5HT3 receptor antagonist (ondansetron) reverses depressive behavior evoked by chronic unpredictable stress in mice: Modulation of hypothalamic–pituitary–adrenocortical and brain serotonergic system

PBB-71975; No of Pages 8 Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh

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Deepali Gupta ⁎, Mahesh Radhakrishnan, Yeshwant Kurhe

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Department of Pharmacy, Birla Institute of Technology & Science, Pilani, Rajasthan, 333031, India

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a r t i c l e

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Article history: Received 12 October 2013 Received in revised form 23 May 2014 Accepted 31 May 2014 Available online xxxx

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Keywords: 5HT3 Receptor antagonist Chronic unpredictable stress Corticosterone Forced swim test Serotonin Sucrose preference test

O R O

a b s t r a c t

Chronic stress is one of the major causes of depression, associated with behavioral and biochemical impairments. 5HT3 receptor antagonists (such as ondansetron) have shown alleviation of depressive symptomology in preclinical and in few clinical studies. However, their effects in chronic stress-induced depressive behavior and the underlying mechanism(s) are yet to be known. In the present study, the effects of a 5HT3 receptor antagonist, ondansetron were evaluated in chronic unpredictable stress (CUS)-evoked depressive behavior. In addition, the possible mechanism was determined by measuring plasma corticosterone (CORT) as a marker of hypothalamic–pituitary–adrenocortical (HPA)-axis activity and serotonin levels in the discrete brain regions. Mice were subjected to a battery of unpredictable stressors for 28 days. Ondansetron (0.05, 0.1 and 1 mg/kg, p.o.) and fluoxetine (10 mg/kg, p.o.) were administered during the last 14 days (day 15–28th) of CUS testing paradigm. The results showed that the 4-week CUS produced significant depressive behavior in mice, which included increased despair effects in forced swim test (FST) and reward-related deficits in sucrose preference test. Biochemical assays demonstrated a significant increase in percentage of plasma CORT and decrease in percentage of serotonin levels in the discrete brain regions of CUS mice. Chronic ondansetron treatment, similar to that of positive control fluoxetine, significantly reversed despair effects in FST and reward-related deficits in sucrose preference test. In addition, ondansetron and fluoxetine treatments significantly increased percentage of serotonin levels in the measured brain regions and attenuated HPA-axis hyperactivity, as evidenced by low percentage of plasma CORT levels in CUS mice. These findings indicate the potential role of ondansetron (a 5HT3 receptor antagonist) in reversing CUS-induced depressive behavior, which is possibly mediated by its modulating effects on the HPA-axis and serotonergic system. Further, the study represents that 5HT3 receptor antagonists can be a potential therapeutic candidate for stress-related depressive disorders. © 2014 Published by Elsevier Inc.

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(Ruhe´ et al., 2006). In addition, effective improvement in depressive symptoms occurs only after several weeks of treatment with currently available clinical drugs (Grunebaum et al., 2004). Therefore, there is a need to develop and discover new agents with high efficacy that can improve the pharmacotherapy of depressive disorder. In rodents, chronic unpredictable stress (CUS) as a model of depression was developed in an attempt to mimic some of the environmental factors contributing to induction of depression in humans (Moretti et al., 2012; Nollet et al., 2013). In addition, several lines of evidences have revealed that CUS resembles a variety of neurochemical, neurobehavioral and neuroendocrine alterations of human depressive disorder (Holsboer, 2000; McEwen, 2008). Animals subjected to a variety of stressors such as deprivation of food and water, wet husk bedding, cage tilting and exposure to inverted light and dark cycle have shown depressive behavior in numerous behavioral testing paradigms (Larsen et al., 2010; Ma et al., 2011; Rasheed et al., 2011). Previous studies have shown that mice exposed to four week stress condition exhibit anhedonia-like behavior in sucrose preference test (Strekalova and

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5HT3 receptor antagonist (ondansetron) reverses depressive behavior evoked by chronic unpredictable stress in mice: Modulation of hypothalamic–pituitary–adrenocortical and brain serotonergic system

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1. Introduction

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Stressful situations such as major life events or the factors related to surroundings severely affect the psychological status of an individual and inability to overcome or adapt to these, often lead to psychosocial disorders such as depression (Paykel, 2003). Depression is a recurrent mental condition that clinically presents with sad mood, anhedonia, feelings of guilt or low self-worth, disturbed sleep or appetite, poor concentration and frequent suicidal tendencies (APA, 2000). According to World Health Organization, almost 1 million deaths occur per year due to suicide which translates 3000 suicide deaths every day (WHO, 2012). Despite a constant increase in the number of antidepressant drugs, the prevalence of depression is increasing, probably due to unclear pathophysiology and inconsistent existing pharmacotherapy

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⁎ Corresponding author. Tel.: +91 9352011573 (mobile); fax: +91 1596244183. E-mail addresses: [email protected] (D. Gupta), [email protected] (M. Radhakrishnan), [email protected] (Y. Kurhe).

http://dx.doi.org/10.1016/j.pbb.2014.05.024 0091-3057/© 2014 Published by Elsevier Inc.

Please cite this article as: Gupta D, et al, 5HT3 receptor antagonist (ondansetron) reverses depressive behavior evoked by chronic unpredictable stress in mice: Modulation of ..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.05.024

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2.1. Animals

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Swiss albino adult male mice were obtained from Hisar Agricultural University, Haryana, India. Mice were housed in polycarbonate cages in a group of 6–7 and maintained in standard laboratory conditions with alternating light and dark cycle of 12 h each, temperature 23 ± 2 °C and humidity conditions 62 ± 5% RH in the housing unit and had free access to food (standard pellet chow feed) and filtered water ad libitum except for the stress protocol. Mice were treated according to the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA, Registration number 417/01/a/CPCSEA) and all experiments were conducted in adherence to the approved protocol of the Institutional Animal Ethics Committee (IAEC) of Birla Institute of Technology & Science, Pilani, India (Protocol number IAEC/RES/ 14/11, August-2011).

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behavior associated with neurological disorders (Dawes et al., 2005; Faris et al., 2006), which highlights the antidepressant potential of the drug in human subjects as well. Therefore, the objective of the present study was to evaluate the effects of a 5HT3 receptor antagonist, ondansetron in CUS model of depression in mice. The possible modulation of the HPA-axis and brain serotonergic system underlying its effect in CUS model was also investigated.

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2.2. Drugs

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Ondansetron hydrochloride was obtained from Indian Pharmaceutical Combine Association Labs, India. Fluoxetine hydrochloride was generously provided by Ranbaxy Research Laboratories, India and used as a positive control in this study. Corticosterone was purchased from Sigma-Aldrich, USA. All other biochemical reagents used were of analytical grade.

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2.3. Experimental design and dosing schedule

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Forty two mice (7 in each group) were randomly divided into six groups: (1) non-CUS mice received vehicle treatment, (2) CUS mice with vehicle treatment, (3) CUS mice with ondansetron (0.05 mg/kg) treatment, (4) CUS mice received ondansetron (0.1 mg/kg), (5) CUS mice with ondansetron (1 mg/kg) treatment and (6) CUS mice with fluoxetine (10 mg/kg) treatment. Ondansetron and fluoxetine were freshly prepared everyday in distilled water before administration. The drugs were administered by oral gavage (p.o.) once a day for 14 days. The doses and dosing schedule of the drugs were selected on the basis of experiments previously performed in our laboratory and literature data confirming the efficacy of the above mentioned protocol (Dulawa et al., 2004; Ramamoorthy et al., 2008).

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2.4. Chronic unpredictable stress protocol

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Mice in CUS groups were subjected to different types of stressors (Table 1), while mice in normal control group were left undisturbed except for the general housekeeping procedure (Strekalova and Steinbusch, 2010; Jindal et al., 2013). CUS procedure was continued for four successive weeks followed by behavioral and biochemical assessment. Behavioral assays were performed 24 h after the last dosing to avoid the acute effects of the drug with one behavioral test was performed on each day to avoid the residual effect of the earlier testing paradigm. Moreover, plasma and the brain samples were collected 24 h after the last behavioral assay to eliminate the effect of acute stress on the biochemical parameters. The detailed protocol of the study is given in the Fig. 1.

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Steinbusch, 2010). The behavioral despair effects in neurobehavioral models such as forced swim test (FST) have also been demonstrated in mice subjected to chronic stress (Dang et al., 2009; Larsen et al., 2010). It is well reported that chronic stress-induced depression is associated with dysregulation of HPA-axis (Guerry and Hastings, 2011). Exposure to chronic stress results in HPA-axis hyperactivity, which can cause damaging effects to neuronal structures, a common sign of stress-related neuropsychiatric disorder such as depression (De Kloet et al., 2005; Goosens and Sapolsky, 2007). HPA-axis hyperactivity, as characterized by increased levels of circulating glucocorticoids is found both in rodents and humans demonstrating depressive behavior. While hypercortisolism is associated with depressed patients (Holsboer, 2001), preclinical studies have revealed that mice subjected to variety of stressors exhibit elevated corticosterone (CORT) levels (Sanchez et al., 2001; Schmidt et al., 2010; Qiu et al., 2013). Furthermore, earlier reports have shown that treatment with certain antidepressants can reverse this depressive symptomology (Vermetten et al., 2006; Lenze et al., 2011; Jindal et al., 2013). In addition, the biochemical studies have linked altered central monoaminergic system with the consequences of CUS (Gamaro et al., 2003; Dranovsky and Hen, 2006). Mice with CUS have shown decreased serotonin levels that result in reduced serotonin-related responses in the brain (Chung et al., 1999; Mineur et al., 2003). The classic hypothesis of depression, which shows that decreased brain monoamine levels or more specifically deficiency of serotonin in the brain results in depressive episodes, gives one of the basic pathophysiological mechanisms of CUS-induced depression in mice (Hirschfeld, 2001; Iversen, 2005;). Further, the correction of this deficiency reflects the plausible mechanism of several antidepressants, effective in CUS-induced depression (Katz et al., 1981; Willner et al., 1987; Owens, 2004;) Therefore, altered HPA-axis and the serotonergic system play an important role in the neuropathology of stress-related depressive disorder and biochemical assessment of these parameters may help in elucidating the efficacy of the therapeutic interventions. In the last few decades, 5HT3 receptors have been identified as a potential target for depression (Rajkumar and Mahesh, 2010; Carr and Lucki, 2011). Several 5HT 3 receptor antagonists have shown antidepressant-like activity in preclinical and few clinical studies (Greenshaw, 1993; Johnson et al., 2003; Rajkumar and Mahesh, 2010). For example, bemesetron and tropisetron have demonstrated antidepressant-like behavior in the mouse FST (Bravo and Maswood, 2006). Also, 5HT3 receptor knockout mice have elicited enhanced depressive behavior (Bhatnagar et al., 2004). Furthermore, various antidepressants in clinical use such as mirtazepine, fluoxetine and reboxetine have demonstrated functional antagonism of 5HT3 receptors in the brain (Anttila and Leinonen, 2001; Eisensamer et al., 2003). Altogether, these findings reflect the potential involvement of 5HT3 receptors in the pathophysiology of depressive disorder. In addition, several neuropharmacological studies have shown the effectiveness of 5HT3 receptor antagonists at much lower doses than those of clinically effective antidepressants (Redrobe and Bourin, 1997; Ramamoorthy et al., 2008). Moreover, reversal of depressive behavior by 5HT3 receptor antagonists has shown to occur within 2–3 weeks of the treatment, which reveals a major advantage over the current clinical pharmacotherapy (Hewlett et al., 2003; Piche et al., 2005). However, the effects of 5HT3 receptor antagonist in alleviating chronic stress-induced depressive behavior are still open for investigation. In the present study, ondansetron (OND), as a reference compound with selective 5HT3 receptor antagonistic activity, was selected on the basis of previous findings in our laboratory that ondansetron treatment alleviates behavioral effects in mice exposed to acute stressors (Ramamoorthy et al., 2008). Moreover, literary data has shown that co-administration of ondansetron enhanced antidepressant activity of selective serotonin reuptake inhibitors in rodent behavioral models (Redrobe and Bourin, 1997). In few clinical studies, ondansetron has shown potential effects in controlling depressive

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Please cite this article as: Gupta D, et al, 5HT3 receptor antagonist (ondansetron) reverses depressive behavior evoked by chronic unpredictable stress in mice: Modulation of ..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.05.024

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the last 4 min of the 6 min test. The mice were subjected to 15 min train- 214 ing session under similar conditions, 24 h before the test. 215

Table 1 The protocol employed in chronic unpredictable stress. Day

Stress type

Duration of stress (hrs)

t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Food and water deprivation Wet bedding Restraint Cage titling (45°) Inversion of light and dark cycle Tail suspension Predator exposure Food and water deprivation Exposure to empty water bottles Cage titling (45°) Isolation Wet bedding Inversion of light and dark cycle Restraint Tail suspension Isolation Exposure to empty water bottles Predator exposure Food and water deprivation Cage titling (45°) Isolation Tail suspension Inversion of light and dark cycle Restraint Food and water deprivation Predator exposure Cage titling (45°) Wet bedding

24 6 3 6 12 3 3 24 12 6 24 6 12 3 3 24 24 3 24 6 24 3 12 3 24 3 6 6

2.6. Forced swim test

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FST was carried out as described elsewhere with slight modifications (Porsolt et al., 1977; Ramamoorthy et al., 2008). Mice were dropped individually into a plexiglass cylinder (height: 30 cm, diameter: 22.5 cm) filled with water to a depth of 15 cm and maintained at 23–25 °C. In this test, after an initial vigorous activity of 2 min, mice acquired an immobile posture which was characterized by motionless floating in the water and making only those movements necessary to keep the head above the water. The duration of immobility (s), was recorded during

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After 24 h of behavioral assay the mice were decapitated and the blood was collected in clean centrifuge tubes containing disodium ethylenediaminetetraacetate (EDTA, as anticoagulant). The tubes were then centrifuged at 12,000 rpm for 20 min at 4 °C. Plasma was separated and stored at −80 °C until the CORT estimations was carried out. CORT assay was performed by the method of Katyare and Pandya (2005) with slight modifications. Plasma (1 ml) was treated with 0.2 ml of freshly prepared chloroform: methanol mixture (2:1 v/v), followed by extraction with 3 ml of chloroform. The chloroform extract was treated with 0.3 ml of sodium hydroxide (0.1 N) and then with 3 ml of 30 N sulfuric acid. The tubes containing the sulfuric acid layer were kept in the dark for 30–60 min and thereafter fluorescence measurements were carried out in an SL-174-spectrofluorometer with excitation and emission wavelengths set at 472 and 533 nm, respectively. The plasma CORT contents were expressed as percentage with respect to non-CUS group (taking non-CUS group values as 100%).

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2.9. Brain serotonin assay

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The concentrations of serotonin in different regions of the brain like midbrain (along with hippocampus), prefrontal cortex and cerebellum were measured by spectrophotofluorometric method. All chemicals used were of analytical grade. n-Butanol was acidified by adding 85 μl concentrated HCl in 100 ml of n-butanol. 0.004% w/v o-Phthalaldehyde

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The effects of CUS and ondansetron treatment were evaluated on spontaneous locomotor activity in mice using an actophotometer. The apparatus consisted of a dark square chamber (30 cm × 30 cm) with inside walls painted black (Boissier and Simon, 1965; Ramamoorthy et al., 2008). Mice were individually placed in the chamber and after an initial 2 min familiarization period, the digital locomotor scores were recorded for the next 8 min period. The chamber was cleaned with dilute alcohol (70% v/v ethyl alcohol) and dried between trails.

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Sucrose preference test was carried out as described previously (Willner et al., 1987). It is conducted in three phases as follows: phase 1 habituation, phase 2 sucrose preference baseline, and phase 3 sucrose preference testing. In phase 1, tap water in the homecage was replaced with 1% w/v sucrose in tap water for 24 h to habituate mice to the novel solution. In phase 2, each mouse was transferred to single cage and was exposed to both tap water and sucrose solution consequently for 3 days to attain the sucrose preference baseline. Sucrose preference was then determined by a two-bottle choice test using standard bottles, one filled with tap water and one with 1% sucrose solution, supplied to mice for 24 h (phase 3). The locations of bottles containing water and sucrose solution (left/right) were counterbalanced across the study. The tap water and sucrose solution intake was quantified by subtracting the final weight of bottles after 24 h exposure period from their initial weight and averaged for 2 days. The preference to consume sucrose solution was then calculated as percentage preference = [(sucrose intake/total intake) × 100].

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Fig. 1. The schematic representation of the study protocol. CUS, chronic unpredictable stress; non-CUS, unstressed mice; FST, forced swim test; BHA, behavioral assay; BIA, biochemical assay; SPT, sucrose preference test; CORT, corticosterone; 5HT serotonin; SLA, spontaneous locomotor activity.

Please cite this article as: Gupta D, et al, 5HT3 receptor antagonist (ondansetron) reverses depressive behavior evoked by chronic unpredictable stress in mice: Modulation of ..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.05.024

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Data were analyzed using GraphPad prism 3.0 software, USA. The results are expressed as mean ± SEM. The data for body weight were analyzed using two-way ANOVA followed by Sidak test. The results of CUS testing paradigm were analyzed using one-way ANOVA followed by post-hoc Tukey's Multiple Comparison Test.

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3. Results

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3.1. Behavioral analysis

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Table 2 Effects of ondansetron (OND, 0.05, 0.1 and 1 mg/kg) and fluoxetine (FLX, 10 mg/kg) on the body weight in mice.

t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13

Non-CUS CUS CUS + OND (0.05) CUS + OND (0.1) CUS + OND (1) CUS + FLX (10)

Body weight (g) Initial (day 0)

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25.14 26.14 25.71 24.98 25.57 26.43

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± ± ± ± ± ±

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treatment (dose, mg/kg)

3.1.2. Spontaneous locomotor activity There was no significant alteration in the spontaneous locomotor scores between CUS and un-stressed mice. Moreover, ondansetron (0.05, 0.1 and 1 mg/kg) and fluoxetine (10 mg/kg) had no significant changes in spontaneous locomotor scores [F (5,36) = 0.928, p N 0.05 vs. non-CUS mice] as summarized in Fig. 2A.

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3.1.1. Body weight Initially, there were no significant changes in the body weights between mice when randomly assigned to different treatment groups (p N 0.05). However, mice subjected to a 4-week CUS procedure exhibited significant decrease in the body weight as compared to non-CUS mice [F (5,36) = 3.959, p b 0.01 vs. non-CUS group]. Although, there was a decrease in the body weight of mice in treatment groups (ondansetron and fluoxetine), the decrease was not statistically significant as compared to non-CUS mice [F (4,30) = 3.959, p N 0.05 vs. nonCUS group], Table 2.

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(OPT) in 10 N hydrochloric acid (HCl), 0.1% w/v L-cysteine in 0.1 N HCl and standard serotonin in millipore water were prepared immediately before use. After 24 h of the last behavioral assay, the mice were decapitated, the brain samples were collected and mid brain (along with hippocampus), prefrontal cortex and cerebellum were dissected as previously described in clean centrifuge tubes on ice bath (Glowinski and Iversen, 1966). The samples were washed and homogenized in 1 ml of cold acidified n-butanol. The homogenates were centrifuged at 3000 rpm for 20 min at 4 °C. 500 μl of the resulting supernatant was mixed with 1 ml of n-heptane and 80 μl of 0.1 N HCl (containing 0.1% L-cysteine). The resultant mixture is mechanically shaken for 5 min and the phases were separated by centrifugation (3000 rpm, 4 °C for 20 min). The 0.1 ml samples of the aqueous phase were pipetted into another centrifuge tubes and 0.6 ml of 0.004% w/v o-phthalaldehyde was added. The tubes were shaken and heated over the boiling water bath for 15 min. The tubes were cooled and fluorescence was measured in micro-cuvettes. Activation and fluorescent wavelengths used were 360 and 465 nm, respectively. The serotonin levels were then determined using calibration curves of respective brain regions and expressed as the percentage with respect to non-CUS group (taking non-CUS group values as 100%) (Curzon and Green, 1970).

% sucrose preference

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D. Gupta et al. / Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx

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0.37 1.27 ⁎ 0.87 0.83 1.00 1.23

Values represent mean ± SEM. Results from post hoc Sidak test are indicated as; *p b 0.05, as compared to unstressed control mice (non-CUS); n = 7/group.

Fig. 2. Effects of ondansetron (OND, 0.05, 0.1 and 1 mg/kg) and fluoxetine (FLX, 10 mg/kg) on spontaneous locomotor activity in actophotometer (A), duration of immobility during FST (B) and % of sucrose preference in sucrose preference test. The columns represent mean values, while error bars show SEM. The Results from post hoc Tukey's Multiple Comparison Test are indicated in the figure; ⁎p b 0.05, ^p b 0.01 as compared to unstressed control mice and #p b 0.05, ##p b 0.01 as compared to CUS control mice; n = 7/group.

3.1.3. Forced swim test After a 4-week CUS procedure, mice elicited pronounced increase in the duration of immobility as compared to non-CUS mice [F (5,36) = 5.746, p b 0.05]. Treatment with the positive control fluoxetine (10 mg/kg), significantly reversed CUS-induced increased duration of immobility [F (5,36) = 5.746, p b 0.01 vs. CUS control] in stressed mice. In addition, ondansetron treatment significantly decreased the duration of immobility in stressed mice [F (5,36) = 5.746, p b 0.05 vs. CUS control for 0.05 mg/kg and p b 0.01 for 0.1 and 1 mg/kg], Fig. 2B.

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3.1.4. Sucrose preference test CUS exposure significantly decreased the percentage of sucrose preference in mice when compared with the unstressed mice [F (5,36) = 8.318, p b 0.01 vs. non-CUS mice]. Chronic treatment with fluoxetine

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Please cite this article as: Gupta D, et al, 5HT3 receptor antagonist (ondansetron) reverses depressive behavior evoked by chronic unpredictable stress in mice: Modulation of ..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.05.024

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3.2.2. Brain serotonin levels The effects of ondansetron and fluoxetine on the brain serotonin content were evaluated. CUS exposure resulted in significant decrease in the percentage of serotonin levels in the mice in discrete areas of the brain including mid brain [F (5,36) = 9.412, p b 0.01 vs. non-CUS group], prefrontal cortex [F (5,36) = 10.36, p b 0.01 vs. non-CUS group] and cerebellum [F (5,36) = 9.852, p b 0.01 vs. non-CUS group] when compared to unstressed mice. Chronic treatment with ondansetron exhibited increased percentage of serotonin in the mid brain (containing hippocampus) [F (5,36) = 9.412, p b 0.05 vs. CUS group for 0.05– 1 mg/kg] and prefrontal cortex [F (5,36) = 10.36, p b 0.05 vs. CUS group for 0.05 mg/kg and p b 0.01 for 0.1 and 1 mg/kg] of chronically stressed mice. However, only ondansetron at 1 mg/kg exhibited increased % of serotonin levels in cerebellum [F (5,36) = 9.852, p b 0.05

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Fig. 3. Effects of ondansetron (OND, 0.05, 0.1 and 1 mg/kg) and fluoxetine (FLX, 10 mg/kg) on % of plasma corticosterone (CORT) levels (A), and the % of brain serotonin contents (B). The columns represent mean values, while error bars show SEM. The Results from post hoc Tukey's test are indicated in the figure; ^p b 0.01 as compared with non-CUS mice and #p b 0.05, ##p b 0.01 as compared with the CUS control mice; n = 7/group.

Depression is probably the most common stress-related neuropsychiatric disorder, which occurs as an outcome of an eventual inability to deal with a stream of dissimilar and unpleasant stimuli imposed by the environment (Lanfumey et al., 2008). In the present study, CUS model which simulates many of the stress-induced human depressive symptomology, with considerable influence on behavioral and biochemical systems, has been used. CUS mice elicited pronounced behavioral derangements with significant alterations in the brain serotonergic and HPA-axis systems. On the other hand, ondansetron (0.05, 0.1 and 1 mg/kg), as a reference compound with selective 5HT3 receptor antagonistic activity, and fluoxetine (10 mg/kg) significantly reversed stressinduced depressive behavior as evidenced by decreased despair effects (duration of immobility) in FST and anhedonia in sucrose preference test. Moreover, ondansetron (0.05, 0.1 and 1 mg/kg) and fluoxetine (10 mg/kg) significantly increased the serotonin levels in the discrete areas of brain and inhibited CUS-induced HPA-axis hyperactivity as indicated by decreased percentage of plasma CORT levels. The chronic exposure to various uncontrollable stressors in an unpredictable manner is a well-documented animal model for the preclinical evaluation of antidepressants (Katz and Schmaltz 1980; Willner, 1997, 2005). CUS is a widely accepted model of depression with high predictive (behavioral changes are reversed by chronic treatment with different classes of antidepressants), constructive (CUS results in anhedonia, the core symptom of the major depressive disorder) and face validities (various observable behavioral and biochemical features of human depression can be demonstrated) (Willner, 1997, 2005; De Kloet et al., 2005; Nestler and Hyman, 2010). Hence, this model can be used to assess the effects of new drug molecules on stress-induced depressive behavior. In the present study, CUS mice exhibited a gradual in body weight progressively during the stress procedure, which is similar to the results of the previous studies (Konkle et al., 2003; Lucca et al., 2008), while mice with ondansetron (0.05, 0.1 and 1 mg/kg), or fluoxetine (10 mg/kg) treatments did not experience a significant loss of body weight due to chronic stress exposure. Weight loss is a symptom of major depressive disorder and a significant decrease in body weight observed after the CUS procedure may be how this symptom is phenotypically expressed (APA, 2000). It is unlikely that blunted weight gain in CUS mice is due to deprivation of food and water during the stress protocol, because the decrease in body weight was insignificant in drug treated groups, which were otherwise exposed to the same stress procedure. Additionally, it is unlikely that the anhedonia seen in CUS mice could account for decreased body weight as it is reported earlier that measure of anhedonia is independent of body weight changes (Willner et al., 1996). The behavioral despair tests such as FST are the most widely accepted models to assess the effects of the compounds on depressive behavior (Cryan et al., 2005). The increased duration of immobility reflects a state of hopelessness which is a core symptom of major depressive disorder in humans (Castagne et al., 2011). It is well reported that, stress produces depressive conditions in mice (like increase in duration of immobility during FST) that can be reversed by chronic treatment with antidepressants (Larsen et al., 2010). In the current finding, CUS mice exhibited pronounced behavioral despair effects as evidenced by increased duration of immobility in FST. Chronic fluoxetine (10 mg/kg) treatment reversed the CUS-induced elevated duration of immobility in mice, demonstrating high predictive validity of CUS model. Chronic treatment with the 5HT3 antagonist ondansetron (0.05, 0.1 and 1 mg/kg), similar to that of the positive control fluoxetine

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3.2.1. Plasma corticosterone levels Effects of ondansetron and fluoxetine treatments were estimated on plasma CORT levels. CUS procedure for four weeks significantly elevated percentage of plasma CORT levels [F (5,36) = 5.822, p b 0.01 vs. non-CUS group]. Chronic ondansetron administration (0.05, 0.1 and 1 mg/kg), on the other hand, significantly decreased the percentage of plasma CORT levels in stressed mice [F (5,36) = 5.822, p b 0.05 vs. CUS group for 0.05 and 1 mg/kg; p b 0.01 vs. CUS mice for 0.01 mg/kg]. Fluoxetine (10 mg/kg) treatment also elicited significant decrease in percentage of plasma CORT levels in stressed mice [F (5,36) = 5.822, p b 0.05 vs. CUS control group], Fig. 3A.

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vs. CUS group] and ondansetron (0.05 and 0.1 mg/kg) had no significant increase in the percentage of the serotonin content in cerebellum. Fluoxetine on the other hand elevated the serotonin content in all the measured regions of the brain (mid brain, prefrontal cortex and cerebellum) (p b 0.01 vs. CUS group), Fig. 3B.

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(10 mg/kg) increased the % of sucrose preference in stressed mice [F (4,30) = 8.318, p b 0.01 vs. CUS control]. Moreover, ondansetron administration, similar to that of the positive control, increased the % of sucrose preference in chronic stressed mice [F (4,30) = 8.318, p b 0.05 vs. CUS control mice for 0.05 mg/kg; p b 0.01 vs. CUS control mice for 0.1 and 1 mg/kg], Fig. 2C.

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may produce significant effects in CUS-induced depressive behavior and may involve modulation of HPA-axis as one of the possible mechanisms. Impairment in monoaminergic activity is another major biochemical alterations associated with CUS-induced depressive disorders (Gamaro et al., 2003; Dranovsky and Hen, 2006). Serotonin, a monoamine neurotransmitter, which primarily regulates mood and emotional behavior, is found to be reduced in rodents as well as in humans exposed to chronic stress (Chung et al., 1999; Mineur et al., 2003). In the current study, CUS resulted in significant decrease in serotonin levels in discrete areas of the brain such as prefrontal cortex, mid brain (containing hippocampus) and cerebellum as compared to unstressed mice, which is thought to be associated with impairment of serotonergic neurotransmission mediated regulation of behavioral activity in stressed mice. This contention is further supported by the observation that intra-hippocampal injection of serotonin has demonstrated to reverse chronic stressinduced depressive behavior (Luo et al., 2008). Ondansetron, similar to that of the positive control fluoxetine, reversed CUS-induced serotonergic deficits in mid brain (along with hippocampus), prefrontal cortex and cerebellum. However, it is important to note that ondansetron only at higher dose of 1 mg/kg elicited significant effect on serotonin levels in cerebellum. The reason of this observation is uncertain, but could be attributed to the dose dependent activity of the test compound; that the lower doses of ondansetron were ineffective to increase the serotonin content up to a significant level. Further investigations are necessary to unpin the plausible reasons. Besides, in accordance with the previous reports, the current finding revealed that the modulation of the brain serotonergic system may be involved in the postulated effects of the test drug in CUS model of depression (Katz et al., 1981; Willner et al., 1987; Owens, 2004). Moreover, previous reports have shown that 5HT3 receptor antagonists increase the synaptic serotonergic neurotransmission, (Rajkumar and Mahesh, 2010) further supporting the present finding. Altogether, the present study showed that, chronic stress elicited pronounced depressive behavior in mice, as indicated by behavioral despair effect in FST and reward-related deficits in sucrose preference test, which reflect hopelessness and anhedonia, respectively, the core symptoms of major depressive disorder in humans. Biochemical assessment demonstrated that CUS resulted in HPA-axis hyperactivity and impaired serotonergic system. Ondansetron (0.05, 0.1 and 1 mg/kg), a 5HT3 receptor antagonist, significantly reversed depressive behavior evoked by CUS in mice. Moreover, reversal of CUS-induced HPA-axis and serotonergic dysregulation following chronic ondansetron (0.05, 0.1 and 1 mg/kg) treatment revealed that modulation of HPA-axis and serotonergic system is involved in the postulated effect of the compound. Therefore, the results of the present finding provide new evidences that the drugs with 5HT3 receptor antagonistic activity may alleviate chronic stress-induced depressive behavior by modulating HPA-axis and brain serotonergic system. However, studies at molecular levels are required to elucidate the further mechanism(s) such as post receptor action and signal transduction pathways, involved in the modulatory effects of 5HT3 receptor antagonists (such as ondansetron) on these regulatory systems.

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(10 mg/kg), reversed CUS-induced despair behavior which is considered to reflect the potential effects of the drug in stress-induced depressive derangements. Besides, to nullify the hypothesis that psychostimulant effects of the drug might be involved in the behavioral activity during FST, the effects of ondansetron and fluoxetine on spontaneous locomotor activity were investigated. Interestingly, stressed mice had no influence on locomotor scores, which is in accordance with the previous reports (Jindal et al., 2013). Also, neither ondansetron (0.05, 0.1 and 1 mg/kg) nor fluoxetine (10 mg/kg) at tested doses affected locomotor scores in mice. This is in agreement with the previous findings that ondansetron and fluoxetine treatments do not have generalized stimulant effects on locomotor activity (Ramamoorthy et al., 2010) Anhedonia, the inability to experience pleasure or reward is one of the cardinal symptoms of major depressive disorder (APA, 2000). This behavior can be easily assessed in rodents by their preference to consume palatable sucrose solution over drinking water. The test is based on the principle that (1) consumption of sucrose solution is a valid measure of reward-related behavior and (2) that CUS causes a generalized decrease in reward-related behavior, rather than a specific effect on responses to sweet taste (Willner, 1997). Earlier reports have demonstrated that CUS exposure results in a significant decrease in preference of sucrose solution over drinking water, which can be reversed by chronic antidepressant treatment (Harkin et al., 2002; Gronli et al., 2005). In the present report, stressed mice exhibited a significant reduction in preference of sucrose consumption over normal drinking water, an indicator of decreased reward behavior in stressed mice. Moreover, the decrease in sucrose consumption cannot be explained by nonspecific changes in fluid consumption (or decreased thirst) as intake of total fluid (drinking water) was unaffected by CUS (data not shown) (Willner, 1997). Ondansetron (0.05, 0.1 and 1 mg/kg) as well as fluoxetine (10 mg/kg) significantly increased the percentage of sucrose preference over drinking water in CUS mice. This indicates the positive influence of ondansetron in reward-related behavior, which reflects the considerable effects of the tested drug in chronic stress-induced anhedonia-like behavior. In addition, the ability of fluoxetine in reversing CUS-induced depressive behavior supports the idea that it has significant effects in stress-related depressive disorder. One of the major pathophysiology of CUS-induced depression is dysregulation of HPA system (Guerry and Hastings, 2011). Under normal physiology, acute stress stimulates hypothalamus to release corticotrophin releasing hormone, which triggers the release of adrenocorticotropic hormone (ACTH) from pituitary gland to stimulate CORT secretion from adrenals. This in turn restores the homeostasis, followed by suppression of stimulated HPA-axis activity by negative feedback regulation of CORT (Lanfumey et al., 2008). However, prolonged activation of HPA-axis (as in case of exposure to persistent stress) results in functional hypertrophy of adrenal glands with subsequent elevation of CORT release, which can cause neurocellular damage resulting in impaired neuronal activity (Lanfumey et al., 2008; Schmidt et al., 2010). This suggests that excessive CORT levels can have severe impact on neurocellular functions in the brain and increased plasma CORT levels can serve as a putative marker of HPA-axis hyperactivity. Previous studies have revealed that chronic stress stimulates HPA-axis activity and increases CORT levels (De Kloet et al., 2005; Goosens and Sapolsky, 2007; Garcia et al., 2009). Similar to the previous findings, in this study CUS mice exhibited significant increase in plasma CORT levels, an indicator of HPA-axis hyperactivity. Subsequently, the plasma CORT levels underwent reduction by chronic treatment with ondansetron (0.05, 0.1 and 1 mg/kg) and fluoxetine (10 mg/kg). This is in accordance with the earlier reports that antidepressants inhibit HPA-axis hyperactivity due to chronic stress (Schmidt et al., 2007). Moreover, previous studies have demonstrated the significant influence of 5HT3 receptor antagonists on ACTH mediated stimulation of CORT release in vitro (Calogero et al., 1995), which further support the current findings. At this juncture, it is worth mentioning that ondansetron

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