Altered expression of neuronal tryptophan hydroxylase-2 mRNA in the dorsal
and median raphe nuclei of three genetically modified mouse models relevant
to depression and anxiety
Ali Jahanshahia,b,c,d,*, Erwan Le Maitrea, Yasin Temelb,c,d, Laurence Lanfumeye, Michel Hamone,
Klaus-Peter Leschf, Rosa M. Torderag, Joaquı ´n Del Rı ´og, Ester Asoh, Rafael Maldonadoh, Tomas Ho ¨kfelta,
Harry W.M. Steinbuschb,d
aDepartment of Neuroscience, Karolinska Institutet, Stockholm, Sweden
bDepartment of Neuroscience, Faculty of Health, Medicine and Life Sciences, School for Mental Health and Neuroscience, Maastricht University, Maastricht, The Netherlands
cDepartment of Neurosurgery, Maastricht University Medical Center, Maastricht, The Netherlands
dEuropean Graduate School of Neuroscience (EURON), The Netherlands
eUMR 894 INSERM-UPMC, Faculty of Medicine Pierre & Marie Curie, Paris, France
fMolecular Psychiatry, Department of Psychiatry, Psychosomatics and Psychotherapy, University of Wu ¨rzburg, Wu ¨rzburg, Germany
gDepartment of Pharmacology, University of Navarra, Pamplona, Spain
hLaboratori de Neurofarmacologia, Departament de Cie `ncies Experimentals i de la Salut, Universitat pompeu Fabra, PRBB, Barcelona, Spain
H I G H L I G H T S
? Our finding shows decreases in TPH2 mRNA expression in the DRN of 5-HTT?/? mice.
? Increases were observed in TPH2 mRNA levels in the DRN of VGLUT+/? mice.
? We also found substantial increase in TPH2 mRNA levels in the DRN of CB1?/? mice.
? TPH2 mRNA expression in different raphe subregions are differentially regulated.
Depression is a leading cause of disability worldwide and a
serious health problem (Greenberg et al., 2003), but the exact
mechanisms underlying its pathophysiology are still not well
Journal of Chemical Neuroanatomy 41 (2011) 227–233
A R T I C L E
I N F O
Received 18 February 2011
Received in revised form 22 May 2011
Accepted 25 May 2011
Available online 15 June 2011
In situ hybridization
Cannabinoid receptor 1
Glutamate vesicular transporter 1
Dorsal raphe nucleus
Median raphe nucleus
A B S T R A C T
Depression and anxiety are among the leading causes of societal burden. Abnormalities in 5-
hydroxytryptamine (5-HT; serotonin) neurotransmission are known to be associated with depressive
and anxiety symptoms. The rostral projections of brainstem dorsal (DRN) and median (MRN) raphe nuclei
are the main sources of forebrain 5-HT. The expression, turnover and distribution of tryptophan hydroxylase
2 (TPH2), the rate-limiting enzyme in 5-HT biosynthesis in the DRN and MRN are complex, in keeping with
the existence of different subpopulations of 5-HT neurons in this area. In the present study, we measured the
expression of TPH2 mRNA in the DRN and MRN using in situ hybridization in three genetically modified
mouse models, all relevant to depression and anxiety, and matched wild-type controls. Our results show
quantitative modifications in TPH2 mRNA expression in the three main subregions of the DRN as well as the
MRN in relation to changes in serotonergic, glutamatergic and endocannabinoid neurotransmission
systems. Thus, there were significant decreases in TPH2 transcript levels in 5-HT transporter (5-HTT)?/?
mutant mice, whereas increases were observed in the vesicular glutamate transporter 1 hemi knock out
(VGLUT1+/?) and cannabinoid receptor 1 mutant (CB1R?/?) mice.
Based on these findings, we suggest that TPH2 mRNA expression is under the influence of multiple
messenger systems in relation to presynaptic and/or postsynaptic feedback control of serotonin
synthesis that, 5-HTT, VGLUT1 and CB1R seem to be involved in these feedback mechanisms. Finally, our
data are in line with previous reports suggesting that TPH2 activity within different raphe subregions is
differentially regulated under specific conditions.
? 2011 Published by Elsevier B.V.
* Corresponding author at: Department of Neuroscience, FHML, Maastricht
University, UNS 50, Box 38, 6229 ER Maastricht, The Netherlands.
Tel.: +31 43 3881174; fax: +31 43 3671096.
E-mail addresses: email@example.com,
firstname.lastname@example.org (A. Jahanshahi).
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understood. One line of evidence suggests that abnormalities in 5-
hydroxytryptamine (5-HT; serotonin) neurotransmission are
associated with depressive and anxiety symptoms (Cowen, 2008).
The brainstem dorsal (DRN) and median (MRN) raphe nuclei,
cell groups B7 and B8, respectively, according to Dahlstrom and
Fuxe (1964), are the main source of forebrain serotonergic
innervation and subsequently 5-HT release (Steinbusch, 1981,
1984; Steinbusch and Nieuwenhuys, 1983; Michelsen et al., 2007,
2008). The DRN and MRN project heavily to several forebrain areas
that modulate emotional and cognitive processes (Kosofsky and
Molliver, 1987). The DRN can be subdivided anatomically into
ventromedial (DRV) and dorsomedial (DRD) subregions over much
of its length, and dorsolateral wings (DRL) in the mid-regions
(Steinbusch et al., 1981). Each of these subregions has been shown
to project to specific parts of the cerebral cortex and subcortical
regions, and consequently, may differentially regulate 5-HT
neurotransmission (O’Hearn and Molliver, 1984; Steinbusch and
de Vente, 1997). Moreover, these brainstem nuclei receive
descending projections from forebrain regions such as prefrontal
cortex (Hajos et al., 1998; Celada et al., 2001; Peyron et al., 1998).
Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in 5-
HT biosynthesis. The discovery of a neuronal isoform, TPH2, by
Walther and Bader (2003) opened up a new way to reliably map
brain 5-HT neurons with immunohistochemistry and in situ
hybridization. Results confirm a wide expression, including cell
bodies in DRN and MRN (Zhang et al., 2004; Clark et al., 2008). In
fact, it is now established that TPH2 is the predominant isoform in
the rodent brain (Gutknecht et al., 2008, 2009). However, early
studies have previously reported that TPH expression, turnover
and distribution in DRN are complex, in line with the existence of
different subpopulations of 5-HT neurons in this area (Weissmann
et al., 1990).
Changes in TPH2 gene and/or protein expression in the brain
have been reported in various mood disorders and have been
validated in animal models (Hiroi et al., 2006; Bach-Mizrachi et al.,
2008; Bonkale and Austin, 2008). TPH2 genetic variants have also
been extensively reported to be associated with major depression
(Zill et al., 2004; Van Den Bogaert et al., 2006; Haghighi et al.,
2008). However, whether these changes might reflect alterations
in possible modulatory effects of other neurotransmitter systems
on 5-HT systems is still largely unknown. To address this question
directly, we measured TPH2 mRNA expression in three different
transgenic mouse models, all related to pathological states of
depression and anxiety, but caused by mutations affecting
different neurotransmitter systems.
One of the models is a 5-HT transporter-deficient mouse (5-
HTT?/?) (Bengel et al., 1998; for review see Murphy and Lesch,
2008). By mediating the 5-HT reuptake in the nerve terminal (and
other parts of the 5-HT neuron), 5-HTT fine-tunes the magnitude
and duration of serotonergic signaling (Lesch and Mossner, 2006;
Canli and Lesch, 2007) which makes it the target for many
antidepressant drugs, including the selective serotonin reuptake
inhibitors (SSRIs) (Owens and Nemeroff, 1998). They exhibit major
adaptive changes in 5-HT neurotransmission, when compared
with their wild-type controls. It has been shown that lack of 5-HTT
depletes 5-HT and its metabolite 5-hydroxyindoleacetic acid by
60–80% in several brain areas such as the brainstem, striatum,
hippocampus and frontal cortex (Bengel et al., 1998; Fabre et al.,
2000). Functional desensitization of 5-HT1A, 5-HT1B autorecep-
tors has been reported in the DRN of the 5-HTT?/? mutants as a
consequence of high extracellular 5-HT levels in the vicinity of the
serotonergic cells in the DRN. However, in terms of their target
areas such as hippocampus and forebrain increased or no changes
were observed in expression of these receptors (Fabre et al., 2000;
Mannoury la Cour et al., 2001). Autoradiographic labeling of 5-
HT2A receptors has also revealed a 30–40% reduction in the density
of these receptors in the cerebral cortex and lateral striatum of 5-
HTT?/? in comparison to the wild type mice (Rioux et al., 1999). In
addition, anxiolytic- and antidepressant-like responses have been
observed in 5HTT?/? mice in behavioral test paradigms such as
the elevated plus maze, tail suspension and forced swim test
(Holmes et al., 2003a,b; Renoir et al., 2008).
The second model consists of mice heterozygous for the
vesicular glutamate transporter 1 (VGLUT1+/?) (Wojcik et al.,
2004). As the first of three vesicular glutamate transporters
(VGLUT1, VGLUT2 and VGLUT3) (Takamori et al., 2000), VGLUT1
has been shown to have a high level of expression in glutamatergic
neurons in the cerebral cortex (Hisano, 2003). By concentrating
glutamate in synaptic vesicles, VGLUT1 mediates glutamate
release from synaptic terminals and facilitates efficient glutama-
tergic transmission (Fremeau et al., 2004; Wilson et al., 2005).
Recent studies have demonstrated that VGLUT1+/? mice exhibit
deficient glutamate transmission (Balschun et al., 2009), depres-
sive-like behavior and neurochemical changes, which are related
to depression and anxiety (Tordera et al., 2007; Garcia-Garcia et al.,
2009). Aberrations in glutamate synthesis and its dysregulation
also appear to play a relevant role in major depression (Krystal
et al., 2002). In keeping with this, recent post-mortem studies
showing decreased cortical VGLUT1 in depressed subjects (Uezato
et al., 2009) together with clinical findings of an excitatory
inhibitory imbalance in the cortex of depressed patients (Sanacora
et al., 2004; Bhagwagar et al., 2007) suggest that decreased
VGLUT1 levels may have clinical implications.
Finally, mice with targeted disruption of the gene encoding
the cannabinoid 1 receptor (CB1R?/?) (Ledent et al., 1999) were
used as an additional model of mood disorders. The endocan-
nabinoid system is a major neuromodulatory system that
contributes to the control of emotional behavior (Maldonado
et al., 2006). The pharmacological and genetic blockade of the
CB1R induces a behavioral state analogous to depression in
experimental animals (Hill and Gorzalka, 2005). Thus, CB1R?/?
responsiveness to reward stimuli (Sanchis-Segura et al., 2004;
Maldonado et al., 2006) and enhanced anxiety levels and
sensitivity to stress (Martin et al., 2002; Aso et al., 2008).
Moreover, the chronic absence
alterations in 5-HT-dependent negative feedback. In particular,
enhanced extracellular 5-HT levels in the prefrontal cortex
decreased 5-HTT binding site density and caused functional
desensitization of the 5-HT1A autoreceptors. As well reduced
5-HT2C receptor expression in different brain regions has
previously been described in these mutants (Aso et al., 2009).
In addition, according to Mato et al. (2007) mice lacking
CB1R exert impaired post-synaptic serotonergic signaling,
suggesting that CB1R?/? mice are useful models to reveal
more regarding the nature of cannabinoid-5-HT interactions in
of CB1R activity induces
2. Experimental procedures
The experiments were carried out on 8–12-week-old male 5-HTT?/?,
VGLUT1+/? and CB1R?/? mice. Corresponding wild-type littermates were used
as controls for each genetic model. All animals used in a given experiment were
matched for age and weight. Mice were housed five per cage in a temperature
(21 ? 1 8C)- and humidity-controlled (55 ? 10%) room with a 12:12-h light/dark
cycle (light on 08:00) with food and water ad libitum. Animal procedures were
conducted according to European ethical guidelines (European Communities Council
Directive 86/609/EEC) and approved by the respective local Ethical Committees
Homozygous male 5-HTT?/? mice and wild-type (WT) littermates (obtained
from Pierre & Marie Curie, Paris, France) born from heterozygous mutants at the
tenth generation (F10) of backcrossing with C57BL/6J mice were used (Renoir
et al., 2008). Genotyping was performed as described by Bengel et al. (1998).
A. Jahanshahi et al. / Journal of Chemical Neuroanatomy 41 (2011) 227–233
Heterozygous VGLUT1 mice (VGLUT1+/?; C57BL/6N) were obtained from S.
Wojcik (Go ¨ttingen, Germany). The targeted knockout allele was generated by
truncation of the coding region of the VGLUT1 gene between the start codon and a
BglII site in the fifth coding exon through homologous recombination in embryonic
stem cells (129/ola background). A colony of WT and VGLUT1+/? mice was bred in
the animal house of the University of Navarra from heterozygous fathers and WT
mothers (Harlan, France). In this laboratory, mice were weaned and genotyped at
the age of 3 weeks. The first heterozygous generation has already been bred for
more than 20 generations in the C57BL/6N background. VGLUT1+/? mice were
studied and compared to their WT littermates. Heterozygous mice exhibited no
apparent phenotypic abnormalities during development and adulthood.
Mice lacking CB1R (obtained from PRBB, Barcelona, Spain) were generated as
previously described by Ledent et al. (1999). In order to obtain homogeneous
genetic background, the first heterozygous generation was bred for 30 generations
on a CD1 background, with selection for the mutant CB1R gene at each generation.
After the 30th generation of backcross, heterozygote–heterozygote mating of CB1R
knockout mice produced WT and CB1R?/? littermates for subsequent experiments.
All mice were sacrificed between 9:00 and 10:00 am, alternating between WT and
KO mice excluding all bias linked to the circadian cycle. Frozen brains from all three
mouse models were sent on dry ice from the respective collaborating university to
the Karolinska Institutet (Stockholm).
2.2. In situ hybridization
2.2.1. Tissue preparation
Mice were sacrificed by decapitation, and the brains were rapidly removed,
frozen in isopentane and stored at ?80 8C until use. Mice brainstem were cut into
20 mm thick sections through the rostro-caudal extent of the DRN and MRN using
cryostat, thaw-mounted on Superfrost slides (Fisher Scientific) and stored at
?20 8C. From the series of 50 sections through the rostro-caudal extent of DRN and
MRN, first of every four sections (12 in total) were subjected to in situ hybridization
(ISH). Therefore, optical density measurements for each subregion were carried out
using 12 sections taken from each brain and average value used for statistics.
Antisense oligoprobes complementary to mouse TPH2 mRNA (50-TCC GTC CAA
ATG TTG TCA GGT GGA TCC AGC CTC ACA ATG GTG GTC-30, position 505; accession
# NM_173391) were synthesized by CyberGene AB (Huddinge, Sweden). The
oligonucleotides were labeled at the 30end using terminal deoxynucleotidyl-
transferase (Amersham, Buckinghamshire, UK) with [a-33P]dATP (NEN, Boston,
MA, USA) to a specific activity of 1–4 ? 106cpm/ng oligonucleotide. The labeled
oligoprobes were purified using ProbeQuant G-50 Micro Columns (Amersham).
Sections were hybridized as described previously (Schalling et al., 1988; Dagerlind
et al., 1992). Briefly, air dried sections were incubated in a hybridization cocktail
(50% formamide, 4xSSC, 1? Denhardt’s solution 1% sarcosyl, 0.02 M phosphate
buffer, PH 7.6, 10% dextran sulfate, 500 mg/ml heat-denatured salmon sperm DNA,
1 ? 107cpm/ml of the labeled probe) in a humidified chamber for 16–18 h at 42 8C.
After hybridization, the sections were washed in 1? SSC for 4? 15 min at 55 8C and
for 30 min at RT, then air-dried and dipped into Kodak NTB 2 emulsion (Kodak,
Rochester, NY) diluted 1:1 with water. After exposure at 4 8C for 72 h, the slides
were developed in Kodak D19, fixed in Kodak Unifix and mounted in glycerol–
phosphate buffer. For specificity control, adjacent sections were incubated with an
excess (100?) of unlabelled probe.
2.3. Quantitative analysis of the TPH2 mRNA expression levels in the dorsal and median
The expression levels of TPH2 mRNA in the DRD, DRV, DRL and MRN were
quantified using an image analysis system (Nikon Microphot-MX microscope
equipped with a dark-field condenser) from digital photos taken with a digital
camera, DXM1200 (Nikon). Densitometric measurements (Image J software version
1.38?; NIH, Bethesda, USA) were obtained from the DRN after delineation of DRD,
DRV and DRL and from the MRN based on a mouse brain atlas (Paxinos and Franklin,
2001). The data are expressed as optical density ratios.
2.4. Statistical analyses
For all experiments, samples from transgenic and wild type mice were treated in
parallel. Data are presented as means and standard errors of means (S.E.M.). The
quantitative data of the TPH2 mRNA expression levels in the DRD, DRV, DRL and the
MRN were analyzed using two-way ANOVA and statistically significant differences
were evaluated further by a Tukey’s post hoc test. All statistical analyses were
performed with SPSS 15.0 version for Windows. p-values lower than 0.05 were
In all of the experiments, there were no significant differences between the right
and left sides of the DRL in any of the groups. Therefore, the data were pooled for
these subregions. Note that the ISH for each strain of mice and corresponding
controls was carried out independently at separate times. Therefore, the baseline of
the optical density signal differs in different sets of experiments.
3.1. Decreased TPH2 mRNA expression in the dorsal and median raphe
nuclei of mice deficient in 5-HT transporter
Quantitative optical density measurements of in situ hybridiza-
tion labeling showed that TPH2 mRNA expression was significantly
decreased in the DRD and DRV subregions of the DRN as well as in the
MRN (p < 0.01, p < 0.05 and p < 0.05, respectively) of homozygous
5-HTT?/? mice (n = 4) compared with WT counterparts (n = 4).
Although a decrease was also noted in the DRL, the difference
between 5-HTT?/? and WT genotypes did not reach the critical level
of statistical significance in this DRN subregion (Fig. 1).
3.2. Enhanced TPH2 mRNA expression in the dorsal and median raphe
nuclei of heterozygous VGLUT1+/? mice and CB1R knock out mutants
A significant increase of TPH2 mRNA expression in the DRD,
DRV, DRL and MRN (p < 0.01, p < 0.01, p < 0.05 and p < 0.05,
respectively) was observed in VGLUT1+/? mice (n = 4) in
comparison to wild-type littermates (n = 3) (Fig. 2).
Similarly, ISH labeling of TPH2 mRNA in the DRD, DRV, DRL and
MRN was at a significantly higher density (p < 0.05) in homozy-
Fig. 1. (A) Optical densitometry of TPH2 mRNA levels in the dorsomedial (DRD), ventromedial (DRV), and dorsolateral (DRL) parts of the dorsal raphe nucleus (DRN) and the
median raphe nucleus (MRN) of wild-type (WT) controls and 5-HTT knock-out (5-HTT?/?) mice. (B) Representative photomicrographs of 20 mm-thick sections at the level of
the DRN and the MRN from a WT and a 5-HTT?/? mouse. Note that 5-HTT?/? mice show a reduction of TPH2 mRNA expression. Each bar is the mean + S.E.M. from 4 mice of
each genotype. Scale bar: 500 mm, *p < 0.05 and **p < 0.01.
A. Jahanshahi et al. / Journal of Chemical Neuroanatomy 41 (2011) 227–233
gous CB1R?/? mutants (n = 5) than in paired wild-type controls
(n = 5) (Fig. 3).
Using three genetically modified mouse models, all character-
ized by altered behaviors in depression-related paradigms, our
results show quantitative differences in TPH2 mRNA expression in
the three main subregions of the DRN as well as in the MRN. These
changes are related to serotonergic or glutamatergic neurotrans-
mission and to endocannabinoid systems. Thus, there were
significant decreases in TPH2 mRNA expression in the 5-HTT?/
? mutant mice, whereas increases in TPH2 mRNA expression were
observed in the VGLUT1+/? and CB1R?/? mutant mice.
4.1. The 5-HTT?/? mouse
It has previously been shown that TPH mRNA levels and activity
can be regulated by the end-product 5-HT. For example, lower 5-
HT levels result in higher TPH mRNA expression in vivo (Park et al.,
1994). There is evidence that 5-HTT?/? mice exhibit marked
changes in 5-HT synthesis and turn-over. In particular, lack of 5-
HTT is associated with a 60–80% depletion of 5-HT and its
metabolite 5-hydroxyindoleacetic acid in several brain areas such
as the brain stem, striatum, hippocampus and frontal cortex as
compared with wild-type controls (Bengel et al., 1998; Fabre et al.,
2000). Desensitization of 5-HT1A, 5-HT1B autoreceptors has been
reported in 5-HTT?/? mutants as a consequence of high
extracellular 5-HT levels in the DRN (Fabre et al., 2000; Mannoury
la Cour et al., 2001). Interestingly, such changes were also observed
in mice after long-term SSRI administration (Landgrebe et al.,
2002), which are accompanied by decreased expression of TPH2
mRNA in DRN and MRN in rodents (Abumaria et al., 2007). In view
of behavioral and biochemical evidence of similar alterations of
serotonergic neurotransmission in mice lacking 5-HTT and in mice
receiving long-term SSRI therapy, it can be proposed that high
concentrations of 5-HT in the extracellular space of 5-HTT?/?
mice are also causally related to the reduced TPH2 mRNA
expression in these mutants. Conversely, high TPH2 protein and
mRNA levels have been reported in the DRN of depressed patients
compared to control subjects, possibly due to a compensatory
response to counteract deficits in extracellular 5-HT concentra-
tions (Bach-Mizrachi et al., 2006). Increased TPH2 mRNA levels in
the DRN have also been observed in rats after adverse experiences
during early life and adulthood (Gardner et al., 2009). However,
further investigations are needed in order to elucidate the precise
relationships between extracellular levels of 5-HT and TPH2 mRNA
expression because changes opposite to those noted above in SSRI-
treated rodents (i.e., increased TPH2 mRNA expression) have also
been reported (Shishkina et al., 2007).
The lack of a significant decrease of TPH2 mRNA level in the DRL
in 5HTT?/? mice in comparison with the other subregions of the
Fig. 2. (A) Optical densitometry of TPH2 mRNA levels in the dorsomedial (DRD), ventromedial (DRV), and dorsolateral (DRL) parts of the dorsal raphe nucleus (DRN) and the
median raphe nucleus (MRN) of wild-type (WT) mice and paired heterozygous VGLUT1+/? mice. (B) Representative photomicrographs of the DRN and the MRN from a WT
and a VGLUT1+/? mouse. VGLUT1+/? mice show increased TPH2 mRNA expression compared to WT. Each bar is the mean + S.E.M. of independent data obtained in 3–4 mice.
Scale bar: 500 mm, *p < 0.05 and **p < 0.01.
Fig. 3. (A) Optical densitometry of TPH2 mRNA levels in the dorsomedial (DRD), ventromedial (DRV), and dorsolateral (DRL) parts of the dorsal raphe nucleus (DRN) and the
median raphe nucleus (MRN) in wild-type (WT) mice and paired CB1R knock-out (CB1R?/?). (B) Representative photomicrographs at the level of DRN and MRN from a WT
and a CB1R?/? mouse. A significant increase of TPH2 mRNA expression is observed in CB1R?/? compared to WT mice. Each bar is the mean + S.E.M. from 5 mice of each
genotype. Scale bar: 500 mm. *p < 0.05.
A. Jahanshahi et al. / Journal of Chemical Neuroanatomy 41 (2011) 227–233
DRN, known to contain fewer serotonergic neurons (Steinbusch,
1981), suggests that the DRL may be less involved in this model. In
agreement with our data, previous studies already reported a
heterogeneous distribution of TPH2 mRNA in the DRN with the
greatest number of TPH2 mRNA-positive neurons in the DRD and
DRV subnuclei (Austin and O’Donnell, 1999; Clark et al., 2006).
Perhaps, the DRL exhibits a lower sensitivity to extracellular 5-HT
levels and consequently does not display any alteration in TPH2
expression. Accordingly, changes in TPH2 expression in the
ventromedial and dorsomedial DRN might play a more predomi-
nant role in modulating serotonergic neurotransmission and
depressive behavior (Bonkale et al., 2006; Lowry et al., 2008).
Nevertheless, it must be taken in account that due to the sample
sizes (n = 4) the statistical analysis might not have sufficient power
to detect a decrease in TPH2 expression in the DRL of 5-HTT?/?
4.2. The VGLUT1+/? mouse
Alterations in glutamate and GABA neurotransmission appear
to play a key role in depression (Krystal et al., 2002). Interestingly,
beside high TPH2 mRNA levels in the raphe nuclei (Bach-Mizrachi
et al., 2008), marked changes in glutamatergic and GABA-ergic
neurotransmission have been observed in depressed patients, e.g.,
increased glutamate and decreased GABA at cortical and hippo-
campal levels (Bhagwagar et al., 2007). However, the underlying
molecular mechanisms responsible for the abnormal glutamate
neurotransmission in the brain of depressed patients remain to be
Interestingly, the VGLUT1+/? mice also exhibit changes in
cortical and hippocampal release of glutamate and GABA together
with increased anxiogenic/depressive-like responses (Tordera
et al., 2007; Garcia-Garcia et al., 2009). VGLUT1 plays a critical
role in refilling synaptic glutamate vesicles (Wilson et al., 2005;
Takamori, 2006) and indeed, a recent study has demonstrated that
(Balschun et al., 2009). Moreover, the lack of VGLUT1 in glial cells
also increases the probability of excitotoxic effects of excessive
extracellular glutamate in brain, which in turn can lead to more
disturbed glutamatergic neurotransmission (Valentine and Sana-
The present study demonstrates that the glutamatergic system
through VGLUT1 affects TPH2 mRNA expression. In particular,
VGLUT1+/? mice exhibited increased TPH2 mRNA levels in the
DRN and MRN that may be related to alterations in the
glutamatergic/GABAergic-5-HT negative feedback system. Specifi-
cally, it has been established that forebrain glutamatergic neurons
send projections to the ventral periaqueductal grey, stimulating
local GABA neurons, which in turn inhibit 5-HT neurons (Arnsten
and Goldman-Rakic, 1984; Sharp et al., 2007). Compromised
glutamatergic signaling due to the reduced VGLUT1s in cortical
prefrontal descending projections to the brainstem, as expected in
VGLUT1+/? mice, would lead to attenuated stimulation of GABA
neurons, and decreased inhibition of the 5-HT neurons and
presumably, increased TPH2 mRNA levels. Moreover, the altered 5-
HT function could contribute to explain the increased vulnerability
of these mice to depressive like-behavior after exposure to chronic
mild stress (Garcia-Garcia et al., 2009). With regard to the
ascending component of the circuitry, 5-HT modulates the
magnitude of responses in many cortical glutamatergic and
GABA-ergic neurons (Araneda and Andrade, 1991; Aghajanian
and Marek, 1999; Zhou and Hablitz, 1999). In addition, it has been
reported that 5-HT controls, in turn, the activity of descending
excitatory inputs through the activation of pyramidal cortical
neurons (Amargos-Bosch et al., 2004). Since the neurochemical
pathways regulating glutamate and GABA signaling are closely
related (Choudary et al., 2005), changes in the levels and the
activity of these neurotransmitters can lead to altered excitation–
inhibition ratios in the cortex (Bhagwagar et al., 2007).
4.3. The CB1R?/? mouse
Previous evidence demonstrates that the endocannabinoid
system through CB1R regulates the activity of the serotonergic
system by modulating different components of serotonergic
feedback (Hill et al., 2006; Bambico et al., 2007; Aso et al.,
2009). In the present study, our data reveal additional alterations
in the serotonergic activity at the levels of 5-HT synthesis in the
absence of the CB1R. Mice lacking CB1R exhibit increased TPH2
mRNA expression in the MRN and all subregions of the DRN
compared to wild-type littermates. Considering the role of TPH2 in
5-HT synthesis and the reciprocal innervation between the
prefrontal cortex and DRN (Jankowski and Sesack, 2004), the
increased expression of TPH2 in the DRN of CB1R?/? mice could
contribute to the enhanced extracellular 5-HT levels in the
prefrontal cortex of these mutants (Aso et al., 2009). Similarly,
the alterations of different components involved in 5-HT depen-
dent negative feedback, such as enhanced extracellular 5-HT levels
in the prefrontal cortex, decreased 5-HTT binding site density,
functional desensitization of the 5-HT1A receptors and reduced 5-
HT2C receptors have been described in CB1R?/? mice (Aso et al.,
2009), as well as the enhancement of the 5-HT2A receptor
excitatory effect on DRN and MRN produced by the lack of CB1R
activity (Gorzalka et al., 2005), could also contribute to the
increased 5-HT extracellular levels in the prefrontal cortex in
CB1R?/? mutants. The synergistic alteration of these multiple
serotonergic components modulated by CB1R on 5-HT signaling
might be a reason for the robust up-regulation of TPH2 mRNA in all
subregions of the DRN/MRN complex in CB1R?/? mice.
In contrast to the expected consequence of the enhanced
serotonergic release, the genetic and pharmacological blockade of
the CB1R induces a depressive-like phenotype (Hill and Gorzalka,
2005). This could be explained by the impairment of other
mechanisms controlling emotional homeostasis beyond 5-HT
neurotransmission in the absence of CB1R, such as the effects of
stress on glucocorticoids and neurotrophic factor levels (Aso et al.,
2008). The alterations observed on 5-HT neurotransmission have
been proposed to be a substrate for counteracting the stress-
induced emotional impairment in mice lacking CB1R since similar
changes have been described after antidepressant treatments
(Lanfumey et al., 2000; Le Poul et al., 2000; Gould et al., 2006).
Nevertheless, an impaired post-synaptic serotonergic signaling has
been reported in CB1R?/? mice (Mato et al., 2007), which could
lead to a reduction in the efficacy of 5-HT for ameliorating their
Taken together, these data suggest that the CB1R gene knock
out should lead to a reduction of the inhibitory effect exerted
through 5-HT1A and 5-HT2C receptors and enhancement of the
excitatory effect through 5-HT2A receptors on DRN and MRN 5-HT
neurons, possibly leading to increased expression of TPH2 mRNA.
Additionally, CB1R?/? mice are characterized by impaired
serotonergic negative feedback (Aso et al., 2009), and CB1R
stimulation in the cortex directly inhibits 5-HT release (Nakazi
et al., 2000), whereas a CB1R antagonist exerts the opposite effect
(Tzavara et al., 2003). Therefore, changes in negative feedback
input to the raphe nuclei from medial prefrontal cortex (Hajos
et al., 1999) due to the absence of CB1R in DRN could compromise
the feedback mechanisms in the raphe nuclei. The synergistic
attenuation of the multiple actions mediated by CB1R on 5-HT
signaling might be a reason why CB1R?/? mice show such a
robust up regulation of TPH2 mRNA in all parts of the DRN/MRN
A. Jahanshahi et al. / Journal of Chemical Neuroanatomy 41 (2011) 227–233
In conclusion, based on the findings of the present study, we
suggest that TPH2 mRNA expression is under the influence of
multiple messenger systems in relation with presynaptic and/or
postsynaptic feedback control of serotonin synthesis. 5-HTT,
VGLUT1 and CB1R seem to be involved in the execution of these
feedback mechanisms. Finally, our data are consistent with
previous reports suggesting that TPH2 activity within different
raphe subregions are differentially regulated under specific
This study was supported by an EC grant (Newmood; LHSM-CT-
2003-503474), the Swedish Research Council (2887), The Mar-
ianne and Marcus Wallenberg Foundation, a FP6 Marie Curie Early
Stage Training Fellwoship (MEST-CT-2005-020589), the French
Institut National de la Sante ´ et de la Recherche Me ´dicale (INSERM),
the University Pierre & Marie Curie, and the German Research
Foundation (SFB 581, SFB TRR 58).
We are grateful to Professor S. Wojcik, University of Go ¨ttingen,
Germany for supplying the original VGLUT1 heterozygous mice.
We are grateful to Prof. D. A. Hopkins, Dalhousie University, Halifax
and Visiting Professor Maastricht University for his commends.
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