Behavioural Brain Research 213 (2010) 161–174 Contents lists available at ScienceDirect Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr Research report Repeated agmatine treatment attenuates nicotine sensitization in mice: Modulation by ␣2-adrenoceptors Nandkishor Ramdas Kotagale, Brijesh Gulabrao Taksande, Avinash Yashwant Gahane, Rajesh Ramesh Ugale, Chandrabhan Tukaram Chopde ∗ Division of Neuroscience, Department of Pharmacology, Shrimati Kishoritai Bhoyar College of Pharmacy, New Kamptee, Nagpur, Maharashtra 441002, India article info abstract Article history: Agmatine [2-(4-aminobutyl)guanidine] is an endogenous amine proposed as a neurotransmit- Received 19 November 2009 ter/neuromodulator that binds to multiple target receptors in brain. Besides, many central and peripheral Received in revised form 24 April 2010 functions, agmatine have been implicated in the process of drug addiction. The purpose of the present Accepted 28 April 2010 study was to examine the effects of centrally injected agmatine on nicotine induced locomotor sensitiza- Available online 5 May 2010 tion in Swiss male mice. Our data shows that repeated injections of nicotine (0.4 mg/kg, sc, twice daily for 7 days) gradually increased locomotion during 7 days development period or after 3 days (nicotine) with- Keywords: drawal phase challenged with nicotine (0.4 mg/kg, sc) on day 11. Mice were pretreated with agmatine Agmatine ␮ Nicotine (40–80 g, icv) or agents known to increase endogenous brain agmatine levels [e.g. an agmatine biosyn- thetic precursor, l-arginine (80 ␮g, icv), ornithine decarboxylase inhibitor, difluoromethyl-ornithine ␣2-Adrenoceptors Locomotor sensitization (50 ␮g, icv), diamine oxidase inhibitor, aminoguanidine (25 ␮g, icv) and agmatinase inhibitor, arcaine (50 ␮g, icv)] 30 min before daily first nicotine injection or during nicotine withdrawal phase. All these treatments attenuated the development as well as incubation of locomotor sensitization to nicotine. Coadministration of agmatine (20 ␮g, icv) and ␣2-adrenoreceptors agonist, clonidine (0.1 ␮g, icv) evoked synergistic inhibition of nicotine sensitization. Conversely, prior administration of ␣2-adrenoceptor antagonist, yohimbine (5 mg/kg, ip) or idazoxan (0.4 mg/kg, ip) reversed the inhibitory effect of agma- tine on nicotine sensitization. There was no significant difference in activity between mice injected with any of these agents/saline alone and saline/saline groups. These data indicate that agmatine attenuates nicotine induced locomotor sensitization via a mechanism which may involve ␣2-adrenergic receptors. Thus, agmatine might have therapeutic implications in the treatment of nicotine addiction and deserve further investigations. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Recently, agmatine [2-(4-aminobutyl) guanidine], an endoge- nous amine has been implicated in the process of drug addiction Nicotine is the major psychoactive constituent of tobacco with [2,46]. Agmatine attenuates ethanol and morphine withdrawal reinforcing and addictive potential in humans. Repeated admin- symptoms [3,67], decreases morphine, cocaine or fentanyl self- istration of nicotine in rodents evokes behavioral sensitization administration [36,61] and blocks locomotor as well as biochemical indicated by gradual increase in locomotor activity [33,34]. Behav- (dopamine release) expression of morphine sensitization [71].It ioral sensitization is thought to be one of the basic mechanisms inhibits the expression of nicotine induced conditioned hyper- underlying development of drug addiction [50]. Behavioral effects locomotion without affecting its either acute locomotor and of nicotine including sensitization are regulated through its inter- sensitizing or discriminative stimulating effects [76]. Agmatine is actions with multiple neurotransmitters/receptor systems in brain formed by decarboxylation of l-arginine by the enzyme arginine areas like ventral tegmental area (VTA), nucleus accumbens (NAc) decarboxylase (l-ADC) and has been suggested to be a putative neu- and prefrontal cortex [10,57,79]. Nicotine stimulates dopamine rotransmitter/neuromodulator in mammals. It is synthesized in the release by directly acting on nicotinic acetylcholine receptors brain, stored in synaptic vesicles in regionally selective neurons, (nAChRs) located on the mesolimbic dopamine neurons leading to accumulated by uptake and degraded by agmatinase [15,45,48]. locomotor sensitization [65,22]. Agmatine binds to ␣2-adrenoreceptors [26], imidazoline binding sites [44,48], blocks N-methyl-d-aspartate (NMDA) receptors [74], nAch receptors [30] and other ligand gated ion channels [72,78]. ∗ Corresponding author. Tel.: +91 7109 288650; fax: +91 7109 287094. It also inhibits nitric oxide synthase (NOS), an enzyme responsi- E-mail address: [email protected] (C.T. Chopde). ble for nitric oxide (NO) formation in brain [4,13]. Agmatine is a 0166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2010.04.049 162 N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174 pleiotropic molecule with many central and peripheral functions. 2.3. Surgery Its systemic administration evokes anxiolytic [25], antidepressant [28,80], antinociceptive [40], anticonvulsive [5], anti-inflammatory Under pentobarbital sodium (60 mg/kg, ip) anesthesia mice were placed in a stereotaxic frame (David Kopf, CA, USA). A guide cannula (C315 G/Spc, Plastic One [54], antiproliferative [19] and neuroprotective [38] properties Inc., Virginia, USA) was implanted bilaterally into the third ventricle (0.8 mm pos- and also facilitates working memory [29]. Agmatine stimulates terior, 1.3 mm lateral to midline and 3.5 ventral to the bregma) according to the release of luteinizing hormone-releasing hormone from hypothala- mouse brain atlas [42]. A 28-gauge stainless steel dummy cannula was inserted to mus [23], catecholamines from adrenal chromaffin cells and insulin occlude the guide cannula when not in use. After surgery each mouse was injected with oxytetracycline injection (25 mg/kg, im, Pfizer Ltd., Chennai) and Neosporin from pancreatic ␤-cells [1]. ointment (Burroughs Wellcome Ltd., Mumbai) was applied to avoid infection. Ani- Chronic nicotine administration increases adrenergic binding mals were then placed individually in home cage and allowed to recover for 7 days. sites in several brain regions [73]. Agmatine and ␣2-adrenergic During this period animals were habituated to the testing environment by transfer- receptors have important functional interactions in behavioral ring them to experimental room and handling daily to treatment schedule. The icv effects of psychoactive agents including potentiation of mor- injections were given via 33 gauge internal cannula (internal diameter 0.18 mm and outer diameter 0.20 mm) (C315 I/Spc), which was attached to a Hamilton microliter phine induced analgesia [12,75] and conditioned place preference syringe (Hamilton, Nevada, USA) via polyethylene tubing (PE-10) (internal diam- [63], attenuation of morphine withdrawal symptoms and sev- eter 0.28 mm; outer diameter 0.61 mm), that extended 0.5 mm beyond the guide eral aspects of drug addiction [61,75]. Moreover clonidine, an cannula. The internal cannula was held in position for another 1 min before being slowly withdrawn to prevent backflow and promote diffusion of drug. ␣2-adrenoceptor agonist, is clinically used for the treatment of After all sensitization testing, dilute India ink was injected (icv) and subjects nicotine addiction and relapse [6,14]. While much has been stud- were sacrificed under an overdose of sodium pentobarbital anesthesia (120 mg/kg, ied on the modulatory influence of agmatine on morphine effects, ip). Brains were removed and cryostat cut into 50-␮m sections, mounted and viewed its regulatory role in behavioral effects of nicotine including sen- using light microscopy to verify cannulae placements. The data of animals with sitization and addiction is poorly understood. In present study, cannula placement of more than 0.5 mm away from coordinates were excluded from we have investigated the effects of intracerebroventricularly (icv) the study (<15%) and data from mice with uniform ink distribution into ventricles were used for statistical analysis. injected agmatine or the agents augmenting the brain agmatine content on nicotine induced motor sensitization. Increasing biosyn- 2.4. Measurement of locomotor activity thesis of endogenous agmatine and blocking its degradation are the approaches to elevate agmatine levels in brain. Biosynthesis Locomotor activity was measured using actophotometer × × of agmatine by l-ADC depends upon the availability of l-arginine (20 cm 20 cm 10 cm) (Techno, India) equipped with six infrared photo l sensors, 2.5 cm apart from each other. Mice were habituated to the actophotometer [60]. -Arginine is also converted into ornithine by arginase and chamber for 30 min before any testing. Baseline locomotor activity of each mouse to NO by an enzyme NOS. Ornithine subsequently turned into was recorded for 20 min as a total count of ambulatory, horizontal and vertical putrescine by l-ornithine decarboxylase (l-ODC) [48]. DFMO (␣- activity. difluoromethyl-ornithine), is an inhibitor of arginase [56] and also stimulator of enzyme l-ADC [17]. Its function as arginase inhibitor 2.5. Effect of agmatine and its modulators on nicotine sensitization l as well as stimulator of -ADC would increase the availability of The procedure outlined by Shim et