Steroids 96 (2015) 95–102

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Effect of a novel 5-HT3 receptor antagonist 4i, in corticosterone-induced depression-like behavior and oxidative stress in mice Deepali Gupta a,⇑, Mahesh Radhakrishnan b, Yeshwant Kurhe a a b

Department of Pharmacy, Birla Institute of Technology & Science, Pilani, Rajasthan 333031, India Faculty Affairs and Professor, Department of Pharmacy, Birla Institute of Technology and Science, Pilani, Rajasthan 333031, India

a r t i c l e

i n f o

Article history: Received 11 August 2014 Received in revised form 21 January 2015 Accepted 23 January 2015 Available online 7 February 2015 Keywords: 5HT3 receptor antagonist HPA-axis Corticosterone Depression Oxidative stress

a b s t r a c t Stress in our daily life severely affects the normal physiology of the biological system. Dysregulation of hypothalamic–pituitary–adrenal (HPA) axis has been implicated in the development of depression-like behavior, which remains under diagnosed and poorly treated. Exogenous corticosterone (CORT) administration has been demonstrated to develop a depression model, which has shown to mimic HPA-axis induced depression-like state in rodents. In the present study, the effect of a novel 5HT3 receptor, 4i was examined on CORT induced depression in mice. CORT (30 mg/kg, subcutaneously) was given for 4-weeks to mice in control group, while mice in drug treated group were given 4i (0.5–1 mg/kg, intraperitoneally)/fluoxetine (as a positive control, 10 mg/kg), for the last 2-weeks of CORT dosing. Repeated CORT dosing caused depression-like behavior in mice as indicated by increased despair effects in forced swim test (FST) and anhedonia in sucrose preference test. In addition, CORT administration induced oxidative load in the brain with significant increase in pro-oxidant (lipid peroxidation and nitrite levels) markers and a substantial decline in anti-oxidant defense (catalase and reduced glutathione levels) system, indicating a direct effect of stress hormones in the induction of the brain oxidative damage. On the other hand, 4i and fluoxetine treatment reversed the CORT induced depressive-like deficits. Furthermore, 4i and fluoxetine prevented CORT induced oxidative brain insults, which may plausibly demonstrate one of the key mechanisms for antidepressant-like effects of the compounds. Thus, the study suggests that 5HT3 antagonist; 4i may be implicated as pharmacological intervention targeting depressive-like anomaly associated with HPA-axis dysregulation. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Stress is crucially related to the pathophysiology of mood disorders. One of the key adaptive responses to stress involves stimulation of hypothalamic–pituitary–adrenal (HPA) axis releasing glucocorticoids (GCs) (cortisol in humans and corticosterone (CORT) in rodents) to the systemic circulation [1]. GCs elicit a spectrum of physiologic changes that help the organism deal with an acute stressor in an effective manner. However, excessive release of GCs can promote psychological dysfunctions such as depression and anxiety [1,2]. The hypothesis linking depression and excess GCs is based on the clinical observations that patients with elevated levels of GCs develop depressive-like symptomology and inversely, depressed patients show impairments in HPA axis functions and high circulating GC levels [3–5]. In addition, earlier studies have shown that high GC levels also correlate with anxiety ⇑ Corresponding author. Mobile: +91 9352011573; fax: +91 1596244183. E-mail addresses: [email protected] (D. Gupta), rmaheshbits@gmail. com (M. Radhakrishnan), [email protected] (Y. Kurhe). http://dx.doi.org/10.1016/j.steroids.2015.01.021 0039-128X/Ó 2015 Elsevier Inc. All rights reserved.

disorders [6,7] and more specifically that, depression shows a strong relationship with high levels of GCs when co-morbid anxiety is present [8]. Accordingly, an animal model has been developed using exogenous CORT administration in rodents to mimic the pathophysiological changes associated with stressful events that can invoke depression-like behavior [6,9]. CORT-induced depression model has advantage over the stress models (such as restraint stress exposure) that it avoid the possibility of potential habituation effects and variation in HPA axis response to stress stimuli [10,11]. Previous reports have shown that repeated CORT administration develops depression-like behavior in mice during forced swim test (FST) [10]. Chronic treatment with CORT has shown anhedonia-like behavior in sucrose consumption test [12]. Furthermore, preclinical studies have revealed that elevated CORT level is sufficient enough to induce anxiety in mice [6]. David et al. have observed that CORT treated mice exhibit anxiety-like behavior in open field and novelty suppressed feeding paradigms [6]. Therefore, chronic corticosterone treatment appears to model an anxious and depression-like state in mice and help to evaluate

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the potential activity of compounds in the management of depressive disorders associated with dysfunctional HPA axis activity. Among the complex interplay of multitudinous pathophysiological factors of depression, oxidative stress has been reported to play a key role [13,14]. The theory of oxidative stress in depression, at its most basic, can be explained by the concept that excessive amount of free radicals are toxic to the neuronal cells and can affect the physiological activity of the brain [15,16]. The accumulated reactive oxygen species (ROS) are highly unstable in nature and have potential to damage cellular proteins, lipids and nucleic acids [15]. The endogenous antioxidant enzymes have free radical scavenging action [16]. However, enhanced accumulation of free radicals due to increased production and/or deficiencies of antioxidant defense results in oxidative stress in the brain that culminate in the development of psychological deficits such as depression [13,17]. Previous reports have shown that repeated CORT treatment results in increased pro-oxidant markers such as lipid peroxidation with subsequent decline in antioxidant enzyme (reduced glutathione and catalase) activities in the brain [18]. This suggests that stress hormones have a causal role in oxidative processes and abnormal activity can have deleterious effects. Furthermore, examining the potential effects of novel compounds on oxidative insults induced by increased GCs activity in correlation with the behavioral response may provide the possible mode of their action and hold important implications for therapeutic interventions. In the last decade, 5-HT3 receptors have been identified as potential targets for the management of depressive disorders. The antagonism of 5-HT3 receptors has demonstrated antidepressant effects [19]. Unique among the serotonin receptors subtype, 5-HT3 receptors belong to ligand gated ion channels superfamily, which may possibly account for the quick onset of the therapeutic effects (unlike conventional antidepressants, which show recovery of depressive symptomology only after 2–3 weeks of consecutive treatment) and potential activity at lower dose ranges [20–24]. 5-HT3 receptors antagonists like tropisetron, bemesetron and ondansetron have shown significant antidepressant-like effects in preclinical as well as in clinical studies [19,20,24–26]. Ramamoorthy et al. have shown that ondansetron decreases duration of immobility in FST [20]. When given concomitantly it potentiates the anti-immobility effects of selective serotonin reuptake inhibitors (SSRIs) [27]. Earlier reports have evidenced the antidepressant and anxiolytic-like effects of novel 5-HT3 receptor antagonists (like MCI-225) in rodent behavioral models [28,29]. Also the recent findings have revealed that novel compounds with potential 5-HT3 antagonistic activity exhibit antidepressant and anxiolytic-like effects [30,31]. In addition, the findings that the different classes of antidepressants are functional antagonists at 5-HT3 receptors convincingly support the potential activity of 5-HT3 antagonists in attenuating depression-like behavior [32]. In our previous study, we have reported the antidepressant and anti-anxiety-like activity of a novel 5-HT3 receptor antagonist, 4i in acute and chronic rodent models [33]. However, the probable activity of 4i in depression associated with dysfunctional HPA axis activity has not been evaluated. Therefore, the present study was designed to examine the effects of 4i in CORT-induced depression in mice and to clarify the possible involvement of putative 5-HT3 receptors in depression associated with abnormal HPA axis functions. Furthermore, the effects of 4i on oxidative stress in the brain were evaluated as a probable mechanism of action.

Animals were group housed in cages and were maintained in standard laboratory conditions with alternating light–dark cycle of 12 h each, temperature 23 ± 2 °C and humidity conditions 62 ± 5% relative humidity in the housing unit. Animals had free access to food (standard pellet chow feed) and filtered water ad libitum. Behavioral testing was done during the light cycle. Animals 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/REV/06, August-2012). 2.2. Drugs 4i, N-(3-Chloro-2-methylphenyl) quinoxalin-2-carboxamide (Fig. 1) was synthesized and its structure was confirmed with Infrared (IR) spectroscopy, Mass spectrometry (MS) and Proton Nuclear Magnetic Resonance (H1NMR) spectroscopy by the Medicinal Chemistry Group, BITS-Pilani, India. The chemical synthesis scheme and analysis data is given in Supplementary material. Fluoxetine (FLX, a selective serotonin reuptake inhibitor) was obtained from Ranbaxy Research Laboratories, India. CORT was purchased from Sigma–Aldrich, Chemicals, USA). 2.3. Study protocol CORT (40 mg/kg) was freshly prepared everyday in 0.25% w/v carboxymethylcellulose and given subcutaneously (s.c.) in a constant volume of 1 mg/kg body weight once daily for 4-weeks (28 consecutive days). Mice in vehicle groups received only vehicle (s.c.) without CORT for the same period. 4i and FLX were dissolved in 0.9% w/v saline and administered intraperitoneally (i.p.) at 10 ml/kg body weight during the last 2-weeks of CORT dosing. The drugs were injected 1 h prior to CORT treatment. All dosing were carried out between 0900 and 1100 h and all behavioral assays were conducted between 0900 and 1100 h to avoid any confounding results that may have been obtained due to conducting experiments during the hours when rodent plasma CORT rises sharply (usually between 1200 and 2000 h). The doses of the drugs were selected on the basis of previous studies [33]. Behavioral assays were carried out 24 h after the last drug/vehicle dosing for three consecutive days, these included spontaneous locomotor activity (SLA), FST and light/dark aversion test. Twenty four hours after the behavioral assay, mice were weighed and decapitated. The brain samples were then stored at 80 °C until biochemical analysis. A timeline of the experiment is depicted in the Fig. 2. 2.4. Spontaneous locomotor activity The effects of CORT, 4i and FLX treatment on SLA were assessed using the actophotometer [20,34]. It consisted of a dark square chamber (30 cm  30 cm) with inside walls painted black. 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 (70% v/v) alcohol and dried between trials.

2. Materials and methods 2.1. Animals Swiss Albino male mice (22–25 g; age: 11–12 weeks) were obtained from Hisar Agricultural University, Haryana, India.

O N

N H

Cl

N Fig. 1. Structure of 4i (N-(3-chloro-2-methylphenyl) quinoxalin-2-carboxamide).

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Day 1 7 Treatment CORT/vehicle dosing

14 21 28 4i/FLX/vehicle dosing

29 30 31 SLA FST L/D

32 Collection of brain

Measurement of oxidative stress parameters Fig. 2. The schematic representation of the study protocol. CORT, corticosterone; FLX, fluoxetine; FST, forced swim test; L/D, light–dark test; SLA, spontaneous locomotor activity.

2.5. Forced swim test FST was carried out as described elsewhere with slight modifications [35]. 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) and number of quadrants crossed (as a measure of swimming behavior) were recorded during the last 4 min of the 6 min test [36]. The mice were subjected to 15 min training session under similar conditions, 24 h before the test. 2.6. Light–dark box test The method of Crawley and Goodwin [37] was adopted with slight modifications [38]. In light–dark model, mice were individually kept in a polypropylene chamber (44  21  21 cm) in which 2/3rd of the light chamber was separated from 1/3rd dark chamber with 13 cm long block having 5 cm high openings. The light chamber was illuminated with a 60 Watt bulb. The animal was keenly observed for latency of the first entry into dark chamber and time spent in the light chamber over a time period of 5 min. The apparatus was cleaned as mentioned above. 2.7. Oxidative stress parameters 2.7.1. Preparation of brain homogenate Animals were sacrificed by decapitation. The brains were quickly removed and washed with ice-cold sterile saline (0.9% w/ v). The whole brain samples were then homogenized with ice-cold 0.1 M phosphate buffer (pH 7.4) 10 times (w/v). The homogenate was centrifuged at 2500g (4 °C) for 15 min to remove cellular debris and aliquots of supernatant were separated and used for oxidative stress indices estimations. 2.7.2. Lipid peroxidation Malondialdehyde (MDA) content, a measure of lipid peroxidation, was measured in whole brain content in the form of thiobarbituric acid reactive substance (TBARS) as per the reported method [39]. Briefly, 0.5 ml of brain homogenate and 0.5 ml of Tris–HCl were incubated at 37 °C for 2 h. After incubation, 1 ml of 10% trichloroacetic acid was added and centrifuged at 1000 rpm for 10 min. To 1 ml of supernatant, 1 ml of 0.67% thiobarbituric acid was added and the tubes were kept in boiling water for 10 min. After cooling 1 ml double distilled water was added and absorbance was measured at 532 nm. TBARS were quantified using an extinction coefficient of 1.56  105 M 1 cm 1. Protein (mg/g of tissue) was estimated by commercial available kit. The brain MDA content was expressed as nmols of MDA per mg of protein.

2.7.3. Nitrite levels Nitrite levels were estimated in the whole brain content using the Greiss reagent which served as an indicator of nitric oxide production [40]. A measure of 500 ll of Greiss reagent (1:1 solution of 1% sulphanilamide in 5% phosphoric acid and 0.1% naphthylamine diamine dihydrochloric acid in water) was added to 100 ll of brain homogenate and absorbance was measured at 546 nm. Nitrite levels were calculated using a standard curve for sodium nitrite. Nitrite levels were expressed as percentage of control (unit activity). 2.7.4. Catalase activity Catalase activity was assessed in the brain by the standard method [41]. Briefly, the assay mixture consisted of 1.95 ml phosphate buffer (0.05 M, pH 7.0), 1.0 ml hydrogen peroxide (0.019 M) and 0.05 ml brain homogenate (10%) in a final volume of 3.0 ml. Changes in absorbance were recorded at 240 nm. Catalase activity was calculated in terms of k min 1 and expressed as mean ± SEM. 2.7.5. Reduced glutathione Reduced glutathione in the brain was estimated according to the method described by Ellman [42]. 1 ml supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold digested at 4 °C for 1 h. The samples were centrifuged at 1200g for 15 min at 4 °C. To 1 ml of this supernatant, 2.7 ml of phosphate buffer (0.1 mol/l, pH 8) and 0.2 ml of 5, 5-dithio-bis (2-nitrobenzoic acid) were added. The color developed was measured immediately at 412 nm (UV-1800 Spectrophotometer, Shimadzu, Japan). Results were expressed as micromole per milligram protein. 2.8. Statistical analysis Data of the present study were analyzed by GraphPad Prism software (version 3.0), USA. All values were expressed as mean ± standard error mean (S.E.M.). The intergroup variation was measured by one way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison post hoc test. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Effect of CORT and 4i on spontaneous locomotor activity The effects of CORT and 4i on the locomotor activity of mice were evaluated and illustrated in Fig. 3. It was found that CORT treatment had no significant effect on the spontaneous locomotor scores [F4,25 = 1.60, p > 0.05] as compared to untreated control mice. Similarly, 4i and FLX (the positive control used in the present study) treatment did not significantly affect the locomotor activity of mice [F4,25 = 1.60, p > 0.05].

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p < 0.05 for 4i (0.5 mg/kg) and p < 0.01 for 4i (1 mg/kg) vs. CORT– CON group]. The number of quadrants crossed was also increased in mice after 4i dosing compared to those given only CORT [F4,25 = 7.50, p < 0.05 for 4i (0.5 mg/kg) and p < 0.01 for 4i (1 mg/ kg) vs. CORT–CON group]. Similarly, chronic treatment of FLX, the positive control used in this study decreased the duration of immobility (s) [F4,25 = 8.53, p < 0.001 vs. CORT–CON group] and increased number of quadrants crossed [F4,25 = 7.50, p < 0.001 vs. CORT–CON group] in comparison to mice treated with CORT alone (Fig. 4A and B).

450 spontaneous locomotor scores

400 350 300 250 200 150 100 50 0 CON

CORT-CON CORT-4i (0.5) CORT-4i (1) treatment (dose, mg/kg)

CORT-FLX (10)

Fig. 3. Effects of CORT, 4i (0.5–1 g/kg) and fluoxetine (FLX, 10 mg/kg) on spontaneous locomotor activity in actophotometer.

3.2. Effect of CORT and 4i on forced swim test As depicted in the Fig. 4, there was a significant increase in the duration of immobility (s) of mice after chronic administration of CORT [F4,25 = 8.53, p < 0.001 vs. CON group]. Furthermore, CORT treatment produced a significant decrease in the number of quadrants crossed by mice when subjected to FST [F4,25 = 7.50, p < 0.01 vs. CON group]. On the other hand, 4i administration resulted in a statistically decrease in the duration of immobility (s) in mice as compared to those received only CORT treatment [F4,25 = 8.53,

The effect of CORT and 4i treatment on mice behavior during light–dark box test is depicted in the Fig. 5. CORT treatment significantly decreased latency (time to leave the light chamber for the first time) as compared to untreated control mice [F4,25 = 4.54, p < 0.05 vs. CON group]. Similarly, the time spent in light chamber was also decreased significantly in CORT treated group [F4,25 = 6.08, p < 0.01 vs. CON group]. On the other hand, 4i treatment at 1 mg/kg dose resulted in the significant increase in latency [F4,25 = 4.54, p < 0.05 vs. CORT–CON group] and time spent in light chamber [F4,25 = 6.08, p < 0.01 vs. CORT–CON group] in mice as compared to CORT treated control mice. While, the lower dose of the test drug 4i (0.5 mg/kg) although increased the latency and time spent in light chamber but not up to a statistically significant level. In addition, the positive control used in the study (FLX) increased

A 70

A 200

#

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80

latency (s)

duraon of immobility (s)

3.3. Effect of CORT and 4i on light–dark box test

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0 CON

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CORT-4i (0.5)

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CORT-CON CORT-4i (0.5) CORT-4i (1) treatment (dose, mg/kg)

CORT-FLX (10)

treatment (dose, mg/kg)

B 180

80

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number of quadrants crossed

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160 me spent in light chamber (s)

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0 CON

CORT-CON

CORT-4i (0.5)

CORT-4i (1)

CORT-FLX (10)

treatment (dose, mg/kg) Fig. 4. Effects of CORT, 4i (0.5–1 g/kg) and fluoxetine (FLX, 10 mg/kg) on duration of immobility (s) (A) and number of quadrants crossed (B) during FST. 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 < 0.05, #p < 0.01 significant as compared to untreated control mice (CON group) and ⁄p < 0.05, ⁄⁄ p < 0.01 significant as compared to CORT treated control mice (CORT–CON group); n = 6/group.

CON

CORT-CON

CORT-4i (0.5)

CORT-4i (1)

CORT-FLX (10)

treatment (dose, mg/kg) Fig. 5. Effects of CORT, 4i (0.5–1 g/kg) and fluoxetine (FLX, 10 mg/kg) on latency (s) (A) and time spent in light chamber (s) (B) during light–dark 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 < 0.01 significant as compared to untreated control mice (CON group) and ⁄p < 0.05, ⁄⁄p < 0.01 significant as compared to CORT treated control mice (CORT–CON group); n = 6/ group.

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D. Gupta et al. / Steroids 96 (2015) 95–102 Table 1 Effects of CORT, 4i (0.5–1 mg/kg) and fluoxetine (FLX, 10 mg/kg) on the pro-oxidant indices and anti-oxidant defense markers in whole brain content of mice. Group

CON CORT–CON CORT-4i (0.5) CORT-4i (1) CORT-FLX (10)

Pro-oxidant markers

Anti-oxidant markers

MDA (nmols/mg protein)

Nitrite (lg/ml)

CAT (U/mg protein)

RGH (lmol/mg protein)

2.835 ± 0.713 6.026 ± 1.682b 3.606 ± 0.582c 3.598 ± 1.086c 2.692 ± 0.364d

35.052 ± 2.421 90.885 ± 11.880b 44.271 ± 5.943d 50.703 ± 13.976c 40.599 ± 4.305d

0.679 ± 0.015 0.154 ± 0.789b 0.420 ± 0.306 0.661 ± 1.456c 0.596 ± 0.874c

0.159 ± 0.015 0.026 ± 0.005a 0.160 ± 0.022c 0.171 ± 0.054c 0.169 ± 0.022c

The results from post hoc Tukey’s Multiple Comparison Test are indicated as; ap < 0.05, bp < 0.01 significant as compared to untreated control mice (CON group) and cp < 0.05, d p < 0.01 significant as compared to CORT treated control mice (CORT–CON group); n = 6/group.

latency [F4,25 = 4.54, p < 0.01 vs. CORT–CON group] and time spent in light chamber [F4,25 = 6.08, p < 0.01 vs. CORT–CON group] in mice as compared to CORT alone treated mice (Fig. 5A and B).

levels [F4,25 = 4.69, p < 0.05 vs. CORT–CON group for 4i (0.5 and 1 mg/kg) and FLX (10 mg/kg)] in the brain of CORT treated mice as compared to those treated with CORT alone.

3.4. Effect of CORT and 4i on oxidative stress in the brain 4. Discussion The effect of CORT and 4i on the oxidative stress parameters (pro-oxidant markers and anti-oxidant defense indices) is illustrated in Table 1. 3.4.1. Lipid peroxidation The CORT treatment significantly increased the MDA content (measured in the form TBARS level) in the brain as compared to untreated control mice [F4,25 = 5.87, p < 0.01 vs. CON group]. The treatment with 4i reversed increased MDA level in the mice brain due to CORT administration [F4,25 = 5.87, p < 0.05 vs. CORT–CON group for 4i (0.5 and 1 mg/kg)]. Similarly, FLX treatment produced a significant reduction in MDA content in the brain as compared to CORT treated control mice [F4,25 = 5.87, p < 0.01 vs. CORT–CON group]. 3.4.2. Nitrite level Repeated CORT administration for 4-weeks resulted in a significant increase in brain nitrite levels as compared to untreated control mice [F4,25 = 6.28, p < 0.01 vs. CON group]. Chronic 4i administration produced decreased nitrite levels in the brain in CORT treated mice as compared to mice treated with CORT alone [F4,25 = 6.28, p < 0.01 vs. CORT–CON group for 4i at 0.5 mg/kg and p < 0.05 vs. CORT–CON group for 4i at 1 mg/kg]. Likewise, positive control FLX also reduced elevated nitrite levels in the brain as compared to CORT treated mice [F4,25 = 6.28, p < 0.01 vs. CORT–CON group]. 3.4.3. Catalase activity Treatment with CORT induced a significant reduction of catalase activity in the mice brain [F4,25 = 4.64, p < 0.01 vs. CON group]. On the other hand, only higher dose of 4i (1 mg/kg) administration resulted in a significant increase in catalase activity in CORT treated mice brain as compared to mice treated with CORT alone [F4,25 = 4.64, p < 0.05 vs. CORT–CON group]. The lower 4i dose used in the present study had no significant increase in catalase activity in the brain of mice when compared with the group treated with the CORT alone. In addition, FLX illustrated a significant increase in catalase activity in the brain in mice given CORT treatment as compared to the mice treated with CORT alone [F4,25 = 4.64, p < 0.05 vs. CORT–CON group]. 3.4.4. Reduced glutathione levels There was a statistically significant reduction of the antioxidant enzyme reduced glutathione level in the brain of mice administered with CORT as compared to untreated control mice [F4,25 = 4.69, p < 0.05 vs. CON group]. In contrast, drug treatment 4i and FLX produced a significant increase in reduced glutathione

The present study demonstrated the antidepressant-like effects of a novel 5-HT3 receptor antagonist 4i in CORT-induced mouse model of depression (when evaluated in FST and light–dark aversion test) reinforcing that 5-HT3 receptors have an important role in the pathophysiology of depression due to dysregulation of HPA axis. Furthermore, the results showed that 4i has significant attenuating effects on increased oxidative stress in the brain in CORT treated mice revealing the significance of oxidative insults in the development of depressive state and diminution of which may provide one of the mode of actions of novel compounds. Animal models are indispensable tools for evaluating antidepressant-like effects of novel compounds. Among them FST is one of the extensively utilized behavioral despair tests to screen antidepressants as well as to investigate the underlying mechanisms of action of antidepressants [43]. The test is based on the observation that animals, when forced to swim in an enclosed space, eventually cease to struggle, surrendering themselves to the exposed conditions. This behavioral despair condition is considered to be a depressive-like state, which simulate the state of hopelessness observed in depressed patients [44]. Drugs with depressant behavior increase, while antidepressants decrease duration of immobility in FST [35]. In the present study, chronic CORT treatment developed a depressive-like condition in mice as indicated by increased duration of immobility and decreased swimming episodes. Similar to the previous studies, the results of the current finding revealed the correlation between depression and abnormal GCs levels [6,9,10,12,45,46]. As expected, FLX used as a positive control inhibited depressive-like despair behavior induced due to chronic CORT administration, which suggests the high predictive validity of the model. Interestingly, the novel 5-HT3 receptor antagonist, 4i reversed the despair behavior in CORT treated mice as illustrated by decreased duration of immobility and increased swimming episodes. Cryan et al. have shown that increase in swimming behavior reflects the serotonergic modulatory activity of the compounds [35]. Drugs which enhance synaptic serotonin mediated neurotransmission in the brain show increased swimming behavior. Therefore decrease in the swimming episodes in CORT treated control mice could be due to impairments of serotonin activity as a result of abnormal GCs functions. Previous reports have shown that increased cortisol levels (due to activated HPA axis in stressful situations) decrease the basal serotonin levels and hence the synaptic neurotransmission [47]. An elevated cortisol level reduces the tonic levels of serotonin in the synaptic cleft and stimulates its reuptake. Taking into account that a deficiency of brain serotonin levels is related to depression, this could presumably contribute to the overt depressive-like behavior in

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chronic CORT treated control mice. On the other hand inhibition of synaptic reuptake of serotonin could be associated with the antidepressant effects the positive control FLX (a SSRI). It has been already mentioned in our earlier reports that, 5-HT3 receptor antagonists facilitate synaptic serotonergic neurotransmission [19]; while other reports have shown that pre-treatment of 5HT3 receptor antagonists potentiate the antidepressant effects of SSRIs [27]. It implies that one of the modes of antidepressant-like action of 4i in CORT-treated mice could be in the direction of improvement of serotonin functions in the brain. It has been well established that depression is often associated with anxiety and that the abnormal GCs activity can also induce anxiety-like behavioral deficits [9], the present study investigated the effects of CORT and 4i treatment on anxiety-like behavior in mice using light–dark aversion test. The model is based on an approach/avoidance conflict of mice between the drive to explore novel areas and an aversion to brightly illuminated environment [48]. Anxiolytic drugs abolish the apparent apprehension of light area and increase the latency time for the first passage from light to dark chamber as well as total time spent in light chamber [38]; while compounds with anxiogenic behavior decrease latency and total time spent in light chamber [48]. In consistent with the previous reports, chronic CORT administration resulted in increased anxiety-like effects in mice subjected to light–dark test with significantly increased aversion and decreased exploratory behavior in light chamber [9]. However, few reports have demonstrated that repeated CORT dosing has no significant effect on anxiety-like behavior in open field and social interaction tests [10]. The inconsistency in the results could be attributed to many factors, among them, the dose of CORT, the duration of CORT dosing, the difference in anxiety models employed and finally due to factors related to each animal species. It is noted that 4i (only at higher dose of 1 mg/kg), similar to FLX, increased exploratory behavior of CORT treated mice in brightly lit light chamber. However, the lower dose of the test compound was ineffective, while it has shown anxiolytic-like effects in untreated mice during the behavioral paradigm [33]. The reason for this could be attributed to the fact that dose was not effective enough in attenuating the CORT induced anxiety in mice. Besides, this suggests that 4i has potential to ameliorate anxiety-like behavior in addition to depression-like state associated with abnormal GCs activity and particularly disinhibition of the HPA axis. While assessing the antidepressant and anxiolytic like effects of compounds based on the despair behavior and exploratory based models, the psychomotor stimulant/sedative effects can lead to false positive/negative results, thus the influence of the test drug on the locomotor activity of the animal is a governing concern [34,43]. In the present study, to eliminate the influence of 4i and CORT on the non-specific motor activity of mice (that could affect the behavior in FST and light–dark aversion test), the effects on the locomotor activity of mice were evaluated. Neither CORT nor did the 4i affected the basal locomotion significantly in actophotometer test. Therefore, it is unlikely that the depressogenic behavior of CORT and antidepressant-like effects of 4i and FLX is an artifact due to changes in locomotor activity. The fact that brain has comparatively greater vulnerability to oxidative damage, increased oxidative stress is one of the key pathogenic mechanisms of depressive disorders [49]. Previous reports have shown that repeated CORT injections lead to elevated oxidative load in the brain that culminates in the development of depressive symptomology [18]. In the present study, chronic CORT administration exhibited increased TBARS and nitrite levels in the brain demonstrating increased oxidation of cellular lipids and generation of ROS. Subsequently, CORT elicited reduced antioxidant enzyme actions as indicated by decreased catalase activity and reduced glutathione levels,

considering that the activities of these enzymes might be markedly decreased for scavenging ROS in the neuronal cells. This contention is supported by the earlier findings that abnormally elevated GCs levels stimulate oxidative stress in the brain [50]. Furthermore, previous reports have documented that GCs may damage hippocampus and other brain areas by generating ROS, which are potentially related to the regulation of mood and behavioral activity [51]. Therefore, the inhibited hippocampal function due to elevated oxidative damage plausibly provides positive correlation between abnormal GCs activity and depression. Thus, increased oxidative stress due to chronic CORT could be related to the depressive-like behavior observed in mice during behavioral testing paradigms. Interestingly, 4i similar to FLX ameliorated CORT induced oxidative load in the brain. This is in line with the earlier reports that several antidepressants reverse oxidative damage in the brain; one of the pathogenic causes of depression, which indicates the possible mechanism of antidepressant like effects of 4i [49]. It is important to note that additional pathological pathways might modulate the depressogenic behavior of CORT or antidepressant-like effects of 4i and FLX. It is evident that enhanced glutamate neurotransmission is associated with the pathogenesis of depression. Stimulated glutamate activity can cause excitotoxicity and neuronal damage in discrete areas of the brain [52]. Moreover, a considerable body of work carried out by in vivo microdialysis has shown that both stress and chronic CORT treatment induce glutamate release and transmission in the brain, which may result in direct damage of cellular proteins and lipids that consequently lead to loss of membrane fluidity and receptor functions [51,53,54]. On the other hand, antidepressant drugs (like FLX, reboxetine, desipramine and venlafaxine) have shown to inhibit stress evoked glutamate release in hippocampus and prefrontal cortex [55–57]. In addition, reports have demonstrated that stimulation of pre-synaptic 5-HT3 receptors facilitate the brain glutamatergic synaptic inputs, while 5-HT3 antagonists markedly suppress the same [58]. Therefore, considering that abnormal GCs functions due to repeated CORT administration may affect glutamate activity and that 4i and FLX may have modulating effects on glutaminergic system and may impose an inhibiting effects on oxidative stress induced brain damage, one could suppose the involvement of aforementioned events in the inducing effects of CORT and attenuating ability of 4i and FLX on depressive-like behavior. However, additional studies are warranted to confirm such hypothesis. Taken together, the finding presented herein point to a hypothesis that dysregulated HPA axis as a result of the excessive CORT, present severe depressive-like behavior that can be attributed, at least in part, to a stimulated brain damage due to enhanced oxidative stress. The present study revealed that 4i, a novel 5HT3 receptor antagonist can reverse CORT induced depressive-like behavior in mice. Furthermore, this effect can be related to the decrease in oxidative stress induced neuronal damage as the results of the study demonstrate that it decreases pro-oxidant markers and increases anti-oxidant defense system in the brain. However, further studies are required to understand the molecular mechanism such as signal transduction pathways and post receptor action and to elucidate the correlation between the behavioral activity and regulatory effects on the oxidative and anti-oxidant defense system produced by the test compound.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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Acknowledgments The authors are thankful to BITS-Pilani and University Grants Commission, India for providing support and research facilities to pursue this work.

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