Vesicular glutamate transporter 3

Transcription

RESEARCH ARTICLE
Vesicular Glutamate Transporter 3-Expressing
Nonserotonergic Projection Neurons Constitute a
Subregion in the Rat Midbrain Raphe Nuclei
Hiroyuki Hioki,1 Hisashi Nakamura,1 Yun-Fei Ma,1 Michiteru Konno,1 Takashi Hayakawa,1
Kouichi C. Nakamura,1,2 Fumino Fujiyama,1 and Takeshi Kaneko1,2*
1
Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST),
Kawaguchi 332-0012, Japan
2
ABSTRACT
We previously reported that about 80% of vesicular glutamate transporter 3 (VGLUT3)-positive cells displayed immunoreactivity for serotonin, but the others were negative
in the rat midbrain raphe nuclei, such as the dorsal (DR)
and median raphe nuclei (MnR). In the present study, to
investigate the precise distribution of VGLUT3-expressing
nonserotonergic neurons in the DR and MnR, we performed double fluorescence in situ hybridization for
VGLUT3 and tryptophan hydroxylase 2 (TPH2). According
to the distribution of VGLUT3 and TPH2 mRNA signals, we
divided the DR into six subregions. In the MnR and the
rostral (DRr), ventral (DRV), and caudal (DRc) parts of the
DR, VGLUT3 and TPH2 mRNA signals were frequently colocalized (about 80%). In the lateral wings (DRL) and core
region of the dorsal part of the DR (DRDC), TPH2producing neurons were predominantly distributed, and
about 94% of TPH2-producing neurons were negative for
VGLUT3 mRNA. Notably, in the shell region of the dorsal
part of the DR (DRDSh), VGLUT3 mRNA signals were abundantly detected, and about 75% of VGLUT3-expressing
neurons were negative for TPH2 mRNA. We then examined
the projection of VGLUT3-expressing nonserotonergic
neurons in the DRDSh by anterograde and retrograde labeling after chemical depletion of serotonergic neurons.
The projection was observed in various brain regions such
as the ventral tegmental area, substantia nigra pars compacta, hypothalamic nuclei, and preoptic area. These results suggest that VGLUT3-expressing nonserotonergic
neurons in the midbrain raphe nuclei are preferentially distributed in the DRDSh and modulate many brain regions
with the neurotransmitter glutamate via ascending axons.
J. Comp. Neurol. 518:668 – 686, 2010.
© 2009 Wiley-Liss, Inc.
INDEXING TERMS: glutamatergic; serotonergic; tryptophan hydroxylase 2; in situ hybridization; anterograde labeling;
retrograde labeling
The dorsal and median raphe nuclei (DR and MnR) are
two of the major serotonin sources and innervate a multitude of targets throughout the central nervous system
(CNS) via their ascending and descending pathways (Steinbusch, 1981, 1984). The serotonin system is involved in
the control of various behavioral and physiological processes and has been implicated in brain dysfunction, especially in mood disorders such as depression (Jacobs and
Azmitia, 1992; Michelsen et al., 2007, 2008).
The DR is located in the rostral pontine and caudal midbrain tegmentum and can be divided into six subregions
based on the cytoarchitecture and the distribution of the
serotonergic neurons, rostral (DRr), dorsal (DRD), ventral
(DRV), lateral (DRL), caudal (DRc), and interfascicular (DRI)
© 2009 Wiley-Liss, Inc.
668
parts (Baker et al., 1990; Lowry et al., 2008; Steinbusch,
1981). The lateral extension is also called the “lateral
wings” and is well developed at the level of the central DR
(Steinbusch, 1981). The DRD can be further divided into
two subregions, the DRD core region (DRDC) and DRD
shell region (DRDSh). The DRDC contains a compact clusGrant sponsor: Ministry of Education, Culture, Sports, Science and
Technology (MEXT); Grant number: 20700315 (to H.H.); Grant number:
19700317 (to K.C.N.); Grant number: 21700380 (to K.C.N.); Grant number: 20020014 (to F.F.); Grant number: 17022020 (to T.K.); 21650083
(to T.K.); Grant sponsor: Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST); Grant number: 1000406000026 (to T.K.).
*CORRESPONDENCE TO: Prof. Takeshi Kaneko, MD, PhD, Department
of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan. E-mail: kaneko@mbs.med.kyoto-u.ac.jp
Received 13 February 2009; Revised 15 June 2009; Accepted 2 October 2009.
DOI 10.1002/cne.22237
Published online October 13, 2009 in Wiley InterScience (www.interscience.
wiley.com).
The Journal of Comparative Neurology 円 Research in Systems Neuroscience
518:668 – 686 (2010)
------------------------------------------------------------------------------------------------------------------------ VGLUT3-expressing nonserotonergic neurons in DR
ter of serotonergic neurons, whereas the DRDSh consists
of scattered serotonergic neurons in the surrounding regions (Abrams et al., 2005; Lowry et al., 2008).
The rat DR has, however, been indicated to contain a
substantial number of nonserotonergic neurons (Descarries et al., 1982). Numerous studies have revealed that
nonserotonergic neurons in the rat DR project to various
brain regions (Aznar et al., 2004; Beitz et al., 1986; Datiche
et al., 1995; Halberstadt and Balaban, 2006, 2007, 2008;
Hay-Schmidt et al., 2003; Kim et al., 2004; Kohler and
Steinbusch, 1982; Ma et al., 1991; O’Hearn and Molliver,
1984; Petrov et al., 1992, 1994; Van Bockstaele et al.,
1993; Villar et al., 1988). These nonserotonergic projection neurons are considered to utilize dopamine (Lindvall
and Bjorklund, 1974; Ochi and Shimizu, 1978; Trulson et
al., 1985), ␥-aminobutyric acid (GABA; Mugnaini and Oertel, 1985; Stamp and Semba, 1995), glutamate (Kaneko et
al., 1989, 1990; Kiss et al., 2002; Ottersen and StormMathisen, 1984; Schwarz and Schwarz, 1992), nitric oxide
(Nakamura et al., 1991; Pasqualotto et al., 1991), and neuropeptides such as CRF (Commons et al., 2003) as a neurotransmitter.
We and other groups have previously reported that vesicular glutamate transporter 3 (VGLUT3), which is responsible for the uptake of glutamate into synaptic vesicles, is
abundantly expressed in the DR and MnR (Fremeau et al.,
2002; Gras et al., 2002; Hioki et al., 2004). The expression
of VGLUT1 and VGLUT2, the other VGLUT isoforms, accounts for almost all acknowledged glutamatergic neuronal populations of the brain, and the two have been utilized
as selective and reliable markers for glutamatergic neurons (Fremeau et al., 2004; Herzog et al., 2004; Kaneko
and Fujiyama, 2002; Takamori, 2006). In contrast, VGLUT3
is expressed mostly in neurons that use transmitters other
than glutamate, such as GABAergic neurons in the neocortex (Fremeau et al., 2002; Hioki et al., 2004) and hippocampus (Somogyi et al., 2004), cholinergic neurons in
the neostriatum (Fremeau et al., 2002; Fujiyama et al.,
2004; Gras et al., 2002; Schafer et al., 2002), and serotonergic neurons in the DR and MnR (Fremeau et al., 2002;
Gras et al., 2002; Hioki et al., 2004; Jackson et al., 2009;
Mintz and Scott, 2006; Schafer et al., 2002; Shutoh et al.,
2008). Although VGLUT3 has not yet been well established
as a marker for glutamatergic neurons, it has been demonstrated that VGLUT3 is responsible for the glutamatergic transmission in the inner hair cells of mouse and zebrafish (Obholzer et al., 2008; Ruel et al., 2008; Seal et al.,
2008) and the rat medullary raphe neurons (Nakamura et
al., 2004). Thus, it is likely that VGLUT3 contributes to
glutamatergic transmission at some synapses in the brain.
In the DR and MnR, although most VGLUT3-expressing
neurons are serotonergic, significant numbers of VGLUT3expressing neurons are negative for serotonin (Gras et al.,
2002; Hioki et al., 2004; Jackson et al., 2009; Mintz and
Scott, 2006). Geisler et al. (2007) investigated the glutamatergic inputs to the ventral tegmental area (VTA) by a
combination of retrograde tracing method and in situ hybridization histochemistry for VGLUTs and demonstrated
that some retrogradely labeled neurons displayed the signals for VGLUT3 in the DR and MnR. However, it has not
been revealed whether these VGLUT3-expressing projection neurons were serotonergic or nonserotonergic, insofar as serotonin immunoreactivity was not determined in
their report. A more recent study reported that some of the
VGLUT3-expressing nonserotonergic neurons, mostly in
the MnR, project to the hippocampal CA1 and medial septum by retrograde tracer injection and triple immunofluorescence labeling for VGLUT3, serotonin, and retrograde
tracer (Jackson et al., 2009). Although the latter study
clearly indicates that some nonserotonergic projection
neurons in the DR and MnR are positive for VGLUT3, it
remains unclear that how VGLUT3-expressing neurons are
distributed in the DR and MnR and whether or not these
neurons might send axons to brain regions other than the
VTA, hippocampal CA1, and medial septum.
In the present study, we attempted to explore the precise distribution of VGLUT3-expressing neurons in the DR
and MnR by in situ hybridization histochemistry and then
to examine the colocalization of VGLUT3 and tryptophan
hydroxylase 2 (TPH2) mRNAs in the DR and MnR. TPH2 is
one of the rate-limiting enzymes for serotonin biosynthesis
and is expressed principally in the CNS (Clark et al., 2006;
Cote et al., 2003; Malek et al., 2005; Patel et al., 2004).
Surprisingly, VGLUT3-expressing nonserotonergic neurons were preferentially distributed in the DRDSh, so we
further investigated the projection targets of VGLUT3expressing nonserotonergic neurons in the DRDSh with
anterograde and retrograde tracing methods after chemical depletion of serotonergic neurons in the DR.
MATERIALS AND METHODS
Animals and primary antibodies
The experiments were conducted in accordance with
the Committee for Animal Care and Use and that for Recombinant DNA Study of Kyoto University. Eighteen adult
male Wistar rats (200 –250 g; Japan SLC, Hamamatsu, Japan), four female guinea pigs (200 g; Japan SLC), and two
female white rabbits (2 kg; Japan SLC) were used in the
present study. All efforts were made to minimize animal
suffering and the number of animals used.
In the present study, we used a rabbit polyclonal antibody to serotonin (5-hydroxytryptamine; 5-HT), mouse
monoclonal antibodies to neuron-specific nuclear protein
(NeuN) and tyrosine hydroxylase (TH), a guinea pig polyclonal antibody to VGLUT3, and a goat polyclonal antibody
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Hioki et al. -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
TABLE 1.
Primary Antibodies Used
Antibody
Immunogen
Host
Manufacturer data
Sigma-Aldrich (St. Louis, MO),
rabbit polyclonal, catalog
No. S5545, lot No.
32K4871
Millipore (Billerica, MA),
mouse monoclonal, catalog
No. MAB377, lot No.
0507004415
Millipore, mouse monoclonal,
catalog No. MAB318, lot
No. 24040251
Hioki et al., 2004
1:1,000
List Biological Laboratories
(Campbell, CA), goat
polyclonal, catalog No.
703, lot No. 7032F
1:1,000 (immunofluorescence)
and 1:5,000
(immunoperoxidase)
Serotonin (5-HT)
Serotonin creatinine sulfate
complex conjugated to
bovine serum albumin
Rabbit,
polyclonal
Neuron-specific
nuclear protein
(NeuN)
Purified cell nuclei from mouse
brain
Mouse,
monoclonal
Tyrosine
hydroxylase (TH)
Purified TH from PC12 cells
Mouse,
monoclonal
Vesicular glutamate
transporter 3
(VGLUT3)
C-terminal 25 amino acids of
rat VGLUT3 with addition of
N-terminal cysteine,
sequence: CQQRESAFEGEEPLSYQNEEDFSETS
B subunit pentamer of cholera
toxin (choleragenoid)
Guinea pig,
polyclonal
Cholera toxin B
subunit (CTb)
Goat,
polyclonal
for cholera toxin B subunit (CTb) as primary antibodies
(Table 1). All antibodies have been characterized previously.
Anti-serotonin serum was developed in rabbit using serotonin creatinine sulfate complex conjugated to bovine
serum albumin (BSA) as the immunogen. The antibody
stains serotonin-containing cells and fibers in the rat brain.
This staining is abolished by preincubation of the antiserum with 500 ␮M serotonin or 200 ␮g/ml serotoninBSA (manufacturer’s product information sheet).
A mouse monoclonal antibody against NeuN was originally made against cell nuclei purified from mouse brain
(clone A60; Mullen et al., 1992). This antibody recognizes
the nuclei and cell bodies of most neuronal cell types but
not glial fibrillary acidic protein (GFAP)-positive cells
throughout the CNS of rodents (Mullen et al., 1992). The
antibody also detects several bands at 46 – 48 kDa on
Western blots with isolated mouse brain nuclei and is
thought to reflect multiple phosphorylated isoforms of
NeuN (Lind et al., 2005; Mullen et al., 1992).
A mouse anti-TH monoclonal antibody was raised
against purified TH from PC12 cells, which recognizes an
epitope on the outside of the regulatory N-terminus of TH.
On Western blots with protein extracts of the rat brain, the
antibody recognizes a single band at 62 kDa, which corresponds to the estimated molecular weight of TH (Shepard
et al., 2006; Wolf and Kapatos, 1989).
A guinea pig polyclonal antibody to VGLUT3 was produced and characterized in a previous study (Hioki et al.,
2004). Briefly, peptide corresponding to the C-terminal 25
amino acids of rat VGLUT3 was synthesized with addition
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Dilution
1:100
1:1,000
1.0 ␮g/ml
of N-terminal cysteine for coupling of the peptide with a
carrier protein. The guinea pigs were immunized with the
conjugate of the peptide and maleimide-activated BSA
(Pierce, Rockford, IL). The antisera were then affinity purified by column chromatography with an antigenconjugated column. In the immunoblotting test with rat
brain extracts, the antibody specifically recognized a single band that was in register with molecular weight of
VGLUT3. When the primary antibody was preincubated
with an excess amount of the antigen peptide, no immunoreactivity was observed on the rat tissue sections.
A goat polyclonal antibody to CTb was used to detect
CTb in the retrograde labeling experiment. The anti-CTb
antibody was made against the B subunit pentamer of
cholera toxin (choleragenoid) as immunogen and did not
bind to any endogenous epitopes in the lower brainstem of
the rat (Pang et al., 2006). In the present study, we also
performed immunostaining for CTb using the sections of
animal brain without injection of CTb and observed no
immunoreactivity in the rat forebrain or midbrain.
Anti-mRFP1 antibody production and
characterization
We produced antibodies against monomeric red fluorescent protein (mRFP1; Campbell et al., 2002; gift from Dr.
Roger Y. Tsien) in rabbits and guinea pigs. The full-length
coding sequence of mRFP1 was introduced into the SmaI
site of pGEX4T2 (GE Healthcare Bio-Sciences, Piscataway,
NJ). Glutathione-S-transferase (GST)-mRFP1 fusion protein
was induced in Escherichia coli. After removing GST with
thrombin protease, mRFP1 was purified according to the
manufacturer’s instruction (GE Healthcare Bio-Sciences).
Two female white rabbits and four female guinea pigs were
immunized by intradermal injections of the purified mRFP1
(2 mg/rabbit, 0.5 mg/guinea pig) in Freund’s complete
adjuvant (BD Biosciences, San Jose, CA) and of the same
amount in incomplete adjuvant 4 weeks later. The sera
were recovered 9 –21 days after the second immunization.
The guinea pig and rabbit antibodies were purified to crude
IgG by ammonium sulfate fractionation (50% saturation)
and by two-step sodium sulfate fractionation (18% and
14%; Johnstone and Thorpe, 1982), respectively. The polyclonal antibodies were further purified by affinity chromatography on a mixture of 0.5 ml Affi-Gel 10 and 0.5 ml
Affi-Gel 15 (Bio-Rad, Hercules, CA) coupled with the antigen protein mRFP1 (2 mg). The specific antibodies were
eluted with 0.1 M glycine-HCl (pH 2.5).
To perform Western blotting, we first prepared the
HEK293 cells expressing mRFP1. The full-length coding
sequence of mRFP1 was inserted into the EcoRV site of
pBSIISK-CMV-WPRE [pBSIISK; pBluescript II SK(⫹); Stratagene, La Jolla, CA; CMV, cytomegalovirus immediateearly promoter; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; Hioki et al., 2007).
Forty-eight hours after the transfection of the plasmid into
the HEK293 cells with Lipofectamine 2000 (Invitrogen,
Carlsbad, CA), whole-cell protein containing mRFP1 was
extracted with CelLytic M (Sigma-Aldrich, St. Louis, MO).
The whole-cell protein solution (10 mg) was reduced by
heating at 100°C for 10 minutes with 0.7% (v/v)
2-mercaptoethanol and 1% (w/v) sodium dodecyl sulfate
(SDS) and electrophoresed in 12% polyacrylamide gel in
the presence of 0.1% (w/v) SDS. Electrophoresed proteins
were further transferred onto a polyvinylidene difluoride
membrane (Millipore, Bedford, MA). After blocking with
Block-Ace (Dainippon Sumitomo Pharma, Osaka, Japan),
the membranes were incubated overnight at room temperature with 1 ␮g/ml rabbit or guinea pig antibody and then
for 1 hour with alkaline phosphatase-conjugated goat antibody to rabbit IgG (0.05 ␮g/ml, AP156A; Millipore) or to
guinea pig IgG (0.1 ␮g/ml, AP108A; Millipore). The antibodies were diluted with 5 mM sodium phosphate (pH
7.4)-buffered 0.9% (w/v) saline (PBS) containing 10% (v/v)
Block-Ace and 0.2% (v/v) Tween-20. The membranes were
finally developed with 0.375 mg/ml nitroblue tetrazolium
and 0.188 mg/ml 5-bromo-4-chloro-3-indolyl phosphate
(NBT/BCIP; Roche Applied Science, Basel, Switzerland) in
0.1 M Tris-HCl (pH 9.5), 0.1 M NaCl, and 50 mM MgCl2.
Anterograde or retrograde labeling of
VGLUT3-expressing neurons in the DR after
chemical depletion of serotonergic neurons
Four milligram of 5,7-dihydroxytryptamine (5,7-DHT;
Sigma-Aldrich) was freshly dissolved in 200 ␮l of 0.9%
(w/v) saline containing 0.1% (w/v) ascorbic acid. Twelve
rats were deeply anesthetized with chloral hydrate (35
mg/100 g body weight) and then injected stereotaxically
into the right lateral ventricle (1.3 mm posterior to the
bregma, 2.0 mm left to the midline, and 3.2 mm deep from
the brain surface) with 10 ␮l of 5,7-DHT solution by pressure through a glass micropipette attached to a Picospritzer III (General Valve Corporation, East Hanover, NJ).
One week after the chemical depletion of serotonergic
neurons with 5,7-DHT, we injected 0.2 ␮l pal-mRFP1Sindbis viral solution (1.0 ⫻ 106 IU/ml in saline; Nishino et
al., 2008) by pressure into the DR (7.6 mm posterior to the
bregma, the midline and 5.6 mm deep from the brain surface; n ⫽ 3). We also injected the virus (1.0 ⫻ 1010 IU/ml
in saline) into the neostriatum of the untreated rats (0.2
mm posterior to the bregma, 3.4 mm left to the midline,
and 3.7 mm deep from the brain surface; n ⫽ 2) to examine
the specificity of rabbit and guinea pig anti-mRFP1 antibodies on tissue sections. The rats were allowed to survive
for 48 hours.
One week after the chemical depletion, we also injected
2% (w/v) CTb (List Biological Laboratories, Campbell, CA)
in 0.1 M sodium phosphate (PB; pH 7.4) iontophoretically
by passing 1-␮A positive current pulses (7-sec on/7-sec
off) for 30 minutes into the medial and lateral preoptic area
(MPA and LPO; 0.7 mm posterior to the bregma, 1.2 mm
right to the midline, and 8.3 mm deep from the brain surface; n ⫽ 3), anterior hypothalamic area (AHA; 1.8 mm
posterior to the bregma, 0.7 mm right to the midline, and
8.1 mm deep from the brain surface; n ⫽ 3), or ventral
tegmental area and substantia nigra (VTA and SN; 4.9 mm
posterior to the bregma, 1.7 mm right to the midline, and
7.8 mm deep from the brain surface; n ⫽ 3). These nine
rats were allowed to survive for 4 days.
Tissue preparation
The rats injected with pal-mRFP1-Sindbis virus or CTb,
and four untreated rats were deeply anesthetized with
chloral hydrate (70 mg/100 g body weight) and perfused
transcardially with 200 ml PBS. The rats were further perfused for 30 minutes with 200 ml 3% (w/v) formaldehyde,
75%-saturated picric acid, and 0.1 M Na2HPO4 (pH 7.0).
The brains were removed, cut into several blocks, and
postfixed with the same fixative for 8 hours at 4°C.
For in situ hybridization histochemistry, we instead used
4% (w/v) formaldehyde in 0.1 M PB as a fixative and postfixed the brain blocks with the same fixative for 3 days at
4°C. After cryoprotection with 30% (w/v) sucrose in PBS,
the brain blocks containing the midbrain raphe nuclei or
the other brain regions were cut into 20- or 40-␮m-thick
frontal sections, respectively, on a freezing microtome.
Sections for the immunoperoxidase reaction were im-
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Hioki et al. -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
mersed in PBS containing 1.0% (v/v) H2O2 for 30 minutes
to remove the endogenous peroxidase reactivity.
In situ hybridization histochemistry
Complementary DNA fragment of VGLUT1 (nucleotides
855–1788; GenBank accession No. XM_133432.2; Watakabe et al., 2006), VGLUT2 (848 –2044; NM_080853.2; Nakamura et al., 2007), or glutamate decarboxylase 67 kDa isoform (GAD67; 276 – 894; NM_008077.4; Tamamaki et al.,
2003) was cloned into a vector pBluescript IIKS(⫹) (Stratagene, La Jolla, CA). cDNA fragment of VGLUT3 (2105–2462;
AJ491795.1; Hioki et al., 2004) or TPH2 (1487–2194;
NM_173839.2) was cloned into pBSIISK. By using the linearized plasmids as template, we synthesized sense and antisense single-strand RNA probes with a digoxigenin (DIG) or
fluorescein (FITC) RNA labeling kit (Roche Applied Science).
The following hybridization procedure was based on that
of the previous report (Liang et al., 2000), with some modifications. Free-floating sections were rinsed twice in 0.1 M
PB and then treated with 0.25% (v/v) acetic anhydride in
0.1 M triethanolamine for 10 minutes. After two washes in
0.1 M PB, the sections were preincubated for 1 hour at
60°C with a hybridization buffer, which consisted of 5⫻
saline sodium citrate (SSC; 1⫻ SSC ⫽ 0.15 M NaCl, and
0.015 M sodium citrate, pH 7.0), 2% (w/v) blocking reagent (Roche Applied Science), 50% (v/v) formamide, 0.1%
(w/v) N-lauroylsarcosine (NLS), and 0.1% (w/v) SDS. The
sections were then hybridized for 20 –24 hours at 60°C
with 1 ␮g/ml DIG-labeled sense or antisense RNA probe in
the hybridization buffer. After two washes in 2⫻ SSC, 50%
(v/v) formamide, and 0.1% (w/v) NLS for 20 minutes at
60°C, the sections were incubated with 20 ␮g/ml ribonuclease A (RNase A) for 30 minutes at 37°C in 10 mM
Tris-HCl (pH 8.0), 1 mM ethylenediamine tetraacetic acid,
and 0.5 M NaCl, followed by two washes with 0.2⫻ SSC
containing 0.1% (w/v) NLS for 20 minutes at 37°C. Subsequently, the sections were incubated overnight at room
temperature with 1:1,000-diluted alkaline phosphatase
(AP)-conjugated anti-DIG sheep antibody (11-093-274910; Roche Applied Science) in 0.1 M Tris-HCl (pH 7.5)buffered 0.9% (w/v) saline (TS7.5) containing 1% Blocking
Reagent. After being washed three times for 10 minutes
with TS7.5 containing 0.1% (v/v) Tween-20 (TNT), the sections were reacted for several hours with NBT/BCIP.
Sense probes detected no signal higher than the background.
For double-fluorescence in situ hybridization, sections
were hybridized with a mixture of 1 ␮g/ml FITC-labeled and 1
␮g/ml DIG-labeled riboprobes. After washes and RNase A
treatment, the hybridized sections were incubated overnight
at room temperature with a mixture of 1:2,000-diluted
peroxidase-conjugated anti-FITC sheep antibody (11-426346-910; Roche Applied Science) and 1:1,000-diluted AP-
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conjugated anti-DIG sheep antibody. After being washed
three times for 10 minutes each with TNT, the sections were
treated with a TSA-Plus dinitrophenol (DNP) kit (PerkinElmer,
Wellesley, MA) for 30 minutes and then with 1:250-diluted
AlexaFluor488-conjugated anti-DNP rabbit antibody (A-11097;
Invitrogen) for 2 hours. The sections were finally reacted with
a 2-hydroxy-3-naphtoic acid-2⬘-phenylanilide phosphate
(HNPP) Fluorescence Detection kit (HNPP/FastRed; Roche
Applied Science) for several hours.
For double labeling with fluorescence in situ hybridization and immunofluorescence, sections were hybridized
with 1 ␮g/ml FITC-labeled VGLUT3 riboprobe, DIG-labeled
TPH2 riboprobe, or DIG-labeled GAD67 riboprobe. The sections hybridized with FITC-labeled VGLUT3 riboprobe were
incubated overnight at room temperature with a mixture of
1:2,000-diluted peroxidase-conjugated anti-FITC sheep
antibody and mouse anti-NeuN antibody or mouse anti-TH
antibody. After two washes with TNT, the sections were
incubated with 5 ␮g/ml AlexaFluor594-conjugated goat
antibody to mouse IgG (A-11032; Invitrogen) for 2 hours.
After being washed three times for 10 minutes with TNT,
the sections were treated with TSA-Plus DNP kit for 30
minutes and then with 1:250-diluted AlexaFluor488conjugated anti-DNP rabbit antibody for 2 hours.
The sections hybridized with DIG-labeled TPH2 or
GAD67 riboprobe were incubated overnight at room temperature with a mixture of 1:1,000-diluted AP-conjugated
anti-DIG sheep antibody and mouse anti-TH antibody or
antiserotonin antibody. After two washes with TNT, the
sections were incubated with 5 ␮g/ml AlexaFluor488conjugated goat antibody to mouse IgG (A-11029; Invitrogen) or rabbit IgG (A-11034; Invitrogen) for 2 hours and
then reacted with HNPP/FastRed for several hours.
Double-immunofluorescence labeling
In the anterograde labeling experiment with pal-mRFP1Sindbis virus, the sections were incubated overnight with a
mixture of 1.0 ␮g/ml affinity-purified rabbit anti-mRFP1
antibody and guinea pig anti-VGLUT3 antibody. After a
rinse with PBS containing 0.3% (v/v) Triton X-100 (PBS-X),
the sections were incubated for 1 hour in PBS-X, 0.25%
(w/v) ␭-carrageenan, and 1% (v/v) normal donkey serum
(PBS-XCD) with a mixture of 5 ␮g/ml AlexaFluor647conjugated goat antibody to rabbit IgG (A-21245; Invitrogen) and 5 ␮g/ml AlexaFluor488-conjugated goat antibody to guinea pig IgG (A-11073; Invitrogen).
In the CTb injection experiment, the adjacent sections
containing the middle DR were incubated overnight with a
mixture of guinea pig anti-VGLUT3 antibody and goat antiCTb antibody in PBS-XCD. After a rinse with PBS-X, the
sections were incubated for 1 hour in PBS-XCD with 10
␮g/ml biotinylated donkey antibody to goat IgG and then
for 1 hour with a mixture of 10 ␮g/ml Cy5-conjugated
donkey antibody to guinea pig IgG (AP193S; Millipore)
and 2 ␮g/ml AlexaFluor488-conjugated streptavidin
(S-11223; Invitrogen) in the presence of 10% (v/v) normal
goat serum.
Immunoperoxidase staining
Some sections were incubated overnight with mouse
anti-TH antibody or goat anti-CTb antibody, followed by 10
␮g/ml biotinylated donkey antibody to mouse IgG (715065-151; Jackson Immunoresearch, West Grove, PA) or
goat IgG (705-065-003; Jackson Immunoresearch) in PBSXCD. The sections were further incubated for 1 hour with
avidin-biotinylated peroxidase complex (ABC-Elite; Vector
Laboratories, Burlingame, CA) in PBS-X. After a rinse with
PBS-X, the sections were reacted for 20 – 40 minutes with
0.02% (w/v) 3,3⬘-diaminobenzidine (DAB)-4HCl (Dojindo,
Tokyo, Japan) and 0.001% (v/v) H2O2 in 50 mM Tris-HCl
(pH 7.6).
Image acquisition
The sections stained with NBT/BCIP or DAB were
mounted onto glass slides, dehydrated, cleared with xylene, and coverslipped. The micrographs were taken with a
QICAM FAST digital monochrome camera (QImaging, Surrey, British Columbia, Canada). Fluorescent sections were
mounted onto glass slides and coverslipped with the aqueous mounting medium Permafluor (Beckman Coulter, Fullerton, CA) or 50% (v/v) glycerol and 2.5% (w/v) triethylenediamine in PBS. The sections of the rats that received
pal-mRFP1-Sindbis virus were observed under a confocal
laser scanning microscope (AOBS-TCS SP2; Leica, Wetzlar, Germany) with a ⫻63 oil-immersion objective lens
(HCX PL Apo, NA ⫽ 1.40; Leica). AlexaFluor488 or AlexaFluor647 was excited with 488- or 633-nm laser beams
and observed through 500 – 610- or 645– 850-nm emission prism windows, respectively. The other fluorescent
sections were observed under an LSM5 Pascal confocal
laser scanning microscope (Carl Zeiss, Oberkochen, Germany) with appropriate laser beams and filter sets for AlexaFluor488 (excitation, 488 nm; emission, 505–530 nm),
fast red and AlexaFluor594 (excitation, 543 nm; emission,
ⱖ560 nm), or Cy5 (excitation, 633 nm; emission, ⱖ650
nm) using a ⫻10 (Plan-Neofluar, NA ⫽ 0.35; Carl Zeiss) or
a ⫻63 water-immersion objective lens (Plan-Neofluar,
NA ⫽ 0.75; Carl Zeiss). Digital images were modified
(⫾30% contrast and brightness enhancement) in Canvas 8
software (ACD Systems, Saanichton, British Columbia,
Canada) and saved as TIFF files. Cytoarchitectonic areas
were determined by using Nissl- or DAPI-stained sections
(Abrams et al., 2005; Lowry et al., 2008; Paxinos and
Watson, 2007).
Cell counting
To investigate the chemical characteristics of VGLUT3expressing neurons in the midbrain raphe nuclei, we examined whether VGLUT3-expressing cells might show the
signals for TPH2 mRNA, GAD67 mRNA, or TH immunoreactivity. We selected 20-␮m-thick single sections from
three rats and ensured the section level by carefully observing the shape and location of the aqueduct, medial
longitudinal fasciculus, and superior cerebellar peduncle
with DAPI counterstaining. We then counted the number of
cells with a clear nucleus. To avoid overcounting of the cell
number, we applied the Abercrombie correction factor
(Abercrombie, 1946; Guillery, 2002). The correction factor
is T/(T ⫻ D), in which T ⫽ section thickness and D ⫽
diameter. We utilized the mean diameters of nuclei of each
cell type as D (n ⫽ 20 cells; VGLUT3, 7.5 ␮m; TPH2, 7.7
␮m; VGLUT3 and TPH2, 7.5 ␮m; GAD67, 7.2 ␮m; TH, 7.2
␮m; NeuN, 7.4 ␮m; see Tables 2, 3).
RESULTS
Production and characterization of antimRFP1 antibody
Antibodies against mRFP1 were raised in rabbits and
guinea pigs and affinity purified with the antigenconjugated column. In the Western blot tests with the supernatant from the homogenate of HEK293 cells expressing mRFP1, anti-mRFP1 rabbit and guinea pig antibodies
detected a single protein band at the position of about
26,000 Da on the membrane (Fig. 1b,c). The immunoreactivity was completely abolished by preincubation of the
antibody with an excess amount of the antigen protein
(Fig. 1d,e). We further examined the specificity of the antibodies on tissue sections. Forty-eight hours after the injection of pal-mRFP1-Sindbis virus into the rat neostriatum, many neurons displayed strong native fluorescence
of mRFP1 (Fig. 1f). When the section was incubated with
anti-mRFP1 antibodies preabsorbed with an excess
amount of the antigen protein and then labeled with AlexaFluor488, no signal for AlexaFluor488 was detected on
the section (Fig. 1f⬘). These results indicate that the rabbit
and guinea pig antibodies specifically recognize the antigen protein mRFP1.
Distribution of TPH2 and VGLUTs mRNAs
By in situ hybridization histochemistry, intense signals
for TPH2 mRNA were observed in the DR, MnR, and B9 cell
group (Fig. 2a) as previously reported (Clark et al., 2006;
Cote et al., 2003; Malek et al., 2005; Patel et al., 2004).
Intense signals for VGLUT1 mRNA were found in the mesencephalic trigeminal nucleus (Me5), pontine tegmental
reticular nucleus of Bechterew (PTR), and pontine nuclei
(Pn; Fig. 2b) as previously reported (Hioki et al., 2003; Pang
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Figure 1. Characterization of rabbit and guinea pig anti-mRFP1 antibodies. a: Protein stain with Coomassie brilliant blue R-250. b– e:
The homogenate of HEK293 cells expressing mRFP1 was electrophoresed in 12% polyacrylamide gel in the presence of SDS, blotted onto
the membrane, and immunostained with the anti-mRFP1 antibodies
raised in rabbits (b,d) or guinea pigs (c,e). When the primary antibodies raised in rabbits (d) or guinea pigs (e) were preincubated with a
100-fold (in mol) excess amount of the antigen, no immunoreactivity
was observed. f,fⴕ: Forty-eight hours after the injection of pal-mRFP1Sindbis virus into the rat neostriatum, many neurons showed strong
native fluorescence for mRFP1 at the injection site (f). The section
was incubated with 1 ␮g/ml rabbit anti-mRFP1 antibody preabsorbed by a 100-fold (in mol) excess amount of the antigen and then
labeled with 5 ␮g/ml AlexaFluor488-conjugated goat antibody to
rabbit IgG (A-11034; Invitrogen). The section was observed under an
epifluorescence microscope (Axiophot; Carl Zeiss) with the appropriate filter sets for AlexaFluor488 (excitation, 450 – 490 nm; emission,
514 –565 nm) and for native fluorescence of mRFP1 (excitation,
530 –585 nm: emission ⱖ615 nm). No signal for AlexaFluor488 was
detected (f⬘). Scale bar ⫽ 40 ␮m.
et al., 2006). Weak to moderate signals for VGLUT2 mRNA
were observed in many regions, including the inferior colliculus (IC), periaqueductal gray (PAG), oral pontine reticular nucleus (PnO), PTR, and Pn (Fig. 2c), consistent with
previous studies (Fremeau et al., 2001; Geisler et al.,
2007; Hioki et al., 2003; Oka et al., 2008). The signals for
VGLUT3 mRNA in the DR and MnR were strong (Fig. 2d),
but those for VGLUT2 in the DR and MnR, if any, were very
scarce and weak (Fig. 2c).
Double fluorescence in situ hybridization for
VGLUT3 and TPH2 mRNAs in the DR and MnR
According to the distribution of VGLUT3 and TPH2
mRNA signals and the previous reports (Abrams et al.,
2004, 2005), we first divided the DR along the rostrocaudal axis into the rostral (DRr), middle (DRm), and caudal
(DRc) parts (Fig. 3a– c). We further subdivided the DRm
into the ventral part (DRV), lateral part (DRL), and core
region (DRDC) and shell region (DRDSh) of the dorsal part
(Fig. 3a–a⬘⬘). VGLUT3 mRNA signals were observed mainly
in the DRr, DRV, DRDSh, DRc, and MnR, whereas the expression of TPH2 mRNA was found in almost all the regions
674
Figure 2. In situ hybridization histochemistry for TPH2 and VGLUTs
mRNAs. a– d: Adjacent sections were hybridized with DIG-labeled
antisense RNA probe for TPH2, VGLUT1, VGLUT2, or VGLUT3 and
then visualized with NBT/BCIP. Sense probes detected no signal
higher than the background. Aq, aqueduct. Scale bar ⫽ 1 mm.
(Fig. 3a–a⬘⬘, Table 2). As previously reported (Abrams et
al., 2005; Lowry et al., 2008), the density of TPH2expressing neurons was remarkably high in the DRDC
compared with the surrounding regions, DRDSh (Figs. 3a⬘,
4b⬘,c⬘). Because the distribution of VGLUT3 and TPH2
mRNA signals in the interfascicular part (DRI) of the DR
was highly similar to that in the DRV, we included the DRI in
the DRV in the following experiments.
We also examined the double labeling for VGLUT3
mRNA and NeuN immunoreactivity, because it has been
reported that VGLUT3 immunoreactivity was found in a
subset of astrocytes by both light microscopy and immunoelectron microscopy (Fremeau et al., 2002). In the
present study, 99.4% ⫾ 0.4% (2,197 of 2,210, total cell
number, n ⫽ 3) of VGLUT3-expressing cells were immunoreactive for NeuN in the DR and MnR (Fig. 3e– e⬘⬘), indicating that VGLUT3 expression was restricted in neuronal
cells in the midbrain raphe nuclei. In addition, 99.2% ⫾
0.5% (5,539 of 5,586) of serotonin-positive neurons
showed signals for TPH2. Inversely, 99.4% ⫾ 0.1% (5,539
of 5,571) of TPH2-expressing neurons were immunoreac-
Figure 3. Double labeling for VGLUT3 and TPH2 mRNAs in the DR and MnR. VGLUT3 and TPH2 mRNAs were visualized with AlexaFluor488 (green)
and FastRed (magenta), respectively, in the DR and MnR. The DR was divided along the rostrocaudal axis into the rostral (DRr), middle (DRm), and
caudal (DRc) parts. We further subdivided the DRm into the ventral part (DRV), lateral part (DRL), and core region (DRDC) and shell region (DRDSh)
of the dorsal part, according to the expressions of VGLUT3 and TPH2 mRNAs (a–a⬘⬘). In the DRr, DRV, DRc, and MnR, the signals for TPH2 and
VGLUT3 mRNAs were frequently colocalized (a– d). TPH2-positive but VGLUT3-negative signals were found mainly in the DRL and DRDC (a–a⬘⬘),
whereas the signals for TPH2 were sparse, and VGLUT3-positive but TPH2-negative signals were dominant in the DRDSh (a–a⬘⬘). e– eⴕⴕ: VGLUT3
mRNA signals and NeuN immunoreactivity were visualized with AlexaFluor488 (green) and AlexaFluor594 (magenta), respectively. Almost all
VGLUT3-expressing neurons were immunoreactive for NeuN. f–fⴕⴕ: Serotonin (5-HT) immunoreactivity and TPH2 mRNA signals were visualized
with AlexaFluor488 (green) and FastRed (magenta), respectively. Almost all TPH2-expressing neurons were positive for 5-HT, and vise versa.
Arrowheads indicate the colocalization. Scale bars ⫽ 200 ␮m in a⬘⬘ (applies to a–a⬘⬘); 200 ␮m in b– d; 20 ␮m in f⬘⬘ (applies to e–f⬘⬘).
tive for serotonin (Fig. 3f–f⬘⬘), indicating that serotonergic
neurons in the midbrain raphe nuclei were sufficiently visualized with the present method.
We then investigated the colocalization of the signals
for VGLUT3 and TPH2 mRNA in the DR and MnR. We also
estimated the total number of neurons in each subregion by counting NeuN-immunoreactive cells with the
adjacent sections and calculated percentages of neurons showing the signals for VGLUT3 and/or TPH2 (Ta-
ble 2). In the DRr, DRV, DRc, and MnR, about 80% of
VGLUT3-expressing neurons were positive for TPH2
mRNA, and vice versa (Fig. 4a–a⬘⬘, Table 2). In the DRDC
and DRL, VGLUT3-expressing neurons were very few
(Table 2), and about 95% of TPH2-producing neurons
were negative for VGLUT3 mRNA (Fig. 4c,d⬘⬘, Table 2). It
was notable that VGLUT3-positive but TPH2-negative
neurons were found very frequently in the DRDSh (Table
2). Although 90.6% of TPH2-producing neurons showed
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Hioki et al. -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
TABLE 2.
Chemical Characterization of VGLUT3-Expressing Neurons in the DR and MnR
NeuN⫹1
VGLUT3⫹ TPH2⫹
VGLUT3⫹ TPH2–
VGLUT3– TPH2⫹
VGLUT3⫹ GAD67⫹
VGLUT3⫹ GAD67–
VGLUT3⫹ GAD67⫹
TPH2⫹
GAD67⫹
TPH2⫹
GAD67–
TPH2–
GAD67⫹
TH⫹3
TPH2/VGLUT3
VGLUT3/TPH2
DRr
DRV
DRDSh
DRDC
DRL
DRc
MnR
147.0 ⫾ 11.4
(100)
56.0 ⫾ 6.02
(38.1 ⫾ 2.7)2
9.2 ⫾ 2.2
(6.3 ⫾ 1.4)
11.1 ⫾ 2.3
(7.5 ⫾ 1.5)
0
(0)
63.5 ⫾ 7.0
(43.2 ⫾ 3.4)
45.3 ⫾ 6.1
(30.9 ⫾ 3.9)
1.7 ⫾ 0.6
(0.8 ⫾ 0.2)
64.3 ⫾ 13.4
(43.6 ⫾ 7.9)
49.5 ⫾ 4.8
(33.7 ⫾ 2.8)
22.1 ⫾ 4.6
(14.5 ⫾ 2.4)
86.0 ⫾ 1.74
83.6 ⫾ 1.7
227.7 ⫾ 13.9
(100)
153.0 ⫾ 13.9
(67.5 ⫾ 9.3)
17.2 ⫾ 2.2
(7.6 ⫾ 1.4)
33.0 ⫾ 3.6
(14.6 ⫾ 2.3)
0
(0)
148.1 ⫾ 12.0
(65.4 ⫾ 8.9)
9.8 ⫾ 3.7
(4.4 ⫾ 1.8)
0.3 ⫾ 0.6
(0.1 ⫾ 0.2)
200.5 ⫾ 22.1
(88.6 ⫾ 14.6)
11.5 ⫾ 3.3
(5.1 ⫾ 1.7)
1.7 ⫾ 1.1
(0.8 ⫾ 0.5)
89.8 ⫾ 1.7
82.3 ⫾ 2.4
182.0 ⫾ 14.3
(100)
36.6 ⫾ 8.3
(20.0 ⫾ 3.3)
111.0 ⴞ 13.2
(61.3 ⴞ 8.6)
3.9 ⫾ 1.5
(2.1 ⫾ 0.7)
0.3 ⫾ 0.6
(0.1 ⫾ 0.2)
161.2 ⫾ 10.1
(89.0 ⫾ 9.9)
13.7 ⫾ 1.9
(7.6 ⫾ 1.1)
0.3 ⫾ 0.6
(0.1 ⫾ 0.2)
37.3 ⫾ 7.6
(20.4 ⫾ 2.7)
17.2 ⫾ 3.0
(9.4 ⫾ 0.9)
14.0 ⫾ 3.4
(7.6 ⫾ 1.3)
24.9 ⴞ 5.9
90.7 ⫾ 2.3
40.1 ⫾ 2.6
(100)
2.2 ⫾ 0.7
(5.4 ⫾ 1.5)
0.5 ⫾ 0.4
(1.2 ⫾ 1.0)
36.8 ⫾ 3.6
(92.4 ⫾ 14.7)
0
(0)
1.2 ⫾ 0.4
(3.0 ⫾ 1.0)
0
(0)
0
(0)
39.0 ⫾ 4.5
(97.0 ⫾ 6.5)
0
(0)
0
(0)
85.0 ⫾ 13.2
5.7 ⴞ 2.3
940
(100)
4.8 ⫾ 2.2
(2.1 ⫾ 0.9)
1.5 ⫾ 0.7
(0.6 ⫾ 0.3)
113.6 ⫾ 9.8
(49.8 ⫾ 6.0)
0
(0)
4.4 ⫾ 0.7
(2.0 ⫾ 0.3)
102.7 ⫾ 9.2
(44.9 ⫾ 2.7)
2.0 ⫾ 1.0
(0.6 ⫾ 0.3)
112.2 ⫾ 11.3
(49.1 ⫾ 5.7)
99.0 ⫾ 16.5
(43.2 ⫾ 6.0)
1.7 ⫾ 0.8
(0.7 ⫾ 0.4)
77.3 ⫾ 2.5
4.2 ⴞ 2.2
228.7 ⫾ 8.1
(100)
77.8 ⫾ 10.2
(60.4 ⫾ 2.9)
23.8 ⫾ 3.3
(18.4 ⫾ 1.1)
9.4 ⫾ 2.6
(7.2 ⫾ 1.4)
0
(0)
109.3 ⫾ 12.4
(85.4 ⫾ 10.8)
15.0 ⫾ 5.6
(11.5 ⫾ 3.3)
0.3 ⫾ 0.6
(0.2 ⫾ 0.3)
80.0 ⫾ 10.0
(63.0 ⫾ 13.1)
12.5 ⫾ 5.1
(9.6 ⫾ 3.1)
0
(0)
76.4 ⫾ 4.8
89.0 ⫾ 4.0
105.4 ⫾ 8.2
(100)
51.9 ⫾ 7.0
(49.4 ⫾ 7.2)
17.7 ⫾ 1.8
(17.0 ⫾ 3.1)
14.7 ⫾ 1.8
(14.1 ⫾ 2.9)
0
(0)
70.1 ⫾ 6.2
(66.5 ⫾ 1.4)
21.8 ⫾ 3.7
(20.7 ⫾ 3.1)
0
(0)
66.2 ⫾ 9.0
(63.1 ⫾ 9.6)
21.6 ⫾ 4.9
(20.4 ⫾ 3.4)
0
(0)
74.4 ⫾ 3.5
77.8 ⫾ 3.4
We calculated the number of neurons in each subregion by counting NeuN-immunoreactive cells in one sections of three rats and display the mean ⫾ SD.
We counted the number of neurons showing the signals for VGLUT3 and/or TPH2 in sections from three rats and display the mean ⫾ SD. We also
estimated the percentages of those neurons, assuming the number of NeuN-immunoreactive cells in the adjacent sections as 100%. The numbers in
parentheses are the mean ⫾ SD of the percentages in three rats.
3
All TH-immunoreactive neurons were negative for other chemical markers, VGLUT3, TPH2, and GAD67.
4
The numbers indicate the mean ⫾ SD of the percentages in three rats.
1
2
the signals for VGLUT3, only 24.9% of VGLUT3expressing neurons were positive for TPH2 mRNA (Fig.
4b– b⬘⬘, Table 2).
tonergic and serotonergic neurons in the midbrain raphe
nuclei do not utilize GABA or dopamine as a neurotransmitter.
Distribution of GABAergic or dopaminergic
neurons in the DR and MnR
Chemical depletion of serotonergic neurons
in the DR
We subsequently examined GAD67 mRNA signals and
TH immunoreactivity in VGLUT3-expressing neurons, because GABAergic and dopaminergic neurons were distributed in the midbrain raphe nuclei. The expressions of
VGLUT3 and GAD67 mRNAs were almost complementary
in the DR (Fig. 5a–a⬘). Almost all VGLUT3-expressing neurons were negative for GAD67 mRNA signals (Fig. 5b– b⬘,
Table 2). We also performed the double-fluorescence labeling for TPH2 and GAD67 mRNAs. As previously reported (Stamp and Semba, 1995), double-labeled neurons
were present only in small numbers (Table 2). We then
examined the colocalization of TH immunoreactivity
and VGLUT3, TPH2, or GAD67 mRNA signals. THimmunoreactive neurons were distributed mainly in the
DRr and DRDSh but were scarce in the DRV and DRL (Table
2). All TH-positive neurons displayed no signal for VGLUT3
(Fig. 5c– d⬘), TPH2, or GAD67 mRNA in the DR and MnR.
These results indicate that VGLUT3-expressing nonsero-
We injected serotonin analogue 5,7-DHT into the lateral
ventricle to deplete serotonergic neurons from the midbrain raphe nuclei. This chemical compound is well known
to be a selective toxin for serotonergic neurons and to
abolish almost all serotonergic neurons in the midbrain
raphe nuclei (Hioki et al., 2004; Reader, 1989). One week
after the injection, the signals for TPH2 mRNA in the DR
had almost completely disappeared (Fig. 6a). VGLUT3expressing neurons were largely decreased in the DR (Fig.
6b, Table 3), probably because many VGLUT3-expressing
neurons were serotonergic in the DR (Table 2). Indeed,
there were no significant differences between the number
of VGLUT3-positive but TPH2-negative neurons in the normal rats and the number of VGLUT3-positive neurons in
the 5,7-DHT-treated rats (Table 3; two-tailed Student’s
t-test, 0.12 ⱕ P ⱕ 0.71). On the other hand, GAD67 mRNA
signals or TH immunoreactivity remained intact in the DR
(Fig. 6c,d). The numbers of GAD67- or TH-positive neurons
676
Figure 4. Double labeling for VGLUT3 and TPH2 mRNAs in the subregions of the DR. a– dⴕⴕ: VGLUT3 and TPH2 mRNAs were visualized
with AlexaFluor488 (green) and FastRed (magenta), respectively. In
the DRV, colocalization of signals for VGLUT3 and TPH2 was frequently observed (arrowheads in a–a⬘⬘) but not in the DRDSh, DRDC,
or DRL. In the DRDSh, many neurons expressed only VGLUT3, and
only a few cells were double positive for VGLUT3 and TPH2 mRNA
signals (arrowheads in b– b⬘⬘). In the DRDC and DRL, most neurons
displayed the signals for TPH2 alone (c– d⬘⬘). Scale bar ⫽ 20 ␮m.
were not significantly different between the normal rats
and the 5,7-DHT-treated rats (Table 3; two-tailed Student’s
t-test, 0.07 ⱕ P ⱕ 0.74 for GAD67, 0.25 ⱕ P ⱕ 0.74 for
TH). These results suggest that deprivation of serotonergic
neurons with 5,7-DHT had no effect on the number of the
nonserotonergic neurons in the DR.
Anterograde labeling of VGLUT3-expressing
neurons in the DRDSh
The projection of VGLUT3-expressing neurons in the
DRDSh was then examined with an anterograde viral
tracer, pal-mRFP1-Sindbis virus (Nishino et al., 2008). This
viral vector expresses mRFP1 with a plasma membranetargeting signal (Furuta et al., 2001; Hioki et al., 2009;
Kameda et al., 2008; Moriyoshi et al., 1996), which is effective for visualizing neuronal processes. After chemical
depletion of serotonergic neurons in the DR and MnR, we
injected pal-mRFP1-Sindbis viral vector into the DRDSh.
As Sindbis virus causes rapid inhibition of host cell protein
Figure 5. Double labeling for VGLUT3 mRNA and GAD67 mRNA or
TH immunoreactivity. a– bⴕ: VGLUT3 and GAD67 mRNAs were visualized with AlexaFluor488 and FastRed, respectively. Almost all of
the GAD67-expressing neurons were negative for VGLUT3 mRNA
(arrows in b– b⬘). c– dⴕ: VGLUT3 mRNA and TH immunoreactivity
were visualized with AlexaFluor488 and AlexaFluor594, respectively.
All TH-immunoreactive neurons displayed no signal for VGLUT3 (arrows in d– d⬘). Scale bars ⫽ 200 ␮m in a⬘ (applies to a,a⬘); 20 ␮m in
b⬘ (applies to b,b⬘); 200 ␮m in c⬘ (applies to c,c⬘); 20 ␮m in d⬘ (applies
to d,d⬘).
synthesis by affecting the synthesis of host mRNAs (Frolov
and Schlesinger, 1994), it was difficult to detect VGLUT3
mRNA by in situ hybridization method in the present study.
Because the anti-VGLUT3 antibody stains not only axonal
terminals but also cell bodies (Hioki et al., 2004), we applied an immunohistochemical method to visualize
VGLUT3-expressing neurons in the following experiments.
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Figure 6. Chemical depletion of serotonergic neurons in the DR. a: One week after the injection of serotonin analogue 5,7-DHT into the right
lateral ventricle, almost all serotonergic neurons were removed from the DR. b: The signals for VGLUT3 mRNA were largely decreased in the DRV,
but those in the DRDSh showed slight decrease. c,d: GAD67 mRNA signals and TH immunoreactivity remained intact in the DR. Scale bar ⫽ 200
␮m.
TABLE 3.
Distribution of VGLUT3-, GAD67-, and TH-Expressing Neurons After Chemical Depletion of Serotonergic Neurons in the DR1
VGLUT3
GAD67
TH
DRr
DRV
DRDSh
DRDC
10.2 ⫾ 1.5
43.6 ⫾ 5.4
24.0 ⫾ 5.5
16.2 ⫾ 2.6
10.8 ⫾ 1.5
2.0 ⫾ 0.4
106.9 ⫾ 12.4
12.5 ⫾ 1.3
13.0 ⫾ 3.0
0
0
0
DRL
1.9 ⫾ 0.8
114.0 ⫾ 7.8
2.5 ⫾ 0.4
DRc
18.9 ⫾ 3.3
13.7 ⫾ 1.9
0
1
After chemical depletion of serotonergic neurons with 5,7-DHT, we counted the number of neurons expressing VGLUT3, GAD67, or TH in one sections of
three rats and display the mean ⫾ SD.
Forty-eight hours after the viral injection, we examined
whether the infected cells were positive for VGLUT3 immunoreactivity. About 90% (60 of 67, total cell number, n ⫽ 3)
of mRFP1-expressing cells were immunoreactive for
VGLUT3 (Fig. 7a– b⬘⬘). mRFP1-immunoreactive axons were
observed mainly in the VTA, substantia nigra pars compacta (SNc), anterior hypothalamic area (AHA), lateral
hypothalamic area, posterior hypothalamic area (PHA),
678
paraventricular hypothalamic nucleus (Pa), dorsomedial
hypothalamic nucleus (DMH), and preoptic area (POA) and
slightly in the ventral pallidum, parafascicular thalamic nucleus, central medial thalamic nucleus, central lateral thalamic nucleus, and periaqueductal gray. In these regions,
VGLUT3 immunoreactivity was mostly restricted to axonal
varicosities (Fig. 7b⬘– e⬘), and VGLUT3-immunoreactive
cell bodies were not observed. Then, we examined the
7b– b⬘⬘), SNc (Fig. 7c– c⬘⬘), AHA (Fig. 7d– d⬘⬘), PHA, DMH,
Pa, and POA (Fig. 7e– e⬘⬘). This indicates that at least
some VGLUT3-expressing nonserotonergic neurons in the
DRDSh are projection neurons.
Retrograde labeling of VGLUT3-expressing
neurons in the DR
Figure 7. Anterograde labeling of VGLUT3-expressing neurons in the
DRDSh with pal-mRFP1-Sindbis virus. a– bⴕⴕ: After the chemical depletion of serotonergic neurons, we injected pal-mRFP1-Sindbis virus
into the DRDSh (n ⫽ 3). Forty-eight hours after the viral injections,
90.2% ⫾ 2.8% (60 of 67 total cell number) of mRFP1-expressing
neurons were immunoreactive for VGLUT3 in the DRDSh (arrowheads in a–a⬘⬘). The cells immunoreactive for both mRFP1 and
VGLUT3 (solid circles) or only mRFP1 (open circles) were superimposed on a drawing for each injection experiment. c–fⴕⴕ: The mRFP1labeled axon varicosities were frequently colocalized with the immunoreactivity for VGLUT3 in the VTA (c– c⬘⬘), SNc (d– d⬘⬘), AHA (e– e⬘⬘),
and POA (f–f⬘⬘). Arrowheads indicate the colocalization. Scale bars ⫽
20 ␮m in b⬘⬘ (applies to b– b⬘⬘); 5 ␮m in f⬘⬘ (applies to c–f⬘⬘).
colocalization of immunoreactivities for mRFP1 and
VGLUT3 under a confocal laser scanning microscope.
Many axonal varicosities labeled with mRFP1 displayed
immunoreactivity for VGLUT3, especially in the VTA (Fig.
To confirm the projection of VGLUT3-expressing nonserotonergic neurons in the DRDSh, we also performed
retrograde labeling of VGLUT3-expressing neurons with
CTb. After chemical depletion of serotonergic neurons
with 5,7-DHT, we iontophoretically injected CTb into
the right side of the POA, AHA, or VTA/SNc (n ⫽ 3, for
each injection experiment). We examined by doubleimmunofluorescence staining whether or not CTblabeled neurons might show immunoreactivity for
VGLUT3 in the DR (Fig. 8a–a⬘⬘) and then counted the
number by using five serial sections, which mostly covered the DRm, for each injection experiment.
In the CTb injection into the POA (Fig. 8b– d), 88.8% ⫾
9.8% (26 of 30, total cell number, n ⫽ 3) of retrogradely
labeled cells displayed immunoreactivity for VGLUT3 in the
DRDSh (Fig. 8b⬘– d⬘). In contrast, only 3.3% ⫾ 5.8% (1 of
40) of CTb-positive cells showed immunoreactivity for
VGLUT3 in the DRL. In the DRV, fewer neurons were labeled with CTb, and 41.7% ⫾ 52.0% (4 of 9) of CTb-positive
cells were immunoreactive for VGLUT3. In the injection
into the AHA (Fig. 9a– c), 86.6% ⫾ 6.2% (31 of 36) of CTblabeled cells showed immunoreactivity for VGLUT3 in the
DRDSh (Fig. 9a⬘– c⬘). In the DRL, only 1.5% ⫾ 2.6% (1 of 43)
of CTb-positive cells showed immunoreactivity for
VGLUT3. In the DRV, 55.5% ⫾ 50.9% (5 of 10) of CTbimmunoreactive cells were positive for VGLUT3. With the
injection into the VTA/SNc (Fig. 10a– c), 91.4% ⫾ 4.8% (71
of 77) of retrogradely labeled cells displayed immunoreactivity for VGLUT3 in the DRDSh (Fig. 10a⬘– c⬘). In the DRV,
the colocalization of immunoreactivities for CTb and
VGLUT3 was also high, and 93.3% ⫾ 11.5% (14 of 15) of
CTb-immunoreactive cells were positive for VGLUT3. On
the other hand, only 9.0% ⫾ 1.8% (3 of 34) of CTb-positive
cells showed immunoreactivity for VGLUT3 in the DRL.
In all the retrograde labeling experiments, CTb- and
VGLUT3-positive nonserotonergic neurons were found
mainly in the DRDSh. Furthermore, there seemed to be no
laterality in the distribution of retrogradely labeled
VGLUT3-positive neurons. By contrast, CTb-positive but
VGLUT3-negative neurons were found predominantly in
the ipsilateral DRL. These projection neurons might be
GABAergic, because as most neurons in the DRL were
positive for either GAD67 or TPH2 mRNA signals (Table 2).
The present anterograde and retrograde labeling studies
indicate that VGLUT3-expressing nonserotonergic neu-
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Figure 8. Retrograde labeling of VGLUT3-expressing neurons in the DR with CTb injection into the POA. b– d: After the chemical depletion of
serotonergic neurons with 5,7-DHT, CTb was iontophoretically injected into the right side of the POA (n ⫽ 3) and then visualized by immunoperoxidase staining. a–aⴕⴕ: CTb and VGLUT3 were labeled with AlexaFluor488 (green) and Cy5 (magenta), respectively. Arrowheads and arrows
indicate the neurons immunoreactive for both CTb and VGLUT3 or only VGLUT3, respectively, in the DRDSh. bⴕ– dⴕ: The cells immunoreactive for
both CTb and VGLUT3 (solid circles) or only CTb (open circles) were superimposed on a drawing by using five major serial sections (20 ␮m
thickness) for each injection experiment. ac, anterior commissure; LPO, lateral preoptic area; MPA, medial preoptic area; ox, optic chiasm; SO,
supraoptic nucleus. Scale bars ⫽ 40 ␮m in a⬘⬘ (applies to a–a⬘⬘); 500 ␮m in d (applies to b– d).
rons in the DR, especially in the DRDSh, send ascending
axons to many brain regions.
DISCUSSION
In the present study, we investigated the distribution
and chemical characteristics of VGLUT3-expressing neurons in the DR and MnR. The signals for VGLUT3 mRNA
were observed mainly in the DRr, DRV, DRDSh, DRc, and
MnR. In the DRr, DRV, DRc, and MnR, about 80% of
680
VGLUT3-expressing neurons displayed the signals for
TPH2, and vice versa. In the DRL and DRDC, VGLUT3expressing neurons were very scarce, and only 5% of TPH2producing neurons were positive for VGLUT3 mRNA. Notably, in the DRDSh, many neurons expressed VGLUT3,
and about 75% of VGLUT3-producing neurons were negative for TPH2 mRNA, indicating that VGLUT3-expressing
nonserotonergic neurons were preferentially distributed in
the DRDSh. By using anterograde and retrograde trac-
Figure 9. Retrograde labeling of VGLUT3-expressing neurons in the DR with CTb injection into the AHA after the chemical depletion of
serotonergic neurons. a– c: Three injection sites. aⴕ– cⴕ: Retrograde labeling in the DR. For details see the legend to Figure 8. AHC, central part
of anterior hypothalamic area; AHP, posterior part of anterior hypothalamic area; f, fornix. Scale bar ⫽ 500 ␮m.
ing methods, we further revealed that these VGLUT3expressing nonserotonergic neurons in the DRDSh project
to many brain regions such as the VTA, SNc, AHA, Pa, and
POA. Although the DR has generally been considered as a
serotonergic nucleus, our findings demonstrate that
VGLUT3-expressing nonserotonergic, probably excitatory,
projection neurons are more numerous in the DRDSh.
Glutamatergic neurons in the DR and MnR
It has been suggested that glutamatergic neurons are
distributed in the rat DR and MnR by using antibodies
against glutamate (Ottersen and Storm-Mathisen,
1984) and phosphate-activated glutaminase (Kaneko,
2000; Kaneko et al., 1989). Ottersen and StormMathisen (1984) demonstrated that glutamate-like immunoreactivity was high in the rat DR and MnR by using
a polyclonal antibody against glutamate. They supposed, however, that the immunoreactivity might reveal
not only the transmitter glutamate but also other
metabolism-related glutamate. Because glutamate is a
general metabolic substrate and serves as the precursor of inhibitory transmitter GABA, glutamate immunoreactivity is not specific to glutamatergic neurons.
The distribution of glutamatergic neurons was also
examined by using a monoclonal antibody against
phosphate-activated glutaminase. Glutaminase is considered as a main synthetic enzyme of the transmitter
glutamate and has been applied as a morphological
marker for glutamatergic neurons and axon terminals in
the CNS. Cell bodies immunoreactive for phosphateactivated glutaminase were found in the rat DR and
MnR, presumably in the DRV, DRDSh, and MnR (Kaneko
et al., 1989). Furthermore, it was reported that most
serotonin-like immunoreactive cells were positive for
phosphate-activated glutaminase in the rat DR and MnR
(Kaneko et al., 1990). However, glutaminase is located
in some GABAergic neurons, such as thalamic reticular
nucleus neurons, where glutaminase probably supplies
the GABA precursor glutamate (Kaneko and Mizuno,
1988). Thus, glutaminase is not a strictly selective
marker for glutamatergic neurons in the CNS.
In the present study, we performed in situ hybridization
histochemistry for VGLUTs and revealed that VGLUT3 was
expressed mainly in the DR and MnR, consistent with the
previous study (Fremeau et al., 2002). Although VGLUT3
shows ⬎70% amino acid identity with and biochemical
characteristics similar to those of VGLUT1 and VGLUT2,
the distribution of VGLUT3 was quite different from those
of VGLUT1 and VGLUT2. VGLUT1 and VGLUT2 are expressed in well-established glutamatergic neurons,
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Figure 10. Retrograde labeling of VGLUT3-expressing neurons in the DR with CTb injection into the VTA/SNc after the chemical depletion of
serotonergic neurons. a– c: Three injection sites. aⴕ– cⴕ: Retrograde labeling in the DR. For details see the legend to Figure 8. ml, medial lemniscus;
SNr, substantia nigra pars reticulata. Scale bar ⫽ 500 ␮m.
whereas VGLUT3 is expressed mostly in neurons using
transmitters other than glutamate (Takamori, 2006). The
question, therefore, has been raised of the contribution of
VGLUT3 to exocytotic release of the neurotransmitter glutamate.
VGLUT3-expressing neurons in the CNS
However, several lines of evidence suggest that some
VGLUT3-expressing cells are glutamatergic. In the neostriatum, almost all cholinergic neurons express VGLUT3 but
not VGLUT1 and VGLUT2. We previously examined the
postsynaptic localization of ionotropic glutamate receptors in VGLUT3-expressing cholinergic synapses by the
postembedding immunogold method for double labeling of
VGLUT3 and glutamate receptors (Fujiyama et al., 2004).
Asymmetric synapses with VGLUT3-immunopositive axon
terminals showed immunoreactivities for AMPA receptor
subunits at the postsynaptic sites. This suggests that glutamate is utilized as a neurotransmitter in VGLUT3expressing cholinergic neurons.
The contribution of VGLUT3 in the glutamate release
was clearly demonstrated in the inner hair cells by using
VGLUT3 knockout mice (Seal et al., 2008). The inner hair
cells utilize glutamate as a neurotransmitter and express
only VGLUT3 among all the VGLUTs. In the knockout mice,
682
the inner hair cells exhibit physiological properties very
similar to those of the wild-type animals. Afferent nerve
terminals of the cells also displayed sodium and potassium
conductances very similar to those of the wild-type animals. However, the afferents of the knockout mice lack
synaptically evoked glutamate currents, indicating a specific, presynaptic defect in the glutamate release. Thus,
VGLUT3 should be necessary for vesicle filling and vesicular release of glutamate in the inner hair cells.
In the medullary raphe regions, VGLUT3-expressing
nonserotonergic neurons mediate thermoregulatory functions, including fever (Nakamura et al., 2004), with the
neurotransmitter glutamate. These VGLUT3-expressing
nonserotonergic neurons innervate thermoregulatory effector organs through sympathetic preganglionic neurons
in the intermediolateral cell column (IML) of the thoracic
spinal cord. Application of glutamate into the IML produced thermogenesis in the interscapular brown adipose
tissue (BAT), whereas microinjection of glutamate receptor antagonists blocked BAT thermogenesis, suggesting
that VGLUT3-expressing nonserotonergic neurons in the
medullary raphe regions mediate thermoregulatory functions via descending glutamatergic pathways. Although
further study is necessary for understanding of the
VGLUT3 function, it is likely that VGLUT3 contributes to
the glutamatergic transmission at some synapses in the
brain, including the midbrain raphe nuclei.
VGLUT3-expressing serotonergic and
nonserotonergic neurons in the DR and MnR
In the present study, we revealed that VGLUT3expressing neurons were distributed mainly in the DRr,
DRV, DRDSh, DRc, and MnR by in situ hybridization histochemistry. About 80% of VGLUT3-expressing neurons in
the DRr, DRV, DRc, and MnR showed signals for TPH2 and
vice versa. Although the expression of VGLUT3 in the serotonergic neurons seems controversial, several reports
suggest the corelease of glutamate and serotonin. Electrical stimulation of the DR evoked not only serotoninmediated inhibition with long latency but glutamatemediated excitation with short latency in the locus
coeruleus (Segal, 1979). It was also reported that mesopontine serotonergic neurons in microcultures produced biphasic responses consisting of fast excitatory
postsynaptic potentials (EPSPs) and slow inhibitory
postsynaptic potentials (IPSPs) and that these fast EPSPs
and slow IPSPs were blocked by glutamate and serotonin
receptor antagonists, respectively (Johnson, 1994; Johnson and Yee, 1995). However, further evidence should be
provided for the corelease of glutamate and serotonin.
We and other groups have previously reported that most
serotonergic neurons were positive for VGLUT3 mRNA or
immunoreactivity but that significant numbers of VGLUT3positive cells were nonserotonergic in the DR and MnR
(Gras et al., 2002; Hioki et al., 2004; Jackson et al., 2009;
Mintz and Scott, 2006). These cells have been assumed to
be glutamatergic neurons (Gras et al., 2002), GABAergic
neurons, or astrocytes (Mintz and Scott, 2006). In the
present study, we demonstrated that almost all VGLUT3expressing nonserotonergic cells were positive for NeuN
but negative for GAD67 and TH. Furthermore, we revealed
that these VGLUT3-expressing nonserotonergic neurons
were preferentially distributed in the DRDSh. These results
suggest that glutamatergic nonserotonergic neurons constitute a subregion, DRDSh, within the DR.
Gervais and Rouillard (2000) investigated the effects of
electrical stimulation of the DR neurons on the spontaneous activity of dopaminergic neurons in the VTA and SNc
with normal and serotonin-depleted rats. Electrical stimulation of the DR neurons elicited two different types of
responses in the VTA and SNc dopaminergic neurons in
the normal rats: inhibition– excitation and excitation–
inhibition responses. After chemical depletion of serotonergic neurons, the inhibition– excitation response was almost completely abolished, without any change in the
excitation–inhibition response in the VTA and SNc dopaminergic neurons. The authors concluded that serotoner-
gic input from the DR is mainly inhibitory and that nonserotonergic afferents from the DR play an excitatory role in
the VTA and SNc dopaminergic neurons. Thus, it is likely
that VGLUT3-expressing nonserotonergic neurons in the
DRDSh directly innervate and modulate the VTA and SNc
dopaminergic neurons with the neurotransmitter glutamate.
Projection of VGLUT3-expressing neurons in
the DR and MnR
The projection of glutamatergic neurons in the DR has
been suggested by using radioactive D-aspartate as a retrograde tracer (Kiss et al., 2002; Schwarz and Schwarz,
1992). In the present study, we revealed that VGLUT3expressing nonserotonergic neurons in the DRDSh project
to many brain regions such as the POA, hypothalamic nuclei, VTA, and SNc by anterograde and retrograde labeling
methods after chemical depletion of serotonergic neurons
with 5,7-DHT. The projection of VGLUT3-expressing neurons in the DR and MnR to the VTA was also recently
demonstrated by a combination of retrograde labeling
techniques and in situ hybridization histochemistry for
VGLUTs (Geisler et al., 2007). The VGLUT3-expressing projection neurons were largely distributed in the DRV and
DRD, presumably in the DRDSh, and less so in the MnR.
Insofar as Geisler et al. reported just the expression of
VGLUT3 mRNA in the retrogradely labeled cells and did not
determined whether these VGLUT3-expressing neurons
were serotonergic or nonserotonergic, the distribution in
the DR and MnR may contain both VGLUT3-expressing
serotonergic and nonserotonergic neurons. However, their
report supports the present observation that VGLUT3expressing nonserotonergic neurons in the DRDSh project
to the VTA. It was also reported that VGLUT3-expressing
nonserotonergic neurons in the DR and MnR project to the
hippocampal CA1 and medial septum (Jackson et al.,
2009). After injection of the retrograde tracer CTb into the
hippocampus or medial septum, Jackson et al. observed
immunoreactivities for CTb, TPH, and VGLUT3 in the DR
and MnR. In both the retrograde labeling experiments,
VGLUT3-positive but TPH-negative neurons were found
mostly in the MnR and less so in the DRL. These reports
and the present results suggest that VGLUT3-expressing
nonserotonergic neurons are heterogeneous with respect
to the projection targets and the distribution in the DR and
MnR. Thus, further study is necessary to elucidate completely the projection of VGLUT3-expressing nonserotonergic neurons in the DR and MnR.
Functional considerations
The dorsal raphe nucleus has been assumed to be topographically organized. Serotonergic neurons in each sub-
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region possess unique afferents, efferents, and functional
properties (Lowry et al., 2008). For instance, serotonergic
neurons in the DRD have been indicated to have an important role in the regulation of anxiety-related responses and
affective disorders, whereas those in the DRL have been
postulated to inhibit stress-induced autonomic and behavioral responses. In the present study, we revealed that the
DRDSh or DRL contained a significant number of VGLUT3positive or -negative nonserotonergic neurons, presumably glutamatergic or GABAergic, respectively (Table 2).
We further demonstrated that these nonserotonergic neurons projected to the same targets, such as the POA, AHA,
and VTA/SNc. Because serotonergic and nonserotonergic
DR neurons have developed local axon collaterals within
the DR (Li et al., 2001), they are considered to exchange
information with each other in the DR. Then, the integrated
information might be separately transferred to the target
regions by nonserotonergic and serotonergic neurons and
involved in the execution of their functions in a cooperative
manner.
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Vesicular glutamate transporter 3

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