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Behavioural Brain Research 262 (2014) 118– 124
Contents lists available at ScienceDirect
Behavioural Brain Research
j ourna l h o mepa ge: www.elsev ier .com/ locate /bbr
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
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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
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N.R. Kotagale et al. / Behavioural Brain Research 262 (2014)
118– 124 121
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).
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122 N.R. Kotagale et al. / Behavioural Brain Research 262 (2014)
118– 124
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
-
N.R. Kotagale et al. / Behavioural Brain Research 262 (2014)
118– 124 123
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.
-
124 N.R. Kotagale et al. / Behavioural Brain Research 262 (2014)
118– 124
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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