Region-Specific Effects of Olanzapine on c-Fos Expression of Chronically Socially Isolated Rats

Accepted Manuscript Research Article Brain sub/regions-specific effects of olanzapine on c-Fos expression of chronically socially isolated rats Andrijana Stanisavljević, Ivana Perić, Peter Gass, Dragos Inta, Undine E. Lang, Stefan Borgwardt, Dragana Filipović PII: DOI: Reference:

S0306-4522(18)30739-5 https://doi.org/10.1016/j.neuroscience.2018.11.015 NSC 18737

To appear in:

Neuroscience

Received Date: Revised Date: Accepted Date:

30 May 2018 9 November 2018 12 November 2018

Please cite this article as: A. Stanisavljević, I. Perić, P. Gass, D. Inta, U.E. Lang, S. Borgwardt, D. Filipović, Brain sub/regions-specific effects of olanzapine on c-Fos expression of chronically socially isolated rats, Neuroscience (2018), doi: https://doi.org/10.1016/j.neuroscience.2018.11.015

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Brain sub/regions-specific effects of olanzapine on c-Fos expression of chronically socially isolated rats Andrijana Stanisavljević1, Ivana Perić1, Peter Gass2, Dragos Inta2,3, Undine E. Lang3, Stefan Borgwardt3, Dragana Filipović1 1

Vinča Institute of Nuclear Sciences, Laboratory for molecular biology and endocrinology,

University of Belgrade, Serbia 2

Department of Psychiatry and Psychotherapy, Central Institute of Mental Health, Medical

Faculty Mannheim, Heidelberg University, Mannheim, Germany 3

Department of Psychiatry (UPK), University of Basel, Switzerland

Corresponding author: Dragana Filipović, 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

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Abstract Olanzapine (Olz) is an atypical antipsychotic used to treat depression, anxiety and schizophrenia, which can be caused by chronic psychosocial stress. c-Fos protein expression has been used as an indirect marker of neuronal activity in response to various forms of stress or pharmacological treatments. We examined the effects of 3 weeks treatment of Olz (7.5 mg/kg/day) on c-Fos protein expression in stress-relevant brain sub/regions, its relationship with isolation-induced behavioural changes, and potential sites of Olz action on control and male rats exposed to 6 weeks of chronic social isolation (CSIS), an animal model of depression. Olz treatment reversed depressive- and anxiety-like behaviours induced by CSIS and suppressed a CSIS-induced increase in the number of c-Fos positive cells in subregions of the dorsal hippocampus, ventral (v) DG, retrosplenial cortex, and medial prefrontal cortex. In contrast, no change in c-Fos expression was seen in the CA3v, amygdala and thalamic, hypothalamic or striatal subregions in Olz-treated CSIS rats, suggesting different brain sub/regions-susceptibility to Olz. An increased number of c-Fos positive cells in the CA1v, amygdala and thalamic, hypothalamic and striatal subregions in controls as well as in the CA1v and subregion of the hypothalamus and nucleus accumbens in Olz-treated CSIS rats was found. Results suggest the activation of brain sub/regions following CSIS that may be involved in depressive and anxiety-like behaviours. Olz treatment showed region-specific effects on neuronal activation. Our data contribute to a better understanding of the mechanisms underlying the CSIS response and potential brain targets of Olz in socially isolated rats.

Key words: depression- and anxiety-like behaviours, social isolation, c-Fos, olanzapine, brain sub/regions

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Abbreviation ABC, avidin-biotinylated peroxidase; AcbC, accumbens nucleus, core; AcbSh, accumbens nucleus, shell; ANOVA, analysis of variance; AP1, activator protein 1 family; CA1d/v, Cornu ammonis, area 1, dorsal/ventral; CA2, Cornu ammonis, area 2; CA3d/v, Cornu ammonis, area 3; dorsal/ventral; Cg1,cingulate cortex; CNS, central nervous system; CPu, caudate putamen; CSIS, chronic social isolation; DAB, 3,3'-Diaminobenzidine tetrahydrochloride hydrate; DGd/v, dentate gyrus dorsal/ventral; DMH, dorsomedial hypothalamic nucleus; dHIPP, dorsal hippocampus; DP, dorsopeduncular cortex; EPSE, extrapyramidal side effects; HTH, hypothalamus; IL, infralimbic corex; i.p., intraperitoneal; MD, major depression; MB, marble burying; mPFC, medial prefrontal cortex; NGS, normal goat serum; NAc, nucleus accumbens; Olz, olanzapine; PBS, phosphate-buffered saline; PCP-4, Purkinje cell protein marker 4; PrL, prelimbic cortex; PVP, paraventricular thalamic nucleus, posterior part; RSC, retrosplenial cortex; RSD, retrosplenial dysgranular cortex; RSGc, retrosplenial granular cortex, c region; RT, room temperature; SP, sucrose preference Veh, vehicle; vHIPP, ventral hippocampus; VMH, ventromedial hypothalamic nucleus;

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INTRODUCTION Chronic exposure to stress may cause behavioural changes resulting from modifications in brain function that affect the neuroendocrine system (Ieraci et al., 2016). Social isolation stress is the most common form of stress that may be involved in the development of depression and anxiety (Deussing, 2006). Due to the complexity of the molecular mechanisms of these diseases, as well as ethical considerations, research in humans is drastically limited and thus the use of valid animal models is essential in psychiatric research. We used chronic social isolation (CSIS), a mild psychosocial stress that has been used as a more natural stressors in rodents, as it has been shown to produce neurochemical, neuroendocrine and behavioural changes in rats similar to those observed in humans with major depression (MD) (Heinrich and Gullone, 2006) and schizophrenia (Möller et al., 2013). Although rodent studies have investigated the impact of social isolation during adolescence (Whitaker et al., 2013; Yorgason et al., 2013), there is much less information about social isolation during adulthood (Filipović et al., 2017b). Moreover, chronic psychosocial stress in adulthood modulates brain structure and function, resulting in cognitive deficits and an increased risk for psychiatric disorders (De Kloet et al., 2005; Lupien et al., 2009). Thus, CSIS may provide essential insights into the mechanisms linking a comprehensive network of factors involved in the etiology of stress-induced depression, thereby improving our understanding of the biological mechanisms of depression in adulthood. Furthermore, differences in CSIS-related behavioural and physiological function of adolescent rodents from that of adults may be attributable to differences in the nature and/or duration of the social isolation, rat strain, and the age of animals at its onset (Serra et al., 2007). Olanzapine (Olz) is an atypical antipsychotic used to treat depression, anxiety and schizophrenia. It has high affinity for multiple receptors, especially 5-hydroxytryptamine (5HT2), dopamine (D1, D2-D4), muscarinic cholinergic (M1-M5) (Horowitz et al., 2003), histaminergic H1 and ɑ1-adrenergic receptors (Robertson and Fibiger, 1996), distributed through cortical, limbic and striatal structures. Clinical studies have revealed that Olz improves cognition, learning and memory in schizophrenia patients (Cuesta et al., 2001; Jann, 2004), and neurocognitive functions in patients with early psychosis (Keefe et al., 2007). This drug reduces the symptoms of mania in bipolar patients via the inhibition of dopamine (Tohen et al., 2000). As a 5-HT2 receptor antagonist (Bymaster et al., 1997), Olz increases the release of serotonin from 4

the presynaptic membrane, which may explain its antidepressant and anxiolytic effects (Jackson et al., 2004). Moreover, in combination with the antidepressant fluoxetine, Olz rapidly increases antidepressant effects in the treatment of therapy-resistant depressed patients (Narasimhan et al., 2007; Brunner et al., 2014). In contrast to typical antipsychotics, Olz is associated with a lower incidence of extrapyramidal side effects (EPSE) common with conventional antipsychotics. In animal models of depression, Olz has shown protection against stress-induced anhedonia (Orsetti et al., 2006). It upregulates BDNF mRNA expression in the rat hippocampus and prevents the decrease of Bcl-2 expression, an anti-apoptotic gene product, in methamphetamine-induced neurotoxicity in the rat caudate putamen (Bai et al., 2003; He et al., 2004). Recently, we demonstrated that chronic Olz treatment alleviates oxidative stress by improving antioxidant defense in the liver of socially isolated rats (Stanisavljevic et al., 2017). Chronic stress-induced behavioural changes in rodents have been associated with changes in immediate-early gene transcription factors, such as c-Fos, in different brain areas (Ieraci et al., 2016). C-Fos is one of the components of activator-protein 1 (AP-1) transcription factor complex, which is involved in cellular processes including differentiation, proliferation, and apoptosis (Herdegen and Waetzig, 2001). Its transcription is activated by various stimuli trough several signaling pathways involving Ca2+ influx trough N-methyl-d-aspartate (NMDA) receptors and voltage-sensitive Ca2+ channels, whereby Ca2+ is the major second messenger regulating immediate-early genes expression in excitable cells (Sheng and Greenberg, 1990). As c-Fos is induced in strongly activated neurons in the brain (Dragunow and Faull, 1989; Gass and Herdegen, 1995) following stimuli presentation, it has been considered an indirect marker for neuronal activation and used to map neuronal pathways in the central nervous system (CNS) (Hoffman et al., 1993). Stress is a strong factor for c-Fos induction in many brain regions, such as the hippocampus, medial prefrontal cortex and amygdala (Westenbroek et al., 2003). The research of Ceccatelli et al. (1989) for the first time introduced c-Fos as a marker of neuronal activity under stress conditions, indicating that c-Fos may be a reliable biological marker for detecting pathogenesis in the CNS (Tae et al., 2016). Nevertheless, numerous pharmacological studies have used c-Fos immunoreactivity for the identification of potential neuroanatomical regions of drug actions. The underlying molecular mechanisms of Olz treatment and CSIS induced depressive-and anxiety-like behaviours remains unclear. More detailed research of brain 5

parameters, neural pathways and target regions affected by depressive like behaviour induced by CSIS and of Olz treatment is needed. The aim of the present study was to investigate the effect of chronic Olz treatment on neuronal activity, assessed by the number of c-Fos positive cells, in stress-relevant brain sub/regions of both vehicle-treated and chronically socially isolated rats. Given that CSIS leads to depression- and anxiety-like behaviours (Perić et al., 2017), modulates brain structure and function in adult, male Wistar rats (Perić et al., 2018 ), we sought to identify the link between CSIS-induced behavioural changes and patterns of c-Fos protein expression in specific brain sub/regions, as well as Olz therapeutic efficacy based on a correlation between its effect on CSIS induced behaviour changes and c-Fos protein expression. We presumed that changes in c-Fos protein expression would give an insight into those brain regions associated with CSIS induced anxiety- and depressive-like behaviours and Olz treatment. Our results show that brain sub/regions showing c-Fos de/activation in adult male rats following CSIS and/or Olz treatment may provide insight into a neural circuit activated in the CSIS model of depression and schizophrenia, as well as the antidepressive and anxiety efficacy of Olz treatment in this circuit.

EXPERIMENTAL PROCEDURES Animals Experiments were performed using adult male Wistar rats (2 -3 months old, body weight 300400g). Animals were housed in groups of four per cage and maintained under conditions of controlled temperature (20 ± 2 °C) and humidity (55 ± 10%) on a 12h light/ 12h dark cycle (lights on between 07:00 and 19:00 h) with free access to food and water, ad libitum. All animal experiments were carried out in agreement with the Ethical Committee for the Use of Laboratory Animals of the Institute of Nuclear Sciences “Vinča”, which follows the guidelines of the registered “Serbian Society for the Use of Animals in Research and Education”, license 323-0701893/2015-05.

Preparation of olanzapine solution Olz (Sizap tablets, containing 10 mg of Olz) was purchased from Alkaloid, Skopje, Macedonia. Solution of Olz was prepared by crushing tablets to fine powder, followed by its solubilization in 6

0.1 M HCl. pH was adjusted to 5.8 with 1M NaOH. Further dissolving of the solution was performed with the aid of ultrasound and filtered through Whatman No. 42 filter paper. Rats received 7.5 mg/kg olanzapine-hydrochloride (hereafter referred as Olz) or vehicle (0.1 M HCl, pH 5.8) daily by intraperitoneal (i.p.) injection. This dose produces plasma levels in rats that correspond to therapeutically valid concentrations in humans (Elsworth et al., 2011).

Study design At the onset of the experiment rats were divided into group-housed, control rats (four rats per cage) and individually housed rats that underwent CSIS stress. CSIS rats were deprived of any visual and tactile contacts with other rats, but had regular auditory and olfactory experiences, according to the model of Garzón and Del Rio (1981). Olz treatment was administered to control and CSIS group of rats (Cont+Olz, CSIS+Olz, n=5-6 rats in each group) during the last 3 weeks of social isolation. Vehicle groups of animals (Cont+Veh, CSIS+Veh, n=5-6 rats in each group) received daily i.p. injections of 0.1M HCl, pH 5.8 (Fig.1.). Rats were tested in behaviour studies, such as sucrose preference (SP) and marble burying (MB). Our previous study showed that three weeks of social isolation in adult male Wistar rats resulted in a reduction in sucrose intake and mobility in the forced swim test, indicative of an impaired sensitivity to reward and anhedonia as well as despair behaviour, important characteristics of depressive-like behaviour (Zlatković et al., 2014). Moreover, socially isolated rats displayed an increased tendency to bury marbles, indicative of anxious behaviour (Perić et al., 2018). Also, CSIS during adulthood in rats compromised hypothalamic–pituitary–adrenal axis activity (Filipović and Pajović, 2009). Since treatment with antidepressants or antipsychotics requires prolonged medication (at least 3 weeks), we focused our study on a model with 3 weeks exposure to social isolation, enough to induce depressive- and anxiety- like behaviours, and 3 weeks of drug treatment in order to achieve its antidepressant- and anxiolytic-like action.

Sucrose preference SP was performed to assess anhedonia in rats, a key characteristic of depression (Willner et al.,1987). Rats were placed in separate plastic cages and given a 2-bottle choice, one filled with tap water and the other with 2% sucrose solution for 3 days, with volume intake controlled for 7

1h. To exclude possible side preferences in drinking behaviour, the position of the two bottles was switched across days. Preference for sucrose solution was calculated as a percentage of consumed sucrose solution of the total volume of liquid intake (sucrose solution + water). The SP test was performed at the start of the study (baseline) and at the end of third and sixth week.

Marble burying MB is used to evaluate anxiety-like behaviour (Ho et al., 2002). Each rat was placed individually in a plastic cage filled with a 2-cm thick lightly compressed layer of bedding, with six glass marbles (2.5 cm in diameter) arranged over the surface along a side wall of every cage. After 30 min of exposure to the marbles, the rats were removed from cage and the number of buried marbles at least two-thirds deep in the bedding was counted. The MB test was performed at the start of the study (baseline) and at the end of the third and sixth week.

Immunohistochemistry At the end of experiment rats were deeply anesthetized with ketamine/xylazine (100/5 mg/kg i.p) and transcardially perfused with physiological saline and then sacrificed by guillotine (Harvard Apparatus, South Natick, MA, USA) decapitation. Brains were rapidly removed and post-fixed overnight in fresh 4% paraformaldehyde (pH 7.4) (Filipović et al., 2013). After 24h, brains were placed in 0.4 % paraformaldehyde (pH 7.4) until processing. Using a vibratome (VT 100 S; Leica Bensheim, Germany) brains were cut into coronal sections of 40-μm thickness and collected in cryoprotective solution. C-Fos immunostaining was performed on free-floating sections. Sections were rinsed with phosphate-buffered saline (PBS) pH 7.4 containing 0.05 % Triton X-100 pH 7.4, treated with 0.6 % hydrogen peroxide at room temperature (RT) for 30 min, thoroughly washed with PBS and preincubated in blocking solution containing 2% normal goat serum (NGS) (Vector Laboratories) and 0.2 % Triton X-100 in PBS for 1 h at RT. Sections were incubated with primary antibody (anti-c-Fos, mouse monoclonal, Santa Cruz 8047, 1:500) prepared in blocking solution overnight at 4°C. After washing three times in PBS, sections were incubated with secondary antibody (Biotinylated anti-mouse IgG, 9200, 1:300) diluted in PBS with 2% NGS for 2h at RT. After thorough washing, sections were subsequently incubated for 20 min at RT with avidin-biotinylated peroxidase (ABC kit, Vector Laboratories) in PBS. 8

Following thorough washing, the peroxidase reaction was developed using nickel-enhanced 3,3'Diaminobenzidine tetrahydrochloride hydrate (Inta et al., 2017) (DAB, D5637 Sigma-Aldrich). Further, the sections were washed, mounted on superfrost™ plus microscope slides, air dried, and cover slipped with Eukitt mounting medium. For determining boundaries of Cornu ammonis, area 2 (CA2), between Cornu ammonis, area 1, dorsal (CA1d) and Cornu ammonis, area 3, dorsal (CA3d) one of the hippocampal subregions, we used Purkinje cell protein marker 4 (PCP4) prepared in 6 % NGS and 0.2 % Triton X-100 in PBS (rabbit anti-PCP4, HPA005792; Sigma, 1:200) and secondary antibody diluted in 10% NGS in PBS (goat anti-rabbit IgG, biotinylated antibody, Vector Laboratories, 1:500).

Counting c-Fos positive cells The number of c-Fos positive cells was quantified in the following sub/regions: dorsal hippocampus (dHIPP), bregma -3.12 mm to -3.60 mm (subregions: CA1d, CA2 and CA3d, Dentate gyrus (DGd); ventral HIPP (vHIPP), bregma -5.16 mm to -6.12mm (subregions: CA1v, CA3v and DGv); lateral/basolateral complex of amygdala, bregma -3.12 mm to -3.60 mm; retrosplenial cortex (RSC), bregma -3.12 mm to -3.60 mm (RSC granular cortex, c region (RSGc) and dysgranular (RSD)); paraventricular thalamic nucleus, posterior part (PVP), bregma -3.12 mm to -3.60mm; dorsomedial hypothalamic nucleus (DMH) and ventromedial hypothalamic nucleus (VMH), bregma -3.00 mm to -3.36 mm; medial prefrontal cortex (mPFC), bregma 3.72 mm to 2.76 mm (subregions: cingulate cortex (Cg1), prelimbic cortex (PrL), infralimbic corex (IL) and dorsopeduncular cortex (DP)); caudate putamen (CPu), nucleus accumbens (NAc) core (AcbC) and nucleus accumbens (NAc) shell (AcbSh), bregma 1.92 mm to 1.22 mm (Paxinos and Watson, 2005). Selected regions are presented in a schematic diagram (Fig. 2.). The boundaries of area of interest in selected rat brain regions were determined by immunostaining with a PCP4 antibody (Filipović et al., 2017a), thionin-stained sections and according to the anterior-posterior coordinates specified by atlas used in this study (Paxinos and Watson, 2005). CA2 pyramidal neurons are positive for PCP4, which effectively dealinates CA3d/CA2 as and CA2/CA1d borders (San Antonio et al., 2014; Filipović et al., 2017a). The subregions of vHIPP were identified in thionin-stained sections, in accordance with Paxinos and Watson (2005) and study of Fanselow and Dong (2010). Thionine-stained coronal sections were 9

used to distinguish anatomical borders of the lateral/basolateral complex of amygdala, RSC, PVP, DMH and VMH relative to other regions. The borders of subregions of mPFC and striatum were distinguished based on boundaries describes in the rat brain atlas (Paxinos and Watson, 2005). The number of c-Fos positive cells was manually calculated inside the borders of the sub/region according to the atlas and it was averaged from four sections per animal of each brain. The images were recorded at 4 x (for selecting the sub/regions) and 10x, 20x magnification for deeper analyses, using a light microscope BTC equipped with BIM 313T digital camera and further analyzed using ImageJ Software (version 1.51 u). To minimize mistakes and avoid double counting, only dark black cells were considered for counting. The number of c-Fos positive cells was counted by two independent observers blind to the experimental condition.

Statistical analyses Behavioural studies: SP and MB tests were analyzed by two-way repeated measures analysis of variance (ANOVA) [the factors were stress (levels: control and CSIS), drug treatment (levels: vehicle and Olz)] and the time as repeated measure [levels: days 0 baseline (baseline), 3 and 6 weeks], followed by Duncan's post hoc test (n=5-6 animal per group). Statistical analysis of the number of c-Fos positive cells within each sub/region was performed by two-way ANOVA [the factors were stress (levels: control and CSIS) and drug treatment (levels: vehicle and Olz)] followed by Duncan's post hoc test used to evaluate divergence among groups. Statistical analysis of percentage (%) changes in c-Fos expression between sub/regions in Olz-treated controls or CSIS compared to vehicle-treated controls, and Olz-treated CSIS compared to CSIS, was performed by one-way ANOVA followed by Duncan's post hoc test. Pearson’s test was used to evaluate correlations (r) between the number of c-Fos cells and behavioural parameters in SP and MB tests in the brain sub/regions where Olz reversed or boosted CSIS induced c-Fos protein expression. Statistical significance was set at p<0.05. The data are expressed as mean ± standard error of the mean (S.E.M.) of 5-6 animals per group.

RESULTS Behavioural tests Olanzapine reversed depressive- and anxiety-like behaviours induced by CSIS in rats 10

The results of SP are presented in Fig 3A. A two-way repeated measures ANOVA revealed significant main effects of CSIS (F1.19 = 21.96, p < 0.001), CSIS × Olz interaction (F1.19 = 10.64, p < 0.01), as well as time (F2.38 = 9.73, p < 0.001), CSIS × time interaction (F2.38 = 12.48, p < 0.001), drug treatment × time interaction (F2.38 = 6.68, p < 0.01) and CSIS × drug treatment x time interaction (F2.38 = 7.94, p < 0.01). Post hoc test revealed a significant decrease in preference for sucrose consumption in the CSIS+Veh at the end of 3 and 6 weeks compared to baseline values (***p < 0.001). The CSIS + Olz group, prior to treatment with Olz, also show a significant decrease in sucrose at the end of 3 weeks compared to baseline ( ***p < 0.001). The CSIS+Olz group showed a significant increase in SP at the end of 6 weeks, compared to the 3week time-point (^^^p < 0.001). Also, a significant increase was observed in CSIS+Olz group compared to CSIS+Veh rats at the end of 6 weeks ( +++p < 0.001). Post hoc test didn’t reveal significant change in Olz-treated controls or vehicle-treated controls. Results revealed that 6 weeks of CSIS led to a reduction in preference for sucrose consumption in rats, and promoted anhedonic-like behaviour; while 3 weeks of Olz treatment (7.5 mg/kg/day) at the 6-week timepoint reversed reduction of sucrose in socially isolated rats. The results of the number of total buried marbles are shown in (Fig.3B). A two-way repeated measures ANOVA revealed significant main effects of CSIS (F1.19 = 121.70, p < 0.001), Olz treatment (F1.19 = 39.93, p < 0.001), CSIS × Olz interaction (F1.19 = 29.84, p < 0.001), as well as time (F2.38 = 54.05, p < 0.001), CSIS × time interaction (F2.38 = 57.89, p < 0.001), drug treatment × time interaction (F2.38 = 24.60, p < 0.001) and CSIS × drug treatment x time interaction (F2.38 = 24.65, p < 0.001). Post hoc test revealed a significant increase in the number of buried marbles in the CSIS+Veh at the end of 3 ( ***p < 0.001) and 6 weeks (***p < 0.001), compared to baseline values. An increase was found in the CSIS+Olz group, prior to treatment with Olz, following 3 weeks compared to baseline (***p < 0.001). Moreover, a significant decrease in the number of buried marbles was found in the CSIS+Olz group (6 weeks), as compared to 3-week time-point (^^^p < 0.001) and to CSIS+Veh animals following 6 weeks ( +++p < 0.001). No significant changes were found in Olz-treated controls or vehicle-treated controls. These results indicate that 6 weeks of CSIS induced anxiety-like behaviours, while Olz treatment (7.5 mg/kg/day) reversed this effect in socially isolated rats.

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The number of c-Fos positive cells followed CSIS and/or Olz treatment Olz treatment modulates the number of c-Fos positive cells in subregions of the hippocampus and medial prefrontal cortex The number of c-Fos positive cells in CA1d, CA2, CA3d and DGd, subregions of rat dHIPP are presented in Fig. 4A. A two-way ANOVA revealed significant main effects of CSIS (F1.20=29.34, p<0.001; F1.20=22.23, p<0.001; F1.20=21.21, p<0.001; F1.20=44.17, p<0.001), Olz treatment (F1.20=27.57, p<0.001; F1.20=13.00; p<0.01; F1.20=27.62, p<0.001; F1.20=8.11; p<0.01) and combined effects of CSIS and Olz (F1.20=24.74, p<0.001; F1.20=22.23, p<0.001; F1.20=23.26, p<0.001) on the number of c-Fos positive cells in CA1d, CA2, CA3d and DGd, respectively. The number of c-Fos positive cells in the vehicle-treated CSIS was increased as compared to vehicletreated controls (***p<0.001), while Olz treatment showed a reduction in CSIS compared to the vehicle-treated CSIS rats (^^^p<0.001), for CA1d, CA2 and CA3d. For DGd, a significant increase in the number of c-Fos positive cells was observed in the vehicle-treated CSIS (***p<0.001) and Olz-treated CSIS (*p<0.05) rats as compared to vehicle-treated controls. Post hoc test showed a significant decrease in Olz-treated CSIS as compared to vehicle-treated CSIS rats (^p<0.05) in DGd subregion. The number of c-Fos positive cells in CA1v, CA3v and DGv, subregions of vHIPP are presented in Fig. 4B. A two-way ANOVA revealed significant main effects of CSIS (F1.18=48.66, p<0.001; F1.18=22.24, p<0.001; F1.18=23.62, p<0.001) on the number of c-Fos positive cells in CA1v, CA3v and DGv, respectively. Also, a two-way ANOVA revelaed a significant main effect of Olz treatment (F1.18=42.70, p<0.001) on the number of c-Fos positive cells in CA1v, and combined effects of CSIS and Olz (F1.18=8.41, p<0.01; F1.18=5.84, p<0.05) on the number of c-Fos positive cells in CA1v and DGv, respecitvely. The number of c-Fos positive cells in the vehicle-treated CSIS was increased as compared to vehicle-treated controls (***p<0.001), for CA1v, CA3v and DGv. A significant increase in the number of c-Fos positive cells in Olz-treated controls (***p<0.001) as compared to vehicle-treated controls, for CA1v, was found. Post-hoc test showed a significant increase in the number of c-Fos positive cells in Olztreated CSIS rats (***p<0.001;* p<0.05) as compared to vehicle treated controls, for both subregions CA1v and DGv, respectively. Also, a significant increase in the number of c-Fos

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positive cells was observed in Olz-treated CSIS as compared to vehicle-treated CSIS rats (^p<0.05), for CA1v, while significant decreased (^p<0.05) was seen in DGv. The numbers of c-Fos positive cells in Cg1 and PrL subregions of mPFC are presented in the Fig. 5. A two-way ANOVA revealed main effect of CSIS (F1.20=148.34, p<0.001; F1.20=78.53, p<0.001), and Olz treatment (F1.20=21.88, p<0.001; F1.20=6.74, p<0.05), as well as CSIS × Olz treatment interaction (F1.20=33.31, p<0.001; F1.20=13.63, p<0.05) in Cg1 and PrL subregions of mPFC, respectively. A significant increase in the number of c-Fos positive cells in vehicle-treated CSIS (***p<0.001) and Olz-treated CSIS (***p<0.001) groups as compared to vehicle-treated controls was found. Post hoc test showed a significant decrease in Olz-treated CSIS compared to vehicle-treated CSIS rats (^^^p<0.001) for both subregions Cg1 and PrL. The number of c-Fos positive cells in the IL and DP of mPFC is shown in Fig. 5. A twoway ANOVA revealed a significant main effect of CSIS (F1.20=31.30, p<0.001) and CSIS × Olz interaction (F1.20=12.97, p<0.01) on the number of c-Fos positive cells in IL region. A significant increase in the number of c-Fos positive cells was observed in the vehicle-treated CSIS (***p<0.001) and Olz-treated CSIS (**p<0.01) as compared to vehicle-treated controls. A significant decrease in Olz-treated CSIS as compared to vehicle-treated CSIS rats (^^p<0.01) was found. A two-way ANOVA revealed a significant CSIS × Olz interaction (F1.20=8.62, p<0.01) on the number of c-Fos positive cells in DP. A significant increase in the number of c-Fos positive cells was observed in the vehicle-treated CSIS as compared to vehicle-treated control rats (**p<0.01), while a decrease was found in Olz-treated controls as compared to Olz-treated CSIS rats (^^p<0.01) in DP.

CSIS and Olz treatment increased the number of c-Fos positive cells in the lateral/basolateral complex of amygdala The number of c-Fos positive cells in the lateral/basolateral complex of amygdala is shown in Fig 6. A two-way ANOVA revealed a significant main effect of CSIS (F1.19=34.56, p<0.001), and CSIS × Olz treatment interaction (F1.19=10.78, p<0.01) on the number of c-Fos positive cells. A significant increase in the number of c-Fos positive cells was observed in the vehicle- treated CSIS (***p<0.001), Olz-treated controls (**p<0.01) and -CSIS rats (***p<0.001), as compared to vehicle-treated controls. 13

Olz treatment decreased the number of c-Fos positive cells in subregions of the RSC in CSIS rats The number of c-Fos positive cells in the RSD and RSGc subregions of RSC is shown in Fig 7. A two-way ANOVA revealed a significant main effect of CSIS (F1.20=192.59, p<0.001; F1.20=189.45, p<0.001), and Olz treatment (F1.20=81.28, p<0.001, F1.20=86.46, p<0.001), as well as CSIS × Olz treatment interaction (F1.20=107.68, p<0.001, F1.20=83.43, p<0.001) on the number of c-Fos positive cells in these two subregions, respectively. Post hoc test showed a significant increase of c-Fos in vehicle-treated CSIS (***p<0.001) and Olz-treated CSIS (**p<0.01) as compared to vehicle-treated controls in RSD. Also, a significant decrease in the number of c-Fos positive cells was observed in Olz-treated CSIS as compared to vehicle-treated CSIS rats (^^^p<0.001). In RSGc, post hoc test showed significant increase in vehicle-treated (***p<0.001) and Olz treated –CSIS (**p<0.001) rats as compared to vehicle-treated control animals. A significant decrease in the number of c-Fos positive cell was found in Olz-treated CSIS as compared to vehicle-treated CSIS rats (^^^p<0.001).

CSIS and/or Olz-treatment increased the number of c-Fos positive cells in the hypothalamus (DMH and VMH) and thalamus (PVP) The number of c-Fos positive cells in the PVP is shown in Fig 8. A two-way ANOVA revealed a significant main effect of CSIS (F1.20=113.5, p<0.001) and Olz treatment (F1.20=8.39, p<0.01) on the number of c-Fos positive cells. Post hoc test showed a significant increase in the Olz-treated controls (**p<0.001), vehicle-treated CSIS (***p<0.001) and Olz-treated CSIS (***p<0.001) as compared to vehicle-treated controls. The number of c-Fos positive cells in the DMH and VMH is shown in Fig 9. A two-way ANOVA revealed a significant main effect of CSIS (F1.20=22.7, p<0.001; F1.20=23.53, p<0.001) and Olz treatment (F1.20=6.23, p<0.05; F1.20=30. 6, p<0.001) on the number of c-Fos positive cells in these brain subregions, respectively. Post hoc test showed a significant increase in Olztreated controls (*p<0.05; **p<0.01), vehicle-treated CSIS (***p≤0.001; **p≤0.01) and Olz-treated CSIS (***p<0.001) rats as compared to vehicle-treated controls in DMH and VMH, respectively.

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Also, a significant increase was found in Olz-treated CSIS as compared to CSIS treated rats (^^^p<0.001) only in VMH.

CSIS and Olz treatment increased the number of c-Fos positive cells in CPu and nucleus accumbens (AcbC, AcbSh) The number of c-Fos positive cells in the dorsolateral/medial CPu and AcbSh and AcbC is shown in Fig.10. A two-way ANOVA revealed main effect of CSIS (F1.20=52.68, p<0.001), and Olz treatment (F1.20=119.86, p<0.001), as well as a CSIS × Olz interaction (F1.20=66.14, p<0.001) on the number of c-Fos positive cells in CPu. A significant increase in the number of c-Fos positive cells was observed in Olz-treated controls (***p<0.001), vehicle-treated CSIS (***p<0.001), and Olz-treated CSIS (***p<0.001), as compared to vehicle-treated control group. A two-way ANOVA revealed a significant main effect of CSIS (F1.20=106.82, p<0.001; F1.20=50.24, p<0.001), and Olz treatment (F1.20=23.86, p<0.001; F1.20=10.25, p<0.01) on the number of c-Fos positive cells in AcbC and AcbSh, respectively. Post hoc test showed a significant increase of c-Fos in the Olz-treated controls (***p<0.001, **p<0.01), vehicle-treated CSIS (***p<0.001) and Olz-treated CSIS (***p<0.001), as compare to vehicle-treated controls, in AcbC and AcbSh, respectively. A significant increase in the number of c-Fos positive cells was also observed in the AcbC subregion for Olz-treated CSIS as compared to vehicle-treated CSIS (^^p≤0.01).

Correlation analysis between c-Fos protein expression and anxiety and depressive-like behaviours Pearson’s correlation tests were applied to the number of c-Fos positive cells and behaviour parameters in SP and MB tests, in rat subregions that showed reduce or increase in c-Fos protein expression in Olz-treated CSIS rats. These correlations are shown in Table 1. A significant negative correlation between c-Fos protein expression and SP behaviour was found in CA1d, CA2, DGd, DGv RSD, RSGc, Cg1, PrL, IL and DP subregions, while significant positive correlations was found in CA1v, AcbC and VMH subregions. A negative significant correlation between c-Fos protein expression and MB behaviour, where Olz reversed CSIS induced c-Fos protein expression, was found in CA1d, CA3d, DGd, 15

DGv, PrL, IL, DP subregions, whereby CA2, RSD, RSGc and Cg1 showed negative but nonsignificant correlations. A significant positive correlation between c-Fos protein expression and MB behaviour was found in CA1v, AcbC and VMH subregions.

CSIS and/or Olz treatment showed different percentage changes of c-Fos expression between brain subregions One-way ANOVA showed significant main effect of Olz treatment (F1.7=56.04, p<0.001) on the percentage of c-Fos expression in the brain subregions of vehicle-treated controls in which Olz treatment increased the number of c-Fos positive cells compared to controls (Fig. 11A). The greatest increase in percentage of c-Fos expression was seen in the CPu (648%) and the lowest in DMH (56%) in Olz-treated rats as compared to controls. Also, a significant increase in CPu as compared to AcbC (***p<0.001) and to AcbSh ( ***p<0.001) was found, while no significant differences between AcbC (p=0.50) and AcbSh (p=0.50) were revealed. Also, no significant differences were seen between subregions of hypothalamus, DMH (p=0.62) and VMH (p=0.62). One-way ANOVA revealed significant main effects of CSIS (F1.19=22.32, p<0.001) on the percentage of c-Fos expression in the brain subregions of vehicle-treated controls, in which CSIS increased the number of c-Fos positive cells compared to controls (Fig. 11B). The largest increase in percentage of c-Fos expression was seen in the CPu (524%), followed by RSD (488%), Cg1 (486%), CA2 (425%), RSGc (424%), while the smallest was seen in DGd (77%), VMH (64%) and DP (51%). Duncan's post-hoc test showed a significant increase in CA2 as compare to CA3d (*p<0.05) and DGd (***p<0.001), as well as, in CA1d and CA3d compared to DGd (***p<0.001). However, a significant increase in percentage of c-Fos expression in CA1d as compared to CA1v ( **p<0.01), and CA3d as compared to CA3v ( ***p<0.001) was found, while between DGd and DGv there was no significant differences (p=0.76). Also, between subregions of vHIPP there was no significant differences in percentage of c-Fos expression. Moreover, a significant increase in percentage of c-Fos expression was revealed in Cg1 as compared to PrL (***p<0.001), IL (***p<0.001) and DP (***p<0.001), while a significant increase in PrL was seen as compared only to DP (**p<0.01). Duncan's post-hoc test revealed no significant differences between RSD (p=0.23) and RSGc (p=0.23), or between DMH (p=0.42) and VMH (p=0.42). Also, a significant increase was revealed in CPu as compared to AcbC ( ***p<0.001) and AcbSh 16

(***p<0.001), while no significant differences in percentage of c-Fos expression between AcbC (p=0.66) and AcbSh (p=0.66) were found. One-way ANOVA revealed significant main effects of CSIS × Olz treatment interaction (F1.10=15.85, p<0.001) on the percentage of c-Fos expression in the brain subregions of CSIS rats in which Olz treatment decreased number of c-Fos positive cells, as compared to CSIS alone (Fig. 11C). The highest decrement in Olz-treated CSIS rats as compared to CSIS alone was seen in CA3d (80%), followed by CA1d (77%) and CA2 (71.4%), while the smallest was in DGv (26.8%) and DGd (20%), for the hippocampal subregions. In subregions of mPFC, the highest percentage was observed in Cg1 (49%), while the lowest was seen in IL (29%). The percentage change of c-Fos expression in RSC was almost identical, 66.4 % for RSD and 65.15 % for RSGc without significant differences between them (RSD p=0.88, and RSGc p=0.88). Duncan's posthoc test revealed significant increase in CA3d, CA1d and and CA2, as compared to dorsal and ventral DG (^^^p<0.001), while there was no significant difference between dorsal and ventral part of DG (p=0.41). Also, we found a significant increase in Cg1 as compared to IL (^p<0.05).

DISCUSSION In the present study we demonstrated that chronic Olz treatment ameliorated depressive- and anxiety like behaviours in CSIS animals, evidenced by the SP and MB tests, in which reversal of these behavioural changes may be associated with the antidepressant and anxiolytic effects of Olz. In addition to its antipsychotic effect, Olz has shown antidepressant efficacy, which may be one of the main strategies for augmentation of antidepressant treatment (Wang and Si, 2013). A notable effect of Olz was demonstrated in the study of Zhang et al. (2005), in which Olz enhanced the efficacy of sucrose as a reinforcer in motivational behaviour in rats and provided relief from the negative symptoms of schizophrenia. Previous data has revealed differences in functions and afferent/efferent connections of dHIPP and vHIPP, where dHIPP appears to be much more implicated in cognitive processing of information, while vHIPP is involved in processing of emotional ones (Strange et al., 2014). In the current study, both dHIPP and vHIPP showed a significant increase in c-Fos expression following CSIS. Despite significant c-Fos expression throughout the hippocampal longitudinal axis, the percentage of c-Fos expression showed subregion-specific effects following CSIS; the 17

CA2 subregion was found to be more sensitive than all other hippocampal subregions, excluding the CA1d subregion. Dysfunction of hippocampal circuitry in the CA2 subregion, which plays a role in social memory (Hitti and Siegelbaum, 2014), appears to be linked to psychiatric diseases. Furthermore, increased c-fos mRNA expression in the dHIPP has been demonstrated following 30 min of restraint stress (Guimarães et al., 1993). In addition, direct and indirect interconnections between HIPP and RSC, which contribute to hippocampal functions in memory and learning, have been characterized (Wyss and Groen, 1992). We found that CSIS had no subregion-specific effects in RSC, revealing similar percentages of c-Fos expression in the RSD and RSGc. Nevertheless, following CSIS, in both the RSGc and RSD, the number of c-Fos positive cells show the same trend and pattern as in the d/vHIPP, which may result from interconnections, especially those between the RSC and CA1d (Wyss and Groen, 1992; Cenquizca and Swanson, 2007), one of the major outputs of the HIPP. A region that plays an important role in the pathophysiology of schizophrenia and depression is the PFC (Koenigs and Grafman, 2009). In the current study, CSIS increased the number of c-Fos positive cells in all mPFC subregions, indicating susceptibility of these subregions to CSIS. This result may indicate the effect of social isolation on working memory and other higher brain functions (Famitafreshi et al., 2015). However, among all of the mPFC subregions, CSIS most affected the Cg1, while the least affected was the DP. Percentage changes of c-Fos expression between mPFC subregions may have resulted from different cell types across subregions and layers of mPFC and their sensitivity to the conditions of CSIS. The amygdala has an important role in regulating emotional and social behaviour (Davis et al., 1994; Sandi et al., 2008), and may be involved in the pathobiology of depression (Sibille et al., 2010). In our study, the increased number of c-Fos positive cells in the lateral/basolateral complex of amygdala following CSIS showed a similar pattern to the d/vHIPP and mPFC, regions that have strong interconnections with the amygdala. Especially significant is the projection between amygdala and vHIPP, with important interconnections between the basolateral part of amygdala and CA1v, which comprises a circuit involved in anxiety and social behaviour (Felix-Ortiz et al., 2014; Yang and Wang, 2017). These data support the findings in our study in which an increased number of c-Fos positive cells was found in the

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lateral/basolateral complex of amygdala and vHIPP in CSIS rats that showed anxiety-like behaviour. Regarding the number of c-Fos positive cells and percentage of c-Fos expression, CSIS revealed a large impact on the striatum. This result may be due to long-lasting effects of social condition and rearing on behaviour, striatal anatomy and physiology, especially in primates and rodents (Báez-Mendoza and Schultz, 2013). Also, a reduction in volume of the striatum in depressed patients has been observed (Baumann et al., 1999; Marais et al., 2009; Fedotova, 2012). Moreover, CSIS caused the strongest neuronal activation in the CPu as compared to AcbC and AcbSh, revealing subregional effects of CSIS on striatal c-Fos expression. No significant differences between the increased percentage of c-Fos positive cells in the AcbC and AcbSh were found. The AcbC and AcbSh (NAc) represent key structures of the ventral striatum involved in motivational and emotional processes. Based on the fact that the NAc receives glutamatergic afferents from several limbic regions, including the vHIPP (Bagot et al., 2015), mPFC (Montaron et al., 1996), basolateral part of amygdala and thalamus (Sesack and Grace, 2010; Floresco, 2015), an increased number of c-Fos positive cells in AcbC and AcbSh of CSIS rats compared to controls might be due to an increase in glutamate in those regions. Our results also revealed an increased number of c-Fos positive cells following CSIS, in the PVP, which is known to be activated following exposure to various stressors (Behrendt, 2012), suggesting sensitivity of this brain region in depressive- and anxiety like behaviours. The increased number of c-Fos positive cells in the PVP exhibited a similar pattern as that observed in the amygdala, AcbC and AcbSh, with no significant difference in percentage of c-Fos expression among these brain sub/regions, indicating strong, primarily glutamatergic connections between the subregions (Haight and Flagel, 2014). Nonetheless, increased neuronal activity in the VMH and DMH following CSIS may be a consequence of input from the HIPP to these subregions (Behrendt, 2012), especially from the vHIPP. Moreover, among subregions of the hypothalamus, there were no significant differences in percentage of c-Fos expression, but a significantly smaller percentage of c-Fos expression relative to hippocampal subregions, with no significance in c-Fos expression between the DMH and CA1v or between hypothalamus subregions and the CA3v and DGd/v.

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Olz treatment may ameliorate the harmful effect of reactive oxygen species induced by different kind of stressors in various tissue (Martins et al., 2008; Todorović et al., 2016). Our results showed that chronic Olz treatment decreased the number of c-Fos positive cells in all dHIPP subregions and DGv in CSIS rats, suggesting that antidepressive and anxiolytic effects of Olz may be mediated by its actions on hippocampal neurotransmission. This is consistent with reports of suppressed stress-induced c-Fos expression in the DGd and CA1d hippocampal regions by benzodiazepines and imipramine (De Medeiros et al., 2005). As Olz decreased the number of c-Fos positive cells only in the DGv, this may be due to a strong role of this subregion in anxiety disorders (Strange et al., 2014). We speculate that Olz achieved anxiolytic effects by antagonizing CSIS induced c-Fos expression in DGv, which also showed a negative correlation between c-Fos expression and burring behaviour in the MB test (r= -95^). Regarding the percentage of c-Fos expression, Olz revealed different effects throughout the longitudinal HIPP axis, with the most pronounced in the dorsal CA field (CA1, CA2, CA3), no effect in the ventral CA field (CA1, CA3), and with similar effects in the DGv and DGd. Taking into account indirect connections between the dHIPP and mPFC, we speculate that the Olz-induced decrease in c-Fos expression in the mPFC, may coincidence with the aforementioned dHIPP changes. In addition, the most affected subregion in the mPFC was the Cg1. Based on the similar pattern of c-Fos expression in the dHIPP and RSC following Olz treatment in CSIS rats, we conclude that this pattern arises from interconnections between these two regions. The current results are in accordance with the research of Fujimura and coworkers (2000), who showed that pharmacologically effective doses of Olz blocked Fos-like protein expression and neurological modifications in the RSC of female rats induced by the NMDA receptor antagonist dizocilpine. Regarding the percentage of c-Fos expression, we found no significant differences in percentage of c-Fos expression between the RSD and RSGc, subregions of the RSC. Given that Olz decreased the number of c-Fos positive cells in the RSGc and RSD, as well as in all subregions of the mPFC, dHIPP and DGv of CSIS rats, these results indicate the significance of the therapeutic effects of Olz in depressive- and anxiety-like behaviours. These data are also corroborated by a high Pearson's correlation coefficient for the aforementioned subregions. In contrast, Olz in CSIS rats did not suppress the number of c-Fos positive cells in the CA3v, amygdala, CPu, AcbSh, PVP or DMH compared to CSIS alone. Moreover, the increased c-Fos 20

expression in the CA1v, VMH and AcbC in Olz-treated CSIS rats suggests synergistic effects of CSIS and Olz. Chronic Olz treatment increased the number of c-Fos in the CA1v, CPu, AcbC, AcbSh, PVP, DMH, VMH and amygdala in control rats, emphasizing the importance of these subregions in the antipsychotic actions of Olz. The most significant effect of Olz treatment on percentage of c-Fos expression was revealed in the CPu, suggesting that the CPu is a critical site for Olz action. The study of Deutch and coworkers (1992) revealed regional effects of atypical antipsychotics (clozapine and remoxipride) on striatal c-Fos expression, suggesting the AcbSh as a locus of antipsychotic effects. In contrast, our results showed no significant differences in percentage of c-Fos expression between the AcbC and AcbSh, indicating no subregional impact of chronic Olz treatment on NAc.

CONCLUSION According to divergent c-Fos expression changes following CSIS and Olz treatment, the antidepressive- and anxiolytic-like effects of Olz action may be related to specific brain sub/regions i.e. dHIPP, DGv, RSC and mPFC. Olz antagonized the expression of c-Fos in these subregions, which correlates with the normalization of behavioural parameters following Olz treatment. Additionally, Olz showed a synergistic effect with social isolation stress in elevating the expression of c-Fos in the CA1v, VMH and AcbC. Whether the Olz induced neuronal activation of these subregions promotes therapeutic effects in depression- and anxiety-like behaviours or results in other pharmacological effects remains to be investigated. The differential distribution of c-Fos in brain regions is useful for mapping the targets of chronic Olz treatment in CSIS, which is important for elucidating the mechanisms and neural circuits underlying the antidepressant and anxiolytic actions of Olz.

CONFLICT OF INTEREST The authors declare no conflict of interests.

Acknowledgments -This work was supported by the Grant of Ministry of Education, Science and Technological Development of the Republic of Serbia (173044) and by grants from 21

Deutsche Forschungsgemeinschaft (IN168/3-1), the Ingeborg Ständer Foundation and the Research Fund of the UPK Basel to D.I.

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Figure captions: Figure 1 Study design timeline. Cont-controls, CSIS-chronic social isolation. Veh-vehicle (rats treated with 0.1M HCl, pH 5.8), Olz-olanzapine (rats treated with 7.5 mg/kg of olanzapine solution daily). For details refer to section “Study design“.

Figure 2 Schematic representations of brain sub/regions of interest analyzed in the present study (Paxinos and Watson, 2005).

Figure 3 Sucrose preference (A) and the number of buried marbles (B) in controls (Cont) and chronic social isolation (CSIS) rats at baseline and following 3 and 6 weeks treated either with (Veh. 0.1M HCl, pH 5.8), or olanzapine (Olz, 7.5 mg/kg/day). Results are expressed as mean ± SEM, n = 5-6 rats in each group. Significant differences between groups obtained from two-way ANOVA analysis followed by post hoc Duncan test are indicated as follows: A sucrose preference (***p<0.001 CSIS+Veh (3 weeks) vs. CSIS+Veh (baseline); ***p<0.001 CSIS+Veh (6 weeks) vs. CSIS+Veh (baseline); *** p<0.001 CSIS+Olz (3 weeks) vs. CSIS+Olz (baseline); ^^^

p<0.001 CSIS+Olz (6 weeks) vs. CSIS+Olz (3 weeks); +++p<0.001 CSIS+Olz (6 weeks) vs.

CSIS+Veh (6 weeks)); B number of buried marbles (***p<0.001 CSIS+Veh (3 weeks) vs. CSIS+Veh (baseline); ***p<0.001 CSIS+Veh (6 weeks) vs. CSIS+Veh (baseline); *** p<0.001 CSIS+Olz (3 weeks) vs. CSIS+Olz (baseline); ^^^p<0.001 CSIS+Olz (6 weeks) vs. CSIS+Olz (3 weeks); +++p<0.001 CSIS+Olz (6 weeks) vs. CSIS+Veh (6 weeks)).

Figure 4 A DAB-stained dorsal hippocampus sections for detecting subregion specific changes of the number of c-Fos positive cells following CSIS and Olz treatment. PCP-4 was used as a CA2 subregion marker. Duncan's post-hoc test showed significant increase in the number of cFos positive cells in CA1d, CA2, CA3d, DGd between CSIS and Cont (***p<0.001). Also, significant increase was seen in DGd between Olz-treated CSIS rats and Cont (*p<0.05). Significant decrease in the number of c-Fos cells was observed in CA1d, CA2 and CA3d between Olz-treated CSIS and CSIS rats (^^^p<0.001), and in DGd between the same groups (^p<0.05). Scale bar 200 um. 28

Figure 4 B DAB-stained vHIPP sections for detecting subregion specific changes of the number of c-Fos positive cells following CSIS and Olz treatment. Thionin-stained section of vHIPP was used to identify borders of subregions of vHIPP. Duncan's post-hoc test showed significant increase in the number of c-Fos positive cells in: CA1v, CA3v, and DGv between CSIS and Cont (***p<0.001); CA1v between Olz-treated controls and Olz-treated CSIS rats as compared to Cont (***p<0.001); CA1v between Olz-tretaed CSIS and CSIS rats (^p<0.05). Also, a significant decrease in DGv between Olz-treated CSIS and CSIS rats (^p<0.05) was found. Scale bar 200 um.

Figure 5 DAB-stained mPFC sections for detecting subregion specific changes of the number of c-Fos positive cells following CSIS and Olz treatment. Duncan's post-hoc test showed increased number of c-Fos positive cells in Cg1, PrL and IL between CSIS and Cont (***p<0.001), and in DP (**p<0.01) between the same groups. Also, a significant increase was observed in Cg1 and PrL between Olz-treated CSIS rats and Cont (***p<0.001), and in IL (**p<0.01), between the same groups. Post-hoc test showed significant decrease in the number of c-Fos in Cg1 and PrL between Olz-treated CSIS and CSIS rats (^^^p<0.001), and in IL and DP (^^p<0.01) between the same groups. Scale bar 200um.

Figure 6 DAB-stained sections of lateral/basolateral complex of amygdala for detecting changes in the number of c-Fos positive cells following CSIS and Olz treatment. Thionin stained was used to identify borders of lateral/basolateral complex of amygdala. Duncan's post-hoc test revealed a significant increase in the number of c-Fos positive cells in the lateral/basolateral complex of amygdala between CSIS (***p<0.001), Olz-treated Cont (**p<0.01), –CSIS (***p<0.001) and Cont. Scale bar 200 um.

Figure 7 DAB stained sections of RSC for detecting subregion specific changes regarding the number of c-Fos positive cells following CSIS and Olz treatment. Thionin stained sections was used to identify the borders between RSGc and RSD. Duncan's post-hoc test showed a significant increase in the number of c-Fos positive cells in RSGc and RSD, between CSIS rats (***p<0.001), Olz-treated CSIS rats (**p<0.01) and Cont, respectively. Post-hoc test revealed a 29

significant decrease in the number of c-Fos positive cells in Olz-tretaed CSIS rats compared to CSIS rats (^^^ p <0.001) in both, RSGc and RSD. Scale bar 200 um.

Figure 8 DAB stained sections of PVP for detecting changes of the number of c-Fos positive cells following CSIS and Olz treatment. Thionin stained section was used to identify the borders of PVP. Duncan's post-hoc test showed a significant increase in the number of c-Fos positive cells between vehicle-treated CSIS (***p<0.001), Olz-treated Cont (**p<0.01), Olz-treated CSIS rats (***p<0.001) and Cont. Scale bar 200 um.

Figure 9 DAB-stained sections of hypothalamus for detecting subregions specific changes regarding the number of c-Fos positive cells, following CSIS and Olz treatment. Thionin staining was used to identify the borders of subregions of hypothalamus, DMH and VMH. Duncan's posthoc test showed a significant increase in the number of c-Fos positive cells in vehicle treatedCSIS rats (***p<0.001, **p<0.01), Olz-treated Cont (*p<0.05, **p<0.01), Olz-treated CSIS rats (***p<0.001, ***p<0.001) as compare to Cont, in DMH and VMH, respectively. Also, a significant increase in the number of c-Fos positive cells was observed in Olz-treaed CSIS rats as compared to vehicle-treated CSIS rats (^^^p<0.001) in VMH. Scale bar 200 um.

Figure 10 DAB-stained sections of striatum for detecting subregions specific changes regarding the number of c-Fos positive cells, following CSIS and Olz treatment. Duncan's post-hoc test showed a significant increase in the number of c-Fos positive cells in vehicle treated-CSIS rats (***p<0.001), Olz-treated Cont (***p<0.001), and Olz-treated CSIS rast (***p<0.001) as compared to Cont, in CPu. Significant increase was seen in vehicle treated-CSIS rats (***p<0.001, ***

p<0.001), Olz-treated Cont (***p<0.001, **p<0.01), Olz-treated CSIS rats (***p<0.001,

***

p<0.001), as compared to Cont, in AcbC and AcbSh, respectively. Also, a significant increase

in Olz-treated CSIS rats as compared to vehicle treated CSIS-rats (^^p<0.01), was revealed only in AcbC. Scale bar 200 um. Figure 11 (A-C) Percentage of c-Fos expression following CSIS and/or Olz treatment in rat’s brain subregions. A) In Olz-treated rats as compared to Cont, significant increase in CPu as 30

compared to AcbC (***p<0.001) and to AcbSh ( ***p<0.001) was found. B) In CSIS rats, as compared to Cont, significant increase in CA2 as compare to CA3d (*p<0.05) and DGd (***p<0.001), as well as in CA1d and CA3d as compared to DGd (***p<0.001) was revealed. Also, significant increase was seen in CA1d as compared to CA1v ( **p<0.01) as well as in CA3d as compared to CA3v (***p<0.001). In mPFC, significant increase in percentage of c-Fos expression was seen in Cg1 as compared to PrL ( ***p<0.001), IL (***p<0.001) and DP (***p<0.001) as well as in PrL compared to DP ( **p<0.01). Increased percentage of c-Fos expression in CPu as compared to AcbC ( ***p<0.001) and AcbSh ( ***p<0.01) was noted. C) In Olz-treated CSIS rats as compared to CSIS, alone, significant increase in CA3d, CA1d and and CA2, as compared to DGd (^^^p<0.001) was found. Also, similar subregion-dependent pattern of c-Fos activation was noted in Cg1 as compared to IL (^p<0.05).

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Table 1. Correlation between c-Fos protein expression and behavioural changes (SP and MB results) in the brain subregions, where Olz reversed or boosted CSIS induced increase in c-Fos protein expression. The statistical symbols are presented as follows ^p<0.05

Region

Subregion

SP

MB

CA1d

r= -0.88^

r= -0.94^

CA2d

r= -0.80^

r= -0.66

CA3d

r= -0.83^

r= -0.81^

DGd

r= -0.86^

r= -0.85^

CA1v

r= 0.94^

r= 0.96^

DGv

r= -0.91^

r= -0.95^

RSD

r= -0.98^

r= -0.73

RSGc

r= -0.83

^

r= -0.75

Cg1

r= -0.80^

r= -0.78

PrL

r= -0.83^

r= -0.82^

IL

r= -0.91^

r= -0.93^

DP

r= -0.91^

r= -0.91^

NAc

AcbC

r= 0.84^

r= 0.84^

HTH

VMH

r= 0.94^

r= 0.93^

HIPP

RSC

mPFC

32

33

34

35

36

37

38

39

40

41

42

Highlights    

Olz reverses depressive- and anxiety-like behaviours induced by CSIS CSIS increases c-Fos protein expression in stress-relevant brain sub/regions Olz shows region specific effects on c-Fos protein expression Olz in controls shows most pronounced changes if c-Fos protein expression in CPu

43