14
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 Article history: Received 19 November 2009 Received in revised form 24 April 2010 Accepted 28 April 2010 Available online 5 May 2010 Keywords: Agmatine Nicotine 2-Adrenoceptors Locomotor sensitization abstract Agmatine [2-(4-aminobutyl)guanidine] is an endogenous amine proposed as a neurotransmit- ter/neuromodulator that binds to multiple target receptors in brain. Besides, many central and peripheral functions, agmatine have been implicated in the process of drug addiction. The purpose of the present study was to examine the effects of centrally injected agmatine on nicotine induced locomotor sensitiza- 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- drawal phase challenged with nicotine (0.4 mg/kg, sc) on day 11. Mice were pretreated with agmatine (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 (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 Nicotine is the major psychoactive constituent of tobacco with reinforcing and addictive potential in humans. Repeated admin- istration of nicotine in rodents evokes behavioral sensitization indicated by gradual increase in locomotor activity [33,34]. Behav- ioral sensitization is thought to be one of the basic mechanisms underlying development of drug addiction [50]. Behavioral effects of nicotine including sensitization are regulated through its inter- actions with multiple neurotransmitters/receptor systems in brain areas like ventral tegmental area (VTA), nucleus accumbens (NAc) and prefrontal cortex [10,57,79]. Nicotine stimulates dopamine release by directly acting on nicotinic acetylcholine receptors (nAChRs) located on the mesolimbic dopamine neurons leading to locomotor sensitization [65,22]. Corresponding author. Tel.: +91 7109 288650; fax: +91 7109 287094. E-mail address: [email protected] (C.T. Chopde). Recently, agmatine [2-(4-aminobutyl) guanidine], an endoge- nous amine has been implicated in the process of drug addiction [2,46]. Agmatine attenuates ethanol and morphine withdrawal symptoms [3,67], decreases morphine, cocaine or fentanyl self- administration [36,61] and blocks locomotor as well as biochemical (dopamine release) expression of morphine sensitization [71]. It inhibits the expression of nicotine induced conditioned hyper- locomotion without affecting its either acute locomotor and sensitizing or discriminative stimulating effects [76]. Agmatine is formed by decarboxylation of l-arginine by the enzyme arginine decarboxylase (l-ADC) and has been suggested to be a putative neu- rotransmitter/neuromodulator in mammals. It is synthesized in the brain, stored in synaptic vesicles in regionally selective neurons, accumulated by uptake and degraded by agmatinase [15,45,48]. 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]. It also inhibits nitric oxide synthase (NOS), an enzyme responsi- 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

Agm - Nic Sensitisation

  • Upload
    niku

  • View
    255

  • Download
    0

Embed Size (px)

DESCRIPTION

hfjh

Citation preview

Page 1: Agm - Nic Sensitisation

R

RM

NRD

a

ARRAA

KAN�L

1

riiiuoaaar(l

0d

Behavioural Brain Research 213 (2010) 161–174

Contents lists available at ScienceDirect

Behavioural Brain Research

journa l homepage: www.e lsev ier .com/ locate /bbr

esearch report

epeated agmatine treatment attenuates nicotine sensitization in mice:odulation by �2-adrenoceptors

andkishor Ramdas Kotagale, Brijesh Gulabrao Taksande, Avinash Yashwant Gahane,ajesh Ramesh Ugale, Chandrabhan Tukaram Chopde ∗

ivision of Neuroscience, Department of Pharmacology, Shrimati Kishoritai Bhoyar College of Pharmacy, New Kamptee, Nagpur, Maharashtra 441002, India

r t i c l e i n f o

rticle history:eceived 19 November 2009eceived in revised form 24 April 2010ccepted 28 April 2010vailable online 5 May 2010

eywords:gmatineicotine2-Adrenoceptorsocomotor sensitization

a b s t r a c t

Agmatine [2-(4-aminobutyl)guanidine] is an endogenous amine proposed as a neurotransmit-ter/neuromodulator that binds to multiple target receptors in brain. Besides, many central and peripheralfunctions, agmatine have been implicated in the process of drug addiction. The purpose of the presentstudy was to examine the effects of centrally injected agmatine on nicotine induced locomotor sensitiza-tion in Swiss male mice. Our data shows that repeated injections of nicotine (0.4 mg/kg, sc, twice daily for7 days) gradually increased locomotion during 7 days development period or after 3 days (nicotine) with-drawal phase challenged with nicotine (0.4 mg/kg, sc) on day 11. Mice were pretreated with agmatine(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(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 thesetreatments 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-adrenoceptorantagonist, 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 withany of these agents/saline alone and saline/saline groups. These data indicate that agmatine attenuatesnicotine 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.

. Introduction

Nicotine is the major psychoactive constituent of tobacco witheinforcing and addictive potential in humans. Repeated admin-stration of nicotine in rodents evokes behavioral sensitizationndicated by gradual increase in locomotor activity [33,34]. Behav-oral sensitization is thought to be one of the basic mechanismsnderlying development of drug addiction [50]. Behavioral effectsf nicotine including sensitization are regulated through its inter-ctions with multiple neurotransmitters/receptor systems in brainreas like ventral tegmental area (VTA), nucleus accumbens (NAc)

nd prefrontal cortex [10,57,79]. Nicotine stimulates dopamineelease by directly acting on nicotinic acetylcholine receptorsnAChRs) located on the mesolimbic dopamine neurons leading toocomotor sensitization [65,22].

∗ Corresponding author. Tel.: +91 7109 288650; fax: +91 7109 287094.E-mail address: [email protected] (C.T. Chopde).

166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2010.04.049

© 2010 Elsevier B.V. All rights reserved.

Recently, agmatine [2-(4-aminobutyl) guanidine], an endoge-nous amine has been implicated in the process of drug addiction[2,46]. Agmatine attenuates ethanol and morphine withdrawalsymptoms [3,67], decreases morphine, cocaine or fentanyl self-administration [36,61] and blocks locomotor as well as biochemical(dopamine release) expression of morphine sensitization [71]. Itinhibits the expression of nicotine induced conditioned hyper-locomotion without affecting its either acute locomotor andsensitizing or discriminative stimulating effects [76]. Agmatine isformed by decarboxylation of l-arginine by the enzyme argininedecarboxylase (l-ADC) and has been suggested to be a putative neu-rotransmitter/neuromodulator in mammals. It is synthesized in thebrain, stored in synaptic vesicles in regionally selective neurons,accumulated by uptake and degraded by agmatinase [15,45,48].

Agmatine binds to �2-adrenoreceptors [26], imidazoline bindingsites [44,48], blocks N-methyl-d-aspartate (NMDA) receptors [74],nAch receptors [30] and other ligand gated ion channels [72,78].It also inhibits nitric oxide synthase (NOS), an enzyme responsi-ble for nitric oxide (NO) formation in brain [4,13]. Agmatine is a
Page 2: Agm - Nic Sensitisation

1 l Brain

pI[[armf

srep[e�niiswictto[tpdsaag(iuaitc�

2

2

irteb(ow

2

DaNrsiflMv

62 N.R. Kotagale et al. / Behavioura

leiotropic molecule with many central and peripheral functions.ts systemic administration evokes anxiolytic [25], antidepressant28,80], antinociceptive [40], anticonvulsive [5], anti-inflammatory54], antiproliferative [19] and neuroprotective [38] propertiesnd also facilitates working memory [29]. Agmatine stimulateselease of luteinizing hormone-releasing hormone from hypothala-us [23], catecholamines from adrenal chromaffin cells and insulin

rom pancreatic �-cells [1].Chronic nicotine administration increases adrenergic binding

ites in several brain regions [73]. Agmatine and �2-adrenergiceceptors have important functional interactions in behavioralffects of psychoactive agents including potentiation of mor-hine induced analgesia [12,75] and conditioned place preference63], attenuation of morphine withdrawal symptoms and sev-ral aspects of drug addiction [61,75]. Moreover clonidine, an2-adrenoceptor agonist, is clinically used for the treatment oficotine addiction and relapse [6,14]. While much has been stud-

ed on the modulatory influence of agmatine on morphine effects,ts regulatory role in behavioral effects of nicotine including sen-itization and addiction is poorly understood. In present study,e have investigated the effects of intracerebroventricularly (icv)

njected agmatine or the agents augmenting the brain agmatineontent on nicotine induced motor sensitization. Increasing biosyn-hesis of endogenous agmatine and blocking its degradation arehe approaches to elevate agmatine levels in brain. Biosynthesisf agmatine by l-ADC depends upon the availability of l-arginine60]. l-Arginine is also converted into ornithine by arginase ando NO by an enzyme NOS. Ornithine subsequently turned intoutrescine by l-ornithine decarboxylase (l-ODC) [48]. DFMO (�-ifluoromethyl-ornithine), is an inhibitor of arginase [56] and alsotimulator of enzyme l-ADC [17]. Its function as arginase inhibitors well as stimulator of l-ADC would increase the availability ofgmatine in brain [32]. Agmatine is degraded to putrescine anduanido-butanoic acid by enzyme agmatinase and diamine oxidaseDAO), respectively [48] and inhibition of these enzymes resultedn augmentation of endogenous agmatine [46]. In present study wesed DAO inhibitor, aminoguanidine [31] and agmatinase inhibitor,rcaine [18,46] to block the agmatine metabolic pathways lead-ng to increase brain agmatine levels [58]. We further assessedhe involvement of �2-adrenoceptors in agmatine effects usinglonidine, �2-adrenoceptor agonist and yohimbine and idazoxan,2-adrenoceptor antagonists.

. Materials and methods

.1. Subjects

Swiss albino male mice weighing 20–25 g were group housed (five per cage)n a temperature and light (12:12 h light:dark cycle, lights on 07.00 h) controlledoom. Food and water were available ad libitum. Animals were allowed for 48 ho acclimatize to the laboratory environment before experiments. All testing werexecuted in accordance with the guidelines for the care and use of laboratory animalsy Committee for the Purpose of Control and Supervision of Experiments on AnimalsCPCSEA) and were approved by Institutional Animal Ethical Committee. All thebservations were made during 09.00–14.00 h to avoid circadian variations. All miceere experimentally naïve.

.2. Drugs

Agmatine sulfate, nicotine hydrogen tartarate, aminoguanidine hemisulfate, DL-FMO, arcaine sulfate, clonidine, yohimbine hydrochloride, idazoxan hydrochloridend l-arginine monohydrochloride were purchased from Sigma–Aldrich, Co., USA.icotine hydrogen tartarate, yohimbine hydrochloride and idazoxan hydrochlo-

ide were dissolved in isotonic saline solution. Nicotine was administered byubcutaneous (sc) route whereas yohimbine and idazoxan were administered byntraperitoneal (ip) route. All other drugs were dissolved in artificial cerebrospinaluid (aCSF) of the following composition (140 mM NaCl, 3.35 mM KCl, 1.15 mMgCl2, 1.26 mM CaCl2, 1.2 mM Na2HPO4, 0.3 mM NaH2PO4, pH 7.4) and administered

ia icv route.

Research 213 (2010) 161–174

2.3. Surgery

Under pentobarbital sodium (60 mg/kg, ip) anesthesia mice were placed in astereotaxic frame (David Kopf, CA, USA). A guide cannula (C315 G/Spc, Plastic OneInc., Virginia, USA) was implanted bilaterally into the third ventricle (0.8 mm pos-terior, 1.3 mm lateral to midline and 3.5 ventral to the bregma) according to themouse brain atlas [42]. A 28-gauge stainless steel dummy cannula was inserted toocclude the guide cannula when not in use. After surgery each mouse was injectedwith oxytetracycline injection (25 mg/kg, im, Pfizer Ltd., Chennai) and Neosporinointment (Burroughs Wellcome Ltd., Mumbai) was applied to avoid infection. Ani-mals were then placed individually in home cage and allowed to recover for 7 days.During this period animals were habituated to the testing environment by transfer-ring them to experimental room and handling daily to treatment schedule. The icvinjections were given via 33 gauge internal cannula (internal diameter 0.18 mm andouter diameter 0.20 mm) (C315 I/Spc), which was attached to a Hamilton microlitersyringe (Hamilton, Nevada, USA) via polyethylene tubing (PE-10) (internal diam-eter 0.28 mm; outer diameter 0.61 mm), that extended 0.5 mm beyond the guidecannula. The internal cannula was held in position for another 1 min before beingslowly withdrawn to prevent backflow and promote diffusion of drug.

After all sensitization testing, dilute India ink was injected (icv) and subjectswere sacrificed under an overdose of sodium pentobarbital anesthesia (120 mg/kg,ip). Brains were removed and cryostat cut into 50-�m sections, mounted and viewedusing light microscopy to verify cannulae placements. The data of animals withcannula placement of more than 0.5 mm away from coordinates were excluded fromthe study (<15%) and data from mice with uniform ink distribution into ventricleswere used for statistical analysis.

2.4. Measurement of locomotor activity

Locomotor activity was measured using actophotometer(20 cm × 20 cm × 10 cm) (Techno, India) equipped with six infrared photosensors, 2.5 cm apart from each other. Mice were habituated to the actophotometerchamber for 30 min before any testing. Baseline locomotor activity of each mousewas recorded for 20 min as a total count of ambulatory, horizontal and verticalactivity.

2.5. Effect of agmatine and its modulators on nicotine sensitization

The procedure outlined by Shim et al. [57] was adapted to sensitize mice tonicotine. The protocols were designed to examine the effects of exogenously admin-istered agmatine or drugs which alter endogenous agmatine concentration in brainon nicotine sensitization. To investigate the effects on the development phase eitheragmatine (40, 80 �g/mouse, icv); DAO inhibitor, aminoguanidine (25 �g/mouse,icv); arginase inhibitor, DFMO (50 �g/mouse, icv); agmatinase inhibitor, arcaine(50 �g/mouse, icv); precursor for agmatine, l-arginine (80 �g/mouse, icv) or aCSF(2 �l/mouse, icv) were administered to the separate group of animals (n = 9) 30 minbefore the daily first dose of nicotine hydrogen tartarate (0.4 mg/kg as free base) orsaline (1 ml/kg, sc) during 7 days of development phase. Drugs were not injectedon days 8, 9 and 10 of experiment. On day 11, all mice received a challenge dose ofnicotine (0.4 mg/kg, sc) or saline (1 ml/kg, sc) and locomotor activity was recordedas mentioned above once daily at 09.00 a.m. for 20 min immediately after every firstinjection of nicotine or saline through days 1–7 and on day 11.

In another set of experiments (n = 9), following repeated injections of nicotine(0.4 mg/kg, sc, twice daily) for 7 consecutive days mice were treated with agmatineor its modulators (as mentioned above) on days 8, 9, 10 of nicotine free period, i.e.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-vidual mice was measured immediately for 20 min.

2.6. Effect of ˛2-adrenoceptor agonist and antagonists on nicotine sensitization

To investigate the effect of �2-adrenoceptor agonist clonidine (0.1,0.2 �g/mouse, icv) or aCSF (2 �l/mouse, icv) alone or its subeffective dose(0.1 �g/mouse, icv) combination with agmatine (20 �g/mouse, icv) were eitheradministered (n = 9) during development of sensitization (days 1–7) or duringnicotine free period (days 8, 9, 10). Treatment protocols were also designed (n = 9)to assess the effects of �2-adrenoceptor antagonists, yohimbine or idazoxan onagmatine induced inhibition of nicotine sensitization. Mice (n = 9) were treatedwith yohimbine (5 mg/kg, ip), idazoxan (0.4 mg/kg, ip) or saline (1 ml/kg, ip) oncedaily 15 min before agmatine (40 �g/mouse, icv) followed by daily injection ofnicotine (0.4 mg/kg, sc) during the development phase of nicotine sensitization orduring nicotine free period. Appropriate control groups (n = 9) were maintained inall the cases. Locomotor counts were monitored for all groups daily for 20 min asper the schedule on day 1 through 7 and on day 11 after nicotine challenge.

2.7. Statistical analysis

Acute effect (day 1) of nicotine on locomotion were analyzed by Unpaired ‘t’-test. Data obtained from development phase (days 1–7) treatment was analyzed byTwo-way analysis of variance (ANOVA) with repeated measures on time followed

Page 3: Agm - Nic Sensitisation

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 (40and 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 locomotorcounts ± 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 byBonferroni 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 (40and 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).

Page 4: Agm - Nic Sensitisation

164 N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174

F n. Micf g/moo AL (SA1

bthmow

3

3

lobsPficFFaattbf(edot

(d

ig. 3. Effects of agmatine (AGM) on nicotine (NIC) induced behavioral sensitizatioor 7 consecutive days and injected with aCSF (NIC/aCSF–NIC) or AGM (40 and 80 �n day 11. The normal group was pretreated with aCSF and challenged with only S–7) (One-way ANOVA followed by Newman–Keuls test).

y post hoc Bonferroni multiple comparison test. Data of 11th day nicotine sensi-ization was analyzed by One-way analysis of variance (ANOVA) followed by postoc Dunnett’s/Newman–Keuls test. Data obtained on day 11, following the treat-ent during withdrawal period (days 8, 9, 10), was analyzed by One-way analysis

f variance (ANOVA) followed by post hoc Dunnett’s/Newman–Keuls test. P ≤ 0.05as considered to be statistically significant.

. Results

.1. Agmatine inhibits nicotine sensitization

Effects of agmatine on the development of nicotine inducedocomotor sensitization are shown in Fig. 1. Acute administrationf the first dose of nicotine (0.4 mg/kg, sc) on day 1 modestlyut significantly increased the locomotion by 45% compared toaline control (aCSF–SAL) group [Unpaired t-test; t = 2.18, df = 12,< 0.05]. On the other hand, repeated nicotine injections twice daily

or 7 consecutive days resulted in a progressive and significantncrease in locomotor response through the entire treatment periodonsistent with sensitization development [Two-way ANOVA –Treatment (1, 72) = 381.87, P < 0.001, FTime (6, 72) = 19.37, P < 0.001,Treatment × Time (6, 72) = 13.26, P < 0.001]. On 7th day the hyper-ctivity in response to nicotine injection was increased by 400%bove the level observed on day 1 [day 7 vs. day 1, Unpaired-test; t = 10.85, df = 12, P < 0.001]. However, repeated saline injec-ions for 7 days had no effect on locomotor activity. As cane seen in Fig. 2, similar to those for mice received nicotineor 7 days, a injection of challenge dose of nicotine on day 11aCSF/NIC–NIC) following nicotine free period (days 8–10) alsoxhibited greater locomotor activity than mice with saline onay 11 (aCSF/NIC–SAL) [Unpaired t-test; t = 5.67, df = 12, P < 0.001]

r from saline–saline (aCSF/SAL–SAL) control [Unpaired t-test;= 15.39, df = 12, P < 0.001].

As shown in Fig. 1, coadministration of agmatine40–80 �g/mouse, icv) with nicotine (0.4 mg/kg, sc) on day 1id not significantly change (AGM–NIC) the acute nicotine induced

e were pretreated with saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) twice dailyuse, icv) during a 3-day withdrawal phase and challenged with NIC (0.4 mg/kg, sc)L/aCSF–SAL) (n = 7). #P < 0.001 vs. SAL/aCSF–SAL. *P < 0.001 vs. NIC/aCSF–NIC (days

(AGM–NIC) hyperlocomotor response as compared to Acsf–NIC[One-way ANOVA post hoc Dunnett’s analysis; F(2, 20) = 0.06,P > 0.05]. Acute injection of agmatine (40–80 �g/mouse, icv) alone(day 1) to saline treated groups (AGM–SAL) did not change animal’sbasal 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-tine before daily first dose (0.4 mg/kg, sc) of nicotine (AGM–NIC)for 7 consecutive days significantly reduced the magnitude of loco-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].

Administration of agmatine (40–80 �g/mouse) to the micebefore nicotine for 7 days significantly attenuated the hyperloco-motor response to nicotine challenge on day 11 [One-way ANOVA,F(5, 41) = 52.63, P < 0.001] after 3 days extinction period comparedto control (aCSF/NIC–NIC). Post hoc Newman–Keuls comparisonshowed that both 40 �g (P < 0.001) as well as 80 �g (P < 0.001) dailytreatments significantly lowered locomotor activity (Fig. 2).

Administration of agmatine alone (AGM/SAL–SAL) withoutnicotine treatment for 7 days did not produce any significantchange in the activity counts compared to (aCSF/SAL–SAL) treatedgroup [FTreatment (2, 108) = 0.83, P = 0.45; FTime (6, 108) = 1.27,P = 0.27 and FTreatment × Time (12, 108) = 0.85, P = 0.59].

Further giving agmatine only during nicotine free period (days8, 9, 10) significantly attenuated (Fig. 3) the locomotor response on11th day to nicotine challenge [F(2, 20) = 11.42, P < 0.001] as com-pared to mice received nicotine (days 1–7) and aCSF (days 8, 9, 10)indicating blockade of consolidation or incubation of sensitization.

3.2. l-Arginine, DFMO, arcaine and aminoguanidine attenuatesnicotine sensitization

As shown in Fig. 4, acute icv injection of l-arginine(80 �g/mouse, l-arginine–NIC) or DFMO (50 �g/mouse,DFMO–NIC) (Fig. 4A) or arcaine (50 �g/mouse, arcaine–NIC)or aminoguanidine (25 �g/mouse, AMG–NIC) (B) on day 1,

Page 5: Agm - Nic Sensitisation

N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161–174 165

F tivityo mouseo t repr* rroni

3naDs

ig. 4. Effects of brain agmatine modulators on nicotine (NIC) induced locomotor acr l-arginine (80 �g/mouse, icv) or DFMO (50 �g/mouse, icv) (A) or arcaine (50 �g/f saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) for 7 consecutive days. Each poin*P < 0.01, ***P < 0.001 vs. aCSF–NIC (days 1–7) (Two-way ANOVA followed by Bonfe

0 min prior to the first nicotine injection (0.4 mg/kg, sc) didot significantly change acute locomotor response to nicotines compared to aCSF–NIC groups [One-way ANOVA post hocunnett’s analysis; F(4, 34) = 2.12, P > 0.05]. However, these curves

tart from lower baseline indicating their tendency towards

during 7-day development phase. Mice were pretreated with aCSF (2 �l/mouse, icv), icv) or aminoguanidine (25 �g/mouse, icv) (B) 30 min before first daily injections

esents the mean locomotor counts ± SEM (n = 7). #P < 0.001 vs. aCSF–SAL. *P < 0.05,multiple comparison test).

blockade. Two-way ANOVA revealed that daily treatment ofl-arginine (80 �g, l-arginine–NIC) or DFMO (50 �g, DFMO–NIC)during 7 days development phase significantly reduced nicotineinduced locomotor activity when compared against control group(aCSF–NIC) [l-arginine: FTreatment (3, 144) = 170.84, P < 0.001; FTime

Page 6: Agm - Nic Sensitisation

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) orl-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 themean 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 < 0.001vs. NIC/aCSF–NIC (days 1–7) (One-way ANOVA followed by Newman–Keuls test).

Page 7: Agm - Nic Sensitisation

l Brain

(P(Pad(Fa(F

(daa

cwlPoao

a(lp4ci((

3cs

mdpnO0negFP

(toFatdw

t(t(

N.R. Kotagale et al. / Behavioura

6, 144) = 23.70, P < 0.001 and FTreatment × Time (18, 144) = 6.34,< 0.001; DFMO: FTreatment (3, 144) = 99.44, P < 0.001; FTime

6, 144) = 22.84, P < 0.001 and FTreatment × Time (18, 144) = 8.78,< 0.001]. The injections of arcaine (50 �g/mouse, icv, arcaine–NIC)nd aminoguanidine (25 �g/mouse, icv, aminoguanidine–NIC)uring 7 days development phase of locomotor sensitizationFig. 4B) exhibited significant effect on locomotor activity [arcaine:Treatment (3, 144) = 130.31, P < 0.001; FTime (6, 144) = 28.13, P < 0.001nd FTreatment × Time (18, 144) = 6.63, 1 P < 0.001; DFMO: FTreatment3, 144) = 120.46, P < 0.001; FTime (6, 144) = 28.14, P < 0.001 andTreatment × Time (18, 144) = 10.53, P < 0.001].

Administration of l-arginine (80 �g, l-arginine–SAL) or DFMO50 �g, DFMO–SAL) or arcaine (50 �g, arcaine–SAL) or aminoguani-ine (25 �g, AMG–SAL) with saline for 7 days did not produceny significant change in the activity counts when compared withCSF–SAL treated group.

As depicted in Fig. 5, treatment of these modulators for 7onsecutive days during development phase followed by 3-dayithdrawal also significantly blocked sensitization to nicotine chal-

enge on 11th day [One-way ANOVA – F(9, 69) = 49.85, P < 0.001].ost hoc Dunnett’s comparison demonstrated the significant effectf l-arginine (80 �g) (P < 0.001) or DFMO (50 �g) (P < 0.001) orrcaine (50 �g) (P < 0.001) or aminoguanidine (25 �g) (P < 0.001)n the locomotor stimulation to nicotine challenge on day 11.

Furthermore as shown in Fig. 6 injections of the agents thatlters the brain agmatine content during 3-day nicotine free perioddays 8, 9, 10) after 7-day induction phase, significantly inhibitedocomotor sensitization to nicotine challenge on day 11 as com-ared to its control group (NIC/aCSF–NIC) [One-way ANOVA – F(5,1) = 24.96, P < 0.001]. Post hoc analysis of the mean locomotorounts showed the significant attenuation of the locomotor activ-ty on 11th day by l-arginine (80 �g) (P < 0.01) or DFMO (50 �g)P < 0.001) or arcaine (50 �g) (P < 0.01) or aminoguanidine (25 �g)P < 0.001).

.3. Effect of ˛2-adrenoceptor agonist, clonidine and itsombination with agmatine on nicotine induced behavioralensitization

Fig. 7A and B shows locomotor activity level for each treat-ent group at day 1 (acute effect) and through 7 days sensitization

evelopment. Acute injections of clonidine (0.2 �g, icv) on day 1,rior (30 min) to nicotine injections did not significantly block theicotine induced acute motor stimulation [F(3, 27) = 3.35, P > 0.05].n other hand (A) pretreatment with clonidine (0.2 �g but not.1 �g) (clonidine–NIC) during the 7-day development phase sig-ificantly blocked development of nicotine sensitization throughntire 7 days period when compared against its control (aCSF–NIC)roup [Two-way ANOVA – FTreatment (5, 216) = 100.11, P < 0.001;Time (6, 216) = 48.19, P < 0.001 and FTreatment × Time (30, 216) = 8.11,< 0.001].

As depicted in Fig. 7B, per se subeffective dose of clonidine0.1 �g, icv) in combination with subeffective dose of agma-ine (20 �g, icv) exhibited synergistic inhibition of developmentf nicotine induced locomotor sensitization [Two-way ANOVA –Treatment (5, 216) = 74.42, P < 0.001; FTime (6, 216) = 30.93, P < 0.001nd FTreatment × Time (30, 216) = 6.33, P < 0.001]. However, adminis-ration of clonidine (0.1–0.2 �g, clonidine–SAL) with saline for 7ays did not produce any significant effect on the motor activityhen compared with aCSF–SAL treated group.

Daily treatment of clonidine (0.2 �g/mouse) (P < 0.001) orhe combination of clonidine (0.1 �g/mouse) and agmatine20 �g/mouse) (P < 0.001) from day 1 to day 7 significantly blockedhe hyperlocomotor response to nicotine challenge on day 11Fig. 8) [F(9, 69) = 46.15, P < 0.001] (Post hoc Newman–Keuls test).

Research 213 (2010) 161–174 167

The effect of clonidine (0.1, 0.2 �g/mouse) and the combinationtreatment of low doses (non-effective if given alone with aCSF–SAL)of clonidine (0.1 �g/mouse) and agmatine (20 �g/mouse) dur-ing withdrawal phase on the locomotor sensitization is depictedin Fig. 9. Clonidine (0.2 �g/mouse, icv) and combined treat-ment of agmatine (20 �g) and clonidine (0.1 �g) during 3-daywithdrawal periods (days 8, 9 and 10) after the 7 days induc-tion phase inhibited nicotine sensitization on 11th day whencompared against control group (NIC/aCSF–NIC) [F(5, 41) = 36.98,P < 0.001]. Post Newman–Keuls comparisons indicated that cloni-dine (0.2 �g – 0.001 vs. NIC/aCSF–NIC) and the combined agmatine(20 �g) and clonidine (0.1 �g) treatment during nicotine freeperiod on days 8, 9 and 10 also significantly attenuated thenicotine sensitization on 11th day when compared against aCSF(NIC/aCSF–NIC) (P < 0.001), agmatine (NIC/AGM–NIC) (P < 0.001) orclonidine (NIC/clonidine–NIC) (P < 0.001) treated group.

3.4. ˛2-Adrenoceptor antagonists reversed the inhibitory effect ofagmatine on nicotine sensitization

Two-way ANOVA revealed that daily treatment of yohimbine(5 mg/kg, ip) (Fig. 10A) or idazoxan (0.4 mg/kg, ip) (Fig. 10B)before agmatine (40 �g/mouse) injections during 7 day devel-opment period of nicotine sensitization significantly reversedthe inhibitory effect of agmatine on nicotine induced locomo-tor sensitization [yohimbine: FTreatment (3, 144) = 19.90, P < 0.001;FTime (6, 144) = 82.63, P < 0.001 and FTreatment × Time (18, 144) = 2.67,P = 0.0006; idazoxan: FTreatment (3, 144) = 44.27, P < 0.001; FTime(6, 144) = 77.49, P < 0.001 and FTreatment × Time (18, 144) = 2.24,P = 0.0045]. This treatment protocol of 7 days followed by 3-daywithdrawal period after which challenged with nicotine on 11thday also showed reversal of agmatine effect on nicotine hyper-locomotion (Fig. 11) [One-way ANOVA, yohimbine: F(7, 55) = 101.7,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 (days8, 9 and 10) after 7 days induction phase exerted an obviousreversal of agmatine effect on nicotine induced behavioral sensiti-zation as tested on day 11 when compared against control animalsthat received nicotine (days 1–7) and agmatine/saline (days 8, 9,10) (Fig. 12) [yohimbine: F(3, 34) = 53.32, P < 0.01; idazoxan: F(3,34) = 32.58, P < 0.001]. However, administration of yohimbine oridazoxan alone in the doses used here did not influence the nicotinesensitization.

4. Discussion

Results of the present study showed that acute injections ofnicotine (day 1) to mice stimulated locomotor activity and follow-ing its repeated daily injections for 7 consecutive days producedlocomotor sensitization. Nicotine induced behavioral sensitizationis consistent with the earlier reports [7,53,66]. In mice acute injec-tions of nicotine generally fails to stimulate locomotor activity.However the modest but significant acute activating effects (day 1)observed here are in concordance with recent reports [7,66]. Sev-eral lines of evidence indicated that nicotine stimulates dopaminerelease by directly acting on the nicotinic receptors located onthe mesolimbic dopaminergic neurons which lead to locomotorhyperactivity or hypoactivity depending on dose and animal strain[9,69]. Behavioral sensitization is thought to be produced by incre-

mental neuroadaptations of neural systems due to repeated useof abused drugs [69]. Nicotine has been shown to increase therelease of agmatine from adrenal chromaffin cells [47,62] whichis implicated in drug addiction [2,60] and several effects of psy-chostimulants [60,71]. The brain regions (amygdala, VTA and NAc)
Page 8: Agm - Nic Sensitisation

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 developmentphase. 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 doseof 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 themean 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 multiplecomparison test).

Page 9: Agm - Nic Sensitisation

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. Micewere 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-wayANOVA 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 pretreatedwith 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-daywithdrawal 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 followedby Newman–Keuls test).

Page 10: Agm - Nic Sensitisation

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 activityduring 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 locomotorcounts ± 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-wayANOVA followed by Bonferroni multiple comparison test).

Page 11: Agm - Nic Sensitisation

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( ohimb( c) durs (n = 7S

iibaa

aaAondit[aO[fiionn3tdiaTt

NIC) challenge on day 11. Mice were pretreated with saline (SAL) (1 ml/kg, ip) or y40 �g/mouse, icv) 30 min before injections of SAL (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) challenge on day 11. Each column represents the mean locomotor counts ± SEMAL + AGM/NIC–NIC (One-way ANOVA followed by Newman–Keuls test).

nvolved in drug addiction has extensive distribution of agmatine,ts transporters and enzymes participated in its degradation andiosynthesis [48]. Therefore we examined whether agmatine playscrucial role in locomotor response to the acute or chronic repeateddministration of nicotine in mice.

This study investigated the effect of exogenously (icv) injectedgmatine and the agents reported to increase endogenous braingmatine content on nicotine induced behavioral sensitization.dministration of agmatine precursor, l-arginine or inhibitionf metabolic pathways might result in augmentation of endoge-ous agmatine levels in brain. In the present study, we usediamine oxidase (DAO) inhibitor, aminoguanidine or agmatinase

nhibitor, arcaine and arginase inhibitor, DFMO to block the agma-ine metabolism leading to increased agmatine levels in brain18,31,46,56,60]. Agmatine is extensively metabolized peripher-lly in liver, kidney and has very short biological half life [15].n the other hand, its half life in spinal cord and brain is 12–18 h

49]. Therefore to avoid peripheral metabolism and to attain suf-cient concentration in brain, agmatine and its modulators were

njected by icv route daily during development phase (days 1–7)r during nicotine withdrawal phase (days 8–10). We found thateither agmatine nor its modulators displayed any effect on sponta-eous motor activity on day 1 (acute response) when administered0 min before first dose of nicotine. On the other hand, daily pre-reatments (days 1–7) with these drugs significantly attenuated the

evelopment of nicotine induced locomotor sensitization. Interest-

ngly none of these drugs by themselves at any dose level causedny change in basal locomotor activity in saline treated animals.hus their effects on behavioral sensitization can not be attributedo locomotor suppression or sedation. It is noteworthy that drugs

ine (5 mg/kg, ip) or idazoxan (0.4 mg/kg, ip) before aCSF (2 �l/mouse, icv) or AGMing 7-day development phase and tested with SAL (1 ml/kg, sc) or NIC (0.4 mg/kg,

). #P < 0.001 vs. SAL + aCSF/SAL–SAL, $P < 0.001 vs. SAL + aCSF/NIC–NIC. *P < 0.001 vs.

employed in the present investigation in addition to their effecton agmatine [64] also possess other activities like NMDA antag-onism (arcaine), NOS inhibition (aminoguanidine) as well as NOprecursor (l-arginine) activity. Nonetheless, the involvement ofimidazoline receptors and other biological targets of agmatinelike NMDA and NOS cannot be ruled out and warrant furtherinvestigation. Aminoguanidine, a DAO inhibitor [31] that inhibitsdegradation of agmatine to guanidine–butanoic acid, is also a selec-tive inducible NOS inhibitor [57]. Based on these findings it seemsplausible that rather than acute dose of agmatine, its daily pretreat-ment for chronic period may be required to block nicotine effect.Thus although premature, it can be anticipated that a reductionin agmatinergic tone in NAc shell may contribute to behavioralsensitization to nicotine. However further behavioral studies usingimmunoneutralization or selective drugs that reduce the agmatineformation may help to clarify its role in nicotine sensitization.

Our findings that agmatine did not block acute nicotine effectsin mice are consistent with the results of Zaniewska et al. [76] inrats. This is the only study that investigated the effect of agmatine(ip) on nicotine evoked behavioral responses in rats. Their pharma-cological analysis indicated lack of effect of agmatine on locomotor,sensitizing or subjective effects but displayed inhibition of condi-tioned hyperlocomotion induced by nicotine. In contrast, our datashows blockade of development of nicotine sensitization. One pos-sible explanation for these discrepancies could be due to different

doses, route, treatment schedule and animal strain. Additional evi-dence supporting our data is that agmatine attenuates ethanolas well as morphine withdrawal [3,27,67], decreases cocaine andfentanyl self-administration [36] and expression of morphine sen-sitization [71] in experimental animals.
Page 12: Agm - Nic Sensitisation

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 behaviorals , sc) t( /moud SAL/a# IC/SAL

irtoiattacitgsaitalrarosar

mbal

ensitization. Mice were pretreated with saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg5 mg/kg, ip) or idazoxan (0.4 mg/kg, ip) before aCSF (2 �l/mouse, icv) or AGM (40 �gay 11. The normal group was pretreated with aCSF and challenged with only SAL (P < 0.001 vs. SAL/SAL + aCSF–SAL, $P < 0.001 vs. NIC/SAL + aCSF–NIC, *P < 0.001 vs. N

Furthermore, in the present study icv injections of agmatine orts modulators during nicotine withdrawal period (days 8, 9, 10)esulted in a pronounced block in locomotor response to nico-ine challenge on day 11. This suggests that exogenous agmatiner increasing endogenous agmatine in brain by different meansnhibits the consolidation or incubation of sensitization. However,cute dose of agmatine (40–80 �g/mouse, icv) given before nico-ine challenge on day 11 (data not shown) does not counteracthe expression of nicotine sensitization. This agrees with previousgmatine studies on nicotine sensitization [76]. Although the psy-hoactive actions of nicotine are mediated centrally though directnteraction with nicotinic receptors (nAchRs) [9], some data existshat implies the role for the other neurotransmitter systems likelutamatergic and adrenergic systems [55,57]. Earlier studies haveuggested that NMDARs are key receptors in expression as wells development of behavioral sensitization and that NO is alsonvolved in development but not in expression of nicotine sensi-ization [57,66]. The diverse central effects of agmatine also aressociated with its ability to bind to �2-adrenoceptors and imidazo-ine binding sites, inhibition of NOS activity and blockade of NMDAeceptors or nicotinic receptors [3,13,30,35,44,74]. The fact thatgmatine acts on so many different biochemical processes, neu-otransmitters or receptors makes it difficult to determine whichf its many effects are critical to suppress nicotine induced sen-itization. Thus suppression of nicotine induced sensitization bygmatine could also be due to its ability to block NMDA or nicotiniceceptors or inhibition of enzyme NOS.

Several lines of experimental evidence indicated the involve-ent of noradrenergic receptors in psychostimulant induced

ehavioral effects including sensitization [16,59,70]. Systemicdministration of the �2-adrenergic receptor agonists clonidine,ofexidine and guanabenz attenuated stress-induced reinstatement

wice daily for 7 consecutive days and injected with SAL (1 ml/kg, ip) or yohimbinese, icv) during a 3-day withdrawal phase and challenged with NIC (0.4 mg/kg, sc) onCSF + aCSF–SAL). Each column represents the mean locomotor counts ± SEM (n = 7).

+ AGM–NIC (One-way ANOVA followed by Newman–Keuls test).

of cocaine-seeking behavior [11] and footshock-induced reinstate-ment of nicotine-seeking behavior in rats [79]. Moreover, chronicadministration of nicotine increases the density of �2-adrenergicbinding sites in some brain regions [73]. Interestingly, severalstudies have identified correlation between agmatinergic neuronsand �2-adrenergic receptors in many brain regions [24,37]. Likeclonidine, agmatine has been shown to bind �2-adrenoceptors[63,75,80]. Agmatine alters the firing rate of locus ceruleus neuronsin vivo [43,52] and induces �2-adrenoceptors dependant antinoci-ception [39]. The potentiating effect of agmatine on morphineinduced analgesia is mediated by �2-adrenoceptors [51,75]. Inview of these findings, we studied the possible involvement of �2-adrenergic receptors in agmatine induced attenuation of nicotinesensitization.

The present work showed that chronic administration of �2-adrenoceptors agonist, clonidine during development and nicotinefree period significantly blocked the nicotine induced increase inlocomotor counts. Moreover, clonidine also augmented the sup-pression of nicotine sensitization by agmatine in the doses thatwere ineffective when administered alone. Thus, our finding isin line with reports of agmatine/clonidine synergism [77] andstrengthens the notion that several biological as well as pharma-cological effects of agmatine are closely linked to �2-adrenergicreceptors activation [63,75,80]. In fact clonidine has been reportedto reduce morphine [8] cocaine [21] and d-amphetamine [68]induced sensitization. It may be recalled that, clonidine is clin-ically used for smoking cessation and attenuation of nicotine

withdrawal symptoms [6,14]. Involvement of �2-adrenergic recep-tors in attenuation of nicotine sensitization by agmatine is furthersupported by the fact that agmatine effect was completely abol-ished by selective �2-adrenergic receptor antagonists, yohimbineand idazoxan, a mixed antagonist of �2/imidazoline I2 recep-
Page 13: Agm - Nic Sensitisation

l Brain

trdiafadosrirataineataat

tvliaafii

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

N.R. Kotagale et al. / Behavioura

or. This is consistent with previous reports that �2-adrenergiceceptor antagonists attenuated several effects of agmatine inrug addiction [63,75]. Thus the results clearly indicate that the

nhibitory effects of agmatine on nicotine sensitization are medi-ted through �2-adrenoceptors. Alternatively the effects observedollowing administration of agmatine and clonidine could be medi-ted by acting on entirely different systems. It is difficult for anyecision on this matter unless explored whether ability of NMDAr nicotinic receptors antagonist to block nicotine induced sen-itization could be reversed by �2-adrenoceptor antagonists. Ouresults suggest that endogenous brain agmatine and its resultantnteraction with �2-adrenergic receptors may play an importantole in nicotine induced sensitization. Moreover, �2-adrenoceptorsgonist, clonidine decreases the dopamine overflow by psychos-imulants [20]. It is important to note that midbrain regions like VTAnd NAc involved in sensitization expresses abundant agmatinemmunoreactivity [41]. Agmatine also diminishes dopaminergiceurotransmission by reducing the levels of dopamine in VTAvoked by morphine withdrawal [71]. Thus, it is reasonable tossume that attenuation of nicotine induced locomotor sensi-ization by agmatine is associated with �2-adrenergic receptorctivation. However, it needs critical investigation whether �2-drenergic receptor activation and inhibition of dopaminergicransmission by agmatine are directly related.

In conclusion, attenuation of nicotine induced locomotor sensi-ization by repeated agmatine administration seems to be mediatedia its interaction with �2-adrenoceptors. However, whether pro-ong exposure to nicotine alters or lowers brain agmatine level ands subsequently responsible for nicotine sensitization is not cleart present. Although it is premature to conclude that endogenousgmatine regulates behavior in nicotine addiction, the findingsrom this study suggest that agmatine or the agents augment-ng endogenous agmatine levels may have therapeutic potentialn nicotine abuse.

eferences

[1] Alberti K, Wood H, Whalley M. Mechanism of action of the monoguani-dine hypoglycemic agents, galegine and agmatine. Eur J Clin Invest 1973;3:208.

[2] Aricioglu-Kartal F, Regunathan S. Effect of chronic morphine treatmenton the biosynthesis of agmatine in rat brain and other tissues. Life Sci2002;71:1695–701.

[3] Aricioglu-Kartal F, Uzbay IT. Inhibitory effect of agmatine on naloxoneprecipitated abstinence syndrome in morphine dependent rats. Life Sci1997;61:1775–81.

[4] Auguet M, Viossat I, Marin JG, Chabrier PE. Selective inhibition of induciblenitric oxide synthase by agmatine. Jpn J Pharmacol 1995;69:285–7.

[5] Bence AK, Worthen DR, Stables JP, Crooks PA. An in vivo evaluation of theantiseizure activity and acute neurotoxicity of agmatine. Behav Brain Res2003;74:771–5.

[6] Buchhalter AR, Fant RV, Henningfield JE. Novel pharmacological approachesfor treating tobacco dependence and withdrawal: current status. Drugs2008;68(8):1067–88.

[7] Celik E, Uzbay IT, Karakas S. Caffeine and amphetamine produce cross-sensitization to nicotine-induced locomotor activity in mice. Prog Neuropsy-chopharmacol Biol Psychiatry 2006;30(1):50–5.

[8] Chen SQ, Zhai HF, Cui YY, Shi J, Le Foll B, Lu L. Clonidine attenuates morphinewithdrawal and subsequent drug sensitization in rhesus monkeys. Acta Phar-macol Sin 2007;28(4):473–83.

[9] Clarke PB, Fu DS, Jakubovic A, Fibiger HC. Evidence that mesolimbic dopamin-ergic activation underlies the locomotor stimulant action of nicotine in rats. JPharmacol Exp Ther 1988;246(2):701–8.

10] Di Chiara G. Role of dopamine in the behavioural actions of nicotine related toaddiction. Eur J Pharmacol 2000;393:295–314.

11] Erb S, Hitchcott PK, Rajabi H, Mueller D, Shaham Y, Stewart J. Alpha-2 adren-ergic receptor agonists block stress-induced reinstatement of cocaine seeking.Neuropsychopharmacology 2000;23(2):138–50.

12] Fairbanks CA, Wilcox GL. Spinal antinociceptive synergism between morphineand clonidine persists in mice made acutely or chronically tolerant to morphine.J Pharmacol Exp Ther 1999;288(3):1107–16.

13] Galea E, Regunathan S, Eliopoulos V, Feinstein DL, Reis DJ. Inhibition of mam-malian nitric oxide synthases by agmatine, an endogenous polyamine formedby decarboxylation of arginine. Biochem J 1996;316:247–9.

[

[

Research 213 (2010) 161–174 173

14] Gourlay S, Forbes A, Marriner T, Kutin J, McNeil J. A placebo-controlledstudy of three clonidine doses for smoking cessation. Clin Pharmacol Ther1994;55(1):64–9.

15] Halaris A, Piletz J. Agmatine: metabolic pathway and spectrum of activity inbrain. CNS Drugs 2007;21(11):885–900.

16] Harris GC, Hedaya MA, Pan WJ, Kalivas P. �-Adrenergic antagonism alters thebehavioral and neurochemical responses to cocaine. Neuropsychopharmacol-ogy 1996;14(3):195–204.

17] Hernandez S, Schwarcz de Tarlovsky S. Arginine decarboxylase in Trypanosomacruzi, characteristics and kinetic properties. Cell Mol Biol (Noisy-le-grand)1999;45:383–91.

18] Huang MJ, Regunathan S, Botta M, Lee K, McClendonYi GB, Pedersen ML, et al.Structure-activity analysis of guanidine group in agmatine for brain agmati-nase. Ann N Y Acad Sci 2003;1009:52–63.

19] Isome M, Lortie MJ, Murakami Y, Parisi E, Matsufuji S, Satriano J. The antiprolif-erative effects of agmatine correlate with the rate of cellular proliferation. AmJ Physiol Cell Physiol 2007;293(2):C705–711.

20] Jentsch JD, Sanchez D, Elsworth JD, Roth RH. Clonidine and guanfacine attenuatephencyclidine-induced dopamine overflow in rat prefrontal cortex: mediat-ing influence of the alpha-2A adrenoceptor subtype. Brain Res 2008;1246:41–6.

21] Jiménez-Rivera CA, Feliu-Mojer M, Vázquez-Torres R. Alpha-noradrenergicreceptors modulate the development and expression of cocaine sensitization.Ann N Y Acad Sci 2006;1074:390–402.

22] Kalivas PW, Stewart J. Dopamine transmission in the initiation and expres-sion of drug- and stress-induced sensitization of motor activity. Brain Res Rev1991;16:223–44.

23] Kalra SP, Pearson E, Sahu A, Kalra PS. Agmatine, a novel hypothalamicamine, stimulates pituitary luteinizing hormone release in vivo and hypotha-lamic luteinizing hormone-releasing hormone release in vitro. Neurosci Lett1995;194(3):165–8.

24] King PR, Gundlach AL, Louis WJ. Quantitative autoradiographic localization inrat brain of �2-adrenergic and non-adrenergic I-receptor binding sites labelledby [3H]rilmenidine. Brain Res 1995;675:264–78.

25] Lavinsky D, Arteni NS, Netto CA. Agmatine induces anxiolysis in the elevatedplus maze task in adult rats. Behav Brain Res 2003;141(1):19–24.

26] Li G, Regunathan S, Barrow CJ, Eshraghi J, Cooper R, Reis DJ. Agma-tine: an endogenous clonidine-displacing substance in the brain. Science1994;263:966–9.

27] Li J, Li X, Pei G, Qin BY. Correlation between inhibitions of morphine with-drawal and nitric-oxide synthase by agmatine. Zhongguo Yao Li Xue Bao1999;20(4):375–80.

28] Li YF, Gong ZH, Cao JB, Wang HL, Luo ZP, Li J. Antidepressant-like effect ofagmatine and its possible mechanism. Eur J Pharmacol 2003;469:81–8.

29] Liu P, Bergin DH. Differential effects of i.c.v. microinfusion of agma-tine on spatial working and reference memory in the rat. Neuroscience2009;159(3):951–61.

30] Loring RH. Agmatine acts as an antagonist of neuronal nicotinic receptors. Br JPharmacol 1990;99(1):207–11.

31] Lu G, Su RB, Li J, Qin BY. Modulation by �-difluoromethyl-ornithine andaminoguanidine of pain threshold, morphine analgesia and tolerance. Eur JPharmacol 2003;478:139–44.

32] McGehee DS, Role LW. Physiological diversity of nicotinic acetylcholine recep-tors expressed by vertebrate neurons. Annu Rev Physiol 1995;57:521–46.

33] Miller DK, Wilkins LH, Bardo MT, Crooks PA, Dwoskin LP. Once weekly admin-istration of nicotine produces long-lasting locomotor sensitization in ratsvia a nicotinic receptor-mediated mechanism. Psychopharmacology (Berl)2001;156(4):469–76.

34] Miller DK, Harrod SB, Green TA, Wong MY, Bardo MT, Dwoskin LP. Lobelineattenuates locomotor stimulation induced by repeated nicotine administrationin rats. Pharmacol Biochem Behav 2003;74(2):279–86.

35] Molderings GJ, Menzel S, Kathmann M, Schlicker E, Göthert M. Dual interac-tion of agmatine with the rat �2D-adrenoceptor: competitive antagonism andallosteric activation. Br J Pharmacol 2000;130:1706–12.

36] Morgan AD, Campbell UC, Fons RD, Carroll ME. Effects of agmatine on the escala-tion of intravenous cocaine and fentanyl self-administration in rats. PharmacolBiochem Behav 2002;72:873–80.

37] Nicholas AP, Pieribone T, Hokfelt VA. Distributions of mRNAs for alpha 2 adren-ergic receptor subtypes in rat brain: an in situ hybridization study. J CompNeurol 1993;328:575–94.

38] Olmos G, DeGregorio-Rocasolano N, Paz Regalado M, Gasull T, AssumpcióBoronat M, Trullas R, et al. Protection by imidazol(ine) drugs and agmatine ofglutamate-induced neurotoxicity in cultured cerebellar granule cells throughblockade of NMDA receptor. Br J Pharmacol 1999;127(6):1317–26.

39] Onal A, Soykan N. Agmatine produces antinociception in tonic pain in mice.Pharmacol Biochem Behav 2001;69(1–2):93–7.

40] Onal A, Delen Y, Ulker S, Soykan N. Agmatine attenuates neuropathic painin rats: possible mediation of nitric oxide and noradrenergic activity in thebrainstem and cerebellum. Life Sci 2004;73:413–28.

41] Otake K, Ruggiero DA, Regunathan S, Wang H, Milner TA, Reis DJ. Regional

localization of agmatine in the rat brain: an immunocytochemical study. BrainRes 1998;787(1):1–14.

42] Paxinos G, Franklin RBJ. The mouse brain in stereotaxic coordinates. San Diego:Academic Press; 1997.

43] Pineda J, Ruiz-Ortega JA, Martín-Ruiz R, Ugedo L. Agmatine does not haveactivity at alpha 2-adrenoceptors which modulate the firing rate of locus

Page 14: Agm - Nic Sensitisation

1 l Brain

[

[

[

[

[

[

[[

[

[

[

[

[

[

[

[

[

[

[[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

74 N.R. Kotagale et al. / Behavioura

coeruleus neurones: an electrophysiological study in rat. Neurosci Lett1996;219(2):103–6.

44] Raasch W, Schafer U, Chun J, Dominiak P. Biological significance of agma-tine, an endogenous ligand at imidazoline binding sites. Br J Pharmacol2001;133:755–80.

45] Regunathan S, Reis DJ. Characterization of arginine decarboxylase in ratbrain and liver: distinction from ornithine decarboxylase. J Neurochem2000;74(5):2201–8.

46] Regunathan S. Agmatine: biological role and therapeutic potential in morphineanalgesia and dependence. AAPS J 2006;8(5):E479–84.

47] Reis DJ, Regunathan S. Agmatine: an endogenous ligand at imidazolinereceptors may be a novel neurotransmitter in brain. J Auton Nerv Syst1998;72(2–3):80–5.

48] Reis DJ, Regunathan S. Is agmatine a novel neurotransmitter in brain? TrendsPharmacol Sci 2000;21:187–93.

49] Roberts JC, Grocholski BM, Kitto KF, Fairbanks CA. Pharmacodynamic and phar-macokinetic studies of agmatine after spinal administration in the mouse. JPharmacol Exp Ther 2005;314:1226–33.

50] Robinson TE, Berridge KC. Addiction. Annu Rev Psychol 2003;54:25–53.51] Roerig SC. Spinal and supraspinal agmatine activate different recep-

tors to enhance spinal morphine antinociception. Ann N Y Acad Sci2003;1009:116–26.

52] Ruiz-Durántez E, Ruiz-Ortega JA, Pineda J, Ugedo L. Effect of agmatine on locuscoeruleus neuron activity: possible involvement of nitric oxide. Br J Pharmacol2002;135(5):1152–8.

53] Sahraei H, Aliabadi AA, Zarrindast MR, Ghoshooni H, Nasiri A, Barzegari-Sorkheh AA, et al. Ascorbic acid antagonizes nicotine-induced place preferenceand behavioral sensitization in mice. Eur J Pharmacol 2007;560(1):42–8.

54] Satriano J, Schwartz D, Ishizuka S, Lortie MJ, Thomson SC, Gabbai F, et al. Sup-pression of inducible nitric oxide generation by agmatine aldehyde: beneficialeffects in sepsis. J Cell Physiol 2001;188(3):313–20.

55] Schoffelmeer ANM, De Vries TJ, Wardeh G, van de Ven HWM, VanderschurenLJMJ. Psychostimulant-induced behavioral sensitization depends on nicotinicreceptor activation. J Neurosci 2002;22(8):3269–76.

56] Selamnia M, Mayeur C, Robert V, Blachicr F. �-Difluoromethylornithine (DFMO)as a potent arginase activity inhibitor in human colon carcinoma cells. BiochemPharmacol 1998;55:1241–5.

57] Shim I, Kim HT, Kim YH, Chun BG, Hahm DH, Lee EH, et al. Role of nitric oxideinhibitors and NMDA receptor antagonist in nicotine-induced behavioural sen-sitization in the rats. Eur J Pharmacol 2002;443:119–24.

58] Slotkin TA, Seidler FJ, Trepanier PA, Whitmore WL, Lerea L, Barnes GA, et al.Ornithine decarboxylase and polyamines in tissues of the neonatal rat: effectsof alpha-difluoromethylornithine, a specific, irreversible inhibitor of ornithinedecarboxylase. J Pharmacol Exp Ther 1982;222:741–5.

59] Su RB, Li J, Gao K, Pei G, Qin BY. Influence of idazoxan on analgesia, tolerance,and physical dependence of morphine in mice and rats in vivo. Acta PharmacolSin 2000;21(11):1011–5.

60] Su RB, Li J, Qin BY. A biphasic opioid function modulator: agmatine. Acta Phar-macol Sin 2003;24:631–6.

61] Su RB, Wang WP, Lu XQ, Wu N, Liu ZM, Li J. Agmatine blocks acquisition andre-acquisition of intravenous morphine self-administration in rats. PharmacolBiochem Behav 2009;92(4):676–82.

62] Tabor CW, Tabor H. Polyamines. Ann Rev Biochem 1984;53:749–90.63] Tahsili-Fahadan P, Firouz-Abadi NY, Khoshnoodi MA, Langroudi RM, Tahaei SA,

Ghahremani MH, et al. Agmatine potentiates morphine-induced conditioned

[

Research 213 (2010) 161–174

place preference in mice: modulation by alpha(2)-adrenoceptors. Neuropsy-chopharmacol 2006;31:1722–32.

64] Taksande BG, Kotagale NR, Tripathi SJ, Ugale RR, Chopde CT. Antidepres-sants like effect of selective serotonin reuptake inhibitors involve modulationof imidazoline receptors by agmatine. Neuropharmacology 2009;57(4):415–24.

65] Tapper AR, McKinney SL, Nashmi R, Schwarz J, Deshpande P, Labarca C, etal. Nicotine activation of �4* receptors: sufficient for reward, tolerance, andsensitization. Science 2004;306:1029–32.

66] Ulusu U, Uzbay IT, Kayir H, Alici T, Karakas S. Evidence for the role of nitricoxide in nicotine-induced locomotor sensitization in mice. Psychopharmacol-ogy (Berl) 2005;178(4):500–4.

67] Uzbay IT, Yesilyurt O, Celik T, Ergün H, Isimer A. Effects of agmatine on ethanolwithdrawal syndrome in rats. Behav Brain Res 2000;107(1–2):153–9.

68] Vanderschuren LJ, Beemster P, Schoffelmeer AN. On the role of noradrenaline inpsychostimulant-induced psychomotor activity and sensitization. Psychophar-macology (Berl) 2003;169(2):176–85.

69] Vezina P, McGehee DS, Green WN. Exposure to nicotine and sensitizationof nicotine-induced behaviors. Prog Neuropsychopharmacol Biol Psychiatry2007;31(8):1625–38.

70] Villégier AS, Drouin C, Bizot JC, Marien M, Glowinski J, Colpaërt F, et al.Stimulation of postsynaptic alpha1b- and alpha2-adrenergic receptors ampli-fies dopamine-mediated locomotor activity in both rats and mice. Synapse2003;50(4):277–84.

71] Wei XL, Su RB, Wu N, Lu XQ, Zheng JQ, Li J. Agmatine inhibits morphine-inducedlocomotion sensitization and morphine-induced changes in striatal dopamineand metabolites in rats. Eur Neuropsychopharmacol 2007;17:790–9.

72] Weng XC, Gai XD, Zheng JQ, Li J. Agmatine blocked voltage-gated cal-cium channel in cultured rat hippocampal neurons. Acta Pharmacol Sin2003;24(8):746–50.

73] Yamanaka K, Oshita M, Muramatsu I. Alteration of alpha and muscarinic recep-tors in rat brain and heart following chronic nicotine treatment. Brain Res1985;348(2):241–8.

74] Yang XC, Reis DJ. Agmatine selectively blocks the N-methyl-d-aspartate sub-class of glutamate receptor channels in rat hippocampal neurons. J PharmacolExp Ther 1999;288:544–9.

75] Yesilyurt O, Uzbay IT. Agmatine potentiates the analgesic effect of morphineby an alpha(2)-adrenoceptor-mediated mechanism in mice. Neuropsychophar-macology 2001;25:98–103.

76] Zaniewska M, McCreary AC, Sezer G, Przegalinski E, Filip M. Effects of agma-tine on nicotine-evoked behavioral responses in rats. Pharmacol Reports2008;60:645–54.

77] Zeidan MP, Zomkowski ADE, Rosa AO, Rodrigues ALS, Gabilan NH. Evidence forimidazoline receptors involvement in the agmatine antidepressant-like effectin the forced swimming test. Eur J Pharmacol 2007;565:125–31.

78] Zheng JQ, Weng XC, Gai XD, Li J, Xiao WB. Mechanism underlying blockadeof voltage-gated calcium channels by agmatine in cultured rat hippocampalneurons. Acta Pharmacol Sin 2004;25:281–5.

79] Zislis G, Desai TV, Prado M, Shah HP, Bruijnzeel AW. Effects of the CRF recep-

tor antagonist D-Phe CRF(12-41) and the alpha2-adrenergic receptor agonistclonidine on stress-induced reinstatement of nicotine-seeking behavior in rats.Neuropharmacology 2007;53(8):958–66.

80] Zomkowski AD, Hammes L, Lin J, Calixto JB, Santos AR, Rodrigues AL. Agma-tine produces antidepressant-like effects in two models of depression in mice.Neuroreport 2002;13:387–91.