact that exposure to
predatory odors is used in some psychiatric disease models, our data can elucidate the mechanisms involved in
differential odor responses and contribute to the development of pharmacotherapeutic interventions for stressrelated disorders.
P1-018 Spontaneous activity governs the dendrite pruning of mitral cells to establish discrete
connectivity
Satoshi Fujimoto1, Marcus N. Leiwe1, Yuko Muroyama2, Reiko Kobayakawa3, Ko Kobayakawa3, Tetsuichiro Saito2,
Takeshi Imai1,4
1
Laboratory for Sensory Circuit Formation, RIKEN Center for Developmental Biology, Kobe, Japan, 2Department of Developmental
Biology, Graduate School of Medicine, Chiba University, Chiba, Japan, 3Institute of Biomedical Science, Kansai Medical University,
Hirakata, Japan, 4Graduate School of Biostudies, Kyoto University, Kyoto, Japan
During the development of continuous sensory maps, such as retinotopic and tonotopic maps, spontaneous
neuronal activity produced by the sensory organs plays an important role in establishing precise topographic
connectivity between pre- and post-synaptic neurons. In contrast, olfactory sensory neurons (OSNs) form a
discrete glomerular map, which is known to be regulated by a directed guidance process instructed by genetic
information and odorant receptor-derived signals. In the olfactory bulb, each mitral cell connects a single primary
dendrite to a specific glomerulus in the discrete glomerular map. However, little has been known as to how mitral
cells establish specific dendritic connectivity to specific glomeruli. Each mitral cell initially connects multiple
primary dendrites to multiple glomeruli, and then prunes all but one primary dendrite during the first postnatal
week. However, the mechanisms of the selective dendrite pruning have remained largely unknown. Here we
revealed that the selective dendrite pruning is regulated by spontaneous neuronal activity, which is produced
independently of OSNs. The dendrite pruning was perturbed by the over-expression of Kir2.1 in mitral cells, but
was not affected by the inhibition of synaptic transmission from OSNs. We also observed spontaneous network
activity in the developing olfactory bulb both in vivo and in vitro using 2-photon calcium imaging. Before the
dendritic pruning period, spontaneous activity was highly correlated among glomeruli; however, the pattern of
spontaneous activity gradually became decorrelated at later stages. Pharmacological experiments revealed that
gap junctions and ionotropic glutamate receptors are required for propagating spontaneous activity at an early
stage. Taken together, our results demonstrate that the sensory stimuli-independent spontaneous network
activity regulates dendritic pruning of mitral cells to establish discrete connectivity between OSNs and mitral
cells.
2016.05.27 11.25.45 Page 9(1)
P1-019 Enhancement of sympathetic nervous system activation by orange aroma inhalation during
sleep
Masako Ohira-Hasegawa1, Takuma Sakamoto2, Takuro Ichikawa2, Kanetoshi Ito3, Shusaku Nomura2
1
Faculty of Education, Shiga University, Otsu, Japan, 2Faculty of Engineering, Nagaoka University of Technology, Nagaoka, Japan,
Corporate Research & Development, Takasago International Corporation, Hiratsuka, Japan
3
Orange aroma has been reported to enhance parasympathetic nervous system activation in the daytime.
However, its effect at the nighttime is not yet known. The objective of this study is to investigate the effect of
orange aroma inhalation during sleep on autonomic nervous system activity. The orange essential oil was
administered to subjects (17 male aged 20-24 years) using an olfactometer during the six-hour sleep period.
Each subject received the odorant with different concentration of 1% and 20%, or scentless air (control) in a
counterbalanced order. Polysomnography (PSG) was recorded during the sleep period and an hour after
awakening. Subjective sleep measures were given at awakening. Comparing to control condition, the heart rate
(HR) during sleep was significantly higher and tend to be higher in 1% and 20% of orange aroma inhalation
(p<0.05 and p<0.10, respectively). PSG analysis revealed that the ratio of Wakening stage (WS) in orange
condition of 1% and 20% was tend to be higher and significantly higher than that of control (p<0.10 and p<0.05,
respectively) and that the ratio of Slow wave sleep (SWS) in Stage 3 in orange condition of 1% and 20% was
significantly lower than that of control (p<0.05 and p<0.05, respectively). Meanwhile, there is no difference in
subjective sleep measures. The higher HR and WS and lower SWS with the orange aroma inhalation indicates
the enhancement of sympathetic nervous system activation during sleep by the odorant. However, the
subjective sleep measures reveals the quality of sleep was not impaired by the aroma administration. Moreover,
the orange aroma was the most by the subjects. This antagonistic and newly found results illustrates a complex
characteristic of orange aroma. Further study should be done for the better understanding.
P1-020 Coordinated electrical activity in the olfactory bulb gates the oscillatory entrainment of
entorhinal networks in neonatal mice
Sabine Gretenkord, Johanna Katharina Kostka, Henrike Hartung, Ileana Livia Hanganu-Opatz
Developmental Neurophysiology, Institute of Neuroanatomy, University Medical Center Hamburg Eppendorf, Hamburg, Germany
Sensory inputs from the environment are critical for survival, yet their impact on higher cognitive performance is
poorly understood. This is especially true when considering the ontogeny of mnemonic and executive
processing within limbic networks. In rodents, most sensory systems are underdeveloped during the early
postnatal period. As a notable exception, olfaction reaches full maturity during intrauterine life and controls
mother-offspring interactions. It is, however, still unknown whether early olfactory inputs drive the development
of limbic networks. Here, we aim at understanding the structural and functional principles underlying the
connectivity and communication between the olfactory bulb (OB) and lateral entorhinal cortex (LEC)-the
gatekeeper of limbic circuitry, during neonatal development. For this, we combine tracing of long-range OB LEC
connectivity with extracellular and patch-clamp recordings in vivo from the OB mitral cell layer and LEC. We
show that discontinuous oscillations with main frequency within the theta band (4 12 Hz) accompany the
respiration-related activity in the OB of neonatal mice. They time the firing and synaptic activity of mitral cells.
The coordinated activity provides tight coupling by synchrony and directed interactions between OB and LEC.
This functional communication relies on early emerging axonal projections of mitral cells to LEC. The data
elucidate the structural and physiological signature of OB-to-LEC communication during early development.
Taking into account that LEC drives prefrontal-hippocampal networks during the neonatal period, the OB
coordinated activity might facilitate the maturation of limbic circuitry.
P1-021 Is olfactory bulb affected in Neurofibromatosis Type 1?
Muzaffer Saglam1, Murat Salihoglu2, Ercan Karabacak3, Hakan Mutlu4, Guner Sonmez1, Hakan Mutlu1
1
Radiology, Gulhane Military Medical Academy, Haydarpasa Training Hospital, Istanbul, Turkey, 2Otorhinolaryngology, Gulhane
Military Medical Academy, Haydarpasa Training Hospital, Istanbul, Turkey, 3Dermatology, Gulhane Military Medical Academy,
Haydarpasa Training Hospital, Istanbul, Turkey, 4Neurology, Gulhane Military Medical Academy, Haydarpasa Training Hospital,
Istanbul, Turkey
Purpose: To investigate olfaction, taste and olfactory bulb volume (OBV) in Neurofibromatosis Type 1 (NF1)
patients. Material and Methods: We evaluated 13 NF1 patients (12 men, 1 woman; mean age 22,3 years) and 30
healthy controls (28 men, 2 women; mean age 22,8 years). OBV measurement, olfactory function and taste tests
were performed in both groups. OBV measurement was performed with a 1.5-T magnetic resonance imaging
2016.05.27 11.25.45 Page 10(1)
system. Psychophysical testing of olfactory function was performed with the validated Sniffin Sticks test that
comprised 3 components; odor threshold, discrimination and identification. Four basic tastants (sweet, sour,
salty, bitter) were used for taste evaluation. NF1 group was compared with control group regarding OBV,
olfactory and taste functions. Results: In NF1 group, the mean right and left OBV were measured to be 82,84+26,17 mm3 and 83,76+-27,63 mm3 respectively. In control group, the mean right and left OBV were measured
to be 52,14+-11,92 mm3 and 53,9+-11,1 mm3 respectively. There was significant difference between both side
OBV of NF1 and controls (P<0,0001). OBV and TDI correlations were investigated and no significant correlation
were found in NF1 group (right; p=-0,088, left; p=0,575). There was no difference between threshold of NF1
group and control group (thresholds: 7,75+-0,43 vs 7,85+-0,56, p=0,766). Discrimination, identification and TDI
scores of NF1 group was significantly lower than control group (discrimination: 12,08+-0,76 vs 13,27+-1,05, p=
0,001; identification: 12,69+-0,63 vs 13,5+-1,01, p=0,011; TDI score: 33,17+-1,75 vs 34,58+-1,37, p=0,016). The
results from taste test showed that there was no difference between two groups for sweet, sour, salty and bitter.
Conclusion: Although NF1 patients have larger olfactory bulbs they have lower scores of odor discrimination,
identification and TDI scores when compared with healthy controls. No taste differences was present between
NF1 patients and healthy controls.
P1-022 Postembryonic development of sex pheromone-receptive olfactory sensory neurons in the
cockroach
Hidehiro Watanabe1, Yukino Koike1, Hiroshi Nishino2, Fumio Yokohari1
1
Department of Earth System Science, Faculty of Science, Fukuoka University, Fukuoka, Japan, 2Research Institute for Electronic
Science, Hokkaido University, Sapporo, Japan
In the cockroach Periplaneta americana , sex pheromone-receptive olfactory sensory neurons (pOSNs)
explosively increase in number during the last-instar stage. At the same time, axon terminals of newly born
pOSNs are recruited into a macro-glomerulus according to antennal locations of sex pheromone-receptive
sensilla; pOSNs arising from sensilla on the more proximal region of the flagellum tend to project progressively
to the more proximal region of the macro-glomerulus, relative to axonal entry point. However, it is still unknown
developmental mechanisms underlying the formation of the topographic organization of pOSNs in the
macro-glomerulus. To tackle this question, elucidating when and where pOSNs emerge in an antenna is
indispensable. First, we compared the sensilla distributions on an identical set of flagellomeres between before
and after final molting, and revealed that sex pheromone-receptive sensilla located in the distal part of the adult
antenna are derived from the different type of sensilla during the last-instar stage. Second, we detected
new-born antennal sensory neurons using EdU assay, which is a nucleoside analog and is incorporated into
DNA during DNA synthesis. Using these techniques, we revealed temporal and spatial dynamics of pOSNs
increment patterns during postembryonic development within the cockroach antenna. Finally, we stained OSNs
housed in a cognate type of sensillum, and identified that the nymphal cockroach equips putative pOSNs in their
antenna. Based on these results, we hypothesized a new model for formation of topographic organization of
pOSNs in the macro-glomerulus.
P1-023 A specific subtype of olfactory bulb interneurons is necessary for odor detection and odorbackground segregation
Hiroo Takahashi1, Sei-ichi Yoshihara1, Yoichi Ogawa2, Ryo Asahina1, Tatsuro Kitano1, Akio Tsuboi1
1
Laboratory for Molecular Biology of Neural System, Nara Medical University, Japan, 2Department of Physiology I, Nara Medical
University, Japan
Neural circuits that undergo reorganization by newborn interneurons in the olfactory bulb (OB) are necessary for
odor detection, odor-background segregation, olfactory memory and innate olfactory responses, including
predator-avoidance, food-finding and sexual behaviors. The OB possesses a battery of interneurons, including
various types of granule cells (GCs); however, the contribution that each type of interneuron makes to the control
of olfactory behavior remains unknown. Here, we investigated the in vivo functional role of oncofetal trophoblast
glycoprotein 5T4, a regulator for the dendritic arborization of 5T4 -expressing GCs (5T4 GCs), which is reduced
in its knockout OB (Yoshihara et al , J Neurosci, 32;2217, 2012). Electrophysiological recordings with acute OB
slices indicated that external tufted cells (ETCs) can be divided into two types, bursting and non-bursting.
Optogenetic stimulation of 5T4 GCs revealed their connection to both bursting and non-bursting ETCs, as well
as to MCs. Interestingly, in 5T4 knockout mice, non-bursting ETCs received fewer inhibitory inputs from 5T4 GC
dendrites than in the wild type, while bursting ETCs and MCs received the similar inputs from 5T4 GCs,
2016.05.27 11.25.45 Page 11(1)
respectively. We are in the process of recording the excitatory inputs into 5T4 GCs in 5T4 knockout OB,
compared with the wild type. Notably, in olfactory behavior tests, 5T4 knockout mice had higher odor-detection
thresholds than the wild type, and were impaired in odor-discrimination learning, such as segregating the food
odor from the irrelevant background odor for finding food. Taken together, our findings suggest that loss of 5T4
attenuates inhibitory inputs from 5T4 GCs to non-bursting ETCs, thus probably leading to superfluous firing of
the ETCs and consequent disturbances in olfactory behaviors. Hence, we demonstrated for the first time that
among various types of OB interneurons, the 5T4 GC subtype is required for odor-detection and discrimination
behaviors (Takahashi et al , revised).
P1-024 The olfactory cortex internally generates exhalation-phased active state without olfactory
sensory input
Kimiya Narikiyo1,2,3, Hiroyuki Manabe1,2,4, Yoshihiro Yoshihara3, Kensaku Mori1,2,3
1
Department of Physiology, The University of Tokyo, Tokyo, Japan, 2Japan Science and Technology Agency, CREST, Tokyo, Japan,
RIKEN Brain Science Institute, Saitama, Japan, 4Department of System Neuroscience, Doshisha University, Kyoto, Japan
3
Sensory inputs in the olfactory system depend on respiration. The olfactory cortex is engaged in on-line
processing of external odor information during inhalation, but is isolated (off-line) from the external world during
exhalation. Previous studies on the olfactory cortex focused on the neural mechanisms of the on-line processing
during the inhalation phase, but little is known about the cortical information processing during the off-line
exhalation phase. To investigate how the neural circuits in the olfactory cortex operate during the exhalation
phase, we recorded local field potentials and neuronal spike activities of anterior olfactory nucleus and piriform
cortex in head-fixed awake rats using liner multi-channel electrode with simultaneous monitoring of respiration.
Local field potential of the olfactory cortex showed respiration-phased slow oscillatory (0.5-3Hz) activity. Current
source density analysis of the slow oscillatory activity revealed a slow current sink in the superficial layer (Layer
I) during the inhalation phase and, surprisingly, a slow current sink in the deep layer (Layer II/III) during the
exhalation phase. Olfactory sensory deprivation by naris occlusion or bilateral olfactory bulbectomy spared both
the inhalation-phased superficial-layer slow current sink and the exhalation-phased deep-layer slow current sink.
The exhalation-phased deep-layer slow current sink accompanied enhanced firing of olfactory cortex neurons,
which remained even after the bilateral olfactory bulbectomy. These results indicate that olfactory cortex
internally generate the active state via synaptic inputs to the deep layer during exhalation phase, which may
represent the internal state of the olfactory cortex.
P1-025 Nrp2+ mitral cells in the main olfactory bulb send axons to the medial amygdala to induce
social behaviors
Kasumi Inokuchi1,2, Haruki Takeuchi2, Fumiaki Imamura3, Ryang Kim4, Hiroyuki Okuno4, Hirofumi Nishizumi1,2,
Haruhiko Bito4, Takefumi Kikusui5, Hitoshi Sakano1,2
1
Faculty of Medical Science, The University of Fukui, Fukui, Japan, 2Department of Biophysics and Biochemistry, The University of
Tokyo, Tokyo, Japan, 3Department of Pharmacology, Pennsylvania State University, Hershey, USA, 4Department of Neurochemistry,
The University of Tokyo, Tokyo, Japan, 5School of Veterinary Medicine, Azabu University, Kanagawa, Japan
The medial amygdala (MeA) is known to mediate innate social behaviors by receiving pheromone inputs from
the accessory olfactory bulb. However, involvement of the main olfactory bulb has also been suggested. Here,
we report that mitral cells localizing to the ventrolateral main olfactory-bulb are Nrp2-positive, and send their
axons to the anterior MeA. Retrograde virus-tracing from the MeA reveals that synaptic connections are indeed
made with the ventrolateral mitral cells. Surprisingly, ectopic expression of human Nrp2 can even bring
dorsal-lineage mitral cells to the ventrolateral bulb, and guide their axons to the anterior MeA. Knockout
experiments demonstrate that repulsive interactions of Nrp2 with Sema3F regulate both migration and targeting
of mitral cells. Furthermore, pup suckling behavior and male ultrasonic vocalization are impaired in the knockout
mice. We, therefore, conclude that a subset of Nrp2-positive mitral cells in the mouse main olfactory system is
responsible for inducing innate social behaviors by sending odor signals from the ventrolateral olfactory bulb to
the anterior MeA.
2016.05.27 11.25.45 Page 12(1)
P1-026 Automatic orientation of spatial visual attention from lateralized olfactory-trigeminal
stimulations
Matthieu Jeremiah Ischer1,2, Sylvain Delplanque1,2, Myriam Esselier1,2, Christelle Porcherot3, Nadine Gaudreau3,
Christophe Mermoud4, Isabelle Cayeux3, David Sander1,2
Swiss Center for Affective Sciences (CISA), University of Geneva, Geneva, Switzerland, 2E3Lab, Department of Psychology,
University of Geneva, Geneva, Switzerland, 3Firmenich, S.A., Geneva, Switzerland, 4Department of Medicine, University of Geneva,
Geneva, Switzerland
1
Almost all perceived volatile compounds stimulate both the main olfactory system and the trigeminal system.
Through the trigeminal system, it is possible to intentionally pay attention to a scent and spatially localize it, but
whether a scent is capable of automatically orienting spatial attention intermodally remains unknown. Yet, i)
there is a common pool of attentional resources shared across sensory modalities, ii) the trigeminal system
enables humans to differentiate a stimulation delivered to the left or the right nostril, and iii) strong trigeminal
stimulations create alert responses and can thus be considered relevant stimuli, which are particularly prone to
capturing an individual s attention. Based on these findings, we predicted that lateralized olfactory trigeminal
information would automatically guide visual spatial attention. In four studies, we used CO2-a gas considered to
be a pure trigeminal stimulant-as lateralized cues in a variant of the visual spatial cueing paradigm. Participants
were required to detect as fast and as accurately as possible the orientation of a visual target following an
olfactory trigeminal cue. In valid trials, cues and targets were presented on the same side (e.g., cue in left nostril
and target in the left visual field), whereas in invalid trials, they were presented on opposite sides (e.g., cue in the
left nostril and target in the right visual field). We also varied the delay between the cue and the target in the 4
different studies. As predicted, an intermodal effect was observed: reaction times in valid trials were faster than
invalid trials. We also found that this effect varied as a function of the delay between the olfactory cue and the
visual target. This series of studies demonstrates, for the first time to our knowledge, the automatic orientation of
visual spatial attention from olfactory-trigeminal stimulation.
P1-027 Effect of fragrance on prefrontal cortex activity during the working memory task: A nearinfrared spectroscopy study
Kao Yamaoka1, Toshiaki Imasu1, Yoshihisa Abe1, Satomi Kunieda2, Chizuru Konagai4, Yoshihiko Koga3
1
Innovation Research Center, FANCL Corporation, Kanagawa, Japan, 2Corporate Research & Development Division, TAKASAGO
INTERNATIONAL CORPORATION, Kanagawa, Japan, 3Department of Neuropsychiatry, Kyorin University School of Medicine,
Tokyo, Japan, 4Department of Food and Nutrition, Nitobe Bunka College, Japan
We evaluated the influence of fragrances on the levels of oxygenated-hemoglobin (oxy-Hb) in the cerebral blood
while the participants performed the working memory (WM) task. The levels of cerebral blood are one of the
indexes of evaluating brain activities. In our study, the participants were exposed to different types of fragrance,
including the odorless control sample, and they were required to carry out the n-back task to measure the WM
function. The levels of oxy-Hb during the task were evaluated using near-infrared spectroscopy (52ch). The
different patterns in levels of oxy-Hb were observed while being exposed to a specific type of fragrance,
compared to others including the control sample. Furthermore, it was shown that the most salient change in the
levels of oxy-Hb was observed at the brain areas that overlapped with the Brodmann area 46, known as the
neural substrates of WM function. These results may suggest that the exposure to a specific type of fragrance
may have an effect on the brain areas that are related to the WM process.
P1-028 Similar perceived odors may induce similar systemic brain activity
Jisub Bae1, Won-Seok Kang2, Ji-Woong Choi3, Bongki Cho1, So Yeun Kim1, Cheil Moon1
1
Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea,
Wellness-convergence Division, Gyeongbuk Institute of Science and Technology, Daegu, South Korea, 3Department of Information
and Communication Engineering, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea
2
How we discriminate odors is an important question. According to previous studies, perceiving distinct odors
begins with activating distinct combination of olfactory receptor (OR) repertoire in olfactory receptor neurons
(ORN). This effect consecutively activates distinct ORN and these distinct ORN activities encode in olfactory
bulb. Encoded odor information finally is processed in the brain so we can perceive and discriminate odors.
However, the questions are still unsolved why different OR repertoires induce different brain activities, and
similar neural encoding of the olfactory bulb induces distinct odor responses. To solve these questions, it is
necessary to study the odor responses in the brain in which the odor information is lastly processed. Thus, this
2016.05.27 11.25.45 Page 13(1)
study focused on how similar perceiving odors process in the brain. We used two odors, 2-acetylpyrazine (AP)
and 2, 3, 5-trimethly pyrazine (TP) that describe as similar perceptual descriptor but activating different OR
repertoire. To measure the direct brain signal, EEG was used to understand how participants process odor in
their brain. Power spectrum and event related spectral perturbation (ERSP) of EEG frequency bands are used to
verify similarity between responses upon AP and TP treatment. Interestingly, AP and TP induce similar patterns
of power spectrum in total electrodes. These results suggest that these odors activated similar regions in the
brain. Also ERSP results of AP and TP suggest that AP and TP induce similar pattern of brain activity in the timescale. These two results emphasize that AP and TP may activate similar olfactory pathway in the brain.
However, total intensities of power spectrum are different between AP and TP. These differences may imply how
people are able to distinguish these two odors. Based on these results, similar perceiving odors may induce
similar systemic brain activity. and odors may be distinguished by different intensity of the brain activities.
P1-029 Flexible olfactory coding by mushroom body output neurons of Drosophila
Toshihide Hige1,2, Yoshinori Aso1, Mehrab N. Modi2, Gerald M. Rubin1, Glenn C. Turner1,2
1
HHMI, Janelia Research Campus, Ashburn, VA, USA, 2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
One of the most important higher-order functions of sensory systems is to flexibly interpret stimulus information
to select appropriate behaviors depending on the animal’s experience and states. In Drosophila olfactory
system, we recently demonstrated such flexible sensory processing at the mushroom bodies, suggesting their
important roles in adaptive sensory-motor transition. However, it has not been clear what kind of synaptic
modulations underlie the flexible odor coding. Here we provide the first demonstration of a long-term synaptic
plasticity at the output synapses of the Drosophila mushroom body, in the context of associative learning. Using
in vivo whole-cell recordings, we show that brief pairing of odor delivery and optogenetic activation of specific
dopamine neurons, which mimics aversive reinforcing stimuli, is sufficient both to induce synaptic plasticity and
to induce memory formation in a behavioral assay. The induction of plasticity strictly depends on the temporal
order of the two stimuli with sub-second precision, replicating the logical requirement for associative learning.
Furthermore, by precisely manipulating individual circuit elements, we reveal that dopamine action is confined to
and distinct across different anatomical compartments of the mushroom body lobes. Finally, we find that overlap
between sparse representations of different odors determines both how stimulus-specific the plasticity is and
how generalizable the odor memories are. Thus, the plasticity we find here not only manifests important features
of associative learning that are apparent at the behavioral level but also provides general insights into how a
sparse sensory code is read out at the stage of sensory-motor transition.
P1-030 ER stress impaired olfactory learning by inhibiting synaptic plasticity via a presynaptic
mechanism in the olfactory bulb
Jia Tong1, Fumino Okutani2, Yoshihiro Murata1, Hideto Kaba1
1
Department of Physiology, Kochi Medical School, Kochi, Japan, 2Department of Occupational Health, Kochi Medical School,
Kochi, Japan
The endoplasmic reticulum (ER) stress links to neuronal death in various neurodegenerative diseases. Behavior
experiments show aversive olfactory learning is prevented by intrabulbar infusion of tunicamycin (TM). Using
electrophysiology, we reveal TM has an inhibitory effect on the induction long-term potentiation at
dendrodendritic synapses in the olfactory bulb (OB). Histological results show intrabulbar infusion with TM
causes upregulation of C/EBP-homologous protein, also known as growth arrest-and DNA damage-inducible
gene 153, that is strong in the mitral cell layer but weak in the granule cell layer. Furthermore, TM causes
disturbance on paired-pulse facilitation ratio dependent on a presynaptic mechanism evoked by paired-pulse
stimulation. These results suggest that ER stress impairs olfactory learning by inhibiting synaptic plasticity via a
presynaptic mechanism in the OB.
P1-031 Exposure to odor generated from the Maillard reaction decreases blood pressure by affecting
the autonomic nervous system
Lanxi Zhou1, Motoko Ohata1, Chisato Owashi1, Katsuya Nagai2, Keizo Arihara1
1
Department of Animal Science, Kitasato University, Aomori, Japan, 2ANBAS Corporation, Osaka, Japan
In a previous study, we investigated the effects of odor generated from the Maillard reaction of protein digests
with xylose on blood pressure. The protein digests were prepared by treating chicken meat with papain. Xylose
2016.05.27 11.25.45 Page 14(1)
and the protein digests were dissolved in sodium carbonate buffer and the pH was adjusted to either 5 or 10.
Samples were then heated at 90 degrees for 240 min. Exposure of Wistar rats to the odor generated by the
reaction at pH 10 significantly decreased their systolic blood pressure (SBP). In this study, we identified the
active odorants that affected blood pressure and their mechanism of action. The potent odorants contributing
most to the odor of the pH 10 sample were identified by aroma extract dilution analysis, and Wistar rats were
exposed to specific concentrations of each of the odorants. Of the four most potent odorants (acetic acid, 2hydroxy-3-methyl-2-cyclopenten-1-one, 2,5-dimethyl-4-hydroxy-3(2H )-furanone [DMHF] and 5-methylpyrazine2-methanol [MPM]), DMHF and MPM significantly decreased SBP. The earliest decrease in SBP was observed
5 min after exposure to DMHF. Application of ZnSO4 to the nasal cavity, which blocks the olfactory nerve,
eliminated this effect, suggesting that DMHF affects SBP through the olfactory system. Furthermore,
parasympathetic gastric vagal nerve activity was elevated and renal sympathetic nerve activity was lowered after
exposure to DMHF, indicating that the mechanism of action of DMHF on SBP involves the autonomic nervous
system.
P1-032 Wiring and information processing in an amphibian olfactory network
Thomas Offner, Thomas Hassenkloever, Sara Joy Hawkins, Lukas Weiss, Ivan Manzini
Neurophysiology and Cellular Biophysics, The University of Goettingen, Goettingen, Germany
The glomerular layer of the main olfactory bulb is characterized by an array of ovoid neuropil agglomerates, the
olfactory glomeruli. They represent the first relay station of olfactory information processing where the axon
terminals of olfactory receptor neurons synapse onto the apical dendrites of mitral/tufted cells. In rodents, each
glomerulus is innervated by the single, unbranched axons of a particular population of olfactory receptor
neurons. Remarkably in larval and adult Xenopus laevis , the axons of olfactory receptor neurons bifurcate and
terminate in one or multiple anatomically distinct glomeruli. Via single cell electroporation and fast two photon
calcium imaging we demonstrated the functional connection of both axon terminals with their respective
glomerulus. In addition, we characterized the exact position and chemosensory map of an amino-acid
responsive cluster in the ventro-lateral main olfactory bulb of larval Xenopus . Despite the unparcellated
morphology of the cluster, we were able to derive the morphology of various glomeruli from the odor induced
activity of mitral/tufted cell apical tufts. Single cell electroporation experiments revealed that the vast majority of
mitral/tufted cells in the ventro-lateral bulb exhibited multiple apical dendrites projecting their tufts into either one
or multiple anatomically distinct glomeruli. The targeting of multiple glomeruli by single neurons from both, the
pre- and postsynaptic side, rules in the possibility of a yet unprecedented olfactory wiring logic. We are currently
using transsynaptic tracing or a combination of single cell electroporation and two photon calcium imaging to
understand the significance of this unique circuitry for vertebrate olfaction in general.
[Supported by Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular
Physiology of the Brain (CNMPB); DFG Priority Programme 1392; German Ministry of Research and Education
(BMBF, project 1364480); DFG Research Projekt MA 4113/3-1]
P1-033 Temporally-coordinated communication between olfactory cortex and orbitofrontal cortex
during slow-wave sleep in rats
Hiroyuki Manabe1,2, Naomi Onisawa1, Kensaku Mori1
1
Department of Physiology, Graduate School of Medicine, The University of Tokyo, Japan, 2Department of Systems Neuroscience,
Graduate School of Brain Science, Doshisha University, Japan
Slow-wave sleep provides a specific time window for off-line communications among wide areas of the
neocortex. During slow-wave sleep, the large scale networks of the neocortex show slow oscillatory activities
that consist of up-state with enhanced neuronal activity and down-state with neuronal silence. Because olfactory
cortex (OC) areas show sharp-wave activities during slow-wave sleep, we examined in freely behaving rats
whether the OC sharp-wave activity occurs in temporal coordination with the slow oscillatory activity in the
orbitofrontal cortex (OFC) during slow-wave sleep. Simultaneous recordings of local field potentials and spike
activities in the anterior piriform cortex (APC) and the OFC showed that during the down-state in the OFC, APC
showed reduced spike activity and suppression of sharp-wave generation. APC sharp-waves tended to occur
during two distinct phases of the OFC up-state: at the initial phase of the up-state shortly after the down-to-up
transition and at the late phase shortly before the up-to-down transition. Because many APC neurons show
synchronized spike activity during APC sharp-waves, we further examined whether OFC neurons show spike
discharges in synchrony with the APC sharp-waves. About 45% of recorded neurons in the OFC showed
2016.05.27 11.25.45 Page 15(1)
enhanced spike activity during the APC sharp-waves. These results suggest that during slow-wave sleep, OC
and OFC communicate preferentially at the specific time windows shortly after the down-to-up transition and
shortly before the up-to-down transition.
P1-034 Valproic acid promotes neural regeneration of olfactory epithelium in adult mice after
methimazole-induced damage
Takao Ogawa, Takeshi Shimizu, Kumiko Takezawa
Department of Otorhinolaryngology, Shiga, Japan
BACKGROUND: Recent experiments have revealed that valproic acid (VPA), a histone deacetylase inhibitor,
has neuroregenerative effects in rodent models of spinal cord and optic nerve injury. VPA has a potential to
provide a new therapeutic strategy for sensorineural olfactory dysfunction. To elucidate the effects of VPA on
regeneration of olfactory sensory neurons, we examined the in vivo effects of oral VPA administration on
recovery from methimazole-induced degeneration of olfactory neuroepithelium in mice. METHODS: Male ICR
mice (10 weeks old) were intraperitoneally injected with methimazole (75 mg/kg), an olfactory toxic reagent, to
induce degenerative changes in the olfactory neuroepithelium. The effects of daily VPA administration on
recovery from methimazole-induced changes were examined histologically. RESULTS: Oral VPA administration
dose dependently enhanced increases in epithelial thickness and number of olfactory marker protein (OMP)
positive cells in the olfactory epithelium during recovery from methimazole-induced degeneration. VPA also
enhanced early increases in the number of Ki-67(+) and growth-associated protein-43(+) cells during the
regeneration of olfactory neuroepithelium. CONCLUSION: VPA administration promotes regeneration of
olfactory sensory neurons in damaged neuroepithelium by stimulating the proliferation and differentiation of
olfactory precursor cells. VPA has been used for several decades to safely treat neurological disorders. VPA
may provide a new therapeutic strategy for the treatment of olfactory dysfunction caused by degeneration of the
olfactory neuroepithelium.
P1-035 Neuroprotective and neuroendocrine effects of essential oils on immortalized hypothalamic
neurons
Masahiro Kawahara1, Akiko Nakamura3, Dai Mizuno2
1
Department of Bio-Analytical Chemistry, Faculty of Pharmacy, Musashino University, Japan, 2Department of Forensic Medicine,
Faculty of Medicine, Yamagata University, Japan, 3Quality of Life Research Institute in Kyushu University of Health and Welfare,
Japan
Aromatherapy and plant-based essential oils are widely used as complementary and alternative therapies for
symptoms including anxiety or neurodegenerative diseases. To evaluate the neuroprotective and
neuroendocrine effects of essential oils, we have developed an in vitro system using immortalized hypothalamic
neurons (GT1-7 cells), which are derived from murine hypothalamic neurons and hold the neuronal
characteristics such as the neurite extension or the secretion of GnRH. Various essential oils were administered
to GT1-7 cells with or without toxic compounds such as hydrogen peroxide, aluminum, zinc. Thereafter, the cell
viability was observed by MTT assay. Among tested 25 oils, several oils were revealed to have neuroprotective
activities against these toxic substances. We found that oils of damask rose, eucalyptus, fennel, geranium,
ginger, attenuated neuronal death induced by hydrogen peroxide. Damask rose oil had protective effects against
aluminum-induced neurotoxicity, while geranium and rosemary oil showed protective activity against zincinduced neurotoxicity. GT1-7 cells express estradiol receptors and tamoxifen, an antagonist of estrogen
receptor, induced apoptotic death. We found that essential oils of geranium and ginger enhanced tamoxifeninduced neurotoxicity. Our in vitro assay system could be useful for the neuropharmacological and endocrine
pharmacological studies of essential oils, and for the evaluation of their efficacy.
P1-036 Transcription factor ATF5 controls the maturation and survival of mouse vomeronasal sensory
neurons
Haruo Nakano, Yoshitaka Iida, Marie Aoki, Makoto Suzuki, Miyu Yamamoto, Takahiro Murase, Mariko Umemura,
Shigeru Takahashi, Yuji Takahashi
School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
The vomeronasal sensory neurons in the vomeronasal organ (VNO) detect pheromones in order to regulate
social behaviors such as mating and aggression. They express G-protein-coupled pheromone receptors (V1rs,
2016.05.27 11.25.45 Page 16(1)
V2rs, Fprs) that have putative seven transmembrane domains. So far, only a few transcription factors are known
to regulate the development of vomeronasal sensory neurons. Activating transcription factor 5 (ATF5) is a
member of the CREB/ATF family of transcription factors, which is highly expressed in olfactory chemosensory
tissues, the main olfactory epithelium and VNO in mice; however, the physiological role of ATF5 in the
vomeronasal sensory system remains unknown. In this study, we found that the differentiation of mature
vomeronasal sensory neurons, assessed by olfactory marker protein expression, was inhibited in ATF5-deficient
VNO. In addition, many apoptotic vomeronasal sensory neurons were evident in the basal region of the
vomeronasal epithelium. We demonstrated that the differentiation, survival and axonal projection of V2r-type
rather than V1r-type vomeronasal sensory neurons were severely inhibited in ATF5-deficient VNO. These
results suggest that ATF5 is one of the transcription factors crucial for the vomeronasal sensory formation.
P1-037 Ion-channel mechanisms specializing firing patterns of olfactory and vomeronasal sensory
neurons
Tomohiro Noguchi, Hitoshi Sasajima, Sadaharu Miyazono, Makoto Kashiwayanagi
Department of Sensory Physiology, Asahikawa Medical University, Hokkaido, Japan
In rodents, the main olfactory system collaborates with the vomeronasal system on processing molecular signals
that regulate their endocrinal and emotional states. However, these two systems may be able to detect
distinguished aspects of the same molecules by using different excitability of their receptor cells. Firing
properties of olfactory sensory neurons (OSNs) are adaptive, while those of vomeronasal sensory neurons
(VSNs) are non-adaptive. Here, we show that differences in intrinsic ionic kinetics of OSNs and VSNs generate
distinctive firing patterns. Kinetics of voltage-gated sodium channels was analyzed by the patch clamp technique
and Hodgkin-Huxley-type numeric calculation. The voltage-dependence of activation in OSNs was 20 mV
deeper than that in VSNs. The half-activation potentials in OSNs was -58 mV, while that in VNSs was -35 mV.
Similarly, the voltage-dependence of inactivation in OSNs was deeper than that in VSNs (the half-inactivation
potentials, -89 mV in OSNs and -56 mV in VSNs). The time constants indicated that activation and inactivation in
OSNs were faster than those in VSNs. These parameters were applied to Hodgkin-Huxley-type simulation for
OSNs and VSNs. Simulation revealed generation of after-depolarization following the action potentials not in
VSNs but in OSNs. This after-depolarization may prevent recovery from the inactivation of the sodium channels
and facilitate firing adaptation. The present results suggest that the difference in firing pattern between OSNs
and VSNs is inherent characteristics and is an essential mechanism for parallel olfactory processing.
P1-038 Short-term impact of a Western diet on the physiology of the peripheral olfactory system
David Jarriault1,2,3, Vincent Canova1,2,3, Julie Paradis1,2,3, Tibor Chomel1,2,3, Xavier Fioramonti1,2,3, Xavier Grosmaitre1,2,3
1
CSGA UMR, Dijon, France, 2UMR, Dijon, France, 3Universite de Bourgogne-Franche-Comte, Dijon, France
Current feeding behaviors contribute to the epidemic levels of obesity and diabetes observed in Europe and
worldwide. Both the quantity and the quality of ingested food are incriminated. Together with other sensory
modalities, olfaction is involved in the control of food intake. Olfactory cues can influence eating behaviors, yet
the nutritional status and diet can also alter olfactory abilities. Patients with metabolic disorders present impaired
olfactory sensitivity which could in turn worsen their eating behaviors.
Here we examined the short-term impact of a Western diet enriched in fat and sugar (High Fat High Sugar,
HFHS) on the anatomy and physiology of the olfactory epithelium of postnatal mice. We used a transgenic line of
mice expressing GFP under the promoter of the SR1 odorant receptor in order to monitor the properties of a
define population of neurons. After 8 weeks of diet, HFHS animals were glucose intolerant with normal
glycaemia and insulinemia, higher adiposity but no overweight compared to control mice. We measured electroolfactogram amplitudes in response to two ligands of the SR1 olfactory receptor: amyl acetate and (R)-(+)carvone. Detection thresholds of amyl acetate estimated from the dose-response curves were higher after 8
weeks of a HFHS diet (medians were 10-5 M for control vs 10-3 M for HFHS, p<0.01). Reconstruction of the cilia
of SR1 olfactory sensory neurons revealed shorter cilia in HFHS mice compared to control animals (4.5±0.3 μm
vs 6.0±0.3 μm, p<0.01).
Our results demonstrate that diet enriched in fat and sucrose can alter the physiology of the olfactory
epithelium on a short term. Anatomical changes of individual olfactory sensory neurons may participate to the
reduced olfactory sensitivity. Olfactory dysfunctions appear early on after exposure to a Western diet.
2016.05.27 11.25.45 Page 17(1)
P1-039 Olfactory receptor pseudo-pseudogenes
Richard Benton1, Lucia Prieto-Godino1, Raphael Rytz1, Benoite Bargeton1, Liliane Abuin1, J. Roman Arguello1,
Matteo Dal Peraro2
1
Center for Integrative Genomics, University of Lausanne, Switzerland, 2Institute of Bioengineering, EPFL, Switzerland
Pseudogenes, numbering 14,000 in the human genome, have long been considered to be non-functional DNA
sequences that arise from protein-coding genes through nonsense or frame-shift mutations. Although certain
pseudogene-derived RNAs have regulatory roles, no clear functions for pseudogene-derived proteins have been
described. Olfactory receptor families contain many pseudogenes, reflecting low selection pressures to maintain
receptors that are either functionally redundant or detect odours no longer relevant for the fitness of a species.
Here we have characterised a pseudogene in the ionotropic glutamate receptor-related (IR) chemosensory
repertoire of Drosophila sechellia, an olfactory specialist endemic to the Seychelles that feeds only on ripe fruit of
Morinda citrofolia. This locus, DsecIR75a, bears a premature termination codon (PTC) that appears to be fixed
in the population. Unexpectedly, DsecIR75a encodes a functional receptor, due to efficient translational readthrough of the PTC. Read-through occurs only in neurons, and is independent of the type of termination codon
but dependent upon sequence downstream of the PTC. We further show that while the intact D. melanogaster IR
75a orthologue detects acetic acid―a chemical cue important for this species to locate and ovipositing on
fermenting food but at trace levels in the host fruit of D. sechellia―DsecIR75a has evolved distinct odour-tuning
properties, through changes in three amino acids in the ligand-binding domain. We discover functional PTCcontaining genes within other olfactory receptor repertoires and species, suggesting that such pseudopseudogenes represent a widespread phenomenon that might include non-olfactory loci.
P1-040 Metabolic conversion of food odorants in mouse nasal mucosa
Chiori Ijichi1, Hidehiko Wakabayashi2, Yuko Kodama1, Yasuko Nogi1, Yutaka Ishiwatari1, Ayumi Nagashima3,4,
Kazushige Touhara3,4
1
Institute for Innovation, AJINOMOTO Co., Inc., Japan, 2Institute of Food Sciences & Technologies, AJINOMOTO Co., Inc., Japan, 3
Volatile compounds are dissolved in the nasal mucosal layer prior to the detection by odorant receptors (ORs)
expressed in olfactory sensory neurons. Previous studies showed that enzymes located in the nasal mucus
immediately metabolized volatile compounds and the metabolites were recognized by ORs. For example,
aldehydes and esters were converted to the corresponding acids and alcohols by mucosal enzymes (ref. 1). We
hypothesized that some odorants in food also underwent enzymatic conversion in mucus while eating, and the
resulting odorant mixture was recognized by ORs, leading to ‘an odor of the food’ that is different from the odor
of the original mixture. In this study, we examined metabolic conversion of 17 food-derived volatile compounds,
which are known to be key flavor components in 227 food samples, by murine mucosal enzymes (ref. 2). The
volatile compounds were treated with the isolated mouse nasal mucosa and the treated sample was analyzed
using GC/MS or LC/MS/MS. It was revealed that some aldehydes were metabolized to alcohols, whereas no
conversion was observed for ketones and carboxylic acids, probably because of lack of the catalytic enzymes for
these compounds in mucus. These results suggest that at least some part of food volatile compounds were
converted in nasal mucus before the detection by ORs. Although most of the studies on food odorants have
focused solely on compounds that can be extracted from foods, the current results suggest the importance of
analyzing enzymatically converted odorants for better understanding of food odor perception in human. Ref. 1.
A. Nagashima and K. Touhara (2010) Enzymatic conversion of odorants in nasal mucus affects olfactory
glomerular activation patterns and odor perception. J. Neurosci., 30(48), 16391-16398 Ref. 2. A. Dunkel et al.,
(2014) Nature’s chemical signatures in human olfaction: A foodborne perspective for future biotechnology
Angew. Chem. Int. Ed., 53, 7124-7143
P1-041 A bioelectronic nose based on human olfactory nanovesicles for sensing cancer-specific
volatile organic compounds
Sang Won Cho1, Tai Hyun Park1,2,3, Hwi Jin Ko2
1
School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea, 2N-BIO Institute, Seoul National
University, Seoul, South Korea, 33Advanced Institutes of Convergence Technology, Suwon, South Korea
There have been many efforts to develop a non-invasive and early diagnosis method of cancer. Recently, the
volatile organic compounds (VOCs) exhaled from cancer patients are accepted as a potential biomarker of
2016.05.27 11.25.45 Page 18(1)
cancer. However, the gas profile of breath is highly variable so that it is difficult to identify the exact biomarker.
Accordingly, we identified cancer-specific VOCs as a biomarker using in vitro cancer system in order to exclude
the variables from condition of patients and finally constructed a bioelectronic nose for the detection of these
biomarkers. VOCs were collected from the headspace gas of lung cancer cell (SK-MES), stomach cancer cell
(SNU-1), normal lung cell (MRC-5), and stomach cell (SNU-NCC-19) and then analyzed by using SPME-GC/
MS. As a result, 2-ethyl-1-hexanol and tert-butyl methyl sulfoxide were detected from headspace gas of SK-MES
and SNU-1 cells, respectively and decided as biomarkers for lung and stomach cancer. Human olfactory
receptor 4D11P was identified as a receptor which is specific for 2-ethyl-1-hexanol and heterologously
expressed in HEK293 cells. Nanovesicles containing 4D11P were produced from the cells and immobilized on
the surface of carbon nanotube for construction of bioelectronic nose. This bioelectronic nose selectively
detected 2-ethyl-1-hexanol at a concentration as low as 10 f M showing a possibility of lung cancer diagnosis in
real-time.
P1-042 Artificial olfactory system based on insect olfactory receptor for detecting water contaminant
geosmin
So-ong Kim1, Manki Son2, Tai Hyun Park1,2,3
1
School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea, 2Interdisciplinary Program for
Bioengineering, Seoul National University, Seoul, South Korea, 3Advanced Institutes of Convergence Technology, Suwon, South
Korea
Animals find chemical cues to get information on food, mates, dangers, pathogens and predators. Animal
olfactory systems have evolved to detect the cues from complex environment. Geosmin is an earthy and muddy
odorant, which is produced by cyanobacteria, and can be used as a potential marker of water contamination.
Recently, olfactory receptor complex of Drosophila melanogaster, dOR56a and Orco, have been studied for the
highly sensitive detection of geosmin. Herein we developed an artificial olfactory system using dOR56a-Orco
heteromeric complex that assesses the level of water contamination. The receptors were expressed in HEK-293
cells and their functionality was evaluated. Olfactory nanovesicles derived from artificial cell surfaces also
showed high sensitivity and selectivity for geosmin. We also compared newly developed artificial systems with
human olfactory receptor-based systems. These artificial sensory systems could be used for the assessment of
water quality.
P1-043 Visualization of odor using ion channel-coupled human olfactory receptor
Dongseok Moon1, Il Ha Jang1, Eun Hae Oh2, Tai Hyun Park1,2,3
1
School of Chemical and Biological Engineering, Seoul National University, South Korea, 2Interdisciplinary Program of
Bioengineering, Seoul National University, South Korea, 3Advanced Institutes of Convergence Technology, Suwon, South Korea
In the human olfactory system, there are 388 kinds of olfactory receptors (ORs) which bind to thousands of
odorants. This system discriminates the combinations of ORs responses respectively. Characterizing
OR-odorant binding is essential for application as biosensors. Therefore, many researchers have been trying to
deorphanize the ORs to understand the odorant recognition patterns by various screening methods. Although
there are conventional screening methods such as calcium imaging and cAMP response element (CRE) reporter
system, these assays are not suited to patterning odorant responses because these are time-consuming,
labor-intensive and complex in signaling pathway. In this study, our group designed a screening method for
visualizing and patterning odorant responses in real-time. 14 human olfactory receptors (hORs) in chromosome
17 were coupled to inward-rectifier K+ (Kir 6.2) channel. Then, the ion-channel-coupled human olfactory
receptors (ICChORs) were expressed in HEK293 cells. When an odorant bind to the hOR of the ICChOR,
conformational change of the hOR occurs and it causes influx of potassium ions into the cell through Kir 6.2
channel. Membrane potential assay can detect membrane potential change of the cell. we used components of
artificial rose scent for screening and patterning the odorant responses as one example. Cells expressing the
ICChORs showed high fluorescent responses when the compounds bind to the hORs. The results show that this
screening tool can be used to visualize and pattern thousands of odorant responses about numerous scents in
real-time.
2016.05.27 11.25.45 Page 19(1)
P1-044 Codification of rose smell using human olfactory receptors in human chromosome 17
Dahoon Min1, Heehong Yang1, Tai Hyun Park1,2
1
Department of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea, 2Advanced Institutes of
Convergence Technology, Suwon, South Korea
Olfaction follows the complex signaling cascades from the peripheral receptors to the brain of humans. At the
molecular level, humans can perceive and discriminate smells with the odorant-olfactory receptors (ORs)
binding pattern. A combinatorial code, which is based on the odorant-ORs binding pattern, could be assigned to
the individual odorants. In this study, the codes of four odorants that are main components of a rose
fragrance-geraniol, citronellol, 2-phenylalcohol, and linalool-were identified. OR gene cluster in human
chromosome 17 (Chr17) can be a representative model to understand the human olfaction. 14 ORs in human
Chr17 were expressed in the HEK-293 cell line. The activation of their binding with four odorants was analyzed
by a luciferase assay, known as a suitable assay for the high-throughput deorphanization of ORs. Assessing
these patterns would be an initial step to make the specific codes of the different odorants that can be useful for
standardization and codification of smells. Furthermore, it could help to understand the human olfactory system.
P1-045 Synergistic action of nectins and cadherins establish the mosaic cellular pattern of the
olfactory epithelium
Sayaka Katsunuma1,2, Hisao Honda3,4, Ken-ichi Nibu2, Hideru Togashi1
1
Division of Molecular and Cellular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School
of Medicine, Kobe, Japan, 2Department of Otolaryngology-Head and Neck Surgery, Kobe University Graduate School of Medicine,
Kobe, Japan, 3Laboratory for Neural Differentiation and Regeneration, Department of Physiology and Cell Biology, Kobe University
Graduate School of Medicine, Japan, 4Laboratory for Morphogenetic Signaling, RIKEN Center for Developmental Biology, Kobe,
Japan
The olfactory epithelium (OE) is a sensory neuroepithelium involved in olfaction. In the apical surface of the OE,
olfactory cells (OCs) and supporting cells (SCs) are arranged in a characteristic mosaic pattern, in which OCs
are enclosed by SCs, established by cellular rearrangements during development. However, the mechanism of
the cellular patterning remains unknown. The morphogenesis of developing epithelium is associated with control
of cell-cell contacts mediated by adhesion receptors. Cadherins are major adhesion receptors, which mediate
homophilic trans-interactions. It is reported that OCs and SCs express different cadherins, but they are not
segregated. Nectins are a family of immunoglobulin-like adhesion receptors and the heterophilic
trans-interactions are much stronger than their homophilic trans-interactions. We have shown that the
heterophilic interaction between nectins play a key role in checkerboard-like cellular patterning of the mouse
auditory epithelium (Togashi et al., Science., 2011). In cell-cell adhesion, nectins first form cell-cell contacts and
recruit cadherin-catenin complex to these contact sites via α-catenin, leading to maturation of adherens
junctions. In this study, we examined the cooperative mechanism between cadherins and nectins in the cellular
patterning using mouse OE. We revealed that OCs expressed nectin-2 and N-cadherin, and SCs express
nectin-2, nectin-3, E-cadherin, and N-cadherin. The heterophilic interaction between nectin-2 on OCs and
nectin-3 on SCs recruit cadherin-catenin complex to the heterotypic junctions. It provides the driving force to
promote cellular intercalation of SCs between attached OCs, resulting in the separation of attached OCs. In
mathematically and culture cell model, we proved cellular patterning leaded by differential adhesiveness on each
junction. In this way, synergistic action of nectins and cadherin-catenin complex makes the differential
adhesiveness depending on types of junctions, which contribute to the mosaic cellular patterning of the OE
(Katsunuma et al., JCB., in press).
P1-046 Evidence of peripheral olfactory adaptation: Electrophysiological recordings of the human
olfactory epithelium
Charlotte Sinding1,2, Anica Schunke1, Pauline Joussain3, Jennifer Summerfield3, Thomas Hummel1
1
Smell & Taste Clinic, Department of Otorhinolaryngology, University of Dresden Medical School, Dresden, Germany., 2Center for
taste and Feeding behavior, INRA, Dijon, France, 3Givaudan UK Ltd, Ashford, UK
Habituation requires a complex filtering system that may occur through different mechanisms, as central
inhibition (central adaptation) or reconformation of olfactory receptors (peripheral adaptation). Habituation has
been shown to result mainly from central adaptation processes. Indeed, to the best of our knowledge, a tangible
proof of peripheral adaptation has never been shown. We tested the hypothesis of recording peripheral
adaptation within 50s of repetitive odorant stimulations with electroolfactography. We tested 4 odorants (eugenol
2016.05.27 11.25.45 Page 20(1)
[1-hydroxy-2-methoxy-4-allyl benzene], manzanate [ethyl 2-methyl pentanoate], Iso E super [dimethyl
myrcetone] and PEA [phenethyl alcohol]), each presented 10 times in a row (1s stimulation and 4s ISI), to 30
subjects. Odor event related potentials (OERP) were recorded by placing an electrode on the ventral part of the
middle turbinate, in the nose. We tested the effect of repetitions on the amplitude of N1 component of mean
OERPs, likely related to the level of activation of the olfactory receptors tuned to the odorant. The success rate
of the measure, i.e. percentage of subjects who had at least one OERP, comprised between 53.3% and 76.7%
for each repetition to each odorant, which is rather high in comparison to other studies reporting OERPs of the
olfactory epithelium. We found a significant decrease of N1 amplitude for eugenol and PEA but not for
manzanate and Iso E Super. These results highlight first, that adaptation occurs in a relatively short time period
of 50s; and secondly, that different odorants do not induce the same level of adaptation. These results suggest
that the structure of the molecule may, at least partly, determine peripheral adaptation.
P1-047 Effect of endoscopic sinus surgery on olfactory disturbance in eosinophilic and noneosinophilic chronic paranasal sinusitis
Kazuki Satou, Nozomu Wakayama, Yuma Yoshioka, Mariko Ishida, Kuwon Sekine, Satoshi Yamaguchi, Shin Kitamura,
Shoji Matsune
Department of Otolaryngology, Nippon Medical School Musashikosugi Hospital, Kanagawa, Japan
(Introduction) While olfactory disturbance (OD) is classified into central and peripheral ones, chronic paranasal
sinusitis is the more major causative disease of OD than common cold, allergic rhinitis and head injury. In
chronic paranasal sinusitis, it is one of the important mechanisms of OD that odorous substance is interrupted to
reach olfactory epithelium and receptors by mucosal swelling along the olfactory cleft, which is called respiratory
OD. Accordingly, recovery from this mucosal stenotic pathology by endoscopic sinus surgery (ESS) possibly
leads this type of OD in chronic paranasal sinusitis to healthy olfaction. On the other hand, eosinophilic chronic
paranasal sinusitis (ECS) has been known as refractory sinusitis with severe eosinophil infiltration into polypoid
and sinus mucosa, of which OD is more persistent and resistant to surgical and conservative therapy than noneosinophilic paranasal sinusitis (non-ECS). (Aim) This study was designed in order to analyze the effect of ESS
on OD in ECS in comparison with non-ECS cases. (Methods) Thirty-one ESS patients ranging from 28 to 71
year-old (18 males and 13 females) were enrolled. These cases were classified into ECS and non-ECS group
based on the diagnostic standard by JESREC study. OD and recovery after ESS were estimated by T&T
olfactometer and intravenous olfaction test before and 3-month after ESS. (Results and Discussion) Persistent
and refractory OD was estimated, in detail, in ECS in comparison with non-ECS based on the new differential
diagnosis by JESREC study. This study is supposed to provide us with new important data and information in
order to establish clinical strategy to manage severe OD in ECS.
P1-048 Anti-stress effect of medium-chain fatty acid via olfactory receptor expressed in the brain
Doyun Kim, JiHoon Kim, JuYi Kim, NaNa Kang, NaHye Lee, YoonGyu Jae, JaeHyung Koo
Department of Brain & Cognitive Sciences, DGIST, Daegu, South Korea
Stress is an important survival factor for animals by responding to threatening environment through the
regulation of multiple neural and endocrine systems. HPA axis is a major part of the neuroendocrine system that
controls stress and regulates neural processes such as mood and emotion. The ligands and GPCRs related to
HPA axis are targets for various pharmacological therapeutic. In our recent study, next generation sequencing
demonstrated the extensive expression of olfactory receptors (ORs) in several tissues including HPA axis. Here,
we presented that unique OR is mainly expressed in the pituitary gland by quantitative real-time RT-PCR
analysis. The histological location and cell type were also identified by immunohistochemistry and some OR (+)
cells were located in the anterior lobe of the pituitary gland with ACTH. The blood ACTH and corticosterone level
were measured in acute restrain stress followed by medium-chain fatty acid (MCFA), in i.p. injection.
Interestingly, MCFA showed inhibitory effect on both the ACTH and the corticosterone release. Furthermore,
anxiety and depression level were assessed by behavioral test in chronic restrain stress with MCFA treatment,
showing decreased anxiety and depression level. Our studies demonstrated a potential role of OR responding to
stress through HPA axis, suggesting that OR may be an important therapeutic target for mood disorder. Key
words: olfactory receptor, OR, stress, mood, MCFA
2016.05.27 11.25.45 Page 21(1)
P1-049 Defective pre-target axon sorting during olfactory map regeneration after olfactory nerve
transections
Aya Murai1,2, Kazunori Nishizaki2, Takeshi Imai1
1
Laboratory for Sensory Circuit Formation, RIKEN Center for Developmental Biology, Kobe, Japan, 2Department of OtolaryngologyHead and Neck Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama,
Japan
The mammalian olfactory system is a highly complex system that can detect numerous odorants. This is
represented in the form ofan olfactory map in the olfactory map. However, olfactory disorders such as dysosmia,
which alter our olfactory perception often occur after traumatic brain injury. To date there are no effective
therapies for dysosmia. In order to produce effective therapies it is necessary to understand the underlying
processes and cellular mechanisms that occur after dysosmia induced by traumatic brain injury. Olfactory
Sensory Neurons (OSNs) are continuously regenerated, and the regenerated OSNs can project to the correct
glomerulus during the normal OSN turnover process. However, after severe OSN damage regenerated OSN
axons are known to connect to wrong glomeruli in the olfactory bulb, a potential cause of the dysosmia. To
obtain mechanistic insights into traumatic dysosmia, we studied the regeneration process using a dysosmia
model, produced by mechanical nerve transection. It has been reported that Nrp1+axons projecting to posterior
OB and Nrp1+ ones projecting to anterior OB are presorted in axon bundles. However, these two types of axons
were intermingled after axon transection; as a result, Nrp1+ axons were mistargeted into the anterior OB.
Similarly, the segregation of class I and class II OSN axons was perturbed after the nerve transection. These
results indicate that pioneer-follower axo-axonic interactions are essential for the projection of regenerating OSN
axons. We also found that the post-synaptic targets of OSNs, mitral cells, had reduced or altered connectivity
after OSN transection, suggesting a role of OSN axons in the maintenance of OB circuits. Based on these
results, possible therapeutic strategies for dysosmia will be discussed.
P1-050 Molecular Dynamics simulations reveal the ligand-dependent activation of hOR7D4
Claire A. de March1, Elise Bruguera1, Jeremie Jacques Topin2, Hiroaki Matsunami1, Jerome Golebiowski2
1
Duke University Medical Center, Durham, NC, USA, 2Institut de Chimie de Nice, UMR CNRS, Universite de Nice Sophia Antipolis,
France
The complexity of the odor chemical space and the large number of odorant receptors (ORs) associated to their
combinatorial activation make understanding odor coding an enormous challenge. More specifically, being able
to predict the behavior of an OR in front of an agonist, an antagonist or a non-agonist remains to be done.[1]
Using a joint approach combining molecular modeling and experimental data on several ORs[2], we have built a
model that can capture the active or inactive state of these proteins when bound to ligands with different
potencies.[3] The methodology is illustrated on a challenging case. By the aim of computational tools combined
with site directed mutagenesis, we predict the activation of human OR7D4 by its strong agonists, androstenone
and androstadienone, and its inactivated form by a non-agonist the (Z)-2-decenal.[4] These models allow us to
investigate the activation mechanism of other mammalian olfactory receptors from the binding cavity to the G
protein binding site, revealing the network of conserved amino acids involved in ORs activation. Such powerful
approaches will help unravel odor-coding in the nervous system and facilitate the understanding of general rules
of neuronal activation induced by an odor. [1] C. A. de March, S. Ryu, G. Sicard, C. Moon and J. Golebiowski,
Flavour and Fragrance Journal 2015, 30, 331-410. [2] C. A. de March, S.-K. Kim, S. Antonczak, W. A. Goddard
and J. Golebiowski, Protein Science 2015, 24, 1543-1548. [3] C. A. de March, Y. Yu, M. J. Ni, K. A. Adipietro, H.
Matsunami, M. Ma and J. Golebiowski, Journal of the American Chemical Society 2015, 137, 8611-8616. [4] A.
Keller, H. Y. Zhuang, Q. Y. Chi, L. B. Vosshall and H. Matsunami, Nature 2007, 449, 468-U466.
P1-051 A full size mathematical model of the early olfactory system of honeybees
Ho Ka Chan, Alan Diamond, Thomas Nowotny
School of Engineering and Informatics, University of Sussex, Brighton, UK
Experimental measurements often can only provide limited data from an animal’s sensory system. As a result,
data driven models, e.g. [1], are similarly limited. However, in order to make biologically relevant predictions it is
important to consider inputs representative of the full input space. Here we present a full size model of the early
olfactory system of honeybees that extrapolates inputs from the limited subset of available experimental
observations. Our model comprise olfactory receptor neurons (ORNs), local neurons (LNs) and projection
2016.05.27 11.25.45 Page 22(1)
neurons (PNs) organized in 160 glomeruli. The ORN response patterns are generated using a set of equations
describing the binding and activation of receptors as in [2]. The parameters for these processes are chosen to
match the statistical distribution of experimental observed quantities in [3] and [4] as well as the statistics of
asymptotic responses observed experimentally [5]. The PN response patterns are generated using a network
model mimicking the early olfactory system of honeybees and assumed that the connectivity between PNs and
LNs is based on the correlation between the activities of their respective glomeruli as in [6]. A rate model,
derived using the leaky integrate-and-fire model, is used to determine the input-output relationship. We
confirmed that response patterns for ORNs and PNs generated by our model can replicate key features of their
biological counterparts experimentally measured in [4] and [7]. Our model can be generalized to the early
olfactory systems of other insects, and hence provides a possible basis for future numerical studies of olfactory
processing in insects. Acknowledgements This work was supported by HFSP, RGP0053/2015 and EPSRC, EP/
J019690/1. References 1. Brain Res. 2013,1536:119-134. 2. Nat Neurosci. 1999 May, 2(5):473-478.3. Brain
Res. 2012,1434:123-135. 4. J Neurosci. 2008, 28(10):2659-2666. 5. Proc Natl Acad Sci U S A. 2014,111(47):
16925-16930. 6. J Neurophysiol. 2005,93(6):3410-3417. 7. Ditzen M. [PhD thesis], 2005.
P1-052 A calcium signaling ‘fingerprint’ in vomeronasal sensory neurons
Maximilian Nagel, David Fleck, Marc Spehr
Department of Chemosensation, RWTH-Aachen University, Aachen, Germany
Recently, a growing number of chemosensory signals were discovered which are detected by the mammalian
vomeronasal organ (VNO). These complex chemical cues regulate social behavior and carry information about
sexual, social and reproductive status of both con- and heterospecific individuals. To date, however, little is
known about both the sensory coding strategies implemented by the VNO and the stimulation-dependent activity
patterns in single vomeronasal sensory neurons (VSNs).
In this study, we used Ca2+ imaging in acute mouse VNO slices to determine the activity of VSNs both on the
population and the individual neuron level. Slices were loaded with the Ca2+-sensitive dye Cal520 AM in a
custom-made circulating oxygenation chamber. This new loading approach significantly increased the yield of
vital VSNs. Precise focal perfusion of a variety of physiologically relevant stimuli-urine, saliva, vaginal secretions,
etc. -allowed us to analyze the neural code of vomeronasal information. In parallel, we analyzed the
spatiotemporal response characteristics in single VSNs.
Together, we present an improved in situ Ca2+ imaging approach that allows for effective VNO ligand screening,
characterization of population response patterns, kinetics analysis of individual Ca2+ transients, as well as
investigation of VSN adaptation and signaling robustness. Thus, on-going experiments aim to provide a
quantitative perspective on vomeronasal coding at the VSN population level as well as a detailed analysis of
Ca2+ signaling events in single neurons.
P1-053 Different processing of amyloid precursor protein in the olfactory system
Jae Yeon Kim, Ameer Rasheed, Seung-Jun Yoo, Bongki Cho, So Yeun Kim, Cheil Moon
Department of Brain & Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea
Abnormal processing of amyloid precursor protein (APP), through sequential cleavages first by beta secretase
and then by gamma secretase complex, leads to excessive production of beta amyloid (Abeta) in the central
nervous system (CNS), that is Alzheimers disease. Specially, gamma secretase complex have presenilin 1 and
2 as the catalytic core and gene mutations markedly trigger Abeta accumulation. Before AD symptoms such as
memory loss or muscle retardation turn up, olfactory dysfunction usually occurs. It means that the olfactory
system is one of the most vulnerable regions in the AD development. But it is not well understood that how
olfactory dysfunction can be induced and whether APP processing really happens in both olfactory epithelium
(OE) and olfactory bulb (OB). To find the features in the olfactory system, we first compared expression pattern
of AD related factors in OE, OB and other brain regions of WT mice. And then, we also checked these
differences in the 10 month aged Tg 2576 mice in which olfactory dysfunction has just started. Here, we showed
that normal APP processing and secretases expression pattern are different in the OE and other regions,
respectively, in WT mice. Remarkably, gamma secretase component mRNAs are highly expressed in the OE,
compared with OB and of those, presenilin 1 and 2 proteins are predominantly expressed in OE. Furthermore, in
Tg 2576 mice, abnormal APP processing is found in the olfactory system and expressions of presenilin 1 and 2
are unregulated between OE and OB. It seems that the unique expression and its change of presenilins might be
mainly associated with the olfactory dysfunction. These findings help to understand the special mechanisms in
2016.05.27 11.25.45 Page 23(1)
OE, further correlation between olfactory deficit and AD.
P1-054 Characterizing the impairment peripheral olfactory system of Tg2576 mice in the preclinical
stages of Alzheimer’s disease
Seung-Jun Yoo1, Ji-Hye Lee1, So Yeun Kim1, Bongki Cho1, Gowoon Son1, Jae Yeon Kim1, Seung-Woon Yu1, Keun-A Chang2,
Yoo-Hun Suh2, Cheil Moon1
1
Department of Brain&Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea,
Department of Pharmacology, School of Medicine, Gachon Medical School, Incheon, South Korea
2
Olfactory dysfunction is a common symptom associated with neurodegenerative diseases including Alzheimers
disease (AD). Although some evidence suggest that peripheral olfactory organs are involved in the olfactory
dysfunction that accompanies AD pathology, the underlying mechanisms have yet to be fully elucidated. Using
behavioral tests, transgenic mice overexpressing a Swedish mutant form of human amyloid precursor proteins
were found to have olfactory impairments from presymptomatic stages. Expression of tyrosine hydroxylases
indicated that specific regions of the olfactory bulb (OB), especially the ventral part exhibited significant
decreases in numbers of dopaminergic neurons in the periglomerular regions. Another factor to notice in ventral
part of OB is that the increasing of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) coincide with
elevation of amyloid-beta (Abeta) oligomer. Additionally, olfactory epithelium, especially the ectoturbinate in
which axons of olfactory receptor neurons (ORNs) have direct connections with ventral OB, exhibited significant
decreases in both thickness and cell number even at presymptomatic stages. This result suggests that Abeta
oligomer toxicity in the OE via mitogen-activated protein kinase (MAPK) p38 pathway may have induced ORN
cell decline and functional impairment of the olfactory system. Collectively, we first demonstrated that the
impairment of the peripheral olfactory system may be the presymptomatic event caused by Abeta oligomer
accumulation in AD, indicating the region specific impaired pattern in the olfactory neural circuit.
P1-055 Effect of cytochrome P450 on the response of olfactory receptor
Masahi Asakawa1, Yosuke Fukutani1, Aulaphan Savangsuksa1, Hiroaki Matsunami2, Masafumi Yohda1
1
Department of Biotechnology and Life Science, Tokyo University of of Agriculture and Technology, Tokyo, Japan, 2Department of
Molecular Genetics and Microbiology, Duke University Medical Center, NC, USA
Olfaction of animals is caused by odor molecules in the air bind to olfactory receptors (ORs) inside nasal cavity.
It was reported the possibility that enzymes secreted in olfactory mucus of mouse convert the odor molecule and
its metabolite binds to olfactory receptor. It was known that cytochrome P450 (CYP) of redox enzymes are
expressed in the olfactory mucosa. In this study, I would like to elucidate of influence of CYPs to the response of
ORs to odorant molecule. I used cell-based assay for measuring the response of ORs in culture cell
co-expressed CYPs expressing in olfactory epithelium. I suggested that CYP1a2 expressed cells could convert
acetophenone to methyl salicylate and the odorant metabolize influenced of response of MOR161-2. In addition
we used in situ hybridization for measuring the response of OR in olfactory epithelium. MOR161-2 could respond
to acetophenone and methyl salicylate in vivo. When the olfactory mucus is cleaned, the response of MOR161-2
to acetophenone is reduced, while the response of MOR161-2 to methyl salicylate is not varied. This study
suggests that CYPs could change the odor molecule response of OR.
P1-056 Fruit odor as a dietary signal for a wild population of capuchin monkeys in Costa Rica
Mizuki Endo1, Mika Shirasu2,3, Rachel E. Williamson4, Omer Nevo5, Amanda D. Melin4,6, Kazushige Touhara2,3,
Shoji Kawamura1
1
Department of Integrated Biosciences, The University of Tokyo, Tokyo, Japan, 2Department of Applied Biological Chemistry,
University of Tokyo, Tokyo, Japan, 3ERATO Touhara Chemosensory Project, JST, Tokyo, Japan, 4Department of Anthropology &
Archaeology, University of Calgary, Calgary, Canada, 5Institute of Evolutionary Ecology and Conservation Genomics, University of
Ulm, Ulm, Germany, 6Cumming School of Medicine, University of Calgary, Calgary, Canada
Recent studies have questioned a simple view that primates are visually-oriented mammals, that do not rely on
other senses when searching for foods or detecting social signals. For example, field studies of Neotropical
primates have revealed that monkeys sniff fruits and reject them more often when fruits are visually cryptic to
background leaves. However, it is not well understood which traits of fruits (i.e. color, odor, softness) provide the
most effective signal for primate food selection. It is also not known which odors are associated with ripeness
and/or edibility. We collected dietary fruits of white-faced capuchin monkeys (Cebus capucinus) inhabiting the
Sector Santa Rosa of the Area de Conservation Guanacaste in Costa Rica. We measured chromaticity,
2016.05.27 11.25.45 Page 24(1)
softness, nutrients and odor composition of the fruit samples. In this presentation, we focus on our analysis of
odor and color. Odorants were collected by silica-based carbon graphite from the fruit samples in closed oven
bags and were identified and quantified by using gas chromatography-mass spectrometry (GC-MS). We found
prominent differences of the total amount (in terms of volatile organic compound: VOC) and composition of
odorants between maturation stages in some fruit species. Our principal component analysis suggests that
some odorants are associated with maturation. In some species, change of odorants appeared to better monitor
the progression of maturation than change of coloration. Although more data are needed, our preliminary
analysis suggests that odor can serve as a better signal of fruit maturation than color for seed-dispersing
primates during their food selection.
P1-057 Insulin signaling is involved in the maturation of newly generated olfactory sensory neurons
Akihito Kuboki1,2, Shu Kikuta1, Takashi Sakamoto1, Kaori Kanaya1, Hironobu Nishijima1, Makiko Hirano1,2, Kenji Kondo1,
Daiya Asaka2, Nobuyoshi Otori2, Hiromi Kojima2, Tatsuya Yamasoba1
1
Department of Otolaryngology, The University of Tokyo, Tokyo, Japan, 2Department of Otolaryngology, Jikei University, Tokyo,
Japan
Insulin is a peptide hormone that regulate glucose metabolism in the periphery, but has different regulatory roles
as extrinsic signals in the brain over that of the periphery. Although the rising evidence of the various roles of the
insulin in the central nervous system, it is not well understood whether insulin signaling affects ongoing
incorporation of newly generated olfactory sensory neurons (OSNs) to maintain homeostasis in the olfactory
epithelium (OE). Here, we examined in adult mice whether insulin signaling affected the dynamic incorporation
of newly generated OSNs 7, 14, and 28 days following injury. The mice were administered streptozotocin (STZ)
to damage pancreatic beta-cells, resulting in hypoinsulinemia. Methimazole, an olfactotoxicity-inducing drug,
was also intraperitoneally injected to ablate OSNs in the STZ-administered mice. The OE following injury in
saline administered-mice (control) instead of the STZ was restored to its pre-injury condition by day 28. Between
days 0 and 7, there were no significant differences in the numbers of olfactory marker protein (OMP)-positive
mature OSNs and apoptotic OSNs between the STZ-administered and the control mice. However, between days
7 and 28, the STZ-administered mice showed remarkably fewer OSNs and mature OSNs, but more apoptotic
OSNs than the control mice. Consistent with fewer mature OSNs in the STZ-administered mice, the expression
of c-fos (a neural activity marker) induced by odor at day 28 following injury were significantly reduced compared
with those in the control mice. Furthermore, insulin administration to the STZ-administered mice during the
period between 7 and 14 days post-injury promoted the recovery of the OE. These results indicate that insulin
signaling is involved in homeostatic regeneration of the OE following injury, and that newly generated OSNs
have a high susceptibility period to insulin signaling for their maturation between days 7 and 14 post-injury.
P1-058 Influences of breathing and chewing on the timing of flavor detection
Satomi Miyaoka1, Yozo Miyaoka2
1
Department of Eating Disorder and Dysphagia, Graduate School of Rehabilitation, Niigata University of Rehabilitation, Murakami,
Japan, 2Department of Health and Nutrition, Niigata University of Health and Welfare, Niigata, Japan
Previous studies have documented two routes for sensing of flavors during food chewing, the orthonasal and
retronasal routes, and suggested the importance of the respiratory airflow via the nose in that sensation.
Moreover, previous studies have documented the role of jaw movement in the sensing of flavors. We developed
a system using the masseter electromyogram to assess the flavor detection time. The involvements of the
respiratory airflow via the nose and jaw movement with flavor detection time are not well defined. Herein, we
aimed to evaluate the roles of the airflow and jaw movement on this timing. Test foods, three fruity gummy
candies, were wrapped in wafer papers to prevent direct contact between food and oral tissues before chewing.
Healthy young adults participated both in experimental and control sessions. Flavor detection time was
determined using the electromyography-based system. The respiratory phases of each participant were
monitored by a thermistor placed on the nostril. Test foods were given randomly and placed in their wrapping
between the molars of the participants, and a command informed the participants to begin chewing. In the
experimental session, the participants were asked to press a signal button as soon as they detected a flavor.
Two-thirds of signals were recorded during the expiratory phase and one-third during the inspiratory phase. The
participants were asked to press the button at arbitrary timings in the control session. Signals were recorded
almost evenly in both respiratory phases. The masseter electromyogram showed that more signals were
recorded during the jaw closing phase in the experimental session, while the average period of the jaw closing
2016.05.27 11.25.45 Page 25(1)
phase was shorter than that of the jaw opening. The present findings suggest active roles of the expiratory
airflow and of jaw closing in the detection of flavors emitted from foods in the oral cavity during chewing.
P1-059 Correlation between olfactory disorder severity and sinonasal CT findings in patients with
chronic rhinosinusitis
Kenzo Tsuzuki, Kengo Hashimoto, Yoriko Yukitatsu, Takahiro Saito, Masafumi Sakagami
Department of Otolaryngology-Head and Neck Surgery, Hyogo College of Medicine, Hyogo, Japan
Background: The study aimed to clarify the correlation between olfactory disorder severity and radiological
findings in patients with chronic rhinosinusitis (CRS) in the preoperative stage. Patients and Methods: Between
2007 and 2014, 272 adult patients with olfactory disorder due to bilateral CRS who were scheduled to undergo
primary endoscopic sinus surgery (ESS) were enrolled. There were 163 men and 109 women. Median age was
51 years (range, 22-80 years). According to the Japanese Epidemiological Survey of Refractory Eosinophilic
Chronic Rhinosinusitis (JESREC) study, the criteria indicate eosinophilic CRS (ECRS) for a total score more
than 11 from the following four items: i) bilateral lesion (3 points); ii) nasal polyps (2 points); iii) ethmoid sinus
dominant or pansinusitis on CT (2 points); and iv) blood eosinophilia (4, 8, or 10 points). Patients were classified
into two groups: ECRS (n=193) and non-ECRS (n=79). To evaluated olfactory disorder, T&T olfactometer
recognition threshold test and intravenous olfaction test, which are covered by health insurance in Japan, were
used. Computed tomography (CT) scores for sinuses and olfactory clefts (OC) were applied. Correlations
between olfactory acuity and CT score were statistically analyzed. Results: Recognition threshold correlated
significantly with CT score in both groups. Recognition threshold and CT score were significantly more severe in
ECRS than in non-ECRS. CT score at OC showed the strongest correlation with recognition threshold. CT
scores for total sinuses and OC in patients having a positive response to the intravenous olfaction test (n=239)
were significantly milder than those in the negative group (n=29), but ethmoid CT score was not. Conclusion:
Olfactory disorder severity showed significant correlation with CT opacification in the preoperative stage.
Olfactory disorder and CT findings were more severe in patients with ECRS than in those with non-ECRS.
P1-060 Characteristic aroma of trees in Chichibu, Japan, a renowned “forest bathing” area
Ryohei Takayama1, Toshio Hasegawa1, Takahisa Yamanaka2, Bujyuro Shimazaki2, Takashi Fujihara1
1
Department of Chemistry, Graduate School of Science and Engineering, Saitama University, Saitama, Japan, 2Chichibu Sap
Production Cooperative Association Ohtaki, Chichibu, Saitama, Japan
The district of Chichibu, located in the west of Saitama Prefecture, which borders Tokyo, has abundant natural
and forested areas contain many tree species. These forests have been included in the Forest Agency of
Japan’s list, One Hundred Forests in Japan for Forest Bathing, compiled based on the Agency’s research into
Japanese forests. The term “forest bathing” refers to a Japanese practice of making relaxed visits to a forest for
pleasure or for the various positive health benefits of being out in nature, and is a common practice among
Japanese. National research institutes have found that volatile compounds in trees decrease stress and lower
heart rate; however, the aroma profiles of many trees have not been clarified. Previously, we reported the aroma
profiles of common Japanese trees by detailed gas chromatography analysis of Japanese cypress, camphor,
and Japanese cedar. In the present study, we examine the aroma profiles of Chamaecyparis pisifera , Lindera
praecox , and Lindera obtusiloba Blume to clarify the effects of forest bathing. All these trees are found in
Chichibu Forest, and have characteristic odors. The odor constituents of the target trees were obtained by
hexane extraction and monolithic material sorptive extraction (MMSE). Trees have many low-boiling point odor
compounds that can be collected by MMSE, which is an extraction technique for headspace odor. The
constituents of the obtained extracts were identified by gas chromatography olfactometry and gas
chromatography-mass spectrometry. We found that both major and minor constituents contributed to the aroma
of Chamaecyparis pisifera , Lindera praecox , and Lindera obtusiloba Blume.
P1-061 Change in odor of patchouli according to drying process
Kazuma Yoshitome1, Toshio Hasegawa1, Mardi Santoso2, Muhammad Abdul Aziz2, Takashi Fujihara1
1
Graduate School of Science and Engineering, Saitama University, Saitama, Japan, 2Department of Chemistry, Institut Teknologi
Sepuluh Nopember, Surabaya, Indonesia
Patchouli (Pogostemon cablin) is a tropical plant cultivated in China, India, Thailand, Indonesia, Malaysia, the
Philippines, West Africa, and Vietnam. Patchouli has long been used for its fragrance, and is noted as an
2016.05.27 11.25.45 Page 26(1)
incense material in Buddhist scriptures. In modern times, patchouli essential oil is an important odor source in
perfume and is widely used in the fragrance industries, as it is one of only four known essential oils with woody
notes (the other three being sandalwood, vetiver, and cedar wood). Patchouli leaves used for their aroma
material are not generally used fresh, but rather dried. We previously found that the odor of fresh patchouli
leaves was different from that of dried leaves, more specifically, fresh patchouli leaves had a grassy odor while
dried leaves had an astringent and earthy odor. Furthermore, dried patchouli leaves had a stronger odor than
fresh leaves. We investigated the effect of the drying process on the odor of patchouli leaves to further reveal
differences in the leaves’ odor. The aroma constituents of fresh and dried patchouli leaves were extracted by
hexane and the constituents were analyzed by gas chromatography-olfactometry (GC-O). There was no clear
difference in the constituents of fresh and dried patchouli extract. We then separated the constituents of hexane
extracts using fractional distillation, and identified each fractions using gas chromatography-olfactometry and
gas chromatography-mass spectrometry. We found various constituents such as isoaromadendrene epoxide
and caryophyllene oxide as the key compounds that effected the change in odor.
P1-062 Investigation of the aroma of green teas derived from Chinese or Assam tea varieties
Yuka Hayakawa1, Toshio Hasegawa1, Piyaporn Chueamchaitrakun2,3, Takashi Fujihara1
1
Graduate School of Science and Engineering, Saitama University, Saitama, Japan, 2Food Technology Program School of AgroIndustry, Mae Fah Luang University, Chiang Rai, Thailand, 3Tea Institute, Mae Fah Luang University, Chiang Rai, Thailand
Tea is a very popular beverage worldwide, and its fascinating, unique aroma is an important characteristic point
for the quality control of tea. Teas are classified into three types by their degree of fermentation: green tea
(unfermented), oolong tea (semi-fermented), and black tea (fermented). These teas are made from Camellia
sinensis var. sinensis (Chinese variety) or Camellia sinensis var. assamica (Assam variety), the former mainly
used for green tea and oolong tea and the latter mainly for black tea. In this study, we investigated the effect of
tea plant variety on the odor of green tea. Because in Japan, only var. sinensis is used in green tea production,
this study examined green tea from Thailand, where both var. sinensis and var. assamica are used. Odorants in
tea leaves were obtained by hexane extraction followed by fractional distillation. The obtained extracts and
fractions were analyzed by gas chromatography-olfactometry and gas chromatography-mass spectrometry. We
found characteristic compounds for both varieties: linalool, α-terpineol, and some acids in var. assamica , and
some hydrocarbons in var. sinensis . The residue obtained by fractional distillation of Thai green tea prepared
from var. assamica had a sweet, roasted, and sour odor in addition to weak green notes, which different from
the matcha-like odor of Japanese green tea that we have reported previously. Thai green tea prepared from var.
sinensis had an intermediate character between Japanese green tea and Thai green tea from var. assamica .
These results indicate that tea plant variety is an important factor in determining the odor of the green tea
produced.
P1-063 Activation of calcium sensing receptors on olfactory sensory neurons modulate renal function
through renal sympathetic nerve activities enhancement in rats
Shih-Pin Lee1, Jia-Hao Zhou2, Jong-Kai Hsiao2, Chiang-Ting Chien1
1
Department of Life Science, National Taiwan Normal University, Taipei City, Taiwan, 2Department of Diagnostic Radiology,
Buddhist Tzu Chi Medical Foundation, New Taipei City, Taiwan
Olfaction is an important external chemical sensing system and kidney is the major organ involved with internal
ionic homeostasis in vertebrates. Under economically consideration, the connection between external chemical
sensing system and internal chemical homeostasis system would be helpful for livings to enhance their
adaptation and/or tolerance to face the environmental chemical changes. In previous researches,
calcium-sensing receptors (CaSRs), one of the G-protein coupling receptors, was expressed on fish olfactory
epithelium and considered as a potential salinity sensor. Through the immunohistofluorescence analysis, we
found that CaSRs was colocalized with CFAP69, one of olfactory neuron specific cilia and flagella associated
proteins, on the topical part of olfactory sensory neuron. In electrophysiology and hemodynamic experiments,
the manganese ions exposure in nasal cavity would enhance the renal sympathetic nerve activities and
decrease the renal artery blood flow rate and renal surface microcirculation. AC-265347 and NPS2143
hydrochloride are the agonist and antagonist of CaSR separately. AC-265347 intranasal administration would
similarly enhance the renal sympathetic nerve activities and decrease the renal artery blood flow rate. NPS2143
hydrochloride intranasal administration would inhibit the influence from manganese intranasal exposure on renal
sympathetic nerve activities and renal artery blood flow rate. In order to understand the changes of renal
2016.05.27 11.25.45 Page 27(1)
function, we discovered the glomerular filtration rate was decreased under the intranasal manganese exposure
in dynamic contrast-enhanced magnetic resonance imaging experiments. Based on our current results, we firstly
found that CaSRs were functional expressed on olfactory sensory neurons. We also further demonstrated that
the olfactory stimulation based on the activation of CaSRs on olfactory sensory neurons would influence the
renal function through the changes of renal sympathetic nerve activities. In the future, we prefer to prove the
connection between the olfaction and hepatic function. This will be helpful to understand the physical reaction
under different olfactory stimulation.
P1-064 Analyzing the effects of volatiles on human psychophysiological aspects in a room with
Japanese cedar interior walls
Eri Matsubara1, Shuichi Kawai2
1
Department of Wood-based Materials, Forestry and Forest Products Research Institute, Tsukuba, Japan, 2Graduate School of
Advanced Integrated Studies in Human Survivability, Kyoto University, Kyoto, Japan
Wood has been used since ancient times as a building material around the world. Studies about the habitability
of rooms with wooden walls have been widely carried out, and the psychophysiological relaxation effects of
interior wood materials through visual and tactile pathways have been reported. Volatile organic compounds
(VOCs) extracted from wood chips and essential oils produce relaxant effects on the autonomic nervous system.
However, few studies have been performed on the psychophysiological responses to odors in a full-scale
wooden room. This study aimed to confirm the effects of VOCs emitted from interior wood walls. The evaluation
indices of the participants are arithmetic performance, subjective assessments of VOCs, salivary stress markers,
and temporal changes in heart rate and autonomic nerve activity. We used Japanese cedar timber dried in a
mild temperature. VOCs in the experimental room used, were collected with a carbon tube for 24 hours and the
VOCs were eluted by acetone and analyzed by the GC-MS system. The VOCs were composed mainly of
delta-cadinene, alpha-muurolene and other kinds of sesquiterpenes. To evaluate cardiac autonomic nervous
activity, we performed a power spectral analysis of R-R intervals. Salivary alpha-amylase was measured using a
salivary amylase monitor and salivary cortisol, secretory immunoglobulin A and chromogranin A were measured
by enzyme immunoassay kits. We found that the inhalation of air containing VOCs emitted from Japanese cedar
interior walls suppresses the increase in salivary alpha-amylase activity and chromogranin A secretion. And the
volatiles emitted from Japanese cedar were pleasant and natural for the participants. In the present study, we
found that VOCs emitted from Japanese cedar walls affect the autonomic nervous system and emotions. We
suggested that an interior containing Japanese cedar can help people maintain an optimum living environment.
P1-065 Large-scale transcriptional profiling of chemosensory neurons identifies receptor-ligand pairs
in vivo
Benoit von der Weid1,2,3, Daniel Rossier1,2,3, Matti Lindup1,2,3, Joel Tuberosa1,2,3, Alexandre Widmer1,2,3, Julien Dal Col1,2,3,
Chenda Kan1,2,3, Alan Carleton2,4, Ivan Rodriguez1,2,3
1
Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland, 2Geneva Neuroscience Center, University of
Geneva, Geneva, Switzerland, 3Institute of Genetics and Genomics of Geneva, University of Geneva, Geneva, Switzerland,
4
Department of Basic Neurosciences, School of Medicine, University of Geneva, Geneva, Switzerland
In mammals, olfactory perception is based on the combinatorial activation of G protein-coupled receptors.
Identifying the full repertoire of receptors activated by a given odorant in vivo , a quest that has been hampered
for over 20 years by technical difficulties, would represent an important step in deciphering the rules governing
chemoperception. We found that odorants induced a fast and reversible concentration-dependent decrease in
the transcription of genes corresponding to activated receptors in intact mice. On the basis of this finding, we
developed a large-scale transcriptomic approach to uncover receptor-ligand pairs in vivo . We identified the
mouse and rat odorant receptor signatures corresponding to specific odorants. Finally, we found that this
approach, which can be used for species for which no genomic sequence is available, is also applicable to nonvertebrate species such as Drosophila .
2016.05.27 11.25.45 Page 28(1)
P1-066 Taste and olfactory functions in hypogonadotrophic hypogonadism patients
Murat Salihoglu1, Ferhat Deniz2, Onuralp Kurt3, Seyid Ahmet Ay2, Aytug Altundag4, Kamil Baskoy2, Hakan Tekeli5,
Muzaffer Saglam6, Arif Yonem2, Thomas Hummel7
1
Department of Otorhinolaryngology, Gata Haydarpasa Training Hospital, Istanbul, Turkey, 2Department of Endocrinology, Gata
Haydarpasa Training Hospital, Istanbul, Turkey, 3Department of Otorhinolaryngology, Erzincan Military Hospital, Erzincan, Turkey,
4
Department of Otorhinolaryngology, Istanbul Surgery Hospital, Istanbul, Turkey, 5Department of Neurology, Gata Haydarpasa
Training Hospital, Istanbul, Turkey, 6Department of Radiology, Gata Haydarpasa Training Hospital, Istanbul, Turkey,
7
Interdisciplinary Center “Smell & Taste”, Department of Otorhinolaryngology, TU Dresden, Dresden, Germany
Intraduction: The aim of this study is to evaluate both retronasal and orthonasal olfaction in patients with
idiopathic hypogonadotrophic hypogonadism (IHH) patiens. Possible differences in taste levels of IHH patients
were also researched. Material and Methods: Participants; consisting of age-gender-education year matched 45
IHH patients and 31 control patients. Psychophysical olfactory test were made using commercially available
Sniffin’ Stick test kit. For retronasal olfactory testing, a standardized, validated test was used. Four basic tastants
with 4 concentrations were used for taste evaluation (taste strip test). Olfactory Bulb Measurement; all
examinations were performed with a 1.5-T magnetic resonance imaging (MRI) Results: Threshold scores of
patients with normosmic IHH was significantly lower than controls but discrimination and identification scores of
patients with normosmic IHH. Retronasal testing of olfaction was lowered only in anosmic IHH group.
Orthonasally hyposmic and normosmic IHH groups were not statistically lower in retronasal olfaction when
compared with controls. Bitter, sweet, sour, salty tastes were indifferent when compared between IHH and
controls. OBV was lower in all groups (normosmic, hyposmic and anosmic IHH) when compared with controls.
OBV found significantly correlated with TDI scores of IHH in both sides. Serum indicators of hypogonadism such
as FSH, LH, Serum Free Testosterone, Serum Total Testosterone and TDI correlation was investigated and no
significant correlations were found. Conclusion: Evaluation of IHH patients under 3 subgroups determined by
their TDI scores as normosmics, hyposmics and anosmics gave us;1-No taste differences was present between
controls and subgroups, 2-retronasal olfaction was reduced only in anosmics. Orthonasally hyposmic groups’
retronasal olfaction found normal. 3-Olfactory bulb volumes are highly correlated with olfaction scores in HH
group.
P1-067 Comparing temporal dominance of sensation and ultra-flash GC-based electronic nose results
for milk chocolates
Koichi Yoshida, Kiyoharu Ikehama
Alpha MOS Japan K.K., Japan
The use of alternative ingredients such as artificial sweeteners and flavors has increased in recent years.
Understanding the temporal changes in the sensory characteristics of food and beverage products is important
to differentiate products from their competitors. A novel method for evaluating the temporality of the sensory
perception, called temporal dominance of sensations (TDS), enables the simultaneous recording of several
attributes over time. However, the technique has some drawbacks, such as the subjectivity and fatigue of the
panelists. Therefore, there is a need for an instrument that can objectively measure the sensory properties of
samples. The present study compared milk chocolate quality assessments made by a trained sensory panel
using TDS with those made by an ultra-flash GC-based electronic nose combined with chemometric analysis.
The electronic nose features very fast analysis (<3 min) with fast column heating rates and highly sensitive with
integrated a solid adsorbent trap to achieve an efficient pre-concentration of volatiles. It was additionally
equipped with an odor and chemical database comprising information on chemical compounds with sensory
attributes and a related bibliography. Results from these analyses demonstrated that the TDS curves
characterized the differences in the dynamics of flavor perception between brands after product consumption.
The electronic nose identified key compounds from aroma profiling that contribute to differentiation between
products and classified them using specific odor descriptors. Furthermore, the elution order of odor compounds
was similar to the sequence of attribute perception in TDS results. In conclusion, TDS is the primary technique
for interpreting the temporal changes in flavors of milk chocolates; the ultra-flash GC-based electronic nose
could be used to predict these changes in a new product development reducing the sensory work, which will also
objectively support the conclusion.
2016.05.27 11.25.45 Page 29(1)
P1-068 Identification of the Origin of Coffee by using E-Nose System considering GC Olfactometry
Analysis
Kyuki Kato1, Motohiko Sugiura1, Toshiyuki Yajima2
1
Tokyo Allied Coffee Roasters CO., LTD, Japan, 2Alpha M.O.S. Japan K.K., Japan
Purpose: Because of the variety of odorants in coffee, an aroma analysis is important for a proper sensory
evaluation. A coffee aroma analysis requires a simple and easy method for product development and quality
control because gas chromatography (GC) is very time consuming. Thus, we focused on the electric nose
system that implemented flash GC for a quick and easy aroma analysis. However, electric nose system is
difficult to analyze by GC olfactometry for retention time of a few minutes. In this study, we attempted to identify
the origin of coffee using the electric nose system and a GC olfactometry analysis. Methods: Four kinds of lightroasted coffee, such as arabica from Brazil, Colombia, and Ethiopia as well as robusta from Vietnam were
analyzed by GC olfactometry. Approximately 40 potent odorants were scored for odor intensity. In addition, 12
samples, including light-, medium-, and dark-roasted of each origin were analyzed using the electric nose
system. The peaks of the 40 odorants analyzed using the electric nose system and the peaks selected by GC
olfactometry were adjusted using the retention index. The odor intensity analyzed by GC olfactometry were used
to revision of the peak areas analyzed using the electronic nose system. The balance of each peak area
considering odor intensity was compared using principal component analysis (PCA). Results: PCA using the
data from the electric nose system tended to identify each roasting level. However, PCA used to standardize the
peak areas considering odor intensity was able to identify the origin of coffee regardless of roasting level.
P1-069 Sex differences of axillary odor in a Japanese population
Ayumi Kyuka1, Kasumi Maeda1, Maki Sawada2, Akiko Sawada2, Tadashi Shimada2, Takeshi Hara2, Mayumi Shimizu1
1
Fundamental Research Institute, Mandom Corp., Osaka, Japan, 2Technical Development Center, Mandom Corp., Osaka, Japan
Introduction The human axilla contains eccrine, apocrine, and sebaceous glands. The compounds secreted
from these glands produce odorous chemical molecules mediated by bacterial breakdown and subsequent
oxidation. In our previous study, we characterized axillary odor intensity and quality in Japanese males using
sensory evaluation. Odor intensity in males decrease with age and the odor quality is classified into seven
categories. To determine the sex differences in the Japanese population, we evaluated and compared axillary
odor in Japanese males and females.
Methods The axillary odor intensity and quality of 87 healthy Japanese males (age, 21-49 years) and 82 healthy
Japanese females (age, 20-55 years) were directly evaluated by four trained assessors. Transepidermal water
loss (TEWL) was measured using a Tewameter TM300 (Courage+Khazaka). Further, the relationship between
axillary odor and physical profiles such as body mass index (BMI), susceptibility to perspiration, and type of
earwax was assessed by using questionnaire data from experimental subjects.
Results In the 20-29 and 40-49 age ranges, odor intensity of males was stronger than that of females. Odor
intensity in males decreased with age. However, a similar tendency was not observed in females. Interestingly,
TEWL tended to be higher in females who had given birth. In the group of female subjects classified as having
dry-type earwax, odor intensity tended to positively correlate with TEWL.
Discussion We found that the change in axillary odor intensity with age showed sex difference. The decreasing
odor intensity in males probably results from a natural decline in metabolism. While in females, the physical
attributes, such as the metabolism of external secretions or the perspiration rate indicated by TEWL value, would
change with age and birth experience. Therefore, odor intensity should be maintained despite age increase in
females.
P1-070 A key male glandular odorant evoking female attractive behavior in Lemur catta
Mika Shirasu1,2, Satomi Ito3, Takashi Hayakawa4,5, Kodzue Kinoshita3, Isao Munechika6, Hiroo Imai3, Kazushige Touhara1,2
1
Department of Applied Biological Chemistry, The University of Tokyo, Tokyo, Japan, 2ERATO Touhara Chemosensory Signal
Project, JST, The University of Tokyo, Tokyo, Japan, 3Department of Cellular and Molecular Biology, Primate Research Institute,
Kyoto University, Aichi, Japan, 4Department of Wildlife Science (Nagoya Railroad Co., Ltd.), Primate Research Institute, Kyoto
University, Aichi, Japan, 5Japan Monkey Centre, Aichi, Japan, 6The Research Institute of Evolutionary Biology, Tokyo, Japan
Ringtailed lemurs (lemur catta , one of the Malagasy strepsirrhine primates) utilize olfactory cues to make
conspecific communications. Male lemurs have well-developed scent glands in the wrist (antebrachial scent
gland) and shoulder (brachial scent gland). The antebrachial gland is mainly composed of apocrine glands and
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secretes small quantities of highly volatile clear fluid, while the brachial gland is composed of sebaceous glands
producing brown greasy paste. Males perform scent marking to show the territory with wrist rubbing and load the
tails to spread the volatiles from the glands and appeal females. Here, we combined ethological and chemical
approaches to elucidate the role of volatiles emitted from the antebrachial gland secretion and to identify key
odorant(s) involved in sexual communication in lemurs. We collected 62 glandular secretions over 2 years from
2 adult male lemurs. Behavioral assay indicated that female lemurs sniffed the male antebrachial gland
secretions in the breeding season longer than those in non-breeding season. We then examined seasonal
changes in volatile components in secretions by gas chromatography mass spectrometry olfactometory
analyses. We found that a volatile long-carbon-chain aldehyde showed a significant increase in the amount
during the breeding season in a testosterone-dependent manner. Moreover, the sniffing duration of female
lemurs towards this compound was significantly longer than that to a control odor or diluent solvent; this
compound attracts female lemurs. These results suggest that the identified compound is a key odorant in the
male scent gland secretion that is involved in sexual communication during the breeding season in lemurs.
P1-071 Intranasal rotenone administration to mice induces the degeneration of dopaminergic neurons
in the olfactory bulb and the substantia nigra
Hitoshi Sasajima, Sadaharu Miyazono, Tomohiro Noguchi, Makoto Kashiwayanagi
Department of Sensory Physiology, Asahikawa Medical University, Asahikawa, Japan
Xenobiotic compounds in the nasal cavity reach the brain by bypassing physiological barriers. Through the
olfactory transport, xenobiotics absorbed at the nasal mucosa are directly delivered to the brain and the
cerebrospinal fluid through the connection between olfactory neurons and the olfactory bulb (OB). Thus olfactory
transport is suspected to be a vulnerable mechanism of the brain against invasion threats of environmental
chemicals which are potentially neurotoxic. The etiology of Parkinson’s disease revealed that the survivability of
dopaminergic neurons is susceptible to mitochondrial dysfunction. Rotenone, an inhibitor of mitochondrial
respiration chain complex-I, induces reactive oxygen species in the cells through incomplete electron transport
system at mitochondria. Dopaminergic neurons are sensitive to oxidative damage which induced by rotenone. In
this study, we focused on the neuronal toxicity of rotenone administered intranasally to mice. The results showed
that rotenone administration attenuated olfactory functions of the mice. We also found that intranasally
administered rotenone induced acute mitochondrial stress at the OB. The repeated administration of rotenone
resulted in a decrease in the number of dopaminergic neurons, which are inhibitory interneurons in the OB.
Moreover, further administration of rotenone resulted in the neurite degeneration of dopaminergic neurons in the
substantia nigra. Taken together, our findings suggest that the inhalation of environmental toxins induces the
neurodegeneration of cranial neurons through olfactory transport, and that olfactory dysfunction will be observed
as an earliest symptom of neurodegeneration caused by inhaled neurotoxins.
P1-072 “Open Essence” odor-identification test card to an elementary low grade school student
Eri Mori, Keisuke Uno, Yoshinori Matsuwaki, Nobuyoshi Otori
Department of Otorhinolaryngology, Jikei University, School of Medicine, Japan
Introduction: Olfactory dysfunction in childhood is associated with congenital dysfunction, traumatic, and
Kallmann’s syndrome and with nasopharyngeal disease, such as adenoid hyperplasia, sinusitis and allergic
rhinitis. The “Open Essence” (“OE”) odor-identification test, which was developed by the National Institute of
Advanced Industrial Science and Technology in Japan and was marked in 2008, is considered useful for
evaluating olfaction in adults but not yet in children. Therefore the aim of present study was to examine the ability
of the “OE” test to evaluate olfaction in young Japanese children. Method: The subjects were 87 elementary
school students from the metropolis of Tokyo (33 children) and the small town of Tsunan (54 children). Of the 87
subjects, 54 were 6 years old and 33 were 7 years old; 39 were boys and 48 were girls. Subjects were excluded
if they had sinonasal or ear problems, cognitional olfactory dysfunction, or developmental disabilities. The ability
of the “OE” test to assess the subjects’ identification of 12 olfactory items was compared among children of
different sex, age, and residence. Result: The mean total score among all subjects was 7.6+ or -1.64 items
(range, 3-12). Scores did not differ significantly with sex or age. Surprisingly, the total score was slightly, but not
significantly, higher for children in Tsunan (p=0.086). The rates of identification of “cooking gas” and “roasted
garlic” were significantly greater for children in Tsunan (p=0.034, p=0.001), but the rate of identification of
“perfume” was significantly greater for children in Tokyo (p=0.004). Among all children the rate of identification
were lowest for “indian ink” and “leather” and highest for “menthol”. Conclusion: These results suggest that, with
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some modification, the ”OE” test can be used to evaluate olfaction in young Japanese children.
P1-073 Treatment of olfactory disorders with theophylline and lipoic acid
Mikyung Ye
Department of Otolaryngology, Catholic University of Daegu, Daegu, South Korea
Objectives: Estimated prevalence of olfactory disorders in general population is 20%, however, effective
treatment has not been formally established. The study aimed to investigate the potential therapeutic effects of
theophylline and alpha-lipoic acid (ALA) in olfactory loss following upper respiratory infection. A recent study
suggests theophylline, an unspecific phosphodiesterase inhibitor increases olfactory sensitivity due to interaction
with the signal transduction in the olfactory epithelium. Possible mechanisms of actions of ALA include the
release of nerve growth factor and antioxidative effects, both of which may be helpful in the regeneration of
olfactory receptor neurons. Methods: A total of 50 patients participated (13men, 37women; mean 52 years); 30
of them were treatment group and 20 were control group. All patients had been previously treated without
success with systemic corticosteroids. In the treatment group, theophylline and ALA were used orally at a dose
of 400mg/day and 600 mg/day respectively and ciclesonide topical nasal spray. Participants in the control group
only used ciclesonide topical nasal spray. They were prescribed for an average period of 3 months. Olfactory
function was evaluated at baseline and again at 3 months, and results were quantified in the form of each patient
TDI (threshold, discrimination, identification) score. Results: In treatment group, 11 patients (37%) showed
remarkable and 10 patients (33%) showed moderate improvement in olfactory function; in contrast, in control
group, 4patients (20%) showed remarkable and 6 patients (30%) showed moderate improvement in olfactory
function. Severe side effects were not observed following oral administration. Recovery of olfactory function
appeared to be more pronounced in hyposmic patients than in anosmic patients. Conclusions: Although the
treatment response was not statistically different with control group, the addition of theophylline and ALA showed
a tendency of greater efficacy in the treatment of postviral olfactory loss.
P1-074 Olfactory airflow after endoscopic sinus surgery: An analysis using virtual surgery and
computational fluid dynamics
Hironobu Nishijima1, Kenji Kondo1, Tsutomu Nomura2, Tatsuya Yamasoba1
1
Department of Otolaryngology, The University of Tokyo, Tokyo, Japan, 2Department of Otolaryngology, Saitama Medical Center,
Saitama Medical University, Saitama, Japan
[Background] Chronic rhinosinusitis with nasal polyps can disturb olfactory airflow, which is potentially one of
the most important factors in olfaction. Endoscopic sinus surgery (ESS) for refractory chronic rhinosinusitis is
known to improve olfactory function. However, the association between a particular ESS technique and
post-operative olfactory airflow has not been studied. The present study used virtual ESS and computational
fluid dynamics analysis to compare olfactory airflow after ESS between conventional ethmoidectomy and
ethmoidectomy with superior meatus enlargement in order to determine which procedure increases
post-operative olfactory airflow.
[Materials and Methods] Nasal computed tomography images from four adult subjects were used to generate
models of the nasal airway. The original pre-operative model was digitally edited as virtual ESS by performing
uncinectomy, ethmoidectomy, antrostomy, and frontal sinusectomy. The two post-operative models were the
ethmoidectomy with normal superior meatus model (ESS model) and the ethmoidectomy with superior meatus
enlargement model (ESS-SM model). Calculated three-dimensional nasal geometries were confirmed using
virtual endoscopy to ensure that they corresponded to the post-operative anatomy observed in the clinical
setting. Steady-state, laminar, inspiratory airflow was simulated, and the velocity, streamline, and mass flow rate
in the olfactory region were compared among the pre-operative and two post-operative models.
[Results and Conclusion] The mean velocity in the olfactory region, number of streamlines bound to the
olfactory region, and mass flow rate were higher in the ESS-SM model than in the other models. Our findings
suggest that ethmoidectomy with superior meatus enlargement might be an effective procedure as it might
increase olfactory airflow, leading to better olfactory perception. We successfully used an innovative approach
involving virtual ESS, virtual endoscopy, and computational fluid dynamics to assess post-operative outcomes
after ESS.
2016.05.27 11.25.45 Page 32(1)
P1-075 A case report of oral steroids efficacy for posttraumatic olfactory dysfunction patients
Masayoshi Tei1, Eri Mori1, Keisuke Uno2, Yoshinori Matsuwaki1, Nobuyoshi Otori1
1
Department of Otorhinolaryngology, The Jikei University School of Medicine, Tokyo, Japan, 2Department of Otorhinolaryngology,
Dokkyo Medical University, Tochigi, Japan
Head trauma is one of the major causes for olfactory dysfunction. The cure rate is around 20%, which is lower
than other olfactory dysfunctions. Treatment varies between institutions, with no established therapy. We have
experienced three cases of successfully treated posttraumatic olfactory dysfunction, which all patients
underwent oral steroid administration.
The first case is a 56 year-old female, who injured head trauma with a fall while drunk. Oral steroid was
administered 9 days after injury with 30mg prednisolone for 5 days. Recognition threshold was improved from
5.8 to -1.0 after 2 years of treatment.The second case is a 21 year-old female, who also injured head trauma
with a fall while drunk. Oral steroid was administered 7 days after injury with 30mg prednisolone for 3 days,
followed by 20mg for 2 days, 10mg for 2 days. Recognition threshold was improved from 5.8 to 0.0 after 1 year
of treatment.The last case is a 60 year-old female, who slipped and injured head trauma with a memory loss.
Oral steroid was administered 9 days after injury with 3mg betamethasone for 1 day, followed by 2mg for 3 days,
1mg for 3 days. Recognition threshold was improved from 5.8 to 4.4 after 1 year of treatment.
Standard treatment for olfactory dysfunction has not yet been established, but the usage of steroid is said to be
effective. Oral administration of steroid during the early periods after the injury in posttraumatic olfactory
dysfunction patients may also be an effective treatment.
P1-076 Olfactory disorders due to allergic rhinitis and chronic rhinosinusitis
Kengo Hashimoto, Kenzo Tsuzuki, Yoriko Yukitatsu, Masafumi Sakagami
Department of Otolaryngology-Head and Neck Surgery, Hyogo College of Medicine, Hyogo, Japan
Objections: Few patients with allergic rhinitis (AR) complain of olfactory disorder. This study aimed to compare
the olfactory disorder between AR and CRS. Methods: Two hundred and thirty-nine patients with AR and CRS,
who underwent bilateral primary sinonasal surgery in our department between March 2012 and April 2015, were
enrolled. The patients consisted of two groups: i) group AR included 77 patients with AR who underwent bilateral
inferior turbinate surgery; and ii) group CRS included 162 patients with bilateral CRS who underwent endoscopic
sinus surgery. Chief complaints, olfactory visual analog scale (VAS), severity of olfactory disorder by the mean
recognition thresholds using T&T olfactometer, and reaction to intravenously injected prosultiamine at the
preoperative stage in both groups were retrospectively analyzed. Results: No patient in the group AR
complained of olfactory disorder. Mean olfactory VAS (73%) in group AR was significantly better than that (40%)
in the group CRS (p<0.001). In the group AR, normosmia (n=27, 35%) and mild hyposmia (n=38, 49%) were
mainly observed. On the other hand, in the group CRS, anosmia (n=67, 41%) were predominantly observed.
Mean T&T recognition thresholds (1.6±1.2) in the group AR was significantly milder than those (4.1±1.8) in the
group CRS (p<0.001). In both groups AR (p<0.01, rs=-0.3471) and CRS (p<0.001, rs=-0.8781) showed
significant correlations between VAS and the mean recognition thresholds. As regards to intravenous olfaction
test, patients without response to prosultiamine were found in 7.1% (11/154) of the group CRS, whereas in 0%
(0/68) of the group AR. Conclusion: Although the frequency and severity of the olfactory disorder in patients with
AR are less and milder than those in patients with CRS, importance of accurate evaluation of olfactory disorder
in patients with AR was suggested.
P1-077 Control of local inflammation ameliorates olfactory system recovery following head injury
Mohammed Omar Al Salihi1, Masayoshi Kobayashi1, Kengo Tamari1, Tomotaka Miyamura1, Kazuhiko Takeuchi1,
Richard M. Costanzo2
1
Department of Otorhinolaryngology-Head and Neck Surgery, Mie, Japan, 2Department of Physiology and Biophysics, Virginia
Commonwealth University School of Medicine, Richmond, VA, USA
The olfactory system has a remarkable capacity for neural regeneration and recovery following injury. Clinically,
however, the prognosis of olfactory dysfunction by head injury is reported to be poor. We investigated factors
that influence the degree of recovery. Using transgenic (OMP-tau-lacZ) mice, we studied mild and severe
olfactory nerve transection (NTx) injury models.. Histological assessment was made for regenerating olfactory
nerves (ON), astrocytes and macrophages. With mild injury we observed less injury-associated tissue and more
ON regeneration. At 42 days recovery, more astrocytes and macrophages were observed in the severe injury
model. Dexamethazone sodium phosphate (DXM)-injected mice, anti-interleukin-6 receptor antibody-injected
2016.05.27 11.25.45 Page 33(1)
mice and tumor necrosis factor-alpha blocker-injected mice with severe injury all showed less injury-associated
tissue, better ON recovery and fewer astrocytes and macrophages. These results indicate that ON recovery
depends on the severity of injury and that treatment with anti-inflammatory drug is effective in improving recovery
outcome during the acute phase of head injury. Patients with head injury usually notice their olfactory
dysfunction several weeks or months after the injury, which may be a factor in poor recovery outcomes.
Therefore, we examined the effect of anti-inflammatory treatment at different time points. In the severe injury
model, DXM injection was started at 7, 14, 28 and 42 days after NTx injury. DXM treatment was effective when
initiated at 7 days but ineffective 14 days or longer after head injury. These findings suggest that different
therapeutic strategies for inhibition of inflammation are needed for traumatic olfactory dysfunction in the chronic
phase of recovery. Finally, we confirmed clinical application of these results by administering steroids to patients
with olfactory dysfunction due to head trauma in the acute phase, resulting in good outcomes.
P1-078 Effects of ethnicity, primary language and acculturation on odor identification
Claire Murphy, Nobuko Kemmotsu, Yurika Enobi
San Diego State University, San Diego, CA, USA
Elegant studies have investigated odor identification in Japanese participants with odors chosen for use in
Japan. Informed by the work of Kobayakawa and colleagues, here we focused on cultural factors that drive
differences in performance on an American odor identification test, with a particular interest in JapaneseAmerican participants. First we studied 71 Japanese-American participants and 71 Caucasian-American
participants, aged 45 to 91 yrs, who completed the San Diego Odor Identification Test. Age and gender were
related to performance in both ethnic groups. Japanese-American participants tended to score higher on odor
identification at younger ages and lower at older ages. In a second study, we investigated performance on the
same measure in 100 bilingual Japanese-Americans. For 50 the primary language was Japanese and for 50 the
primary language was English. Hierarchical regression analyses examined the effects of predictor variables: 1)
primary language spoken, 2) acculturation level as measured on the Suinn-Lew Asian Self-Identity Acculturation
Scale (SL-ASIA test), 3) years of residence in the US and 4) years of education in English. Age and gender were
entered into the model at step 1, predictor variables at step 2. Interestingly, in those whose primary language
was Japanese, there were no gender differences; whereas in those whose primary language was English,
females performed at a higher level than males. Primary language spoken and acculturation were more
predictive than years of residence in the US or years of education in English. Acculturation was not a significant
factor in the group whose primary language was Japanese. In those whose primary language was English the
SL-ASIA scores predicted odor identification. A deeper understanding of what drives cultural differences in
chemosensory perception is becoming increasingly important as we consider olfactory function as a contributing
biomarker for detection of neurodegenerative disease. Supported by NIH grant AG004085-26.
P1-079 Subjective olfaction and responses to food-related odors
Antti Knaapila1, Oskar Laaksonen1, Markus Virtanen1, Hanna Lagstrom2, Mari Sandell3
1
Food Chemistry and Food Development, Department of Biochemistry, University of Turku, Turku, Finland, 2Child and Youth
Research Institute, University of Turku, Turku, Finland, 3Functional Foods Forum, University of Turku, Turku, Finland
Whether self-rated olfactory acuity influences responses to food odors is unclear. The aim of the study was to
clarify relationships among subjective olfactory experience (collected using an online survey) and
psychophysical responses to food-related olfactory stimuli (collected at a sensory laboratory). We studied how
self-rated olfactory function (0-10) was associated with experienced disturbance from everyday odors (0-5),
Affective Impact of Odor scale (AIO, Wrzesniewski et al. 1999), and responses to 12 olfactory stimuli
(cinnamaldehyde, cis-3-hexenol, beta-ionone, eugenol, 1-octen-3-ol, thymol, allyl-isothiocyanate, anethol,
S-(+)-carvone, limonene, allyl-disulphide, and vanillin). The stimuli were rated for their orthonasally perceived
intensity (5-point scale), pleasantness (9-point scale), and identity (free naming, scoring judged by the
researchers) by 126 individuals (93 women and 33 men; aged 25-61 years, mean age 39 years). Self-rated
olfactory function correlated with the disturbance from everyday odors (r=0.30; p=0.001), AIO score (r=0.32;
p<0.001), mean perceived intensity of the odors (r=0.31; p=0.001), and identification score (r=0.20; p=0.034),
but not with the mean pleasantness of the odors. People who rated their olfactory acuity above average (>=6)
also rated the disturbance from everyday odors as higher (p=0.001) and regarded odors in general as more
important for liking/disliking foods/places/cosmetics/people (i.e., scored higher in AIO; p=0.002). They also rated
the sampled olfactory stimuli, on average, as more intense (p<0.001) and scored higher in odor identification
2016.05.27 11.25.45 Page 34(1)
(naming; p=0.001). Our results suggest that the self-assessed olfactory acuity is associated not only with
experienced affective impact of odors, but also with psychophysically measured responses to food-derived
olfactory stimuli.
P1-080 Does the number of residual teeth affect to the olfactory functions in human?
Chizuru Utsugi1, Makoto Kashiwayanagi2, Tadashi Katayama3, Mamoru Machino1,3, Kazuyo Muramoto4
1
Sayama General Hospital, Saitama, Japan, 2Department of Sensory Physiology, Asahikawa Medical University, Asahikawa, Japan,
Division of Oral Diagnosis Depertment of Theraphic and Diagnostic Dentistry, Meikai University, Saitama, Japan, 4Division of
Physiology, Department of Human Development and Fostering, Meikai University, Saitama, Japan
3
Many reports suggested that an adequate mastication was important for the development and improvement of
the higher brain function. We previously reported that a restriction of mastication impaired neurogenesis in the
subventricular zone and olfactory functions in mice. As a progressive investigation, we examined the correlation
between the number of residual teeth and the olfactory function for human subjects. Subjects were selected from
dental patients without dysosmia and dementia over 60 years of age, and divided them into two groups on the
basis of the number of the residual teeth; less than 14 teeth (low group) and over 14 teeth (high group). The
olfactory function was examined using the odor stick identification test for Japanese (OSIT-J). We analyzed
differences in OSIT-J score about the following items; 1) The number of residual teeth (low group vs. high
group), 2) The age classes (groups aged 60s, 70s and 80s), 3) The sex differences. The number of correct
answer of the OSIT-J in the low group (The total number of the residual teeth) was significantly lower than that in
the high group. The OSIT-J score in the 80s was significantly smaller than that in the 60s. The sex-related
difference in OSIT-J score was not significant. These results suggest that the restriction of mastication impaired
the olfactory function in human as well as mice. The mastication is very important to maintain the olfactory
functions.
P1-081 Development of taste sensing system
Yohichiro Kojima
Department of Electrical and Electronic Engineering, Hokkaido University of Science, Sapporo, Japan
Taste sensors, which have been developed by Toko et al., use various types of artificial lipid membranes to
simulate biological taste sense receptors and are applied for evaluating the taste of food products from these
electrical characteristics in response to taste solutions. Therefore, this taste sensor is being used by many
research institutions and companies. On the other hand, we have developed various chemical sensors, such as
ion-electrode sensors and surface plasmon resonance (SPR) sensors, and have reported their validity for taste
identification and quality control. In particular, the taste sensor equipped with an ion-electrode sensor combines
multiple ion electrodes to detect a specific ion, and can also detect various substances because the combined
electrodes show some reaction even to substances other than the target ions. In this study, various attributes of
taste substances can be determined using taste sensor with lipid membrane and multiple ion electrodes;
therefore, taste sensors can be developed using output patterns obtained from these sensors. We discussed the
applicability of these sensors to the evaluation of the taste of a beverage on the market, with the aim of applying
a taste sensing system to the food industry.
P1-082 Plasma osmolality/fluid volume sensing and drinking behavior via TRPV1 and 4 channels in
mice
Yoichi Ueta, Takanori Matsuura, Yasuhito Motojima, Hiromichi Ueno, Mitsuhiro Yoshimura, Takashi Maruyama,
Hirofumi Hashimoto, Hideo Ohnishi
Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
The detection of plasma osmolality and body fluid volume and sensation of thirst related to drinking behavior
may be mediated in part by TRPV1 and TRPV4 channels. In the present study, we examined the effects of
osmotic and non-osmotic stimuli that cause drinking behavior on water intake in TRPV1-deficient (TRPV1-/-) and
TRPV4-deficient (TRPV4-/-) mice in comparison with wild type (WT). Intraperitoneal injection of hypertonic
saline (HS) and water deprivation (WD) for 24 hours was performed as osmotic stimuli. Subcutaneous injection
of 20% polyethylene glycol (PEG) and intracerebroventricular injection of AII was also performed as non-osmotic
stimuli. HS and WD caused drinking behavior and drank similar accumulating water volume in all types of mice.
PEG-induced water intake in TRPV4-/- mice was significantly smaller than that in TRPV1-/- and WT mice. AII-
2016.05.27 11.25.45 Page 35(1)
induced water intake in TRPV1-/- mice was significantly smaller than that in TRPV4-/- and WT mice. These
results suggest that TRPV1 and TRPV4 may be involved potently in AII- and hypovolemia-induced drinking
behavior, respectively.
P1-083 Longitudinal changes of olfaction and taste during pregnancy
Minori Shibata1,2, Hideaki Suzuki2, Eiji Shibata3
1
Health Center, University of Occupational and Environmental Health, Kitakyushu, Japan, 2Department of OtorhinolarhingologyHead & Neck Surgery, University of Occupational and Environmental Health, Kitakyushu, Japan, 3Department of Obstetrics and
Gynecology, University of Occupational and Environmental Health, Kitakyushu, Japan
The alteration of olfaction and taste during pregnancy, especially in the first trimester, is well-known
phenomenon. Sharpened olfaction may cause nausea and vomiting. Furthermore, taste change may affect food
preferences and consumption. The mechanism of this phenomenon remains to be delineated. In addition,
detailed data on olfaction and taste during pregnancy is limited.
One hypothesis is that dramatic changes in reproductive hormone levels may modulate olfaction and taste
sensitivity during pregnancy. We also hypothesize that the change in serum trace elements such as Zn, which
are associated with taste sensation, may cause this phenomenon. Serum trace elements are described as being
associated with many diseases. Pregnancy is also associated with changes in trace element serum level.
To evaluate the difference of olfaction and taste during pregnancy, we performed detailed serial clinical
assessments (1st, 2nd, 3rd trimester and postpartum) of T&T olfactometry, electrogustmetry, and the taste disc
method (standard clinical tests in Japan) in 37 pregnant subjects who visited the Department of Obstetrics and
Gynecology of University of Occupational and Environmental Health from 2012 to 2014. Interview sheets were
used to survey aversive or favorite odors and tastes, appetite and nausea. Taste-associated serum trace
elements, reproductive hormones, appetite-related peptides were also examined at each time period.
We present a part of our findings here, many subjects were consciously aware of keen olfaction and taste in
the 1st trimester. They were averse to the smell of just cooked hot rice (Japanese staple food) and cigarette
smoke, and they preferred to eat fried potato, potato chips and yogurt in the 1st trimester. Other obtained results
have been discussed in this presentation.
P1-084 The perception of environmental signals that initiate the major pelago-benthic life cycle
transition in the sponge, Amphimedon queenslandica
Tahsha Say, Sandie Degnan
School of Biological Science, The University of Queensland, Brisbane, Australia
Sophisticated chemosensory abilities are crucial for almost all aspects of life under the sea, with even
microscopic larvae relying on chemosensation to guide their major transition into the adult stage of their life
cycle. The settlement and metamorphosis of a free-swimming larva into a benthic adult is orchestrated by finescale chemical signals emanating from the environment. Larval settlement and metamorphosis is most often
irrevocable, thus highlighting the importance of the settlement “decision” for selecting the precise location in
which an adult will live out the rest of it’s life. Even sea sponges, one of the most ancient and morphologically
simple of all animals, produce larvae that coordinate sophisticated photosensory and chemosensory behaviours
to identify a suitable adult habitat. Despite the universality of such behaviours, we have only a limited
understanding of the molecular mechanisms underlying chemical detection in the marine environment. Our
research combines behavioural and molecular analyses to determine what role the surprisingly large and
diverse repertoire of cell-surface G-protein coupled receptors (GPCRs) encoded by the genome of the sponge
Amphimedon queenslandica may play in mediating the perception of chemical settlement inducers. Our growing
body of transcriptome data highlights the particular importance of several representatives from three different
GPCR families, Rhodopsin, Glutamate and Adhesion, during the larval “exploration” phase of the sponge life
cycle. We also have generated transcriptomes from individual sponge larvae maintained under constant light,
constant dark or a natural day-night cycle, to elucidate the complex mechanisms linking phototactic and
chemotactic behaviours that govern the morphogenetic changes following settlement. The morphological
simplicity and basal position of sponges in the animal tree makes them an ideal comparative organism for
understanding how chemical communication operates in the marine environment, and may provide new
perspectives on the origin and evolution of chemosensation.
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P1-085 Specificities of chemical senses among sensory modalities: Synchrony perception for
olfactory-visual, visual-gustatory, olfactory-gustatory and match-mismatch combinations
Tatsu Kobayakawa, Naomi Gotow
National Institute of Advanced Industrial Science and Technology (AIST), Japan
Vision is a physical sense, whereas olfaction and gustation are chemical senses. Active sensing might function
in vision, olfaction, and gustation, whereas passive sensing might function in vision and olfaction but not
gustation. To investigate whether each sensory property affected synchrony perception, participants in this study
performed temporal judgment for three cross-modal combinations using visual (red LED), olfactory (coumarin),
and gustatory (NaCl) stimuli. We calculated the half-width at half-height (HWHH) and point of subjective
simultaneity (PSS) on the basis of temporal distributions of simultaneity rates in each combination, and
compared HWHH and PSS among three cross-modal combinations. Although HWHH did not differ among three
cross-modal combinations, HWHH exhibited a higher value in cross-modal combinations involving one or two
chemical stimuli than in combinations of two physical stimuli. The PSS of the olfactory-visual combination was
approximately equal to the point of objective simultaneity (POS), whereas the PSS of visual-gustatory and
olfactory-gustatory combinations were shifted greatly relative to the POS. These results related to synchrony
perception in cross-modal combinations suggest specificities of chemical senses (especially gustation) among
sensory modalities. On the other hand, participants in next study performed temporal judgement for olfactory
(soy source flavor) and gustatory (NaCl) stimuli as match condition. At that case, HWHH in match condition
showed significant higher value than that in mismatch condition, while PSS showed N.S. between conditions.
This means that under match condition, in other words, familiar combination to participants, they had lower
temporal separation between olfaction and gustation. And this would imply matched olfaction and gustation
combination constructs flavor object , and is difficult to separate.
P1-086 Dissection of neural circuitry mediating CO2-evoked escape behavior in the larval zebrafish
Tetsuya Koide, Yoshihiro Yoshihara
RIKEN Brain Science Institute, Saitama, Japan
Avoidance of carbon dioxide (CO2) is a vital reaction commonly preserved in various animal species from worms
to mammals for their survival. In the present study, we aim to elucidate the neural circuit mechanism underlying
CO2 avoidance in larval zebrafish. First, we observed that the application of CO2 to larval zebrafish elicits a
robust escape response. Next, by taking advantage of the optical transparency of zebrafish larvae, we
performed in vivo whole-brain Ca2+ imaging in GCaMP-expressing transgenic zebrafish to map neural substrates
governing CO2-evoked escape response. Upon CO2 application to the nose, ensembles of neural activation were
reproducibly observed in multiple brain regions, including the olfactory bulb (OB), the trigeminal ganglion (TG),
the forebrain, the midbrain, and the hindbrain. Because the fish nose receives two distinct types sensory
information, olfactory and trigeminal, we laser-ablated either the OB or the TG and performed Ca2+ imaging and
behavioral analysis to examine each contribution to the escape response. As a result, the CO2-evoked neural
activation in the hindbrain and escape behavior were markedly decreased in the TG-ablated fish, but not in the
OB-ablated fish. These findings suggest that the trigeminal pathway is responsible for CO2-evoked escape in
larval zebrafish and provides a framework for dissection of multiple neural pathways linking sensory inputs to
behavioral outputs.
P1-087 Functional dissociation in sweet taste receptor neurons between and within taste organs of
Drosophila
Vladimiros Thoma1, Stephan Knapek2, Shogo Arai3, Marion Hartl2, Hiroshi Kohsaka4, Pudith Sirigrivatanawong3, Ayako Abe1,
Koichi Hashimoto3, Hiromu Tanimoto1
1
Graduate School of Life Sciences, Tohoku University, Japan, 2Max-Planck Institute for Neurobiology, Japan, 3Graduate School of
Information Sciences, Tohoku University, Japan, 4Department of Complexity Science and Engineering, Graduate School of Frontier
Sciences, the University of Tokyo, Japan
Finding food sources is essential for survival. Insects detect nutrients with external taste receptor neurons.
Drosophila possesses multiple taste organs that are distributed throughout its body. However, the role of
different taste organs in feeding remains poorly understood. By blocking subsets of sweet taste receptor
neurons, we show that receptor neurons in the legs are required for immediate sugar choice. Furthermore, we
identify two anatomically distinct classes of sweet taste receptor neurons in the leg. The axonal projections of
2016.05.27 11.25.45 Page 37(1)
one class terminate in the thoracic ganglia, whereas the other projects directly to the brain. These two classes
are functionally distinct: the brain-projecting neurons are involved in feeding initiation, whereas the thoracic
ganglia-projecting neurons play a role in sugar-dependent suppression of locomotion. Distinct receptor neurons
for the same taste quality may coordinate early appetitive responses, taking advantage of the legs as the first
appendages to contact food.
P1-088 The monosodium glutamate to growing ADHD model rat decreases the aggressiveness
mediated by vagus nerve
Hideki Hida, Yoshihiro Yokoyama, Haruka Nagai, Ruriko Nishigaki, Yoshitomo Ueda, Sachiyo Misumi
Dept Neurophysiology & Brain Science, Nagoya City University of Graduate School Medical Science, Nagoya, Japan
Attention-deficit hyperactivity disorder (ADHD) is characterized by hyperactivity, impulsivity and inattention.
Dysfunction of mesocorticolimbic dopaminergic system such in the amygdala and prefrontal cortex is reported in
ADHD relating to emotional disorder. Spontaneously hypertensive rat (SHR) is known as an ADHD model. As
environmental stimuli during the period of development could modify emotional behavior such social behavior, a
taste substance of umami, monosodium L-glutamate (MSG), was used as a simple stimulus to examine how
environmental stimuli contributes to the formation of emotion during the period of development in ADHD model
rat. It revealed that oral intake of MSG (0.6% solution) for 5 weeks from postnatal day 25 (P25) to P60 caused
decreased aggressiveness in isolated SHR. However, the mechanism of MSG effect on enhanced social
behavior in adulthood is still unknown. To investigate the effect of brain-gut communication via vagus nerve, the
vagotomy was performed at the stomach level in P25 followed by MSG intake for 5 weeks. The vagotomy with
MSG administration failed to decrease aggressiveness: the number of attack became to the same level as H2Otreated control group. To investigate the critical period in MSG effect on social behavior, SHR rats (P25) were
housed in a isolated condition (one rat per cage) treated with MSG for various period until P60: early-treated
group (P25-P40), late-treated group (P40-P60), all-period group (P25-P60) and non-treated group. Early-treated
group decreased the number of riding (parameter of aggression to unfamiliar rat) compare with control group,
which is the same level as all-period group. However, no significant difference was found between late-treated
group and non-treated group. Data suggest that MSG effect on aggressiveness was mediated by the afferent of
vagus nerve with a critical period of P25-P40. Supported by JSPS KAKENHI No 26430020 to HH, and No
26860851 to SM.
P1-089 Ingestion of dried-bonito broth (dashi) facilitates the expression
immunoreactive neurons in the brain, and affects emotional behaviors in mice
of
parvalbumin-
Jargalsaikhan Undarmaa1, Hiroshi Nishimaru1, Jumpei Matsumoto1, Yusaku Takamura1, Tomoya Nakamura1, Etsuro Hori1,
Takashi Kondoh2, Taketoshi Ono1, Hisao Nishijo1
1
System Emotinal Science, University of Toyama, Toyama, Japan, 2Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, Japan.
Emerging evidence suggests that traditional diets and nutrition have a significant impact on brain development,
and could contribute to the promotion of mental health and prevention of psychiatric disorders in children and
adolescents. Moreover, deficits in parvalbumin (PV)-immunoreactive and/or GABAergic neurons are closely
associated with various psychiatric disorders in children and adolescents. To investigate the possible neural
mechanisms of diet involvement in mental health, we analyzed the effects of dried-bonito dashi (Japanese fish
broth) (DBD) on PV-immunoreactive neurons and emotional behaviors in young mice. Male mice after weaning
were fed DBD for more than 4 weeks, and tested with a resident-intruder test for aggressiveness and a forced
swimming test for depression-like symptoms. After the behavioral testing, PV-immunoreactive neurons in the
brain were immunohistochemically analyzed. The results indicated that DBD intake decreased aggressiveness
and depression-like symptoms, and increased the densities of PV-immunoreactive neurons in the medial
prefrontal cortex (mPFC), amygdala, hippocampus, and superior colliculus (SC). These behavioral changes
were correlated with the densities of PV-immunoreactive neurons in the mPFC, amygdala, and hippocampus.
However, subdiaphragmatic vagotomy did not affect the effects of DBD on emotional behaviors, although it
nonspecifically decreased the densities of PV-immunoreactive neurons. The results suggest that DBD might
modulate emotional behaviors by promoting PV-immunoreactive and/or GABAergic neuronal activity through
parallel routes. The present results highlight a new mechanism for diet involvement in brain functions, and
suggest that DBD might have therapeutic potential for the promotion of mental health.
2016.05.27 11.25.45 Page 38(1)
P1-090 Effects of AgRP neuron-derived neuropeptides on high-fat/high-sugar diet selection in mice
Ou Fu, Masataka Narukawa, Takumi Misaka, Kenichiro Nakajima
Graduate School of Agricultural and Sciences, The University of Tokyo, Tokyo, Japan
Obesity is one of severe health problems in the world. Not only how much food we eat, but also what kind of food
we eat is one of the important factors to improve our health status. However, the relationship between appetite
and food preference has not been fully elucidated. Agouti-related peptide (AgRP)-expressing neurons located in
the arcuate nucleus of the hypothalamus play a key role in triggering appetite. Interestingly, AgRP neurons
release two chemically and functionally different orexigenic peptides: neuropeptide Y (NPY), and AgRP. While
NPY induces acute increase in food intake for several hours, AgRP induces chronic increase in food intake over
several days. To clarify the roles of AgRP and NPY on food selection, we evaluated the effects of these
neuropeptides on food choice behavior in mice with two typical palatable diets: fat-rich diet and sugar-rich diet.
High lard and high sucrose diets were simultaneously provided to the mice administrated AgRP or NPY by single
intraventricular injections. The mice treated with NPY showed stronger preference toward high sucrose diet over
high fat diet within several hours compared to the control mice injected with saline. This change was back to the
normal within a few days after NPY injection. By contrast, we did not observe such changes in sugar preference
in the mice injected with AgRP. These results suggest that, although both AgRP and NPY induce orexigenic
responses, they affect food selection behavior differently. We are currently conducting several sensory assays
including brief access taste tests to investigate whether taste sensitivities change in the above mice.
P1-091 Exploring amino acid sensing in Drosophila
Makoto I. Kanai, Greg S. B. Suh
NYU School of Medicine, USA
L amino acids (L-AA) are precursors for proteins, which govern all of the biological processes. Hence, the
appropriate levels of protein and L-AAs must be monitored and maintained in organism. While, feeding behavior
is intensively studied, AA feeding behavior is relatively less studied. To have better understanding of the
detection and ingestion of essential L-AAs. We first established the behavior assay that allow us to investigate
how essential L-AAs are detected and selected by Drosophila melanogaster. Using this assay, we found that 1;
AA deprivation results in a decrease of AA concentration of the body. 2; AA deprived flies prefer L-AAs to D-AAs.
3; Even taste-blind flies retain the preference for L-AAs. 4; AA deprived flies can rapidly distinguish L-AAs from
D-AAs. 5. TOR pathway, not GCN2 pathway, is required for appropriate AA selection. We are currently
searching for neurons in the brain that are essential for mediating this behavior.
P1-092 Vitamin C deficiency affects ingestive behavior and taste nerve responses in rats
Toshiaki Yasuo, Takeshi Suwabe, Noritaka Sako
Department of Oral Physiology, Asahi University School of Dentistry, Gifu, Japan
Some mammals including human beings cannot synthesize Vitamin C (VC), so it must be obtained from diet.
There are few reports about the behavioral mechanisms of ingesting VC. The aim of this study was to examine
how the VC deficient rats select VC. For that aim, we performed behavioral and the electrophysiological
experiments using Osteogenic Disorder Shionogi/Shi Jcl-od/od rats, which cannot synthesize VC. The
preference ratios for various concentrations of VC solutions were evaluated before and after deprivation of VC
by using two-bottle preference test. The number of licks for VC and NaCl were counted before and after
deprivation of VC. The whole chorda tympani nerve (CTN) responses were also recorded in deficient and normal
rats. In the two-bottle preference test, the rats avoided the 10-100 mM of VC solutions during the VC nondeficient period, but this aversive behavior was reduced during deficiency. However, preference ratios for 0.1-3
mM of VC solutions were not changed during the VC deficiency. There were no significant difference in the lick
ratios between deficient and normal rats in all tested stimuli except 150 mM NaCl. The magnitudes of the CTN
responses to VC, HCl, citric acid, acetic acid, tartaric acid and NaCl in deficient rats were significantly smaller
than those in normal rats. In contrast, the responses to sucrose, quinine HCl and monopotassium glutamate in
the deficient rats were not significantly different than those in the normal rats. These data suggest that rats with
adequate VC can avoid the 10 mM or more of VC solutions by sensing VC as a tastant, but this avoidance is
reduced when VC deficiency occurs. These data suggest also that the ingestive behavior for VC during VC
deficiency may be related to the reduction of CTN responses to sour tastants and VC.
2016.05.27 11.25.45 Page 39(1)
P1-093 Intensity, recognition and hedonic ratings for the binary taste stimuli in human and rats
Yoshihisa Katagawa3, Shigeki Yamada2, Toshiaki Yasuo1, Takeshi Suwabe1, Keika Gen2, Noritaka Sako1
1
Department of Oral Physiology, Asahi University School of Dentistry, Gifu, Japan, 2Department of Dentistry for Disability and Oral
Health, Asahi University School of Dentistry, Gifu, Japan, 3Department of Oral Anatomy, Asahi University School of Dentistry, Gifu,
Japan
There were small studies to detect intensity, recognition and hedonic ratings for the binary taste mixtures in
human and animals. In our present study, therefore, we conducted behavioral studies in both human and rats. In
human study, subjects evaluated intensity and hedonic ratings for the binary taste mixtures by using Labeled
Magnitude Scale (Green et al. , 1993) and Labeled Hedonic Scale (Lim et al. , 2009). In animal study, preference
and recognition for the taste stimuli were measured by using two bottle preference test and conditioned taste
aversion test in Wistar/ST rats.
As results of human study, when unfavorable pure taste stimuli were mixed with sweetener, hedonic ratings
for those mixtures were higher than those for unfavorable pure taste stimuli, and intensity for quality of
unfavorable taste stimuli in mixtures were lower than those for unfavorable pure taste stimuli. In animal study, we
found that rats had an ability to recognize and remember the preferable and unfavorable components of binary
mixtures as well as preferable and unfavorable binary taste mixtures.
P1-094 Neuronal activities concerning the conditioned taste aversion with immune reactions
Chizuko Inui-Yamamoto1,2, Fuminori Sugihara2, Ting Chen2, Yoshichika Yoshioka2, Satoshi Wakisaka1
1
Department of Oral Anatomy and Developmental Biology, Osaka University Graduate School of Dentistry, Japan, 2Biofunctional
Imaging Laboratory, WPI IFReC, Osaka University, Japan
It is well known lipopolysaccharides (LPS) is produced by infected bacteria and triggers several acute phase
responses. Some reports show that rodents can acquire aversion to the taste stimulus paired with LPS.
However, the brain mechanisms in the conditioned taste aversion (CTA) with immune reactions and in its
retrieval remain obscure. Therefore, to elucidate the brain mechanism in the retrieval of CTA with LPS (LPSCTA), we tried to visualize the brain activities using the manganese enhanced MRI. C57BL/6N male mice were
trained to drink distilled water via an intraoral cannula. On the conditioning day, they were injected
intraperitoneally saline (Saline-group), LiCl (LiCl-group), or LPS (1 mg/kg) (LPS-group) 15 min after drinking
saccharin solution for 8 min. On the test day, all mice were given saccharin solution and injected 20 mg/kg MnCl
2 to the stomach 30 min before MRI scanning. They were fixed in the animal cradle of the MR apparatus under
anesthesia (2.5% sevoflurane) and scanned by 11.7 T. The results showed that the signal intensity of the central
nucleus of the amygdala (CeA) in the LPS- and LiCl-groups were higher than that in the Saline-group. The signal
intensity of the dorsomedial hypothalamus (DMH) in the LPS-group was higher than that in other groups. The
activities of the CeA, which is involved in the visceral input, are the same as the previous study in rats using LiCl.
LPS may evoke the visceral sensation as the same as LiCl, so Toll-like receptor 4 for LPS is localized to the
gastrointestinal mucosa. In addition, it is known that LPS induces body temperature changes accompanied by
the inflammatory reaction. The hypothalamus including the DMH has a role of the body temperature control.
Therefore, there is a possibility that the activation of DMH in the retrieval of LPS-CTA might cause body
temperature changes.
P1-095 An examination of the motivational taste properties of L-glutamate in wild-type and T1R1+3
double knock-out mice
Ginger Blonde, Alan Spector
Program in Neuroscience & Department of Psychology, Florida State University, Tallahassee, FL, USA
We recently demonstrated that mice lacking either subunit of the L-amino acid receptor T1R1+3 can detect high
concentrations of monosodium glutamate (MSG) in the presence of inosine 5’-monophosphate (IMP, 2.5mM),
with the sodium component reduced by amiloride (A, 100μM). To investigate the hedonic characteristics of this
stimulus complex (M+A+IMP), we tested knock-out (KO) mice missing both T1R1+3 subunits and wild-type (WT,
both n=13) mice in a battery of behavioral tests. Food-deprived mice were first trained and tested in gustometers
with a concentration series of Maltrin-580, a maltodextrin, in a brief-access (10-s trials) test that minimizes
postingestive feedback. Similar tests followed using M+A+IMP and M+A. The brief-access series was repeated
following both short-term (five 30-min sessions) and long-term (48h) exposures to 0.2M M+A+IMP. Additional
brief-access tests were conducted with NaGluconate replacing MSG. Finally, progressive ratio tests (ratio=3),
2016.05.27 11.25.45 Page 40(1)
which assess appetitive behavior while minimizing satiation, were conducted using 8% Maltrin-580 and 0.4M M+
A+IMP. Although KO mice took significantly fewer licks than WT mice, Maltrin-580 elicited robust concentrationdependent responding in both groups in brief-access tests. In contrast, M+A+IMP elicited very weak licking
responses across the concentration range in WT mice, and KO mice were virtually unresponsive to this stimulus.
Both groups initially showed no concentration-dependent licking to M+A, implicating IMP as an important
component for consummatory responses to M+A+IMP. WT and KO mice initiated a similar number of trials
during MSG tests, and initiated significantly fewer for NaGluconate, suggesting that appetitive (approach)
behavior was maintained by the glutamate anion. In the progressive ratio task, both groups completed a similar
number of ratios for M+A+IMP. Overall, these results suggest that the T1R1+3 heterodimer is necessary for M+A
+IMP to elicit relatively weak taste-guided consummatory licking in brief-access tests, but is unnecessary for
appetitive responses to be expressed to this “umami” stimulus complex in mice.
P1-096 The role of the neural projections from the basolateral amygdala to the bed nucleus of the stria
terminalis on the retrieval of conditioned taste aversion
Tadashi Inui, Tsuyoshi Shimura
Graduate School of Human Sciences, Osaka University, Osaka, Japan
The basolateral amygdala (BLA) is suggested to be involved in the retrieval of conditioned taste aversion (CTA).
The projective neurons from the BLA to the bed nucleus of the stria terminalis (BNST) are activated by the
presentation of a conditioned aversive taste. In order to investigate the functional role of the projections, we
examined the effects of pharmacological manipulations of the BLA and BNST on animal’s behaviors during the
CTA retrieval. Wistar rats were implanted with guide cannulae into the BLA or BNST, and received a pairing of
saccharin (conditioned stimulus, CS) with an i.p. administration of lithium chloride. On the retrieval test day, the
rats received microinjections of GABAA receptors agonist muscimol into the BLA or antagonist bicuculline into
the BNST, just before the CS presentation. We recorded the rat’s approach and licking behaviors to the CS in an
experimental chamber. The muscimol injections into the BLA decreased the number of the approaches without
licking, indicating that the rats did not hesitate to consume the CS solution. These treatments also increased
total licks and the duration of licking burst, meaning the reduction of taste aversion. These results imply,
therefore, that the BLA is involved in both of approach and aversion to the CS. On the other hand, the bicuculline
injections into the BNST shortened latency to contact with the CS solution whereas they had no influence on the
CS intake, suggesting the involvement of the BNST in approach, but not aversive, behaviors to the CS. The
differences in the rat’s behaviors between the BLA and BNST manipulations provide the possibility that the
neural projections from the BLA to BNST play a role in the approach behaviors to a CTA, but not the
consumption of it, during the retrieval of CTA.
P1-097 Evolution of bitter taste receptor genes in birds and vampire bats
Huabin Zhao
Department of Ecology, College of Life Sciences, Wuhan University, Wuhan, China
The bitter taste serves as an important natural defence against the ingestion of poisonous foods. To test the
evolutionary malleability of bitter taste receptor genes (Tas2rs) in animals, we chose to study vampire bats and
birds, because vampire bats are obligate blood feeders, and birds have long been believed to have an inferior
taste system. We found vampire bats to have a significantly greater percentage of pseudogenes than other bats,
suggestive of a reduction of bitter taste function in vampire bats. After examining 48 genomes representing all
but 3 avian orders, we identified the total number of bitter taste receptor genes from each bird, ranging from 1 in
the domestic pigeon to 12 in the bar-tailed trogon, with an average of 4. This finding suggested that a much
smaller Tas2r gene repertoire exists in birds than in other vertebrates. Furthermore,we uncovered a positive
correlation between the number of putatively functional Tas2rs and the abundance of potential toxins in avian
diets. Because plant products containmore toxins than animal tissues and insects release poisonous defensive
secretions, we hypothesized that herbivorous and insectivorous birds may demand more functional Tas2rs than
carnivorous birds feeding on noninsect animals.Our analyses appear to support this hypothesis and highlight the
critical role of taste perception in birds.
2016.05.27 11.25.45 Page 41(1)
P1-098 Zinc deficiency induced reduction of taste cell proliferation and apoptosis in rat circumvallate
papillae
Akiyo Kawano1,2, Shiho Honma1,3, Hitoshi Niwa2, Satoshi Wakisaka1
1
Department of Oral Anatomy and Developmental Biology, Osaka University Graduate School of Dentistry, Osaka, Japan,
Department of Dental Anesthesiology, Osaka University Graduate School of Dentistry, Osaka, Japan, 3Department of Oral Health
Scienses, Faculty of Nursing and Health Care, Baika Women’s University, Osaka, Japan
2
Low zinc is one of the reasons to cause taste disorder. Previous studies have shown that the turnover of taste
cells is delayed in zinc-deficient rats. In the present study, we examined proliferation and apoptosis of taste buds
in circumvallate papillae of zinc-deficient rats. Furthermore, we investigated whether the changes of
zinc-deficient rats were rescued after feeding a normal diet. Rats were divided into three groups: fed a normal
diet for 8 weeks (normal rats); fed a zinc-deficient diet for 4 weeks (zinc-deficient rats); fed a zinc-deficient diet
for 4 weeks, followed by a normal diet for 4 weeks (rescued rats). The number of taste cells in the taste buds of
normal rat and zinc-deficient rat is no significant difference. The positive rate of proliferating cell nuclear antigen
(PCNA), a marker of proliferating cells, was significantly decreased from 17.5% in the taste buds of normal rats
to 10.7% in those of zinc-deficient rats. The positive rate of Bcl-2-assosiated X protein (Bax), a marker of
apoptotic cells, was significantly decreased from 29.4% in the taste buds of normal rats to 20.9% in those of
zinc-deficient rats. In rescued rats, both the PCNA and Bax-positive rate returned to that observed in normal
rats. The present results indicate that low-zinc caused the prolonged life span of taste bud, and that these
changes in zinc-deficient rats were rescued after feeding normal diet.
P1-099 Expansion of a bitter taste receptor family in Helicoverpa armigera
Wei Xu1,2, Alexie Papanicolaou1, Huijie Zhang1, Alisha Anderson1
1
CSIRO, Canberra, Australia, 2Murdoch University, Western Australia, Australia
Gustatory receptors form the interface between a taste system and the environment. In Lepidoptera the
gustatory system has been shown to have multiple roles including adapting to host plants. Using genome and
transcriptome sequencing we identify novel gustatory receptor (GR) genes from the polyphagous pest
Helicoverpa armigera. These GRs include a significantly expanded bitter receptor family (180 GRs) that can be
further divided into three categories based on polypeptide lengths, gene structure and amino acid sequence.
Type 1 includes bitter GR genes that possess introns, like the majority of bitter GRs found in other lepidopteran
species. Type 2 includes long intronless bitter GRs, while Type 3 comprises short intronless bitter GRs. We use
topological and calcium imaging studies to investigate the function of this expanded gene family and
hypothesise that this expansion may be functionally tied to its polyphagous behavior. Understanding the
molecular basis of polyphagy may provide opportunities for the development of novel effective pest control
strategies.
P1-101 Umami taste and health status
Aytug Altundag
Department of Otorhinolaryngology, Istanbul Cerrahi Hastanesi, Istanbul, Turkey
Umami Taste and Health Status There have been many researches about umami and other four basic tastes
from the time umami taste identified and still emerges as an issue that being discussed. The changes in four
basic tastes due to many circumstances and painful diseases have been investigated in many studies, whereas
there has been limited studies on umami because of its various perceptions on tongue and palatal area, also
because of cultural differences in the taste of umami. Today, because of the intense contribution to the
perception of the taste of umami flavor, that brings a high level of impact on quality of life as well. Which leads to
problems in the perception of taste in the mouth exceptions made changes in the perception of umami taste
changes in the salivary secretions are provided with umami taste stimulations Laryngopharyngeal reflux disease
has effects such as dental erosion and halitosis but also has effects on the taste of umami taste perception that
occur deterioration in these patients also appears to be a decreased quality of life parameters. However, the
change in the anatomy of the oral cavity causing snoring surgery method performed anterior palatoplasy also
arises changes in the sensitivity of the umami taste. In particular, the stimulation must necessarily be informed in
detail and will consist of patients with postoperative changes in the perception of taste umami taste and a
reduction in sensitivity may occur after resections in the palate.
2016.05.27 11.25.45 Page 42(1)
P1-102 Role of the C-terminal region of T1r3 in the membrane trafficking of taste receptor T1r2/T1r3
Yuko Kusakabe1, Masayo Okano1, Yumiko Shindo1, Atsuko Yamashita2, Takayuki Kawai1
1
Food Function Division, National Food Research Institute, NARO, Tsukuba, Ibaraki, Japan, 2Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan
G protein-coupled receptors (GPCR) are involved in the regulation of numerous physiological functions. GPCR
has several functions including reception of stimuli and subsequent signal transduction. The C-terminal domain
of a typical structure of GPCR type A and B contains a helix 8 motif and palmitoylated cysteine for membrane
localization. However, less information is available about the C-terminal domain of the taste receptor T1r3, which
belongs to type C GPCR family. In this study, we focused on the function of the C-terminal domain of the T1r3,
and compared the functions of C-termini of human, mouse, and medaka fish T1r3. We compared the amino acid
sequences of the C-terminal domains of human, mouse, and medaka fish T1r3s, and found that each has a helix
8 motif and a conserved cysteine residue. We created C-terminal deletion mutants for each T1r3 by deletion just
after the conserved cysteine residues, transfected them in HEK293 cells, and observed their membrane
trafficking by immunostaining under non-permeabilized conditions. Interestingly, all mutants stained slightly,
while wild-type T1r3s were clearly stained. Then, we created mutants by deleting different C-terminal regions of
T1r3, and found that the region responsible for membrane trafficking is different in all the three animal species.
There is a possibility that helix 8 has a role in the membrane trafficking of human and mouse T1r3, but not for
medaka fish T1r3. We also found that mutations in the conserved cysteine residues did not influence their
membrane trafficking. These results suggest that the membrane trafficking system of T1r3 might be different
from those of GPCR type A and B.
P1-103 TRPV1-ANO1 interaction enhances HOT taste?
Yasunori Takayama1,2, Makoto Tominaga1,2
1
Division of Cell Sig, OIIB (NIPS), Aichi, Japan, 2Department of Physiological Science, SOKENDAI, Aichi, Japan
Capsaicin receptor TRPV1 is activated by various noxious stimuli, and the stimuli are converted into electrical
signals in primary sensory neurons. It is believed that cation influx through TRPV1 causes depolarization,
leading to the activation of voltage-gated sodium channels, followed by action potential generation. However, we
reported that the capsaicin-evoked action potential is induced by two components: a cation influx-mediated
depolarization due to TRPV1 activation and a subsequent anion efflux-mediated depolarization via activation of
anoctamin 1 (ANO1), a calcium-activated chloride channel, due to the entry of calcium through TRPV1.
Intracellular chloride concentrations are maintained at a high level because of low expression of potassiumchloride co-transporter type 2 in DRG neurons. Therefore, depolarization is evoked by chloride efflux through
ANO1 activation followed by action potential generation. In fact, capsaicin-activated inward currents were
significantly inhibited by a specific ANO1 antagonist, T16Ainh-A01 (A01) in mice DRG neurons. In addition,
capsaicin-evoked action potential generation was inhibited by A01. Furthermore, capsaicin-induced pain-related
behaviors in hind paw were significantly reduced by the concomitant administration of A01. It was also reported
that TRPV1 is expressed with ANO1 in TG neurons. Therefore, the TRPV1-ANO1 interaction could be involved
in hot taste in mouth.
P1-104 Taste substance binding to the ligand-binding domains of T1r taste receptor heterodimer
Atsuko Yamashita1, Eriko Nango2, Shuji Akiyama3, Saori Maki-Yonekura2, Yuji Ashikawa2, Yuko Kusakabe4,
Elena Krayukhina5, Takahiro Maruno5, Susumu Uchiyama5, Nipawan Nuemket1, Koji Yonekura2, Madoka Shimizu4,
Nanako Atsumi1, Norihisa Yasui1, Takaaki Hikima2, Masaki Yamamoto2, Yuji Kobayashi5
1
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Japan, 2RIKEN SPring-8 Center,
Japan, 3Research Center of Integrative Molecular System, Institute for Molecular Science, Japan, 4National Food Research
Institute, NARO, Japan, 5Graduate School of Engineering, Osaka University, Japan
Sweet and umami taste substances are perceived by T1r taste receptors expressed in taste buds. In human, the
heterodimer of T1r2 and T1r3 recognizes sugars, while the heterodimer of T1r1 and T1r3 recognizes
L-glutamate. T1r proteins possess a large ligand-binding domain (LBD) at the extracellular side. The T1r LBDs
are response for ligand recognition, binding, and resultant receptor activation, and thus play crucial roles for
taste receptor functions. So far, preparation of the purified sample of T1r LBDs have been hampered by
difficulties in heterologous expression, and thus the detailed analyses of structure and protein-ligand interactions
have been impractical. To overcome the bottlenecks, we extensively screened the expression conditions for
T1r LBDs, and found a combination of specific T1r genes and an expression host, amenable to large-scale
2016.05.27 11.25.45 Page 43(1)
expression and sample preparation. The purified T1r LBD proteins are found to form a stable heterodimer,
analyzed by analytical ultracentrifugation and electron microscopy. We confirmed that the sample retains the
ability of ligand binding, by thermodynamic analysis. The Förster resonance energy transfer analysis indicated
the conformational change of T1r LBD heterodimer upon taste substance binding. Solution X-ray scattering
analysis of T1r LBD heterodimer in the presence and absence of a taste substance revealed two different
conformational state, a compact one and a spread one, which may correspond to distinct T1r receptor functional
states.
P1-105 Molecular analysis of the taste receptors in sea otter that have not been adapted to life in the
sea
Mitsuru Ebihara1, Yui Ohshima1, Ryosuke Tanii1, Yukiko Nishioka1, Yukihito Wakai2, Chisato Nakano2
1
Department of Food Science, Ishikawa Prefectural University, Ishikawa, Japan, 2Toba Aquarium, Mie, Japan
It has been reported that most of marine mammals such as Pinnipedia, Cetartiodactyla and Sirenia lost their
ability to perceive taste in their food. It was strongly suggested that loss of taste perception is essential to marine
mammals to survive in the sea, because of salty sea water. Their behavior to catch fishes are characterized by
swallowing with sea water. Sea otter, however, get their food in the sea and come up on the sea, then eat them
by chewing, suggesting that they may perceive its taste (even not all of tastes). To determine whether or not sea
otter perceive any taste, we cloned umami, sweet, bitter, salt, sour and fat receptor genes. As results, most of
taste receptor genes in sea otter could be active. In addition to the above observation, dolphin, dugong and seal
went back to the sea 50, 50 and 25 million years ago, but sea otter fell behind them. It was just 5 million years
ago when sea otter went back to the sea. It was good enough time for dolphin, dugong and seal to adapt to life in
the sea, meaning changing body design and losing taste perception but might not for sea otter. Sea otter could
be only one marine animal that can perceive taste of their food.
P1-106 Functional Expressions of the Bitter Taste Receptor Tas2r108 in the Submandibular Glands
SuYoung Ki1, KyungNyun Kim1,2, KiMyung Chung1,2, YoungKyung Cho1,2, HaJung Choi1
1
Department of Physiology and Neuroscience, College of Dentistry, Gangneung-Wonju National University, South Korea, 2Research
Institute of Oral Sciences, Gangneung-Wonju National University, South Korea
Mammals have 3 pairs of major salivary glands; the submandibular, parotid, and sublingual glands. Saliva
secretion of these glands is modulated by taste perception. Salivary glands are composed mainly of acinar and
ductal cells. Primary saliva is secreted by acinar cells and is modified during ductal flow. Our previous studies
found that bitter taste receptors were expressed in the submandibular glands by RT-PCR. Further, we showed
that several bitter taste substances elicited increase of Ca2+ activity in the murine submandibular glands acinar
cells with dose-dependent manner. The objective of the present study was to confirm which bitter taste receptor,
out of the murine 35 bitter taste receptors, is important using qPCR and in situ hybridization (ISH). Male 42-60
days old DBA2 mice were used. Quantitative PCRs were carried out for expression levels analysis of bitter taste
receptors. Results of qPCR from the submandibular glands revealed that Tas2r108 was expressed at the
highest levels. Digoxigenin (DIG) labeled-Tas2r108 cRNA probes were generated from mRNAs of the
submandibular glands. These probes were transcribed in anti-sense and sense orientation using T7 RNA
polymerase. To estimate integrity and optimal diluting concentration of these probes, dot blot hybridization was
performed. ISH for Tas2r108 in the submandibular glands was performed. Dot blot hybridization data
demonstrated that DIG labeled-Tas2r108 cRNA anti-sense probes detected specifically Tas2r108 cDNA. ISH
results showed that the anti-sense probes labeled acinar and ductal cells in the submandibular glands, whereas
no staining was visible in sense controls. The expression levels of Tas2r108 were higher in acinar cells than in
ductal cells. These results suggest that Tas2r108 may be associated with primary saliva secretion than with
ductal modification of saliva in the submandibular glands.
P1-107 Functional Characterization of TAS2R38 Bitter Taste Receptors to Phenylthiocarbamid (PTC) in
Colobine Monkeys
Laurentia Henrieta Permita Sari Purba1, Kanthi Arum Widayati1, Sarah Nila1, Kei Tsutsui2, Nami Suzuki-Hashido2,
Takashi Hayakawa2, Bambang Suryobroto1, Hiroo Imai2
1
Department Biology, Bogor Agricultural University, Bogor, West Java, Indonesia, 2Molecular Biology Section, Primate Research
Institute, Kyoto University, Inuyama, Aichi, Japan
Bitter perception evokes innate avoidance behavior in animals because it usually associated with toxic
2016.05.27 11.25.45 Page 44(1)
molecules. Various bitter taste sensitivities in mammals may reflect the species-specific diet during mammalian
evolution. Bitter perception in mammals is mediated by G-protein coupled receptors named TAS2Rs. One of
TAS2R receptors is TAS2R38 encoded by TAS2R38 gene that recognize phenylthiocarbamide (PTC). In
primates, TAS2R38 gene is observed to be polymorphic. Mutant TAS2R38 receptors in many primates had been
shown to have reduced sensitivity to PTC. Four species of captive colobines ate bitter apples-containing PTC in
contrast with macaque that strongly avoid to ate the apples. The non-avoidance behavior might be caused by
some non-synonymous mutations in their TAS2R38 gene. Here, we investigate functional characteristic of TAS2
R38 receptors in Asian Colobinae. Calcium imaging analysis in cells expressing these TAS2R38s support the
observed behavior that colobines had lower sensitivities to PTC than macaque did. This suggest the possible
mechanism underlying the adaptation to eat leaves by colobines.
P1-108 Structural and enzymatic characterization of a Drosophila melanogaster glutathione
transferase overexpressed in the sensory organs after exposure to bitter molecules in food
Daniel Gonzalez, Stephane Fraichard, Jean-Francois Ferveur, Yves Artur, Loic Briand, Jean Marie Heydel, Fabrice Neiers
Centre des Sciences du Gout et de l’Alimentation, Universite de Bourgogne Franche Comte, Dijon, France
Glutathione transferases (GSTs) are ubiquitous key detoxification enzymes that catalyze the conjugation of
glutathione to a large variety of xenobiotic chemical including odorants and sapid molecules. A previous study in
Drosophila melanogaster demonstrated the existence of a GST specifically expressed in the antenna and
involved in signal termination by catalyzing biotransformation of odorant molecules. Genomic analysis allows
identifying forty GSTs in Drosophila melanogaster but their biochemistry is poorly documented. Here we
demonstrated that among the GSTs, dmelGSTD2-2 was strongly and preferentially overexpressed in the
sensory organs (antennae, proboscis, legs, wings) after an exposure to bitter molecules supplemented in food.
In regard to these aspects we shed light on the existence of a gustatory GST. We expressed, purified and
performed enzymatic characterization revealing that dmelGSTD2-2 is able to interact and biotransform bitter
compounds (such as caffeine or isothiocyanates). Moreover, we solved the crystal structure of this dmelGSTD22 which is the first of this enzyme class and also one of the first in insect. DmelGSTD2-2 was solved at 1.5
angstrom resolution and appears as a dimer. The biochemical function in Drosophila melanogaster of this
enzyme toward bitter compounds is discussed in the light of the in vivo study supplemented by in silico binding
analysis and enzymatic assay with a panel of bitter molecules.
P1-110 Sweet and bitter responses of human taste receptors to ethyl α-D-glucoside, a distinctive sweet
substance in sake
Ren Yabiku1, Akihiro Nakao1, Kana Nakajima1, Masafumi Tokuoka1, Yutaka Kashiwagi1, Liquan Huang2, Kenji Maehashi1
1
Department of Fermentation Science, Tokyo University of Agriculture, Tokyo, Japan, 2Monell Chemical Senses Center,
Philadelphia, USA
Ethyl α-D-glucoside (α-EG) is a sweet substance present in sake as a major component after ethanol and
glucose. It is produced through transglucosylation from maltose and ethanol by α-glucosidase of Aspergillus
oryzae during the sake brewing process. It is known that α-EG tastes sweet with a bitter aftertaste; therefore, it is
believed that α-EG contributes to the unique flavor of sake. In this study, we examined the sweet and bitter
responses of human taste receptors to α-EG using a cell-based assay. HEK293F/G16gust44 cells were
transfected with mouse rhodopsin-tagged hT2R genes constructed in pcDNA5FRT and stable cell lines
expressing hT2Rs were generated. Using these cells, bitter responses were measured through calcium imaging.
For measuring sweet responses, the cells were transiently transfected with hT1R2 and hT1R3 constructed in a
separate pcDNA3.1, and calcium imaging was conducted using the same method as for bitter response. The
results showed that sweet-receptor-expressing cells responded to α-EG at 100 mM that is similar to those for
other sweeteners such as sucrose and glucose. Among five hT2R-expressing cells tested, hT2R45 mostly
responded to &alpha-EG with an EC50 of 50 mM, and did not respond to any other tested glucosides, such as
methyl &alpha-D-glucoside, phenyl β-D-glucoside, and salicin. Therefore, it can be concluded that hT2R45 is a
bitter receptor for α-EG as a ligand. This also indicates that &alpha-EG activates not only sweet receptor but
also bitter receptors to elicit both sweet and bitter tastes, respectively. The distinctive sweet-bitter taste of α-EG
is attributed to its ability to activate multiple taste receptors.
2016.05.27 11.25.45 Page 45(1)
P1-111 Bitter compounds delayed gastric emptying and induced intestinal smooth muscle relaxation in
a pig model
Minghai Fu1, Mary-Louise Manchadi1, Nadia De Jager1, David Val-Laillet2, Sylvie Guerin2, Eugeni Roura1
1
The University of Queensland, Brisbane, Australia, 2INRA, St Gilles, France
Bitter compounds induced innate aversion which was reflected in loss of appetite and decreased fat deposition
in a pig model reported by our group. However, the mechanism remains unclear. We hypothesized that bitter
compounds would reduce gastric emptying and slow down intestinal peristalsis in the pig. Based on previous invivo trial and known molecular mechanisms, caffeine and quinine were the two bitter compounds selected. Both
compounds are known agonists of human bitter taste receptors (hT2R) with high homology with the porcine T2
Rs: Tas2r4, Tas2r7, Tas2r10, Tas2r20 and Tas2r39. Real-time PCR was used to study the expression of
porcine bitter taste receptor genes in the stomach and duodenum that related to the tested bitter compounds.
The results showed high expression both in the stomach and duodenum and that the expression level was most
significant in the epithelium relative to the smooth muscle layer. The non-invasive scintigraphy imaging data
showed that caffeine and quinine mixed feed significantly (P<0.01 and P<0.05 respectively) decreased gastric
emptying compared to control pigs. In addition, an ex-vivo intestinal smooth muscle contraction study illustrated
that the two bitter compounds induced (P<0.001) dose-response relaxation in the duodenum. To conclude,
dietary bitter compounds significantly reduced gastric emptying and gut motility. These effects may partially
explain previous results showing a reduction in appetite and fat deposition in a pig model. The potential of bitter
compounds to help prevent obesity in humans is worth further investigation.
P1-112 Expression of umami taste-related genes in the tongue: A pilot study for genetic taste
diagnosis
Noriaki Shoji1, Shizuko Satoh-Kuriwada1, Masahiro Tsuchiya1,2, Hisayuki Uneyama3, Misako Kawai3, Takashi Sasano1
1
Division of Oral Diagnosis, Tohoku University Graduate School of Dentistry, Sendai, Japan, 2Tohoku Fukushi University, Sendai,
Japan, 3Institute for Innovation, Ajinomoto Company Inc., Kawasaki, Tokyo, Japan
Umami taste is important for maintaining not only good health but also the quality of life. A wide variety of test is
used to discriminate between normal and abnormal umami taste sensation. However, it is not necessarily useful
for patients who cannot express their sensation to the tastant, such as patients with dementia, because of the
subjective nature of the test. Against this background, we evaluated the expression of the umami receptor genes
in the tongue to develop an objective umami taste test. Tissue samples were collected from healthy volunteers
by scraping the foliate papillae of the tongue. Immunocytochemistry staining of gustducin, a taste-cell-specific G
protein, and gene expression analysis by real-time polymerase chain reaction of β-actin , gustducin (GNAT3 ),
and umami receptors (T1R1 , T1R3 , and mGluR1 ) were performed. Further, amplified PCR products were
analyzed by DNA sequencing to confirm the expected sequences. Changes in umami receptor expression
following application of umami substances onto the tongue were analysed. Results showed: (i) Gustducinpositive cells were observed in the samples, indicating the presence of taste cells; (ii) Gene expression of βactin , GNAT3 , T1R1 , and T1R3 was detected in all seven samples tested, while that of mGluR1 was detected
in four samples; (iii) Sequence analysis by NCBI Blast showed that each polymerase chain reaction product had
a 99% rate of identification of its targeted sequence, indicating that the designed specific primers enabled to
amplify the targeted gene expression without mispriming and/or primer-dimer artefacts; (iv) Stimulation of the
tongue by monosodium glutamate significantly upregulated the gene expression levels of T1R1 and T1R3 ,
indicating that this method can detect alterations in umami-related gene expression. In conclusion, evaluation of
the expression of the umami receptor genes T1R1 and T1R3 in the tongue could be clinically useful for
objective genetic diagnosis of umami taste disorders.
P1-113 The anatomy of mammalian sweet taste receptors
Jean-Baptiste Cheron, Jerome Golebiowski, Serge Antonczak, Sebastien Fiorucci
Institut de Chimie de Nice, Faculte des Sciences, Universite de Nice-Sophia Antipolis, Nice, France
All sweet-tasting compounds are detected by a single G-protein coupled receptor (GPCR), the heterodimer T1R
2-T1R3, for which no experimental structure is available. The sweet taste receptor is a class C GPCR, and the
recently published crystallographic structures of metabotropic glutamate receptor (mGluR) 1 and 5 provide a
significant step forward for understanding structure-function relationships within this family. In this article, we
critically align the sequences of class C GPCRs that recapitulate available structural and site-directed
2016.05.27 11.25.45 Page 46(1)
mutagenesis data. We provide a homology model of the human sweet taste receptor that is consistent with
experimental constraints. The T1R2-T1R3 structure gives an atomic point of view and a robust starting point for
initiating mechanistic or structural studies on the receptor; for example, the rational design of new sweeteners or
sweet taste modifiers. We highlight key residues involved in orthosteric and allosteric ligand binding and those
responsible for receptor activation, notably at the protein-protein interface and in the transmembrane domain.
The sequence of the sweet taste receptor is highly conserved in mammals, suggesting a similar structure and a
conserved activation mechanism. Subtle variations in key residues explain the phenotype of rodents, who do not
perceive primate-specific sweeteners.
P1-114 Localization of T2R in taste microvilli is critical for initiation of bitter taste avoidance behavior
Atstuko Yamashita1, Kaori Kondo2,3, Sachiko Iseki3, Takashi Kondo2, Masato S. Ota1,3
1
Graduate school of Human Science and Design, Japan Women’s University, Tokyo, Japan, 2Graduate School of Medical and
Dental Science, Tokyo Medical and Dental University, Tokyo, Japan, 3Laboratory of Developmental Genetics, Riken IMS,
Yokohama, Japan
BACKGROUND: The bitter taste avoidance behavior (BAB) is known to play a fundamental role to protect
animals from potential toxic compounds which usually indicate a bitter taste, but the molecular mechanism of
development BAB remains unknown. METHODS: The sucking nipple test was performed by spreading bitter
substances on the mother’s nipples and observed the nursing action of mouse infants from postnatal one day
(PD1) to PD9. Histological analysis of developing taste papillae was followed to observe morphological changes
of the tongue epithelium from PD1 to PD9. In addition, double fluorescent immunostaining was also performed in
similar stage of developing tongue epithelium with specific antibodies for, actin filament, ZO-1 and Type 2 taste
receptor 119 (Tas2R119) of a bitter taste receptor. Taste cell microvilli were modified by oral administration of
Cytochalasin D, an inhibitor of actin fiber polymerization, to examine the change of localization of Tas2R119.
RESULTS: The sucking nipple test indicated that about 40% of the mouse infants began to show BAB at PD7
and maintained BAB for thiamine. They did not exhibit clear BAB for sucrose and water. Histological analysis
indicated taste pores and spindle-shaped cells in taste buds after PD3, therefore gross morphology of taste buds
were nearly complete around at PD3 in mice infants. Actin immunoreactive taste microvilli were observed at the
ZO-1 immunoreactive taste pore at PD7, which indicated colocalization of Tas2R119 immunoreactive small
molecules. When mice were treated by oral administration of Cytochalasin D, some of them exhibited reduction
of BAB and also loss of Tas2R immunoreactivity at the tip of microvilli. CONCLUSION: These results suggested
that polymerization of actin filament is important for localization of Tas2R119 at the tip of taste microvilli and it is
critical event for the initiation of BAB.
P1-115 Renin-angiotensin system (RAS) components are expressed in the taste organ
Noriatsu Shigemura1, Yuzo Ninomiya2
1
Section of Oral Neuroscience, Graduate School of Dental Science, Kyushu University, Fukuoka, Japan, 2R&D Center for Taste and
Odor Sensing, Kyushu University, Fukuoka, Japan
Renin-angiotensin system (RAS) plays a major role in the regulation of body fluid and sodium homeostasis.
Angiotensin II (AngII) is the most powerful biologically active product of the RAS. We recently revealed that AngII
suppresses amiloride-sensitive salt taste responses and enhances sweet taste responses via the angiotensin II
type 1 receptor (AT1) expressed in taste cells. However, the underlying molecular mechanisms on such taste
modulations remain unknown. Here we examined the expression of RAS components producing AngII, renin
(Ren), angiotensinogen (Agt) and angiotensin converting enzyme (Ace), in mouse taste tissues. We found that
all of RAS components were present in taste buds, and co-expressed with αENaC (a salt taste receptor) or T1r3
(a sweet taste receptor component). The expression level of Ren in taste cells increased significantly after water
deprivation. These results suggest that taste organ exhibits their own RAS activity, and the specific modulation
of taste sensitivity via AngII signaling is regulated by the taste specific RAS (temporal feed-forward regulation) in
addition to the classical circulating RAS (continuous negative feedback regulation). Such reciprocal regulation of
peripheral taste sensitivity by the two RASs may play important roles in sodium/calorie homeostasis.
2016.05.27 11.25.45 Page 47(1)
P1-116 Immunohistochemical localization of serotonin in taste buds of circumvallate papillae in mice
Norihiro Fujimoto, Yukako Hayashi, Hidenori Shimizu, Ryo Kitada
Graduate School of Agriculture, Kyoto University, Kyoto, Japan
The taste receptive cells are divided into two types; Type II taste cells are activated by sweet, umami and bitter
stimuli and Type III cells are activated by sour and salty stimuli. Although Type III cells form classical synapses
onto the gustatory nerve fibers, Type II cells do not have that. The mechanism of signal transduction from Type II
cells has not been elucidated. It is proposed that activated Type II cells by tastants release ATP as a transmitter.
Serotonins which are thought as a transmitter are released from Type III cells and activate gustatory nerve
fibers. Previous our research elucidated that Type III cells are further divided into two types; taste receptive cells
and ATP responsive cells. Thus in order to elucidate mechanism of signal transduction from taste cells to nerve
fibers, we investigated whether both types of Type III cells have serotonins. Immunohistochemical analyses are
carried out with the mouse circumvallate papillae. Fourteen percent of total cells expressing Gα-gustducin,
typical Type II cell marker, expressed serotonin. Seventy four point seven percent of total cells expressing
NCAM, typical Type III cell marker, expressed serotonin. Sixteen point eight percent of total cells expressing
CAR-4, typical taste receptive Type III cells marker, expressed serotonin. Hence the taste receptive Type III cells
may not release serotonin as a transmitter.
P1-117 Construction of cells expressing taste receptor genes
Ryosuke Tanii, Yui Ohshima, Yukiko Nishioka, Mitsuru Ebihara
Department of Food Science, Ishikawa Prefectural University, Ishikawa, Japan
Taste receptors, the sensory molecules for tastes, have interesting vital roles for most of mammals. To
investigate the function of taste receptors, cells which express taste receptors must be useful and essential. It’s,
however, reported that T1R2 and T1R3 heterodimer was not functional, if they have no chance to interact with
each other in golgi. Interaction could be essential to membrane transfer and proper conformation of these sweet
receptors. Interestingly this was not found in sweet receptors derived from mouse. It has been strongly
suggested that mechanism of taste receptor activation is different between species. We have cloned and
reported gene structure of umami receptors in many wild animals, such as lion, giraffe and sea otter, but
functional assay was not carried out. It was because there was no establish cell line to do functional assay. In
this study, we tried to establish cell line for functional assay of taste receptors. To establish stable cell line
expressing taste receptors, we used target integration system based on pseudo attB sequences on
chromosomes. Whenever the transfer genes which is taste receptor genes are integrated on chromosome, it
would be inserted in the same locus of the same chromosome by using this target integration system. We will
discuss availability of this system in detail.
P1-118 In vivo anti-obese effect of grains of paradise extract
Hiroyuki Hattori1, Tohru Mitsunaga1, Siaw Onwona Agyeman2
1
Faculty of Applied Biological Sciences, The University of Gifu, Gifu, Japan, 2Institute of Agriculture, The Tokyo University of
Agriculture and Technology, Tokyo, Japan
Aframomum melegueta is widely distributed in West Africa. The seeds of the plant, called Grains of Paradise
(GP), have been traditionally used as spice for flavouring foods or remedy for diarrhea, intestinal health, and
bacterial infection in Africa. GP extract has been reported to exhibit antioxidant, hepatoprotective, and antiulcer
effects. However the pharmacological investigation of the plant is not well studied so far. In the present study, we
examined anti-obese effect of GP extract and tried to isolate and identify the active ingredients in GP.
Dried GP powder was extracted with methanol. The methanol extract was successively extracted with hexane,
ether, ethyl acetate and methanol. Hexane and ether soluble parts were separated by silica gel column
chromatography. Twelve vanilloids were isolated using PHPLC with reversed phase column and identified using
1
H-, 13C-NMR and MALDI-TOF-MS. Among them, 1-(4’-hydroxy-3’-methoxyphenyl)-undecan-3-ol and 1-(4’hydroxy-3’-methoxyphenyl)-3-octen-5-one were identified as novel compounds.
After preliminary breeding male mice aged 5-week-old fed with normal diet (ND), they were fed with high fatty
diet (HFD) containing 2% GP. The mice aged 12 weeks were dissected to obtain the serums, livers, and fats.
The total cholesterol (TC), triglyceride (TG), and HDL cholesterol (HDL-C) in the serums and livers were
determined.
GP suppressed the weight gain of HFD mice to the ND level, and significantly decreased the weight of livers
2016.05.27 11.25.45 Page 48(1)
and fats surrounding testes. Furthermore GP seed also reduced the concentration of TC and TG in the serums
and livers. GP intake resulted in potently prevention of fat accumulation and improvement of the lipid metabolism
on liver in HFD mice.
P1-119 Lineage tracing of Sox2-expressing cells in the oral epithelium
Makoto Ohmoto, Ichiro Matsumoto
Monell Chemical Senses Center, Philadelphia, USA
Oral epithelial cells including taste cells and non-gustatory epithelial cells are maintained through continuous
turnover. They arise from local epithelial stem cells. Recent lineage tracing and ex vivo culture analyses
contributed to partially identify the taste stem/progenitor cells located in the basal region of oral epithelia. Sox2 is
also expressed in the basal cells of oral epithelia, and Sox2+ cells are stem/progenitor cells of non-gustatory
tongue epithelial cells. However, it remains elusive if they also give rise to taste cells. To test whether Sox2+ cells
can supply taste cells, we performed lineage tracing analyses. About 21 months after the tamoxifen injection in
mice with Sox2-CreERT2 Cre-driver and Rosa26-tdTomato reporter alleles, almost all oral epithelial cells
including taste cells in the posterior and anterior tongue and soft palate were labeled with tdTomato. Considering
the half-lives of taste cells (about 20 days or shorter), these results suggest that Sox2+ cells are taste stem cells
that can continuously give rise to taste cells for a long term. In mice with Rosa26-Confetti as a reporter allele,
about 6 months after the tamoxifen injection, a cluster of cells including taste cells and neighboring epithelial
cells expressed the same fluorescent proteins and most of single taste buds were labeled with different colors,
while epithelial cells were randomly labeled with different colors at three days after the tamoxifen injection.
These results indicate that Sox2+ stem cell is multipotent to give rise to all types of taste and non-gustatory
epithelial cells, and that taste cells in a single taste bud are derived from multiple Sox2+ stem cells.
P1-120 Expression and functionality of bitter taste receptors on ovarian and prostate cancer cells
Louis Martin, Brooklyn Kennedy, Elaine Nguyen, Denis J. Dupre
Department of Pharmacology, Dalhousie University, Halifax, Canada
Mammals evolved to sense their environment, and ingested food is sensed by specialized sensory cells located
in taste buds in the oral cavity, capable of detecting one of the basic taste qualities. Bitter taste is detected in
humans by members of the bitter taste receptor (TAS2R) subfamily. Since plant poisonous metabolites are often
bitter, sensing bitterness provides protection against poison consumption. Many of the drugs used in therapy to
kill cancer cells are bitter compounds. While these bitter pills suffer from low patient compliance due to aversion
to their taste, the fact that drugs have bitter taste receptors as off-targets could also be beneficial: taste receptors
are also expressed in extra-oral tissues. For example, extra-orally expressed TAS2Rs were shown to have
important roles in airway bronchodilation, where their stimulation with various bitter compounds causes potent
muscle relaxation, suggesting that inhaled bitter compounds could be used for treatment of airway diseases.
Recently, it was shown that mammary epithelial cells also express Tas2Rs, and that the expression of some Tas
2Rs is downregulated in breast cancer cells. We analyzed a series of online cancer databases for genetic
alterations and noticed Tas2R gene amplification was very high in breast and ovarian cancer, while few
mutations or deletions were present, when compared to most other cancers. Drug resistance in cancer cells is
often linked to amplification of genes that change absorption of chemotherapeutic agent by cells. Interestingly,
noscapine, a Tas2R14 ligand, sensitizes chemoresistant ovarian cancer cells to paclitaxel. It is possible that Tas
2Rs could have an important role in the development of new therapeutic strategies. We characterized the
expression of Tas2R subtypes expressed in ovarian and prostate cancer cell lines and demonstrated their
functionality.
P1-121 The expression of taste receptor hTAS2Rs during pregnancy, after delivery and in suckling
infants
Tetsuya Takao1,2, Chieko Kogomori1, Mika Ishihara2, Saki Ueno2, Iwao Maekawa3, Mieko Aoki4, Kyoichi Takao5, Seigo Shiga1
1
Graduate School of Human Life Science, Showa Women’s University, Tokyo, Japan, 2Department of Health Science, Showa
Women’s University, Tokyo, Japan, 3Maekawa Obstetrics and Gynecology Clinic, Shizuoka, Japan, 4Food Nutrition, Sanyo Gakuen
College, Okayama, Japan, 5Medical School, Nihon University, Tokyo, Japan
The incidence of taste changes during pregnancy and suckling infants feel the taste has long been known. But,
the expression of the hTAS2Rs in the pregnancy and suckling infant were not clarified. Pregnant females (n=9)
2016.05.27 11.25.45 Page 49(1)
were tested in the pregnancy periods of 8-11 weeks (EP), 12-15 weeks(MP), 27-30 weeks (LP), and 37-40
weeks (AD), as well as in 4 weeks after their delivery (4D, n=8), and tested in suckling infants of 1-2 months after
birth (n=8). Sample tissues were scraped from their foliate papilla and the expression characteristics of hTAS2R
isoforms (1, 3, 4, 5, 7, 8, 9, 10, 13, 14, 16, 38, 39, 40, 42, 43, 44, 45, 47, 48, 49) were defined by the RT-PCR
method. The average age of the pregnant subjects was 29.8±5.2 years old, the BMI before pregnancy was 19.7
±1.1 kg/m2. The average period of gestation was 39.6±1.2 weeks. The average weight of the suckling infants
at birth was 2.99±3.95kg. The average weight of 1 to 2 months after birth was 4.19±0.66kg (n=7). While the
average number of hTAS2R isoforms expressed in the pregnant subjects were as follows: 3.9±3.9 in EP; 7.1±
3.4 in MP; 5.3±3.8 in LP; 4.7±4.2 in AD; and 7.3±5.4 in 4D. In 4D, the expression of hTAS2R8, 9, 13 is
decreased in the 1st trimester, then recovered in mid pregnancy, and decreased again in the late pregnancy and
the delivery period. The average number of hTAS2R isoforms expressed in the suckling infants was 2.5±1.2.
Among them hTAS2R14 expressed in 75% and hTAS2R47 expressed in 50% of the suckling infants. The
expression of hTAS2Rs isoforms changed with the period of the pregnancy. There was very little expression
variety of hTAS2Rs isoforms in suckling infants.
P1-122 Clinical analysis of patients with taste disorder in Mie University Taste Clinic−The conflict with
self-rating test and taste tests−
Kohei Nishida, Masayoshi Kobayashi, Hiroyuki Morishita, Noriko Hamaguchi, Masako Kitano, Kazuhiko Takeuchi
Department of Otorhinolaryngology-Head and Neck Surgery, The University of Mie, Mie, Japan
BACKGROUND We reported clinical analysis of patients with taste disorder in our taste clinic in this meeting 12
years ago and found that results of the taste tests,the filter paper disk (FPD) taste tests and electrogustometry
(EGM), did not correspond to patient’s self-rating taste function after treatment of the disorder. We analyzed
patients who presented to the clinic in recent 5 years to examine if these differences are still in problem.
METHODS A total of 224 patients with taste dysfunction (2010 Apr-2015 Mar) were recruited from our taste
clinic (85 men and 139 women, age: 18-94 years, median age: 63years), we evaluated their specific taste
disorder symptoms by interview and otolaryngological examination. They were required to report grades of their
levels of taste function (5 step evaluation: from 1=normal (cured), to 5=ageusia), EGM and FPD taste test. The
treatments were internal use of poraprezinc, vitaminB12, iron preparation, gargle by povidone iodine, minor
tranquilizers and herbal medicine. Evaluation was made as cured, improved and no change by using three
measurements; self-rating, EGM and FPD. RESULTS The FPD taste test did not correspond in recovery rate
(cured+improved) to patients’ self-rating taste function. Receiver operating characteristics (ROC) curve analysis
for 24 cases of normal level of taste function in the FPD taste test, the threshold was 4.625 (sensitivity 0.792,
specificity 0.404). CONCLUTION The present study revealed that results of the FPD taste test does not
correspond to patients’self-rating taste function. We need further reviewing to determine the acceptance
standard by the FPD. Cut-off level of taste function between normal subjects and patients is recommended 4 or
more in the FPD taste test based on our statistical analyses using standard deviation and ROC curve.
P1-123 Effect of heat treatment on the taste properties and chemical modifications of Maillard reacted
peptides
Yutaka Inoue1,2, Tadayoshi Katsumata2, Hirohito Watanabe3, Fumitaka Hayase1
1
Department of Agricultural Chemistry, Meiji University, Kanagawa, Japan, 2Food Research & Development Laboratory, MC Food
Specialties Inc., Ibaraki, Japan, 3Department of Life Science, Meiji University, Kanagawa, Japan
Maillard reacted peptides (MRPs) obtained by reacting a peptide fraction (1000-5000 Da) purified from soy
protein hydrolysate with monosaccharides are thought to affect the chorda tympani response to NaCl and MSG
by acting on TRPV1t and T1R1+T1R3, respectively (Katsumata et al. , 2008). This current study was conducted
to investigate the influence of heat-treatment time on taste properties and chemical modifications of MRPs. We
prepared three kinds of MRPs heated with glucose at 95℃ for either 1hr, 4hr, or 7hr, and conducted a sensory
evaluation to assess their respective contributions to mouthfulness in consommé soup, using the scoring
method described by Ogasawara et al. (2006). The results showed that the addition of each MRP significantly
enhanced mouthfulness in consommé soup. Information about the extent of the early Maillard reaction between
the glucose and the peptide amino group of each MRP was obtained by quantifying the amount of N-(2furoylmethyl) amino acids (FMAAs) in the respective MRP acid hydrolysates by using RP-HPLC with UV/MSdetection. The detection of N-(2-furoylmethyl)-lysine (FM-Lys) in each MRP indicated glucose-induced glycation
at the amino groups of peptide-bound lysine. In a further study, the soy peptides were acetylated with acetic
2016.05.27 11.25.45 Page 50(1)
anhydride, and then the N-acetylated MRP was prepared by reacting the acetylated soy peptides with glucose at
95℃ for 1hr. The amount of FM-Lys in the N-acetylated MRP was lower than that in any of the MRPs. The
results of sensory evaluation revealed that the addition of the N-acetylated MRP did not significantly enhance
mouthfulness. These results suggest that glycation at the amino group of peptide-bound lysine in MRPs is
important for mouthfulness enhancing activity.
P1-124 Nerve-independent induction of taste buds in organ culture of fetal tongues
Kotaro Honda, Yasuhiro Tomooka
Department of Biological Science and Technology, Graduate School of Industrial Science and Technology, Tokyo University of
Science, Tokyo, Japan
A taste bud consists of eosinophobic epithelial cells expressing CK8 surrounded by lingual eosinophilic epithelial
cells expressing CK14 in mice. During early development and regeneration, taste buds depend on gustatory
innervation though canonical Wnt signaling. However, it is unknown how Wnt signaling activation plays a role in
taste bud morphogenesis.The present study examined whether taste buds were generated nerve-independently
in vitro . It has been believed that nerve-independent activation of canonical Wnt pathway is unable to induce
taste buds in vitro. Interestingly, a very effective GSK3β inhibitor (activation of canonical Wnt signaling pathway)
induced many eosinophobic cells looking like taste buds when fetal mouse tongues were organ-cultured in a
method developed for teeth bioengineering. Filiform papillae decreased in number and almost all lingual
epithelial cells expressed Sox2, a taste epithelial marker. Eosinophobic cells covered the basal lamina of fetal
mouse tongues at 28.8-53.6% and expressed CK8, a taste cell marker. Some of CK8-expressing cells had the
shape of spindle, suggesting that taste buds were generated in organ culture without innervation.The GSK3β
inhibitor prevents β-catenin destruction and increases the amount of subcellular β-catenin.
Immunohistochemistry of organ-cultured tongues revealed that a few eosinophobic cells expressing CK14
showed β-catenin nuclear translocation, while most of eosinophobic cells expressed β-catenin in membrane.
This result suggested that the cytoskeletal transition during the morphogenesis of taste buds was correlated with
β-catenin nuclear translocation. Only activation of canonical Wnt siglaning pathway may be sufficient for taste
bud morphogenesis in vitro .
P1-125 Arousal as the link between carbonation and taste quality modulation
Johan N. Lundstrom1,2,3, Bruce Bryant2, Paul M. Wise2
1
Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden, 2Monell Chemical Senses Center, Philadelphia, PA USA,
Psychology, University of Pennsylvania, PA, USA
3
Oral irritation signals the presence of potentially dangerous materials. Why humans comprise one of few species
that like and even actively seek out oral irritation in food and liquids is still a much debated topic with little existing
data. There is, however, a clear link between arousal and liking, explained as misattribution of arousal, and
previous demonstration that inhaling CO2 increases physical and psychological arousal. In line with this, we
hypothesized that carbonation (CO2) might increase physical arousal, which in turn results in a greater liking. In
Experiment 1, we assessed whether ingesting carbonated water increased physical arousal (skin conductance;
SC) more than ingesting still water. Carbonation significantly raised SC responses compared to still water. In
Experiment 2, we assessed whether self-rated anxiety is affected by ingesting carbonated water and whether
Experiment 1 could be replicated. Both self-rated anxiety and SC were significantly higher after ingesting
carbonated water compared to still water. In Experiment 3, we assessed how carbonation-induced anxiety
affects taste quality. Participants ingested a weak sweet solution, either carbonated or still. For each solution,
subjects ingested 33cc, divided in 9 sips. Carbonation decreased perceived sweetness and increased perceived
bitterness, but did not affect liking. Bitterness ratings were positively correlated with ingestion time (sips) and
arousal level. Sweetness was not correlated with these variables. As in previous experiments, carbonated
beverages induced significantly higher levels of both self-rated arousal and SC. These three experiments
demonstrate that drinking carbonated beverages increases both physical arousal and subjective anxiety.
Moreover, CO2 intake is related to change in taste quality, an effect that is modulated by physical arousal for
bitter but not sweet taste sensations.
2016.05.27 11.25.45 Page 51(1)
P1-126 Advanced applications of taste sensor to evaluate for beverages and detect the bitterness
suppression effect for pharmaceutical samples
Zhiqin Huang1, Xiao Wu1, Hideya Onitake1, Yusuke Tahara1, Rui Yatabe2, Kiyoshi Toko1,2
1
Graduate School of Information Science and Electrical Engineering, Kyushu University, Fukuoka, Japan,
Research and Development Center for Taste and Odor Sensing, Kyushu University, Fukuoka, Japan
2
Sensory tests are widely used in food and pharmaceutical industries to evaluate the taste of foods, beverages
and pharmaceuticals. However, the results of sensory tests are low of reproducibility and objectivity. There are
also some risks such as toxic substances and side effects when evaluating pharmaceuticals. The prototype of a
taste sensor for objective evaluation such as the discrimination and quantification of tastes has been developed
since around 1990. It was then improved by using lipid/polymer membranes. The lipid/polymer membrane is
composed of lipids and plasticizers mixed with polyvinyl chloride. By adjusting the components in the membrane,
the taste sensor with global selectivity is developed. Global selectivity is defined as the decomposition of
characteristics of chemical substances into taste qualities and their quantification, rather than the discrimination
of individual substances. In this study, we used the taste sensor to evaluate the bitterness of beers. We also
detected the bitterness suppression effect caused by bitterness masking substance. The taste of beers of
different brands from different countries were evaluated using the taste sensor. The output was showed on a
taste map with sourness intensity as X-axis and bitterness intensity as Y-axis. It provides the visible information
of the taste as well as the discrimination of beers. We also detected the bitterness suppression effect caused by
a commercial bitterness masking substance (BMI-40, Kao Corporation). 0.1mM quinine hydrochloride was used
as the standard bitterness solution. The CPA (the change in membrane potential caused by adsorption) value
which represents the bitterness intensity decreases greatly with BMI-40 concentration increasing. The result
indicates that the bitterness suppression effect can be detected by the taste sensor.
P1-127 Analysis of cell lines established from lingual epithelia and taste buds of p53-/- mice
Emiri Nakahima, Takahiro Kakefuda, Kotaro Honda, Yasuhiro Tomooka
Department of Biological Science and Technology, Tokyo University of Science, Tokyo, Japan
A tongue is covered with a stratified epithelium equipped with taste buds. In our laboratory, immortalized
clonal cell lines were established from lingual epithelia and taste buds; 20A and 24-5 cells were from lingual
epithelia and TBD-a5 and TBD-c1 were from taste buds.
Both 20A and 24-5 cell lines express stem cell markers of non-gustatory epithelia, Bmi1 and Tcf3 , suggesting
that the cell lines are multipotent. In 3D culture, both 20A and 24-5 cell lines formed stratified epithelia without
basement membrane. In 3D co-culture with TMD (tongue mesenchyme-derived) cells, both 20A and 24-5 cell
lines formed stratified epithelia with clear basement membrane.
Both TBD-a5 and TBD-c1 lines express GLAST, gustducin, T1R3, and NCAM. In collagen gel culture, TBD-a5
cells form a luminal structure with cilia. TBD-a5 cells developed microvilli-like structures when they were cultured
with serum-free medium at air-iquid interface.
Moreover, our study attempted to induce lingual epithelial structure with tastes buds from lingual epithelial cell
lines in vitro .
P1-128 Alteration in gustatory sensation of patients undergoing cancer treatment
Misako Kawai1, Setsuko Kuwahara2,3, Masahiro Sunaga4, Hiroki Matsubara5, Noriko Aoki3
1
Institute for Innovation, Ajinomoto Co., Inc., Japan, 2College of Nursing and Nutrition, Shukutoku University, Japan, 3Nutrition
Management Office, National Cancer Center Hospital, Japan, 4Nutrition Management Office, National Hospital Organization
Yokohama Medical Center, Japan,
5
Nutrition Management Office, Funabashi Municipal Medical Center, Japan
BACKGROUND AND AIMS: Many patients receiving chemo- and/or radiotherapies for cancer experience
difficulties in oral intake of foods because of adverse effects of the therapies such as nausea and vomiting, oral
mucositis, and alterations in food tastes. However, because taste alteration itself is not lethal, it has been
ignored in cancer therapy and there are many unclear points. We conducted a survey on taste alterations
caused by cancer therapy by asking clinical dietitians who were coping with menu planning for cancer patients.
METHODS: We sent a questionnaire about dietitians’ experience of taste alterations of cancer patients to
nutritional management sections of 397 Designated Cancer Care Hospitals in Japan. We also asked about smell
alterations of cancer patients because people sometimes confuse taste with flavor, a combined sensation of
taste and smell. Methods for adjustment of diet in response to taste and smell alterations (TSAs) of cancer
2016.05.27 11.25.45 Page 52(1)
inpatients were further investigated. RESULTS: Approximately two-thirds of the hospitals responded, as follows:
1) Common types of taste dysfunctions reported by dietitians were hypo/ageusia (96%), hypergeusia (86%), and
parageusia (69%). Saltiness tended to decrease and sweetness showed an increase. 2) Types of smell
dysfunctions included hyperosmia (94%) and smell aversions (94%), but there was a lower rate of hypo/anosmia
(29%). 3) There was no regular coping method that was common for taste alterations. For smell alterations,
serving chilled foods, and changes of the staple diet were done frequently to avoid aversive smells.
CONCLUSION: Details about TSAs due to adverse effects of cancer therapies are still unclear, and are
challenging issues for dietitians even in leading cancer hospitals in Japan. Establishment of appropriate
evaluation methods for TSA with coping methods is important for improvement of both, nutritional status and
quality of life of cancer patients.
P1-129 Taste detection and recognition thresholds in Japanese patients with Alzheimer-type dementia
Sayuri Yamamoto1, Takao Ogawa1, Naoya Irikawa1, Akihiko Shiino2, Ikuo Tooyama3, Takeshi Shimizu1
1
Department of Otorhinolaryngology, Shiga University of Medical Science, Shiga, Japan, 2Department of Biomedical MR Science
Center, Shiga University of Medical Science, Shiga, Japan, 3Department of Molecular Neuroscience Reserch Center, Shiga
University of Medical Science, Shiga, Japan
Background: Alzheimer-type dementia (AD) is characterized pathlogically by massive neuronal loss in brain and
the taste cortex is suggested to be affected in AD patients. However, there are only a few studies reported on the
gustatory function of AD patients, and the conclusions of these researches are inconsistent. Material and
Methods: This study included 22 prospective patients with mild to moderately severe Alzheimer-type dementia
(AD) (mean age: 84.0 years-old), and 49 non-demented elderly volunteers (mean age: 71.0 years-old). The
subjects for the non-demented groups were divided into two groups: 1) younger-old aged adults groups (60-73
years-old, mean age: 68.5, n=28), 2) older-old aged adults groups, (74-93 years old, mean age: 83.0, n=21). The
gustatory function was investigated using the filter paper disc method and the electrogustometry. Results: The
gustatory function measured by the filter paper disc method was significantly impaired in patients with AD, as
compared with age-matched non-demented subjects. There were no differences in the gustatory functions
measured by the filter paper disc method between the younger-old aged group and the older-old aged group. On
the other hand, as for the electrogustrometric thresholds, there were no differences between the AD patients
group and the age-matched non-demented group. Conclusion: Filter paper method demonstrates that the
gustatory function in AD patients was impaired. The decrease of the gustatory function during the elderly stages
may be a predictive factor for AD.
P1-130 Development of an umami taste sensitivity test and its clinical use−patients with umami
specific tatste diorder and their loss of appetite
Shizuko Satoh-Kuriwada1, Noriaki Shoji1, Mikaso Kawai2, Hisayuki Uneyama2, Takashi Sasano1
1
Department of Oral Medicine and Surgery, The University of Tohoku, Miyagi, Japan, 2Institute for Innovation, Ajinomoto Co,
Kanagawa, Japan
There is a close relationship between perception of umami-taste, which has become recognized as the fifth
taste, and the human physical condition. We have developed a clinical test for umami-taste sensitivity using a
filter paper disc with a range of six monosodium glutamate (MSG) concentrations. We recruited 28 patients with
taste disorders (45-48 years) and 184 controls with no taste disorders (102 young; 18-25 years, and 82 older;
65-89 years participants). Filter paper disc (5 mm diameters) was soaked in aqueous MSG solutions (1, 5, 10,
50, 100 and 200 mM), then placed on three oral sites innervated by different taste nerves. The lowest
concentration participants correctly identified was defined as the recognition threshold (RT) for MSG. This test
showed good reproducibility for inter- and intra-observer variability. We found that: (1) No significant difference in
RT was found between healthy young and healthy older individuals at any measurement site. (2) The RT of
patients with taste disorders was higher before treatment than that of the healthy controls. After treatment the RT
in these patients improved to the same level as that of the healthy controls. Thus, this umami taste sensitivity
test could discriminate abnormal umami taste sensations from normal. Next, we applied this method and
measured the taste sensitivity of the five basic tastes for 44 patients with taste disorders (41-85 years). We
found that, 16 percent of the patients showed higher RT only for the umami-taste, whereas the other four basic
tastes were all within normal levels. These patients were all elderly, and complained of appetite loss and poor
physical conditions. After treatment, patients regained their appetite and good health, accompanied with the
recovery of the umami-taste RT. Consequently, newly developed umami-taste sensitivity test is very useful for
2016.05.27 11.25.45 Page 53(1)
the assessment and treatment for the taste disorders.
P1-131 Bortezomib alters sour taste sensitivity in mice
Akihiro Ohishi, Kentaro Nishida, Kazuki Nagasawa
Department of Environmental Biochemistry, Kyoto Pharmaceutical University, Kyoto, Japan
Chemotherapy-induced taste disorder is one of the critical issues which reduces patients’ quality of life in cancer
therapy. However, the effective coping method and underlying mechanism with the symptom remain unknown.
Bortezomib, a proteasome inhibitor, is a key agent in multiple myeloma therapy. Although it induces a taste
disorder, there is little information on its characteristics and underlying mechanism. In this study, to know the
effect of bortezomib on taste sensitivity, we investigated the behavior and taste perception system of
bortezomib-administered mice. Bortezomib (1 mg/kg) was administered to mice subcutaneously twice a week for
4 weeks. Brief-access test was performed on the next day of bortezomib administration and used five basic taste
solutions (sucrose, citric acid, quinine hydrochloride, sodium chloride and mono sodium glutamate). Taste bud
morphology and number of taste cells were investigated by HE staining and immunohistochemistry (IHC),
respectively. Expression levels of mRNA and protein were measured by real-time PCR and IHC or western blot,
respectively. Among five taste solutions, lick ratio (lick numbers for taste solution per 10 sec/water per 10 sec)
for sour taste solution decreased from day 16 and later. The change of lick ratio between control and
bortezomib-administered mice was not detected 3 or more days after the cessation of bortezomib administration.
In bortezomib-administered mice, there was no change in the morphology of taste buds, number of type III taste
cells, or expression of sour taste receptors PKD1L3, PKD2L1 and TRPV1 on day 29 as compared with the case
of control mice. These results suggest that although there is no apparent change in the sour taste perception
system in bortezomib-administered mice, sour taste sensitivity is increased and this alteration is reversed on
cessation of its administration.
P1-132 Perceived 6-n-propylthiouracil (PROP) bitterness is associated with the dietary sodium intake
in female Japanese college students
Yuichi Suzuki1, Hiroko Inoue2, Toshiko Kuwano3, Kimiko Yamakawa-Kobayashi3
1
Department of Health and Nutrition, Faculty of Human Sciences, Sendai-Shirayuri Women’s College, Japan, 2Department of
Nutrition and Health Sciences, Faculty of Food and Nutritional Sciences, Toyo University, Japan, 3School of Food and Nutritional
Sciences, The University of Shizuoka, Japan
Objective Despite the negative health consequences of a high sodium consumption, humans consume well
above the recommended levels. This study examines whether or not the dietary intake of sodium was affected
by individual variation of the perceived bitterness of 6-n-propylthiouracil (PROP), and examines the relationship
between the perceived bitterness of PROP and the preferred NaCl concentration of the broth.
Methods This study utilized a cross-sectional design. Female students (20-22 years old) were recruited from the
university community. Genotypes of A49P and I296V polymorphism of the TAS2R38 bitter taste receptor were
determined for each subject. Samples containing NaCl, PROP or broth in 5-mL portions were evaluated by
sensory testing. The participants completed a food record for each diet. A one-way repeated-measure analysis
of variance and Tukey HSD comparison were used to assess significant differences among three TAS2R38
genotypes, i.e., PV/PV, PV/AI and AI/AI. Pearson’s product moment correlation was used to determine any
association between the taste intensity rating and dietary intake, and the association between the taste intensity
rating and liking for NaCl in the broth.
Results The individuals perceiving PROP to be more bitter had consumed a greater amount of dietary sodium.
In contrast, there was no significant positive correlation between an individual’s perceived saltiness and the
dietary sodium intake. Those who perceived PROP to be more bitter preferred a broth containing a higher
concentration of NaCl. All of these correlations were apparent even after those subjects with TAS2R38 AI/AI
homozytotes (PROP non-taster) had been excluded.
Conclusion The results of this study suggest that a factor affecting the bitter rating to PROP other than the AI/AI
homozygotes of TAS2R38 contributes to the variation in sodium intake and the preference for salty food.
2016.05.27 11.25.45 Page 54(1)
P1-133 Sucrose release from agar gels and sensory perceived sweetness
Kaoru Kohyama1, Fumiyo Hayakawa1, Katsuyoshi Nishinari2
1
National Agriculture and Food Research Organization, Tsukuba, Japan, 2School of Food and Pharmaceutical Engineering, Hubei
University of Technology, Wuhan, China
Sweetness resulting from sucrose released from agar gels of varying concentrations of agar (0.5, 1.0, and 1.5%
w/w) and sucrose (10%-50% w/w) was evaluated. The intensity of sweetness as perceived by subjects was
investigated during consumption of the gels. A time-intensity method was applied: the maximum intensity and
area under the time-intensity curve were higher in gels with lower concentrations of agar as expected from
previous studies. The intensity of sweetness as estimated by the area under the time-intensity curve increased
with sucrose concentration, but the maximum intensity tended to reach a plateau above a certain sucrose
concentration. The manifestation of the maximum sweetness was delayed with increasing concentrations of
sucrose and agar. The total duration of the perception of sweetness was longer in gels with higher sucrose
concentrations but did not depend on the concentration of agar in the range examined. Instrumental
characterization of these gels was also conducted. Young’s modulus, fracture stress, fracture strain, and energy
obtained from a conventional uniaxial compression test of sample gels increased with sucrose concentration and
the modulus, stress, and energy were higher in gels with higher agar concentration. Although mechanical
characteristics such as fracture stress, strain, and energy, Young’s modulus increased with increasing sucrose
concentration, the sweetness intensity tended to a plateau; therefore, physiological and psychological factors
experienced during oral processing should be taken into account to understand the perceived sweetness
intensity.
P1-134 Instruction on the concept of umami taste improved umami sensitivity score
Lili Cao1, Minatsu Kobayashi2, Akiko Watanabe1, Misako Kawai1
1
Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, Japan, 2Department of Food Science, Faculty of Home Economics, Otsuma
Women’s University, Tokyo, Japan
Background: We developed a method for evaluating the sensitivity of sweet, salty, and umami tastes by a
whole-mouth tasting method. Subjects taste five-level concentrations of test solutions for each taste, identify the
taste perceived, and rate the confidence of the perception. Although umami is one of the five basic tastes, some
individuals find it difficult to determine umami taste because of a lack of concept of umami taste. We studied the
effect of instruction on the concept of umami taste on umami sensitivity score in our taste tests. Methods: The
subjects included 291 female university students residing in Tokyo or in the suburbs of Tokyo. The subjects were
divided into three groups: group A was given no instruction, group B was given an oral explanation of umami
taste, and group C tasted 3 mL of 10.9 mM monosodium L-glutamate solution after an oral explanation of umami
taste. A taste sensitivity score (0-3) was given to the answer for each test solution according to the taste quality
and confidence of perception. The sum of scores for five test solutions (0-15) was regarded as the taste
sensitivity. ANOVA was used to compare the differences in taste sensitivity among the three groups. Bonferroni
test was used to compare the difference in taste sensitivity for each group. Results: The umami sensitivity score
of group C(6.83±4.05) was significantly higher than that of groups A (5.42±4.10) and B (5.38±4.09). Further,
for umami test solution at the lowest concentration, the umami sensitivity score of group C was much higher than
that of groups A and B. Conclusion: Our results suggest that developing a concept for umami taste through oral
instruction and tasting improved the umami sensitivity score among young Japanese subjects.
2016.05.27 15.17.09 Page 1(1)
ＫｙｏｒｉｎＷＰＳ／３５０３３−５２２２／ｉｓｏｔｐ１７−１３−３＿／ｋｙ７８６３１７５２２２１０００２９９９
Poster Session
P2-001 Child odors and daily caregiving: A questionnaire survey of affective and instrumental roles
Masako Okamoto1,2, Mika Shirasu1,2, Rei Fujita1,2,3, Yukei Hirasawa1,2, Kazushige Touhara1,2
1
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo,
Japan, 2ERATO Touhara Chemosensory Signal Project, JST, The University of Tokyo, Tokyo, Japan, 3Technical Research Institute,
Research & Development Center, T. Hasegawa Co., Ltd, Japan
Background: Odors emitted from the young serve as important cues for caregiving in nonhuman animals.
However, similar role for odors in human child-raring has been understudied. To explore whether parents aware
of, and reactive to odors from their offspring in daily caregiving, we conducted a series of questionnaire surveys
targeting parents with children under age 6. Methods: First, we collected episodes on experiencing odors/sniffing
various parts of child body (n=507). Prevalence of experiencing events described in those episodes was
examined in a separate survey (n=384). Based on those results, the Child Odor in Parenting scale (COPs) was
developed, and used in the main survey (n=888). Results: Concurrent validity of the COPs was supported by
positive correlations with olfactory-related and with child care-related scales, and lack of correlation with a
measure of social desirability bias. Responses to the COPs demonstrated that parents, especially mothers with
infants, are aware of odors from their offspring, and actively seek them in daily childcare. Factor mixture analysis
indicated that child odors have two roles: Affective, inducing a loving feeling and affectionate sniffing, and
Instrumental, conveying specific needs of cleaning care. The head was the most frequent source of Affective
experiences, and the bottom of Instrumental ones. Each was experienced by more than 90% of mothers with a
child below age 1. Affective experiences declined as the child grew older, while for Instrumental, the age-effect
varied across body parts. Conclusion: These findings suggest that child odors likely play both affective and
utilitarian roles in human parenting, and that their nature and significance change during the development of
child.
P2-002 Action of vasopressin on chemical signal processing at the reciprocal synapses between mitral
cells and granule cells in the mouse accessory olfactory bulb through the V1 receptors
Mutsuo Taniguchi, Toshiharu Namba, Hideto Kaba
Department of Physiology, Kochi Medical School, Kochi University, Japan
Central vasopressin facilitates social recognition and modulates numerous complex social behaviors in
mammals. Recent analysis of transgenic rats engineered with an enhanced green fluorescent protein reporter
for vasopressin synthesis identified new population of vasopressin neurons in the accessory olfactory bulb
(AOB). The AOB has been demonstrated to be a critical site for mating-induced mate recognition (olfactory
memory) in female mice. The effect of vasopressin, however, on the synaptic transmission between dendrites in
the AOB of female mice is largely unknown.
To address this issue, evoked synaptic currents were measured from mitral cells in slice preparations
prepared from 23- to 36-day-old Balb/c mice. To evoke dendrodendritic inhibition, a depolarizing voltage step
from -70 mV was applied to a mitral cell under the whole-cell configuration. We have demonstrated that
vasopressin significantly reduced the IPSCs in Mg2+-free solution.
In the present study, to determine the contribution of different vosopressin receptor subtypes (V1a and V1b) to
reduce the IPSCs, effects of antagonists for V1Rs on the IPSCs were tested. The suppressive effect of
vassopressin on the IPSCs was diminished by an antagonist for V1a receptors, Manning compound, while an
antagonist for V1b receptors, SSR149415 unaffected the effects of it. The present results suggest that
vasopressin modulates reciprocal transmission between mitral cells and granule cells through vasopressin V1a
receptors. We are further studying on the involvement of vasopressin receptors in the GABAergic transmission
from granule cells to mitral cells by analyzing the miniature IPSCs in the presence of antagonists for
glutamatergic transmission, CNQX and AP5.
P2-003 Maintenance of long-term potentiation in the accessory olfactory bulb: Cellular mechanisms of
olfactory recognition memory
Yoshihiro Murata, Hideto Kaba
Department of Physiology, Kochi Medical School, Kochi, Japan
Olfactory recognition memory strongly affects the development of the individual’s social and emotional behavior.
2016.05.27 15.17.09 Page 2(1)
One of the most characterized contexts is that some olfactory cues in urine of an unfamiliar male mouse block
pregnancy of the female exposed within 24 hours after mating; those of the mating male do not. The inability of
the mating male to disrupt the pregnancy is caused by the memory of his pheromones formed by the female.
Previous reports have shown that the olfactory recognition memory depends on new protein synthesis during the
sensitive period and can last for 30-50 days. The olfactory recognition memory is based on the neural changes
in the accessory olfactory bulb (AOB), the first relay in the vomeronasal system; the thickening of the excitatory
synapse is observed in the female following mating. Microcircuits in the AOB include the prominent reciprocal
dendrodendritic synapse between mitral cells, a single class of projection neurons, and granule cell
interneurons. At the AOB synapse, long-term potentiation (LTP) is expected to underlie the olfactory recognition
memory. To elucidate the cellular mechanisms, we focused on the possible mechanisms to maintain LTP at the
AOB synapse. A protein synthesis inhibitor anisomycin blocked the late-phase of tetanus-induced LTP, and had
no effects on the early phase. We also found that an actin polymerization inhibitor cytochalasin D affected the
late-phase of LTP, and that an actin polymerization inducer jasplakinolide facilitated LTP induction. The results
suggest that both new protein synthesis and actin polymerization are required for LTP maintenance at the AOB
synapse.
P2-004 Impairment of DCG-IV-induced long-term potentiation and mate recognition memory in mGluR
2-deficient mice
Hideto Kaba, Guang-Zhe Huang
Department of Physiology, Kochi Medical School, Kochi University, Kochi, Japan
Female mice learn to recognize the urinary chemosignals of the male with which they mate. This mate
recognition memory is of critical biological importance, because it prevents any subsequent exposure to the
mating male’s chemosignals from initiating neuroendocrine mechanisms that would terminate pregnancy.
Chemosignals from strange males, for which no memory has been formed, activate the vomeronasal system,
thereby causing pregnancy block prior to blastocyst implantation. The neural changes underlying this mate
recognition memory occur in the accessory olfactory bulb (AOB), the first relay in the vomeronasal system,
depend upon mating-induced release of noradrenaline in the AOB and involve changes at the reciprocal
synapses between mitral and granule cells. We have shown that the activation of mGluR2, a metabotropic
glutamate receptor, in the AOB permits the formation of a chemosignal-specific memory that faithfully reflects
the mate recognition memory formed at mating. To further investigate the role of mGluR2 in memory formation,
we have analyzed mGluR2-deficient mice by behavioral and electrophysiological techniques. Homozygous
mutant mice showed a significantly higher pregnancy block rate when reintroduced to the stud than did the wildtype mice. Bath application of DCG-IV, a group II mGluR agonist, under spaced and long-term theta stimulation
(10 Hz) of mitral cell axons in coronal slices of the AOB induced N-methyl-D-aspartate receptor-independent
long-term potentiation (LTP) at the mitral-to-granule cell glutamatergic synapses in wild-type mice. The
DCG-IV-induced LTP was blocked by the group II mGluR antagonist, LY341495. The DCG-IV-induced LTP was
impaired in homozygous mutant mice. These results demonstrate that mGluR2 in the AOB is important in the
formation of the mate recognition memory.
P2-005 Post-prandial triglycerides influence brain response to food odors in a D2R-dependent manner
Yuko Nakamura1, Xue Sun2, Maria G. Veldhuizen1,3, Serge Luquet4, Dana M. Small1,3,5
1
The John B. Pierce Laboratory, New Haven, CT, USA, 2Department of Psychology, University of Pennsylvania, Philadelphia, PA,
USA, 3Department of Psychiatry, Yale School of Medicine, New Haven, CT, USA, 4University of Paris Diderot, Sorbonne Paris Cite,
Paris, France, 5Department of Psychology, Yale University, New Haven, CT, USA
Heightened food cue reactivity is a well-known risk factor for obesity that is linked to alterations in dopamine
signaling in animal models (Robisnson et al., 2015). In humans the efficacy with which nutrient consumption
diminishes brain response to a flavor cue is correlated with pre to post meal differences (i.e. excursions) in
plasma triglyceride (TG) levels (Sun et al., 2015) possibly because it acts as a signaling molecule in mesolimbic
dopamine neurons (Cansell et al. 2014). Here we used fMRI to evaluate whether meal-induced TG excursions
modulate meal-induced changes in brain response to food versus nonfood aromas and we test whether this
differs in carriers (A1+, n=13) versus non-carriers (A1-, n=16) of the A1 allele of the TaqIA polymorphism, as it is
associated with decreased dopamine 2 receptor density (Jonsson et al., 1999). TG excursion was positively
correlated with response to food versus nonfood odors in the ventromedial prefrontal cortex (vmPFC), globus
pallidus, and hippocampus in A1+ while the opposite correlation was found for A1-individuals. Importantly, the
2016.05.27 15.17.09 Page 3(1)
association in the vmPFC was independent of other energy-related signals including insulin, glucose, free fatty
acids, calorie intake and reported liking. In addition response in the nucleus accumbens at the post-meal scan
was also differentially associated with TG levels in A1+ vs. A1-. These findings suggest that the influence of TG
on brain reactivity to food aromas in humans is dopamine-dependent. Alteration of TG signals in conditions of
chronically elevated TG levels could lead to adaption in dopamine neural substrates encoding reward to promote
uncontrolled eating habits.
P2-006 Partners body odor lowers stress discomfort
Amy R. Gordon1, Pehr Granqvist2, Karolina Vestbrant2, Lillian Dollinger2, Mats J. Olsson1, Johan N. Lundstrom1,3,4
1
Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden, 2Department of Psychology, Stockholm University,
Stockholm, Sweden, 3Monell Chemical Senses Center, Philadelphia, PA, USA, 4Department of Psychology, University of
Pennsylvania, Philadelphia, PA, USA
Somatic symptoms for a range of diseases and overt stress responses are commonly reduced in the presence
of ones partner, an effect thought to be mediated by the initiation of more adaptive coping strategies. In children,
this is not limited to an individuals physical presence; the mere presence of a caretakers body odor reduces
stress hormones and renders children less pain sensitive, calmer, and more social. We hypothesized that the
presence of a partners body odor would make adults better able to cope with a stressful situation and that this
effect would be moderated by individual differences in adult attachment styles between partners. Thirty-nine
women and their partners participated in the study by providing body odor samples collected by sleeping in a
clean t-shirt as well as providing ratings of adult, intra-couple attachment. In a blocked design (each block 5
min), their partners body odor, their own body odor, the odor of a clean t-shirt, and the highly familiar odor of rose
were presented to women while they were stressed by random presentations of weak electric shocks. Measures
of physical arousal (skin conductance) were assessed continuously, and levels of perceived discomfort were
assessed before and after each block. The electric shocks significantly elevated arousal in all blocks. There was
a significant main effect of odor on perceived discomfort (p<.001) where a partners odor, relative to other odors,
significantly lowered discomfort (all ps<.02) and the rose odor significantly elevated discomfort (all ps<.002).
There was no significant effect of attachment style, length of relationship, or ability to identify the odors on any
measure. These data demonstrate that the mere presence of a partners body odor significantly reduces
perceived discomfort within a stressful situation.
P2-007 From perception to metacognition: A comparative study between auditory and olfactory
functions in early blind, late blind, and sighted individuals
Stina Cornell Kaernekull1, Artin Arshamian1,2,3, Mats E. Nilsson1, Maria Larsson1
1
Gosta Ekman Laboratory, Department of Psychology, Stockholm University, Stockholm, Sweden, 2Division of Psychology,
Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden, 3Center for Language Studies and Donders
Institute for Brain, Cognition, and Behavior, Radboud University, Nijmegen, The Netherlands
Research from at least the last two decades have documented that loss of vision can change perceptual and
cognitive abilities in the remaining sensory modalities. Few studies have compared such compensatory effects
in blind individuals between modalities using similar perceptual and cognitive tasks. In this study, threshold,
discrimination, identification, episodic recognition, judgments of learning (JOL), and mental imagery for the
auditory and olfactory senses were assessed in early blind (n=15), late blind (n=15), and sighted (n=30)
participants. The largest compensatory effects in blind participants were demonstrated for discrimination and
episodic recognition of sounds, and especially in the early blind. Although there were similar trends of group
differences for some of the olfactory tasks, there were no significant differences. Moreover, early blind
participants showed better metacognitive abilities in predicting both olfactory and auditory memory. Blind
participants, especially the early blind, also had better imagery abilities of odors and sounds than the sighted. In
conclusion, there were clear modality and task related differences in compensatory effects, and onset age of
blindness was an important factor. The larger compensatory effects observed for the auditory sense might be
related to increased attentional capacity and training, as this sense is crucial for everyday orientation and
functioning in the blind population.
This work was supported by a program grant from The Swedish Foundation for Humanities and Social Sciences
(M14-0375:1) to Maria Larsson entitled “Our Unique Sense of Smell”.
2016.05.27 15.17.09 Page 4(1)
P2-008 Bioluminescence imaging reveals dynamic presentation and reception of pheromonal cues that
mediate attraction in Drosophila melanogaster
Damien Mercier1,2, Yoshiko Tsuchimoto1, Hokto Kazama1
1
Brain Science Institute, RIKEN, Japan, 2Graduate School of Science and Engineering, Saitama University, Japan
The fruit fly Drosophila melanogaster wears odors with complex profiles believed to mediate pheromone
communication. The chemical composition of the fly cuticle is heavily studied and some odorants, such as
cis-vaccenyl acetate (cVA), are shown to influence fly’s behavior. In particular, cVA, is known for playing a major
role in social behaviors including courtship and aggression. However, when and how a fly is presenting or
receiving these chemical cues under ethological settings remains elusive.
To study the dynamics of pheromone presentation and reception by animals, we developed a method to monitor
responses of identified neurons to natural stimuli in freely behaving adult Drosophila. Neural activity was probed
by detecting the bioluminescence emitted from a genetically encoded calcium indicator, while the fly’s behavior
was recorded by a camera.
Using the Gal4-UAS system, we expressed the calcium indicator in Or67d-positive olfactory receptor neurons
that have been shown to respond exclusively to cVA. Strikingly, we found that these pheromone-sensing
neurons remained unresponsive to other male’s cuticle, on which cVA is reported to be present, and rather
responded strongly to markings placed by males in the landscape. These markings were locally and transiently
attractive for the flies. Genetic mutation and targeted expression of tetanus-toxin demonstrated that this
attraction is largely mediated by Or67d-positive neurons.
These results suggest that this actively placed marking may enhance social behaviors by both creating a local
attraction landmark that increases the chance of interaction and by activating the pheromone-sensing neurons
that trigger courtship and aggression.
P2-009 Functional neural processing of emotional chemosignals communicating aggression in
humans
Smiljana Mutic, Rea Rodriguez-Raecke, Jessica Freiherr
Diagnostic and Interventional Neuroradiology, Uniklinik RWTH Aachen, Germany
Human aggression chemosignals emitted from potentially dangerous conspecifics seem to elicit an
anxiety-oriented behavioral adaptation in a odor recipient. The underlying central nervous processing sites might
involve emotional, attentional, social and also olfactory neural networks. The aim of this study was to evaluate
how aggression chemosignals perceived through the nose alter brain activation in healthy subjects. Male and
female normosmic participants (N=23) were exposed to aggression and exercise chemosignals of healthy body
odor donors (N=17). In the fMRI experiment, they completed an emotional higher order processing task
(i.e., emotional stroop task with the emotional word categories anger, anxiety, happiness and neutral).
Functional imaging data show an involvement of social information processing areas rather than olfactory
processing areas. Aggression chemosignals modulate higher order processing by an involvement of
consciousness-related processing sites (e.g., thalamus and basal ganglia). We observe differential modulations
with regards to anxiety, anger and happiness word processing. Sites related to attentional processing
(e.g., cingulate cortex) and emotional salience (e.g., amygdala) were activated. The results provide evidence
that emotional chemosensory stimuli communicating aggression are involved in the modulation of attentional
and emotional neural correlates during higher order processing.
P2-010 Effect of autonomic nerve activity in rat by inhalation of essential oil flavor from Cryptomeria
japonica
Tomoka Hayashi1, Megumi Ogawa1, Eri Matsubara2, Tohru Mitsunaga1
1
Faculty of Applied Biological Science, The University of Gifu, Gifu, Japan, 2Forestry and Forest products Research Institute,
Ibaraki, Japan
Cryptomeria japonica , a representing Japanese conifer, includes a number of monoterpenes. Volatile
components from C. japonica woods were reported to suppress the sympathetic nerve activity due to an
experiment of physiological and psychological response in human. Additionally, inhalation of C. japonica leaf
essential oil suppressed the sympathetic nerve activity and activated parasympathetic nerve activity in rats. In
this study, we fractionated the essential oil in C. japonica leaf and woods in order to identify the effective
ingredients on the autonomic nervous activity.
2016.05.27 15.17.09 Page 5(1)
Essential oils of leaf and wood were fractionated on a silicagel open column chromatography to obtain Fr.1-6.
Analyses of the volatile constituents in Fr.1-6 were determined using the GC-MS system. Then, brown adipose
tissue sympathetic nerve activity (BAT-SNA) of anesthetized rats were measured. The sympathetic nerve was
taken from the dominated brown adipose tissue, and BAT-SNA was observed by converting electrical signals
obtained from the nerve.
Fr.1 contains 79.4% monoterpene hydrocarbons, for example sabinene and α-pinene as the major compounds.
On the other hand, Fr.6 contains 93.7% oxygenated sesquiterpenes, for example elemol as the major
compounds. Additionally, Fr.1 obtained from the leaf essential oil includs a large amount of highly volatile
monoterpene hydrocarbons suppressed the sympathetic nerve activity. In contrast, Fr.6 contains a less volatile
oxygenated sesquiterpenes activated the sympathetic nerve activity. These results indicated that the essential
oil from C. japonica leaf has a both characteristics of activating sympathetic and parasympathetic nerve.
Furthermore, a component which is suppressed the sympathetic nerve activity and another component which is
activated the sympathetic nerve activity are mixed in leaf essential oil.
The wood essential oil was also analysed by GC-MS. It contains 97.0% sesquiterpenes, for example δ-cadinene
as a major compound. Furthermore, the wood essential oil suppressed BAT-SNA.
P2-011 Suppression of physiological stress response under a short-term calculation task by orange
essential oil
Kanetoshi Ito1, Keita Yoshimura2, Masaki Nakachi2, Lekamalage Sugeeswari Lekamge2, Masako Ohira-Hasegawa3,
Shusaku Nomura2
1
Corporate Research & Development, Takasago International Corporation, Hiratsuka, Japan, 2Faculty of Engineering, Nagaoka
University of Technology, Nagaoka, Japan, 3Faculty of Education, Shiga University, Ohtsu, Japan
Aroma therapy is frequently introduced for the purpose of stress release in a context of complementary and
alternative medicine. However, the psychophysiological effect of each aroma is not thoroughly investigated. In
this study, the effect of orange aroma inhalation under a short-term stressor on autonomous nervous system
(ANS) activity was investigated. Sixteen healthy male university students aged from 20 to 24 participated in the
study. In a within-subjects experimental design, subjects were asked to conduct 30 min of simple calculation
task as a short-term stressor with inhaling each one of aroma condition, which is 1% and 20% of orange
essential oil, and scentless air (control) in a counter-balanced order. Aroma administration for the subject was
controlled with a proprietary developed olfactometer. Electrocardiogram (ECG) was recorded for the whole
experiment period, which consists of 10 min of initialization period, 30 min of calculation task, and 15 min of
recovering period. Subjects were instructed to stay in calm on a chair at the initialization and the recovering
period. The heart rate (HR) during the task period was significantly lower in 1% orange aroma condition than that
in 20% and control conditions. The high frequency component (HF) of the heart rate variability significantly
higher in 1% condition than that in 20% and control conditions. Orange oil was preferred by all subjects but there
is no statistical difference in the level of preference between 1% and 20% of the orange aroma. The results
indicate the suppression of ANS stress response by orange aroma inhalation, but it was limited in the
concentration of 1%. Because the subjective preference of the odorant was not differed by the concentration, the
concentration of the orange aroma may play a key role to decide whether the odorant has an efficacy of
alleviating physiological stress response.
P2-012 Comparison of brain activation by the olfaction of orthonasal and retronasal odor-routes
Miyuki Koma-Takayanagi1, Kanetoshi Ito1, Kaori Takahashi1, Li-qun Wang2, Mitsuo Tonoike3, Shinya Kuriki2
1
Corporate Research & Development, Takasago International Corporation, Hiratsuka, Japan, 2Tokyo Denki University, Tokyo,
Japan, 3Department of Medical Engineering, Faculty of Health Science, Aino University, Japan
Odors often bring about desirable effects on human psychological and physiological states. Some odors give
rise to emotional responses of positive or negative feeling. Additionally, same odorant can have different odor
qualities between orthonasal and retronasal olfaction. However, it is unclear how the information of two olfaction
is processed in the human brain. The aim of this study is to assess the influence of orthonasal and retronasal
odors by psychophysical estimation and fMRI. Twenty one healthy females underwent fMRI scanning during
which they performed a block design task. Each of the participants was provided with three odors of chocolate
fragrance (chocolate), soap fragrance (soap), and Dipropylene glycol (DPG) in separate blocks conducted in a
random order. We expected that chocolate and soap evoked pleasant emotion, and DPG was scentless serving
as a control. We delivered each of the odors via the orthonasal and retronasal routes with an olfactometer.
2016.05.27 15.17.09 Page 6(1)
Recorded fMRI data were analyzed to compare the images acquired under above conditions. The brain regions
activated by orthonasal and retronasal routes were very different, even for the same odor. It seems that these
two routes transferred different information in the brain. In orthonasal chocolate, significant activation was
observed in the parahippocampal gyrus and the medial frontal gyrus. In retoronasal chocolate, significant
activation was observed in the caudate, putamen and thalamus. The caudate is known to represent the
magnitude of reward, and the thalamus is in an extensive nerve network sending signals to all around structures
of the brain including the cerebral cortex. Thus, it is suggested that the odor of retoronasal chocolate was
recognized as a reward with interaction of taste and somatic sensation. In conclusion, our findings suggest that
distinct olfactory neural pathways operate, depending on the way of odor administration and whether the odor
represents foods.
P2-013 What do olfactory preferences tell us about personality
Neta Reshef, Kobi Snitz, Noam Sobel
Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel
Olfactory receptor genetics imply that each individual may have a unique nose. We have recently developed a
highly sensitive perceptual test that may capture this variability. Using perceptual similarity matrices we
generated individually specific olfactory perceptual profiles we termed olfactory fingerprints. We initially found
that such fingerprints are indicative of a person’s human leucocyte antigen (HLA) genetic makeup (Secundo et
al., PNAS 2015). Previous evidence has implied that transient emotions and basic personality traits may be
related in some way to olfactory perception. Here we set out to test the hypothesis that olfactory fingerprints are
telling of basic personality traits. We used 12 odorants and 39 verbal descriptors to generate olfactory
fingerprints for 51 subjects (25M, mean age=25.7 std=3.45). All subjects then completed a battery of personality
tests that included the Big 5, love attitude scale, AQ, HDYT, and more. We next separated the data into training
(n=31) and testing (n=20) sets, and tested various models for predicting personality traits from olfactory
fingerprints. Using a regularized least-squared approach (Lasso) we observed only trends towards correlations
between particular personality traits and olfactory fingerprints. For example, the trait of attentional control was
linked to olfactory fingerprints at r=0.368, p=0.092 in the testing set. Such trends imply a possible link that should
be further probed using a larger cohort. Successfully characterizing human personality traits from an
independent task (olfactory fingerprints) may have significant diagnostic value for psychiatric disease and
conditions.
P2-014 Does the nose smell what the eyes see? Olfactory and visual influences on multimodal social
interactions
Jessica Michelle Gaby, Vivian Zayas
Department of Psychology, Cornell University, NY, USA
How does body odor affect interpersonal judgments during social interactions? Though much is known about
factors that influence interpersonal judgments of liking, up until this point, surprisingly little research has
examined how body odors affect social judgments in real life interactions. For example, first impressions based
on facial photographs strongly predict liking when two people meet in real life. Does olfactory information have a
similar effect? Natural body odor contains cues that are important in social judgments: gender and sexual
orientation, health, and emotional state, among others. However, most social olfactory research has focused on
natural axillary odors, devoid of any outside fragrance. These samples bear little resemblance to the way that we
interact with body odor in real life. In particular, they fail to consider what we term diplomatic odor-the modified
body odor that most people present in daily life, which includes influences from fragrances, deodorants, and
dietary choices. In this study, we assess the extent to which olfactory and visual first impressions, based
respectively on diplomatic odor samples and facial photographs, predict liking of the same individuals in a live,
multimodal interaction. We asked our (female only) participants to make snap judgments of faces and olfactory
judgments of t-shirts from each of our other participants. Afterward, participants met each other in a speed
dating-based “speed friending” paradigm, and completed ratings based on their live interactions. Preliminary
analysis of approximately half our data (study in progress) shows that visual judgments of liking strongly
predicted liking in the speed friending task (p=.001), but that olfactory judgments of liking did not (p=.793).
Interestingly, visual judgments also predicted olfactory judgments (p=.004). These preliminary findings suggest
that visual and olfactory-based judgments provide converging signals of liking, but that in multimodal
interactions, visual judgments take precedence.
2016.05.27 15.17.09 Page 7(1)
P2-015 True blood: Mammalian blood odor component modulates physiological and behavioral
responses in humans
Artin Arshamian1,2,3, Matthias Laska4, Amy R. Gordon1,5, Danja Porada1, Harald Melin1, Mats J. Olsson1, Johan N. Lundstrom1,5,6
1
Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden, 2Center for Language Studies and Donders
Institute for Brain, Cognition, and Behavior, Radboud University, Nijmegen, The Netherlands, 3Gosta Ekman laboratory, Department
of Psychology, Stockholm University, Sweden, 4Department of Physics, Chemistry and Biology, Linkoping University, Linkoping,
Sweden,
5
Monell Chemical Senses Center, Philadelphia, PA, USA, 6Department of Psychology, University of Pennsylvania, Philadelphia, PA,
USA
In mammals, the smell of blood can elicit a variety of adaptive responses such as directional cues for localizing
wounded prey in predators and flight behavior and increased vigilance in prey species. In non-mammal
vertebrates, chemical signals (e.g., alarm signals) can be conserved across taxonomic groups. Similar findings
in mammals are scarce, although it was recently demonstrated that trans 4,5-epoxy(E)-2-decenal (TED), a
single compound from mammalian blood odor, induced behavioral responses that were identical to those
triggered by real mammalian blood odor across four carnivore species.
Although it has been suggested that many food odors can be characterized by relatively few monomolecular
“key compounds”, neither non-human animals nor humans usually respond to a single odorant in the same
manner as they do to the naturally occurring odor mixture. Thus, TED seems to be unique in that it retains the
blood signature independent of the complete blood odor mixture and can convey a predator food signal across
mammalian taxa. Moreover, TED also seems to elicit freezing responses in prey species, such as mice.
Altogether, these data suggest that the blood odor component may be important for a wide range of mammals.
Thus, we hypothesized that akin to non-human animals, TED would be a biologically relevant odor in humans,
here operationalized as initiating a unique arousal and behavioral response. To address this issue, we
measured 33 participants’ galvanic skin response (GSR), heart rate (HR) and reaction time to a visual search
task of emotional facial stimuli while presenting TED, a control odor, and clean air. TED significantly increased
GSR but not HR, while decreasing detection time of emotional stimuli compared to the control odor and air.
These results suggest that the blood odor component may be an interspecific signal of special ecological value
that is conserved across mammalian species.
P2-016 Identification of a specific area of olfactory cortex involved in stress hormone responses to
predator odors
Kunio Kondoh1, Zhonghua Lu1, Xiaolan Ye1, David P. Olson2, Bradford B. Lowell2, Linda B. Buck1
1
Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, USA, 2Beth Israel Deaconess Medical
Center and Harvard Medical School, Boston, USA
Animals sense environmental dangers that challenge their survival and respond with behavioral and
physiological changes to eliminate the challenge. The olfactory system detects chemical cues emitted from
predators that stimulate an instinctive fear response. In addition to characteristic behaviors, this response
includes increases in blood stress hormones that mobilize multiple body systems to escape impending danger.
These are controlled by corticotropin-releasing hormone (CRH) neurons in the hypothalamus of the brain. To
explore the neural circuits that convey predator odor signals from the nose to CRH neurons, we made
neurotropic viruses that allowed visualization of neurons either one or multiple synapses upstream of CRH
neurons. We discovered that neurons presynaptic to CRH neurons are present in multiple areas of the olfactory
cortex (OC), which receives signals from the nose, as well as in vomeronasal amygdala (VA) areas that receive
olfactory information from the vomeronasal organ. However, only one minor area in the OC, the
amygdalo-piriform transition area (AmPir), contained neurons upstream of CRH neurons that were activated by
volatile predator odors. Chemogenetic stimulation of AmPir activated CRH neurons and induced an increase in
blood stress hormone, mimicking an instinctive fear response. Moreover, chemogenetic silencing of AmPir
markedly reduced the stress hormone response to predator odors without affecting a fear behavior. These
findings suggest that AmPir plays a key role in generating hormonal fear responses to volatile predator cues that
signal danger.
2016.05.27 15.17.09 Page 8(1)
P2-017 Stress modulates odor-specific sensory perception and alters social behavior
Tomohiro Tanaka, Norah Koblesky, Sandeepa Dey, Lisa Stowers
Department of Neuroscience, The Scripps Research Institute, USA
Sensory responses are modulated by internal state. This is partly due to fluctuations of hormones and
neuropeptides. Stress is thought to modulate neuronal activity central nervous system which controls behavior,
and is well described for large contribution of hormones released from hypothalamic-pituitary-adrenal axis and
from axon terminals of locus coeruleus neurons. It has not been elucidated whether stress alters behavior by
directly modulating the sensory activity in the olfactory organs. In mice, many conspecific cues and predator
odors evoke activity of vomeronasal sensory neurons (VSNs), triggering social and fear behavior, respectively.
We use calcium imaging and pS6 immunostaining to show that female/male urine evoked activity of VSNs is
reduced following acute restraint stress. Concomitantly, attraction behavior towards conspecific cues is
significantly suppressed by stress. Predator odor-activated VSNs are also reduced following acute stress,
however the attenuation of the activity is not complete and avoidance behavior towards predator odor is not
canceled by stress. We find that different repertoires of stress hormone receptors are enriched in activated
VSNs in an odor-specific manner, by using phosphorylated ribosome immunoprecipitation of mRNA from VSNs
of odor-stimulated mice followed by quantitative PCR. Functional analysis of these receptors will be further
discussed. Our results point to a possible role of stress receptors co-expressed in the VSNs, which directly
modulates sensory perception and alters behavior.
P2-018 Long-term odor experience induces limited generalization in perception
Haruko Sugiyama1, Saho Ayabe-Kanamura2
1
Kansei Science Research, Kao Corporation, Tokyo, Japan, 2Faculty of Human Sciences, Division of Psychology, University of
Tsukuba, Japan
INTRODUCTION: Even familiar or everyday odors could induce misidentification, misrecognition or inconsistent
perception. The purpose of this study was to investigate whether perception of a long-term experienced odor
would be specific to it or be generalized among similar odors, and to know what was behind change of
perception. METHODS: One standard fragrance for a long-term experience and six modified odors by adding
three different amounts of floral or green fragrances to the standard were used. Other two standard fragrances
and each modified odors were prepared for universality, and 104 women were assigned to one of three standard
fragrance groups. The study consisted of three phases. In the first phase, in a testing room, participants
performed three tasks, 1) grouping three standard odors, 2) judging similarities among the seven odors (one
standard and six modified), and 3) evaluating characteristics of each odor. In the second phase, the participants
were asked to smell the assigned standard odor six times per day for five weeks at home. Finally, again in the
testing room, they smelled the seven odors and judged whether they had smelled each in the experience period.
And they performed the same three tasks of the first phase. RESULTS & DISCUSSION: After long-term
experience, only a-little-modified odors were recognized as the experienced standard odor. MDS analysis of
similarity between the seven odors showed that the standard odor and a-little-modified odors are laid more close
in a two-dimensional space than before the standard odor was experienced. These results indicate that
perception of the experienced odor could be generalized at some level. Interestingly, characteristics of each
odor showed no significant change between before and after experience. It seems to be difficult to indicate
variance of odor perception via linguistic evaluations.
P2-019 Olfactory attention modulates gamma band activity in healthy humans
Archana K. Singh, Masako Okamoto, Kazushige Touhara
Aims: Attention is a cognitive process that requires self-control to focus on the stimuli of our interest. However,
the role of attention is still not clearly established for olfaction. Specifically, there is a lack of information on how
the attention to an odor modulates gamma band activity, i.e., the spectral power in 30-80 Hz frequency band,
which has been linked with the cognitive processing of a stimulus (e.g., in vision). In this EEG study, we
examined the changes in the gamma-band activity during two olfactory tasks with different levels of attention.
Methods: Eighteen healthy subjects participated. The odor of 2-phenylethyl alcohol (a rose-like smell) was
presented to the nose during inspiration using an olfactometer and a breathing sensor. We computed the
gamma band activity from the wavelet-decomposed EEG signals time-locked to the odor stimulus. A bootstrap
2016.05.27 15.17.09 Page 9(1)
test was used for the statistical comparison of spectral-power between attended and ignored odor tasks.
Results: Several instances of significantly enhanced gamma-band synchronization (p<0.05, fdr corrected) were
observed in the early as well as late stages of olfactory neural processing, consistent with the findings from
visual attention studies*. Conclusion: These results lend support to the commonly held notion that attention to a
stimulus enhances its neural processing. As the experimental reports of spectral-power associated with human
olfactory attention are scarce, this study may provide a useful direction for future research on the topic.
Acknowledgement: ERATO Touhara Chemosensory Signal Project from JST, Japan Ref: *Tallon-Baudry,C.,et
al. (2005). Attention modulates gamma-band oscillations differently in the human lateral occipital cortex andf
usiform gyrus. Cereb. Cortex
P2-020 Wine experts remember wines better than novices, and this is not mediated by language
Ilja Croijmans, Asifa Majid
Centre for Language Studies, Radboud University, Nijmegen, The Netherlands
Naming smells can improve memory for those odors. This is surprising given that, people in the West are poor at
naming odors, yet memory for smells is remarkably good. Wine experts have been found to be better at
remembering smells (e.g. Parr, Heatherbell & White, 2002). In addition, when experts are asked to describe wine
and wine-related smells, they are more precise than novices (Lawless, 1984). Could wine experts’ superior
memory for odors be mediated by language? And is this memory advantage restricted to odors from the experts’
domain of expertise, or does this advantage generalize to other odors too? Twenty-four wine experts and 24
novices were invited to remember the smell of wines, wine-related odors, and common odors. Half of the
participants were just asked to remember the smells, whereas the other half were asked to name the smells
during encoding too. After ten minutes, participants’ recognition memory was tested. All participants were then
also asked to name the odors. Wine experts were more consistent in describing wines and wine-related odors.
Experts also remembered wine odors better than novices, but were no better at remembering any of the other
odors. However, experts did not benefit from naming odors or wines compared to just smelling them when they
memorized the odors. In addition, there was no correlation between experts’ ability to name odors at encoding
and their subsequent memory for those odors. To conclude, wine experts’ odor memory advantage is restricted
to their domain of expertise, and it appears experts’ odor memory is not linguistically mediated.
P2-021 Differential serotonergic modulation across the main and accessory olfactory bulb
Zhenbo Huang1, Nicolas Thiebaud1, Debra Ann Fadool1,2
1
Program in Neuroscience, Department of Biological Science, The Florida State University, Florida, USA, 2Institute of Molecular
Biophysics, The Florida State University, Florida, USA
Because mitral cells of the main olfactory bulb (MOB) typically project one apical dendritic tuft into a single
glomerulus, whereas those of the accessory olfactory bulb (AOB) extend several apical dendrites to multiple
glomeruli, differential anatomical features across the two systems may lead to functionally distinct information
coding. Recent studies have demonstrated that MOB vs. AOB mitral cells have distinct intrinsic membrane
properties but differential neuromodulation has not been well explored. Herein, we investigated a
widely-distributed CNS modulator, serotonin (5-HT), for its ability to modulate the biophysical properties of mitral
cells, using an in vitro , brain slice approach in postnatal 15-30 day mice. Using a whole-cell configuration in
current-clamp mode, bath application of 20 μm 5-HT elicited one of three responses for MOB mitral cells,
namely, cells were either excited (73%, n=115), inhibited (9%, n=15), or exhibited a mixed response-first
inhibition followed by excitation (12%, n=19). For the prevailing excitatory responses, 5-HT elicited a 4.6±3.1
(Mean±SD) fold increase in firing frequency, which could be blocked by 5-HT2A receptor antagonist spiperone.
In contrast, mitral cells of the AOB were predominantly inhibited by 5-HT (83%, n=95). For these inhibitory
responses, 83% appeared to be indirectly inhibited through 5-HT2 receptors on GABAergic interneurons based
upon gabazine sensitivity-a GABAA receptor antagonist-and 17% were insensitive to gabazine, suggesting
involvement of 5-HT1 receptors via direct inhibition. These data suggest that serotonergic pathways may be
largely excitatory for MOB mitral cells (membrane capacitance, 117.1±24.2 pF; input resistance, 80.6±21.7
MΩ) but predominantly inhibitory for AOB mitral cells (membrane capacitance, 84.3±29.9 pF; input resistance,
162.4±67.2 MΩ).
2016.05.27 15.17.09 Page 10(1)
P2-022 Identification of the olfactory signaling molecules
Woochan Choi, Kyuhyung Kim
Department of Brain and Cognitive Sciences, DGIST, Daegu, South Korea
Animals must recognize and discriminate among thousands of chemicals in order to generate the correct
behavioral response. Understanding basic design of a sensory system in simple animals gives the opportunity to
elucidate detailed molecular and neural mechanisms underlying sensory responses in higher animals. C.
elegans detects a large number of odorants via chemsensory neurons and elicit a multitude of olfactory
behaviors (Bargmann, 1993, Cell). Due to the established connectome, the olfactory system of C. elegans
provides an excellent model system in which to explore the circuit mechanisms underlying olfactory processing
at the single cell resolution. Previous genetic and behavioral experiments have identified set of signaling genes
including olfactory receptors in C. elegans, but the knowledge is still limited. Here we are trying to construct a
comprehensive map of odorants and their receptors in C. elegans. We screened volatile chemicals that are not
tested previously, and found that animals respond to a set of chemicals including acetic acid, acetoin, DMTS,
formaldehyde, 1-propanol, or 2-propanol. We further identified that the AWC neurons are required for
chemotactic responses to these chemicals. We then performed candidate gene searches and found that lsk46
mutants exhibit a specific defect in chemotactic response to 1-propanol, and altered Ca2+ response of AWC to
1-propanol, indicating a unique role of lsk46 mutation in the 1-propanol chemotactic response. We are currently
identifying the molecular lesion of the lsk46 mutation.
P2-023 Behavioral assessment of mouse olfaction using a new experimental device
Masayuki Harita1, Hideaki Shiga1, Tomoko Hiraba1, Yukari Nakamura1, Takuya Noda1, Kentaro Yamada1, Hideaki Ninomiya2,
Takaki Miwa1
1
Department of Otorhinolaryngology, Kanazawa Medical University, Ishikawa, Japan, 2Department of Medical Resarch Institute,
Kanazawa Medical University, Ishikawa, Japan
Aim
Behavioral assessment experiments for mouse olfaction have been conducted widely since the 1950s.
However, many past experiments required significant preparation time to create appropriate assessment
conditions as well as time to set up or calibrate intricate devices. Such complex devices often make it more
difficult to extract data. For example, it has already been proven that mice display aversive behavior towards an
odor called 2,4,5-trimethylthiazoline (TMT), also known as fox odor, but the established experimental device and
behavioral testing methods have yet to be determined due to device complexities. In the current study, we
created and used a new, simplified device to examine the olfactory behaviors in mice.
Method
We created a simple device with two cylinders connected by a passage, which enabled mice to move freely
from one cylinder to the other. We measured the behaviors of mice against different odors. Specifically, we filled
each cylinder with a different odor: the control cylinder with the odor of distilled water, and the cylinder with TMT.
We video-recorded the respective mice behaviors, and conducted time measurements using software that
automatically plotted their movements. We also investigated how the olfaction of mice is disturbed by
Methimazole, how mice behave in the recovery process, and when they resume detecting odors.
Results
The results of our experiment demonstrated that there was no significant difference between the time mice
spent in the TMT cylinder and the control cylinder, even though the mice were able to move freely between the
two cylinders.
Conclusion
The current study was an experiment to examine how a new, simple device could be used to investigate
mouse olfaction behaviors. Based on our results, we see potential in continuing to construct simplified devices
that will improve better experimental methods and ultimately provide valuable data.
2016.05.27 15.17.09 Page 11(1)
P2-024 When specific anosmia fails the smell test: Behavioral and cerebral correlates of a subjectively
unperceived odorant
Geraldine Coppin1,2, Aline Pichon1,2,3, Corrado Corradi DellAcqua3,4, Sylvain Delplanque1,2, Patrik Vuilleumier1,3, David Sander1,2
1
Swiss Center for Affective Sciences, Switzerland, 2Laboratory for the Study of Emotion Elicitation and Expression, Department of
Psychology, University of Geneva, Switzerland, 3Laboratory for Neurology and Imaging of Cognition, Department of Neurosciences
and Clinic of Neurology, University Medical Centre, Geneva, Switzerland, 4Theory of Pain Laboratory, Department of Psychology,
University of Geneva, Switzerland
We investigated how the unconscious processing of an odorant can change the behavioral and cerebral effects
of another consciously perceivable odorant. We tested individuals with the inability to perceive a particular
odorant, namely androstenone, while being presented with an odorant to which androstenone was added, as
compared to the very same odorant without androstenone. Using a triangle test, we selected females anosmic to
androstenone. First, participants were asked to rate the familiarity, intensity and pleasantness of six odorants
(androstenone alone, androstenone paired with a smell the participant really liked, this really liked smell, another
pleasant smell, an unpleasant smell and an odorless control). Second, participants were fMRI-scanned while
being presented with these stimuli in a cued sniffing paradigm. Participants had to indicate after each trial
whether they perceived an odorant or not. Results showed that participants failed to perceive any difference
between androstenone and an odorless control in terms of intensity, familiarity or pleasantness. There was no
evidence of a difference in the sensitivity index between these two conditions. However, we found activations in
the post-central gyrus and superior frontal gyrus in response to androstenone versus the odorless control, which
we interpret as evidence for a trigeminal stimulation in the anosmic individuals. The pairing androstenone+very
pleasant smell was not perceived as more intense, familiar, or pleasant, than the very pleasant smell alone, but
the sensitivity index was higher. Moreover, we found increased responses in the anterior cingulate cortex,
anterior insula, orbitofrontal cortex, piriform cortex and superior frontal gyrus to the pairing in comparison to the
very pleasant smell alone. This suggests that adding an unperceivable odorant to a perceivable odorant may
affect the cerebral processing of the latter by activating an alert network linked to the trigeminal perception, as
well brain regions typically involved in affective processing.
P2-025 Histochemical localization of a glycan structure governed by molecular clock in the mouse
olfactory system
Daisuke Kondoh, Motoki Sasaki, Nobuo Kitamura
Department of Basic Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan
Glycosylation affects functional properties of proteins and lipids, and plays critical roles in neuronal formation in
the central nervous system. We previously reported the daily fluctuation of several sugar chain structures within
the mouse olfactory bulb and the molecular mechanism of diurnal α1-2-fucosylation governed by the molecular
clock. As several studies have shown that α1-2 fucose (α1-2Fuc) glycan mediates neuronal functions and
morphology, including neurite outgrowth and synaptic plasticity, this glycan structure may be a potential factor
for circadian rhythm in the activity of the olfactory system. About a dozen histochemical studies of Ulex
europaeus agglutinin-I (UEA-I) that detects α1-2Fuc glycan have localized α1-2Fuc in the mouse olfactory
system, but the findings are diverse. We therefore histochemically analyzed in detail the olfactory pathway (the
olfactory epithelium, bulb and cortex) of adult male and female ICR and C57BL/6J mice using UEA-I. Ulex
europaeus agglutinin-I reacted with most receptor cells arranged at basal regions of the olfactory epithelium.
The olfactory nerve and glomerular layer of the main olfactory bulb were speckled with positive UEA-I staining,
and positive fibers were scattered from the glomerular to the internal plexiform layer. We identified interneurons
and projection neurons, i.e. tufted and mitral cells, as the origins of the UEA-I-positive fibers in the main olfactory
bulb. The lateral olfactory tract and rostral migratory stream were also positive for UEA-I. On the other hand, the
olfactory cortex was negative for UEA-I. Previous studies have focused only on the primary pathway, but the
present findings indicated that α1-2Fuc glycan is located in the primary and secondary, but not the ternary,
pathways of the olfactory system and in local circuits of the main olfactory bulb.
2016.05.27 15.17.09 Page 12(1)
P2-026 Main olfactory system-mediated social behaviors in mice
Tomohiko Matsuo1, Tatsuya Hattori2, Akari Asaba2, Naokazu Inoue3,4, Nobuhiro Kanomata5, Takefumi Kikusui2,
Reiko Kobayakawa1, Ko Kobayakawa1
1
Department of Functional Neuroscience, Institute of Biomedical Science, Kansai Medical University, Osaka, Japan, 2CompanionAnimal Research, School of Veterinary Medicine, Azabu University, Kanagawa, Japan, 3Research Institute for Microbial Diseases,
Osaka University, Osaka, Japan, 4Department of Cell Science, Institutes for Biomedical Sciences, School of Medicine, Fukushima
Medical University, Fukushima, Japan, 5Department of Chemistry and Biochemistry, Waseda University, Tokyo, Japan
Pheromones induce social behaviors through the main olfactory system (MOS) and vomeronasal system (VNS).
However, the functional contributions of each subsystem in social behavior remain unclear. To genetically
dissect roles of the MOS and VNS in social behavior, conditional knockout mouse lines that led to a
loss-of-function confined in the dorsal MOS were analyzed. These mutant mice showed normal sniffing behavior
and VNS activity. However, multiple social behavior defects were observed: attraction to female urine and the
accompanying ultrasonic vocalizations, chemoinvestigatory preference, aggression, maternal behaviors, and
risk-assessment behaviors in response to an alarm pheromone. Our results demonstrated an MOS-mediated
pathway that processes semiochemical information, independent of the VNS. The mouse odorant receptor
family comprises more than 1,000 intact odorant receptors (ORs) and are expressed in the main olfactory
epithelium. To isolate ORs that regulate social behavior, we cloned 1,038 intact OR genes and each OR was
expressed in HEK293T-derived HANA3A cells. Using this heterolougous system, we identified several candidate
ORs that bind the mouse alarm pheromone.
P2-027 Local sodium spikes in dendritic spines of olfactory bulb granule cells
Vanessa Rupprecht1, Wolfgang Georg Bywalez1,2, Dinu Patirniche2, Martin Stemmler2, Andreas Herz2, Balazs Rozsa3,
Denes Palfi3, Veronica Egger1
1
Institute of Zoology, Regensburg University, Regensburg, Germany, 2Department of Biology II, Ludwig-Maximilians-University,
Munich, Germany, 3Two-Photon Imaging Center, Institute of Experimental Medicine, Budapest, Hungary
Olfactory bulb granule cells (GCs) are interconnected with lateral dendrites of mitral cells (MCs) via reciprocal
dendrodendritic synapses; postsynaptic GC Ca2+-signals are strictly localized to the reciprocal spines. We
hypothesized that these spines can function as independent mini-neurons based on local activation of voltagegated Na+-channels (Navs). To mimic unitary inputs we performed two-photon uncaging (TPU) of glutamate at
single GC spines and imaged the spine Ca2+-signals (∆F/F)TPU while recording the uncaging-evoked EPSPs
(uEPSPs) at the GC soma.
Blocking Navs with TTX resulted in a strong reduction of (∆F/F)TPU in most spines (to 0.63±0.20 of control, mean
±S.D., n=34). uEPSP amplitudes were only slightly decreased whereas their rise time became slower and their
duration longer. The extra depolarization provided by Navs boosts Ca2+-entry mainly via high voltage-activated
Ca2+-channels, since blockade of NPQ type Ca2+-channels with ω-conotoxin MVIIC (CTX) decreased (∆F/F)TPU as
much as TTX (n=24). CTX applied after TTX reduced (∆F/F)TPU only marginally (n=14), similar to the reverse
experiment (TTX after CTX; n=8). Notably, blockade of NMDA receptors post TTX application still had a
considerable effect on (∆F/F)TPU (to 0.11±0.06 of control, n=14), indicating that NMDAR-mediated Ca2+-entry is
not boosted by Navs. As to the contribution of K+-channels, a NEURON model supports our indirect evidence for
a Nav-activated slowly repolarizing current that fits the properties of delayed-rectifier K+-channels.
To prove that local sodium spikes are indeed capable of mediating GABA release from GC spines, we recorded
from MCs in voltage clamp while performing TPU of glutamate along proximal MC lateral dendrites in the
presence of 1 mM Mg2+. The resulting small evoked currents could be blocked by Bicuculline (n=5) and were
often sensitive to TTX (5 out of 6 experiments), confirming independent processing by the reciprocal spines.
P2-028 Activation of the vasopressin 1a receptor is necessary for the induction of synaptic plasticity in
the accessory olfactory bulb of male mice
Toshiharu Namba1, Mutsuo Taniguchi1, Yoshihiro Murata1, Fumino Okutani2, Hideto Kaba1
1
Department of Physiology, Kochi Medical School, Nankoku, Kochi, Japan, 2Department of Occupational Health, Kochi Medical
School, Nankoku, Kochi, Japan
Central vasopressin enhances social recognition and controls many complex social behaviors in mammals.
Previous studies have reported the presence of vasopressin receptors and neurons in the accessory olfactory
bulb (AOB). However, their functions are largely unknown. The AOB has been demonstrated to be a critical site
for pheromonal learning in female mice. Therefore, we investigated the effect of vasopressin on synaptic
2016.05.27 15.17.09 Page 13(1)
plasticity of glutamatergic transmission from mitral to granule cells in the AOB of male mice. The maximal initial
slope of lateral olfactory tract (LOT)-evoked field EPSPs of granule cells was measured to monitor the strength
of glutamatergic transmission. Sub-threshold LOT stimulation, consisting of a 100 Hz, 100-pulse train applied
twice at a 3-min interval, induced only short-term potentiation that decayed back to its control value. In contrast,
the pairing of the sub-threshold LOT stimulation and bath application of vasopressin produced robust long-term
potentiation (LTP) that lasting for at least 3 hours. Manning compound, an antagonist of the vasopressin 1a
receptor, blocked the expression of LTP. On the other hand, SSR149415, an antagonist of the vasopressin 1b
receptor, failed to block the expression of LTP. These results suggest that vasopressin facilitates the induction of
LTP at the mitral to granule cell synapse through the activation of vasopressin 1a receptor in the AOB.
P2-029 Dissection of neural circuit mediating olfactory alarm reaction in zebrafish
Miwa Masuda1,2, Sayoko Ihara2,3, Tetsuya Koide1, Nobuhiko Miyasaka1, Noriko Wakisaka1, Keiichi Yoshikawa3,
Kazushige Touhara2,3, Yoshihiro Yoshihara1,2
1
RIKEN Brain Science Institute, Japan, 2ERATO Touhara Chemosensory Signal Project, JST, Japan, 3Department of Applied
Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan
Various fresh-water fishes including zebrafish show a robust alarm response to putative alarm pheromones
released from injured skin of conspecifics. This behavior is important for fishes in risk avoidance even without
visible presence of predators. However, the identity of alarm pheromones and the molecular, cellular and neural
circuit mechanisms mediating the alarm reaction remain largely unknown. In this study, we first developed an
automatic analysis system to quantify alarm response. Upon application of conspecific skin extract, most
zebrafish showed burst swimming followed by freezing at the bottom of the tank. This alarm reaction was not
observed in olfactory epithelium-removed zebrafish, indicating the essential role of the olfactory system. To
identify neurons activated by alarm pheromones, we next histochemically examined ERK phosphorylation and/
or c-fos mRNA expression as makers for neuronal activation at different levels along the olfactory circuitry in skin
extract-stimulated zebrafish. In the olfactory epithelium, phosphorylated ERK (pERK) was present in various
populations of olfactory sensory neurons with heterogeneous morphologies. In the olfactory bulb, however,
strong pERK signals were observed in three discrete glomeruli specifically. Interestingly, one of the three
glomeruli responded only to skin extract of zebrafish; another glomerulus to zebrafish and goldfish; the
remaining one to zebrafish, goldfish and medaka, suggesting that, species-specific and superorder-specific
chemical cues may be involved in eliciting alarm response in a complex environment. In higher olfactory centers,
c-fos induction was detected in restricted nuclei of the telencephalon, preoptic area, and hypothalamus. Finally,
we biochemically purified two fractions containing putative alarm pheromones from zebrafish skin extract. The
mixture of these two fractions significantly induced an alarm reaction in zebrafish, while each of the fractions
alone did not. These results suggest that a neural circuit mechanism for coincidence detection of two pheromone
components underlies the expression of the alarm reaction in zebrafish.
P2-030 Visualization of neural activity with appetitive or non-appetitive olfactory stimulation in fly brain
Toru Maeda1, Tomoyosi Nisimura2, Miwako Tamotsu1, Mamiko Ozaki1
1
Department of Biology, Kobe University, Kobe, Japan, 2Department of Liberal Arts, Nihon University, Tokyo, Japan
Taste and olfaction are crucial sensory modalities for insects to distinguish between nutritious foods and harmful
poisons, and thus regulate many aspects of feeding behavior. When the blowflies, Phormia regina , feed sweet
nectar on the flower, they must sense the flower odor. They have innate flavor preference to certain odors
enhancing their feeding motivation. In P. regina , many reports about gustatory system and feeding behavior
have appeared. Fly has two kinds of olfactory organs, antennae and maxillary palps, and they received different
odorants which inhibited and promoted feeding behavior to sweet substances via antennae and maxillary palps
respectively. However, it is not known that how olfactory information integrates with gustatory information
resulting in proper modulation of feeding behavior. In order to search neural route of olfactory information, we
used pERK antibody which binds to phosphorylated ERK, classical MAPK, which deeply concern with signaling
in neuron cell. By using pERK antibody we investigated where neurons were activated in brain by stimulating two
kinds of olfactory organs with appetitive or non-appetitive odorants. In our result, different active area of neurons
was visualized in the brain between two kinds of olfactory organs which received same odorant. In addition,
there was different neuronal activity in the brain between appetitive and non-appetitive odorants which was
received with same olfactory organ. Especially, significant difference was observed in the pattern of activated
glomerulus in antennal lobe, the primary olfactory center. And in mushroom body, the memory center, only part
2016.05.27 15.17.09 Page 14(1)
of α-lobe was activated in case of stimulated with non-appetitive odorant but broader region was visualized in
case of stimulated with appetitive odorant. It is thought that these results were valuable to clarify mechanism of
olfactory effect on feeding behavior from the side of neurophysiology.
P2-031 Identification of a rat tear protein evoking anxiety and decreased locomotor activity in mice via
the vomeronasal organ
Mai Tsunoda, Kazushige Touhara
Applied Biological Chemistry, Graduated School of Agricultural and Life Sciences, The University of Tokyo, Japan
Communications between animals are regulated by a variety of chemical cues emitted from body fluids. Recent
works have revealed that a tear fluid is one of the important source of cues that regulate intraspecies
communication through the vomeronasal organ (VNO). However, it has been unknown whether tear-derived
chemical cues mediate only intraspecies communication. In this study, we aimed to understand a novel function
of tear fluids in interspecies communication by focusing on tear fluids of rats, a predator of mice. It was revealed
that rat tear fluids contained some stimulants that induced phosphorylation of ribosomal protein S6 in the VNO
and c-fos expression in the accessory olfactory bulb (AOB). We purified the stimulants from rat tear fluids by
activity-based fractionation. Amino-terminal peptide sequence and genome analysis revealed that the stimulant
was a 18 kDa protein, we named P18, whose function was unknown. P18 was expressed in the extraorbitallacrimal gland in a testosterone-dependent manner and secreted specifically into male rat tear fluids.
Recombinant P18 induced c-Fos expression in the mouse AOB, the MePV (medial amygdaloid mucleus
posterior part) and the VMHdm (ventromedial hypothalamic nucleus dorsomedial part). In the VMHdm, P18
induced c-Fos expression in the steroidogenic factor-1 positive neurons that were involved in defensive
behavior. P18, however, did not induce avoidance behavior, risk assessment behavior nor aggressive biting
behavior, which are known to be induced by a predator odor. In contrast, P18 induced anxiety and decreased
locomotor activity. These results suggest a possibility that P18 is not a conventional avoidance cue, but a
substrate that evokes emotional changes to prepare for the next appropriate behavior in mice.
P2-032 Olfactory representation from the periphery to higher brain centres in Drosophila melanogaster
Yoichi Seki1,2, Hany K. M. Dweck1, Juergen Rybak1, Dieter Wicher1, Silke Sachse1, Bill S. Hansson1
1
Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany, 2School of Life Sciences,
Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
Odor information is processed through multiple receptor-glomerular channels in the first order olfactory center,
the antennal lobe (AL), whereafter it is reformatted into higher brain centers and eventually perceived by the fly.
To reveal the logic of olfactory processing, it is fundamental to map odor representations from the glomerular
channels and follow the flow of information into higher brain centers. We used in vivo whole cell patch-clamp
recordings in Drosophila melanogaster and characterized odor response profiles of AL projection neurons (PNs)
originating from 31 glomeruli, which cover ca. 60% of all glomeruli. We revealed that combinatorial odor
representations are generally conserved from olfactory sensory neurons (OSNs) to PNs and transformation of
odor tuning curves are glomerulus-dependent. In the AL, we found three clusters of neighboring glomerular
groups that likely are important for the representation of odor valence. The functional maps are converted into
higher brain centers, where two separate representations form integration sites for attractive and aversive odors
respectively, both in the mushroom body and the lateral horn. Our results thus fill the gap between the spatial
odor map in the AL and the odor representation in higher brain centers leading to odor perception underlying
innate odor-guided behavior.
P2-033 Chemical communication for agression or aversion in the ant
Mamiko Ozaki
Department of Biology, Kobe University, Kobe, Japan
Social insects exhibit complex societies characterized by self-organized division of labor, as well as conflict of
interests among colony members. Pheromones play a major role in regulating cooperative behavior, division of
labor and conflict resolution. Especially, cuticular hydrocarbons (CHCs) convey various individual-information,
and thus important chemical signals for their societies. Therefore, it is plausible that specific expansion of
odorant receptor genes for CHCs perception underlie sophisticated communication required for complex social
organization. By conducting genome sequencing of the ant Camponotus japonicus followed by extensive
2016.05.27 15.17.09 Page 15(1)
manual annotation, we found over 400 chemosensory receptor genes (GRs, ORs and IRs) in this species. We
also conducted comparative antennal RNA-seq analyses among the workers, alate queens and males, and
revealed dozens of odorant receptors (ORs) showing female biased expression in their antennae. Phylogenetic
analyses indicated that these female-biased ORs shape the specific cluster in their phylogeny. We also found
these ORs are expanded only in social lineages of Hymenoptera (wasps, bees, and ants), especially in ants.
These results suggest evolution of specific clade of ORs are crucial for social organization, and we will discuss
the possible function of these OR genes.
P2-034 A social chemosignal may modulate aggressive behavior in humans
Eva Mishor, Daniel Amir, Shani Agron, Aharon Ravia, Noam Sobel
Several main olfactory receptor types and subfamilies have been specifically implicated in rodent social
chemosignaling, these types and subfamilies are largely retained in humans as well. One such subfamily is the
mammalian-specific OR37, whose subtypes A, B and C are activated by the long-chain aliphatic aldehydes
Pentadecanal, Hexadecanal and Heptadecanal, respectively. OR37 glomeruli are clustered in a small ventral
domain of the olfactory bulb, a region implicated in processing socially relevant cues. Rodents release typical
OR37 ligands in bodily secretions, and exposure to these ligands reduces stress responses in conspecifics. This
has lead to the suggestion that OR37 ligands may act as promoters of social buffering (Klein et al., 2015, Bautze
et al., 2012). Humans express an ortholog receptor (2S2), and similarly secrete these ligands. Hexadecanal for
example can be found in human feces, breath and skin.
With this in mind, we set out to test the hypothesis that Hexadecanal (HEX) (C16H32O) may modulate
aggressive behavior in humans. We conducted a double-blind between-subjects design experiment in which
subjects were exposed to either HEX masked in Eugenol or to Eugenol alone. Subjects then engaged in an
aggression-provoking social interaction, and subsequently, were given the opportunity to discharge aggression
in the form of a noise blast game. The measured noise blasts’ volume multiplied by duration served as an
aggressive-behavior index. In addition, we collected self-ratings of mood and salivary testosterone and cortisol.
Preliminary results indicated that women exposed to HEX induced stronger and longer noise blasts than
unexposed women (mean HEX=5094±426.5, N=10, CONT=2679±185.8 (N=12), t(12.38)=5.19, p<0.0005).
Conversely, men showed reduced aggressive behavior when exposed to HEX (mean HEX=3364±361.2, CONT
=6156±664.8, t(6.34)=3.69, p<0.01). These preliminary findings suggest that Hexadecanal, a compound
secreted in various human excretions, may act as a chemosignal to modulate aggression.
P2-035 Correlation analysis among chemical structure, olfactory receptor activation profile, and odor
character or similarity of eugenol or vanillin-related odorants
Yasuko Nogi1, Chiori Ijichi1, Masayuki Sugiki1, Yusuke Ihara1, Yuko Kodama1, Wakana Saikawa1, Kei Yamada1,
Yutaka Ishiwatari1, Sayoko Ihara2,3, Yoshihito Niimura2,3, Kazushige Touhara2,3
1
Institute for Innovation, AJINOMOTO Co., Inc., Japan, 2Department of Applied Biological Chemistry, Graduate School of
Agricultural and Life Sciences, The University of Tokyo, Japan, 3ERATO Touhara Chemosensory Signal Project, JST, The University
of Tokyo, Japan
Eugenol and vanillin are similar in structure, but they are known to have different odor characters, such as spicy
and vanilla, respectively. This is likely due to a difference in the combination of activated olfactory receptors
(ORs). In other words, the slight difference in the functional group in eugenol and vanillin resulted in activation of
overlapping but distinct ORs, and thus, in different odor qualities. Previous studies have mainly focused on
structure-activity relationship of single ORs but not on the correlation between activated ORs and odor character
or similarity. In this study, to investigate the relationship among odorant structure, activated OR profile, and odor
quality, we performed screening of human ORs that recognized 34 odorants structurally related to eugenol and
vanillin, and analyzed correlation between the activated OR profile and the odor character and similarity
obtained by sensory evaluation. It was found that the 34 odorants activated mainly six ORs (OR10G4, OR10G7,
OR5K1, OR2J2, OR5P3, and OR1A1) among all human ORs. We then analyzed relationship among the
following parameters such as molecular structure, OR activation pattern and odor description or degree of odor
similarity. As a result, rough correlation was observed between the OR profile and structural classification.
Partial correlation was also found between the OR profile and odor description or similarity. These results
indicate that in some cases, the activation pattern of ORs could be predicted by structural information of an
odorant molecule and in addition, its odor could also be predicted by the activated OR profile. Our result that
2016.05.27 15.17.09 Page 16(1)
odor character has some correlation with the chemical structure and OR activation pattern may provide a new
strategy upon designing a flavor in the future.
P2-036 Towards a computational biomimetic nose
Caroline Bushdid1, Jeremie Jacques Topin1, Claire A. de March2, Jerome Golebiowski1,3
1
Institut de Chimie de Nice, Universite de Nice Sophia-Antipolis, Nice, France, 2Department of Molecular Genetics and
Microbiology, Duke University Medical Center, Durham NC, USA, 3Department of Brain & Cognitive Sciences, DGIST, Daegu,
South Korea
The actual consensus states that the code to olfactory perception is a combinatorial one. According to this
theory, each one of our odorant receptors (ORs) is differentially activated when interacting with an odorant
molecule.
Using a computational approach, we here seeked to test whether ligand/receptor affinity can predict OR
activation leading to odor perception. Virtual screening of odorants with similar olfactory notes was performed on
the odorant receptor repertoire built by molecular modeling.
Models for all expressed ORs in the whole human olfactory mucosa [1] were obtained on the basis of a
validated homology alignement with known GPCR class A structures. [2] This database of ORs was tested
against molecules having different molecular structures, but belonging to the same olfactory families. We chose
for example molecules having minty smells (R-carvone and menthone), or having been described as smelling
earthy (geosmin and 2-ethyl fenchol). Optimal docking scores were obtained for each molecule and on each
receptor.
By computationally screening the cases in which ORs get activated by certain ligands, we established the
fingerprint associated with each odorant on all ORs. The aim of this study is to assess if docking experiments on
in silico modeled ORs are predictive enough to obtain a good basis for establishing a reliable structure-odor
relationship. [3]
[1] Verbeurgt C. et al. Profiling of Olfactory Receptor Gene Expression in Whole Human Olfactory Mucosa
PLoS ONE 9 (2014) e96333
[2] DeMarch C.A. et al. G Protein-Coupled Odorant Receptors: from sequence to structure Prot. Science 214
(2015) 1543-1548
[3] DeMarch C.A. et al. Structure-odor relationships reviewed in the postgenomic era FFJ 30 (2015) 342-361
P2-037 Natural variation and manipulation of genes underlying free-flight odor tracking in Drosophila
Benjamin Houot1,2, Alain Robichon3, Jean-Francois Ferveur1,2
1
CNRS, France, 2Universite de Bourgogne Franche Comte, France, 3Institut Sophia Agrobiotech, France
Insect ability to fly has likely evolved with the acquisition of adaptive mechanisms allowing individuals to escape
from terrestrial predators and migrate to new ecological niches. The biological basis of flight has been
investigated in insects with a special emphasis, in Drosophila species, on its developmental, sensory-motor,
behavioral and aerodynamic determination.Also, the impact of visual, mechanosensory and olfactory cues on
free-flight has been deeply investigated in D.melanogaster laboratory strains. We measure, in a wind tunnel,
how odorant attractiveness can be modulated according to (i) natural variation and to targeted alteration of (ii)
wing chemosensory sensilla, or of (iii) tissues involved in pheromonal communication. The comparison between
sexes in four species and three D. melanogaster populations reveals an important divergence for several flight
aspects (lifting-up, flying, landing). Moreover, the genetic manipulation of wing chemosensory organs or of
pheromone-related tissues, not always of neuronal nature, specifically affects one or several of these free-flight
aspects. Our data also pinpoints a putative sexual dimorphism in the chemosensors of the anterior wing margin.
These sex-, population-, species- and tissue-specific variations indicate that the modulation of olfactory-induced
flight results of a complex evolutionary adaptation determined by the subtle balance of gene expression in a
variety of tissues.
P2-038 Expression analysis of Bcl11b in Xenopus tropicalis olfactory epithelium
Kanako Nakayama1, Hikoyu Suzuki2, Masato Nikaido2, Takayuki Enomoto3, Junji Hirota1,3
1
Department of Bioengineering, Tokyo Institute of Technology, Yokohama, Japan, 2Department of Bioscience, Tokyo Institute of
Technology, Yokohama, Japan, 3Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama, Japan
Each species has developed a distinct repertoire of odorant receptors (ORs) during evolution to adapt lifestyle
2016.05.27 15.17.09 Page 17(1)
and environment. During metamorphosis of Xenopus tropicalis , the main olfactory epithelium (MOE) is
remodeled and divided into principal cavity (PC: air nose) and middle cavity (MC: water nose). In adult X.
tropicalis , terrestrial-specific class II OR genes are exclusively expressed in the PC, whereas the expression of
fish-like class I OR genes is restricted to the MC. Bcl11b/Ctip2 gene encodes a zinc finger transcription factor.
Because Bcl11b is specifically expressed in class II OR-expressing olfactory sensory neurons in mice, it is
conceivable that Bcl11b has important roles in the expression of terrestrial specific class II ORs. Here, to
investigate the function of Bcl11b in the evolutionary process of olfactory system from aquatic to terrestrial, we
examined the expression of Bcl11b in the X. tropicalis olfactory epithelium. In X. tropicalis , XtBcl11b was
predominantly expressed in the PC and co-expressed with class II OR genes, indicating an important role for
class II OR genes expression in adult and metamorphic X. tropicalis olfactory system. In this paper, we also
reported the expression of zBcl11b in the Danio rerio olfactory epithelium and propose the hypothesis that Bcl
11b has a key role for adaptation to terrestrial olfaction.
P2-039 Lhx2 and emx2 determine odorant receptor expression frequency in mature olfactory sensory
neurons
Timothy S. McClintock, Guangfan Zhang, William Titlo
Department of Physiology, University of Kentucky, Lexington, KY, USA
Homeodomain-like sites are functional elements of odorant receptor (OR) gene control regions. Lhx2 and Emx2
are the most likely transcription factors acting at these elements because they bind proximal promoters; and in
the case of Lhx2, OR enhancers as well. To avoid the loss of mature olfactory sensory neurons (OSNs) that
confounds the assessment of germ line knockouts of Lhx2 and Emx2, we restricted the deletion of Lhx2 and
Emx2 to immature and mature mouse OSNs. Loss of Lhx2 in immature OSNs decreases 676 OR mRNAs.
These decreases are due to reduced frequencies of expression rather than reduced amounts of OR mRNA per
OSN. Deletion of Emx2 gives much smaller effects and more often results in increased expression frequencies
of ORs. Deletion of both Lhx2 and Emx2 in immature OSNs has a distinct additive, rather than synergistic, effect;
reducing the frequency of expression of 755 OR mRNAs. Contrary to expectations about the stability of OR
expression in OSNs, conditional deletion of Lhx2 in mature OSNs is very similar to deletion in immature OSNs,
decreasing the frequency of expression of 765 ORs, but also causing increased expression frequencies of
several ORs. Of 1,098 OR mRNAs measured, only 44 are insensitive to the loss of Lhx2 across all three deletion
genotypes tested. Lhx2 controls expression of nearly all OR genes via a mechanism that not only helps
determine which OR gene is chosen for expression in each immature OSN, but also is necessary to maintain
expression of the chosen OR. Our data suggests the hypothesis that Emx2 acts differently, probably by helping
to control the availability of OR genes for expression.
P2-040 Trigeminal modulation of olfactory signal processing
Federica Genovese, Hanke Gwendolyn Bauersachs, Ines Graesser, Janina Kupke, Marion Thews, Frank Moehrlen,
Stephan Frings
Department of Molecular Physiology, University of Heidelberg, Heidelberg, Germany
Olfactory perception can be described as the integration of olfactory and trigeminal systems, as most odors can
act as irritants, and almost every irritant has an odor. Peptidergic trigeminal fibers have been found in close
association with the olfactory system. In the main olfactory epithelium, fibers that contain calcitonin gene-related
peptide (CGRP) are found among olfactory sensory neurons (OSNs) and between epithelial supporting cells.
CGRP inhibits the odor response of OSNs, an effect that may cause the reduced odor perception during
trigeminal co-stimulation. In the olfactory bulb, the function of CGRP-positive fibers is not well understood. We
performed a study of trigeminal innervation of the olfactory system and traced the trajectory of the ethmoid
branch of the trigeminal nerve from the trigeminal ganglia through the main olfactory bulb to the nasal cavity. A
detailed examination of the trigeminal innervation of the glomerular layer revealed a close proximity of
CGRP-positive fibers with juxtaglomerular interneurons that expressed calbindin, calretinin or tyrosine
hydroxylase, but not those expressing parvalbumin. Serial section of the mouse olfactory bulb showed that half
of all glomeruli showed this pattern of innervation and may be considered possible targets for trigeminal
neuromodulation. CGRP release from bulbar fibers was induced by agonists of TRPA1 and TRPV1. Results of
functional studies suggested that CGRP influenced signal processing in the bulb through an inhibitory effect on
periglomerular microcircuits. This became evident through a CGRP-induced reduction of c-Fos immunosignals
in interneurons of the glomerular layer. Furthermore, the spontaneous activity pattern of mitral cells, the output
2016.05.27 15.17.09 Page 18(1)
neurons of the olfactory bulb, was altered upon application of CGRP to acute slices. Our data point to a
modulatory role of trigeminal neuropeptides in the first steps of olfactory signal processing.
P2-041 Morphological characteristics of neuron-like cells in the vomeronasal organ of human fetuses
Shigeru Takami1,2, Sawa Horie3, George Matsumura4
1
Sakai Electron Microscopy Application Laboratory, Saiatama, Japan, 2Department of Physiology, Iwate Medical University School
of Dentistry, Iwate, Japan, 3Department of Anatomy, Kawasaki Medical School, Okayama, Japan, 4Department of Anatomy, Faculty
of Medicine, Kyorin University, Tokyo, Japan
The vomeronasal organ (VNO) of five-month-old fetuses was examined immunohistochemically by the use of an
antiserum to protein gene product 9.5 (PGP). The PGP antiserum labeled abundant cells within the vomeronasal
epithelium (VE), nerve fiber bundles in its lamina propria, and cells associated with these bundles.
PGP-immunoreactive (ir) vomeronasal epithelial cells were classified into three subtypes. Type I cells, about
44% of the total cells observed, did not have any processes and tended to be located in the basal layer of the
VE. Type II cells, about 37%, had a single apical process that projected toward the lumen, ending at the
epithelial surface. Type III cells sent a prominent process mainly toward the basement membrane, and occupied
about 19% of the total cells observed. In the lamina propria, PGP-ir cells were also observed. Some of them
were present in nerve fiber bundles and contained processes parallel to the bundles. In addition, PGP-ir nerve
fiber bundles and cells associated with them were even present in the portion of the nasal septal mucosa that
was very close to the brain. The present results strongly suggest that the VE in human fetuses at mid-gestation
is a neuroepithelium and that the VE may produce migrating cells toward the brain.
P2-042 Evolution of odorant receptor gene family in social Hymenoptera
Masaru K. Hojo1, Shuji Shigenobu2,3, Kenichi Ishii4, Katsushi Yamaguchi2, Midori Sakura1, Mamiko Ozaki1
1
Department of Biology, Kobe University, Kobe, Japan, 2NIBB Core Research Facilities, National Institute for Basic Biology,
National Institutes of Natural Sciences, Okazaki, Japan, 3Department of Basic Biology, Graduate University for Advanced Studies,
Okazaki, Japan, 4The Salk Institute for Biological Studies, CA, USA
Social insects exhibit complex societies characterized by self-organized division of labor, as well as conflict of
interests among colony members. Pheromones play a major role in regulating cooperative behavior, division of
labor and conflict resolution. Especially, cuticular hydrocarbons (CHCs) convey various individual-information,
and thus important chemical signals for their societies. Therefore, it is plausible that specific expansion of
odorant receptor genes for CHCs perception underlie sophisticated communication required for complex social
organization. By conducting genome sequencing of the ant Camponotus japonicus followed by extensive
manual annotation, we found over 400 chemosensory receptor genes (GRs, ORs and IRs) in this species. We
also conducted comparative antennal RNA-seq analyses among the workers, alate queens and males, and
revealed dozens of odorant receptors (ORs) showing female biased expression in their antennae. Phylogenetic
analyses indicated that these female-biased ORs shape the specific cluster in their phylogeny. We also found
these ORs are expanded only in social lineages of Hymenoptera (wasps, bees, and ants), especially in ants.
These results suggest evolution of specific clade of ORs are crucial for social organization, and we will discuss
the possible function of these OR genes.
P2-043 Broadly tuned Drosophila olfactory receptor responses allow recognition of a wide range of
volatiles
Thomas Nowotny1, Marien de Bruyne2, Amalia Z. Berna3, Coral G. Warr2, Stephen C. Trowell3
1
School of Engineering and Informatics, University of Sussex, Brighton, UK, 2School of Biological Sciences, Monash University,
Clayton, Australia, 3Food and Nutrition, CSIRO, Canberra, Australia
Driven by the motivation of using Drosophila receptors as biosensors in technical applications we investigated
the suitability of the olfactory receptor neuron (ORN) responses for recognising a variety of volatiles. We
performed in vivo sensillar recordings on Drosophila melanogaster exposed to two sets of volatiles, a “wine set”
of 36 odours from fermenting fruit and an “industrial set” of 35 volatiles related to chemical hazards, including
fires, explosives, or illegal drugs. Individual ORN types were identified and their responses were encoded by the
mean firing rate above or below baseline. We then used machine learning to assess how well the chemicals in
each set could be individually recognised based on the recorded Drosophila ORN responses. We found to our
surprise that the ORN responses led to the same accuracy of odour recognition in both sets, even though the
wine set is ecologically highly relevant to flies, and the industrial set is not. Drosophila receptors hence appear
2016.05.27 15.17.09 Page 19(1)
highly capable of distinguishing chemicals that they have not evolved to process. We then evaluated which
combination of receptor types led to the best recognition performance. In contrast to our previous work with
metal oxide sensors [1], we found that Drosophila receptors achieved the best recognition accuracy if the data
from all 20 available receptor types were used. The results presented here are published in the Bioinspiration
and Biomimetics Journal [2].
[1] Nowotny et al., Sens. Actuators B 187 (2013) 471-480.
[2] Nowotny et al., Bioinspir. Biomim. 9 (2014) 046007.
P2-044 Caloric restriction in mice reduces basal cell proliferation and neuroepithelial regeneration
following olfactotoxic mucosal damage
Hitoshi Iwamura, Kenji Kondo, Tatsuya Yamasoba
Department of Otolaryngology-Head and Neck Surgery, The University of Tokyo, Graduate School of Medicine, Tokyo, Japan
Caloric restriction (CR) effects on mouse olfactory neuroepithelial cell dynamics were investigated. Eight-weekold male C57BL/6 mice were fed either a control (104 kcal/week) or 36% calorie restricted (67 kcal/week) diet.
The uninjured olfactory neuroepithelium cytoarchitecture and its regeneration after exposure to methimazole, an
olfactotoxic chemical, were compared between mice fed with control and CR diets. The effect of the timing of CR
intervention on neuroepithelial regeneration after injury was also examined. In uninjured olfactory mucosa, the
neuroepithelial thickness and number of olfactory marker protein (OMP)-positive olfactory receptor neurons
(ORNs) in septal olfactory mucosa were not significantly different between groups at 1 month, but there were
significantly fewer 5-bromodeoxyuridine (BrdU)-positive proliferating basal cells in the CR group. At 3 months,
the CR group showed a significantly thinner neuroepithelial thickness containing significantly fewer
OMP-positive ORNs and BrdU-positive proliferating basal cells. Methimazole-induced mucosal injury increased
the number of BrdU-positive basal cells in both groups, but the induction was lower with CR. Final
neuroepithelium recovery was less complete in mice kept under CR before methimazole administration than
those under CR after the administration. In conclusion, CR may be disadvantageous to olfactory neuroepithelial
maintenance and renewal, particularly after injury.
P2-045 Odorant reception in the diamondback moth Plutella xylostella
Guirong Wang, Mengjing Sun, Jiao Zhu, Paolo Pelosi
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural
Sciences, Beijing, China
Olfaction is critical for insect survival, mediating key behaviors such as host preference, mate choice, and
oviposition site selection. We identified the olfactory gene subfamilies of the important vegetable pest, the
diamondback moth Plutella xylostella through de novo transcriptomic analysis. These genes include 25 OBPs,
54 ORs, 16 IRs and 15 CSP. We used fluorescence spectrophotometer to measure the binding affinity of PBP to
sex pheromone and its analogs. We also used heterologous expression system Xenopus oocyte combined twoelectrode voltage clamp to deorphanized PRs in Plutella xylostella. However, electrophysiological and
behavioral assays didnot match with molecular studies. Further investigation revealed that PBP increased the
sensitivity of PR to sex pheromone components. Behavioral assays suggested larvae of the diamondback moth
Plutella xylostella were attracted to their natural sex pheromone and to their major sex pheromone component.
However, all three PBPs were detected not to be expressed in the larval antennae. Instead, two general
odorant-binding proteins, abundantly expressed in the three major sensilla basiconica of the larval antenna,
involved in sex pheromone component reception. We also provided evidence that GOBP2 was a narrowly tuned
binding protein, whose affinity could be easily switched from linear pheromones to branched plants terpenoids.
P2-046 Lysine-Specific Demethylase-1 is required for maintenance of monogenic OR expression
Rutesh Vyas, Diane Meredith, Robert P. Lane
Department of Molecular Biology and Biochemistry, Wesleyan University, CT, USA
The proper function of the mouse olfactory system depends on developing specialized sensory neurons that
each respond to a narrow range of odorant chemistry. In mice, this specialization is accomplished by the
expression of only one odorant receptor (OR) gene per cell from a genome repertoire of 1400 OR genes. Recent
evidence suggests epigenetic mechanisms contributing to monogenic OR transcription, including a role for
2016.05.27 15.17.09 Page 20(1)
nuclear compartmentalization and regulation of histone methylation in the establishment of monogenic OR
expression. The lysine-specific demethylase-1 (LSD1) is a versatile protein utilized in repressing and activating
functions in various developmental contexts, by functioning in H3K4 and H3K9 histone demethylation pathways,
respectively. In the context of OR regulation, previous work has implicated LSD1 as functioning in regulating
monogenic OR expression by restricting its H3K9 (activating) histone demethylation activity to only one OR per
cell. Here, we show that mouse LSD1 protein is compartmentalized in the nucleus during a narrow
developmental window within the earliest post-mitotic cells of the olfactory sensory neuronal lineage when OR
selections are thought to be occurring; these LSD1 compartments appear to interact with one or a small number
of OR loci at a time, consistent with possibly playing a role in mutually exclusive selection. We have used RNAi
to knockdown LSD1 expression to undetectable levels within an olfactory-placode derived cell line (OP6). We
show that LSD1 depletion disrupts monogenic OR transcription in these cells, suggesting that LSD1 is normally
required to prevent multiple OR activation events per cell. Therefore, our data provides new and surprising
insights into a possible function of LSD1 in OR regulation, suggesting a role in preventing multi-genic OR
expression (i.e., a generic repressive role), thereby challenging previous models that propose a function in
effectuating mono-genic OR expression via an exclusive activating function.
P2-047 An investigation into the differences in damage of the olfactory epithelium between female and
male mice after administration of methimazole
Kentaro Yamada1, Hideaki Shiga1, Hideaki Ninomiya2, Jyunpei Yamamoto1, Takuya Noda1, Masayuki Harita1,
Yukari Nakamura1, Tomoko Hiraba1, Takaki Miwa1
1
Department of Otorhinolaryngology, Kanazawa Medical University, Ishikawa, Japan, 2Medical Resarch Institute, Kanazawa
Medical University, Ishikawa, Japan
Introduction Upper respiratory tract infection (URTI) is the second largest cause of olfactory dysfunction.
Furthermore, olfactory dysfunction after URTI is common in middle-aged and elderly women. However, the
reason why olfactory dysfunction after URTI often occurs in middle-aged and elderly women has not been
shown. In this study, we aimed to assess whether the olfactory epithelial damage is more severe in female mice
rather than in male mice. Materials and methods Thirty Bulb-c female and male mice (8 weeks of age; young
adult mice) were assessed. Methimazole is shown to induce olfactory epithelium damage. The heads of three
subsets of mice were resected respectively at 2w, 4w and 6w after intraperitoneal administration of methimazole
(75mg/kg). Samples were paraffin embedded, and the slices were stained with olfactory marker protein (a
marker of mature olfactory receptor cells) by immunohistochemistry. The thickness of the olfactory epithelium in
the nasal septum was measured under microscope. Results There were no significant differences between the
male and female mice in the thickness of the olfactory epithelium at 2w, 4w and 6w after administration of
methimazole. Conclusion Our results suggest that there is no difference in damage of the olfactory epithelium
between young adult female mice and male mice. We are considering additional study.
P2-048 Effect of Kampo medicine Tokishakuyakusan on olfactory epithelium in olfactory impaired mice
Takuya Noda1, Hideaki Shiga1, Hideaki Ninomiya2, Yukari Nakamura1, Tomoko Hiraba1, Masayuki Harita1, Kentaro Yamada1,
Junpei Yamamoto1, Takaki Miwa1
1
Department of Otorhinolaryngology, Kanazawa Medical University, Japan, 2Medical Research Institute, Kanazawa Medical
University, Japan
Olfactory impaired patients with rhinosinusitis or allergic rhinitis respond well to treatment with intranasal or oral
corticosteroids. However, treatments with corticosteroids are not as effective in olfactory impaired patients after
upper respiratory tract infections. In Japan, the herbal formula, Tokishakuyakusan (TSS), is often administered
to URTI patients with olfactory dysfunction. TSS is usually prescribed for treatment of dysmenorrhea,
menopausal disorders, and anemia. In this study, we investigated the effect of TSS in mice peripheral olfactory
systems after olfactotoxic drug induced damage. 10 female, 8 week old, BALB/C mice were used. We induced
olfactory dysfunction in them by intraperitoneal administration of methimazole (75mg/kg). After one week, the
mice were divided into two groups: one control group and one group fed with TSS. The Heads of all mice were
dissected one week after starting TSS feeding. We investigated the thickness of the olfactory epithelium in mice.
We also examined the expression of olfactory marker protein (OMP) and doublecortin (DCX) in the mice nasal
epithelium. We found that the thickness of the olfactory epithelium was significantly reduced by Methimazole, but
the olfactory epithelium in the TSS feeding group was significantly thicker than in the control group. Also, OMP
expression was significantly more in the TSS feeding group than in the control group. There was no significant
statistical difference regarding DCX expression. The results of this study suggest that Tokishakuyakusan
2016.05.27 15.17.09 Page 21(1)
activates regeneration of olfactory neurons after olfactotoxic drug induced damage in mice.
P2-049 Regulation of insect odorant receptors
Fabio Miazzi, Merid N. Getahun, Shannon B. Olsson, Bill S. Hansson, Dieter Wicher
Max Planck Institute for Chemical Ecology, Jena, Germany
Flying insects have a remarkably sensitive olfactory system that allows to detect faint and turbulent odor traces.
This ability relies on the olfactory receptors class of odorant receptors (ORs) the occurrence of which is
restricted to winged insects. Insects ORs are ligand-gated ion channels composed of two seven-transmembrane
domain proteins, an odorant-specific protein OrX and a ubiquitous coreceptor (Orco). The sensitivity of the ORs
is adjusted according to the previous background odor concentration. A sensitization on demand represents a
short term memory which lasts from a couple of seconds. Sensory neurons expressing the olfactory receptor
class of ionotropic receptors cannot regulate the sensitivity to odors. The regulation of OR sensitivity contributes
to the capability of OR expressing neurons to detect odors over a wide range of concentrations. Part of the
sensitization process is the activation of Orco proteins which are known to form homomeric ion channels
activated by cAMP and permeable to cations including calcium. We here demonstrate that OR sensitization
depends on calmodulin signalling. Inhibition of calmodulin disrupted the sensitization in the outer dendritic
segment of the sensory neurons. Furthermore, using flies expressing the cAMP reporter Epac1-camps in
olfactory sensory neurons we show that odor stimulation of ORs leads to cAMP production. In parallel, odor
stimulation enhances Orco phosphorylation by PKC which was previously found to be required for
cAMP-induced activation of Orco. On the other hand, mutation of PKC sites in Orco or mutations in PKC
subtypes reduced the sensitivity and dynamic ranges of OR expressing neurons in vivo and thus the
competence for odor localization of flies during flight. We conclude that intracellular regulation of OR sensitivity is
necessary for efficient odor localization, which suggests a mechanistic advantage for the evolution of the OR
complex in flying insects. Supported by Max Planck Society and DFG (SPP 1392).
P2-050 The guanylyl cyclase D-expressing olfactory subsystem is upregulated in mice lacking the
cyclic nucleotide-gated channel A2 subunit
Weihong Lin, Parisa Rangghran, Ashmita Adhikari, Tatsuya Ogura
Department of Biological Sciences, University of Maryland, Baltimore, USA
The main olfactory epithelium (MOE) consists of several subsets of olfactory sensory neurons (OSNs) that differ
in sensory transduction mechanisms. While the vast majority of OSNs utilizes the canonical cAMP signaling
transduction pathway, sensory detection in the guanylyl cyclase D-expressing (GC-D+) OSNs does not involve
cAMP. The GC-D+ OSNs project axons caudally to necklace glomeruli (NG) and are important for sensing CO2
and for acquiring socially transmitted food preferences. Whether activities of these olfactory subsystems
influence or compensate each other is largely not known. Using the cyclic nucleotide-gated channel A2 subunit
knockout mice (CNGA2-KO) to abolish the canonical sensory signaling, we investigated activity-dependent
impacts of the canonical OSNs on the GC-D+ olfactory subsystem. We found that CNGA2 knockout leads to a
significant increase in the number of GC-D+ OSNs visualized by immunolabeling of a cGMP-specific
phosphodiesterase 2A (PDE2A) in the MOE. Concomitantly, the size and number of NG are also increased
significantly and some NG also shift anteriorly as compared with the wild-type mice. Additionally, there is a
striking increase in ectopic projection of PDE2A immunoreactive axons to regions where canonical glomeruli are
located. These ectopically projected axons exhibit a higher level of immunoreactivity of vesicular glutamate
transporter 2 than the surrounding CNGA2-null axons, indicating that these ectopically projected axons make
synaptic connections and are active. Furthermore, some of the ectopic glomeruli are also associated with
relatively normal expression of the sensory activity dependent marker tyrosine hydroxylase. Together, our data
provide strong evidence for the upregulation of the non-canonical GC-D+ subsystem in the absence of canonical
olfactory signaling activity. Our data also imply that canonical afferent activity helps to restrict GC-D+ axons to
caudal olfactory bulb and prevents their ectopic projection. Supported by NIH/NIDCD DC009269 and 012831 to
WL.
2016.05.27 15.17.09 Page 22(1)
P2-051 Structure-function relationshio of two soluble proteins involved in chemoreception
Durige Wen2, Xing Li1, Masaru Hojo3, Mamiko Osaki3, Tatsuo Iwasa1,2
1
Division of Engineering, Muroran Institute of Technology, Muroran, Japan, 2Division of Production Systems Engineering, Muroran
Institute of Technology, Muroran, Japan, 3Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan.
The sense of smell begins with the binding of small chemicals called odorants to the odorant receptors on the
cilia membrane of olfactory neurons. Given that mucus layers cover these cilia and most odorants are
hydrophobic, it would appear difficult for odorants to reach the receptor. A large amount of soluble proteins
named odorant-binding proteins (OBP) or chemosensory proteins (CSP) were found in the lymph or mucus
surrounding the chemoreceptor cells. Because these proteins can bind to hydrophobic compounds like odorants
and pheromones, they can transport the compounds to the relevant receptors and modulate the receptor
functions. These proteins are called peri-receptor proteins (PRPs). This study aims to elucidate the physiological
role of PRPs in chemoreception. Here, we cloned, expressed, and analyzed two kinds of PRPs, namely
CjapCSP1 found from the sensillum of the carpenter ant, Camponotus japonica , and Cp-Lip1 found from the
olfactory organ of the Japanese common newt, Cynops pyrrhogaster . These proteins were expressed using the
Escherichia coli expression system and purified by the tag-affinity column followed by Q-column.
MALDI-TOFMS analysis of both purified proteins resulted in a single peak. The secondary structure of the
proteins was investigated using circular dichroism (CD) measurements. Proteins that belong to the CSP group
have α-helix-rich structures and those that belong to OBP have β-strand-rich structures. The CD spectra of both
purified proteins showed the characteristic features of the corresponding structure indicating that they retained
their native structures. Fluorescence-quenching experiments were carried out on both proteins. The fluorescent
probe, 1-NPN, was replaced with (z)-9-tricosene, and bis-ANS was replaced with IBMP in CjapCSP1 and
Cp-Lip1, respectively, indicating the odorant-binding capacity of both proteins. We produced a series of mutants
of these proteins, and their properties will be presented and discussed in the poster.
P2-052 Identification of putative chemosensory G-protein coupled receptors in Crown-of-thorns
starfish, Acanthaster planci
Rebecca E. Roberts1, Scott F. Cummins1, Mike R. Hall2
1
Department of Science and Engineering, University of the Sunshine Coast, Sippy Downs, Australia, 2Australian Institute of Marine
Science, Townsville, Queensland, Australia
Chemosensation is critical in marine environments, particularly for invertebrates such as Echinoderms, which
have poorly developed other senses. Recent advances in genomics and transcriptomics have facilitated
breakthroughs in understanding chemosensation in aquatic environments, potentially yielding novel biological
control methods for invasive species including the Crown-of-thorns starfish (COTS), Acanthaster planci. COTS
is a coral predator which undergoes cyclic population outbreaks and can destroy entire coral reefs. In this study,
we have identified putative chemosensory rhodopsin-like G-coupled protein receptors (GPCRs) within the
genome and olfactory organ transcriptomes of COTS, many of which cluster within the COTS genome. Tube
feet and terminal sensory tentacles contain the highest proportion of chemoreceptors and in situ hybridisation
shows specific transcript localisation within their sensory epithelia. This is the first study to identify
chemosensory receptors in a starfish and our results provide a basis for future research which may enable the
development of a biological control for COTS, preventing further outbreaks of this species on the Great Barrier
Reef and other reefs globally.
P2-053 Comparison of ionic currents between human and newt olfactory epithelium cells
Kengo Tamari1,2, Masayoshi Kobayashi2, Kazuhiko Takeuchi2, Tetsuro Yamamoto3
1
College of Liberal Arts and Sciences, Mie University, Mie, Japan, 2Department of Otorhinolaryngology-Head and Neck Surgery,
Mie University Graduate School of Medicine, Mie, Japan, 3Department of Neurophysiology, Mie University Graduate School of
Medicine, Mie, Japan
Many electrophysiological studies of olfactory epithelium cell have been performed only with non-human
materials such as newt. In order to confirm that isolated olfactory epithelium cell of other kinds such as newt can
adapt for studies of ionic currents in human olfaction, we tried to test whether human nasal tissues surgically
extracted in the ordinary clinical setting contains olfactory receptor cells and it is possible to analyze their
electrophysiological properties. First we examined the olfactory function of the treated-patient and revealed that
olfactory functions were not severely affected after surgery by usual clinical estimations. The surgically extracted
olfactory tissues were treated to dissociate epithelial cells as usual in the case of newt tissues and checked
2016.05.27 15.17.09 Page 23(1)
histologically by double fluorescent staining using olfactory marker protein and 4’, 6-diamidino-2-phenylindole
(DAPI). The dissociated cells were checked by characteristic morphological features of olfactory epithelium cells
(bipolar cell, an apical dendritic knob and size of 5-10 μm in diameter). The cells with such features were
targeted for whole cell patch-clamp recording. The whole cell clamping showed following features; Transient
inward currents were activated at -40 mV, reached a peak at about -30 mV. Sustained outward currents were
activated at -40 mV and suppressed by TEA (tetraethyl ammonium). These electrophysiological findings were
similar to those obtained in our previous studies on newt. These facts suggested the possibility that isolated
olfactory epithelium cell of non-human animal model such as newt can be available for analyzing human
olfactory mechanisms.
P2-054 Ultrastructure of the olfactory sensory unit consisting of 100 receptor neurons for nestmatenonnestmate discrimination in Camponotus japonicus
Yusuke Takeichi1, Kouji Yasuyama2, Naoyuki Miyazaki3, Kazuyoshi Murata4, Masaru K. Hojo1, Kanako Inoue5, Mamiko Ozaki1
1
Department of Biology, Kobe University, Hyogo, Japan, 2Department of Biology, Kawasaki Medical School, Okayama, Japan,
Institute for Protein Research, Osaka University, Osaka, Japan, 4National Institute of Physiological Science, Aichi, Japan,
5
Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Osaka, Japan
3
It has been reported that the workers of Japanese carpenter ant, Camponotus japonicus , discriminate their
nestmates from non-nestmates with the cuticular hydrocarbon (CHC) patterns using the sensilla basiconica on
the antennae. This type of sensilla consists of about 100 olfactory receptor neurons (ORNs). In this study, we
investigated the ultrastructure of the sensilla basiconica using serial block face scanning electron microscope
(SBF-SEM). In the distal end of the sensilla basiconica, we could not find dendritic processes of the ORNs
beneath the olfactory pores, thus we recognized here as a dendritic processes-free space. Based on the serial
images from the middle to the basement of the sensilla basiconica, we constructed its 3D model. It showed a
twisting bundle of the dendritic processes having no branches but characteristic swellings. At these swellings,
adjacent cell membranes of dendritic processes were closely associated each other. In a sensilla basiconica, the
total number of swellings are 388, 75% of all of the swellings were closely adjacent to other dendritic processes.
Every dendritic process had 1-7 swellings in the sensillum. Thus the total interaction points were estimates at
933 regions. We supposed some of these interaction points among ORNs were not only morphological but also
functional for signal transduction. If it is the case, they might affect the functional mechanism of the sensilla
basiconica as a sensory unit to detect a difference in the CHC patterns.
P2-055 Transcriptomic deconstruction of mammalian olfaction: From human to mouse
Luis R. Saraiva1,2,3, Ximena Ibarra-Soria2, Fernando R.M. Aguilera2, Mona Khan4, Laura Van Gerven5, Mark Jorissen5,
Peter Mombaerts4, John C. Marioni2,3, Darren W. Logan2,6
1
Department of Experimental Genetics, Sidra Medical & Research Center, Qatar Foundation, Doha, Qatar,
Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton-Cambridge, UK, 3European Bioinformatics Institute (EMBLEBI), European Molecular Biology Laboratory, Wellcome Genome Campus, Hinxton-Cambridge, UK, 4Max Planck Research Unit
for Neurogenetics, Frankfurt, Germany,
5
Department of ENT-HNS, UZ Leuven, Belgium, 6Monell Chemical Senses Center, Philadelphia, USA
2
The importance of sensing the molecular environment is reflected in the genetic investment in encoding olfactory
receptors (ORs), which constitute the largest mammalian gene family. The OR repertoire is largely
species-specific, and shaped by the nature and necessity of chemosensory information for survival in each
species’ niche. In addition to differences in the ORs, the morphology, size, neural projections and organization of
chemosensory epithelia vary remarkably across mammals, suggesting differences in wider gene expression
networks. By combining RNA-seq with FACS in a hierarchical fashion from whole olfactory mucosa (WOM) to
single olfactory sensory neurons (OSNs), we have identified the complete transcriptional profile of mouse OSNs,
and their heterogeneity at the single cell level. But 25 years after the discovery of the ORs, the interspecific
molecular heterogeneity of the olfactory system still remains largely unknown. To study the evolutionary
dynamics of gene expression in the olfactory system among species with different chemosensory niches, we
performed RNA-seq of the WOM of six species of rodents, carnivores and primates (including humans). Our
comparative transcriptome-wide analysis reveals a high degree of molecular conservation across 95 million
years of mammalian evolution. We found that ORs are expressed across a large dynamic range in these six
species. RNA abundances correlate well with the number of OSNs expressing an OR. Combining RNA-seq with
a phylogeny-based method that classifies ORs into orthologous gene groups (OGGs), we found that
phylogenetic conservation does not imply conservation in OR gene expression: we find numerous examples of
2016.05.27 15.17.09 Page 24(1)
highly-expressed ORs specific for a single species or order. Of the 73 highly conserved one-to-one OGGs, one
only case was the OR orthologue highly expressed in all species analysed. This experimental strategy has
identified OR genes that may have been selected for different niches, contributing to a better understanding of
the evolution of olfaction.
P2-056 Differential localization of GPCRs to cilia of neurons in the olfactory bulb
Jeremy C. McIntyre
Department of Neuroscience, Center for Smell and Taste, University of Florida, USA
Primary cilia are signaling centers for numerous extracellular cues. In the olfactory system it is well known that
odorant receptors, 7-transmembrane GPCRs, localize to the cilia of olfactory sensory neurons where they
function to detect volatile chemicals. Neurons found in the central nervous system also posses a primary cilium,
where other GPCRs are known to be localized. However, the roles of neuronal cilia and the GPCRs localized
there in regulating neuronal function remains unclear. Recent work has demonstrated the neuronal cilia are
important for regulating dendritic aborization, while several of the GPCRs are receptors for neuromodulatory
cues that may be involved in regulating neuronal responses to stimulation. To address their role in neuronal
function, analysis of cilia and ciliary GPCRs, across the olfactory bulb (OB) was performed. Antibody staining for
AC3 and ARL13b revealed cilia present on interneurons surrounding glomeruli, mitral and tufted cells as well as
granule interneurons. To identify potential signaling pathways the localization of several GPCRs, SSTR3, MCHR
1, DRD1, HTR6, CHRM2, CHRM3, and CHRM5, was tested. Of these receptors, SSTR3 and MCHR1 were
detected in cilia on olfactory bulb neurons. SSTR3 was present in cilia on mitral and granule cells but not
glomerular neurons, while MCHR1 localized primarily to granule cells and glomerular interneurons. SSTR3 and
MCHR1 exhibited minimal co-localization in neuronal cilia. Ciliary localization of SSRT3 and MCHR1 was lost in
BBS4 knockout mice, suggesting both specificity in staining and that ciliopathies could also affect olfactory
function through alterations in the OB. The localization of these receptors to granule interneurons suggests a
mechanism by which the function of these cells can be modulated through hormonal regulation. Further study
will help to elucidate the functional roles of primary cilia on OB neurons.
P2-057 Studies on the cis-element for mouse class I odorant receptor genes using the Bacillus subtilis
genome vector
Tetsuo Iwata1, Shinya Kaneko2, Takafumi Ogawa3, Chizuru Kobayashi3, Yuh Shiwa4, Takayuki Enomoto1,
Hirofumi Yoshikawa4,5, Junji Hirota1,3
1
Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama, Japan, 2Department of Molecular
Bioscience, Tokyo Institute of Technology, Yokohama, Japan, 3Department of Bioengineering, Tokyo Institute of Technology,
Yokohama, Japan, 4Genome Research Center, NODAI Research Institute, Tokyo University of Agriculture, Tokyo, Japan,
5
Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan
Mouse odorant receptors (ORs) are phylogenetically classified into class I and class II. Class II OR (terrestrial
specific) genes are constitute approximately 90% of mouse OR genes and are distributed throughout the mouse
genome. Some cis- acting elements for class II OR genes have been identified. In contrast, class I OR gene
family, which consists of 160 genes, forms an approximately 3 Mb gene cluster on chromosome 7. The ciselement for this family has not yet been identified. In addition, deficiency of LIM-homeobox protein 2 (Lhx2), a
key transcription factor for class II OR genes, is not essential for class I OR genes expression, suggesting that
the different mechanisms regulate the expressions of the class I and II OR genes.
In this study, we applied the B. subtilis genome (BGM) vector system, which is a novel DNA cloning tool, to
explored the cis- acting element for class I OR genes. First, we cloned large mouse genomic DNA fragments
containing class I OR genes to the BGM vector using previously established bacterial artificial chromosome
(BAC) clones. We constructed two transgenes by inserting a reporter gene IRES-tauEGFP downstream of the
stop codon of a class I OR gene. We fused the two transgenes by mediating the inversion technique. We purified
the transgenes from the BGM vector, and generated transgenic mice. Transgenic mice carrying the enlarged
transgene expressed the reporter gene. Our studies demonstrate that the BGM vector system enables various
genetic modifications of large DNA fragments, and provide the first experimental evidence of a cis- acting
element for class I OR gene expression. We will report further functional analyses of the cis- acting element for
class I OR genes.
2016.05.27 15.17.09 Page 25(1)
P2-058 Odorant receptor expression is perturbed in mice following recovery from genetically-mediated
lesion
Jessica Brann1, Xinmin Zhang2, Eleonora Spinazzi3, Claire Franklin1, Lis Paulina1, Natasha Kharas3, Clara Altomare3,
Stuart Firestein3
Department of Biology, Loyola University Chicago, USA, 2BioInfoRx, USA, 3Department of Biological Sciences, Columbia
University, USA
1
A repository of basal neural stem cells in the olfactory epithelium (OE) generates excitatory projection neurons
that extend long axons from the OE to the olfactory bulb. Each sensory neuron expresses a single odorant
receptor (OR), conferring an identity required for odorant detection and appropriate axon targeting to the OB. It
has long been known that these stem cells generate sensory neurons, but their ability to faithfully recapitulate
the OE after lesion is relatively unexplored. In particular, aging may alter OR gene choice in newborn neurons.
Here we probe the ability of the stem cell to generate a diverse array of sensory neurons expressing the
appropriate repertoire of odorant receptors in aged animals. To this end we generated a line of mice, iDTR+
OMPCre/+, whereby a Cre-mediated excision of a STOP cassette renders mature neurons sensitive to
diphtheria toxin (DT) via activation of the DT receptor. This method permits a specific and reversible ablation of
mature (OMP-expressing) neurons upon DT administration. We administered either DT or saline to male mice of
several age groups (2-18 months) for six days. RNAs were harvested 30 days following ablation, to allow for
recovery of the OE. Results reveal that age does not affect the cohort of OR genes expressed following recovery
from lesion in DT injected mice. In addition we observe that age does not affect OR expression in saline injected
mice. However, the OR repertoire does significantly change following ablation and these effects are observed at
all ages tested. These results provide evidence that the regenerative potential of the neuronal stem cell is not
altered by age per se, as a wide array of sensory neurons are generated. However, lesion induced ablation of a
large number of sensory neurons disrupts the typical OR expression patterns observed in intact mice.
P2-059 PhOR3 contributes to the reception of DEET in body lice, Pediculus humanus corporis
Takuma Iwamatsu1,2, Hidefumi Mitsuno3, Daisuke Miyamoto1, Takeshi Sakurai3, Ryohei Kanzaki1,3
1
Department of Advanced Interdisciplinary Studies, The University of Tokyo, Tokyo, Japan, 2JSPS Research Fellow, Tokyo, Japan,
Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan
3
The control of the body lice, Pediculus humanus corporis , is an important issue for human health and welfare
because lice act as vectors of serious diseases. The continuous use of pyrethroid insecticides has led to the
emergence of insecticide-resistant lice. Therefore, insect repellent is effective in controlling body lice. Although
DEET, which is the standard insect repellent, was reported to be an effective repellent for body lice, its repellent
effect was not as strong as on other insects. Thus, the understanding of molecular mechanisms underlying
repellent effects of DEET is important for developing more effective repellent. DEET was previously reported to
directly activate olfactory receptor (OR136) in Culex quinquefasciatus . In body lice 10 genes were discovered as
candidate ORs (PhORs) in their genomes. Among 10 PhORs, one gene encodes a full-length OR co-receptor
(PhOrco) and 6 genes encode full-length ORs. However, it is unknown whether the PhOR is responsible for
reception of DEET in body lice. Here, we report that one PhOR, named PhOR3, contributes to DEET reception
in body lice. First, behavioral analysis indicated that DEET induces avoidance behavior in females but not in
males. Thus, PhORs predominantly expressed in female is likely to be involved in DEET reception. Sequence
analysis revealed that among 6 PhORs, PhOR4 contained a nonsense mutation. Next, in comparison of the
transcripts level of 5 PhORs between female and male lice by RT-PCR, PhOR3 was predominantly expressed in
female, whereas other PhORs were expressed in both sexes. Finally, functional analysis of 5 PhORs with
Xenopus oocytes revealed that oocytes co-expressing PhOR3 and PhOrco strongly responded to DEET among
90 tested odorants. Other PhORs did not respond to DEET. These results suggested that PhOR3 contributes to
DEET reception, and is a candidate OR for avoidance behavior to DEET in female body lice.
P2-060 Masking effect of katsuo-dashi odor on sour taste as measured by salivary hemodynamic
responses
Kazushi Yoshida1, Kana Iizumi1, Akiko Nakamura1, Kenji Haraguchi1, Tsukasa Saito1, Kensaku Mori1,2
1
R&D Center, T. HASEGAWA CO.,LTD., Kanagawa, Japan, 2The University of Tokyo, Japan
Sour taste-induced saliva secretion is mediated by reflex pathways in the brainstem and, based on perceived
sourness, can be modulated by signals likely transmitted from the forebrain. Therefore, to assess the masking
effect of an odor on a sour taste, we monitored salivary hemodynamic responses of the parotid glands to sour
2016.05.27 15.17.09 Page 26(1)
stimuli using near-infrared spectroscopy (NIRS). We focused on the masking effect of (4Z ,7Z )-trideca-4,7-dienal
(TDD), the key aroma-active compound in dried bonito, on sour taste because dried bonito soup stock, termed
katsuo-dashi , effectively masks sour tastes. First, we determined that the magnitude of salivary responses
positively correlated with the concentration of an acetic acid solution. Then, we found that the magnitude of
salivary responses to a 1% acetic acid solution with TDD was lesser than that to a 1% acetic acid without TDD.
With regard to sensory evaluation, the addition of TDD to the 1% acetic acid solution decreased the perceived
intensity of sourness of and the aversiveness to the acidic stimulus. These results indicate that the decrease in
salivary hemodynamic responses to the acetic acid solution positively correlated with the decrease in subjective
assessment of sourness of and aversiveness to the sour stimulus. Further, these findings suggest that a salivary
hemodynamic signal, as measured using NIRS, is a useful indicator of perceived sourness of and aversiveness
to sour stimuli. Because one of the important functions of saliva secretion is to buffer the acidity of sour solutions
based on perceived sourness, we speculate that the decreased salivary hemodynamic responses induced by
the addition of TDD to sour stimuli reflects the decrease in the perceived sourness of the solution.
P2-061 Development of neurospheres from olfactory receptor neuron precursors using a recombinant
protein
Samhwan Kim1, Seung-Jun Yoo1, Bongki Cho1, Seong-Kyun Choi2, Won Bae Jeon2, Cheil Moon1
1
Department of Brain and Cognitive Sciences, Daegy Gyeongbuk Institute of Science and Technology, Daegu, South Korea,
Division of NanoBio Technology, Daegy Gyeongbuk Institute of Science and Technology, Daegu, South Korea
2
Neurospheres are spherical free-floating cell clusters and composed of neural progenitor cells and stem cell
population so that can be differentiated into neurons by environmental modulations. However, the neurosphere
culture system is sensitive to experimental conditions such as cell density and medium composition. In this
research, we have introduced a new method for stable and reproducible generation of neurospheres, and
characterized the neurospheres using primary cultures of rat olfactory receptor neurons (ORNs). In order to
establish stable and reproducible neurospheres, a recombinant protein (REP) was employed. The REP was
designed for inducing fibronectin-integrin signaling pathway. Neurospheres were produced by incubating freshly
dissociated rat ORN precursors on REP coated plates. ORN neurospheres formed on the plates were firstly
characterized if stemness is maintained by immunocytochemistry (ICC) using stemness and proliferation
markers. We next examined a physiological function of ORNs by performing calcium imaging in differentiated
ORNs after neurospheres were moved to laminin coated plates. Also, electrical signals of ORNs activated by
odorant stimulation were measured using micro electrode array (MEA) to verify a physiological function of
ORNs. Expressions of nestin and Ki67 indicate that ORN neurospheres have innate characteristics of stemness
and proliferation. And elevation of intracellular calcium levels upon odorant stimulation in ORNs after
differentiation from neurospheres suggests that differentiated ORNs have innate function in odor detection.
Moreover, we found that different activating patterns in distinct odors and similar activating patterns in same
odors by analyzing electrical signals measured by MEA. Due to the introduction of REP, stable and reproducible
neurosphere generation was realized. ORNs derived from neurospheres were steadily activated by odors, and
which neurospheres maintain functions of ORNs during cultivation. Our results may facilitate ORN-based
bioelectronic nose development for chemical detection and advanced researches for understanding odor
encoding process in the mammalian olfactory system.
P2-062 Biomimetic chemical vapor sensor using agarose gel as mucus
Satoshi Fujii1, Aiko Nobukawa1,2, Toshihisa Osaki1,2, Nobuo Misawa1, Koki Kamiya1, Yuya Morimoto2, Shoji Takeuchi1,2
1
Kanagawa Academy of Science and Technology (KAST), Japan, 2Institute of Industrial Science (IIS), The University of Tokyo,
Japan
We have developed a biomimetic device for chemical vapor sensor. In olfactory system of living organism,
odorant molecules are absorbed by the mucus and captured by the receptor, which sends electrical signals to
nerve. In this study, we used agarose gel as an absorbent of volatile molecules, mimicking the muscus. Lipid
bilayer was formed by contacting the buffer solution and agarose gel, based on the reported technique called the
droplet contact method (1,2). Then, alpha-hemolysin was incorporated in the lipid bilayer. DNA aptamer
designed to bind with the target volatile molecule was included in agarose gel, and the chambers filled with the
buffer and the gel were connected to a patch-clamp amplifier for electrical recordings. Under a voltage clamp,
the DNA is able to translocate through the nanopore of alpha-hemolysin. When the chemical vapor, omethoate,
was applied to the environment of this device, 1) the agarose gel absorbed the vaporized omethoate, 2) the DNA
2016.05.27 15.17.09 Page 27(1)
aptamer selectively captured the omethoate, 3) built up a molecular complex, and 4) inhibited the ionic current
through a nanopore. We obtained two types of signals, the spike-like signals and the long-and-deep blocking.
The spike-like signals were due to single DNA molecule translocations, while the long-and-deep blocking
represented the clogging of the omethoate-DNA aptamer complex at the nanopore. We proposed a chemical
vapor sensor that would directly detect volatile molecule from vapor by designing the air-water interface using
the agarose gel. This work will be of use in the future for developing an on-site chemical vapor sensor. (1)
Funakoshi et al., Anal Chem., 2006, 78, 8169-8174. (2)Nobukawa et al., Proc. MicroTAS, 2015, 1577-1579.
P2-063 Therapy with combination tablet of antihistamine and vasoconstrictor for the control of nasal
obstruction and hyposmia in patients with allergic rhinitis
Motohiko Suzuki, Yoshihisa Nakamura, Shingo Murakami
Department of Otorhinolaryngology, Nagoya City University, Nagoya, Japan
Introduction: Many people are suffering from allergic rhinitis all over the world. Nasal obstruction and hyposmia
are major symptom caused by allergic rhinitis. Recently, combination tablet of antihistamine and vasoconstrictor
was released in Japan, and is paid attention to for the control of nasal obstruction and hyposmia in patients with
allergic rhinitis. However, it has been not reported to use combination tablet of antihistamine and vasoconstrictor
for long period, 8 weeks. And it had been controversy whether combination tablet of antihistamine and
vasoconstrictor was effective for the control of olfactory function in patients with allergic rhinitis. Therefore, we
examined the effect and safely of long use of combination tablet of antihistamine and vasoconstrictor in this
study. Methods: Ten patients, who visited Nagoya City University, were included in this study. Patients, include
in this study, received combination tablet of antihistamine (Fexofenadine 60mg) and vasoconstrictor
(Isoephedrine Hydrochloride 120mg) twice a day for 8 weeks, and received no drug for 2 weeks after therapy
with combnation tablet of antihistamine and vasoconstrictor. Results and Discussion: Side effects were not seen
in all patients after use for 8 weeks. Combination tablet of antihistamine and vasoconstrictor significantly
inhibited sneezing, nasal discharge, nasal obstruction, and olfactory dysfunction. The strongest correlation was
seen between olfactory dysfunction and nasal discharge.
P2-064 Improving the odorant sensitivity of olfactory receptor-expressing yeast with accessory
proteins
Yosuke Fukutani1, Ryohei Tamaki1, Kentaro Ikegami1, Hiroaki Matsunami2, Masafumi Yohda1
1
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan, 2Department of
Molecular Genetics and Microbiology, Duke University Medical Centre, Durham, North Carolina, USA
Olfaction depends on the selectivity and sensitivity of olfactory receptors. We have try to improve the
bioluminescence-based signaling assay system to create a practical application of a biomimetic odor sensor
using an engineered yeast-expressing olfactory receptors (ORs). Using the yeast endogenous pheromone
receptor (Ste2p) as a model GPCR, we determined the suitable promoters for the firefly luciferase (luc) reporter
and GPCR genes. Previous attempts at constructing a mammalian olfactory receptor-based artificial odorant
sensing system in the budding yeast Saccharomyces cerevisiae suffered from low sensitivity and activity. This
result may be at least in part due to poor functional expression of olfactory receptors and/or limited solubility of
some odorants in the medium. In this study, we examined the effects of two types of accessory proteins,
receptor transporting protein 1 short (RTP1s) and odorant binding proteins (OBPs), in improving odor-mediated
activation of olfactory receptors expressed in yeast. We found that RTP1s enhanced the membrane expression
and ligand-induced responses of some olfactory receptors. Usage of recombinant OBP of the silkworm moth
Bombyx mori enhanced the sensitivity of a mouse olfactory receptor. Our results suggest that different classes
of accessory proteins can confer sensitive and robust responses of olfactory receptors expressed in yeast.
Inclusion of accessory proteins may be essential in the future development of practical olfactory receptor-based
odorant sensors. Furthermore, we are attempting the structural analyze of RTP family proteins to understand
deeply about expression mechanism of OR. In this session, we will show the preliminary data on structural
analysis of RTP1s.
2016.05.27 15.17.09 Page 28(1)
P2-065 Continuous odour detection combining an e-nose with a spiking neural model based on the
insect olfactory system
Alan Diamond1, Michael Schmuker1, Amalia Berna2, Stephen C. Trowell2, Thomas Nowotny1
1
School of Engineering and Informatics, University of Sussex, UK, 2CSIRO, Black Mountain Laboratories, Canberra, Australia
In previous e-nose work, we have shown that odour identification can be improved by capturing aspects of the
temporal characteristics of sensor responses. For example, encoding odour responses as a set of timed sample
points relative to odour onset, is a straightforward approach which performs better than dynamic filters such as
the exponential moving average of the derivative. However, this approach nominally requires using precise
odour onset signals relative to which sample times can be determined; yet signals of this kind are generally
unavailable in real world situations, such as continuously operating odour detectors or animals’ environments.
Here we propose that using delays to create relatively timed samples could obviate the need for an onset signal.
Studies of the insect olfactory system suggest that axonal delays from spatially located receptors may indeed be
leveraged by neural circuitry to encode odour information. For example, varying spike transmission delays in the
Acheta Domesticus cricket have been shown to modulate the neural coding of odours.
To investigate the viability of our hypothesis, we started from an abstracted model of the insect antennal lobe,
added three different parallel delays between inputs and projection neurons, and implemented an algorithm
which identifies active periods to demarcate evaluation time windows. Comparative spike counts during an
evaluation window decided the identity of input odours. We trained and tested the model on a dataset comprising
200 responses of a Fox e-nose to a set of 20 analytes. We found that the continuous signals from just 4 out of 12
sensors were sufficient to identify the chemicals with an accuracy of 92%, compared to 70% when using just the
maximum response of each sensor.
P2-066 Artificial odor cluster map of odorant molecular parameters and odor maps in rat olfactory
bulbs
Liang Shang1, Chuanjun Liu2, Yoichi Tomiura1, Kenshi Hayashi1
1
Department of Electronics, Graduate School of Information Science and Electrical Engineering, Kyushu University, Fukuoka,
Japan, 2Division of Odor Sensor, Research and Development Center for Taste and Odor Sensing, Kyushu University, Fukuoka,
Japan
The study of the relationship of olfactory bulb patterns to odorant molecule parameters for understanding the
mechanisms of mammalian olfactory is helpful for developing odor sensors. In this study, glomerular activity
patterns in rat olfactory bulbs and odorant molecule parameters were analyzed to explore the relationship
between odorant molecule parameters and activity patterns. Activity patterns for 321 odorants were obtained
from the database of Leon laboratory (http://gara.bio.uci.edu/) and 52 molecule parameters were calculated by
ChemBio3D. Firstly, correlation coefficient between the brightness on each pixels and each parameter of
odorant odor maps were calculated. Correlation coefficient maps for each molecule parameter were established,
and hierarchical clustering analysis were applied to find the similar parameters effected on odor maps. The
cluster results show that all parameters had been divided for 7 clusters, and a relatively similar pattern had been
found in each cluster. Besides, analysis of variance (ANOVA) were applied to find the response area for function
groups in odor maps. T-distributed stochastic neighbor embedding (t-SNE) was applied for establishing artificial
odor cluster map of odor pattern datasets. In t-SNE map, odorant molecular from chemical category named
primary alcohol, alcohol (not phenol), aromatic ester (not lactone), aliphatic ester (not alicyclic), aromatic
aldehyde, aliphatic ketone (not alicyclic), aliphatic or alicyclic ketone and aliphatic ester multiple O-containing
functional groups (not lactone) were clustered in a group, respectively. However, odorant molecular from
chemical category named small aliphatic ester, aliphatic or alicyclic hydrocarbon, aromatic and carboxylic acid
were clustered in two groups, respectively. It suggested that the response area of glomerular activity patterns
selected by ANOVA would be acceptable. The artificial odor cluster map would be applied for evaluation of odor
quality in agriculture.
2016.05.27 15.17.09 Page 29(1)
P2-067 Identification of unique flavor compounds derived from ‘SORACHI ACE’ hop, known as a
popular ‘flavor hop’ among the U.S. craft brewers
Ayako Sanekata1, Atsushi Tanigawa2, Kiyoshi Takoi2, Yutaka Itoga3, Tomohiro Sano2, Ichiro Matsumoto2, Takashi Kaneko1,
Yasuyuki Nakayama2, Takeshi Nakamura1
Frontier Laboratories of Value Creation, SAPPORO BREWERIES LTD., Shizuoka, Japan, 2Product & Technology Innovation
Department, SAPPORO BREWERIES LTD., Shizuoka, Japan, 3Bioresources Research & Development Department, SAPPORO
BREWERIES LTD., Hokkaido, Japan
1
Hops are an important natural ingredient of beer, contributing to not only beer bitterness but also beer flavor. In
recent years, new varieties of hops, which have various characteristic flavors and are mainly grown in the United
States and Australia, have attracted research attention all over the world. Among such varieties, many craft
brewers are interested in ‘SORACHI ACE’ hop. This hop imparts a variety-specific flavor, for example woody,
pine-like, citrus, dill-like and lemon grass-like, to finished beers. However, the flavor compound that contributes
to its characteristic aroma, especially the woody, pine-like flavor, has not been sufficiently investigated. We
investigated the specific flavor compounds derived from ‘SORACHI ACE’ hop by Solid Phase Micro ExtractionGas Chromatography-Mass Spectrometry (SPME-GC-MS) and Gas Chromatography-Olfactometry (GC-O)
analysis. We also used two-dimensional GC-MS to find key flavor compounds. As a result, we identified the
unique volatile compound only in the test-beer brewed with ‘SORACHI ACE’ hop at a significant level.
Furthermore, we investigated the sensory characteristics of these unique compounds derived from ‘SORACHI
ACE’ hop by a model sensory evaluation (reconstitution and omission test).
P2-068 The development of “KOKU” providing flavor
Satomi Kunieda1, Nobuhisa Mabuchi2
1
Corporate Research & Development Division/Takasago International Corporation, Hiratsuka, Japan, 2Flavor Division/Takasago
International Corporation, Hiratsuka, Japan
Japanese cuisine “WASHOKU” was added to the Intangible Cultural Heritage list by UNESCO 2013. In the
aftermath, a lot of people are showing a great deal of interest in Japanese food or cooking and so on. For
example, we Japanese use “KOKU” as one of the feature expression of flavors on a daily basis, and this unique
taste (flavor) expression is widely shared by people all over the world to introduce “WASHOKU” culture.
However, it is not easy to explain the “KOKU” concept or meaning in a clear manner. Takasago’s flavors are
designed to provide the preferred quality taste reminiscent of “KOKU”, i.e., flavorful, rich and pleasant aftertaste
to many market products. The challenge is to explain the unique concept of “KOKU” to those unfamiliar with the
term but yet can experience its contribution to taste quality and delicious imagery. In this study, we first
investigated how Japanese consumers define “KOKU” in their own words. We then confirmed the relationship
“KOKU” and embodying its contribution to intensifying aroma, taste and chemesthesis perception of foods. We
will highlight a prepared model of beef Consomme soup types which confirmed the “KOKU” effect through
sensory evaluation by a consumer panel and by experts.
P2-069 Identification of a protein involved in volatile sensing in plants
Ayumi Nagashima1,2, Takumi Higaki3, Ken Ishigami1, Hidenori Watanabe1, Seiichiro Hasezawa3, Kazushige Touhara1,2
1
ERATO Touhara Chemosensory Signal Project, JST, The University of Tokyo, Japan, 3Department of Integrated Biosciences,
Graduate School of Frontier Sciences, The University of Tokyo, Japan
Accumulated evidences in field studies suggest that plants neighboring to herbivore-infested plants acquire
higher levels of resistance to damages. The current understanding is that it is due to volatile organic compounds
(VOCs), released from wounded plants, which trigger defensive responses in neighboring healthy plants. The
molecular basis for sensing VOCs in plants, however, is poorly understood. Here, we show identification of a 120
kDa protein that has a VOC-binding activity, involved in VOC sensing in tobacco. During the course of screening
for volatiles that induce stress-responsive gene expression in tobacco BY-2 cell, we found that some
sesquiterpenes induced stress-responsive gene expression in 3-6 hours of incubation. Also in tobacco plants,
we observed the same sesquiterpenes induced the gene expression regardless of light conditions in 6-8 hours of
exposure. These results provide evidence that plants sense VOCs, and motivated us to analyze the
mechanisms underlying volatile sensing using tobacco as a model system. By pull-down assays, we identified a
120 kDa protein (P-120) that was specifically bound to the sesquiterpenes derivative-linked beads. Importantly,
when P-120 was overexpressed in BY-2 cell line, the level of gene expression by the sesquiterpenes was
2016.05.27 15.17.09 Page 30(1)
reduced. In contrast, the overexpression of P-120 mutant proteins enhanced the gene expression. These results
suggest that the interaction of P-120 and sesquiterpenes lead to the induction of target gene and thus shed light
on the mechanism of VOC sensing in plants.
P2-070 Assessing olfactory function in healthy korean children using the cross-cultural smell
identification test and butanol threshold test
Woo yong Bae, Dong won Jung, Tae Kyoung Koh
Department of Otorhinolaryngology-Head and Neck Surgery, College of Medicine, Dong-A University, Busan, South Korea
Olfaction is used to detect environmental risk, is involved in the digestion of foods for survival purposes, and
performs various additional roles crucial to the propagation of the species. The loss of sensory function impacts
upon normal development and learning although evaluation methods for hearing and vision currently exist for
young children, the optimal means of indexing olfactory function in this population is subject to conjecture.
Olfactory loss is caused by various factors such as inherited traits, allergies, upper airway infection,
rhinosinusitis, head injury, and endocrinopathy exposure to such factors in young children affects their overall
development. Several studies have suggested clinically feasible methods of evaluating olfactory function in
young children, but no such reports have concerned Korean children. The CC-SIT is a clinically feasible
instrument with a high rate of concordance with the olfactory threshold test when applied to Korean adults. We
herein assess the suitability of both the BTT and CC-SIT forevaluating olfactory function in young Korean
children.
P2-071 Biosensors based on odorant binding proteins and major urinary proteins for security
applications
Krishna C. Persaud1, Khasim Cali1, Mara Bernabei1, Jimena Ricatti2, Carla Mucignat2
1
School of Chemical Engineering and Analytical Science, The University of Manchester, Manchester, UK, 2Department of Molecular
Medicine, The University of Padova, Padova, Italy
Odorant Binding Proteins and Major Urinary Proteins are small soluble proteins that contain binding sites that
are selective to a range of small molecules. They have high conformational stability, making them of interest as
biorecognition elements for new biosensors. Using in silico mutagenesis and docking screening techniques, the
binding pockets of major urinary proteins from the mouse and wild type mosquito Anopheles Gambiae Odorant
Binding Protein (OBP1) were modified to produce theoretical variants with enhanced sensitivity to selected
explosives. A subset of mutants and wild type proteins were expressed and their binding properties to target
analytes were assessed using a fluorescent competitive binding assay in solution. Some of these proteins do
indeed show enhanced sensitivity compared to wild type proteins. When immobilised on to quartz crystal
microbalances (QCM) it was found that they remain stable for many months and are able to sensitively detect
analytes in the vapour phase. Selected proteins were incorporated into an array of sensors which were tested
against traces of target explosives. During field trials at an airport, the device positively detected traces of TNT,
PETN and NH4NO3. This opens an new approach for designing biosensors that target specific analytes in the
vapour phase.
P2-072 Odor character prediction from mass spectra of chemical utilizing data of sensory evaluation
using binary verbal descriptors
Yuji Nozaki, Takamichi Nakamoto
Tokyo Institute of Technology, Japan
Sensory evaluation is widely used to measure humans’ impression of the sense of smell or taste by verbal
descriptors. However, it is quite difficult to realize large-scale evaluation since it requires significant amount of
time and resources. Therefore, we have been engaged in a research to establish computational methods to
predict odor impression of a chemical from its physicochemical property. The ingredients catalog published from
Sigma-Aldrich (Flavors and Fragrances) was used as sensory evaluation data. 1266 chemical ingredients were
described as binary on 150 descriptors. Accordingly, dataset is much sparser than those obtained by semantic
differential method. Thus, prediction would be more difficult especially using linear method such as partial least
squares regression. Mass spectrum of 999 chemicals out of 1266 chemicals listed in the catalog were obtained
from NIST’s Chemical web book. Their peaks between 51-250 mass-to-charge ratio were used. Multi-layer
neural network can learn a generalized projection function from mass spectra onto sensory data to predict odor
2016.05.27 15.17.09 Page 31(1)
impression of an unknown chemical. However, since sensory data matrix which has samples in rows and
descriptors in columns are highly dimensional and sparse, dimensionality reduction is necessary for accurate
prediction. Therefore, we firstly applied Principal Component Analysis to the sensory data to obtain the 1st and
the 2nd principal components, which explain 12% of cumulative variance of original dataset. They were used as
target values of our model composed of 200-120-30-10-2 network. In the result of cross validation, our predictive
model marks R>.6 correlation coefficient between original values and predicted values, it means, a fundamental
part of odor character can be predicted from physicochemical property of chemical even sensory data are
described by binary descriptors. We will soon improve the capability of the system by utilizing autoencoders for
efficient dimensionality reduction.
P2-073 Spatially parallelized lock-in measurement technique for cell-based odor biosensor
Yuji Sukekawa1, Totok Mujiono1, Takamichi Nakamoto1, Hidefumi Mistuno2, Ryohei Kanzaki2
1
Tokyo Institute of Technology, Tokyo, Japan, 2The University of Tokyo, Tokyo, Japan
Recently, various studies on odor sensors compatible with real organisms, which have high selectivities and
sensitivities, are being conducted. We have developed odor sensing system with biological cells expressing
insect olfactory receptors and fluorescent proteins. Odor information is obtained as change of brightness value
of the cell image, and the lock-in measurement technique contributed to the improvement of the limit of
detection. In this study, we aimed to make the system more robust against noise using signal processing
methods which enables in real time spatially parallelized lock-in measurements of multiple areas where an
image is segmented.
In our sensor system, a cultured cell (Sf21 ) expressing Drosophila melanogaster olfactory receptor (Or13a),
which selectively respond to 1-octen-3-ol, and Ca2+ indicator fluorescent protein (GCaMP6s) are worked as a
transducer. The receptor activated by their ligand induces influx of Ca2+ dependent on the concentration of
ligands. Then, fluorescent intensity change of GCaMP6s occurs. Namely, the cells convert odorant information
into fluorescence. This response is measured as brightness change of image taken by a camera. When the
modulated excitation light signal is used as a reference signal, lock-in technique can be applied. Then the
response with high signal to noise ratio can be obtained in real time.
As the experiments, the images with 480x640 pixels acquired from the system every 1 second were processed
with a software program when cells with Or13a were exposed to ligand, 1-octen-3-ol. We confirmed that the
lock-in measurement could be correctly applied to area divided images.
In conclusion, the result suggests that the spatially parallelized lock-in measurement technique is available for
cell images. Since we confirmed the fundamental principle using software, we will extend it to real-time
processing using application specific circuits.
P2-074 Development of a new odor identification test for Japanese elderly people: A comparison of
olfactory functions between elderly and young adults
Eriko Sugiyama1, Saho Ayabe-Kanamura2, Yuki Okabe1, Yukako Konoike1, Yuko Miyake1
1
Institute of Food Sciences & Technologies, Ajinomoto Co., Inc., Japan, 2Faculty of Human Sciences, Division of Psychology,
University of Tsukuba, Japan
Objective: Odors are important factors of tastes of food, and it is considered that age-related olfactory
impairment causes a reduction in palatability and appetite. In this study, we compared the olfactory function of
elderly with young adults by using a newly developed odor identification test which comprised specifically food
odors.
Methods: Based on the classification of food odors, 16 odors representative for each clusters were selected.
To adopt four-alternative forced-choice paradigm for the test, other three odors to each of 16 odors were also
selected from the same cluster, next cluster, and far cluster. Moderate concentrations of each 16 flavor
compounds, which can reproduce the 16 odors, were determined through a pre-evaluation by 22 healthy adults.
And then 105 young (20s to 40s) and 108 elderly (60s to 80s) adults were investigated with the new odor
identification test and the results were compared with the existing odor test (Open Essence ).
Results and Discussion: 1: Average rates of correct answer of each odor in elderly adults were 60.2-94.4%
(61.0-98.1% in young adults). 2: Among wrong answers in the elderly adults, alternatives from the same cluster
were mostly selected (10.6% in average), they from the next cluster were secondly selected (5.6% in average).
3: Elderly adults showed significantly lower correct answer rates in 10/16 odors than young adults. 4: A total
number of correct answer in 16 odors (test score) was positively high correlated with Open Essence score in
2016.05.27 15.17.09 Page 32(1)
both elderly and young adults. 5: Based on the distribution of scores in young adults, participants with 11 or less
scores (37.0% of the elderly and 10.5% of the young adults) were classified to having an olfactory impairment.
It is suggested that our newly developed food odor identification test may be useful to investigate olfactory
changes with age.
P2-075 Characteristics of odor perception in olfactory conditioning
Tomoko Matsubasa1,2, Saho Ayabe-Kanamura3
1
TOKYO GAS CO., LTD., Tokyo, Japan, 2Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba,
Japan, 3Faculty of Human Sciences, University of Tsukuba, Tsukuba, Japan
In this study, we examined whether odor perception was changed by olfactory conditioning, and how the
perception has changed.
Methods: Each participant was presented two pairs of odor and sound. One pair comprised neutral odor and
negative sound to make participants aversive conditioning. Another pair did neutral odor and neutral sound as
control. We investigated whether odor perception was changed from neutral to unpleasant experimentally.
Before and after conditioning, participants were presented odors and evaluated subjective intensity,
pleasantness, familiarity and arousal. And at the same time startle eyeblink reflex were measured. In addition,
we presented odor to participants for 5 consecutive minutes, and asked to evaluate subjective odor intensity
throughout while odor was presented.
Results and Discussion: Hedonic and arousal ratings of odor didn’t change after conditioning. In this
experimental method, olfactory conditioning might not be effective in change of subjective odor assessments.
We compared the data of startle eyeblink reflex immediately after odor presentation, there was also no
significant difference between before and after olfactory conditioning. It was not clear that the olfactory
conditioning could modify the perceptual change, by neither subjective odor assessment nor startle eyeblink
reflex. However, in the result of continuously odor intensity, there was a difference between aversive conditioned
odor and controlled odor. In sum of subjective odor intensity for each 50sec, time-course transition of
conditioning trial was larger than that of control trial(p<.05). It was indicated that the adaptation time of aversive
conditioned odor was slower than that of controlled odor. Thus, olfactory conditioning, even if it does not come
into effect in subjective perception, could make influence to odor adaptation.
P2-076 Development of a new odor identification test for Japanese elderly people: Selection and
classification of food odors
Saho Ayabe-Kanamura1, Eriko Sugiyama2, Yuki Okabe2, Yukako Konoike2, Yuko Miyake2
1
Faculty of Human Sciences, Division of Psychology, University of Tsukuba, Tsukuba, Japan, 2Institute of Food Sciences &
Technologies, Ajinomoto Co., Inc., Japan
Objectives: Odors are important factors of tastes of food, and it is considered that age-related olfactory
impairment causes a reduction in palatability and appetite. Several convenient odor identification tests have
been developed either in the world wide or in the cultural specific. The ability of odor identification is influenced
by olfactory environment of everyday life. Therefore a domestic odor identification test is needed to evaluate the
identifiability of everyday odor. In this study, we developed a new odor identification test to investigate a decline
of olfaction, especially odor sensation of foods, for Japanese elderly people. Methods: At first, from the
interviews to 22 elderly adults in 60s-90s, we collected the descriptors of various kinds of food odors which can
be relatively clearly imaged. Then these collected descriptors were printed on a card one by one. Based on the
similarity of odor images, 40 participants in 20s-40s were asked to sort these cards. Half of participant was
asked to make groups freely, and remaining half was instructed to make around 20-30 groups. Results and
Discussion: We selected 96 food odor descriptors from both previous researches and interviews. The average
number of groups which was made by 40 participants was 32 (no difference between instructions to sort). A
cluster analysis was applied for this data and 96 food odors were classified into 16 odor groups. Using this
classification, we are developing a new odor identification test for Japanese elderly people.
P2-077 Odour characteristics of wild edible nordic mushrooms
Heikki Aisala1,2, Auri Raittola1, Oskar Laaksonen1, Mari Sandell2
1
Food Chemistry and Food Development, Department of Biochemistry, University of Turku, Turku, Finland, 2Functional Foods
Forum, University of Turku, Turku, Finland
Nordic wild edible mushrooms have an annual harvest of billions of kilograms, but only a fraction of this harvest
2016.05.27 15.17.09 Page 33(1)
is utilized as food. The many bioactive compounds of mushrooms make this natural resource a great option for
improving the wellbeing of people, as well as a valuable food ingredient for value added products for forestbased bioeconomy. There is anecdotal evidence of the highly varying odour profiles of edible mushroom species
in guidebooks, but the scientific examination of these sensory differences using analytical sensory research
methods is still scarce.
This study was conducted to explore the aroma properties that differentiate Nordic edible wild mushrooms from
each other and from cultivated mushrooms. The study results demonstrated that 1) human subjects differentiate
the odour of wild mushrooms from cultivated species, and 2) the perceived odour properties of fresh and
processed mushrooms are different.
Projective Mapping combined with Ultra Flash Profiling was applied in our sensory laboratory (ISO 8589,
University of Turku). Both wild (n=4) and cultivated mushrooms (n=5) were used as samples. An A3 sized (297
mm×420 mm) paper was used and samples were given on separate beakers in randomized order. Untrained
volunteer subjects (n=53) were asked to classify mushroom samples according to their odor sensation. The
responses were digitized, and principal component analysis (PCA) with auto-scaled data was used to study
respondent distributions and sample grouping. Full cross-validation was used. Multivariate analyses were
carried out with Unscrambler X (version 10.3, Camo Software, Oslo, Norway).
Based on results, the mushroom samples separated into three groups in both coordinate and descriptor-wordbased models: wild mushrooms, processed cultivated mushrooms and fresh cultivated mushrooms. Odour
descriptors linked to these groups were intense, fresh and fruity; forest and mushroom; and mild and woody,
respectively.
P2-078 Birhinal odor source localization in the open space
Lenka Martinec Novakova1,2,4, Marketa Sobotkova1, Anna Kernerova2, Jan Vodicka3, Jan Havlicek1,4
1
Department of Zoology, Faculty of Science, Charles University in Prague, Czech Republic, 2Department of Anthropology, Faculty
of Humanities, Charles University in Prague, Czech Republic, 3Faculty of Health Studies, University of Pardubice, Czech Republic,
4
National Institute of Mental Health, Klecany, Czech Republic
Olfactory sources are difficult to localize, particularly those of lesser nasal pungency. The aim of the present
study was to investigate whether individuals are able to localize odors of differential nasal pungency in the open
space. The procedure consisted in placing a swivel chair on a plastic mat (260 cm in diameter) on which a
degree circle was marked. A blindfolded participant with a hearing protector was seated in the centre. On each
of the 6 trials, the participant was allowed to rotate the chair within the maximum of 60 seconds and indicate the
position of the odor source, presented from angles differing by a minimum of 30 degrees. 104 normosmics and
20 anosmics have participated in the task. Data analysis was performed in Oriana, a tool for calculating statistics
for circular and radial data. Anosmics and normosmics alike were able to localize odorants of relatively greater
nasal pungency (formic acid, indole, and skatole) significantly above the chance level. Moreover, normosmics
were also able to localize some odorants of relatively lesser nasal pungency (e.g. coumarin). The preliminary
results show that individuals are able to localize odors of both greater and, to a certain degree, lesser nasal
pungency and we propose this procedure as an alternative to the more established odor localization methods,
especially in studies utilizing real-life settings.
P2-079 Loss of olfactory function predicts mortality irrespective of dementia conversion: 10-year
follow-up of an age-varied sample
Maria Larsson1, Ingrid Ekstrom1, Sara Sjolund1, Steven Nordin2, Annelie Nordin Adolfsson3, Rolf Adolfsson3,
Lars-Goran Nilsson1, Jonas K. Olofsson1,4
1
Gosta Ekman Laboratory, Psychology Department, Stockholm University, Stockholm, Sweden, 2Department of Psychology, Umea
University, Umea, Sweden, 3Department of Clinical Sciences, Umea University, Umea, Sweden, 4Swedish Collegium for Advanced
Study, Uppsala, Sweden
The objective of this study was to examine the association between performance in odor identification and future
mortality in a community cohort of adults aged between 40 and 90 years. We assessed olfactory performance
with a 13-item-version of the Scandinavian Odor Identification Test (SOIT). The results showed that during
follow-up (mean=9.4 years, standard deviation=2.23), 411 of 1774 (23.2%) participants died. In a Cox model, the
association between higher SOIT score and mortality was highly significant (hazard ratio [HR]=0.74, per point
interval, 95% confidence interval [CI]=0.71-0.77, p<0.001). The effect was attenuated, but remained significant
2016.05.27 15.17.09 Page 34(1)
after controlling for age, sex, education, and health and cognitive variables that were also associated with an
increased risk of mortality (HR=0.92, 95% CI=0.87-0.97, p=0.001). Controlling for dementia conversion prior to
death did not attenuate the association between SOIT score and mortality (HR=0.92, 95% CI=0.87-0.97,
p=0.001). Similar results were obtained for olfactory sensitivity as assessed by self-report. Overall, the present
findings show that poor odor identification performance is associated with an increased likelihood of future
mortality in middle-aged and older adults, after controlling for social, cognitive, and medical risk factors. Most
importantly, controlling for the development of dementia did not attenuate the association between odor
identification and mortality, suggesting that olfactory decline might mark deteriorating health also irrespective of
dementia.
This work was supported by a program grant from The Swedish Foundation for Humanities and Social
Sciences (M14-0375:1) to Maria Larsson entitled “Our Unique Sense of Smell ”.
P2-080 Post-operative olfaction in patients who underwent olfactory mucosal autografts for spinal
cord injury: Two case reports
Hitoshi Akazawa1,3, Masaki Hayama1, Yohei Maeda1, Takashi Shikina3, Yuichiro Onishi2, Koichi Iwatsuki2, Toshiki Yoshimine2,
Hidenori Inohara1
1
Department of Otorhinolaryngology-Head and Neck Surgery, The University of Osaka, Osaka, Japan, 2Department of
Neurosurgery, The University of Osaka, Osaka, Japan, 3Department of Otorhinolaryngology, Ikeda City Hospital, Osaka, Japan
Olfactory mucosal autograft (OMA) for human chronic spinal cord injury was performed by Lima et al. in Portugal
in 2001 for the first time. Olfactory mucosa which contains the neural stem cell and olfactory ensheathing cell is
thought to contribute to the repair of spinal cord injury. In the surgical procedure of OMA, pieces of olfactory
mucosa are harvested, cut into small pieces and grafted into the site of spinal cord injury after the removal of
scar tissue. Olfactory mucosal autografts have been also performed at our institute since 2008. Lima et al.
reported that olfaction returned to normal in all patients. But, no pre- and post-operative olfactory test results in
OMA cases have been reported at all. We report here olfactory function by T&T olfactometer in two patitents at
pre- and post-operative periods of OMA for chronic spinal cord injury.
P2-081 The smell of trust: Body odor may convey information about trustworthiness
Daniel Amir, Yaara Endevelt-Shapira, Noam Sobel
Social chemosignaling serves an important function in human communication. However, the exact nature of the
information conveyed in this form of communication remains largely unknown. Here we test the hypothesis that a
person’s body odor carries information about their trustworthiness. We first used the trust game, a commonly
applied behavioral economics paradigm, to screen 53 participants (25F, mean age 26±3.4 years) for
trustworthiness. We then selected 10 men who spanned the trustworthiness distribution to serve as body-odor
donors and sleep with a t-shirt. Finally, 35 additional subjects (21F, 25.2±3.1) smelled the t-shirts and rated
them for perceived trustworthiness, pleasantness and intensity. Actual trustworthiness was significantly
correlated with perceived trustworthiness among men (rho=0.63, p<0.05), and with pleasantness among women
(rho=0.71, p<0.05). Moreover, odors from untrustworthy donors elicited a galvanic skin response with a shorter
time-to-peak (TTP) compared with odors from trustworthy donors (mean TTP untrustworthy=4.01±0.53, TTP
trustworthy=5.62±0.57, t(21)=2.08, p<0.05). We conducted two replications of this study: One with new body
odor samples from the same donors (n=45, 21F, 27.6±3.8), and one with the original samples (n=41, 20F, 25.1
±3.2). In these replications the original correlations were not observed, yet when subjects from all experiments
were combined, the two least and two most trustworthy body odors were perceived differently: Among men, a
nearly significant difference in average trustworthiness rankings was observed (average rank untrustworthy=
5.33±0.24, rank trustworthy=6.13±0.3, W=-1.91, p=0.056, Wilcoxon signed-rank test). Among women, a
significant difference in average pleasantness rankings was found (average rank untrustworthy=5.19±0.2, rank
trustworthy=6.63±0.27, W=-3.59, p<0.0005, Wilcoxon signed-rank test). These results imply that body odor
conveys information about trustworthiness, which men and women process differently.
2016.05.27 15.17.09 Page 35(1)
P2-082 Are TRPA1 channels involved in the detection of chemical irritants by the earthworm,
Lumbricus terrestris ?
Wayne L. Silver, Albert H. Kim, Eui Young Kim
Department of Biology Wake Forest University, Winston-Salem, NC, USA
Charles Darwin said of the earthworm: Without the work of this humble creature, who knows nothing of the
benefits he confers upon mankind, agriculture, as we know it, would be very difficult, if not wholly impossible
(1881). Earthworms keep the soil aerated and full of nutrients. They are often used as biomarkers for healthy
soil; the more worms present, the healthier the soil. Chemicals in the soil that are repellent to earthworms could
therefore impact the ability of an area to grow plants. However, little is known about how earthworms detect
aversive compounds. Ally isothiocyanate (AITC) is used as a chemical expellant for earthworm sampling to help
quantify the role of earthworms in ecosystems. Thus, earthworms can detect and are repelled by a known
transient receptor potential cation channel, subfamily A, member 1 (TRPA1) agonist. In other organisms, from
roundworms to humans, TRPA1 channels respond to chemical irritants and are associated with pain and
temperature sensation. We have recently begun investigating whether earthworms also use TRPA1 channels to
detect AITC and other chemical irritants. Chemically receptive neurons in the earthworm skin epithelium send
their axons through segmental nerves to the ventral nerve cord. Using standard whole-nerve
electrophysiological recordings, we demonstrate that earthworm epithelial receptor neurons respond to AITC
delivered to the epithelium, but not capsaicin. Immunohistochemical experiments demonstrate that some
earthworm epithelial cells are positive for TRPA1 immunoreactivity. Behaviorally, earthworms avoid AITC and
cinnamaldehyde (TRPA1 agonists) in a T maze assay. Earthworms do not respond to capsaicin (TRPV1
agonist). Therefore, it appears that the earthworm, may express TRPA1 channels that detect chemical irritants,
but not TRPV1 channels. We are currently conducting calcium imaging experiments and degenerate PCR to test
this hypothesis.
P2-083 Interaction between taste and olfactory signals of a diurnally delivered palatable food
contributes to shift to daytime feeding patterns in mice
Yasunobu Yasoshima, Tsuyoshi Shimura
Division of Behavioral Physiology, Osaka University, Suita, Japan
Rodents are nocturnal animals and normally consume food during dark nighttime. Previous studies indicate that
the normal nocturnal feeding patterns in rodents can be shifted to a diurnal pattern when a palatable food is
delivered during limited daylight hours. In line with the findings, our previous study showed that the feeding
patterns in mice could be shifted to a diurnal pattern when a high-sucrose containing chow was delivered during
daytime. However, it remains unsolved what triggers their feeding behavior during the resting hours when mice
receive the diurnally scheduled delivery of the sweet chow. We assumed that interaction between an olfactory
cue and the taste of the sweet chow played a role in the shift in feeding patterns because the odor of the sweet
chow is different from the familiar odor of normal chow. To address the assumption, we tested the effects of
olfactory disruption on the shift in feeding patterns. Diurnal intake of the sweet chow gradually increased when
ad libitum fed-mice received a 2-h access to the sweet chow during daytime for 10 days. After the 10-day
access, approaching behavior to the sweet chow was greater than that to normal chow even when
consummatory bite of the sweet chow was blocked. In separate mice, dissection of the olfactory nerves from the
olfactory bulb significantly impaired the diurnal increase of the sweet chow intake. Our data suggest that the odor
of the sweet chow plays as a cue signal to trigger the diurnal intake of the sweet chow. Mice may develop
preference for the sweet chow with association between the taste palatability of the sweet chow and its odor
during the diurnal delivery period, resulting in shift to the diurnal feeding patterns.
P2-084 Catecholamines facilitate fuel expenditure and protect against obesity via a novel network of
the gut-brain axis in transcription factor Skn-1 -deficient mice
Yoshiro Ishimaru1, Shota Ushiama1, Masataka Narukawa1, Misako Yoshioka1, Chisayo Kozuka2, Naoki Watanabe3,
Makoto Tsunoda4, Naomi Osakabe3, Tomiko Asakura1, Hiroaki Masuzaki2, Keiko Abe1,5
1
Department of Applied Biological Chemistry, The University of Tokyo, Tokyo, Japan, 2Division of Endocrinology, Diabetes and
Metabolism, Hematology, Rheumatology, University of the Ryukyus, Okinawa, Japan, 3Department of Bio-science and Engineering,
Shibaura Institute of Technology, Saitama, Japan, 4Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo,
Japan, 5Kanagawa Academy of Science and Technology, Kanagawa, Japan
Taste signals and nutrient stimuli sensed by the gastrointestinal tract are transmitted to the brain to regulate
2016.05.27 15.17.09 Page 36(1)
feeding behavior and energy homeostasis. This system is referred to as the gut-brain axis. We provide novel
evidence that both brush cells and type II taste cells are eliminated in the gastrointestinal tract of transcription
factor Skn-1 knockout (KO) mice. Despite unaltered food intake, Skn-1 KO mice have reduced body weight with
lower body fat due to increased energy expenditure. In this model, 24-hr urinary excretion of catecholamines
was significantly elevated, accompanied by increased fatty acid β-oxidation and fuel dissipation in skeletal
muscle and impaired insulin secretion driven by glucose. These results suggest the existence of novel
brain-mediated energy homeostatic pathways originating from brush cells and taste cells in the gastrointestinal
tract and ending in peripheral tissues, including the adrenal glands. The discovery of food-derived factors that
regulate both types of cells may open new avenues for the treatment of obesity and diabetes.
P2-085 Absence of TRPV2 impaired BAT thermogenesis in mice
Wuping Sun1,2, Kunitoshi Uchida1,2, Yoshiro Suzuki1,2, Minji Kim3, Nobuyuki Takahashi3, Tsuyoshi Goto3,
Shigeo Wakabayashi4, Teruo Kawada4, Yuko Iwata4, Makoto Tominaga1,2
1
Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), Okazaki,
Japan, 2Department of Physiological Sciences, SOKENDAI, Okazaki, Japan, 3Division of Food Science and Biotechnology,
Graduate School of Agriculture, Kyoto University, Kyoto, Japan, 4Department of Molecular Physiology, National Cerebral and
Cardiovascular Center Research Institute, Osaka, Japan
Brown adipose tissue (BAT), a major site for mammalian non-shivering thermogenesis, could be a target for
prevention and treatment of human obesity. Transient receptor potential vanilloid 2 (TRPV2), a Ca2+-permeable
non-selective cation channel, plays vital roles in the regulation of various cellular functions. However, the
involvement of TRPV2 in physiological function of mouse BAT remains unexplored. This study aimed to clarify
the expression and physiological function of TRPV2 in mouse BAT. TRPV2 was expressed in brown adipocytes
at mRNA, protein and functional levels. mRNA levels of thermogenic genes and increases in the gene
expression following β-adrenergic receptor activation were significantly lower in cultured adipocytes and BAT
from TRPV2 knockout (TRPV2KO) mice compared with wild type (WT) mice. Furthermore, TRPV2KO mice have
significantly heavier white adipose tissue and larger sizes of brown adipocytes. And TRPV2KO mice showed
cold intolerance and less increases in in vivo BAT temperature to β3-adrenergic receptor stimulation. In addition,
TRPV2KO mice showed significantly heavier body weight and fat upon high fat diet treatment. In conclusion,
TRPV2 might be involved in thermogenesis by modulating the expression levels of genes related to
thermogenesis and energy metabolism.
P2-086 Timing of post-exercise nutrient ingestion: Effects on gastric emptying and splanchnic blood
flow
Masaki Fujimoto, Yoshiyuki Fukuba, Nao Harada, Kohei Eguchi, Masako Yamaoka Endo, Hideaki Kashima
School of Health Sciences, Prefecturaal University of Hiroshima, Hiroshima, Japan
Eight healthy young subjects performed the supramaximal intermittent cycling exercise for 30 min, and then
ingested nutrient drink containing carbohydrate/protein at the timing of either 5 min (PE5) or 30 min (PE30) after
the cessation of exercise. As a control (Con), same drink ingestion without exercise was performed. The blood
flows of celiac and superior mesenteric arteries (BFCA, BFSMA) and gastric emptying (GE) were continuously
assessed throughout the protocol (mainly post-exercise 1 hour) by ultrasonography. After ingestion in PE5,
compared to those in PE30 and Con, GE was consistently slower, BFCA was not increase enough, and BFSMA
delayed to increase. These results indicate that the timing of post-exercise nutrient ingestion impacts the
following GE and splanchnic BF responses, and consequentially affects the digestive/absorptive function.
P2-087 Role of ENaC in initiation of swallows in anesthetized rats
Takanori Tsujimura, Kojun Tsuji, Shogo Sakai, Taku Suzuki, Jin Magara, Makoto Inoue
Division of Dysphagia Rehabilitation, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
Purpose: Mechanical stimulation to pharyngolaryngeal regions can readily evoke swallowing reflex which plays a
role in airway protection. Because disordered laryngopharyngeal sensation is significantly related to the
development of aspiration pneumonia, it is important to know how laryngeal sensory system regulates swallow
initiation. The amiloride-sensitive epithelial sodium channels (ENaCs) have been implicated in a sensor of
sodium salt taste and mechanotransduction. The aim of this study was to investigate the role of ENaC in
initiation of mechanically evoked swallows. Materials and Methods: Urethane-anesthetized male Sprague
Dawley rats were used. Swallows were identified by supra- and infrahyoid electromyographic bursts
2016.05.27 15.17.09 Page 37(1)
accompanied by transient decreases in upper airway (UA) flow or increases in esophageal pressure. Amiloride
analogues (amiloride, benzamil, dimethylamirolide), ASIC inhibitors (mambalgine-1, diminazene) and
gadolinium were applied topically (0.3-30 nmol) to the vocal folds. Swallowing threshold was measured for von
Frey filament or electrical stimulation to vocal folds (n=4-6 in each group). The number of swallows by capsaicin
application (0.03 nmol) to vocal folds or UA distension were also measured (n=5 and 6, respectively). Results:
The mechanical threshold of swallowing was increased by application of amiloride analogues and gadolinium in
a dose-dependent manner, not by ASIC inhibitors. The increased swallowing threshold by amiloride analogues
and gadolinium was recovered after saline washout. The number of swallows by UA distention was significantly
decreased following benzamil application (Control; 8.2±0.7 vs. 30 nmol Benzamil; 1.0±0.5). However,
benzamil unchanged swallow-related muscle activities in UA-evoked swallows. Benzamil did not affect the
initiation of swallows evoked by capsaicin and electrical stimulation. Conclusion: We speculate that ENaC is
involved in initiation of mechanically evoked swallows in larynx.
P2-088 Addition of ornithine to miso soup increases its preference in mice
Haruno Mizuta, Takashi Yamamoto
Department of Nutrition, Faculty of Health Sciences, Kio University, Nara, Japan
Miso soup with low sodium content has been recommended to prevent excessive intake of sodium ions,
resulting in reduced palatability. Palatability of various food stuffs including miso soup and beef consomme are
known to be enhanced by addition of monosodium glutamate (MSG). During the course of our study on
searching other substances that have similar palatability-increasing effects as MSG, we came across ornithine
as a candidate of such an enhancer. Ornithine, a non proteinogenic amino acid, works in the liver together with
arginine and citrulline for the disposal of excess nitrogen and is also used as a potential medical
supplementation to expect attenuation of fatigue and treatment of cirrhosis. Using adult C57/B6 male mice, we
employed the conventional 48-h two bottle preference test for water or miso soup with and without ornithine,
arginine or citrulline. The taste of ornithine itself tended to be aversive judging from the lowered intake of
ornithine-containing water than that of plain water. However, preferences were significantly increased when
ornithine was added to miso soup at concentrations ranging from 3 to 100 mM. Addition of arginine or citrulline to
miso soup also increased preferences, but was less effective than ornithine. In the next step, additive effects of
ornithine to single or combinations of basic taste solutions were examined. Addition of ornithine to a mixture of
NaCl, sucrose and MSG increased the preference, but this addition to single or binary mixtures of these
solutions showed no such preference enhancing effects. The present study has shown that ornithine has unique
properties of palatability enhancing effects. We are currently recording taste nerve responses using different
receptor antagonists to elucidate the background mechanism. Supported by Kakenhi (25350165 to TY)
P2-089 The gustatory response is accelerated by the olfactory information in rat insular cortex
Naoko Mizoguchi1, Masayuki Kobayashi2, Kazuyo Muramoto1
1
Division of Physiology, Department of Human Development and Fostering, Meikai University School of Dentistry, Saitama, Japan,
Department of Pharmacology, Nihon University School of Dentistry, Tokyo, Japan
2
Gustation and olfaction are essential sensation to identify what we eat. These chemical senses are considered
to be integrated in the central nervous system. Recently, the insular cortex (IC) is found to receive inputs of
olfaction in addition to gustation. However, little has been known the mechanisms of integration of these
chemical senses in the IC.
The present in vivo optical imaging study aimed to examine the spatiotemporal dynamics of excitatory
propagation induced by simultaneous stimulation (5 train pulses at 50 Hz) of the chorda tympani nerve (CT) and
olfactory bulb (OB). The first series of experiment, we examined the cortical regions responding to electrical
stimulation of CT or ventral OB. We found that the principal responding region located in the rostral part of the
dysgranular IC (DI) with the CT stimulation. On the other hand, ventral OB stimulation evoked excitatory
propagation that spread in the piriform cortex (PC) and the agranular region (AI), which locates ventrally
adjacent to DI.
In the second series of experiment, we performed CT stimulation in combination with ventral OB stimulation.
Ventral OB stimulation was applied at three timings: simultaneous, 150 ms before, and 150 ms after CT
stimulation. Simultaneous stimulation of CT and ventral OB additively increased the response-amplitude in AI,
and shorten the time to peak of excitation in DI without changing its signal amplitude. Ventral OB stimulation 150
ms before or after CT stimulation evoked the response that peaked just after CT stimulation in DI and after OB
2016.05.27 15.17.09 Page 38(1)
stimulation in AI, suggesting that DI principally responses to inputs from CT, and in contrast, AI is more sensitive
to inputs from OB than CT.
Our data suggest that gustatory and odor information is at least partially integrated in AI and DI, which may
contribute to detect flavor of food.
P2-090 Two distinct neuronal groups for the NaCl responses in the parabrachial nucleus in rats
Tatsuko Yokota, Katsunari Hiraba
Department of Physiology, Aichi-Gakuin University, Nagoya, Japan
In the previous study, for the rostral nucleus of the solitary tract (rNST) in the medulla oblongata, the first-order
taste relay, we have reported that NaCl/HCl (NH)-best neurons could be distinguished from the NaCl (N)-best
neurons not only by the taste magnitude and the response duration, but also by their distribution along the
rostrocaudal (RC) axis. In the parabrachial nucleus (PBN) of the pons, the second-order taste relay, functional
distribution has been assumed for the subdivisions, but differences in the spatial distribution and the accuracy in
the taste responses between the N-best and the NH-best neurons remains unknown. In the present study, we
recorded extracellular single unit activities in the PBN neurons using multi-barrel glass micropipettes while under
urethane anesthesia. Taste solutions were applied to the tongue and the oral cavity. Thirty-seven taste-sensitive
neurons were classified into 15 NaCl (N)-best, 11 NH-best, 7 HCl (H)-best, 3 sucrose (S)-best, 1 NSH-best and 0
quinine (Q)-best. To investigate the fidelity of taste responses, the coefficient of variation (CV) was calculated
from the taste responses in individual trials. The mean CV of the N-best neurons was significantly smaller than
that in the NH-best neurons. The steady responsiveness in the N-best neurons in the PBN was similar as the
N-best neurons in the rNST in our previous study. These response properties for the N-best and the NH-best
neurons to NaCl appear to be kept from the rNST to the PBN. In histological preparations, most of the N-best
neurons (12/13 neurons) were localized on the medial subdivision, whereas the NH-best neurons (9/10 neurons)
were found on the brachium conjunctivum. These results suggest that taste information for NaCl may be
transmitted separately by different neuronal groups (i.e. the N-best and the NH-best neurons) from the rNST to
the PBN.
P2-091 Acquisition and retention of sweet taste memory in rats with bilateral transection of chorda
tympani nerves
Sena Chung, Wei-Peng Jin, Jong-Ho Lee, Jeong Won Jahang
Dental Research Institute, Department of Oral & Maxillofacial Surgery, Seoul National University School of Dentistry, Seoul, South
Korea
Some patients in dental clinics appeal not only altered taste perception but also negative emotion after lingual
nerve damages. We have previously reported that bilateral transections of the lingual and chorda tympani
nerves (Nx) lead to the development of depression-like behaviors in rats. Furthermore, the retrieval of hedonic
taste memory was blunted in Nx rats. In this study, we have examined if the disrupted sweet taste memory is
mainly due to the loss of chorda tympani nerves rather than the loss of lingual nerves. In the experiment 1, male
SD rats underwent 7 days of drinking training with 3-bottles (one sucrose bottle with visual cue, two water bottles
without visual cues) and then received bilateral transections of chorda tympani nerves (CTx) or sham surgery.
CTx and sham rats were subjected to drinking test with three water bottles (one with visual cue, two without
visual cues) after a week of post-operational recovery. Water consumption from cued bottle was significantly
increased in sham rats, but not in CTx, compared to non-cued bottles. In the experiment 2, drinking training was
performed for 9 consecutive days after a week of recovery from CTx or sham surgery, and then the drinking test
was done on the following day. Sham rats consistently drank more sucrose than water during the whole training
period. However, sucrose consumption of CTx rats did not differ from water during the first two days of training
and thereafter it was increased for the rest of period. On the test day, water consumption from the cued bottle
was increased in sham rats compared to both the non-cued bottles, but in CTx only to one of them. Results
suggest that taste sensory loss from the anterior 2/3 of tongue may affect both the acquisition and retention of
hedonic taste memory.
2016.05.27 15.17.09 Page 39(1)
P2-092 Palatability of three basic tastes in a cross-species approach
Aurelie De Ratuld1, R Kyle Palmer2, Magali Fournier1, Emilie Leclerc1, Daniel L. Long2
1
Research Center, Diana Petfood, Elven, France, 2OpertechBio Inc., Philadelphia, PA, USA
Taste sensitivities across mammalian species are thought to depend on feeding strategies shaped by evolution,
supported by growing evidences obtained from wild species occupying different environmental and behavioral
niches. Surprisingly little has been documented of the taste sensitivities of domestic cats and dogs, companion
animals of substantial social and economical interest. Consequently we have conducted a series of palatability
tests focusing on 3 basic taste categories (salty, sour, bitter) in cats and dogs, and compared the results to those
obtained in similar studies of an omnivorous mammal Rattus norvegicus . Twelve tastants, 4 from each basic
taste category, each were examined at 3 concentrations. Palatability in cats and dogs was evaluated by monadic
consumption of tastant solutions relative to water, and in rats by a high throughput operant taste system. In the
salty category, NaCl (100 mM) was appetitive, whereas KCl was aversive to all 3 species, other ionic
compounds tested in this category being species-dependent. Among sour stimuli, organic acids (citric, lactic,
ascorbic) were aversive at 100 mM to all species. In contrast the inorganic phosphoric acid was neutral to rats at
all concentrations, appetitive to cats at 1 mM, and aversive to both cats and dogs at 10 mM. Species-specific
differences were most evident in the bitter category: although quinine was commonly rejected, responses to
denatonium benzoate, naringin, and L-phenylalanine were observed to be both species- and concentrationdependent. Our data reveal for the first time some commonalities in the taste sensitivities across cats, dogs and
rats that could be relevant to the development of food for companion animals.
P2-093 Preference of the flavor alters autonomic nervous activity and chewing pattern
Shiho Honma1,2, Satoshi Wakisaka1
1
Department of Oral Anatomy and Developmental Biology, Osaka University Graduate School of Dentistry, Osaka, Japan,
Department of Oral Health Sciences, Baika Women’s University, Osaka Japan
2
Ingestion behavior, which is comprised of chewing and swallowing, is an important factor controlling quality of
life. Many studies revealed that the characters of foods, such as texture, taste and flavor, affect the chewing and
swallowing. A little is known, however, on the effects of preference of flavor on the ingestion behavior. In the
present study, we examine the effects of preference of flavor on autonomic nervous activity and on chewing
pattern during ingestion behavior by monitoring the electorocardiogram (ECG) and the surface electromyogram
(EMG). We investigated chewing behavior during ingestion of the two types of gum, favorable flavor gum and
unfavorable flavor gum in adult male human subjects (25-35 years old). ECG was monitored autonomic nerve
activity from heart rate variability (HRV) analyzing R-R intervals, and surface EMG was monitored for
masticatory muscle movements. The experiments are approved by the Ethics Committee of the Osaka
University Graduate School of Dentistry. Specifically we focused on the autonomic nervous activity during
post-chewing stage and correlated it to individual chewing patterns. Four types of autonomic nervous activity
were observed; 1; sympathetic nervous activity was getting dominant. 2; parasympathetic nervous activity was
getting dominant. 3; both sympathetic and parasympathetic nervous activities were shown as almost the same
degree. 4; hard to analyze because of the behavior was shown randomly. In group 1, masticatory muscles
activity tend to increase, however some subjects and some parameters in the chewing patterns was decreased.
In group 2, masticatory muscles activity tend to decrease. In group 3, both increasing and decreasing muscle
activities were observed. In group 4, we could not find the definitive alterations of autonomic nervous activity and
mastication activity. (Supported by JSPS KAKENHI Grant Number 15eK11445 to SH.)
P2-094 Serotonin 5-HT2A receptor expressed in the insulin pathway regulating the feeding behavior in
Drosophila
Yuta Mabuchi1, Nobuaki Tanaka2
1
Department of Biological Sciences, Hokkaido University, Sapporo, Japan, 2Creative Research Institution, Hokkaido University,
Sapporo, Japan
The biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which acts as a messenger substance, is found
throughout the insect nervous system. In the fruit fly, Drosophila melanogaster , it has already been shown that
one 5-HT receptor subtype, the 5-HT2A receptor can modulate feeding behaviors and circadian rhythms.
However, the molecular mechanisms linking 5-HT2A receptor activation to behaviors are not well understood. It
also remains unclear how serotonin works in the neural network. We thus made polyclonal antibodies raised
2016.05.27 15.17.09 Page 40(1)
against the artificial peptide of the 5-HT2A receptor. Contradictory to the previous work based on the observation
of an enhancer trap strain for the 5-HT2A receptor gene in Drosophila , the antibodies immunolabeled not only the
ellipsoid body neurons, but also median bundle neurons, insulin producing cells, and neurons in the
subesophageal ganglion. We confirmed that the polyclonal antibodies recognize the 5-HT2A receptor as the
antigen by genetic methods: with the antibodies the induced ectopic expression of 5-HT2A receptor in specific
neurons was detected, in contrast, no signal was seen in a 5-HT2A receptor-deficient strain. To analyze the
functions of each class of the 5-HT2A receptor-expressing neurons in feeding behaviors, we screened for 6000
Janelia GAL4 lines which express GAL4 in the subset of 5-HT2A receptor-expressing neurons and four lines were
identified. Offspring of the GAL4 line and UAS-5HT2A-RNAi line decreased the expression level of the 5-HT2A
receptor protein in the specific subset of neurons. We are currently analyzing how 5-HT2A receptors regulate
feeding behaviors via the insulin pathway by using the RNAi knockdown lines.
P2-095 Conditioned flavor preferences with umami solutions in weanling rats
Kayoko Ueji, Takashi Yamamoto
Department of Health and Nutrition, Kio University, Nara, Japan
Our previous study showed that weanling rats could establish conditioned flavor preferences when sweet tasting
substance, such as glucose, sucrose and saccharin, wear used as associative rewarding stimuli. The present
study aims to examine whether these animals can acquire a flavor preference when umami substances are used
as rewarding stimuli. Three-week-old Wistar male rats were trained in a flavor learning task. Half of the rats
received grape-flavored water on odd-numbered days and cherry-flavored umami solution (0.05M MSG, 0.1M
MSG,0.01M IMP or a mixture of 0.05M MSG and 0.01M IMP) on even-numbered days. The remaining rats
received grape-flavored umami solution on odd-numbered days and cherry-flavored water on even-numbered
days. During this acquisition session, the liquid was presented to each rat for 15 min daily for 6 consecutive
days. In the following test session, each rat was presented with cherry-flavored water and grape-flavored water
simultaneously for 15 min daily for 4 consecutive days. Flavor preference were acquired only when the mixture
was used and were retained when re-tested in adulthood at the age of 20 weeks. However, they failed to show
flavor preferences when either MSG or IMP was used. Both the weanling and 20-week-old rats significantly
preferred all the 4 umami solutions to water. The present study suggests that synergistically increased umami
taste is responsible for this long-lasting flavor preference rather than postingestive effects, since it is reported
that synergism between MSG and IMP dese not occur in the gut. In conclusion, conditioned flavor preferences
are established on the basis of umami-flavor association in weanling rats, and surprisingly this acquired
preferences continued to adulthood.
P2-096 Genetic identification of candidate gustatory second-order neurons that convey appetitive taste
information to the motor/reward systems
Takaaki Miyazaki1,2,3, Tzu-Yang Lin1, Emiko Suzuki3, Chi-hon Lee1, Mark Stopfer1, Kei Ito2
1
National Institute of Child Health and Human Development, NIH, Bethesda, MD, USA, 2Institute of Molecular and Cellular
Biosciences, The University of Tokyo, Tokyo, Japan, 3National Institute of Genetics, Shizuoka, Japan
Sugar stimuli detected by the taste sensory system elicit reflective feeding behaviors in many animals, and also
provide reward cues for associative learning. In Drosophila , where a relatively simple nervous system and a
plethora of genetic tools enable mapping neural circuits and testing their functions, gustatory sensory neurons
(GSNs) send sugar information directly from the periphery to the brain. Previous studies reported that specific
subpopulations of brain neurons are required for commanding the feeding behavior sequence and integrating
sugar reward to the associative memory system. However, neural circuits that link the GSNs and those motor/
reward systems are still unknown. To identify such neurons, we conducted an anatomical screen for gustatory
second-order neurons that receive synaptic inputs from appetitive GSNs. After screening about 5,000 GAL4
lines, we identified 32 candidate lines that sparsely label neurons whose neural fibers project to axon terminals
of the GSNs. As a secondary screen, we expressed two complementary fragments of GFP in axonal terminals of
the GSNs and candidate neuron dendrites to detect potential contacts between them by reconstituted GFP
fluorescence. Among the 16 resulting strains, some visualized neurons that send neural fibers to the dendrites of
the motor command neurons for feeding behavior. Neurons labeled with several other lines project to the input
sites of octopaminergic neurons, which reportedly convey reward signal in associative learning. These
observations suggest that the labeled neurons receive sensory inputs from the GSNs and transmit information to
the feeding-motor and reward systems, respectively. We are analyzing the distribution of input and output
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synaptic sites using tagged acetylcholine receptor subunit (Dα7) and presynaptic marker (Syt), respectively, to
check the direction of information in the identified neurons. Our results provide ways to genetically manipulate
gustatory second-order neurons to dissect the information processing steps required for appropriate control of
gustatory motor/reward systems.
P2-097 Numerical studies on the expression of cell-type marker proteins in mouse foliate and
circumvallate taste buds
Takahiro Ogata1,2, Yoshitaka Ohtubo1
1
Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Fukuoka, Japan, 2ASTEC, Co, Ltd,
Fukuoka, Japan
The responsiveness of taste nerves depends on the types of taste nerves that innervate taste buds. We
investigated the number of immunoreactive cells per taste bud in mouse foliate and circumvallate taste buds,
and compared the cell density (number of immunoreactive cells per taste bud divided by the maximal
cross-sectional area of the taste bud) and co-expression ratio of each cell-type marker among the taste buds
distributed in the mouth. Taste bud cells in the peeled lingual epithelia were immunohistochemically stained with
cell-type-specific markers and were optically sliced with a confocal laser-scanning microscope, rather than by
conventional slicing, to count whole cells. Morphological analyses showed that size of foliate taste buds was
significantly smaller than that of the others. The number of taste bud cells and each immunoreactive cell per
taste bud increased as a linear function of the maximal taste bud area. The total cell density was significantly
lower in foliate taste buds than in the others. The density of SNAP-25 immunoreactive cells (type III cells) was
significantly higher in circumvallate taste buds than that in the others. The co-expression ratio of
Gγ13-immunoreactive cells relative to PLCβ2- or IP3R3-immunoreactive cells (type II cells) was significantly
higher in circumvallate and foliate taste buds than in fungiform taste buds. These results showed that the density
and co-expression ratio of type II and III cells depend on the location of taste buds. These differences may affect
the respective taste nerve responses.
P2-098 Identify the impact of the fat content in the diet on fat taste receptors
Dongli Liu1,2, Andrew Costanzo1, Nicholas Archer2, Caryl Nowson3, Konsta Duesing2, Russell Keast1
1
Centre for Advanced Sensory Science, Deakin University, VIC, Australia, 2CSIRO, Food & Nutrition Flagship, NSW, Australia, 3
Institute for Physical Activity and Nutrition, Deakin University, VIC, Australia
Obesity has become a worldwide epidemic which has been associated with genetic and environmental
contributors. Among the environmental factors, fat intake constitutes a large proportion. The tendency to
consume excess fat in the obese has been associated with attenuated taste sensitivity to fatty acid. Besides, the
sensitivity was reported to decrease after a high-fat diet. The genetic mechanism for the taste sensitivity-diet
relationship was explained in the animal models by the reduced expression of CD36, a putative fat taste receptor
with nanomolar affinity to a range of lipid-based ligands. The objective of this study is to explore the impact of the
dietary fat content on the expression of fat taste receptor genes in humans. Ten monozygotic twin pairs (age
18-65) were recruited through the Australian Twin Registry with BMI values ranging from 18 to 35 kg/m2. Each
twin was randomly assigned to a high (>30%) or low (<15%) fat dietary intervention for 8 weeks. After the
intervention, fungiform papillae biopsy was conducted and RNA was extracted from these samples. The
expression level of the candidate fat taste receptor genes CD36, GPR120, GPR43, GPR84 as well as the
delayed rectifying potassium channel genes such as KV1.2, KV2.2 were analysed by the real time RT-PCR and
compared between the high-fat and low-fat diet with paired t-test. Based on the assumption that the expression
level of the targeted genes of the monozygotic twins is almost the same at the baseline time point, the difference
of each twin pairs after the dietary intervention represents the influence of fat intake on the fat taste gene
expression. The statistical significance was set at P<0.05 and the results will be presented at the conference.
P2-099 Evaluation of peripheral taste system among the different life stages
Masataka Narukawa1, Azusa Kurokawa1, Rie Kota1, Keiko Abe1,2, Takumi Misaka1
1
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan, 2Kanagawa Academy of Science and
Technology, Kanagawa, Japan
Taste sensation is greatly influenced by physiological conditions such as nutritional state, disease, and fatigue.
For example, zinc deficiency affects taste sensitivity, obesity suppresses sucrose evoked neural activity, and
fatigue also influences taste sensitivities. Previous studies investigating the relationship between aging and taste
2016.05.27 15.17.09 Page 42(1)
palatability have shown that the latter could be modified by aging in both humans and rodents. However, the
effects of aging on peripheral taste perception have not been studied in depth. To investigate the effects of aging
on peripheral taste perception, we have compared taste palatability, gustatory nerve response, expression of
taste related molecules, and cell turnover rate in the taste buds between adolescent and senescent mice.
Behavioral assays revealed changes in preference ratios for bitter, salty, and sweet tastes. When gustatory
nerve responses were examined, an increase was observed in responses to sweet and salt tastes in senescent
mice. Although there was no apparent difference in the expression levels of representative taste receptors
between both age groups, the mRNA expression of gustducin and Plcβ2, which are the downstream signaling
effectors of taste receptors, decreased slightly but significantly in senescent mice. Furthermore, the turnover rate
of taste buds tended to decrease in senescent mice. These results indicate that aging affects taste perception,
accompanied by changes of peripheral taste organs. This work was supported in part by Council for Science,
Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP),
“Technologies for creating next-generation agriculture, forestry and fisheries”.
P2-100 Morphological diversity of taste fibers in the mouse tongue
Tao Huang, Robin F. Krimm
Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, USA
In most sensory systems, neurons were initially classified into different types based on their morphology.
Nothing is known about the branching characteristics of individual taste neurons or whether they can be
organized into distinct morphological types. A popular view of taste coding is that the neurons reflect the
response characteristics of taste receptor cell types, which would require little branching. To determine if this is
the case, we traced the peripheral branching patterns of single taste neurons, from where they enter the tongue
to their terminations in the taste bud, using sparse cell genetic labeling. We analyzed total length, branch-points
and ends, and the number of taste buds innervated. None of these traits were distributed normally, indicating
that taste neurons exist as morphological subtypes. Based on the numbers of branch ends, 40 taste fibers were
classified as sparsely-(32%), moderately-(40%), or heavily-branched (28%). Sparsely branched fibers had 1-5
terminal branches innervating between 1-3 separate taste buds, while heavily branched fibers had 13 to 27
separate terminal branches innervating as many as 7 fungiform taste buds. Heavily branched fibers typically
branched more within each taste bud than nerve fibers with fewer branches (F=9.80, p<0.001); one heavily
branched neuron had 13 separate branches innervating a single taste bud. Given this impressive diversity in the
branching characteristics of individual taste nerve fibers, it is unlikely that all gustatory neurons simply reflect the
input from a single taste receptor cell type. Instead, gustatory neurons exist as morphological subtypes, each of
which may code gustatory information differently. Current views of gustatory neural coding must consider why
some neurons innervate only a few taste receptor cells while others appear to gather information from a large
number of taste receptor cells across multiple taste buds. Supported by NIH grant DC001706.
P2-101 Long chain fatty acids have a bitterness-masking effect on quinine and other nitrogenous bitter
substances by formation of insoluble binary complexes
Haruyuki Yamashita, Tohru Terada, Reiko Ueda, Yoshiro Ishimaru, Keiko Abe, Tomiko Asakura
Department of Applied Biological Chemistry, The University of Tokyo, Tokyo, Japan
Bitterness-masking compounds were identified in a natural, white mold cheese. An oily fraction was extracted
from the cheese and further fractionated by using silica gel column chromatography. Four fractions obtained
were characterized by thin layer chromatography and nuclear magnetic resonance (NMR) spectroscopy. The
fatty acid containing fraction was found to have the highest bitterness-masking activity on quinine hydrochloride.
The bitterness-masking activity was quantitated using a method based on subjective equivalents. At 0.5 mM,
sodium salt of the fatty acids mixture, which had a composition similar to that of cheese, suppressed the
bitterness of 0.0080% quinine hydrochloride to be equivalent to that of 0.0049%−0.0060% and 0.5 mM sodium
oleate to that of 0.0032%−0.0038% solution. The binding potential and thermodynamic parameters between
sodium oleate and quinine hydrochloride were estimated by isothermal titration calorimetry (ITC). The interaction
was detected with quinine hydrochloride but not caffeine. To clarify the mechanism of bitterness-masking
between bitter substances and fatty acids, we investigated the interactions between sodium oleate and 22 bitter
substances using ITC. The hydrochloride salts of quinine, promethazine, propranolol, berberine and yohimbine
interacted with fatty acids containing 12 or more carbon atoms. Of these compounds quinine, promethazine and
propranolol strongly interacted. The 1H-NMR spectra of these substances obtained in the presence of the
2016.05.27 15.17.09 Page 43(1)
sodium salts of the fatty acids in dimethyl sulfoxide, revealed the formation of hydrogen bonds between the
nitrogen atoms of the bitter substances and the carboxyl groups of the fatty acids. When aqueous solutions of
sodium laurate and quinine hydrochloride were mixed, an equimolar complex formed as insoluble
heterogeneous needle-like crystals. These results suggested that fatty acids interact directly with bitter
substances through hydrogen bonds and hydrophobic interactions to form insoluble binary complexes, which
mask bitterness.
P2-102 Electrophysiological recordings from the tongue for the objective evaluation of individual
variations of 6-n-propylthiouracil (PROP) sensitivity
Melania Melis1, Giorgia Sollai1, Danilo Pani2, Piero Cosseddu2, Roberto Crnjar1, Annalisa Bonfiglio2,
Iole Tomassini Barbarossa1
1
Department of Biomedical Sciences, University of Cagliari, Italy, 2Department of Electrical and Electronic Engineering, University of
Cagliari, Italy
Several studies have focused on identifying the factors that lead to divergent conclusions about the role of the
PROP phenotype in taste perception, food preferences, and nutrition. One of the major issues is the difficulty in
obtaining an objective measure of the taste phenotype. Here we present direct measures of the gustatory
system activation, by electrophysiological recordings from the tongue, in thirty subjects classified by PROP
taster status and genotyped for TAS2R38. The potentials generated by PROP stimulation were recorded by
means of differential electrophysiological derivations between two silver electrodes, one in contact with the
ventral surface of the tongue and one in perfect adhesion with the dorsal surface. The signals were recorded by
a polygraph for human use and visualized in real time on PC. The electrotastegrams (ETGs) were analyzed by
Clampfit 10.0 software and validated by comparing the voltage amplitude of signals with the perceived PROP
bitterness intensity, PROP taster status and TAS2R38 genotypes. The ETGs showed that PROP stimulation
evoked negative monophasic potentials characterized by a fast initial rise followed by a slow decline. These
potentials represent the measure of the summated voltage change resulting from the response of stimulated
taste cells. The amplitude of potentials was highly correlated with the perceived PROP bitterness intensity (p<
0.0001), with voltage reaching as high as 120 mV in super-taster who were homozygous for the tasting variant
(PAV) of TAS2R38. The depolarization amplitude, as well as depolarization rate, were associated with PROP
taster status and TAS2R38 diplotypes. The direct relationship between the potentials recorded and the
parameters defining PROP phenotype show that ETG is a simple and reliable technology for the quantitative
measure of the peripheral taste function, which may find application as an objective investigation tool in studies
aimed at evaluating eating behavior and taste function impairment.
P2-103 Chemosensory basis for feeding acceptance of different host plants in the larvae of the
Corsican swallowtail Papilio hospiton
Giorgia Sollai, Maurizio Biolchini, Paolo Solari, Iole Tomassini Barbarossa, Roberto Crnjar
Department of Biomedical Sciences, University of Cagliari, Italy
Papilio hospiton is an oligophagous species, endemic of the islands of Corsica and Sardinia, using various
Apiaceae and Rutaceae as host plants, but no plants outside of these families. In Sardinia it uses Ferula
communis as an almost exclusive host plant, but when this is unavailable, Ferula arrigonii and Ruta lamarmorae
are the plants of choice; in Corsica, it also uses other endemic host plants (Pastinaca latifolia). We previously
found that the lateral sensillum has two deterrent GRNs (L and M2), one phagostimulant (M1) and one salt
neuron (S), while the medial sensillum has two phagostimulant GRNs (L and M1), one deterrent (M2) and one
salt neuron (S). In this work we studied the sensitivity of GRNs to saps of F. communis, F. arrigonii, R.
lamarmorae and P. latifolia and evaluated the relationship between taste sensitivity to different host-plants and
larval growth rate on each of them. The spike activity was recorded from medial and lateral taste sensilla
stimulated with plant saps, and GRN response patterns were cross compared in the light of a different feeding
acceptance. The spike responses of GRNs are statistically different. Same neurons are activated by all plant
saps, but the phagodeterrent GRNs show a higher activity in response to F. arrigonii and R. lamarmorae than to
F. communis and P. latifolia. Behavioral trials show that the time to pupation was significantly longer when larvae
were reared on F. arrigonii and R. lamarmorae than on F. communis and P. latifolia. These results suggest that
the different activity of the phagodeterrent GRNs can inhibit food acceptance and extend the duration of the
larval stage.
2016.05.27 15.17.09 Page 44(1)
P2-104 In vivo juxtacellular labeling of rat geniculate ganglion neurons
Yusuke Yokota, Robert M. Bradley
Biologic & Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, Michigan, USA
Primary afferent neurons of the chorda tympani nerve (CT) convey information about sensory properties of food
to the central nervous system. CT fibers have cell bodies in the geniculate ganglion (GG) that respond to
chemical, thermal and mechanical properties of oral stimuli. We have recorded in vivo from GG neurons during
natural stimulation of the tongue and measured receptive field size, sensory response properties, fiber response
latency and conduction velocity. Our results add to demonstrated heterogeneous GG properties while detailing
differences in receptive field characteristics, conduction velocity of the afferent fibers and additional response
properties that include sensitivity to cooling stimuli. We hypothesize that single GG neurons with known
neurobiological characteristics have a specific termination pattern in the rostral nucleus of the solitary tract
(rNST). To identify the brainstem terminal projections of single neurons, we are using the technique of
juxtacellular labeling of GG neurons. The GG was exposed using a ventral approach and a glass extracellular
recording electrode containing Neurobiotin was used to isolate a single CT neurons. Once isolated, current was
passed through the electrode to iontophorese the label into the neuron. After a post-ejection survival period rats
were perfused with phosphate buffered paraformaldehyde, the GG and brainstem removed and horizontal
sections prepared. To date we have successfully labeled 30 GG neurons. Of these the label filled either the
central or peripheral process, but recently we have filled both the central and peripheral processes. Most of
these neurons had a main process originating from the cell body, which branched into central and peripheral
processes. We are currently tracing the central termination patterns in rNST. With juxtacellular labeling of GG
neurons we have the ability to define central termination details of individual taste ganglion cells.
P2-105 Pharyngeal taste function in mediating feeding behavior in Drosophila
Yu-Chieh Chen, Anupama Dahanukar
Interdepartmental Neuroscience Graduate Program, University of California, Riverside, USA
In Drosophila, sweet and bitter tastes are mediated mainly by gustatory receptors (Grs) expressed in gustatory
receptor neurons (GRNs). GRNs are housed within taste sensilla that are distributed in various body parts,
including the labellum, pharynx, distal segments of the legs (tarsi), wing margins, and ovipositor. Although
extensive studies have described taste coding in the labellum and tarsi, little is known about the molecular and
cellular basis of pharyngeal taste. Given that the pharynx lies in an anatomical position where it may serve as the
last checkpoint for food ingestion, exploration of pharyngeal taste is imperative for a comprehensive
understanding of how gustatory inputs from different peripheral taste organs are integrated and translated into
appropriate feeding behaviors. To specifically study the function of pharyngeal GRNs in feeding behaviors, we
took advantage of pox-neuro (poxn) mutants, in which the external taste bristles are transformed into
mechanosensory bristles but internal pharyngeal taste organs are retained. Using binary choice feeding assays,
we found that poxn mutants are capable of making similar feeding choices as wild-type flies, suggesting that
pharyngeal input is sufficient for driving appropriate behavioral responses. Moreover, poxn mutants show
behavioral responses to a wide range of tastants representing sweet, bitter, salt, amino acid, and acid
categories, suggesting that the pharynx has broad taste receptivity. Notably, the degree of aversiveness of bitter
compounds is correlated with pharyngeal taste sensitivity to these compounds rather than their toxicity.
Together, our results highlight the key role of this organ in food evaluation. Next, we constructed a
Gr-to-pharyngeal GRN map with Gr-Gal4 lines, which revealed a distinct molecular organization as compared to
that observed in external taste organs. By selectively manipulating subsets of pharyngeal neurons, our initial
results reveal several classes of pharyngeal GRNs in mediating attractive and aversive feeding behaviors.
P2-106 Identifying the functional bitter taste receptors and their antagonist in chickens
Bapon Dey, Fuminori Kawabata, Yuko Kawabata, Shotaro Nishimura, Shoji Tabata
Laboratory of Functional Anatomy, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
Taste preferences and aversions determine what subjects eat and drink and thus impact on their health and
disease risk. Bitterness is usually believed to guard against toxicity, but some nutritious feedstuffs from plants
contain bitter compounds that cause an aversive reaction without being poisonous at physiological
concentrations. The elucidation of the sense of taste in chicken offers opportunities to create and improve
feedstuffs for chickens and to contribute to the understanding of mammalian sense of taste in comparison with
2016.05.27 15.17.09 Page 45(1)
the functioning of mammalian taste receptors. There are three putative chicken bitter taste receptors, T2R1, T2R
2 and T2R7 that were identified by genome information and cell-based assays. Previously, we have shown that
chicken T2R1 is the functional bitter taste receptor through behavioral test. Thus, in this study, we focused on
the sensitivities of the other two bitter receptors T2R2 and T2R7, by using these agonists in behavioral tests. We
had tested six agonists of chicken T2R2 and T2R7. Furthermore, we also constructed chicken T2R1 and
T2R7-expressing cells for searching of new antagonist for these receptors. In the behavioral drinking study, the
intakes of chicken T2R2 agonist solutions were not different from that of water in the 10 min drinking test. On the
other hand, the intakes of T2R7 agonist solutions were significantly lower in compared to water. By using
calcium imaging methods, we found that 6-methoxyflavanone can inhibit the activity of both chicken T2R1 and
T2R7, which is the first identified antagonist of chicken bitter receptors. These results suggest that T2R7 is one
of the functional bitter taste receptors as same as T2R1 but T2R2 is not functioned, and 6-methoxyflavanone is
the antagonist for these two functional chicken bitter taste receptors, T2R1 and T2R7.
P2-107 Thermostabilizing effects of disordered regions in human T1R3 transmembrane-domain
Tomoya Nakagita1, Takumi Misaka2, Takuya Kobayashi1
1
Department of Cell Biology, Kyoto University, Kyoto, Japan, 2Department of Applied Biological Chemistry, The University of Tokyo,
Tokyo, Japan
[Background] T1Rs, members of a taste receptor type 1 family in class C G protein-coupled receptor (GPCR),
are well-known receptors of umami and sweet taste. Although there is an issue whether GPCR functions as a
monomer or dimer, T1Rs are supposed to function as heterodimers, and play different physiological roles in
human when bound to different members of T1Rs; T1R1/T1R3 heterodimer is a receptor for umami whereas T1
R2/T1R3 for sweet taste. Transmembrane-domain (TMD) of T1R3 is known to interact with positive allosteric
modulator (cyclamate/neohesperidin dihydrochalcone) and negative allosteric modulator (lactisole/gymnemic
acid). However, the molecular mechanisms of these allosteric modulators remain unknown. This encouraged us
to determine crystal structures of T1R3 TMD with different binding ligands. [Method] T1R3 TMD construct was
designed based on two crystal structures of chimeric metabotropic glutamate receptors (mGluRs) solved with
fusion partners, which are the only class C GPCR family structures reported thus far. Both ends of T1R3 TMD
were truncated and extended for improving expression and thermostability. EGFP was fused to the C-terminus
for fluorescence-detection size-exclusion chromatography (FSEC) analysis. [Result] First, we could obtain a
chimeric T1R3 TMD construct fused with bRIL at the N-terminus according to a method described in mGluR1
paper, and establish a protocol for expression and purification of T1R3 TMD. Next, we tried further optimization
of T1R3 TMD construct by extending both ends of the domain. Then, using some of them, we could successfully
express as good as mGluR1-like one. In poster session, we would like to discuss the roles of both ends of T1R3
TMD and future perspectives. This study was performed by grant of Cross-ministerial Strategic Innovation
Promotion Program (SIP), Council for Science, Technology and Innovation, Cabinet Office, Government of
Japan.
P2-108 Analysis of sweet taste responses via SGLT1 in mouse chorda tympani and glossopharyngeal
nerves
Keiko Yasumatsu-Nakano1,2, Yuki Ito2,3, Tadahiro Ohkuri2, Shusuke Iwata2, Robert F. Margolskee4, Yuzo Ninomiya1,2
1
Division of Sensory Physiology, Research and Development Center for Taste and Odor Sensing, Kyushu University, Fukuoka,
Japan, 2Section of Oral Neuroscience, Graduate School of Dental Science, Kyushu University, Japan, 3Mitsubishi Shoji Foodteck
Co., Ltd., Japan, 4Monell Chemical Senses Center, USA
Several studies indicated that there are T1r-independent mechanisms for detecting sugars. Recently, plausible
candidates of sugar sensors for this mechanisms were reported to express mouse gustatory tissues. Among
them, a sodium-glucose cotransporter (SGLT) 1 was shown to fulfil a role as an intestinal sensor in
enteroendocrine cells in addition to mediation of glucose absorption in intestinal epithelial cells. In the present
study, we investigated the function of SGLT1 as sweet taste receptor in taste tissues by examining nerve
responses to sugars, sugar alcohols, artificial sweeteners, amino acids with and without NaCl in the chorda
tympani (CT) and the glossopharyngeal (GL) nerves of C57BL/6J mice. Significant enhancement of responses
were observed in responses to sucrose, glucose, and sorbitol when added 10 mM NaCl to the solutions but not
in responses to KCl, MPG, citric acid, quinine sulfate, SC45647, and polycose in both CT and GL nerves. These
enhancement were abolished by lingual application of phloridzin, a competitive inhibitor of SGLTs. In single fiber
recording, a subset of fibers best responded to sucrose showed significant reduction of impulse frequencies by
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lingual application of phloridzin in responses to glucose and sorbitol. These results suggest that the
enhancement of sweet taste occurs via SGLT1 when NaCl exists.
P2-109 An analysis of transduction mechanism of sour and salty tastes in clonal cell lines derived from
murine taste buds
Eri Katsumata1, Yukari Mitsuhashi1, Miyako Nishiyama1, Yasuhiro Tomooka3, Takenori Miyamoto1,2
1
Faculty of Science, Laboratory of Behavioral Neuroscience, Japan Women’s University, Japan, 2Division of Material and Biological
Sciences, Graduate School of Science, Japan Women’s University, Japan, 3Department of Biological Science and Technology,
Faculty of Industrial Science and Technology, Tokyo University of Science, Japan
We have recently established clonal cell lines (TBD cells) from taste buds of a p53 -deficient mouse.
Immunocytochemical analysis demonstrated that the cell lines are positive to NTPDase2 as Type I cell marker,
gustducin and T1R3 as Type II cell markers or NCAM as Type III marker. In addition, it was also demonstrated
by RT-PCR that TB cell lines express several receptor molecules for tastants including sour, salty, sweet and
bitter tastes such as T1Rs, T2Rs and PKD2L1, transduction-related molecules such as PLCβ2, SNAP25, HCN4,
ion channels including epithelial Na2+ channel (ENAC), voltage-dependent Na2+ channel (VNAC) and
voltage-dependent Ca2+ channel (VCAC). However, it was revealed that TBD cells has responded only to sour
and salt tastes, which are thought to be mediated by ionotropic type of transduction mechanism, but not to
sweet, bitter and umami taste, which are mediated by metabotropic transduction mechanism using Ca2+-imaging.
A transient increase of [Ca2+]i was elicited by sour stimulation in TBD cells and the magnitude of responses to
organic acid were much larger than that of inorganic acid over the range of pH tested as reported in vivo .
Removal of extracellular Ca2+ and perfusion with a nonspecific VCAC blocker, verapamil suppressed the citric
acid response of TBD cells, indicating that Ca2+-responses to sour stimulation of TBD cells are mediated by influx
of extracellular Ca2+ through VCACs as shown in previous studies. Furthermore, in most of TBD cells, the
verapamil-sensitive [Ca2+]i elevation was elicited not only by NaCl but also by neutralized arginine, which
produces salty taste in addition to bitter and umami tastes in mice, but not by basic arginine at all. These results
suggest that TBD cells is useful tool as in vitro model for study of sour and salty transduction.
P2-110 Development and maintenance of taste epithelium: Taste buds and surrounding cells
Hirohito Miura1, Ayumi Nakayama1, Yuko Kusakabe2, Shuitsu Harada1
1
Department of Oral Physiology, Kagoshima University, Kagoshima, Japan, 2National Food Research Institute, NARO, Ibaraki,
Japan
Taste buds in mice are maintained by continuous cell renewal depending on taste nerves throughout life, and
denervation causes a disappearance of taste buds in about 10 days. Here we show that the terminal
differentiation of keratinocytes surrounding taste buds is also regulated by taste nerves. Sprr2a, a cross-linked
envelope protein of keratinocytes, was selectively expressed in the upper layer of the epithelium surrounding
taste buds in the circumvallate papilla (CV) where the epithelium is organized into taste pores. Sprr2a
expression occurred along with the taste bud development and disappeared about 10 days after the transection
of IXth cranial nerve innervating taste buds in the CV. In addition, the expression of cytokeratin 13 in the
epithelium adjacent to taste buds was maintained at a very low level under the control of taste nerves. The nerve
dependent control of these molecules around taste buds likely reflects that taste buds and surrounding
epithelium share precursor cells. However, coordinated development of taste buds and surrounding epithelium
remained unclear. We recently reported Sox2 was intensely expressed in the surrounding epithelium during
development into the adult stage, while the intense Sox2 expression was originally reported to be restricted to
and critical for the taste bud cell lineage. Here we also show the expression patterns of Sox2 and Prox1 from the
embryonic to adult stage and discuss embryonic differentiation of taste bud cells and surrounding cells. This
work was supported by KAKENHI to H.M. (26462818) and A.N. (22791793).
P2-111 Evolution of gustatory sensing mechanism for host selection in swallowtail butterflies
Katsuhisa Ozaki1, Masasuke Ryuda2, Masaaki Kotera3, Ai Muto4, Hiroshi Yoshikawa1
1
JT Biohistory Research Hall, Osaka, Japan, 2Analytical Research Center for Experimental Sciences, Saga University, Saga, Japan,
School of Life Science and Technology, Tokyo Institute of Technology, Tokyo, Japan, 4Graduate School of Biological Sciences,
Nara Institute of Science and Technology, Nara, Japan
3
One of the keys factors to success in specialized phytophagous insects is the ability to efficiently find their host
plant. Swallowtail butterflies belonging to the family of Papilionidae selectively utilize a limited number of plants
2016.05.27 15.17.09 Page 47(1)
from a single or a few families as their host. Female butterflies lay eggs on their host plants only if they detect
specific chemicals through their foreleg chemosensilla while drumming reaction on the leaf surface. Here we
identified a gustatory receptor involved in the recognition of an oviposition stimulant, synephrine, from the
butterfly Papilio xuthus using a combination of in silico , in vitro and in vivo approaches. The receptor, PxutGr1,
specifically responded to synephrine and the sensitivities of tarsal taste sensilla to synephrine in
electrophysiological approach and the oviposition behavior assay were strongly reduced by RNAi. These
observations indicate that PxutGr1 represents a key factor in host specialization in P. xuthus. We perforne
extended search other receptor genes involved in host selection by transcriptome analysis using next generation
sequencer from several species of butterflies. We have elucidated the mechanism underlying the regulation of
oviposition behavior by multiple oviposition stimulants using electrophysiological recordings. We are going to
discuss the evolution of swallowtail butterflies that were driven by changing in the mechanism of host
recognition.
P2-112 Gustatory receptor gene repertoires linked to host range variation in Nymphalid butterflies
Hiromu Suzuki1, Katsuhisa Ozaki2, Takashi Makino1, Hironobu Uchiyama3, Shunsuke Yajima3, Masakado Kawata1
1
Graduate School of Life Science, Tohoku University, Sendai, Japan, 2JT Biohistory Research Hall, Takatsuki, Japan, 3NODAI
Genome Research Center, Tokyo University of Agriculture, Tokyo, Japan
Herbivore insects show a considerable variation in the degree of host plant specialization. Insects can be
classified as specialists or generalists based on the number of taxa they utilize. However, genetic basis
underlying differences in host ranges of herbivores has not been studied well. Butterflies are ideal taxa to
address this question, because of their well-described ecology and rich information on host plant use. Female
butterflies have to select suitable larval host plants during oviposition. Interestingly, they can perceive secondary
metabolites on plants with their forelegs, and discriminate the right hosts based on those chemical cues. This
chemoreception process is crucial for reproductive success of butterflies, and may therefore be determining their
host ranges.
Here we searched for chemosensory gene families expressing in female butterfly legs, which are likely to play
a role in butterfly oviposition behavior, and investigated the relationship between chemosensory genes and host
ranges in butterflies. For our study, 3 species with different host ranges were chosen from tribe Nymphalini. We
performed RNA-seq, and successfully detected gustatory receptor (GR), olfactory receptor (OR), ionotropic
receptor (IR) and odorant-binding protein (OBP) gene families. Among all these families, only number of GR
genes was significantly larger in generalist than in specialist species. Difference in GR counts was mainly due to
an excess of putative bitter receptor genes, which may be involved in detection of plant secondary metabolites.
Our results suggest that generalist butterflies have many GR genes so that they detect a diverse array of
secondary metabolites during host selection process. The detected pattern of GR counts may indicate that
duplication in this gene family was a key event in evolution to broader host preferences.
P2-113 In vivo imaging of trigeminal ganglion neuron responses to γ EVG, capsaicin, allyl
isothiocyanate (AITC) and menthol in mice
Joseph M. Breza1, Michael H. Berger3, Yutaka Maruyama4, Nirupa Chaudhari2, Stephen D. Roper2
1
Department of Psychology, Eastern Michigan University, MI, USA, 2Department of Physiology & Biophysics and Program in
Neuroscience, Miller School of Medicine, University of Miami, Miami, USA, 3Miller School of Medicine, University of Miami, Miami,
USA, 4Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, Japan
The small peptide γ-glutamyl-valyl-glycine (γEVG) and plant-derived substances such as capsaicin, allyl
isothiocyantate (AITC), and menthol enhance flavor or add pungency to oral stimuli. For instance, γEVG
modifies specific oral sensations (kokumi ); warm foods/beverages seem hotter after capsaicin; and menthol
stimulates cooling. We have begun to explore the neural basis of these effects using confocal Ca2+ imaging of
trigeminal ganglion cells in mice that express the Ca-indicator GCaMP3 in sensory neurons. Mice were deeply
anesthetized and cortical tissue aspirated via a lateral window in the cranium to expose area V3 of the trigeminal
ganglion. V3 contains neurons that innervate the oral cavity. Aqueous solutions of γEVG, capsaicin, AITC, and
menthol, and distilled H2O at 2o to 47o were delivered as 10 sec oral lavages via a catheter inserted through the
esophagus. Imaging fields of 200 to 800 trigeminal neurons showed that capsaicin stimulated 0.2-0.5% of the
cells at 10 μM and 2-5% of cells at 100 μM (16 mice). Menthol activated ~0.4% of trigeminal neurons at 1 mM
and 2.5% at 10 mM (6 mice). AITC (20 mM) stimulated 2-3% of cells (4 mice). γEVG (1-3 mM) excited 0.5% of
neurons (4 mice). Each of these stimuli elicited responses in distinct populations of trigeminal neurons. Neurons
2016.05.27 15.17.09 Page 48(1)
stimulated by γEVG, cold (2o), and capsaicin were all small-diameter cells (means=18.1±0.7, 20.2±0.8, and
18.8±1.0 μm, respectively), significantly<average trigeminal neuron diameter (24.4±0.4 μm). Capsaicin
enhanced responses to subsequent stimulation with warm (37o) and hot (47o) aqueous lavage. Our data replicate
what is commonly experienced when consuming pungent spices and demonstrate that thermal perceptions from
chemical stimuli can be generated at peripheral sites independent of cortical integration and processing. The
preparation promises to be a powerful tool for studying somatosensations, including pain, generated during
eating and swallowing.
P2-114 Glycolimia in burning mouth syndrome
Hanif Kamran, Matteo Perrone, Alan Hirsch
Smell & Taste Treatment and Research Foundation, USA
Objective: while pain with Burning Mouth Syndrome (BMS) is reduced with ingestion of food and artificial
sweetener, glycolimia with BMS has not heretofore described. Methods: Case Reports: A fifty seven year old
postmenopausal female presented with a three year history of burning mouth on the tip of the tongue and taste
disturbances, precipitated by multiple dental extractions. She rates her taste as 2% and has a foul bitter glue
taste. This taste is 10/10 in severity, 24/7. This would blend with foods she ingests causing the taste to be
distorted and cacogeusic. Thus are made better with chewing gum, and eating of sweet foods. Before being
afflicted she disliked sweet foods, but now eats each day a gallon of chocolate mint ice cream and three king
size Mr Goodbar (380 calories/bar), and 45 piece of bubble gum. Results: Chemosensory testing: Olfactory:
Brief Smell Identification: 7 (hyposmia). Alcohol Sniff: 6 (Anosmia). Pocket Smell: 3 (normosmia). Retronasal
Smell Index: 7 (normal). Gustatory: 6-n-propylthiouracil disc: 5 (abnormal). Taste Threshold: normogeusia to
sodium chloride, Hydrochloric acid, and phenylthiocarbanol. Hypogeusia to sucrose and urea. Taste quadrant:
whole mouth taste weakness to quinine hydrochloride and carbonated water. Application of 1 gram of Splenda
(a combination of dextrose, maltodextrin, and sucralose) led to temporary total elimination of bitter taste and
burning mouth from 10/10 to 0/10. Discussion: Possible mechanism include exogenous sweet acting to inhibit
endogenous bitter taste, her salivary consistency thus reducing any disordered somesthetic effect of the saliva.
It may be worth to screen people with glycolimia (i.e. diabetics) for burning mouth syndrome, the management of
which may help treat their underlying condition.
P2-115 Unilateral nasal obstruction affects taste cells in circumvallate papillae in rats
Ershu Ren1, Ippei Watari1, Jui-Chin Hsu1, Rieko Ono1, Emina Wakasugi-Aoyama1, Mariko Mizumachi-Kubono1,
Katarzyna Anna Podyma-Inoue2, Tetsuro Watabe2, Takashi Ono1
1
Orthodontic Science, Division of Oral Health Sciences, Graduate School of Medical and Dental Science, Tokyo Medical and
Dental University (TMDU), Japan, 2Section of Biochemistry, Department of Bio-matrix, Graduate School of Medical and Dental
Sciences, Tokyo Medical and Dental University (TMDU), Japan
/Introduction/ Mouth breathing caused by nasal obstruction affects the growth and development of craniofacial
structures, muscle function in the stomatognathic system, and saliva secretion. Hypoxia inducible factor 1 alpha
(HIF1a) is a modulator of the response to hypoxia. We previously reported that nasal obstruction causes
changes in fungiform and circumvallate papillae in the rat. However, few studies have addressed the causeeffect relationship between nasal obstruction and gustatory function. In the present study, we examined HIF1a
expression in circumvallate papillae in a rat model of unilateral nasal obstruction. /Materials and Methods/
Neonatal male Wistar rats were divided randomly into control and experimental groups. Rats in the experimental
group underwent unilateral nasal obstruction by cauterization of the external nostril at 8 days old. Arterial oxygen
saturation (SpO2) was measured weekly thereafter. At 12 weeks of age, the rats were euthanized and the
tongue tissues were removed, fixed overnight in paraformaldehyde solution, and embedded in paraffin. Serial
sections (5 micro meter thick) of circumvallate papillae were prepared, and immunohistochemical staining was
performed using anti-HIF1a antibody. Histological sections were observed under a light microscope.ResultsSpO
2 was significantly lower in experimental rats than in control rats from 6 to 12 weeks of age. Furthermore, HIF1a
expression in the taste buds of the rats in the experimental group seemed to be higher than that in control rats.
/Discussion and Conclusions/ To our knowledge, this is the first report to identify HIF1a expression in taste buds.
It is possible that unilateral nasal obstruction caused the taste bud deterioration via HIF1a produced in
circumvallate papilla. However, further research is necessary to elucidate the molecular mechanism underlying
the changes in rat circumvallate papillae under unilateral nasal obstruction.
2016.05.27 15.17.09 Page 49(1)
P2-116 Expression of GLP-1 and GLP-1 receptor in rat circumvallate papillae during growth period
Beauboeuf Roody1, Ippei Watari1, Jui-Chin Hsu1, Mariko Mizumachi-Kubono1, Katarzyna Anna Podyma-Inoue2,
Tetsuro Watabe2, Takashi Ono1
1
Orthodontic Science, Division of Oral Health Sciences, Graduate School of Medical and Dental Science, Tokyo Medical and
Dental University (TMDU), Japan, 2Section of Biochemistry, Department of Bio-matrix, Graduate School of Medical and Dental
Sciences, Tokyo Medical and Dental University (TMDU), Japan
/Introduction/ The modulation of taste sensing in taste buds is crucial for gustatory function. It has been reported
that bioactive peptides closely related to glucose homeostasis, such as leptin, affect taste sensing. However,
little is known about the development of the mechanisms involved in modulating taste sensitivity. Here, we used
immunohistochemistry to determine the expression of glucagon-like peptide-1 (GLP-1) and its receptor (GLP-1
R) in rat circumvallate taste buds during the growth period. /Materials and Methods/ Male Wistar rats (3, 7 and
12 weeks old) were euthanized, and tongue tissue was removed and fixed overnight in paraformaldehyde
solution. The tissue was then embedded in paraffin, and serial sections (5 micro meter thick) of circumvallate
papillae were made. To evaluate GLP-1 and GLP-1R expression in taste buds, immunohistochemical staining
was performed using anti-GLP-1 and anti-GLP-1R antibodies (Abcam, Cambridge, MA, USA). Sections were
observed using a light microscope. /Results/ GLP-1 and GLP-1R immunoreactivity was observed in taste cells in
the circumvallate papillae at all ages studied. /Discussion/ In agreement with previous studies in mice, we
detected GLP-1 and GLP-1R in rat taste cells. Moreover, we observed GLP-1 and GLP-1R expression during
the growth period. Since GLP-1 signaling is known to modulate taste sensing, further studies are needed to
explore the function of GLP-1 in taste cells during the growth stage.
P2-117 Mutual suppressions of the taste responses between sweet and bitter compounds in some fly
species
Tadashi Nakamura, Shiro Komiyama, Atsushi Nakamura
Department of Engineering Science, The University of Electro-Communications, Tokyo, Japan
Animals determine whether they can intake chemicals as their foods or not, depending of responses of their
taste receptors. In flies, small amount of bitter compounds contaminated in the sucrose solution suppresses their
intake of sucrose solution, which may be due to the suppression of the sugar taste responses by bitter
compounds. Recently in fruit flies, it was reported that the suppression takes place either through the interaction
between sugar receptors with odorant binding protein (OBP) in the endolymph in their chemosensilla or through
the synaptic control of sugar receptor cells by the neuronal signal originated from the bitterness receptor cells.
On the other hand, while it is also known that bitter taste responses appear to become mild by the mixing of
certain amount of sucrose, mechanisms for which have not been well described yet. Here, we tried to see the
suppression of the bitter taste response by the sucrose in blowflies Phormia regina, and fruit flies Drosophila
melanogaster. Using the tip-recording method on the chemosensillum at the tarsus which was cut at the tibia
and held at the tip of the glass capillary electrode containing physiological solutions, we recorded taste
responses of chemosensilla to solutions of bitter compounds (quinine or strychnine) alone, sucrose alone, and
mixtures of bitter compounds and sucrose in various concentrations. We observed that responses to low
concentration of the bitter compounds were suppressed by the addition of sucrose, while the suppression of the
responses to high concentration of bitter compounds was obscure. Possibility of the competitive inhibition
between bitter compounds and sucrose on the bitterness receptors will be discussed.
P2-118 Effects of acetic, succinic and citric acids on the response of the salt receptor cells of the fly,
Phormia regina : An electrophysiological study by tip-recording
Naoko Kataoka-Shirasugi, Yumi Sako, Taisaku Amakawa
Graduate school, Human Development and Environment Kobe University, Kobe, Japan
We examined the influence of three types of organic acids with different numbers of carboxyl groups, i.e., acetic
acid, succinic acid and citric acid, on the salt response of the labellar salt receptor cells of the blowfly, Phormia
regina . We have previously reported that the salt responses to 0.01, 0.05 and 0.1 M NaCl aqueous solutions
were enhanced by low concentrations of acetic, succinic and citric acid, although each organic acid did not
induce the salt response by itself. However it is depressed by high concentrations, measured by tip-recording
method (Murata Y., Kataoka-Shirasugi N. and Amakawa T., Chemical Senses, 27, 57-65 (2002)). In this
investigation, we also found that the most enhanced salt responses (MESRs) for acetic acid were obtained at
2016.05.27 15.17.09 Page 50(1)
less than 0.3 M NaCl, although they were not observed at 0.5 and 0.7 M NaCl. The MESRs induced by 0.01−
0.1 M NaCl plus citric and succinic acids were hardly observed when higher concentration of NaCl ( 0.2 M) plus
each acid was applied, respectively. We also found that the salt concentrations affected the degree of
enhancement in the salt response intensity by the organic acids. Specifically, the degree of enhancement in
intensity declined in proportion to NaCl concentration. This suggests that the greater ionic strength of the
stimulus might hinder distinguishing the action of the organic acids. Additionally, pH values at the MESR for
NaCl plus organic acids were different among the three kinds of organic acids at all concentrations of NaCl. This
result indicates the possibility of the involvement of other molecular species dissociated or undissociated
molecules of organic acids, than hydrogen ions in the enhancement of salt response in the blowfly.
!
P2-119 Comparison of sweet taste sensitivity between Japanese monkey and human
Emiko Nishi, Kei Tsutsui, Hiroo Imai
Molecular Biology Section, Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Aichi,
Japan
Sweet taste is a signal that the food contains carbohydrate. It has been reported by behavior test, that most of
the primates including human prefer sweet foods and that the sweet taste sensitivity to some sugars in natural
foods is different among the species. Because sweet taste receptor Tas1R2/Tas1R3 has various amino acid
replacements among species, we hypothesized that these mutations affect the sweet taste sensitivity of animals.
In this research, we compared sweet taste sensitivity of Japanese monkeys (Mf) with human (Hs) on both
molecular and behavioral level. In molecular experiments, first we determined the sequence of Mf Tas1R2/Tas1
R3. Then, we transfected Mf and Hs Tas1R2/Tas1R3 with Ggust44 to HEK293T cell and measured the
response to natural sugars by detecting the change of intracellular Ca2+ concentration using a fluorescent dye.
Surprisingly, Mf Tas1R2/Tas1R3 expressing cells showed high sensitivity to some of the natural sugars which
Hs Tas1R2/Tas1R3 expressing cells showed a little response. Now we are conducting behavioral experiments
to confirm whether Japanese monkeys show high sweet taste sensitivity in behavior too.
This is a first report that has measured the macaque’s sweet taste receptor function, and showed the possibility
that Japanese monkey might have a higher sensitivity to some sugars than human have. This high sensitivity
can be helpful for Japanese monkeys to find foods that contain potentially high nutrients in wild. Also, this result
could be the clue to find which amino acids in Tas1R2/Tas1R3 are responsible to receive sugars, which are
unclear with human sweet taste receptor because of the low sensitivity.
P2-120 The role of oral sweet taste in regulating postprandial gastric emptying and gastrointestinal
blood flow in humans
Hideaki Kashima, Kohei Eguchi, Kanae Miyamoto, Masaki Fujimoto, Masako Endo Yamaoka, Yoshiyuki Fukuba
Department of Health Science, Prefectural University of Hiroshima, Hiroshima, Japan
The aim of this study was to explore the effect of oral sweet taste sensation of glucose ingestion on the following
gastric emptying (GE) and splanchnic blood flow (SBF). Eight subjects ingested 4 times apart 5.5 min of total
200 mL of 15% glucose solution containing 100 mg of 13 C-sodium acetate after rinsing with 25 ml of either 2.5%
roasted green tea (Con) or 2.5% gymnemic acid solution (Gym) which is known to inhibit sweet taste sensation.
GE and SBF were assessed by the carbon-labeled breath test and ultrasonography, respectively. The time to
attain peak values of SBF, and GE were slowerfor Gym than those for Con. It was suggested that oral sweet
taste sensation itself plays a certain role in controlling postprandial gastrointestinal function
P2-121 Expression of nicotinic acetylcholine receptors (nAChRs) in cultured human fungiform (FF)
taste papillae (HBO) cells
Vijay Lyall1, Jie Qian1, Shobha Mummalaneni1, Andrew I. Spielman2, Mehmet Hakan Ozdener3
1
Department of Physiology & Biophysics, Virginia Commonwealth University, Richmond, VA, USA, 2NYU College of Dentistry, New
York, NY, USA, 3Monell Chemical Senses Center, Philadelphia, PA, USA
We have previously shown that neural and behavioral responses to nicotine are modulated by nAChR agonists
and antagonists (Oliveira-Maia et al. PNAS 106: 1596-1601, 2009; Ren et al. PLOS ONE 10:e0127936, 2015).
Here, we investigated the expression and localization of nAChRs in HBO cells using molecular techniques.
Using specific human primers for various nAChRs and RT-PCR the mRNAs for chrna3, chrna4, chrna5, chrna6,
and chrnb4 were detected in HBO cells. In addition, we detected the mRNAs of scnn1a, scnn1b, scnn1g, T1R1,
2016.05.27 15.17.09 Page 51(1)
T1R3, PLCβ2, TRPM5 and T2R38 taste receptors. Western blot experiments confirmed the presence of α4, α5,
and β2 nAChR proteins in HBO cells. In addition, we detected scnn1a and scnn1g proteins in HBO cells.
Immunofluorescence studies showed only a subset of HBO cells bind to α3 and α5 antibodies. The α3 nAChR
subunit co-localized in TRPM5 positive HBO cells. In HBO cells, α- and γ-ENaC antibodies were localized mainly
in the intracellular compartment of a subset of cells. In Western blot experiments, incubating HBO cells with 250
nM and 500 nM nicotine for 24h induced a differential increase in nAChRs expression. A similar nicotine-induced
differential increase in nAChR expression was also observed in mouse STC-1 cells. Exposing HBO cells loaded
with Fura-2 to acetylcholine (200 μM) or nicotine (200 μM) induced an increase in cell Ca2+ as indicated by an
increase in F340/F380. We conclude that HBO cells express functional nAChRs. Thus, HBO cells are an excellent
model for investigating the expression, localization and function of novel taste receptors. Supported by NIDCD
grant DC-011569 (VL)
P2-122 Application of e-Tongue to analysis of seafood: Assessment of difference in taste
characteristics among squid species
Noriko Ishida1, Ken Tohata1, Yuko Murata1, Chieko Abe2, Masaaki Habara2, Hidekazu Ikezaki2
1
National Research Institute of Fisheries Science, Yokohama, Japan, National Research Institute of Fisheries Science, Yokohama,
Japan, 2Intelligent Sensor Technology, Inc., Kanagawa, Japan
Decapod squid are popular seafoods in Japan. Especially, Japanese common squid Todarodes pacificus and
neon flying squid Ommastrephes bartramii are widely consumed. Since the catch of these squid has been
recently decreasing, the underutilized purpleback flying squid Sthenoteuthis oualaniensis has potential for
commercial use. However, purpleback flying squid is thought to be less delicious, so we studied its taste
characteristics and compared them to other squids. First, we developed an extraction method by boiling and
centrifugation, and then analyzed extracts using a taste sensing system. As a first step in e-Tongue analysis, we
developed a preparative method for squid. Frozen squid were thawed and meat divided into mantle, fin, and
tentacles, and then cut into 5mm strips. Two portions of 10g meat strips from different squid were blended
together for one sample meat for further analysis. The sample meat (20g) was added to 80mL water, boiled for
10 min, then chilled for 30 min, and centrifuged at 7000xg for 10 min. The obtained supernatant was filtered and
analyzed. These sample extracts were analyzed using the taste sensing system (SA402B, Intelligent Sensor
Technology, Inc., Japan), and for the nucleic acid and amino acid compositions. The taste sensing system had
five taste-sensors to measure bitterness (C00), sourness (CA0), saltiness (CT0), umami (AAE), and astringency
(AE1). Each sensor was designed to selectively respond to a single basic taste and to correlate its sensitivity
with human gustatory sensation. Differences in taste-sensor response among body parts: fin and tentacles had
stronger saltiness than mantle for Japanese common squid and purpleback flying squid. Furthermore,
differences among species: neon flying squid had stronger saltiness and aftertaste of umami than the other two
squids. The purpleback flying squid had stronger umami than the other two squids. Taste characteristics are
concluded to differ among the squids
P2-123 Evaluation of bitterness masking effect using fabricated sensors for artificial sweeteners
Tomohiro Hattori1, Xiao Wu1, Hideya Onitake1, Tamami Haraguchi2, Yusuke Tahara1, Rui Yatabe3, Miyako Yoshida2,
Masato Yasuura1, Hirotaka Okazaki1, Takahiro Uchida2, Hidekazu Ikezaki4, Kiyoshi Toko1
1
Graduate School of Information Science and Electrical Engineering, Kyushu University, Japan, 2Department of Clinical
Pharmaceutics, Mukogawa Women’s University, Japan, 3Research and Development Center for Taste and Odor Sensing, Kyushu
University, Japan, 4Intelligent Sensor Technology, Inc., Japan
In the development of medicines, it is an important role to evaluate taste of medicines by including bitterness
masking effect. There are some problems to take the medicine in the human sensory test. We have developed a
taste sensor based on membrane potential changes of lipid/polymer membranes first in the world. In this study,
we demonstrated an evaluation method for the taste of medicines using the taste sensor. Evaluation of
bitterness masking requires a sensor for artificial sweeteners, and hence we have developed the taste sensor for
artificial sweeteners. In this study, we optimized the quantities and types of the lipid and plasticizer for the
fabrication of sensors with high selectivity and sensitivity. We evaluated the masking effect of bitterness by
measuring mixture of quinine hydrochloride as bitterness of medicine and aspartame or saccharine sodium as
artificial sweeteners using a bitterness sensor and the fabricated sensors for artificial sweeteners. As a result,
the bitterness-prediction formulas were derived by a regression analysis of the outputs of the sensors. Moreover,
a good correlation was obtained between bitterness scores of the human sensory test and the formulas. These
results show a possibility of evaluation of the bitterness suppression effect due to artificial sweeteners using the
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taste sensor.
P2-124 Activation of the calcium-sensing receptor by physiological concentrations of glucose
Yuko Nakagawa, Johan Medina, Masahiro Nagasawa, Itaru Kojima
IMCR, Gunma University, Japan
Glucose is a major fuel in the body and regulates various cellular functions. However, cell-surface receptor for
physiological concentrations of glucose has not been identified. Here we show that the calcium-sensing receptor
(CaSR) is activated by physiological concentrations of glucose. In HEK293 cells stably expressing CaSR,
elevation of the ambient glucose concentration from 3 to 5 mM induced an elevation of cytoplasmic Ca2+ ([Ca2+]c).
The effect of glucose was detected at 4 mM, and was the maximal at 5 mM. A non-metabolizable glucose
analogue 3-O-methylglucose reproduced the effect of glucose. Both sucrose and sucralose elevated [Ca2+]c but
slightly higher concentrations were required compared to glucose. In the absence of extracellular calcium,
glucose was not able to increase [Ca2+]c. Glucose also decreased cytoplasmic cAMP concentration. In PT-r
parathyroid cells and in Madin-Darby canine kidney cells, both of which express endogenous CaSR, glucose
elevated [Ca2+]c. Hence, physiological concentration of glucose acts as a positive allosteric modulator of CaSR
and may modulate cellular functions in CaSR-expressing cells.
P2-125 Taste Sensor measurement is a potent tool for evaluation of Japanese dashi soup
Yukako Hayashi1, Takuji Takahashi2, Yasuki Matsumura1, Toru Fushiki3
1
Graduate School of Agriculture, Kyoto University, Uji, Japan, 2Traditional Kyoto-style Cuisine Kinobu, Kyoto, Japan, 3Faculty of
Agriculture, Ryukoku University, Otsu, Japan
It is no room for doubt that “Dashi” is indispensable for Japanese cuisine. The typical dashi is prepared from
kelp, dried bonito flakes, shitake mushroom, and dried sardine. The composition of the Dashi soup from
combination of kelp and dried bonito flakes is simple, mainly three amino acids, a nucleotide, and general
minerals. The combination of these components elicits umami taste. The taste sensing system (TS-5000Z,
Intelligent Sensor Technology, Inc., Kanagawa, Japan), which has five sensors using lipid/polymer membranes,
evaluates dashi solutions as the scores of 8 kinds of taste (saltiness, sourness, umami, bitterness, astringency,
aftertaste of bitterness, aftertaste of astringency and aftertaste of umami) using change in membrane potential
caused by adsorption of substances. Although the principal of the taste sensing system is not based on
physiological mechanism, the system is used for the analysis of beers, curry, tea, and so on. Dashi soup is
commercially available as styles of powder, concentrated, and non-concentrated. In this study, the evaluation of
dashi soups prepared by a chef at a Japanese restaurant and commercially available dashi soups with taste
sensor was carried out. The chef-prepared dashi soups exhibited distinct pattern from commercially available
dashi soups. Interestingly, there were no significant differences among umami scores between all dashi soups.
Furthermore, dashi soups are be assessed by the experts using the sensory evaluation. Together with these
results, we now show that the taste sensing system could discriminate among dashi soups made from kelp and
dried bonito.
P2-126 Genetic diversity of bitter taste receptors and chemical ecology of bitter plant foods in wild
chimpanzees
Takashi Hayakawa1,2, Eiji Inoue3, Yasuka Toda4, Hodaka Matsuo1, Naruki Morimura5, Miho Inoue-Murayama5,
Chie Hashimoto1, Takumi Misaka4, Hajime Ohigashi6, Tetsuro Matsuzawa1,2, Hiroo Imai1
1
Primate Research Institute, Kyoto University, Aichi, Japan, 2Japan Monkey Centre, Aichi, Japan, 3Faculty of Science, Toho
University, Chiba, Japan, 4Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan, 5Wildlife Research
Center of Kyoto University, Kyoto, Japan, 6Faculty of Biotechnology, Fukui Prefectural University, Fukui, Japan
Mammals avoid ingesting poisonous compounds using the sense of bitter taste when they choose and eat
plants. We previously revealed that nucleotide sequences of 28 bitter taste receptor (TAS2R) genes in
chimpanzee (Pan troglodytes ) are evolutionarily diversified among subspecies, suggesting that each
subspecies may genetically adapt to region-specific poisonous plants. In this study, we investigated ecological
background of such TAS2R diversification of chimpanzees through genetic and functional analysis of TAS2Rs
and chemical analysis of bitter plant foods in wild chimpanzees. First, we determined genetic diversity of two
selected TAS2Rs, TAS2R38 and TAS2R46, in a wild West African chimpanzee (P. t. verus ) population in
Bossou (Guinea) and two wild East African chimpanzee (P. t. schweinfurthii ) populations in Mahale (Tanzania)
and Kalinzu (Uganda), using DNA from noninvasively collected feces. As a result, subspecies differentiation was
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observed in both TAS2R38 and TAS2R46 genes of wild chimpanzees as previously reported in captive
chimpanzees. For example, pseudogenized alleles of TAS2R38, which cause less sensitivity to bitterness of
phenylthiocarbamide, were found only in West African chimpanzees. Next, cellular assay confirmed that
chimpanzee TAS2R46 is a functional receptor as well as TAS2R38, using candidate ligands to which human
TAS2R46 responds; therefore, genetic diversity of chimpanzee TAS2R46 may affect bitter plant ingestion in wild
chimpanzees. Finally, we analyzed concentration of a poisonous but antiparasitic sesquiterpene lactone in
leaves of Vernonia species, which are broadly available in chimpanzee habitats. Chimpanzees in Mahale ingest
Vernonia plants, but chimpanzees in Bossou have not been reported to ingest them. We found that leaves of V.
amygdalina in Mahale contain higher concentration of the sesquiterpene lactone than other Vernonia species in
Bossou. These results indicated that genetic and functional diversity of TAS2Rs and region-specific food choices
of bitter plants could be organically linked with each other.
P2-127 Effects of intraoral stimulation with capsaicin on salivary secretion and neural activity
Yoko Kono1, Ayako Kubota2, Mika Matsushima3, Masato Taira1, Kumiko Sugimoto4
1
Cognitive Neurobiology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan,
Behavioral Dentistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan,
3
Master of Medical Administration Course, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan, 4Basic Oral Health Engineering, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental
University, Tokyo, Japan
2
Aim: Though it has been reported that intake of pungent component increases gastric mucus secretion and
enhances metabolism, the influence of intraoral stimulation with it on oral and systemic conditions has not been
elucidated substantially. Therefore, we investigated the effect of oral stimulation with capsaicin on oral condition
and neural activities by measuring changes in salivary secretion and electroencephalogram (EEG) induced by
capsaicin stimulation. Methods: Eighteen healthy adults participated in this study and the detection threshold for
capsaicin and the recognition thresholds for five basic tastes were measured from them first. Five ml of each
taste and capsaicin solution with basically fourfold concentration of measured threshold and deionized water
(DW) was held in subject’s mouth for 1 min and then spat out with secreted saliva. The increased weight was
taken as the amount of secreted saliva. The concentration of secretory immunoglobulin A (SIgA), the dominant
substance of mucosal immune activity, of saliva was measured using the assay kit. In addition, salivary
α-amylase activity which is known to increase with sympathetic nervous activity was measured before and after
capsaicin stimulation. From recorded EEG, the emotional changes were analyzed using Emotion Spectrum
Analysis System (Brain Functions Laboratory, Inc., Japan). This study was approved by the Ethical Committee
of our university. Results: Mean salivary secretion rate during capsaicin stimulation was much higher than DW
and five basic tastes. The secreted amount of SIgA per minute and salivary α-amylase activity was elevated by
capsaicin stimulation. Regarding emotional changes, capsaicin and quinine demonstrated similar change
pattern showing increased curiosity and tiredness accompanied by decreased calm. Conclusion: This study
demonstrated that intraoral stimulation with capsaicin increased salivary flow, SIgA amount and α-amylase
activity, suggesting that capsaicin may be effective for improvement of oral condition accompanied by
sympathetic elevation.
P2-128 A comprehensive psychophysical investigation of sweet taste function
Julia Yu Qing Low1, Robert McBride1, Katie Lacy2, Russell Keast1
1
Centre of Advanced Sensory Science, School of Exercise and Nutrition Sciences, Deakin University, Melbourne, Australia, 2Centre
for Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Deakin University, Melbourne, Australia
Sweet is one of the five taste primaries and is activated by sugars and non-nutritive sweeteners. The aim of this
study was to investigate measures of sweet taste function [detection threshold (DT), recognition threshold (RT),
and suprathreshold intensity ratings] across multiple sweeteners. Sixty participants, 18-52 years of age (mean
age in years=26, SD=±7.8; 28 male, 32 female), were recruited to participate in the study. DT and RT were
collected for caloric sweeteners (glucose, fructose, sucrose, erythritol) and high intensity sweeteners (HIS)
(sucralose, Rebaudioside A). Sweet intensity for all sweeteners was measured using a general Labeled
Magnitude Scale. There were strong correlations between DT and RT of all four caloric sweeteners (r =0.620.90, P <0.001), and moderate correlations between DT and RT of both of the HIS (r =0.39-0.48, P <0.05);
however, weaker correlations were observed between the DT/RT of the caloric and HIS (r =0.26-0.48, P <0.05).
The DT and RT of glucose and fructose were not correlated with DT/RT of sucralose (P <0.05). In contrast, there
were strong correlations between the sweetness intensity ratings of caloric and HIS (r =0.70-0.96, P <0.001).
2016.05.27 15.17.09 Page 54(1)
This suggests that partially independent mechanisms for the DT/RT of caloric sweeteners, and HIS. At
suprathreshold level, however, the strong correlation between caloric and HIS through weak, moderate and
strong intensity supports commonality in sweet taste mechanism for the perceived intensity range.
P2-129 Hypogeusia with dietary supplements: Report of a case
Tadashi Ishimaru, Hitomi Ishimaru
Hyotan-machi ENT Clinic, Kanazawa, Japan
Recently, a lot of types of dietary supplements were provided on the commercial market. Side effects of these
substances were not well known. We experienced a case of the forskolin induced hypogeusia. A 42 years old
female patient with connective tissue disease consulted otolaryngology of a general hospital with complaining
hypogeusia for several months. The result of blood testing indicated hypo zinc serum (40 μg/dl). Oral use
polaprezinc was administrated on her for one month. Taste test was not performed at that hospital. Because her
complain of taste dysfunction was not recovered in spite of that polaprezinc was administrated for one month,
she consulted us. We asked detail of her recent eating habit. She used the dietary supplement of forskolin for
half year. Results of taste test indicated senses of basic 4 tastes were lost on her tongue sensed with chorda
tympani nerve. There was no complaining about smell sense. After 1 month later, the use of forskolin was
inhibited, complain of hypogeusia was disappeared though results of taste test had not been recovered
completely. Forskolin stimulate adenylate cyclase and increase intra-cellar cyclic AMP level. Olfaction was
normal and not influenced by forskolin in this patient. When forskolin increase the concentration of cyclic AMP in
sensory cells, both olfaction and gustation may be affected because cyclic AMP works as second messenger not
only in the taste cell but also in the olfactory cell. Mechanism of hypogeusia may not be thought as direct effect
to taste cells by forskolin. In this case, serum zinc level is decreased by reduced absorption on the digestive
organ caused by forskolin. Then, hypo zinc serum might caused hypogeusia.
P2-130 The importance of nutrition support for the taste disorder−Nutrient conditions of the patient
with taste disorder−
Chikako Hibino, Shizuko Satoh-Kuriwada, Takashi Sasano
Division of Oral Diagnosis, Tohoku University Graduate School of Dentistry, Japan
Background The decline of taste sensation is related to malnutrition especially in the elderly people. However
there are few reports regarding the nutrient condition of the taste disorder. We investigated how the nutrient
intake is in the patients with taste disorder. Methods Participants were 34 patients with taste disorder, visited in
the Tohoku University Hospital.(62.2±15.0 years).We assessed each patient’s nutritional status following the
MNAⓇ(Mini Nutritional Assessment),and the FFQg(food frequency questionnaire based on food groups).The
patients’ tendency of nutrient intake was judged based on the national health and nutrition survey in 2013 of
Japanese Ministry of Health,Labor and Welfare. Result MNAⓇ:Malnutrition was observed in 7 patients (20%).
Risk of malnutrition was observed in 21 patients (62%). Decrease in the amount of food was observed in 19
patients (56%). Weight loss was observed in 18 patients (53%).Under BMI 20kg/m2 was observed in 11 patients
(32%).FFQg: The averages of each item are as follows. Energy intake: 1567±412 kcal, (83% of the standard
values (SV)), protein intake: 52.6±15.4 g (75% of the SV), carbohydrate intake: 214.5±59.3 g (82% of SV), zinc
intake: 6.4±1.9 mg (79% of the SV), cereals intake: 295.5±95.3 g (67% of the SV), fish and shellfishes intake:
41.8±27.9 g (53% of the SV), meats intake: 44.5±29.0 g (51% of the SV), eggs intake: 23.3±19.0 g (68% of
the SV), confectioneries intake: 60.7±44.8 g (240% of the SV). Sixteen patients (47%) take many supplements,
instead of their meal. Conclusions Adequate meal should be necessary to regenerate healthy taste cells and to
maintain taste sensation, however many patients with taste disorder take an unbalanced diet. This result
indicates that nutritional support is necessary for the treatment process of the taste disorder.
P2-131 What causes bitter taste phantoms?
Saori Funayama1, Kayoko Ito1, Makoto Inoue2
1
Oral Rehabilitation, Niigata University Medical and Dental Hospital, Niigata, Japan, 2Division of Dysphagia Rehabilitation, Niigata
University Graduate School of Medical and Dental Sciences, Niigata, Japan
Phantogeusia is a perception of taste that occurs in the absence of a tastant. The most common chief complaint
is bitter. The etiology of bitter phantogeusia is unknown, but a bitter substance in saliva, for example
magnesium, some substances that are components of medication, as well as psychological stress, may induce
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bitter taste phantoms. The aim of this study was to investigate the cause of bitter phantogeusia. Participants
were 9 female patients (50-81 years old) with bitter taste phantom and 19 normal controls (11 female and 8
male, 23-44 years old). Questionnaires including medical history, medication and bitter taste sensation, and
psychological tests using the General Health Questionnaire (GHQ30) and the Self-rating Depression Scale
(SDS) were performed. Magnesium, cortisol and chromogranin A in unstimulated saliva and magnesium,
copper, iron, vitamin B12 and zinc in serum were also measured. As a bitterness masking test, participants
gargled with Benecoat BMI-60, which is a specific inhibitor for bitterness. Mean value of all parameters was
compared between patient and control groups. Total GHQ30 and SDS scores were significantly higher in the
patient group than in the control group. There were four participants with an allergy to pollen dust in the patients
group and one in the control group. Two of the nine patients had a higher salivary magnesium level, although
their serum magnesium level was normal. Another four patients took medication with adverse effects of
dysgeusia. The number of positive participants for the bitterness masking test was three in the patient group and
zero in the control group. Based on these findings, salivary magnesium level, medication with adverse effect of
dysgeusia, and medical history of allergy may be factors influencing the phantom sensation of bitterness.
Furthermore, a bitterness masking test and various psychological tests may be useful for the diagnosis of
phantogeusia.
P2-132 Improving the perceived texture and feelings of nursing care foods using a pseudo-chewing
sound: A study in healthy elderly subjects
Hiroshi Endo, Shuichi Ino, Waka Fujisaki
National Institute of Advanced Industrial Science and Technology (AIST), Japan
Elderly individuals whose ability to chew and swallow have declined can only eat unpleasant food whose texture
is modified to be very soft and monotonous, leading to a poor appetite. To address this problem, we sought to
influence the perception of food texture by providing a chewing sound. Although we do not usually pay attention
to the sounds emitted when we chew food, biting and chewing sounds can influence the perception of food
texture, especially the perception of crispness and crunchiness. Because chewing sounds are barely emitted by
texture-modified diets, they had to be artificially generated in a manner that was synchronous with chewing
behavior. To achieve synchrony, we used the electromyogram (EMG) of the masseter. The provided pseudochewing sound resembled the crunchy sound emitted by root vegetables. We investigated whether the
perceived sensations and impressions of nursing care foods were altered by the pseudo-chewing sound. Thirty
healthy elderly participants took part in the experiment. They evaluated six kinds of nursing care foods (three
kinds of pureed foods and three kinds of minced foods) and filled out questionnaires to rate the taste and texture
of sample foods, as well as the perceived feelings they evoked. When the pseudo-chewing sound was provided,
participants were more likely to evaluate a food as having the property of stiffness. Moreover, additional effects
were also observed, and some observed effects could be attributed to an effect inherent to crispy and crunchy
sounds. Therefore, presentation of the pseudo-chewing sound is a useful technique for helping people
experience varied food textures, even if the actual texture of their food is dull.
P2-133 Fibre enrichment decreases bitter and salty taste sensitivity in a solid food model
Pridhuvi Thavaraj1, Michael Gidley2, Delma Greenway3, Eugeni Roura4
1
Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, Australia, 2Queensland Alliance
for Agriculture and Food Innovation, The University of Queensland, St. Lucia, Australia, 3School of Agriculture and Food Science,
The University of Queensland, St. Lucia, Australia, 4Queensland Alliance for Agriculture and Food Innovation, The University of
Queensland, St. Lucia, Australia
Consuming dietary fibre (DF) is associated with benefits that range from reducing plasma lipid and glucose
levels to increasing satiation and delaying the rate of gastric emptying. The effect of hydrocolloids leading to
diminished taste and aroma in liquid or semi-solid foods has been reported, however, little is known about how
much DF enrichment affects taste perception and taste thresholds levels in solid foods. Since taste perception
has far more implications such as determining food intake and selection for nutrients at subsequent meals, it is
important to investigate from that point of view, how fibre enrichment influences the perception of other tastes.
Although there are many means to measure taste perception, detection thresholds have shown to be a reliable
and repeatable tool. The objective of this study was to assess the effect of 20% replacement of insoluble and
soluble DF, on taste threshold levels of the 5 primary tastes, measured using an ascending forced choice test
method. Dehydrated flour chips enriched with either 20% beta-Glucan or 20% wheat bran was compared to
2016.05.27 15.17.09 Page 56(1)
100% flour controls chips. Taste perception in all tastes decreased as a result of fibre replacement and was
statistically significant (P<0.01) for bitter and salt tastes. No differences were observed on taste thresholds
between the two DF sources. Overall these results suggest that DF enrichment on sensory aspects of foods is
not homogeneous across different tastes and requires further investigation. Since DF has the potential to
decrease the perception of the sweet and salt tastants, which can lead to known health conditions, this finding
has the potential to improve healthy formulations of fibre-enriched foods and most importantly to prevent the
excess intake of sodium or sucrose in fibre enriched foods.
P2-134 Flavor improvement of reduced-fat peanut butter and custard cream by the addition of a
kokumi peptide, γ-glutamyl-valyl-glycin
Shuichi Jo1, Misa Nishida1, Motonaka Kuroda1, Tohru Kouda2, Naohiro Miyamura1
1
Institute of Food Sciences and Technologies, Ajinomoto Co., Inc., Tokyo, Japan, 2Institute for Innovation, Ajinomoto Co., Inc,
Tokyo, Japan
Recent studies have demonstrated that kokumi substances, which enhance basic tastes and modify
mouthfeels, are perceived through the calcium-sensing receptor (CaSR). We have discovered a potent kokumi
peptide, γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) by screening a compound library, using a cell-based assay for
CaSR and a sensory evaluation. It is known that the addition of γ-Glu-Val-Gly to chicken consomme significantly
enhances mouthfeels, and therefore likely to be the key part of the sensation evoked by the addition of fatcontaining food materials such as dairy fat emulsion. However, the effect of the peptide in fat-reduced foods still
remains unclear. In this study, the sensory effect of γ-Glu-Val-Gly on reduced-fat peanut butter and custard
cream was investigated.
First, a descriptive analysis was conducted between full-fat and reduced-fat peanut butter with well-trained
panelists (n=20). The score of the full-fat sample was significantly higher than that of the reduced-fat sample in
peanut flavor (p<0.01), thick flavor (p<0.05), aftertaste (p<0.01), continuity of taste (p<0.01), and oiliness (p<
0.01). Second, for these five attributes, we examined the effect of 40 ppm γ-Glu-Val-Gly on the sensory profile of
reduced-fat sample (n=19) and found significant enhancement of thick flavor (p<0.01), aftertaste (p<0.05) and
oiliness (p<0.05) in the reduced-fat peanut butter.
In addition, the effect of γ-Glu-Val-Gly on reduced-fat custard cream was examined with the same method (n=
19). The addition of γ-Glu-Val-Gly at 40 ppm significantly enhanced the intensity of thick flavor (p<0.05),
continuity (p<0.05) and tended to enhance the intensity of aftertaste (p<0.1).
These results demonstrate that the addition of γ-Glu-Val-Gly enhanced the sensations lowered by fatreduction in peanut butter and custard cream, suggesting that γ-Glu-Val-Gly improves the sensory quality of fatreduced foods and contributes to food technology for fat-reduction without compromising palatability.
P2-135 The quantitative sensory evaluation of taste intensity by half-tongue test −salt enhancement−
Yasutaka Shoji, Shiori Ando, Toshio Miyazawa
Functional Products R&D Laboratory, Ogawa & Co., Ltd., Tokyo, Japan
Sensory evaluation is the one of the most important methods to measure the sensory perception of human. In
psychophysical experiment, the forced choice methods are widely used, but are difficult to obtain the quantitative
data about the intensity of perception. On the other hand, quantitative descriptive tests are more informative than
the forced choice tests, but require subjects train to obtain objective data. Therefore, the aim of this study is to
provide the simple and objective method to quantitate the sensory intensity.
To measure the taste enhancing effect quantitatively, the half-tongue test (spatial forced choice) was used.
Sodium chloride (NaCl) was used as a tastant, and spilanthol derived from a herb, Spilanthes acmella , as a
taste modifying compound. One side of the subject’s tongue was pretreated by sub-threshold amount of
spilanthol and the other side by water. Then, subjects sipped NaCl solution to choose the side that yielded
higher intensity of saltiness. In control experiment, deionized water was used instead of NaCl solution.
As a result, significant enhancement of saltiness by spilanthol was observed. Importantly, in the control
experiment, it was confirmed that spilanthol alone did not elicit noticeable sensation in the absence of NaCl. In
addition, the concentration of NaCl which elicits equivalent saltiness was estimated by psychometric function.
These results suggest that the amount of spilanthol was low enough not to induce spilanthol derived sensations,
but was effective to enhance saltiness. This was consistent with a result of our recent research demonstrated
that spilanthol sensitizes response of taste cell to NaCl. Excessive intake of NaCl is known to lead diseases such
as hypertension. Our result suggests that spilanthol may help to reduce salt in foods without changing sensory
2016.05.27 15.17.09 Page 57(1)
properties.
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ＫｙｏｒｉｎＷＰＳ／３５０３３−５２２２／ｉｓｏｔｐ１７−１３−４＿／ｋｙ３１６９２９５２２２１０００３０００
Poster Session
P3-001 Biphasic modulation of mitral cells by preproglucagon neurons in the olfactory bulb
Nicolas Thiebaud, Debra Ann Fadool
Department of Biological Science, Program in Neuroscience, The Florida State University, Tallahassee, USA
Mitral cells (MCs) constitute the principal relay of olfactory information between olfactory sensory neurons and
higher processing areas of the brain. We previously characterized neuromodulation of MCs by the incretin
(gastrointestinal) hormone glucagon-like peptide-1 (GLP-1), which increased the firing frequency of evoked
action potentials (APs). Using a transgenic mouse model expressing YFP under the control of preproglucagon
(PPG), the precursor of GLP-1, we have discovered a population of GLP-1 producing neurons located in the
inner part of the granule cell layer (GCL). The PPG-neurons are characteristic of deep-short axon cells (dSACs)
or Cajal cells having stellar dendrites and one axon projecting to the MC layer and the external plexiform layer.
To optogenetically activate this class of dSACs, we crossed PPG-Cre mice to floxed ChannelRhodopsin-2 mice.
The resultant progeny were used in acute slice preparations of the olfactory bulb whereby light-activation
parameters for PPG-neurons were empirically determined by voltage-clamp. Light-activation of the dSACs
evoked simultaneous inhibitory (IPSCs) and excitatory (EPSCs) postsynaptic currents in MCs that were
abolished after application of glutamatergic inhibitors. While recording alternatively from granule cells, light
elicited glutamate-induced EPSCs, supporting a di-synaptic origin of IPSCs recorded from the MCs. This was
further corroborated by confocal localization of the glutamate transporter VGLUT2 in the dSAC synaptic
terminals. A biphasic inhibition-excitation control of AP firing in MCs was found recording under current-clamp
conditions as a result of light-activation of the dSACs. Taken together, these results demonstrate that this class
of dSACs/PPG-neurons constitutes a unique population of glutamatergic neurons within the GCL that form a
local microcircuit controlling MC activity. This work was supported by 14POST20380615 fellowship from the
American Heart Association (AHA) and R01 DC013080 from the NIH/NIDCD.
P3-002 Search strategies in complex olfactory environments
Brian J. Jackson1, Sujean Oh1, Venkatesh Gopal2, Agnese Seminara3, David H. Gire1
1
Department of Psychology, University of Washington, Seattle, WA, USA, 2Department of Physics, Elmhurst College, Elmhurst, IL,
USA, 3CNRS, Universite Nice Sophia Antipolis, Laboratoire de physique de la matiere condensee, Parc Valrose, Nice, France
In most terrestrial environments odor molecules are transported by turbulent flow as fluctuating plumes. While
many animals, including rodents, use these airborne cues to find food and avoid predators, this is made difficult
by the dynamic, intermittent nature of odor plumes. The ability of rodents to form internal representations of their
environment could allow them to apply learned spatial information to dynamic odor plumes, creating a map that
would greatly enhance odor-guided navigation. To investigate how rodents use learned spatial information to
follow odor plumes we have constructed an automated open field arena. This arena uses computer-controlled
robotics to place odor sources and food rewards at user-defined locations. Multiple high speed cameras
precisely capture movement while animals search for food across complex odor landscapes with all experiments
done under red light to eliminate visual cues. Odor plumes are visualized through the use of particle imaging
velocimetry combined with numerical simulations. Using this system, we can control both the amount of
information that an animal has about its environment as well as the complex mixtures of odors that it encounters
while navigating in search of food. In a first set of experiments we investigated how rodents use learned spatial
information during odor-guided searches by manipulating the amount of information that rats had regarding
probable locations of weak odor sources (sugar pellets). Rats trained on a reliable set of pellet locations that
varied little from day to day (high spatial information) learned to focus their search to small regions with high
probability of odor plume contact. Those trained on unpredictable locations (low spatial information) did not
exhibit this focused search strategy and had greater difficulty locating pellets. Our results demonstrate that rats
adaptively apply learned spatial information to odor-guided searches to efficiently locate food using turbulent
odor plumes.
2016.05.27 17.38.25 Page 2(1)
P3-003 Involvement of the mouse olfactory tubercle in adaptation of learned odor-induced foodseeking behavior
Koshi Murata1,2,3, Masamichi Mikame1,2, Wataru Murofushi1,2, Kazuto Kobayashi4, Kensaku Mori1,2, Masahiro Yamaguchi1,2
1
Department of Physiology, The University of Tokyo, Tokyo, Japan, 2JST-CREST, Tokyo, Japan, 3Department of Morphological and
Physiological Sciences, University of Fukui, Fukui, Japan, 4Department of Molecular Genetics, Fukushima Medical University,
Fukushima, Japan
Olfaction is one of the key sensations to induce food-seeking behavior. However, it is unclear how the central
olfactory system transforms odor cues into food-seeking behavior. We previously demonstrated that learned
odor-induced food-seeking behavior accompanies c-Fos expression of dopamine receptor D1-expressing cells
in the anteromedial olfactory tubercle (OT) of mice. Here, we examined the role of D1-expressing cells in the
anteromedial OT in learned odor-induced food seeking behavior by the immunotoxin-mediated cell ablation
system. We trained mice to associate an odor (amyl acetate) with the location of sugar reward before the cell
ablation. Then we injected immunotoxin locally into the medial OT and induced ablation of about 50% of the
D1-expressing cells in the medial OT. After the cell ablation, the mice were exposed to the sugar-associated
odor, which was hidden under the bedding of the test cage without sugar reward. One session consists of 3 min
of odor exposure and the session was repeated four times by changing the location of the odor in each session.
On the first session, the D1 cell-ablated mice showed approaching behavior to the odor as rapidly as the
sham-operated control mice, and both groups of mice spent most of the time for food seeking behavior toward
the odor cue. On later sessions, while the control mice changed their behavior to spend less time for food
seeking toward the odor, the D1 cell-ablated mice continued to spend much time for food seeking toward the
odor. These results indicate that the D1 cell-ablated mice showed impairment in quitting learned odor-induced
food seeking behavior when they did not obtain expected food, and suggest that the anteromedial OT plays an
important role in adaptively changing odor-induced food seeking behavior according to the alteration in the
odor-food reward relationship.
P3-004 Physiological and molecular phenotyping of interneurons in the glomerular layer of the mouse
olfactory bulb
Oliver Robert Braubach1,2,3, Tuce Tombaz1,3, Ryota Homma2,3, Yunsook Choi1,2, Thomas Bozza4,5, Lawrence Baruch Cohen1,2,3
1
Center for Functional Connectomics, Korea Institute of Science and Technology, Seoul, South Korea, 2Department of Cellular and
Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA, 3NeuroImaging Cluster, Marine Biological
Laboratory, Woods Hole, MA, USA, 4Department of Neurobiology, Northwestern University, Evanston, IL, USA, 5Visiting Scientist
Program, HHMI Janelia Farm Research Campus, Ashburn VA, USA
Olfactory glomeruli are innervated by diverse interneurons, which control glomerular excitability and cross-talk.
These interneurons are very diverse and it has been difficult to understand how they contribute to olfactory
information processing. We studied the population activity of glomerular layer interneurons during different levels
of olfactory and optogenetic stimulation. These experiments were initially conducted in transgenic mouse lines
that express GFP in GABAergic (GAD65 and GAD67) and dopaminergic interneurons. Interneurons were
bulk-loaded with AM calcium dyes, and their stimulus-evoked activities measured via acute in vivo two-photon
calcium imaging. Most interneuron types responded preferentially to odor stimuli delivered at high
concentrations. Interneuron responses were also coupled to responses in adjacent glomeruli during short odor
presentations (2-5 sec), but became decoupled and more diverse during prolonged odor exposures (20 sec).
We repeated these experiments in mice that express channelrhodopsin in a single glomerulus. Optogenetic
activation of a single glomerulus at different intensities and durations replicated our findings from odor
stimulation experiments, suggesting that the diverse interneuron signals seen in olfactory experiments are of
intraglomerular origin. Finally, we combined our calcium imaging experiments with post-mortem
immunocytochemistry targeted against the calcium-binding proteins calretinin, calbindin, S100 and neurocalcin.
Consistent with previous results, we found that calcium binding proteins are expressed in subpopulations of
GABAergic and dopaminergic interneurons. We are now studying if molecularly distinct interneuron
subpopulations comprise some of the different interneuron pools that we identified based on our calcium imaging
experiments.
2016.05.27 17.38.25 Page 3(1)
P3-005 Effects of malodors on human stress systems
Yukei Hirasawa1,2, Masako Okamoto1,2, Mika Shirasu1,2, Kazushige Touhara1,2
1
Japan, 2ERATO Touhara Chemosensory Signal Project, JST, The University of Tokyo, Tokyo, Japan
We are surrounded by various malodors. Malodors can be a serious nuisance, and negatively affect our
wellbeing. There are at least three mechanisms by which these nuisance odors affect human health. First, some
odorants are toxic, and/or cause irritation, and the exposure gives negative health impacts. Second, even though
odorants themselves are not toxic, they accompany pollutants, such as bacteria endotoxin or non-odorous toxic
chemicals. Finally, people complain of health damage even when odorants are not toxic. This is possibly
because malodors cause unpleasant feelings and induce stress. However, influence of non-toxic malodors on
human stress systems has been largely unknown. In the current study, we aimed to examine whether malodors
triggered stress responses in humans. A total of eighty-three healthy subjects participated in the experiment.
They smelled various odors, including non-toxic malodors at various concentrations. We examined changes in
activities of the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic-adrenal-medullary (SAM) axis, two
major systems that regulate stress responses in humans, upon malodor exposure. We used salivary cortisol and
salivary α-amylase as the surrogate markers of the HPA and SAM axes, respectively. Subjective ratings on odor
pleasantness, intensity, and mood were also collected. Relationships among perceived odor quality, self-rated
mood, and reactions of the HPA and SAM axes to odors were examined. We discuss possible psychobiological
mechanisms of the effects of non-toxic malodors on the human stress responses based on these results.
P3-006 Study on the associative structures of learned flavor preference in rats with higher-order
conditioning paradigms
Takuya Onuma1,2, Nobuyuki Sakai1
1
Department of Psychology, Tohoku University, Japan, 2Division for International Advanced Research and Education, Tohoku
University, Japan
Previous studies have shown that rats preferred an odor that had been paired with saccharin solution to an odor
that had been paired with quinine solution (learned flavor preference). It was unclear whether the odors were
associated with the emotional aspects of gustatory information (i.e., positive and/or negative hedonics) or with
the qualitative aspects (i.e., sweetness and/or bitterness). This study aimed to examine this question using
higher-order conditioning paradigms: second-order conditioning (SOC) and sensory preconditioning (SPC).
Fifty-two adult Wistar rats were divided into SOC (n=26) and SPC (n=26) groups. Food flavors, purchased from
the Japanese market, such as melon (0.05%), lemon (0.1%), vanilla (0.1%) and almond (0.1%), were randomly
used as odors A, B, C, and D in each rat. The SOC rats were exposed to 0.005M saccharin solution with odor A
and 0.02M quinine solution with odor C in the first 5 days of learning, and exposed to water with odor AB mixture
and water with odor CD mixture in the next 5 days of learning. The order of these two learning sessions was
reversed in the SPC rats. We hypothesized that if odor was associated with the emotional (or qualitative)
aspects, the SOC (or SPC) rats would prefer odor B to odor D. Our results showed that the SOC rats preferred
odor B to odor D, whereas the SPC rats did not show any such preference. This suggests that the emotion
evoked by gustation was associated with odor.
P3-007 Development of the olfactory system during pupal diapause in a butterfly
Mikael A. Carlsson
Department of Zoology, Stockholm University, Stockholm, Sweden
Diapause is a an essential part of the life cycle of many insects in order to survive harsh environmental
conditions. The butterfly Pieris napi enters pupal diapause in response to shorter day length at the late larval
stages. Whereas direct pupal development takes approximately ten days from pupation to eclosion, diapause
may last several months. How different structures develop during diapause is, however, largely unknown, i.e. if
development completely arrests and if so at what stage. Here, development of the olfactory system was studied
by immunohistochemical methods. The antennal lobe and the presynaptic region of the mushroom bodies
develop equally in larvae, during prepupal stage and until the third day of pupation in both directly developing
and diapause-destined animals. However, from pupal day 3 the development of the olfactory structures in
diapausing pupae remain at a constant size until the animals have been exposed to 7 days at 10 degr C and 4
days at 20 degr C. At the same time glomeruli become visible (visible at day six in directly developing butterflies).
2016.05.27 17.38.25 Page 4(1)
The characteristic layered structure of the optic medulla, on the other hand, is already present at the third pupal
day in both groups. Thus, our results indicate that while the optic system may be needed during diapause the
olfactory system only starts developing after the termination of the diapause.
P3-008 How do pheromones affect plant odorant responses in antennal lobe projection neurons? A
calcium imaging study in the moth Helicoverpa armigera
Elena Ian, Nicholas Hagen Kirkerud, Bente Gunnveig Berg
Norwegian University of Science and Technology
Moths possess a highly specialized olfactory system comprised of two segregated pathways dedicated to
processing information about plant odors and pheromones, respectively. The male moth faces the challenge of
seeking out the right female in a highly dynamic odor world. Her pheromone blend, which is a limited resource
serving as guidance for him, will reach his antennae in intermittent pockets of odor filaments mixed with
biologically relevant volatiles from the host plants. In the study presented here, we hypothesized that the
antennal lobe activation pattern of the male moth is subjected to fine-tuning to plant volatiles that repeatedly
co-occur with the pheromones. Such an effect could serve as a signal facilitator and a backup if the pheromone
trail is temporarily lost. We performed calcium imaging of the uni-glomerular projection neurons in the antennal
lobe (analog to mitral cells in the vertebrate olfactory bulb) to investigate how pheromones influence plant odor
responses. Increased response specificity to the plant odorants that had been subsequently presented together
with pheromones compared to those that were not would indicate a form of perceptual learning that drives
attention towards relevant odors. The underlying neurobiological mechanisms might be based on rapid plastic
changes in the circuit of local interneurons and projection neurons and/or efferent modulation of the antennal
lobe.
P3-009 Relation between pleasantness of odor-evoked memories and preference of the odor
Noriko Takahashi1, Rika Haga2, Tomoko Kohno3, Noboru Ichinose1, Nobuyuki Sakai4
1
Flavor & Fragrance Technology Research Laboratories, Lion Corporation, Tokyo, Japan, 2Beauty Care Research Laboratories,
Lion Corporation, Tokyo, Japan, 3Living Care Research Laboratories, Lion Corporation, Tokyo, Japan, 4Graduate School of Arts and
Letters, Tohoku University, Sendai, Japan
It is said that there is a close relation between smells and memories, which is often called as “Proustian
Phenomenon”. In this phenomenon, our memories are triggered by both pleasant and unpleasant odors. Studies
concerning the relationships between odor and memory reported that the person who remembered an
autobiographical memory from the past experiences claimed feelings of nostalgia with considerable probability.
Also, the person who felt the odor as pleasant or liked especially reported nostalgia by the high percentage. The
aim of this research is to clear not only nostalgia but also pleasantness evoked by an odor, thus we investigated
relationships between the pleasantness of odor-evoked memory and preference of the odors. Five hundred
people, consisted by both men and women in their twenties to sixties, participated in this study. Eight typical
scents, such as grapefruit, freshly washed sheet, forest mist, chocolate, flower garden, soda water, fragrant olive
and camphor stored in the chest are used as odor stimuli in this study. Two hundred fifty three females in their
twenties to fifties were asked to sniff the odors and to evaluate their preference for the odor at first. Then they
were asked to evaluate the strength of nostalgia, remembrance of the memory, and pleasantness of the
memory. The results were the following; 1) In all odors, the person who reported odor-evoked good memory
tended to evaluated higher preferences for the odor than the person who reported odor-evoked bad memory.
There was positive correlation between pleasantness of the memories and preferences for the odors. 2)
Relationships between nostalgia of the odor-evoked memories and preferences for the odors did not show such
a higher correlation. These results suggested that preferences of odor were more strongly linked to
pleasantness of memory than feelings of nostalgia.
P3-010 Imaging of second-order olfactory neurons in a standard brain atlas
Nicholas Hagen Kirkerud, Elena Ian, Bente Gunnveig Berg
Norwegian University of Science and Technology, Norway
Descriptive morphological studies are a prerequisite for explaining functional neural networks. Thus, in order to
explore basic principles characterizing chemosensory information processing we study the central olfactory
pathways of the moth. Generally, odor information is conveyed from the primary olfactory center of the moth
2016.05.27 17.38.25 Page 5(1)
brain, the antennal lobe, via projection neuron axons passing along several parallel tracts. These antennal-lobe
tracts target two main regions in the protocerebrum, the calyces of the mushroom bodies and the lateral horn.
Recently, we reported about the distinct projection patterns of each tract, demonstrating that all of them target
the lateral horn, whereas mainly one innervates the calyces. The findings also included a new tract, not
previously identified in the moth. In the study presented here, we have performed intracellular iontophoretic
staining combined with confocal microscopy for characterizing individual antennal lobe projection neurons
forming these pathways. Totally, we identified output neurons confined to one of five different tracts. Further, by
means of the visualization software AMIRA we reconstructed and registered the stained neurons into a standard
brain atlas. This allowed us to compare the termination areas of individual projection neurons both across and
within the distinct tracts. The data from this anatomical study will improve the basis for future analyses regarding
the function of parallel olfactory pathways.
P3-011 Cholinergic enhancement of olfactory bulb odor responses increases odor salience and drives
behavioral dishabituation
Max Fletcher, Mounir Bendahmane, Cameron Ogg
Univeristy of Tennessee Health Science Center, USA
The olfactory bulb (OB) receives cholinergic input from the basal forebrain (BF) and expresses a variety of
cholinergic receptors. Acetylcholine (ACh) is known to be necessary for proper olfactory perception, however the
mechanisms are largely unknown. To explore the effects of cholinergic modulation on odor processing, we used
transgenic mice expressing calcium indicators in OB M/T cell populations in intact, anesthetized mice. Overall,
we find that OB ACh release via electrical BF stimulation or pharmacological manipulation increases the gain of
glomerular odor responses over a wide odor concentration range. Further, pharmacological experiments
demonstrate that this effect is mediated through the activation of OB m2 AChRs. Overall, we find this
mechanism increases individual glomerular sensitivity to odors and decreases glomerular activation thresholds
and could serve to enhance responsivity to weak or degraded input under certain conditions. We are currently
investigating the role this cholinergic-mediated gain control plays in modulating olfactory habituation at both the
physiological and behavioral levels. Data from our lab and others suggest that habituation of MT odor responses
is due to reduced OSN input. Thus, controlling the gain of MT cell responses could modulate the magnitude of
adaptation. We hypothesized that OB ACh release could reinstate reduced OB odor responses after OSN
adaptation, a process known as dishabituation. We find that BFS following prolonged odor presentations
recovers habituated OB odor responses to near baseline levels. Based on this, we tested whether OB ACh
release leads to similar behavioral effects in a habituation/dishabituation paradigm using mice expressing ChR2
in cholinergic neurons. Preliminary data suggest that OB ACh release leads to increased investigation of
habituated odorants, an effect not seen in wildtype littermates. Overall, these studies establish a novel functional
role for olfactory ACh release, whereby odor representation and salience can be rapidly and dynamically
modulated.
P3-012 Functional connectivity of perinatal and adult born granule cells following localized
optogenetic stimulation in the mouse olfactory bulb
Marta Pallotto, Kevin L. Briggman
National Institute of Neurological Disorders and Stroke, National Institute of Health, Bethesda, Maryland, USA
Granule cells (GCs) are the main GABAergic interneurons of the olfactory bulb (OB). They are produced from
birth to adulthood and modulate the activity of mitral and tufted cells (M/TCs), with whom they make dendrodendritic synapse which represent GCs only output. MCs and TCs are functionally distinct cell types and process
different aspects of olfactory information, forming two different sub-circuits. The role of GCs in the OB has been
extensively investigated. However, whether the time of birth of these interneurons contributes differently to the
inhibition of MC and TC output neurons is unknown. The aim of this work is to investigate, in adult mice, the
functional connectivity of GCs with MCs and TCs, in particular as a function of the time of birth of GCs. We use
Pdch::CRE transgenic mice, in which injections of a light activated ion channel, fl-ChETA AAV, allow expression
specifically in M/TCs. In OB slices, whole field light stimulation elicits axon potentials in MCs. Using a micromirror device (DMD), we are able to selectively stimulate specific areas of the OB of different size and shapes
with high temporal and spatial resolution. We are able to light stimulate OB layers as well as single MCs, while
recording axon potentials from MCs. In the same mouse we inject AAV vectors encoding genetic encoding
calcium indicators (GECIs, as GCaMP6s and Ruby-GCaMP6s) at postnatal day 3 (p3) and p40, to label and
2016.05.27 17.38.25 Page 6(1)
identify perinatal and adult-born GCs, respectively. Whole field light stimulation of M/TCs elicit calcium transients
in both perinatal and adult born GCs. We are in the process of investigating whether perinatal and adult-born
GCs respond differently to localized light stimulation. These experiments will help us to investigate the specificity
of connectivity between MCs, TCs and adult-born and perinatal born GCs, and therefore develop a better
understanding of adult neurogenesis.
P3-013 Function of contra-lateral interactions between olfactory bulbs
Florence Marianne Kermen, Emre Yaksi
Kavli Institute for System Neuroscience, Centre for Neural Computation, Faculty of Medicine, Trondheim, Norway
Similar to vision or audition, the peripheral olfactory system consists of a pair of sensory organs. Olfactory
information coming from a given nostril is sent to the ipsilateral olfactory bulb and then transmitted to the
olfactory cortices. This suggests an independent and somehow redundant encoding of olfactory information by
two parallel olfactory channels. Interestingly, previous anatomical studies showed that each olfactory bulb
receives top-down projections from the olfactory cortices of the contralateral hemispheres. However, the extent
of these contralateral inputs as well as their functional consequences on odor processing is not studied in detail.
Using electroporation of dextran-coupled dyes and transgenic fish lines, we showed that the two olfactory bulbs
are directly connected in addition to the projections from the cortical areas. Moreover our results suggest that
these direct connections between olfactory bulbs are topographically organized and mostly terminating at the
superficial layers of the olfactory bulb. On the contrary, the top-down projections from the olfactory cortices are
rather diffuse and mostly terminating at the inhibitory granule cells layer. Our results suggest that these different
types of contralateral connections are layer specific and thus could target different cell populations (excitatory
projection neurons or inhibitory interneurons). Using biphoton calcium imaging and intracellular recordings in a
brain-explant preparation of adult zebrafish, we showed that information coming from one olfactory bulb can
significantly alter odor responses in the contralateral olfactory bulb, eliciting a balance of excitation and
inhibition. We are currently exploring the contribution of these contralateral inputs in improving signal to noise
ratio and odor discriminability.
P3-014 Sniffing activates olfactory cortex in individuals with congenital anosmia
Tali Weiss1, Sagit Shushan1,2, Aharon Ravia1, Or Pinchasov1, Yehudah Roth2, Noam Sobel1
1
Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel, 2Department of Otolaryngology-Head and Neck
Surgery, Edith Wolfson Medical Center, Holon, Israel
Individuals diagnosed with congenital anosmia fail to detect odors and have no recollection of smell. The
mechanisms underlying this impairment remain poorly understood. Anosmia may reflect damaged peripheral
mechanisms in olfactory epithelium or damaged central mechanisms in olfactory bulb and cortex. We set out to
test the brain response to sniffing alone in congenital anosmia. Whereas sniffing in normosmic individuals
activates both somatosensory and olfactory cortex, we hypothesized that sniffing in congenitally anosmic
individuals would activate somatosensory cortex alone. We used 3-Tesla fMRI to measure the blood
oxygenation level dependent signal response during an event-related modified sniffing task: subjects were cued
by an auditory tone to sniff for 2 seconds once every 20-24 seconds (jittered) for 30 times. After each trial
subjects pressed a button denoting whether an odor was present or not. Subjects were misleadingly instructed in
advance that very low concentration odorants would be delivered, yet in practice no odorants were actually
delivered. We studied 10 subjects with congenital anosmia (4 F, mean age=31.3±3.8) and 10 matched
normosmic controls (4 F, mean age=27.7±1.9). We observed no differences in the brain response to sniffing in
anosmia and normosmia. Both groups activated extensive portions of olfactory cortex in ventral temporal lobe
and orbitofrontal cortex in response to sniffing (P<0.01, Bonferroni correction). That olfactory cortex responds to
sniffing in congenital anosmia implies a potential brain response to odors in these subjects that may remain
without awareness.
P3-016 The search for human social chemosignals: Characterizing volatiles emanating from the human
ear
Shani Agron, Tali Weiss, Sagit Shushan, Smadar Cohen-Atsmoni, Noam Sobel
The study of human social chemosignaling has concentrated on sweat as a primary chemosignal carrier. Sweat,
2016.05.27 17.38.25 Page 7(1)
however, is not the only path for excretion of volatile organic compounds (VOCs). For example, recently we
identified a social chemosignal in human emotional tears that leads to reductions in testosterone and related
behavior in men who sniff it. A similar mating behavior inhibitor was later identified in juvenile mouse tears. The
human ear extracts cerumen (earwax), a rich source of VOCs. Moreover, the human ear is considered an
erogenous organ across cultures. Finally, polymorphism in a gene central to human odor production (ABCC11)
leads to polymorphism in cerumen phenotypes. A recent comprehensive study of cerumen VOCs identified
several compounds (Prokop-Prigge et al., 2014). To further extend this effort we obtained cerumen from 13
healthy individuals (6 F, age=36.3±17.1) by way of suction from the ear canal. We subjected cerumen
headspace to VOC analysis using gas chromatography mass spectrometry (GC-MS). In brief, we incubated
cerumen samples with PDMS-covered stir bars (Gerstel Twister) at 37oc, overnight. Then the Twister was
desorbed in a Thermal desorption Unit (TDU, Gerstel GmbH, Germany). In addition to compounds previously
identified by Prokop-Prigge et al (2014), we identified an additional novel cerumen VOC: 2-hexanone. It now
remains to be tested whether previously identified cerumen VOCs, or this novel compound, function as human
social chemosignals.
P3-017 Neural investigation of the Crown-of-thorns starfish reveals radial nerve excretion sites
Meaghan Smith1, Utpal Bose1, Tianfang Wang1, Bronwyn Rotgans1, Mike R. Hall2, Scott F. Cummins1
1
Department of Sience and Engineering, University of the Sunshine Coast, Australia, 2Australian Institute of Marine Sciences,
Australia
The Crown-of-thorns starfish (COTS; Acanthaster planci) are mobile coral predators that are prevalent on the
Great Barrier Reef (GBR) and reefs across the Indo-Pacific. They are highly fecund echinoderms, a feature that
has contributed to extensive population outbreaks, causing detrimental damage to coral reefs. In fact, the past
three decades have seen a steady decline in over half of all coral cover on the GBR, with 37% of this due to
COTS. Whilst equipped with basic visual sense, COTS primarily navigate their environment using
chemosensory mechanisms. To help elucidate the neural mechanism of chemosensation in COTS, we
performed an ultrastructure investigation followed by multi-omics analysis of the COTS radial nerve cord, a
tissue that controls the numerous arms and chemosensory tentacles. Scanning electron micrographs revealed
that the ventral surface of the radial nerve is completely covered in bulbous structures. Subsequent transmission
electron micrographs showed that these novel formations are protrusions of the ectoneural region of the radial
nerve and are populated with cilia and large granular secretory vesicles. Within the radial nerve cord, a
repertoire of neurotransmitters was identified including neuropeptides and G-protein coupled receptors.
Neuropeptides match to both known and novel precursors, some of which may be unique to starfish and
possibly even the COTS. Our analysis provides the first exploration into the radial nerve cord of this coral
predator, allowing for identification of species-specific features that may reveal vulnerabilities to be target for
pest control.
P3-018 The influence of androstadienone on neural stress reactions in depression
Ka Chun Chung1, Bruce Turetsky2, Jessica Freiherr3,4, Birgit Derntl1,5,6
1
Department for Psychiatry, Psychotherapy and Psychosomatic, RWTH University Hospital, Aachen, Germany, 2Department of
Psychiatry, Medical School, University of Pennsylvania, Philadelphia, USA, 3Diagnostic and Interventional Neuroradiology, RWTH
University Hospital, Aachen, Germany, 4Fraunhofer Institute for Process Engineering and Packaging IVV, Freising, Germany, 5JARA
Brain, Juelich-Aachen Research Alliance, Translational Brain Medicine, Germany, 6Department of Psychiatry and Psychotherapy,
University of Tuebingen, Germany
Introduction: Vulnerability to stress is seen as a major risk factor for developing and maintaining depression.
Androstadienone (ANDR; Wyart et al., 2007), a synthetic male steroid, has been shown to be a modulator for
stress reactions at neural, hormonal and behavioral levels in healthy females and males depending on their
stress sensitivities to social threat (Chung et al., 2016a, 2016b). The current study will provide the first look at the
influence of ANDR on psychosocial stress responses in depressive patients.
Methods: In this fMRI study, 66 healthy and 21 depressive heterosexual individuals were measured twice, one
time using ANDR masked with musk, another time using pure musk (placebo) while performing the Montreal
Imaging Stress Task (Dedovic et al., 2005), a mental arithmetic task with social evaluative feedback. BOLDactivity (Siemens 3T; TR=2000ms, TE=28ms, 34 slices, 3.3mm, gap 10%), cortisol, psychophysiological data,
subjective stress reaction and behavioral performance were assessed.
Results/Discussion: No group differences in MONEX score (Freiherr et al., 2012), pleasantness, intensity and
familiarity of ANDR emerged (all ps .075). Regarding behavior, significant treatment x group interactions
!
2016.05.27 17.38.25 Page 8(1)
emerged: Patients performed worse under exposure to ANDR compared to placebo. ANDR decreased negative
mood and anxiety in patients. Regarding neural activation, a significant treatment effect occurred with stronger
activations of dorsolateral prefrontal cortex (BA 8, 9) and left dorsal anterior cingulate cortex in the placebo>
ANDR contrast. Moreover, a treatment-by-group interaction emerged, as controls showed stronger activation of
DLPFC (BA 9, 47) compared to patients in the stress condition.
Taken together, this is the first study to investigate the interaction of ANDR and stress induction in depression.
Our findings suggest that cognitive control during social threat processing is impaired in depression when
socially salient chemosensory signals, i.e. ANDR, are present.
Acknowledgement: Funded by START Programme (Medical Faculty RWTH Aachen University, Project
691302).
P3-019 Identification of urinary pheromones releasing the flehmen response in the domestic cat
Minoru Maita1, Jana Caspers2, Tetsuro Yamashita1, Stefan Schulz2, Masao Miyazaki1
1
Department of Biological Chemistry and Food Sciences, Iwate University, Iwate, Japan, 2Department of Life Science, Technical
University of Braunschweig, Braunschweig, Germany
Pheromones release innate behavior without learning and prior experiences in animals. Behavioral responses in
several mammalian species suggest that most mammals use pheromones. However, only a few pheromones
have been chemically characterized in a limited numbers of mammalian species. This is because pheromones
are usually found at a very low concentration in secretions containing thousands of compounds. Here, we report
chemical structures and in vivo origins of urinary pheromones releasing an innate behavior known as the
flehmen response in the domestic cat. When cats sniff the urine deposited by other cats, they exhibit the flehmen
response; the cats raise the head and hold the mouth partially open with an enraptured expression for a few
seconds. Urinary pheromones releasing the flehmen response were purified from male cat urine by liquid
chromatography using the response as a bioassay indicator. The pheromones were concentrated into a total
lipid fraction extracted from the urine. The lipid was fractionated by normal-phase and reversed phase
chromatography. Bioassays showed that the pheromones were enriched into free fatty acid (FFA) fraction. The
bioactivity of the FFA fraction was inactivated by treatment with trimethylsilyldiazomethane that modified FFA to
FFA methyl esters, strongly suggesting that pheromones are FFAs. GC-MS detected some branched chain
FFAs at minor peaks with long-chain FFAs in the FFA fraction. Finally we found that two branched chain FFAs
release the flehmen response in cats. Although these compounds were minor compounds in the FFA fraction of
cat urine, most of urinary triglycerides had them. Furthermore, such triglycerides were detectable in lipid droplets
that are physiologically accumulated in cat kidneys. These findings will improve our understanding of chemical
structures of mammalian pheromones and biosynthetic pathways.
P3-020 The effects of low levels of odors on behavior in rat
Rieko Hojo, Yukie Yanagiba, Masao Tsuchiya, Akinori Yasuda
National Institute of Occupational Safety and Health, Japan
We examined the effects of odors on behavior in rat. Several kinds of chemicals with low levels were exposed to
male Wistar rats. Odors used in the present study were acetone, which is frequently detected in workplace, 2phenyl ethyl alcohol (2PA), which is smell of rose, and 2, 4, 5-trimethyl-3-thiazoline (TMT), to which rat innately
shows an aversion. General activity, emotion, attention, motor function, and learning and memory function of rat
were examined with several kinds of behavioral tests. Results before and after exposure of each behavioral test
were compared. It is clarified that odors affected emotion, general activity and learning and memory function of
rat. In addition, it is possible that pattern of behavioral modification was different depending on odors. As a result,
it was clarified that odors used in the present study affected emotion, general activity and learning and memory
function of rat. Patterns of behavioral modifications derived from acetone exposure were similar to those of TMT.
The smell of acetone can be abominable for rat same as TMT. We concluded that behavior can be changed by
odor even if the level of odor is quite low. It may be necessary to consider novel standard values of chemicals in
workplace for efficiency of working performance, not a health problem.
2016.05.27 17.38.25 Page 9(1)
P3-022 Activities of the medulla and the limbic system synchronized with respiration during olfactory
stimuli−fMRI study−
Yuri Masaoka1, Masaki Yoshida2, Nobuyoshi Koiwa3, Keiko Watanabe1, Akira Yoshikawa1, Masahiro Ida4, Ikuo Homma5,
Masahiko Izumizaki1
Department of Physiology, Showa University School of Medicine, Tokyo, Japan, 2Department of Ophthalmology, Jikei Medical
University, Tokyo, Japan, 33 Department of Health and Science, University of Human Arts and Sciences, Saitama, Japan,
4
Department of Radiology, Stroke Center, Ebara Tokyo Hospital, Japan, 5Tokyo Ariake University of Medical and Health Sciences,
Tokyo, Japan
1
Olfaction is the only sense that is directly dependent on a basic physiological process, relying on respiratory
activity for the delivery of odorants to chemoreceptors in the nasal cavity. The link between respiration and
olfaction is evidenced explicitly by observed synchronization between respiratory cycles and neural activation of
the olfactory circuit during odor perception in both animals, and humans. Respiratory center in the medulla
generates the basic respiratory rhythm, however, the respiratory rhythm is modulated by inputs from higher
olfactory-related areas. It is unknown how interact between olfactory-related areas and the brainstem respiratory
rhythm generator for changing respiratory rhythm during olfactory stimuli. In this study, we performed
simultaneous recordings of respiration and brain activities using functional Magnetic Resonance Imaging (fMRI)
during resting state and olfactory stimuli to identify synchronization of the inspiration onsets and bold signals in
medulla, pons, parabrachial nucleus as well as higher centers such as piriform cortex, amygdala and
hippocampus. Respiratory flow and cardiac signal were simultaneously recorded during the scan and
physiological noises from fMRI-BOLD signal measurement were retrospectively removed (Drifter, SPM8).
Respiratory flow was used to identify the time series of inspiration onsets. Subjects were scanned during resting
and olfaction state for 10 minutes each. During resting state, dorsal and ventral medulla were activated with
synchronization of inspiration onsets. Olfactory stimuli activated the amygdala, hippocampus and parabrachial
nucleus in addition to dorsal medulla, and while activated less in ventral medulla. Olfactory stimuli caused
emotional change with activations of amygdala and hippocampus which modulated respiratory frequency, and
these respiratory change may relayed through activation of parabrachial nucleus and -dorsal medulla
connections.
P3-023 Transformation of odor representations in the Drosophila mushroom body facilitates both
discrimination and categorization
Keita Endo, Yoshiko Tsuchimoto, Hokto Kazama
Laboratory for Circuit Mechanisms of Sensory Perception, RIKEN BSI, Saitama, Japan
The function of the olfactory system is not only to discriminate myriads of odorants, but also to categorize a set
of odorants as a unitary odor object, because most odors in the nature are mixtures of many odorous chemicals
and have a relevant meaning for animals when presented as a whole. To understand the circuit mechanisms
involved in odor categorization, we compared representations of various odorants and mixtures in the primary
olfactory center, the antennal lobe (AL), with those in the secondary center, the mushroom body (MB), in the
Drosophila brain, using functional calcium imaging.
We found that in general, different odorants were represented more distantly from each other in the MB than in
the AL, conferring downstream neurons more discriminability. Yet in the MB, the representations of mixtures with
different mixing-ratio were closer to each other, while more distant from their components regardless of the
mixing-ratio, allowing downstream neurons to categorize mixtures as a unitary odor object. Decoding the mixture
representations by linear classifiers revealed that the representations in the MB can be categorized as a mixture
more accurately than those in the AL.
The distinct mixture representation in the MB was attributed to individual neurons that changed their responses
to mixtures distinctly from their components. Decoding mixture representation of MB with only neurons that
changed responses gradually from components to mixtures resulted in a drop of the categorization accuracy to
the level of the AL, indicating that odor categorization requires neurons that respond to mixtures distinctively in
the MB.
The distinctive responses of the MB neurons to mixtures were simulated by a model MB with global inhibition,
even without modulating individual synaptic strengths between the AL and the MB neurons.
2016.05.27 17.38.25 Page 10(1)
These results suggest that odor representations are transformed in the MB to facilitate both discrimination and
categorization.
P3-024 The sniffing brain−linking cognitive performance with nasal respiration
Ofer Perl, Tali Weiss, Liron Pinchover, Nofar Mor, Lavi Secundo, Elad Cohen, Lee Sela, Noam Sobel
Department of Neurobiology, The Weizmann Institute of Science, Rehovot, Israel
The primary role of respiration is to maintain life. However, the respiratory cycle also plays an important role in
sensory perception. Most terrestrial mammals rely on chemosensory sampling through rhythmic nasal
inhalation, namely sniffing. Olfaction, however, is not the only sense orchestrated by the respiratory cycle, and
respiratory phase-locking of diverse active sensing mechanisms is common across species. Whether humans
exhibit such respiratory-driven modulation of sensing or mentation remains unknown. With this in mind, we set
out to ask whether respiration mode affects performance of simultaneously conducted cognitive tasks. First, we
asked whether humans subliminally opt to engage in cognitive tasks during a specific respiratory phase. To
answer this we monitored nasal respiration of subjects as they engaged in cognitive tasks, self-initiated by the
subjects. An analysis of respiratory phase revealed a reduction in intra-subject variance, time-locked with task
initiation, implying a highly-repetitive individual strategy. To control for this effect, we then used the subjects’ own
respiration to trigger a lexical decision task time-locked with inhale and exhale onset. Reaction time for non-word
trials was lower for words presented during nasal inhalation compared with exhalation. This modulatory effect
however, was not evident in oral respiration, suggesting a unique role for nasal inhalation in cognitive processing
(ANOVA nasal/oral X word/non-word X inhale/exhale : F(1,39)=5.16, p<0.03). Finally, we recently replicated this
paradigm within the fMRI environment to explore the neural correlates associated with these effects. Results
from this part of the study will be presented at the meeting. Our results suggest that human brain function
fluctuates between two modes of activation driven by respiration, with inhalation being associated with improved
acquisition and processing of information.
P3-025 On the way to blood: Odor-guided behavior in kissing bugs
Florencia Campetella1, Rolf Beutel2, Bill S. Hansson1, Silke Sachse1
1
Department of Evolutionary Neuroethology, MPI for Chemical Ecology, Jena, Germany, 2Friedrich Schiller University, Jena,
Germany
Finding a blood meal is all it takes for a kissing bug to develop into the adult stage. To achieve this, these
insects, belonging to the family of Triatomines (Reduviidae, Heteroptera) and vectors of Chagas disease, relay
on physical and chemical cues. In order to find a blood source, kissing bugs explore an olfactory space, where
they encounter attractive, repellant or neutral stimuli. Blends and single compounds constitute the dimensions of
this space, which to date remains uncharacterized. Even more, how this sensory information is processed by the
nervous system of these insects remains a mystery. In the present work we first unveil which olfactory cues are
used in the context of odor-guided behavior; we assigned a hedonic value to each of the chemical compounds
tested. We next asked how this information is detected and processed by their peripheral nervous system. We
present evidence indicating specific morphological structures dedicated to olfactory detection, which were
further characterized by electrophysiological means. These olfactory sensilla were classified in different types,
according to their response profile to our broad odor panel. We show that specific sensilla types respond to
attractive odorants (i.e. vertebrate volatiles). As is the case with other insect species, these bugs have dedicated
circuits involved in olfactory processing, which trigger specific behaviors, such as host finding. Our results
constitute the first step towards the understanding of olfactory coding in Triatomines.
P3-026 Defining a function of olfactory bulb processing via comparison of input and output
Douglas Anthony Storace, Lawrence B. Cohen
Yale University, USA
The goal of the present study was to identify the function(s) of the olfactory bulb by comparing its output with its
input. The input olfactory receptor neurons were anatomically targeted via nasal infusion with an organic calcium
sensitive dye. In the same preparation, genetically encoded voltage or calcium indicators were targeted to the
output mitral and tufted cells using Cre-dependent AAV transduction in a transgenic mouse (Pcdh21) that
expresses Cre recombinase in mitral and tufted cells. Wide-field epifluorescence imaging was used to measure
odor-evoked activity in glomeruli across 2 log units of odorant concentration in freely breathing anesthetized
2016.05.27 17.38.25 Page 11(1)
mice. Remarkably, the output maintained a relatively stable representation of odor quality over the tested
concentrations, while the number of activated input glomeruli and the input amplitude markedly declined. This
provides the first direct evidence that the mammalian olfactory bulb participates in generating the perception of
concentration invariance of odor quality. These imaging methods should also be useful for determining the
transform from input to output in other regions of the mammalian brain.
P3-027 Feasibility of the functional MRI as a objective assessment of olfactory dysfunction after
traumatic brain injury
Donghyuk Im, Sung Hwan Ahn, Jin Kook Kim
Department of Otorhinolaryngology-Head and Neck Surgery, Konkuk University School of Medicine, Seoul, South Korea
Objectives The objective of this study to assess the feasibility of functional MRI as an objective tool in the
measurement of patients with olfactory dysfunctions after sustaining traumatic brain injury, and to compare the
activation area of the functional MRI images between normal and patient groups. Materials and Method This
study was conducted on 18 patients who had suffered olfactory dysfunction after experiencing traumatic brain
injury from 2012 to Feb. 2016 and 10 subjects of the control group with normal olfactory functions. The functional
MRI using a devised olfactometer composed of Citraval 1M/β-mercaptoethanol and Blood Oxygenation level
Dependent signals to localize activation areas within the brain. Results In the early group, the POC(Primary
olfactory cortex) activation rate was 18.18%. and 7 out of 11 patients showed OPOC(Outside POC) activation. In
the delayed group, the POC activation rate was 0% and the 4 out of 7 patients showed OPOC activation. In the
control group, the POC activation rate was 30% and every subject showed outside POC activation. When there
was damage to the frontal lobe, POC, OPOC area activation was seen but no POC, OPOC activation was to be
detected in the occipital-damaged group. When intracranial injury was associated, activation was barely initiated
compared to non-intracranial injury. When comparing the amount of brain tissue loss, more tissue loss seemed
to result in less activation. There was no significant difference regardless of the type of reagent used thus,
implying that activation area was related to area damaged and the amount of brain tissue loss, rather than the
reagent used. Conclusion The functional MRI showed different signal activation within the brain area between
the patient and control group, suggesting the possibility of functional MRI as a compatible tool for objective
olfactory function evaluation in patients suffering olfactory dysfunction after traumatic brain injuries.
P3-028 Defining a human olfactory network based on resting-state functional connectivity
Thomas Campbell Arnold1, Ivan de Araujo2, Wen Li1
1
Psychology, Florida State University, Tallahassee, USA, 2John Pierce Laboratory, Yale University, New Haven, USA
Recent application of resting-state functional magnetic resonance imaging (rs-fMRI) has unveiled several key
inherent networks in the human brain.
While networks for the physical senses (sensorimotor, auditory, and visual) have been defined with this new
technique, networks for chemical senses of olfaction and gustation are still unknown. Here, we explored the
olfactory network by analyzing inter-regional correlation between rs-fMRI timeseries.
Rs-fMRI data (to date, N=229, each containing 4,800 scans) were obtained from a large data set provided by the
Human Connectome Project. We examined regions known to be involved in or related to olfaction, including
orbital frontal cortex (OFC, including anterior, posterior and olfactory OFC), anterior and posterior piriform cortex
(APC/PPC), amygdala, anterior and posterior hippocampus (AH/PH), insula (divided into eight subdivisions to
roughly equate the size to other regions), and thalamus (divided into four subdivisions). Three modules (defined
by strong internal correlations and weak external correlations) emerged from the analysis-a thalamus module,
an insula module and importantly, an olfactory module (encompassing olfactory OFC, piriform cortex, amygdala
and hippocampus). Representing the olfactory network, the olfactory module was characterized by these
specific connections: the APC connection to olfactory OFC and PPC; PPC connection to APC and amygdala;
and amygdala connection to PPC and both AH and PH (p’s =.001-.05; the other inter-region connections did not
survive t-tests of significance). Besides these intra-module connections, the olfactory module was linked to the
insula via APC and olfactory OFC, which connected with dorsal anterior insula (a region implicated in human
olfaction). Lastly, connection between the olfactory network and the thalamus appeared indirect and was
mediated by the dorsal anterior insula that linked the dorsal medial thalamus with APC and olfactory OFC. Our
findings thus provided insights into the olfactory network while validating current knowledge of olfactory
functional neuroanatomy.
2016.05.27 17.38.25 Page 12(1)
P3-029 Discovery and characterization of a novel olfactory connection in the human
Jennifer J. Stamps1,2, Jay Deng3,4, Kaan Yagmurlu5
1
Department of Food Science and Human Nutrition, The University of Florida, Gainesville, FL, USA, 2Department of Neurology, The
University of Florida, Gainesville, FL, USA, 3Department of Neuroscience, The University of Gainesville, FL, USA, 4EnCor
Biotechnologies, Inc., Gainesville, FL, USA, 5The Department of Neurosurgery, The Barrow Neurological Institute, Phoenix, AZ,
USA
Projections from the olfactory bulb (OB) were primarily mapped out in rat and nonhuman primate studies (Price
JL, 1973; Carmichael, Clugnet, Price, 1994). Those studies focused on the lateral olfactory tract. Three
nonhuman primate studies of the medial olfactory tract (mOT) in the 1970s concluded that the mOT does not
receive secondary olfactory fibers from the olfactory bulb and that after projecting to the anterior olfactory
nucleus and the olfactory tubercle, the entire secondary olfactory axons enters the lateral olfactory tract (Heimer,
1975; Rosene and Heimer, 1977; Turner, Gupta, Mishkin, 1978). Since those early, nonhuman primate studies,
a characterization of the mOT in humans have not been reported (Nieuwenhuys, Voogd, Van Huijzen, 2008).
After careful dissection of the human basal forebrain, we observed the olfactory tract bifurcating and the mOT
traversing above the anterior perforated substance and then ending completely into the Nucleus Accumbens
(NAc). We then conducted immunohistochemistry with four human specimens and confirmed that the
overwhelming majority, and perhaps all, of the mOT terminates and disperses its axons from the OB into the
NAc. While the lateral OT provides a direct connection between olfaction and memory and emotion networks, it
appears that in humans, the mOT provides a direct connection between olfaction and our pleasure network. We
are continuing to characterize this uniquely human pathway.
P3-030 Effect of changing waving patterns of maxillipeds during olfactory search by crayfish
Hanako Ishida, Ryuichi Takemura, Hiroshi Ishida
Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan
Crayfish generate jet-like water currents by waving the flagella of their maxillipeds during food search. The
generated jets induce an inflow that is considered to help collect odors from the surroundings to their olfactory
organs. When crayfish change the directions of the jets by altering the way of waving their maxillipeds, the
direction of the inflow is changed accordingly. Our recordings of the maxillipeds’ activity of crayfish during food
search have shown that crayfish mostly wave the maxillipeds on both sides when they walk straight. It has been
also found that, when crayfish are turning to one side, they mostly wave the maxillipeds only on the side opposite
to the turning direction. Our objective here is to investigate the significance of this change in the waving pattern
of the maxillipeds in terms of odor sample collection. A robotic device equipped with a pump and electrochemical
sensors is used to simulate the jet generation and chemical reception by crayfish. The device can generate jets
in several different directions. The odor sample collection efficiency can be evaluated by placing an odor source
and the device in a water tank and observing the responses of the chemical sensors. Results of the experiments
using this device have shown that changing the waving pattern of the maxillipeds while turning is beneficial in
food search. Suppose that a crayfish is turning to the left. When the maxillipeds only on the right side are waved,
a flow is generated from the left to the right side of the crayfish. Water samples are drawn to the olfactory organs
of the crayfish intensively from the left side. Therefore, if a food exists in the turning direction, the crayfish can
notice its presence at an earlier timing by waving the maxillipeds on one side than waving those on both sides.
P3-031 Implicit measures of flavor preference as a result of repeated taste and smell exposure
Peter de Kok1, Harold Bult1, Joost Wegman2, Esther Aarts2
1
NIZO Food Research, Ede, The Netherlands, 2Centre of Cognitive Neuroimiging, Donders Institute for Brain, Cognition, and
Behavior, Radboud University Nijmegen, The Netherlands
Neuroimaging literature, mostly based on functional Magnetic Resonance Imaging (fMRI), has demonstrated the
involvement of centers in the basal ganglia in the experience and the anticipation of reward. Responses in these
tissues are involuntary and immediate. Hence, anticipated reward is a primary, non-verbal response that reflects
how much a person wants the stimulus shown. This relation was shown for non-food, food and food-related
stimuli. In recent years, we have evaluated a number of event-related (electro-) physiological measures on their
sensitivity to measure reward anticipation. To evaluate the effects of food flavor on reward anticipation, subjects
consumed five differently flavored unbranded tomato sauces during three consecutive exposure sessions. Each
flavor version was labeled with an abstract colored label and subjects learned to associate the label with the
flavor. After this exposure phase, labels were shown to the same subjects, both during fMRI sessions as well as
2016.05.27 17.38.25 Page 13(1)
during sessions measuring event-related EEG, pupil dilation, near-infra-red spectroscopy (NIRS) measures of
the intra-cranial blood oxygenation level and response latencies of a joystick approach-avoidance task. Positive
correlations were observed between hemodynamic responses in parts of the basal ganglia (fMRI) and
alternative physiological measures in response to food labels. Notably, aspects of event-related EEG potentials
(ERP) 300 to 500 ms after stimulus presentation were predictive of anticipated reward responses in the basal
ganglia. From these learnings, an automated system was developed to measure anticipated food reward
responses in a real-time fashion allowing the immediate evaluation of anticipated reward responses to different
product versions as follows: 1. individual wanting responses can be assessed after taste and smell exposure,
and linked to target consumer groups. 2. The nature of the wanting responses makes it independent of cultural
background since no language or customary behaviors are required to generate a response.
P3-032 The Apis mellifera GABAa receptor (Rdl) in the olfactory circuit in the honey bee brain
Irina T. Sinakevitch, Brian H. Smith, Ying Wang
School of Life Science, Arizona State University, Tempi, USA
Processing of olfactory information in insect brain begins in the antennal lobe (AL), where GABAergic inhibition
modifies processing in potentially different ways to pass information to higher brain centers. Our current interest
involves understanding how inhibition is involved in extraction of temporal features of odor stimuli that enable
foreground-background segregation. GABA acts via GABAa receptors, one subunit of which-Resistance to
Dieldrin (Rdl)-has been well characterized in behavior, anatomical localization and knockdown experiments in
the fruit fly. The aim of our study is characterization of the honey bee ortholog receptor AmelRdl (BEEBASE GB
40975) and its role in tracking target odors against background odors. First, we have developed antibodies
against the AmRDL receptor using the conjugated peptides in N (extracellular) and C (intracellular) domains of
the receptor and characterized their specificities using western-blot analyses, preadsorption procedures and
dsiRNA knockdown experiments. These anti-AmRDL antibodies were also used to examine the expression of
AmRDL in the ALs of fixed brain sections from forager bees. The anti-AmRDL was expressed in all glomeruli of
theAL but not in the aglomerular neuropil, suggesting that the AmRDL/GABAa receptor plays an important role
in synaptic boutons of each glomeruli. The axons from olfactory sensory receptor neurons and projection
neurons do not express the receptor. However, their branches in the glomeruli could express of AmRDL in
boutons post-synaptic to GABAergic processes. The widespread nature of anti-AmRDL staining in AL glomeruli
suggests that the RDL subunit codes for a major component of honey bee GABAa receptor and plays an
important role in inhibitory modulation of odor responses in honey bee.
P3-033 Functional analysis of ctcf in odorant receptor gene expression in mice
Chizuru Kobayashi1, Tetsuo Iwata2, Niles Galjart3, Junji Hirota1,2
1
Department of Bioengineering, Tokyo Institute of Technology, Yokohama, Japan, 2Center for Biological Resources and Informatics,
Tokyo Institute of Technology, Yokohama, Japan, 3Department of Cell Biology and Genetics, Erasmus MC, Rotterdam, The
Netherlands
The CCCTC-binding factor (CTCF), a zinc finger transcription factor, is a key molecule to organize the global
chromatin architecture to regulate gene expression, including clustering genes, such as β-globin and Pcdh
(Protocadherin ) genes. In the β-globin locus, CTCF-based cell-type specific interactions in erythroid cells are
required to establish the developmentally regulated β-globin gene expression by the locus control region (LCR).
In the Pcdh locus, CTCF-mediated chromatin conformational changes regulate the stochastic and combinatorial
expression of the three clusters of Pcdh genes in each neuron to promote neuronal diversity.
In the olfactory system, individual olfactory sensory neuron expresses single olfactory receptor (OR) gene in
monogenic and monoallelic manner. Several studies have demonstrated that some enhancer regions function
as LCR to establish the singular OR gene expression from an OR cluster. Thus, it is conceivable that
CTCF-mediated chromatin organization contributes OR gene expression as in β-globin and Pcdh locus.
In this study, to elucidate the function of CTCF, we analyzed effects of loss of CTCF function on OR gene
expression in mice.
2016.05.27 17.38.25 Page 14(1)
P3-034 Expression divergence of chemosensory genes between Drosophila sechellia and its sibling
species and its implications for host shift
Meng-Shin Shiao1,2, Jia-Ming Chang3,4,5, Wen-Lang Fan1,3, Mei-Yeh Jade Lu1, Cedric Notredame3,4, Shu Fang1, Rumi Kondo7,
Wen-Hsiung Li1,8
Biodiversity Research Center, Academia Sinica, Taipei, Taiwan, 2Research Center, Faculty of Medicine Ramathibodi Hospital,
Mahidol University, Bangkok, Thailand, 3Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and
Technology, Barcelona, Spain, 4Universitat Pompeu Fabra (UPF), Barcelona, Spain, 5Institute of Human Genetics (IGH),
Montpellier, France, 6Whole-Genome Research Core Laboratory of Human Diseases, Chang Gung Memorial Hospital, Keelung,
Taiwan, 7Department of Biology, Ochanomizu University, Tokyo, Japan, 8Department of Ecology and Evolution, University of
Chicago, USA
1
Drosophila sechellia relies exclusively on the fruits of Morinda citrifolia , which are toxic to most insects,
including its sibling species Drosophila melanogaster and Drosophila simulans . Although several odorant
binding protein (Obp ) genes and olfactory receptor (Or ) genes have been suggested to be associated with the
D. sechellia host shift, a broad view of how chemosensory genes have contributed to this shift is still lacking. We
therefore studied the transcriptomes of antennae, the main organ responsible for detecting food resource and
oviposition, of D. sechellia and its two sibling species. We wanted to know whether gene expression, particularly
chemosensory genes, has diverged between D. sechellia and its two sibling species. Using a very stringent
definition of differential gene expression, we found a higher percentage of chemosensory genes differentially
expressed in the D. sechellia lineage (7.8%) than in the D. simulans lineage (5.4%); for upregulated
chemosensory genes, the percentages were 8.8% in D. sechellia and 5.2% in D. simulans . Interestingly, Obp50
a exhibited the highest upregulation, an approximately 100-fold increase, and Or85c −previously reported to be
a larva-specific gene−showed approximately 20-fold upregulation in D. sechellia . Furthermore, Ir84a
(ionotropic receptor 84a ), which has been proposed to be associated with male courtship behavior, was
significantly upregulated in D. sechellia . We also found expression divergence in most of the chemosensory
gene families between D. sechellia and the two sibling species. Our observations suggest that the host shift of
D. sechellia was associated with the enrichment of differentially expressed, particularly upregulated,
chemosensory genes.
P3-035 Analysis of active components in proestrous urine from female rats
Hitomi Akutsu1, Masaharu Kamo2, Jiro Hitomi3, Tomoyuki Saino1
1
Department of Anatomy (Cell Biology), Iwate Medical University, Iwate, Japan, 2Division of Cell Biosignal Sciences, Department of
Biochemistry, Iwate Medical University, Iwate, Japan, 3Department of Anatomy (Human Embryology), Iwate Medical University,
Iwate, Japan
Female rats’ urine is supposed to contain a sexual pheromone because that urine stimulate the cells of
accessory olfactory bulb and brain. However pheromone molecule and pheromone effect derived from female
are surrounded by mystery. To investigate the pheromone activity of female rats’ urine, we performed Ca2+
imaging by use of vomeronasal organ (VNO) slice separated from male rats with an exposure of female rats’
urine. The urine was sampled at proestrus when female rats showed mating behavior and diestrus to compare
the pheromone activity depend on the estrous cycle. Vomeronasal sensory neurons (VSNs) respond to the
exposure of the both urines with the increases of intracellular Ca2+ concentration ([Ca2+]i). We found that the
patterns of [Ca2+]i increase were divided broadly into two categories. One was a transient increase that VSNs
increased [Ca2+]i rapidly and got back it to the baseline level soon, the other was a persistent increase that VSNs
increased [Ca2+]i and kept it on the elevated level for several tens of seconds. Proestrous urine seemed to have a
specific activity which could induce persistent increases in more number of VSNs than diestrous urine. To
identify the factor that activate VSNs in proestrous urine, the components of proestrous and diestrous urine were
separated and compared with HPLC. Although we tried to use the reversed phase chromatography with C18
column under isocratic elution of 0.1% formic acid as a mobile phase, clear differences were not found. Then
hydrophilic interaction chromatography was performed by use of HILIC column under gradient elution (from
100% to 85%) of mobile phase B: 97% acetonitrile/10 mM ammonium formate. The small differences of
chromatograph between the proestrous urine and the diestrous urine were observed. It was supposed that
proestrous urine contained a hydrophilic substance which has a specific activity.
2016.05.27 17.38.25 Page 15(1)
P3-036 The molecular evolution and the expression of vomeronasal receptors 1 in common marmoset
Keiko Moriya-Ito1, Hikoyu Suzuki2, Takashi Hayakawa3, Kimiko Hagino-Yamagishi1, Masato Nikaido2
1
Animal Research Division, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan, 2Department of Biological Science,
Tokyo Institute of Technology, Tokyo, Japan, 3Primate Research Institute, Kyoto University, Kyoto, Japan
Vomeronasal organ (VNO) is known as a chemical sensor for detecting pheromones. However, VNO is absent
or vestigial in some species, one of which is represented by primate. VNOs exist in prosimians and New World
monkeys but not in Old World monkeys and hominoids including human. We are interested in the evolutionary
changes of pheromonal processing in primates. In prosimians, VNOs are well developed like rodents, and many
nocturnal animals maintain both vomeronasal receptor 1 (V1R) genes and vomeronasal receptor 2 (V2R) genes.
In contrast, VNO of some lineages of New World monkeys are reduced or vestigial, and intact V1R genes are
dramatically decreased and all V2R genes are pseudogenes in New World monkeys. It seems that their VNOs
are degenerating in their evolution. We focus on common marmoset (Callithrix jacchus ), a diurnal small New
World monkey. Given that common marmoset is expected to retain only a minimal subset of pheromone
processing via VNO, the marmoset can be a powerful model to understand the process of VNO regression in
primate evolution. We identified the V1R gene repertoire in the latest version of marmoset whole genome
sequences. The number of intact and disrupted V1R genes were 7 and about 50. V1Rs expression and
molecular evolutionary analysis revealed that the V1Rs generated by gene duplication in New World monkeys
were expressed in only the VNO. Some of other V1Rs were expressed in both VNO and olfactory epithelium
(OE), and the others were not expressed in VNO or OE. These V1R genes have high homology to intact
hominoids’ V1Rs. We speculate that species’ specific chemicals are mainly received by VNO and general
chemicals like hormones are received by both VNO and OE in common marmoset.
P3-037 Gene expression of odorant receptors in the olfactory organ of the Chinese soft-shelled turtle
Shoko Nakamuta1, Masao Miyazaki2, Yoshio Yamamoto1, Nobuaki Nakamuta1
1
Laboratory of Veterinary Anatomy, Iwate University, Iwate, Japan, 2Department of Biological Chemistry and Food Sciences, Iwate
University, Iwate, Japan
In general, the olfactory organ of semi-aquatic turtle is constituted by the upper and the lower chamber epithelia
that contain ciliated and microvillous olfactory receptor cells, respectively. In the olfactory organ of the Chinese
soft-shelled turtle (Pelodiscus sinensis ), however, ciliated olfactory receptor cells are localized not only in the
upper chamber epithelium but also in the lower chamber epithelium. Bioinformatics analysis identified more than
1000 odorant receptor genes in the genome of soft-shelled turtles. These odorant receptor genes were
suggested to be expressed in the ciliated olfactory receptor cells of both upper and lower chamber epithelia.
Thus, we investigated the tissue distribution of odorant receptor genes in the olfactory organ of soft-shelled
turtle. Turtle odorant receptor genes examined in this study were divided into three groups: those expressed
mainly in the upper chamber epithelium, those expressed mainly in the lower chamber epithelium, and those
expressed equally in the both epithelia. By categorizing these odorant receptor genes into class I and class II
odorant receptors according to the deduced amino acid sequences, we found that tissue distribution of most
class I or class II odorant receptors are different between the upper and the lower chamber epithelia, although
some of them were expressed equally in both epithelia. These results suggest that the upper and lower chamber
epithelia in the olfactory organ of soft-shelled turtle are equipped with partially overlapping but distinct functions.
P3-038 Pheromone binding protein is required for sensitive detection of sex pheromones in the
silkmoth Bombyx mori
Takeshi Sakurai1, Yusuke Shiota1, Takaaki Daimon2, Hidefumi Mitsuno1, Ryohei Kanzaki1
1
Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan, 2National Institute of
Agrobiological Sciences, Ibaraki, Japan
Male moths detect sex pheromones emitted by conspecific females with high sensitivity and specificity by the
olfactory sensilla on the antennae. Pheromone binding proteins (PBPs) are highly enriched in sensillum lymph of
pheromone sensitive olfactory sensilla, and supposed to contribute to sensitivity of pheromone detection by
enhancing solubility of hydrophobic pheromone molecules into sensillum lymph and/or protecting them from
enzymatic degradation during transport to sex pheromone receptor proteins on the dendritic membrane of
olfactory receptor neurons (ORNs). PBPs are also suggested to participate in discrimination of pheromone
components because they exhibit different binding affinities to each pheromone components. However,
functional role of PBPs in moth sex pheromone detection in in vivo is still obscure, partly due to the lack of
2016.05.27 17.38.25 Page 16(1)
loss-of-function mutants of PBPs in moths. In the silkmoth, Bombyx mori , female moths emit two sex
pheromone components, bombykol and bombykal. A pair of ORNs specifically tuned to either bombykol or
bombykal co-localizes in the trichodeum sensilla whose sensillum lymph contains at least one type of PBP,
named BmPBP1. Here, we show that the targeted disruption of BmPBP1 gene significantly reduced
electrophysiological responses of male antennae to bombykol and bombykal. We generated a BmPBP1 gene
knockout silkmoth line by transcription activator-like effector nuclease mediated gene targeting.
Electroantennogram analysis revealed that the response peak amplitude of BmPBP1-knockout male antennae
to bombykol and bombykal was significantly reduced compared to wild type male antennae. Furthermore, single
sensillum recordings of trichodeum sensilla showed that the disruption of BmPBP1 lowered sensitivity of
bombykol or bombykal-sensitive ORNs but did not affect selectivity of these ORNs. Our results indicate that
BmPBP1 is involved in detection of both bombykol and bombykal and plays an important role in the sensitivity of
silkmoth sex pheromone detection system.
P3-039 Effects of single olfactory receptor deletion on odor preference
Nao Horio1, Keiichi Yoshikawa1,2, Yoshihiro Yoshihara3,4, Kazushige Touhara1,4
1
Japan, 2Kansei Science Research Laboratory, Kao Corporation, Tochigi, Japan, 3Laboratory for Neurobiology of Synapse, RIKEN
Brain Science Institute, Saitama, Japan, 4ERATO Touhara Chemosensory Signal Project, JST, The University of Tokyo, Tokyo,
Japan
The olfaction plays an important role in innate behaviors. Odorants are recognized by multiple olfactory
receptors (ORs), and typical ORs are functionally redundant. In humans, there are several cases in which
genetic variation in a single OR has a large influence on the preference to the cognate odorant. However it is
unclear whether the activation of a single OR is enough to induce preference to the cognate odorant in mice.
(Z)-5-tetradecen-1-ol (Z5-14:OH) was identified as a male-specific natural ligand for the mouse olfactory
receptor 288 (Olfr288). Female mice spent more time investigating the Z5-14:OH-spiked castrated male urine
than control castrated urine, suggesting that Z5-14:OH is an attractant to females. To examine an effect of single
receptor deletion on odor preference, we generated Olfr288 knockout (KO) mice and performed the odor-finding
test and the odor-preference test. The odor-finding test showed that the detection threshold was one order of
magnitude higher in Olfr288-KO mice than wild-type (WT) mice. The odor-preference test demonstrated that Olfr
288-KO mice did not show preference to Z5-14:OH at a concentration that WT mice did, suggesting that the
activation of a single OR, Olfr288, can elicit preference behavior to Z5-14:OH. We next investigated the case of
muscone, a sex pheromone in musk deer. Muscone is recognized by two ORs, MOR215-1 and MOR214-3, in
mice, and attracts male mice. The detection threshold was two or three orders of magnitude higher in
MOR215-1-KO mice than WT mice. We found that both MOR215-1-KO and WT mice exhibited preference to
high concentrations of muscone, whereas only WT mice showed preference to low concentrations of muscone.
These data suggest that the activation of single OR, either MOR215-1 or MOR214-3, can elicit preference
behavior to muscone. This study demonstrates a clear link between a single OR and odor preference behavior in
mice.
P3-040 On the function of Trpm5 in olfactory sensory neurons
Martina Pyrski1, Eugenia Eckstein1, Andreas Schmid1, Ulrich Boehm2, Frank Zufall1
1
Center for Integrative Physiology and Molecular Medicine, University of Saarland, School of Medicine, Homburg, Germany, 2
Department of Pharmacology and Toxicology, University of Saarland, School of Medicine, Homburg, Germany
We have begun to systematically investigate the function of the transient receptor potential channel Trpm5 in
olfactory sensory neurons (OSNs) and the role of Trpm5-expressing cells in the olfactory system of the mouse.
We have carefully analyzed a Trpm5-IRES-Cre knockin strain that enables the mapping and characterization of
Trpm5-expressing cells using a binary genetic strategy (Kusumakshi et al., Chem. Senses 2015:413-425). To
monitor Cre recombinase activity, these mice were bred with a fluorescent ROSA26 reporter strain to generate
Trpm5-IC/eR26-τGFP double knockin mice. In these mice, τGFP expression reports the history of activity of the
Trpm5 promoter, thus labeling both cells that used to express Trpm5 as well as cells that acutely express Trpm5
at the time of analysis. To monitor acute protein expression, we have performed immunohistochemistry using
previously validated anti-Trpm5 antisera. One key result emerging from these studies is the finding that Trpm5
protein seems to be transiently expressed in an OMP-positive subpopulation of OSNs located in the embryonic
olfactory epithelium. To obtain insight into the function of Trmp5-expressing OSNs, we have performed Ca2+
2016.05.27 17.38.25 Page 17(1)
imaging measurements in Trpm5-IRES-Cre mice crossed with R26-GCaMP3 mice. For these experiments, we
developed a novel wholemount preparation of the main olfactory epithelium enabling in situ Ca2+ monitoring of
genetically labeled cells in adult mice. All Trpm5-expressing OSNs responded to forskolin with a Ca2+ rise,
indicating an intact cAMP signaling cascade in these neurons. By applying a number of chemostimuli, we found
that Trpm5-expressing OSNs comprise a heterogeneous group of OSNs many of which seem to be capable of
detecting a range of general odorant cues.
Supported by grants from the Deutsche Forschungsgemeinschaft.
P3-041 The perplexing roles of olfactory marker protein in olfactory transduction
Michele Dibattista1, Johannes Reisert2
1
Department of Basic Medical Sciences, Neuroscience and Sensory Organs, University of Bari, Bari, Italy, 2Monell Chemical
Senses Center, Philadelphia, USA
Olfactory marker protein (OMP) is a small cytosolic protein expressed in mature olfactory receptor neurons
(ORNs). Since its discovery over 30 years ago its mechanistic contribution to olfactory transduction has been
puzzling. In ORNs lacking OMP the odor response is greatly prolonged and OMP is a key component in
speeding up the kinetics of the olfactory second messenger cAMP by a hitherto unknown mechanism. But cAMP
does not only change in response to odorant stimulation but, as different olfactory receptors (ORs) have different
basal activity, also in the absence of odorants. We investigated the mechanisms of OMP in an OR specific
manner. We generated two OMP KO lines that also expressed GFP with either the low basal activity mOR-EG
OR or the higher basal activity M71 OR. We found that OMP functions in an OR dependent manner by altering
basal cAMP levels to a greater extent in ORNs expressing a quiet OR (mOR-EG) compared to a noisier (M71)
OR. Interestingly, lack of OMP abolishes differences in basal cAMP levels between ORNs expressing different
ORs, suggesting that one of OMP’s role is to reduce basal activity of adenylyl cyclase. In addition, OMP speeds
up signal transduction for ORNs to better synchronize their output with high-frequency stimulation and to
perceive brief stimuli. In summary, OMP plays a key regulatory role in ORN physiology by controlling multiple
aspects of the odorant response.
P3-042 Synergistic control of olfactory receptor trafficking to the cell surface membrane in
heterologous cells
Kentaro Ikegami1,2, Ruchira Sharma2, Elise Bruguera2, Masafumi Yohda1, Hiroaki Matsunami2
1
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan, 2Department of
Molecular Genetics and Microbiology, Duke University Medical Center, Durham, USA
Cell surface trafficking of olfactory receptors (ORs) in olfactory sensory neurons (OSNs) is fundamental for their
function. A vast majority of ORs are not trafficked to the cell surface when expressed alone in heterologous cells.
Previous findings show that receptor transporting protein 1 (RTP1) and RTP2, accessory proteins specifically
expressed in the olfactory system, enhance OR trafficking. We conducted a transcriptome analysis of the
olfactory epithelium of RTP1/2 double knockout mice, which showed that 10% of ORs were overrepresented
(oORs) while a large majority of ORs were underrepresented (uORs). FACS studies in the heterologous system
using a large set of oORs and uORs revealed that only some oORs show robust cell surface expression without
the RTPs. We hypothesized that certain amino acid (AA) residues in the oORs are responsible for this enhanced
cell surface trafficking in the absence of RTPs. To test this, we chose a closely related pair of ORs as a model:
Olfr541, a uOR, which was poorly expressed on the cell surface and Olfr539, an oOR, which was efficiently
trafficked to the cell surface. To identify AA residues important for OR trafficking we generated a series of
chimera ORs replacing parts of Olfr539 with corresponding regions of Olfr541 and showed that the middle region
of the oOR, which contained 16 AA changes, was important for RTP independent cell surface trafficking. We
generated point mutations for all 16 AAs in Olfr539 and found that two single mutants, G154C and V209G,
displayed decreased cell surface trafficking. Conversely, double mutation of C154G and G209V of Olfr541
conferred cell surface trafficking ability without the RTPs. Hence we found that two AAs in the transmembrane
domain 4 (TM4) and TM5 determined RTP-independent OR trafficking. We propose that multiple AA residues
synergistically control cell surface trafficking of ORs.
2016.05.27 17.38.25 Page 18(1)
P3-043 Receptor repertoire for aversive odorants
Xiaoyang Serene Hu1, Yue Jiang1, Kentaro Ikegami2
1
Molecular Genetics and Microbiology, Duke University, USA, 2Biotechnology and Life Science, Tokyo University of Agriculture and
Technology, Tokyo, Japan
Predator and conspecific derived olfactory cues can trigger hardwired innate fear responses. While the field has
made progress in identifying functional compartments of the olfactory system mediating this behavior, it is still
largely unclear which odorant receptors (ORs) are involved in the detection of such cues, partly due to difficulty
in the tracking of OR activation upon odor stimulation. Our recently developed method using phospho-S6
ribosomal protein immunoprecipitation (pS6-IP) combined with RNA-Sequencing allows us to screen the entire
mouse OR repertoire via mRNA profiling of the active olfactory sensory neurons in vivo. In this project, we
improved the accuracy of the pS6-IP/RNA-Sequencing method to identify ORs responding to 2,3,5-Trimethyl-3thiazoline (TMT), a component of fox odor and TMT’s synthetic analog 2-methyl-2-thiazoline (2MT) which
triggers robust fear-related responses. Furthermore, we expanded the known OR repertoire for 2-sec-butyl-4,5dihydrothiazole (SBT), which is released by dominant and endangered conspecifics. To improve the sensitivity
of OR gene expression analysis, we refined definitions of mouse OR transcripts by including their untranslated
regions and utilized a pS6 monoclonal antibody for improved efficiency and consistency. Using this method, we
identified a diverse set of 40, 20, and 34 ORs responding to 1% TMT, 2MT and SBT, respectively (FDR
corrected p<0.05). We found several ORs to be activated across the aversive analogs, while other receptors are
specific to single odorants. This study will aid in our basic understanding of receptor coding for aversive odors.
P3-044 Direct transport of insulin to the olfactory bulb of mice by nasal administration
Yukari Nakamura1, Hideaki Shiga1, Hideaki Ninomiya2, Takuya Noda1, Kentaro Yamada1, Masayuki Harita1, Tomoko Hiraba1,
Junpei Yamamoto1, Takaki Miwa1
1
Department of Otorhinolaryngology, Kanazawa Medical University, Japan, 2Medical Research Institute, Kanazawa Medical
University, Japan
Purpose:
The side effects of systemic medicines for Alzheimer type dementia have become a problem. Impaired insulin
sensitivity is related to cognitive impairment in patients with Alzheimer type dementia. Intranasal insulin delivery
improves memory, however it is not known whether intranasal insulin transport takes place along the olfactory
nerve pathway. The purpose of this study was to determine how nasally administered insulin is transported to
the olfactory bulb and brain in normal mice.
Materials and Methods:
Male ICR mice aged 8 weeks were housed in a 22-・C air-conditioned room with a 12:12-h light-dark cycle and
freely provided with food and water. Ten microliters of FITC-labeled human recombinant insulin (FITC-insulin)
saline solution (Sigma-Aldrich Japan, Tokyo, Japan) was carefully instilled into the left nasal cavity of each
mouse (N=5) via a microinjection pipette; sneezing was prevented by using anesthesia (intraperitoneal
administration of pentobarbital sodium, 0.05 mg/g). Tissue samples were obtained from the mouse heads 30 min
after FITC-Insulin nasal administration. Frozen sections were assessed under fluoroscopic microscope.
Results:
FITC-insulin was detected in the posterior part of nasal cavity, olfactory bulb and frontal cerebellum 30 min after
intranasal administration of FITC-insulin in the mice.
Conclusions:
Our results suggest that intranasal insulin delivery to the brain occurs along the olfactory nerve in vivo.
P3-045 Probability of odorant receptor gene choice in mousensor transgenic mice
Raena Mina1,2, Paul Feinstein1,2, Charlotte D’Hulst2
1
Biology Neuroscience Department City University of New York Graduate Center, USA, 2Biology Department Hunter College, USA
In the mouse, an olfactory sensory neuron (OSN) chooses to express only one out of 2000 possible odorant
receptor (OR) alleles. The mechanisms behind OR gene choice still remain poorly understood. Using a new
technology, called MouSensor, we have shown that the multimerization of a short sequence known to be
essential in OR gene choice increases significantly the number of OSNs expressing a defined specified OR
coding sequence (CDS) along with a fluorescent protein from transgenic minigenes. To further assess how this
2016.05.27 17.38.25 Page 19(1)
enhancer sequence affects the probability of gene choice, we looked at the effect of the enhancer on OR
expression in the context of competition. Therefore, we have developed transgenic MouSensors for different OR
CDSs and looked at OR expression patterns in the main olfactory epithelium (MOE). Through confocal imaging,
we compared the number of labeled OSNs in offspring of crosses between specific MouSensor transgenic lines
and gene-targeted strains in which the endogenous OR locus is tagged with a fluorophore. One of our M71
MouSensor lines (alpha) has 25 times more total number of labeled OSNs than endogenous OR M0R23. In
comparison one of our MOR23 MouSensor (alpha) lines has 250 times more total number of labeled OSNs than
endogenous OR MOR23. When expressing both in the same animal, the massive increase in OSN population
expressing MOR23 seems to cannibalize probability of choice for the M71 MouSensor line expression and
endogenous MOR23. Some OR coexpression between enhancer OR labeled cells is also observed, implying
that the presence of the enhancer may break the one OR per OSN rule in certain occasions. This study provides
insight on how probability of gene choice is regulated.
P3-046 Olfactory receptor accessory proteins RTP1 and RTP2 play a crucial role in receptor gene
choice, development and odor detection
Ruchira Sharma1, Yoshi Ishimaru2, Ian Davison3, Kentaro Ikegami4, Hiroaki Matsunami1
1
Duke University Medical Center, USA, 2University of Tokyo, Japan, 3Boston University, USA, 4Tokyo University of Agriculture and
Technology, Japan
Receptor transporting proteins (RTP1 and RTP2), specifically expressed in the olfactory sensory neurons
(OSNs), have been shown to enhance the cell surface expression of olfactory receptors (ORs) when expressed
in heterologous cells. We have generated RTP1 and RTP2 double knockout mice (RTP1,2-/-) to test the function
of the RTPs in vivo. RTP1,2-/- mice show no gross morphological defects. Localization of a specific OR, M71 is
lost in RTP1,2-/- OSN dendrites and RTP1,2-/- mice do not form glomeruli for this OR but are able to form
glomeruli for beta2 adrenergic receptor, an non-OR GPCR whose expression has been shown to substitute that
of an OR. Fewer mature OSNs in RTP1,2-/- mice along with diminished EOG responses to odors indicates lower
olfactory capacity in these animals. Strikingly, although the expression of the vast majority of ORs is diminished
in RTP1,2-/- mice (uORs), some ORs are overexpressed in the mutant (oORs), suggesting a biased OR choice
in the absence of the RTPs. Consistent with previous findings, uORs are not efficiently trafficked to the cell
surface in HEK293T cells in the absence of RTPs, while some oORs show robust cell surface expression even
without the RTPs. Expression patterns of nATF5 (indicator of the unfolded protein response and ongoing OR
gene choice) and LSD1 (histone demethylase required for OR transcription initiation) are vastly expanded in the
OSNs of the knock out mice. OSNs expressing uORs in RTP1,2-/- mouse show mor frequent co-localization with
nATF5. Lineage tracing experiments demonstrated reduced OR gene choice stability in OSNs expressing M71
in the RTP1,2-/- animals. We hypothesize that mutant OSNs that express uORs are unstable in OR gene choice,
while OSNs that choose to express oORs are stabilized, which coincides with cell surface trafficking of the OR
proteins and down regulation of nATF5 and LSD1.
P3-047 The comprehensive expression profile of vomeronasal type 1 receptors in the domestic cat
Nao Matsuda1, Shoko Nakamuta2, Tetsuro Yamashita1, Nobuaki Nakamuta2, Masao Miyazaki1
1
Department of Biological Chemistry and Food Sciences, Iwate University, Iwate, Japan, 2Laboratory of Veterinary Anatomy, Iwate
University, Iwate, Japan
Most mammals detect pheromones with the vomeronasal organ (VNO), which expresses vomeronasal type 1 (V
1R) and type 2 receptors (V2R) on vomeronasal sensory neurons. Each vomeronasal sensory neuron
expresses just one V1R or V2R gene. Bioinformatics analyses have identified V1R and V2R genes in the
genomes of several mammalian species, revealing that the numbers of V1R and V2R genes differ markedly
among species. The mouse genome has 191 V1R and 121 V2R genes, while the human genome has only 5 V1
R genes. However, little is known about whether VRs are expressed equally in the VNOs. Here, we report the
comprehensive expression profile of V1R genes in the VNOs of the domestic cat whose genome contains 23 V1
R genes and V2R pseudogenes only. In situ hybridization was used to detect 16 of the 23 V1R genes to count
the numbers of vomeronasal sensory neurons expressing each V1R in the VNOs of female cats. We examined
approximately 800 neurons in a VNO tissue section: 1 of the 16 V1Rs was expressed in more than 50 neurons; 3
of the 16 V1Rs were observed in 20 to 50 neurons; 11 of the 16 V1Rs were observed in fewer than 20 neurons;
and 1 of the 16 V1Rs was not expressed in any neuron. These observations indicate that the numbers of
vomeronasal sensory neurons expressing each V1R differ markedly in the VNOs depending on the varieties of
2016.05.27 17.38.25 Page 20(1)
the V1Rs. We postulate that the V1R that was expressed in the largest number of neurons may be important for
chemical communication in female cats.
P3-048 Enzymatic conversion of odorants in human olfactory cleft mucus
Yumi Motoyama1, Kiyomi Sakakibara1, Kunihiro Nishimura2, Tetsuya Ogawa2, Takao Imaeda1
1
Toyota Central R&D Labs., Inc., Japan, 2Dept. of Otorhinolaryngology, Aichi Medical University, Japan
Odorants are recognized by using multiple combinations with odorant receptors(ORs) and transformation into
discrete spatial patterns of olfactory glomerular activity. Human have approximately 400 intact odorant
receptors, and it has been thought that the odorant−OR matrices obtained by receptor assays would reveal the
entire view of the odor coding map. However matching human odorant receptors to ligands has seen limited
success. On the other hand, emerging evidence showed that odorants with functional group such as aldehydes
and esters are targets of metabolic enzymes in the mouse mucus, resulting in their conversion to the other
compounds, and the conversion assumed to be fast enough to affect olfactory perception. Here we determined
whether human olfactory cleft mucus has enzymatic activity that converts odorants to other compounds like
mouse mucus, and examine whether the conversion affect ORs activation patterns by receptor assays, if such
conversion occurs. Acetyl isoeugenol, Acetyl eugenol, p-Cresyl acetate and Phenyl acetate were well converted
to the corresponding alcohol with human olfactory cleft mucus, whereas the other ester odorants were converted
poorly, similar to the conversion with rabbit mucus. All of these odorants which were well converted have Phenyl
acetate in their chemical construction. And these activity inhibited by carboxylesterase inhibitor. We also
revealed that the response of OR1A1 which recognize p-Cresyl acetate decreased when it was stimulated with
rabbit mucus. In this study we found that the odorants which have Phenyl acetate structure are caused
enzymatic conversion to other odorants by human olfactory cleft mucus, and this enzymatic event could affect
the odorant−OR matrices in vitro receptor assays.
P3-049 Differential expression of axon-sorting molecules in mouse olfactory sensory neurons
Naoki Ihara1, Ai Nakashima1, Yuji Ikegaya1,2, Haruki Takeuchi1,3
1
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan, 2Center for Information and Neural Networks,
National Institute of Information and Communications Technology, Osaka, Japan, 3Japan Science and Technology Agency (JST),
PRESTO, Saitama, Japan
In the mouse olfactory system, the axons of olfactory sensory neurons that express the same type of odorant
receptor (OR) converge to a specific set of glomeruli in the olfactory bulb (OB). It is widely accepted that
expressed OR molecules instruct glomerular segregation by regulating the expression of axon-sorting
molecules. Although the relationship between the expression of axon-sorting molecules and OR types has been
analyzed in detail, those between the expression of axon-sorting molecules remain to be elucidated. Here we
collected the expression profiles of four axon-sorting molecules from a total of 1,799 glomeruli, corresponding to
the total number of glomeruli in the OB. These molecules demonstrated position-independent mosaic
expressions, but their patterns were not identical in the OB. Comparing their expressions identified positive and
negative correlations between several pairs of genes even though they showed various expressions.
Furthermore, the principal component analysis revealed that the factor loadings in the principal component 1,
which explain the largest amount of variation, were most likely to reflect the degree of the cyclic nucleotide-gated
(CNG) channel dependence on the expression of axon-sorting molecules. Thus, neural activity generated
through the CNG channel is a major component in the generation of a wide variety of expressions of axonsorting molecules in glomerular segregation.
P3-050 Activity-dependent mechanisms of olfactory map formation
Ai Nakashima1, Naoki Ihara1, Yuji Ikegaya1, Haruki Takeuchi1,2
1
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan,
Japan Science and Technology Agency (JST), PRESTO, Saitama, Japan
2
In the mouse olfactory system, olfactory sensory neurons (OSNs) express only one functional odorant receptor
(OR) out of 1000 OR genes. OSNs expressing the same type of OR target their axons into a specific set of
glomeruli in the olfactory bulb. It is now widely accepted that OR play an instructive role in segregating like OSN
axons into a single glomerular structure. Previous studies have shown that spontaneous neural activity in the
OSNs instructs glomerular segregation by regulating the expression of axon-sorting molecules e.g. Kirrel2/3,
2016.05.27 17.38.25 Page 21(1)
OL-pc, Sema7A. To date, the relationship between OR species and expression of axon-sorting molecules are
examined in detail. However, little is known about how the OR identity is encoded within the sequence of action
potentials to establish OR-specific expression pattern of axon-sorting molecules. Here we analyzed the neural
activity of OSNs using a genetically-encoded calcium indicator (GECI), and examined the relationship between
neural activity and OR species. OSN-specific expression of GECI, GCaMP6f allowed us to monitor the calcium
spikes that accompany spontaneous neural activity. Calcium imaging using GCaMP6f revealed that OSNs show
various patterns of spontaneous calcium spikes. Furthermore, imaging with OR-defined neurons indicated that
spike patterns of calcium are OR-specific and dramatically different between OR species. Given that
axon-sorting molecules require calcium for their expression, neural activity in the OSNs is converted into specific
patterns of calcium spikes to generate OR-specific expression pattern of axon-sorting molecules.
P3-051 Examining the variable influence of population structure on odor perception
Casey Trimmer1, Jason R. Willer2, Andreas Keller3, Leslie B. Vosshall3, Nicholas Katsanis2, Hiroaki Matsunami2,
Joel Mainland1
1
Monell Chemical Senses Center, Philadelphia PA, USA, 2Duke University, NC, USA, 3The Rockefeller University, NY, USA
Correlating functional variation in the olfactory receptor (OR) gene family with differences in human odor
perception is a challenge due to the combinatorial nature of the olfactory code and the size of the human OR
repertoire. One way to get around this issue is by examining cases in which loss-of-function of a single OR has a
significant role in odor perception. We examined the association between the perceived intensity and valence of
68 odors and the genotype of 400 ORs in 332 human subjects. We find significant associations between 10
odors (15% of the 68 tested) and 16 different ORs (p<0.05 following FDR correction). In addition, our results
indicate that population structure is a significant factor in the perception of some odors. For example, the
perceived valence of vanillin is significantly correlated with population structure (vanillin valence rank vs. the top
principal component calculated from all SNP data, r2=0.077, p=2.66e-07), and self-reported Caucasians tend to
rate this odor to be significantly more pleasant than African-Americans (t(150)=-4.35, p=2.49e-05). After
correcting for this structure in our analysis, no individual OR is significantly associated with vanillin perception,
suggesting that overall genetic architecture is more important in this phenotype than the genotype of a particular
OR. We observe similar results for the perceived intensity of 2-decenal (r2=0.068, p=1.37e-06) and the perceived
valence of octyl acetate (r2=0.055, p=1.65e-05). In contrast, the perception of other odors, such as bourgeonal,
appear to be largely unaffected by population structure (r2=0.0044, p=0.23). These results suggest that genetic
studies of odor perception may be significantly influenced by the structure of the study population, and that
genetic ancestry may be an important contributor to the perception of some odors.
P3-052 Expression of G proteins in the olfactory receptor neurons of the mole
Tomoaki Nakada, Mina Iwahashi, Shota Kaizu, Makoto Yokosuka
Department of Veterinary Medicine, Nippon Veterinary and Life Science University, Japan
Talpid moles are small blind insectivores that live in dark underground tunnels. Their navigation and food
searching are highly depend on the somatosensory and olfactory functions. However, there are few studies on
their olfactory systems. In the present study, we investigated the expression of G protein α subunits in the
olfactory system of the small Japanese mole, Mogera imaizumii by immunostaining with antibodies against Gαolf,
Gαo and Gαi2. The main olfactory epithelium is located in the dorsomedial region of the nasal cavity. In this
region, the Gαolf- and Gαo-expressing sensory neurons are located in the apical and basal zone of the olfactory
epithelium, respectively. In the vomeronasal organ which is located in the ventromedial part of the rostral nasal
cavity, the sensory neurons mostly express Gαi2. These Gαi2-immunopositive neurons project their axons toward
the accessory olfactory bulb. We will discuss the observations in the ground of diversification and evolution of
the mammalian olfactory system.
P3-053 Four olfactory marker protein genes derived from salmon-specific whole genome duplication
Hikoyu Suzuki1, Mika Sakamoto1,2, Masato Nikaido3
1
Nihon Biodata Corporation, Kanagawa, Japan, 2Department of Disease Prevention Science, Graduate School of Humanities and
Sciences, Ochanomizu University, Tokyo, Japan, 3Department of Biological Sciences, Graduate School of Bioscience and
Biotechnology, Tokyo Institute of Technology, Tokyo, Japan
Olfactory Marker Protein (OMP) is known as a specific marker of mature olfactory sensory neurons (OSNs) in
2016.05.27 17.38.25 Page 22(1)
vertebrates. OMP is thought to be encoded by a single-copy gene, however, we previously reported that teleost
fish retain two copies of OMP, OMP1 and OMP2, derived from teleost-specific whole genome duplication
(WGD). Generally, WGD-derived paralogs have been retained by subfunctionalization and/or
neofunctionalization. Indeed, the expression patterns of teleost OMP1 and OMP2 was clearly differentiated:
OMP1 is mainly expressed in the olfactory sensory neurons (OSNs) in the deep layer of the olfactory epithelium
(OE), OMP2 is expressed in a minor population of the OSNs in the superficial layer of the OE and also
expressed in retinal horizontal cells. Here, we report that four copies of OMP exist in Atlantic salmon (Salmo
salar). It is well known that another WGD occurred independently in salmon lineage. Consistently, our
phylogenetic analysis exhibited that two of them belong to OMP1 clade, and other two belong to OMP2 clade.
We thus named them OMP1a, OMP1b, OMP2a, and OMP2b, respectively. We also noticed a long branch of
OMP1b, which suggests positive selection or relaxation of purifying selection. Interestingly, we found a
nonsynonymous mutation, which cause leucine to proline substitution, in Eph2B-receptor-like loop. This domain
is highly conserved among orthologs and paralogs. Furthermore, Smith et al. previously suggested that Eph2Breceptor-like loop is structurally important for OMP function as a molecular switch. We supposed that OMP1b is
on the way to neofunctionalization or pseudogenization. Such evolution patterns of OMP1b might be due to the
OE-specific expression patterns of OMP1. In contrast, OMP2 might be easy to avoid such evolution patterns
because OMP2 was expressed in OE and retina.
P3-054 Progresses in human olfactory receptors deorphanization and characterizations
Pierre Chatelain, Alex Veithen, Magali Philippeau, Francoise Wilkin, Yannick Quesnel
ChemCom, Brussels, Belgium
The discovery by Buck and Axel (1991) of a family of odorant receptors paved the way to the comprehensive
description of the sense of smell. Hence the systematic deorphanization and characterization of odorant
receptors is a key step towards the elucidation of the properties of olfaction and the development of applications.
To date 273 odorant receptors have been shown to be expressed in the human olfactory epithelium; nearly half
of them (approximately 130) have been deorphanized and characterized in multiple assays formats in
heterologous cells. One thousand different odorant molecules have been thoroughly characterized allowing the
establishment of profiles of activation currently analyzed with respect to organoleptic and physic-chemical
properties. This allows the deciphering of complex mixtures, the search for modulators (antagonist and/or
enhancer) for specific odorant receptors and could lead to the design of new odorants and/or their use with
respect to yet unrevealed properties. The process putted in place to achieve this goal will be described together
with recent examples of deorphanization, profiling and modulations.
P3-055 A food odorant influences epigenetic state through histone deacetylase inhibition and delays
neurodegeneration in Huntington’s disease model
Sachiko Haga-Yamanaka, Sarah Perry, Anandasankar Ray
University of California Riverside, CA, USA
Animals detect volatile chemicals in the environment primarily using transmembrane receptors expressed in
olfactory receptor neurons. Activation of the receptors induces neuronal excitation and the information is
conveyed to higher brain centers where olfactory perception is generated. The system adapts its sensitivity after
the onset of the initial detection, which allows the animal to tolerate exposure to odorants in the environment
without being constantly stimulated. Although adaptation causes a decreased sensitivity to a given odorant, it
still remains in the animal’s environment. It is not known whether continued exposure to chemicals impact the
physiological condition of the animal.We have recently discovered that an odorant can also modulate the
chromatin state within the nucleus and affect gene expression by directly acting as an inhibitor of histone
deacetylases (HDACs). This odorant inhibits human class I HDACs and promotes a dose-dependent increase in
histone acetylation in a human cell line. Interestingly, exposure to a food-level concentration of the odor for only
5 days substantially increases the level of histone acetylation in the nucleus. It also substantially alters gene
expression in the Drosophila olfactory organs. Differentially modulated genes affected by the odor exposure
overlaps partially with ones affected by known HDAC inhibitors. Orally administered HDAC inhibitors can
improve several disease conditions, including neurodegeneration. Remarkably, exposure to just the odorant
vapor dramatically slows down the progression of neurodegeneration in a Huntington’s disease model in
Drosophila, as has been reported previously with orally administered HDAC inhibitors. Discovery of a food odor
HDAC inhibitor raises serious questions about the effects of odorants on health and also opens possibilities of
2016.05.27 17.38.25 Page 23(1)
new types of therapeutics that are natural, affordable, and already approved for human consumption.
P3-056 Elimination of a ligand gating site generates a supersensitive olfactory receptor
Kanika Sharma1, Gaurav Ahuja1, Ashiq Hussain1,2, Sabine Balfanz3, Arnd Baumann3, Sigrun I. Korsching1
1
Institute of Genetics, Biocenter, University at Cologne, Cologne, Germany, 2Current address: Max Planck Institute for
Neurobiology, Martinsried, Germany, 3Institute of Complex Systems (ICS-4), Research Center Julich, Julich, Germany
Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled
multitude of odor molecules facing a smaller but still impressively large number of cognate olfactory receptors.
Sometimes a single functional group of the ligand determines the specificity of the ligand-receptor interaction, in
other cases an ensemble of molecular features is recognized. We have recently deorphanized an olfactory
receptor of the trace amine-associated receptor family, TAAR13c, as a specific and sensitive receptor for a
bifunctional compound, the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c
interaction. Several predicted binding residues were exchanged by site-directed mutagenesis, and after
heterologous expression the functionality and pharmacological properties of the resulting receptors were
compared with wildtype TAAR13c. We observed a binding site for cadaverine at the external surface of the
receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity.
Unexpectedly, site-directed mutagenesis of the external binding site resulted in supersensitive receptors.
Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby
downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological
sensitivity to socially relevant odors.
P3-057 Decoding spatial map of olfactory receptor expression in the amphibian MOE
Adnan S. Syed, Sigrun I. Korsching
Institute of Genetics, University of Cologne, Cologne, Germany
The sense of smell helps animals to evade predators, localize prey and recognize suitable mates. In mammals,
olfactory receptor families are segregated into different olfactory organs, main olfactory epithelium (MOE) and
vomeronasal organ (VNO). In contrast, olfactory receptor families of teleost fish are intermingled in a single
sensory surface. To what extent such differences influence the coding and discrimination abilities of the
respective olfactory systems is unclear, and the evolutionary path toward such segregation is unknown. The
analysis of amphibians, which are early diverging tetrapods compared to mammals, may shed light on this
transition from shared sensory surface to segregated subsystems. In the Xenopus laevis olfactory system,
olfactory receptors (ORs), vomeronasal receptors type 1 (V1Rs) and trace amine associated receptors (TAARs)
are exclusively expressed in the MOE. Interestingly, vomeronasal receptors type 2 (V2Rs) have a bimodal
expression-ancestral V2R genes (basal positions in the phylogenetic tree) are expressed exclusively in the MOE
and later diverging V2R genes are exclusively expressed in the VNO (Syed et al. 2013). For this study, we
focused on the organization of sensory inputs in the MOE of X. laevis. We cloned several genes belonging to
TAAR, V2R, V1R, OR gene families, as well as olfactory marker protein (OMP) and transient receptor potential
channel (TRPC2) which are known markers for ciliated and microvillous neurons, respectively. We report here
that OMP, ORs, and TAARs are expressed in an apical zone, partially overlapping, but clearly distinct from the
basal zone of TRPC2 and V2R-expressing cells. Results of this study shed light on the olfactory regionalization
of X. laevis, which is an excellent model system to study the molecular driving forces governing the evolution of
vertebrate olfactory system.
P3-058 Characterization and fast stability evaluation of various milk samples using an electronic nose
and tongue
Kiyoharu Ikehama1, Hiroaki Sato2, Koichi Yoshida1
1
Alpha M.O.S. Japan K.K., Tokyo, Japan, 2Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of
Agriculture, Hokkaido, Japan
Plant-based milks such as almond and coconut milks have recently attracted attention as substitutes for cow’s
milk. Plant-based milks contain no lactose; therefore, people who are lactose intolerant can drink them without
any problems. These are the perfect drinks for people with a diet-oriented and health-conscious lifestyle
because they do not contain milk fat.In this study, we characterized various plant-based milks and established a
fast evaluation method to characterize sample stability with aging. Commercial cow’s milk and four plant-based
2016.05.27 17.38.25 Page 24(1)
milks (almond, coconut, rice, and soy) were used as samples. They were stored at two different temperatures for
three days and seven days after opening. These samples, which were unopened products, were then examined
using a ultra-fast gas chromatography-based electronic nose and an electronic tongue. Data acquired by each
system was studied with multivariate statistics using the retention index of the detected peaks for the electronic
nose and 7 sensors in the electronic tongue as explanatory variables. Differences among samples were
compared. The data from the electronic tongue showed that the Euclidean distance between the unopened
products and aged sample correlated with storage temperature. Moreover, their peaks both increased and
decreased with aging were detected on the chromatograms for the electronic nose. A search of volatile
compounds was performed using a library of odor and retention indices, including various chemical information
and odor descriptors. Aldehydes and ketones were found in the peaks that increased with aging. These
compounds indicated that fat oxidation increased with aging.
P3-059 Development of cell-based sensor array for targeting multiple odorants based on insect
odorant receptors
Maneerat Termtanasombat1, Hidefumi Mitsuno1, Nobuo Misawa2, Shinya Yamahira3, Satoshi Yamaguchi1,
Teruyuki Nagamune3, Ryohei Kanzaki1
1
Research Center for Advanced Science and Technology, The University of Tokyo, Japan, 2Artificial Cell Membrane Systems
Group, Kanagawa Academy of Science and Technology, Japan, 3Department of Chemistry and Biotechnology, The University of
Tokyo, Japan
The olfactory system of living organisms has evolved to accurately discriminate numerous odors using only
limited types of odorant receptors (ORs). Existing odorant sensors were generally developed by arraying several
sensing elements such as coated materials-conjugated quartz crystal microbalances. However, it is difficult for
the sensors to identify target odorants with a clear response pattern due to the poor odorant selectivity of each
element. Recently, we developed novel odorant sensor elements with high sensitivity and selectivity based on
insect ORs expressed Sf21 cells, which respond to target odorants by displaying an increased fluorescence
intensity. However, a method that utilizes the odorant sensor cell to discriminate multiple odorants has not been
established. Here, we report a technique for creating an odorant sensor chip with an array pattern of several cell
lines expressing different ORs. In this study, cell immobilizing reagents, Biocompatible Anchor for Membrane
(BAM), and a patterned Polydimethylsiloxane film were used to create an array of four cell lines expressing ORs
of Drosophila melanogaster (Or13a, Or56a) and Bombyx mori (BmOR1, BmOR3). First, we observed that the
four cell lines were separately aligned in the array and remained tightly immobilized on a BAM-modified surface
during applying odorant stimuli under flow. Next, the sensitivity and selectivity of the odorant sensor cells were
verified to be unaffected by BAM. Finally, our sensor array showed odorant-specific response pattern, which can
be visualized as an increased fluorescence intensity in the area of Or13a, Or56a, BmOR1, and BmOR3 cell lines
when 1-octen-3-ol, geosmin, Bombykol, and Bombykal were applied respectively. Therefore, we successfully
developed a new methodology for creating cell-based odorant sensor array that enables us to discriminate
multiple target odorants. Our method could be expanded to target a large range of odorants for usage in various
applications such as food administration and disease diagnosis.
P3-060 Development of a sensitive and selective cell-based sensor for detecting mold odorants based
on insect odorant receptors
Hidefumi Mitsuno1, Maneerat Termtanasombat1, Takeshi Sakurai1, Yuko Nakajima1, Nobuo Misawa2, Ryohei Kanzaki1
1
Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan, 2Artificial Cell Membrane
Systems Group, Kanagawa Academy of Science and Technology, Kanagawa, Japan
Highly sensitive and specific odorant sensing technology can be utilized for various applications such as
detection of mold contamination in foods and waters. Current practical odorant sensors have been developed
based on metal-oxide semiconductors and quartz crystal microbalances. However, it is difficult to develop the
sensing elements with high sensitivity and selectivity. To overcome these problems, we attempt to develop
cell-based odorant sensors focusing on insect’s odorant receptors (ORs), which are expressed in olfactory
sensory neurons, for selectively detecting various types of environmental odorants. Until now, we have
demonstrated that Sf21 cells co-expressing silkmoth’s pheromone receptors and co-receptor, Orco, along with
fluorescent calcium indicator proteins are available as odorant sensor elements with high degree of sensitivity
and selectivity. However, we have not yet led this to the development of a practical odorant sensor for detection
of target odorants. Here we report the development of a cell-based odorant sensor that sensitively and
selectively detects mold odorants using insect’s ORs. We used two Drosophila melanogaster ORs (Or56a;
2016.05.27 17.38.25 Page 25(1)
geosmin, Or13a; 1-octen-3-ol) to establish the Sf21 cell lines. Or56a and Or13a cell lines selectively detected
geosmin and 1-octen-3-ol by increasing their fluorescence intensities. Both cell lines dose-dependently
responded to target odorants with lower detection limits of 300 nM, which corresponds to several tens of ppb in
solution. Interestingly, Or56a cell line specifically detected geosmin even in the presence of background odors.
In addition, we constructed a compact odorant sensor chip by integrating these cell lines into microfluidic
channels, demonstrating that the chip can detect two mold odorants as a pattern of increased fluorescence
intensity both in the presence and absence of background odors. These results represent the first step towards
practical cell-based odorant sensors that enable us to detect target odorants with high degree of sensitivity and
selectivity.
P3-061 Multiple episodic evolution events in V1R receptor genes of East-African cichlids
Masato Nikaido1, Hikoyu Suzuki1, Yoko Satta2, Semvua Isa Mzighani3, Christian Sturmbauer4, Kimiko Hagino-Yamagishi5,
Norihiro Okada1
1
School of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan, 2The Graduate University for Advanced Studies
(Sokendai), Japan, 3Tanzania Fisheries Research Institute, Tanzania, 4Department of Zoology, Karl-Franzens-University Graz,
Austria, 5Tokyo Metropolitan Institute of Medical Science, Japan
Fish use olfaction to detect a variety of nonvolatile chemical signals, and thus this sense is key to survival and
communication. However, the contribution of the olfactory sense to social, especially reproductive, interactions
in cichlids is still controversial. To obtain insights into this issue, we investigated the genes encoding V1Rs,
possible candidates for reproductive pheromone receptors, among East African cichlids. Interestingly, we found
an excess of non-synonymous over synonymous substitutions in four of six V1R genes in multiple cichlid
lineages. First, we found that highly dimorphic V1R2 allele groups were shared among the cichlids inhabiting all
East African Great Lakes emerged through the episodic accumulation of the nonsynonymous substitutions prior
to the radiation of the Lake Tanganyika species flock. We further detected such episodic events in V1R1 of the
tribe Tropheini, and in V1R3 and V1R6 of the tribe Trematocarini. The excess of nonsynonymous substitutions in
these examples were indicated as dN per dS more than 1, which were all statistically significant by Fisher exact
test. Furthermore, we speculate that the amino acid changes in these episodic events are likely the signature of
functional switch because they occurred in the putative ligand-binding pocket. Our finding of the occurrence of
multiple episodic events and the unexpected gene diversity in one unique gene family is suggestive of the
contribution of the V1R to the species diversification as well as the social interaction in cichlids.
P3-062 Chemosensory stimuli modulate feeding behavior and glucose kinetics after glucose-loading
Tadataka Tsuji1,2, Bakhshishayan Sanam2, Kumiko Kida2, Susumu Tanaka2, Koichi Satoh1, Mikihiko Kogo2,
Takashi Yamamoto3
1
Department of Oral and Maxillofacial Surgery, Saiseikai Matsusaka General Hospital, Japan, 2The First Department of Oral and
Maxillofacial Surgery, Graduate School of Dentistry, Osaka University, Japan, 3Department of Health and Nutrition, Faculty of Health
Science, Kio University, Japan
Chemosensory stimuli including tastes and flavors modulate various physiological activities related to energy
homeostasis. We suggested the importance of these chemical senses in glucose kinetics by imposing
interventions during glucose intake in humans. The specific odor stimuli directly affect the levels of feedingrelated neuropeptides in the hypothalamus in rats, resulting in the changes in the feeding pattern. The aim of this
study was to examine how any interventions associated with odor during and/or before ingestion affect feeding
behavior and blood glucose (BG) after ingestion. We thereby recorded the feeding pattern with
electromyography (EMG) activities of the masticatory muscles in the anosmic rats and applied the conventional
glucose tolerance test to rats through disruption and various stimulation of these chemical senses. After the
anosmic treatment, the feeding time for 2 g food was shortened with a longer latency to the start of eating and
the cumulative food intake over 4hrs was decreased compared to those before the treatment. EMG analyses of
the masticatory muscles during feeding pellets showed that the EMG bursts, which correspond to rhythmical jaw
movements, became larger. These behavioral changes indicate fast eating with powerfully gnawing and chewing
after the anosmic treatment. The experimentally-impaired olfactory function induced an evident downward shift
in the BG curve together with changing of sweet palatability by two-bottle preference test, consistent with the
previous reports. Addiction of grapefruit odor stimuli before ingestion, a representative flavor of enhanced
sympathetic nerve, dramatically changed the BG curve. More specifically, the effect of grapefruit odor was due
to neural message from nasal cavity, consistent with that of limonene odor. These results suggest that favorable
chemical senses before and during ingestion are the important factors to maintain BG after ingestion as well as
2016.05.27 17.38.25 Page 26(1)
feeding pattern. An attractive fragrance could be utilized as a fascinating means of BG control.
P3-063 Comparative analyses of olfactory receptor genes in primates suggest a shift from olfaction to
vision at the ancestor of haplorrhines
Atsushi Matsui1,2, Yoshihito Niimura1,2, Kazushige Touhara1,2
1
Olfactory receptors (ORs) recognize odor molecules in the environment. It is well known that OR genes
comprise of the largest multigene family in mammals. The numbers of OR genes are highly variable among
species; for example, humans and chimpanzees have approximately 400 functional OR genes, whereas mice
and rats have >1,000. Primates are classified into two suborders, strepsirrhines and haplorhines. Strepsirrhines
are characterized by the presence of the rhinarium, which is useful for detecting odors. Moreover, most
strepsirrhines are nocturnal except for some lemurs, while haplorhines are predominantly diurnal with the
exceptions of tarsiers and owl monkeys. Our previous study did not support the ‘color vision-priority hypothesis’,
in which the degeneration of OR gene repertoires coincided with the acquisition of trichromatic vision at the
ancestor of catarrhines. The purpose of this study is to examine when and how the reduction of OR genes
repertoires occurred in the primate evolution. To answer this question, we newly identified the entire sets of OR
genes from the draft genome sequences of primates, including strepsirrhines and haplorhines with both
nocturnal and diurnal habits, and compared the orthologous gene groups of functional OR genes among them.
The analyses showed that the numbers of OR genes in strepsirrhines are much larger than those in haplorhines.
We also found that the change of OR gene repertoires had occurred suddenly at the ancestor of haplorhines
during a short period. Nocturnal primates tend to retain larger OR gene repertoires than closely related diurnal
species, but the differences are much smaller than those between haplorhines and strepsirrhines. These
observations suggest that the degeneration of OR gene repertories was synchronized with the hypertrophy of
eyes and the reduction of olfactory bulbs in haplorhines, and the ancestor of haplorhines might have dynamically
shifted to utilize visual system from olfaction.
P3-064 A biosynthetic pathway of a species-specific sulfurous odor emitted from the urine of the
domestic cat
Ayami Futsuta, Wataru Hojo, Tamako Miyazaki, Ken-ichi Kimura, Tetsuro Yamashita, Masao Miyazaki
Iwate Univerisity, Iwate, Japan
Many mammals use species-specific chemicals for scent communication such as species-recognition. It is
thought that each mammal developed their biosynthetic pathways, and then co-opted their secretions for scent
communication. However little is known how each mammal primary acquired their biosynthetic pathway during
evolution. Cats excrete large amount of felinine, a non-proteinogenic amino acid, into the urine. Felinine is
decomposed into volatile 3-mercapt-3-methylbutanol (MMB), which gives cat urine distinctive sulfurous odor.
Urinary felinine excretion level in other mammals than cats, including mice and humans, is under 0.05% of its
excretion level in cats. Here we report how the domestic cat acquired the felinine biosynthetic pathway during
evolution. In vitro assays demonstrated that the felinine biosynthetic pathway begins with dephosphorylation of
dimethylallyl diphosphate (DMAPP), an end-product of the mevalonate pathway for cholesterol biosynthesis.
Cats express an enzyme for upregulation of the dephosphorylation at high levels, as compared to mice. The
dephosphorylated products are conjugated with glutathione to produce 3-methylbutanol glutathione, a felinine
precursor. The glutathione conjugate is decomposed into felinine by a major urine protein with peptidase activity.
Nutritional experiments showed that the felinine biosynthetic pathway enables cats to consume a lipid-rich diet
without increasing serum cholesterol levels. Cholesterol and felinine productions were up- and down-regulated
by feeding lipid-restricted diets to cats, respectively. These observations strongly suggest that cats primary
developed the felinine biosynthetic pathway to adapt an animal-based, lipid-rich diet, and then co-opted its high
excretion for scent communication. These findings improve our understating of how mammals primary acquired
the biosynthetic pathways of species-specific compounds that later evolved to scent signals.
2016.05.27 17.38.25 Page 27(1)
P3-065 Statistical analysis for clustering of areas on the olfactory bulb and estimation of the physicochemical properties detected by glomeruli in each area
Kohei Omori1, Yoichi Tomiura1, Kenshi Hayashi2
1
Department of Informatics, Kyushu University, Fukuoka, Japan, 2Department of Electronics, Kyushu University, Fukuoka, Japan
The odorants are received by the olfactory receptors on the olfactory receptor cells and activate their cells. The
activations of the cells are conveyed to glomeruli in the olfactory bulb, and activate neurons in the glomeruli. An
olfactory receptor receives a partial structure of an odorants, and all olfactory receptors connected to a
glomerulus receive the same partial structure of an odorants. Furthermore, the location of every glomerulus in
the olfactory bulb does not have individual difference. Therefore, a glomerulus in a particular location of the
glomerular layer in the olfactory bulb detects particular physico-chemical properties. Some researchers have
clarified this correspondence between physico-chemical properties and locations in the glomerular layer.
However, properties or areas in the glomerular layer they clarified were coarse. We clarified this correspondence
by statistically analyzing the activation pattern images of the glomerular layer in the rat olfactory bulb for various
odorants, which are put on the site http://gara.bio.uci.edu/index.jsp. First, we constructed the statistical model to
generate these images. This model has parameters that express the probabilities of generating each type from
each pixel or the probabilities of generating each brilliance from each pair of image and type. The type of the
pixel will correspond to the physico-chemical properties that the glomerulus in the pixel detect. Next, we
estimated the parameters using the activation pattern images by Gibbs sampling. We found clusters of pixels
(that is to say, the type for each pixel) and found probability that the pixel with the type t is brilliant for each image
and each type t. Finally, we estimated the physico-chemical properties corresponding to each type based on the
correlation between the probability that the pixels with type t is brilliant and whether the odorant has property p
for each p and each t.
P3-066 The effect of aromatherapeutic foot massage
Yoshihiko Akakabe1, Shoko Hatano2
1
Department of Biological Chemistry, Yamaguchi University, Japan, 2Cherryfield House, Japan
Aromatherapy is one of the effective relaxation methods, which leads to reduce stress, calm down, and improve
blood circulation. The aim of this study was to examine the effects of aromatherapeutic massage. The subjects
were treated below the knees by a sweet almond oil and by the career oil containing a black pepper oil,
respectively. Skin temperature of the calf and the toes were measured using an infrared camera before and after
treatment. The skin temperature of the calf elevated significantly after treating with the black pepper oil for 20
minutes as compared with the control, although the warming sensation in the legs was not significantly changed.
Increase in moods and decrease in swelling sensation were also observed as the result of treatment with the
black pepper oil. We found the foot massage with black pepper oil caused to improve blood and lymphatic
circulation, suggesting on the reduction of swelling and the recovery of physical fatigue.
P3-067 Temporal chemical profiling of the headspace gas emitted from domestic cat urine and their
olfactory discrimination ability
Chiharu Suzuki, Tetsuro Yamashita, Masao Miyazaki
Department of Biological Chemistry and Food Sciences, Iwate University, Iwate, Japan
Territorial animals deposit their urine and feces on their territories. These excretions contain thousands of
metabolites some of which may be used as scent signals for species, sex, age, and individual recognitions.
There are lots of studies that analyzed chemical profiles of animal excretions in several species. These studies
have suggested that compounds identified species-, sex-, and age-specifically and varied among individuals are
important for scent communication. In these studies, fresh and frozen-preserved excretions have been analyzed,
rather than excretions after ageing periods of several hours or days, which animals usually encounter in their
territories. Therefore, it is necessary to analyze not only fresh excretions but also aging excretions for
understanding of territorial behavior using scent signals in animals. Here, we report temporal changes of volatile
compounds emitted from urine of domestic cats, and their olfactory discrimination ability toward these volatiles.
Cottons were infiltrated with cat urine, and then the headspace gas emitted from the cottons before and after
air-drying were analyzed in comprehensive two-dimensional gas chromatography (GC×GC)-mass spectrometry
(MS). GC×GC-MS detected over five hundreds of volatile compounds in both headspace gas emitted from fresh
and air-drying urine. Chemical profiles of the headspace gas differed markedly between fresh and air-drying
2016.05.27 17.38.25 Page 28(1)
urine. There was a tendency that contents of compounds with low-boiling point increased in the air-drying urine.
In behavioral assays, cats habituated to fresh urine showed dishabituation to air-drying urine. These results
indicate that there are different in chemical contents between fresh and aging cat urine, and cats can distinguish
the differences by olfaction. Our findings and experimental strategies will improve to understand of territorial
behaviors in animals.
P3-068 Olfactory stimulation is effective in treatment of anosmic patients
Fumino Okutani1,2, Hiroaki Ito2, Taisuke Kobayashi2, Masamitsu Hyodo2
1
Department of Occupational Health, Kochi Medical School, Nankoku, Japan, 2Department of Otolaryngology, Kochi Medical
School, Nankoku, Japan
A number of studies have shown that rehabilitative training is effective for motor recovery after ischemic brain
damage. Mechanism underlying rehabilitation is thought to be synaptic plasticity with morphological and
functional changes. Long-tem potentiation is a well-known phenomenon observed in synapses with facilitated
transmission induced by intense electrical stimulation on presynaptic neurons. For sensory impairment,
however, we have not established effective rehabilitative training methods. Although patients with cognitive
disorder like agnosia including Wernicke aphasia are treated by rehabilitative training, a wide variety of
methodology is subscribed dependent on severity or stages of diseases. “Olfactory training” invented as a
therapy for anosmic patients in Germany has been reported to be effective for postinfectious olfactory
dysfunction. This is comprised of 4 odors as rose, lemon, eucalyptus, and clove for 15 seconds exposure by 2
times every morning and evening. Therefore we tried to use odors which are more familiar to Japanese people
for olfactory stimulation therapy. Since odorants should vapor persistently for a few months in conventional
environments and not be hazardous, we chose rose, vanilla, lemon, and banana odorants absorbed in pieces of
cotton each in small bottles. We consider that olfactory stimulation is capable of excitation of olfactory system,
inducing synaptic plasticity with potentiated transmission. So far we obtained the preliminary results showing
that olfactory stimulation is effective in olfactory improvement of anosmic or hyposmic patients due to viral
infection or brain trauma. This treatment method has other positive effects that encourages and supports
patients to get better by themselves as well as self-evaluation of their olfactory function.
P3-069 Development of olfactory phenotyping pipeline in genetically engineered mouse
Jin Won Kim1, Bora Kim3, Sang-Chul Park1, Hyo Jin Chung1, Jin Kook Kim5, Chang-Hoon Kim1,2,3,4, Joo-Heon Yoon1,2,3,4
1
Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, South Korea, 2The Airway Mucus Institute,
Yonsei University College of Medicine, Seoul, South Korea, 3Research Center for Human Natural Defense System, Yonsei
University College of Medicine, Seoul, South Korea, 4Korea Mouse Sensory Phenotyping Center, South Korea, 5Department of
Otorhinolaryngology-Head and Neck Surgery, Konkuk University College of Medicine, Seoul, South Korea
Mouse is the most popular animal model in olfactory research covering anatomy, physiology, genetics, neural
connection, and functional phenotype. Among them, behavior test is essential and sole to determine olfactory
phenotype. So far, mouse olfactory behavior test has not been standardized and could be affected by
inter-observer variations. Also, other specific additional tests would be necessary to confirm olfactory function.
So, we tried to develop a clearly organized pipeline of phenotyping mouse olfactory function that can be
assessed chronologically. After selecting a specific mouse model, we waited until postnatal 12 weeks to ensure
olfactory function be full-developed. At 12 weeks, mouse olfactory behavior test, including preference test and
avoidance test, was performed. In our setting, the Smart 3.0 video-tracking software (Harvard apparatus) and
video camera system was used to track automatically mouse behavior in the standard breeding cage. After three
times of habituations without odorant, we tested with two odorants (peanut butter; preference test, 2MB;
avoidance test) and water (control), and behavior was recorded for 3 minutes and analyzed by software. For
preference test, investigation time was measured and time spent in odorant zone was measured for avoidance
test. At 16 weeks, we performed functional MRI using MnCl2 to investigate olfactory bulb and its tract. Mnenhancement was observed at the interface between olfactory nerve layer and olfactory turbinate, and as time
passes, highlighting of outer layers of OB where olfactory glomeruli and mitral cells are located was noted. After
fMRI, mouse was sacrificed for electrophysiologic test, and basic histopathologic study. Our novel protocols for
examining mouse olfactory function including behavior test, fMRI, electrophysiologic study and basic
histopathologic study seem to be accurate and well-organized. We suggest that our pipeline can be utilized as a
possible standard method to search for features of olfactory phenotype in mouse model.
2016.05.27 17.38.25 Page 29(1)
P3-070 The relationship between olfactory dysfunction and the number of eosinophils in nasal polyp
tissues in surgical cases
Tomoko Hiraba, Yukari Nakamura, Takuya Noda, Kentaro Yamada, Junpei Yamamoto, Kanako Teraguchi, Hideaki Shiga,
Takaki Miwa
Department of Otorhinolaryngology, Kanazawa Medical University, Japan
Aims: Olfactory dysfunction is one of the common symptoms in patients with chronic rhinosinusitis (CRS). We
previously investigated the clinical characteristics and prognosis of olfactory dysfunction in the patients with
eosinophilic chronic rhinosinusitis (ECRS) and non-eosinophilic chronic rhinosinusitis (NECRS). In the present
study, we examined the relationship between olfactory dysfunction and the number of eosinophils in nasal polyp
tissues in patients with ECRS and NECRS. Patients and Methods: This study included 100 chronic rhinosinusitis
(CRS) patients with olfactory dysfunction treated with endoscopic sinus surgery (ESS) in our hospital from June
2009 to September 2014. The patients were divided into ECRS and NECRS groups using the criteria of the
Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC) study. We
evaluated the olfactory function at both pre- and post-ESS. We counted the number of eosinophils in the nasal
polyp tissues removed at ESS at three locations and calculated the average value. Results: The number of
eosinophils in nasal polyp tissues from all NECRS patients (N=20) was less than 70. Comparatively, the number
of eosinophils from the majority of ECRS patients (N=80) was more than 70. There was a significant correlation
between blood eosinophil count and nasal polyp tissues eosinophil count. Blood eosinophils correlated with
disease severity as determined by JESREC criteria, but the number of eosinophils in nasal polyp tissues did not
correlate with disease severity in the ECRS group. The number of eosinophils in the nasal polyp tissues did not
correlate with the severity of preoperative olfactory dysfunction. Postoperative olfactory function showed more
improvement in patients with severe eosinophil infiltration in nasal polyp. Conclusion: The results of the current
study suggest that the number of eosinophils in nasal polyp tissues does not affect olfactory function in patients
with CRS.
P3-071 Vapor detection and discrimination with a panel of odorant receptors expressed in
heterologous cells
Hitoshi Kida1, Aashutosh Vihani2, Joel Mainland3, Hiroaki Matsunami2,4
1
Department of Mechanical Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan, 2Department of
Neurobiology, Duke Institute for Brain Sciences, Durham, USA, 3Monell Chemical Senses Center, Philadelphia, USA, 4Department
of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, USA
Mammals are capable of detecting and discriminating a large number of volatile chemicals. Fundamentally, this
relies on the interaction of odorant molecules and olfactory receptors (ORs). Here, we identified ORs responding
to a set of odorants and developed an OR-based odorant sensor system, capable of detection and
discrimination of odorants in the vapor phase. First, we conducted a large-scale cell-based screening using the
majority of mouse ORs against eight odorants: eugenol, acetophenone, cyclohexanone, heptanal, 2-heptanone,
limonene, N-amyl acetate, and methyl benzoate. To measure the response of these ORs, we leveraged the
established luciferase reporter gene assay in Hana3A cells. In this assay, cells were directly stimulated by
various odorants in liquid phase and responses were ranked. This screening resulted in identification of a set of
high affinity ORs for the tested odorants. To test whether ORs expressed in heterologous cells are activated in
vapor phase, we selected a diverse set of 28 ORs robustly responding to at least one of the tested odorants, as
well as three well studied ORs. We expressed individual ORs in 96 well plates in triplicates and measured
responses against vapor phase odor stimulation ranging from 10-10 dilution to undiluted odorants using the
GloSensor assay system, capable of measuring cAMP levels in real time. Significant responses were recorded
at concentrations as low as 10-6 dilution, and robust responses were recorded as low as 10-4 dilution. Each OR
showed a unique response profile to the odorant panel. Lastly, we tested structurally analogous odorants to
eugenol and acetophenone, showing discrimination between structural analogs using activation patterns of the
set of ORs. Our results form a basis for OR-based volatile sensor arrays.
P3-072 The olfactory implant system: A novel method to treat anosmia
Daniel H. Coelho, Tennessee Park, Mostafa Abdel-Hamid, Richard M. Costanzo
Virginia Commonweatlh University School of Medicine, USA
The ability to directly stimulate areas of the brain to activate a sensory percept is not without precedent.
2016.05.27 17.38.25 Page 30(1)
Cochlear implants used for the restoration of hearing and other sensor-brain interfaces are an increasingly
popular modality for those with sensorineural loss. Those principles that have made cochlear implantation so
successful can be applied to treat anosmia. In our research laboratories we have been exploring the use of
direct electrical stimulation of the olfactory bulb as a novel method to bypass damaged olfactory nerves. This
poster will present a proposed olfactory implant system (US patent pending, PCT/US2014/023945) and provide
physiologic rationale for its function. Though not yet commercially available, neuroprostheses like these are
likely to be a dominant technique in the treatment of anosmia in the coming years.
P3-073 Enhancement of synchronized neural activity in the endopiriform nucleus of rats raised under
abnormal oral conditions
Hiroshi Yoshimura
Department of Molecular Oral Physiology, Tokushima University, Tokushima, Japan
Endopiriform nucleus (EPN) is located deep to the piriform cortex, and has neural connections with not only
neighboring sensory areas but also subcortical areas where emotional and nociceptive information is processed.
Although function of the EPN has been difficult to understand, recent findings suggest that the EPN participate in
olfactory and gustatory information processing. Well-balanced oral condition might play an important role in
stability of brain activities. When the oral condition is impaired, several areas in the brain might be affected. In
the present study, we investigated whether abnormal conditions of oral region influence neural activities in the
EPN. Orthodontic appliance that generates continuous force and chronic pain-related stress was fixed to
maxillary incisors of rats, and raised. Field potential recordings were made from the EPN of brain slices. We
previously reported that the EPN has an ability to generate membrane potential oscillation. In the present study,
we have applied the same methods to assess activities of neuron clusters in the EPN. In the case of normal rats,
stable field potential oscillations were induced in the EPN by application of low-frequency electrical stimulation
under the medium with caffeine. In the case of rats with the orthodontic appliance, stable field potential
oscillations were also induced, but both duration of oscillatory activities and wavelet number were increased.
The enhanced oscillations were depressed by blockade of NMDA receptors. Thus, impairment of oral health
under application of continuous orthodontic force and chronic pain-related stress enhanced neural activities in
the EPN, in which up-regulation of NMDA receptors may be concerned. These findings suggest that noxious
conditions of oral region may have influences on neural activities related to olfactory and gustatory information
processing in the EPN.
P3-074 The relationship between aroma component compositions of several citrus oils and the physiopsychological effects on humans
Chiaki Hara1, Kenichi Tomi2, Kosuke Shimizu3, Shinya Kanzaki2, Yutaka Tsuchihashi1, Takahiro Hayashi2
1
Department of Life Environment, Koshien Junior College, Hyogo, Japan, 2Department of Agricultural Science, Kindai University,
Nara, Japan, 3Experimental Farm, Kindai University, Wakayama, Japan
Limonene is a major component of citrus oils that is considered to be responsible for their physio-psychological
effects. However, the effects of some citrus oils are different from those of limonene, which suggests that other
minor components have physio-psychological effects. The purpose of this study was to determine the
relationship between minor components and their physio-psychological effects on humans.
We performed gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS) to analyze
essential oils extracted by cold-pressing from 10 taxa of citrus fruits obtained from Yuasa Farm, Experiment
Orchard of Kindai University. Next, we performed principal component analysis to select some citrus oils having
characteristic aroma component compositions. Based on the results, we classified them into 5 groups. We
investigated the physio-psychological effects of citrus oils selected from each group; grapefruit (Citrus paradisi
Macf.), Shunkokan (C. shunkokan Hort. ex Tanaka.), Kikudaidai (C. canalicurata Hort. ex Tanaka.), Fukuhara
orange (C. sinensis (L.) Osbeck.), and Keraji (C. keraji Hort. ex Tanaka.). The panel consisted of 13 healthy
adults (7 males and 6 females, mean age 21.6±0.3 years) attended the physio-psychological evaluation. The
psychological effect was evaluated by the Japanese UWIST Mood Adjective Checklist (JUMACL). The
physiological effects were determined by examining the autonomic nervous system activity and salivary amylase
activity.
The results suggested that grapefruit oil had a stimulant effect and other citrus oils tended to show sedative
effects judging from the autonomic nervous system activity.
This study suggested that the minor components affect the physiological effects of citrus oils.
2016.05.27 17.38.25 Page 31(1)
P3-075 Probing the olfactory code using antagonists
Marissa Lyn Kamarck, Nicolle R. Murphy, Paul M. Wise, Joel D. Mainland
The olfactory system recognizes each odor using a unique subset of odorant receptors. Although the specific
pattern of odorant receptors activated by an odorant code for the odorant’s identity, there are few, if any, explicit
predictions relating odorant receptor activity patterns to olfactory perception. Given that the receptor activation
patterns encode the odorant identity and intensity, antagonizing these receptors should alter perception of the
odorant. Here we targeted a trace-amine associated receptor (TAAR) due to previous work indicating that
genetic knockout of a single TAAR eliminated innate aversion to a predator odor (Dewan et al., 2013). Human
TAAR5, activated by trimethylamine (TMA), is the only human TAAR with a published ligand (Wallrabenstein et
al., 2013). Using a cell-based luciferase assay, we first identified an in vitro antagonist for hTAAR5 (p<0.0001),
and then tested the antagonist in a psychophysical paradigm. Using an olfactometer, we presented subjects with
one of the following odors: TMA alone, the antagonist alone, or TMA with the antagonist. Subjects were asked to
rate the intensities of the odor qualities in each mixture. TMA-alone and antagonist-alone were not different in
intensity (p=0.217). The antagonist decreased the perceived intensity of TMA relative to both TMA-alone (p<
0.01) and TMA mixed with an intensity-matched control odor (p<0.05). Antagonist odors promise to be a
powerful tool for examining the contribution of individual receptors to odor perception.
P3-076 Sensing odor mixtures
Terry E. Acree, Geraldine Prevost, Charlotte Maxa, Manon Gros
Food Science, Cornell University, Ithaca, New York, USA
The perception of odor mixtures is shaped by two established ideas. First the number of identifiable component
stimuli is small (1-4) especially during brief encounters. This limitation has been observed many times during the
last 30 years with various olfactory stimuli including cross modal studies with tastants, indicating a top-down
process that is not unique to olfaction (Laing1986, Wilson 2006). Secondly, the number of sensible or key
odorants reported in any given food is less than 230. Although, hundreds of chemicals with odor in the pure state
have been found in foods (Nijssen 1963-2011), less than 1000 have been detected in all foods (Arn 1997,
Dunkle 2014) and less than 10 of these are key odorants in any particular food. Using GCO and a new
psychophysical technology, Sniff Olfactometry (Wyckoff 2016), we have studied the behavior of binary mixtures
of key odorants in 70 ms encounters. Results confirm that odorant mixture sensations are not synthetic but
analytic and that the range of odorant ratios, that are analyzable, is less than 10 to 1. The impact of these results
on ideas of odor image formation will be discussed.
P3-077 Assessment of olfactory stimulation protocols in the framework of psychophysiology
measurements
Jeremie Jacques Topin1, Claire A. de March2, Jerome Golebiowski1
1
Institut de Chimie de Nice, UMR CNRS, Universite de Nice Sophia Antipolis Nice, France, 2Duke University Medical Center,
Durham, USA
There is a growing interest for using odor stimulations as an alternative to pharmacological treatment for patients
suffering from anxiety. Benzodiazepine, antidepressants or anticonvulsants have important drawbacks such as
dependence liability, sedation or sexual dysfunction. Moreover, a significant number of people do not meet all
the criteria for a diagnosis of general anxiety disorder and thus do not receive any treatment. Essential oils are
frequently cited as efficacious and well-tolerated alternatives. For example, it has been shown that the use of
orange or lavender scent leads to a reduction of anxiety level of dental patients. However, the assessment of
physiological measurements upon odor stimulations through a standardized protocol is still to be done.
Methodological issues include gender choice, time of stimulation or evaluation of anxiety in animal models.
In the present study, the effect of odor stimulation on the autonomous nervous system was assessed. A group of
16 people was stimulated with four different pure compounds belonging to the “green odor” family and several
physiological parameters were monitored: breathing frequency, heartbeat and skin conductance. In addition, a
survey was conducted to assess the edibility, relaxing and/or stimulating effect, as well as the familiarity of
odorants. In particular, the influence of the stimulation on the results was evaluated by varying the mode of
delivery and the length of exposure. The final protocol optimizes the consistency between physiological
measurements and surveys.
2016.05.27 17.38.25 Page 32(1)
P3-078 A computational approach towards estimating the number of discriminable odors
Aharon Ravia, Kobi Snitz, Lavi Secundo, Noam Sobel
The number of discriminable stimuli is a fundamental property in the characterization of a sensory system. In
vision, there are 2-7 million discriminable colors and in audition there are ~300,000 discriminable tones.
However, the number of odors humans can discriminate is controversial, with estimates ranging from 6,500 to 1
trillion (Bushdid, Magnasco, Vosshall & Keller, 2014; Gerkin & Castro, 2015; Meister, 2015) different odors. Here
we used a computational model to build a set of odors which are all distinct from each other. In order to derive
estimation for this number, an understanding of the stimulus space and its mapping to the sensory system is
needed. We applied a previously developed model that predicts similarity and discriminability between odor
mixtures (Odors Perceptual Similarity by Snitz et al. 2013, Olfactory White by Weiss et al., 2012). This model
takes into account only the physico-chemical properties of the molecules. We chose a set of 128 odorants and
looked at all the mixtures that consist of 10 to 30 odorants out of them. We then generated different sets of
mixtures and looked for the maximal set of pairwise discriminable odors. As the number of possible mixtures is
extremely large, and the heuristic we used to build this set was not optimal we expected this estimation to be a
strict lower bound. Initial results, assuming that odors are given at the same intensities estimated the size of this
subset at 1,000-10,000 odors. In color vision, light intensity is responsible for at least two orders of magnitude in
the estimation of the number of discriminable colors. We predict at least the same in olfaction, and hypothesize
that incorporating the parameter of intensity will increase this estimation to 100,000-1,000,000. We will present
continued development of this model to incorporate odor intensity.
P3-079 Activation of OR1A1 attenuates hepatic lipid accumulation via activation of PKA-CREB-HES1
signaling axis
Chunyan Wu, Su Hyeon Hwang, Yaoyao Jia, Bo-Ram Mok, Sung-Joon Lee
Department of Biotechnology, Graduate School of Life Sciences & Biotechnology, College of Life Sciences and Biotechnology,
Korea University, Seoul, South Korea
Olfactory receptors are widely expressed in non-olfactory tissues, however, the molecular mechanism of actions
of ectopically expressed olfactory receptors are largely unknown. Here we demonstrate that OR1A1 is
significantly expressed in the plasma membrane of cultured hepatocytes. OR1A1 activation by (-)-carvone, a
known OR1A1 ligand, increased the levels of cAMP, but not intracellular calcium or inositol phosphates, thereby
inducing protein kinase A activity with subsequent phosphorylation of CREB and upregulation of the
CREB-responsive gene HES-1, a corepressor of PPAR-γ in hepatocytes. In (-)-carvone-stimulated cells, the
repression of PPAR-γ reduced the expression of the target gene, mitochondrial glycerol-3-phosphate
acyltransferase, which encodes a key enzyme involved in triglyceride synthesis. Intracellular triglyceride level
and lipid accumulation were reduced in cells stimulated with (-)-carvone, effects that were diminished following
the loss of OR1A1 function. In high-fat diet-fed mice, (-)-carvone administration significantly reduced plasma
triglyceride concentrations, adiposity, and hepatic steatosis, and improved glucose and insulin tolerance. These
results demonstrate that activation of OR1A1 attenuates cellular lipid accumulation via activation of protein
kinase A signaling pathway. The (-)-carvone, an OR1A1 ligand, reduces lipid accumulation in the liver and
adipose tissue.
P3-080 The neural networks underlying the liking and wanting responses to food odors are modified in
bulimia nervosa
Tao Jiang1, Robert Soussignan2, Benoist Schaal2, Daniel Rigaud2, Edward Carrier3, Jean Pierre Royet1
1
Lyon Neuroscience Research Center, CNRS-Inserm-Universite Lyon1, Lyon, France, 2Center fo Smell, Taste, and Food Science, Universite Bourgogne-Inra, Dijon, France, 3Clinque St Vincent de Paul, Lyon, France
With a high mortality and the poorly understood etiology, any advance in the appreciation of the neural
substrates and psychobiological processes underlying Bulimia nervosa (BN) may constitute step towards
prevention and treatment. Current research indicates that the brain reward responses to food stimuli are critical
in controlling food intake. Thus, we investigated the reward processing and related neural circuits in BN in using
food odor stimuli as adequate sensory probes of the reward processes. Event-related fMRI was used to
compare responses to food odors in 12 healthy (Control) and 13 BN women. Each participant was scanned
during two sessions over two consecutive days, one in the preprandial and the other in the postprandial state.
2016.05.27 17.38.25 Page 33(1)
During each session, BOLD responses to liking and wanting judgments were acquired in two separate runs. The
analyses showed that food odors activate not only the reward circuit but also the areas involved in executive and
cognitive functions such as the dorsolateral prefrontal cortex (DLPFC). The food odors elicited differential neural
activation in BN and Control, and that is metabolic state-dependent. In the hunger state, BN showed higher
activation in vision-related areas (occipital cortex), while the Controls had higher activation in reward-related
areas (insula, striatopallidal regions, and substantia nigra) and in semantic-related areas (angular and
supramarginal areas). In the satiety state, BN had increased activation in other areas of the reward circuit
(ventral medial prefrontal cortex, anterior cingulate cortex), while Control had higher activation in bilateral
DLPFC. These findings suggest that the neural networks involved in the processing of external and internal
signals related to food and metabolism are modified in BN. While BN tend to maintain an elevated sensory
“attention” to food cues, they appear to mobilize their reward circuit in a way that does not match their inner
state.
P3-081 Morphogenesis of lateral line neuromast in the trunk of Polypterus
Yasuyo Shigetani, Tohru Yano, Masataka Okabe
Department of Anatomy, the Jikei University School of Medicine, Tokyo, Japan
Polypterus, the most basal extant actinopterygian fish in molecular phylogeny, reminds us of an extinct primitive
antinopterygii or teleostei. Fossil records of these extinct genera reveal that there are no apparent openings on
the surface of the scale for the lateral line neuromast. The lateral line neuromast is the mechanosensory or
electrosensory receptor, which is distributed to the cranial and lateral body regions from a part of the cranial
nerves. A wide variety of shapes are known in the lateral line in existing bony fishes, and they undergo 3 rounds
of whole-genome duplication. In terrestrial tetrapods, on the other hand, the lateral line is thought to be a
vestigial organ. Therefore, we investigated the morphogenesis of the lateral line neuromast in Polypterus as a
representative model of the primitive actinopterygians. Initial neuromast cells appeared at neural as the placode
and the neural crest cells to coalesce into apparent rosette structures in the bistratal epidermis. At the larval
stage, the lateral line neuromasts, hair cells of which bulged out from the surfaces of the epidermis, projected
their axons inward lateral line nerve. When mineralization starts in a few underlying scales in the rear, the
posterior margin of the scale transiently appeared, and the neurogenesis appeared to be avoiding calcium
deposits for a short period. The neuromasts in the adult were macroscopically seen where pigment cells
gathered, and the axon bundle from the neuromast threaded the vascular cavities of the lateral line scale
inwards and finally pieced it. Therefore, the lateral line neuromasts along the body existed superficially for life
and did not exhibit a distinct feature of the canal structure, even after mineralization, which indicates that the
lateral line neuromast in the trunk of Polypterus is a superficial pit organ.
P3-082 Orexin modulates neuronal activities in mesencephalic trigeminal sensory neurons via orexin
receptor-2 in rats
Kiyomi Nakayama, Shiro Nakamura, Ayako Mochizuki, Tomio Inoue
Department of Oral Physiology, Showa University School of Dentistry, Tokyo, Japan
Orexins (hypocretins) are a pair of neuropeptides expressed by a specific population of neurons in the lateral
hypothalamic area. The orexin neurons are activated by taste stimulation with saccharin as well as motivated
behaviors and active waking, and they regulate food intake, wakefulness, and reward seeking. To examine the
role of orexin in the control of jaw movements in accompany with food intake in mammals, whole-cell patchclamp recordings from mesencephalic trigeminal sensory neurons (MesV neurons), which are critical
components of the circuits controlling oral-motor activity, particularly mastication, were performed on brainstem
slice preparations from Wistar rats aged between postnatal days 8 and 12. Bath application of both orexin A (500
nM) and orexin B (500 nM) induced small membrane depolarization from the resting potential in the absence and
presence of tetrodotoxin. Moreover, an orexin receptor-2 (OX2R) agonist, [Ala11, D-Leu15]-orexin B (ALOXB, 500
nM) depolarized the membrane potential, while orexin A-induced depolarization was significantly reduced in the
presence of an OX2R antagonist, TCS-OX2-29 (10 μM) in MesV neurons (n =6, P=0.001). In contrast, an OX1R
antagonist, SB334867 (10 μM) did not significantly reduce orexin A-induced depolarization (n =4, P=0.967). The
burst discharges in MesV neurons were reduced dose-dependently by bath application of orexin A and ALOXB.
Although TCS-OX2-29 (1 μM) antagonized the inhibition of the spike discharge by 200 nM orexin A and 200 nM
ALOXB, SB334867 (1 μM) did not significantly reduce the rate of inhibition of the spike discharge induced by
orexin A. Persistent sodium currents, which have been suggested to contribute to the production of such burst
2016.05.27 17.38.25 Page 34(1)
discharges, were also reduced by orexin A and ALOXB. These results suggest that orexin inhibits activity in
MesV neurons via OX2R.
P3-084 Blockade of swallow initiation evoked by capsaicin following co-application of QX-314 and
capsaicin in anesthetized rats
Kojun Tsuji, Takanori Tsujimura, Shogo Sakai, Taku Suzuki, Jin Magara, Makoto Inoue
Division of Dysphagia Rehabilitation, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
Purpose: Capsaicin is known to be an irritant component to facilitate swallow initiation in mammals. However,
mechanisms of capsaicin-sensitive receptors/fibers on naturally evoked swallows remains unclear. The aim of
the present study was (1) to develop an animal model of impairment of swallow initiation using QX-314 and
capsaicin (QX-314/capsaicin) and (2) to investigate the role of capsaicin-sensitive laryngeal nerves in
mechanically evoked swallows. Materials and Methods: Urethane-anesthetized male Sprague Dawley rats were
used. Swallowing reflex was identified by the electromyographic burst of suprahyoid and thyrohyoid muscles.
Following topical application of capsaicin alone or together with QX-314 to the vocal folds, the number of
swallows by capsaicin application to the vocal folds was measured at 5, 30 and 60 min after initial procedure.
Mechanical threshold of swallow initiation evoked by von Frey filament to vocal folds was also measured at all
time points. Results: The number of swallows was not changed among the time points in capsaicin alone model
(5 min; 19.9±2.7, 30 min; 22.0±6.5, 60 min; 20.2±6.6). The initiation of swallows in QX-314/capsaicin model
was strongly suppressed at 5 min and was back in a time dependent manner (5 min; 0.7±0.3, 30 min; 13.2±
4.1, 60 min; 18.2±3.6). Mechanically evoked swallows were not affected at any time in both models.
Conclusion: Co-application of QX-314/capsaicin inhibits swallow initiation evoked by capsaicin but capsaicin
sensitive nerves are less involved in initiation of mechanically evoked swallows in larynx.
P3-085 Orexins excite parasympathetic preganglionic neurons in the superior salivatory nucleus
innervating the salivary glands
Yoshihiro Mitoh1, Tadasu Sato2, Masako Fujita1, Motoi Kobashi1, Hiroyuki Ichikawa2, Ryuji Matsuo1
1
Department of Oral Physiology, Okayama University, Okayama, Japan, 2Division of Oral and Craniofacial Anatomy, Tohoku
University, Sendai, Japan
Introduction: Orexin-A and -B are exclusively synthesized by lateral hypothalamic (LH) neurons and are
implicated in the regulation of feeding and arousal. Previous neuroanatomical studies have revealed that the
superior salivatory nucleus (SSN) neurons innervating the submandibular and sublingual salivary glands, which
is the primary parasympathetic center, receives direct projections from LH neurons. The aim of this study is to
examine the effects of orexins on excitability of SSN neurons. Methods: We examined the responsiveness to
orexins and orexin receptor subtypes (OXR-1 and -2) in brainstem slice including SSN neurons retrogradely
labeled with a fluorescent tracer from the chorda-lingual nerve in rats using whole-cell patch-clamp and
immunohistochemical method. Results & Discussion: In immunohistochemical study, SSN neurons showed
immunoreactivities for OXR-1 (n=172/317 cells) or OXR-2 (n=124/307 cells), indicating that SSN neurons were
innervated by orexinergic LH neurons. In electrophysiological study, many SSN neurons responded to orexins,
and all of the responses were excitatory. In voltage-clamp recordings, SSN neurons exhibited inward currents in
bath-applied orexins at -70 mV, but the responsiveness to orexin-A (n=18/24) was higher than that to orexin-B (n
=6/16). In current-clamp recordings, most SSN neurons (n=15/17 cells) generate depolarization in bath-applied
orexin-A and about half of the neurons were accompanied with repetitive action potentials. The membrane
potentials (V) to a series of current (I) steps were examined before and during bath-applied orexin-A. The I-V
relationship indicated an increase in input resistance with a reversal potential at approximately -80 mV, which
was close to the equilibrium potential (-85 mV) of K+ under our recording conditions. These results suggest that
SSN neurons are excited by orexins via OXR-1 and OXR-2 with closure of K+ channels. Orexins may contribute
to abundant salivary secretion during feeding.
P3-086 Relationship between chemical structure and cooling properties of menthol derivatives by
sensory evaluation
Seiji Fujikawa, Yoshiyuki Wada, Katsuya Sakuma, Toshio Miyazawa
Functional Products R&D Laboratory, Ogawa & Co., Ltd., Chiba, Japan
Menthol is widely used cooling agent in gum, confectionery, beverages, oral care and other products. Menthol
2016.05.27 17.38.25 Page 35(1)
exhibits a pleasant cooling sensation, but also has some undesirable properties such as burning, tingling
sensation and bitterness. In order to improve such disadvantages of menthol, a considerable number of menthol
derivatives have been synthesized. There are many reports about their cooling properties, but reports related to
the effect which a certain resembling chemical structure gives to cooling properties are not so many.
At this time, to investigate the relationship between chemical structure and cooling properties of menthol
derivatives, we performed two sensory evaluations for six commercially available menthol derivatives that are
common in having four carbon atoms in the side chain moiety. First, to compare the cooling strength, the cooling
thresholds in water were determined by two-alternative forced choice (brank, coolant). Then, to examine the
expression of the cooling sensation, time/intensity courses were measured for the above menthol derivatives.
The measurements were performed in two medium, i.e. aqueous solution (hydrophilic condition) and chewing
gum (lipophilic condition).
As a result, cooling thresholds were higher than menthol by derivatization and tended to be lower by
functionalization of the side chain moiety. In addition, time to perceive cooling sensation tended to be slower
than menthol by derivatization.
For the future work, we will evaluate the cooling activity of above menthol derivatives by a cell physiological
method, and examine the correlation between those physiological data and these sensory evaluation results.
We expect that these results lead to the development of new cooling agents.
P3-087 Two mechanisms for the detection of non-nutritive compounds by gustatory neurons in the
mouthparts of the buff-tailed bumble bee
Sebastien Kessler, Carolyn Ma, Ashwin Miriyala, Geraldine Wright
Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK
Generalist pollinators like the buff-tailed bumble bee (Bombus terrestris) eat floral nectar and pollen. Floral
nectar is a reward for pollinators, but it sometimes contains toxic compounds. Recent studies indicate that
bumblebees have difficulty detecting toxins in sucrose solution, and few studies have investigated their sense of
taste. Using a new behavioural taste assay we show that 1mM quinine hydrochloride added to a 1M sucrose
solution completely inhibits feeding by forager bumble bees. However, such a strong deterrent effect was not
observed when 1mM nicotine hydrogen tartrate (NHT) or 100mM KCl were added to the same 1M sucrose
solution. Using electrophysiological recordings from the sensilla chaetica on the gallea of bumble bees, we show
that 100mM KCl and 1mM NHT elicit excitatory responses from putative salt and/or bitter gustatory receptor
neurons (GRNs). Quinine also elicited action potentials but only when the galeal sensilla were stimulated with a
saturated solution (10mM). In contrast, 1mM quinine mixed in 1M sucrose had a strong inhibitory effect on the
excitatory responses of sugar-sensitive GRNs whereas no significant effect was found when 100mM KCl or 1
mM NHT were added to the same 1M sucrose solution. Together, these results suggest that the suppression of
the sugar response plays a stronger role in the detection and avoidance of toxins by bees than the activation of a
specific bitter neuron.
P3-088 A gut hormone, peptide YY, inhibits a palatability-induced consumption of sucrose in mice
Erina Yamaguchi, Yasunobu Yasoshima, Tsuyoshi Shimura
Division of Behavioral Physiology, Graduate School of Human Sciences, Osaka University, Osaka, Japan
It is well known that gut peptides such as cholecystokinin (CCK), glucagon like peptide-1 (GLP-1) and peptide
YY (PYY) suppress homeostatic food intake. Previous studies suggested that CCK and GLP-1 also affected
reward-related behavior and taste evaluation. However, it remains unknown whether PYY affects voluntary
consumption of and/or taste reactivity to a palatable taste substance in the absence of homeostatic hunger. To
address these issues, we examined the effect of peripheral administration on both licking behavior and taste
reactivity to a sucrose solution in mice. Mice were accommodated to a 4-h daytime presentation of a 0.5 M
sucrose solution for 14 days. On day 15, mice received an intraperitoneal injection of PYY followed by 4 h
sucrose access. Licking behavior on days 14 and 15 was compared. The result showed that a large number of
licks to sucrose was observed during initial 5-min of the sucrose access; however, PYY reduced the number of
licks during the initial period. In separate mice, orofacial taste reactions to oral stimulation with sucrose were
measured after acclimation to the sucrose solution for 2 days. Ingestive taste responses to 0.3 M sucrose were
observed when mice received saline injection and a series of brief (30 s) intraoral infusions of the sucrose
solution with 2 min interval for 30 min. However, the ingestive responses to the sucrose were decreased when
the same mice received PYY administration. Aversive taste responses increased when mice were given PYY
2016.05.27 17.38.25 Page 36(1)
administration. These results suggest that PYY decreases the taste reward and palatability of the sweetness of
sucrose so as to decrease the palatability-induced consumption of sugar.
P3-089 Ultrasonic vocalizations in rats accompanied by chemical stimuli-induced emotional behaviors
Aya Terashima, Tsuyoshi Shimura
Department of Human Sciences, Osaka University, Osaka, Japan
Taste and smell are indispensable for survival or reproduction. These chemical stimuli usually evoke positive or
negative emotion to animals. It is documented that rats emit 50-kHz ultrasonic vocalizations (USVs) during
positive and 22-kHz USVs during negative emotional states. However, little is known about the relationship
between USVs and chemical stimuli-induced emotional behaviors. Thus we examined the expression of USVs
accompanied by taste or olfactory stimulus-induced behavior in rats. Rats were daily trained to drink a taste
solution for 20 minutes after 18-hours water deprivation. After the consumption of the taste solution was
stabilized, we recorded USVs using Batbox Duet (UK) immediately before and after the presentation of the taste
solution. When rats were daily presented with 0.5 mM saccharin, the expression of 50-kHz USVs prominently
increased during the period before the presentation of the solution. On the other hand, when rats were daily
presented with 0.1 M NaCl, the number of 50-kHz USVs before the presentation of the solution was smaller than
that of trials with saccharin presentation. The USVs in this experiment may be related to the anticipation of taste
reward. In a separate experiment, we examined whether the pattern of USVs changed by different bedding
odors in rats. The number of 50-kHz USVs were highest in the bedding with own smell followed by that in the
fresh bedding and lowest in the bedding with stranger’s scent. This result suggests that the USVs reflect different
affective states evoked by olfactory stimuli. Collectively, these findings demonstrate that the pattern of USVs in
rats is related to taste- and olfactory-induced emotional behaviors.
P3-090 The architecture and information processing in brainstem bitter taste-relaying neurons defined
by genetic tracing
Makoto Sugita, Kuniyo Yamamoto, Chikara Hirono, Yoshiki Shiba
Department of Physiology and Oral Physiology, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan
Bitter taste detection is mediated by taste receptor cells that coexpress multiple T2Rs, and provides mammals
with a sensory input to evoke aversive and displeasing responses. To gain insight into how bitter taste
information is translated in the brain into appropriate behavioral responses, we combined the genetic tracing of
bitter taste representation in the brain with electrophysiological and immunohistochemical analyses. We
visualized the neuronal circuitries for bitter by selectively expressing the fluorescent transneuronal tracer, tWGADsRed, in mT2R5-expressing cells in mice. In the solitary tract nucleus, a first-order central relay, the neurons
labeled by tWGA-DsRed originating from mT2R5-expressing cells are posteriorly located. In the parabrachial
nuclei that receive input from the solitary tract nuclei, the tWGA-DsRed-labeled neurons were rostrally located in
the external lateral parabrachial nuclei, and caudally located in the medial parabrachial nuclei. The expression of
Zif268, an immediate early gene, was induced in the tWGA-DsRed-labeled neurons in the solitary tract nuclei by
oral stimulation with the bitter substance cycloheximide, but not with sweet substance or by intragastric
administration of cycloheximide, suggesting that the tracer-labeled neurons may be selectively activated by oral
stimulation with the bitter substance. Analyses of cellular characteristics of tWGA-DsRed-labeled neurons
indicated that the tracer-labeled neurons in the solitary tract nuclei receive glutamatergic synaptic input via the
AMPA receptor, which is enhanced by the satiety peptide cholecystokinin. However, the tWGA-DsRed-labeled
neurons in the solitary tract nuclei are heterogeneous, and can be classified into catecholamine and proopiomelanocortin neurons. Interestingly, the tWGA-DsRed-labeled neurons in the medial, and the external
lateral parabrachial nuclei exhibited the differential responsivities to noradrenaline and α-MSH. Our data reveal
the heterogeneity of bitter-taste relaying neurons both in the solitary tract nuclei and in the parabrachial nuclei,
which may interact with the homeostatic systems of feeding control.
P3-091 Effect of NaCl on amino acid taste preferences in C57BL/6 mice
Yuko Murata1, Alexander A. Bachmanov2
1
National Research Institute of Fisheries Science, Japan Fisheries Research and Education Agency, Yokohama, Japan, 2Monell
Chemical Senses Center, Philadelphia, PA, USA
NaCl is an important seasoning widely used in various foods. In addition to contributing saltiness, NaCl also
2016.05.27 17.38.25 Page 37(1)
modifies taste of other nutrients, including amino acids. NaCl was reported to enhance sweetness of Gly, L-Ala
and L-Ser (Ugawa et al., 1992), and to suppress bitterness of L-Phe and L-Ile (Ogawa et al., 2004) in humans.
Here, we analyzed the effects of NaCl on the taste of amino acids in C57BL/6J mice by measuring preferences
in the 2-bottle choice tests for 0-50mM L-Arg, L-Glu, Gly, L-Val, L-Leu, and L-Met mixed with 0-200mM NaCl.
These 6 amino acids were chosen because they differ in palatability and taste qualities: Mice had strong
aversion for Arg (probably due to its bitterness) and Glu (probably due to its acidity), strong preference for Gly
(probably due to its sweetness), and weak or no preference for Val, Leu and Met. Effects of adding NaCl
depended on amino acid and concentration. The strongest increase in palatability was observed when NaCl was
added to Arg. NaCl did not affect preference scores for Glu or Gly, but at some concentrations it moderately
increased preference scores for Val and Leu and slightly decreased preference scores for Met. These results
suggest that the effects of NaCl on amino acid preferences depend more on taste quality than on hedonic
perception of each amino acid: NaCl increased palatability of Arg, Val and Leu probably by inhibiting their
bitterness, but NaCl did not affect palatability of aversive and acidic Glu or attractive and sweet Gly. Supported
by Fisheries Research Agency (Yokohama, Japan) research grant (YM) and NIH grant R01DC00882 (AAB).
P3-092 The feed-forward suppression of NaCl intake induced by mating in SPR KO fruit fly
Akira Furuyama1, Tadayuki Kojima2
1
Department of Oral Function and Molecular Biology, Ohu University, School of Dentistry, Japan, 2Department of Oral and
Maxillofacial Surgery, Ohu University School of Dentistry, Japan
Female fruit flies eat more after mating. This effect is mediated by the male-derived sex peptide (SP or Acp70A )
and the SP receptor (SPR ) in the female nervous system. The existence of an SPR -independent system that
induces post-mating responses in feeding behavior has not been elucidated sufficiently. In this study, we used
SPR KO (SPR -deficient or SPR -RNAi) flies to investigate the effect of mating on the intake of sucrose, sodium
chloride (NaCl), and an amino acid mixture. In the wild type females, intake of sucrose and the amino acid
mixture increased after mating; these post-mating responses were not observed in the SPR KO females. NaCl
intake increased in the wild type females after mating, but decreased in the SPR KO females after mating.
These results suggest the existence of an SPR -independent mechanism that specifically suppresses NaCl
intake after mating. Our study revealed that excessive intake of NaCl after mating, induced by the SP-SPR
system, is harmful to flies, and therefore additional post-mating circuitry might be necessary to reduce NaCl
intake. Further research is required to verify whether the previously reported post-mating circuitry is involved in
this feed-forward suppression of NaCl intake.
P3-093 A neural circuit for memory-dependent Na+ Chemotaxis dissected in caenorhabditis elegans
Lifang Wang1, Hirofumi Sato1, Yohsuke Satoh1, Masahiro Tomioka2, Hirofumi Kunitomo1, Yuichi Iino1,2,3
1
Department of Biological Sciences, The University of Tokyo, Japan, 2Molecular Genetics Research Lab, Graduate School of
Science, The University of Tokyo, Japan, 3CREST, JST, Japan
Animals show various behaviors in response to environmental chemicals, which are often plastic depending on
previous experiences. C. elegans, which senses chemicals with a limited number of sensory neurons, is an ideal
model for analyzing the role of each neuron in innate and learned behaviors. ASE neurons (ASE -Left neuron
and ASE -Right neuron), which play major roles for salt chemotaxis, have lateralized functions: ASER neuron is
activated by decreases in salt concentrations and ASEL neuron is activated by increases in salt concentrations.
So far, our lab and others have focused on the roles of ASER and its signaling pathways in salt chemotaxis
plasticity, but whether and how ASEL regulates the plastic behaviors in salt chemotaxis remains less explored.
In this study, chemotaxis assay and optogenetics combined with calcium imaging and behavioral assay were
used to investigate ASEL ’s roles in worm behaviors and to dissect the neural circuit underlying Na+ chemotaxis.
Our results indicated that worms’ behavior changed depending on previous experience, and this
memory-depending behaviors in Na+ chemotaxis were generated by different neural circuits downstream of
ASEL neuron: I) when worms were transferred from salt-free precondition to salt-present condition, ASEL sent
signal to the secondary interneuron AIY to inhibit turning, so worms showed no salt gradient preference; II) when
worms were in salt-present precondition, or transferred from salt-free precondition to salt-free condition, ASEL
sent signal to all three secondary interneurons, AIY , AIA , and AIB , to activate forward movement and inhibit
turning, driving the animal to higher salt concentrations.
2016.05.27 17.38.25 Page 38(1)
P3-094 The central processing of salt taste quality induced by neutralizing a basic amino acid, arginine
and its enhancement with inosine monophosphate
Ema Suzuki1, Yuka Nakazato1, Hanae Nakano1, Takayuki Sako2, Takenori Miyamoto1
1
Laboratory of Behavioral Neuroscience, Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s
University, Tokyo, Japan, 2Department of Food and Nutrition, Faculty of Human Science and Design, Japan Women’s University,
Tokyo, Japan
A basic amino acid, arginine (Arg) shows complex taste qualities including sweet, bitter and umami.
Furthermore, additive salty taste is induced by neutralization of arginine (nArg). The sensory rating experiments
revealed that human sensitivity to salty taste of nArg is divided into the sensitive and insensitivity types: sensitive
subjects showed gradual increase of the sensitivity to saltiness and induced by nArg in the dose-dependent
manner and its enhancement by inosine monophosphate (IMP), whereas insensitive subjects showed lower
sensitivities to all concentration range with decrement of sensitivities to higher concentration of nArg and smaller
enhancement by IMP. Both the dose-dependent increase of bitterness and the dose-dependent decrease of
pleasantness induced by nArg were suppressed by addition of IMP to nArg. The measurement of change of
oxy-hemoglobin (oxy-Hb) using a near-infrared spectroscopy (NIRS) also represented similar tendency to the
rating experiments in the prefrontal brain region. Significant correlations between the rating values and changing
amount of oxy-Hb for saltiness and bitterness as well as pleasantness induced by nArg appeared chiefly in the
lateral area of the prefrontal brain region, but the correlated area moved from the lateral area to the central area
after addition of IMP to nArg. These results suggest a possibility that enhancement of saltiness and
pleasantness are due to suppression of bitterness by synergic enhancing effect by addition of IMP to nArg
through the interaction between different processing pathways for nArg and IMP respectively in the prefrontal
brain region.
P3-095 Genetics of amino acid taste and appetite
Alexander A. Bachmanov1, Natalia P. Bosak1, John I. Glendinning2, Masashi Inoue1,3, Xia Li1,6, Satoshi Manita1,3,7,
Stuart A. McCaughey1,8, Yuko Murata1,4, Danielle R. Reed1, Michael G. Tordoff1, Gary K. Beauchamp1,5
1
Monell Chemical Senses Center, Philadelphia, USA, 2Department of Biology, Barnard College, Columbia University, New York,
USA, 3Laboratory of Cellular Neurobiology, Department School of Life Sciences, Tokyo University of Pharmacy and Life Science,
Tokyo, Japan, 4National Research Institute of Fisheries Science, Yokohama, Japan, 5Department of Psychology and School of
Veterinary Medicine, University of Pennsylvania, Philadelphia, USA, 6Cincinnati Childrens Hospital, Cincinnati, USA, 7Department of
Physiology, Division of Medicine, University of Yamanashi, Yamanashi, Japan, 8Center for Medical Education, IUSM-Muncie at Ball
State University, USA
Our studies have shown that inbred mouse strains differ in voluntary amino acid consumption, and these
differences depend on sensory and nutritive properties of amino acids. Like humans, mice perceive some amino
acids as having sweet (sucrose-like) taste and others as having umami (glutamate-like) taste. Mouse strain
differences in the consumption of some sweet-tasting amino acids (D-phenylalanine, D-tryptophan and
L-proline) are associated with polymorphisms of a taste receptor gene, Tas1r3, and involve differential
peripheral taste responsiveness. Strain differences in the consumption of some other sweet-tasting amino acids
(glycine, L-alanine, L-glutamine and L-threonine) do not depend on Tas1r3 polymorphisms and so must be due
to allelic variation of other, as yet unknown, genes involved in sweet taste. Strain differences in the consumption
of L-glutamate may depend on postingestive rather than taste mechanisms. Thus, genes and physiological
mechanisms responsible for strain differences in consumption of each amino acid depend on the nature of its
taste and post-ingestive properties. Overall, mouse strain differences in amino acid taste and appetite have
complex genetic architecture: In addition to the Tas1r3 gene, these differences depend on other genes likely
involved in determining the taste and postingestive effects of amino acids. Identification of these genes may lead
to discovery of novel mechanisms that regulate amino acid taste and appetite.
P3-096 Effects of zinc deficiency on salivary secretion and salivary carbonic anhydrase activity in rats.
Tomoko Goto, Yuka Onuma, Hitoshi Shirakawa, Michio Komai
Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
It is well known that zinc deficiency leads to taste disorder (decreased taste sensitivity), a symptom that can be
reversed by zinc administration. We previously reported that long-term zinc deficiency significantly reduced taste
sensitivity, carbonic anhydrase (CA) activity in the submandibular gland, and salivary secretion in rats. However,
it remained unclear what is the onset timing of the decrease of salivary secretion and salivary CA activity after
2016.05.27 17.38.25 Page 39(1)
feeding of the zinc-deficient diet. Therefore, we monitored salivary secretion and CA activities in the saliva and
submandibular gland in zinc-deficient rats over time. Sprague-Dawley rats (male, 4 weeks old) were divided into
four groups: zinc-deficient (Zn-Def), marginal zinc-deficient (Low-Zn), zinc-sufficient (Zn-Suf) and pair-fed
(Pair-fed). The amounts of zinc in each diet of Zn-Def, Low-Zn and Zn-Suf were 2.1, 4.1 and 33.7 mg/kg diet,
respectively. Pair-fed rats that were fed with zinc-sufficient diet, were pair-fed against the Zn-Def rats one day
later. Salivary secretion and CA activity in the submandibular gland in Zn-Def and Low-Zn rats were significantly
lower than that of Zn-Suf rats after 42 days of feeding. However, CA activity in the saliva in the Zn-Def rats was
found to be significantly lower than that in other three groups of rats after 21 days of feeding. These results
suggest that CA activity in the saliva can be more sensitive index of zinc-deficiency in rats comparing to salivary
secretion or CA activity in the submandibular glands.
P3-097 Spilanthol modulates the responses of the chorda tympani and lingual trigeminal nerves
responses in SD rats
Kyosuke Yoshida1, Michio Komai1, Tomoko Goto1, Hitoshi Shirakawa1, Toshio Miyazawa2, Yasutaka Shoji2, Bruce Bryant3
1
Graduate School of Agricultural Science, Tohoku University, Sendai, Japan, 2Functional Products R&D Laboratory, Ogawa & Co.,
Ltd./Urayasu, Japan, 3Monell Chemical Senses Center, Philadelphia PA, USA
Spilanthol is the pungent component, mainly contained in the plants such as Spilanthes oleracea. Recently, we
have shown that this compound acts as a salt taste enhancer in rodents taste nerve recording and human
sensory evaluation test. We have examined the influence of spilanthol on salty taste response by recording the
chorda tympani (CT) nerve response in SD rats. This time, we are going to add the effect of
hydroxyl-alpha-sanshool which is a analogue compound of spilanthol, mainly contained in Zanthoxylum
piperitum. Firstly, we determined the threshold concentration of these compounds to cause the CT nerve
responses, and found out that there was a significant difference between spilanthol and hydroxyl-alpha-sanshool
(5 times higher than spilanthol). Furthermore, we have also undertaken the lingual trigeminal nerve responses to
these two compounds in SD rats, and found out that spilanthol caused firing of both of the menthol-sensitive
and -insensitive nerve fibers. Further analyzed data on the CT and trigeminal nerves responses will be added in
this presentation.
P3-098 The ATP-gated K+ channel mediates taste responses to sugars in mice
Masafumi Jyotaki, Karen Yee, Robert F. Margolskee
Monell Chemical Senses Center, Philadelphia, PA, USA
The sweet taste receptor T1r2+T1r3 is well established as the primary receptor for non-caloric sweeteners.
Indeed, T1r3 knockout (KO) mice lack behavioral and taste nerve responses to non-caloric sweeteners.
However, T1r3 KO mice display near normal responses to multiple sugars arguing for T1r3-independent
pathway(s) for sugar taste. We previously demonstrated that T1r3+ taste cells selectively express glucose
transporters (GLUTs), sodium-glucose co-transporter-1 (SGLT1) and subunits of the ATP-gated K+ channel (K
ATP) metabolic sensor [e.g. sulfonylurea receptor-1 (SUR-1) and inwardly rectifying potassium channel (Kir6.1)].
In the present study we used gustatory nerve recordings of wildtype (WT), T1r3 KO and SUR1 KO mice to
assess their responses to sugars and non-caloric sweeteners. By immunohistochemistry we confirmed that SUR
1 was selectively expressed in T1r3+ taste cells from WT mice, but was absent from the taste cells of SUR1-KO
mice. Chorda tympani nerve responses to glucose and sucrose were markedly lower in SUR1-KO vs. WT mice.
In contrast, the nerve responses of SUR1-KO mice to the non-caloric sweetener sucralose were comparable to
those of WT mice. There were no significant difference between WT and SUR1 KO mice in their nerve
responses to umami, bitter, salty and sour tasting stimuli. Our results indicate that responses to glucose and
some other sugars are mediated by both the T1r2+T1r3 pathway and the T1r-independent pathway comprised
of GLUTs, SGLT1, and KATP.
P3-099 Simultaneous recording of multiple single-fiber activities in the chorda tympani nerve with a
stereotrode method
Yui Kano, Tetsuhiro Kobayashi, Hiroyuki Minezumi, Hiroyoshi Miyakawa, Masashi Inoue
Laboratory of Cellular Neurobiology, Department of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
The chorda tympani (CT) nerve innervates the anterior tongue, and carries taste information to the brain.
Despite the recent studies using genetically modified mice provide the evidence for the labeled-line model, it has
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been shown that afferent nerve fibers in the CT nerve are broadly tuned to taste modalities. The previous taste
nerve fiber studies largely depended on the surgical techniques and it is possible that the identification of the
“single” fiber activity would have been based on subjective criteria. In order to solve the problem, we have
modified the stereotrode method used in the CNS studies and developed a novel method that provides a high
degree of reliability to identify and distinguish single fiber activity. Wistar rats and C57BL/6 mice were used for
nerve recoding experimens. The Animal was anesthetized and the CT nerve was dissected free from its junction
with lingual nerve to the tympanic bulla from surrounding connective tissue and transected. The desheathed CT
nerve was teased into several fine strands and each strand was placed on a glass platform and two electrodes
made from glass and/or nichrome wire were inserted into the strand. Multiple fiber activities were recorded from
the each electrode. Taste stimuli were applied to the anterior tongue. Off-line spike sorting was carried out with
an analysis method that combines feature extraction with multimodality-weighted principal component analysis
and clustering by variational Bayes for Student’s t mixture model. To evaluate the cluster quality, we used both L
ratio and Isolation Distance and set the criteria values as 0.1 and 4.00, respectively. Using these methods, we
have successfully isolated reliable 2-5 single units from a two-channel multi fiber record. This novel method may
facilitate to obtain useful results to understand how the gustatory information is coded and represented in the
taste nerve.
P3-100 Degeneration of fungiform and circumvallate papillae following molar extraction and unilateral
nasal obstruction in rats
Ippei Watari, Jui-Chin Hsu, Rieko Ono, Yukiha Funaki, Satoshi Kokai, Takashi Ono
Orthodontic Science, Division of Oral Health Sciences, Graduate School of Medical and Dental Science, Tokyo Medical and Dental
University (TMDU), Tokyo, Japan
/Introduction/ Tooth loss or mouth breathing associated with nasal airway obstruction cause malocclusion, which
leads to various oral complications. However, few studies have investigated the relationship between
malocclusion and gustatory function. We evaluated the changes in tongue papillae caused by molar extraction
and unilateral nasal obstruction in rats. /Materials and Methods/ In the first experiment, 7 week old male Wistar
rats were divided into control and experimental groups. All maxillary molars were extracted in rats of the
experimental group at the age of 7 weeks, and sham operations were conducted in the control group. In the
second experiment, 8 day old male Wistar rats were divided into control and experimental groups. Unilateral
nasal obstruction was produced by nasal mucosa incineration, and sham operations were conducted in rats of
the control group. In first studies, rats were euthanized at 1, 2, or 4 weeks after the procedures. In second study,
rats were euthanized at 8 weeks after the procedures. We then analyzed the morphological changes of the
circumvallate papillae and taste buds by immunohistochemical staining using anti cytokeratin 8 antibody and
those of the fungiform papillae by methylene blue staining. /Results/ Rats in the experimental group in the first
study showed significantly fewer fungiform papillae, narrower circumvallate papillae, smaller taste bud areas,
and fewer taste buds than did rats in the control group. These findings suggests a possible synergic relationship
between occlusion and gustatory function. On the other hand, rats in the experimental group in the second study
showed significantly fewer fungiform papillae and smaller taste bud volumes, smaller total taste bud area in
circumvallate papillae than did rats in the control group. /Conclusion/ These findings suggest that molar
extraction and nasal obstruction may influence gustatory function via morphologic changes in the taste papillae
and taste buds on the tongue.
P3-101 Augmented transcriptomics of taste cells by RNA-Seq analysis of single cells and pooled taste
buds
Sunil Kumar Sukumaran, Brian C. Lewandowski, Alexander A. Bachmanov, Robert F. Margolskee
Whole mRNA transcript sequencing known as RNA sequencing or RNA-Seq, is now the method of choice for
analyzing gene expression in cells or tissues. We have combined single taste cell RNA amplification with
RNA-Seq to monitor gene expression from four different types of mouse taste cells labeled with green
fluorescent protein (GFP): Tas1r3+ and Gustducin+ Type II cells, Gad1+ Type III cells, and Lgr5+ stem cells. We
have also done RNA-Seq on many individual physiologically identified Type III taste cells. Cluster and correlation
analysis and other quality control metrics, along with expression of known markers specific for each cell type
confirmed the high quality of our data set. We found that all three Tas1rs were frequently co-expressed in
Tas1r3-GFP+ cells. Multiple Tas2rs were expressed in any single Gustducin+ cell. Surprisingly, Tas1rs were
2016.05.27 17.38.25 Page 41(1)
frequently co-expressed with Tas2rs in Gustducin+ cells. RNA-Seq libraries were also prepared from pooled
fungiform and circumvallate papillae taste bud cells harvested by laser microdissection. This data set was use to
generate taste cell specific gene models using de novo transcriptome assembly. Novel isoforms for many genes
were identified by this approach, including alternative spliced and polyadenylated isoforms of many Tas2rs and
Tas1r3. Many of these novel isoforms were also present in the single cell RNA-Seq data, in particular the 3’
isoforms; the latter a reflection of the 3’ end bias in these libraries. Thus, while single cell RNA-Seq provides
unparalleled resolution, the unbiased data from pooled taste cells allows us to identify novel taste specific
isoforms of genes and augments the analysis of the single cell RNA-Seq data.
P3-102 Expression patterns and functional analysis of GPR120 and CD36 in oral and gastrointestinal
tissues of chicks
Yuko Kawabata, Minako Mizobuchi, Fuminori Kawabata, Ryo Sawamura, Shotaro Nishimura, Shoji Tabata
Since chicken is the model organism of birds and is one of the most important industrial animals, the studies of
chemical senses of chicken are thought to be important. We have previously reported that chicken GPR120 is
expressed in oral tissues and GPR120 is activated by oleic acid and linoleic acid. In this study, we analyzed the
expression patterns of GPR120 and CD36 in chicken oral and gastrointestinal tissues by RT-PCR, and
searched for new agonists of GPR120 by Ca2+ imaging. We observed the broadly expressions of GPR120 and
CD36 mRNAs in oral and gastrointestinal tissues of chicks. Interestingly, although CD36 was expressed in the
rostrum, the expression of GPR120 was not rich. By Ca2+ imaging, we found that palmitic acid and α-linolenic
acid are agonists of chicken GPR120. These results imply that chickens detect fat taste via oral GPR120 and
CD36, and fatty acids via gastrointestinal GPR120 and CD36. Furthermore, we constructed a stable HEK293T
cell line expressing chicken GPR120. The cell line will be useful for further studies.
P3-103 Functional analysis of the extracellular calcium-sensing receptor (CaSR) in chicken oral tissues
Hikaru Omori, Yuko Kawabata, Fuminori Kawabata, Shotaro Nishimura, Shoji Tabata
Calcium is one of the essential ingredients for vertebrate. Especially, chickens need calcium because of the
much calcium turnover during making eggshell and extremely rapid initial growth of bones. Although it is well
known that chickens prefer calcium-rich diets when offered a choice, it is unclear how they detect the presence
of calcium in feeds. In this study, we focused on the calcium-sensing receptor (CaSR), which regulates calcium
homeostasis by sensing slight changes of blood Ca2+ in parathyroid, and which is also expressed in taste cells of
mice. Firstly, we investigated the expressions of chicken CaSR (cCaSR ) in oral tissues, kidney, and small
intestine by RT-PCR. We observed the expressions of cCaSR in these tissues. Next, we cloned the cCaSR
gene from kidney because of the high expressions in kidney, and we made the HEK293T cells transfected with
the cloned cCaSR (cCaSR cells) or empty vector (mock cells) by electroporation. Then, we searched the
agonists and antagonists of cCaSR by using Ca2+ imaging technics in these cells. cCaSR was activated by
extracellular Ca2+ dose-dependently. In addition, NPS2143 weakly inhibited the response of cCaSR by
extracellular Ca2+. These results suggest that cCaSR can sense extracellular Ca2+, and that NPS2143 is a partial
antagonist of cCaSR. Taken together, it is possible that chickens detect calcium taste in oral tissues.
P3-104 Differences of acidic sensitivity of TRPV1 between chickens and mice
Ruojun Liang, Yuko Kawabata, Fuminori Kawabata, Shotaro Nishimura, Shoji Tabata
It is important to understand the pain transduction systems of chickens for making animal products more
efficiently. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel to transduce
pungent, heat and acidic signals to brain. Previously, we found that chicken TRPV1 (cTRPV1) is hard to be
desensitized by acidic stimulus compared with mouse TRPV1 (mTRPV1). The purpose of this study was to
reveal the mechanisms. Because calmodulin (CaM) binding site was reported as one of the important structures
for TRPV1 desensitization, we made the chimeric cTRPV1 that was changed the CaM binding site of chicken
with that of mouse (cTRPV1-mCaM). Furthermore, we also compared the acidic responses of native TRPV1 in
chicken dorsal root ganglion (DRG) cells with that of mouse. Whole-cell patch-clamp recordings were carried out
in cTRPV1-mCaM-transfected HEK293T cells. DRG neuron cells isolated from chick and mouse were cultured
2016.05.27 17.38.25 Page 42(1)
at 37℃ and 5% CO2. Cultured DRG cells were used for whole-cell patch clamp recordings within 24 h. Acidic
stimulus were repeated twice to observe the desensitization, and the cells were checked by capsaicin at last for
selecting the capsaicin-sensitive cells only. The whole-cell patch clamp revealed that low pH stimuli activated
both of cTRPV1 and mTRPV1, but the peak current of cTRPV1 was lower than that of mTRPV1, and the
desensitization of cTRPV1 was less than that of mTRPV1. The results of cTRPV1-mCaM showed that the
desensitization of mutant cTRPV1 was similar with that of mTRPV1. Besides, the native TRPV1 expressing cells
in the chicken DRG were also hard to be desensitized by low pH unlike mouse. These results suggest that there
are the differences of pain transduction systems between chickens and mice caused by the mutation of CaM
biding site of TRPV1. These results imply that chickens feel pain repeatedly by acidic stimulus without
desensitization.
P3-105 The relationship between continuous dietary casein restriction and development of taste
organs
Katsura Ueda1, Yoshifumi Matsuda1, Chizuko Inui-Yamamoto1,2, Michiko Nakatsuka1, Shunji Kumabe1, Isao Tamura1
1
Department of Oral Anatomy, Osaka Dental University, Hirakata, Japan, 2Department of Oral Anatomy and Developmental Biology,
Osaka University Graduate School of Dentistry, Suita, Japan
Malnutrition in developmental and growth stages brings various unfavorable alterations in several organs in our
body including taste organs. We previously revealed that continuous dietary sodium restriction during
developmental stage induced some alterations in taste preference and the properties of taste cells constructing
taste buds. However, the effects of the deficiency of other nutrients on taste sense and the peripheral taste
organs are still unclear. In this study, to investigate the relationship between continuous dietary casein restriction
and development of taste organ, we performed two-bottle taste preference test for the solutions of basic tastes
against distilled water by using rats exposed to continuous dietary casein restriction, fed by casein restricted
food (5% casein) from embryonic day 3 through developmental stage to adulthood. We could not observe
apparent difference in the preference for salty taste. But we could find some difference in the preference for
saccharin and sodium inosinate (IMP), in spite of the preference for sucrose and monosodium glutamate do not
show obvious difference between two groups. In addition, we provided immunohistochemistry for the molecules
taking part in peripheral taste transmission (PLCβ2, G-α gustducin and NCAM) on taste buds in circumvallate
papillae of normal and casein restricted rats. The size of taste buds seems to be reduced by dietary casein
restriction, but immunoreaction for these molecules do not show apparent alteration between two groups.
P3-106 Inhibition of gastrointestinal TRPV1 by TRPV1 antagonists enhanced energy expenditure in
mice
Jun Hai, Fuminori Kawabata, Yuko Kawabata, Shotaro Nishimura, Shoji Tabata
Transient receptor potential vanilloid 1 (TRPV1) is known to a nociceptive cation channel, and can be activated
by heat, protons, and chemical ligands such as capsaicin. TRPV1 is not expressed only in primary afferent
neurons but also in gastrointestinal tracts. It has been reported that the activation of gastrointestinal TRPV1 by
TRPV1 agonists such as capsaicin or capsiate enhances energy expenditure. On the other hand, because
administrations of some TRPV1 antagonists increase body temperature in humans and mice, we hypothesized
that TRPV1 antagonists can also change energy metabolisms. In this study, we examined whether the inhibition
of gastrointestinal TRPV1 changes energy metabolisms in mice by using respiratory gas analysis system. In
addition, to reveal the contributions of three different modes of TRPV1 inhibition (for heat, protons, and
capsaicin), we investigated the influence of three different TRPV1 antagonists on energy expenditure. Here, we
showed intragastric administration of a nonselective antagonist of TRPV1 (for heat, protons, and capsaicin), and
a selective one, which affects two modes of TRPV1 activation (by heat and protons), enhanced carbohydrate
oxidation and energy expenditure. Besides, another selective antagonist, that blocks only one mode of TRPV1
activation (by capsaicin), did not enhance energy expenditure. Taken together, these results suggest that the
inhibition of gastrointestinal TRPV1 by blocking two modes of TRPV1 activation (by heat and or protons) may
enhance energy expenditure. It is possible that the enhancement of energy expenditure by TRPV1 agonists
such as capsaicin and capsiate may not be caused by the TRPV1 activation only, but the subsequent
desensitization of TRPV1, since TRPV1 agonists can cause desensitization.
2016.05.27 17.38.25 Page 43(1)
P3-107 Preferences for sugars and T1r2-independent sweet taste molecules in chickens
Momoko Higashida, Yuko Kawabata, Fuminori Kawabata, Shotaro Nishimura, Shoji Tabata
Laboratory of Functional Anatomy, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
Although it has been reported that chickens didn’t respond to sugars in behavioral tests, precise analysis about
sweet taste of chickens has not been reported. Because T1r2 gene is lost in chickens, chickens don’t have the
major sweet taste receptor, T1r2/T1r3 heterodimer. However, there is also evidence of T1r-independent sweet
taste in mammals such as glucose transporters (GLUTs), a sodium-glucose cotransporter (SGLT1), and two
components of the ATP-gated K+ (KATP) metabolic sensor. Thus, in this study, we focused on these
T1r2-independent sweet taste molecules in chickens. In addition, we investigated whether chickens sense sweet
substances such as glucose, sucrose, stevioside, and saccharin. Rhode Island Red strain 0-2 week-old chicks
were used. Firstly, we examined the expression of glucose transporters (GLUT2 , GLUT8 , GLUT9 , and SGLT
1 ), the components of KATPchannel (SURs , Kir6.1 , and Kir6.2 ), T1r3 , and TRPM5 in the chicken palate, where
taste buds are rich, by RT-PCR. In behavioral testes, after water deprivation for 23.5 hours, the chicks were
given water (control) or sweeteners for 30 min. In palate, all of the genes examined by present study were
expressed except GLUT2 . Furthermore, we observed that chicks slightly preferred glucose, sucrose, and
stevioside and disliked saccharin and high dose sucrose. These results suggest that chicks sense sweet taste
slightly via T1r2-independent pathway in oral tissues.
P3-108 Diversity of bitter taste receptor genes in turtles
Yukiko Nishioka, Yui Ohshima, Ryosuke Tanii, Mitsuru Ebihara
Department of Food Science, Ishikawa Prefectural University, Ishikawa, Japan
The detection of bitter-tasting compounds is crucial for most animals to prevent them from ingesting toxic food. It
is commonly known that bitter taste receptor (T2R) genes are coded in a single exon. But exon-intron structure
of reptiles could be different from those of the other animals. Recently draft genome sequence of aquatic turtle,
such as Chelonia mydas and Pelodiscus sinensis revealed that they also have intron sequences in their bitter
taste receptor genes. But decipherment of terrestrial tortoise genome has not yet been conducted. In this study,
we analyzed the gene structure of T2Rs of Geochelone sulcata , which belong to terrestrial tortoises. To isolate T
2R genes from Geochelone sulcata , degenerated PCR was performed. Primers for degenerated PCR was
designed based on conserved sequences of known T2R genes from Chelonia mydas , Pelodiscus sinensis , and
Chrysemys picta bellii . Here we successfully cloned, at least, five T2R7 (functional: 1, pseudogene: 4), T2R9
(functional), two T2R39 (functional: 1, pseudogene: 1), and three T2R40 (functional: 2, pseudogene: 1).
Previously it’s been reported that some of T2R genes from Chrysemys picta bellii have intron(s), but no intron
was found in those from Geochelone sulcata . Surprisingly intron1 of T2R7 in Chrysemys picta bellii might
encode amino acids in counterpart sequences of Geochelone sulcata with only one base insertion, suggesting
that it could be exon in Geochelone sulcata . These results showed the possibility that T2R7 in Chrysemys picta
bellii acquired intron after it diverged from Geochelone sulcata . Association between adaptation to life in sea
water and this diversity is discussed.
P3-109 Sensitivity of larval taste sensilla to adult oviposition stimulants in Asian swallowtail butterfly−
Papilio xuthus
Yasutaka Yoshizawa, Hiroshi Yoshikawa, Katsuhisa Ozaki
JT Biohistory Research Hall, Osaka, Japan
A Caterpillar of Asian swallowtail butterfly, papilio xuthus (Lepidoptera: papilionidea), is oligophagous insect
selectively feed on the leaves of rutaceae spp. Host-plant selection in the caterpillar, contact chemoreception by
use of medial and lateral styloconic sensilla located on the maxillary galea is especially important for decision of
food choice, acceptance or rejection. Host-plant selection is usually studied for the adult stage exclusively, (e.g.,
chemical compounds eliciting oviposition behavior), While little is known about the larval stage in papilio.
Congruence of chemical profiles between larval feeding stimulants and oviposition stimulants has been shown in
several butterfly taxa. Thus, these oviposition stimulants were assumed to be received in the caterpillar and
related to feeding behavior. To investigate this possibility, we performed an Electrophysiological study using
tip-recording techniques to characterize electrophysiological property of taste sensilla that house gustatory
receptor neurons.First, We tasted adult oviposition stimulants for p.xuthus. Among six compounds tested, four
and two compounds elicited action potential firing in a dose-dependant manner on each medial and lateral
2016.05.27 17.38.25 Page 44(1)
styloconic sensilla, respectively. Next, three compounds that elicit oviposition behavior for two related papilio
species, P.polytes and P.protener, but not elicit for p.xuthus, were tested. The medial styloconic sensilla
responded to two compounds and the lateral styloconic sensilla was all of the three. Our electrophysiological
analysis suggests that some conspecific and relative adult female oviposition stimulants are involved in not only
adult egg-raying behavior but also caterpillar food selection.
P3-110 Regulation of CALHM1 channel by protein S-palmitoylation
Akiyuki Taruno1, Hongxin Sun1, Makiko Kashio1, Yoshinori Marunaka1,2
1
Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Department of Bio-Ionomics,
Kyoto Prefectural University of Medicine, Kyoto, Japan
Calcium homeostasis modulator 1 (CALHM1) is a pore-forming subunit of a voltage-gated ATP-permeable ion
channel. CALHM1 channels are expressed in sweet-, bitter- and umami-sensing taste cells and mediate release
of adenosine triphosphate (ATP), the primary neurotransmitter carrying taste information, from taste cells to
gustatory nerve terminals. Although membrane potential and extracellular divalent cations have been
demonstrated to regulate CALHM1 channel, other modes of regulatory mechanisms remain to be identified.
Here we show that CALHM1 channel is regulated by S-palmitoylation on its cysteine residues. Because cysteine
residue is a key element for posttranslational modifications of proteins, we first compared function and cell
surface expression of 8 CALHM1 Cys-to-Ser mutants with those of wild-type CALHM1 by replacing single
cysteine residue in transmembrane or intracellular domains with serine. Many of the single Cys-to-Ser mutations
significantly affected function and cell surface targeting of CALHM1, suggesting that posttranslational
modifications on cysteine residues are involved in the regulation of the channel. Acyl-biotin exchange assay and
metabolic labelling with [3H]-palmitate demonstrated that CALHM1 is a substrate for S-palmitoylation. We
identified multiple palmitoylation sites of CALHM1 within its intracellular loop and carboxy-terminus, C100 and C
207, respectively. The palmitoylation-deficient mutant CALHM1 (C100/207S) caused higher cytotoxicity and
larger ATP release induced by low extracellular Ca2+ concentration than wild-type CALHM1, and treatment with
2-bromopalmitate, an inhibitor of S-palmitolation, in cells transfected with wild-type CALHM1 phenocopied the C
100/207S mutation, demonstrating that deficiency of S-palmitoylation activates CALHM1 channel. The cell
surface localization of CALHM1 was facilitated by the C100/207S mutation. Collectively, we conclude that
S-palmitoylation serves as a negative regulator of CALHM1 channel by slowing its trafficking to the cell surface.
Since S-palmitoylation is a reversible and rapid lipid modification of proteins, this work shed light on the possible
roles of S-palmitoylation on taste physiology.
P3-111 Intracellular acidification is involved in full activation of the sweet taste receptor by miraculin
Keisuke Sanematsu1, Masayuki Kitagawa1, Ryusuke Yoshida1,2, Satoru Nirasawa3, Noriatsu Shigemura1, Yuzo Ninomiya1,4
1
Graduate School of Dental Science, Kyushu University, Fukuoka, Japan, 2OBT Research Center, Kyushu University, Fukuoka,
Japan, 3Japan International Research Center for Agricultural Sciences, Tsukuba, Japan, 4Research and Development Center for
Taste and Odor Sensing, Kyushu University, Fukuoka, Japan
Miraculin (MCL) is a glycoprotein inducing sweet taste by acidification in humans, but not in mice. This
sweet-inducing effect of MCL is more intense when acidification occurs with weak acids as opposed to strong
acids. Here, we focused on the molecular mechanisms underlying the MCL-induced activation of the sweet taste
receptor. Utilizing a sweet receptor assay, HEK293 cells expressing hTAS1R2+hTAS1R3 displayed a pH
dependence with citric acid (weak acid) being right shifted to that with HCl (strong acid) after application of MCL.
Using mixed-species pairings of human and mouse sweet receptor subunits and chimeras, we confirmed that
the amino-terminal domain of hTAS1R2 is required for the sweet-inducing effect of MCL. Replacement of
histidine residues to alanine in both the intracellular and extracellular region of hTAS1R2 reduced or abolished
the sweet-inducing effect of MCL. Stronger intracellular acidification of HEK293 cells was induced by citric acid
than by HCl. The sweet-inducing effect of MCL was proportional to intracellular pH regardless of acid types.
These results strongly suggest that intracellular acidification is required for full activation of the sweet taste
receptor by MCL.
2016.05.27 17.38.25 Page 45(1)
P3-112 Adeno-associated virus-mediated gene transduction of taste cells in vivo
Makiko Kashio1, Akiyuki Taruno1, Hongxin Sun1, Kenta Kobayashi2, Hiromi Sano3, Atsushi Nambu3, Yoshinori Marunaka1,4
1
Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Section of Viral Vector
Development, National Institute for Physiological Sciences, Okazaki, Japan, 3Division of System Neurophysiology, National Institute
for Physiological Sciences, Okazaki, Japan, 4Department of Bio-Ionomics, Kyoto Prefectural University of Medicine, Kyoto, Japan
In taste cells, many signaling molecules cooperate together to code and transmit information on modality and
intensity of taste to the nervous system. Viral-mediated gene transduction techniques have been commonly
used to study and control function of a gene of interest in vivo . However, an effective method of gene
transduction for taste cells “in vivo ” is not yet established, although adenovirus and herpes simplex virus type I
vectors have been demonstrated to successfully transduce a subpopulation of isolated taste cells. Here, we
have established a highly efficient recombinant adeno-associated virus (AAV) vector-mediated transduction
method in taste cells of a living mouse. First, we screened six AAV serotypes along with a recombinant lentivirus
for their ability to transduce taste cells using EGFP as a reporter gene. Seven days after viral injection into
subepithelial layer of the anterior region of the tongue, EGFP expression in taste cells of fungiform papillae was
observed only in animals injected with AAV-DJ, a synthetic serotype. The time course of AAV-DJ-mediated gene
transduction efficiency was then quantified. Effective transduction appeared as early as 2 days, peaked around
7 days and lasted at least four weeks after injection. Because taste cells are classified into three types by distinct
patterns of gene expression, taste cell types were determined for cells transduced by AAV-DJ. EGFP expression
was observed in PLCβ2+ and AADC+ taste cells as well as in PLCβ2-/AADC- cells, suggesting that AAV-DJ does
not discriminate taste cell types. In conclusion, AAV-DJ will be a promising tool to transfer foreign gene into all
types of taste cells in vivo to study physiology and molecular basis of taste.
P3-113 Serotonin immunopositive basal cells of taste bud in ray fined fish
Takanori Ikenaga1, Tatsufumi Nakamura1, Toshinao Ineno2, Sadao Kiyohara1
1
Department of Chemistry and Bioscience, Graduate School of Science and Engineering, Kagoshima University, Kagoshima,
Japan, 2Freshwater Branch Facility, Miyazaki Prefectural Fisheries Research Institute, Miyazaki, Japan
Taste buds, end organ of gustatory sense in vertebrates, were constitutes with elongated support cells and taste
cells in mammals. Some of the taste cells are serotonin immunopositive. Immature cells, which will become to
the taste cells, were localized in basal region of the taste buds. In contrast, it is known that amphibian and teleost
fish have matured cells on the base of their taste buds. These basal cells are serotonin immunopositive. In some
teleost fish including catfish, taste buds are distributed not only in gustatory organs but also in non-gustatory
organs. Existence of serotonin immunopositive basal cells of the taste buds in the non-gustatory organs is not
well understood. In this study, first we investigated the basal cells of the taste buds in sea catfish, Plotosus
japonicus . The serotonin immunopositive basal cells of the taste buds were observed in both gustatory organs,
including the barbels and palatal organ, and non-gustatory organs including fins and trunk region. Most of taste
buds of Plotosus had 1 basal cell with minor exception. These results suggest that there are no regional
differences about existence of the serotonergic basal cells in the taste buds in Plotosus . Next, to evaluate
diversity and evolution of these cells, we observed cellular organization of the taste buds in ancient species of
ray fined fish, including gars, sturgeons, and bichirs. In these fishes, serotonin immunopositive basal cells were
also observed in the taste buds. Shape and position of basal cells in these fish were similar to those in Plotosus .
However, numbers of basal cells of each taste bud were 2-5 in gars, 2-6 in sturgeons, and over 9 in bichirs.
These results suggest that serotonergic basal cells of taste buds are common in ray fined fish and may have
functional significance in ancestral species than in teleost.
P3-114 Visualization of taste buds reveals sensory degradation in obese mice
Kinya Seo, Asuka Sakata, Satoshi Nishimura
Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan
Taste sense in obesity can be altered, which may in turn affect food consumption. To assess this hypothesis, we
developed a technique based on in vivo two-photon microscopy to visualize three-dimensional micromorphology of intact taste buds with functional taste perceptions and surrounding blood circulation in obese and
lean mice. The obese mice treated with a high fat diet showed remarkable decline of calcium fluorescent signals
(GCaMP3 or Rhod2-AM) in taste buds in response to sugar and umami stimulations, which accompanied a
change in the flow rate of blood circulation. Behavioral observation of food intake showed that obese mice had
2016.05.27 17.38.25 Page 46(1)
reduced ability to distinguish these tastants compared with lean mice. These results are supported by molecular
analyses in which expression levels of some taste receptors and their downstream signaling were significantly
downregulated. In conclusion, we demonstrate that diet-induced obesity deteriorates taste perception in taste
buds, which likely affect the amount of food intake, thus promote obesity in a vicious circle.
P3-115 Analysis of peptidergic signaling-related gene expression in RNA-seq data of individual type II
and III mouse taste cells
Brian C. Lewandowski, Sunil K. Sukumaran, Robert F. Margolskee, Alexander A. Bachmanov
Information from taste cells is sent to gustatory brain regions that underlie taste perception and help drive
feeding related behaviors. The body’s metabolic state is also a key driver of feeding related behaviors. There is
growing evidence that signaling pathways related to metabolic state and energy homeostasis, including
peptidergic hormones, can influence the activity of taste cells. Taste cells are known to produce and/or detect a
number of peptide hormones and neuropeptides, including glucagon-like peptide-1, cholecystokinin and
neuropeptide Y. However, a global survey of the genes and receptors for peptidergic signaling in individual taste
cells has not been conducted. In this study, we used next generation RNA sequencing (RNA-seq) of single taste
cells isolated from the circumvallate papillae of C57BL/6 wild-type and transgenic mice to examine and compare
the expression of peptidergic signaling-related genes in several subpopulations of taste cells: Tas1r3-expressing
type II taste cells, gustducin-expressing type II taste cells, and sour-responsive type III taste cells. Our single-cell
RNA-seq data met high quality control standards and accurately detected differential expression of known
marker genes for type II (e.g., Trpm5, PlcB2, Tas1r’s) and type III (e.g., Ncam1, Pkd2l1, Gad1) taste cells.
Analysis of peptidergic signaling-related genes revealed expression of both known and previously unreported
peptides and their receptors in taste cells. For example, we detected transcripts for adiponectin (Adipor1, Adipor
2) and thrombin (F2r, F11r) receptors in both type II and type III taste cells, for somatostatin receptor 3 (Sstr3) in
type II cells, for apelin (Apln) in gustducin-expressing and sour-responsive taste cells, and for apelin receptor
(Aplnr) in Tas1r3-expressing type II cells. Our analysis of peptidergic signaling-related gene expression should
greatly benefit the effort to understand the crosstalk between the peripheral sensory mechanisms of taste and
the internal signaling pathways associated with energy homeostasis and metabolic state.
P3-116 Gli3 acts as negative regulator of taste bud maintenance
Yumei Qin, Sunil K. Sukumaran
The hedgehog signaling pathway is a principal regulator of cell proliferation and differentiation in numerous
tissues and is known to be active in taste papillae and taste buds. Sonic hedgehog (shh) has established roles in
the regulation of fungiform development, but the downstream transcriptional effectors of hedgehog signaling in
taste organ maintenance are largely unknown. Gli3 is a key transcriptional effector in shh signaling pathway, but
its distribution in the adult tongue and its role in taste bud maintenance were previously unknown. We used RNA
sequencing of single taste cells and bioinformatics (transcriptomics) to determine that Gli3 was more highly
expressed in type II taste cells and stem cells than in type III taste cells. Quantitative RT-PCR and double
immunofluorescence microscopy showed that Gli3 was selectively expressed in T1r3-expressing type II taste
cells and Lgr-5-expressing taste stem cells, but not in 5-HT-expressing type III taste cells or Glast-expressing
type I taste cells. To determine what role Gli3 might have in taste cell function, we generated
Lgr5-EGFP-CreERT2, flox-p flanked Gli3 transgenic mice. Taste behavioral tests in Lgr5-EGFP CreERT2/Gli3 f/f
mice showed that in comparison to wildtype, Gli3 conditional knockout mice were more sensitive to sweet
compounds, less sensitive to bitter and sour compounds, and similarly responsive to umami and salty
compounds. Recording from the glossopharyngeal nerves showed that Gli3 conditional knockout mice are much
more responsive to sweet and bitter compounds than are wildtype mice, and similar responses to umami and
salty compounds, consistent with the results from behavioral tests. Moreover, in Gli3 conditional knockout mice,
significant increases were observed in the size of the taste buds and the number taste receptor cells per taste
bud. In sum our results suggest that Gli3 is an important negative regulator of taste bud maintenance.
Acknowledgements: Supported by NIDCD/NIH grant R01DC014105 to RFM.
2016.05.27 17.38.25 Page 47(1)
P3-117 Evaluation of individual bitterness sensitivities to phenylthiocarbamide and 6-npropylthiouracil associated with sex difference and self-reported bitterness sensitivity to grapefruit
Hiroki Saito, Michiko Yamaki, Yukiko Numakura, Maiko Iribe, Asumi Ishikura, Kunio Isono, Tomoko Goto,
Hitoshi Shirakawa, Michio Komai
Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
PTC (PhenylthiocarFbamide) and PROP (6-n-propylthiouracil) are the ligands of TAS2R38. It is well known that
the combination of three SNPs in TAS2R38 results in three major genotypes and can be a critical determinant of
bitterness sensitivities to PTC and PROP: AVI/AVI (non-taster), AVI/PAV (medium-taster) and PAV/PAV
(super-taster). However, factors causing difference of individual bitterness sensitivities to PTC and PROP are
not well understood. In this study, we attempted to explore the new factors affecting individual bitterness
sensitivities to PTC/PROP except for TAS2R38. Bitterness sensitivities to PTC/PROP were evaluated at a single
concentration with gLMS method. SNP analysis was conducted sequencing target DNA sites with PCR and
direct sequence. Healthy university students (age: 20-25 years old, 112 male and 205 female) attended this
study as subjects. Analyzing the relationships between bitterness sensitivities to PTC/PROP and questionnaires
about dietary habit and preference, we found that sex difference and self-reported bitterness sensitivity to
grapefruit had a relation to bitterness sensitivities to PTC/PROP. In addition, PROP-sensitivity was more
influenced by sex difference effect than PTC-sensitivity though self-reported bitterness sensitivity showed a
stronger correlation with PTC-sensitivity than PROP-sensitivity. In order to exclude the contribution of TAS2R38,
we first divided subjects into three groups by TAS2R38 SNP genotype and then subdivide them by another
factor. As a result, only among AVI/PAV subjects, bitterness sensitivities to PTC/PROP and self-reported
bitterness sensitivity to grapefruit showed strongly significant correlation; subjects who had higher bitterness
sensitivity to grapefruit showed higher bitterness sensitivities to PTC/PROP. This result indicates the possibility
that factors affecting the bitterness sensitivity to grapefruit also affect the bitterness sensitivities to PTC/PROP.
P3-118 Contribution of bitter receptor TAS2Rs and Glutathione S-transferases on preference and
bitterness of the cruciferous vegetables
Michiko Yamaki, Hiroki Saito, Kunio Isono, Tomoko Goto, Hitoshi Shirakawa, Michio Komai
Graduate School of Agricultural Science,Tohoku University, Sendai, Japan
Individual differences in the bitter taste sensitivity and preference to cruciferous vegetables are known to be
associated with disease. Cruciferous vegetables are suggested to reduce risk for cancer since they contain
isothiocyanates that activate the metabolic process by detoxification enzyme glutathione S-transferase (GST).
Null variations in GSTM1 and GSTT1 have been shown to be associated with diseases. Bitter-taste reception by
type 2 taste receptors (TAS2Rs) is another important defense mechanism against food toxins as it helps avoid
the intake of harmful substances before they are metabolized by detoxification systems. Variations in some TAS
2Rs have been shown to be associated with the taste sensitivity to natural and artificial bitter substances. In this
study we investigated how genetic variations in TAS2Rs and GSTs affect bitter taste and preference to
cruciferous vegetables by questionnaires and taste sensory tests using Japanese subjects. TAS2R genotypes
were analyzed by direct sequencing. Null variations of GSTM1 and GSTT1 were analyzed by gel electrophoresis
after amplifying the genomic region uncovering the deletion. About twenty per cent of the subjects were PTC
non-tasters for TAS2R38 and the ratio was considerably smaller than those for Caucasian while about fifty per
cent of the subjects were GSTM1-null or GSTT1-null and the ratio was considerably greater than those for
Caucasian. No significant difference were found for the average bitterness sensitivity to cruciferous vegetables
among TAS2R38 genotypes in the sensory tests while bitterness of the vegetables obtained from questionnaires
is apparently dependent on both GSTM1 and GSTT1 genotypes with a tendency of higher bitterness evaluations
in subjects carrying GSTM1-null and GSTT1-null genotypes.
P3-119 The pilot study: Umami taste induced saliva secretion in Thai older adults
Nattida Chotechuang1, Matichon Lokkumlue2, Chidsanu Changsiripun3, Kanet Wongravee4, Chanida Palanuvej5
1
Department of Food Tecnology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand, 2Department of Nutrition and
Dietetics, Faculty of Allied Health Science, Chulalongkorn University, Bangkok, Thailand, 3Department of Orthodontics, Faculty of
Dentistry, Chulalongkorn University, Bangkok, Thailand, 4Department of Chemistry, Faculty of Science, Chulalongkorn University,
Bangkok, Thailand, 5College of Public Health Sciences, Chulalongkorn University, Bangkok, Thailand
Saliva serves as an important lubricant that protects the oral cavity, aids the formation of food boli and facilitates
swallowing and speech. However, hyposalivation and xerostomia or dry mouth, affecting appetite and nutrition,
2016.05.27 17.38.25 Page 48(1)
are common clinical problems among the elderly. This pilot study (N=25) aimed to investigate whether food
tastes and odor stimulated saliva secretion in older Thai adults. Healthy older adult volunteers held 3 ml of
solution for each of sour and umami tastes-having “moderate” taste intensity-for 30 seconds in their mouths or
instead smelled black pepper oil on filter paper for 30 seconds. Saliva was then collected every 30 seconds for
10 minutes by spitting, and measuring. Water stimulation and resting saliva were also measured. Based on the
results, umami taste stimulation significantly increased total saliva secretion and saliva flow rate within 10
minutes more than water stimulation and resting (p<0.05). No changes were observed with sour taste
stimulation and black pepper oil stimulation. The data suggest that food taste, and especially umami taste, could
improve saliva secretion in Thai elderly.
P3-120 Rapid expansion of phenylthiocarbamide (PTC) non-tasters among Japanese macaques
Nami Suzuki-Hashido1, Takashi Hayakawa1,2, Atsushi Matsui1, Yasuhiro Go3, Yoshiro Ishimaru4, Takumi Misaka4, Keiko Abe4,
Hirohisa Hirai1, Yoko Satta5, Hiroo Imai1
1
Primate Research Institute, Kyoto University, Aichi, Japan, 2Japan Monkey Centre, Aichi, Japan, 3Center for Novel Science
Initiatives, National Institute of Natural Sciences, Aichi, Japan, 4Graduate School of Agricultural and Life Sciences, The University of
Tokyo, Tokyo, Japan, 5Department of Evolutionary Studies of Biosystems, The Graduate University for Advanced Studies,
Kanagawa, Japan
Bitter taste receptors, TAS2Rs, allow mammals to detect and avoid ingestion of toxins in food. Thus, TAS2Rs
play an important role in food choice and are subject to complex natural selection pressures. To understand the
adaptive evolutional process of TAS2R in wild mammals, we investigated genetic variations of TAS2R38 , a
gene encoding bitter taste receptor for phenylthiocarbamide (PTC), in 594 Japanese macaques (Macaca
fuscata ) from 17 local populations. We identified a PTC non-taster TAS2R38 allele in Japanese macaques that
was caused by a loss of the start codon. Using cellular and behavioral experiments, we found that this allele lost
its receptor function for perceiving PTC. Interestingly, this PTC non-taster allele was only found in a limited local
population (the Kii area), at a frequency of 29%. To understand evolutional backgrounds of this allele, we
conducted TAS2R38 flanking analysis and population genetic analysis. The nucleotide sequences of the
non-taster allele including flanking regions (of about 10 kb) from 23 chromosomes were identical, suggesting
that a non-taster allele arose and expanded in the Kii population during the last 13,000 years. Genetic analyses
of non-coding regions in Kii individuals and neighboring populations indicated that the non-taster allele
frequency (29%) in the Kii population could not be explained by demographic history, suggesting that positive
selection resulted in a rapid increase in PTC non-tasters. The loss-of-function that occurred at the TAS2R38
locus presumably provided a fitness advantage to Japanese macaques in the Kii population. Because TAS2R38
ligands are contained in cruciferous and citrus plants which are digested by Japanese macaques, this functional
change is perhaps related to feeding habit specificity.
P3-121 Comprehensive structural analysis of halogenated sucrose derivatives
Zetryana Puteri Tachrim, Lei Wang, Yasuyuki Hashidoko, Makoto Hashimoto
Graduate School of Agriculture, Hokkaido University, Sapporo, Japan
The halogenated sucrose derivatives at the primary positions are known as important synthesis intermediate,
particularly for chlorinated products, commonly used as artificial sweetener. To date, Appel reaction with carbon
tetrahalides and triphenylphosphine is one of the famous reagents for selective halogenation only on sucrose
primary hydroxyl groups. In general, the reactivity of sucrose primary alcohols toward halogenation is
comparatively followed the order of 6 and/or 6’-, then 1’ position. The selectivity has been reported over two
decades ago. Monohalogenation has been archived with limited amounts of Appel reagents, but selectivity has
not been identical for each manuscripts. We reconsider structural elucidation for monohalogenated sucrose
derivatives with 1D and 2D NMR. The monohalogenated sucroses were prepared with Appel reagents on 6 and
6’ position, which cannot be separated with chromatography. The mixture was subjected to acetylaion to
separate with column chromatography in diether ether-hexane systems. The separation cannot be archived in
ethyl acetate or dichloromethane systems. The purified mono-halogenated sucrose derivative was subjected to
detail H-NMR analysis. The peracetylated monohalo derivatives on 6 position can be observed geminal
coupling. On the other hand, the peracetylated monohalo derivatives on 6’position cannot be detected geminal
coupling. 1’ monohalogenated sucrose was also prepared by organic synthesis. The identifications for three
monohalogenated sucrose derivatives were confirmed with 2D NMR. Comprehensive structural elucidations for
monohalogenated sucrose dissolve the confused results for selective reactivity on sucrose primary alcohols.
2016.05.27 17.38.25 Page 49(1)
P3-122 Evaluation of Japanese sake taste with metal goblets using taste sensors
Misaki Orihara1, Kazuyuki Koide2, Yo Ohta1, Ganzaya Perenlei3, Masatoshi Kubota1, Motoni Kadowaki1, Shinobu Fujimura1
1
Graduate School of Science and Technology, Niigata University, Niigata, Japan, 2Food Science Center, Niigata University, Niigata,
Japan, 3Niigata Agriculture Research Institute Livestock Research Center, Niigata, Japan
Objectives: Niigata Prefecture is famous in its high quality Japanese sake and metal tableware production in
Japan. Generally, pottery tablets were used in Japan. While, drinking sake in metal goblet feels milder taste than
glasser porcelain, but there was no scientific evidence. Therefore, in this study, we compared tastes of
Japanese sake between stored in metal goblet and pottery goblet using the taste sensor and sensory evaluation.
Materials and Methods: Experiment 1: five basic tastes and richness of 16 brands Japanese sake in Niigata
were evaluated by taste sensor with 5 sensor probes (AAE, CA0, CT0, C00 and AE1) and BT0 sensor probe.
Experiment 2: Japanese sake was stored in 5 kinds of goblet (tin, tin and copper-plated metal, gold-plated metal,
titanium or pottery) for 10 minutes and were mearsured by taste sensor. Furthermore, in order to elucidate the
reason of taste differences, we measured sake pH and electric conductivity. After that, we perforemed sensory
evaluation of Japanese sake between pottery goblet and tin goblet. Sensory panel member was 24 trained man
and women who 20s age. Results and discussions: As a result of experiment 1, 16 brands can be classified
depending on the intensity of sour and salty tastes. Experiment 2: Differences in aftertaste was observed in
metal goblets, however, there were no differences in sake pH and electrical conductivity. These results suggest
that aftertaste is one of the factors in metal goblets with Japanese sake.
P3-123 Generation time of cells in each subnucleus of the brainstem gustatory relay nuclei in rats
Takeshi Suwabe, Toshiaki Yasuo, Noritaka Sako
Department of Oral Physiology, Division of Oral Functional Sciences and Rehabilitation, School of Dentistry, Asahi University, Gifu,
Japan
In the central taste pathway, the rostral nucleus of the solitary tract (rNST) is the first relay located in the medulla.
In rodents and lagomorphs, the rNST conveys gustatory information to the parabrachial nucleus (PBN) in the
pons. These gustatory relay nuclei are reportedly composed of cells generated between embryonic day (E) 11
and E14 in rats. However, little is known about distribution patterns of E11-, E12-, E13- and E14-generated cells
in the nuclei. We have investigated percentages of the cells in each subnucleus of the rNST and PBN. To label
cells generated at E11, E12, E13 and E14, a thymidine analog 5-ethynyl-2’-deoxyuridine (EdU) was
intraperitoneally injected into pregnant dams at gestational day 11, 12, 13 or 14. EdU-labeled cells in the rNST
and PBN were counted in confocal images of brainstem slices made from pups at P21-P25 and P80-90. E11and E12-generated cells are differentially distributed in the rNST and PBN. In these nuclei, the percentage of E
11-generated cells becomes high in the region reported to be responsive to mechanical stimuli to the oral cavity
(the rostral lateral and the external lateral/external medial subnuclei in the rNST and PBN, respectively),
whereas there are many E12-generated cells in the region known to be responsive to taste stimuli (the rostral
central and the central medial/ventral lateral subnuclei in the rNST and PBN, respectively). This result indicates
the possibility that cellular distribution and function in the rNST and PBN are determined by generation time of
cells.
P3-124 Taste-modulator effect of the yeast extract derived from Candida Utilis -III: Suppression effect
of bitterness by the addition of the fraction from the yeast extract
Toshihide Nishimura1, Yuji Ohno1, Shinnosuke Furuya1, Ai Egusa1, Yoshie Yasumatsu2, Kenichi Ason2, Sakiko Ikeda2,
Hirotaka Yamashita2
1
Department of Applied Biological Science, Nippon Veterinary and Life Science University, Tokyo, Japan, 2KOHJIN Life Science
Co. Ltd., Japan
We found that the addition of yeast extract derived from Candida Utilis to basic tastes can modulate the taste
sensation. The intensity of bitterness has been suppressed by the addition of this yeast extract to bitter solution.
In this research, we examined the effect of bitterness suppression by the fraction from yeast extract using
sensory evaluation and the calcium imaging method with STC1 (JCRB1053) cells in which bitter taste receptor
was expressed. Yeast extract derived from Candida Utilis was fractionated to five fractions such as the fraction I
(MW more than 13000), the fraction II (MW 13000-6000), the fraction III (MW 6000-3000), the fraction IV (MW
3000-1000), and the fraction V (MW less than 1000) by membrane filtration. A trained 12 panelists conducted
sensory evaluation for paired comparison tests at room temperature. About 30 mL of sample solution was
served at 25 degrees Celsius to panelists. They evaluated each taste profile such as intensity in maximum taste,
2016.05.27 17.38.25 Page 50(1)
top taste, after taste, and overall taste of 0.001% quinine solution. The yeast extract prepared was added to
each standard solution at 0.1% of final concentration. All statistical treatments were conducted using the method
reported by Roessler et al. As a result, it was found that the intensity in maximum, top or overall taste of
bitterness was significantly suppressed by the addition of the fraction II or IV derived from the yeast extract to
quinine solution. The analyses by calcium imaging method with STC1 cells also showed that the addition of
these fractions to quinine solution reduced the response of cells by quinine solution. This result corresponded to
the suppression effect by the fraction II or IV on bitterness in sensory evaluation.
P3-125 Taste-modulator effect of the yeast extract derived from Candida Utilis -II: Enhancement effect
of sweetness by the addition of the fraction from the yeast extract
Sakiko Ikeda1, Yuji Ohno2, Ai Egusa2, Yoshie Yasumatsu1, Kenichi Ason1, Hirotaka Yamashita1, Toshihide Nishimura2
1
KOHJIN Life Science Co.,Ltd, Japan, 2Nippon Veterinary and Life Science University, Tokyo, Japan
We found that the addition of yeast extract derived from Candida Utilis to basic tastes can modulate the taste
sensation. The intensity of bitterness has been suppressed by the addition of this yeast extract to bitter solution.
In this research, we examined the effect of bitterness suppression by the fraction from yeast extract using
sensory evaluation and the calcium imaging method with STC1 (JCRB1053) cells in which bitter taste receptor
was expressed.
Yeast extract derived from Candida Utilis was fractionated to five fractions such as the fraction I (MW more
than 13000), the fraction II (MW 13000- 6000), the fraction III (MW 6000-3000), the fraction IV (MW 3000-1000),
and the fraction V (MW less than 1000) by membrane filtration. A trained 12 panelists conducted sensory
evaluation for paired comparison tests at room temperature. About 30 mL of sample solution was served at 25
degrees Celsius to panelists. They evaluated each taste profile such as intensity in maximum taste, top taste,
after taste, and overall taste of 0.001% quinine solution. The yeast extract prepared was added to each standard
solution at 0.1% of final concentration. All statistical treatments were conducted using the method reported by
Roessler et al.
As a result, it was found that the intensity in maximum, top or overall taste of bitterness was significantly
suppressed by the addition of the fraction II or IV derived from the yeast extract to quinine solution. The analyses
by calcium imaging method with STC1 cells also showed that the addition of these fractions to quinine solution
reduced the response of cells by quinine solution. This result corresponded to the suppression effect by the
fraction II or IV on bitterness in sensory evaluation.
P3-126 Taste-modulator effect of the yeast extract derived from Candida Utilis -I: The effect on five
basic tastes by addition of the yeast extract
Yoshie Yasumatsu1, Yuji Ohno2, Ai Egusa2, Kenichi Ason1, Sakiko Ikeda1, Hirotaka Yamashita1, Toshihide Nishimura2
1
KOHJIN Life Science Co.,Ltd., Japan, 2Nippon Veterinary and Life Science University, Tokyo, Japan
Yeast extract is well known to be a functional material used for seasonings, supplement, pharmaceutical drug,
and cosmetics. Recently, it was found that the addition of yeast extract derived from Candida Utilis was able to
enhance the intensity of sweetness in dairy products. However, there is few research on the compound involved
in this effect and the mechanism for taste modulation. In this research, we inves

ISOT2016 17th International Symposium on Olfaction and Taste

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