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Home , Acetylcholine, Agmatine

Behavioural Research 213 (2010) 161–174

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Behavioural Brain Research

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Research report Repeated treatment attenuates 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 , Shrimati Kishoritai Bhoyar College of Pharmacy, New Kamptee, Nagpur, Maharashtra 441002, India

article info abstract

Article history: Agmatine [2-(4-aminobutyl)] is an endogenous 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- (80 ␮g, icv), decarboxylase inhibitor, difluoromethyl-ornithine ␣2-Adrenoceptors Locomotor sensitization (50 ␮g, icv), inhibitor, aminoguanidine (25 ␮g, icv) and inhibitor, arcaine (50 ␮g, icv)] 30 min before daily first nicotine 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 , (0.1 ␮g, icv) evoked synergistic inhibition of nicotine sensitization. Conversely, prior administration of ␣2-adrenoceptor antagonist, (5 mg/kg, ip) or (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 with [2,46]. Agmatine attenuates ethanol and morphine withdrawal reinforcing and addictive potential in humans. Repeated admin- symptoms [3,67], decreases morphine, 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- ( 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 / systems in brain formed by of l-arginine by the arginine areas like (VTA), (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 receptors brain, stored in synaptic vesicles in regionally selective , (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 channels [72,78]. ∗ Corresponding author. Tel.: +91 7109 288650; fax: +91 7109 287094. It also inhibits 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 (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 [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], from adrenal chromaffin cells and 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). 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. 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 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 al. [57] was adapted to sensitize mice to agmatine in brain [32]. Agmatine is degraded to putrescine and nicotine. The protocols were designed to examine the effects of exogenously admin- guanido-butanoic acid by enzyme agmatinase and diamine oxidase istered agmatine or drugs which alter endogenous agmatine concentration in brain (DAO), respectively [48] and inhibition of these resulted on nicotine sensitization. To investigate the effects on the development phase either agmatine (40, 80 ␮g/mouse, icv); DAO inhibitor, aminoguanidine (25 ␮g/mouse, in augmentation of endogenous agmatine [46]. In present study we icv); arginase inhibitor, DFMO (50 ␮g/mouse, icv); agmatinase inhibitor, arcaine used DAO inhibitor, aminoguanidine [31] and agmatinase inhibitor, (50 ␮g/mouse, icv); precursor for agmatine, l-arginine (80 ␮g/mouse, icv) or aCSF arcaine [18,46] to block the agmatine metabolic pathways lead- (2 ␮l/mouse, icv) were administered to the separate group of animals (n =9)30min ing to increase brain agmatine levels [58]. We further assessed before the daily first dose of nicotine tartarate (0.4 mg/kg as free base) or saline (1 ml/kg, sc) during 7 days of development phase. Drugs were not injected the involvement of ␣2-adrenoceptors in agmatine effects using ␣ on days 8, 9 and 10 of experiment. On day 11, all mice received a challenge dose of clonidine, 2-adrenoceptor agonist and yohimbine and idazoxan, nicotine (0.4 mg/kg, sc) or saline (1 ml/kg, sc) and locomotor activity was recorded ␣2-adrenoceptor antagonists. as mentioned above once daily at 09.00 a.m. for 20 min immediately after every first injection of nicotine or saline through days 1–7 and on day 11. In another set of experiments (n = 9), following repeated injections of nicotine 2. Materials and methods (0.4 mg/kg, sc, twice daily) for 7 consecutive days mice were treated with agmatine or its modulators (as mentioned above) on days 8, 9, 10 of nicotine free period, i.e. 2.1. Subjects withdrawal phase. On day 11, they received a challenge dose of nicotine (0.4 mg/kg, sc) or saline (1 ml/kg, sc) or aCSF (2 ␮l/mouse, icv) and locomotor activity of indi- Swiss albino male mice weighing 20–25 g were group housed (five per cage) vidual mice was measured immediately for 20 min. in a temperature and light (12:12 h light:dark cycle, lights on 07.00 h) controlled room. Food and water were available ad libitum. Animals were allowed for 48 h 2.6. Effect of ˛2-adrenoceptor agonist and antagonists on nicotine sensitization to acclimatize to the laboratory environment before experiments. All testing were executed in accordance with the guidelines for the care and use of laboratory animals To investigate the effect of ␣2-adrenoceptor agonist clonidine (0.1, by Committee for the Purpose of Control and Supervision of Experiments on Animals 0.2 ␮g/mouse, icv) or aCSF (2 ␮l/mouse, icv) alone or its subeffective dose (CPCSEA) and were approved by Institutional Animal Ethical Committee. All the (0.1 ␮g/mouse, icv) combination with agmatine (20 ␮g/mouse, icv) were either observations were made during 09.00–14.00 h to avoid circadian variations. All mice administered (n = 9) during development of sensitization (days 1–7) or during were experimentally naïve. nicotine free period (days 8, 9, 10). Treatment protocols were also designed (n =9)

to assess the effects of ␣2-adrenoceptor antagonists, yohimbine or idazoxan on agmatine induced inhibition of nicotine sensitization. Mice (n = 9) were treated 2.2. Drugs with yohimbine (5 mg/kg, ip), idazoxan (0.4 mg/kg, ip) or saline (1 ml/kg, ip) once daily 15 min before agmatine (40 ␮g/mouse, icv) followed by daily injection of Agmatine sulfate, nicotine hydrogen tartarate, aminoguanidine hemisulfate, DL- nicotine (0.4 mg/kg, sc) during the development phase of nicotine sensitization or DFMO, arcaine sulfate, clonidine, yohimbine hydrochloride, idazoxan hydrochloride during nicotine free period. Appropriate control groups (n = 9) were maintained in and l-arginine monohydrochloride were purchased from Sigma–Aldrich, Co., USA. all the cases. Locomotor counts were monitored for all groups daily for 20 min as Nicotine hydrogen tartarate, yohimbine hydrochloride and idazoxan hydrochlo- per the schedule on day 1 through 7 and on day 11 after nicotine challenge. ride were dissolved in isotonic saline solution. Nicotine was administered by subcutaneous (sc) route whereas yohimbine and idazoxan were administered by 2.7. Statistical analysis intraperitoneal (ip) route. All other drugs were dissolved in artificial cerebrospinal fluid (aCSF) of the following composition (140 mM NaCl, 3.35 mM KCl, 1.15 mM Acute effect (day 1) of nicotine on locomotion were analyzed by Unpaired ‘t’-

MgCl2, 1.26 mM CaCl2, 1.2 mM Na2HPO4, 0.3 mM NaH2PO4, pH 7.4) and administered test. Data obtained from development phase (days 1–7) treatment was analyzed by via icv route. Two-way analysis of variance (ANOVA) with repeated measures on time followed N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174 163

Fig. 1. Effects of agmatine (AGM) on nicotine (NIC) induced locomotor activity during 7-day development phase. Mice were pretreated with aCSF (2 ␮l/mouse, icv) or AGM (40 and 80 ␮g/mouse, icv) 30 min before first daily injections of saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) for 7 consecutive days. Each point represents the mean locomotor counts ± SEM (n = 7). Ф < 0.05 vs. aCSF–SAL (day 1) (Unpaired ‘t’-test), #P < 0.001 vs. aCSF–SAL. *P < 0.01, **P < 0.001 vs. aCSF–NIC (days 1–7) (Two-way ANOVA followed by Bonferroni multiple comparison test).

Fig. 2. Effects of agmatine (AGM) on locomotor activity in response to nicotine (NIC) challenge on day 11. Mice were pretreated with aCSF (2 ␮l/mouse, icv) or AGM (40 and 80 ␮g/mouse, icv) 30 min before injections of saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) during 7-day development phase and tested with SAL (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) challenge on day 11. Each column represents the mean locomotor counts ± SEM (n = 7). #P < 0.001 vs. aCSF/SAL–SAL, $P < 0.001 vs. aCSF/NIC–SAL (Unpaired ‘t’-test). *P < 0.001 vs. aCSF/NIC–NIC (One-way ANOVA followed by Newman–Keuls test). 164 N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174

Fig. 3. Effects of agmatine (AGM) on nicotine (NIC) induced behavioral sensitization. Mice were pretreated with saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) twice daily for 7 consecutive days and injected with aCSF (NIC/aCSF–NIC) or AGM (40 and 80 ␮g/mouse, icv) during a 3-day withdrawal phase and challenged with NIC (0.4 mg/kg, sc) on day 11. The normal group was pretreated with aCSF and challenged with only SAL (SAL/aCSF–SAL) (n = 7). #P < 0.001 vs. SAL/aCSF–SAL. *P < 0.001 vs. NIC/aCSF–NIC (days 1–7) (One-way ANOVA followed by Newman–Keuls test).

by post hoc Bonferroni multiple comparison test. Data of 11th day nicotine sensi- (AGM–NIC) hyperlocomotor response as compared to Acsf–NIC tization was analyzed by One-way analysis of variance (ANOVA) followed by post [One-way ANOVA post hoc Dunnett’s analysis; F(2, 20) = 0.06, hoc Dunnett’s/Newman–Keuls test. Data obtained on day 11, following the treat- P > 0.05]. Acute injection of agmatine (40–80 ␮g/mouse, icv) alone ment during withdrawal period (days 8, 9, 10), was analyzed by One-way analysis of variance (ANOVA) followed by post hoc Dunnett’s/Newman–Keuls test. P ≤ 0.05 (day 1) to saline treated groups (AGM–SAL) did not change animal’s was considered to be statistically significant. basal activity [One-way ANOVA post hoc Dunnett’s analysis; F(2, 20) = 1.68, P > 0.05]. On the other hand repeated injections of agma- 3. Results tine before daily first dose (0.4 mg/kg, sc) of nicotine (AGM–NIC) for 7 consecutive days significantly reduced the magnitude of loco- 3.1. Agmatine inhibits nicotine sensitization motor sensitization [FTreatment (5, 216) = 65.21, P < 0.01; FTime (6, 216) = 19.90, P < 0.01 and FTreatment × Time (30, 216) = 6.57, P < 0.01]. ␮ Effects of agmatine on the development of nicotine induced Administration of agmatine (40–80 g/mouse) to the mice locomotor sensitization are shown in Fig. 1. Acute administration before nicotine for 7 days significantly attenuated the hyperloco- of the first dose of nicotine (0.4 mg/kg, sc) on day 1 modestly motor response to nicotine challenge on day 11 [One-way ANOVA, but significantly increased the locomotion by 45% compared to F(5, 41) = 52.63, P < 0.001] after 3 days extinction period compared saline control (aCSF–SAL) group [Unpaired t-test; t = 2.18, df = 12, to control (aCSF/NIC–NIC). Post hoc Newman–Keuls comparison ␮ ␮ P < 0.05]. On the other hand, repeated nicotine injections twice daily showed that both 40 g(P < 0.001) as well as 80 g(P < 0.001) daily for 7 consecutive days resulted in a progressive and significant treatments significantly lowered locomotor activity (Fig. 2). increase in locomotor response through the entire treatment period Administration of agmatine alone (AGM/SAL–SAL) without consistent with sensitization development [Two-way ANOVA – nicotine treatment for 7 days did not produce any significant change in the activity counts compared to (aCSF/SAL–SAL) treated FTreatment (1, 72) = 381.87, P < 0.001, FTime (6, 72) = 19.37, P < 0.001, group [FTreatment (2, 108) = 0.83, P = 0.45; FTime (6, 108) = 1.27, FTreatment × Time (6, 72) = 13.26, P < 0.001]. On 7th day the hyper- activity in response to nicotine injection was increased by 400% P = 0.27 and FTreatment × Time (12, 108) = 0.85, P = 0.59]. above the level observed on day 1 [day 7 vs. day 1, Unpaired Further giving agmatine only during nicotine free period (days t-test; t = 10.85, df = 12, P < 0.001]. However, repeated saline injec- 8, 9, 10) significantly attenuated (Fig. 3) the locomotor response on tions for 7 days had no effect on locomotor activity. As can 11th day to nicotine challenge [F(2, 20) = 11.42, P < 0.001] as com- be seen in Fig. 2, similar to those for mice received nicotine pared to mice received nicotine (days 1–7) and aCSF (days 8, 9, 10) for 7 days, a injection of challenge dose of nicotine on day 11 indicating blockade of consolidation or incubation of sensitization. (aCSF/NIC–NIC) following nicotine free period (days 8–10) also exhibited greater locomotor activity than mice with saline on 3.2. l-Arginine, DFMO, arcaine and aminoguanidine attenuates day 11 (aCSF/NIC–SAL) [Unpaired t-test; t = 5.67, df = 12, P < 0.001] nicotine sensitization or from saline–saline (aCSF/SAL–SAL) control [Unpaired t-test; t = 15.39, df = 12, P < 0.001]. As shown in Fig. 4, acute icv injection of l-arginine As shown in Fig. 1, coadministration of agmatine (80 ␮g/mouse, l-arginine–NIC) or DFMO (50 ␮g/mouse, (40–80 ␮g/mouse, icv) with nicotine (0.4 mg/kg, sc) on day 1 DFMO–NIC) (Fig. 4A) or arcaine (50 ␮g/mouse, arcaine–NIC) did not significantly change (AGM–NIC) the acute nicotine induced or aminoguanidine (25 ␮g/mouse, AMG–NIC) (B) on day 1, N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174 165

Fig. 4. Effects of brain agmatine modulators on nicotine (NIC) induced locomotor activity during 7-day development phase. Mice were pretreated with aCSF (2 ␮l/mouse, icv) or l-arginine (80 ␮g/mouse, icv) or DFMO (50 ␮g/mouse, icv) (A) or arcaine (50 ␮g/mouse, icv) or aminoguanidine (25 ␮g/mouse, icv) (B) 30 min before first daily injections of saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) for 7 consecutive days. Each point represents the mean locomotor counts ± SEM (n = 7). #P < 0.001 vs. aCSF–SAL. *P < 0.05, **P < 0.01, ***P < 0.001 vs. aCSF–NIC (days 1–7) (Two-way ANOVA followed by Bonferroni multiple comparison test).

30 min prior to the first nicotine injection (0.4 mg/kg, sc) did blockade. Two-way ANOVA revealed that daily treatment of not significantly change acute locomotor response to nicotine l-arginine (80 ␮g, l-arginine–NIC) or DFMO (50 ␮g, DFMO–NIC) as compared to aCSF–NIC groups [One-way ANOVA post hoc during 7 days development phase significantly reduced nicotine Dunnett’s analysis; F(4, 34) = 2.12, P > 0.05]. However, these curves induced locomotor activity when compared against control group l start from lower baseline indicating their tendency towards (aCSF–NIC) [ -arginine: FTreatment (3, 144) = 170.84, P < 0.001; FTime 166 N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174

Fig. 5. Effects of brain agmatine modulators on locomotor activity in response to nicotine (NIC) challenge on day 11. Mice were pretreated with aCSF (2 ␮l/mouse, icv) or l-arginine (80 ␮g/mouse, icv) or DFMO (50 ␮g/mouse, icv) (A) or arcaine (50 ␮g/mouse, icv) or aminoguanidine (25 ␮g/mouse, icv) 30 min before injections of saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) during 7-day development phase and tested with SAL (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) challenge on day 11. Each column represents the mean locomotor counts ± SEM (n = 7). #P < 0.001 vs. aCSF/SAL–SAL. *P < 0.001 vs. aCSF/NIC–NIC (One-way ANOVA followed by Newman–Keuls test).

Fig. 6. Effects of brain agmatine modulators on nicotine (NIC) induced behavioral sensitization. Mice were pretreated with saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) twice daily for 7 consecutive days and injected with aCSF (NIC/aCSF–NIC) or l-arginine (80 ␮g/mouse, icv) (NIC/l-arginine–NIC) or DFMO (50 ␮g/mouse, icv) (NIC/DFMO–NIC) or arcaine (50 ␮g/mouse, icv) (NIC/arcaine–NIC) or aminoguanidine (25 ␮g/mouse, icv) (NIC/aminoguanidine–NIC) during a 3-day withdrawal phase and challenged with NIC (0.4 mg/kg, sc) on day 11. The normal group was pretreated with aCSF and challenged with only SAL (SAL/aCSF–SAL) (n = 7). #P < 0.001 vs. SAL/aCSF–SAL. *P < 0.01, **P < 0.001 vs. NIC/aCSF–NIC (days 1–7) (One-way ANOVA followed by Newman–Keuls test). N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174 167

␮ (6, 144) = 23.70, P < 0.001 and FTreatment × Time (18, 144) = 6.34, The effect of clonidine (0.1, 0.2 g/mouse) and the combination P < 0.001; DFMO: FTreatment (3, 144) = 99.44, P < 0.001; FTime treatment of low doses (non-effective if given alone with aCSF–SAL) ␮ ␮ (6, 144) = 22.84, P < 0.001 and FTreatment × Time (18, 144) = 8.78, of clonidine (0.1 g/mouse) and agmatine (20 g/mouse) dur- P < 0.001]. The injections of arcaine (50 ␮g/mouse, icv, arcaine–NIC) ing withdrawal phase on the locomotor sensitization is depicted and aminoguanidine (25 ␮g/mouse, icv, aminoguanidine–NIC) in Fig. 9. Clonidine (0.2 ␮g/mouse, icv) and combined treat- during 7 days development phase of locomotor sensitization ment of agmatine (20 ␮g) and clonidine (0.1 ␮g) during 3-day (Fig. 4B) exhibited significant effect on locomotor activity [arcaine: withdrawal periods (days 8, 9 and 10) after the 7 days induc- = FTreatment (3, 144) 130.31, P < 0.001; FTime (6, 144) = 28.13, P < 0.001 tion phase inhibited nicotine sensitization on 11th day when and FTreatment × Time (18, 144) = 6.63, 1 P < 0.001; DFMO: FTreatment compared against control group (NIC/aCSF–NIC) [F(5, 41) = 36.98, (3, 144) = 120.46, P < 0.001; FTime (6, 144) = 28.14, P < 0.001 and P < 0.001]. Post Newman–Keuls comparisons indicated that cloni- ␮ FTreatment × Time (18, 144) = 10.53, P < 0.001]. dine (0.2 g – 0.001 vs. NIC/aCSF–NIC) and the combined agmatine Administration of l-arginine (80 ␮g, l-arginine–SAL) or DFMO (20 ␮g) and clonidine (0.1 ␮g) treatment during nicotine free (50 ␮g, DFMO–SAL) or arcaine (50 ␮g, arcaine–SAL) or aminoguani- period on days 8, 9 and 10 also significantly attenuated the dine (25 ␮g, AMG–SAL) with saline for 7 days did not produce nicotine sensitization on 11th day when compared against aCSF any significant change in the activity counts when compared with (NIC/aCSF–NIC) (P < 0.001), agmatine (NIC/AGM–NIC) (P < 0.001) or aCSF–SAL treated group. clonidine (NIC/clonidine–NIC) (P < 0.001) treated group. As depicted in Fig. 5, treatment of these modulators for 7 consecutive days during development phase followed by 3-day 3.4. ˛ -Adrenoceptor antagonists reversed the inhibitory effect of withdrawal also significantly blocked sensitization to nicotine chal- 2 agmatine on nicotine sensitization lenge on 11th day [One-way ANOVA – F(9, 69) = 49.85, P < 0.001]. Post hoc Dunnett’s comparison demonstrated the significant effect Two-way ANOVA revealed that daily treatment of yohimbine of l-arginine (80 ␮g) (P < 0.001) or DFMO (50 ␮g) (P < 0.001) or (5 mg/kg, ip) (Fig. 10A) or idazoxan (0.4 mg/kg, ip) (Fig. 10B) arcaine (50 ␮g) (P < 0.001) or aminoguanidine (25 ␮g) (P < 0.001) before agmatine (40 ␮g/mouse) injections during 7 day devel- on the locomotor stimulation to nicotine challenge on day 11. opment period of nicotine sensitization significantly reversed Furthermore as shown in Fig. 6 injections of the agents that the inhibitory effect of agmatine on nicotine induced locomo- alters the brain agmatine content during 3-day nicotine free period tor sensitization [yohimbine: F (3, 144) = 19.90, P < 0.001; (days 8, 9, 10) after 7-day induction phase, significantly inhibited Treatment F (6, 144) = 82.63, P < 0.001 and F × (18, 144) = 2.67, locomotor sensitization to nicotine challenge on day 11 as com- Time Treatment Time P = 0.0006; idazoxan: F (3, 144) = 44.27, P < 0.001; F pared to its control group (NIC/aCSF–NIC) [One-way ANOVA – F(5, Treatment Time (6, 144) = 77.49, P < 0.001 and F × (18, 144) = 2.24, 41) = 24.96, P < 0.001]. Post hoc analysis of the mean locomotor Treatment Time P = 0.0045]. This treatment protocol of 7 days followed by 3-day counts showed the significant attenuation of the locomotor activ- withdrawal period after which challenged with nicotine on 11th ity on 11th day by l-arginine (80 ␮g) (P < 0.01) or DFMO (50 ␮g) day also showed reversal of agmatine effect on nicotine hyper- (P < 0.001) or arcaine (50 ␮g) (P < 0.01) or aminoguanidine (25 ␮g) locomotion (Fig. 11) [One-way ANOVA, yohimbine: F(7, 55) = 101.7, (P < 0.001). P < 0.001; idazoxan: F(7, 55) = 80.11, P < 0.001]. In analogy with above the injections of yohimbine (5 mg/kg) or idazoxan (0.4 mg/kg) during 3-day withdrawal period (days 3.3. Effect of ˛ -adrenoceptor agonist, clonidine and its 2 8, 9 and 10) after 7 days induction phase exerted an obvious combination with agmatine on nicotine induced behavioral reversal of agmatine effect on nicotine induced behavioral sensiti- sensitization zation as tested on day 11 when compared against control animals that received nicotine (days 1–7) and agmatine/saline (days 8, 9, Fig. 7A and B shows locomotor activity level for each treat- 10) (Fig. 12) [yohimbine: F(3, 34) = 53.32, P < 0.01; idazoxan: F(3, ment group at day 1 (acute effect) and through 7 days sensitization 34) = 32.58, P < 0.001]. However, administration of yohimbine or development. Acute injections of clonidine (0.2 ␮g, icv) on day 1, idazoxan alone in the doses used here did not influence the nicotine prior (30 min) to nicotine injections did not significantly block the sensitization. nicotine induced acute motor stimulation [F(3, 27) = 3.35, P > 0.05]. On other hand (A) pretreatment with clonidine (0.2 ␮g but not 0.1 ␮g) (clonidine–NIC) during the 7-day development phase sig- 4. Discussion nificantly blocked development of nicotine sensitization through entire 7 days period when compared against its control (aCSF–NIC) Results of the present study showed that acute injections of group [Two-way ANOVA – FTreatment (5, 216) = 100.11, P < 0.001; nicotine (day 1) to mice stimulated locomotor activity and follow- FTime (6, 216) = 48.19, P < 0.001 and FTreatment × Time (30, 216) = 8.11, ing its repeated daily injections for 7 consecutive days produced P < 0.001]. locomotor sensitization. Nicotine induced behavioral sensitization As depicted in Fig. 7B, per se subeffective dose of clonidine is consistent with the earlier reports [7,53,66]. In mice acute injec- (0.1 ␮g, icv) in combination with subeffective dose of agma- tions of nicotine generally fails to stimulate locomotor activity. tine (20 ␮g, icv) exhibited synergistic inhibition of development However the modest but significant acute activating effects (day 1) of nicotine induced locomotor sensitization [Two-way ANOVA – observed here are in concordance with recent reports [7,66]. Sev- FTreatment (5, 216) = 74.42, P < 0.001; FTime (6, 216) = 30.93, P < 0.001 eral lines of evidence indicated that nicotine stimulates dopamine and FTreatment × Time (30, 216) = 6.33, P < 0.001]. However, adminis- release by directly acting on the nicotinic receptors located on tration of clonidine (0.1–0.2 ␮g, clonidine–SAL) with saline for 7 the mesolimbic dopaminergic neurons which lead to locomotor days did not produce any significant effect on the motor activity hyperactivity or hypoactivity depending on dose and animal strain when compared with aCSF–SAL treated group. [9,69]. Behavioral sensitization is thought to be produced by incre- Daily treatment of clonidine (0.2 ␮g/mouse) (P < 0.001) or mental neuroadaptations of neural systems due to repeated use the combination of clonidine (0.1 ␮g/mouse) and agmatine of abused drugs [69]. Nicotine has been shown to increase the (20 ␮g/mouse) (P < 0.001) from day 1 to day 7 significantly blocked release of agmatine from adrenal chromaffin cells [47,62] which the hyperlocomotor response to nicotine challenge on day 11 is implicated in drug addiction [2,60] and several effects of psy- (Fig. 8)[F(9, 69) = 46.15, P < 0.001] (Post hoc Newman–Keuls test). chostimulants [60,71]. The brain regions (amygdala, VTA and NAc) 168 N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174

Fig. 7. Effects of ␣2-adrenoceptor agonist, clonidine alone (A) and in combination with agmatine (B) on nicotine (NIC) induced locomotor activity during 7-day development phase. Mice were pretreated with aCSF (2 ␮l/mouse, icv) or clonidine (0.1 and 0.2 ␮g/mouse, icv) or clonidine (0.1 ␮g/mouse, icv) in combination with non-effective dose of agmatine (20 ␮g/mouse, icv) 30 min before first daily injections of saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) for 7 consecutive days. Each point represents the mean locomotor counts ± SEM (n = 7). #P < 0.01, ##P < 0.001 vs. aCSF–SAL. *P < 0.01, **P < 0.001 vs. aCSF–NIC (days 1–7) (Two-way ANOVA followed by Bonferroni multiple comparison test). N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174 169

Fig. 8. Effects of ␣2-adrenoceptor agonist, clonidine alone and in combination with agmatine on locomotor activity in response to nicotine (NIC) challenge on day 11. Mice were pretreated with aCSF (2 ␮l/mouse, icv) or clonidine (0.1 and 0.2 ␮g/mouse, icv) or clonidine (0.1 ␮g/mouse, icv) in combination with non-effective dose of agmatine (20 ␮g/mouse, icv) 30 min before injections of saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) during 7-day development phase and tested with SAL (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) challenge on day 11. Each column represents the mean locomotor counts ± SEM (n = 7). #P < 0.001 vs. aCSF/SAL–SAL, *P < 0.001 vs. aCSF/NIC–NIC (One-way ANOVA followed by Newman–Keuls test).

Fig. 9. Effects of ␣2-adrenoceptor agonist, clonidine alone and in combination with agmatine on nicotine (NIC) induced behavioral sensitization. Mice were pretreated with saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) twice daily for 7 consecutive days and injected with aCSF (NIC/aCSF–NIC) or clonidine (0.1 and 0.2 ␮g/mouse, icv) (NIC/clonidine–NIC) or clonidine (0.1 ␮g/mouse, icv) in combination with non-effective dose of agmatine (20 ␮g/mouse, icv) (NIC/Clonidine + AGM–NIC) during a 3-day withdrawal phase and challenged with NIC (0.4 mg/kg, sc) on day 11. The normal group was pretreated with aCSF and challenged with only SAL (SAL/aCSF + aCSF–SAL) (n = 7). #P < 0.001 vs. SAL/aCSF + aCSF–SAL, *P < 0.001 vs. NIC/aCSF + aCSF–NIC, $P < 0.001 vs. NIC/clonidine + aCSF–NIC, @P < 0.001 vs. NIC/aCSF + AGM–NIC (One-way ANOVA followed by Newman–Keuls test). 170 N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174

Fig. 10. Effects of ␣2-adrenoceptor antagonists, yohimbine (Fig. 9A) and idazoxan (Fig. 9B) on the influence of agmatine (AGM) on nicotine (NIC) induced locomotor activity during 7-day development phase. Mice were pretreated with saline (SAL) (1 ml/kg, ip) or yohimbine (5 mg/kg, ip) or idazoxan (0.4 mg/kg, ip) before aCSF (2 ␮l/mouse, icv) or AGM (40 ␮g/mouse, icv) 30 min before first daily injections of SAL (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) for 7 consecutive days. Each point represents the mean locomotor counts ± SEM (n = 7). #P < 0.001 vs. SAL + aCSF–SAL, !P < 0.05, !!P < 0.01, !!!P < 0.001 vs. SAL + aCSF–NIC (days 1–7) *P < 0.05, **P < 0.01, ***P < 0.001 vs. SAL + AGM–NIC (Two-way ANOVA followed by Bonferroni multiple comparison test). N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174 171

Fig. 11. Effects of ␣2-adrenoceptor antagonists, yohimbine (Fig. 9A) and idazoxan (Fig. 9B) on the influence of agmatine (AGM) on locomotor activity in response to nicotine (NIC) challenge on day 11. Mice were pretreated with saline (SAL) (1 ml/kg, ip) or yohimbine (5 mg/kg, ip) or idazoxan (0.4 mg/kg, ip) before aCSF (2 ␮l/mouse, icv) or AGM (40 ␮g/mouse, icv) 30 min before injections of SAL (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) during 7-day development phase and tested with SAL (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) challenge on day 11. Each column represents the mean locomotor counts ± SEM (n = 7). #P < 0.001 vs. SAL + aCSF/SAL–SAL, $P < 0.001 vs. SAL + aCSF/NIC–NIC. *P < 0.001 vs. SAL + AGM/NIC–NIC (One-way ANOVA followed by Newman–Keuls test). involved in drug addiction has extensive distribution of agmatine, employed in the present investigation in addition to their effect its transporters and enzymes participated in its degradation and on agmatine [64] also possess other activities like NMDA antag- biosynthesis [48]. Therefore we examined whether agmatine plays onism (arcaine), NOS inhibition (aminoguanidine) as well as NO a crucial role in locomotor response to the acute or chronic repeated precursor (l-arginine) activity. Nonetheless, the involvement of administration of nicotine in mice. imidazoline receptors and other biological targets of agmatine This study investigated the effect of exogenously (icv) injected like NMDA and NOS cannot be ruled out and warrant further agmatine and the agents reported to increase endogenous brain investigation. Aminoguanidine, a DAO inhibitor [31] that inhibits agmatine content on nicotine induced behavioral sensitization. degradation of agmatine to guanidine–butanoic acid, is also a selec- Administration of agmatine precursor, l-arginine or inhibition tive inducible NOS inhibitor [57]. Based on these findings it seems of metabolic pathways might result in augmentation of endoge- plausible that rather than acute dose of agmatine, its daily pretreat- nous agmatine levels in brain. In the present study, we used ment for chronic period may be required to block nicotine effect. diamine oxidase (DAO) inhibitor, aminoguanidine or agmatinase Thus although premature, it can be anticipated that a reduction inhibitor, arcaine and arginase inhibitor, DFMO to block the agma- in agmatinergic tone in NAc shell may contribute to behavioral tine leading to increased agmatine levels in brain sensitization to nicotine. However further behavioral studies using [18,31,46,56,60]. Agmatine is extensively metabolized peripher- immunoneutralization or selective drugs that reduce the agmatine ally in liver, kidney and has very short biological half life [15]. formation may help to clarify its role in nicotine sensitization. On the other hand, its half life in and brain is 12–18 h Our findings that agmatine did not block acute nicotine effects [49]. Therefore to avoid peripheral metabolism and to attain suf- in mice are consistent with the results of Zaniewska et al. [76] in ficient concentration in brain, agmatine and its modulators were rats. This is the only study that investigated the effect of agmatine injected by icv route daily during development phase (days 1–7) (ip) on nicotine evoked behavioral responses in rats. Their pharma- or during nicotine withdrawal phase (days 8–10). We found that cological analysis indicated lack of effect of agmatine on locomotor, neither agmatine nor its modulators displayed any effect on sponta- sensitizing or subjective effects but displayed inhibition of condi- neous motor activity on day 1 (acute response) when administered tioned hyperlocomotion induced by nicotine. In contrast, our data 30 min before first dose of nicotine. On the other hand, daily pre- shows blockade of development of nicotine sensitization. One pos- treatments (days 1–7) with these drugs significantly attenuated the sible explanation for these discrepancies could be due to different development of nicotine induced locomotor sensitization. Interest- doses, route, treatment schedule and animal strain. Additional evi- ingly none of these drugs by themselves at any dose level caused dence supporting our data is that agmatine attenuates ethanol any change in basal locomotor activity in saline treated animals. as well as morphine withdrawal [3,27,67], decreases cocaine and Thus their effects on behavioral sensitization can not be attributed fentanyl self-administration [36] and expression of morphine sen- to locomotor suppression or sedation. It is noteworthy that drugs sitization [71] in experimental animals. 172 N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174

Fig. 12. Effects of ␣2-adrenoceptor antagonists, yohimbine (Fig. 9A) and idazoxan (Fig. 9B) on the influence of agmatine (AGM) on nicotine (NIC) induced behavioral sensitization. Mice were pretreated with saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) twice daily for 7 consecutive days and injected with SAL (1 ml/kg, ip) or yohimbine (5 mg/kg, ip) or idazoxan (0.4 mg/kg, ip) before aCSF (2 ␮l/mouse, icv) or AGM (40 ␮g/mouse, icv) during a 3-day withdrawal phase and challenged with NIC (0.4 mg/kg, sc) on day 11. The normal group was pretreated with aCSF and challenged with only SAL (SAL/aCSF + aCSF–SAL). Each column represents the mean locomotor counts ± SEM (n = 7). #P < 0.001 vs. SAL/SAL + aCSF–SAL, $P < 0.001 vs. NIC/SAL + aCSF–NIC, *P < 0.001 vs. NIC/SAL + AGM–NIC (One-way ANOVA followed by Newman–Keuls test).

Furthermore, in the present study icv injections of agmatine or of cocaine-seeking behavior [11] and footshock-induced reinstate- its modulators during nicotine withdrawal period (days 8, 9, 10) ment of nicotine-seeking behavior in rats [79]. Moreover, chronic resulted in a pronounced block in locomotor response to nico- administration of nicotine increases the density of ␣2-adrenergic tine challenge on day 11. This suggests that exogenous agmatine binding sites in some brain regions [73]. Interestingly, several or increasing endogenous agmatine in brain by different means studies have identified correlation between agmatinergic neurons inhibits the consolidation or incubation of sensitization. However, and ␣2-adrenergic receptors in many brain regions [24,37]. Like acute dose of agmatine (40–80 ␮g/mouse, icv) given before nico- clonidine, agmatine has been shown to bind ␣2-adrenoceptors tine challenge on day 11 (data not shown) does not counteract [63,75,80]. Agmatine alters the firing rate of locus ceruleus neurons the expression of nicotine sensitization. This agrees with previous in vivo [43,52] and induces ␣2-adrenoceptors dependant antinoci- agmatine studies on nicotine sensitization [76]. Although the psy- ception [39]. The potentiating effect of agmatine on morphine choactive actions of nicotine are mediated centrally though direct induced analgesia is mediated by ␣2-adrenoceptors [51,75].In interaction with nicotinic receptors (nAchRs) [9], some data exists view of these findings, we studied the possible involvement of ␣2- that implies the role for the other systems like adrenergic receptors in agmatine induced attenuation of nicotine glutamatergic and adrenergic systems [55,57]. Earlier studies have sensitization. suggested that NMDARs are key receptors in expression as well The present work showed that chronic administration of ␣2- as development of behavioral sensitization and that NO is also adrenoceptors agonist, clonidine during development and nicotine involved in development but not in expression of nicotine sensi- free period significantly blocked the nicotine induced increase in tization [57,66]. The diverse central effects of agmatine also are locomotor counts. Moreover, clonidine also augmented the sup- associated with its ability to bind to ␣2-adrenoceptors and imidazo- pression of nicotine sensitization by agmatine in the doses that line binding sites, inhibition of NOS activity and blockade of NMDA were ineffective when administered alone. Thus, our finding is receptors or nicotinic receptors [3,13,30,35,44,74]. The fact that in line with reports of agmatine/clonidine synergism [77] and agmatine acts on so many different biochemical processes, neu- strengthens the notion that several biological as well as pharma- rotransmitters or receptors makes it difficult to determine which cological effects of agmatine are closely linked to ␣2-adrenergic of its many effects are critical to suppress nicotine induced sen- receptors activation [63,75,80]. In fact clonidine has been reported sitization. Thus suppression of nicotine induced sensitization by to reduce morphine [8] cocaine [21] and d-amphetamine [68] agmatine could also be due to its ability to block NMDA or nicotinic induced sensitization. It may be recalled that, clonidine is clin- receptors or inhibition of enzyme NOS. ically used for smoking cessation and attenuation of nicotine Several lines of experimental evidence indicated the involve- withdrawal symptoms [6,14]. Involvement of ␣2-adrenergic recep- ment of noradrenergic receptors in psychostimulant induced tors in attenuation of nicotine sensitization by agmatine is further behavioral effects including sensitization [16,59,70]. Systemic supported by the fact that agmatine effect was completely abol- administration of the ␣2- clonidine, ished by selective ␣2-adrenergic receptor antagonists, yohimbine lofexidine and guanabenz attenuated stress-induced reinstatement and idazoxan, a mixed antagonist of ␣2/imidazoline I2 recep- N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174 173

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