Molecular and Cellular Neuroscience 74 (2016) 87–95

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Molecular and Cellular Neuroscience

journal homepage: www.elsevier.com/locate/ymcne

Protein biomarkers of susceptibility and resilience to stress in a rat model

of depression
Johan Palmfeldt a,1, Kim Henningsen b,⁎,1, Stine Aistrup Eriksen b, Heidi K. Müller b, Ove Wiborg b
a
Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University Hospital, University of Aarhus, Aarhus N, Denmark
b
Translational Neuropsychiatry Unit, Department of Clinical Medicine, University of Aarhus, Risskov, Denmark

a r t i c l e i n f o a b s t r a c t

Article history: The molecular etiologies of psychological stress and major depressive disorder (MDD) are highly complex and
Received 24 February 2015 many brain regions are involved. The prefrontal cortex (PFC) has gained attention in depression research due
Revised 24 February 2016 to its role in cognition including working memory and decision-making, which are impaired in MDD. The aim
Accepted 18 April 2016
of the present study was to identify differentially regulated synaptosomal proteins from PFC in stress-exposed
Available online 20 April 2016
animals. The well-established chronic mild stress (CMS) rodent model was applied and three groups of rats
Keywords:
were studied: unstressed controls, stress-susceptible and stress resilient. Large-scale proteomics based on rela-
Chronic mild stress model of depression tive iTRAQ quantification was applied on three synaptosomal Percoll gradient fractions and 27 proteins were
Prefrontal cortex found to undergo significant differential regulation. Gradient fraction two (F2) contained the highest amounts
Synaptosome of synaptosomal proteins and is therefore recommended to be included in proteomic studies onwards, in addi-
Biomarkers tion to the traditionally used fractions F3 and F4.
Glial fibrillary acidic protein The regulated proteins corroborate previous studies on depression regulated proteins; including GFAP, HOMER1
and glutamatergic transmission (vesicular transporter 1, VGLUT1). However, additional functionalities were rep-
resented – especially in stress-resilient rats – such as oxidative stress protection (peroxiredoxins PRDX1 and
PRDX2), Na/K-transporter ATP1A2 and respiratory chain subunits (UQCRC1 and UQCRFS1), which illustrate
the biochemical complexity behind the stress phenotypes, but may also aid in the development of novel treat-
ment strategies.
© 2016 Elsevier Inc. All rights reserved.

1. Introduction
Novel molecular markers of depression are important for obtaining
in-depth understanding of the biology behind depression and aid the
development of improved treatments of depression. Although depres-
sion is a highly complex and heterogeneous disorder it can be modeled
in rodent models, such as the chronic mild stress (CMS) rat model. The
CMS model is a highly validated model of depression and therefore is a
suitable model for studying this disease (Willner, 1997; Christensen
et al., 2013; Wiborg, 2013; Henningsen et al., 2012a). The CMS rodent
model has the advantages, as compared to clinical studies, that it allows
Abbreviations: ATP1A2, sodium/potassium-transporting ATPase subunit alpha-2;
CMS, chronic mild stress; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid;
GFAP, glial fibrillary acidic protein; HOMER1, homer protein homolog 1; iTRAQ, isobaric
tags for relative and absolute quantitation; MDD, major depressive disorder; PFC, prefron-
tal cortex; PKC, protein kinase C; PRDX1, peroxiredoxin 1; PRDX2, peroxiredoxin 2;
PRKCG, protein kinase C gamma type; UQCRC1, cytochrome b-c1 complex subunit 1, mi-
tochondrial; UQCRFS1, cytochrome b-c1 complex subunit Rieske, mitochondrial;
VGLUT1, vesicular glutamate transporter 1.
⁎ Corresponding author.
E-mail address: kimhenni@gmail.com (K. Henningsen).
1
Contributed equally to this work.
full control of experimental settings and subjects investigated, and pro-
vides unrestrained access to tissue samples. The model is based on a
variable chronic stress regime, which induces a behavioral and physio-
logical condition that mimics a clinical depression. Chronic exposure
to stress always causes a response, but some individuals appear to be
less prone to the debilitating effects of stress (Hjemdal et al., 2010). By
using the CMS model we have consistently reported that CMS exposure
causes a non-uniform effect on sucrose intake in rats (Henningsen et al.,
2012a; Bergstrom et al., 2008; Christensen et al., 2011) and based on
this behavioral measure, the CMS-exposed rats can be divided into sub-
groups. Rats decreasing their sucrose intake are defined as anhedonic-
like, and rats not decreasing their sucrose intake are defined as resilient.
Thus, the CMS model can be used to study the biological underpinning
of stress-susceptibility.
The prefrontal cortex (PFC) has long been implicated in depression
research due to its role in cognition. Dysfunction of the PFC, and associ-
ated circuitries, is assumed to partly underlie the impairment of cogni-
tive function and executive processes, including working memory and
decision-making, which constitute common symptoms of major de-
pressive disorder (MDD) (Lewicka, 1997; Pelosi et al., 2000).
A malfunction in PFC-related cognitive abilities can be mediated
through altered neurotransmitter release in the PFC, and changes in

http://dx.doi.org/10.1016/j.mcn.2016.04.001
1044-7431/© 2016 Elsevier Inc. All rights reserved.
88 J. Palmfeldt et al. / Molecular and Cellular Neuroscience 74 (2016) 87–95
neurotransmitter release in turn involve changes in neurotransmitter
turnover processes, such as exocytosis and endocytosis. To study the bi-
ology of neurotransmitter release, one can use synaptosomes, which are
artificial structures obtained by homogenization and gradient centrifu-
gation of neuronal tissue (Dunkley et al., 2008). Synaptosomes contain
the complete presynaptic terminal, mitochondria, and parts of the post-
synaptic terminal (Dunkley et al., 1986, 2008). Synaptosomes are there-
fore suitable for studying changes in synaptic protein expression levels.
Although the molecular biology of depression has been an area of
scientific interest for decades, a detailed molecular fingerprint of de-
pression remains to be elucidated (Krishnan and Nestler, 2008). Given
this situation, a proteomic screening approach was chosen for the pres-
ent study.
In large-scale discovery proteomics it is often beneficial to fraction-
ate the proteome to get increased analytical depth. In the present
study, synaptosome fractionation was used to enrich the sample for
proteins with neural function. A discontinuous Percoll gradient is com-
monly used to prepare synaptosomes from brain tissue synapses, and
five relatively distinct bands (F1, F2, F3, F4 and F5) can be obtained for
further analysis (Fig. 1) (Dunkley et al., 2008). Synaptosome fraction-
ation is mostly used for obtaining viable synaptosomes to study trans-
mitter release. Activity studies have shown that viable synaptosomes
are predominantly located in fraction F3 and F4 and only to a lesser ex-
tent in fraction F2 (Dunkley et al., 2008). However, the proteomes of
these fractions remains to be thoroughly characterized.
The major aim of the present study was to look for changes in pro-
tein expression profiles in synaptosomes extracted from the PFC of
rats exposed to CMS, comparing anhedonic-like rats, resilient rats and
non-stressed controls.
A secondary aim of the present study was to conduct a proteomic
comparison of fraction F2, F3, and F4, to clarify whether the fractions
that are commonly used to study viable synaptosomes also hold the
highest level of synaptic proteins.
We report a list of 27 proteins found to undergo significant differen-
tial regulation. Of specific interest, the glial fibrillary acidic protein, was
found to be downregulated in the anhedonic-like group, confirming
earlier studies and suggesting stress susceptibility to be associated
with a glial deficit.
Fig. 1. Discontinuous Percoll gradient consisting of four different concentrations of Percoll
(3, 10, 15 and 23%) layered on top of each other. The homogenised prefrontal cortices
were layered on top of the gradient and centrifuged at 20,000 ×g for 5 min to produce
four fractions. The four fractions, annotated F2, F3, F4 and F5, are located in the
interfaces of the specific Percoll gradients.
2. Materials and methods
2.1. Animals
5–6 weeks old male Wistar rats were purchased from Taconic M&B,
Denmark. The animals were singly housed, food and water was avail-
able ad libitum, and the animals were kept on a standard 12-h light/
dark cycle except when one of these parameters was changed according
to the stress regime. All procedures involving animals were accepted by
the Danish National Committee for Ethics in Animal Experimentation
(2008/561-447).
2.2. Sucrose consumption test
Animals were adapted for five weeks to consume a palatable sucrose
solution (1.5%). In this period, the sucrose test was performed twice a
week during the first three weeks and once a week during the last
two weeks. The animals were food and water deprived 14 h prior to
the sucrose test. The test consisted of a one-hour period of free access
to a bottle of the sucrose solution. The sucrose consumption was mea-
sured by weighing the bottles pre- and post-testing. Baseline sucrose
consumption was calculated for each animal and is defined as the
mean sucrose consumption from the last three sucrose tests prior to
stress start. During the stress period, the sucrose consumption test
was performed once a week.
2.3. Chronic mild stress
Based on the baseline sucrose consumption animals were divided
into two matched groups (Equal mean baseline ± 0.5 g), control and
CMS groups, and placed in separate rooms. The CMS group was exposed
to eight weeks of chronic mild stressors. Briefly, this procedure
consisted of seven different stressors and lasted from 10 to 14 h. The
mild stressors consisted of interchanging periods of intermittent illumi-
nation, stroboscopic light, grouping, food and/or water deprivation,
soiled cage, 45° cage tilting and no stressors according to the week
schedule illustrated below (Table 1). The control group was left unchal-
lenged except for 14 hour food and water deprivation before sucrose
consumption test.
During grouping, animals were housed with different partners, the
individual rat alternatively being a resident or an intruder. Following
exposure to stress for four weeks, rats were categorised as anhedonic-
like (defined as a N 30% within-subject decrease in sucrose intake) or
stress-resilient (defined as a b 10% within-subject decrease in sucrose
intake). Rats with sucrose consumption not corresponding to either cat-
egory were excluded from the experiment.
2.4. Tissue preparation
A preliminary study was conducted to examine the efficiency of the
synaptosome fractionation protocol as a tool to fractionate and enrich a
sample for a subtype of proteins. Three rats were decapitated and brains
were removed and PFC immediately dissected. The PFCs were pooled
and processed as described for the treatment study below.
For the CMS study, three groups of animals; control (n = 9),
anhedonic-like (n = 9) and stress-resilient (n = 9), were decapitated
and brains were removed and PFC immediately dissected. This was per-
formed by removing the olfactory bulb and making a coronal section of
the brain to isolate the PFC according to Paxinos and Watson, (1998).
Immediately hereafter the tissue was snap-frozen on dry ice and stored
at −80 °C until further processing. Within each group three PFCs were
pooled resulting in three subgroups; control (n = 3), anhedonic-like
(n = 3) and stress-resilient (n = 3), to ensure sufficient amounts of tis-
sue sample for synaptosomal fractionation.
 J. Palmfeldt et al. / Molecular and Cellular Neuroscience 74 (2016) 87–95
Table 1
The chronic mild stress regime. The stressors are applied according to this weekly schedule during the entire regime.
Time of day Monday Tuesday
Morning (8.00 AM) Intermittent illumination Water deprivation
Afternoon (6.00 PM) No stress Cage tilting
2.5. Synaptosome preparation
Isolation of synaptosomal fractions was performed by centrifugation
through a discontinuous Percoll gradient containing four layers accord-
ing to the method developed by Peter Dunkley, Neil Sims and Mary Mc-
Kenna (Dunkley et al., 1986, 2008).
The three pooled PFCs were homogenised in 5 mL ice cold 0.32 M su-
crose buffer [1.0 mM EDTA, 5.0 mM Tris-HCL pH 7.4, complete EDTA-
free protease inhibitor cocktail tablet, 0.28 mM DTT], using a prechilled
15-ml Potter-Elvehjem type Teflon-glass tissue grinder (19 × 84 mm,
chamber clearance 0.1–0.15 mm) and electrical laboratory stirrer at
700 rpm/min. The homogenate was centrifuged at 3200 rpm (800 ×g)
for 10 min at 4 °C to remove nuclei and cell debris as a pellet, leaving
the bulk of the synaptosomes in the supernatant. The supernatant was
collected loaded on top of two discontinuous Percoll gradients (Fig 1).
From the top down, the Percoll gradient comprised of 2 mL each of 3%,
10%, 15% and 23% Percoll (vol/vol) in 1.28 M sucrose buffer [4 mM
EDTA, 20 mM Tris-HCL, pH 7.4]. The gradients were centrifuged at
20,000 rpm (31,400 ×g), 4 °C for 5 min (excluding time for acceleration
and deceleration time) using a Sorvall Evolution RC Superspeed Centri-
fuge (Thermo Scientific) with a SA300 rotor head, producing four visible
fractions in the interfaces between the Percoll layers termed (from top
down): F2, F3, F4 and F5 (Fig. 3B). According to the literature an addi-
tional fraction, F1 will appear between the interfaces of 0% and 3%
Percoll if the tissue/buffer-ratio is higher and contains mostly mem-
branes (Dunkley et al., 1986, 2008). An F1 fraction was not seen in the
present study. Each faction was carefully collected using a disposable
glass Pasteur pipette and washed twice to remove Percoll by centrifuga-
tion at 16,000 rpm (20,000 ×g) for 15 min in 8 mL ice cold 0.32 M su-
crose buffer [1.0 mM EDTA, 5.0 mM Tris-HCL, pH 7.4], leaving the
fraction as a pellet in the bottom of the tube. The two corresponding
fractions produced from each homogenate were pooled and stored at
−80 °C until further processed.
2.6. Peptide labeling and fractionation
The labeling procedure was performed three times corresponding to
biological triplicates of each of the three synaptosomal fractions. For
each triplicate samples from three animals were pooled (33 μg of pep-
tides each) and each of the resulting 100 μg peptide sample was labeled
with a specific iTRAQ reagent in accordance with the manufacturer's in-
structions (Applied Biosystems). The subsequent analytical steps were
performed as previously described (Henningsen et al., 2012b). Labeled
peptides were mixed and purified on a Strong Cation Exchange (SCX)
chromatography column followed by elution with volatile buffer con-
taining 5% ammonia and 30% methanol. Pure peptides were dried and
resuspended in denaturing buffer. The peptides were separated on an
Immobiline Drystrip gel (GE Healthcare, Uppsala, Sweden) using iso-
electric focusing with 3500 V for 10 h on a Multiphor II unit (Pharmacia
Biotech AB, Uppsala, Sweden). The pH range was 3–10 and the gel strip
was subsequently cut into ten pieces for peptide extraction in two steps
with 0.5% trifluoracetic (TFA) acid in 5% acetonitrile (ACN). Peptides
were vacuum-dried, re-dissolved in 0.5% TFA in 5% ACN, and purified
on PepClean C-18 Spin Columns (Pierce, Rockford, IL, USA) according
to manufacturer's protocol and subsequently analyzed by nanoLC-MS/
MS analysis.
89
Wednesday Thursday Friday Saturday Sunday
Stroboscopic light No stress Sucrose test Food deprivation Cage tilting
Soiled cage Water/food deprivation Grouping Cage tilting Soiled cage
2.7. Nano-liquid chromatography and mass spectrometry (MS) analysis
The peptide mixtures were separated by nano-liquid chromatogra-
phy (EASY nano LC from Proxeon, Odense, Denmark) coupled to mass
spectrometry (LTQ-Orbitrap, Thermo Fisher Scientific, Waltham, USA)
through a nano-electrospray source with stainless steel emitter
(Proxeon). The peptides were separated on a reverse phase column,
75 μm in diameter and 100 mm long, packed with 3.5 μm Kromasil
C18 particles (Eka Chemicals, Bohus, Sweden) at a flow of 300 nL/min
using a 100 min gradient of ACN in 0.4% acetic acid, starting with 5%
and ending with 35% ACN. The mass spectrometry detection was full
scan (m/z 400–2000) with Orbitrap detection at resolution R =
60.000 (at m/z 400) followed by up to four data-dependent MS/MS
scans, with linear ion trap (LTQ) detection of the most intense ions. Dy-
namic exclusion of 25 s was employed as well as rejection of charge
state + 1. Pulsed Q dissociation (PQD) fragmentation was performed
with activation time of 0.1 s, normalized collision energy of 33, and
activation Q of 0.7. The mass spectrometry proteomics data have been
deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014)
via the PRIDE partner repository with the dataset identifiers
PXD001775, PXD001776, PXD001777 and 10.6019/PXD001775 to
10.6019/PXD001777.
2.8. Database searches and statistics
The raw data files were processed using extract_msn.exe (Thermo
Fisher Scientific, released 2/15/2009) to generate peak lists of the tan-
dem spectra. The processed data were searched with Mascot (www.
matrixscience.com) version 2.2.04 (Matrix Science, London, UK),
which was used for protein identification and iTRAQ reporter quantifi-
cation. In each study, the ten different peptide fractions were MS-ana-
lyzed in duplicate and all generated peak lists were merged. The
merged files were searched against the SwissProt (release 2012_03
with 7724 Rattus sequences) using the MudPIT scoring algorithm of
Mascot. Full scan tolerance was 5 ppm and MS/MS tolerance was
0.70 Da. Setting of trypsin digestion was cleavage at C-terminal of lysine
and arginine except before proline, and up to two missed cleavages
were accepted. Fixed modifications were those originating from iTRAQ
protocol: iTRAQ-4plex of lysine and N-terminal and methylthio modifi-
cation of cysteines, whereas oxidation of methionine and iTRAQ-4plex
of tyrosine were set as variable modifications. The significance level of
protein identifications was set to 0.001 in Mascot, which was confirmed
by false discovery rates (FDR ranging 0.0010–0.0013) from searches
against a randomized decoy database. Throughout the manuscript, the
HGNC symbol (http://www.genenames.org/) was used to refer to pro-
tein hits. iTRAQ values were reported for proteins with six or more mea-
sured iTRAQ scan values from peptides with expectation values of 0.02
or below. iTRAQ quantitation was performed in Proteome Discoverer
(Thermo Scientific) where normalization to summed intensities was
applied to compensate for possible variation in starting material. The
iTRAQ-ratios between the experimental groups were calculated for
each protein from the three independent iTRAQ studies giving indepen-
dent triplicate values (e.g. three separate values of A/C values). Only
proteins detected from at least two peptides were included in the statis-
tical calculations. A low number of proteins with different direction of
expression in the three fractions (F2, F3 and F4), compared across treat-
ment groups, were excluded from further analyses since those data
90 J. Palmfeldt et al. / Molecular and Cellular Neuroscience 74 (2016) 87–95
might not reflect the biological situation of the original tissue. Ratios for
each protein were reported as significantly different from 1.0 if they
passed two tests: 1) a fold change criterion of a factor 1.2 was applied
(based on two times the global standard error 2 × 0.1 = 0.2) and 2) a
two-tailed Student's t-test for equal variance data.
2.9. Western blotting
Aliquots of synaptosomal fractions were mixed with SDS-sample
buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 0.02%
bromophenol blue, and 125 mM dithiothreitol, incubated at 50 °C for
10 min and analyzed by SDS-polyacrylamide gel electrophoresis using
10% precast NuPAGE gels (Invitrogen, CA, USA) with a MOPS buffer sys-
tem. Proteins were transferred onto nitrocellulose membranes using
the iBlot dry blotting system (Invitrogen, CA, USA). The membranes
were blocked with 5% dry milk in TBS-T (50 mM Tris-HCl, pH 8.0,
150 mM NaCl, and 0.5% Tween 20) for 1 h at RT and probed with the pri-
mary antibodies: rabbit polyclonal anti-GFAP (1:10,000) (ab7260,
Abcam) and goat polyclonal anti-actin (1:3000) (sc-1616, Santa Cruz
Biotechnology), overnight at 4 °C followed by incubation with the ap-
propriate HRP-conjugated secondary antibody for 2 h at RT: goat anti-
rabbit antibody (1:10,000) or rabbit anti-goat antibody (1:10,000)
(both obtained from Pierce, IL, USA). Immunoreactive bands were visu-
alized using ECL Select Western Blotting Detection Reagent (GE
Healthcare, UK). The chemiluminescent signals were captured on a
KODAK Image Station 440 and relative intensities were quantified by
the KODAK 1D3.6 Image Analysis Software. Group means were analyzed
for statistical significance using the Student's t-test.
2.10. Bioinformatics
The DAVID Bioinformatics Database (Huang da, 2009a) and the Pan-
ther Classification System (Mi et al., 2013) were used to infer bioinfor-
matics of the identified proteins.
3. Results
3.1. Sucrose intake
Sucrose consumption was applied to assess stress-induced
anhedonic-like and resilient behavior of rats. To monitor the stress dy-
namics, a one-hour sucrose consumption test was conducted on all
rats once a week for a total of eleven weeks. Baseline sucrose consump-
tion was defined as the mean sucrose consumption during three sucrose
tests conducted before stress initiation. Rats were allocated into exper-
imental groups based on their baseline sucrose intake. The data of the
groups was normally distributed (as tested by Q-Q-plots). Moreover,
no statistically significant difference between the groups, according to
student's t-test, indicated that they were well matched (data not
shown). Three weeks exposure to CMS (n = 224) resulted in signifi-
cantly decreased sucrose consumption when compared to unchal-
lenged controls (n = 79) (p b 0.00001). Fig. 2a shows the mean group
sucrose intake indexed to baseline values after three weeks of stress.
55% of the stressed rats showed a N 30% decrease in sucrose intake
and were defined as anhedonic-like. On the other hand, 24% did not de-
crease in sucrose intake and were designated stress resilient. Unchal-
lenged rats did not decrease in sucrose intake. A subset of the controls
(U-Control), anhedonic-like (CMS-A) and resilient (CMS-R) rats (nine
in each group) were used for the proteome analysis. Fig. 2b shows the
seven-week mean sucrose intake (indexed to baseline) of the rats
used for proteome analysis.
3.2. Proteomic comparison of Percoll fractions
An initial aim was to evaluate the ability of the synaptosome frac-
tionation method as a tool to separate and enrich a sample preparation
for proteins of synaptic function. A preliminary study was conducted to
investigate this issue by estimating the relative protein expression
levels of Percoll fractions, F2, F3, and F4 of three protein categories; syn-
apse, cytoskeleton and mitochondrion (Fig. 3). The qualitative investi-
gation and subsequent quantitative measurements was conducted on
PFC samples from three control rats. Proteins were only selected if
they were quantified in all three fractions and this selection criterion re-
sulted in inclusion of 202 proteins. The protein functional annotation
was inferred from the David Informatics Resources (Huang da, 2009b).
Three annotation terms were selected for a descriptive analysis of the
fractions. These were mitochondrion (n = 78), cytoskeleton (n = 33),
and synapse (n = 25).
Proteins annotated to the mitochondrion had increased expression
level in the F4 fraction, while proteins annotated to the cytoskeleton
and the synapse had increased levels in the F2 fraction.
3.3. Protein overlap in fractions F2, F3, and F4
To evaluate the differences in protein composition of fraction F2, F3
and F4, the 400 most abundant proteins in each fraction of the main
study were plotted in a Venn diagram (Fig. 4). 270 of the 400 proteins
were detected in all three fractions. The pairwise overlap was higher
for the neighboring gradient fractions with F2 vs F3 and F3 vs F4 both
having N 50 proteins in specific overlap, whereas F2 vs F4 only had 20
specifically overlapping proteins.
3.4. Quantitative proteomic comparisons between the stress-response
phenotypes
Quantitative proteomic comparisons between the two stress-
response phenotypes, anhedonic and resilient and the control group,
were performed in the three synaptosomal fractions, F2, F3 and F4. Alto-
gether, 27 proteins were identified as regulated according the selection
criteria (Table 2).
The list of significant proteins (Table 2) was uploaded to the DAVID
Bioinformatics Database (Huang da, 2009b). The database was used to
gather bioinformatics of the individual proteins and search for potential
enriched protein functions and/or protein pathways, when comparing
experimental groups. The functional annotation clustering tool identi-
fied 18 annotation clusters. These clusters could be functionally
assigned as follows; cytoskeleton (n = 5), signaling (n = 6), metabo-
lism (n = 4) and mitochondrion (n = 3). The pathway analysis showed
“actin cytoskeleton regulation” as the highest ranking pathway with 4
associated proteins; ACTN1, TUBA4A, ARPC2 and CFL1.
3.5. Glial fibrillary acidic protein (GFAP) as a marker of stress-induced
anhedonia
Due to a previously reported role of GFAP in depression (Johnston-
Wilson et al., 2000) and as a validation of the iTRAQ experiment, a west-
ern blot experiment was conducted on the samples from the synapto-
some fractions used in the iTRAQ study. GFAP was detected in fraction
2 and 3, but not in fraction 4, which corresponds to what was seen in
the iTRAQ experiments. The results in fraction 2 showed a lower GFAP
expression in the anhedonic group as compared to the control and resil-
ient group respectively, however only significant in the anhedonic vs.
resilient comparison (p = 0.001). The data further showed a significant
difference between the control and resilient group (p = 0.03) (Fig. 5).
The results for fraction 3 showed no difference (data not shown).
These results confirm what was seen in the iTRAQ study.
4. Discussion
This paper describes the identification of molecular biomarkers as-
sociated with stress susceptibility or resilience in a chronic mild stress
rat model of depression. 27 proteins passed the statistical criteria,
 J. Palmfeldt et al. / Molecular and Cellular Neuroscience 74 (2016) 87–95 91

Fig. 2. A. Three weeks of exposure to chronic mils stress resulted in significantly decreased sucrose consumption when compared to unchallenged controls (n = 79–224). 2B shows the
sucrose consumption of the rats used for the proteome analysis; an unchallenged control group, an anhedonic group and a resilient group. Data are shown as mean sucrose intake indexed
to baseline.

shedding light on altered biological/biochemical functions in prefrontal
cortex (PFC) during anhedonia and resilience. The major protein
markers with putative links to depression are outlined below.
4.1. Protein findings
The full list of differentially expressed proteins is shown in Table 2.
All the proteins in Table 2 were investigated for a potential relation to
stress and depression, using the PubMed database. Interestingly, the
highest number of protein hits was found in the F2 fraction, supporting
a notion also to include this fraction in future studies. The proteins be-
long to multiple functional categories. Several cytoskeletal proteins
and metabolic enzymes were differentially expressed, however these
proteins are too ubiquitous in various tissues/diseases to provide specif-
ic information on synapse function in PFC during depression. Although
the vast majority of proteins are of neuronal origin, two protein hits
are directly attributed to glia (GFAP and PEA15). This is not surprising,
given the anatomical and communicational proximity of astrocytes
and neurons.
4.1.1. GFAP, a specific marker of stress-induced anhedonia
The proteomic analysis showed that the level of GFAP was signifi-
cantly lower expressed in the anhedonic group as compared to the
control and resilient groups respectively. This result was partially con-
firmed by a western blot experiment, as the difference between
anhedonic and control groups almost reached standard statistical signif-
icance levels (p = 0.09).
The GFAP protein is the main intermediate filament protein in astro-
cytes. It is involved in a wide range of functions, such as cell motility,
astrocyte-neuron interactions, vesicle trafficking etc. (Middeldorp and
Hol, 2011).
Clinical studies have found GFAP levels to be decreased in depressed
subjects in cortical regions (Johnston-Wilson et al., 2000;
Miguel-Hidalgo et al., 2000; Si et al., 2004) and likewise in the hippo-
campus (Muller et al., 2001). Moreover, clinical studies have shown re-
duced cortical glial cell density and cell number in depressed subjects
(Cotter et al.; Cotter et al.; Ongur et al.). These studies suggest that
glial loss may, in part, underlie the cognitive symptoms of depression

Fig. 3. The relative protein expression levels of Percoll fractions, F2, F3, and F4 of representative proteins from the three categories mitochondrion (n = 78), cytoskeleton (n = 33), and
synapse (n = 25).
92 J. Palmfeldt et al. / Molecular and Cellular Neuroscience 74 (2016) 87–95

et al., 2010). These studies point to the glutamate-modulating role of

glia cells as central to the pathological role. Future studies should be con-
ducted, on susceptible vs. resilient rats, to elucidate this issue further.

Fig. 4. Venn diagram showing overlap between identified proteins of the three
synaptosome fractions (F2, F3 and F4). The 400 top proteins from each fraction were
included in the figure, of which 270 were identified in all three fractions.
and in turn point to a glial pathology hypothesis of depression
(Rajkowska & Miguel-Hidalgo).
The findings in the present study support an association between a
stress-induced anhedonic condition and decreased GFAP expression and
accordingly a lack in this effect on stress resilient animals. Given the ex-
tensive number of functions in which GFAP plays a role it is not straight-
forward to deduct the functional impact of the association between the
stress response and GFAP-levels found in the present study. However,
since GFAP is relatively specific for astrocytes (Sofroniew and Vinters,
2010), a down-regulation of GFAP is likely to be correlated to a down-
regulation of astrocytes or astrocyte processes. The causative pattern
and the possibility for pharmacological manipulation have been investi-
gated using rodent models (Banasr & Duman; Banasr & Duman Banasr
4.1.2. Protein kinase C gamma type (PRKCG). A stress marker in the PFC
The results showed that PRKCG was downregulated in the anhedon-
ic (F2) and the resilient group (F2 and F3) compared to the control
group. This indicates that decreased PRKCG in the PFC is an effect of
stress per se and not associated with an anhedonic state. The potential
role of PRKCG, or the PKC family in general, in stress response and psy-
chiatric disease has received much attention (Timmermans et al., 2013;
Machado-Vieira et al., 2010). PRKCG may through its participation in
synaptic strength, glutamate signaling, and long-term potentiation, be
associated with the cognitive symptoms of psychiatric disease. Howev-
er, the relationship is highly complex and requires further studies before
any documented conclusions can be made. The present study supports a
role for PRKCG in the PFC response to chronic stress and our findings
further suggest that this role is not specific to a depression-like condi-
tion, but rather an effect of stress per se.
This is in line with our previous findings of an impaired working
memory in both stress-susceptible and resilient rats (Henningsen
et al., 2009).
4.1.3. Homer protein homolog 1 (HOMER1), associated with stress
resilience
HOMER1 was found to be upregulated in the resilient rats compared
to the anhedonic rats, and thus pointing to HOMER1 being involved in
stress resilience. A downregulation of Homer1 (Homer1 gene) in the
PFC has previously been reported in rats exposed to CMS (Orsetti
et al., 2008). Moreover, a genome-wide association study (GWAS) has
implicated HOMER1 in the etiology of MDD (Rietschel et al., 2010).
The functional role of HOMER1 has been studied in HOMER1 knockout
(KO) mice. Deletion of HOMER1 enhances the expression of certain
depression- and anxiety-related behaviors (Lominac et al., 2005).
Adeno-associated virus (AAV)-mediated restoration of HOMER1 to the

Table 2
The table shows the differentially regulated proteins found in the synaptosome gradient fractions F2, F3, and F4. The differentially expressed proteins in the comparisons between resilient
(R), anhedonic (A) and control (C) animals were detected in the PFC synaptosome fractions and passed the statistical criteria (p b 0.05 and fold change N 1.2).

Protein name Abbreviation Accession # Fraction Ratio p-Value Comparison

Myristoylated alanine-rich C-kinase substrate MARCKS P30009 F2 1.24 0.02 A/C

Thymosin beta-4 TBETA4 P62329 F2 1.87 0.04 A/C
Ankyrin repeat and sterile alpha motif. protein 1B AIDA1 P0C6S7 F2 0.72 0.01 A/C
Glial fibrillary acidic protein GFAP P47819 F2 0.50 0.01 A/C
Protein kinase C gamma type PKC P63319 F2 0.59 0.03 A/C
Cell adhesion molecule 2 IGSF4D Q1WIM2 F2 0.75 0.04 A/C
Vesicular glutamate transporter 1 VGLUT1 Q62634 F2 0.81 0.02 A/C
Alpha-actinin-1 ACTN1 Q9Z1P2 F2 0.78 0.04 A/C
Malate dehydrogenase, cytoplasmic MDH1 O88989 F4 1.21 0.02 A/C
Tubulin alpha-4A chain TUBA4A Q5XIF6 F4 0.81 0.04 A/C
Cytochrome b–c1 complex subunit Rieske, mitochondrial UQCRFS1 P20788 F2 1.29 0.03 R/A
Dipeptidyl aminopeptidase-like protein 6 DPP6 P46101 F2 1.31 0.002 R/A
Glial fibrillary acidic protein GFAP P47819 F2 1.42 0.02 R/A
Homer protein homolog 1 HOMER1 Q9Z214 F2 1.24 0.02 R/A
Hemoglobin subunit beta-2 HGB P11517 F2 0.70 0.02 R/A
Peroxiredoxin-1 PRDX1 Q63716 F2 0.83 0.04 R/A
Actin-related protein 2/3 complex subunit 2 ARPC2 P85970 F3 0.82 0.03 R/A
Reticulon-4 RTN4 Q9JK11 F3 0.83 0.02 R/A
Sodium/potassium-transporting ATPase subunit alpha-2 ATP1A2 P06686 F4 1.35 0.04 R/A
Acyl-CoA-binding protein DBI P11030 F4 0.75 0.03 R/A
Adaptin ear-binding coat-associated protein 1 NECAP1 P69682 F4 0.79 0.04 R/A
Aldehyde dehydrogenase, mitochondrial ALDH2 P11884 F4 0.82 0.01 R/A
Cofilin-1 CFL1 P45592 F4 0.80 0.04 R/A
Dihydropyrimidinase-related protein 2 DPYSL2 P47942 F4 0.82 0.01 R/A
Isocitrate dehydrogenase [NADP], mitochondrial IDH2 P56574 F4 0.79 0.03 R/A
Peroxiredoxin-2 PRDX2 P35704 F4 0.72 0.04 R/A
Astrocytic phosphoprotein PEA-15 PEA15 Q5U318 F2 0.67 0.02 R/C
Protein kinase C gamma type PRKCG P63319 F2/F3 0.60/0.61 0.02 R/C
Cytochrome b–c1 complex subunit 1, mitochondrial UQCRC1 Q68FY0 F4 1.23 0.03 R/C
Tubulin alpha-4A chain TUBA4A Q5XIF6 F4 0.75 0.04 R/C
 J. Palmfeldt et al. / Molecular and Cellular Neuroscience 74 (2016) 87–95
Fig. 5. Validation by Western blot. A) Representative Western blot picture depicting protein expression of glial fibrillary acidic protein (GFAP) in pooled samples of Control, Anhedonic
(CMS-A) and resilient (CMS-R). The cytoskeletal protein β-actin was used as reference protein and loading control. To the right are quantitative values from the densitometric analysis
of the blot pictures (n = 3).
PFC of KO mice reverses the genotypic differences, indicating that
HOMER1 expression is important in the stress-response mechanism
(Lominac et al., 2005).
4.1.4. Stress-induced anhedonia correlates with reduced VGLUT1 expression
The vesicular glutamate transporter 1 (VGLUT1) was down-
regulated in the anhedonic group, compared to the control group. This
result is in line with previous reports of reduced VGLUT1 expression
being associated with increased stress susceptibility in VGLUT1-
heterozygous mice (Lominac et al., 2005). Other studies have also impli-
cated vesicular glutamate transporters in depression pathology. We
have previously reported a decreased level of VGLUT2 in hippocampal
tissue of anhedonic rats (Henningsen et al., 2012a). A decreased
VGLUT1 expression in hippocampal and occipital cortex was reported
in the learned helplessness model of depression (Zink et al., 2010). Fi-
nally, differential expression of glutamate transporters was reported
in hippocampal tissue from post-mortem MDD patients (Medina et al.,
2013). Although compared to the present study, this was tissue from a
different region and the regulation in an opposite direction, the study
points to altered glutamate vesicular transporters as a key marker of
depression.
The overall picture of glutamate vesicular transporters and their role
in a stress/depression-related context appears to be complicated. None-
theless, glutamate regulation, and the glutamate system in general, has
received a lot of focus in recent years and this study further underscores
the relevance of following this path.
4.1.5. Sodium/potassium-transporting ATPase
The expression level of the ATP1A2 Sodium/potassium-transporting
ATPase subunit alpha-2 was increased in resilient versus anhedonic an-
imals. ATP1A2 is important for maintenance of Na+ and K+ gradients
across the cell membrane and lack of the transporter has been shown
to cause impaired glutamate uptake (Ikeda et al., 2003). Furthermore,
mice heterozygous for ATP1A2 displayed increased anxiety-related be-
havior, reduced locomotor activity, and impaired spatial learning
(Moseley et al., 2007). The observed increase in resilient animals, in
the present study, might play a role in counteracting these negative
effects.
4.1.6. Mitochondrial respiration and oxidative stress
The mitochondrial respiratory chain has been shown to be compro-
mised in animal models of depression, including specific effects on com-
plex III in cortex (Rezin et al., 2008). In the present study the core
complex III proteins UQCRFS1 and UQCRC1, both cytochrome b–c1 com-
plex subunits, were upregulated in resilient animals compared to
93
anhedonic and control, respectively. A causal link between UQCRC1
overexpression and enhanced complex III activity has been established
in neuroblastoma cells (Kriaucionis et al., 2006), suggesting that
UQCRC1 in resilient animals might confer improved complex III activity.
Oxidative stress is, like mitochondrial functions, commonly reported
to be linked to depression, and in the present study two proteins with
antioxidant function (peroxiredoxins, PRDX1 and PRDX2) were down-
regulated in resilient vs anhedonic animals, possibly indicating that re-
silient animals were less challenged by oxidative stress.
4.2. Rat model of depression
The use of animal depression models is widespread and offers a pre-
clinical alternative to study the biology of depression. A clear advantage
is that the environment can be fully controlled and that the experimen-
tal subjects can be comprehensively monitored. The most obvious dis-
advantage is that translational value is not an absolute measure and
therefore conclusions based on results gathered from animal depression
models are constrained to the limitations of the model used.
The CMS model fulfills all commonly used validity criteria, namely;
etiological-, construct-, face-, and predictive validity and therefore has
a strong translational value (Wiborg, 2013; Willner et al., 1992). Com-
paring results across depression models should be done with caution.
At present “depression model” is an unrestricted term that includes en-
vironmental, genetic and lesion-based models. Moreover, there is no
consensus in the literature as to how many of the validity criteria a
model has to fulfill, to be called a depression model. Finally, within the
individual depression models, the experimental approach is also highly
diverse and this diversity therefore should be considered before com-
paring results.
4.3. Other studies looking at stress-related PFC molecular biomarkers
The present study, to our knowledge, is the first study aimed to iden-
tify protein biomarkers in PFC synaptosomes of rats exposed to CMS. A
previous study by Y. Yang et al. was also aimed at identifying stress-
associated PFC protein biomarkers in rats (Liu et al., 2014). Compared
to the present study, there were no overlaps in differentially regulated
proteins. The explanation probably should be found in the methodolog-
ical differences, ie stress paradigm, biological focus (synaptosomes) and
analytical method applied. The Yang study was based on a different ver-
sion of the CMS model, using severe stressors like 48-hour food depriva-
tion, cold swim stress, and tail pinching. The severe stress paradigm,
might also explain why no resilient rats was seen in their study. In addi-
tion to the proteomic studies, a study by Orsetti, et al. was aimed at
94 J. Palmfeldt et al. / Molecular and Cellular Neuroscience 74 (2016) 87–95
identifying genetic biomarkers in the PFC of rats exposed to CMS
(Orsetti et al., 2008). Comparing their results to the present, the specific
overlap in significantly regulated genes and proteins biomarkers are
limited to the Homer 1 gene, mentioned above. Moreover, both studies
identify a target in the Thymosin beta (Thymosin beta-like 1 gene and
Thymosin beta-4 protein) family and a subunit of ATPase 1 (Atp1a1
gene and ATP1A2 protein). The relatively low overlap could again be at-
tributed to differences in the stress paradigm. In addition, it is well
known that protein and gene expression levels are not necessarily con-
verging, possibly due to fact that post-transcriptional, translational and
protein degradation regulation influence the protein levels (Vogel and
Marcotte, 2012). The present study, with focus on synaptosomal chang-
es, contains novel findings complementing previous studies, and thus
improves the understanding of the brain plasticity during CMS.
4.4. Synaptosome fractionation
The synaptosome fractionation method was used to increase the
yield of synaptic proteins. The literature on this method describes the
F4 fraction and in parts the F3 fraction as particular useful in the study
of the synaptosome functions (Dunkley et al., 2008; Bai and
Witzmann, 2007). The results of the present study however point to
the F2 fraction as the fraction holding the highest level of synaptic pro-
teins. Although F2 synaptosomes likely have low functional activity due
to low mitochondrial content, inclusion of F2 is highly relevant in prote-
omics studies, since it will lead to enhanced data on synapse proteins.
Synaptosome fractionation appears as an appropriate tool for
fractioning a proteome into less complex entities and thereby increas-
ing proteome coverage. More thorough knowledge about the individual
fractions, for example from the present study, benefit targeted studies
of proteins central for synapse function including future studies on bio-
markers and functional effects of post translational protein
modifications.
5. Conclusions
Synaptosomal fractionation is an optimal technique for fractioning
and enriching brain samples for synaptic proteins. In studies of func-
tional synaptosomes only a single subfraction (F4) is usually studied,
however, here we illustrate that for proteomic investigations F2 and
F3 also are also highly valuable fractions due to their high content of
synaptic proteins.
Through a differentiated view of the stress-response the present
study discriminates an altered protein profile in the depression-like
state, from alterations caused by stress exposure per se. The study de-
scribes depression-linked alterations in the prefrontal cortex, e.g. gluta-
matergic signaling (vesicular glutamate transporter 1, VGLUT1), ion
regulators (Na/K-transporting ATPase, ATP1A2, and calcium-binding
HOMER1), respiratory chain subunits (UQCRFS1 and UQCRC1) and a
regulator of cell interactions and intracellular transport (GFAP). These
alterations are specific to the pathological stress response, and interest-
ingly they diverge for resilient rats and unchallenged control rats. The
reported protein data from depression-like and resilient specimen high-
lights that multiple functionalities are altered in the course of resiliency
– in addition to glutamatergic signaling − and these data may shed
more light on pathophysiological mechanisms underlying MDD and
aid in the development of novel pharmacological treatment regimens.
Acknowledgements
The authors would like to thank the Lundbeck Foundation (R108-
A10711) and The John and Birthe Meyer Foundation for financial
support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.mcn.2016.04.001.
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