Chronic stress and impaired glutamate function elicit a depressive-like phenotype and
common changes in gene expression in the mouse frontal cortex
Tordera R.M1., Garcia-García A.L.* 1, Elizalde N.*1, Segura V2. Aso E. 3, Venzala E. 1,
Ramírez MJ1 and Del Rio J 1.
1. Department of Pharmacology, University of Navarra, 31080 Pamplona, Spain
2. CIMA. Center for Applied Medical Research
3. Department of Experimental and Health Sciences. University Pompeu Fabra
* These authors contributed equally to this work
Dr. R. M. Tordera,
University of Navarra
Email: firstname.lastname@example.org Tel: 0034 948 425600 Fax: 0034 948 425649
Major depression might originate from both environmental and genetic risk factors. The
environmental chronic mild stress (CMS) model mimics some environmental factors
contributing to human depression and induces anhedonia and helplessness. Mice
heterozygous for the synaptic vesicle protein (SVP) vesicular glutamate transporter 1
(VGLUT1) have been proposed as a genetic model of deficient glutamate function linked to
depressive-like behaviour. Here, we aimed to identify, in these two experimental models,
gene expression changes in the frontal cortex, common to stress and impaired glutamate
Both VGLUT1+/- and CMS mice showed helpless and anhedonic-like behavior. Microarray
studies in VGLUT1+/- mice revealed regulation of genes involved in apoptosis,
neurogenesis, synaptic transmission, protein metabolic process or learning and memory. In
addition, RT-PCR studies confirmed gene expression changes in several glutamate, GABA,
dopamine and serotonin neurotransmitter receptors. On the other hand, CMS affected the
regulation of 147 transcripts, some of them involved in response to stress and
oxidoreductase activity. Interestingly, 52 genes were similarly regulated in both models.
Specifically, a dowregulation in genes that promote cell proliferation (Anapc7), cell growth
(CsnK1g1), cell survival (Hdac3), inhibition of apoptosis (Dido1) was observed. Genes
linked to cytoskeleton (Hspg2, Invs), psychiatric disorders (Grin1, MapK12) or an
antioxidant enzyme (Gpx2) were also downregulated. Moreover, genes that inhibit the MAPK
pathways (Dusp14), stimulate oxidative metabolism (Eif4a2) and enhance glutamate
transmission (Rab8b) were upregulated.
We suggest that these genes could form part of the altered “molecular context” underlying
depressive-like behaviour in animal models. The clinical relevance of these findings is
Increasing evidence suggests that major depression might originate from both
environmental and genetic risk factors. This observation early prompted the need to
better refine and develop mouse-specific models for analysing depression. For
instance, the chronic mild stress (CMS) model of depression was developed (Willner,
2005) in an attempt to mimic some of the environmental factors contributing to the
induction of depressive disorders in humans (Kessler, 1997; Kendler et al., 1999;
2001; Monroe et al., 2006).
CMS reproduces core clinical symptoms such as long-lasting anhedonia and
helplessness (Elizalde et al., 2008). In addition, CMS induces neuroadaptive
changes that could be addressing clinical findings with depressed patients (Gould et
al., 2007; Sanacora et al., 2004; Rajkowska., 2000; Lucassen et al., 2006; Frodl et
Recent clinical (Uezato et al., 2009) and preclinical studies (Tordera et al., 2007;
García-García et al., 2009) have linked decreased levels of the synaptic vesicle
protein (SVP) vesicular glutamate transporter 1 (VGLUT1) to depressive like
behaviour. Specifically, decreased VGLUT1 levels in the frontal cortex of depressed
subjects have been reported (Uezato et al., 2009) in post-mortem studies. In
addition, recent studies with the VGLUT1 heterozygous mice (VGLUT1+/-) suggest
that decreased VGLUT1 levels affects glutamate transmission (Balschun et al., 2009)
and induces depressive-like behavior comorbid with anxiety and impaired recognition
memory (Tordera et al., 2007). Moreover, VGLUT1+/- mice show decreased cortical
and hippocampal levels in GABA as well as an increased vulnerability to depressive-
like behaviour after chronic stress (García-García et al., 2009).
Here, we aimed to study, using microarray technology, how decreased VGLUT1
levels (VGLUT1+/- mice) or an environmental model (chronic mild stress) affect
behaviour and gene expression in the frontal cortex.
Firstly, anhedonic and helpless behaviour in both VGLUT1+/- and CMS models was
comparatively studied. Secondly, we evaluated differences in gene expression in
VGLUT1+/- mice compared to WT. From all the significant genes, we selected some
candidates to validate by RT-PCR considering their relationship with different
neurotransmitter systems and the coincidence with another genetic animal model of
depression, the CB1 KO mice (Aso et al., 2010, Special Issue). Differences in gene
expression in the CMS model were also evaluated. Subsequently, gene expression
changes common to the VGLUT1+/- and CMS models were selected and their
possible involvement in depressive-like behaviour was discussed.
2. Experimental procedures
Heterozygous VGLUT1 male mice (VGLUT1+/-) C57BL⁄6N were bred from
heterozygous fathers (Dr S. Wojcik, Gottingen, Germany) and WT mothers (Harlan,
France). The VGLUT1-/- knock-out 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) (Wojcik et al., 2004). These mice show a progressive neuropathological
phenotype and increased lethality rate at 2–3 weeks after birth. Mice were weaned
and genotyped at the age of 3 weeks. VGLUT1+/- heterozygous mice exhibited no
apparent phenotypic abnormalities during development and adulthood.
Heterozygous VGLUT1 and WT male mice (C57BL/6) (8-10 weeks of age) were
housed in individual cages and allowed for 2 weeks to habituate before beginning
experimentation. Food and water were available ad libitum for the duration of the
experiments unless otherwise specified. Animals were maintained in a temperature
(21 ± 1 ºC) and humidity-controlled room (55 ± 2 %) on a 12 h light-dark cycle (lights
on at 08:00 h).
Experimental procedures and animal husbandry were conducted according to the
principles of laboratory animal care as detailed in the European Communities Council
Directive (2003/65/EC), Spanish legislation (Real decreto 1201/2005) and approved
by the Ethical Committee of University of Navarra.
2.2 Experimental design
Both WT and VGLUT1+/- mice were divided into control and CMS groups
(n=15mice/group, four groups, n=60 in total). Chronic mild stress (CMS) procedure
was applied for six weeks (Elizalde et al., 2008). Anhedonic-like behavior was
evaluated by weekly monitoring of sucrose intake in both control and CMS mice
during the 6 weeks of the stress procedure. Over the last week of CMS, a battery of
behavioural tests including motor activity, novel object recognition, test for anxiety
(elevated plus maze) and depression (forced swimming test) were performed. The
behavioural phenotype of these groups have been previously shown (García-García
et al., 2009). Tests were performed from 9:00-1:00 p.m. Animals were sacrificed 76
h after the last test (forced swimming test) and 24 h after the last stress procedure.
Mice were killed by cervical dislocation, brains were rapidly removed and the
prefrontal cortex (around 15 mg) was rapidly dissected according to standard
procedures and frozen in dry ice. Samples were stored at -80°C until RNA was
2.3 Chronic mild stress procedure
The following unpredictable mild stressors (2-3 in any 24 h period) were randomly
applied for 6 weeks (Elizalde et al., 2008): stroboscopic illumination (8h), intermittent
bell (10 db, 1s/10s) or white noise (4 h), rat odour (8h), cage tilt 45º (8 h), soiled
bedding (6 h), paired housing (2h) overnight illumination, removal of nesting material
(12 h) and confinement (1 h). Once a week, anhedonic-like behaviour was evaluated
by weekly monitoring of sucrose intake (Elizalde et al., 2008). During the 15 h of
duration of the sucrose intake test (from 6 p.m to 9 a.m.) no stressors were applied.
2.4 Sucrose intake test
Anhedonic like behaviour was evaluated by weekly monitoring of sucrose intake
(Elizalde et al., 2008). Mice were first trained to drink a sucrose solution by exposing
them to two standard drinking bottles, one containing 2.5 % sucrose and the other
tap water, for every other night during one week. After this preliminary phase, mice
were food deprived and exposed to the sucrose solution and water from 6:00 p.m.
until 09:00 h in the morning, once a week during 6 weeks. The intake baseline for the
sucrose solution was established, which corresponded to the average of three
WT and VGLUT1+/- mice were subdivided into two groups (CMS and non-stressed
controls) matched for sucrose consumption and body weight. Mice were weekly
given a 15-h exposure to the sucrose solution and tap water as described above and
during this test no stressors were applied. The position of the 2 bottles (right/left) was
varied randomly from trial to trial. Body weight measurements were taken weekly and
relative sucrose intake was calculated as absolute intake (g) per body weight.
2.5 Forced swimming test
Mice were individually placed into glass cylinders (height 24 cm, diameter 13 cm)
containing water (14 cm, 22–23°C). Immobility, indicative of helpless behaviour, was
recorded during the last 4 min of the 6 min testing period and is considered a
measure of helpless behaviour.
2.6 RNA extraction
Isolation of total RNA was carried out according to manufacturer’s instructions
(NucleoSpin RNA II kit, Macherey-Nagel). Total RNA was isolated separately from
each individal cortex. The frozen prefrontal cortex samples were lysed and dounze-
homogenized in the presence of a highly denaturing ß-mercaptoethanol-containing
buffer, which immediately inactivates RNases. Ethanol was added to provide
appropriate binding conditions, and the sample was then applied to an RNeasy Mini
spin column, where the total RNA binded to the membrane and contaminants were
washed away. RNA was then eluted in 30–60 μL RNase-free water. The eluates
were stored at –80°C.
2.7 Microarray hybridization
RNA quality control was checked with the Agilent Bioanalyzer (Agilent Technologies
Inc, Santa Clara, CA, USA) and the RNA concentration was evaluated by using a
NanoDrop™ Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).
Microarray hybridizations were outsourced to ServiceXS B.V. (BZ Leiden, The
Netherlands). Nine animals out of 15 in each group (control and CMS, WT and
VGLUT1+/- mice) were used for large-scale gene expression analysis. Selection of
the animals was based on the RNA quality from the frontal cortex extracts. RNA from
groups of three animals was pooled to reduce noise and individual variability, giving
three samples per group, which were treated as such for statistical analysis. The
Illumina TotalPrep™ RNA Amplification Kit
(Ambion Inc, Austin, TX, USA) was used
to synthesize biotine labeled cRNA. Then, 750 ng of biotin labeled cRNA were
hybridized onto the MouseRef-8 v2.0 Expression BeadChips (Illumina Inc., San
Diedo, CA, USA) accordingly to manufacturer’s instructions. The used multi-sample
array format allows to assay approximately 25,600 transcripts and to profile eight
samples simultaneously on a single MouseRef-8 v2.0 Expression BeadChip.
2.8 Validation of the differentially expressed genes by RT-PCR
TaqMan Low Density Arrays (TLDA) microfluidic card technology from Applied
Biosystems (Foster City, CA, USA) was used to validate the differential expression of
selected genes (Table 1) in 6 independent samples per group. Each reaction well
contained all reagents specific for a given assay. For each tissue sample, 100 ng
reverse-transcribed RNA were diluted to 50 μL with sterile water, combined with an
equal volume of TaqMan Universal PCR Master Mix (2×; Applied Biosystems, Foster
City, CA, USA), mixed by inversion, and spun briefly in an Eppendorf® 5415C
microcentrifuge (Brinkmann Instruments, Westbury, NY, USA). After TLDAs were
brought to room temperature, 100 μL master mix were loaded into each port
connected to reaction wells. TLDAs were placed in Sorvall®/Heraeus® Custom
Buckets (Applied Biosystems, Foster City, CA, USA) and centrifuged in a Sorvall
Legend™ centrifuge (Kendro Scientific, Asheville, NC, USA) for 1 min at 331×g
followed closely by a second 1-min centrifugation at 331 g. Cards with excess
sample in the fill reservoir were spun for an additional 1 min. Immediately following
centrifugation, the cards were sealed with a TaqMan LDA Stylus Staker (Applied
Biosystems, Foster City, CA, USA), and the loading ports excised. The final volume
in each well after centrifugation was <1.5 μL; thus, the final reverse-transcribed RNA
concentration was approximately 1.5 ng/reaction. Quantitative RT-PCR amplifications
were run on an ABI Prism® 7900HT Sequence Detection System (Applied
Biosystems, Foster City, CA, USA) with a TaqMan LDA cycling block and an
automation accessory upgrade. Thermal cycling conditions were 2 min at 50°C, 10
min at 95°C, followed by 40 cycles of denaturation at 95°C for 15 s and annealing
and extension at 60°C for 1 min. Each test sample was processed in duplicate on
individual TLDA cards, thus allowing four samples to be processed on each card.
2.9 Analysis of quantitative RT-PCR Data
RT-PCR TaqMan instrumentation monitors gene-specific products with fluorescent
dye chemistry. A cycle threshold (CT) for each reaction is the number of cycles at
which the reaction crosses a selected threshold. The threshold is defined as a
straight line drawn above noise/baseline and positioned within the linear region of the
semi-log amplification plot. The fewer cycles required to reach threshold fluorescence
intensity, the lower the CT value and the greater the initial amount of input target.
Results for each target on TLDAs were quantified concurrently using the same
baseline and threshold for a target gene in order to limit interplate errors in the
Samples were analyzed by the double delta CT (ΔΔCT) method. Delta CT (ΔCT)
values represent normalized target genes levels with respect the internal control.
Normalization was based on a single reference gene (GAPDH). Delta CT (ΔΔCT)
values were calculated as the ΔCT of each test sample (stressed wild-type or non-
stressed/stressed CB1 knockout mice) minus the mean ΔCT of the calibrator samples
(non-stressed wild-type) for each target gene. The fold change was calculated using
the equation 2(−ΔΔCT).
2.10 Data normalization and analysis
Behavioural studies. Immobility in the forced swimming test was analyzed using
Student t-test analysis. For the sucrose intake test one way ANOVA repeated
measures followed by Student t-test for individual weeks was applied.
Gene expression studies. Data normalization and gene expression analysis was
carried out with the R/Bioconductor package beadarray (Dunning et al., 2007). The
data normalization was performed by using the quantile normalization algorithm
(Bolstad et al., 2003). The statistical analysis were based on the Propagating
Uncertainty in Microarray Analysis (PUMA) method that allows to identify differentially
expressed genes by combining biological replicates and to perform a principal
component analysis (PCA) (Rattray et al, 2006). The filtering criterion used to define
statistically significant differentially expressed genes was the minimum Pplr (MinPplr)
lower than 0.001.
Functional enrichment analysis of Gene Ontology (GO) categories
(http://www.geneontology.org/) and KEGG pathways
(http://www.genome.ad.jp/kegg/) was carried out using standard hypergeometric test
(Huang et al, 2009). To identify biologically relevant clusters of interrelated genes a
stringency criterion of p<0.01 were adopted in DAVID software (Huang et al, 2009).
The functional interpretation and biological knowledge extraction was complemented
through the use of Ingenuity Pathway Analysis (Ingenuity Systems,
www.ingenuity.com), which database includes manually curated and fully traceable
data derived from literature sources. Additionally, the lists of differentially expressed
genes were browsed to find genes encoding neurotransmitter metabolizing enzymes,
neurotransmitter receptors or receptor subunits, neurotrophic factors and others
genes related to nervous system.
3.1. Forced swimming and sucrose intake tests
Mice body weight was not affected neither by CMS or genotype. CMS mice
decreased sucrose intake (solution (g)/body weight) from the fourth week of CMS
until the end of this procedure (Fig. 1A). VGLUT1+/- mice decreased also sucrose
intake measured as the average of sucrose intake on three consecutive weeks (Fig.
In the forced swimming test (FST), increased immobility times were observed in CMS
exposed mice (Fig. 1B). On the other hand, VGLUT1+/- heterozygous mice showed a
significant increase in the immobility time in the FST compared to their WT littermates
3.2 Patterns of altered gene expression in VGLUT1+/- mice compared to WT
We considered statistical significance levels of MinPplr<0.001 when comparing
patterns of gene expression on microarray among the different groups. In addition,
significant levels of MinPplr<0.005 were also considered when differences in gene
expression levels were higher than 1.4 or lower than 0.7 times of WT control levels.
The levels of 1046 gene transcripts were significantly affected by genotype in the
prefrontal cortex at basal conditions being 583 dowregulated and 463 upregulated
(see clustering analysis results in supplemental material, figure 1s).
Following the The DAVID 2008 Functional Annotation Tool was used to identify
enriched biological pathways in the lists of differentially expressed genes obtained
according to KEGG pathways and GO categories. After this functional analysis a set
of enriched KEGG pathways including apoptosis, neurogenesis, synaptic
transmission, protein metabolic process or learning and memory were detected
(summarized in Table 2).
From all the significant genes, we selected some candidates to validate considering
their relationship with different neurotransmitter systems and the coincidence with the
results obtained by analyzing differential gene pattern in another animal model of
depression, the CB1 KO mice (Aso et al., 2009). In total, the gene expression of 20
transcripts was evaluated by quantitative RT-PCR (Table1). Among them, RT-PCR
confirmed the differential expression of several genes involved in GABA and
glutamate transmission, including a dowregulation of VGLUT1, ionotropic kainate
glutamate receptor 5 (Grik5) and an upregulation of GABA-A receptor subunits
(Gabrg2 and Gabra3), GABA-B receptor subtype 1 (Gabbr1), glycine receptor beta
subunit (Glrb), member of the Ras oncogene family 8 (Rab8b) and member A of the
ras homolog gene family (Rhoa). In addition, a dowregulation of the dopamine
receptor 1a (Drd1a) and an upregulation of the 5-hydroxytryptamine receptor 1A
(Htr1a) and adenilate cyclase 8 (Adyc8) were also confirmed (Table 1).
3.3 Patterns of altered gene expressions common to the chronic mild stress
and VGLUT1+/- model
Similarly, 147 transcripts showed significantly altered expression levels after
exposure to CMS; 94 were downregulated and 41 were upregulated. (See clustering
analysis results in supplemental material, figure 2s). CMS affected the regulation of
147 transcripts, some of them involved in response to stress and oxidoreductase
activity. We identified 52 genes similarly regulated by both the CMS and the
VGLUT1+/- models being 37 downregulated and 15 upregulated. Among them,
genes playing an important role on cell proliferation and survival, apoptosis, oxidative
metabolism, glutamate transmission or cytoskeleton was included (Table 3, and see
hierarchical clustering image in Figure 2). Specifically, a dowregulation in genes that
promote cell proliferation (Anapc7; anaphase promoting complex subunit 7), cell
growth (CsnK1g1; casein kinase 1 gamma 1), cell survival (Hdac3; histone
deacetylase 3), inhibition of apoptosis (Dido1; death induced-obliterator 1) was
observed. Other genes linked to cytoskeleton (Hspg2; perlecan, Invs; inversin),
psychiatric disorders (Grin1; glutamate receptor, ionotropic, MapK12; mitogen-
activated protein kinase 12) or an antioxidant enzyme (Gpx2; glutathione peroxidase
2) were also downregulated. In addition, an upregulation of genes that inhibit the
MAPK pathways (Dusp14; dual specificity phosphatase 14), stimulate oxidative
metabolism (Eif4a2; eukaryotic translation initiation factor 4A2) and enhance
glutamate transmission (Rab8b; member RAS oncogene family 8) was detected. On
the other hand, genes supposed to be neuroprotective such as an inhibitor of
apoptosis (Pip5k1a; phosphatidylinositol-4-phosphate 5-kinase, type 1 alpha), an
stimulator of cell differentiation (Nedd9; neural precursor cell expressed
developmentally down-regulated gene 9) as well as genes linked to antioxidant
activity (Prnp; prion protein) or cytoskeleton (Catna1; catenin alpha 1) were also
Here, we firstly show that in agreement with previous studies carried out in our
laboratory (García-García et al., 2009; Elizalde et al., 2008) both the genetic
(VGLUT1+/- mice) and the environmental (chronic mild stress) model of depression
showed helpless and anhedonic-like behavior.
The vesicular glutamate transporter 1 (VGLUT1) is the major isoform in cortical and
hippocampal regions (Takamori et al., 2000; Fremeau et al., 2001), where it plays a
key role in the vesicular uptake and synaptic transmission of glutamate (Wojcik et al.,
2004: Fremeau et al., 2004; Balschun et al., 2009) in telencephalic areas. Thus, the
anhedonic and helpless behaviour shown by these mice could be linked to
decreased glutamate transmission in those areas in which VGLUT1 is the major
isoform. 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 might
have functional and clinical implications.
Microarray studies in VGLUT1+/- mice revealed regulation of genes involved in
apoptosis, neurogenesis, synaptic transmission, protein metabolic process or
learning and memory. Importantly, given that glutamate transmission is reduced in
both VGLUT1+/- (Balschun et al., 2009) and VGLUT2+/- mice (Moechars et al.,
2006) and that both complementary isoforms show identical pharmacological
properties (Takamori et al., 2001) it would be interesting to identify which of these
differentially expressed genes are common in both genetic models. These studies
would add relevant information like which gene changes are directly linked to
impaired glutamate transmission and which could be more related to developmental
in a particular brain area.
We selected some candidates to validate by RT-PCR considering their relationship
with different neurotransmitter systems and the coincidence with the results obtained
by analyzing differential gene pattern in another animal model of depression, the CB1
KO mice (Aso et al., In this Issue). Changes in several glutamate, GABA, dopamine
and serotonin neurotransmitter receptors were confirmed. Interestingly, VGLUT1+/-
ampal levels of GABA without
mice showed an upregulation of GABA-A receptor subunits (Gabrg2 and Gabra3),
GABA-B receptor subtype 1 (Gabbr1), glycine receptor beta subunit (Glrb) and a
downregulation of the ionotropic kainate glutamate receptor 5 (Grik5). Some of these
changes might have a clinical relevance for different psychiatric disorders. For
instance, associations between a polymorphism in GRIK5 receptor subunit (Gratacos
et al., 2009) and an upregulation of the Gabra3 (Massat et al., 2002) to bipolar
disorder have been described.
We suggest that the upregulation of the different GABA subunits and receptors could
be a compensatory effect for decreased GABA levels in the synaptic cleft. Indeed,
previous studies in our laboratory show that VGLUT1 heterozygous mice exhibit
normal levels of glutamate but low cortical and hippoc
alterations of the GAD65 synthesizing enzyme (García-García et al., 2009).
Moreover, these mice show a downregulation of the excitatory aminoacid transpoter
1 (EAAT1) (García-García et al., 2009). It has been suggested that a downregulation
of EAAT1 could limit the glial glutamate uptake from the synaptic cleft and limit the
synthesis of GABA in the GABAergic neuron (Mathews and Diamond, 2003).
Nowadays, there is growing evidence confirming the implication of the GABAergic
dysfunction (Krystal et al., 2002; Brambilla et al., 2003; Tunnicliff and Malatynska.,
2003). Clinical studies have shown that depressed patients have reduced GABA
levels in cortex demonstrated by proton magnetic resonance spectroscopy (MRS)
(Sanacora et al., 1999, 2004), plasma (Tunnicliff and Malatynska., 2003) and
cerebrospinal fluid. Moreover, preclinical studies show decreased GABA levels in
animal models of depression (Brambilla et al., 2003; Gronli et al., 2007; Sanacora et
al., 2007; Garcia-Garcia et al., 2009).
On the other hand, the upregulation of both Rhoa and Rab8b could be also a
compensatory effect for a decreased glutamate release in the VGLUT1 dependent
glutamatergic terminals. Both proteins are involved in the glutamate synaptic streght
and in the release of AMPA receptors to the dendritic spines (Gerges et al., 2004).
Further studies should investigate whether AMPA transmission is affected in these
mice. Taken together, the RT-PCR studies further support the idea of an altered
balance between the excitatory (glutamate) and inhibitory (GABA) transmission in
Among the rest of neurotransmitter receptors, a dowregulation of the dopamine
receptor 1a (Drd1a) agree with the CB1 KO model (Aso et al., 2010, in this special
The chronic mild stress model has been extensively studied at the behavioural
(Strekalova et al., 2004;2006) molecular (Airan et al., 2007; Gronli et al., 2007;
Banasr et al., 2008; García-García et al., 2009) or cellular (Warner-Schmidt and
Duman 2006; Jayatissa et al., 2008) levels. In addition to core symptoms of
depression, such as long-lasting anhedonia (Elizalde et al., 2008), CMS in
neuroadaptive changes that could be addressing clinical findings with depressed
patients (Gould et al., 2007; Sanacora et al., 2004; Rajkowska., 2000; Lucassen et
al., 2006; Frodl et al., 2008). CMS affected the regulation of 147 transcripts, some of
them involved in response to stress and oxidoreductase activity. Here, we aimed to
identify in these two experimental models (CMS and VGLUT1+/- mice), gene
expression changes common to stress and impaired glutamate function.
Specifically, a dowregulation in genes that promote cell proliferation (Anapc7)
(Gieffers et al., 2001), cell growth (CsnK1g1) (Kusuda et al., 2000), cell survival
ese changes agree different hypothesis that link major depression to
. Yet, the functional relevance of these changes for major depression would
e further supported by gene expression studies on other genetic models.
(Hdac3) (Xia et al., 2007), inhibition of apoptosis (Dido1) (Fütterer et al., 2005) was
observed. Genes linked to cytoskeleton (Hspg2, Invs) (Farach-Carson et al., 2008;
Nürnberger et al., 2004), psychiatric disorders (Grin1, MapK12) (Georgi et al., 2007;
Qi et al., 2006) or an antioxidant enzyme (Gpx2) (Ranjekar et al., 2003) were also
downregulated. Moreover, genes that inhibit the MAPK pathways (Dusp14), (Kingler
et al., 2008) stimulate oxidative metabolism (Eif4a2) (Cheyssac et al., 2008) and
enhance glutamate transmission (Rab8b) (Gerges et al., 2004) were upregulated.
either structural changes such as an increased neuronal death, neurite atrophia or
molecular changes that lead to a failure in synaptic function and plasticity (Duman et
On the other hand, perhaps as compensatory mechanims, genes supposed to be
neuroprotective such as an inhibitor of apoptosis (Pip5k1a; phosphatidylinositol-
4phosphate 5-kinase, type 1 alpha), (Bassi et al., 2008) an stimulator of cell
differentiation (Nedd9; neural precursor cell expressed developmentally down-
regulated gene 9) (Sasaki et al., 2005) as well as genes linked to antioxidant activity
(Prnp; prion protein) (Rachidi et al., 2003) or cytoskeleton (Catna1; catenin alpha 1)
(Park et al., 2002) were upregulated.
Summing up, we have identified here genes similarly regulated by both
environmental (CMS) and genetic model (VGLUT1+/- heterozygous mice) that could
form part of the altered “molecular context” underlying depressive-like behaviour in
these models and might provide new insights into the molecular basis of clinical
We thank Timothy Hinsley and Andrew Brass for their excellent bioinformatics
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re legends Figu
eterozygous and wild type (WT) mice on the sucrose intake test (C) and on the
calculated. *p<0.05 vs corresponding control or WT mice (One-way ANOVA repeated
measures for the sucrose intake test during CMS, and Student t-test for the rest of
Figure 2. Hierarchical clustering analysis of gene expression profiles shows similar
patterns of differentially expressed transcripts in the prefrontal cortex for WT mice
exposed to CMS and VGLUT1+/- mice.
re 1. Performance of WT-CMS and WT control mice in the sucrose intake (A)
in the forced swimming test (B). In addition, performance of VGLUT1
forced swimming test (D). Values show the mean ± SEM (n=15 mice/group). In
- mice, the average of the sucrose intake of three consecutive weeks was
Figure 1S. Hierarchical cluster analysis of expression profiles corresponding to the
ssed transcripts in the prefrontal cortex of VGLUT1+/- compared to differentially expre
Figure 2S. Hierarchical cluster analysis of expression profiles corresponding to the
differentially expressed transcripts in the prefrontal cortex of WT mice exposed to
CMS compared to WT controls.
Author Disclosure Download full-text
Role of funding source
was supported by the EU Framework 6 Integrated Project NEWMOOD
SHM-CT-2004-503474), the Ministry of Science and Innovation (SAF2008-02217, (L
Dr Rosa M. Tordera, Natalia Elizalde and Alvaro García-García performed the
behavioural experiments and RNA extractions. In addition, Dr Rosa M. Tordera
carried out the data collection analysis of gene expression and wrote the manuscript
first draft. Dr Ester Asó carried out the quantitative RT-PCR experiments. Dr Victor
Segura contributed to the microarray data interpretation. Elisabet Venzala carried out
the genotyping. Dr Maria J. Ramírez contributed to the microarray data discussion
and interpretation. Prof. Joaquin Del Rio oversaw the project and contributed to the
final draft of the paper. All authors contributed and agreed to the final draft of the
Conflict of interest
All the authors declare no conflict of interest.
Spanish Government) and a fellowship from the Spanish Government (Department of
Education) to N. Elizalde.