European Journal of Pharmacology 720 (2013) 115–120
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
Agmatine attenuates chronic unpredictable mild stress induced behavioral alteration in mice Brijesh G. Taksande, Dharmesh S. Faldu, Madhura P. Dixit, Jay N. Sakaria, Manish M. Aglawe, Milind J. Umekar, Nandkishor R. Kotagale n Division of Neuroscience, Department of Pharmacology, Shrimati Kishoritai Bhoyar College of Pharmacy, New Kamptee, Nagpur 441002, Maharashtra, India
art ic l e i nf o
a b s t r a c t
Article history: Received 2 May 2013 Received in revised form 19 October 2013 Accepted 23 October 2013 Available online 29 October 2013
Chronic stress exposure and resulting dysregulation of the hypothalamic pituitary adrenal axis develops susceptibility to variety of neurological and psychiatric disorders. Agmatine, a putative neurotransmitter has been reported to be released in response to various stressful stimuli to maintain the homeostasis. Present study investigated the role of agmatine on chronic unpredictable mild stress (CUMS) induced behavioral and biochemical alteration in mice. Exposure of mice to CUMS protocol for 28 days resulted in diminished performance in sucrose preference test, splash test, forced swim test and marked elevation in plasma corticosterone levels. Chronic agmatine (5 and10 mg/kg, ip, once daily) treatment started on day15 and continued till the end of the CUMS protocol significantly increased sucrose preference, improved self-care and motivational behavior in the splash test and decreased duration of immobility in the forced swim test. Agmatine treatment also normalized the elevated corticosterone levels and prevented the body weight changes in chronically stressed animals. The pharmacological effect of agmatine was comparable to selective serotonin reuptake inhibitor, fluoxetine (10 mg/kg, ip). Results of present study clearly demonstrated the anti-depressant like effect of agmatine in chronic unpredictable mild stress induced depression in mice. Thus the development of drugs based on brain agmatinergic modulation may represent a new potential approach for the treatment of stress related mood disorders like depression. & 2013 Elsevier B.V. All rights reserved.
Keywords: Agmatine Depression Anhedonia CUMS Splash test FST
1. Introduction Chronic stress and the resulting dysregulation of the hypothalamic pituitary adrenal (HPA) axis enhance vulnerability to a variety of neurological disorders. Several neurotransmitters/neuromodulators and their interactions in CNS regulate response to stress (Grønli et al., 2005). However the exact mechanism behind deleterious effect of chronic stress exposure is yet to be elucidated. Agmatine, a biogenic amine and putative neurotransmitter, has been implicated in stress response and related disorders (Aricioglu et al., 2003). Agmatine is biosynthesized from amino acid Larginine by arginine decarboxylase, stored in synaptic vesicles, accumulated by uptake, released by depolarization and inactivated by agmatinase. Agmatine metabolized to putrescine and guanido-butanoic acid by an enzyme agmatinase and diamine oxidase respectively (See review Uzbay, 2012).
n
Corresponding author. Tel.: þ 91 7109 288650; fax; þ 91 7109287 094. E-mail address: [email protected] (N.R. Kotagale).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.10.041
Agmatine interact with α2 adrenergic, imidazoline, NMDA receptors and possesses nitric oxide synthase inhibitory activity in brain (see review Uzbay, 2012). Although the physiological role of agmatine in normal brain is largely unknown, the exogenous agmatine has exhibited several intriguing neurally relevant functions of potential therapeutic importance. Systemic administration of agmatine reduced neuronal injury produced by focal ischemia, spinal cord injury and hypoxic ischemia (Lu et al., 2006), diminishes chemically and electrically induced convulsions (Demehri et al., 2003). It also produces anxiolytic (Lavinsky et al., 2003), antidepressant (Zomkowski et al., 2002), antinociceptive (Önal et al., 2003), anti-convulsant (Bence et al., 2003), anti-inflammatory (Satriano et al., 2001), antiproliferative (Regunathan and Reis, 1997) and neuroprotective effects (Olmos et al., 1999). Agmatine has been proposed as an adjuvant in the treatment of several chronic pain syndromes (Paszcuk et al., 2007). In addition, several stressful pathological conditions like inflammation, ischemia and lipopolysacarides (LPS) injection increased agmatine levels in brain. On the other hand, simultaneous treatment with exogenous agmatine attenuated repeated immobilization induced elevated corticosterone levels and glutamate efflux in brain nuclei associated with modulation of stress response (M.Y. Zhu et al., 2008; M. Zhu et al., 2008). Further, several nuclei of hypothalamus and
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pituitary gland have shown abundant agmatine immunoreactivity (Otake et al., 1998) and its co-localization with neuropeptides (Gorbatyuk et al., 2001). Several studies have reported the neuroprotective effects of agmatine against cell damage caused by glucocorticoids and glutamate in primary neuronal cultures of the hippocampus (Iyo et al., 2006; Wang et al., 2006; M.Y. Zhu et al., 2008; M. Zhu et al., 2008). These studies have focused agmatine as an endogenous neuromodulator of stress and suggested that agmatine homeostasis may play an important role in modulation of stress. Endogenous levels of agmatine increased during stressful conditions in compensatory manner, however not high enough to modulate the harmful effect of stressor or inflammation (M.Y. Zhu et al., 2008; M. Zhu et al., 2008). Hence, exogenous administration which restored the agmatine levels can exhibit anti-inflammatory, antiproliferative and neuroprotective effects in rodent. Therefore, one might postulate that as one element of self-protection mechanisms in the brain, agmatine synthesis is trigged by stress through activation of ADC expression, which in turn increases endogenous agmatine levels as an initial protective response to stress. However, the effect of exogenous agmatine on stress induced depression and related behavioral alteration remained unexplored. This study was therefore designed to evaluate the role of agmatine in chronic unpredictable mild stress-induced neurobehavioral effects and biochemical changes in mice. Chronic unpredictable mild stress (CUMS) exposed animal exhibits several neurobehavioral alteration, resembling the symptoms of chronic human depression and widely employed for preclinical screening of antidepressants. In the present study we examined the effect of chronic agmatine treatment on CUMS induced anhedonia, motivational behavior, despair, body weight changes and elevated corticosterone levels in mice.
2. Material and methods 2.1. Subjects Adult albino Swiss mice of either sex weighing 25–30 g were housed in polypropylene cages in a temperature (25 72 1C), relative humidity (50–70%) and maintained on a 12:12 h light/ dark cycle (lights on 07:00–19:00 h). Food and water were provided ad libitum except during specific experimental protocols. Control, stressed and treatment group were tested in identical environmental conditions. Cages were changed once per week and water bottlers were changed three times per week. All experimental procedures were carried out under strict compliance with Institutional Animal Ethical Committee according to guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests; Government of India; New Delhi. 2.2. Drug solutions and administrations Agmatine sulfate was purchased from Sigma-Aldrich, USA while fluoxetine was received as a gift sample from SUN pharmaceuticals, Baroda, India. Both the drugs were dissolved in saline just before the experiments and administered intraperitoneally (ip) in a volume of 1 ml/kg. Normal saline (0.9% w/v NaCl) was used as control. 2.3. Chronic unpredictable mild stress The chronic unpredictable mild stress (CUMS) protocol was designed to maximize the unpredictable nature of stressor. Restrained stress, continuous overnight lighting, tail pinch, shaker stress, tilt-cage
and overnight soiled cage were applied once a week over a period of 4 weeks. Immediately after completion of each stress session, the animals were returned to their home cage and transferred to standard laboratory condition. The duration of CUMS and the nature of different stressor are based on the available literature (Ducottet and Belzung, 2004; Ducottet et al., 2003; Katz et al., 1981; Lu et al., 2006; Willner et al., 1987). Separate group of mice subjected to CUMS were injected with saline (1 ml/kg, ip) or fluoxetine (10 mg/kg, ip) or agmatine (2.5, 5, and 10 mg/kg, ip) from day-15 onwards in between 09:00 and 10:00 daily. Unstressed group was injected with saline and handled daily but was not subjected to any stressors. In order to avoid the immediate effect of stressor and pharmacological treatment all the behavioral test were carried out 24 h after the last stressor except sucrose preference test (SPT). SPT was carried out on days 0, 14, 21 and 28. The body weight of animals was measured daily. Separate group of animal was used for every behavioral paradigm. 2.3.1. Sucrose preference test (SPT) Sucrose preference test was performed as described earlier (Jindal et al., 2013; Liu et al., 2013) with some modifications. As agmatine enhanced caloric intake in animals (Taksande et al., 2011) and sucrose consumption can be altered by food and water deprivation (Matthews et al., 1995), we did not apply food and water deprivation in CUMS protocol. SPT was conducted on day 0, 14, 21 and 28 of CUMS protocol. Briefly, 72 h before the test, mice were habituated to drink 1% sucrose solution (w/v) and subsequently exposed to two bottles (1% sucrose solution in one bottle and tap water in another bottle). After habituation mice were housed in individual cages. Mice were permitted ad libitum access to 100 ml of sucrose solution (1% w/v) and 100 ml of tap water. We have also minimized the effect of side preference in drinking behavior by switching the bottle in the middle of the test (Strekolova and Steinbusch, 2010). In addition, we have monitored the sucrose preference for 3 h to minimized the % error commonly observed with little amount of sucrose consumed in 1 h sucrose preference test. After 3 h of exposure the consumption volumes of sucrose solution and tap water were recorded and the sucrose preference was calculated as [(sucrose consumption)/(water consumptionþ sucrose consumption)] 100. Mice showing basal sucrose preference less than 60% were considered anhedonic and not used in the study. 2.3.2. Splash test The splash test is pharmacological validated animal model exhibiting motivational behavior of animals. CUMS decreases grooming behavior in splash test and this phenomenon is considered to be similar with apathy observed in depressive patients. Animals were isolated in home cages for 24 h and 10% sucrose solution is squirted on the dorsal coat of mice and the latency to initiate a grooming behavior as well as the frequency of grooming was recorded by independent observers unaware of the treatment for a period of 5 min where grooming (nose/face grooming, head washing and body grooming) latency is considered as self-care and grooming frequency is considered as motivational behavior. 2.3.3. Forced swim test The procedure was quite similar as described by Porsolt et al., (1977) with some modification. Mice were introduced individually in plexiglas cylinder (21 cm height 12 cm internal diameter) containing fresh water till a height of 9 cm at 257 1 1C and forced to swim for a 6 min test session. The immobility time was measured by trained observer blind to the treatment. A mouse was considered immobile when it remained floating motionless in
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water except making any necessary movement to keep its head above water. 2.3.4. Corticosterone levels Immediately after the completion of behavioral tests on day-29, blood was withdrawn from the retro-orbital plexus of the mice in the eppendorff tubes previously rinsed with sodium citrate and centrifuged at 13,000g for 15 min at 4 1C. Separated plasma was stored at 20 1C for corticosterone estimation. A quaternary gradient HPLC system equipped with Crestpak C18T-5 column and PDA detector (MD2010 plus) (Jasco, Japan) was used for quantification of plasma corticosterone. The biochemical estimation on HPLC was carried according to the method described by Woodward and Emery (1987) and Sheikh et al. (2007) with minor modifications. Briefly, 50 ml of plasma was extracted with 1 ml of DCM–Ether mixture (DCM:Ether—50:50) on mechanical shaker for 15 min. Supernatant (50 ml) was transferred to eppendorff tube and evaporated under the slow stream of nitrogen. After complete evaporation, 1 ml of mobile phase (Water:Methanol—80:20) was added and 20 ml of this was injected into the HPLC (Flow rate1 ml/min and estimated at 243 nm). 2.4. Statistical analysis The data were expressed as mean 7S.E.M. The results of splash test, forced swimming test, open field were analyzed by one-way ANOVA followed by the Newman–Keul test. Sucrose preference and body weight changes were analyzed by two-way ANNOVA with post-hoc Bonferroni mean comparisons. Corticosterone levels were analyzed by unpaired t test. Results of statistical tests with Po 0.05 were considered significant.
3. Results 3.1. Effect of chronic administration of agmatine in stress induced anhedonia Fig. 1 shows the effect of chronic unpredictable mild stress on sucrose preference test. Chronic exposure of animal to unpredictable mild stress significantly decrease the sucrose preference at day-14 (P o0.001) and the effect was continued upto day-28 (P o0.001) as compared to unstressed animals [FStress Time (3, 30) ¼75.59, P o0.001; FStress(1, 30) ¼ 3775.72, Po 0.001; FTime (3, 30) ¼18.88, P o0.001- Two way ANOVA]. The sucrose preference on day-28 was significantly lower as compared to day-14 (P o0.001). Post-hoc Bonferroni mean comparisons revealed significant difference in sucrose preference between the groups on day-14 (P o0.001) and day-28 (P o0.001). Agmatine (5 and 10 mg/kg) [FTreatment Time (12, 75) ¼11.66, P o0.001; FTreatment (4, 75) ¼ 62.71, P o0.001; FTime (3, 75) ¼ 35.35, Po 0.001] and fluoxetine (10 mg/kg) [FTreatment Time (6, 45) ¼20.94, P o0.001; FTreatment (2, 45)¼ 174.63, P o0.001; FTime (3, 45) ¼9.72, Po 0.001] treatment from day-15 onwards completely reversed the anhedonia induced by CUMS paradigm on day28. Post-hoc Bonferroni mean comparisons revealed that the sucrose preference in the stressed animals treated with agmatine (5 and 10 mg/kg) and fluoxetine (10 mg/kg, ip) were significantly different as compared to stressed mice treated with vehicle on day-28. However no significant effect of treatment was observed on day-21. The difference in the % sucrose preference on day 21 and 28 in the animal treated with agmatine (10 mg/kg) was not statistically significant as compared to fluoxetine (10 mg/kg, ip) treatment. Administration of agmatine and fluoxetine to nonstressed group did not produce any effect on sucrose preference
Fig. 1. Effects of agmatine and fluoxetine treatment on sucrose preference test. Mice (n¼ 6) were subjected to chronic unpredictable mild stress (CUMS) and injected with saline (1 ml/kg, ip) or fluoxetine (10 mg/kg, ip) or agmatine (2.5– 10 mg/kg, ip) from day-15 onwards and sucrose preference was evaluated on days 0, 14, 21 and 28. Control group received saline (1 ml/kg, ip) under the same schedule and was not subjected to stress. Each point indicate mean sucrose preference (%) 7S.E.M. $P o0.001 vs. saline treated non-stressed animals; n Po 0.05 nnP o 0.001 vs. saline treated CUMS animals (two way ANOVA post-hoc Bonferroni mean comparisons).
test as compared to vehicle treated non-stressed group (data not shown).
3.2. Effect of agmatine in stress induced alterations in self-care and motivational behavior Chronic agmatine (5 and 10 mg/kg) and fluoxetine (10 mg/kg) treatment from third week onward of CUMS protocol restored the motivational and self-care behavior in the splash test (Fig. 2). One way ANOVA followed by post-hoc Newman–Keuls comparisons demonstrated that agmatine [5(Po 0.001 and P o0.001) and 10 mg/kg (Po 0.001 and P o0.001) respectively] significantly decreased the grooming latency [F (4, 29) ¼22.08, Po 0.001] [Fig. 2A] and increased grooming frequency [Fig. 2B] as compared to vehicle treated stressed animals. Similarly fluoxetine (10 mg/kg) [(P o0.001and P o0.001) respectively] treatment to separate group of animals showed significant decrease in grooming latency [F (2, 17) ¼94.95, P o0.001] and increase in grooming frequency [F (2, 17) ¼ 89.33, P o0.001]. Agmatine (2.5–10 mg/kg) and fluoxetine (10 mg/kg) treatment to non-stressed mice did not affect the behavior in splash test (data not shown).
3.3. Effect of agmatine in FST As shown in Fig. 3, CUMS exposure significantly increases immobility duration in stressed animals as compared to nonstressed group (Po 0.001). Repeated administration of agmatine (5 and 10 mg) dose dependently reduces the % immobility time in mice exposed to CUMS by 23% (P o0.05) and 42% (P o0.001) respectively as compared to vehicle treated stressed animal [F (4, 29) ¼10.08, Po0.001]. Agmatine at the doses used here did not influence the immobility duration in non-stressed mice (data not shown). Fluoxetine treatment at dose of 10 mg/kg to a separate group of mice also reduced immobility time in CUMS exposed animal by 46% (P o0.001) [F (2, 17) ¼51.51, Po 0.001].
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Fig. 3. Effects of agmatine and fluoxetine treatment on immobility time in forced swim test (FST). Mice (n¼ 6) were subjected to chronic unpredictable mild stress (CUMS) and injected with saline (1 ml/kg, ip) or fluoxetine (10 mg/kg, ip) or agmatine (2.5–10 mg/kg, ip) from day-15 onwards and immobility time [(seconds (s))] was determined 24 h after the last stressor. Control group received saline (1 ml/kg, ip) under the same schedule and was not subjected to stress. Each bar indicate mean immobility time (s)7S.E.M. $Po0.001vs saline treated non-stressed animals; n Po0.05, nn Po0.001 vs. saline treated CUMS animals (one way ANOVA post-hoc Newman–Keul mean comparisons).
Fig. 2. Effects of agmatine and fluoxetine treatment on motivational behavior in splash test. Mice (n¼ 6) were subjected to chronic unpredictable mild stress (CUMS) and injected with saline (1 ml/kg, ip) or fluoxetine (10 mg/kg, ip) or agmatine (2.5–10 mg/kg, ip) from day-15 onwards and grooming latency [(seconds (s))] (Fig. 2A) and frequency (Fig. 2B) were evaluated 24 h after the last stressor. Control group received saline (1 ml/kg, ip) under the same schedule and was not subjected to stress. Each bar indicate mean grooming latency (s)/grooming frequency7 S.E.M. $Po 0.01, $$P o0.001 vs. saline treated non-stressed animals; n Po 0.001 vs. saline treated CUMS animals (one way ANOVA post-hoc Newman– Keul mean comparisons).
3.4. Effect of agmatine on % body weight change in CUMS induced depression Body weight was monitored daily during the experimental protocol. Two-way ANOVA showed a significant reduction in the body weight of the CUMS exposed animals as compared to nonstressed mice [FStress Time (3, 30) ¼11.98, Po0.001; FStress (1, 30) ¼ 44.91, P o0.001; FTime (3, 30) ¼6.59, P o0.01]. Post hoc analysis by Bonferroni multiple comparison showed significant difference in % body weight change on day-21 (P o0.01) and 28 (Po0.05). No significant change in the body weight was observed in the mice that received repeated agmatine treatment and subjected to CUMS when compared against saline treated non-stressed animals [FTreatment Time (15, 90) ¼0.86, P ¼0.61; FTreatment (5, 90) ¼7.46, P o0.001; FTime (3, 90) ¼1.49, P ¼0.22]. As depicted in Fig. 4, fluoxetine (10 mg/kg) significantly prevented reduction in body weight in animals subjected to CUMS protocol. Two way ANOVA indicated insignificant change in the body weight in fluoxetine (10 mg/kg) treated animals as compared to saline injected mice exposed to CUMS [FTreatment Time (6, 45) ¼2.84, P o0.05; FTreatment (2, 45) ¼25.23, P o0.001; FTime (3, 45)¼ 0.57, P ¼0.64]. No significant difference was determined between the stressed animals injected with agmatine (10 mg/kg) and fluoxetine (10 mg/kg) (post-hoc Bonferroni mean comparisons).
Fig. 4. Effects of agmatine and fluoxetine treatment on changes in body weight. Mice (n¼ 6) were subjected to chronic unpredictable mild stress (CUMS) and injected with saline (1 ml/kg, ip) or fluoxetine (10 mg/kg, ip) or agmatine (2.5–10 mg/kg, ip) from day-15 onwards and body weights were recorded on day 0, 14, 21 and 28. Control group received saline (1 ml/kg, ip) under the same schedule and was not subjected to stress. Each point indicate mean changes in body weight (%) 7S.E.M. nP o0.05, nnP o 0.01 vs. saline injected non-stressed animals (two way ANOVA post-hoc Bonferroni mean comparisons).
3.5. Agmatine attenuates CUMS induced increased plasma corticosterone levels in mice HPLC analysis demonstrated the significant higher levels of plasma corticosterone in the animals subjected to CUMS for the period of 28 days (Po 0.001) as compared to non-stressed control group (t ¼13.93; df ¼4, unpaired t test). As depicted in Fig. 5, plasma corticosterone levels were significantly lower and comparable to the non-stressed group in agmatine [5 (P o0.01) and 10 mg/kg (Po 0.001)] (F (4, 14) ¼30.63, (P o0.001)) and fluoxetine (P o0.001) [F (2, 8) ¼74.75, (P o0.001)] treated mice.
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Fig. 5. Effects of agmatine and fluoxetine treatment on plasma corticosterone levels. Mice (n¼ 3) were subjected to chronic unpredictable mild stress (CUMS) and injected with saline (1 ml/kg, ip) or fluoxetine (10 mg/kg, ip) or agmatine (2.5–10 mg/kg, ip) from day-15 onwards. 24 h after the last stressor and blood samples were withdrawn to determine corticosterone level. Control group received saline (1 ml/kg, ip) under the same schedule and was not subjected to stress. Each bar indicate mean plasma corticosterone (ng/ml) 7 S.E.M. $Po 0.001vs saline treated non-stressed animals; nP o0.01, nnP o 0.001 vs. saline treated CUMS animals (one way ANOVA posthoc Newman Keul mean comparisons).
4. Discussion Stressful life experiences are important etiological factors in the onset and maintenance of depressive episode (Lee et al., 2002). In view of this, an animal model of CUMS induced depression has been developed to replicate the pathogenesis of depressive illness. Number of experimental evidence suggests that CUMS exposure to animals induce alteration in behavioral and biochemical parameters resembling symptoms of clinical depression (Luo et al., 2008). Consistent with several earlier findings, the results of the present study showed that mice when subjected to chronic exposure to a variety of mild stressor regimen induced significant reduction in sucrose preference test, grooming behavior in splash test, body weights and increased immobility in forced swim test and plasma corticosterone level as compared to non-stressed mice. Sucrose preference test is an indicator of anhedonia like behavioral change (Willner, 2005). The results of the present study showed that the long term treatment of agmatine significantly suppressed anhedonia induced by CUMS protocol suggesting the potential antidepressant like effect of agmatine. The antidepressant effect was specific to stress exposure as agmatine did not influence the sucrose consumption in normal non-stressed group. Similarly, agmatine treatment did not influence the water intake in naïve animals. The anti-depressant like effect of agmatine was compared to selective serotonin reuptake inhibitor—fluoxetine with the only difference that agmatine has shown delayed onset of action. The sucrose consumption was normalized by fluoxetine treatment on day-21 whereas it was evident on day-28 in agmatine treated group. This could be attributed to lower doses of agmatine used in the present study and their differential mechanism of action. Further, intraperitoneally injected agmatine extensively metabolized peripherally in liver and kidney and has very short biological half life (Halaris and Plietz, 2007). The delayed onset of action and significant weight gain is a possible side effect with SSRI and other antidepressant including tricyclic antidepressant and MAO inhibitors (Ruetsch et al., 2005). In our study, although agmatine exhibited delayed onset of action as compared to fluoxetine, it did not induced significant changes in body weight of animal after chronic administration. Moreover, unlike other antidepressant, it did not affect the locomotor counts of animal. Thus agmatinergic modulation may offer
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several advantages and may be useful in combination with other antidepressant in the treatment of resistant depression. The splash test is direct measure of self-care and motivational behavior. Chronic stress exposure to mice resulted in increase in latency and decreased frequency of grooming behavior, indicating loss of self-care and motivational behavior in mice subjected to CUMS procedure. The disturbance in grooming behavior is considered to mimic apathy observed in clinical depression (Willner, 2005). Agmatine and fluoxetine treatment prevented the effect of CUMS on splash test. Indeed, similar effects have been already reported for fluoxetine and CRF II antagonist (Ducottet et al., 2003; Santarelli et al., 2003). Chronic stress has been shown to dramatically increase the immobility time of mice in FST (Zhou et al., 2007). Similarly our CUMS protocol also increased immobility time in forced swim test in mice. Chronic treatment with agmatine and fluoxetine attenuated the CUMS associated reduction in immobility time. These behavioral observations demonstrated that agmatine produced an antidepressant like effect in chronically stressed mice. In parallel with our observation, simultaneous treatment with exogenous agmatine attenuated repeated immobilization induced architectural alteration in hippocampus and prefrontal cortex such as elevated plasma corticosterone levels and high glutamate influx (M.Y. Zhu et al., 2008; M. Zhu et.al., 2008). Agmatine also attenuated stress and lipopolysaccharide induced hyperthermia in rats (Aricioglu and Regunathan, 2005). Moreover Bernstein et al. (2012) have shown up-regulation of the agmatine degrading enzyme agmatinase in patients with unipolar and bipolar depression suggesting that reduction of endogenous brain agmatine level may play a central role in the pathogenesis of depression. Thus, it can be inferred from our results and available literature that endogenous agmatine system may play an important role in adaptation response to chronic stress in order to maintain brain homeostasis. In the present study, the CUMS exposure led to significant reduction in body weights compared to saline treated nonstressed animals. The results are in agreement with previous data showing that chronic mild stress lead to reduced body weight in exposed animals (Wang et al., 2008). Fluoxetine and agmatine treatment significantly prevented the body weight reduction induced by CUMS protocol. It is important to note that, body weight reduction not only provides face validity to CUMS procedure but also consider as a diagnostic criterion for a major depressive episode in DSM-IV. Thus agmatine treatment might offer additional advantages in the treatment of stress related disorders associated with anorexia and significant loss of body weights. However, body weight changes can also be considered as a confounding variable in sucrose consumption and could lead to false results in CUMS protocol. Our results of anhedonia should not be attributed to body weight changes as absolute sucrose consumption may be influenced by body weight gain, sucrose preference may not be influenced and consider as a better index of anhedonia (Matthews et al., 1995). HPA axis plays a critical role in eliciting physiological responses to various stressful stimuli (Pan et al., 2006). Acute stress activates HPA axis with simultaneous increase in levels of corticosterone in rodents or cortisol in human being. Sustained activation of HPA axis is associated with an abnormally high blood glucocorticoid level, which may eventually lead to pathological conditions such as depression (Johnson et al., 2006). Thus, the normalization of HPA axis and glucocorticoid levels may be critically involved in the therapeutic action of antidepressant drug. Normalization of the HPA system has been shown to be a prerequisite for stable remission of the disease (Holsboer, 2000), which has been shown to occur during successful antidepressant treatment (Barden et al., 1995), again supporting the importance of the stress hormone system in the development and maintenance of affective disorders. In accordance with several earlier findings, we found that
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CUMS significantly elevated corticosterone levels in mice plasma, and chronic agmatine treatment prevented these alterations. Thus preventive effects of agmatine on CUMS associated behavioral alterations might be linked with reduction in the corticosterone levels. Although we have not investigated specific mechanism, target receptor and enzymes like α2 adrenoceptor, imidazoline receptor, NMDA receptor or nitric oxide might be involved in antidepressant like effect of agmatine. Further more specific studies are required for better understanding of this proposed mechanism. To conclude, the results of present study demonstrated antidepressant like effect of agmatine in chronic unpredictable mild stress induced depression in mice. Chronic agmatine (5 and 10 mg/ kg, ip, once daily) treatment started on day-15 and continued till the end of the CUMS protocol significantly increased sucrose preference, improved self-care and motivational behavior in splash test, decreased duration of immobility in forced swim test and also normalized the body weight changes and elevated corticosterone plasma levels in mice. Thus the development of drugs based on modulation of agmatinergic system in brain may represent a new potential approach for the treatment of stress related neurological disease like depression. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2013.10. 041. References Aricioglu, F., Regunathan, S., 2005. Agmatine attenuates stress- and lipopolysaccharideinduced fever in rats. Physiol. Behav. 85, 370–375. Aricioglu, F., Regunathan, S., Piletz, J., 2003. Is agmatine an endogenous factor against stress? Ann. NY. Acad. Sci. 1009, 127–132. Barden, N., Reul, J.M., Holsboer, F., 1995. Do antidepressants stabilize mood through actions on the hypothalamic–pituitary–adrenocortical system? Trends Neurosci. 18, 6–11. Bence, K., David, R., Stables, J.P., Crooks, P.A., 2003. An in vivo evaluation of the antiseizure activity and acute neurotoxicity of agmatine. Pharmacol. Biochem. Behav. 74, 771–775. Bernstein, H.G., Stich, C., Jäger, K., Dobrowolny, H., Wick, M., Steiner, J., Veh, R., Bogerts, B., Laube, G., 2012. Agmatinase, an inactivator of the putative endogenous antidepressant agmatine, is strongly upregulated in hippocampal interneurons of subjects with mood disorders. Neuropharmacology 62, 237–246. Demehri, S., Homayoun, H., Honar, H., Riazi, K., Vafaie, K., Roushanzamir, F., Dehpour, A.R., 2003. Agmatine exerts anticonvulsant effect in mice: modulation by alpha 2-adrenoceptors and nitric oxide. Neuropharmacology 45, 534–542. Ducottet, C., Belzung, C., 2004. Behavior in the elevated plus-maze predicts coping after subchronic mild stress in mice. Physiol. Behav. 81, 417–426. Ducottet, C., Griebel, G., Belzung, C., 2003. Effects of the selective nonpeptide corticotropin-releasing factor receptor 1 antagonist antalarmin in the chronic mild stress model of depression in mice. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 27, 625–631. Gorbatyuk, O.S., Milner, T.A., Wang, G., Regunathan, S., Reis, D.J., 2001. Localization of agmatine in vasopressin and oxytocin neurons of the rat hypothalamic paraventricular and supraoptic nuclei. Exp. Neurol. 171, 235–245. Grønli, J., Murison, R., Fiske, E., Bjorvatn, B., Sørensen, E., Portas, C.M., Ursin, R., 2005. Effects of chronic mild stress on sexual behavior, locomotor activity and consumption of sucrose and saccharine solutions. Physiol. Behav. 84, 571–577. Halaris, A., Plietz, J., 2007. Agmatine metabolic pathway and spectrum of activity in brain. CNS Drugs 21 (11), 885–900. Holsboer, F., 2000. The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology 23, 477–501. Iyo, A.H., Zhu, M.Y., Ordway, G.A., 2006. Expression of arginine decarboxylase in brain regions and neuronal cells. J. Neurochem. 96, 1042–1050. Jindal, A., Mahesh, R., Bhatt, S., 2013. Etazolate, a phosphodiesterase 4 inhibitor reverses chronic unpredictable mild stress-induced depression-like behavior and brain oxidative damage. Pharmacol. Biochem. Behav. 105, 63–70. Johnson, S.A., Fournier, N.M., Kalynchuk, L.E., 2006. Effect of different doses of corticosterone on depression-like behavior and HPA axis responses to a novel stressor. Behav. Brain Res. 168, 280–288. Katz, R.J., Roth, K.A., Carroll, B.J., 1981. Acute and chronic stress effects on open field activity in the rat: implications for a model of depression. Neurosci. Biobehav. Rev. 5, 247–251.
Lavinsky, D., Arteni, N.S., Netto, C.A., 2003. Agmatine induces anxiolysis in the elevated pluse maze task in adults rat. Behav. Brain Res. 141, 19–24. Lee, A.L., Ogle, W.O., Sapolsky, R.M., 2002. Stress and depression: possible links to neuron death in the hippocampus. Bipolar Disord. 4, 117–128. Liu, Y., Jia, G., Gou, L., Sun, L., Fu, X., Lan, N., Li, S., Yin, X., 2013. Antidepressant-like effects of tea polyphenols on mouse model of chronic unpredictable mild stress. Pharmacol. Biochem. Behav. 104, 27–32. Lu, X.-Y., Kim, C.S., Frazer, A., Zhang, W., 2006. Leptin: a potential novel antidepressant. Proc. Natl. Acad. USA 103, 1593–1598. Luo, D., An, S., Zhang, X., 2008. Involvement of hippocampal serotonin and neuropeptide Y in depression induced by chronic unpredicted mild stress. Brain Res. Bull. 77, 8–12. Matthews, K., Forbes, N., Reid, I.C., 1995. Sucrose consumption as an hedonic measure following chronic unpredictable mild stress. Physiol. Behav. 57, 241–248. Olmos, G., DeGregorio-Rocasolano, N., Paz Regalado, M., Gasull, T., Assumpcio Boronat, M., Trullas, R., 1999. Protection by imidazol(ine) drugs and agmatine of glutamate induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor. Br. J. Pharmacol. 127, 1317–1326. Önal, A., Delen, Y., Ülker, S., Soykan, N., 2003. Agmatine attenuates neuropathic pain in rats: possible mediation of nitric oxide and noradrenergic activity in the brainstem and cerebellum. Life Sci. 73, 413–428. Otake, K., Ruggiero, D.A., Regunathan, S., Wang, H., Milner, T.A., Reis, D.J., 1998. Regional localization of agmatine in the rat brain: an immunocytochemical study. Brain Res. 787, 1–14. Paszcuk, A.F., Gadotti, V.M., Tibola, D., Quintão, N.L., Rodrigues, A.L.S., Calixto, J.B., Santos, A.R., 2007. Anti-hypernociceptive properties of agmatine in persistent inflammatory and neuropathic models of pain in mice. Brain Res. 1159, 124–133. Pan, Y., Zhang, W.-Y., Xia, X., Kong, L.-D., 2006. Effects of icariin on hypothalamic– pituitary–adrenal axis action and cytokine levels in stressed Sprague–Dawley rats. Biol. Pharm. Bull. 29, 2399–2403. Porsolt, R.D., Le Pichon, M., Jalfre, M., 1977. Depression: a new animal model sensitive to antidepressant treatments. Nature 266, 730–732. Regunathan, S., Reis, D.J., 1997. Stimulation of imidazoline receptors inhibits proliferation of human coronary artery vascular smooth muscle cells. Hypertension 30, 295–300. Ruetsch, O., Viala, A., Bardou, H., Martin, P., Vacheron, M.N., 2005. Psychotropic drugs induced weight gain: a review of the literature concerning epidemiological data, mechanisms and management. Encephale 31 (4), 507–516. Santarelli, L., Saxe, M., Gross, C., Surget, A., Battaglia, F., Dulawa, S., Weisstaub, N., Lee, J., Duman, R., Arancio, O., 2003. Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 301, 805–809. Satriano, J., Schwartz, D., Ishizuka, S., Lorie, M.J., Thomson, S.C., Gabbai, F., 2001. Supression of inducible nitric oxide generation by agmatine aldehyde: beneficial effects in sepsis. J. Cell. Physiol. 188, 313–320. Sheikh, N., Ahmad, A., Siripurapu, K., Kuchibhotla, V., Singh, S., Palit, G., 2007. Effect of Bacopa monniera on stress induced changes in plasma corticosterone and brain monoamines in rats. J. Ethnopharmacol. 111, 671–672. Strekolova, T., Steinbusch, H.W.M., 2010. Measuring behavior in mice with chronic stress depression paradigm. Prog. Neuropsychopharmacol. Biol. Psychiatry 34, 348–351. Taksande, B., Kotagale, N., Nakhate, K., Mali, P., Kokare, D., Hirani, K., Subhedar, N., Chopde, C., Ugale, R., 2011. Agmatine in the hypothalamic paraventricular nucleus stimulates feeding in rats: involvement of neuropeptide Y. Br. J. Pharmacol. 164, 704–718. Uzbay, T.I., 2012. The pharmacological importance of agmatine in the brain. Neurosci. Biobehav. Rev. 36, 502–519. Wang, W.P., Iyo, A.H., Miguel-Hidalgo, J., Regunathan, S., Zhu, M.Y., 2006. Agmatine protects against cell damage induced by NMDA and glutamate in cultured hippocampal neurons. Brain Res. 1084 (1), 210–216. (21). Wang, D., An, S.C., Zhang, X., 2008. Prevention of chronic stress-induced depression-like behaviour by inducible nitric oxide inhibitor. Neurosci. Lett. 433, 59–64. Willner, P., 2005. Chronic mild stress (CMS) revisited: consistency and behavioural– neurobiological concordance in the effects of CMS. Neuropsychobiology 52, 90–110. Willner, P., Towell, A., Sampson, D., Sophokleous, S., Muscat, R., 1987. Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant. Psychopharmacology 93, 358–364. Woodward, C.J., Emery, P.W., 1987. Determination of plasma corticosterone using high-performance liquid chromatography. J. Chromatogr. 419, 280–284. Zhou, Q.G., Hu, Y., Hua, Y., Hu, M., Luo, C.X., Han, X., Zhu, X.J., Wang, B., Xu, J.S., Zhu, D.Y., 2007. Neuronal nitric oxide synthase contributes to chronic stress‐induced depression by suppressing hippocampal neurogenesis. J. Neurochem. 103, 1843–1854. Zhu, M.Y., Wang, W.P., Cai, Z.W., Regunathan, S., Ordway, G., 2008. Exogenous agmatine has neuroprotective effects against restraint‐induced structural changes in the rat brain. Eur. J. Neurosci. 27, 1320–1332. Zhu, M., Wang Huang, J., Feng, Y.Z., Regunathan, S., Bissette, G., 2008. Repeated immobilization stress alters rat hippocampal and prefrontal cortical morphology in parallel with endogenous agmatine and arginine decarboxylase levels. Neurochem. Int. 53, 346–354. Zomkowski, A.D., Hammes, L., Lin, J., Calixto, J.B., Santos, A.R.S., Rodrigues, A.L.S., 2002. Agmatine produces antidepressant-like effects in two models of depression in mice. Neuroreport 13, 387–391.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
Agmatine attenuates chronic unpredictable mild stress induced behavioral alteration in mice Brijesh G. Taksande, Dharmesh S. Faldu, Madhura P. Dixit, Jay N. Sakaria, Manish M. Aglawe, Milind J. Umekar, Nandkishor R. Kotagale n Division of Neuroscience, Department of Pharmacology, Shrimati Kishoritai Bhoyar College of Pharmacy, New Kamptee, Nagpur 441002, Maharashtra, India
art ic l e i nf o
a b s t r a c t
Article history: Received 2 May 2013 Received in revised form 19 October 2013 Accepted 23 October 2013 Available online 29 October 2013
Chronic stress exposure and resulting dysregulation of the hypothalamic pituitary adrenal axis develops susceptibility to variety of neurological and psychiatric disorders. Agmatine, a putative neurotransmitter has been reported to be released in response to various stressful stimuli to maintain the homeostasis. Present study investigated the role of agmatine on chronic unpredictable mild stress (CUMS) induced behavioral and biochemical alteration in mice. Exposure of mice to CUMS protocol for 28 days resulted in diminished performance in sucrose preference test, splash test, forced swim test and marked elevation in plasma corticosterone levels. Chronic agmatine (5 and10 mg/kg, ip, once daily) treatment started on day15 and continued till the end of the CUMS protocol significantly increased sucrose preference, improved self-care and motivational behavior in the splash test and decreased duration of immobility in the forced swim test. Agmatine treatment also normalized the elevated corticosterone levels and prevented the body weight changes in chronically stressed animals. The pharmacological effect of agmatine was comparable to selective serotonin reuptake inhibitor, fluoxetine (10 mg/kg, ip). Results of present study clearly demonstrated the anti-depressant like effect of agmatine in chronic unpredictable mild stress induced depression in mice. Thus the development of drugs based on brain agmatinergic modulation may represent a new potential approach for the treatment of stress related mood disorders like depression. & 2013 Elsevier B.V. All rights reserved.
Keywords: Agmatine Depression Anhedonia CUMS Splash test FST
1. Introduction Chronic stress and the resulting dysregulation of the hypothalamic pituitary adrenal (HPA) axis enhance vulnerability to a variety of neurological disorders. Several neurotransmitters/neuromodulators and their interactions in CNS regulate response to stress (Grønli et al., 2005). However the exact mechanism behind deleterious effect of chronic stress exposure is yet to be elucidated. Agmatine, a biogenic amine and putative neurotransmitter, has been implicated in stress response and related disorders (Aricioglu et al., 2003). Agmatine is biosynthesized from amino acid Larginine by arginine decarboxylase, stored in synaptic vesicles, accumulated by uptake, released by depolarization and inactivated by agmatinase. Agmatine metabolized to putrescine and guanido-butanoic acid by an enzyme agmatinase and diamine oxidase respectively (See review Uzbay, 2012).
n
Corresponding author. Tel.: þ 91 7109 288650; fax; þ 91 7109287 094. E-mail address: [email protected] (N.R. Kotagale).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.10.041
Agmatine interact with α2 adrenergic, imidazoline, NMDA receptors and possesses nitric oxide synthase inhibitory activity in brain (see review Uzbay, 2012). Although the physiological role of agmatine in normal brain is largely unknown, the exogenous agmatine has exhibited several intriguing neurally relevant functions of potential therapeutic importance. Systemic administration of agmatine reduced neuronal injury produced by focal ischemia, spinal cord injury and hypoxic ischemia (Lu et al., 2006), diminishes chemically and electrically induced convulsions (Demehri et al., 2003). It also produces anxiolytic (Lavinsky et al., 2003), antidepressant (Zomkowski et al., 2002), antinociceptive (Önal et al., 2003), anti-convulsant (Bence et al., 2003), anti-inflammatory (Satriano et al., 2001), antiproliferative (Regunathan and Reis, 1997) and neuroprotective effects (Olmos et al., 1999). Agmatine has been proposed as an adjuvant in the treatment of several chronic pain syndromes (Paszcuk et al., 2007). In addition, several stressful pathological conditions like inflammation, ischemia and lipopolysacarides (LPS) injection increased agmatine levels in brain. On the other hand, simultaneous treatment with exogenous agmatine attenuated repeated immobilization induced elevated corticosterone levels and glutamate efflux in brain nuclei associated with modulation of stress response (M.Y. Zhu et al., 2008; M. Zhu et al., 2008). Further, several nuclei of hypothalamus and
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pituitary gland have shown abundant agmatine immunoreactivity (Otake et al., 1998) and its co-localization with neuropeptides (Gorbatyuk et al., 2001). Several studies have reported the neuroprotective effects of agmatine against cell damage caused by glucocorticoids and glutamate in primary neuronal cultures of the hippocampus (Iyo et al., 2006; Wang et al., 2006; M.Y. Zhu et al., 2008; M. Zhu et al., 2008). These studies have focused agmatine as an endogenous neuromodulator of stress and suggested that agmatine homeostasis may play an important role in modulation of stress. Endogenous levels of agmatine increased during stressful conditions in compensatory manner, however not high enough to modulate the harmful effect of stressor or inflammation (M.Y. Zhu et al., 2008; M. Zhu et al., 2008). Hence, exogenous administration which restored the agmatine levels can exhibit anti-inflammatory, antiproliferative and neuroprotective effects in rodent. Therefore, one might postulate that as one element of self-protection mechanisms in the brain, agmatine synthesis is trigged by stress through activation of ADC expression, which in turn increases endogenous agmatine levels as an initial protective response to stress. However, the effect of exogenous agmatine on stress induced depression and related behavioral alteration remained unexplored. This study was therefore designed to evaluate the role of agmatine in chronic unpredictable mild stress-induced neurobehavioral effects and biochemical changes in mice. Chronic unpredictable mild stress (CUMS) exposed animal exhibits several neurobehavioral alteration, resembling the symptoms of chronic human depression and widely employed for preclinical screening of antidepressants. In the present study we examined the effect of chronic agmatine treatment on CUMS induced anhedonia, motivational behavior, despair, body weight changes and elevated corticosterone levels in mice.
2. Material and methods 2.1. Subjects Adult albino Swiss mice of either sex weighing 25–30 g were housed in polypropylene cages in a temperature (25 72 1C), relative humidity (50–70%) and maintained on a 12:12 h light/ dark cycle (lights on 07:00–19:00 h). Food and water were provided ad libitum except during specific experimental protocols. Control, stressed and treatment group were tested in identical environmental conditions. Cages were changed once per week and water bottlers were changed three times per week. All experimental procedures were carried out under strict compliance with Institutional Animal Ethical Committee according to guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests; Government of India; New Delhi. 2.2. Drug solutions and administrations Agmatine sulfate was purchased from Sigma-Aldrich, USA while fluoxetine was received as a gift sample from SUN pharmaceuticals, Baroda, India. Both the drugs were dissolved in saline just before the experiments and administered intraperitoneally (ip) in a volume of 1 ml/kg. Normal saline (0.9% w/v NaCl) was used as control. 2.3. Chronic unpredictable mild stress The chronic unpredictable mild stress (CUMS) protocol was designed to maximize the unpredictable nature of stressor. Restrained stress, continuous overnight lighting, tail pinch, shaker stress, tilt-cage
and overnight soiled cage were applied once a week over a period of 4 weeks. Immediately after completion of each stress session, the animals were returned to their home cage and transferred to standard laboratory condition. The duration of CUMS and the nature of different stressor are based on the available literature (Ducottet and Belzung, 2004; Ducottet et al., 2003; Katz et al., 1981; Lu et al., 2006; Willner et al., 1987). Separate group of mice subjected to CUMS were injected with saline (1 ml/kg, ip) or fluoxetine (10 mg/kg, ip) or agmatine (2.5, 5, and 10 mg/kg, ip) from day-15 onwards in between 09:00 and 10:00 daily. Unstressed group was injected with saline and handled daily but was not subjected to any stressors. In order to avoid the immediate effect of stressor and pharmacological treatment all the behavioral test were carried out 24 h after the last stressor except sucrose preference test (SPT). SPT was carried out on days 0, 14, 21 and 28. The body weight of animals was measured daily. Separate group of animal was used for every behavioral paradigm. 2.3.1. Sucrose preference test (SPT) Sucrose preference test was performed as described earlier (Jindal et al., 2013; Liu et al., 2013) with some modifications. As agmatine enhanced caloric intake in animals (Taksande et al., 2011) and sucrose consumption can be altered by food and water deprivation (Matthews et al., 1995), we did not apply food and water deprivation in CUMS protocol. SPT was conducted on day 0, 14, 21 and 28 of CUMS protocol. Briefly, 72 h before the test, mice were habituated to drink 1% sucrose solution (w/v) and subsequently exposed to two bottles (1% sucrose solution in one bottle and tap water in another bottle). After habituation mice were housed in individual cages. Mice were permitted ad libitum access to 100 ml of sucrose solution (1% w/v) and 100 ml of tap water. We have also minimized the effect of side preference in drinking behavior by switching the bottle in the middle of the test (Strekolova and Steinbusch, 2010). In addition, we have monitored the sucrose preference for 3 h to minimized the % error commonly observed with little amount of sucrose consumed in 1 h sucrose preference test. After 3 h of exposure the consumption volumes of sucrose solution and tap water were recorded and the sucrose preference was calculated as [(sucrose consumption)/(water consumptionþ sucrose consumption)] 100. Mice showing basal sucrose preference less than 60% were considered anhedonic and not used in the study. 2.3.2. Splash test The splash test is pharmacological validated animal model exhibiting motivational behavior of animals. CUMS decreases grooming behavior in splash test and this phenomenon is considered to be similar with apathy observed in depressive patients. Animals were isolated in home cages for 24 h and 10% sucrose solution is squirted on the dorsal coat of mice and the latency to initiate a grooming behavior as well as the frequency of grooming was recorded by independent observers unaware of the treatment for a period of 5 min where grooming (nose/face grooming, head washing and body grooming) latency is considered as self-care and grooming frequency is considered as motivational behavior. 2.3.3. Forced swim test The procedure was quite similar as described by Porsolt et al., (1977) with some modification. Mice were introduced individually in plexiglas cylinder (21 cm height 12 cm internal diameter) containing fresh water till a height of 9 cm at 257 1 1C and forced to swim for a 6 min test session. The immobility time was measured by trained observer blind to the treatment. A mouse was considered immobile when it remained floating motionless in
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water except making any necessary movement to keep its head above water. 2.3.4. Corticosterone levels Immediately after the completion of behavioral tests on day-29, blood was withdrawn from the retro-orbital plexus of the mice in the eppendorff tubes previously rinsed with sodium citrate and centrifuged at 13,000g for 15 min at 4 1C. Separated plasma was stored at 20 1C for corticosterone estimation. A quaternary gradient HPLC system equipped with Crestpak C18T-5 column and PDA detector (MD2010 plus) (Jasco, Japan) was used for quantification of plasma corticosterone. The biochemical estimation on HPLC was carried according to the method described by Woodward and Emery (1987) and Sheikh et al. (2007) with minor modifications. Briefly, 50 ml of plasma was extracted with 1 ml of DCM–Ether mixture (DCM:Ether—50:50) on mechanical shaker for 15 min. Supernatant (50 ml) was transferred to eppendorff tube and evaporated under the slow stream of nitrogen. After complete evaporation, 1 ml of mobile phase (Water:Methanol—80:20) was added and 20 ml of this was injected into the HPLC (Flow rate1 ml/min and estimated at 243 nm). 2.4. Statistical analysis The data were expressed as mean 7S.E.M. The results of splash test, forced swimming test, open field were analyzed by one-way ANOVA followed by the Newman–Keul test. Sucrose preference and body weight changes were analyzed by two-way ANNOVA with post-hoc Bonferroni mean comparisons. Corticosterone levels were analyzed by unpaired t test. Results of statistical tests with Po 0.05 were considered significant.
3. Results 3.1. Effect of chronic administration of agmatine in stress induced anhedonia Fig. 1 shows the effect of chronic unpredictable mild stress on sucrose preference test. Chronic exposure of animal to unpredictable mild stress significantly decrease the sucrose preference at day-14 (P o0.001) and the effect was continued upto day-28 (P o0.001) as compared to unstressed animals [FStress Time (3, 30) ¼75.59, P o0.001; FStress(1, 30) ¼ 3775.72, Po 0.001; FTime (3, 30) ¼18.88, P o0.001- Two way ANOVA]. The sucrose preference on day-28 was significantly lower as compared to day-14 (P o0.001). Post-hoc Bonferroni mean comparisons revealed significant difference in sucrose preference between the groups on day-14 (P o0.001) and day-28 (P o0.001). Agmatine (5 and 10 mg/kg) [FTreatment Time (12, 75) ¼11.66, P o0.001; FTreatment (4, 75) ¼ 62.71, P o0.001; FTime (3, 75) ¼ 35.35, Po 0.001] and fluoxetine (10 mg/kg) [FTreatment Time (6, 45) ¼20.94, P o0.001; FTreatment (2, 45)¼ 174.63, P o0.001; FTime (3, 45) ¼9.72, Po 0.001] treatment from day-15 onwards completely reversed the anhedonia induced by CUMS paradigm on day28. Post-hoc Bonferroni mean comparisons revealed that the sucrose preference in the stressed animals treated with agmatine (5 and 10 mg/kg) and fluoxetine (10 mg/kg, ip) were significantly different as compared to stressed mice treated with vehicle on day-28. However no significant effect of treatment was observed on day-21. The difference in the % sucrose preference on day 21 and 28 in the animal treated with agmatine (10 mg/kg) was not statistically significant as compared to fluoxetine (10 mg/kg, ip) treatment. Administration of agmatine and fluoxetine to nonstressed group did not produce any effect on sucrose preference
Fig. 1. Effects of agmatine and fluoxetine treatment on sucrose preference test. Mice (n¼ 6) were subjected to chronic unpredictable mild stress (CUMS) and injected with saline (1 ml/kg, ip) or fluoxetine (10 mg/kg, ip) or agmatine (2.5– 10 mg/kg, ip) from day-15 onwards and sucrose preference was evaluated on days 0, 14, 21 and 28. Control group received saline (1 ml/kg, ip) under the same schedule and was not subjected to stress. Each point indicate mean sucrose preference (%) 7S.E.M. $P o0.001 vs. saline treated non-stressed animals; n Po 0.05 nnP o 0.001 vs. saline treated CUMS animals (two way ANOVA post-hoc Bonferroni mean comparisons).
test as compared to vehicle treated non-stressed group (data not shown).
3.2. Effect of agmatine in stress induced alterations in self-care and motivational behavior Chronic agmatine (5 and 10 mg/kg) and fluoxetine (10 mg/kg) treatment from third week onward of CUMS protocol restored the motivational and self-care behavior in the splash test (Fig. 2). One way ANOVA followed by post-hoc Newman–Keuls comparisons demonstrated that agmatine [5(Po 0.001 and P o0.001) and 10 mg/kg (Po 0.001 and P o0.001) respectively] significantly decreased the grooming latency [F (4, 29) ¼22.08, Po 0.001] [Fig. 2A] and increased grooming frequency [Fig. 2B] as compared to vehicle treated stressed animals. Similarly fluoxetine (10 mg/kg) [(P o0.001and P o0.001) respectively] treatment to separate group of animals showed significant decrease in grooming latency [F (2, 17) ¼94.95, P o0.001] and increase in grooming frequency [F (2, 17) ¼ 89.33, P o0.001]. Agmatine (2.5–10 mg/kg) and fluoxetine (10 mg/kg) treatment to non-stressed mice did not affect the behavior in splash test (data not shown).
3.3. Effect of agmatine in FST As shown in Fig. 3, CUMS exposure significantly increases immobility duration in stressed animals as compared to nonstressed group (Po 0.001). Repeated administration of agmatine (5 and 10 mg) dose dependently reduces the % immobility time in mice exposed to CUMS by 23% (P o0.05) and 42% (P o0.001) respectively as compared to vehicle treated stressed animal [F (4, 29) ¼10.08, Po0.001]. Agmatine at the doses used here did not influence the immobility duration in non-stressed mice (data not shown). Fluoxetine treatment at dose of 10 mg/kg to a separate group of mice also reduced immobility time in CUMS exposed animal by 46% (P o0.001) [F (2, 17) ¼51.51, Po 0.001].
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Fig. 3. Effects of agmatine and fluoxetine treatment on immobility time in forced swim test (FST). Mice (n¼ 6) were subjected to chronic unpredictable mild stress (CUMS) and injected with saline (1 ml/kg, ip) or fluoxetine (10 mg/kg, ip) or agmatine (2.5–10 mg/kg, ip) from day-15 onwards and immobility time [(seconds (s))] was determined 24 h after the last stressor. Control group received saline (1 ml/kg, ip) under the same schedule and was not subjected to stress. Each bar indicate mean immobility time (s)7S.E.M. $Po0.001vs saline treated non-stressed animals; n Po0.05, nn Po0.001 vs. saline treated CUMS animals (one way ANOVA post-hoc Newman–Keul mean comparisons).
Fig. 2. Effects of agmatine and fluoxetine treatment on motivational behavior in splash test. Mice (n¼ 6) were subjected to chronic unpredictable mild stress (CUMS) and injected with saline (1 ml/kg, ip) or fluoxetine (10 mg/kg, ip) or agmatine (2.5–10 mg/kg, ip) from day-15 onwards and grooming latency [(seconds (s))] (Fig. 2A) and frequency (Fig. 2B) were evaluated 24 h after the last stressor. Control group received saline (1 ml/kg, ip) under the same schedule and was not subjected to stress. Each bar indicate mean grooming latency (s)/grooming frequency7 S.E.M. $Po 0.01, $$P o0.001 vs. saline treated non-stressed animals; n Po 0.001 vs. saline treated CUMS animals (one way ANOVA post-hoc Newman– Keul mean comparisons).
3.4. Effect of agmatine on % body weight change in CUMS induced depression Body weight was monitored daily during the experimental protocol. Two-way ANOVA showed a significant reduction in the body weight of the CUMS exposed animals as compared to nonstressed mice [FStress Time (3, 30) ¼11.98, Po0.001; FStress (1, 30) ¼ 44.91, P o0.001; FTime (3, 30) ¼6.59, P o0.01]. Post hoc analysis by Bonferroni multiple comparison showed significant difference in % body weight change on day-21 (P o0.01) and 28 (Po0.05). No significant change in the body weight was observed in the mice that received repeated agmatine treatment and subjected to CUMS when compared against saline treated non-stressed animals [FTreatment Time (15, 90) ¼0.86, P ¼0.61; FTreatment (5, 90) ¼7.46, P o0.001; FTime (3, 90) ¼1.49, P ¼0.22]. As depicted in Fig. 4, fluoxetine (10 mg/kg) significantly prevented reduction in body weight in animals subjected to CUMS protocol. Two way ANOVA indicated insignificant change in the body weight in fluoxetine (10 mg/kg) treated animals as compared to saline injected mice exposed to CUMS [FTreatment Time (6, 45) ¼2.84, P o0.05; FTreatment (2, 45) ¼25.23, P o0.001; FTime (3, 45)¼ 0.57, P ¼0.64]. No significant difference was determined between the stressed animals injected with agmatine (10 mg/kg) and fluoxetine (10 mg/kg) (post-hoc Bonferroni mean comparisons).
Fig. 4. Effects of agmatine and fluoxetine treatment on changes in body weight. Mice (n¼ 6) were subjected to chronic unpredictable mild stress (CUMS) and injected with saline (1 ml/kg, ip) or fluoxetine (10 mg/kg, ip) or agmatine (2.5–10 mg/kg, ip) from day-15 onwards and body weights were recorded on day 0, 14, 21 and 28. Control group received saline (1 ml/kg, ip) under the same schedule and was not subjected to stress. Each point indicate mean changes in body weight (%) 7S.E.M. nP o0.05, nnP o 0.01 vs. saline injected non-stressed animals (two way ANOVA post-hoc Bonferroni mean comparisons).
3.5. Agmatine attenuates CUMS induced increased plasma corticosterone levels in mice HPLC analysis demonstrated the significant higher levels of plasma corticosterone in the animals subjected to CUMS for the period of 28 days (Po 0.001) as compared to non-stressed control group (t ¼13.93; df ¼4, unpaired t test). As depicted in Fig. 5, plasma corticosterone levels were significantly lower and comparable to the non-stressed group in agmatine [5 (P o0.01) and 10 mg/kg (Po 0.001)] (F (4, 14) ¼30.63, (P o0.001)) and fluoxetine (P o0.001) [F (2, 8) ¼74.75, (P o0.001)] treated mice.
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Fig. 5. Effects of agmatine and fluoxetine treatment on plasma corticosterone levels. Mice (n¼ 3) were subjected to chronic unpredictable mild stress (CUMS) and injected with saline (1 ml/kg, ip) or fluoxetine (10 mg/kg, ip) or agmatine (2.5–10 mg/kg, ip) from day-15 onwards. 24 h after the last stressor and blood samples were withdrawn to determine corticosterone level. Control group received saline (1 ml/kg, ip) under the same schedule and was not subjected to stress. Each bar indicate mean plasma corticosterone (ng/ml) 7 S.E.M. $Po 0.001vs saline treated non-stressed animals; nP o0.01, nnP o 0.001 vs. saline treated CUMS animals (one way ANOVA posthoc Newman Keul mean comparisons).
4. Discussion Stressful life experiences are important etiological factors in the onset and maintenance of depressive episode (Lee et al., 2002). In view of this, an animal model of CUMS induced depression has been developed to replicate the pathogenesis of depressive illness. Number of experimental evidence suggests that CUMS exposure to animals induce alteration in behavioral and biochemical parameters resembling symptoms of clinical depression (Luo et al., 2008). Consistent with several earlier findings, the results of the present study showed that mice when subjected to chronic exposure to a variety of mild stressor regimen induced significant reduction in sucrose preference test, grooming behavior in splash test, body weights and increased immobility in forced swim test and plasma corticosterone level as compared to non-stressed mice. Sucrose preference test is an indicator of anhedonia like behavioral change (Willner, 2005). The results of the present study showed that the long term treatment of agmatine significantly suppressed anhedonia induced by CUMS protocol suggesting the potential antidepressant like effect of agmatine. The antidepressant effect was specific to stress exposure as agmatine did not influence the sucrose consumption in normal non-stressed group. Similarly, agmatine treatment did not influence the water intake in naïve animals. The anti-depressant like effect of agmatine was compared to selective serotonin reuptake inhibitor—fluoxetine with the only difference that agmatine has shown delayed onset of action. The sucrose consumption was normalized by fluoxetine treatment on day-21 whereas it was evident on day-28 in agmatine treated group. This could be attributed to lower doses of agmatine used in the present study and their differential mechanism of action. Further, intraperitoneally injected agmatine extensively metabolized peripherally in liver and kidney and has very short biological half life (Halaris and Plietz, 2007). The delayed onset of action and significant weight gain is a possible side effect with SSRI and other antidepressant including tricyclic antidepressant and MAO inhibitors (Ruetsch et al., 2005). In our study, although agmatine exhibited delayed onset of action as compared to fluoxetine, it did not induced significant changes in body weight of animal after chronic administration. Moreover, unlike other antidepressant, it did not affect the locomotor counts of animal. Thus agmatinergic modulation may offer
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several advantages and may be useful in combination with other antidepressant in the treatment of resistant depression. The splash test is direct measure of self-care and motivational behavior. Chronic stress exposure to mice resulted in increase in latency and decreased frequency of grooming behavior, indicating loss of self-care and motivational behavior in mice subjected to CUMS procedure. The disturbance in grooming behavior is considered to mimic apathy observed in clinical depression (Willner, 2005). Agmatine and fluoxetine treatment prevented the effect of CUMS on splash test. Indeed, similar effects have been already reported for fluoxetine and CRF II antagonist (Ducottet et al., 2003; Santarelli et al., 2003). Chronic stress has been shown to dramatically increase the immobility time of mice in FST (Zhou et al., 2007). Similarly our CUMS protocol also increased immobility time in forced swim test in mice. Chronic treatment with agmatine and fluoxetine attenuated the CUMS associated reduction in immobility time. These behavioral observations demonstrated that agmatine produced an antidepressant like effect in chronically stressed mice. In parallel with our observation, simultaneous treatment with exogenous agmatine attenuated repeated immobilization induced architectural alteration in hippocampus and prefrontal cortex such as elevated plasma corticosterone levels and high glutamate influx (M.Y. Zhu et al., 2008; M. Zhu et.al., 2008). Agmatine also attenuated stress and lipopolysaccharide induced hyperthermia in rats (Aricioglu and Regunathan, 2005). Moreover Bernstein et al. (2012) have shown up-regulation of the agmatine degrading enzyme agmatinase in patients with unipolar and bipolar depression suggesting that reduction of endogenous brain agmatine level may play a central role in the pathogenesis of depression. Thus, it can be inferred from our results and available literature that endogenous agmatine system may play an important role in adaptation response to chronic stress in order to maintain brain homeostasis. In the present study, the CUMS exposure led to significant reduction in body weights compared to saline treated nonstressed animals. The results are in agreement with previous data showing that chronic mild stress lead to reduced body weight in exposed animals (Wang et al., 2008). Fluoxetine and agmatine treatment significantly prevented the body weight reduction induced by CUMS protocol. It is important to note that, body weight reduction not only provides face validity to CUMS procedure but also consider as a diagnostic criterion for a major depressive episode in DSM-IV. Thus agmatine treatment might offer additional advantages in the treatment of stress related disorders associated with anorexia and significant loss of body weights. However, body weight changes can also be considered as a confounding variable in sucrose consumption and could lead to false results in CUMS protocol. Our results of anhedonia should not be attributed to body weight changes as absolute sucrose consumption may be influenced by body weight gain, sucrose preference may not be influenced and consider as a better index of anhedonia (Matthews et al., 1995). HPA axis plays a critical role in eliciting physiological responses to various stressful stimuli (Pan et al., 2006). Acute stress activates HPA axis with simultaneous increase in levels of corticosterone in rodents or cortisol in human being. Sustained activation of HPA axis is associated with an abnormally high blood glucocorticoid level, which may eventually lead to pathological conditions such as depression (Johnson et al., 2006). Thus, the normalization of HPA axis and glucocorticoid levels may be critically involved in the therapeutic action of antidepressant drug. Normalization of the HPA system has been shown to be a prerequisite for stable remission of the disease (Holsboer, 2000), which has been shown to occur during successful antidepressant treatment (Barden et al., 1995), again supporting the importance of the stress hormone system in the development and maintenance of affective disorders. In accordance with several earlier findings, we found that
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CUMS significantly elevated corticosterone levels in mice plasma, and chronic agmatine treatment prevented these alterations. Thus preventive effects of agmatine on CUMS associated behavioral alterations might be linked with reduction in the corticosterone levels. Although we have not investigated specific mechanism, target receptor and enzymes like α2 adrenoceptor, imidazoline receptor, NMDA receptor or nitric oxide might be involved in antidepressant like effect of agmatine. Further more specific studies are required for better understanding of this proposed mechanism. To conclude, the results of present study demonstrated antidepressant like effect of agmatine in chronic unpredictable mild stress induced depression in mice. Chronic agmatine (5 and 10 mg/ kg, ip, once daily) treatment started on day-15 and continued till the end of the CUMS protocol significantly increased sucrose preference, improved self-care and motivational behavior in splash test, decreased duration of immobility in forced swim test and also normalized the body weight changes and elevated corticosterone plasma levels in mice. Thus the development of drugs based on modulation of agmatinergic system in brain may represent a new potential approach for the treatment of stress related neurological disease like depression. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2013.10. 041. References Aricioglu, F., Regunathan, S., 2005. Agmatine attenuates stress- and lipopolysaccharideinduced fever in rats. Physiol. Behav. 85, 370–375. Aricioglu, F., Regunathan, S., Piletz, J., 2003. Is agmatine an endogenous factor against stress? Ann. NY. Acad. Sci. 1009, 127–132. Barden, N., Reul, J.M., Holsboer, F., 1995. Do antidepressants stabilize mood through actions on the hypothalamic–pituitary–adrenocortical system? Trends Neurosci. 18, 6–11. Bence, K., David, R., Stables, J.P., Crooks, P.A., 2003. An in vivo evaluation of the antiseizure activity and acute neurotoxicity of agmatine. Pharmacol. Biochem. Behav. 74, 771–775. Bernstein, H.G., Stich, C., Jäger, K., Dobrowolny, H., Wick, M., Steiner, J., Veh, R., Bogerts, B., Laube, G., 2012. Agmatinase, an inactivator of the putative endogenous antidepressant agmatine, is strongly upregulated in hippocampal interneurons of subjects with mood disorders. Neuropharmacology 62, 237–246. Demehri, S., Homayoun, H., Honar, H., Riazi, K., Vafaie, K., Roushanzamir, F., Dehpour, A.R., 2003. Agmatine exerts anticonvulsant effect in mice: modulation by alpha 2-adrenoceptors and nitric oxide. Neuropharmacology 45, 534–542. Ducottet, C., Belzung, C., 2004. Behavior in the elevated plus-maze predicts coping after subchronic mild stress in mice. Physiol. Behav. 81, 417–426. Ducottet, C., Griebel, G., Belzung, C., 2003. Effects of the selective nonpeptide corticotropin-releasing factor receptor 1 antagonist antalarmin in the chronic mild stress model of depression in mice. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 27, 625–631. Gorbatyuk, O.S., Milner, T.A., Wang, G., Regunathan, S., Reis, D.J., 2001. Localization of agmatine in vasopressin and oxytocin neurons of the rat hypothalamic paraventricular and supraoptic nuclei. Exp. Neurol. 171, 235–245. Grønli, J., Murison, R., Fiske, E., Bjorvatn, B., Sørensen, E., Portas, C.M., Ursin, R., 2005. Effects of chronic mild stress on sexual behavior, locomotor activity and consumption of sucrose and saccharine solutions. Physiol. Behav. 84, 571–577. Halaris, A., Plietz, J., 2007. Agmatine metabolic pathway and spectrum of activity in brain. CNS Drugs 21 (11), 885–900. Holsboer, F., 2000. The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology 23, 477–501. Iyo, A.H., Zhu, M.Y., Ordway, G.A., 2006. Expression of arginine decarboxylase in brain regions and neuronal cells. J. Neurochem. 96, 1042–1050. Jindal, A., Mahesh, R., Bhatt, S., 2013. Etazolate, a phosphodiesterase 4 inhibitor reverses chronic unpredictable mild stress-induced depression-like behavior and brain oxidative damage. Pharmacol. Biochem. Behav. 105, 63–70. Johnson, S.A., Fournier, N.M., Kalynchuk, L.E., 2006. Effect of different doses of corticosterone on depression-like behavior and HPA axis responses to a novel stressor. Behav. Brain Res. 168, 280–288. Katz, R.J., Roth, K.A., Carroll, B.J., 1981. Acute and chronic stress effects on open field activity in the rat: implications for a model of depression. Neurosci. Biobehav. Rev. 5, 247–251.
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