Zebrafish Behavioral Profiling Links Drugs to Biological Targets and

Transcription

REPORTS
formatics, Indiana University, Bloomington, IN 47405, USA.
Department of Computer Science, University of Illinois at
Urbana-Champaign, Urbana, IL 61801, USA. 47Department of
Structural Biology and Bioinformatics, University of Geneva
Medical School, CH-1211 Geneva, Switzerland. 48Research
School of Biology, Australian National University, Canberra,
Australian Capital Territory 2601, Australia. 49Roy J. Carver
Center for Comparative Genomics and Department of Biology,
University of Iowa, Iowa City, IA 52242, USA. 50Department of
Genetics and Biochemistry, Clemson University, Clemson, SC
29634, USA. 51Institute of General Zoology, University of Jena,
D-7743 Jena, Germany. 52Faculdade de Medicina de Ribeirão
Preto, Departamento de Genética, Universidade de São Paulo,
Ribeirão Preto, São Paolo 14049-900, Brazil. 53Department of
Entomology, Commonwealth Scientific and Industrial Research
Organisation, Canberra, Australian Capital Territory 2601,
Australia. 54Institute of Evolutionary Biology–School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JT, UK.
55
Vector Group, Liverpool School of Tropical Medicine, Liverpool
L3 5QA, UK. 56Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, Santa
Cruz, CA 95064, USA. 57Reese Consulting, 157/10 Tambon Ban
Deau, Amphur Muang, Nong Khai, 43000, Thailand. 58Department of Biology, Western Washington University, Bellingham,
WA 98225, USA. 59Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. 60Department
of Organismic and Evolutionary Biology, Harvard University,
Cambridge, MA 02138, USA. 61School of Marine and Tropical
Biology and Centre for Comparative Genomics, James Cook
University, Townsville, Queensland 4811, Australia. 62Department of Evolutionary Biology and Animal Ecology, University of
Freiburg, 79104 Freiburg, Germany. 63School of Biology, University of St Andrews, St Andrews KY16 9TH, UK. 64Department
of Computer Science, Royal Holloway, University of London,
46
Zebrafish Behavioral Profiling Links
Drugs to Biological Targets and
Rest/Wake Regulation
Jason Rihel,1*† David A. Prober,1*‡ Anthony Arvanites,2 Kelvin Lam,2
Steven Zimmerman,1 Sumin Jang,1 Stephen J. Haggarty,3,4,5 David Kokel,6
Lee L. Rubin,2 Randall T. Peterson,3,6,7 Alexander F. Schier1,2,3,8,9†
A major obstacle for the discovery of psychoactive drugs is the inability to predict how small
molecules will alter complex behaviors. We report the development and application of a
high-throughput, quantitative screen for drugs that alter the behavior of larval zebrafish. We found
that the multidimensional nature of observed phenotypes enabled the hierarchical clustering of
molecules according to shared behaviors. Behavioral profiling revealed conserved functions of
psychotropic molecules and predicted the mechanisms of action of poorly characterized compounds.
In addition, behavioral profiling implicated new factors such as ether-a-go-go–related gene (ERG)
potassium channels and immunomodulators in the control of rest and locomotor activity. These
results demonstrate the power of high-throughput behavioral profiling in zebrafish to discover and
characterize psychotropic drugs and to dissect the pharmacology of complex behaviors.
M
ost current drug discovery efforts focus
on simple in vitro screening assays.
Although such screens can be successful, they cannot recreate the complex network
interactions of whole organisms. These limitations are particularly acute for psychotropic drugs
because brain activity cannot be modeled in vitro
(1–3). Motivated by recent small-molecule screens
that probed zebrafish developmental processes
(4–7), we developed a whole organism, highthroughput screen for small molecules that alter
larval zebrafish locomotor behavior. We used an
348
automated rest/wake behavioral assay (3, 8) to
monitor the activity of larvae exposed to small
molecules at 10 to 30 mM for 3 days (Fig. 1A)
(3). Multiple behavioral parameters were measured, including the number and duration of rest
bouts, rest latency, and waking activity (i.e., activity not including time spent at rest) (Fig. 1B)
(3). We screened 5648 compounds representing
3968 unique structures and 1680 duplicates and
recorded more than 60,000 behavioral profiles.
Of these, 547 compounds representing 463 unique
structures significantly altered behavior relative
15 JANUARY 2010
VOL 327
SCIENCE
Egham, Surrey TW20 0EX, UK. 65Institut für Mikrobiologie und
Genetik, Universität Göttingen, 37077 Göttingen, Germany.
66
Division of Insect Sciences, National Institute of Agrobiological Science, Tsukuba, Ibaraki 305-8634, Japan. 67Department of Biology and Biocenter Oulu, University of Oulu, 90014
Oulu, Finland. 68Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717, USA.
69
Department of Cellular Biology, University of Georgia, Athens,
GA 30602, USA. 70Department of Biotechnology, Chemistry,
and Food Science, Norwegian University of Life Sciences, N-1432
Ås, Norway.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail: werr@mail.rochester.edu (J.H.W.); stephenr@bcm.tmc.edu (S.R.)
‡Current address: Verhaltensbiologie, Universität Osnabrück,
49076 Osnabrück, Germany.
§Current address: Departament de Genètica i de Microbiologia,
Universitat Autònoma de Barcelona, 8193 Bellaterra, Spain.
||Current address: Weill Cornell Medical College, New York, NY
10065, USA.
¶Current address: Department of Epidemiology, University of
Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Supporting Online Material
www.sciencemag.org/cgi/content/full/327/5963/343/DC1
Materials and Methods
SOM Text
Figs. S1 to S25
Tables S1 to S57
References
22 June 2009; accepted 24 November 2009
10.1126/science.1178028
to controls, according to a stringent statistical
cutoff (3).
Because the alterations in behavior were multidimensional and quantitative, we assigned a
behavioral fingerprint to each compound and
applied clustering algorithms to organize molecules according to their fingerprints (Fig. 2A
and figs. S1 to S3). This analysis organized the
data set broadly into arousing and sedating compounds and identified multiple clusters corresponding to specific phenotypes (Fig. 2, B to
F; Fig. 3, A to C; Fig. 4, B and C; and figs. S1
to S4). Clustering allowed us to address three
questions: (i) Do structural, functional, and behavioral profiles overlap? (ii) Does the data
set predict links between known and unknown
small molecules and their mechanisms of action? (iii) Does the data set identify unexpected
1
Department of Molecular and Cellular Biology, Harvard
University, Cambridge, MA 02138, USA. 2Harvard Stem
Cell Institute, Harvard University, Cambridge, MA 02138,
USA. 3Broad Institute of MIT and Harvard, Cambridge, MA
02142, USA. 4Stanley Center for Psychiatric Research,
Broad Institute of MIT and Harvard, Cambridge, MA 02142,
USA. 5Center for Human Genetic Research, Massachusetts
General Hospital, Boston, MA 02114, USA. 6Developmental
Biology Laboratory, Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA.
7
Department of Medicine, Harvard Medical School, Boston,
MA 02115, USA. 8Division of Sleep Medicine, Harvard
Medical School, Boston, MA 02215, USA. 9Center for Brain
Science, Harvard University, Cambridge, MA 02138, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
schier@fas.harvard.edu (A.F.S.); rihel@fas.harvard.edu (J.R.)
‡Present address: Division of Biology, California Institute
of Technology, Pasadena, CA 91125, USA.
www.sciencemag.org
Downloaded from http://science.sciencemag.org/ on May 11, 2016
Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Universidade de São Paulo, Ribeirão Preto, São Paolo
14040-901, Brazil. 26Department of Biological Sciences,
Vanderbilt University, Nashville, TN 37235, USA. 27Josephine
Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, MA 02536,
USA. 28European Molecular Biology Laboratory, 69117
Heidelberg, Germany. 29Institute for Evolution and Biodiversity,
University of Münster, 48143 Münster, Germany. 30Laboratory
of Zoophysiology, Ghent University, B-9000 Ghent, Belgium.
31
The Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia. 32Department of
Microbiology and the Center for Computational Biology, Montana State University, Bozeman, MT 59715, USA. 33Instituto de
Física de São Carlos, Departamento de Física e Informática,
Universidade de São Paulo, São Carlos, São Paolo 13560-970,
Brazil. 34Subtropical Insects Research Unit, United States Department of Agriculture–Agricultural Research Service (USDAARS), U.S. Horticultural Research Lab, Fort Pierce, FL 34945,
USA. 35School of Biological Sciences, University of Liverpool,
Liverpool L69 7ZB, UK. 36School of Biology, Georgia Institute of
Technology, Atlanta, GA 30332, USA. 37Department of Biology
and Biochemistry, University of Houston, Houston, TX 77204,
USA. 38Bee Research Lab, USDA-ARS, Beltsville, MD, 20705,
USA. 39Australian Research Council Centre of Excellence for
Coral Reef Studies, James Cook University, Townsville, Queensland 4811, Australia. 40Department of Biology, Indiana University, Bloomington, IN 47405, USA. 41Institute of Physiology,
Rheinisch-Westfaelische Technische Hochschule (RWTH) Aachen
University, D-52074 Aachen, Germany. 42Department of Molecular Biology, Umeå University, S-901 87 Umeå, Sweden.
43
Department of Entomology, University of Nebraska, Lincoln,
NE 68583, USA. 44Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya 464-8601, Japan. 45School of In-
candidate pathways that regulate rest/wake
states?
Cluster analysis revealed several lines of evidence that molecules with correlated behavioral phenotypes often shared annotated targets or
therapeutic indications (Fig. 2, B to F, and figs.
S1 to S4). First, drug pairs were more likely to
be correlated if the compounds shared at least
one annotated target (median correlation when
sharing one target, 0.561 versus 0.297 when
sharing zero targets; fig. S5, A and B). Second,
analysis of 50 different structural and therapeutic classes revealed that drugs belonging to the
same class produced highly correlated behaviors in nearly all cases (fig. S5C and fig. S6)
(3). For example, several structurally diverse
selective serotonin reuptake inhibitors (SSRIs)
similarly reduced waking, and sodium channel
agonist insecticides induced large increases in
waking activity (fig. S5C and fig. S6). Third,
behavioral profiling uncovered the polypharmacology of drugs with multiple targets. For
example, the profile of the dopamine reuptake
inhibitor and muscarinic acetylcholine receptor
antagonist 3a-bis-(4-fluorophenyl) methoxytropane correlated only with drugs that also shared
both properties, such as the anti-Parkinson’s drug
trihexyphenidyl (fig. S7) (3). Fourth, modulators
of the major neurotransmitter pathways often
induced locomotor and rest/wake effects in zebrafish larvae similar to those seen in mammals (figs.
S8 to S15) (3). For example, a2-adrenergic receptor agonists (e.g., clonidine) were sedating, whereas
b-adrenergic agonists (e.g., clenbuterol) were arousing, as in mammals (fig. S8). These analyses
indicate that compounds with shared biological
targets yield similar and conserved phenotypes in
our high-throughput behavioral profiling.
Detailed analyses revealed that the clustering
of well-known and poorly characterized drugs
could predict targets for compounds whose mode
of action has been unclear (Fig. 3). For example,
Cl
O
O
Cl
Cl
Cl
Cl
NH
O
N
Cl
A
Cl
Cl
O Cl
HO
ing infection, a role for the immune system in
normal vertebrate sleep/wake behavior has not
been described (14). Behavioral profiling revealed that a diverse set of anti-inflammatory
compounds increased waking activity during the
day with much less effect at night (Fig. 4B and
fig. S17). These anti-inflammatory compounds
included the steroidal glucocorticoids, the nonsteroidal anti-inflammatory drugs (NSAIDs),
phosphodiesterase (PDE) inhibitors, and other
compounds with anti-inflammatory properties,
including the immunosuppressant cyclosporine
and the mood stabilizer valproic acid. Taken
together, these data suggest that inflammatory
signaling pathways not only induce sleep during
infection (14) but also play a role in setting
normal daytime activity levels.
4) Ether-a-go-go–related gene (ERG) potassium channel blockers selectively increased waking activity at night without affecting total rest
(Fig. 4C and fig. S18). This phenotype was induced by compounds with divergent therapeutic
indications (e.g., the antimalarial halofantrine,
the antipsychotic haloperidol, the antihistamine
terfenadine); however, these drugs also inhibit
the ERG channel and can cause the heart rhythm
disorder long QT syndrome (15, 16). Rank-sorting
all the screened compounds by their fingerprints’ mean correlation to the ERG-blocking
cluster resulted in a significant enrichment of
known ERG blockers in the top ranks (Fig. 4D).
Moreover, the specific ERG inhibitor dofetilide
increased nighttime activity, whereas structurally
related non-ERG blocking compounds, including the antihistamines fexofenadine and cetirizine,
did not (fig. S18B). Finally, this phenotype was
not caused by general misregulation of potassium
channels, because psora-4, a drug that blocks the
related shaker potassium channel Kv1.3, induced
a distinct phenotype (fig. S18A). These results
suggest that ERG potassium channels play a role
in regulating wakefulness at night that is distinct
N
H
Cl
NH2 O
HO
4 dpf
B6
Drug 1
Drug 2
Drug 3
Drug 4
Drug 5
Drug 6
Drug 7
Drug 8
Activity (seconds/minute)
Fig. 1. Larval zebrafish locomotor activity assay. (A) At 4 days post-fertilization
(dpf), an individual zebrafish larva is pipetted into each well of a 96-well plate
with small molecules. Automated analysis
software tracks the movement of each
larva for 3 days. Each compound is tested
on 10 larvae. (B) Locomotor activity of a
representative larva. The rest and wake
dynamics were recorded, including the
number and duration of rest bouts [i.e., a
continuous minute of inactivity (8)], the
timing of the first rest bout after a light
transition (rest latency), the average waking activity (average activity excluding rest
bouts), and the average total activity. Together, these measurements generate a
behavioral fingerprint for each compound.
the pesticide amitraz coclustered with a2-adrenergic
agonists (Fig. 3A), consistent with reports that
amitraz causes clonidine-like side effects in mammals and binds to a2-adrenergic receptors (9).
Similarly, sinapic acid methyl ether coclustered
with N-methyl-D-aspartate (NMDA) receptor antagonists (Fig. 3B), which suggests that the mild
anxiolytic effect of sinapic acid in mice is due
to NMDA receptor antagonism rather than gaminobutyric acid (GABA) receptor activation,
as proposed (10). Indeed, several sinapic acid
analogs are known to block NMDA-induced excitotoxicity in vitro (11, 12). Finally, MRS-1220,
an adenosine A3 receptor antagonist (13), clustered with monoamine oxidase (MAO) inhibitor
antidepressants (Fig. 3C). To directly test whether
MRS-1220 inhibits MAO, we performed an in
vitro activity assay and found a median inhibitory
concentration (IC50) of ~1 mM (Fig. 3D). Thus,
behavioral profiling in zebrafish larvae can predict and identify targets of poorly characterized
compounds.
In addition to revealing a conserved neuropharmacology between zebrafish and mammalian
rest/wake states (figs. S8 to S15) (3), behavioral
profiling identified additional pathways involved
in rest/wake behaviors:
1) L-type calcium channel inhibitors of the
verapamil class increased rest with minimal effects on waking activity (fig. S16). This is likely
a direct effect on rest regulation, because average waking activity and associated muscle activity were unaffected.
2) Cluster analysis identified two structurally
related podocarpatrien-3-ones that specifically increased rest latency (Fig. 4A). These and other
compounds also revealed that total rest, rest latency, and waking activity can be disassociated,
indicating that these processes can be regulated
by distinct mechanisms (3).
3) Although inflammatory cytokine signaling has long been known to promote sleep dur-
SCIENCE
VOL 327
Rest Latency
(17 minutes)
5
Active
ActiveBout
Bout
(4
(4 minutes)
minutes)
4
3
Rest Bout
(6 minutes)
2
Lights out
1
0
www.sciencemag.org
REPORTS
0
5
15 JANUARY 2010
10
15
20
25
Minutes
30
35
40
349
Rest
O
3.5
3
2.5
2
1.5
1
0.5
0
5
4
2
0
20
25
30
35
40
45
H
N
O
OH
55
O
44
33
22
Normalized Rest
Normalized Waking Activity
3C
15
C L -701324
2C/3B
2E
10
Cl
2B /4B
2D
5
11
00
D DDT
Cl
Cl
Cl
55
5
4
44
Cl
33
3
Cl
22
2
11
1
00
0
F
F
3.5
3
2.5
2
1.5
1
0.5
0
3.5
3
2.5
2
1.5
1
0.5
0
F
E Halofantrine
55
3.5
3
2.5
2
1.5
1
0.5
0
Cl
44
N
33
OH
Cl
22
11
00
F Methoxyverapamil
O
5
3A
2
O
N
O
O
3
O
N
-3
-2
-1
0
1
2
3
1
0
D
100
alpha2 agonist
alpha2 agonist
alpha2 agonist
not annotated
alpha2 agonist
alpha2 agonist
alpha2 agonist
alpha2 agonist
alpha2 agonist
L-701324
NMDA antagonist
L-701324
NMDA antagonist
L-701324
NMDA antagonist
Sinapic acid methyl ether not annotated
Dizocilpine
NMDA antagonist
Dizocilpine
NMDA antagonist
Dizocilpine
NMDA antagonist
C
MRS-1220
Phenelzine
R(+)-UH-301
Phenelzine
Tranylcypromine
VOL 327
MRS-1220
80
Pargyline
60
40
20
0
1E10-9
1E10-8
1E10-7
1E10-6
1E10-5
Log [Inhibitor] (M)
A3 receptor inhibitor
MAO inhibitor
5HT-receptor agonist
MAO inhibitor
MAO inhibitor
demonstrates a conserved vertebrate neuropharmacology, and identifies regulators of rest/wake
states. Our findings have two major implications for the fields of neurobiology, pharma-
15 JANUARY 2010
Time
Target
Name
Guanabenz
Guanabenz
Guanabenz
Amitraz
Guanfacine
Clonidine
Guanfacine
Guanabenz
UK 14304
B
3.5
3
2.5
2
1.5
1
0.5
0
120
MAO-B activity (%)
Waking
Activity
Activity
Total
Time
Rest
Latency
# Rest
Bouts
Rest
Total
A
2F
4
SCIENCE
cology, and systems biology. First, behavioral
profiling has the potential to complement traditional drug discovery methodologies by combining the physiological relevance of in vivo
www.sciencemag.org
Waking
Activity
B Valproate
-O
1
from the role of shaker channels in regulating sleep
in flies and mice (17, 18).
As applied here, behavioral profiling reveals
relationships between drugs and their targets,
350
Waking Activity
3
S.D.
Fig. 3. Predicting primary and secondary biological targets for poorly
characterized compounds.
(A) The pesticide amitraz coclusters with a2adrenergic agonists. (B)
Sinapic acid methyl ether
coclusters with NMDA antagonists. (C) MRS-1220
coclusters with MAO inhibitors. (D) MRS-1220
inhibits MAO-B activity
in an enzymatic assay with
an IC50 of ~1 mM. Pargyline is a known MAO-B
inhibitor (19). The clusters
include repeats from different chemical libraries.
Activity Total
Rest Total
A
Rest Bout
Length
Fig. 2. Hierarchical clustering reveals the diversity
of drug-induced behaviors.
(A) Behavioral profiles are
hierarchically clustered to
link compounds to behaviors. Each square of the
clustergram represents the
average relative value in
standard deviations (yellow, higher than controls;
blue, lower than controls)
for a single behavioral
measurement. Dark bars
indicate specific clusters
analyzed in subsequent
figures. (B to F) Normalized waking activity and
rest graphs are plotted
for behavior-altering compounds (red trace; average of 10 larvae) and
representative controls
(10 blue traces; average
of 10 larvae each). Compounds that altered behavior include the mood
stabilizer and antiepileptic drug sodium valproate
(B), the psychotomimetic NMDA antagonist L701324 (C), the sodium
channel agonist pesticide
DDT (D), the antimalarial
halofantrine (E), and the
calcium channel blocker
methoxyverapamil (F).
# Rest Bouts
Rest Bout
Length
Rest Latency
REPORTS
Night
DMSO 1
O
O
DMSO 2
O
O
Fig. 4. Unexpected regulators of zebrafish
rest/wake states. (A) Podocarpatrien-3-one
analogs increase rest latency, the time from
light transition to the first rest bout, relative to controls. Error bars represent SEM.
(B) Many wake-promoting anti-inflammatory
and immunomodulating compounds cocluster (blue, NSAIDs; green, glucocorticoids; pink,
PDE inhibitors; yellow, miscellaneous antiinflammatories; white, no anti-inflammatory C
annotation). See fig. S17 for an extended
list. (C) A cluster of ERG-blocking compounds specifically increases waking activity
at night. (D) Rank-sorting the data set by correlation to the ERG blocking
cluster results in a significant enrichment of ERG blockers in the top ranks
[P < 10–13 by the Kolmogorov-Smirnov statistic (3)]. Black lines indicate
known ERG blockers; red indicates high correlation, green indicates low
assays with high-throughput, low-cost screening (3). Future screens can be expanded to include many more uncharacterized compounds
and to assay additional phenotypes, including
those associated with human psychiatric disorders. In this way, behavioral profiling can
characterize large classes of compounds and reveal differences in effectiveness, potential side
effects, and combinatorial properties that might
not be detected in vitro. Second, behavioral profiling allows for the systematic dissection of
the pharmacology of complex behaviors. Our
screen profiled the effects of dozens of neurotransmitter pathways and identified small molecules that regulate discrete aspects of rest/wake
states. Future experiments can test drug combinations to identify synergistic or antagonistic effects among psychotropic compounds and to
build interaction maps. High-throughput behavioral profiling thus may enable application
of the logic and approaches of systems biology
to neuropharmacology and behavior.
Diflunisal
Piroxicam
Flecainide
Flunisolide
Atrazine
Capsazepine
Cyclosporin A
CONH
Oxethazaine
Spectinomycin
Tetrahydrotrimethylhispidin
Theaflavin
Equilin
SKF 94836
Aminophenazone
Diplosalsalate
Fosfosal
Clobetasol
Fenoprofen
Nicotine
(-)-Nicotine
Fenoprofen
5-aminopentanoic acid
Catechin tetramethyl ether
Pentamidine
Valproate
Betamethasone
Mefloquine
Clobetasol
Flumethasone
Desoxycorticosterone
Ethylene glycol analog
Dexamethasone
1
548
correlation to the ERG cluster. This analysis also detected potential indirect
regulators of ERG function, such as the organophosphate coumaphos
(marked with an asterisk), which causes long QT through an unknown mechanism (20).
References and Notes
SCIENCE
Name
Mean Correlation
Haloperidol
0.943
Halofantrine
0.943
Amperozide
0.929
Terfenadine
0.918
Clozapine
0.913
Promethazine
0.911
Mianserin
0.892
Oxatomide
0.884
Roxithromycin
0.868
Mianserin
0.866
Bepridil
0.865
Meclozine
0.859
0.858
Coumaphos
Desmethylclozapine 0.855
Amoxapine
0.839
Clemastine
0.833
Methiothepin
0.832
Astemizole
0.813
Clozapine
Halofantrine
Amperozide
Haloperidol
Terfenadine
1. Y. Agid et al., Nat. Rev. Drug Discov. 6, 189 (2007).
2. M. N. Pangalos, L. E. Schechter, O. Hurko, Nat. Rev.
Drug Discov. 6, 521 (2007).
3. See supporting material on Science Online.
4. R. T. Peterson, M. C. Fishman, Methods Cell Biol. 76, 569
(2004).
5. R. D. Murphey, H. M. Stern, C. T. Straub, L. I. Zon,
Chem. Biol. Drug Des. 68, 213 (2006).
6. T. E. North et al., Nature 447, 1007 (2007).
7. T. E. North et al., Cell 137, 736 (2009).
8. D. A. Prober, J. Rihel, A. A. Onah, R. J. Sung, A. F. Schier,
J. Neurosci. 26, 13400 (2006).
9. P. G. Jorens, E. Zandijk, L. Belmans, P. J. Schepens,
L. L. Bossaert, Hum. Exp. Toxicol. 16, 600 (1997).
10. B. H. Yoon et al., Life Sci. 81, 234 (2007).
11. A. Matteucci et al., Exp. Brain Res. 167, 641 (2005).
12. M. B. Wie et al., Neurosci. Lett. 225, 93 (1997).
13. K. A. Jacobson et al., Neuropharmacology 36, 1157
(1997).
14. L. Imeri, M. R. Opp, Nat. Rev. Neurosci. 10, 199 (2009).
15. U. Langheinrich, G. Vacun, T. Wagner, Toxicol. Appl.
Pharmacol. 193, 370 (2003).
16. E. Raschi, V. Vasina, E. Poluzzi, F. De Ponti, Pharmacol. Res.
57, 181 (2008).
17. C. L. Douglas et al., BMC Biol. 5, 42 (2007).
18. C. Cirelli et al., Nature 434, 1087 (2005).
www.sciencemag.org
Rank
1
2
3
5
7
8
11
13
21
24
26
33
* 34
36
43
47
48
59
VOL 327
19. C. J. Fowler, T. J. Mantle, K. F. Tipton, Biochem.
Pharmacol. 31, 3555 (1982).
20. E. Bar-Meir et al., Crit. Rev. Toxicol. 37, 279
(2007).
21. We thank J. Dowling, D. Milan, and G. Vanderlaan for
suggestions and reagents and D. Schoppik, G. Uhl, and
I. Woods for critical reading of the manuscript. Supported
by a Bristol-Myers Squibb postdoctoral fellowship of the
Life Sciences Research Foundation (J.R.), a Helen Hay
Whitney Foundation postdoctoral fellowship (D.A.P.), a
NIH Pathway to Independence grant (D.A.P.), the Stanley
Medical Research Institute (S.J.H.), the Harvard Stem Cell
Institute (L.L.R.), NIH grants MH086867 and MH085205
(R.T.P.), and grants from NIH and the McKnight
Endowment Fund for Neuroscience (A.F.S.). L.L.R. is a
founder of iPierian Inc., a biotechnology company, and is
a member of its scientific advisory board.
Day
D
Waking
Activity
Activity
Total
Rest
Latency
Rest Bout
Length
B
240
200
160
120
80
40
0
70
60
50
40
30
20
10
0
# Rest
Bouts
Rest Latency (Minutes)
A
Rest
Total
REPORTS
SOM Text
Figs. S1 to S18
Table S1
References
8 October 2009; accepted 11 December 2009
10.1126/science.1183090
15 JANUARY 2010
351
Zebrafish Behavioral Profiling Links Drugs to Biological
Targets and Rest/Wake Regulation
Jason Rihel, David A. Prober, Anthony Arvanites, Kelvin Lam,
Steven Zimmerman, Sumin Jang, Stephen J. Haggarty, David Kokel,
Lee L. Rubin, Randall T. Peterson and Alexander F. Schier (January
14, 2010)
Science 327 (5963), 348-351. [doi: 10.1126/science.1183090]
Behavioral Profiling
The complexity of the brain makes it difficult to predict how a drug will affect behavior without
direct testing in live animals. Rihel et al. (p. 348) developed a high-throughput assay to assess the
effects of thousands of drugs on sleep/wake behaviors of zebrafish larvae. The data set reveals a broad
conservation of zebrafish and mammalian sleep/wake pharmacology and identifies pathways that
regulate sleep. Moreover, the biological targets of poorly characterized small molecules can be
predicted by matching their behavioral profiles to those of well-known drugs. Thus, behavioral profiling
in zebrafish offers a cost-effective way to characterize neuroactive drugs and to predict biological
targets of novel compounds.
This copy is for your personal, non-commercial use only.
Article Tools
Permissions
Visit the online version of this article to access the personalization and
article tools:
http://science.sciencemag.org/content/327/5963/348
Obtain information about reproducing this article:
http://www.sciencemag.org/about/permissions.dtl
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week
in December, by the American Association for the Advancement of Science, 1200 New York
Avenue NW, Washington, DC 20005. Copyright 2016 by the American Association for the
Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Editor's Summary
Supporting Online Material for
Zebrafish Behavioral Profiling Links Drugs to Biological Targets and Rest/Wake
Regulation
Jason Rihel,* David A. Prober, Anthony Arvanites, Kelvin Lam, Steven Zimmerman, Sumin Jang,
Stephen J. Haggarty, David Kokel, Lee L. Rubin, Randall T. Peterson, Alexander F. Schier*
*To whom correspondence should be addressed. E-mail: schier@fas.harvard.edu (A.F.S.); rihel@fas.harvard.edu (J.R.)
Published 15 January 2010, Science 327, 348 (2010)
DOI: 10.1126/science.1183090
This PDF file includes:
SOM Text
Figs. S1 to S18
Table S1
References
Table of Contents
Materials and Methods, pages 2-4
Supplemental Text 1-7, pages 5-10
Text 1. Psychotropic Drug Discovery, page 5
Text 2. Dose, pages 5-6
Text 3. Therapeutic Classes of Drugs Induce Correlated Behaviors, page 6
Text 4. Polypharmacology, pages 6-7
Text 5. Pharmacological Conservation, pages 7-9
Text 6. Non-overlapping Regulation of Rest/Wake States, page 9
Text 7. High Throughput Behavioral Screening in Practice, page 10
Supplemental Figure Legends, pages 11-14
Figure S1. Expanded hierarchical clustering analysis, pages 15-18
Figure S2. Hierarchical and k-means clustering yield similar cluster architectures, page
19
Figure S3. Expanded k-means clustergram, pages 20-23
Figure S4. Behavioral fingerprints are stable across a range of doses, page 24
Figure S5. Compounds that share biological targets have highly correlated behavioral
fingerprints, page 25
Figure S6. Examples of compounds that share biological targets and/or structural
similarity that give similar behavioral profiles, page 26
Figure S7. Cluster analysis of multi-target compounds, page 27
Figure S8. Comparison of the effects of adrenergic drugs on rest/wake states in
zebrafish to the effects in mammals, page 28
Figure S9. Comparison of the effects of serotonin drugs on rest/wake states in
Figure S10. Comparison of the effects of dopamine drugs on rest/wake states in
Figure S11. Comparison of the effects of GABAergic drugs on rest/wake states in
Figure S12. Comparison of the effects of melatonin drugs on rest/wake states in
Figure S13. Comparison of the effects of histamine drugs on rest/wake states in
Figure S14. Comparison of the effects of adenosine drugs on rest/wake states in
Figure S15. Comparison of the effects of glutamate drugs on rest/wake states in
Figure S16. The L-type calcium channel inhibitor, YS-035, dose dependently increases
rest with minimal effects on waking activity, page 36
Figure S17. Examples of immunomodulators that co-cluster as daytime-waking activity
enhancers, page 37
Figure S18. ERG inhibitors, but not non-ERG blocking anti-histamine analogs, dose
1
dependently increase waking activity at night, page 38
Table S1. Summary of dose response experiments for selected compounds indicates
fingerprints are stable across several concentrations, page 39
Supplemental References, pages 40-43.
Small molecule exposure. Larval zebrafish were raised on a 14 hour/10 hour light/dark
cycle at 28.5oC. At four days post fertilization (dpf), a single larva was pipetted into
each of 80 wells of a 96-well plate (7701-1651; Whatman, Clifton, NJ) containing 650 µL
of standard embryo water (0.3 g/L Instant Ocean, 1 mg/L methylene blue, pH 7.0).
Larvae were then exposed to small molecules beginning at ~96 hours post fertilization
(hpf) by directly pipetting a 10 mM stock solution (in DMSO) into each well (10 animals
per compound) for a final chemical concentration of ~10-30 μM and no more than 0.3% DMSO. This concentration of DMSO has no effect on behavior. Small molecules were
obtained from the following libraries and screened at the following final concentrations:
1) Spectrum library from Microsource Discovery at ~30 µM (Gaylordsville, CT); 2)
Prestwick library from Prestwick Chemical at ~15 µM (Illkirch, France); 3)
neurotransmitter (cat#2810), ion channel (cat#2805), and orphan ligand libraries
(cat#2825) from Biomol International at ~15 µM (now Enzo Life Sciences, International;
Plymouth Meeting, PA);; 4) Sigma LOPAC library at ~10 μM (Sigma-Aldrich, St. Louis,
MO). Behavioral recording began at 11 PM, approximately 110 hpf.
Locomotor activity analysis. The behavioral assay is adapted from (S1). Locomotor
activity was monitored for 60 hours using an automated video tracking system
(Videotrack; Viewpoint Life Sciences, Montreal, Quebec, Canada) with a Dinion onethird inch Monochrome camera (model LTC0385; Bosch, Fairpoint, NY) fitted with a
fixed-angle megapixel lens (M5018-MP; Computar) and infrared filter. The movement
of each larva was recorded using the Videotrack quantization mode, and data from two
cameras were collected in alternating minutes by one computer. The 96-well plate and
camera were housed inside a custom-modified Zebrabox (Viewpoint, LifeSciences) that
was continuously illuminated with infrared lights and illuminated with white lights from 9
AM to 11 PM. The 96-well plate was housed in a chamber filled with circulating water to
maintain a constant temperature of 28.5oC. The Videotrack threshold parameters for
detection used were: detection threshold, 40; burst, 25; freeze, 4; bin size, 60 seconds.
Data analysis. The data was processed using custom PERL, Visual Basic Macros for
Microsoft (Seattle, WA) Excel, and Matlab (version R2007a; The Mathworks, Inc). Any
one minute bin with less than 0.1 second of total movement was defined as one minute
of rest; a rest bout was defined as a continuous string of rest minutes (S1). Rest
latency was defined as the length of time from lights on or off to the start of the first rest
bout. Total activity was defined as the average amount of detected activity in seconds,
including all rest bouts. Waking activity was defined as the total amount of detected
activity, excluding all one minute periods of rest. These parameters were calculated for
each experimental day and night, standardized to camera-matched DMSO controls, and
2
expressed as +/- standard deviations from controls for hierarchical clustering (see
below). We calculated the inter-assay variability as a coefficient of variation (standard
deviation of all DMSO controls/mean of DMSO controls*100) for each measurement.
For non-normal distributions, including rest length, rest latency, and wakingactivity, the
coefficient of variation was calculated following a Box-Cox transformation to a normal
distribution. The coefficient of variations (expressed as a %) are as follows: total rest,
day 1, 42.3%, day 2, 42.1%, night 1, 24.4%, night 2, 19.9%; rest bouts, day 1, 27.3%,
day 2, 21.9%, night 1, 16.7%, night 2, 11.3%; rest bout length, day 1, 14.6%, day 2,
12.9%, night 1, 15.6%, night 2, 11.3%; rest latency, day 1, 35.7%, day 2, 45.8%, night
1, 13.2%, night 2, 20.0%; total activity, day 1, 35.5%, day 2, 28.1%, night 1, 36.3%,
night 2, 29.9%; waking activity, day 1, 15.5%, day 2, 17.9%, night 1, 18.9%, night 2,
15.6%. Much of the variability is due normal inter-individual variation.
To generate time series data for average rest per 10 minutes and average waking
activity per 10 minutes (red traces, e.g. Figure 2B-F), these two parameters were
averaged across all 10 replicate animals in 10 minute intervals and then normalized to
camera matched controls by dividing each 10 minute rest and 10 minute waking activity
measure by the mean daytime rest and activity for camera matched controls,
respectively. For comparison, 10 representative sets of DMSO controls (10 larvae each
set; e.g. blue traces in Figure 2B-F) are plotted in each time series. The same DMSO
controls are shown in all graphs for clarity.
To identify compounds that significantly altered behavior, each parameter was
compared to camera-matched DMSO controls by Student’s t-test. Compounds were
chosen for further analysis if either: 1) the compound altered the same parameter in the
same direction at a p value < 10-5 for two consecutive days or nights, 2) the compound
affected a behavioral parameter at p<10-10 within the first 24 experimental hours, or 3)
the compound altered rest latency at p<0.001 for two consecutive days or nights but
had little effect on other parameters. These parameters were chosen to select the
largest manageable set of small molecules with the strongest and most consistent
effects on behavior while minimizing false positives and compounds with spurious shortterm behavioral alterations. Biological targets were assigned to compounds based on
annotations in the Pharmaprojects, Drugbank, Drugs@FDA, and Leadscope Marketed
Drugs databases, as well as information supplied by the library vendors and detailed
manual literature searches for each compound.
Hierarchical and k-means clustering analysis. Clustering analysis was performed with
Cluster 3.0 (adapted by Michiel de Hoon from Cluster, written by Michael Eisen, (S2))
and visualized using TreeView (written by Michael Eisen, (S2)). Hierarchical clustering
was performed using Pearson uncentered correlation and average linkage. The
parameters were weighted as follows: total rest, 1.0; rest bouts, 0.8; rest bout length
0.3, rest latency, 1.0; total activity and waking activity, 1.0. K-means clustering was
performed using k=24 and Euclidean distance. To determine the relative position of the
k-clusters, the average vector for each cluster was calculated, and these 24 vectors
were hierarchically clustered using Euclidean distance.
3
Correlation Analysis: All correlation analysis was performed in Matlab (version R2007a;
The Mathworks, Inc). The weighted, uncentered Pearson’s correlation coefficients were
calculated for all compound pairs. Compounds were assigned to pharmacological
classes as described above. Compounds were considered to share targets if both their
target assignment and activity (i.e. agonist or antagonist) overlapped for at least one
target.
Rank Correlation Analysis: To perform rank correlation analysis for adrenergic
antagonists and ERG blocking drugs, the average weighted uncentered Pearson’s correlation coefficient between each compound of the identified cluster and every other
small molecule was calculated. Each small molecule was then assigned a rank based
on their mean correlation, with rank 1 being the highest mean correlation. Compounds
that showed ERG or adrenergic antagonist activity were identified by their
pharmacological assignments, and the Kolmogorov-Smirnov statistic was used to
assign the p value for whether ERG or adrenergic antagonist compounds were enriched
among the higher ranking compounds. The rank correlations were graphically
represented in Treeview (S2).
Dose Correlation Analysis. Compounds were ordered from Sigma-Aldrich (St. Louis,
MO), dissolved in DMSO to a stock concentration of 30 or 45 mM and tested in the
locomotor activity assay at final concentrations of 0.15, 0.45, 1.5, 4.5, 15, and 45 µM, or
0.1, 0.3, 1.0, 10, and 30 µM (0.3% DMSO final concentration). Each concentration was
tested on 10-20 larvae. To generate the fingerprint for each concentration, the
behavioral parameter measurements were standardized to camera-matched DMSO
controls and expressed as +/- standard deviations from controls. Two-tailed t-test
assessments (Bonferroni corrected for multiple comparisons) determined statistically
significant differences of each measurement relative to camera-matched controls. The
correlation matrices were generated by calculating the weighted, uncentered Pearson’s correlation coefficient between the fingerprints for all concentration pairs. The
fingerprint, significance, and correlation matrix heat maps were generated using Matlab
(version R2007a; The Mathworks, Inc.).
MAO inhibitor assay. MAO-B activity was assayed as described (S3).
4
Supplemental Texts 1-7
Supplemental Text 1. Psychotropic Drug Discovery
The limitations of in vitro drug screening are particularly acute for the development of
drugs that modulate the central nervous system (CNS). Surveys of drug discovery
success show that CNS drugs take longer to develop (12.6 years versus 6.3 years for
cardiovascular drugs) and fail to reach the market more often (7% success rate versus
15%) than other therapeutic indications (S4). The CNS poses a particular challenge to
drug discovery because the brain cannot be modeled in vitro. Indeed, most modern
psychotropic drugs were first developed for non-psychotropic uses and serendipitously
discovered to have behavior-altering side effects (e.g. the MAO inhibitor, iproniazid, was
developed to treat tuberculosis and found to alter mood, and the tricyclic antidepressant
imipramine was first used as an anti-histamine, (S5)). Furthermore, CNS drugs tend to
produce undesirable side effects, including dizziness, tremors, lethargy, and seizures,
which are difficult to predict at early stages of the drug discovery pipeline. Also, the
mechanism of action for many CNS drugs, including bipolar medications,
antidepressants, and antipsychotics, is poorly understood and in many cases may
depend on a compound’s complex poly-pharmacological actions across multiple
neurotransmitter systems (S6-7). Indeed, efforts to increase efficacy and reduce side
effects through the development of newer generations of drugs with more selective
target profiles has to date achieved only limited success (S8-9).
Alternatives or complements to in vitro target-based screens include phenotype-based,
whole organism screens. Such screens have several advantages. First, wholeorganism screens need not be limited to single, well-validated targets. In fact, whole
organism screens can identify efficacious compounds with complex mechanisms of
action. Second, unwanted side effects, an obstacle in any drug discovery pipeline, can
be detected at earlier stages, even during the primary screening process. Third,
compounds that produce a behavioral effect are bio-available, another major stumbling
block to in vitro based drug discovery pipelines. In principle, whole organism
phenotypic screening could replace serendipity, but the requirement for large numbers
of phenotyped animals is usually cost and time prohibitive. Behavioral screening in
zebrafish larvae offers a cost-effective model system for psychotropic drug
characterization and discovery.
Supplemental Text 2. Dose
Choosing the compound concentration is a challenge for any large-scale small molecule
screen. Effective doses can vary among compounds, but it is impractical in most cases
to screen at multiple concentrations. The decision to perform our screen in the 10-30
µM range was based on the following considerations. First, we were guided by several
previous studies. Developmental screens in zebrafish used concentrations of 10-50 µM
(S10-12), single compound behavioral studies in zebrafish were done at 1-100 µM
(S13-19), and the connectivity map cell-based screen was performed at 10 µM (S20,
21). Second, we performed a pilot screen of 96 compounds and found that only 10% of
5
the compounds were overtly toxic at the 30 μM range, a percentage deemed acceptable
for screening. Third, dose response curves on selected compounds indicated that a
concentration of 10-30 µM was an effective range with consistent behavioral effects for
most compounds (Table S1 and Fig. S4). Specifically, we found that fingerprints for the
same compound were strongly correlated (25/29 cases) across the statistically effective
concentration range (Fig. S4). These results show that the behavioral fingerprints are
broadly stable across multiple doses.
Supplemental Text 3. Therapeutic Classes of Drugs Induce Correlated Behaviors
To test the potential of the clustering analysis, we determined whether compounds that
share targets are more likely to produce similar phenotypes (Fig. S5A-B). Expanding
this correlation analysis to 50 categories of drugs, we found, with few exceptions, that
the correlations among related classes of compounds were much larger than chance
(Fig. S5C). In many cases, every intra-class pair-wise correlation was larger than the
zero target correlation (see Figure Legend S5 for definitions of category abbreviations; +
indicates agonist, - indicates antagonist), including the ADR-A2+ (median correlation =
0.853; range = 0.352-0.974), SNRI (median = 0.775; range = 0.453-0.956), 5HT-1A+
(median = 0.791; range = 0.463-0.958), NEURO- (median = 0.759; range = 0.5870.894), DOPA-D1+ (median = 0.885; range = 0.766-0.927), and CHA-NA+ (median =
0.833; range = 0.519-0.985) classes (Fig S5C). In other examples, intra-class pair-wise
correlations were much larger except for a few outliers, including the SSRI (median =
0.767 ; range = 0.262-0.963), 5HT-1A- (median = 0.769; range = -0.02-0.930 ), 5HT-2(median = 0.698; range = 0.187-0.977), DOPA-D2+ (median = 0.727; range = 0.0720.982), GABA- (median = 0.722; range = -0.05-0.972), HIST-H1-(ERG) (median =
0.722; range = 0.229-0.911), and NMDA- (median = 0.651; range = -0.419-0.971)
classes (Fig. S5C). Not all classes had higher correlations than the no target class,
including the adrenergic reuptake class (ADR-U; median = 0.257; range = -0.50-0.959)
and the sodium channel antagonist class (CHA-NA-; median = -0.031; range = -0.513 –
0.913). This may reflect the heterogeneity of secondary targets for the compounds of
these classes.
Supplemental Text 4. Polypharmacology.
Many compounds have multiple, potentially non-overlapping targets. In such cases,
one would expect that not all compounds that share targets produce similar phenotypes
(Fig. S5A-C). For example, outliers of the correlation analysis might reflect the difficulty
in assigning compounds to single classes when in fact they have multiple targets. This
observation can be used to identify potential polypharmacological effects (Figs. S5C
and S7). For example, NMDA antagonist (NMDA-) outliers included compounds that
also bind to σ-opioid receptors (e.g. 3-methoxymorphinan), the GABA antagonist
(GABA-) outlier tert-butyl-bicyclophosphorothionate (TBPS) also potently blocks chloride
channels, the serotonin receptor 1A antagonist (5HT-1A-) outlier S(-)UH-301 is also a
dopamine D2/3 receptor agonist, and the dopamine D2 receptor agonist (DOPA-D2+)
outlier pergolide is also a serotonin receptor agonist (Fig S5C). Our correlation analysis
can also uncover higher order associations among drugs with multiple targets, such as
the high correlation between drugs that share dopamine reuptake inhibition and
acetylcholine antagonism (Figure S7A-B). Furthermore, the clustering analysis can
6
reveal unexpected associations between annotated drug classes. For example, a
cluster of known α1-adrenergic receptor antagonists (ifenprodil, terazosin, and 5-methylurapidil) also cluster with several serotonin receptor modulators, including NAN-190,
RU-24969, WAY-100635, spirotraxine, and buspirone (Fig. S7C-D). Intriguingly,
buspirone and spirotraxine do not strongly bind to α1-adrenergic receptors in vitro yet
can show in vivo adrenergic activity in specific pharmacological contexts (22). These
results show that behavioral profiling can uncover associations between multi-target
drugs whose properties are not fully predicted by in vitro studies.
Supplemental Text 5. Pharmacological Conservation (Figs. S8-15).
We systematically compared the behavioral effects of agonists and antagonists of the
major neurotransmitter systems between zebrafish and mammals. In most cases,
confidence in the conservation between zebrafish and mammals is enhanced when
multiple compounds known to affect the same target elicit similar phenotypes and when
agonist/antagonist pairs elicit opposing phenotypes. We discuss each class in more
detail below.
Adrenaline (Fig. S8). In zebrafish, α2-adrenergic agonists decreased wakefulness with
little effect on rest, while activation of β-adrenergic receptors selectively reduced total
rest at night. Conversely, β-adrenergic receptor antagonists increased total rest. α1adrenergic antagonists gave a “mixed” phenotype of increased waking activity but also
increased rest. These effects are broadly consistent with those observed in mammals
(S23-35).
Serotonin (Fig. S9). The analysis of serotonin-regulating drugs is complicated because
most, if not all, serotonin-modulating drugs have considerable affinity for non-serotonin
receptors, especially dopaminergic and adrenergic receptors. Thus, the drugs chosen
for the table in Figure S9 combine serotonin-signaling selectivity, consistency of
phenotype, and reproducibility (i.e. multiple hits within the screen of the same or similar
compounds). As often as possible, the drugs shown have known mammalian
phenotypes (S36-55). The complexity of the phenotypes we observed with serotonergic
agonists and antagonists is similar to the controversial and contradictory nature of the
mammalian literature pertaining to these drugs. This may reflect the difficulty in
isolating the drugs’ effects on serotonin signaling from other secondary effects, differences between short-term and long-term exposure, or complex signaling
interactions among different populations of serotonergic neurons in the brain. For
example, in mammals agonists and antagonists of serotonin receptor 1A both cause
short-term reduction of sleep and increase in wakefulness followed by longer-term
increases in sleep and reduced activity (S36-40, S45-47). In long-term experiments,
both classes of drugs increased rest in zebrafish. Only buspirone had an observed
short-term increase in waking during the first night of the experiment (black arrow in Fig.
S9). Serotonin receptor 2/3 antagonists have paradoxical effects in mammals,
increasing both exploratory behavior and sleep (S38, S44, S49-52). Similarly, both
waking activity and total rest were increased in zebrafish. Finally, SSRIs consistently
decreased waking activity and increased rest in zebrafish; in human patients, SSRIs
7
often improve insomnia in depressed patients but can have biphasic effects on sleep,
with long-term decreases in sleep in some mammalian models (S53-55).
Dopamine (Fig. S10). With one notable exception, dopamine receptor agonists and
antagonists produce similar phenotypes in mammals and zebrafish (S56-67). For
example, the D2-receptor agonists consistently reduced waking activity and increased
rest in zebrafish, as they do at low concentrations in mammals (S60-63). This effect
was receptor subtype selective, as the D2/D3 receptor agonists quinelorane and
quinpirole only reduced waking activity (S64, S65). As another example of
conservation, D2-receptor antagonists, several of which are used as anti-psychotics,
increase waking activity and rest in zebrafish and mammals (S57, S62, S67). Previous
work indicated that antipsychotics produce locomotor defects in zebrafish larvae (S1516). The only clear examples of non-conserved pharmacology in our study were the
dopamine D1 agonists (Fig. S10, marked in gray), all of which had little effect on waking
activity but greatly increased total rest in zebrafish. In all mammalian species tested,
dopamine D1 agonists increased waking (S56-59). This could indicate a distinct role for
the zebrafish orthologs of D1 receptors in the control of behavior or that these dopamine
D1 agonist drugs have altered target selectivity in zebrafish.
GABA (Fig. S11). While many of the GABA-regulating benzodiazepine drugs were toxic
at the concentrations used in our screen, the GABA-A agonists GBLD-345 and
avermectin B1 increased total rest and had only a small effect on waking activity (S68).
Although only represented by a single hit in the screen, the GABA-B agonist CGP13501 (Fig. S11, marked in gray) slightly increased waking activity at night with very
modest increases in rest during the day, even though this drug is hypnotic in mammals
(S69). In contrast, many GABA-A antagonists dramatically increased waking activity,
possibly reflecting pro-convulsant effects of these drugs on zebrafish larvae, which is
consistent with the mammalian literature (S70-72) and with previous work in zebrafish
(S13, S17, S19). Tracazolate, an anxiolytic acting as an allosteric modulator of GABAA receptors, modestly increased waking activity in zebrafish; in mammals, tracazolate
can both increase or decrease locomotor activity (S73, S74). Intriguingly, the
neuroactive steroid GABA agonists allopregnanolone, pregnanolone, and alfadolone
increased both total rest and waking activity specifically at night (S75-77). As
neuroactive steroids have additional functions in mammalian brains, including
modulation of NMDA receptors (S75), this mixed phenotype may reflect the
polypharmacology of these compounds.
Melatonin (Fig. S12). Although only a few examples were present in our screen,
melatonin-modulating drugs gave results that are consistent with those observed in
mammals (S78-82) and previous work in zebrafish (S14). Melatonin slightly increased
rest in our long-term experiments. In addition, the melatonin agonist 8-methoxy-1propionamidotetralin decreased waking activity and increased rest. An exception was
the agonist IIK7 (S78), which slightly increased waking activity at night and had small
effects on rest.
8
Histamine (Fig. S13). Histamine antagonists identified in our screen tended to increase
rest, decrease waking activity, or both. This increased rest in zebrafish is consistent
with the mammalian literature, although it should be noted that anti-histamine drugs that
do not cross the blood-brain barrier in humans, such as loratadine, tend to be less
sedating than the older generation of drugs that do cross the blood-brain barrier (S8387). Our work is also consistent with previous work on anti-histamines in zebrafish
larvae (S13, S18). Notably, the anti-histamine diphenylpyraline increased waking
activity, consistent with reports of psychostimulatory effects in rodents (S87). Also, as
noted in the main text and Figures 4C-D and S18, anti-histamines that block the human
ether-a-go-go related potassium channel (ERG) consistently increased waking activity
at night, with modest effects on rest in zebrafish larvae.
Adenosine (Fig. S14). Consistent with mammals, adenosine receptor agonists
increased rest in zebrafish larvae, while adenosine receptor A1 antagonists increased
waking activity and reduced rest (S88-93). Although behavioral effects of the adenosine
A3 antagonist MRS-1220 are unknown in mammals, it decreased waking activity and
increased rest in zebrafish. This phenotype is consistent with its inhibitory effects on
MAO (Figure 3D).
Glutamate (Fig. S15). All NMDA receptor antagonists identified in the screen
dramatically increased waking activity during the day and night, consistent with the
effects observed in mammals (S94, S95). Muscarinic glutamate receptor 5 (mGluR5)
antagonists selectively increased rest, with little effect on waking activity. Antagonists of
mGluR5 can block drug-induced hyperlocomotion, down-regulate bursting of prefrontal
cortical neurons in awake rats, and have anxiolytic properties in rodents (S96, S97).
Supplemental Text 6. Non-overlapping Regulation of Rest/Wake States.
Many pharmacological agents selectively altered waking activity, rest latency, or total
rest. For example, verapamil related L-type calcium channel inhibitors increased rest
with no effect on waking activity (Fig. S16), while β-adrenergic agonists, including
clenbuterol and fenoterol, decreased rest only (Fig. S8). Non-competitive NMDA
receptor antagonists, including the psychotomimetics dizocilpine (MK-801) and L701324, predominantly increased waking activity with minimal effects on total rest (Figs.
2C and S15), the D2/D3 agonist, ropinirole, predominantly decreased waking activity
only (Fig. S10), and podocarpatrien-3-one analogs predominantly altered only rest
latency (Fig. 4A). These single parameter effects can also be selective for day versus
night. For example, many anti-inflammatory compounds only increased waking activity
during the day (Figs. 2B, 4B and S17), while ERG-blocking drugs increased waking
activity at night (Figs. 2E, 4C-D, S18). Finally, some compounds can affect waking
activity and total rest in opposite directions (e.g. increase both waking activity and total
rest). For example, the α1-adrenergic antagonists ifenprodil, the antimalarial
quinacrine, and the steroidal GABA agonist alfadolone, increased both waking activity
and total rest (Figs S7C, S11). These observations suggest that the behavioral
parameters of waking activity, total rest, and rest latency during the day and the night
can be under the control of distinct pharmacological mechanisms.
9
Supplemental Text 7. High Throughput Behavioral Screening in Practice
Behavioral profiling in zebrafish is highly efficient. Our current zebrafish behavior room
(~200 sq. ft.) has 16 cameras that can each observe 80 wells of a 96-well plate, for a
total of 1280 larvae. Using our original screening method of 10 larvae per compound
and including 10 control wells for each camera, we can screen 112 compounds (16
cameras x 7 drugs per camera) every three days, or 224 compounds per week. Using
the latest videotracking software, which can track all 96 wells of the plate, and cutting
the number of larvae per drug to eight expands our capacity to 352 compounds per
week (16 cameras X 11 drugs per camera X 2 runs per week). A single technician can
set up the entire behavioral room in less than 4 hours, including fish husbandry, larval
pipetting, drug dispensing, and software setup (an 8 hour commitment per week).
Expanding the screen to 115 cameras and 5 technicians (a total commitment of 40
hours per week) would increase the throughput to more than 10,000 compounds per
month and more than 100,000 per year.
10
Supplemental Figure and Table Legends
Figure S1. Expanded hierarchical clustering analysis. This is an expanded version of
Figure 2A in which the name of each chemical in the clustergram can be seen. Each
row represents a different chemical, and each column represents a behavioral
measurement. From left to right, these measurements are: the rest total, the number of
rest bouts, the rest bout length, the rest latency, the total activity, and the waking
activity. The black bars indicate night measurements; the white bars indicate day
measurements. Yellow indicates that the value is increased relative to controls; blue
indicates that the value is decreased, as in Figure 2A. The measurements are
normalized as standard deviations from control values.
Figure S2. Hierarchical and k-means clustering yield similar cluster architectures. Each
row represents a different chemical, and each column represents a behavioral
Figure S3. Expanded k-means clustergram. This is an expanded version of Figure S2
in which the name of each chemical in the clustergram can be seen. Each row
represents a different chemical, and each column represents a behavioral
Figure S4. Behavioral fingerprints are stable across a range of doses. Select
compounds were tested for behavioral effects at multiple concentrations (see Table S1
for additional examples and summary data). First, the fingerprints for each dose were
calculated as standard deviations from DMSO controls (‘Fingerprint’, left panels). In general, these fingerprints are stable across multiple concentrations until the dose is too
low to elicit a phenotype. To assess the stability of single drug fingerprints over multiple
concentrations, the Pearson uncentered correlation coefficient was calculated pairwise
between each dose (‘correlation matrix’, right panels). With the exception of pergolide
and several other ergoline derivatives, which have distinct fingerprints at high versus
low concentrations, these examples have relatively stable fingerprints from the highest
dose tested (45 μM) to the lowest effective doses (150 nM for NAN-190 and
11
betamethasone, 450 nM for ropinirole, 1.5 µM for clonidine and buspirone), as indicated
by the high correlations (red boxes). See Materials and Methods for additional
information.
Figure S5. Compounds that share biological targets have highly correlated behavioral
fingerprints. (A) The distribution of all pair-wise correlation coefficients (Pearson
weighted, uncentered; see Materials and Methods) for compounds that share at least 1
target (blue) is shifted to higher values compared to compounds that share 0 targets
(magenta). This skewing is strong despite the fact that these compounds can have
many divergent targets in addition to the shared target. Each pair of compounds is
represented once, even if they share multiple targets. (B) Box-whisker plot comparing
correlations between compounds with 0 shared targets, compounds with at least 1
shared target, and repeats of the same compound from different chemical libraries. For
each measure, the blue box represents the interquartile range between the 25th and 75th
percentiles; the red line marks the sample median; the whiskers extend from the box
ends to values within 1.5 times the interquartile distance; red crosses mark outlier
values beyond the 1.5 interquartile distance from the box ends. (C) Box-whisker plots
of correlation coefficients within biologically related classes of compounds. For
comparison, the mean correlation of compounds that share 0 targets is plotted as a
green line (correlation = 0.266). The number of compounds used for comparisons within
each class is indicated (N). See supplemental text 3 for a detailed discussion of specific
classes. For all classes, a (-) sign indicates an inhibitor/antagonist and a (+) sign
indicates an agonist. The classes are: α-adrenergic-receptor 1, ADR-A1; α-adrenergic
receptor-2, ADR-A2; β-adrenergic, ADR-B; noradrenaline reuptake inhibitor, ADR-U-;
serotonin/noradrenaline reuptake inhibitor, SNRI; selective serotonin reuptake inhibitor,
SSRI; serotonin receptor-1A, 5HT-1A; serotonin receptor-1D, 5HT-1D; serotonin
receptor-2, 5HT-2; serotonin receptor-2/3, 5HT-2/3; serotonin receptor 4, 5HT-4;
monoamine oxidase inhibitor, MAO-; neurotransmitter inhibitor (reserpine class),
NEURO-; dopamine receptor, DOPA; dopamine D1 receptor, DOPA-D1; dopamine D2
receptor, DOPA-D2; dopamine D2/3 receptor, DOPA-D2/3; dopamine D3 receptor,
DOPA-D3; dopamine D4 receptor, DOPA-D4; γ-butyric acid receptor, GABA; histamine
H1 receptor, HIST-H1; ether-a-go-go-related potassium channel, ERG; adenosine
signaling, ADS; metabotropic glutamate receptor 5, mGLUR5; N-methyl-D-aspartic acid
receptor, NMDA; acetylcholinesterase inhibitor, ACH-ES- IRev, irreversible; Rev,
reversible; muscarinic acetylcholine receptor; ACH-M; nicotinic acetylcholine receptor,
ACH-N; acetylcholine synthesis, ACH-SY; L-type calcium channel; CHA-CA-L; sodium
channel, CHA-NA; peroxisome proliferator-activated receptor, alpha, PPAR-A.
Figure S6. Examples of compounds that share biological targets and/or structural
similarity that give similar behavioral profiles. (A) Structurally divergent selective
serotonin reuptake inhibitors (SSRIs) similarly decrease waking activity and increase
rest. (B) Structurally related insecticides dramatically increase waking activity
independent of the time of day. The graphs represent the normalized waking activity
and total rest for behavior-altering compounds (red trace; average of 10 larvae) and
representative controls (10 blue traces; average of 10 larvae each).
12
Figure S7. Cluster analysis of multi-target compounds. A dopamine reuptake inhibitor
(DOPA-U) with muscarinic acetylcholine antagonist (ACH-M-) activity does not correlate
well with (A) compounds with only DOPA-U activity or (B) compounds with only ACH-Mactivity. It does, however, show high correlation with other compounds known to share
both properties (B). (C) α1-adrenergic receptor antagonists (marked in blue) form a
large cluster with serotonin modulators (marked in green) that are known to also have
α1- adrenergic receptor antagonist activity in vivo. (D) Rank-correlations of compounds
across the entire dataset compared to the α1-adrenergic receptor antagonist cluster of
ifenprodil, terazosin, and 5-methyl-urapidil enriches for compounds with α1-adrenergic
receptor antagonism. In the rank-graph, the black lines indicate compounds with known
α1-adrenergic antagonism; red indicates high correlation, green indicates low
correlation. Compounds with α1-adrenergic antagonism are highly enriched in the top
ranks (p < 10-23; see Materials and Methods).
Figure S8-15. Comparison of the effects of neuroactive drugs on rest/wake states in
zebrafish to the effects in mammals. The graphs represent the waking activity and rest
for representative agonists and antagonists of each class (red trace; average of 10
larvae) and DMSO controls (10 blue traces; average of 10 larvae each). Each plotted
example is typical for the entire class of compounds. The tables provide a more
comprehensive list of agonists and antagonists of each receptor subtype. The tables
summarize the overall direction and magnitude of the observed effects of the drugs on
zebrafish wake and rest behavior (up arrow, increased; down arrow, decreased; a
dashed line indicates no change; a small arrow indicates a smaller relative effect, while
a large arrow indicates a larger relative effect). See supplemental text 5 for a more
detailed discussion of each phenotype.
Figure S16. The L-type calcium channel inhibitor, YS-035, dose dependently increases
rest with minimal effects on waking activity. (A) Waking activity and rest graphs are
shown for three structurally related L-type calcium channel inhibitors (red trace =
average of 10 larvae) and representative DMSO controls (10 blue traces = average of
10 larvae). (B) YS-035 increases the time spent at rest during both the day (red) and
night (blue) in a dose dependent fashion. Each point indicates the average minutes of
rest per hour for 10 larvae at each YS-035 concentration. The error bars represent +/SEM. (C) YS-035 has little or no effect on waking activity at all concentrations tested,
suggesting that muscle function is not dramatically perturbed. Each point indicates the
average seconds of waking activity every 10 minutes during the day (red) or night (blue)
for 10 larvae at each YS-035 concentration. Error bars represent +/- SEM (B, C).
Figure S17. Examples of immunomodulators that co-cluster as daytime-waking activity
enhancers. (A) The table lists anti-inflammatory agents that selectively increased
daytime waking activity (expanded examples from Figure 4B). (B) Waking activity and
rest graphs are shown for compounds from each major class (red trace = average of 10
larvae) and representative controls (10 blue traces = average of 10 larvae).
Figure S18. ERG inhibitors, but not non-ERG blocking anti-histamine analogs, dose
dependently increase waking activity at night. (A) Waking activity and rest graphs are
13
shown for two ERG-blocking compounds (red trace = average of 10 larvae) and
representative controls (10 blue traces = average of 10 larvae). Both compounds
increased waking activity at night without affecting rest. Psora-4, which blocks Kv1.3
shaker channels, increased nighttime waking activity and also strongly decreased rest,
a phenotype distinct from ERG blockers. Thus, the ERG phenotype is not the result of
general potassium channel misregulation. (B) Dose response curves for ERG blocking
and ERG non-blocking analogs are shown. Each point indicates the waking activity at
night (seconds per minute) averaged for 10 larvae. Cisapride and dofetilide, which have
ERG blocking activity, dose-dependently increased nighttime wakefulness, while the
non-ERG blocking histamine H1 receptor antagonists fexofenadine and cetirizine had
no effect on wakefulness. Another non-ERG blocking anti-histamine, loratadine,
reduced waking activity. Error bars represent +/- SEM. The 0 M concentration is a
DMSO control.
Table S1. Summary of dose response experiments for selected compounds indicates
fingerprints are stable across several concentrations. This table is an expansion of the
dose response fingerprints shown in Fig. S4. Note that only ergoline-derived
compounds (bottom of table) have complex dose dependent effects (i.e. different
nontoxic fingerprints at high versus low concentrations). This complex dose
dependence is likely due to the high affinity that many ergoline drugs have for multiple
receptor families and sub-types, including dopamine, serotonin, and adrenergic
receptors (for example, see (S98)).
14
Waking
Activity
Activity
Total
Rest
Latency
# Rest
Bouts
Rest
Bout
Length
Rest
Total
Figure S1
NAME
Senecrassidol 6-Acetate
Glycine
MG-624
Physcion
Budesonide
Diclofenac Sodium
14-Methoxy-4,4-Bisnor-8,11,13-Podocarpatrien-3-one
5-beta-12-Methoxy-4,4-Bisnor-8,11,13-Podocarpatrien-3-one
Enoxolone
11-alpha-Acetoxykhivorin
Dinitolmide
Chlorthalidone
3-beta-Acetoxydeoxodihydrogedunin
Kynurenine
Estrone Acetate
Betamethasone
Clenbuterol Hydrochloride
Telmisartan
SB 206553 Hydrochloride
Psora-4
Fenoterol Hydrobromide
Ornithine alpha-Ketoglutarate
Chlorophyllide Cu Complex Sodium Salt
Oxiconazole Nitrate
Sulfaguanidine
Benzyl Penicillin Potassium
Retinoic Acid p-Hydroxyanilide
Pimozide
Chlorpromazine Hydrochloride
Mitotane
SB 228357
Maprotiline Hydrochloride
Amitryptiline Hydrochloride
R(-)-Isoproterenol (+)-Bitartrate
Carbenoxolone Sodium
I-OMe-Tyrphostin AG 538
(-)-Eseroline Fumarate
Bacitracin
Leflunomide
Meclizine Hydrochloride
Pancuronium Bromide
Fenoldopam
Chloroethylclonidine Dihydrochloide
Mecamylamine Hydrochloride
1-Benzyl-1-Methyl-4-Cyclopentylmethoxycarbonylpiperidinium Bromide
Tegaserod Maleate
Austricine
Azobenzene
Diflunisal
Piroxicam
Flecanide
Flunisolide
Atrazine
Capsazepine
Cyclosporin A
2-Cyclooctyl-2-Hydroxyethylamine Hydrochloride
Oxethazaine
Spectinomycin Hydrochloide
Theaflavin Digallate
Equilin
SKF 94836
Aminophenazone
Diplosalsalate
Fosfosal
Clobetasol Propionate
Fenoprofen
Nicotine Ditartrate
(-)-Nicotine
Fenoprofen Calcium Salt Dihydrate
5-Aminopentanoic Acid Hydrochloride
Catechin Tetramethylether
Pentamidine Isethionate
Valproate Sodium
Betamethasone
Mefloquine Hydrochloride
Flumethasone
Desoxycorticosterone Acetate
Ethylene Glycol-Bis(2-Aminoethylether)-N,N,N,N-Tetraacetic Acid
Dexamethasone
Propentofylline
Bopindolol Malonate
Betamethasone Valerate
Bretylium Tosylate
Pancuronium Bromide
Cepharanthine
Ursolic Acid
Benzamil Hydrochloride
Cefoxitin Sodium Salt
Papaverine Hydrochloride
Medrysone
6-alpha-Methylprednisolone
6,7-Dichloroquinoxaline-2,3-dione
L-689560
7-Chlorokynurenic Acid
SDZ 220-581
IEM-1460
Finasteride
Bufexamac
Ro 20-1724
Cisapride
(+)-Tubocurarine Chloide
Hydrocortisone
L-701324
L-701324
L-701324
Sinapic Acid Methyl Ether
Dizocilpine Maleate
(-)-MK-801 Hydrogen Maleate
(+)-MK-801
Allopregnanolone
Tetrandrine
Diphenylpyraline Hydrochloride
Spiperone
TG003
Hexylresorcinol
Monobenzone
2,4-Dihydroxychalcone 4-Glucoside
Astemizole
3-(1H-Imidazol-4-yl)Propyl Di(p-Fluorophenyl)Methyl Ether Hydrochloride
Dichlorodiphenyltrichloroethane
Tracazolate Hydrochloride
1,4-PBIT Dihydrobromide
Menadione
(-)-Mk-801 Hydrogen Maleate
S,S,S,-Tributylphosphorotrithioate
Endosulfan
Coumaphos
Amoxapine
Megestrol Acetate
L-701252
Finasteride
Rutilantinone
Terfenadine
N-Desmethylclozapine
Mianserin Hydrochloride
ODQ
Clorgyline Hydrochloride
Amoxapine
Activity 1
NMDA+
ACH-NBACTARACHARACH-
Activity 2
Activity3 Activity 4
LIPCOR+
DIUR+
ESTR+
ARACHADR-B-2+
ANG-2-15HT-2-B/CCHA-KADR-B+
FUNGBACTBACTAPOP+
DOPA-D-2DOPA-D-2ESTR5HT-2-B/CADR-UADR-UADR-B-2+
ADR-U-
LIPCOR+
5HT-2-ADOPA-D-1CHA-NA?
GluR+
ADR-A-1- DOPA-U5HT1-2- HIST-H-1-
5HT-U-(weak)
5HT-UCHA-KADR-B-1+
5HT-U-(weak)
KI-IGF-1OPI-M+
ACH-(weak)
BACTDH-HYOKI-GFPLHISTACHACH-NDOPA-D-1-A+ ADR-A-2+
ADR-A-1ADR-A-2+
ACH-NACH-T5HT-4+
5HT-2-BLP-P-A-2SY-PGSY-PGCHA-NAARACHHERBVANIMNEURT-
IM-
PPAR-G+
ALOX5-
COXCOXLIPCOR+
IM-
GluR+
IM-
GluR+
IM-
GluR+
IMIM-
GluR+
GluR+
IM-
GluR+
LIPCOR+
IMNA/K-ATPase-
GluR+
CHA-Na+
PNMT-
BACTNf-kB-?
ESTR+
ES-P-C-3ES-P-C-?
ES-P-CARACHSY-PGACH-N+
ACH-N+
SY-PGDNAHDACARACHCALMARACHARACHCOR-M+
ACE-2ARACHES-P-CADR-B-1ARACHADR-BACH-NAPOPARACHCHA-NA-CaSY-CWES-P-C-4ARACHARACHEAA-GEAA-GEAA-GEAA-GEAA-GRED-5ASY-PGES-P-C-45HT-4+
ACH-NARACHEAA-GEAA-GEAA-GEAA-GEAA-GEAA-GGABA-A+
CHA-CAHIST-H-1DOPA-D-2KI-CLK-
LIPCOR+
COXCOX-
GABA+
LIPCOR+
LIPCOR+
LIPCOR+
MR+
CHEL+
LIPCOR+
ADS-/+
NF-KBLIPCOR+
IMIM-
GluR+
LIPCOR+
LIPCOR+
NMDANMDANMDANMDAAMPA-R-
IMIM-
GluR+
GluR+
COX-
OX-C-
BACT-
NMDA-
ACH+(indirect) CHA-KACH+
5HT-3LIPCOR+
IMNMDANMDANMDA-
GluR+
NMDANMDANMDACHA-CACHA-K-(Ca+)
5HT-1-A-
5HT-2-A-
SY-PGHIST-1HIST-H3-/+
CHA-NA+
GABA-ANO-SVITA+
EAA-GACHGABA-AACHADR-UESTR+
EAA-GRED-5ABACTHIST-15HT-2-CADR-UCYC-GUMAO-AADR-U-
CHA-KINSECTGABA-A+
NMDA-
5HT-UPROG+
NMDAANDRCHA-KACH-M1+
ADR-A-25HT-U-
HIST-
5HT-2-A/2-C-
15
Figure S1(Cont)
NAME
Loxapine Succinate
Propazine
Bepridil Hydrochloride
1S,9R-Hydrastine
Dacthal
Chlordane
7-Oxocallitrisic Acid, Methyl Ester
Ibudilast
Roxithromycin
LY-367265
Naftopidil Dihydrochloride
Clozapine
Halofantrine Hydrochloride
Amperozide Hydrochloride
Haloperidol
Terfenadine
Fiduxosin Hydrochloride
Strobane
Oxatomide
3-alpha-Bis-(4-Fluorophenyl)Methoxytropane
Dihydroergotamine Mesylate
Methiothepin Maleate
Mianserin
Methoxychlor
Toxaphene
Heptachlor
Aldrin
Bromo-3-Hydroxy-4-(Succin-2-yl)-Caryolane gamma-Lactone
Cyproterone Acetate
Promethazine Hydrochloride
Tyrphostin AG 494
8-Cyclopentyl-1,3-Dipropylxanthine
Flutamide
Derrusnin
Pregnenolone
Deptropine Citrate
Clemastine
Spermine Tetrahydrochloride
Homopterocarpin
Aconitine
Cinnarazine
Verapamil Hydrochloride
Benztropine
(1S,9R)-Beta-Hydrastine
Disulfoton
Naltriben Methanesulfonate
Benzanthrone
Vinpocetine
Oxymetazoline Hydrochloride
3-Isobutyl-1-Methylxanthine
2-[[B-(4-Hydroxyphenol)Ethyl]Aminoethyl]-1-Tetralone
Niloticin
Meclozine Dihydrochloride
CGP-13501
Mesulergine Hydrochloride
Thioridazine Hydrochloride
Hydroxyprogesterone Caproate
Nicolsamide
2-Methoxyestradiol
Tolnaftate
Danazol
(1-[(4-Chlorophenyl)phenyl-methyl]-4-methylpiperazine)
Beclomethasone Dipropionate
Astemizole
Icilin
Oxaprozin
Ro 25-6981 hydrochloride
Droperidol
Ethynodiol Diacetate
Bromocryptine Mesylate
Androsterone Acetate
3,4-Dimethoxyflavone
Trihexyphenidyl Hydrochloride
Vincamine
Cephaeline Dihydrochloride Heptahydrate
LY-53857
Amodiaquine Dihydrochloride
Risperidone
Doxazosin Mesylate
Alcuronium Chloride
L-765314
Prazosin
Benperidol
Saquinavir Mesylate
Butylparaben
Ketanserin Tartrate
Nomegestrol Acetate
PD 168077
Quinacrine Hydrochloride
Ifenprodil Tartrate
Phenamil
Epiandrosterone
Loperamide
L-701252
BWB-70C
Strophanthidinic Acid Lactone Acetate
Domperidone
Spiperone
Spiperone Hydrochloride
Pancuronium Bromide
Amsacrine Hydrochloride
Dihydroergotamine Methanesulfonate
Medroxyprogesterone Acetate
WB 4101 Hydrochloride
3-Methoxymorphinan Hydrochloride
L-741626
AMI-193
Spiroxatrine
AMI-193
trans-Dehydroandrosterone
DO 897/99
Triptophenolide
Azaperone
WB 4101 Hydrochloride
R(+)-Terguride
Azaperone
SB 205384
Alfadolone Acetate
3-Methyl-6-(3-[Trifluoromethyl]Phenyl)-1,2,4-Triazolo[4,3-B]Pyridazine
S(-)-Lisuride
Dihydroergocristine
4-Androstene-3,17-dione
Pirenperone
Ketanserin Tartrate
LY-165163
Astemizole
Chlormadinone Acetate
Totarol
Metergoline Phenylmethyl Ester
Acetopromazine Maleate Salt
Metergoline
BP554
R-(-)-Fluoxetine Hydrochloride
Methapyrilene Hydrochloride
Paroxetine Hydrochloride Hemihydrate
GR 4661
Doxazosin Mesylate
TCPOBOP
Activity 1
DOPA-D-1HERBCHA-CAGABA-AHERBGABA-AES-P-C-4PRT-50S5HT-2-AADR-A-15HT-2CHA-K5HT-2DOPA-D-1-2HIST-1ADR-A-1CHA-NA+
HIST-1DOPA-U5HT-1-D+
5HT-1-2ADR-UCHA-NA+
CHA-NA+
GABA-AGABA-AANDRHIST-1KI-GFEPADS-A-1ANDRGABA-AHISTHIST-1EAA-GCHA-NA+
CHA-CACHA-CAACH-MGABA-AACHOPI-D-
Activity 2
DOPA-D-2-
5HT-2- (indirect?)
CHA-NA-
CHA-K-
INSECT-
ATPP-Ca/MgATPP-Na/K-
NF-kB5HT-UADR-U-?
DOPA-D-2-
5HT-2-?
ADR-
DOPA-D-2NMDACHA-K-
HIST-H-1-
CHA-K-
INSECTACH-M5HT-1-A+
DOPA-D-2ADR-A-2INSECTINSECTINSECTINSECT-
ADR-A-2-ADOPA-D-2/D-3
HIST-
5HT-2-A/2-C-
ATPP-Ca/MgATPP-Na/KATPP-Ca/MgATPP-Na/K-
ACH-M-
5HT-2-
calmodulin-
ACH-MACH-M-?
EAA-G+
NMDA-
HIST-H-1-
DOPA-D-2-ACH-M-
DOPA-U-
HIST-
INSECT-
CHA-NAADR-A-1+
ES-P-CADR-A-1-
ES-P-C-1ADR-A-2+
ADS-
HISTGABA-B+
DOPA-D-2+
DOPA-D-2ESTR+
KI-TYRHIF-1FUNGLHRHHISTARACHHIST-1TRP-M8+
SY-PGEAA-GDOPA-D-2PROG+
DOPA-D-2+
ANDR+
AHRACH-M-1ADR-A-15HT-4+?
5HT-2-
ACH5HT-2-C5HT-2ESTR+
ADRPR+
CHA-K-
IM-
GluR+
ESTRLIPCOR+
CHA-K-
COXNMDA-NR2-BCHA-KESTR+
CREB+
SSTATIN
CHA-CA-?
CHA-NA-
DOPA-D-2- 5HT-2ADR-A-1ACH-MADR-A-1ADR-A-1DOPA-D-2- CHA-KPROTTNF-alpha+
ESTR?
5HT-2DOPA?
ES-P-C-4PROG+
ANDRDOPA-D-4+
ACH-M+
Lipase-A-2ADR-A-1EAA-GCHA-NAESTRCHA-CAOPI+
EAA-GNMDAOX-LP-5ATPP-Na/KDOPA-D-2DOPA-D-2- 5HT-1-ADOPA-D-2- 5HT-1-AACH-NTO-25HT-1-D+
5HT-1-A+
DEC-ORANDRADR-A-1EAA-GNMDADOPA-D-25HT-2DOPA-D-25HT-1-ADOPA-D-25HT-2DOPA-D-2ESTR+
ANDR+
DOPA-D-2/D-3-DOPA-D-3+
ADR-B-
DOPAADR-A-15HT-2-BDOPAGABA-A+
GABA-A+
GABA-A+
DOPA-D-2+
DOPA-D-2+
ESTR5HT-25HT-25HT-1-A+
HIST-1ANDRBACT5HT-1-DDOPA5HT-1-D5HT-1-A+
5HT-UHIST-15HT-U5HT-1-D+
ADR-A-1CAR+
ACH-
HIST-
HIST-
PROL-
OX-LP-5NMDACHA-K(GIRK)GABA-A+
5HT-2-A5HT-2-AADR-A-2-ADOPA-D-2/D-3
ESTR+
PR+
OPI-S+
DOPA-U-?
ADR-A-2GABA-A5-HA-1-A ADR-A-1
DOPA+/partial
HISTACH-
5HT-2-BADR-A+/ANDR+
DOPA-D-2-?
DOPA?
DOPA-D-2CHA-KESTR+
5HT-2-
PR+
5HT-2-
DOPA+
5HT-2-
DOPA+
16
Figure S1(Cont)
NAME
Prednicarbate
CNS-1102
Yohimbine Hydrochloride
Opipramol Dihydrochloride
DCEBIO
Cefamandole Sodium
(+)-Hydrastine
Fusaric Acid
MRS-1220
Phenelzine Sulfate
R(+)-UH-301 Hydrochloride
Phenelzine Sulfate
Tranylcypromine Hydrochloride
L-750667
Dl-p-Chlorophenylalanine Methyl Ester Hydrochloride
(+/-)-Vesamicol Hydrochloride
Loratadine
1-(3-Chlorophenyl)Piperazine Dihydrochloride
Gedunol
1,3-Diethyl-8-Phenylxanthine
Zardaverine
Dioxybenzone
Ciclopirox Olamine
L-703606 Oxalate
Oxibendazole
Niflumic Acid
Cyclopiazonic Acid
Tetradecylthioacetic Acid
Fenofibrate
Clioquinol
Spermine
Tert-Butyl-Bicyclo[2.2.2]Phosphorothionate
Indomethacin Morpholinylamide
Dihydrogeduninic Acid, Methyl Ester
Chol-11-Enic Acid
Diosmetin
Syrosingopine
Apomorphine Hydrochloride
6-(5H)-Phenanthridinone
Lysergol
R(-)Apomorphine Hydrochloride Hemihydrate
Indole-3-Carbinol
Clofibrate
Avermectin B1
Thioxolone
CGS-12066A Maleate
Dihydrocapsaicin
Suprofen Methyl Ester
Pramoxine Hydrochloride
Quipazine Dimaleate
Pergolide Methanesulfonate
Fluspirilen
CY 208-243
7-Hydroxy-DPAT
1-[1-(2-Benzo[B]Thienyl)Cyclohexyl)]Piperidine
BRL 15572
Mexiletine Hydrochloride
Adenosine
Gitoxin
1-Benzo[B]Thien-2-Yl-N-Cyclopropylmethylcyclohexanamine
Tetrac
Haloperidol
Triflupromazine Hydrochloride
Metergoline
Imipramine Hydrochloride
RX 821002 Hydrochloride
CV-3988
Totarol-19-Carboxylic Acid
Clozapine
Amitriptyline Hydrochloride
Norcyclobenzaprine
Estradiol
Chrysin
GW 9662
Chrysene-1,4-Quinone
S(-)-3PPP Hydrochloride
1-(2-Methoxyphenyl)-4-(4-Succinimidobutyl)Piperazine
WAY-100635 Maleate
NAN-190 Hydrobromide
Quinazolin-5(6H)-One Analog
5-Methyl-Urapidil
Terazosin Hydrochloride
Quinacrine Dihydrochloride
Ajmalicine Hydrochloride
RU 24969
Ifenprodil Tartrate
Ifenprodil Tartrate
ODQ
Quinelorane Dihydrochloride
WAY-100635 Maleate Salt
Buspirone
PD 168077 Maleate
Dihydroergocristine Methanesulfonate
Hycanthone
H-89
Nicergoline
MK-912
Omeprazole
(+/-)-Methoxyverapamil Hydrochloride
2-Methyl-6-(Phenylethynyl)Pyridine
Spiroxatrine
GR 103691
Droperidol
LY 235959
Coralyne Chloride Hydrate
Trifluoperazine Hydrochloride
Trifluoperazine Dihydrochloride
YS-035 Hydrochloride
B-HT 920
2,Beta-Dihydroxychalcone
Estrone
Phenazopyridine Hydrochloride
Mesoridazine
Reserpine
PD 169316
BMY 7378 Dihydrochloride
P-MPPF Dihydrochloride
(+/-)-Verapamil Hydrochloride
Rescinnamin
Verapamil
Prometryn
Cortexolone Maleate
Reserpine
Nicergoline
Dihydroergocristine
Risperidone
Testosterone Propionate
Piribedil Maleate
Lisuride
Carvedilol Tartrate
Risperidone
Phloretin
Domperidone Maleate
Activity 1
Activity 2
ARACHEAA-GADR-A-25HT-2CHA-K+
SY-CWGABA-ADOPA-BHADS-A3MAO5HT-1-A+
MAOMAODOPA-D-45HT-SYACH-SYHIST-15HT-1-2+
LIPCOR+
NMDA-
IM-
OPI-S+
HIST-H-1- DOPA-D-2-
ADS-A-1ES-P-C-3UVPERMNK-1NEMOES-P-C-10
SY-PGCHA-CA-iPPAR-A+
PPAR-A+
FUNGEAA-GGABA-ASY-PG-
ES-P-C-IV-
P450NEURTDOPA+
PARP-
GluR+
BACT-
DOPA-D-2?
5HT-2-A?
CHA-K(A5)5HT-3Nf-kB
ATPP-Na/KMICROTUBECOX-
AE2-
EAA-G+
NMDA-
COX-1-2-
COMT-
TH-
DOPA+
COMT-
TH-
PPAR-A+
GABA-A+
ANC-25HT-1-B+
VAN+
SY-PG-
LP-P-A-2+
5HT-1-D+
COX-1-
COX-2-
5HT-2-A/3+
DOPA-D-2+
DOPA-D-2+
DOPA-D-2DOPA-D-1+
DOPA-D-3+ DOPA-D-1-2/4+
DOPA-U5HT-1-DCHA-NAADR-BADS+
ATPP-NA/KDOPA-UTHYR+
DOPA-D-1-2- NMDACHA-KDOPA-D-1/D-1-5HT-2-BACH-M5HT-1-D5HT-2DOPA+
5HT-UADR-UADS-A-1ADR-A-2PAF5HT-2DOPA-D-25HT-UADR-U5HT-2-APROG+
ESTR+
DIUR+
PPAR-GIL-4BACTDOPA-D-25HT-1-A5HT-1-ADOPA-D-4+
5HT-1-AADR-A-1ADR-A-1ADR-A-15HT-1-AADR-A-1ACH-M+
Lipase-A-2ACH-M+
Lipase-A-2SY-CW5HT-1-A/2-C+
ADR-A-1EAA-GADR-A-1EAA-GCYC-GUDOPA-D-2/3+
5HT-1-ADOPA-D-4+
5HT-1-A+
DOPA-D-2DOPA-D-4+
DOPA-D-2+ ADR-A+/KI-PKAADR-A-1ADR-A-2ATPP-H/KCHA-CAEAA-G5HT-1-ADOPA-D-3DOPA-D-2EAA-GDNADOPA-1-2DOPA-1-2CHA-CADOPA-D-2+
mGluR5DOPA-D-2-
ADR-
HIST-H-1-
SXR+
OX-LP-5OX-LP-5NMDANMDA-
CHA-K(GIRK)CHA-K(GIRK)-
5HT-2-
ADR-A-2-
NMDAADR-1ADR-1-
ATPase-KATPase-K-
ADR-A-2+
5HT-3-
ESTR+
DOPA-D-2- 5HT-2-A-(indirect?)
ADR-(indirect?)
NEURTKI-P385HT-1-A+/ADR-A-1-DADR-A-2-C5HT-1-ACHA-CAACECHA-CAHERBGlucoRNEURTADR-A-1DOPA-D-2+ ADR-A+/5HT-2DOPA-D-2- 5HT-2ADR-B- HISTANDR+
ESTR+
DOPA-D-3/D-2+
5HT-2-A/CDOPA-D-2+ 5HT-2-BADR-BADR-1DOPA-D-2- 5HT-2ADR-B- HISTCHA-CACHA-K-?
5HT-UADR-UADS-A-1DOPA-D-2-
17
Figure S1(Cont)
NAME
MDL 72832
8-Methoxy-2-Propionamidotetralin
Bromocriptine Mesylate
N-Methylquipazine
3beta-Chloroandrostanone
(-)-Quinpirole
GR 127935 Hydrochloride
Dioxybenzone
Clemizole Hydrochloride
Perphenazine
Paroxetine Hydrochloride
MDL 73005EF
p-Iodoclonidine Hydrochloride
Ethynylestradiol 3-Methylether
Indirubin-3-Oxime
ARC 239 Dihydrochloride
Prochlorperazine Dimaleate
Trazodone Hydrochloride
Desipramine Hydrochloride
Chloropyramine Hydrochloride
Prazosin Hydrochloride
Cinanserin
Diphenhydramine Hydrochloride
Harmane Hydrochloride
Trimeprazine Tartrate
N-Butyl-N-Ethyl-2-(1-Naphthyloxy)Ethanamine
17alpha-Hydroxyprogesterone
Norethynodrel
3-Hydroxyflavone
Piperacetazine
Zimelidine Dihydrochloride Monohydrate
R(+)-6-Bromo-APB Hydrobromide
Methoxyverapamil
(+/-)-Chloro-APB Hydrobromide
SIB 1757
Benfluorex Hydrochloride
S-(-)-Eticlopride Hydrochloride
1-[1-(2-Benzo[B]Thienyl)Cyclohexyl]Pyrrolidine
Ethinyl Estradiol
Chlorpheniramine Maleate
2-[(4-Phenylpiperazin-1Yl)Methyl]2,3-Dihydroimidazo[1,2C]Quinazolin-5(6H)-One
6-Nitroquipazine
Scoulerine
Dextromethorphan Hydrobromide Monohydrate
Perphenazine
Vincamine
Promazine Hydrochloride
NAN-190
L(-)-Vesamicol Hydrochloride
(+)-Chlorpheniramine Maleate
Fluvoxamine Maleate
Zimelidine Dihydrochloride
(+/-)-SKF 82958
SKF 83565 Hydrobromide
S(-)-UH-301 Hydrochloride
6-Nitro-Quipazine Maleate
SKF 86466
Quipazine
Clovanediol Diacetate
GBLD-345
(-)-trans-(1S,2S)-U-50488 Hydrochloride
Naftifine Hydrochloride
(+/-)-SKF 38393, N-Allyl-, Hydrobromide
Dydrogesterone
Brinzolamide
(+/-)-Brompheniramine Maleate
Dyclonine Hydrochloride
beta-Caryophyllene Alcohol
Biochanin A, Dimethyl Ether
Picrotoxinin
Estradiol Cypionate
Chlorzoxazone
Ticlopidine Hydrochloride
BIO
Iofetamine Hydrochloride
6-Methoxy-1,2,3,4-Tetrahydro-9H-Pyrido[3,4B] Indole
SIB 1757
Enilconazole
Pelletierine Hydrochloride
Nefopam Hydrochloride
S(-)-DS 121 Hydrochloride
Hymecromone
3-[2-[4-(2-Methoxyphenyl)Piperazin-1-Yl]Ethyl]-1,5-Dimethylpyrimido[5,4-B]Indole-2,4-Dione
Ropinirole Hydrochloride
Dipropyl-6,7-ADTN
Fluvoxamine Maleate
R(-)-SCH-12679 Maleate
SCH 23390
Quipazine Maleate
YS-035
Fluoxetine Hydrochloride
Tropanyl 3,5-Dimethylbenzoate
Metixene Hydrochloride
Oxybutynin Chloride
2-Chloroadenosine
Strophanthidin
Atracurium Besylate
Guanabenz Acetate
Guanabenz Acetate
Guanabenz Acetate
Amitraz
Arecaidine Propargyl Ester Hydrobromide
Methomyl
Guanfacine Hydrochloride
Clonidine
Guanabenz Acetate
Uk 14304
R-(-)-Desmethyldeprenyl Hydrochloride
2,6-Dimethoxyquinone
Baicalein
Clonidine Hydrochloride
Tranylcypromine Sulfate
Cinnarizine
Thyroxine
(-)-Huperzine A
Tizanidine Hydrochloride
Propachlor
Amoxapine
Physostigmine Sulfate
SU 6656
Activity 1
5HT-1-A+
ML+
DOPA-D-2+
5HT-3+
DOPA-D-2/3+
5HT-1-B/DUVHIST-1DOPA-D-1-25HT-U5HT-1-A+
ADR-A-2+
PROG+
KI-CDADR-A-2-BDOPA-D-25HT-1-2ADR-UHIST-H-1ADR-A-15HT-25HT-UGABA-A
HIST-H-15HT-1-A+
ESTRESTR-
Activity 2
CREB+
SSTATIN
5HT-1-D+?
ADR-A-1ADR-A-1ESTR+
KI-GSK5HT-1-AADR-A-1ADR-A-15HT-U-
HIST-H-1- 5HT-25HT-2+(high
5HT-Udoses)
ACH-M- ADR-B-
HIST-H-15HT-2-A+
CHA-
PR+
PR+
DOPA-D-1/D-2-?
5HT-2-B-?
5HT-UDOPA-D-1+
CHA-CADOPA-D-1+
HIST-1EAA-GmGluR5RED-HMG-?
DOPA-D-2/D-3DOPA-UPROG+
ESTR+
5HT-UADR-UADR-A-15HT-UGABA-A+
ADR-A-1EAA-GNMDADOPA-D-1-2- ADR-A-1CHA-CAADR-A-1CHA-NADOPA-1-2/4- 5HT-2-A/C5HT-1-AADR-A-1ACH-SY5HT-UADR-U5HT-U5HT-UDOPA-D-1+ DOPA-D-2+
DOPA-D-1+
5HT-1-A+/ADR-A-1-D5HT-1-ADOPA-D-2- ADR-A-15HT-UADR-A-25HT-2-A/3+
GABA-A+
OPI-K+
EPOX-SQDOPA-D-1+
PROG+
ANC-25HT-UCHA-NA-
ADR-A-1?
SXR+
HIST-H-1-
ADR-A-1- 5HTOPI-S+
DOPA-U-?
ACH-M-
ADR-A-1-
HIST-H-1-
ADR-A-2-CHIST-H-1- 5HT-2-
BZD+
FUNG-
ADR-U-
ESTR+
GABAESTR+
CHA-K-(Ca)P2Y-R-12KI-GSK-3-
PLAT-ADKI-PDK1-
MAOCHA-NAEAA-GSY-CW-
mGluR5FUNG-
HIST-H-1-
GLY-RESTR+
5HT-U5HT-UDOPA-D-3-
ADR-UDOPA-U-
ADR-A-1+
DOPA-D-3/2+
DOPA-D-2+
5HT-UDOPA-D-1DOPA-D-15HT-2-A/3+
CHA-CAADR-A-1MAO-A5HT-U5HT-3ACH-MACH-MADS+
ATPP-Na/KACH-N/MADR-A-2+
ADR-A-2+
ADR-A-2+
ADR-A-2+
ACH-M-2+
ACHADR-A-2+
ADR-A-2+
ADR-A-2+
ADR-A-2+
ADR-A-2+
MAO-BBACTPO-RDNAADR-A-2+
MAOCHA-CATHYR+
ES-ACHADR-A-2+
HERBADR-A-1ADR-UES-ACHKI-SRC-
5HT
ADS-A-1ADR-U- COX-
INSECT-
DOPA-D-2?
HIST-H-1-
5HT-2-A?
DOPA-D-2-ACH-M-
ADR-U-?
5HT-U-
5HT-2-?
18
Cluster #
S.D.
Waking
Activity
Rest
Total
# Rest
Bouts
Rest Bout
Length
Rest
Latency
Activity
Total
Hierarchical Clustering
-3
-2
-1
0
1
2
3
Waking
Activity
Rest
Total
# Rest
Bouts
Rest Bout
Length
Rest
Latency
Activity
Total
Figure S2
K-Means Clustering (k=24)
1
2
3
4
5
6
7
8-9
10
11
12
13
14
15
16
17
18
24
|
19
Waking
Activity
Rest
Latency
Activity
Total
# Rest
Bouts
Rest Bout
Length
Rest
Total
Figure S3
NAME
1
2
3
4
5
6
7
Activity 1
Activity 2
SB 206553 Hydrochloride
Fenoterol Hydrobromide
Clenbuterol Hydrochloride
Retinoic Acid p-Hydroxyanilide
Betamethasone
Prednicarbate
Budesonide
Diclofenac Sodium
Monobenzone
Spectinomycin Hydrochloride
Sulfaguanidine
Benzyl Penicillin Potassium
Physcion
Leflunomide
Estrone Acetate
I-OMe-Tyrphostin AG 538
(-)-Eseroline Fumarate
Kynurenine
Chlorophyllide Cu Complex Na Salt
Enoxolone
Chlorthalidone
Dinitolmide
SU 6656
Physostigmine sulfate
5-beta-12-Methoxy-4,4-Bisnor-8,11,13-Podocarpatrien-3-one
14-Methoxy-4,4-Bisnor-8,11,13-Podocarpatrien-3-one
3-beta-Acetoxydeoxodihydrogedunin
11-alpha-Acetoxykhivorin
Carbenoxolone Sodium
Glycine
Senecrassidol 6-Acetate
5HT-2-B/CADR-B+
ADR-B-2+
ADR-UADR-UAPOP+
ARACHARACHARACHARACHSY-PGBACTBACTBACTBACTDH-HYOESTR+
KI-IGF-1OPI-M+
Cisapride
Tegaserod Maleate
Ethylene Glycol-Bis(2-Aminoethylether)-N,N,N,N-Tetraacetic Acid
Benztropine
MG-624
(+)-Tubocurarine Chloride
Pancuronium Bromide
Mecamylamine Hydrochloride
(-)-Nicotine
Nicotine Ditartrate
1-Benzyl-1-Methyl-4-Cyclopentylmethoxycarbonylpiperidinium Br
Chloroethylclonidine Dihydrochloride
Bretylium Tosylate
Bopindolol Malonate
Desoxycorticosterone Acetate
Flunisolide
Hydrocortisone
6alpha-Methylprednisolone
Betamethasone Valerate
Ursolic Acid
Dexamethasone
Medrysone
Betamethasone
Flumethasone
Cefoxitin Sodium Salt
Mefloquine Hydrochloride
Tetrandrine
Flecanide
Benzamil Hydrochloride
Pentamidine Isethionate
Fenoldopam
Propentofylline
Diplosalsalate
Fosfosal
SKF 94836
Ro 20-1724
Equilin
Allopregnanolone
Valproate Sodium
Atrazine
Diphenylpyraline Hydrochloride
Cyclosporin A
Austricine
2-Cyclooctyl-2-Hydroxyethylamine Hydrochloride
Theaflavin Digallate
Finasteride
Bufexamac
Diflunisal
Fenoprofen
Fenoprofen Calcium Salt Dihydrate
Piroxicam
Capsazepine
5-Aminopentanoic Acid Hydrochloride
Catechin Tetramethylether
Aminophenazone
Oxethazaine
Sinapic Acid Methyl Ether
IEM-1460
6,7-Dichloroquinoxaline-2,3-Dione
7-Chlorokynurenic Acid
SDZ 220-581
L-689560
5HT-4+
5HT-4+
ACE-2ACH-MACH-NACH-NACH-NACH-NACH-N+
ACH-N+
ACH-TADR-A-1ADR-BADR-B-1COR-M+
ARACHARACHARACHARACHARACHARACHARACHARACHARACHARACHARACHSY-CWCALMCHA-CACHA-NACHA-NA-CaDNADOPA-D-1-A+
ES-P-CES-P-C-?
ES-P-CES-P-C-3ES-P-C-4ES-P-C-4ESTR+
GABA-A+
HDACHERBHIST-H-1IMLP-P-A-2NEURTNf-kB-?
RED-5ASY-PGSY-PGSY-PGSY-PGSY-PGVAN-
ACH+(indirect)
5HT-2-BCHEL+
DOPA-U-
CHA-K-
ACH+
5HT-3-
COXCOXCOXCOXCOXCHA-Na+
OX-C-
EAA-GEAA-GEAA-GEAA-GEAA-G-
AMPA-RNMDANMDANMDANMDA-
NMDA-
(-)-MK-801Hydrogen Maleate
L-701324
L-701324
L-701324
Dizocilpine Maleate
(+)-MK-801
Spiperone
Azobenzene
EAA-GEAA-GEAA-GEAA-GEAA-GEAA-GDOPA-D-2-
NMDANMDANMDANMDANMDANMDA5HT-1-A-
SB 228357
R(-)-Isoproterenol (+)-Bitartrate
Amitryptiline Hydrochloride
Amoxapine
Telmisartan
Cefamandole Sodium
TCPOBOP
Cinnarazine
Psora-4
Pimozide
Mitotane
Oxiconazole Nitrate
Ornithine alpha-Ketoglutarate
5HT-2-B/CADR-B-2+
ADR-UADR-UANG-2-1SY-CW CAR+
CHA-CACHA-KDOPA-D-2ESTRFUNG-
N-Desmethylclozapine
Coumaphos
S,S,S,-Tributylphosphorotrithioate
Fiduxosin Hydrochloride
Mianserine Hydrochloride
Bepridil Hydrochloride
Aconitine
Loxapine Succinate
Ibudilast
Endosulfan
5HT-2-CACHACHADR-A-1ADR-UCHA-CACHA-NA+
DOPA-D-1ES-P-C-4GABA-AMAO-A-
ACH-M1+
Amoxapine
ODQ
L-701252
Megestrol Acetate
1S,9R-Hydrastine
Chlordane
Dacthal
Propazine
7-Oxocallitrisic Acid, Methyl Ester
ADR-UCYC-GUEAA-GESTR+
GABA-AGABA-AHERBHERB-
5HT-U-
INSECT-
ATPP-Ca/MgATPP-Na/K-
Methiothepin Maleate
Mianserin
Cyproterone Acetate
(1S,9R)-Beta-Hydrastine
Pregnenolone
Astemizole
Bromo-3-Hydroxy-4-(Succin-2-Yl)-Caryolane Gamma-Lactone
5HT-1-2ADR-UANDRGABA-AGABA-AHIST-1-
DOPA-D-2ADR-A-2-
HIST-
5HT-U-(weak)
5HT-U-(weak)
LIPCOR+
LIPCOR+
LIPCOR+
IMIM-
KI-GFPL-
ALOX5-
GluR+
GluR+
ACH-(weak)
DIUR+
KI-SRCES-ACH-
NMDA+
HIST-
ADR-A-2+
NA/K-ATPaseMR+
LIPCOR+
LIPCOR+
LIPCOR+
LIPCOR+
LIPCOR+
LIPCOR+
LIPCOR+
LIPCOR+
LIPCOR+
LIPCOR+
LIPCOR+
BACT-
IMIMIMIMIMIMIMIMIMIMIM-
GluR+
GluR+
GluR+
GluR+
GluR+
GluR+
GluR+
GluR+
GluR+
GluR+
GluR+
CHA-K-(Ca+)
ADR-A-2+
ADS-/+
CHA-CAGABA+
PNMT-
ADR-B-1+
5HT-U5HT-UPPAR-G+
BACT-
5HT-2-A-
CHA-K-
HIST-H-1-
DOPA-D-2-
ACH-M-
5HT-2-ACHA-NA?
ADR-A-1-
DOPA-U-
ADR-A-2CHA-NA-
HISTCHA-K-
5HT-2-A/2-C-
DOPA-D-2-
5HT-2- (indirect?)
NMDAPROG+
5HT-2-A/2-C-
CHA-K-
20
8
9
10
11
Waking
Activity
Rest
Latency
Activity
Total
# Rest
Bouts
Rest Bout
Length
Rest
Total
Figure S3(Cont)
Activity 1
Activity 2
Dichlorodiphenyltrichloroethane
Methoxychlor
Strobane
Toxaphene
Aldrin
Heptachlor
NAME
CHA-NA+
CHA-NA+
CHA-NA+
CHA-NA+
GABA-AGABA-A-
INSECTINSECTINSECTINSECTINSECTINSECT-
ATPP-Ca/Mg- ATPP-Na/KATPP-Ca/Mg- ATPP-Na/K-
Disulfoton
(+)-Hydrastine
ACHGABA-A-
INSECT-
Dihydroergotamine Mesylate
Amperozide Hydrochloride
Clozapine
Cephaeline Dihydrochloride Heptahydrate
Chlormadinone Acetate
Flutamide
Halofantrine Hydrochloride
Haloperidol
Chlorpromazine Hydrochloride
3-alpha-Bis-(4-Fluorophenyl)Methoxytropane
(-)-MK-801 Hydrogen Maleate
Spermine Tetrahydrochloride
Epiandrosterone
CGP-13501
2-Methoxyestradiol
Deptropine Citrate
Astemizole
Astemizole
Clemastine
Oxatomide
Promethazine Hydrochloride
Terfenadine
3-(1H-Imidazol-4-Yl)Propyl Di(P-Fluorophenyl)Methyl Ether
Tyrphostin AG 494
Danazol
1,4-PBIT Dihydrobromide
Roxithromycin
Finasteride
Menadione
2,4-Dihydroxychalcone 4-Glucoside
Benzanthrone
Derrusnin
Hexylresorcinol
Homopterocarpin
5HT-1-D+
5HT-25HT-25HT-4+?
ANDRANDRCHA-KDOPA-D-1-2DOPA-D-2DOPA-UEAA-GEAA-GESTRGABA-B+
HIF-1HISTHIST-1HIST-1HIST-1HIST-1HIST-1HIST-1HIST-H3-/+
KI-GFEPLHRHNO-SPRT-50SRED-5AVITA+
5HT-1-A+
DOPA-D-2DOPA-D-2-
ADR-A-2-A
DOPA-D-2/D-3
ADR-
HIST-H-1-
ESTR+
PR+
NMDADOPA-D-1ACH-MNMDAEAA-G+
CHA-CA-
CHA-K5HT1-2-
MDL 72832
Metergoline Phenylmethyl Ester
Metergoline
GR 4661
LY-367265
R(+)-Terguride
LY-53857
R-(-)-Fluoxetine Hydrochloride
Vincamine
Oxymetazoline Hydrochloride
Yohimbine Hydrochloride
Beclomethasone Dipropionate
Rutilantinone
Acetopromazine Maleate Salt
Azaperone
Triflupromazine Hydrochloride
Haloperidol
L-741626
Thioridazine Hydrochloride
Mesulergine Hydrochloride
CNS-1102
trans-Dehydroandrosterone
Tolnaftate
SB 205384
5HT-1-A+
5HT-1-D5HT-1-D5HT-1-D+
5HT-2-A5HT-2-B5HT-25HT-U5HT-UADR-A-1ADR-A-1ADR-A-1ADR-A-1+
ADR-A-2ARACHBACTCHA-CADOPADOPADOPA-D-1/D-1DOPA-D-1-2DOPA-D-2DOPA-D-2DOPA-D-2+
EAA-GESTR+
FUNGGABA-A+
GABA-A+
HERBHISTHISTHISTHIST-1HIST-1KI-CLKKI-TYROPI-DTRP-M8+
ADR-A-1?
5HT-25HT-2-
LY-165163
BP554
Metergoline
Dihydroergotamine Methanesulfonate
Ketanserin Tartrate
Pirenperone
Ketanserin Tartrate
Paroxetine Hydrochloride Hemihydrate
Trihexyphenidyl Hydrochloride
Quinacrine Hydrochloride
Pancuronium Bromide
Doxazosin Mesylate
WB-4101 Hydrochloride
Ifenprodil Tartrate
Nicergoline
3,4-Dimethoxyflavone
Androsterone Acetate
Totarol
Phenamil
Azaperone
Spiperone Hydrochloride
Benperidol
Spiperone
Droperidol
DO 897/99
Risperidone
S(-)-Lisuride
Dihydroergocristine
Dihydroergocristine
PD 168077
Ro 25-6981 Hydrochloride
Butylparaben
Hydroxyprogesterone Caproate
Alfadolone Acetate
Reserpine
Saquinavir Mesylate
Nomegestrol Acetate
Ethynodiol Diacetate
Oxaprozin
5HT-1-A+
5HT-1-A+
5HT-1-D5HT-1-D+
5HT-25HT-25HT-25HT-UACH-M-1ACH-M+
ACH-NADR-A-1ADR-A-1ADR-A-1ADR-A-1AHRANDR+
BACTCHA-NADOPADOPA-D-2DOPA-D-2DOPA-D-2DOPA-D-2DOPA-D-2/D-3DOPA-D-2DOPA-D-2+
DOPA-D-2+
DOPA-D-2+
DOPA-D-2+
DOPA-D-4+
EAA-GESTR?
ESTR+
GABA-A+
NEURTPROTPROG+
PROG+
SY-PG-
DOPA-D-2-
Spiroxatrine
AMI-193
AMI-193
WB-4101 Hydrochloride
Amoxapine
8-Cyclopentyl-1,3-Dipropylxanthine
Strophanthidinic Acid Lactone Acetate
Vinpocetine
Medroxyprogesterone Acetate
Domperidone
3-Methoxymorphinan Hydrochloride
L-701252
4-Androstene-3,17-Dione
Tracazolate Hydrochloride
Loperamide
BWB70C
5HT-1-A5HT-25HT-2ADR-A-1ADR-UADS-A-1ATPP-Na/KCHA-NADEC-ORDOPA-D-2EAA-GEAA-GESTRGABA-AOPI+
OX-LP-5-
3-Methyl-6-(3-[Trifluoromethyl]Phenyl)-1,2,4-Triazolo[4,3-B]Pyridazine
Prometryn
Meclozine Dihydrochloride
Meclizine Hydrochloride
1-[(4-Chlorophenyl)Phenyl-Methyl]-4-Methylpiperazine
Methapyrilene Hydrochloride
Terfenadine
TG 003
Nicolsamide
Naltriben Methanesulfonate
Icilin
Niloticin
Triptophenolide
12
13
HIST-H-1-
NMDAGABA-A+
ACH-MCHA-KCHA-KACH-M-?
ACH-MCHA-K-
5HT-2-
calmodulin-
ESTRNF-kBANDR-
DOPA+
DOPA+
5HT-UDOPA+/partial
ADR-U-
ADS-A-1-
ADR-U-?
ADR-U-?
CHA-NAADR-A-2+
5HT-2-?
5HT-2-?
LIPCOR+
IM-
HIST5HT-2-BNMDA-
ACHACH-MCHA-K-
5HT-25HT-2-CNMDAANDR+
ADR-
GluR+
CHA-K-
GABA-A-
ACHACH-
CHA-K-
5HT-25HT-1-A+
DOPA?
DOPA-D-2-?
DOPA?
DOPA+
ADR-A-2-A
Lipase-A-2-
OX-LP-5-
EAA-G-
NMDA-
DOPA-D-2/D-3
CHA-K(GIRK)-
CHA-CA-?
HIST5HT-1-ACHA-K5HT-1-ACHA-KDOPA-D-3+
5HT-25HT-2-BADR-A+/ADR-A+/CREB+
ACH5HT-2-A5HT-2-A5-HA-1-A
ADR-B-
ADR-A-1
HIST-
5HT-25HT-2SSTATIN
NMDA-NR2-BESTR+
PR+
TNF-alpha+
ANDRESTR+
COX-
PROL-
DOPA-D-2DOPA-D-2DOPA-D-2-
ADR-A-2-
5HT-U-
ES-P-C-1ANDRNMDANMDAANDR+
GABA-A+
ESTR+
PR+
OPI-S+
DOPA-U-?
21
Waking
Activity
Rest
Latency
Activity
Total
# Rest
Bouts
Rest Bout
Length
Rest
Total
Figure S3(Cont)
NAME
14
Activity 1
1-(2-Methoxyphenyl)-4-(4-Succinimidobutyl)Piperazine
Spiroxatrine
WAY-100635 Maleate Salt
RU-24969
CINANSERIN
Opipramol Dihydrochloride
Norcyclobenzaprine
Alcuronium Chloride
Quinacrine Dihydrochloride Dihydrate
Pancuronium Bromide
5HT-1-A5HT-1-A5HT-1-A5HT-1-A/2-C+
5HT-25HT-25HT-2-AACH-MACH-M+
ACH-M+
ACH-N2-[4-(2-Methoxyphenol)Piperazin-1-Yl]Methyl-6-Methyl2,3Dihydroimidazo[1,2C]Quinazolin-5(6H)One
ADR-A-1ADR-A-12-[[B-(4-Hydroxyphenol)Ethyl]Aminoethyl]-1-Tetralone
ADR-A-1Doxazosin Mesylate
ADR-A-1Ifenprodil Tartrate
ADR-A-1Ifenprodil Tartrate
ADR-A-1L-765314
ADR-A-1Nicergoline
ADR-A-1Prazosin
ADR-A-15-Methyl-Urapidil
ADR-A-2MK-912
ADR-A-2RX 821002 Hydrochloride
ANDR+
Testosterone Propionate
APOPCepharanthine
ATPP-H/KOmeprazole
ATPP-NA/KGitoxin
CHA-CA(+/-)-Methoxyverapamil Hydrochloride
CHA-NAMexiletine Hydrochloride
CYC-GUODQ
DNACoralyne Chloride Hydrate
DOPA-BHFusaric Acid
DOPA-D-2Domperidone Maleate
DOPA-D-2Droperidol
DOPA-D-2Risperidone
DOPA-D-2+
Dihydroergocristine Methanesulfonate
DOPA-D-3GR 103691
DOPA-U1-Benzo[B]Thien-2-Yl-N-Cyclopropylmethylcyclohexanamine
EAA-G2-Methyl-6-(Phenylethynyl)Pyridine
EAA-GLY-235959
ES-P-C3-Isobutyl-1-Methylxanthine
ES-P-C-4Papaverine Hydrochloride
SY-CWAjmalicine Hydrochloride
TO-2Amsacrine hydrochloride
Activity 2
DOPA-D-2DOPA-D-4+
ADR-A-2-
OPI-S+
HIST-H-1-
Lipase-A-2Lipase-A-2-
OX-LP-5OX-LP-5-
EAA-GEAA-G-
NMDANMDA-
DOPA-D-2-
CHA-K(GIRK)CHA-K(GIRK)-
5HT-1-A-
ESTR+
NF-KB-
IM-
ADR-B-
5HT-2ADR-A+/-
ADR-B5HT-2-
HIST-
mGluR5NMDAADS-
Amodiaquine Dihydrochloride
Hycanthone
15
Trazodone Hydrochloride
P-MPPF Dihydrochloride
WAY-100635 Maleate
MDL-73005EF
GR 127935 Hydrochloride
Clozapine
N-Methylquipazine
(+)-Chlorpheniramine Maleate
Diphenhydramine Hydrochloride
Fluvoxamine Maleate
Paroxetine Hydrochloride
Zimelidine Dihydrochloride Monohydrate
Terazosin Hydrochloride
Vincamine
5HT-1-25HT-1-A5HT-1-A5HT-1-A+
5HT-1-A+/5HT-1-B/D5HT-25HT-3+
5HT-U5HT-U5HT-U5HT-U5HT-U5HT-UADR-A-1ADR-A-1ADR-A-13-[2-[4-(2-Methoxyphenyl)Piperazin-1-Yl]Ethyl]-1,5Dimethylpyrimido[5,4-B]Indole-2,4-Dione ADR-A-1+
ADR-A-2+
p-Iodoclonidine Hydrochloride
ADR-A-2-BARC 239 Dihydrochloride
ADR-BCarvedilol Tartrate
ADR-UDesipramine Hydrochloride
CHA-CA(+/-)-Verapamil Hydrochloride
CHA-CAMethoxyverapamil
CHA-CAPhloretin
CHA-CAVerapamil Hydrochloride
CHA-CAYS-035
CHA-CAYS-035 Hydrochloride
DOPA+
Apomorphine Hydrochloride
DOPA-1-2Trifluoperazine Dihydrochloride
DOPA-1-2Trifluoperazine Hydrochloride
DOPA-D-1/D-2-?
Piperacetazine
DOPA-D-1+
(+/-)-SKF 38393, N-Allyl-, Hydrobromide
DOPA-D-1-2Perphenazine
DOPA-D-1-2Perphenazine
DOPA-D-2Mesoridazine
DOPA-D-2Prochlorperazine Dimaleate
DOPA-D-2Risperidone
Quinelorane Dihydrochloride
DOPA-D-2/3+
S(-)-Lisuride
DOPA-D-2+
DOPA-D-2+
B-HT 920
DOPA-D-2+
Dipropyl-6,7-ADTN
Piribedil Maleate
DOPA-D-3/D-2+
17alpha-Hydroxyprogesterone
ESTRESTRNorethynodrel
ESTR+
Estrone
GABA-A
Harmane Hydrochloride
GABA-A+
Scoulerine
GlucoRCortexolone Maleate
HIST-1HIST-H-1Chloropyramine Hydrochloride
Indirubin-3-Oxime
KI-CDKI-P38PD 169316
KI-PKAH-89
NEURTReserpine
P450Diosmetin
PPAR-GGW 9662
PROG+
Ethynylestradiol 3-Methylether
UVDioxybenzone
ADR-A-1-
5HT-2+(high doses)
5HT-U-
DOPA-D-4+
ADR-A-1ADR-A-1-D5HT-1-D+?
DOPA-D-2-
ADR-A-2-C-
1-(3-Chlorophenyl)Piperazine Dihydrochloride
NAN-190
S(-)-UH-301 Hydrochloride
Buspirone
R(+)-UH-301 Hydrochloride
Quipazine
Quipazine Dimaleate
Dl-p-Chlorophenylalanine Methyl Ester Hydrochloride
6-Nitroquipazine
Chlorpheniramine Maleate
Fluvoxamine Maleate
6-Nitroquipazine Maleate
Zimelidine Dihydrochloride
Rescinnamin
L(-)-Vesamicol Hydrochloride
5HT-3ADR-A-1-
ADR-
ADR-UHIST-H-1-
HIST-H-1-
ADR-U-
ADS-A-1-
HIST-H-1-
CHA-NA5HT
5HT-1-AADR-15HT-U-
ACH-M-
ADR-B-
CHA-K-?
COMTADR-1ADR-15HT-2-B-?
THATPase-KATPase-K-
ADR-A-1ADR-A-15HT-2-A-(indirect?)ADR-(indirect?)
ADR-A-1HIST-H-15HT-25HT-2ADR-BHIST5HT-2-BADR-A-2+
5HT-3-
5HT-2-A/CPR+
PR+
5HT-2-A+
ADR-A-1-
CHAADR-A-1-
5HT-
KI-GSK-
IL-4ESTR+
2beta-Dihydroxychalcone
3-Hydroxyflavone
Phenazopyridine Hydrochloride
Totarol-19-Carboxylic Acid
16
2-[(4-Phenylpiperazin-1-Yl)Methyl]-2,3-Dihydroimidazo[1,2-C]Quinazolin-5(6H)-One
SKF 86466
Verapamil
Promazine Hydrochloride
(+/-)-Chloro-APB Hydrobromide
(+/-)-SKF 82958
R(+)-6-Bromo-APB Hydrobromide
SKF 83565 Hydrobromide
S-(-)-Eticlopride Hydrochloride
L-750667
PD 168077 Maleate
1-[1-(2-Benzo[B]Thienyl)Cyclohexyl]Pyrrolidine
Dextromethorphan Hydrobromide Monohydrate
SIB 1757
Syrosingopine
6(5H)-Phenanthridinone
Ethinyl Estradiol
Benfluorex Hydrochloride
Suprofen Methyl Ester
Dihydrogeduninic Acid, Methyl Ester
5HT-1-2+
5HT-1-A5HT-1-A5HT-1-A+
5HT-1-A+
5HT-1-A+/5HT-2-A/3+
5HT-2-A/3+
5HT-SY5HT-U5HT-U5HT-U5HT-U5HT-UACEACH-SYADR-A-1ADR-A-2CHA-CADOPA-1-2/4DOPA-D-1+
DOPA-D-1+
DOPA-D-1+
DOPA-D-1+
DOPA-D-2DOPA-D-2/D-3DOPA-D-4DOPA-D-4+
DOPA-UEAA-GEAA-GNEURTPARPPROG+
RED-HMG-?
SY-PG-
DOPA-D-2ADR-A-1-D-
ADR-A-2-C-
ADR-U-
HIST-H-1-
5HT-2-A/C-
ACH-M-
ADR-A-1-
ADR-A-1-
HIST-H-1-
5HT-2-
NMDAmGluR5-
OPI-S+
DOPA-U-?
ESTR+
SXR+
COX-1-
COX-2-
DOPA-D-2+
22
Waking
Activity
Rest
Latency
Activity
Total
# Rest
Bouts
Rest Bout
Length
Rest
Total
Figure S3(Cont)
NAME
17
18
19
20
21
22
23
24
Activity 1
NAN-190 Hydrobromide
N-Butyl-N-Ethyl-2-(1-Naphthyloxy)Ethanamine
CGS-12066A Maleate
Quipazine Maleate
Tropanyl 3,5-Dimethylbenzoate
(+/-)-Brompheniramine Maleate
Amitriptyline Hydrochloride
Fluoxetine Hydrochloride
Nefopam Hydrochloride
Methomyl
Metixene Hydrochloride
Oxybutynin Chloride
Arecaidine Propargyl Ester Hydrobromide
Atracurium Besylate
Amitraz
Clonidine
Clonidine
Guanabenz Acetate
Guanabenz Acetate
Guanabenz Acetate
Guanabenz Acetate
Tizanidine Hydrochloride
UK 14304
2-Chloroadenosine
Adenosine
MRS-1220
Brinzolamide
Strophanthidin
2,6-Dimethoxyquinone
Chrysene-1,4-Quinone
Cinnarizine
Chlorzoxazone
Chrysin
R(-)-SCH-12679 Maleate
SCH 23390
S(-)-3PPP hydrochloride
(-)-Quinpirole
S(-)-DS 121 Hydrochloride
Ropinirole Hydrochloride
SIB 1757
Naftifine Hydrochloride
(-)-Huperzine A
Biochanin A, Dimethyl Ether
Estradiol Cypionate
Picrotoxinin
GBLD-345
Propachlor
Trimeprazine Tartrate
BIO
6-Methoxy-1,2,3,4-Tetrahydro-9H-Pyrido[3,4B] Indole
Phenelzine sulfate
Phenelzine Sulfate
Tranylcypromine Hydrochloride
Tranylcypromine Sulfate
R-(-)-Desmethyldeprenyl Hydrochloride
8-Methoxy-2-Propionamidotetralin
(-)-trans-(1S,2S)-U-50488 Hydrochloride
Ticlopidine Hydrochloride
CY-3988
Baicalein
Dydrogesterone
Estradiol
Enilconazole
Tetrac
Thyroxine
3beta-Chloroandrostanone
beta-Caryophyllene Alcohol
Clovanediol Diacetate
Hymecromone
Iofetamine hydrochloride
Pelletierine Hydrochloride
Pramoxine Hydrochloride
5HT-1-A5HT-1-A+
5HT-1-B+
5HT-2-A/3+
5HT-35HT-U5HT-U5HT-U5HT-U5HT-UACHACH-MACH-MACH-M-2+
ACH-N/MADR-A-1ADR-A-2+
ADR-A-2+
ADR-A-2+
ADR-A-2+
ADR-A-2+
ADR-A-2+
ADR-A-2+
ADR-A-2+
ADR-A-2+
ADR-A-2+
ADR-A-2+
ADS+
ADS+
ADS-A3ANC-2ATPP-Na/KBACTBACTCHA-CACHA-K-(Ca)CHA-NACHA-NADIUR+
DOPA-D-1DOPA-D-1DOPA-D-2DOPA-D-2/3+
DOPA-D-2+
DOPA-D-3DOPA-D-3/2+
EAA-GEPOX-SQES-ACHESTR+
ESTR+
GABAGABA-A+
HERBHIST-1HIST-H-1KI-GSK-3MAOMAOMAOMAOMAOMAO-AMAO-BML+
OPI-K+
P2Y-R-12PAFPO-RDNAPROG+
PROG+
SY-CWTHYR+
THYR+
Fluspirilen
DOPA-D-2DOPA-D-2+
BRL 15572
CY 208-243
7-Hydroxy-DPAT
1-[1-(2-Benzo[B]Thienyl)Cyclohexyl)]Piperidine
Dihydrocapsaicin
5HT-1-DDOPA-D-1+
DOPA-D-2+
DOPA-D-3+
DOPA-UVAN+
Clioquinol
FUNG-
Cyclopiazonic Acid
Tert-Butyl-Bicyclo[2.2.2]Phosphorothionate
Oxibendazole
Indomethacin Morpholinylamide
CHA-CA-iES-P-C-10
GABA-ANEMOSY-PG-
(+/-)-Vesamicol Hydrochloride
1,3-Diethyl-8-Phenylxanthine
Ciclopirox Olamine
DCEBIO
Spermine
Zardaverine
Loratadine
L-703606 Oxalate
Fenofibrate
Tetradecylthioacetic acid
Niflumic Acid
Dioxybenzone
Gedunol
ACH-SYADS-A-1PERMCHA-K+
EAA-GES-P-C-3HIST-1NK-1PPAR-A+
PPAR-A+
SY-PGUV-
Thioxolone
R(-)Apomorphine Hydrochloride Hemihydrate
Avermectin B1
Clofibrate
Indole-3-Carbinol
Lysergol
ANC-2DOPA+
GABA-A+
PPAR-A+
Activity 2
ADR-A-15HT-1-D+
ADR-UADR-U-
HIST-H-1-
ADR-UDOPA-UINSECT-
ADS-A-1ADR-U-
COX-
HIST-H-1-
DOPA-D-2-
ACH-M-
CREB+
SSTATIN
mGluR5FUNG-
ESTR+
GLY-RBZD+
KI-PDK1-
DOPA-D-2?
DOPA-D-2?
5HT-2-A?
5HT-2-A?
PLAT-AD-
ESTR+
FUNG-
SXR+
DOPA-D-1-2/4+
MICROTUBECOX-1-2-
ES-P-C-IVATPP-Na/K-
Nf-kB
EAA-G+
NMDA-
CHA-K(A5)-
COX-
AE2-
COMT-
TH-
LP-P-A-2+
Chol-11-Enic Acid
23
Correlation
Matrix
NAN190
NAN190
3 S.D.
2
2
1
1
3
0
0
4
-1
-2
-3 S.D.
-1
-2
-3
4
5
4
5
6
-0.5
-0.5
0.15
3
1.5
2
0.45
1
betamethasone
1
betamethasone
3
1
45
15
0
4.5
2
2
1
3
0
4
-1
-2
-3 S.D.
1
1
correlation
3 S.D.
2
1
2
0.5
0.5
3
-1
5
-2
6
15
µM
45
15
4.5
1.5
0.45
0.15
-3
20
1.5
4
0
0
0.45
5
0.15
3
4
5
-0.5
-0.5
6
0.15
2
0.45
1
4.5
1.5
µM
45
15
waking
activity
activity
total
6
rest
latency
# rest
bouts
10
rest bout
length
5
ropinirole
ropinirole
3
3 S.D.
2
1
1
1
45
15
0
0
4.5
3
2
3
4
-1
1
1
2
0.5
0.5
-1
4
5
0.15
6
0
0
1
2
3
4
5
6
-0.5
-0.5
0.15
µM
0.45
clonidine correlation stablity
clonidine-dosestable
3
3 S.D.
2
1
1
1
45
15
0
0
4.5
3
1.5
4
0.45
5
0.15
6
2
2
3
4
-1
1
1
correlation
waking
activity
1.5
0.45
1.5
-3 S.D.
-3
20
activity
total
15
rest
latency
# rest
bouts
µM
45
15
4.5
1.5
0.45
0.15
10
rest bout
length
rest
total
5
4.5
-2
-2
6
45
15
5
1
2
0.5
0.5
-1
3
1
1
0
0
4.5
3
0.5
0.5
4
5
0.15
6
-0.5
-0.5
1
2
0.5
0.5
3
1.5
4
0.45
5
0.15
6
0
1
2
3
4
5
6
0
-0.5
-0.5
0.15
µM
0.45
waking
activity
activity
total
rest
latency
rest bout
length
rest
total
# rest
bouts
-3-3 S.D.
4.5
1.5
-2-2
20
6
1
4.5
0
-1-1
15
5
1
45
15
45
15
11
10
4
pergolide
3 S.D.
22
0
3
0.15
2
0.45
1
1.5
µM
3
0
0
correlation
1.5
0.45
4.5
waking
activity
20
activity
total
rest
latency
rest bout
length
rest
total
# rest
bouts
15
-3-3 S.D.
1
2
pergolide
5
6
1
45
15
-2-2
µM
45
45
15
15
4.5
4.5
1.5
1.5
0.45
0.45
0.15
0.15
5
1
45
15
-1-1
10
4
correlation
2
0.15
waking
activity
1
buspirone
3 S.D.
2
2
5
-0.5
-0.5
µM
3
45
45
15
15
4.5
4.5
1.5
1.5
0.45
0.45
0.15
0.15
0
0
0.45
-3 S.D.
-3
20
activity
total
15
rest
latency
10
rest bout
length
rest
total
# rest
bouts
5
4.5
1.5
-2
-2
6
45
15
5
buspirone
Buspirone
0
0
0.45
6
2
Ropinirole
0.5
0.5
3
µM
1
Clonidine
2
4.5
1.5
0.15
waking
activity
20
activity
total
15
rest
latency
10
rest bout
length
rest
total
µM
45
15
4.5
1.5
0.45
0.15
# rest
bouts
5
1
4.5
6
45
15
45
15
5
rest
total
Betamethasone
NAN-190
2
Pergolide
1
1
3
1
correlation
Fingerprint
µM
45
15
4.5
1.5
0.45
0.15
correlation
Figure S4
24
ADR-BADR-B+
ADR-U-
3
3
10
5HT-1D5HT-1D+
4
3
NEURO-
4
HIST-H1-
8
CHA-NACHA-NA+
PPAR-A+
5
3
CHA-CA-L8
6
ACH-N+
ACH-SY-
7
2
2
ACH-N-
3
>=1 target
2
ACH-MACH-M+
7
ACH-ES-Rev
1
3
0 targets
3 ACH-ES-IRev
NMDA-
3
18
ADSmGLUR5-
2
1
ADS+
2
2
Correlation
5
9HIST-H1-(ERG)
GABA-
3
3
GABA+
DOPA-U-
2
4
8
DOPA-D4+
3
0 Targets Shared
>=1 Target Shared
9
DOPA-D3-
DOPA-D2+
12
DOPA-D3+
DOPA-D2/3-
0.8
2
DOPA-D2/3+
2
DOPA-D2-
24
0.6
2
DOPA-D1DOPA-D1+
7
0.4
8
0.2
DOPA-
MAO-
8
0
DOPA+
5HT-4+
3
Correlation
2
5HT-2B/C-
2
Percent
6
3
5HT-2/3+
-0.2
3
5HT-2-
5HT-1A+/-
2
-0.4
13
5HT-1A5HT-1A+
7
-0.6
9
SSRI
ADR-A2+
12
C.
-0.8
SNRI
ADR-A2-
4
0
-1
10
ADR-A1-
24
Correlation
A.
8
Class
N
Figure S5
B.
1
0.8
0.6
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
repeats
(2 sources)
3
1
1
0.5
0
-0.5
25
Figure S6
A.
Waking Activity
Rest
Average Waking Activity ,3584,6-Nitroquipazine
3
SSRIs
3.5
Normalized Sleep/10 minutes
N+
4
2.5
6-nitroquipazine
O
Sleep,3584,6-Nitroquipazine
4
3
2
2
1.5
1
1
3
2.5
2
2
N
1.5
1
1
0.5
0
0
0
5
3
10
15
20
25
30
35
Experimental Time (Hours)
40
45
0
50
Average Waking Activity ,1552,*(D)Zimelidine Dihydrochloride
Normalized Rest
2
2
1.5
1
1
0.5
0
0
5
10
15
20
25
30
35
40
45
50
Average Waking Activity ,491,(+)-Chlorpheniramine Maleate
3
3
2.5
2
2
1.5
1
1
0.5
15
20
25
30
35
40
45
50
Br
Sleep,1552,*(D)Zimelidine Dihydrochloride
3
N
3
2.5
2
2
1.5
1
1
0.5
0
0
0
5
10
15
20
25
30
35
40
45
N
50
Sleep,491,(+)-Chlorpheniramine Maleate
4
4
Cl
3.5
N
3
3
2.5
2
2
1.5
1
1
0.5
0
0
0
5
10
15
20
25
30
35
40
45
0
0
50
0
5
10
Average Waking Activity ,666,Fluvoxamine Maleate
3
15
20
25
30
35
40
45
50
N
Sleep,666,Fluvoxamine Maleate
4
3.5
2
2
1.5
1
1
0.5
3
3
F
2.5
0
5
10
15
20
25
30
35
40
45
2
1.5
1
1
0
0
50
F
2
F
0.5
0
0
O
4
2.5
10
3.5
3
fluvoxamine
5
4
2.5
0
0
4
3
chlorpheniramine
zimelidine
N
HN
0.5
0
0
5
10
15
20
25
30
35
Time
40
45
N
O
NH2
50
Time
B.
5
5
Cl
strobane
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
toxaphene
Cl
Cl
Cl
Cl
2
Cl
Cl
2
1.5
1
1
0.5
0
0
0
5
10
15
20
25
30
35
40
45
Cl
5
aldrin
Average Waking Activity ,4783,Strobane
5
4.5
4
4
Cl
3.5
3
Cl
Cl
2.5
2
2
Cl
1.5
1
1
0.5
0
0
0
5
10
15
20
25
30
35
40
45
50
Cl
Cl
endosulfan
Average Waking Activity ,4907,*(D)Toxaphene
5
5
4.5
4
4
3
3
2.5
O
2
2
1.5
O
S
0
0
0
5
10
15
20
25
30
35
Time
40
45
50
3
3
2.5
2
2
1.5
1
0.5
0
0
0
5
10
15
20
25
30
35
40
45
50
40
45
50
40
45
50
Average Waking Activity ,4804,Aldrin
5
5
4.5
4
4
3.5
3
3
2.5
2
2
1.5
1
1
0.5
0
0
0
5
10
15
20
25
30
35
Average Waking Activity ,4782,Endosulfan
5
5
4.5
4
4
Cl
3.5
0.5
Cl
Cl
3
3.5
1
Cl
Cl
50
1
Cl
2.5
1
Cl
4
Cl
3
Cl
3
4
Cl
3.5
Cl
Cl
4.5
Cl
Cl
Cl
4
4
Cl
Cl
heptachlor
4.5
Cl
Average Waking Activity ,4797,Heptachlor
5
Average Waking Activity ,4915,*(H)Chlordane
5
chlordane
O-
3
3.5
3
3
2.5
2
2
1.5
1
1
0.5
Cl
O
Cl
0
0
0
5
10
15
20
25
30
35
Time
Cl
26
Figure S7
A.
B.
5
3.5
0
4
1
1.5
2
2.5
3
3.5
6
4
4.5
3α-bis-(4-fluorophenyl)
methoxytropane
1-benzo[B]thien-2-ylN-cyclopropyl methyl
cyclohexanamine
1-[1-(2-benzo[B]thienyl)
cyclohexyl]pyrrolidine
cyclohexyl]piperidine
4.5
0.5
-0.2
0.2
trihexyphenidyl
5
0
metixene
DOPA-U/
ACH-M-
0.2
0.2
6
-0.2
0
0
oxybutynin
7
-0.4
1
7
1
0.4
0.4
2
3
4
DOPA-U
2
3
5
4
5
6
DOPA-U/ACH-M-
7
-0.2
-0.2
-0.4
-0.4
7
ACH-M-
Waking
Activity
Activity
Total
Rest
Latency
Rest Bout
Length
# Rest
Bouts
D.
Rest
Total
C.
6
Name
NAN-190
Quinazolin-5(6h)-one
5-methyl-urapidil
Terazosin
Quinacrine
Quinacrine
Ajmalicine
RU-24969
Ifenprodil
Ifenprodil
ODQ
Quinelorane
WAY-100635
Buspirone
PD 168077
Dihydroergocristine
Hycanthone
H-89
Nicergoline
MK-912
Target/Indication
5-HT1A antagonist
alpha-1 antagonist
alpha-1 antagonist
alpha-1 antagonist
antimalarial
antimalarial
not annotated
5-HT1A agonist
alpha-1 antagonist
alpha-1 antagonist
guanylyl cyclase
dopamine agonist
5-HT1A antagonist
5-HT1A agonist
dopamine agonist
alpha-1 antagonist
not annotated
PKA antagonist
alpha1-antagonist
alpha1-antagonist
Rank
1
2
3
4
6
7
8
10
13
15
16
17
18
20
21
23
26
27
30
32
33
36
37
38
42
43
45
47
49
54
55
Name
Mean Correlation
Quinacrine
0.851
1.
Ifenprodil
0.848
WAY-100635
0.848
Droperidol
0.835
L-765314
0.831
Ajmalicine
0.828
MK-912
0.826
Buspirone
0.822
5-methyl-urapidil
0.818
Ifenprodil
0.817
Vincamine
0.817
H-89
0.813
WAY-100635
0.813
ARC 239
0.811
P-MPPF
0.810
Dihydroergocristine 0.809
NAN-190
0.802
RU-24969
0.802
NAN-190
0.797
Prazosin
0.793
M77
0.793
Quinazolin-5(6h)-one 0.789
Perphenazine
0.789
548
Nicergoline
0.785
BMY-7378
0.781
Quinacrine
0.781
Amodiaquine
0.778
Domperidone
0.770
Terazosin
0.769
Spiroxatrine
0.755
Risperidone
0.750
27
Correlation
0.2
3
4
oxybutynin
0.4
alcuronium
metixene
4
2.5
0.4
trihexyphenidyl
2
0.6
3
alcuronium
DOPA-U
0.6
0.6
benztropine
benztropine
0.8
1.5
0.8
0.6
deptropine
1
1
1
0.8
2
methoxytropane
3
DOPA-U/ ACH-M-
0.5
0.8
deptropine
ACH-M-
DOPA-U/
ACH-M-
2
methoxytropane
1-benzo[B]thien-2-ylN-cyclopropyl methyl
cyclohexanamine
cyclohexyl]pyrrolidine
cyclohexyl]piperidine
1
methoxytropane
1
1
Figure S8
Adrenergic Signaling
Waking Activity
α2-adenergic agonists
(Clonidine)
3
2
2
1.5
1
1
2.5
0.5
0
0
10
15
20
25
30
35
40
45
2
1
1
0.5
0
3
3
2.5
5
10
15
20
25
30
35
40
45
50
α1 antagonists
(Ifenprodil)
Average Waking Activity ,915,Ifenprodil Tartrate
2
2
1.5
1
1
10
15
20
25
30
35
40
45
50
Sleep,5356,Clenbuterol Hydrochloride
2
2
1.5
1
0
0
0
0
10
15
20
25
30
35
40
45
10
3
15
20
25
30
35
40
45
50
Sleep,915,Ifenprodil Tartrate
3
2
β-adrenergic antagonists
(Carvedilol)
3
Average Waking Activity ,4571,Carvedilol Tartrate
3
1
0
5
10
3
15
20
25
30
35
40
45
50
Sleep,4571,Carvedilol Tartrate
3
2
1.5
1
1
0.5
2
2
1.5
5
10
15
20
25
30
35
Time
40
45
50
1
0
0
0
R(-)-Isoproterenol
Clenbuterol
Fenoterol
-
-
Nicergoline
Prazosin
Ifenprodil
5-methyl-urapidil
Doxazosin
L-765314
Beta
1
0.5
0
Clonidine
Guanabenz
Guanfacine
Tizanidine
UK 14304
sedating,
analgesia
(S23-S27)
stimulant,
insomnia
(S28-S30)
Alpha1
1
2.5
2
Selected
References
Antagonists
2
1.5
0
50
Effects in
mammals
Beta
5
2.5
0
5
Rest
1
0.5
0.5
0
Wake
Alpha2
5
2.5
2.5
0
3
0.5
0
1
3
0
0
Agonists
1
0
2
1.5
0
2
0
50
Average Waking Activity ,5356,Clenbuterol Hydrochloride
3
2.5
5
β-Adrenergic agonists
(Clenbuterol)
3
0
2
1.5
0.5
Normalized Rest
0
Sleep,3739,Clonidine
3
2.5
Rest
3
Average Waking Activity ,3739,Clonidine
3
5
10
15
20
25
30
35
Time
40
45
50
Bopindolol
Carvedilol
increased wake,
mixed effects
on REM sleep
-
reduced activity,
sedation
(S30-33)
(S34, S35)
28
Figure S9
Serotonin Signaling
Waking Activity
3
2.5
2
1.5
1
0.5
0
2
2
1.5
1
1
0.5
0
0
10
15
20
25
30
35
40
45
1.5
1
1
10
15
20
25
30
35
40
45
20
25
30
35
40
45
50
Rest
-
5HT1D agonists
(Dihydroergotamine)
Average Waking Activity ,692,Dihydroergotamine Methanesulfonate
1.5
1.5
1
1
5HT-2/3
5
10
15
20
25
30
35
40
45
50
Sleep,692,Dihydroergotamine Methanesulfonate
0
0
5
10
15
20
25
30
35
40
45
5HT-2/3 agonists
(Quipazine)
Average Waking Activity ,3616,Quipazine
3
3
1.5
1
3.5
3
2.5
2
1.5
1
0.5
0
(S36-40)
short term increases in
waking followed by general
deactivation
(S41,42)
migraine treatment
potentiates 5HT-1A induced
locomotion in guinea pig
S43
N-methylquipazine
Quipazine
-/
short-term sleep
suppression; long term
increase in SWS
-/
reduced locomotion;
initial sleep suppression
followed by long term SWS
increase
(S36,45-47)
short-term insomnia in cats
S48
increased sleep; increased
exploration
(S38, S49-52)
(S38,S44)
5HT-1A
2
0
0
50
short term increases in
waking; long term increases
in SWS (humans, rats, cats)
Antagonists
2.5
0.5
0
-
1
3
2
Selected
References
5HT-1D
2
3.5
2
5HT-1B/D
Dihydroergotamine
GR 46611
2.5
3.5
3
2.5
2
1.5
1
0.5
0
Effects in mammals
-
CGS-12066A
Sleep,610,Cgs-12066A Maleate
0
0
50
5
0.5
5
10
15
20
25
30
35
40
45
50
Sleep,3616,Quipazine
3.5
MM-77
NAN-190
P-MPPF
S(-)-UH-301
Spiroxatrine
WAY-100635
3
2.5
2
1
1
0.5
0
0
0
5
10
15
20
25
30
35
40
45
50
5HT-1A antagonists
(NAN-190)
3
Average Waking Activity ,3727,Nan-190
3
2
2
1.5
1
1
0.5
0
0
0
5
10
15
20
25
30
35
40
45
50
5HT-1D antagonists
(Metergoline)
3
Average Waking Activity ,2503,Metergoline Phenylmethyl Ester
3
2.5
5HT-1D
2.5
Metergoline
2
1.5
5HT-2/3
1
0.5
0
0
3.5
3
2.5
2
1.5
1
0.5
0
2.5
Normalized Rest
2
1.5
5
10
15
20
25
30
35
40
45
50
Sleep,3727,Nan-190
3.5
Cinanserin
Ketanserin
LY-53857
Norcyclobenzaprine
3
Reuptake
Inhibitors
2.5
2
1.5
1
0.5
0
0
3.5
3
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
40
45
50
45
50
Sleep,2503,Metergoline Phenylmethyl Ester
3.5
6-Nitroquipazine
Fluoxetine
Fluvoxamine
Paroxetine
Zimelidine
improves insomnia in
patients; REM suppression;
biphasic effects on SWS in
rats/cats -- up short term,
down long term
(S53-55)
3
15
0.5
0
3
10
3
2
3
5
3.5
2
2.5
1
3.5
3
2.5
2
1.5
1
0.5
0
0.5
2
2
1.5
1
1
0.5
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
40
45
50
5-HT-2/3 antagonists
(Ketanserin)
Average Waking Activity ,1248,Ketanserin Tartrate
3
3
2
2
1
1
0.5
0
10
15
20
25
30
35
40
Sleep,1248,Ketanserin Tartrate
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
40
45
50
Serotonin Reuptake
Inhibitors (Fluvoxamine)
3
Average Waking Activity ,666,Fluvoxamine Maleate
3
0
0
3.5
3
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
40
45
50
Sleep,666,Fluvoxamine Maleate
3.5
3
2.5
2
2
1.5
1
1
0.5
0
3.5
3
2.5
2
1.5
1
0.5
0
5
3
1.5
0
0
0
3.5
2.5
1.5
2.5
2
0
0
50
Average Waking Activity ,610,Cgs-12066A Maleate
3
5
5HT1B/D agonists
(CGS-12066A)
3
0
2.5
Wake
0.5
0
0
BMY-7378
BP-554
Buspirone
PAPP
RU-24969
3.5
2.5
0
Sleep,3696,Buspirone
Average Waking Activity ,3696,Buspirone
3
0
Agonists
5HT-1A
5HT-1A-receptor agonists
3.5
(Buspirone)
3
3
0
Rest
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
Time
40
45
50
0
0
5
10
15
20
25
30
35
Time
40
45
50
29
Figure S10
Dopamine Signaling
Waking Activity
D1- agonists
(SKF-83565)
3
Average Waking Activity ,1496,Skf 83565 Hydrobromide
3
2
1
1
0.5
0
0
3
10
15
20
25
30
35
40
45
50
D2- agonists
(Apomorphine)
50
50
3.5
3
2.5
2
1.5
1
0.5
0
1.5
1
1
0
10
15
20
25
30
35
40
45
Average Waking Activity ,1399,Ropinirole Hydrochloride
2
1
1
0.5
5
10
15
20
25
30
35
40
45
D4-receptor agonists
(PD-168077)
3
50
Average Waking Activity ,3574,Pd 168077
3
2
2
1.5
1
1
0.5
0
0
5
10
15
20
25
30
35
40
45
50
D1-antagonists
(SCH-12679)
3
Average Waking Activity ,1274,R(-)-Sch-12679 Maleate
3
2
2
1.5
1
1
0.5
Apomorphine
Bromocriptine
Dipropyl-6,7-ADTN
1
5
10
15
20
25
30
35
40
45
50
5
10
15
20
25
30
35
40
45
50
D2-antagonists
(Benperidol)
3
Average Waking Activity ,2913,Benperidol
3
Selected
References
increased waking, reduction
in sleep (rat and monkeys),
and increased grooming
(S56-59)
biphasic; at low
concentrations, reduces
waking and locomotor
activity, and increases
sleep. At high doses, the
opposite.
(S60-63)
Pergolide
reduced waking; no
significant changes in sleep
(S42,S64)
non-stereotyped shuffling in
rats
S65
D2/3-Receptor
-/
Quinelorane
2.5
2
Quinpirole
1.5
1
Ropinirole
5
10
15
20
25
30
35
40
45
50
D4-Receptor
PD 168077
Sleep,3574,Pd 168077
Antagonists
3.5
3
D1-Receptor
2.5
2
R(-)-SCH-12679
1.5
SCH-23390
1
-
decreased waking,
increased sleep
(S56-59,
S61,S66)
0.5
0
0
3.5
3
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
40
45
50
D2-Receptor
Benperidol
Droperidol
Sleep,1274,R(-)-Sch-12679 Maleate
3.5
3
extra-pyramidal effects
(movement disorders);
increased sedation
Domperidone
2.5
(S57,S61,
S66)
L-741626
2
1.5
D3-Receptor
1
0
0
3.5
3
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
40
45
50
40
45
50
40
45
50
DS121
GR 103691
-/
short term increase in
waking followed by longterm increases in sleep
amount
S65
Sleep,2913,Benperidol
3.5
3
2.5
2
2
1.5
1
1
0.5
0
Effects in mammals
(-)-3PPP
Sleep,1399,Ropinirole Hydrochloride
0.5
0
/-
D2-Receptor
1.5
3.5
3
2.5
2
1.5
1
0.5
0
2.5
N-Allyl-SKF-38393
SKF 83565
Sleep,2347,R(-)Apomorphine Hydrochloride Hemihydrate
2
0
0
45
0.5
0
2.5
40
3
2
0
20
25
30
35
Rest
3.5
1.5
0
15
2.5
0
0
2.5
5
D2/3-agonists
(Ropinirole)
3
0
10
0.5
0
3
0
5
3
2
0
R(+)-6-Bromo-APB
1
3.5
0.5
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
40
45
50
D3 antagonists
(DS121)
3
Average Waking Activity ,885,S(-)-Ds 121 Hydrochloride
3
0
0
3.5
3
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
Sleep,885,S(-)-Ds 121 Hydrochloride
3.5
3
2.5
1.5
3.5
3
2.5
2
1.5
1
0.5
0
2
2
2
1.5
1
1
0.5
0
2
0
0
5
Average Waking Activity ,2347,R(-)Apomorphine Hydrochloride Hemihydrate
3
2.5
0
Chloro-APB
CY 208-243
2.5
0.5
0
Wake
D1-Receptor
Sleep,1496,Skf 83565 Hydrobromide
3
2
0
Agonists
3.5
1.5
0
3.5
3
2.5
2
1.5
1
0.5
0
Normalized Rest
2.5
Rest
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
Time
40
45
50
0
0
5
10
15
20
25
30
35
Time
30
Figure S11
GABAergic Signaling
Waking Activity
GABA-A agonist
(GBLD-345)
3
3
2
2
1.5
1
1
0.5
0
0
20
25
30
35
40
45
1
1
0.5
5
10
15
20
25
30
35
40
45
GABA-A-antagonist
(1S,9R-Hydrastine)
Average Waking Activity ,4354,"*(S)1S,9R-Hydrastine"
3
20
25
30
35
40
45
50
2
1.5
1
1
0.5
0
5
10
15
20
25
30
35
40
45
GABA agonist/
antagonist
(Tracazolate)
3
Average Waking Activity ,3474,*(D)Tracazolate Hydrochloride
3
2.5
50
CGP-13501
Rest
-
/-
Effects in mammals
Selected
References
anxiolytic; sedative
S68
hypnotic
S69
Sleep,410,Cgp-13501
Antagonists
GABA-A
2
1S,9R-Hydrastine
1.5
1
5
10
15
20
25
30
35
40
45
50
Endosulfan
Heptachlor
Chlordane
3.5
3
2.5
2
1.5
1
0.5
0
2
50
increased locomtor activity;
induction of fighting;
pro-convulsant
(S70-72)
Heptachlor
Sleep,4354,"*(S)1S,9R-Hydrastine"
3.5
Aldrin
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
35
40
45
50
dose dependant
Mixed (+/-) Modulator anxiolytic;
increase or decrease in
Tracazolate
- locomotor activity
Neuroactive Steroid GABA Regulators
Allopregnanolone
- stimulant; anxiolytic; ataxic;
Pregnanolone
3.5
3
2.5
2
1.5
1
0.5
0
Alfadolone
Sleep,3474,*(D)Tracazolate Hydrochloride
3.5
depressant
(S73,74)
(S75-77)
hypnotic
3
2
2
1.5
1
1
0.5
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
40
45
50
Steroidal GABA agonist
(Alfadolone)
3
Average Waking Activity ,3397,Alfadolone Acetate
3
2.5
2
0
0
3.5
3
2.5
2
1.5
1
0.5
0
2
1.5
1
1
0.5
5
10
15
20
25
30
35
40
45
50
40
45
50
Sleep,3397,Alfadolone Acetate
3.5
3
15
2.5
0
0
Normalized Rest
0
3
0
10
0.5
2.5
0
GABA-B
5
3
1.5
0
1
3.5
2
0
1.5
3.5
3
2.5
2
1.5
1
0.5
0
2
0
GBLD-345
Avermectin B1
2
0
0
50
15
Average Waking Activity ,410,Cgp-13501
3
10
GABA-B- agonist
(CGP-13501)
3
5
Wake
GABA-A
2.5
0.5
0
2.5
0
Agonists
Sleep,3384,Gbld345
3.5
2.5
3.5
3
2.5
2
1.5
1
0.5
0
Average Waking Activity ,3384,Gbld345
3
Rest
2.5
2
1.5
1
0.5
0
0
0
5
10
15
20
25
30
35
Time
40
45
50
0
0
5
10
15
20
25
30
35
Time
31
Figure S12
Melatonin Signaling
Waking Activity
Melatonin Agonist (8-methoxy3 2-propionamidotetralin)
3.5
Average Waking Activity ,1952,8-Methoxy-2-Propionamidotetralin
2
1
1
0
0
3
3
2.5
5
10
15
20
25
30
35
40
45
50
Melatonin Agonist
(IIK7)
Average Waking Activity ,919,Iik7
2
2
1.5
1
1
0.5
0
0
0
5
10
15
20
25
30
35
40
45
Sleep,1952,8-Methoxy-2-Propionamidotetralin
Selected
References
increase in sleep propensity
in dirunal species; circadian
phase shifting
(S78-81)
Antagonists
1
0
0
3.5
3
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
40
45
50
40
45
50
40
45
50
K 185
DH 97
/-
blocks melatonin effects on
behavior
S82
Sleep,919,Iik7
3.5
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
35
Sleep,1970,DH 97
3.5
3
3
2.5
2
2
1.5
1
1
0.5
0
/-
Effects in mammals
2
1.5
3.5
3
2.5
2
1.5
1
0.5
0
Average Waking Activity ,1970,DH 97
Rest
-
IIK7
2.5
0.5
50
Melatonin Antagonist
(DH 97)
3
0
Normalized Rest
0.5
2
1.5
3
2.5
2
1.5
1
0.5
0
Wake
8-methoxy-2propionamidotetralin
Melatonin
3
2.5
Agonists
3.5
3
0
Rest
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
Time
40
45
50
0
0
5
10
15
20
25
30
35
Time
32
Figure S13
Histamine Signaling
Waking Activity
H1-receptor antagonist
(Clemizole)
3
Average Waking Activity ,2615,Clemizole hydrochloride
3
H1-(no HERG
block)
Sleep,2615,Clemizole hydrochloride
3
2
1
1
0.5
Chloropyramine
2.5
2
Clemizole
1.5
Wake
0
Loratadine
1
0
5
10
15
20
25
30
35
40
45
3
Average Waking Activity ,5651,Diphenylpyraline Hydrochloride
3
2
1.5
1
1
0.5
0
0
5
10
15
20
25
30
35
40
45
50
H1-antagonist(+HERG)
(Clemastine)
3
Average Waking Activity ,5553,Clemastine
3
15
20
25
30
35
40
45
50
sedation (less pronounced
in loratadine)
(S83-86)
-
Trimeprazine
Diphenylpyraline
3.5
3
2.5
2
1.5
1
0.5
0
Selected
References
psychostimulant
S87
H1- (HERG Blockers)
Sleep,5651,Diphenylpyraline Hydrochloride
Astemizole
3
2.5
Clemastine
2
Meclizine
1.5
1
0.5
0
0
5
10
15
20
25
30
35
40
45
50
Oxatomide
Promethazine
/-
some sedation in 1st
generation anti-histamines;
less pronounced in second
generation anti-histamines
Terfenadine
3.5
3
2.5
2
1.5
1
0.5
0
Sleep,5553,Clemastine
3
2
2
1.5
1
1
0.5
0
10
Effects in mammals
3.5
2.5
0
5
3.5
2
0
0
0
50
H1-antagonist
(psychostimulant)
(Diphenylpyraline)
Rest
-/
0.5
0
Normalized Rest
Antagonists
3.5
2
2.5
3.5
3
2.5
2
1.5
1
0.5
0
1.5
2.5
Rest
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
Time
40
45
50
0
0
5
10
15
20
25
30
35
40
45
50
Time
33
Figure S14
Adenosine Signaling
Waking Activity
Agonist
(2-chloroadenosine)
3
Average Waking Activity ,2146,2-Chloroadenosine
3
2
2
1
1
0.5
0
5
10
15
20
25
30
35
40
45
A1-antagonist
(8-cyclopentyl-1,3dipropylxanthine)
3
Average Waking Activity ,599,"8-Cyclopentyl-1,3-Dipropylxanthine"
3
2
2
1.5
1
1
0.5
0
0
0
5
10
15
20
25
30
35
40
45
A3-antagonist
(MRS-1220)
3
Average Waking Activity ,2210,Mrs 1220
3
2.5
50
2
50
2
1
1
0.5
0
Effects in mammals
Selected
References
increased sleep; decreased
locomotor activity
(S88-92)
A1
1
3.5
3
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
40
45
50
1,3-diethyl-8phenylxanthine
8-cyclopentyl-1,3dipropylxanthine
A3
Sleep,599,"8-Cyclopentyl-1,3-Dipropylxanthine"
3.5
MRS-1220
3
(S88,93)
locomotor stimulant
-/
unknown behavioral effects
2.5
2
1.5
1
0.5
0
0
3.5
3
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
40
45
50
40
45
50
Sleep,2210,Mrs 1220
3.5
3
1.5
0
2
1.5
0
0
2.5
-
Rest
Antagonists
2.5
0.5
Normalized Rest
0
Adenosine
2-chloroadenosine
Sleep,2146,2-Chloroadenosine
Wake
3
1.5
0
Rest
3.5
2.5
3.5
3
2.5
2
1.5
1
0.5
0
Agonists
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
35
Time
40
45
50
0
0
5
10
15
20
25
30
35
Time
34
Figure S15
Glutamate Signaling
3
1
1
0.5
5
10
15
20
25
30
35
40
45
mGluR 5 antagonist
(MPEP)
3
Average Waking Activity ,1990,2-Methyl-6-(phenylethynyl)pyridine
3
2.5
2
2
1.5
50
1
1
0.5
0
0
2
Normalized Rest
2
0
2.5
5
10
15
20
25
30
35
Time
40
Effects in mammals
Selected
References
anxiolytic; hyperlocomotion;
anti-convulsant
(S94,95)
anxiolytic; can block drug
induced hyperlocomotion
(S96,97)
2
CNS-1102
1.5
1
0
0
45
50
-/
IEM 1460
0.5
5
10
15
20
25
30
35
40
45
50
L-689560
L-701252
3.5
3
2.5
2
1.5
1
0.5
0
L-701324
Sleep,1990,2-Methyl-6-(phenylethynyl)pyridine
3.5
MK-801
3
SDZ 220-581
2.5
2
1.5
mGluR5
1
0.5
0
Rest
DCQX
7-chlorokynurenic
acid
3
1.5
0
3.5
3
2.5
2
1.5
1
0.5
0
Sleep,3659,"L-701,252"
3.5
2.5
0
Rest
Wake
NMDA
Average Waking Activity ,3659,"L-701,252"
3
Waking Activity
NMDA antagonist
(L-701252)
Antagonists
0
0
5
10
15
20
25
30
35
Time
40
45
50
MPEP
SIB 1757
-/
35
Figure S16
A.
Waking Activity
O
4
5
O
3
4
3
2
2.5
22
11
2
1.5
1
1
0
5
10
15
20
25
30
35
40
45
0
50
Methoxyverapamil
Methoxy-verapamil
Average Waking Activity ,2351,Verapamyl Hydrochloride
5
55
4
44
5
O
4
4
O
4.5
O
O
3
N
3.5
3
33
2
22
3
O
N
2
2.5
2
1.5
1
11
1
0.5
00
0
0
5
10
15
35
40
20
25
30
35
40
45
45
0
50
Verapamil
5
5
4
4
33
5
2
2
1.5
1
1
0.5
0
0 0
0
5
5
10
15
3
O
N
O
3
O
N
2.5
22
11
2
2
1.5
1
1
0.5
40
45
50
40
45
50
4
3.5
3
3
2.5
2
2
1.5
1
1
0.5
0
0 0
0
55
10
15
20
25
30
35
4
5
0
5
10
15
20
25
30
35
40
45
50
0
43
3
4
3.5
3
2
2
11
3
2
2.5
2
1.5
1
1
0.5
0
0
0
5
10
15
20
25
30
35
40
45
50
0
Time
C.
YS-035 Rest
YS-035
YS-035 Waking Activity
60
60
66
66
50
50
55
40
40
44
Day
30
30
33
Night
20
20
22
10
10
11
Average Waking Activity
(seconds/minute)
Average Waking Activity
minutes Rest
rest per
Minutes
perhour
Hour
(seconds/minute)
30
Sleep,83,Methoxy Verapamil
4
Time
B.
25
20
25
30
35
O
4
0
3
4.5
3.5
00
3
2.5
4
5
4
4.5
0.5
0
4
3.5
3.5
00
Sleep,1546,Ys-035 Hydrochloride
4
N
4.5
5
5
5
4
4
4
33
O
O
Normalized
Rest
Normalized
Rest
YS-035
Rest
55
44
33
22
11
00
00
0uM
0
100nM-7
100nM
1x10
300nM-7
300nM
3x10
1uM -6
1uM
1x10
3uM -6
3uM
3x10
10uM -5 3x10
30uM-5
10uM
30uM
1x10
Concentration (Molar)
0uM
0
100nM-7
1x10
300nM-7
3x10
1uM -6
1x10
3uM -6
3x10
10uM -5 3x10
30uM-5
1x10
36
Figure S17.
Glucocorticoid
Betamethasone
O
Average Waking Activity ,318,Betamethasone
O
HO
5
5
4.5
4
4
3.5
3
4
F
3.5
O
3
3
2.5
2
2
1.5
1
3
2.5
2
2
1.5
1
1
1
0.5
0.5
0
0
0
0
0
5
10
15
20
25
30
35
40
45
50
Immunomodulator (NFAT signaling)
cyclosporin A
10
15
20
25
30
35
40
45
50
PDE inhibitor
Fosfosal
4
O
3.5
OH
4
O
3.5
3
3
2.5
2
2
1.5
1
1
0
5
10
15
20
25
30
35
40
45
50
Time
NSAID
Average Waking Activity ,4460,FENOPROFEN
5
5
O
Fenoprofen
4.5
4
OH
Normalized Rest
0
4
3
3
2.5
2
2
1.5
1
1
2
1.5
1
1
0
0
0
5
10
15
20
25
30
35
40
45
50
Time
Sleep,4460,FENOPROFEN
4
4
3
3
2.5
2
2
1.5
1
1
0.5
0.5
0
3
2
3.5
O
3.5
0
3
2.5
0.5
0.5
4
0
Sleep,4662,Fosfosal
4
Average Waking ActivityP,4662,Fosfosal
5
0
5
10
15
20
25
30
35
40
45
0
0
50
0
5
10
15
20
25
30
35
40
45
50
Time
Time
Immunomodulator
HO
O
5
O
O
Cyclosporin A
N
Average Waking Activity ,2764,Cyclosporin
A
4.5
O H
N
N
N
4
4
3.5
H
N
N
H O
3.5
4
O
N
H
N
N
O
N
O
O
O
3
3
2.5
2
2
1.5
1
1
0
0
3
3
2.5
2
2
1.5
1
1
0.5
0.5
0
Sleep,4664,Cyclosporine
4
N
O
5
Other Anti-inflammatory agents
aminophenazone
capsazepine
catechin tetramethyl ether
cepharanthine
pentamidine
theaflavin digallate
valproate sodium
5-aminopentanoic acid
5
Time
4.5
Non-Steroidal Anti-Inflammatory drugs (NSAIDs)
bufexamac
diflunisal
fenoprofen
piroxicam
0
Time
5
PDE inhibitors
diplosalsalate
fosfosal
papaverine
propentofylline
Ro 20-1724
SKF-94836
Sleep,318,Betamethasone
4
OH
O
Glucocorticoids
betamethasone
clobetasol
desoxycorticosterone
dexamethasone
flumethasone
flunisolide
hydrocortisone
medrysone
6α-methylprednisolone
ursolic acid
B.
A.
5
10
15
20
25
30
35
40
45
Time
Waking Activity
50
0
0
0
5
10
15
20
25
30
35
40
Time
Rest
37
45
50
Figure S18
Waking Activity
A.
5
4
F
Halofantrine
F
Rest
4
F
3
Cl
N
3
Cl
2
OH
2
1
1
0
5
4
Terfenadine
OH
OH
N
3
2
1
0
5
4
Psora-4
3
2
1
0
4
O
O
O
O
2
2
3
4
3
3
3.5
Normalized Rest
0
1
1
0
0
Time
Time
2.5
3.5
1.5
3
1
2.5
0.5
2
0
1.5
3.5
3.5
3.5
3
3
3
2.5
2.5
2.5
Average Night Waking Activity
(seconds/minute)
B.2
2
2
2
1.5
1.5
1.5
1
1
1
0.5
0
0
Fexofenadine
Citirizine
ERGLoratadine
blockers
Cisapride
Dofetilide
Non-ERG
blockers
Fexofenadine
Cetirizine
Citirizine
0.5
0.5
0
0
Loratadine
1uM
0.5
10uM
30uM
Cisapride
Dofetilide
0
0
1
3uM
0
1uM -61uM
1x10
3uM
3uM -6
3x10
10uM 10uM
-5
1x10
30uM
30uM-5
3x10
38
Table S1
Drug Name
Clonidine
Ropinirole
NAN-190
MK-801
Betamethasone
Chloro-APB
Buspirone
Cyclosporin A
Ketanserin
Nicergoline
Quipazine
Warfarin
Fluvoxamine
Valproic Acid
Carvedilol
2-Chloroadenosine
Phenelzine
Trazodone
Clozapine
Amoxapine
Cyclobenzaprine
Fluoxetine
Haloperidol
Loxapine
Papaverine
Pergolide
Bromocriptine
Dihydroergotamine
Metergoline
Highest NonToxic Dose
(30/45 µM max)
45 µM
45 µM
30 µM
45 µM
45 µM
45 µM
45 µM
45 µM
15 µM
15 µM
45 µM
45 µM
45 µM
45 µM
4.5 µM
45 µM
45 µM
45 µM
15 µM
30 µM
15 µM
15 µM
15 µM
15 µM
3.0 µM
45 µM
15 µM
45 µM
15 µM
Lowest
Effective Dose
1.5 µM
0.45 µM
0.10 µM
0.45 µM
0.15 µM
0.45 µM
4.5 µM
2 µM
0.15 µM
1.5 µM
1.5 µM
4.5 µM
1.5 µM
15 µM
0.45 µM
45 µM
45 µM
1.5 µM
1.5 µM
3.0 µM
4.5 µM
1.5 µM
1.5 µM
0.45 µM
1.0 µM
0.45 µM
0.15 µM
0.15 µM
15 µM
Correlated Dose Range
1.5-45 µM
0.45-45 µM
0.10-30 µM
0.45-45 µM
0.15-45 µM
0.45-45 µM
4.5-45 µM
2-45 µM
0.15-45 µM
4.5-45 µM
0.15-45 µM
4.5-45 µM
0.45-45 µM
0.45-45 µM
0.45-4.5 µM
0.15-45 µM
4.5-15 µM
1.5-15 µM
1.5-15 µM
1.0-10 µM
1.5-15 µM
1.5-4.5 µM
0.15-1.5 µM
0.15-4.5 µM
0.30-3.0 µM
4.5-45 µM; 0.15-1.5 µM
1.5-15 µM; 0.15-1.5 µM
4.5-45 µM; 0.15-1.5 µM
4.5-15 µM; 0.15-1.5 µM
39
Supplemental References
S1.
S2.
S3.
S4.
S5.
S6.
S7.
S8.
S9.
S10.
S11.
S12.
S13.
S14.
S15.
S16.
S17.
S18.
S19.
S20.
S21.
S22.
S23.
S24.
S25.
S26.
S27.
S28.
S29.
S30.
D. A. Prober, J. Rihel, A. A. Onah, R. J. Sung, A. F. Schier, J Neurosci 26, 13400
(Dec 20, 2006).
M. B. Eisen, P. T. Spellman, P. O. Brown, D. Botstein, Proc Natl Acad Sci
U S A 95, 14863 (Dec 8, 1998).
A. Holt, M. M. Palcic, Nat Protoc 1, 2498 (2006).
M. N. Pangalos, L. E. Schechter, O. Hurko, Nat Rev Drug Discov 6, 521 (Jul,
2007).
T. A. Ban, Dialogues Clin Neurosci 8, 335 (2006).
Y. Agid et al., Nat Rev Drug Discov 6, 189 (Mar, 2007).
A. L. Hopkins, Nat Chem Biol 4, 682 (Nov, 2008).
J. Geddes, N. Freemantle, P. Harrison, P. Bebbington, Bmj 321, 1371 (Dec 2,
2000).
D. Fischer-Barnicol et al., Neuropsychobiology 57, 80 (2008).
T. E. North et al., Nature 447, 1007 (Jun 21, 2007).
T. E. North et al., Cell 137, 736 (May 15, 2009).
C. K. Kaufman, R. M. White, L. Zon, Nat Protoc 4, 1422 (2009).
C. Renier et al., Pharmacogenet Genomics 17, 237 (Apr, 2007).
I. V. Zhdanova, S. Y. Wang, O. U. Leclair, N. P. Danilova, Brain Res 903, 263
(Jun 8, 2001).
N. J. Giacomini, B. Rose, K. Kobayashi, S. Guo, Neurotoxicol Teratol 28, 245
(Mar-Apr, 2006).
W. Boehmler et al., Genes Brain Behav 6, 155 (Mar, 2007).
S. C. Baraban, M. R. Taylor, P. A. Castro, H. Baier, Neuroscience 131, 759
(2005).
N. Peitsaro, M. Sundvik, O. V. Anichtchik, J. Kaslin, P. Panula, Biochem
Pharmacol 73, 1205 (Apr 15, 2007).
S. Berghmans, J. Hunt, A. Roach, P. Goldsmith, Epilepsy Res 75, 18 (Jun,
2007).
J. Lamb et al., Science 313, 1929 (Sep 29, 2006).
J. Lamb, Nat Rev Cancer 7, 54 (Jan, 2007).
D. Centurion et al., Eur J Pharmacol 535, 234 (Mar 27, 2006).
D. W. Gil et al., Anesthesiology 110, 401 (Feb, 2009).
J. P. Makela, I. T. Hilakivi, Med Biol 64, 355 (1986).
D. Rotiroti, R. Silvestri, G. B. de Sarro, G. Bagetta, G. Nistico, J Psychiatr Res
17, 231 (1982).
A. G. Hayes, M. Skingle, M. B. Tyers, Neuropharmacology 25, 391 (Apr, 1986).
P. A. Van Zwieten, Am J Cardiol 61, 6D (Feb 24, 1988).
C. W. Berridge, R. L. Stellick, B. E. Schmeichel, Behav Neurosci 119, 743 (Jun,
2005).
C. W. Berridge, S. O. Isaac, R. A. Espana, Behav Neurosci 117, 350 (Apr, 2003).
L. Leinonen, D. Stenberg, Physiol Behav 37, 199 (1986).
40
S31. I. Hilakivi, A. Leppavuori, P. T. Putkonen, Eur J Pharmacol 65, 417 (Aug 8,
1980).
S32. J. P. Makela, I. T. Hilakivi, Pharmacol Biochem Behav 24, 613 (Mar, 1986).
S33. R. Vetrivelan, H. N. Mallick, V. M. Kumar, Neuroscience 139, 1141 (2006).
S34. E. S. Paykel, R. Fleminger, J. P. Watson, J Clin Psychopharmacol 2, 14 (Feb,
1982).
S35. M. Hirohashi et al., Arzneimittelforschung 40, 735 (Jul, 1990).
S36. A. A. Bjorkum, R. Ursin, Brain Res Bull 39, 373 (1996).
S37. J. A. Lerman, K. I. Kaitin, W. C. Dement, S. J. Peroutka, Neurosci Lett 72, 64
(Dec 3, 1986).
S38. J. B. Lucot, L. S. Seiden, Pharmacol Biochem Behav 24, 537 (Mar, 1986).
S39. F. Marrosu, C. A. Fornal, C. W. Metzler, B. L. Jacobs, Brain Res 739, 192 (Nov
11, 1996).
S40. J. L. Evenden, Br J Pharmacol 112, 861 (Jul, 1994).
S41. B. Bjorvatn, R. Ursin, J Sleep Res 3, 97 (Jun, 1994).
S42. J. M. Monti, H. Jantos, Prog Brain Res 172, 625 (2008).
S43. M. F. O'Neill, G. J. Sanger, Eur J Pharmacol 370, 85 (Apr 9, 1999).
S44. S. P. Bailey, J. M. Davis, E. N. Ahlborn, Int J Sports Med 14, 330 (Aug, 1993).
S45. I. Belcheva, S. Belcheva, V. V. Petkov, C. Hadjiivanova, V. D. Petkov, Gen
Pharmacol 28, 435 (Mar, 1997).
S46. J. M. Monti, H. Jantos, Behav Brain Res 151, 159 (May 5, 2004).
S47. G. Griebel, R. J. Rodgers, G. Perrault, D. J. Sanger, Psychopharmacology (Berl)
144, 121 (May, 1999).
S48. M. Sallanon, C. Buda, M. Janin, M. Jouvet, Eur J Pharmacol 82, 29 (Aug 13,
1982).
S49. P. Bo, M. Patrucco, F. Savoldi, Farmaco [Sci] 42, 91 (Feb, 1987).
S50. R. Kirov, S. Moyanova, Int J Neurosci 93, 257 (Apr, 1998).
S51. S. D. Gleason, H. E. Shannon, Eur J Pharmacol 341, 135 (Jan 12, 1998).
S52. C. Dugovic, A. Wauquier, J. E. Leysen, R. Marrannes, P. A. Janssen,
Psychopharmacology (Berl) 97, 436 (1989).
S53. R. H. Pastel, J. D. Fernstrom, Brain Res 436, 92 (Dec 8, 1987).
S54. L. Sommerfelt, R. Ursin, Behav Brain Res 45, 105 (Nov 26, 1991).
S55. M. Weber et al., Psychopharmacology (Berl) 203, 753 (May, 2009).
S56. E. Ongini, M. G. Caporali, M. Massotti, Life Sci 37, 2327 (Dec 16, 1985).
S57. P. Bo, E. Ongini, A. Giorgetti, F. Savoldi, Neuropharmacology 27, 799 (Aug,
1988).
S58. J. M. Monti, M. Fernandez, H. Jantos, Neuropsychopharmacology 3, 153 (Jun,
1990).
S59. M. Trampus, N. Ferri, M. Adami, E. Ongini, Eur J Pharmacol 235, 83 (Apr 22,
1993).
S60. J. M. Monti, M. Hawkins, H. Jantos, L. D'Angelo, M. Fernandez,
Psychopharmacology (Berl) 95, 395 (1988).
S61. G. Bagetta, M. T. Corasaniti, M. C. Strongoli, S. Sakurada, G. Nistico,
Neuropharmacology 26, 1047 (Aug, 1987).
S62. W. Kropf, K. Kuschinsky, Neuropharmacology 30, 953 (Sep, 1991).
S63. J. Micallef et al., Br J Clin Pharmacol (Feb 9, 2009).
41
S64.
S65.
S66.
S67.
S68.
S69.
S70.
S71.
S72.
S73.
S74.
S75.
S76.
S77.
S78.
S79.
S80.
S81.
S82.
S83.
S84.
S85.
S86.
S87.
S88.
S89.
S90.
S91.
S92.
S93.
S94.
S95.
S96.
S97.
S98.
J. M. Monti, H. Jantos, M. Fernandez, Eur J Pharmacol 169, 61 (Oct 4, 1989).
J. J. Clifford, J. L. Waddington, Neuropsychopharmacology 22, 538 (May, 2000).
D. E. Casey, Psychopharmacology (Berl) 107, 18 (1992).
M. F. Olive, W. F. Seidel, D. M. Edgar, J Pharmacol Exp Ther 285, 1073 (Jun,
1998).
S. Spinosa Hde, S. R. Stilck, M. M. Bernardi, Vet Res Commun 26, 309 (Jun,
2002).
D. Wirtshafter, T. R. Stratford, M. R. Pitzer, Behav Brain Res 59, 83 (Dec 31,
1993).
S. Jamaluddin, M. K. Poddar, Pol J Pharmacol 53, 21 (Jan-Feb, 2001).
S. K. Jamaluddin, M. K. Poddar, Neurochem Res 26, 439 (Apr, 2001).
M. Anand, S. Mehrotra, K. Gopal, R. N. Sur, S. V. Chandra, Toxicol Lett 24, 79
(Jan, 1985).
S. Pellow, S. E. File, Behav Brain Res 23, 159 (Feb, 1987).
J. B. Patel, J. B. Malick, Eur J Pharmacol 78, 323 (Mar 12, 1982).
R. P. Irwin et al., Neurosci Lett 141, 30 (Jul 6, 1992).
M. Lancel et al., J Pharmacol Exp Ther 282, 1213 (Sep, 1997).
A. A. Palmer, M. N. Miller, C. S. McKinnon, T. J. Phillips, Behav Neurosci 116,
126 (Feb, 2002).
S. P. Fisher, D. Sugden, Neurosci Lett 457, 93 (Jun 26, 2009).
D. A. Golombek, E. Escolar, D. P. Cardinali, Physiol Behav 49, 1091 (Jun, 1991).
A. Cagnacci, J. A. Elliott, S. S. Yen, J Clin Endocrinol Metab 75, 447 (Aug, 1992).
F. Wang et al., Pharmacol Biochem Behav 74, 573 (Feb, 2003).
B. Guardiola-Lemaitre, Ann Pharm Fr 63, 385 (Nov, 2005).
M. Haria, A. Fitton, D. H. Peters, Drugs 48, 617 (Oct, 1994).
B. G. Bender, S. Berning, R. Dudden, H. Milgrom, Z. V. Tran, J Allergy Clin
Immunol 111, 770 (Apr, 2003).
S. M. Stahl, CNS Spectr 13, 1027 (Dec, 2008).
H. L. Haas, O. A. Sergeeva, O. Selbach, Physiol Rev 88, 1183 (Jul, 2008).
G. B. Lapa, T. A. Mathews, J. Harp, E. A. Budygin, S. R. Jones, Eur J Pharmacol
506, 237 (Jan 4, 2005).
H. M. Marston et al., J Pharmacol Exp Ther 285, 1023 (Jun, 1998).
R. A. Barraco, V. L. Coffin, H. J. Altman, J. W. Phillis, Brain Res 272, 392 (Aug 8,
1983).
R. W. McCarley, Sleep Med 8, 302 (Jun, 2007).
J. M. Krueger, Curr Pharm Des 14, 3408 (2008).
R. Szymusiak, I. Gvilia, D. McGinty, Sleep Med 8, 291 (Jun, 2007).
R. F. Bruns, J. J. Katims, Z. Annau, S. H. Snyder, J. W. Daly,
Neuropharmacology 22, 1523 (Dec, 1983).
W. Adriani et al., Exp Brain Res 123, 52 (Nov, 1998).
J. Kotlinska, S. Liljequist, Psychopharmacology (Berl) 135, 175 (Jan, 1998).
H. Homayoun, B. Moghaddam, Cereb Cortex 16, 93 (Jan, 2006).
M. Pietraszek, Z. Rogoz, S. Wolfarth, K. Ossowska, J Physiol Pharmacol 55, 587
(Sep, 2004).
T. Kvernmo, J. Houben, I. Sylte, Curr Top Med Chem 8, 1049 (2008).
42
www.sciencemag.org
Table of Contents
Figs. S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18
Table S1
43

Zebrafish Behavioral Profiling Links Drugs to Biological Targets and

Transcription

Similar documents

AB SCIEX Triple Quad™ 3500 Instrument Systemhandbuch

GEBRAUCHSANWEISUNG / INSTRUCTION MANUAL