Agmatine attenuates chronic unpredictable mild stress-induced anxiety, depression-like behaviours and cognitive impairment by modulating nitrergic signalling pathway

Accepted Manuscript Research report Agmatine attenuates chronic unpredictable mild stress-induced anxiety, depression-like behaviours and cognitive impairment by modulating nitrergic signalling pathway Nitin B. Gawali, Vipin D. Bulani, Malvika S. Gursahani, Padmini S. Deshpande, Pankaj S. Kothavade, Archana R. Juvekar PII: DOI: Reference:

S0006-8993(17)30111-7 http://dx.doi.org/10.1016/j.brainres.2017.03.004 BRES 45302

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Brain Research

Received Date: Revised Date: Accepted Date:

20 July 2016 1 March 2017 3 March 2017

Please cite this article as: N.B. Gawali, V.D. Bulani, M.S. Gursahani, P.S. Deshpande, P.S. Kothavade, A.R. Juvekar, Agmatine attenuates chronic unpredictable mild stress-induced anxiety, depression-like behaviours and cognitive impairment by modulating nitrergic signalling pathway, Brain Research (2017), doi: http://dx.doi.org/10.1016/ j.brainres.2017.03.004

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Agmatine attenuates chronic unpredictable mild stress-induced anxiety, depression-like

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behaviours and cognitive impairment by modulating nitrergic signalling pathway

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Nitin B. Gawali, Vipin D. Bulani, Malvika S. Gursahani, Padmini S. Deshpande, Pankaj S.

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Kothavade, Archana R. Juvekar*

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Pharmacology Research Lab 1, Department of Pharmaceutical Sciences and Technology,

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Institute of Chemical Technology, Nathalal Parekh Marg, Matunga (E), Mumbai-400019,

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India.

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*Corresponding Author:

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Name

: Prof. (Mrs.) Archana R. Juvekar

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Address

: Professor in Pharmacology and Physiology,

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Department of Pharmaceutical Sciences and Technology,

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Institute of Chemical Technology,

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Matunga, Mumbai- 400 019, India.

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Phone numbers

: +91 22 3361 2215

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Fax

: +91 22 3361 1020

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E-mail address

: [email protected]; [email protected]

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Abstract

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Agmatine, a neurotransmitter/neuromodulator, has shown to exert numerous effects on the

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CNS. Chronic stress is a risk factor for development of depression, anxiety and deterioration

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of cognitive performance. Compelling evidences indicate an involvement of nitric oxide

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(NO) pathway in these disorders. Hence, investigation of the beneficial effects of agmatine

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on chronic unpredictable mild stress (CUMS)-induced depression, anxiety and cognitive

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performance with the involvement of nitrergic pathway was undertaken. Mice were subjected

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to a battery of stressors for 28 days. Agmatine (20 and 40 mg/kg, i.p.) alone and in

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combination with NO modulators like L-NAME (15 mg/kg, i.p.) and L-arginine (400 mg/kg

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i.p.) were administered daily. The results showed that 4-weeks CUMS produces

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significant depression and anxiety-like behaviour. Stressed mice have also shown a

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significant high serum corticosterone (CORT) and low BDNF level. Chronic treatment with

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agmatine produced significant antidepressant-like behaviour in forced swim test (FST) and

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sucrose preference test, whereas, anxiolytic-like behaviour in elevated plus maze (EPM) and

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open field test (OFT) with improved cognitive impairment in morris water maze (MWM).

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Furthermore, agmatine administration reduced the levels of acetylcholinesterase and

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oxidative stress markers. In addition, agmatine treatment significantly increased the BDNF

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level and inhibited serum CORT level in stressed mice. Treatment with L-NAME (15 mg/kg)

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potentiated the effect of agmatine whereas L-arginine abolished the anxiolytic, antidepressant

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and neuroprotective effects of agmatine. Agmatine showed marked effect on depression and

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anxiety-like behaviour in mice through nitrergic pathway, which may be related to

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modulation of oxidative–nitrergic stress, CORT and BDNF levels.

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Key words: Agmatine; BDNF; HPA axis; CUMS; depression; anxiety.

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1. Introduction

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Mood and anxiety disorders have a large variety of similar pathophysiological characteristics

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and co-occur in nearly 50-60% of clinical subjects (File, 1996). The pathophysiology of

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depressive illness and anxiety is thought to involve both endogenous predisposing factors and

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a dysregulated response to stress (McEwen, 2000). Chronic stress increases corticosterone

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secretion, which causes dysregulation of hypothalamic–pituitary–adrenocortical (HPA) axis

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and also triggers oxidative stress which ultimately leads to impairment of hippocampus-

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dependent learning and memory processes (Sato et al. 2010). Oxidative damage induced by

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chronic unpredictable

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pathogenesis of depression, anxiety and cognitive dysfunctions

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disorders such as immunosuppression, diabetes mellitus, peptic ulceration to hypertension

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and ulcerative colitis (Bhattacharya and Muruganandam, 2003). Unpredictable stressors have

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a greater negative impact than predictable stressors due to their uncertainty (Bondi et al.,

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2008). Therefore, CUMS has been developed as an experimental model of depression and

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anxiety (Mineur et al., 2006, Ruan et al. 2014, Zhu et al., 2014).

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Compelling evidences from both animal and human studies have led to the hypothesis that

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the pathophysiology of depression and neurobiology of stress is associated with

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hyperactivation of HPA axis and alteration in BDNF level which is a common feature of

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stress-related psychiatric diseases such as depression and anxiety (De Kloet et al., 2005;

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McEwen, 2008). BDNF is involved in the pathogenesis of chronic stress-induced depression

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and anxiety disorders and plays a critical role in synaptic plasticity and memory processes

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(Middeldorp et al., 2010; Cowansage et al., 2010). Moreover, clinical studies reported that

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depressive patients have a low serum BDNF level as compared to control subjects (Castren et

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al., 2007) and treatment with antidepressants restore the normal level/functioning of BDNF

mild stress (CUMS) has been postulated to be involved in the plus a variety of other

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(Sen et al., 2008). The functioning of HPA axis and BDNF level therefore, plays an important

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role in the pathogenesis of stress-related depression and anxiety disorders.

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Nitric oxide (NO) is a neurotransmitter and neuromodulator that regulates key functions in

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the central nervous system. Nitric oxide (NO) is produced from L-arginine by a group of NO

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synthases (NOSs). There are three main isoforms, each with a specific distribution profile;

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neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) (Stuehr, 1999).

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Ample evidences indicate the involvement of nitrergic system in the pathogenesis of mood

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and anxiety disorders (Spiacci et al., 2008). Administration of NOS inhibitors, such as

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aminoguanidine, a selective iNOS inhibitor; 7-nitroindazole, a selective nNOS inhibitor; and

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NG-nitro-L arginine methyl ester (L-NAME), a non-selective NOS inhibitor has been shown

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to offer antidepressant and anxiolytic effects (Volke et al., 2003; Montezuma et al., 2012;

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Gilhotra and Dhingra, 2009). Several studies reported that NO plays a role in the mechanism

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of action of some antidepressant and anxiolytic drugs currently in use (Krass et al., 2011;

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Zhang et al., 2010). Agmatine, 4-(aminobutyl) guanidine, is structurally analogous to the

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nitric oxide synthase (NOS) substrate L-arginine. Agmatine, a cationic amine formed by

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decarboxylation of L-arginine by the mitochondrial enzyme arginine decarboxylase (ADC),

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is widely but unevenly distributed in mammalian tissues (Regunathan & Reis, 2000). Nitric

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oxide (NO) is produced from L-arginine by a group of NO synthases (NOSs). Produced NO

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plays directly or indirectly regulation of the central and peripheral nervous system, which

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modulates a variety of physiological processes such as learning and memory, circadian

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rhythms, immune system, anxiety and depression (Chen et al., 1997; Harkin et al., 1999;

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Masood et al., 2003). Agmatine has been shown to be a competitive inhibitor of both nNOS

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and iNOS (Auguet et al., 1995, Galea et al., 1996).

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Neuropeptides are attractive therapeutic targets for depression and anxiety disorders.

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Agmatine has been recognized as an important neuromodulator and/or neurotransmitter in the

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brain which binds with high affinity to α2-adrenoceptors, imidazoline binding sites, inhibits

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NMDA receptors and competitively inhibits nitric oxide synthase (Reis and Regunathan,

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2000; Halaris and Piletz, 2007). Studies have highlighted a prominent role of agmatine in

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anxiety and depression (Lavinsky et al., 2003; Mohseni et al., 2017; Zomkowski et al., 2002).

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Moreover, agmatine has the ability to modulate pro- and anti-oxidative balance in the

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hippocampus, which might also underlie its behavioural effects (Freitas et al., 2014). In

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addition,

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neuroprotective properties and also facilitates working memory (Demehri et al., 2003; Önal et

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al., 2003; Satriano et al., 2001; Olmos et al., 1999; Moosavi et al., 2012). Additionally,

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treatment with agmatine diminished repeated immobilization induced by elevated

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corticosterone levels and glutamate efflux in brain nuclei associated with modulation of stress

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response (Zhu et al., 2008a, b). In stressful condition endogenous agmatine level are

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increased in compensatory manner, however not high enough to modulate the harmful effect

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of stressor or inflammation (Zhu et al., 2008a, b). Hence, exogenous administration which

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restored the agmatine levels can exhibit anti-stress and neuroprotective effects in rodent.

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However, the potential beneficial effects of agmatine in CUMS model and underlying

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mechanism(s) are yet to be explored. Hence, the present study was designed to examine the

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effect of chronic agmatine treatment on CUMS induced depression, anxiety and deterioration

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of cognitive performance and the involvement of nitrergic pathway. Furthermore, the

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potential mechanisms underlying CUMS induced modulation of oxidative–nitrergic stress,

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HPA axis and BDNF levels in hippocampus were also studied.

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2. Result

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2.1. Effects of agmatine on Morris water maze test and its modulation by L-NAME

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The change in the escape latency time (ELT) to reach the hidden platform was observed in

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the acquisition trials. Although there was a downward trend in escape latency time in session

agmatine

exhibited

anticonvulsant,

antinociceptive,

anti-inflammatory,

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for four days but mean latency was significantly prolonged in the CUMS group as compared

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to the control group, indicating a poorer learning performance. Agmatine (20 and 40 mg/kg)

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treatment for 28 days shortened ELT as compared to CUMS group. Further, combination of

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agmatine (20mg/kg) with L-NAME (15mg/kg) showed significant improvement in the

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learning performance (fig. 2A) [F (5, 45) = 7.213, P˂ 0.001)]. Platform was removed on day

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28 to estimate the retention of memory. CUMS control group failed to recollect the location

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of the platform, thus spending less time in the target quadrant when compared to the control

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group. However, agmatine (20 and 40 mg/kg) treatment significantly increased the time spent

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in the target quadrant as compared to CUMS control, indicating improvement in cognitive

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performance [F (5, 45) = 22.19, P˂ 0.001)]. Agmatine (20 mg/kg) with L-NAME (15 mg/kg)

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together significantly increased the time spent in target quadrant (fig. 2B), however, L-ARG

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(400 mg/kg) abolished the effect of agmatine.

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2.2. Effect of agmatine on elevated plus maze (EPM) task and its modulation by L-

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NAME

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In the EPM test, CUMS subjected mice showed a noteworthy (P< 0.001) decrease in

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percentage of open arm entries and percentage of open arm time as compared to control mice.

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Treatment with agmatine (20 and 40 mg/kg) significantly increased percentage of open arm

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entries [F (5, 45) = 24.53, P< 0.001, fig. 3A] and percentage of open arm time [F (5, 45) =

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17.15, P< 0.001, fig. 3B]. Further, pre-treatment of L-NAME (15 mg/kg) with sub-effective

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dose of agmatine (20 mg/kg) significantly potentiated their protective effects in EPM.

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However, pre-treatment of L-arginine (400 mg/kg) with agmatine (20 mg/kg) significantly

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reversed the protective effect of agmatine (20 mg/kg).

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2.3. Effects of agmatine and its modulation by L-NAME in open field performance task

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As shown in fig. 4A and 4B, results obtained in the OFT revealed statistically differences

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between the groups in crossings [F (5, 45) = 17.83, P< 0.001] and rearing [F (5, 45) = 17.44,

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P< 0.001]. Post hoc Tukey's Multiple Comparison Test analysis revealed that the CUMS

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mice showed decreased activity in crossing (P< 0.001) and rearing (P< 0.001) in comparison

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to the control mice in OFT. Treatment with agmatine (20 and 40 mg/kg) reversed the above

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mentioned behavioural alterations as compared to the CUMS group i.e. crossing (P< 0.001)

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and rearing (P˂ 0.001). Further, pre-treatment of L-NAME (15 mg/kg) with sub-effective

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dose of agmatine (20 mg/kg) significantly potentiated their protective effects in open field

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task. However, pre-treatment of L-arginine (400 mg/kg) with agmatine (20 mg/kg)

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significantly reversed the protective effect of agmatine (20 mg/kg).

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2.4. Effects of agmatine and its modulation by L-NAME on immobility period

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CUMS caused a significant increase in the immobility time during the FST as compared to

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vehicle treated group. Treatment with agmatine (20 and 40 mg/kg) significantly shortened the

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immobility time during the final 5 min of FST when compared to CUMS control [F (5, 45) =

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24.33, P< 0.001; fig. 5A]. Post hoc Tukey's Multiple Comparison Test analysis indicated that

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treatment with agmatine (20 and 40 mg/kg) reduced the immobility time compared to the

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CUMS treated group (P< 0.05, P< 0.001). L -Arginine (400 mg/kg) treatment with sub-

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effective dose of agmatine (20 mg/kg) reversed the effect of agmatine (P< 0.001). However,

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L-NAME (15 mg/kg) treatment with agmatine (20 mg/kg) significantly (P< 0.001) produced

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synergistic effect on FST activity (shortened immobility period).

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2.5. Effects of agmatine and its modulation by L-NAME on sucrose preference test

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CUMS animals showed a significant reduction in sucrose consumption as compared to

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vehicle treated group. Treatment with agmatine (20 and 40 mg/kg) improved sucrose

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consumption as compared to CUMS treated group [F (5, 45) = 41.35, P< 0.001; fig. 5B]. Post

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hoc Tukey's Multiple Comparison Test analysis indicated that treatment with agmatine (20

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and 40 mg/kg) increased of sucrose preference as compared to the CUMS treated group (P<

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0.05, P< 0.001). Further, L-NAME (15 mg/kg) treatment with sub effective dose of agmatine

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(20 mg/kg) potentiated its sucrose consumption whereas L -Arginine (400 mg/kg) treatment

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significantly (P< 0.001) reversed the protective effect of agmatine.

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2.6. Effect of agmatine and its modulation by L-NAME on lipid per-oxidation (MDA),

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reduced glutathione (GSH) and nitrite levels

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CUMS mice showed a significant increase in oxidative damage as evidenced by a rise in

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MDA, nitrite levels and depletion of reduced GSH levels as compared to vehicle control

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group. Chronic treatment with agmatine (20 and 40 mg/kg) significantly attenuated the

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oxidative damage (reduced MDA, nitrite levels and restoration of reduced GSH) as compared

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to CUMS group. Based on this evidence we observed that the levels of the endogenous

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antioxidant GSH (fig. 6A) decreased in the hippocampus of animals of CUMS when

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compared to controls [F (5, 45) = 27.07, P< 0.001]. Treatment with L-arginine (400 mg/kg)

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caused a significant reduction in GSH levels as compared to the control (P< 0.001), whereas

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treatment of L-NAME (15 mg/kg) with sub-effective dose of agmatine (20 mg/kg)

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potentiated the antioxidant like effect agmatine.

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The TBARS level was significantly increased in the brain of stressed mice as compared to the

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normal control mice [F (5, 45) = 45.68, P< 0.001, fig. 6B]. The administration of L-NAME

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with sub-effective dose of agmatine (20 mg/kg) significantly prevented the increases in lipid

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per-oxidation induced by CUMS (P< 0.001). Fig. 6C shows that nitrite levels significantly

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enhanced in CUMS group [F (5, 45) = 20.37, P< 0.001] as compared to vehicle treated group.

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Agmatine (20 and 40 mg/kg), reversed CUMS-induced increased nitrite levels (P< 0.001).

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Treatment of L-NAME (15 mg/kg) with sub-effective dose of agmatine (20 mg/kg)

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potentiated the nitric oxide effect of agmatine. However, L-arginine (400 mg/kg) treatment

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with sub-effective dose of agmatine (20 mg/kg) reversed the effect of agmatine.

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2.7.Effect of agmatine on brain acetylcholine levels and its modulation by L-NAME

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Acetylcholinesterase enzyme activity in the hippocampus was significantly (P< 0.001)

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increased after 28 days of chronic unpredictable stress when compared with the vehicle

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control group (fig. 7). Agmatine (20 and 40 mg/kg) treatment attenuated acetylcholinestrase

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activity which was significant as compared to CUMS group [F (5, 45) = 29.78, P< 0.001].

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However co-administration of agmatine (20 mg/kg) with L-NAME (15 mg/kg) potentiated

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the attenuation effect of agmatine (20 mg/kg). However, L-arginine (400 mg/kg) treatment

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with sub effective dose of agmatine (20 mg/kg) significantly (P< 0.05) reversed the effect of

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agmatine.

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2.8. Effect of agmatine and its modulation by L-NAME on serum corticosterone

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(CORT) levels

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CUMS group of animals showed a significant increase in serum CORT levels as compared to

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vehicle control group. Post hoc Tukey's Multiple Comparison Test analysis indicated that

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treatment with agmatine (20 and 40 mg/kg) reduced the CORT level compared to the CUMS

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group [F (5, 45) = 27.97, P˂ 0.001) (P< 0.05, P< 0.001, fig. 8). In addition, treatment of L-

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NAME (15 mg/kg) with sub effective dose of agmatine (20 mg/kg) significantly attenuated

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the increased serum CORT level as compared to CUMS. Further, L-arginine (400 mg/kg)

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treatment with agmatine (20 mg/kg) abolished the effect of agmatine.

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2.9. Effect of agmatine and its modulation by L-NAME on hippocampal BDNF level

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Fig. 9 illustrates the effect of the agmatine (20 and 40 mg/kg) treatment on BDNF level in

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hippocampus. CUMS significantly decreased the level of BDNF as compared to vehicle

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treated group. The treatment with agmatine (20 and 40 mg/ kg) increased BDNF levels as

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compared to CUMS group [F (4, 45) = 39.40, P< 0.001]. BDNF level was significantly

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decreased after CUMS administration in HC (P< 0.001) of mice which was significantly

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restored by agmatine (20 and 40 mg/kg) (P< 0.05, P< 0.001). In addition, treatment of L-

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NAME (15 mg/kg) with sub effective dose of agmatine (20 mg/kg) significantly increased

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BDNF level in hippocampus as compared to CUMS. Furthermore, L-arginine (400 mg/kg)

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treatment abolished the effect agmatine.

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3. Discussion

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In the present study, animals were either unstressed or exposed for 4 weeks to CUMS and

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subsequently tested in the battery of behavioural paradigms, including anxiety, depression

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and memory impairment. In this context, our results revealed that CUMS-exposed animals

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exhibited several behavioural alterations, resembling the symptoms of depression, anxiety

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disorders and cognitive dysfunctions. These behavioural changes were associated with down

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-regulation of BDNF, alteration in nitroso-oxidative stress and HPA axis dysregulation.

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Agmatine administration caused significant improvement in the behavioural activity, the

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findings further corroborated by reversal of CUMS-induced chemical alterations.

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A large body of evidences point towards the association between CUMS and development of

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depressive and cognitive impairment (De Kloet et al., 2005; Song et al., 2006; Zhu et al.,

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2014). The CUMS model is a promising and valuable animal model of depression and has

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been widely used to understand the pathophysiology of depression, as it has an edge over

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genetic models in closely mimicking human depression (Bondi et al., 2008; De Kloet et al.,

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2005; Kumar et al., 2011). In addition, animal studies have shown that CUMS also induces

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anxiety-like behaviour in mice (Zhu et al., 2014), and therefore can be effectively used as a

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model for screening of antidepressant and anxiolytic-like potential of new drug molecules.

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The involvement of nitrergic system in the pathogenesis of CUMS-induced depression is

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evident from the potentiation and inhibition of agmatine by pre-treatment with L-NAME and

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L-arginine respectively.

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It has been demonstrated that nNOS, mainly responsible for NO production in the nervous

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system plays a key role in depression (Zhou and Zhu, 2009) and anxiety disorders (Zhang et

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al., 2010). Although nNOS is richly expressed throughout the limbic system (Bredt et al.,

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1991), the hippocampal nNOS is primarily responsible for stress-related depressions.

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Furthermore, several studies have demonstrated transcriptional regulation of nNOS by

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glucocorticoids in the hippocampus, highlighting its importance in the stress response (Zhou

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et al., 2011). Recent studies in several animal paradigms have demonstrated that inhibitors of

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NOS significantly modulate stress-related behaviours. Paroxetine, a commercially available

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antidepressant and a selective serotonin reuptake inhibitor has been shown to possess NOS

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inhibition capability (Finkel et al., 1996).

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Sucrose preference test is a valid behavioural indicator to assess anhedonia response in

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rodents (Strekalova et al., 2006). Anhedonia is a key symptom of human depression episodes

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as per DSM-IV criteria. Exposure to stress causes a decrease in sucrose consumption in

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animal models of depression (Willner et al., 1992). In the present study, agmatine increased

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the preference to and amount of sucrose consumption in stressed mice, indicating decreased

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anhedonia. This result is consistent with earlier findings in CUMS-depressed mice exposed to

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sucrose (Taksande et al., 2013). Results from study by Peng et al. 2012 showed that treatment

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with NOS inhibitor in stressed mice positively encouraged sucrose consumption and may be

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support the anti-anhedonic response in agmatine-treated animals.

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The FST is the most frequently used to determine depression-like behaviour in rodents after

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exposure to various stressors (Takeda et al., 2006). In this report, mice subjected to chronic

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stress exhibited increase duration of immobility in FST and the results were supported by

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previous reports (Kumar et al., 2011). Similarly, chronic administration of agmatine with L-

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NAME expectedly decreased the duration of immobility and increased the swimming

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episodes in stressed mice.

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Anxiety is also a frequent and well known consequence of chronic stress with stress having a

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positive correlation with anxiety-like behaviour. We investigated anxiety-like activity in two

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most validated rodent models namely, the EPM and the OFT (Lister, 1987; Belzung and

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Griebel, 2001). In our experiments, stressed mice showed a significant decrease in percentage

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of open arm entries and percentage of the time spent on the open arms which was reversed

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with chronic agmatine treatment in the EPM test. Previous studies reported that OFT is useful

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to assess exploration, locomotor activity and anxiety-like behaviour of rats or mice (Keeney

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and Hogg, 1999) and the number of crossings and rearing measured during the OFT reflects

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an animal's exploratory behaviour (Martin et al., 2013). In the current investigation,

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depressed mice showed significantly decreased activity in crossing and rearing, whereas

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treatment with agmatine with L-NAME significantly reversed the above mentioned

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behavioural alterations. Our results are in line with previous reports, clearly indicate a

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possible anti-anxiety like behaviour in depressed mice (Lavinsky et al., 2003). This effect is

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thought to be a result of inhibition of NO by agmatine and L-NAME (Joung et al., 2012;

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Spiacci et al., 2008).

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Stress has a direct impact on cognitive behaviour and memory in the long-run. In the present

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study, chronic unpredictable stress resulted in significant impairment of cognitive tasks in

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MWM as compared to control animals (Utkan et al., 2012). Treatment with agmatine

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shortened escape latency time and significantly increased the time spent in the target quadrant

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as compared to CUMS control, indicating improvement in cognitive performance. These

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findings are in line with the earlier reports from Zarifkar et al. (2010) who showed that

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agmatine prevents LPS-induced memory impairment.

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It is well-known that the central nervous system is extremely sensitive to peroxidative

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damage due to its rich content of oxidizable substrates, high oxygen tension and low

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antioxidant capacity (Metodiewa and Kośka, 2000; Zafir et al., 2009). Several studies have

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shown that chronic stress markedly enhances the generation of ROS/RNS which saturates

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neuronal antioxidant defense system and surpasses the antioxidant's detoxifying capacity in

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the brain, thereby, rendering neurons vulnerable to the deleterious effect of ROS and RNS

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(Poon et al., 2004), paving way for the neurological complications. Enhanced oxidative stress

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is the brain is linked with the etiology of the aging, neurodegeneration, development of

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cognitive impairment, anxiety, depressive like behavior and other psychiatric disorders

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(Filipović et al., 2016; Poon et al., 2004; Shukla et al., 2011). Further, pharmacological

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interventions which are capable of abolishing oxidative stress are known to improve

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behavioral dysfunction in experimental animals as well in clinical patients (Pandya et al.,

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2013). As a result, we examined the effects of CUMS on key antioxidant enzymes of brain in

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hippocampus. In our study, increased TBARS levels (proportional to lipid per-oxidation) and

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reduced brain GSH level indicated an alteration in antioxidant brain defences in CUMS mice

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which were restored by treatment with agmatine. This finding is in line with similar findings

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by Freitas et al. 2014, reporting agmatine-induced attenuation of oxidative stress. Converging

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lines of evidence suggest that depressed patients show elevated nitrite level (Suzuki et al.,

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2001) and therefore reduced nitroso-oxidative stress by agmatine may afford anti-depressive

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benefit. In consistent with our results, there are a number of studies which demonstrated that

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under stressful conditions, NOS plays a more determinant role in the pathophysiology of both

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depressive and anxiety-like behaviours (Montezuma et al., 2012; Joung et al., 2012).

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Acetylcholine (ACh) is an essential neurotransmitter which is required for proper functioning

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of cholinergic transmission and the central cholinergic system plays a vital role in the

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regulation of cognitive function, and memory loss is closely associated with dysfunction of

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the cholinergic system, including alterations in neurotransmitters and their receptors (Nijholt

319

et al. 2004). In addition to other alterations, stress is known to cause changes in the AChE

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levels (Bhutada et al., 2012). Body of evidences has shown that the dysfunction of

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cholinergic transmission system is suggested as a mechanism for pathogenesis of depression

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and Wiklund et al. (1993) have demonstrated that activation of pre and post-synaptic

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cholinergic receptors plays an essential role in the stimulation of NO synthesis. Agmatine

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treatment effectively restored the AChE levels in rats subjected to streptozotocin-induced

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diabetic. Similarly, in the present study, CUMS caused a significant increase in the

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acetylcholinesterase enzyme activity showing deficits in memory process but later was

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restored by chronic agmatine treatment, thereby implicating retrieval and retention of

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memory processes. Several reports have demonstrated that NO donor agents may inhibit the

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AChE activity and subsequently enhance the acetylcholine level in cholinergic synapses. It is

330

also mentioned that scopolamine, a cholinergic receptor antagonist, inhibits NOS and NO

331

production, which lead to a decrease in serum and hippocampal nitrite concentration (Azizi-

332

Malekabadi et al. 2012, Saeedi Saravi et al., 2016).

333

In addition to the effect of agmatine on behavioural abnormalities in CUMS model, we also

334

investigated influence on HPA axis activity by measuring serum CORT level. Research has

335

confirmed that CUMS causes hyperactivation of HPA axis and increases the serum CORT

336

levels, which may subsequently lead to depression (De Kloet et al., 2005; McEwen, 2008;

337

Vreeburg et al., 2010). A central feature of the HPA stress response is the synthesis and

338

secretion of glucocorticoids (corticosterone in mice) from the adrenal cortex. Additionally,

339

glucocorticoids secreted during stressful events are known to influence memory consolidation

340

and retrieval (Roozendaal, 2002). In the present investigation, CUMS animals showed a

341

significant increase in serum corticosterone levels as compared to the control group. Chronic

342

administration of agmatine with L-NAME reduced the CORT levels, similar to results

343

already published (Taksande et al., 2013). Interestingly, studies have shown that neuronal and

344

endothelial NOS are involved in the NOS-inhibitor induced impairment in corticosterone

345

secretion (Okada et al., 2002). In addition, it has been shown that nitric oxide regulates

346

activity of HPA axis that has an impact on the synthesis of stress hormones such as

347

glucocorticoids (Tsuchiya et al., 1997). Furthermore, nNOS in the hippocampus represses GR

14

348

expression and is involved in the modulation of HPA axis, explaining the positive effect of

349

agmatine on CORT levels (Liu et al., 2013).

350

BDNF has been known to be involved in the pathogenesis of chronic stress-induced

351

depression and anxiety disorders (Middeldorp et al., 2010). In the present study, we observed

352

a significant decrease in BDNF levels in CUMS mice. However, treatment with agmatine

353

with L-NAME potentiated the neurogenesis process and increased BDNF levels. These

354

results are consistent with the findings from Freitas et al. (2016) who found activation of the

355

BDNF signalling pathway and upregulation of hippocampal neurogenesis on treatment with

356

agmatine. In addition, a recent study pointed out that the depressive-like behaviour induced

357

by chronic unpredictable mild stress significantly decreased BDNF protein levels in the CA1

358

and CA3 regions of the hippocampus, which was reversed by the inhibition of neuronal nitric

359

oxide synthase (Yazir et al., 2012).

360

In conclusion, our results emphasized that CUMS produces a cognitive deficit in mice similar

361

to that seen in patients with major depressive disorder, as well as a state of anxiety that also

362

occurs in depression. In addition, CUMS also significantly increased the oxidative stress

363

markers and decreased the antioxidant enzymes activity. Moreover stressed mice showed a

364

significant high CORT level and low BDNF level. Chronic treatment with agmatine with L-

365

NAME produced anxiolytic- and antidepressant-like effects behaviour in normal and stressed

366

mice, indicates the anxiolytic- and antidepressant-like effects potential of agmatine. In

367

addition, agmatine reversed the hyperactivation of the HPA axis activity and increased the

368

level of BDNF. These observations suggest that the modifications of the behavioural deficits

369

in CUMS seem to depend on reducing HPA axis activity and restoring BDNF level, which

370

often assumed to be a common property of clinically effective antidepressants (Fig. 10).

371

These results imply the prominent role of agmatine in management of depression and anxiety

372

and may further support the clinical development of agmatine.

15

373

4. Experimental Procedure

374

4.1. Animals

375

In the current study, we used adult male Swiss albino mice weighing 20–24 g. They were

376

procured from National Institute of Biosciences, Pune, India. The animals were housed in

377

opaque polypropylene cages (28 × 21 × 14 cm) and maintained in temperature and humidity-

378

controlled holding facility 25 ± 20 C under 12:12 h light/dark cycle (07:00–14:00 h). All

379

mice received food with free access to rodent chow (Amrut rat and mice feed, Sangli, India)

380

and water. Each experimental group comprised of eight animals. In experiment, 48 mice were

381

randomly assigned to six groups [control, stress control, agmatine (20 and 40 mg/kg),

382

agmatine + L-NAME and agmatine + L-arginine]. The animals were acclimatized for 7 days

383

before use in the experiments. Figure 1 illustrated the schematic design of the experiments.

384

The animal studies were approved by the Institutional Animal Ethics Committee (IAEC),

385

constituted for the purpose of control and supervision of experimental animals by Ministry of

386

Environment and Forests, Government of India, New Delhi, India. Animals were naïve to

387

drug treatment and experimentation at the beginning of all studies. All behavioural

388

experiments were carried out between 09:00 and 14:00 h. All efforts were made to minimize

389

animal suffering and to reduce the number of animals used.

390

4.2. Drugs

391

Agmatine sulfate, NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) and L-

392

Arginine (L-ARG) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All other

393

chemicals used for biochemical estimations were of analytical grade. Mouse ELISA kit for

394

corticosterone and BDNF were purchased from Krishgen Biosystems, Mumbai, India. All

395

drugs were dissolved in 0.9% NaCl. Drug solutions were prepared freshly before the

396

experiments to produce a total injection volume of 1.0 ml/kg body weight. All drugs were

397

administered (between 8.30 and 09:30 a.m.) by intraperitoneal route daily 30 min before

16

398

CUMS procedure for 28 days. The doses of agmatine, L-NAME and L-ARG were selected

399

according to the previous studies (Gawali et al., 2016; Ceren et al., 2016; Lavinsky et al.,

400

2003; Zomkowski et al., 2002).

401

4.3. Experimental design

402

Forty eight male Swiss albino mice animals were randomly divided into six experimental

403

groups containing eight animals in each group. Group 1: control unstressed mice receiving

404

vehicle (by intraperitoneal); Group 2: control animals received CUMS along with an

405

equivalent volume of vehicle; Groups 3–4: CUMS-treated animals receiving agmatine (20

406

and 40 mg/kg; i.p.) daily 30 min before induction of stress; Groups 5: CUMS-treated animals

407

receiving pre-treatment of L-NAME (15 mg/kg; i.p.) 15 min before agmatine (20 mg/kg;

408

i.p.); Group 6: CUMS-treated animals receiving pre-treatment of L-ARG (400 mg/kg; i.p.) 15

409

min before agmatine (20 mg/kg; i.p.). Pictogram of the entire protocol is represented in

410

Figure 1.

411

4.4. Unpredictable chronic mild stress procedure

412

The CUMS protocol was designed to maximize the unpredictable nature of stressor. This

413

animal model of stress consists of chronic exposure to variable unpredictable stressors and

414

was performed with some modifications and illustrated in Table 1. All procedures were

415

carried out in isolated rooms adjacent to the housing room, requiring minimal handling or

416

transport of the mice. After each stressor, animals were kept in a recovery room for 1–2 h,

417

following which they were placed in clean cages with fresh bedding and returned to the

418

housing facility. Briefly, mice were exposed to a random pattern of mild stressors once per

419

day over a period of 28 days (Ducottet et al., 2003). The order of stressors used was

420

illustrated in Table 1. These stressors were randomly scheduled over a 4 week period. Normal

421

control mice were undisturbed except for necessary housekeeping procedures.

422

4.5. Behavioural studies

17

423

4.5.1. Morris water-maze test

424

Morris water maze (MWM) is used to test memory (Morris, 1984). Animal dislikes

425

swimming and hence when placed in a large pool of water its tendency is to escape it by

426

searching for a platform. MWM consisted of large circular pool (122 cm in diameter, 50 cm

427

in height, filled to a depth of 30 cm with water at 22–23 °C). The water was made opaque

428

with milk powder. The tank was divided into four equal quadrants. A submerged platform

429

(10 ×10 cm2), painted white was placed in the middle of the target quadrant of this pool, 1cm

430

below surface of water. The position of platform was kept unaltered throughout the training

431

session. Several brightly coloured cues external to the maze was placed in the room for

432

spatial orientation. The position of the cues remained unchanged throughout the study. The

433

water maze task was carried out from day 24th to 27th. The mice received four consecutive

434

daily training trials in the following 4 days, with each trial having a ceiling time of 120 s. For

435

each trial, individual mouse was gently put into the water at one of four starting positions, the

436

sequence of which being selected randomly and allowed 120 s to locate submerged platform.

437

Then, it was allowed to stay on the platform for 20 s. If animal failed to find the platform

438

within 120 s, it was guided gently onto platform and allowed to remain there for 20 s.

439

Acquisition trial - Each mouse was subjected to four trials on each day. A rest period of 1 h

440

was allowed in between each trial. Four trials per day were repeated for four consecutive

441

days. Starting position on each day to conduct four acquisition trials was changed. Mean

442

escape latency time (ELT) calculated for each day during acquisition trials was used as an

443

index of acquisition.

444

Retrieval trial - On fifth day (day 28th) the platform was removed. Animal was placed in

445

water maze and allowed to explore the maze for 120 s. Mean time spent in all three

446

quadrants, i.e. Q1, Q2 and Q3 were recorded and the time spent in the target quadrant, i.e. Q4

447

in search of missing platform provided an index of retrieval. Care was taken that relative

18

448

location of water maze with respect to other objects in the laboratory serving as prominent

449

visual clues was not disturbed during the total duration of study.

450

4.5.2. Elevated plus maze

451

The anxiety-like behaviour was investigated using the EPM apparatus. The test was

452

performed essentially as described previously (Lister, 1987). In brief, the apparatus consisted

453

of a wooden maze with two enclosed arms (30 × 5 × 15 cm) and two open arms (30 × 5 ×

454

0.25 cm) that extended from a central platform (5 × 5 cm) to form a plus sign. The plus maze

455

apparatus was elevated to a height of 25 cm and placed inside a sound-attenuated room. The

456

trial was started by placing a mouse on the central platform of the maze facing its head

457

towards an open arm. The behavioural parameters recorded during a 5 min test period;

458

percentage open arm entries and percentage time spent in open arm. Entry into an arm was

459

considered valid only when all four paws of the mouse were inside that arm. The apparatus

460

was thoroughly cleaned with 70% ethanol after each trial.

461

4.5.3. Open-field test (OFT)

462

This test is used to estimate possible effects of drug treatment on spontaneous locomotor

463

activity. An acrylic transparent box (72 × 72 × 36 cm3) with its floor divided into 16 equal

464

sized squares (18 × 18 cm2) was used. Four squares were considered as the centre, and the 12

465

squares along the walls were considered as the periphery. Each mouse was put in the centre

466

of the box, and number of central and peripheral crossings (crossing the sector with four

467

paws), rearing movements (raising the forepaws) and the immobility time of mice were

468

observed for 5 min by a video camera.

469

4.5.4. Forced swimming test (FST)

470

FST is used to evaluate the immobility time as the absence of escape-oriented behaviour,

471

which is an important symptom of depression. In this task the immobility period of the

472

animal (during 6 min) was registered and the greater this time, the lower the animal’s

19

473

motivation to escape, representing a depressive-like behaviour (Porsolt et al., 1977). Animals

474

were placed individually in acrylic cylinder (25 cm high, 10 cm in diameter and 15 cm in

475

depth) containing water maintained at 25±2 °C. Mice were placed in an inescapable cylinder

476

for 6 min during the test session and video recorded but immobility time was counted for the

477

last 5 min. Mice were considered immobile when they ceased struggling, remained floating

478

motionless, and only made those movements necessary to keep their head above the water.

479

Mice were unable to escape or touch the bottom of the cylinder.

480

4.5.5. Sucrose preference test

481

The mice were tested for sucrose consumption as described earlier (Mao et al., 2009) with

482

slight modifications. Before the test, mice were trained to adapt sucrose solution (1%, w/v)

483

by placing two bottles of sucrose solution in each cage for a period of 24 h; then one bottle of

484

sucrose solution was replaced with water for 24 h. After the adaptation, mice were deprived

485

of water and food for 24 h. Animals were housed individually throughout the test duration

486

and were presented simultaneously with two bottles in the home cage, one containing a 1%

487

sucrose solution, respectively. To prevent the preference to position, the location of the two

488

bottles was varied during this period. After 24 h, the volumes of consumed sucrose solution

489

and water were recorded. Then percentage of sucrose consumption was calculated as ratio of

490

the amount of sucrose solution to that of total solution (sucrose and water) ingested within 24

491

h.

492

4.5.6. Serum sample collection

493

After the last behavioural test (day 31), mice were euthanized and blood was collected.

494

Samples were allowed to clot for 30 min at 37 °C before centrifugation for 10 min at 3000 ×g

495

at 4 °C. The serum was isolated and stored at −80 °C for corticosterone (CORT)

496

determination.

497

4.5.7. Dissection and homogenization

20

498

After the blood withdrawal, animals were sacrificed by cervical dislocation (day 31);

499

hippocampus (HC) region of the brain was quickly removed, weighed, and rinsed

500

immediately with ice-cold normal 0.9% NaCl. A 10% (w/v) tissue homogenates were

501

prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000 ×

502

g for 15 min and stored at - 80 °C until use.

503

4.5.8. Evaluation of reduced glutathione

504

Reduced glutathione (GSH) levels were determined to estimate endogenous defence

505

mechanism against oxidative stress. The method was based on Ellman’s reagent (DTNB)

506

reaction with free thiol groups (Ellman, 1959). The brain areas (HC) were diluted in EDTA

507

0.02 M buffer (10% w/v) and added to a 50% trichloroacetic acid solution. After

508

centrifugation (10,000 rpm, 5 min), the supernatant of the homogenate was collected and

509

mixed with 0.4 M tris–HCl buffer, pH 8.9 and 0.01M 5,5-dithiobis-(2-nitrobenzoic acid)

510

(DTNB). The assay mixture contained 0.1ml of supernatant, 2.7 ml of tris–HCl buffer of pH

511

8.9 and 0.2 ml of 0.01 M DTNB. The resultant yellow colour was immediately read at 412

512

nm using a spectrophotometer (Gen5 data analysis software Synergy H1, BIO-TEK

513

Instruments, MN, USA). Results were calculated based on a standard glutathione curve.

514

4.5.9. Evaluation of lipid peroxidation

515

The malondialdehyde (MDA) content, a quantitative measurement of lipid per-oxidation was

516

assayed in the form of thiobarbituric acid reactive substances (TBARS) by the method of

517

Draper (1993). It serves as an index of reactive oxygen species production. In this 0.1 ml of

518

supernatant was incubated with 0.5 ml Tris hydrochloric acid (0.1 M, pH 7.4) for 2 h. To this,

519

1 ml of trichloroacetic acid (10% w/v) was added and centrifuged at 1000 ×g for 10 min. To

520

1 ml supernatant, 1 ml (0.67% w/v) thiobarbituric acid (TBA) was added and kept in the

521

boiling water bath for 10 min, immediately kept cold in a bath of ice and then 1 ml distilled

522

water was added. The amount of lipid peroxidation products was measured by reaction with

21

523

thiobarbituric acid at 532 nm using the spectrophotometer (Gen5 data analysis software

524

Synergy H1, BIO-TEK Instruments, MN, USA).

525

4.5.10. Evaluation of nitrite level

526

The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide

527

was assayed spectrophotometrically according to the previously published report (Gawali et

528

al., 2016). Briefly, 60 µl of sample supernatant was mixed with 5 µl of nicotinamide adenine

529

dinucleotide phosphate (NADPH), 10 µl of flavin adenine dinucleotide (FAD) and 5 µl of

530

nitrate reductase for 1 h at 37oC in the dark. Zinc sulphate was added to precipitate the

531

proteins. After centrifuging (6000×g), equal volumes of supernatant (100 µl) and Greiss

532

reagent (100 µl) (1 : 1 mixture of 1% sulfanilamide in 3% orthophosphoric acid and 0.1%

533

naphthyl ethylene diamine) were mixed and the mixture was incubated for 10 min at room

534

temperature in the dark. The plates were then read at 540 nm by UV spectrophotometer, and

535

NOx was calculated by using a sodium nitrite standard curve standard curve.

536

4.5.11. Estimation of acetyl cholinesterase (AChE) activity

537

AChE is a marker of loss of cholinergic neurons in the brain region. The quantitative

538

measurement of acetylcholinesterase levels in the hippocampal homogenates were assessed

539

as described by Ellman et al. (1961). The assay mixture contained 0.05 ml of supernatant, 3

540

ml of 0.01 M sodium phosphate buffer (pH 8.0), 0.10 ml of 0.75 mM acetyl thio-choline

541

iodide (AcSCh) and 0.10 ml of Ellman reagent (5,50-dithiobis[2-nitrobenzoic acid] 10 mM.

542

The change in absorbance was measured for 2 min at 30 s intervals at 412 nm

543

spectrophotometer (Gen5 data analysis software Synergy H1, BIO-TEK Instruments, MN,

544

USA). Results were expressed as micromoles of acetylthiocholine iodide hydrolyzed per min

545

per mg of protein.

546

4.5.12. Assessments of BDNF and corticosterone level

22

547

The concentration of BDNF was determined using commercially available immunoenzymatic

548

assay (ELISA). The ELISA was performed according to the manufacturer’s protocol

549

(Krishgen Biosystems, India). To determine the alteration in the HPA axis, we measured

550

serum CORT level. Measurement of serum CORT was performed using a commercially

551

available enzyme-linked immunosorbent assay (ELISA) kit (IBL, USA) according to the

552

manufacturer instructions.

553

4.5.13. Statistics

554

Data from behavioural and neurochemical determinations are presented as mean ± S.E.M.

555

(standard errors of the mean). All the Statistical analysis of data was carried out by one-way

556

analysis of variance (ANOVA), followed by post hoc Tukey's Multiple Comparison Test

557

except morris water-maze test which was analyzed by two-way ANOVA with post-hoc

558

Bonferroni mean comparisons. Differences were considered statistically significant if the P <

559

0.05. The statistical program used was GraphPad Prism 5.0 Version for Windows, GraphPad

560

Software (San Diego, CA, USA).

561

Conflict of Interest

562

The authors declare no conflicts of interest.

563

Acknowledgement

564

The authors are thankful to University Grants Commission (UGC), New Delhi for providing

565

financial support and Institute of Chemical Technology, Mumbai for provided the research

566

facility for completing work. Authors would like to thank Ms. Sarayu Pai for her assistance in

567

the preparation of manuscript and technical support.

568 569 570 571

23

572 573 574

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817

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818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836

34

837

Figure captions

838

Fig. 1. Diagrammatic representation of the entire study protocol. Mice were subjected to a

839

battery of stressors for 28 days. Agmatine (20 and 40 mg/kg, i.p.) alone and in combination

840

with NO modulators like L-NAME (15 mg/kg, i.p.) and L-arginine (400 mg/kg i.p.) were

841

administered daily. The schematic representation of the experimental protocol indicating both

842

treatment and the time of behavioural testing are depicted. Mice were sacrificed by

843

decapitation 6 h after completion of behavioural testing on day 31, blood samples were

844

collected and brain was dissected and stored at −80°C for subsequent biochemical analysis.

845

Fig. 2. Effect of agmatine and its combination with nitric oxide modulators on escape latency

846

time (ELT) (A) and time spent in target quadrant (B) in Morris water maze test. The ELT was

847

analysed by using two-way ANOVA followed by Bonferroni post hoc test whereas time

848

spend in target quadrant were analysed by one-way ANOVA followed by Tukey’s post hoc

849

test. For statistical significance in escape latency time, aP< 0.05 saline is compared with

850

CUMS; bP < 0.05 CUMS is compared with the dose of agmatine 20 mg/kg; cP< 0.05 CUMS

851

compared is with the dose of agmatine 40 mg/kg; dP< 0.05 CUMS compared with the dose of

852

agmatine 20 mg/kg + L-NAME (15 mg/kg). Values are expressed as the mean ± S.E.M from

853

8 animals. For statistical significance in spent in target quadrant,

854

with control group; ***P < 0.001 and *P < 0.05 as compared with CUMS control.

855

Fig. 3. Depicts the percentage of open arm entries (A) and percentage of open arm time in

856

elevated plus maze (B). Values are expressed as the mean ± S.E.M from 8 animals and were

857

analyzed using one-way ANOVA followed by Tukey’s post hoc test. For statistical

858

significance,

859

compared with CUMS control.

860

Effect of agmatine and its combination with nitric oxide modulators on open-field test

861

performance in CUMS mice. Fig. 4. depicts the counts of crossing (A) and rearing (B) in

###

P < 0.001 as compared

###

P < 0.001 as compared with control group; ***P < 0.001 and *P < 0.05 as

35

862

mice after 28 days of drug treatment. Values are expressed as the mean ± S.E.M from 8

863

animals and were analyzed using one-way ANOVA followed by Tukey’s post hoc test. For

864

statistical significance,

865

0.05 as compared with CUMS control.

866

Fig. 5. Effects of agmatine and its combination with nitric oxide modulators on immobility

867

time in the forced swimming test (A) and percent sucrose preference (B) in CUMS mice.

868

Values are expressed as the mean ± S.E.M from 8 animals and were analyzed using one way

869

ANOVA followed by Tukey's post hoc test. For statistical significance,

870

compared with control group; ***P < 0.001 and *P < 0.05 as compared with CUMS.

871

Fig. 6. Effect of agmatine and its combination with nitric oxide modulators on oxido-

872

nitrosative stress parameters in hippocampus (A) Reduced glutathione, (B) Lipid per-

873

oxidation and (C) Nitric oxide level. Agmatine treatment attenuates the oxidative-nitrosative

874

stress in the hippocampus (HC) of CUMS mice. Further, treatment of L-NAME (10 mg/kg)

875

with sub-effective dose of agmatine (20 mg/kg) potentiated the antioxidant like effect

876

agmatine. Values are expressed as the mean ± S.E.M from 8 animals and were analyzed using

877

one way ANOVA followed by Tukey's post hoc test.

878

group; ***P < 0.001 and *P < 0.05 as compared with CUMS.

879

Fig. 7. Effect of agmatine and its combination with nitric oxide modulators on

880

acetylcholinesterase activity in CUMS mice. Values are expressed as the mean ± S.E.M from

881

8 animals and were analyzed using one-way ANOVA followed by Tukey’s post hoc test. For

882

statistical significance,

883

0.05 as compared with CUMS control.

884

Fig. 8. Effect of agmatine and its combination with nitric oxide modulators on serum

885

corticosterone (CORT) levels. Agmatine inhibits the production of CORT in the

886

hippocampus of mice, determined by ELISA. Values are expressed as the mean ± S.E.M

###

###

P < 0.001 as compared with control group; ***P < 0.001 and *P <

###

###

P < 0.001 as

P < 0.001 as compared with control

P < 0.001 as compared with control group; ***P < 0.001 and *P <

36

887

from 8 animals and were analyzed using one way ANOVA followed by Tukey's post hoc test.

888

###

889

CUMS.

890

Fig. 9. Effect of agmatine and its combination with nitric oxide modulators on BDNF levels

891

in the hippocampus. Agmatine increased BDNF levels as compared to CUMS group in the

892

hippocampus of mice, determined by ELISA. Values are expressed as the mean ± S.E.M

893

from 8 animals and were analyzed using one way ANOVA followed by Tukey's post hoc test.

894

###P < 0.001 as compared with control group; ***P < 0.001 and *P < 0.05 as compared with

895

CUMS.

896

Fig. 10 Schematic illustration for the different targets or pathways involved in the

897

antidepressant-like effect of agmatine. The present results showed that anxiolytic and

898

antidepressant-like effect of agmatine in a CUMS model. Agmatine showed marked effect on

899

depression and anxiety-like behaviour in mice through nitrergic pathway, which may be

900

related to modulation of oxidative–nitrergic stress, CORT and BDNF levels.

P < 0.001 as compared with control group; ***P < 0.001 and *P < 0.05 as compared with

901 902 903 904 905 906 907 908 909 910 911

37

912 913

Figures Fig. 1.

914 915 916

Fig. 2.

917

(A)

918 919

38

920

(B)

921 922

Fig. 3

923

(A)

924

39

925

(B)

926 927 928

Fig. 4

929

(A)

930 40

931

(B)

932 933 934

Fig. 5.

935

(A)

936

41

937

(B)

938 939 940

Fig. 6.

941

(A)

942 42

943

(B)

944 945

(C)

946

43

947

Fig. 7.

948 949

Fig. 8.

950 951

44

952

Fig. 9.

953 954

955

Fig. 10.

956 957

45

958

959

960 961

Table 1 Schedule of stressors used in the 28 days of chronic unpredictable mild stress procedure

962

Day 1

10-min Tail pinch in restrainers

963

Day 2

7 h cage tilt

964

Day 3

24 h food deprivation

965

Day 4

24 h water deprivation

966

Day 5

Overnight illumination

967

Day 6

24 h soiled cage

968

Day 7

2 h physically restraint

969

Day 8

24 h exposure to a foreign object

970

Day 9

Foot shock for 45 min*

971

Day 10

24 h isolation housing

972

Day 11

Overnight illumination

973

Day 12

10-min tail pinch in restrainer

974

Day 13

24 h soiled cage

975

Day 14

2 h physically restraint

976

Day 15

24 h food deprivation

977

Day 16

10-min cold water swim

978

Day 17

24 h isolation housing

979

Day 18

24 h water deprivation

980

Day 19

Foot shock for 45*

981

Day 20

24 h exposure to a foreign object

982

Day 21

2 h physically restraint

983

Day 22

C-7 h cage tilt 46

984

Day 23

24 h soiled cage

985

Day 24

2 h physically restraint

986

Day 25

24 h soiled cage

987

Day 26

24 h water deprivation

988

Day 27

Overnight illumination

989

Day 28

24 h isolation housing

990

* Foot shock (1 mA, 1 second duration, average 1 shock/min for 45 min)

991

992

47

993 994 995

Highlights
Unpredictable chronic mild stress induces depressive and anxiety like behaviours along with memory deficits in mice.

996


Agmatine attenuated CUMS-induced depression and anxiety-like behaviour.

997


Treatment of agmatine alleviated CUMS evoked oxidative stress and reduced levels

998 999 1000 1001 1002

of acetylcholinesterase.
Agmatine up-regulated the BDNF level and inhibited corticosterone level in CUMS mice.
Anxiolytic and antidepressant action of agmatine may be mediated through nitrergic pathway.

1003 1004

48