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Behavioural Brain Research 262 (2014) 118– 124

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Behavioural Brain Research

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Research report

Agmatine attenuates nicotine induced conditioned place preferencein mice through modulation of neuropeptide Y system

Nandkishor R. Kotagalea, Sonali Walkea, Gajanan P. Shelkarb, Dadasaheb M. Kokareb,Milind J. Umekara, Brijesh G. Taksandea,∗a Division of Neuroscience, Department of Pharmacology, Shrimati Kishoritai Bhoyar College of Pharmacy, New Kamptee, Nagpur 441002, Indiab Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur 440033, India

h i g h l i g h t s

• Agmatine attenuated the acquisition of nicotine-induced CPP.• NPY and [Leu31, Pro34]-NPY potentiated the inhibitory effect of agmatine.• BIBP3226 blocked the effect of agmatine on nicotine induced CPP.• Agmatine modified NPY-immunoreactive profile induced by nicotine.

a r t i c l e i n f o

Article history:Received 21 September 2013Received in revised form 6 January 2014Accepted 10 January 2014Available online 16 January 2014

Keywords:NicotineCPPAgmatineNeuropeptide Y

a b s t r a c t

The purpose of the present study was to examine the effect of agmatine on nicotine induced conditionedplace preference (CPP) in male albino mice. Intra-peritoneal (ip) administration of nicotine (1 mg/kg)significantly increased time spent in drug-paired compartment. Agmatine (20 and 40 mg/kg, ip) co-administered with nicotine during the 6 days conditioning sessions completely abolished the acquisitionof nicotine-induced CPP in mice. Concomitant administration of neuropeptide Y (NPY) (1 pg/mouse, icv)or [Leu31, Pro34]-NPY (0.1 pg/mouse, icv), selective NPY Y1 receptor agonist potentiated the inhibitoryeffect of agmatine (10 mg/kg, ip) on nicotine CPP. Conversely, pretreatment with NPY Y1 receptor antago-nist, BIBP3226 (0.01 ng/mouse, icv) blocked the effect of agmatine (20 mg/kg, ip) on nicotine induced CPP.In immunohistochemical study, nicotine decreased NPY-immunoreactivity in nucleus accumbens shell(AcbSh), bed nucleus of stria terminalis, lateral part (BNSTl), arcuate nucleus (ARC) and paraventricularnucleus (PVN). Conversely, administration of agmatine prior to the nicotine significantly reversed theeffect of nicotine on NPY-immunoreactivity in the above brain nuclei. This data indicate that agmatineattenuate nicotine induced CPP via modulation of NPYergic neurotransmission in brain.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Nicotine, a major psychoactive constituent of tobacco inducesconditioned place preference (CPP) and facilitates intracranialself-stimulation in experimental animals [5,14]. It mediates pos-itive reinforcing effects via the activation of central nicotinicacetylcholine receptors (nAChRs). Behavioral effects of nico-tine including addiction are regulated through its interactionswith multiple neurotransmitters receptor systems in differentbrain areas [10,47]. However, the molecular mechanism respon-sible for its rewarding effect and its dependence is still poorlyunderstood.

∗ Corresponding author. Tel.: +91 7109 288650; fax: +91 7109 287094.E-mail address: [email protected] (B.G. Taksande).

Neuropeptide Y (NPY), a 36 amino acids peptide, binds to fivereceptor subtypes (Y1, Y2, Y4, Y5 and y6) and plays a crucial role infeeding [7], depression [15], convulsion [24] and anxiety [6]. Fur-thermore, several studies have suggested a role of NPY in addictionto drugs of abuse, including nicotine. NPY is abundantly expressedin numerous brain areas involved in regulation of addictive processincluding ventral tegmental area (VTA) and nucleus accumbens(Acb) [11]. Number of studies has suggested the existence ofinteraction between nicotine and NPYergic systems. Nicotineadministration dose-dependently increased NPY mRNA levels andNPY immunoreactivity in hypothalamic nuclei and down regulatesNPY Y1 receptors [23]. Moreover, NPY and NPY Y1 receptor agonistattenuated abdominal constriction following nicotine withdrawal[33]. Recently, Nakhate et al. [27] demonstrated that, NPY in thehypothalamic arcuate nucleus (ARC) and paraventricular nuclei(PVN), plays an important role in the regulation of acute, chronic

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N.R. Kotagale et al. / Behavioural Brain Research 262 (2014) 118– 124 119

and withdrawal effects of nicotine mediated feeding behaviourin rats via NPY Y1 receptors. These evidences raise the possibilitythat endogenous NPY system may mediate the rewarding effect ofnicotine.

Agmatine, an endogenous amine formed by decarboxylationof L-arginine and proposed as neurotransmitter in brain [13,32].Agmatine has multireceptorial affinity including !2-adrenoceptors,imidazoline binding sites, N-methyl-D-aspartate (NMDA) recep-tors, nitric oxide synthase (NOS), nAChRs and other ligand gated ionchannels [42]. Agmatine is a pleiotropic molecule with many cen-tral and peripheral functions. Its systemic administration evokesanxiolytic, antidepressant, antinociceptive, anticonvulsive, anti-inflammatory, antiproliferative and neuroprotective properties,and facilitates working memory [20,21,40,41]. The role of agma-tine in processing of drug addiction is fairly well established. Itattenuates the symptoms of ethanol and morphine withdrawal[1,42], decreased morphine, cocaine or fentanyl self-administration[25,37] and blocks morphine-induced striatal dopamine changesin rats [43]. Agmatine inhibits the expression of nicotine inducedconditioned hyper locomotion without affecting its either acutelocomotor sensitizing or discriminative stimulating effects [45].Repeated administration of agmatine attenuated the developmentas well as expression of locomotor sensitization to nicotine [20].Recently, we have reported that agmatine significantly increasesNPY expression in hypothalamic nuclei [41] and some of its phar-macological effects are mediated through NPYergic system inbrain [21]. However, their mutual interaction, if any, in relationto nicotine addiction remains unexplored. In the present studywe tried to characterize the interaction between agmatine andNPY in nicotine induced CPP in mice. Furthermore, the effect ofagmatine on endogenous NPY-containing elements in the nucleusaccumbens shell (AcbSh), bed nucleus of stria terminalis, lateralpart (BNSTl), ARC and PVN was studied employing immunohisto-chemistry. These neuroanatomical areas contain an abundance ofagmatine and NPY and are reported to involve in the regulation ofrewarding effects of nicotine.

2. Materials and methods

2.1. Animals

Male albino mice (25 ± 2 g) housed (3 per cage) under controlledenvironmental condition at 24 ± 1 ◦C and 12:12 h light/dark cycle(lights on at 07:00 am) with ad libitum food and water (exceptduring experiments) were used. All experimental procedures wereapproved by the Institutional Animal Ethical Committee and werecarried out under the strict compliance with guidelines given byCommittee for the Purpose of Control and Supervision of Experi-ments on Animals (CPCSEA), Ministry of Environment and Forests,Government of India, New Delhi. All the experiments were con-ducted in the light phase.

2.2. Drugs

Nicotine hydrogen tartarate, agmatine sulphate, NPY, NPYY1 receptors agonist, [Leu31, Pro34]-NPY and NPY Y1 recep-tors antagonist, BIBP3226 {(N2-diphenylacetyl)-N-[(4-hydroxy-phenyl)-methyl]-D-arginine amide)} were purchased from Sigma-Aldrich Co. (Saint Louis, MO, USA). NPY, [Leu31, Pro34]-NPY andBIBP3226 were dissolved in artificial cerebrospinal fluid (aCSF:140 mM NaCl, 3.35 mM KCl, 1.15 mM MgCl2, 1.26 mM CaCl2, 1.2 mMNa2HPO4 and 0.3 mM NaH2PO4, pH 7.4) containing 0.1% bovineserum albumin (BSA) and injected by intracerebroventricular (icv)route. Agmatine as well as nicotine were administered by intraperi-toneal (ip) route in saline. The doses of the nicotine, agmatine, NPY,

[Leu31, Pro34]-NPY and BIBP3226 were selected on the basis of pre-liminary work carried out in our laboratory and available literature[2,20].

2.3. Place preference apparatus

Place preference studies were conducted using biased pro-cedure and three compartment CPP model, consisting of twoequal sized plexiglass compartments (A and B) (20 × 20 × 20 cm)separated by grey central area (8 × 20 × 20 cm) (C) connected tochamber A and B by guillotine door (7 × 10 cm) equipped withautomated time [seconds (s)] measurement sensor system (VJInstruments, Nagpur, India). Two compartments (A and B) weredistinguished using visual and sensory texture cues. Compartment‘A’ was painted in vertical black strip with smooth white colourfloor and the compartment ‘B’ was painted with horizontal blackstrip and rough textured with white colour floor.

2.4. Experimental design

CPP paradigm consisted of three phases, (1) habituation and pre-conditioning for 2 days, (2) conditioning for 6 days and (3) post-conditioning testing on day 9.

2.5. Habituation and pre-conditioning (2 days)

On the first day (habituation) and second day (pre-conditioning)of the trials, each mouse was separately placed into the central com-partment with guillotine doors open and allowed to move freelybetween both the compartments for 15 min. The time spent in eachcompartment during pre-conditioning (second day) was recorded.Placement of all the four paws was considered as entry in the com-partments. Animals showing strong unconditioned aversion (lessthan 33% of the session time) or preference (more than 67% of thesession time) for any compartment were discarded from the experi-ment. The compartment A/B in which animals spent more time wasconsidered as preferred compartment and compartment with lesstime was referred as non-preferred compartment for respectiveanimal. After pre-conditioning, animals were divided in differentgroups on random basis.

2.6. Conditioning phase (6 days)

Conditioning phase consisted of 12 sessions held in six consec-utive days (two sessions per day each of 45 min) during which eachmouse was confined to the compartment by isolating the compart-ment using guillotine door. During the first daily session (09:00am), half of the animals from each group received the drug and/ornicotine in non-preferred compartment and the other half receivedvehicle in the preferred compartment. Treatment and compart-ment were reversed during the second session conducted after 4 h.On the next day, these sessions were reversed i.e. procedural treat-ment of the second session of previous day was carried during firstsession of the next day. This alternating treatment protocol wascontinued for 6 days of the experiment.

2.7. Post-conditioning phase (9th day)

The 15 min flooring preference tests were conducted on the 9th

day (24 h after the last conditioning sessions without any drugtreatment) in a drug free state under the conditions identical topre-conditioning. The guillotine door was opened and each mousewas placed in the central compartment and allowed free access toboth the compartments. Time spent (s) in each compartment wasrecorded and the results were compared with pre-conditioning.The difference between the time spent in the drug paired

120 N.R. Kotagale et al. / Behavioural Brain Research 262 (2014) 118– 124

compartment during post- and pre-conditioning was consideredas change in preference score.

2.8. ICV cannulation

Mice were anesthetized with a mixture of ketamine (50 mg/kg;Ketmin® 50, Themis Medicare Ltd., India) and xylazine (10 mg/kg;Xylaxin®, Indian Immunologicals Ltd., India), given by ip routeand placed in a stereotaxic instrument (David Kopf Instruments,Tujunga, CA, USA). Under aseptic conditions, a guide cannula pre-pared in-house [19] was implanted into the right lateral ventricleusing stereotaxic coordinates, −0.8 mm posterior, −1.0 mm lateralto midline and −2.3 mm ventral with respect to bregma [28]. Guidecannulae were secured to the skull with stainless steel screws andfixed with rapidly polymerizing dental acrylic cement (DPI-RR ColdCure, acrylic powder, Dental Product of India, Mumbai, India). Fol-lowing hardening of acrylic cement, the animal was removed fromstereotaxic frame and a 30-gauge dummy cannula was insertedto occlude the guide cannula when not in use. After surgery, theanimals were placed individually in cages and allowed to recoverfor 7 days during which they were handled to condition for futureexperimental procedures. Animals were treated with oxytetracy-cline (25 mg/kg, intramuscular) and neosporin ointment to avoidany infection. The icv injections were given by 30-gauge internalcannula attached to a Hamilton microliter syringe via polyethylenetubing (PE-10) that extended 0.25 mm beyond the guide cannula.The internal cannula was held in position for another 1 min beforebeing slowly withdrawn to prevent back flow and promote the dif-fusion of drug. At the end of the experiment, diluted India ink wasadministered by same route and mice were euthanized by an over-dose of thiopentone sodium (70 mg/kg, ip). Data from only thoseanimals with uniform distribution of ink in the ventricles wereconsidered for statistical analysis.

2.9. Immunohistochemistry and morphometric analysis

The different groups of mice (n = 5) were deeply anesthetized(Thiopentone sodium, 70 mg/kg, ip) and trans-cardially perfusedwith heparin-containing phosphate-buffered saline (PBS) followedby 4% paraformaldehyde in 0.1 M phosphate buffer. The brains ofthese animals were removed and post-fixed in same fixative forovernight, cryoprotected in 30% sucrose solution, embedded inpolyvinylpyrrolidone (PVP) and serially sectioned in the coronalplane at 30 "m thickness using a cryostat (Leica, Germany) andcollected in PBS. The sections were processed for NPY immuno-labeling with NPY-antibody using streptavidin-biotin-peroxidasemethod as described earlier [41]. The immunoreactive (ir) area(intergrated density units) covered by NPY-ir cells as well as fiberswas measured from predetermined microscopic image in coronalsections passing through AcbSh, BNSTl, ARC and PVN of controland treated mice. The images were captured using Leica DM2500microscope (Germany) and morphometricaly analysed with ImageJsoftware. Five measurements were taken from predetermined fieldof all sub-regions on either side of each brain. The rectangular areademarcated in respective figures from which the NPY-ir cells aswell as fibers were evaluated. The data of each nucleus from allanimals in each group were pooled separately and the mean ± SEMwas calculated.

2.10. Experimental protocols

2.10.1. Effect of agmatine on nicotine induced CPPMice (n = 6) were injected with saline (0.25 ml/mouse, ip) or

nicotine (0.5, 0.75, 1 mg/kg, ip) during conditioning phase to exam-ine the effect on the time spent in the paired chamber in CPP

apparatus as per above mentioned procedure and to determine thedose of nicotine that induce preference.

Additional group (n = 6) of animals received the injectionsof agmatine (10, 20, 40 mg/kg, ip) or saline (0.25 ml/mouse, ip)30 min before the administration of nicotine (1 mg/kg, ip) or saline(0.25 ml/mouse, ip) prior to their placement in drug paired com-partment during conditioning phase under schedule describedabove.

2.10.2. Effect of NPY Y 1 receptor agonists and its combinationwith agmatine on nicotine induced CPP

In these experiments, different group of animals were injected(n = 5–7) with NPY (1 and 2 pg/mouse, icv), [Leu31, Pro34]-NPY (0.1and 0.2 pg/mouse, icv) or aCSF (5 "l/mouse, icv) during condition-ing phase prior to placement in CPP apparatus under the scheduledescribed earlier.

Separately, mice from different groups (n = 5−7) received NPY(1 pg/mouse, icv), NPY Y1 receptor agonist [Leu31, Pro34]-NPY(0.1 pg/mouse, icv) 15 min before administration of agmatine(10 mg/kg, ip) or saline (0.25 ml/mouse, ip) and injected withnicotine (1 mg/kg, ip) or saline (0.25 ml/mouse, ip) 30 min afteragmatine treatment during conditioning phase.

2.10.3. Influence of NPY Y1 receptor antagonist on the effect ofagmatine on nicotine induced CPP

In order to investigate the interaction of agmatine and NPYY1 receptors on nicotine induced CPP, separate groups of ani-mals (n = 5−7) received NPY Y1 receptor antagonist, BIBP3226(0.005 and 0.01 ng/mouse, icv) or aCSF (5 "l/mouse, icv) and agma-tine (20 mg/kg, ip) or saline (0.25 ml/mouse, ip) 30 min before theadministration of nicotine (1 mg/kg, ip) or saline (0.25 ml/mouse,ip) and placed in treatment paired compartment during condition-ing phase as described earlier.

2.11. Statistical analysis

The change in preference was calculated as the differencebetween time spent in the treatment paired compartment duringpost- and pre-conditioning. Results were presented as mean changein preference (s) ± SEM. Data were analyzed by one-way analysis ofvariance (ANOVA) followed by post hoc Dunnett’s or Bonferroni’smultiple comparison tests. P < 0.05 was considered as significant.

3. Results

3.1. Effect of nicotine on CPP

Administration of nicotine significantly increased the placepreference for drug paired compartment (Fig. 1). Application ofone-way ANOVA showed that nicotine in dose dependent mannerdevelops place preference to the drug paired compartment [F (3,23) = 12.04; P < 0.001]. Post hoc Dunnett’s test reveals that nicotine(1 mg/kg, ip) showed significant place preference (P < 0.01) as com-pared to the saline treated rats. However, lower doses of nicotine(0.5 and 0.75 mg/kg, ip) failed to produce any significant (P > 0.05)conditioning in animals and no preference to either compartmentwas observed. Saline treatment in the conditioning compartmentdid not produce any preference or aversion.

3.2. Agmatine attenuate nicotine induced place preference

Agmatine administered 30 min prior to the nicotine treatmentdecreased the effect of nicotine on CPP (Fig. 2). Application ofone-way ANOVA showed that agmatine (20 and 40 mg/kg, ip) sig-nificantly decreased the place preference to the nicotine (1 mg/kg,ip) paired compartment [F (7, 47) = 7.37; P < 0.0001]. Post hoc


Fig. 1. Effect of nicotine on conditioned place preference. Animal received nicotine[0.5, 0.75 and 1 mg/kg, intraperitoneal (ip)] or saline (0.25 ml/mouse, ip) consecutivefor 6 days in non-preferred compartment and tested for time spent in seconds (s) inthe compartments under the described schedule. Each bar indicate the mean changein preference (s) (Post-conditioning time – Pre-conditioning time) ± SEM (n = 6).*P < 0.01 vs. saline treated mice (one-way ANOVA followed by post hoc Dunnett’stest).

Bonferroni’s multiple comparison test showed that agmatine (20and 40 mg/kg, ip) significantly decreased the effect of nicotine onCPP as compared to the saline treated rats (P < 0.01 and P < 0.001,respectively). However, lower dose of agmatine (10 mg/kg, ip) wasineffective (P > 0.05). Agmatine per se at these doses did not showplace preference or aversion to any compartment.

3.3. NPY or NPY Y1 receptor agonist potentiated the effect ofagmatine on nicotine CPP

Fig. 3 depicts the effect of NPY or [Leu31, Pro34]-NPY on the pref-erence in saline or nicotine paired compartment. Administrationof NPY (1 and 2 pg/mouse, icv) or NPY Y1 receptor agonist [Leu31,Pro34]-NPY (0.1 and 0.2 pg/mouse, icv) in saline (0.25 ml/mouse, ip)or nicotine (1 mg/kg, ip) paired compartment did not show any sig-nificant change in the preference as compared to respective controlanimals (0.25 ml/mouse, ip).

Fig. 2. Effect of agmatine on nicotine induced place preference in mice. Animalsreceived saline [0.25 ml/mouse, intraperitoneal (ip)] or agmatine (10, 20, 40 mg/kg,ip) 30 min before nicotine (1 mg/kg, ip) consecutive for 6 days in non-preferredcompartment during conditioning sessions. Each bar indicate the mean change inpreference in seconds (s) (Post-conditioning time – Pre-conditioning time) ± SEM(n = 6). $P < 0.001 vs. saline treatment, *P < 0.001, **P < 0.0001 vs. saline + nicotinetreated mice (one-way ANOVA post hoc Bonferroni’s multiple comparison test).

Fig. 3. Dose related response of NPY and [Leu31, Pro34]-NPY on place preference.Mice were injected with aCSF [5 "l/mouse, intracerebroventricular (icv)] or NPY(1 and 2 pg/mouse, icv) or [Leu31, Pro34]-NPY (0.1 and 0.2 pg/mouse, icv) 30 minbefore saline [0.25 ml/mouse, intraperitoneal (ip)] or nicotine (1 mg/kg, ip) consec-utive for 6 days in non-preferred compartment during conditioning sessions. Eachbar indicate the mean change in preference in seconds (s) (Post-conditioning time– Pre-conditioning time) ± SEM (n = 5–7). *P < 0.001 vs. aCSF + saline treated mice(one-way ANOVA followed by post hoc Bonferroni’s multiple comparison test).

Administration of per se ineffective dose of NPY (1 pg/mouse, icv)and NPY Y1 receptor agonist [Leu31, Pro34]-NPY (0.1 pg/mouse, icv)in combination with agmatine (10 mg/kg, ip) during conditioningphase exhibited synergistic attenuation of nicotine induced placepreference in mice [F (4, 31) = 13.29; P < 0.001 and F (4, 32) = 13.54;P < 0.001 respectively] (Fig. 4). The dose of NPY, [Leu31, Pro34]-NPYand agmatine alone used in this experiment did not have any effecton place conditioning.

Fig. 4. Effect of co-administration of NPY, NPY Y1 receptor agonist [Leu31, Pro34]-NPY and agmatine on nicotine induced place preference in mice. Animals receivedaCSF [5 "l/mouse, intracerebroventricular (icv)] or NPY (1 pg/mouse, icv) or [Leu31,Pro34]-NPY (0.1 pg/mouse, icv) 15 min prior to agmatine [10 mg/kg, intraperitoneal(ip)] and were injected with nicotine (1 mg/kg, ip) or saline (0.25 ml/mouse, ip)30 min following last injections in the 6 days non-preferred compartment duringconditioning sessions. Each bar indicate the mean change in preference in seconds(s) (Post-conditioning time – Pre-conditioning time) ± SEM (n = 5–7). $P < 0.0001 vs.aCSF + saline + saline, #P < 0.0001 vs. aCSF + saline + nicotine, *P < 0.001, **P < 0.0001vs. aCSF + agmatine + nicotine treated mice (one-way ANOVA followed by post hocBonferroni’s multiple comparison test).


Fig. 5. Influence of NPY Y1 receptor antagonist, BIBP3226 pre-treatment on the effect of agmatine on nicotine-induced place preference in mice. Animals (n = 5−7) receivedNPY Y1 receptor antagonist, BIBP3226 [0.005 and 0.01 ng/mouse, intracerebroventricular (icv)] or aCSF (5 "l/mouse, icv) and agmatine [20 mg/kg, intraperitoneal (ip)] orsaline (0.25 ml/mouse, ip) 30 min before the administration of nicotine (1 mg/kg, ip) or saline (0.25 ml/mouse, ip) consecutive for 6 days in non-preferred compartmentduring conditioning session. Each bar indicate the mean change in preference in seconds (s) (Post-conditioning time – Pre-conditioning time) ± SEM (n = 6). *P < 0.001 vs.aCSF + saline + saline, #P < 0.01 vs. aCSF + saline + nicotine, $P < 0.01 vs. aCSF + agmatine + nicotine treated animals (one-way ANOVA followed by post hoc Bonferroni’s multiplecomparison test).

3.4. NPY Y1 receptor antagonist, BIBP3226 blocked the effect ofagmatine on nicotine CPP

The animals injected with BIBP3226 (0.005 and 0.01 ng/mouse,icv) prior to nicotine (1 mg/kg, ip), did not showed any significantchanges on nicotine induced CPP [F (2, 17) = 0.19; P = 0.82]. How-ever, BIBP3226 (0.01 ng/mouse, icv) significantly reversed the effectof agmatine (20 mg/kg, ip) on nicotine CPP [F (4, 30) = 6.20; P < 0.01].In addition, no place preference or aversion was seen in BIBP3226(0.005 and 0.01 ng/mouse, icv) treated animals when comparedwith vehicle treated control mice [F (2, 20) = 0.66; P = 0.53] (Fig. 5).

The locomotor activity of all the drugs used in present study wasassessed in open field test. We did not find any significant changesin the locomotor activity as compared to the control animals (datanot shown).

3.5. Agmatine restored the nicotine induced changes in NPY-ir

The change in the immunoreactive profile and the morpho-metric analysis of NPY-ir cells as well as fibers in AcbSh, BNSTl,ARC and PVN of saline, nicotine and agmatine + nicotine treatedmice is shown in Fig. 6. Significant decrease in NPY-ir was noticedin the AcbSh (P < 0.001), BNSTl (P < 0.0001), ARC (P < 0.01) andPVN (P < 0.01) following CPP induction by nicotine as compared tosaline treated mice. However, the administration of agmatine priorto nicotine significantly (AcbSh, P < 0.001; BNSTl, P < 0.0001; ARC,P < 0.01 and PVN, P < 0.001) restored the NPY-ir cells and fibers ascompared to the nicotine treated mice.

4. Discussion

Present investigation studied the effect of agmatine on theacquisition of nicotine induced place preference in mice. Con-sistent with earlier findings present study demonstrated thatnicotine (1 mg/kg, ip) induces significant place preference inmice without any influence on basal locomotor activity [4,34].The dose of nicotine was selected according to the narrow doseranges reported in literatures that produces CPP in rodents [3]. CPPparadigm represents an animal model to assess the rewarding and

reinforcing effect of psychostimulants [22]. CPP model used in thisstudy demonstrate that the rodents spent more time in the initiallyless preferred compartment after paired with psychostimulant likenicotine [39]. Agmatine at any doses tested here did not show anyeffects on place conditioning. However, pretreatment of animalswith agmatine during conditioning sessions significantly inhibitedacquisition of nicotine induced CPP. These doses of agmatine didnot significantly alter the spontaneous locomotor activity [20,45];thus, the effect of agmatine on nicotine CPP was specific and couldnot be attributed to any locomotor component. Nicotine has beenshown to increase release of agmatine from adrenal chromaffincells [31,38]. The brain regions (VTA/Acb/amygdala) having abun-dant agmatine and its target receptors are shown to be involvedin the drug addiction [20,32]. In view of this, several studies havealready established a role of agmatine in attenuation of ethanol andmorphine withdrawal syndrome [1,37,44]. It also decreases cocaineand fentanyl self-administration and inhibited morphine CPP [25].Recently we have demonstrated the inhibitory effect of agmatineon nicotine induced behavioural sensitization in mice [20].

Agmatine and NPY show considerable similarities with refer-ence to their anatomical distribution and physiological properties.Both these neuromodulators are released during stress [26,46],they enhance feeding [17,29] and are critically implicated in theregulation of anxiety [9,40], depression [12,21] and drug addic-tion [20,35,37]. Although agmatine is reported to increase NPYimmunoreactivity in hypothalamic nuclei in rats [41], their func-tional interaction with reference to rewarding effects of nicotineremains largely unclear.

In the present study, NPY or [Leu31, Pro34]-NPY, at subeffectivedoses, significantly increased the inhibitory influence of agmatineon nicotine induced place preference. Our data correlates with pre-viously described ability of NPY, NPY Y1 receptor agonist [Leu31,Pro34]-NPY to modulate the behavioural effect of nicotine [27,33].The participation of NPYergic system in the different actions ofnicotine is well documented [23]. Activation of post-synaptic NPYY1 receptor was found in brain regions associated with neurobi-ological responses to psychostimulants [35]. In this background,we determined the effect of agmatine on nicotine CPP in pres-ence of NPY Y1 receptor antagonist, BIBP3226. It is interesting


Fig. 6. Photomicrograph showing NPY-immunoreactive (NPY-ir) cells (arrows) as well as fibers (concave arrowheads) in the nucleus accumbens shell (AcbSh; A,B and C),bed nucleus of stria terminalis, lateral part (BNSTl; D, E and F), arcuate nucleus (ARC; G, H and I) and paraventricular nucleus (PVN; J, K and L) of mice following saline (A, D, Gand J), nicotine (B, E, H and K) and agmatine + nicotine (Agm + Nic; C, F, I and L). Diagram M represent semiquantitative measurement of NPY-ir cells as well as fibers in AcbSh,BNSTl, ARC and PVN of sham, nicotine, and agmatine + nicotine treated animals. Note a significant decreased in NPY-ir in AcbSh, BNSTl, ARC and PVN of nicotine treated mice.These changes were restored by treatment of agmatine prior to the nicotine. ac, anterior commissure; aca, anterior commissure, anterior part; AcbC, nucleus accumbenscore; acp, anterior commissure, posterior part; AHC, anterior hypothalamic area, central part; BNSTM, bed nucleus of the stria terminalis, medial part; DMH, dorsomedialhypothalamic nucleus; 3V, third ventricle; LSV, lateral septal nucleus, ventral part; LV, lateral ventricle; PS, parastrial nucleus; Rch, retrochiasmatic area; StA, strial part of thepreoptic area; VMH, ventromedial hypothalamic nucleus and VP, ventral pallidum. *P < 0.01, **P < 0.001, ***P < 0.0001 vs. sham animals and @P < 0.01, @@P < 0.001, @@@P < 0.0001vs. nicotine treated animals. Scale bar = 50 "M.

to note that, the inhibitory effect of agmatine on nicotine CPPwas reversed by NPY Y1 receptor antagonist, BIBP3226. NPY hasemerged as a neurotransmitter that plays a major role in the rewardprocess [30]. Intra-Acb injections of NPY produced place prefer-ence [7]. Intra- amygdala infusion of BIBP3226 attenuated operant

self-administration of ethanol [35] and produced conditioned placeaversion in rats [18]. While NPY mediated place preference wasblocked by cis-flupenthixol (dopamine antagonist) [16], NPY infu-sion into the AcbSh increased extracellular levels of dopamine [36]suggesting involvement in reward.


To confirm the involvement of NPYergic system in the CPPof nicotine, immunohistochemical study of NPY was carried out.While, nicotine significantly reduced NPY-ir in the AcbSh, BNSTl,ARC and PVN, pretreatment of agmatine restored it. These resultsare inconsistent with the previous reports that chronic cigarettesmoke exposure decreased hypothalamic NPY-ir [8] and up-regulate NPY Y1 receptor density [23]. The decreased in NPY-irfollowing nicotine treatment may be a cause of increased releaseof the peptide which may responsible for the CPP of nicotine. Theseresults put forth the possibility that the effect of agmatine may beunderlined through the NPYergic system.

It is important to note that agmatine is one of the fewneurotransmitters with multi-receptorial affinity and diversephysiological functions. Besides !2-adrenoceptor, agmatine alsointeracts with other neurotransmitter systems like 5-hydroxytryptamine-1A receptors, imidazoline receptors, NMDA recep-tors and NOS that directly or indirectly modulate the effect ofpsychostimulants [13,32]. Therefore, their involvement cannotbe completely ruled out and needs further investigation. Takentogether, the results of present study clearly demonstrated aninhibitory influence of agmatine on nicotine CPP and NPYergicmodulation. This study suggests agmatine-NPY interaction mayplay an important role in smoking cessation and nicotine addiction.

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Behavioural Brain Research - VJ Instruments · 2020. 7. 4. · Agmatine attenuates nicotine induced...

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