Indian Journal of Experimental Biology Vol. 46, March 2008, pp 164-170 Possible involvement of nitric oxide (NO) signaling pathway in the anti- depressant-like effect of MK-801(dizocilpine), a NMDA receptor antagonist in mouse forced swim test Ashish Dhir & SK Kulkarni* Pharmacology Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India Received 27 November 2007; revised 15 January 2008 L-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) is an important signaling pathway involved in depression. With this information, the present study aimed to study the involvement of this signaling pathway in the antidepressant-like action of MK-801 (dizocilpine; N-methyl-d-aspartate receptor antagonist) in the mouse forced-swim test. Total immobility period was recorded in mouse forced swim test for 6 min. MK-801 (5-25 μg/kg., ip) produced a U- shaped curve in reducing the immobility period. The antidepressant-like effect of MK-801 (10 μg/kg, ip) was prevented by pretreatment with L-arginine (750 mg/kg, ip) [substrate for nitric oxide synthase (NOS)]. Pretreatment of mice with 7-nitroindazole (7-NI) (25 mg/kg, ip) [a specific neuronal nitric oxide synthase inhibitor] produced potentiation of the action of subeffective dose of MK-801 (5 μg/kg, ip). In addition, treatment of mice with methylene blue (10 mg/kg, ip) [direct inhibitor of both nitric oxide synthase and soluble guanylate cyclase] potentiated the effect of MK-801 (5 μg/kg, ip) in the forced-swim test. Further, the reduction in the immobility period elicited by MK-801 (10 μg/kg, ip) was also inhibited by pretreatment with sildenafil (5 mg/kg, ip) [phosphodiesterase 5 inhibitor]. The various modulators used in the study and their combination did not produce any changes in locomotor activity per se and in combination with MK-801. MK-801 however, at higher doses (25 μg/kg, ip) produced hyperlocomotion. The results demonstrated the involvement of nitric oxide signaling pathway in the antidepressant-like effect of MK-801 in mouse forced-swim test. Keywords: Cyclic guanosine monophosphate, Forced-swim test, L-arginine, Nitric oxide, MK-801 (dizocilpine), Phosphodiesterase 5 inhibitor An abnormality in glutamate function has been norepinephrine)4. Also, Berman et al.5 reported the implicated in the neural substrate of depressive first placebo-controlled, double-blind study assessing disorders1,2. Glutamate injected into the nucleus the therapeutic effects of a single infusion dose of accumbens dose-dependently decrease swimming ketamine in unipolar depression. time in forced-swim test2. An extensive library of MK-801 [(+)-5-methyl-10, 11-dihydro-5H-dibenzo noncompetitive N-methyl-d-aspartate (NMDA) [a, d] cyclohepten-5, 10-imine maleate)] or antagonists (e.g., MK-801 or dizocilpine, memantine, dizocilpine, a potent, noncompetitive antagonist of the ketamine) that reduce glutamatergic transmission at NMDA receptor6 have been extensively studied for the NMDA receptor have demonstrated use in treatment of diseases with excitotoxic antidepressant-like effects in animal models, components, such as stroke, traumatic brain injury, including forced-swim and tail suspension tests, and neurodegenerative diseases such as Huntington's, inescapable stressors, and in learned helplessness3. An Alzheimer's, and amyotrophic lateral sclerosis. It has acute administration of neramexane (novel NMDA shown effectiveness in protecting neurons in cell receptor antagonist) enhanced the antidepressant-like culture and animal models of excitotoxic neuro- effect of imipramine (tricyclic antidepressant), degeneration7-9. Studies have also demonstrated the fluoxetine (selective serotonin reuptake inhibitor), or antidepressant effect of MK-801 in various animal venlafaxine (dual reuptake inhibitor of serotonin and models of despair10. Although the antidepressant-like —————— effect of MK-801 in various animal models of *Correspondent author depression have been reported, little is known Telephone: +91-172-2534114, 2541142 concerning its possible mechanism of action. Fax: +91-172-2541142 L-arginine-nitric oxide (NO)-cyclic guanosine E-mail: [email protected] DHIR & KULKARNI: MK-801 & NITRIC OXIDE MODULATION 165 monophosphate (cGMP) is an important signaling standard laboratory conditions and maintained on pathway that is reported to be involved in natural light and dark cycle, and had free access to depression11. Nitric oxide, a messenger molecule in food and water. Animals were acclimatized to the brain, synthesized from L-arginine by nitric oxide laboratory conditions before the experiment. Each synthase (NOS), have been implicated in animal was used only once. All the experiments were neurotransmission, synaptic plasticity, learning, carried out between 0900 and 1500 hrs. The perception of pain, aggression and depression12. experimental protocols were approved by the Recent evidences have shown that the reduction of Institutional Animal Ethics Committee (IAEC) and nitric oxide levels within the hippocampus can induce conducted according to the Indian National Science antidepressant-like effects, thus implicating Academy (INSA) Guidelines for the use and care of endogenous hippocampal nitric oxide in the experimental animals. neurobiology of stress and depression13. Several of physiological actions of nitric oxide are mediated Experimental Procedure- through its interaction with the heme iron of soluble Forced Swim Test (FST)—The test procedure was guanylate cyclase (sGC), leading to enzyme activation carried out according to the method described by 14 and consequent increase in cGMP . It is proposed Porsolt20 and validated in the laboratory21-25. In brief, that, in response to activation of the NMDA receptor, mice were individually forced to swim inside a nitric oxide is synthesized from L-arginine by rectangular glass jar (25 ×12 × 25 cm3 containing 15 12,15 NOS . Various studies have shown the possibility cm of water maintained at 23˚-25˚ C). After the initial that various NOS inhibitors can be used as a strategy 2-3 min of vigorous activity the animals showed to enhance the antidepressant effect of various drugs period of immobility by floating with minimum including memantine (non-competitive NMDA movements. An animal is considered to be immobile 16 receptor antagonist) . However, none of the studies whenever it remained floating passively in the water have explored the involvement of nitric oxide in a slightly hunched but upright position, its nose signaling pathway in the antidepressant-like effect of above the water surface. The total immobility time for 17 MK-801. In a recent study, Dhir and Kulkarni have the period of 6 min was recorded with the help of demonstrated the involvement of L-arginine-nitric stop-watch23. oxide-cGMP signalling pathway in mediating the Measurement of locomotor activity— antidepressant activity of venlafaxine in mouse Locomotor activity (ambulations) was measured forced-swim test. It was suggested that NOS- by using a computerized actophotometer inhibitors when administered prior to venlafaxine (IMCORP, India). An array of 16 infrared treatment enhances its antidepressant action. emitter, detector pairs measured animal activity Similarly, the importance of nitric oxide signaling along a single axis of motion, the digital data pathway has been elucidated in the antidepressant 18 being displayed on the front panel meters as action of bupropion, a dopamine reuptake inhibitor ambulatory movements. Mice were allowed to or berberine chloride, an alkaloid obtained from 19 acclimatize to the observation chamber for a Berberis aristata . Since nitric oxide pathway is period of 2 min. Locomotion was expressed in involved in the pathophysiology of depression, the terms of total photobeams counts per 5 min per present study has been undertaken to investigate the animal26,27. participation of the L-arginine-NO-cGMP signaling Drugs and treatment—The following drugs pathway in the antidepressant-like effect of an acute were used: MK-801 (Sigma Chemicals, MO, USA), administration of MK-801 in the mouse forced-swim L-arginine (Loba-Chemie, Mumbai, India), methylene test. blue (S.D.-fine Chem Ltd., Gujarat, India), 7- nitroindazole (7-NI) (Tocris Bioscience, Missouri, Materials and Methods USA), and sildenafil (Panacea Biotec, New Delhi, Animals—Male albino mice (Laca strain) weighing India). All the drugs were dissolved in 0.9% w/v between 22-30 g bred in Central Animal House NaCl except 7-nitroindazole which was (CAH) facility of the Panjab University, Chandigarh, dissolved in few drops of Tween 80 and volume India were used. The animals were housed under was adjusted with distilled water. The doses of 166 INDIAN J EXP BIOL, MARCH 2008 the drugs used and time duration were selected shaped curve in reducing the immobility period in according to the previous studies28 and as reported in mouse FST. MK-801 (5 and 10 μg/kg, ip) did not the literature16,18,21,29. Different doses were alter the locomotor activity in mice (Table 1). administered ip in a fixed volume of 1 ml/100g However, MK-801 at higher dose (25 μg/kg, ip) body weight 30 min before the animals were increased the locomotor activity as evident from subjected to test. The possible participation of the increase in ambulatory movements [F=5.459, P<0.05] L-arginine-NO-cGMP pathway in the antidepressant (Table 1). A dose of 10 μg/kg, ip (dose effective in effect of MK-801 was investigated. Mice were forced-swim test and not affecting locomotor activity) pretreated with L-arginine,