Subregion-specific Protective Effects of Fluoxetine and Clozapine on Parvalbumin Expression in Medial Prefrontal Cortex of Chronically Isolated Rats

Accepted Manuscript Research Article Subregion-specific protective effects of fluoxetine and clozapine on parvalbumin expression in medial prefrontal cortex of chronically isolated rats Nevena Todorović, Bojana Mićić, Marija Schwirtlich, Milena Stevanović, Dragana Filipović PII: DOI: Reference:

S0306-4522(18)30732-2 https://doi.org/10.1016/j.neuroscience.2018.11.008 NSC 18730

To appear in:

Neuroscience

Received Date: Revised Date: Accepted Date:

14 June 2018 18 October 2018 8 November 2018

Please cite this article as: N. Todorović, B. Mićić, M. Schwirtlich, M. Stevanović, D. Filipović, Subregion-specific protective effects of fluoxetine and clozapine on parvalbumin expression in medial prefrontal cortex of chronically isolated rats, Neuroscience (2018), doi: https://doi.org/10.1016/j.neuroscience.2018.11.008

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Subregion-specific protective effects of fluoxetine and clozapine on parvalbumin expression in medial prefrontal cortex of chronically isolated rats

Nevena Todorovića, Bojana Mićića, Marija Schwirtlichb, Milena Stevanovićb,c,d, Dragana Filipovića

a

Laboratory of Molecular Biology and Endocrinology, Institute of Nuclear Sciences “Vinča”,

University of Belgrade, Serbia b

Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade,

Serbia c

University of Belgrade - Faculty of Biology, Belgrade, Serbia

d

Serbian Academy of Sciences and Arts, Belgrade, Serbia

Filipović Dragana, Ph.D. Laboratory of Molecular Biology and Endocrinology Institute of Nuclear Sciences “Vinča”, University of Belgrade P.O.Box 522-090, 11001 Belgrade, Serbia Tel/fax +381 (11) 6455-561 E-mail: [email protected] www.vinca.rs

1

Abstract Dysregulation of GABAergic system is becoming increasingly associated with depression, psychiatric disorder that imposes severe clinical, social and economic burden. Special attention is paid to the fast-spiking parvalbumin positive (PV+) interneurons, GABAergic neurons which are highly susceptible to redox dysregulation and oxidative stress and implicated in a variety of psychiatric diseases. Here we analyzed the number of PV+ and cleaved caspase-3 positive (CC3+) cells in the rat medial prefrontal cortical (mPFC) subregions following chronic social isolation (CSIS), an animal model of depression and schizophrenia. Also, we examined potential protective effects of antidepressant fluoxetine (FLX) and atypical antipsychotic clozapine (CLZ) on the number of these cells in mPFC subregions, when applied parallel with CSIS in doses that correspond to therapeutically effective ones in patients. Immunofluoresce analysis revealed decreased number of PV+ cells in cingulate cortex area 1, prelimbic area (PrL), infralimbic area (IL) and dorsal peduncular cortex of the mPFC in isolated rats, which coincided with depressiveand anxiety-like behaviors. In addition, CSIS-induced increase in the number of CC3+ cells was detected in aforementioned subregions of mPFC. Treatments with either FLX or CLZ prevented behavioral changes, decrease in PV+ and increase in CC3+ cell numbers in PrL and IL subregions in isolated rats. These results indicate the importance of intact GABAergic signaling in these areas for resistance against CSIS-induced behavioral changes, as well as subregionspecific protective effects of FLX and CLZ in mPFC of CSIS rats.

Keywords Parvalbumin, medial prefrontal cortex, chronic social isolation, depression, fluoxetine, clozapine

2

Abbreviation ANOVA, analysis of variance; BA25, Brodmann Area 25; CC3+ cells, cleaved caspase-3 positive cells; Cg1, cingulate cortex; CLZ, clozapine; CSIS, chronic social isolation; DAPI, 4'-6diamidino-2-phenylindole; DP, dorsal peduncular cortex; FLX, fluoxetine; GABA, γaminobutyric acid; GSH, glutathione; HCl, hydrochloric acid; IL, infralimbic cortex; mPFC, medial prefrontal cortex; NaOH, sodium hydroxide; NGS, normal goat serum; PFA, paraformaldehyd; PrL, prelimbic cortex; PV+ interneurons, parvalbumin positive interneurons; TBS, tris-buffered saline; Veh, vehicle.

3

INTRODUCTION Depression is complex, multifactorial and heterogeneous disorder that imposes severe clinical, social and economic burden. GABA (γ-aminobutyric acid) hypothesis of depression proposes that GABAergic dysfunction is causally related to this disorder (Brambilla et al., 2003; Luscher et al., 2011). This hypothesis has increasing support in numerous preclinical and clinical studies which evidence that GABAergic deficit participate in the pathophysiology of depressive and anxiety disorders (Möhler, 2012). GABAergic inhibitory interneurons play a vital role in modulating cortical output and plasticity (Wonders and Anderson, 2006). They are widely known as locally projecting neurons, but long-range GABA-releasing neurons are found as well (Freund, 2003; Melzer et al., 2012; Basu et al., 2016). GABAergic interneurons are heterogeneous and may be classified upon the calcium-binding proteins, receptors, neuropeptides or transcription factors they express (Somogyi, 2010; Kelsom and Lu, 2013). Parvalbumin (PV) is the calcium-binding protein which regulate calcium signaling and homeostasis (Schwaller, 2010). PV positive (PV+) interneurons, which account for ~40% of the total cortical GABAergic interneurons in rodents (Rudy et al., 2011) include at least two distinct morphological subtypes: cortical basket cells and minor population of chandelier neurons (Markram et al., 2004). These neurons are fast-spiking interneurons involved in orchestrating cortical oscillations, maintaining the balance between excitation and inhibition, as well as mediating variety of behaviors (Bartos et al., 2007; Sohal et al., 2009). One PV+ interneuron forms synapses with great number of projecting neurons, so dysfunction of even small number of these interneurons may result with desynchronization of large cortical areas. Increasing body of data associate dysfunction of cortical PV+ interneurons with a variety of neurological and psychiatric diseases (Marín, 2012). Fluoxetine (FLX), a selective serotonin reuptake inhibitor, prolongs serotonergic signaling, but several lines of evidence suggest that antidepressant effects of this drug are partially mediated by enhancing GABA neurotransmission (Pehrson and Sanchez, 2015). Clozapine (CLZ), an atypical antipsychotic, is mainly used in the treatment of schizophrenia, but it may also be effective in treatment-resistant depressive disorders (Li et al., 2015). Recent study demonstrated that treatment with CLZ is associated with an increase in GABAB mediated inhibitory neurotransmission (Kaster et al., 2015). 4

Chronic social isolation (CSIS) is an animal model of depression that meets face, construct and predictive criteria for validity (Abelaira et al., 2013). CSIS is psychosocial stress that causes depressive- and anxiety-like behaviors in adult, male Wistar rats in as few as 21 days (Zlatković et al., 2014a) and is also widely used as a neurodevelopmental animal model of schizophrenia (Möller et al., 2013). Besides, CSIS was shown to compromise antioxidative defense and trigger proinflammatory signaling in the prefrontal cortex and hippocampus (Todorović and Filipović, 2017a, 2017b), as well as to reduce number of PV+ cells in hippocampal subregions (Filipović et al., 2013). In addition, we demonstrated that CSIS predisposed frontal cortex to caspase-3 activation (Filipović et al., 2011). Here we examined the effect of CSIS on the number of PV+ cells and cleaved caspase-3 positive (CC3+) cells in the medial prefrontal cortex (mPFC), mood regulating brain area highly implicated in depressive symptomatology (Riga et al., 2014). Given that mPFC subregions contribute differently to the development of psychiatric disorders and antidepressant response (Peters et al., 2009; Riga et al., 2014), we analyzed the changes within cingulate cortex, area 1 (Cg1), prelimbic (PrL), infralimbic (IL) area and dorsal peduncular cortex (DP) separately, as well as in all mPFC subregions combined. We found that CSIS decreased PV+ and increased CC3+ cells numbers in all analyzed subregions. Furthermore, we demonstrated that treatment with antidepressant FLX and antipsychotic CLZ, which paralleled stress exposure, prevented these changes in PrL and IL.

EXPERIMENTAL PROCEDURES Animals Thirty-six 2.5 months old male Wistar rats (300-350 g) were used in this study. They were kept under standard conditions (temperature 21-23 °C, 12h/12h light/dark cycle). Commercial rat pellets and water were available ad libitum. Ethical Committee for the Use of Laboratory Animals of the Institute of Nuclear Sciences “Vinča”, Belgrade, Serbia approved all experimental procedures (Application No. 02/11). Experiments were conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Animal suffering and the number of animals used were reduced to the minimum.

5

Preparation of drugs solutions Flunisan tablets containing 20 mg of fluoxetine-hydrochloride and Leponex tablets containing 25 mg of CLZ, as well as corresponding standards, were purchased from Hemofarm, Serbia and Novartis Pharmaceuticals UK, respectively. Flunisan and Leponex tablets were crushed and dissolved in distilled, sterile water and 1 N HCl, respectively, as described previously (Todorović and Filipović, 2017a). Solutions were filtered using Whatman No. 42 filter paper and concentrations of FLX and CLZ were determined using Ultra Performance Liquid Chromatography analysis (Kovacevic et al., 2006). Based on drugs concentrations and weekly measured body weights, the volumes of drugs solutions needed to be administered to each animal to achieve target doses were calculated.

Study design The animals were divided into 6 experimental groups (Table 1). Non-stressed rats were housed in standard conditions (4 animals/cage), while CSIS rats were housed individually for 21 days, without visual and tactile contacts with other animals. FLX and CLZ were administered to nonstressed (No stress + FLX and No stress + CLZ) and stressed (CSIS + FLX and CSIS + CLZ) rats, daily by intraperitoneal (i.p.) injections, for 21 days. Drug treatments were applied simultaneously with the stress exposure. Injection site varied between right and left side of the rat, to minimize the tissue damage. Doses of FLX (15 mg/kg/day) and CLZ (20 mg/kg/day), chosen to emulate the therapeutic doses given to patients, were determined based on the literature data (Halim et al., 2004; Czeh et al., 2006; Larsson et al., 2015). This treatment regimens were shown to produce serum drug concentrations that correspond to the concentrations measured in patients who received therapeutically effective doses (Zlatković et al., 2014b).

Behavioral assessments Sucrose preference test and marble burying test were used to assess anhedonia- and anxiety-like behavior, respectively. Behavioral tests were conducted prior experimental protocol (one day prior CSIS started, 0d), and at the end of the experiment (on the 21st day of treatment, 21d). Both

6

tests were performed on the same day, whereby marble burying test followed the sucrose preference test. Animals were individually housed during behavioral testing. Sucrose preference test. The rat’s appetite for a palatable substance is widely used to estimate anhedonia-like behavior (Willner et al., 1987). Two bottles, one containing tap water, and the other 1% sucrose solution were placed in each cage for 1 h. Sucrose preference was calculated using the formula Sucrose preference = [consumed sucrose solution/total liquid consumed (sucrose solution + water)] × 100.

Marble burying test. The rats were tested for anxiety-like behavior by marble burying test. Burying behavior is rodent specific defense reaction to foreign object in the cage which is suppressed by anxiolytic drugs, and therefore is indicative of anxiety (Ho et al., 2002; Farley et al., 2010). Six marbles made of glass (2.5 cm in diameter) were placed evenly across clean sawdust bedding in the cage. After 30 min, the buried marbles (at least two-thirds of the surface covered with bedding), were counted blindly to group conditions. The results are presented as a mean number of buried marbles ± SEM.

Tissue processing and staining Rats were sacrificed 24 h after the last drug administration. They were anesthetized with ketamine/xylazine (100/5 mg/kg i.p.), transcardially perfusied with physiological saline followed by 50 ml of 4% paraformaldehyde (PFA, pH 7.4) (Sigma–Aldrich, St Louis, MO), and decapitated by guillotine (Harvard Apparatus, South Natick, MA, USA). The brains were isolated and post-fixed in 4% PFA overnight at 4°C. Serial coronal 40-µm sections were cut on a vibratome (VT 100 S; Leica, Bensheim, Germany) and stored in cryoprotectant at -20°C until staining. Staining procedures were performed on the free-floating brain sections. Every sixth section (at least 3 sections per rat, bregma 3 – 3.72) was stained against PV by immunofluorescence, and cleaved caspase-3 (CC3) by 3, 3’-diaminobenzidine (DAB). Samples from each experimental group were processed in parallel to avoid any non-specific effect of the staining procedure. Sections were washed in TBS containing 0.05% Triton X-100 (TBS-T), pH 7

7.4., preincubated in blocking solution containing 3% normal goat serum (NGS; Vector Laboratories, Burlingame, USA) and 0.3% Triton X-100 in TBS for 1 h at room temperature. Afterwards, the sections were incubated overnight at 4°C with primary antibody (anti-PV, polyclonal rabbit, SWANT #PV 25, 1:1000), prepared in blocking solution (3% NGS). After washing in TBS-T, the sections were incubated with Alexa-Fluor 555-conjugated goat anti-rabbit secondary antibodies (1:1000) (Molecular Probes, Invitrogen, Eugene, USA) in TBS containing 3% NGS for 2 h at room temperature, in dark. After three washes in TBS, sections were mounted on Superfrost Plus microscope slides (Thermo Scientific, USA), dried and coverslipped with fluorescent mounting medium (Dako, Agilent Technologies, Denmark) containing DAPI (4'-6diamidino-2-phenylindole, 10 l/ml) for nuclei labeling. DAPI staining was used to confirm the cellular nature of the signal obtained after PV labeling (Fig. 1). A negative control (secondary antibody control), performed on the brain sections as described above, with primary antibody omitted, was run in parallel. Images were captured using widefield epifluorescence – microscope Leica DMI6000B, Digital Camera DFC and Leica Microsystems LAS AF-TCS SP8 software (Leica Microsystems). Objective used in this study was HC PL FLUOTAR 10x0.30 DRY objective; resolution 1392x1040 pixels. DAB staining against CC3 was performed as follows. Sections were rinsed in PBS containing 0.05% Triton X-100 (PBS-T), pH 7.4 and incubated in 0.6% H2O2 (Sigma–Aldrich) in PBS-T for 30 min at RT, to inhibit endogenous peroxidase. After washing and blocking with 3% NGS in PBS containing 0.3% TritonX-100 pH 7.4 for 1 h, sections were incubated with the primary antibodies (anti-cleaved caspase-3, polyclonal rabbit, Cell Signaling Technology #9661, USA, 1:500, prepared in blocking solution – 3% NGS), overnight at 4°C. On the second day, the sections were washed and incubated with goat anti-rabbit biotinylated antibodies (Jackson ImmunoResearch,UK, 1:500) for 40 min at RT, followed by incubation with ABC reagents for 20 min at RT (ABC kit, Vector Laboratories, USA). Color was developed with DAB reagent. The sections were rinsed in tap water, mounted on Superfrost Plus microscope slides, dried and coverslipped with Eukitt (Sigma Aldrich, USA). Images were captured using a BTC microscope (objective 10x) equipped with BIM 3135 digital camera. Dark-stained cell bodies were counted.

8

Quantification of labeled cells Images were taken along the x and y axes of the sections (around 40 images per section). Images acquired were stitched digitally using Image Composite Editor Software (Microsoft Research, USA). The appropriate image of the rat brain atlas (Paxinos and Watson, 2005) was digitally overlaid to delineate the brain section into regions in Adobe Photoshop (version CS3) (Adobe, USA) (Fig. 2). PV+ and CC3+ cells were counted in the following subregions: Cg1, PrL, IL and DP of mPFC. The number of clearly defined PV+ or CC3+ cells, in all examined subregions, were obtained using Fiji’s (ImageJ) manual cell counting tool. Counting, with the experimenter blind to the group conditions, was done in duplicate, in both cerebral hemispheres. Values obtained from both counting in both hemispheres were averaged and used for the statistical analysis.

Statistical Analysis The Shapiro-Wilk test was used to check for normal distribution and Levene’s test to check for homogeneity of variances. When both conditions were confirmed, a two-way repeated measures analysis of variance (ANOVA) (STATISTICA Release 7) was used to determine the significant changes in investigated behaviors [the factors were drug treatment (levels: Veh, FLX and CLZ), stress (levels: No stress and CSIS) and within-subject factor time (levels: 0d and 21d), followed by a Duncan’s post-hoc test for SP and MB test (6 animals per condition). The changes in the number of PV+ and CC3+ cells were analyzed using two-way ANOVA, [the factors were drug treatment (levels: Veh, FLX and CLZ) and stress (levels: no stress and CSIS). Duncan’s post-hoc test was used to evaluate differences between groups. Statistical significance was set at p < 0.05. The results are presented as mean ± SEM of 5 – 6 rats per condition.

RESULTS The effects of CSIS and/or FLX or CLZ treatment on behavior A two-way repeated measures ANOVA revealed a significant main effect of CSIS x drug interaction (F2.30 = 3.80, p < 0.05), as well as a significant main effects of time x drug (F2.30 = 9.25, p < 0.001) and time x CSIS x drug interaction (F2.30 = 3.95, p < 0.05) on the sucrose preference. Post-hoc tests showed significant decrease in sucrose preference after 21d compared 9

to baseline value (0d) in Veh-treated CSIS rats (p < 0.001) (Fig. 3A) (6 animals per condition). Besides, significantly higher sucrose preference was noted in FLX- and CLZ-treated CSIS rats compared to Veh-treated CSIS rats at the conclusion of the experiment (21d) (p < 0.001, p < 0.01, respectively). A two-way repeated measures ANOVA revealed a significant main effect of CSIS (F1.30 = 6.49, p < 0.05) and CSIS x drug (F2.30 = 4.97, p < 0.05), as well as a significant main effect of time (F1.30 = 7.61, p < 0.01) and time x CSIS interaction (F1.30 = 6.49, p < 0.05) on the marble burying. Significant increase in the number of buried marbles was noted after 21d of CSIS (p < 0.001) (Fig. 3B) (6 animals per condition). At the conclusion of the experiment (21d), significantly decreased number of buried marbles was seen in CSIS rats treated with FLX or CLZ compared to Veh-treated CSIS rats (p < 0.001, p < 0.01, respectively). Drugs did not cause significant behavioral changes in non-stressed rats (sucrose preference: pFLX = 0.47, pCLZ = 0.81; marble burying pFLX = 0.79, pCLZ = 0.82).

The effects of CSIS and/or FLX or CLZ treatment on the number of PV+ cells in the mPFC subregions A two-way ANOVA revealed a significant main effect of CSIS (F1.27 = 35.36, p < 0.001) on the number of PV+ cells in Cg1 subregion. Post-hoc test showed significant decrease in the number of PV+ cells in CSIS rats treated with Veh, FLX or CLZ, compared to Veh-treated non-stressed rats (p < 0.001, p < 0.01) (Fig. 4A) (5 – 6 animals per condition: No Stress + Veh – 5; No stress + FLX – 6; No stress + CLZ – 5; CSIS + Veh – 6; CSIS + FLX – 6; CSIS + CLZ – 5). In addition, a significant decrease was seen in CLZ-treated CSIS compared to CLZ-treated nonstressed rats (p = 0.001). With regard to PrL, a significant main effect of CSIS (F1.29 = 8.07; p < 0.01) and CSIS × drug interaction (F2.29 = 4.75; p < 0.05) were found. Post-hoc test revealed significantly decreased number of PV+ cells in Veh-treated CSIS compared to Veh-treated nonstressed animals (p < 0.001) (Fig. 4B) (5 – 6 animals per condition: No Stress + Veh – 6; No stress + FLX – 6; No stress + CLZ – 6; CSIS + Veh – 6; CSIS + FLX – 5; CSIS + CLZ – 6), as well as significantly increased number of these cells in FLX- and CLZ-treated CSIS compared to Veh-treated CSIS rats (p < 0.01). In IL subregion, a two-way ANOVA revealed a significant main effect of CSIS (F1.27 = 5.02; p < 0.05), drug (F2.27 = 10.51; p < 0.001) and CSIS × drug 10

interaction (F2.27 = 7.17; p < 0.01) on the number of PV+ cells. Post-hoc test showed significant decrease in the number of these cells in Veh-treated CSIS compared to Veh-treated non-stressed rats (p < 0,001) (Fig. 4C) (5 – 6 animals per condition: No Stress + Veh – 6; No stress + FLX – 5; No stress + CLZ – 6; CSIS + Veh – 5; CSIS + FLX – 5; CSIS + CLZ – 6), as well as significant increase in FLX- and CLZ-treated CSIS compared to Veh-treated CSIS rats (p ≤ 0,001). Finally, a two-way ANOVA revealed a significant main effect of CSIS (F1.26 = 9.53; p < 0.01) and drug (F2.26 = 5.59; p < 0.01) on the number of PV+ cells in DP subregion. Again, posthoc test showed significantly decreased number of examined cells in Veh-treated CSIS compared to Veh-treated non-stressed animals (p < 0.05) (Fig. 4D) (5 – 6 animals per condition: No Stress + Veh – 5; No stress + FLX – 5; No stress + CLZ – 6; CSIS + Veh – 5; CSIS + FLX – 5; CSIS + CLZ – 6). Besides, a significant decrease was seen in CLZ-treated CSIS compared to CLZtreated non-stressed rats (p = 0.01). Drugs did not cause significant changes in the number of PV+ cells in any examined area when applied to non-stressed rats (Cg1: pFLX = 0.16, pCLZ = 0.55; PrL: pFLX = 0.64, pCLZ = 0.32; IL: pFLX = 0.14; pCLZ = 0.19; DP: pFLX = 0.13; pCLZ = 0.07). A two-way ANOVA revealed a significant main effect of CSIS (F1.26 = 16.71, p < 0.001) and CSIS × drug interaction (F2.26 = 6.68; p < 0.01) on the number of PV+ cells in all tested subregions combined. Post-hoc test showed significant decrease in the number of PV+ cells in CSIS rats treated with Veh and FLX compared to Veh-treated non-stressed rats (p < 0.001, p < 0.05, respectively) (Fig. 5) (5 – 6 animals per condition: No Stress + Veh – 5; No stress + FLX – 5; No stress + CLZ – 6; CSIS + Veh – 6; CSIS + FLX – 5; CSIS + CLZ – 5), as well as significantly increased number of these cells in FLX- and CLZ-treated CSIS compared to Vehtreated CSIS rats (p < 0.01) . Drugs did not cause significant changes in the number of PV+ cells in non-stressed rats (pFLX = 0.18, pCLZ = 0.37). The effects of CSIS and/or FLX or CLZ treatment on the number of CC3+ cells in the mPFC subregions A two-way ANOVA revealed a significant main effect of CSIS on the number of CC3+ cells in Cg1 subregion of mPFC (F1.28 = 32.63, p < 0.001). Significantly increased number of these cells was noticed in Cg1 subregion of CSIS rats treated with Veh, FLX or CLZ (p < 0.001) compared to Veh-treated non-stressed rats (Fig. 6A) (5 – 6 animals per condition: No Stress + Veh – 6; No stress + FLX – 5; No stress + CLZ – 6; CSIS + Veh – 5; CSIS + FLX – 6; CSIS + CLZ – 6). A 11

significant increase was also seen in FLX-treated CSIS compared to FLX-treated non-stressed rats (p < 0.01). With regard to PrL, significant main effects of CSIS (F1.28 = 11.86, p < 0.01) and CSIS × drug interaction (F2.28 = 4.11; p < 0.05) on the number of CC3+ cells were found. Posthoc test showed a significant increase in the number of these cells in Veh-treated CSIS compared to Veh-treated non-stressed animals (p < 0.001), as well as a significant decrease in FLX- and CLZ-treated compared to Veh-treated CSIS rats (p < 0.05, p < 0.01, respectively) (Fig. 6B) (5 – 6 animals per condition: No Stress + Veh – 5; No stress + FLX – 5; No stress + CLZ – 6; CSIS + Veh – 6; CSIS + FLX – 6; CSIS + CLZ – 6). With regard to IL, significant main effects of CSIS (F1.27 = 23.95; p < 0.001), drug (F2.27 = 7.92; p < 0.01) and CSIS × drug interaction (F2.27 = 15.83; p < 0.001) on the number of CC3+ cells were found. The number of these cells was significantly increased in Veh-treated CSIS compared to Veh-treated non-stressed rats (p < 0.001), as well as significantly decreased in FLX- and CLZ-treated CSIS compared to Vehtreated CSIS rats (p < 0.001) (Fig. 6C) (5 – 6 animals per condition: No Stress + Veh – 6; No stress + FLX – 5; No stress + CLZ – 5; CSIS + Veh – 6; CSIS + FLX – 5; CSIS + CLZ – 6). A significant increase was observed in FLX-treated CSIS compared to FLX-treated non-stressed rats (p < 0.05). Finally, a two-way ANOVA revealed a significant main effect of CSIS (F1.29 = 7.12; p = 0.01) on the number of CC3+ cells in DP subregion. Post-hoc test showed significantly increased number of examined cells in Veh-treated CSIS compared to Veh-treated non-stressed animals (p = 0.01) (Fig. 6D) (5 – 6 animals per condition: No Stress + Veh – 6; No stress + FLX – 6; No stress + CLZ – 6; CSIS + Veh – 6; CSIS + FLX – 5; CSIS + CLZ – 6). Drugs did not significantly affect the number of CC3+ cells when applied to non-stressed rats (Cg1: pFLX = 0.37; PrL: pFLX = 0.82, pCLZ = 0.54; IL: pFLX = 0.86; pCLZ = 0.15; DP: pFLX = 0.86; pCLZ = 0.16), except CLZ which significantly increased their number in Cg1 (p < 0.05). In addition, a two-way ANOVA revealed a significant main effect of CSIS (F1.25 = 31.93, p < 0.001) and CSIS × drug interaction (F2.25 = 3.77; p < 0.05) on the number of CC3+ cells in all tested subregions combined. Significantly increased number of these cells was found in CSIS rats treated with Veh, FLX and CLZ compared to Veh-treated non-stressed rats (p < 0.001, p < 0.05) (Fig. 7) (5 – 6 animals per condition: No Stress + Veh – 5; No stress + FLX – 5; No stress + CLZ – 5; CSIS + Veh – 5; CSIS + FLX – 5; CSIS + CLZ – 6). Post-hoc test also revealed a significant decrease in FLX- and CLZ-treated CSIS compared to Veh-treated CSIS rats (p < 12

0.05), as well as a significant increase in FLX-treated CSIS compared to FLX-treated nonstressed rats (p < 0.01) in the number of examined cells. Drugs did not cause significant changes in the number of CC3+ cells in non-stressed rats (pFLX = 0.41, pCLZ = 0.24). DISCUSSION A growing body of evidence implicates dysregulation of GABAergic system in the pathophysiology of depression (Kendell et al., 2005; Varga et al., 2017). Selective loss of GABAergic interneurons in certain cortical areas (Rajkowska et al., 2007; Maciag et al., 2010), as well as deficits in GABA synthesis (Thompson et al., 2009) were found in individuals with mood disorders, including depression. In addition, it was noted that more marked GABAergic deficits were associated with more severe symptoms and resistance to treatment (Levinson et al., 2010). In line with previous knowledge, results of present study showed that CSIS, an animal model of depression and schizophrenia, resulted to a decrease in PV+ and increase in CC3+ cells numbers in subregions of mPFC, parallel with depressive- and anxiety-like behaviors. Treatment with antidepressant FLX or antipsychotic CLZ, applied in doses equivalent to clinically effective ones, prevented behavioral changes, reduction of PV+ and increase of CC3+ cells numbers in PrL and IL subregions of CSIS rats. CSIS caused anhedonia, decreased ability to experience pleasure, reflected in decreased sucrose preference. Furthermore, CSIS resulted in anxiety-like behavior manifested by increased burying behavior. This is consistent with our previous results revealing that CSIS induces anhedonia- and anxiety-like, as well as despair-like behavior in the forced swim test (Zlatković et al., 2014a). FLX and CLZ prevented behavioral changes in CSIS rats, as expected based on our previous results (Todorović and Filipović, 2017a) and literature data (Bruins Slot et al., 2008; Brenes and Fornaguera, 2009; Rong et al., 2010; Vardigan et al., 2010; Chatterjee et al., 2012; Yang et al., 2014; Chen et al., 2015). With regard to PV + cells number, the greatest change was observed in IL subregion, where CSIS reduced the number of PV+ cells by 33%, compared to basal value. Interestingly, in this subregion, the highest increase of CC3+ cells number (+43%) was noted. This subregion, the rodent correlate of human Brodmann Area 25 (BA25), projects to numerous brain regions involved in visceral and neuroendocrine stress control such as central nucleus of amygdala (Mcklveen et al., 2015). Increase in metabolic activity of BA25, reversible by various 13

antidepressants, was noted in patients with depression (Hamani et al., 2011). Further, deep brain stimulation of BA25 normalized metabolic hyperactivity in this region in treatment-resistant depressed patients and ameliorated depressive symptoms. The underlying mechanisms are incompletely understood, but activation of inhibitory GABAergic afferents in BA25 likely mediate that action (Mayberg et al., 2005). In addition, an animal study revealed that pharmacological inactivation of the IL cortex showed an antidepressant-like effect in the forced swim test, assessed by decreased immobility behavior (Slattery et al., 2011). Thus, IL subregion has important role in the pathophysiology of depression in humans, and depressive-like states in animal models. In line with this, results of present study showed that this subregion was most sensitive to changes in the number of PV+ cell due to CSIS. It can be assumed that decrease in the number of functional PV+ inhibitory interneurons may, at least in part, be responsible for the hyperactivity seen in this brain area in depression. Decreased number of PV+ cells was also noted in PrL subregion of CSIS rats. PrL projects to brain areas involved in limbic and cognitive functions like dorsal striatum and basolateral amygdala. Besides, it projects to nucleus accumbens which is highly implicated in the pathophysiology of depression (Mcklveen et al., 2015). Stern et al. (2010) showed that inactivation of the PrL cortex during elevated plus-maze testing increased the exploration of open-arms, substantiating its role in anxiety regulation. Decreased number of PV+ cells in this subregion coincided with anxiety-like behavior noted in CSIS rats in marble burying test. Reduced number of PV+ cells may have resulted with increased PrL activity which contributed to anxiety-like behavior observed in CSIS rats. The finding that pharmacological activation of the PrL induced anxiety-like behaviors in mice, corroborate this assumption (Saitoh et al., 2014). Most studies regarding the effect of chronic stress on behavior, as well as the pathophysiology of depression in general, are focused on IL and PrL subregions, while the importance of Cg1 and DP is poorly understood. Results of this study showed that these two subregions were also sensitive to psychosocial stress, since CSIS decreased the number of PV+, and increased the number of CC3+ cells in them as well. Interestingly, 15 weeks of post-weaning isolation did not cause significant changes in the number of PV+ cells, nor in total number of neurons and glial cells (Kaalund et al., 2013). These inconsistencies indicate that duration of

14

isolation, as well as the age of isolated rats represent important determinantes of the stress effects. The involvement of mPFC PV+ interneurons in the development of adaptive and maladaptive behavior, as well as in the pathogenesis of mood disorders was pointed out earlier. Donato et al. (2013) reported that alterations in the differentiation state of hippocampal PV+ GABAergic interneurons are involved in experience-related plasticity in the adult mice. Recently was shown that selective suppression of PV+ interneurons in the mPFC promoted helplessness in mice, behavioral state that resemble features of human depression (Perova et al., 2015). The reduced numbers of PV+ cells in mPFC subregions in CSIS rats that display depressive- and anxiety-like behaviors, found in present study, complement these previous findings. Reduced PV-immunoreactivity following CSIS is probably a consequence of increased apoptosis, judged by the elevated number of CC3+ cells in all examined brain subregions of mPFC. In line with this, mitochondria-related proapoptotic signaling was shown previously in the PFC of CSIS rats (Filipović et al., 2011). It should be noted that increased CC3immunoreactivity may be a consequence of cell death that affects not only PV+ cells, but also other cell types in mPFC. Thus, further immunohistochemical studies are needed to confirm effects specific for PV+ cells. Low intracellular calcium buffering capacity due to low PV results with calcium overload that is particularly neurotoxic and leads to the activation of enzymes that degrade proteins, membranes and nucleic acids causing cell death and tissue damage (Berliocchi et al., 2005). One of the factors that could contribute to the PV+ reduction observed in this study is redox dysregulation. PV+ interneurons are especially vulnerable to oxidative stress due to fastspiking nature and high metabolic requirements that result in increased production of ATP and reactive oxygen species in mitochondria (Gulyás et al., 2006). Thus, fast-spiking neurons need potent antioxidation mechanisms to counterbalance increased production of pro-oxidants. Steullet et al. (2010) reported that genetically compromised glutathione (GSH) synthesis affects the morphological and functional integrity of PV+ interneurons, indeed in hippocampus. We previously found that CSIS compromised prefrontal cortical GSH-dependent defense and promoted susceptibility to oxidative stress (Todorović and Filipović, 2017a). These disturbances in antioxidant defense may contribute to changes in PV-immunoreactivity observed in this study. 15

FLX treatment prevented CSIS-induced decrease in the number of PV+ cells, as well as increase in the number of CC3+ cells in PrL and IL, but not in Cg1 and DP subregions. Protective effect of this antidepressant on PV+ interneurons was shown previously in the hippocampus (Czeh et al., 2005; Godavarthi et al., 2014; Filipović et al., 2018), while in this study, for the first time is demonstrated its subregion-specific protective effect in mPFC. This brain region, particularly cingulate, prelimbic and infralimbic areas, are richly innervated by serotonergic neurons of the dorsal and median raphe nuclei (Puig and Gulledge, 2011). Both pyramidal neurons and interneurons in rat prefrontal cortical areas express 5-HT1A and 5-HT2A receptors (Santana et al., 2004). FLX was found to induce a concentration-dependent increase in the excitability of PV+ fast-spiking interneurons, but to have little effect on pyramidal neurons in rat PFC (Zhong and Yan, 2011). These findings suggest that fast-spiking interneurons are the major target of FLX. CLZ also showed protective effects in PrL and IL by preventing decrease in PV+, and increase in CC3+ cells number in CSIS rats. There are only scarce literature data on the effects of this antipsychotic on PV+ interneurons. Cahir et al. (2005) showed that 3 weeks of CLZ treatment (25 mg/kg/dan) did not affect the number of PV+ cells in PFC and hippocampus in control, non-stressed rats. Accordingly, results of this study showed no significant difference in the number of PV+ cells in any of examined subregions in CLZ-treated non-stressed, compared to Veh-treated non-stressed rats. Interestingly, results of this study showed that CLZ caused an increase in the number of CC3+ cells in Cg1 subregion of non-stressed rats. In vitro study showed that CLZ is capable of inducing apoptosis in granulocytes (Husain et al., 2006). Given that CLZ did not affect the number of PV+ cells in Cg1, examined antipsychotic exerted apoptotic effect on some other cells in this subregion. Protective effects of FLX and CLZ on the number of PV+ cells in PrL, which parallel their anxiolytic effects, complement the literature data concerning implication of this mPFC subregion in anxiety regulation, which was discussed earlier. In conclusion, results of this study indicate that impaired cortical inhibition in mPFC, as assessed by decreased number of PV+ cell, may contribute to behavioral changes seen in CSIS rats. Besides, they showed that FLX and CLZ were protective in subregion-specific manner. Both FLX and CLZ prevented reduction of PV+ cells number in PrL and IL subregions in CSIS 16

rats, as well as behavioral changes related to anhedonia and anxiety. These results indicate that intact GABAergic signaling in these brain areas is important for resistance against CSIS-induced changes in behavior. Acknowledgment Work of D. Filipović, N. Todorović and B. Mićić was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia [grant number 173044]. Work of M. Stevanović and M. Schwirtlich was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia (grant number 173051). D.F. designed the study, supervised the work and reviewed the manuscript. N.T. and B.M. carried out the experiments. N.T. analyzed the data and drafted the manuscript. M. Stevanović and M. Schwirtlich performed microscopy and reviewed the manuscript. All authors contributed to and have approved the final manuscript.

Declarations of interest: none

17

References: Abelaira HM, Réus GZ, Quevedo J (2013) Animal models as tools to study the pathophysiology of depression. ev Bras Psi uiatr (S o Paulo, Brazil 1999) 35 Suppl 2:S112-20. Bartos M, Vida I, Jonas P (2007) Synaptic mechanisms of synchronized gamma oscillations in inhibitory interneuron networks. Nat Rev Neurosci 8:45–56. Basu J, Zaremba JD, Cheung SK, Hitti FL, Zemelman B V, Losonczy A, Siegelbaum SA (2016) Gating of Hippocampal Activity, Plasticity and Memory by Entorhinal Cortex Long-Range Inhibition. Science (80- ) 351:6269. Berliocchi L, Bano D, Nicotera P (2005) Ca2+ signals and death programmes in neurons. Philos Trans R Soc Lond B Biol Sci 360:2255–2258. Brambilla P, Perez J, Barale F, Schettini G, Soares JC (2003) GABAergic dysfunction in mood disorders. Mol Psychiatry 8:721–737. Brenes JC, Fornaguera J (2009) The effect of chronic fluoxetine on social isolation-induced changes on sucrose consumption, immobility behavior, and on serotonin and dopamine function in hippocampus and ventral striatum. Behav Brain Res 198:199–205. Bruins Slot LA, Bardin L, Auclair AL, Depoortere R, Newman-Tancredi A (2008) Effects of antipsychotics and reference monoaminergic ligands on marble burying behavior in mice. Behav Pharmacol 19:145–152. Cahir M, Costello I, King DJ, Reynolds GP (2005) Chronic haloperidol or clozapine treatment does not alter parvalbumin immunoreactivity in the rat frontal cortex or hippocampus. Neurosci Lett 373:57–60. Chatterjee M, Jaiswal M, Palit G (2012) Comparative evaluation of forced swim test and tail suspension test as models of negative symptom of schizophrenia in rodents. ISRN Psychiatry 2012:595141. Chen C, Wang L, Rong X, Wang W, Wang X (2015) Effects of fluoxetine on protein expression of potassium ion channels in the brain of chronic mild stress rats. Acta Pharm Sin B 5:55– 61. Czeh B, Simon M, Schmelting B, Hiemke C, Fuchs E (2006) Astroglial plasticity in the hippocampus is affected by chronic psychosocial stress and concomitant fluoxetine treatment. Neuropsychopharmacology 31:1616–1626. Czeh B, Simon M, van der Hart MG, Schmelting B, Hesselink MB, Fuchs E (2005) Chronic stress decreases the number of parvalbumin-immunoreactive interneurons in the hippocampus: prevention by treatment with a substance P receptor (NK1) antagonist. Neuropsychopharmacology 30:67–79. Donato F, Rompani SB, Caroni P (2013) Parvalbumin-expressing basket-cell network plasticity induced by experience regulates adult learning. Nature 504:272–276. Farley S, Apazoglou K, Witkin JM, Giros B, Tzavara ET (2010) Antidepressant-like effects of an AMPA receptor potentiator under a chronic mild stress paradigm. Int J Neuropsychopharmacol 13:1207–1218. 18

Filipović D, Stanisavljević A, Jasnić N, Bernardi E, Inta D, Perić I, Gass P (2018) Chronic Treatment with Fluoxetine or Clozapine of Socially Isolated Rats Prevents SubsectorSpecific Reduction of Parvalbumin Immunoreactive Cells in the Hippocampus. Neuroscience 371:384–394. Filipović D, Zlatković J, Gass P, Inta D (2013) The differential effects of acute vs. chronic stress and their combination on hippocampal parvalbumin and inducible heat shock protein 70 expression. Neuroscience 236:47–54. Filipović D, Zlatković J, Inta D, Bjelobaba I, Stojiljkovic M, Gass P (2011) Chronic isolation stress predisposes the frontal cortex but not the hippocampus to the potentially detrimental release of cytochrome c from mitochondria and the activation of caspase-3. J Neurosci Res 89:1461–1470. Freund TF (2003) Interneuron Diversity series: Rhythm and mood in perisomatic inhibition. Trends Neurosci 26:489–495. Godavarthi SK, Sharma A, Jana NR (2014) Reversal of reduced parvalbumin neurons in hippocampus and amygdala of Angelman syndrome model mice by chronic treatment of fluoxetine. J Neurochem 130:444–454. Gulyás AI, Buzsáki G, Freund TF, Hirase H (2006) Populations of hippocampal inhibitory neurons express different levels of cytochrome c. Eur J Neurosci 23:2581–2594. Halim ND, Weickert CS, McClintock BW, Weinberger DR, Lipska BK (2004) Effects of chronic haloperidol and clozapine treatment on neurogenesis in the adult rat hippocampus. Neuropsychopharmacology 29:1063–1069. Hamani C, Mayberg H, Stone S, Laxton A, Haber S, Lozano AM (2011) The subcallosal cingulate gyrus in the context of major depression. Biol Psychiatry 69:301–308. Ho Y-J, Eichendorff J, Schwarting RKW (2002) Individual response profiles of male Wistar rats in animal models for anxiety and depression. Behav Brain Res 136:1–12. Husain Z, Almeciga I, Delgado JC, Clavijo OP, Castro JE, Belalcazar V, Pinto C, Zuñiga J, Romero V, Yunis EJ (2006) Increased FasL expression correlates with apoptotic changes in granulocytes cultured with oxidized clozapine. Toxicol Appl Pharmacol 214:326–334. Isaacson JS, Scanziani M (2011) How inhibition shapes cortical activity. Neuron 72:231–243. Kaalund SS, Riise J, Broberg B V., Fabricius K, Karlsen AS, Secher T, Plath N, Pakkenberg B (2013) Differential expression of parvalbumin in neonatal phencyclidine-treated rats and socially isolated rats. J Neurochem 124:548–557. Kaster TS, de Jesus D, Radhu N, Farzan F, Blumberger DM, Rajji TK, Fitzgerald PB, Daskalakis ZJ (2015) Clozapine potentiation of GABA mediated cortical inhibition in treatment resistant schizophrenia. Schizophr Res 165:157–162. Kelsom C, Lu W (2013) Development and specification of GABAergic cortical interneurons. Cell Biosci 3:19. Kendell SF, Krystal JH, Sanacora G (2005) GABA and glutamate systems as therapeutic targets in depression and mood disorders. Expert Opin Ther Targets 9:153–168. Kovacevic I, Pokrajac M, Miljkovic B, Jovanovic D, Prostran M (2006) Comparison of liquid 19

chromatography with fluorescence detection to liquid chromatography-mass spectrometry for the determination of fluoxetine and norfluoxetine in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 830:372–376. Larsson MK, Schwieler L, Goiny M, Erhardt S, Engberg G (2015) Chronic Antipsychotic Treatment in the Rat – Effects on Brain Interleukin-8 and Kynurenic Acid. Int J Tryptophan Res 8:49–52. Levinson AJ, Fitzgerald PB, Favalli G, Blumberger DM, Daigle M, Daskalakis ZJ (2010) Evidence of Cortical Inhibitory Deficits in Major Depressive Disorder. Biol Psychiatry 67:458–464. Li X-B, Tang Y-L, Wang C-Y, de Leon J (2015) Clozapine for treatment-resistant bipolar disorder: a systematic review. Bipolar Disord 17:235–247. Luscher B, Shen Q, Sahir N (2011) The GABAergic deficit hypothesis of major depressive disorder. Mol Psychiatry 16:383–406. Maciag D, Hughes J, O’Dwyer G, Pride Y, Stockmeier C, Sanacora G, ajkowska G (2010) Reduced Density of Calbindin Immunoreactive GABAergic Neurons in the Occipital Cortex in Major Depression: Relevance to Neuroimaging Studies. 67:465–470. Marín O (2012) Interneuron dysfunction in psychiatric disorders. Nat Rev Neurosci:107–120. Markram H, Toledo-Rodriguez M, Wang Y, Gupta A, Silberberg G, Wu C (2004) Interneurons of the neocortical inhibitory system. Nat Rev Neurosci 5:793–807. Mayberg HS, Lozano AM, Voon V, McNeely HE, Seminowicz D, Hamani C, Schwalb JM, Kennedy SH (2005) Deep brain stimulation for treatment-resistant depression. Neuron 45:651–660. Mcklveen JM, Myers B, Herman JP (2015) The Medial Prefrontal Cortex: Coordinator of Autonomic, Neuroendocrine, and Behavioral Responses to Stress. J Neuroendocr 27:446– 456. Melzer S, Michael M, Caputi A, Eliava M, Fuchs EC, Whittington MA, Monyer H (2012) Longrange-projecting GABAergic neurons modulate inhibition in hippocampus and entorhinal cortex. Science 335:1506–1510. Möhler H (2012) The GABA system in anxiety and depression and its therapeutic potential. Neuropharmacology 62:42–53. Möller M, Du Preez JL, Viljoen FP, Berk M, Emsley R, Harvey BH (2013) Social isolation rearing induces mitochondrial, immunological, neurochemical and behavioural deficits in rats, and is reversed by clozapine or N-acetyl cysteine. Brain Behav Immun 30:156–167. Paxinos G, Watson C (2005) The rat brain in stereotaxic coordinates. J Chem Inf Model. Pehrson A, Sanchez C (2015) Altered γ-aminobutyric acid neurotransmission in major depressive disorder: a critical review of the supporting evidence and the influence of serotonergic antidepressants. Drug Des Devel Ther 2015:603–624. Perova Z, Delevich K, Li B (2015) Depression of Excitatory Synapses onto Parvalbumin Interneurons in the Medial Prefrontal Cortex in Susceptibility to Stress. J Neurosci 35:3201–3206. 20

Peters J, Kalivas PW, Quirk GJ (2009) Extinction circuits for fear and addiction overlap in prefrontal cortex. Learn Mem 16:279–288. Puig MV, Gulledge AT (2011) Serotonin and Prefrontal Cortex Function: Neurons, Networks, and Circuits. Mol Neurobiol 44:449–464. ajkowska G, O’Dwyer G, Teleki Z, Stockmeier CA, Miguel-Hidalgo JJ (2007) GABAergic Neurons Immunoreactive for Calcium Binding Proteins are Reduced in the Prefrontal Cortex in Major Depression. Neuropsychopharmacology 32:471–482. Riga D, Matos MR, Glas A, Smit AB, Spijker S, Oever MC Van Den (2014) Optogenetic dissection of medial prefrontal cortex circuitry. 8:1–19. Rong H, Wang G, Liu T, Wang H, Wan Q, Weng S (2010) Chronic mild stress induces fluoxetine-reversible decreases in hippocampal and cerebrospinal fluid levels of the neurotrophic factor S100B and its specific receptor. Int J Mol Sci 11:5310–5322. Rudy B, Fishell G, Lee S, Hjerling-Leffler J (2011) Three groups of interneurons account for nearly 100% of neocortical GABAergic neurons. Dev Neurobiol 71:45–61. Saitoh A, Ohashi M, Suzuki S, Tsukagoshi M, Sugiyama A, Yamada M, Oka JI, Inagaki M, Yamada M (2014) Activation of the prelimbic medial prefrontal cortex induces anxiety-like behaviors via N-Methyl-D-aspartate receptor-mediated glutamatergic neurotransmission in mice. J Neurosci Res 92:1044–1053. Santana N, Bortolozzi A, Serrats J, Mengod G, Artigas F (2004) Expression of serotonin1A and serotonin2A receptors in pyramidal and GABAergic neurons of the rat prefrontal cortex. Cereb Cortex 14:1100–1109. Schwaller B (2010) Cytosolic Ca2 Buffers. Cold Spring Harb Perspect Biol:1–20. Slattery D A, Neumann ID, Cryan JF (2011) Transient inactivation of the infralimbic cortex induces antidepressant-like effects in the rat. J Psychopharmacol 25:1295–1303. Sohal VS, Zhang F, Yizhar O, Deisseroth K (2009) Parvalbumin neurons and gamma rhythms enhance cortical circuit performance. Nature 459:698–702. Somogyi P (2010) Hippocampus – intrinsic organisation. Handb Brain Microcircuits:148–164. Stern CAJ, Do Monte FHM, Gazarini L, Carobrez AP, Bertoglio LJ (2010) Activity in prelimbic cortex is required for adjusting the anxiety response level during the elevated plus-maze retest. Neuroscience 170:214–222. Steullet P, Cabungcal J-H, Kulak A, Kraftsik R, Chen Y, Dalton TP, Cuenod M, Do KQ (2010) Redox Dysregulation Affects the Ventral But Not Dorsal Hippocampus: Impairment of Parvalbumin Neurons, Gamma Oscillations, and Related Behaviors. J Neurosci 30:2547– 2558. Thompson M, Weickert CS, Wyatt E, Webster MJ (2009) Decreased glutamic acid decarboxylase67 mRNA expression in multiple brain areas of patients with schizophrenia and mood disorders. J Psychiatr Res 43:970–977. Todorović N, Filipović D (2017a) Prefrontal cortical glutathione-dependent defense and proinflammatory mediators in chronically isolated rats: Modulation by fluoxetine or clozapine. Neuroscience 355. 21

Todorović N, Filipović D (2017b) The antidepressant- and anxiolytic-like effects of fluoxetine and clozapine in chronically isolated rats involve inhibition of hippocampal TNF-α. Pharmacol Biochem Behav 163. Vardigan JD, Huszar SL, McNaughton CH, Hutson PH, Uslaner JM (2010) MK-801 produces a deficit in sucrose preference that is reversed by clozapine, D-serine, and the metabotropic glutamate 5 receptor positive allosteric modulator CDPPB: relevance to negative symptoms associated with schizophrenia? Pharmacol Biochem Behav 95:223–229. Varga Z, Csabai D, Miseta A, Wiborg O, Czéh B (2017) Chronic stress affects the number of GABAergic neurons in the orbitofrontal cortex of rats. Behav Brain Res 316:104–114. Willner P, Towell A, Sampson D, Sophokleous S, Muscat R (1987) Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant. Psychopharmacology (Berl) 93:358–364. Wonders CP, Anderson SA (2006) The origin and specification of cortical interneurons. Nat Rev Neurosci 7:687–696. Yang C, Guo X, Wang GH, Wang HL, Liu ZC, Liu H, Zhu ZX, Li Y (2014) Changes in tau phosphorylation levels in the hippocampus and frontal cortex following chronic stress. Bra ilian J Med Biol es ev Bras Pes ui m dicas e biol gicas Soc Bras Biof sica . [et al] 47:237–244. Zhong P, Yan Z (2011) Differential regulation of the excitability of prefrontal cortical fastspiking interneurons and pyramidal neurons by serotonin and fluoxetine. PLoS One 6. Zlatković J, Todorović N, Bošković M, Pajović SB, Demajo M, Filipović D (2014a) Different susceptibility of prefrontal cortex and hippocampus to oxidative stress following chronic social isolation stress. Mol Cell Biochem 393:43–57. Zlatković J, Todorović N, Tomanović N, Bošković M, Djordjević S, La arević-Pašti T, Bernardi E, Djurdjević A, Filipović D (2014b) Chronic administration of fluoxetine or clo apine induces oxidative stress in rat liver: a histopathological study. Eur J Pharm Sci 59:20–30.

22

Figure captions: Fig. 1. The confirmation of cellular nature of the signal obtained after PV staining in the prelimbic area of medial prefrontal cortex of vehicle-treated non-stressed rat. PV (red) positive cells overlaid with DAPI nuclear staining (blue). Images were obtained using 20x objective. Scale bar: 100 µm. PV = parvalbumin; DAPI = 4'-6-diamidino-2-phenylindole. Fig. 2. Schematic coronal section of rat medial prefrontal cortical subregions (bregma, +3.72 mm), according to Paxinos and Watson (2005), left. Representative image of coronal section of rat medial prefrontal cortex (approximately bregma, +3.72 mm), of vehicle-treated non-stressed rat, immunostained for parvalbumin (red), right. The section was imaged with 10x objective along the x and y axes, and ~40 images obtained were stitched together. Stitched picture was overlaid with the schematic coronal section shown on the left. Scale bar: 500 µm. Cg1 = cingulate cortex, area 1; PrL = prelimbic area; IL = infralimbic area; DP = dorsal peduncular cortex; PV = parvalbumin. Fig. 3. Sucrose preference (A) and number of buried marbles (B) in non-stressed (NS) and isolated (CSIS) rats treated with vehicle (Veh, 0.9% NaCl), fluoxetine-hydrochloride (FLX, 15 mg/kg/day) or clozapine (CLZ, 20 mg/kg/day) at 0d and 21d of the experimental protocol. The results are presented as mean ± SEM of 6 rats per condition. ***p < 0.001 indicates significant difference between values measured at 0d and 21d for CSIS + Veh; ^^^p < 0.001 CSIS + FLX (21d) vs CSIS + Veh (21d); ^^p < 0.01 CSIS + CLZ (21d) vs CSIS + Veh (21d).

Fig. 4. PV+ cells in Cg1 (A), PrL (B), IL (C) and DP (D) of medial prefrontal cortex of nonstressed (NS) and isolated (CSIS) rats treated with vehicle (Veh, 0.9% NaCl), fluoxetinehydrochloride (FLX, 15 mg/kg/day) or clozapine (CLZ, 20 mg/kg/day). The number of PV+ cells within each subregion (left); diagrams with the marked regions of interest (middle); and representative images of PV+ cells in the respective subregions, obtained using 10x objective (right). The percentages above the CSIS + Veh bars represent the decrease in the number of PV+ cells in this group compared to Veh-treated non-stressed rats. The results are presented as mean ± SEM of 5 – 6 rats per condition. Symbols indicate significant difference: *p < 0.05, **p < 0.01, ***

p < 0.001 from Veh-treated non-stressed rats; ^^p < 0.01, ^^^p < 0.001 from Veh-treated CSIS; 23

##

p = 0.01, ###p = 0.001 between CSIS + CLZ and No stress + CLZ. Scale bar: 200 µm. PV =

parvalbumin; Cg1 = cingulate cortex, area 1; PrL = prelimbic area; IL = infralimbic area; DP = dorsal peduncular cortex.

Fig. 5. The number of PV+ cells in medial prefrontal cortex (Cg1, PrL, IL and DP combined) of non-stressed (NS) and isolated (CSIS) rats treated with vehicle (Veh, 0.9% NaCl), fluoxetinehydrochloride (FLX, 15 mg/kg/day) or clozapine (CLZ, 20 mg/kg/day). The results are presented as mean ± SEM of 5 – 6 rats per condition. Symbols indicate significant difference: *p < 0.05, ***

p < 0.001 from Veh-treated non-stressed rats; ^^p < 0.01 from Veh-treated CSIS rats. PV =

parvalbumin, mPFC = medial prefrontal cortex.

Fig. 6. CC3+ cells in Cg1 (A), PrL (B), IL (C) and DP (D) of medial prefrontal cortex of nonstressed (NS) and isolated (CSIS) rats treated with vehicle (Veh, 0.9% NaCl), fluoxetinehydrochloride (FLX, 15 mg/kg/day) or clozapine (CLZ, 20 mg/kg/day). The number of CC3+ cells within each subregion (left); diagrams with the marked regions of interest (middle); and representative images of CC3+ cells in the respective subregions, obtained using 10x objective (right). The percentages above the CSIS + Veh bars represent the increase in the number of CC3+ cells in this group compared to Veh-treated non-stressed rats. The results are presented as mean ± SEM of 5 – 6 rats per condition. Symbols indicate significant difference: *p < 0.05, **p < 0.01, ***p < 0.001 from Veh-treated non-stressed rats; ^p < 0.05, ^^p < 0.01, ^^^p < 0.001 from Veh-treated CSIS; #p < 0.05, ##p < 0.01 between CSIS + FLX and No stress + FLX. Scale bar: 200 µm. CC3 = cleaved caspase-3; Cg1 = cingulate cortex, area 1; PrL = prelimbic area; IL = infralimbic area; DP = dorsal peduncular cortex.

Fig. 7. The number of CC3+ cells in medial prefrontal cortex (Cg1, PrL, IL and DP combined) of non-stressed (NS) and isolated (CSIS) rats treated with vehicle (Veh, 0.9% NaCl), fluoxetinehydrochloride (FLX, 15 mg/kg/day) or clozapine (CLZ, 20 mg/kg/day). The results are presented as mean ± SEM of 5 – 6 rats per condition. Symbols indicate significant difference: *p < 0.05, ***

p < 0.001 from Veh-treated non-stressed rats; ^p < 0.05 from Veh-treated CSIS rats; ##p <

24

0.01, between CSIS + FLX and No stress + FLX. CC3 = cleaved caspase-3, mPFC = medial prefrontal cortex.

25

Table 1. Experimental groups Stress

Drug

No stress Chronic isolation

0.9% NaCl

Fluoxetine-hydrochloride (15 mg/kg/day)

Clozapine (20 mg/kg/day)

No stress + Veh CSIS + Veh

No stress + FLX CSIS + FLX

No stress + CLZ CSIS + CLZ

CSIS = chronic social isolation; Veh = vehicle; FLX = fluoxetine; CLZ = clozapine.

26

Highlights: 

FLX and CLZ prevented depressive- and anxiety-like behaviors in CSIS rats



CSIS decreased number of PV+ interneurons in Cg1, PrL, IL and DP subregions of mPFC



CSIS increased number of CC3+ cells in Cg1, PrL, IL and DP subregions of mPFC



FLX and CLZ showed subregion-specific protective effects against decrease in PV+ and increase in CC3+ cells numbers

27

Graphical abstract

28