Effects of fluoxetine on plasticity and apoptosis evoked by chronic stress in rat prefrontal cortex.
ABSTRACT The prefrontal cortex is the brain region sensitive to detrimental effects of stress and even mild stress can rapidly impair its function. Aside from initiating proadaptive neuroplastic changes in the prefrontal cortex, chronic stress may also increase vulnerability of cortical neurons to apoptosis. Understanding the mechanism of plasticity and apoptotic processes is of immense importance for therapy of stress-related psychiatric disorders. In this study we tested whether molecular alterations in the prefrontal cortex, which occurred upon chronic social isolation, could be influenced by a prolonged fluoxetine treatment. We analyzed the expression of synaptic plasticity and apoptotic molecular markers in the prefrontal cortex of young-adult male Wistar rats exposed to 6-week social isolation with and without fluoxetine treatment during the last 3 weeks. Compartmental redistribution of NFκB transcription factor, involved in regulation of plasticity and apoptosis, was also examined. The level of synaptosomal polysialic neural cell adhesion molecule (PSA-NCAM) was increased in the prefrontal cortex of isolated rats as compared to untreated controls. Treatment with fluoxetine reduced the PSA-NCAM level only in isolated animals. In addition, mitochondrial Bax protein was elevated by chronic social isolation, while fluoxetine failed to abolish this effect. In spite of elevated Bcl-2 in the mitochondria, the calculated Bax/Bcl-2 ratio and concomitant absence of NFκB activation pointed to initiation of apoptotic signaling in the prefrontal cortex. The results imply that fluoxetine influences plasticity in the prefrontal cortex of chronically isolated rats and fails to prevent stress-induced initiation of apoptosis in this brain structure.
- SourceAvailable from: Liisa A M Galea[Show abstract] [Hide abstract]
ABSTRACT: Depression is a devastating and prevalent disease, with profound effects on neural structure and function; however the etiology and neuropathology of depression remain poorly understood. Though antidepressant drugs exist, they are not ideal, as only a segment of patients are effectively treated, therapeutic onset is delayed, and the exact mechanism of these drugs remains to be elucidated. Several theories of depression do exist, including modulation of monoaminergic neurotransmission, alterations in neurotrophic factors, and the upregulation of adult hippocampal neurogenesis, and are briefly mentioned in the review. However none of these theories sufficiently explains the pathology and treatment of depression unto itself. Recently, neural plasticity theories of depression have postulated that multiple aspects of brain plasticity, beyond neurogenesis, may bridge the prevailing theories. The term "neural plasticity" encompasses an array of mechanisms, from the birth, survival, migration, and integration of new neurons to neurite outgrowth, synaptogenesis, and the modulation of mature synapses. This review critically assesses the role of adult hippocampal neurogenesis and the cell adhesion molecule, PSA-NCAM (which is known to be involved in many facets of neural plasticity), in depression and antidepressant treatment.Neural Plasticity 01/2013; 2013:805497. · 2.86 Impact Factor
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ABSTRACT: Recently, it has been proposed that abnormalities in neuronal structural plasticity may underlie the pathogenesis of major depression, resulting in changes in the volume of specific brain regions, including the hippocampus (HIP), the prefrontal cortex (PC), and the amygdala (AMY), as well as the morphology of individual neurons in these brain regions. In the present survey, we compile the data regarding the involvement of the neural cell adhesion molecule (NCAM) protein and its polysialylated form (PSA-NCAM) in the pathogenesis of depression and the mechanism of action of antidepressant drugs (ADDs). Elevated expression of PSA-NCAM may reflect neuroplastic changes, whereas decreased expression implies a rigidification of neuronal morphology and an impedance of dynamic changes in synaptic structure. Special emphasis is placed on the clinical data, genetic models, and the effects of ADDs on NCAM/PSA-NCAM expression in the brain regions in which these proteins are constitutively expressed and neurogenesis is not a major factor; this emphasis is necessary to prevent cell proliferation and neurogenesis from obscuring the issue of brain plasticity.Pharmacological reports: PR 11/2013; 65(6):1471-1478. · 1.97 Impact Factor
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ABSTRACT: Mesocorticolimbic (MCL) dopaminergic system has an essential role in the rewarding action of opiates and is proved to be influenced by stress. Additionally, both morphine and stress can induce apoptosis. In this study, we investigated the effects of morphine-induced conditioned place preference (CPP) in the presence and absence of stress on the changes of apoptotic factors (Bax/Bcl-2 ratio, caspase-3 activation and PARP degradation) in the MCL system. Male Wistar rats were divided into two saline- and morphine-treated supergroups, each of which consisted of control, acute stress (AS) and subchronic stress (SS) subgroups. In all groups, the CPP paradigm was performed; thereinafter alternations of apoptotic factors in the nucleus accumbens, amygdala, striatum and prefrontal cortex were measured. The results indicated that in the saline-treated animals, AS and SS increased apoptotic factors significantly in the mentioned areas (except for the Bax/Bcl-2 ratio after AS in the Str). In addition, in these animals, conditioning scores decreased after SS but not after AS. In the morphine-treated animals, AS and SS increased apoptotic factors remarkably (except for the Bax/Bcl-2 ratio after AS and SS in the Str and caspase-3 activation after AS in the NAc) and also decreased conditioning scores. Our findings suggest that in the saline- or morphine-treated animals, AS and SS can increase the apoptotic factors in the MCL system and it is more prominent in the morphine-treated animals.Pharmacology Biochemistry and Behavior 06/2014; · 2.82 Impact Factor
Neuropharmacology and analgesia
Effects of fluoxetine on plasticity and apoptosis evoked by chronic stress
in rat prefrontal cortex
Ana Djordjevica,n, Jelena Djordjevicb, Ivana Elakovic ´a, Miroslav Adzicb, Gordana Matic ´a,
Marija B. Radojcicb
aDepartment of Biochemistry, Institute for Biological Research ‘‘Siniˇ sa Stankovic ´’’, University of Belgrade, 142 Despot Stefan Blvd., 11000 Belgrade, Serbia
bLaboratory of Molecular Biology and Endocrinology, Institute of Nuclear Sciences VINCA, University of Belgrade, 11000 Belgrade, Serbia
a r t i c l e i n f o
Received 3 April 2012
Received in revised form
10 July 2012
Accepted 27 July 2012
Available online 21 August 2012
a b s t r a c t
The prefrontal cortex is the brain region sensitive to detrimental effects of stress and even mild stress
can rapidly impair its function. Aside from initiating proadaptive neuroplastic changes in the prefrontal
cortex, chronic stress may also increase vulnerability of cortical neurons to apoptosis. Understanding
the mechanism of plasticity and apoptotic processes is of immense importance for therapy of stress-
related psychiatric disorders. In this study we tested whether molecular alterations in the prefrontal
cortex, which occurred upon chronic social isolation, could be influenced by a prolonged fluoxetine
treatment. We analyzed the expression of synaptic plasticity and apoptotic molecular markers in the
prefrontal cortex of young-adult male Wistar rats exposed to 6-week social isolation with and without
fluoxetine treatment during the last 3 weeks. Compartmental redistribution of NFkB transcription
factor, involved in regulation of plasticity and apoptosis, was also examined. The level of synaptosomal
polysialic neural cell adhesion molecule (PSA-NCAM) was increased in the prefrontal cortex of isolated
rats as compared to untreated controls. Treatment with fluoxetine reduced the PSA-NCAM level only in
isolated animals. In addition, mitochondrial Bax protein was elevated by chronic social isolation, while
fluoxetine failed to abolish this effect. In spite of elevated Bcl-2 in the mitochondria, the calculated
Bax/Bcl-2 ratio and concomitant absence of NFkB activation pointed to initiation of apoptotic signaling
in the prefrontal cortex. The results imply that fluoxetine influences plasticity in the prefrontal cortex of
chronically isolated rats and fails to prevent stress-induced initiation of apoptosis in this brain
& 2012 Elsevier B.V. All rights reserved.
Chronic stress is recognized as one of the most potent
etiological factors of mood disorders (Rygula et al., 2005; Willner,
1997). Among chronic stressors, those with psychosocial compo-
nents are considered particularly important since social stress in
animals evokes symptoms that resemble those found in depressed
patients (Fuchs and Flugge, 2004). Recently, ‘‘neuroplastic’’ hypoth-
esis of depression has evolved suggesting that alterations in
plasticity of neuronal networks represent a relevant factor in the
etiology of mood disorders (Castren, 2005).
Successful adaptation to stress involves alterations in expression
of some synaptic membrane proteins such as polysialylated isoform
of the neural cell adhesion molecule (PSA-NCAM) (Sandi, 2004) and
syntaxin-1 (Davis et al., 1998; Halim et al., 2003). PSA-NCAM
promotes plasticity through its large negatively charged PSA,
postulated to be a spacer that reduces adhesion forces between
cells, allowing their dynamic changes (Gascon et al., 2007). Several
authors (Cerqueira et al., 2007; Cook and Wellman, 2004; Radley
et al., 2004) have suggested that stress may lead to changes in the
strength or efficacy of synaptic transmission, which rely on action of
regulatory synaptic proteins like syntaxin-1 (Sudhof, 2004). Fluox-
etine, a member of the selective serotonin reuptake inhibitor group,
was shown to restore structural plasticity in the adult rat visual
cortex (Maya Vetencourt et al., 2008) and increase PSA-NCAM
expression in the medial prefrontal cortex (Varea et al., 2007a).
In addition to adaptive plasticity, apoptosis has also been
proposed to be a mechanism contributing to structural alterations
observed in stress-related mood disorders both in humans and
animal models (reviewed in McKernan et al., 2009). The apoptotic
process is controlled by proapoptotic (Bax) and antiapoptotic
(Bcl-2) proteins in outer mitochondrial membrane (Cory and
Adams, 2002; Lindsten et al., 2005). An increase in the ratio of
proapoptotic vs. antiapoptotic proteins in mitochondria is asso-
ciated with vulnerability to apoptotic activation and cell death
(Lindsten et al., 2005). The prefrontal cortex is thought to
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European Journal of Pharmacology
0014-2999/$-see front matter & 2012 Elsevier B.V. All rights reserved.
nCorresponding author. Tel.: þ381 11 207 83 18; fax:þ 381 11 276 14 33.
E-mail address: firstname.lastname@example.org (A. Djordjevic).
European Journal of Pharmacology 693 (2012) 37–44
be particularly affected during chronic stress and depression
(Pittenger and Duman, 2008) and it was previously shown that
social isolation promotes proapoptotic signaling in the prefrontal
cortex (Djordjevic et al., 2010) and predisposes the frontal cortex
to the detrimental release of cytochrome-c from the mitochondria
(Filipovic et al., 2011).
Both antidepressants and stress can affect a number of factors
involved in cell survival pathways controlling Bcl-2 expression
(Manji et al., 2001), among which is the nuclear factor kappa B
(NFkB) (Kucharczak et al., 2003). NFkB is considered an important
stress sensor and it was recently implicated in the prevention of
neuronal apoptosis (Mattson, 2005) where upon activation NFkB
translocates to the nucleus and regulates its target genes (O’Neill
and Kaltschmidt, 1997).
Stress and antidepressants are involved in the regulation
of plastic and apoptotic responses through synaptic plasticity
markers, PSA-NCAM and syntaxin-1, and apoptotic proteins, Bax
and Bcl-2, which are partly regulated by NFkB. Hence, we
analyzed the expression of these proteins in the prefrontal cortex
of young-adult male Wistar rats subjected to chronic social
isolation and fluoxetine treatment, alone and in combination.
The results revealed that antidepressant treatment of stressed
animals partially stabilized the plastic response evoked by stress,
but failed to revert it to the control level. Additionally, dominance
of proapoptotic Bax protein over antiapoptotic Bcl-2 in mitochon-
dria suggested that both social isolation and fluoxetine, separately
or in combination, increased vulnerability to apoptotic activation
in the prefrontal cortex. The observed apoptotic signalization
coincides with lack of NFkB activation, a transcription factor with
known antiapoptotic activity in neurons.
2. Material and methods
A fluoxetine-hydrochloride reference standard was kindly
gifted by Galenika a.d., Belgrade, and Flunirins capsules (contain-
ing 20 mg of fluoxetine-hydrochloride) were purchased from the
same company. Polyvinylidene difluoride (PVDF) membrane
(Immobilon-P membrane) and PSA-NCAM antibody were obtained
from Millipore Corporation (USA). Enhanced chemiluminescence
(ECL) reagent pack containing Rabbit IgG, HRP-linked whole anti-
body and ECL Mouse IgG, HRP linked whole antibody were
obtained from Amersham Pharmacia Biotech, UK. Rabbit polyclonal
anti-b-actin (ab8227) was obtained from Abcam (USA), while
Mouse monoclonal anti-mHsp70 was obtained from Affinity Bior-
eagents. Mouse monoclonal antibodies anti-Bcl-2, anti-Bax and
obtained from Santa Cruz Biotechnology (USA).
2.2. Preparation of fluoxetine-hydrochloride solution
The capsules of Flunirins were used to obtain purified fluoxetine
as described previously (Djordjevic et al., 2011; Elakovic et al., 2010).
The concentration of fluoxetine-hydrochloride in the purified pre-
paration was determined by colorimetric method essentially
described by Prabhakar et al. (1999) using pure fluoxetine-
hydrochloride (Elli Lilly, Reference Standard Lot 00IPD5, p¼100%,
v¼0.07%) as the standard. Fluoxetine-hydrochloride preparation
was diluted with distilled water to the final concentration of
5 mg/ml and was administered intraperitoneally at a daily dose of
5 mg/kg body mass.
2.3. Animals and treatment
Young-adult male Wistar rats, bred in our laboratory, were
2-2.5 months old at the beginning of the experiment. All animals
were kept at 2272 1C, with a 12 h light/dark cycle (lights on at
07:00 h), with food and drinking water available ad libitum. The
animals were divided into four experimental groups housed
either four per cage (ControlþVehicle and ControlþFluox groups)
or individually (StressþVehicle and StressþFluox groups). Indivi-
dual housing for 6 weeks was used as a model of chronic social
isolation stress during which animals had normal auditory and
olfactory experiences, but were deprived of any visual or tactile
contact with other rats. Fluoxetine-hydrochloride was adminis-
tered intraperitoneally at a daily dose of 5 mg/kg b.w. at 09:00 h
during a 3-week period. Drug dose was chosen based on previous
reports (Detke et al., 1997; Djordjevic et al., 2011, 2012; Elakovic
et al., 2010, 2011). Fluoxetine treatment was applied to Controlþ
Fluox group, and to StressþFluox group during the last 3 weeks of
isolation. Both Vehicle groups received distilled water under the
same conditions as matching fluoxetine-treated groups. All
experiments were repeated two times, each time with eight
animals per group (32 animals per experiment). All animal
procedures were complied with the European Communities
Council Directive (2010/63/EU) and were approved by the Ethical
Committee for the Use of Laboratory Animals according to the
guidelines of the EU registered Serbian Laboratory Animal Science
2.4. Preparation of cytoplasmic, nuclear and mitochondrial extracts
Animals were sacrificed by rapid decapitation 24 h after the
last fluoxetine injection. The prefrontal cortices were excised,
weighed and homogenized (1:2 w/v) in ice-cold 20 mM Tris–HCl
(pH 7.2) buffer containing 10% glycerol, 50 mM NaCl, 1 mM EDTA,
1 mM EGTA, 2 mM DTT, protease and phosphatase inhibitors. All
further procedures were carried out at 4 1C. To obtain crude
nuclear pellets, the homogenates were centrifuged 10 min at
2000g and the supernatants were further centrifuged at 20,000g
for 30 min to provide crude mitochondrial pellets. The resulting
supernatants of this centrifugation were ultracentrifuged at
105,000g for 1 h to obtain final supernatants used as cytoplasmic
fractions. The crude mitochondrial pellets were washed in 0.5 ml
of homogenization buffer and centrifuged at 20,000g for 30 min.
Mitochondrial pellets were then lysed in the buffer containing
50 mM Tris–HCl (pH 7.4), 5% glycerol, 1 mM EDTA, 5 mM DTT,
protease inhibitors and 0.05% Triton X-100, and incubated on ice
for 1.5 h with frequent vortexing. The resulting fractions were
used as final mitochondrial extracts. Crude nuclear pellets were
washed in 0.5 ml of homogenization buffer, and centrifuged
10 min at 2000g at 4 1C. The final nuclear pellets were weighed,
resuspended (1:1 w/v) in the same buffer supplied with 0.5 M
KCl, incubated for 1 h in ice-bath with frequent vortexing and
centrifuged for 10 min at 8000g at 4 1C. The supernatants were
used as nuclear extracts (Moutsatsou et al., 2001; Spencer et al.,
2.5. Preparation of crude synaptosomal pellets
Crude synaptosomal pellets were isolated according to Sandi
et al. (2001). Briefly, frozen tissues were homogenized in 10
volumes of ice-cold 0.32 M sucrose and 5 mM HEPES buffer
(pH 7.4) that contained protease inhibitors. Homogenates were
centrifuged at 1000g for 5 min at 4 1C. The obtained supernatants
were then centrifuged at 15,000g for 15 min and the pellets were
resuspended in Krebs buffer according to Lynch and Voss (1991)
containing protease inhibitors and 1% Nonidet P-40.
A. Djordjevic et al. / European Journal of Pharmacology 693 (2012) 37–44
2.6. Western blot detection of PSA-NCAM, syntaxin-1, NFkB/p65,
Bax and Bcl-2 proteins
Protein concentration in all samples was determined by the
method of Lowry et al. (1951). The samples were mixed with
denaturing buffer according to Laemmli (1970), boiled, and 60 mg
of proteins were subjected to electrophoresis on 10% (Bax and
Bcl-2) or 7.5% (PSA-NCAM, syntaxin-1) sodium dodecyl sulfate-
polyacrylamide gels. After electrophoresis, proteins were trans-
ferred onto PVDF membrane (Immobilon-P membrane, Millipore)
using a blot system (Transblot, BioRad). Membranes were blocked
with 5% non-fat dry milk for 1 h at room temperature and
incubated with the respective primary and secondary antibodies.
Signal was developed by enhanced chemiluminescence reagent
(ECL, Pierce) and the membranes exposed to X-ray film. Protein
molecular mass standards (PageRulerTMPlus Prestained Protein
Ladder, Fermentas) were used for calibration. Antibodies (Santa
Cruz Biotechnology) against syntaxin-1 (1:1000), Bax (1:1000)
and Bcl-2 (1:1000), NFkB (1:1000), and PSA-NCAM (1:1000,
Millipore) were used to detect these proteins. Loading controls
for cytoplasmic and mitochondrial compartments were obtained
by rabbit polyclonal anti-b-actin antibody (ab8227, 1:5000,
Abcam) and mouse monoclonal anti-mHsp70 antibody (MA3-
028, 1:5000, Affinity Bioreagents), respectively. Blots were devel-
oped with ECL Rabbit IgG, HRP-linked whole antibody or with ECL
Mouse IgG, HRP-linked whole antibody (Amersham). Densitome-
try of protein bands on X-ray film was performed by Image
J analysis PC software.
2.7. Statistical analysis
Data are presented as mean7SEM. For Western blot analysis
3 measurements for each repeated experiment were performed.
To determine the effects of stress and fluoxetine treatment, as
well as their interaction, two-way ANOVA followed by the post-
hoc Tukey test was used. The statistical significance was accepted
at Po0.05. Separate effects of the stress and fluoxetine factors, as
well as their interaction, are given in the Section 3.
3.1. PSA-NCAM and syntaxin-1 protein level in prefrontal cortex
At the level of PSA-NCAM protein, two-way ANOVA revealed
significant effect of stress factor [F(1,32)¼27.87, Po0.001]. Further-
more, PSA-NCAM was significantly elevated under chronic social
isolation in respect to ControlþVeh group (Figs. 1A,
After fluoxetine treatment PSA-NCAM level in stressed rats was
increased in respect to ControlþVeh group (Fig. 1A,nnPo0.01) and
significantly decreased in respect to StressþVeh group (Fig. 1A,
yPo0.05). In addition, syntaxin-1 was significantly decreased in
control animals treated with fluoxetine in respect to ControlþVeh
group (Fig. 1B,nnnPo0.001) and StressþFluox group (#Po0.05).
3.2. Bax and Bcl-2 protein level in the cytoplasm and mitochondria
of prefrontal cortex
The main effects of stress [F(1,21)¼15.35, Po0.001] and
fluoxetine [F(1,21)¼8.82, Po0.01] factors at the level of cytoplas-
mic Bax were detected by Two-way ANOVA (Fig. 2). At the level of
mitochondrial Bax the main effects of stress [F(1,21)¼25.93,
Po0.001] and fluoxetine [F(1,16)¼60.31, Po0.001] were also
found. As shown in Fig. 2A, post-hoc test revealed a significant
increase of Bax in the cytoplasm of control animals treated with
fluoxetine in respect to both ControlþVeh group (nPo0.05) and
StressþFluox group (##Po0.01). Mitochondrial Bax was increased
in all experimental groups in respect to ControlþVeh group
respect to ControlþFluox and StressþVeh groups (##Po0.05 and
A main effect of stress factor [F(1,30)¼9.91, Po0.01] and
fluoxetine [F(1,30)¼27.69, Po0.001] on cytoplasmic Bcl-2 pro-
tein was found by Two-way ANOVA. Post-hoc test showed Bcl-2
elevation in both groups of fluoxetine treated rats in respect to
ControlþVeh group (Figs. 2C,
as its significant elevation in StressþFluox group in respect to
nnnPo0.05) and in StressþFluox group in
nnnPo0.001), as well
PSA-NCAM (% of control)
Syntaxin (% of control)
s+vs+f s+v s+f
Fig. 1. PSA-NCAM and syntaxin-1 protein level in the prefrontal cortex synaptosomes. Representative Western blots and relative quantification of PSA-NCAM protein
(A) and syntaxin-1 protein (B) in the synaptosomes of control and stressed rats treated with fluoxetine. Data are presented as mean7SEM. Significant between group
differences, obtained from two-way ANOVA analysis followed by post hoc Tukey test are indicated as follows:
ControlþVeh group;yPo0.05 StressþFluox vs. StressþVeh.
nnnPo0.001 treated experimental groups vs.
A. Djordjevic et al. / European Journal of Pharmacology 693 (2012) 37–44
StressþVeh group (Figs. 2C,
main effects of stress [F(1,23)¼18.06, Po0.001] and fluoxetine
[F(1,23)¼42.23, Po0.001] were detected by Two-way ANOVA,
while post-hoc test found significant increase of Bcl-2 in Con-
trolþFluox group in respect to ControlþVeh group (nPo0.05) and
in StressþFluox group in respect to all other groups (nnnPo0.001;
In addition, the relative ratios of Bax to Bcl-2 in the cytoplasmic
and mitochondrial compartments were calculated (Fig. 3). The
ratio values above one indicated the dominance of Bax, while
values below one, the prevalence of Bcl-2 protein in the particular
compartment. Two-way ANOVA showed the main effect of stress
factor [F(1,19)¼50.41, Po0.001] and the interaction of stress and
fluoxetine factors [F(1,19)¼5.09, Po0.05] on the calculated ratios
in the cytoplasm. Post-hoc test detected significant decrease of
cytoplasmic Bax/Bcl-2 ratio for both stressed groups in respect to
ControlþVeh group (nnPo0.01 andnnnPo0.001) and for Stressþ-
Fluox group in respect to ControlþFluox group (##Po0.01). Two-
way ANOVA detected the main effects of stress [F(1,19)¼7.94,
Po0.05] and fluoxetine [F(1,19)¼11.11, Po0.01], as well as the
yyPo0.01). In mitochondria, the
interaction of these factors [F(1,19)¼22.53, Po0.001] on mito-
chondrial Bcl-2. In addition, post-hoc test revealed significant
increase of mitochondrial Bax/Bcl-2 ratio in all treated groups in
respect to ControlþVeh group (nnPo0.01 andnnnPo0.001) (Fig. 3).
3.3. NFkB protein (p65 subunit) level in different cellular
compartments of the prefrontal cortex
Two-way ANOVA showed the interaction of the factors
[F(1,21)¼7.79, Po0.05], while the post-hoc test revealed only
significant increase of cytoplasmic NFkB/p65 level in StressþVeh
group in respect to ControlþVeh group (Fig. 4,
nuclei, we did not observe any significant change in the NFkB/p65
level irrespectively of the treatment.
nPo0.05). In the
The main results of the present study revealed increased level
of synaptosomal PSA-NCAM plasticity marker in isolated rats, and
Bcl2 (% of control)
Bax (% of control)
Bax (% of control)
Bcl2 (% of control)
s+vs+fc+v s+v s+f
c+vs+v s+fc+v s+vs+f
Fig. 2. Bax and Bcl-2 protein level in the prefrontal cortex cytoplasm and mitochondria. Western blot analysis and relative quantification of cytoplasmic (A, C) and
mitochondrial (B, D) Bax (A, B) and Bcl-2 (C, D) protein demonstrating the effect of fluoxetine in the hippocampus of control and stressed rats. Data are presented as
mean7SEM. Significant differences between groups, obtained from two-way ANOVA analysis followed by post hoc Tukey test are given as:
nnnPo0.001 between treated experimental groups and ControlþVeh group;yyPo0.01 between StressþFluox and StressþVeh groups and##Po0.01 between StressþFluox
and ControlþFluox groups.
A. Djordjevic et al. / European Journal of Pharmacology 693 (2012) 37–44
its reduction by fluoxetine treatment. Analysis of apoptotic
proteins showed increased mitochondrial Bax level in stressed
animals. Fluoxetine failed to abolish this effect in spite of elevated
Bcl-2, as judged by an increased mitochondrial Bax/Bcl-2 ratio,
while nuclear NFkB remained unchanged.
The prefrontal cortex is a target of chronic stress, which was
shown to induce morphological changes of rat medial prefrontal
cortex by regressing apical dendrites of pyramidal cells (Banasr
et al., 2007; Cook and Wellman, 2004). Dendritic reorganization is
often accompanied by changes in cell adhesion molecules such
as PSA-NCAM, a glycoprotein involved in synaptic remodeling
(Sandi, 2004). We have already shown PSA-NCAM elevation after
6 weeks of chronic stress in the hippocampus (Djordjevic et al.,
2012), and in the present study, under the same stressful condi-
tions, we also found its increased level in the prefrontal cortex.
The studies on other stress models also reported changes in
PSA-NCAM. Nacher et al. (2004) found increased number of
PSA-NCAM immunoreactive cells in the piriform cortex of adult
rats subjected to chronic restraint stress, while predator stress
and swimming were found to reduce synaptic NCAM expression
in the prefrontal cortex (Sandi et al., 2005). Interestingly, earlier
results from our laboratory (Djordjevic et al., 2010) revealed
decreased level of prefrontal cortex PSA-NCAM after 3 weeks of
chronic social isolation probably depicting time-course depen-
dence of synaptic markers regulation.
As for the effect of antidepressant treatment on synaptic
plasticity, recent studies have demonstrated that chronic fluox-
etine treatment induces dendritic spine remodeling of pyramidal
neurons in rat somatosensory cortex (Guirado et al., 2009) and
affects both PSA-NCAM and synaptophysin expression in the
hippocampus, amygdala and different cortical subregions (Varea
et al., 2007b). In the present study, when applied to control
animals, fluoxetine did not affect synaptosomal PSA-NCAM level.
In contrast to our result, Varea et al. (2007a) found increased
PSA-NCAM level in the medial prefrontal cortex of rats treated
with fluoxetine for 14 days. The discrepancy could be explained
by different doses of the drug used in these studies.
When applied to isolated animals fluoxetine partially pre-
vented the stress induced elevation of PSA-NCAM, leaving it
above the level observed in the control group (Fig. 1). This
observation might represent potential ability of fluoxetine to
stimulate removal of PSA, thus stabilizing the plastic response,
since it is known that stable synapses predominantly contain
non-sialylated NCAM (Gascon et al., 2007). The balance between
NCAM and PSA-NCAM levels might be critical for regulation of
activity-dependent structural modifications at synaptic level
(Bruses et al., 2002; Gascon et al., 2007). Interestingly, the effect
of fluoxetine on PSA-NCAM in the prefrontal cortex synaptosomes
was not as efficient as previously observed in the hippocampus
(Djordjevic et al., 2012). Since the decrease in PSA content is
correlated with its specific removal by the enzyme endoneura-
minidase or by decreased activity of the enzyme polysialyltrans-
ferase, which catalyzes its synthesis (Angata and Fukuda, 2003;
Rutishauser, 2008), the observed discrepancy could be regarded
as a consequence of differentially regulated activity of one or both
of these enzymes. In any case, it is likely that the observed PSA-
NCAM changes reflect reorganization of neuronal circuitry after
antidepressant treatment of stressed rats, since synaptic density
and synaptogenesis remained unaffected, as judged by the
unchanged level of syntaxin-1. Supporting this assumption, PSA-
NCAM expression in the prefrontal cortex is restricted to inter-
neurons or processes that project to this region (Varea et al.,
2007a) and is not present in recently generated cortical neurons
(Varea et al., 2011).
Syntaxin-1, a presynaptic membrane-bound protein is consid-
ered a reliable marker of synaptogenesis (Calakos and Scheller,
1994). As shown in Fig. 1, in the prefrontal cortex synaptosomes of
animals subjected to long-term fluoxetine treatment, syntaxin-1
level was lower than in untreated controls, which was opposite to
our previous finding in the hippocampus (Djordjevic et al., 2012).
Getz et al. (2011) demonstrated that chronic exposure to fluoxetine
inhibited synapse formation and reduced synaptic strength in
Lymnaea neurons, while at the structural level this antidepressant
altered the expression and localization of presynaptic protein
synaptophysin, another reliable
(Calakos and Scheller, 1994). In addition, Varea et al. (2007b) did
not find significant changes in synaptophysin expression in the
Veh FluoxVeh Fluox
Veh FluoxVeh Fluox
Fig. 3. Relative ratios between Bcl-2 and Bax in the prefrontal cortex cytoplasm
and mitochondria of control and stressed Wistar rats treated with fluoxetine.
Significant differences between groups obtained by two-way ANOVA followed by
post hoc Tukey test are indicated as follows:
treated experimental groups and ControlþVeh group;##Po0.01 between Stressþ-
Fluox and ControlþFluox groups.
Veh Fluox Veh Fluox
Veh Fluox Veh Fluox
c+f s+v s+f
c+v c+f s+v s+f
NFκB (% of control)
Fig. 4. NFkB protein (p65 subunit) level in cytoplasm and nuclei of the prefrontal
cortex. Representative Western blots and relative quantification of p65 subunit of
NFkB in the prefrontal cortex cytoplasm and nuclei of control and stressed rats
treated with fluoxetine. Data are presented as mean7SEM (as described under
Section 2.7). Significant differences between groups, obtained by two-way ANOVA
followed by post hoc Tukey test are
groups and ControlþVeh group.
nPo0.05 between treated experimental
A. Djordjevic et al. / European Journal of Pharmacology 693 (2012) 37–44
hippocampus after fluoxetine treatment, while Rapp et al. (2004)
found its elevation in all hippocampal subdivisions.
In accordance with our previous results in the rat hippocampus
(Djordjevic et al., 2012), the level of syntaxin-1 in the cortical
synaptosomes was unaltered by isolation stress, and in stressed
animals by fluoxetine. Muller et al. (2011) found elevated level of
syntaxin-1 in the prefrontal cortex of rats subjected to chronic
restraint stress, while Yamada et al. (2002) did not detect any
significant changes in syntaxin-1 level after electroconvulsive
treatment or antidepressants. Furthermore, Gao et al. (2006) and
Muller et al. (2011) found no changes in the levels of syntaxin-1 in
the hippocampus of rats subjected to chronic restraint stress. The
opposing results could be explained by different types of stressors
and regions examined. Unchanged syntaxin-1 level in the pre-
frontal cortex of isolated rats suggest that the effects of stress
could rather be ascribed to posttranslational modifications of
NCAM then to the changes in the number or density of synapses.
In vivo imaging studies revealed selective volume reduction in
the prefrontal cortex of depressed patients (Drevets, 2000). Post
mortem studies of depressed patients mostly did not find strong
relationship between apoptosis and depression (Reif et al., 2006),
but some histological data showed reduction in neuronal size and
glial cell numbers in specific subregions of the frontal cortex (Cotter
et al., 2001; Rajkowska et al., 1999). In the study of Kim et al. (2010)
an increased ratio of Bax/Bcl-2 and increased caspase-3 protein and
mRNA levels were detected in post mortem brain of patients with
bipolar depression. Since human studies are mainly restricted to
post mortem brains, different animal models have been used to
study cell death following chronic stress (for review see Duman,
2004). Animal experiments showed that chronic stress results in
reduced spine density in the medial prefrontal cortex (Cook and
Wellman, 2004; Radley et al., 2006), while dexamethasone treat-
ment results in neuronal loss and atrophy (Cerqueira et al., 2005). It
has also been established that stress may modulate apoptotic
signaling in the hippocampus and entorinal cortex (Lucassen et al.,
2001; Sapolsky, 1996) and that social stress may initiate apoptosis
in the prefrontal cortex (Adzic et al., 2009; Djordjevic et al., 2010;
Filipovic et al., 2011). In the present study we also observed elevated
proapoptotic mitochondrial Bax protein with no significant change
of Bcl-2 under chronic social isolation. The data concerning the level
of antiapoptotic protein Bcl-2 after exposure to stress are contra-
dictory: some authors reported a decrease of Bcl-2 expression
(Lindsten et al., 2005), while some reported its increase under stress
conditions (Bravo et al., 2009). However, calculated ratio between
Bax and Bcl-2 (Fig. 3), as an index of vulnerability to apoptosis
(Jarskog et al., 2004), suggested the activation of apoptotic signaling.
In our previous study on hippocampus, chronic isolation stimulated
antiapoptotic signaling through dominance of Bcl-2, which was even
potentiated by fluoxetine (Djordjevic et al., 2012). However, the
same stress stimulated initiation of proapoptotic signaling in pre-
frontal cortex mitochondria, by elevating Bax/Bcl-2 ratio, which was
not reverted by fluoxetine treatment. This discrepancy may be
ascribed to different glutamatergic transmission, which is central
to many forms of neuroplasticity and apoptosis (Pittenger and
Duman, 2008), and is known to be altered by stress in the
hippocampus (Lowy et al., 1993) and prefrontal cortex (Bagley and
Moghaddam, 1997; Moghaddam et al., 1994). Namely, even mod-
erate glutamate exposure over prolonged periods can lead to
apoptotic cell death in neurons (Banasiak et al., 2000) and the study
of Moghaddam (1993) provided direct evidence that glutamate
levels were significantly higher in the prefrontal cortex than in
other observed regions after stress, making this brain region more
sensitive to apoptosis.
The effect of antidepressants on apoptosis appears to be
complex and depends presumably on cell type. Most studies
report that antidepressant treatment prevents apoptosis through
mitochondrial dependent pathway (Lucassen et al., 2004; Nahon
et al., 2005), while several authors demonstrated proapoptotic
activity of antidepressants (Lirk et al., 2006; Piccotti et al., 2005; Qi
et al., 2002). There are reports that fluoxetine resulted in increased
levels of apoptosis in vitro (Koch et al., 2003), but it is not
established whether changes in Bax expression are sufficient to
induce apoptotic changes in vivo. Our results (Fig. 2B) showed
significant increase of Bax and Bcl-2 in both cytoplasm and
mitochondria upon fluoxetine treatment, but calculated ratio
revealed significant predominance of Bax in mitochondria, suggest-
ing the initiation of apoptosis, since redistribution of Bax from
cytoplasmic to mitochondrial compartment is a hallmark of this
process (Cao et al., 2001; Davies, 1995). Levkovitz et al. (2005)
observed increased apoptosis in rat glioma and human neuroblas-
toma cell lines following treatment with fluoxetine, and Serafeim
et al. (2003) showed that fluoxetine induces apoptosis in Burkitt
lymphoma cells. As mentioned previously, antiapoptotic effects of
antidepressants may be exerted via the anti-apoptotic actions of
Bcl-2, as increased Bcl-2 protein and mRNA levels in the frontal
cortex following chronic antidepressant treatment have already
been reported (Chiou et al., 2006a, 2006b; Drzyzga et al., 2009).
The finding that fluoxetine treatment of both unstressed and
stressed animals enhanced mitochondrial Bcl-2 level might repre-
sent potentiation of antiapoptotic response and thus provide
increased protection from deleterious effects of chronic stress. In
addition, chronic administration of fluoxetine to stressed animals
also increased mitochondrial Bax level (Fig. 2B) but the calculated
ratios between Bax and Bcl-2 in the mitochondria (Fig. 3) revealed
no significant change in respect to previous state established by
the chronic stress. Overall, it seems that evoked Bcl-2 response was
not sufficient to counteract Bax mediated initiation of proapoptotic
response in the prefrontal cortex set by the chronic stress.
Finally, we analyzed the activation and nuclear translocation of
NFkB, since it is implicated in both plasticity and apoptosis
(Kucharczak et al., 2003) and found that stress had significant
effect on NFkB level, while fluoxetine remained ineffective. The
possible explanation may lie in the fact that fluoxetine elevates
the antioxidants (reviewed in Schreck et al., 1992), which then
may inhibit NFkB. It is known that NFkB transactivates several
antiapoptotic genes in neurons and several signals that inhibit its
activity are generated in neurons undergoing apoptosis. Although
exact mechanisms by which neurons undergo apoptosis under the
conditions of NFkB inhibition are poorly understood, data obtained
on primary cultures of rat cortical neurons demonstrated that
NFkB inhibition induced rapid mitochondrial release of cyto-
chrome c, caspase-9 and -3 activation, membrane blebbing and
nuclear fragmentation (Chiarugi, 2002). In addition, inhibition of
nuclear NFkB was previously shown to induce DNA fragmentation
in central nervous system (Taglialatela et al., 1998).
In conclusion, the results of this study show that both chronic
social isolation and fluoxetine affected plasticity in the prefrontal
cortex. In addition, chronic isolation stimulated initiation of
proapoptotic signaling in mitochondria, by elevating Bax/Bcl-2
ratio, which was not reverted by fluoxetine treatment. The
observed apoptotic signalization was paralleled by the absence
of NFkB activation, known for its neuronal antiapoptotic role.
Therefore, although influencing plasticity in the prefrontal cortex
of chronically isolated rats, fluoxetine failed to prevent alterations
in apoptotic signaling in this brain structure.
This work was supported by the Ministry of Education, Science
and Technological Development of the Republic of Serbia, Grants
III41029 and III41009.
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