Behavioural Brain Research 213 (2010) 161174

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Behavioural Brain Research

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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

a r t i c l e

i n f o

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

a b s t r a c t
Agmatine [2-(4-aminobutyl)guanidine] is an endogenous amine proposed as a neurotransmitter/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 sensitization 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) withdrawal phase challenged with nicotine (0.4 mg/kg, sc) on day 11. Mice were pretreated with agmatine
(4080 g, icv) or agents known to increase endogenous brain agmatine levels [e.g. an agmatine biosynthetic precursor, l-arginine (80 g, icv), ornithine decarboxylase inhibitor, diuoromethyl-ornithine
(50 g, icv), diamine oxidase inhibitor, aminoguanidine (25 g, icv) and agmatinase inhibitor, arcaine
(50 g, icv)] 30 min before daily rst 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 agmatine on nicotine sensitization. There was no signicant 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 administration of nicotine in rodents evokes behavioral sensitization
indicated by gradual increase in locomotor activity [33,34]. Behavioral 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 interactions 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: chopdect@hotmail.com (C.T. Chopde).
0166-4328/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbr.2010.04.049

Recently, agmatine [2-(4-aminobutyl) guanidine], an endogenous 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 selfadministration [36,61] and blocks locomotor as well as biochemical
(dopamine release) expression of morphine sensitization [71]. It
inhibits the expression of nicotine induced conditioned hyperlocomotion 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 neurotransmitter/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 responsible for nitric oxide (NO) formation in brain [4,13]. Agmatine is a

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pleiotropic molecule with many central and peripheral functions.

Its systemic administration evokes anxiolytic [25], antidepressant
[28,80], antinociceptive [40], anticonvulsive [5], anti-inammatory
[54], antiproliferative [19] and neuroprotective [38] properties
and also facilitates working memory [29]. Agmatine stimulates
release of luteinizing hormone-releasing hormone from hypothalamus [23], catecholamines from adrenal chromafn cells and insulin
from pancreatic -cells [1].
Chronic nicotine administration increases adrenergic binding
sites in several brain regions [73]. Agmatine and 2 -adrenergic
receptors have important functional interactions in behavioral
effects of psychoactive agents including potentiation of morphine induced analgesia [12,75] and conditioned place preference
[63], attenuation of morphine withdrawal symptoms and several aspects of drug addiction [61,75]. Moreover clonidine, an
2 -adrenoceptor agonist, is clinically used for the treatment of
nicotine addiction and relapse [6,14]. While much has been studied on the modulatory inuence of agmatine on morphine effects,
its regulatory role in behavioral effects of nicotine including sensitization and addiction is poorly understood. In present study,
we have investigated the effects of intracerebroventricularly (icv)
injected agmatine or the agents augmenting the brain agmatine
content on nicotine induced motor sensitization. Increasing biosynthesis of endogenous agmatine and blocking its degradation are
the approaches to elevate agmatine levels in brain. Biosynthesis
of agmatine by l-ADC depends upon the availability of l-arginine
[60]. l-Arginine is also converted into ornithine by arginase and
to NO by an enzyme NOS. Ornithine subsequently turned into
putrescine by l-ornithine decarboxylase (l-ODC) [48]. DFMO (diuoromethyl-ornithine), is an inhibitor of arginase [56] and also
stimulator of enzyme l-ADC [17]. Its function as arginase inhibitor
as well as stimulator of l-ADC would increase the availability of
agmatine in brain [32]. Agmatine is degraded to putrescine and
guanido-butanoic acid by enzyme agmatinase and diamine oxidase
(DAO), respectively [48] and inhibition of these enzymes resulted
in augmentation of endogenous agmatine [46]. In present study we
used DAO inhibitor, aminoguanidine [31] and agmatinase inhibitor,
arcaine [18,46] to block the agmatine metabolic pathways leading to increase brain agmatine levels [58]. We further assessed
the involvement of 2 -adrenoceptors in agmatine effects using
clonidine, 2 -adrenoceptor agonist and yohimbine and idazoxan,
2 -adrenoceptor antagonists.
2. Materials and methods
2.1. Subjects
Swiss albino male mice weighing 2025 g were group housed (ve per cage)
in a temperature and light (12:12 h light:dark cycle, lights on 07.00 h) controlled
room. Food and water were available ad libitum. Animals were allowed for 48 h
to acclimatize to the laboratory environment before experiments. All testing were
executed in accordance with the guidelines for the care and use of laboratory animals
by Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) and were approved by Institutional Animal Ethical Committee. All the
observations were made during 09.0014.00 h to avoid circadian variations. All mice
were experimentally nave.

2.2. Drugs
Agmatine sulfate, nicotine hydrogen tartarate, aminoguanidine hemisulfate, DLDFMO, arcaine sulfate, clonidine, yohimbine hydrochloride, idazoxan hydrochloride
and l-arginine monohydrochloride were purchased from SigmaAldrich, Co., USA.
Nicotine hydrogen tartarate, yohimbine hydrochloride and idazoxan hydrochloride were dissolved in isotonic saline solution. Nicotine was administered by
subcutaneous (sc) route whereas yohimbine and idazoxan were administered by
intraperitoneal (ip) route. All other drugs were dissolved in articial cerebrospinal
uid (aCSF) of the following composition (140 mM NaCl, 3.35 mM KCl, 1.15 mM
MgCl2, 1.26 mM CaCl2, 1.2 mM Na2 HPO4 , 0.3 mM NaH2 PO4 , pH 7.4) and administered
via icv route.

2.3. Surgery
Under pentobarbital sodium (60 mg/kg, ip) anesthesia mice were placed in a
stereotaxic frame (David Kopf, CA, USA). A guide cannula (C315 G/Spc, Plastic One
Inc., Virginia, USA) was implanted bilaterally into the third ventricle (0.8 mm posterior, 1.3 mm lateral to midline and 3.5 ventral to the bregma) according to the
mouse brain atlas [42]. A 28-gauge stainless steel dummy cannula was inserted to
occlude the guide cannula when not in use. After surgery each mouse was injected
with oxytetracycline injection (25 mg/kg, im, Pzer Ltd., Chennai) and Neosporin
ointment (Burroughs Wellcome Ltd., Mumbai) was applied to avoid infection. Animals 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 transferring them to experimental room and handling daily to treatment schedule. The icv
injections were given via 33 gauge internal cannula (internal diameter 0.18 mm and
outer diameter 0.20 mm) (C315 I/Spc), which was attached to a Hamilton microliter
syringe (Hamilton, Nevada, USA) via polyethylene tubing (PE-10) (internal diameter 0.28 mm; outer diameter 0.61 mm), that extended 0.5 mm beyond the guide
cannula. The internal cannula was held in position for another 1 min before being
slowly withdrawn to prevent backow and promote diffusion of drug.
After all sensitization testing, dilute India ink was injected (icv) and subjects
were sacriced under an overdose of sodium pentobarbital anesthesia (120 mg/kg,
ip). Brains were removed and cryostat cut into 50-m sections, mounted and viewed
using light microscopy to verify cannulae placements. The data of animals with
cannula placement of more than 0.5 mm away from coordinates were excluded from
the study (<15%) and data from mice with uniform ink distribution into ventricles
were 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 photo
sensors, 2.5 cm apart from each other. Mice were habituated to the actophotometer
chamber for 30 min before any testing. Baseline locomotor activity of each mouse
was recorded for 20 min as a total count of ambulatory, horizontal and vertical
activity.
2.5. Effect of agmatine and its modulators on nicotine sensitization
The procedure outlined by Shim et al. [57] was adapted to sensitize mice to
nicotine. The protocols were designed to examine the effects of exogenously administered agmatine or drugs which alter endogenous agmatine concentration in brain
on nicotine sensitization. To investigate the effects on the development phase either
agmatine (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 min
before the daily rst dose of nicotine hydrogen tartarate (0.4 mg/kg as free base) or
saline (1 ml/kg, sc) during 7 days of development phase. Drugs were not injected
on days 8, 9 and 10 of experiment. On day 11, all mice received a challenge dose of
nicotine (0.4 mg/kg, sc) or saline (1 ml/kg, sc) and locomotor activity was recorded
as mentioned above once daily at 09.00 a.m. for 20 min immediately after every rst
injection of nicotine or saline through days 17 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 agmatine
or 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 individual 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 either
administered (n = 9) during development of sensitization (days 17) or during
nicotine free period (days 8, 9, 10). Treatment protocols were also designed (n = 9)
to assess the effects of 2 -adrenoceptor antagonists, yohimbine or idazoxan on
agmatine induced inhibition of nicotine sensitization. Mice (n = 9) were treated
with yohimbine (5 mg/kg, ip), idazoxan (0.4 mg/kg, ip) or saline (1 ml/kg, ip) once
daily 15 min before agmatine (40 g/mouse, icv) followed by daily injection of
nicotine (0.4 mg/kg, sc) during the development phase of nicotine sensitization or
during nicotine free period. Appropriate control groups (n = 9) were maintained in
all the cases. Locomotor counts were monitored for all groups daily for 20 min as
per 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 ttest. Data obtained from development phase (days 17) treatment was analyzed by
Two-way analysis of variance (ANOVA) with repeated measures on time followed

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163

Fig. 1. Effects of agmatine (AGM) on nicotine (NIC) induced locomotor activity during 7-day development phase. Mice were pretreated with aCSF (2 l/mouse, icv) or AGM (40
and 80 g/mouse, icv) 30 min before rst daily injections of saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) for 7 consecutive days. Each point represents the mean locomotor
counts SEM (n = 7). < 0.05 vs. aCSFSAL (day 1) (Unpaired t-test), # P < 0.001 vs. aCSFSAL. *P < 0.01, **P < 0.001 vs. aCSFNIC (days 17) (Two-way ANOVA followed by
Bonferroni multiple comparison test).

Fig. 2. Effects of agmatine (AGM) on locomotor activity in response to nicotine (NIC) challenge on day 11. Mice were pretreated with aCSF (2 l/mouse, icv) or AGM (40
and 80 g/mouse, icv) 30 min before injections of saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) during 7-day development phase and tested with SAL (1 ml/kg, sc) or NIC
(0.4 mg/kg, sc) challenge on day 11. Each column represents the mean locomotor counts SEM (n = 7). # P < 0.001 vs. aCSF/SALSAL, $ P < 0.001 vs. aCSF/NICSAL (Unpaired
t-test). *P < 0.001 vs. aCSF/NICNIC (One-way ANOVA followed by NewmanKeuls test).

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

by post hoc Bonferroni multiple comparison test. Data of 11th day nicotine sensitization was analyzed by One-way analysis of variance (ANOVA) followed by post
hoc Dunnetts/NewmanKeuls test. Data obtained on day 11, following the treatment during withdrawal period (days 8, 9, 10), was analyzed by One-way analysis
of variance (ANOVA) followed by post hoc Dunnetts/NewmanKeuls test. P 0.05
was considered to be statistically signicant.

3. Results
3.1. Agmatine inhibits nicotine sensitization
Effects of agmatine on the development of nicotine induced
locomotor sensitization are shown in Fig. 1. Acute administration
of the rst dose of nicotine (0.4 mg/kg, sc) on day 1 modestly
but signicantly increased the locomotion by 45% compared to
saline control (aCSFSAL) group [Unpaired t-test; t = 2.18, df = 12,
P < 0.05]. On the other hand, repeated nicotine injections twice daily
for 7 consecutive days resulted in a progressive and signicant
increase in locomotor response through the entire treatment period
consistent with sensitization development [Two-way ANOVA
FTreatment (1, 72) = 381.87, P < 0.001, FTime (6, 72) = 19.37, P < 0.001,
FTreatment Time (6, 72) = 13.26, P < 0.001]. On 7th day the hyperactivity in response to nicotine injection was increased by 400%
above the level observed on day 1 [day 7 vs. day 1, Unpaired
t-test; t = 10.85, df = 12, P < 0.001]. However, repeated saline injections for 7 days had no effect on locomotor activity. As can
be seen in Fig. 2, similar to those for mice received nicotine
for 7 days, a injection of challenge dose of nicotine on day 11
(aCSF/NICNIC) following nicotine free period (days 810) also
exhibited greater locomotor activity than mice with saline on
day 11 (aCSF/NICSAL) [Unpaired t-test; t = 5.67, df = 12, P < 0.001]
or from salinesaline (aCSF/SALSAL) control [Unpaired t-test;
t = 15.39, df = 12, P < 0.001].
As shown in Fig. 1, coadministration of agmatine
(4080 g/mouse, icv) with nicotine (0.4 mg/kg, sc) on day 1
did not signicantly change (AGMNIC) the acute nicotine induced

(AGMNIC) hyperlocomotor response as compared to AcsfNIC

[One-way ANOVA post hoc Dunnetts analysis; F(2, 20) = 0.06,
P > 0.05]. Acute injection of agmatine (4080 g/mouse, icv) alone
(day 1) to saline treated groups (AGMSAL) did not change animals
basal activity [One-way ANOVA post hoc Dunnetts analysis; F(2,
20) = 1.68, P > 0.05]. On the other hand repeated injections of agmatine before daily rst dose (0.4 mg/kg, sc) of nicotine (AGMNIC)
for 7 consecutive days signicantly reduced the magnitude of locomotor 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 (4080 g/mouse) to the mice
before nicotine for 7 days signicantly attenuated the hyperlocomotor response to nicotine challenge on day 11 [One-way ANOVA,
F(5, 41) = 52.63, P < 0.001] after 3 days extinction period compared
to control (aCSF/NICNIC). Post hoc NewmanKeuls comparison
showed that both 40 g (P < 0.001) as well as 80 g (P < 0.001) daily
treatments signicantly lowered locomotor activity (Fig. 2).
Administration of agmatine alone (AGM/SALSAL) without
nicotine treatment for 7 days did not produce any signicant
change in the activity counts compared to (aCSF/SALSAL) treated
group [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 (days
8, 9, 10) signicantly attenuated (Fig. 3) the locomotor response on
11th day to nicotine challenge [F(2, 20) = 11.42, P < 0.001] as compared to mice received nicotine (days 17) and aCSF (days 8, 9, 10)
indicating blockade of consolidation or incubation of sensitization.
3.2. l-Arginine, DFMO, arcaine and aminoguanidine attenuates
nicotine sensitization
As shown in Fig. 4, acute icv injection of l-arginine
(80 g/mouse,
l-arginineNIC)
or
DFMO
(50 g/mouse,
DFMONIC) (Fig. 4A) or arcaine (50 g/mouse, arcaineNIC)
or aminoguanidine (25 g/mouse, AMGNIC) (B) on day 1,

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165

Fig. 4. Effects of brain agmatine modulators on nicotine (NIC) induced locomotor activity during 7-day development phase. Mice were pretreated with aCSF (2 l/mouse, icv)
or l-arginine (80 g/mouse, icv) or DFMO (50 g/mouse, icv) (A) or arcaine (50 g/mouse, icv) or aminoguanidine (25 g/mouse, icv) (B) 30 min before rst daily injections
of saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) for 7 consecutive days. Each point represents the mean locomotor counts SEM (n = 7). # P < 0.001 vs. aCSFSAL. *P < 0.05,
**P < 0.01, ***P < 0.001 vs. aCSFNIC (days 17) (Two-way ANOVA followed by Bonferroni multiple comparison test).

30 min prior to the rst nicotine injection (0.4 mg/kg, sc) did
not signicantly change acute locomotor response to nicotine
as compared to aCSFNIC groups [One-way ANOVA post hoc
Dunnetts analysis; F(4, 34) = 2.12, P > 0.05]. However, these curves
start from lower baseline indicating their tendency towards

blockade. Two-way ANOVA revealed that daily treatment of

l-arginine (80 g, l-arginineNIC) or DFMO (50 g, DFMONIC)
during 7 days development phase signicantly reduced nicotine
induced locomotor activity when compared against control group
(aCSFNIC) [l-arginine: FTreatment (3, 144) = 170.84, P < 0.001; FTime

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Fig. 5. Effects of brain agmatine modulators on locomotor activity in response to nicotine (NIC) challenge on day 11. Mice were pretreated with aCSF (2 l/mouse, icv) or
l-arginine (80 g/mouse, icv) or DFMO (50 g/mouse, icv) (A) or arcaine (50 g/mouse, icv) or aminoguanidine (25 g/mouse, icv) 30 min before injections of saline (SAL)
(1 ml/kg, sc) or NIC (0.4 mg/kg, sc) during 7-day development phase and tested with SAL (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) challenge on day 11. Each column represents the
mean locomotor counts SEM (n = 7). # P < 0.001 vs. aCSF/SALSAL. *P < 0.001 vs. aCSF/NICNIC (One-way ANOVA followed by NewmanKeuls 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/aCSFNIC) or l-arginine (80 g/mouse, icv) (NIC/l-arginineNIC) or DFMO (50 g/mouse, icv) (NIC/DFMONIC)
or arcaine (50 g/mouse, icv) (NIC/arcaineNIC) or aminoguanidine (25 g/mouse, icv) (NIC/aminoguanidineNIC) 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/aCSFSAL) (n = 7). # P < 0.001 vs. SAL/aCSFSAL. *P < 0.01, **P < 0.001
vs. NIC/aCSFNIC (days 17) (One-way ANOVA followed by NewmanKeuls test).

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(6, 144) = 23.70, P < 0.001 and FTreatment Time (18, 144) = 6.34,
P < 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,
P < 0.001]. The injections of arcaine (50 g/mouse, icv, arcaineNIC)
and aminoguanidine (25 g/mouse, icv, aminoguanidineNIC)
during 7 days development phase of locomotor sensitization
(Fig. 4B) exhibited signicant effect on locomotor activity [arcaine:
FTreatment (3, 144) = 130.31, P < 0.001; FTime (6, 144) = 28.13, P < 0.001
and FTreatment Time (18, 144) = 6.63, 1 P < 0.001; DFMO: FTreatment
(3, 144) = 120.46, P < 0.001; FTime (6, 144) = 28.14, P < 0.001 and
FTreatment Time (18, 144) = 10.53, P < 0.001].
Administration of l-arginine (80 g, l-arginineSAL) or DFMO
(50 g, DFMOSAL) or arcaine (50 g, arcaineSAL) or aminoguanidine (25 g, AMGSAL) with saline for 7 days did not produce
any signicant change in the activity counts when compared with
aCSFSAL treated group.
As depicted in Fig. 5, treatment of these modulators for 7
consecutive days during development phase followed by 3-day
withdrawal also signicantly blocked sensitization to nicotine challenge on 11th day [One-way ANOVA F(9, 69) = 49.85, P < 0.001].
Post hoc Dunnetts comparison demonstrated the signicant effect
of l-arginine (80 g) (P < 0.001) or DFMO (50 g) (P < 0.001) or
arcaine (50 g) (P < 0.001) or aminoguanidine (25 g) (P < 0.001)
on the locomotor stimulation to nicotine challenge on day 11.
Furthermore as shown in Fig. 6 injections of the agents that
alters the brain agmatine content during 3-day nicotine free period
(days 8, 9, 10) after 7-day induction phase, signicantly inhibited
locomotor sensitization to nicotine challenge on day 11 as compared to its control group (NIC/aCSFNIC) [One-way ANOVA F(5,
41) = 24.96, P < 0.001]. Post hoc analysis of the mean locomotor
counts showed the signicant attenuation of the locomotor activity 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).

3.3. Effect of 2 -adrenoceptor agonist, clonidine and its

combination with agmatine on nicotine induced behavioral
sensitization
Fig. 7A and B shows locomotor activity level for each treatment group at day 1 (acute effect) and through 7 days sensitization
development. Acute injections of clonidine (0.2 g, icv) on day 1,
prior (30 min) to nicotine injections did not signicantly block the
nicotine induced acute motor stimulation [F(3, 27) = 3.35, P > 0.05].
On other hand (A) pretreatment with clonidine (0.2 g but not
0.1 g) (clonidineNIC) during the 7-day development phase signicantly blocked development of nicotine sensitization through
entire 7 days period when compared against its control (aCSFNIC)
group [Two-way ANOVA FTreatment (5, 216) = 100.11, P < 0.001;
FTime (6, 216) = 48.19, P < 0.001 and FTreatment Time (30, 216) = 8.11,
P < 0.001].
As depicted in Fig. 7B, per se subeffective dose of clonidine
(0.1 g, icv) in combination with subeffective dose of agmatine (20 g, icv) exhibited synergistic inhibition of development
of nicotine induced locomotor sensitization [Two-way ANOVA
FTreatment (5, 216) = 74.42, P < 0.001; FTime (6, 216) = 30.93, P < 0.001
and FTreatment Time (30, 216) = 6.33, P < 0.001]. However, administration of clonidine (0.10.2 g, clonidineSAL) with saline for 7
days did not produce any signicant effect on the motor activity
when compared with aCSFSAL treated group.
Daily treatment of clonidine (0.2 g/mouse) (P < 0.001) or
the combination of clonidine (0.1 g/mouse) and agmatine
(20 g/mouse) (P < 0.001) from day 1 to day 7 signicantly blocked
the hyperlocomotor response to nicotine challenge on day 11
(Fig. 8) [F(9, 69) = 46.15, P < 0.001] (Post hoc NewmanKeuls test).

167

The effect of clonidine (0.1, 0.2 g/mouse) and the combination

treatment of low doses (non-effective if given alone with aCSFSAL)
of clonidine (0.1 g/mouse) and agmatine (20 g/mouse) during withdrawal phase on the locomotor sensitization is depicted
in Fig. 9. Clonidine (0.2 g/mouse, icv) and combined treatment of agmatine (20 g) and clonidine (0.1 g) during 3-day
withdrawal periods (days 8, 9 and 10) after the 7 days induction phase inhibited nicotine sensitization on 11th day when
compared against control group (NIC/aCSFNIC) [F(5, 41) = 36.98,
P < 0.001]. Post NewmanKeuls comparisons indicated that clonidine (0.2 g 0.001 vs. NIC/aCSFNIC) and the combined agmatine
(20 g) and clonidine (0.1 g) treatment during nicotine free
period on days 8, 9 and 10 also signicantly attenuated the
nicotine sensitization on 11th day when compared against aCSF
(NIC/aCSFNIC) (P < 0.001), agmatine (NIC/AGMNIC) (P < 0.001) or
clonidine (NIC/clonidineNIC) (P < 0.001) treated group.
3.4. 2 -Adrenoceptor antagonists reversed the inhibitory effect of
agmatine 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 development period of nicotine sensitization signicantly reversed
the inhibitory effect of agmatine on nicotine induced locomotor 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-day
withdrawal period after which challenged with nicotine on 11th
day also showed reversal of agmatine effect on nicotine hyperlocomotion (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 (days
8, 9 and 10) after 7 days induction phase exerted an obvious
reversal of agmatine effect on nicotine induced behavioral sensitization as tested on day 11 when compared against control animals
that received nicotine (days 17) 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 or
idazoxan alone in the doses used here did not inuence the nicotine
sensitization.
4. Discussion
Results of the present study showed that acute injections of
nicotine (day 1) to mice stimulated locomotor activity and following its repeated daily injections for 7 consecutive days produced
locomotor sensitization. Nicotine induced behavioral sensitization
is consistent with the earlier reports [7,53,66]. In mice acute injections of nicotine generally fails to stimulate locomotor activity.
However the modest but signicant acute activating effects (day 1)
observed here are in concordance with recent reports [7,66]. Several lines of evidence indicated that nicotine stimulates dopamine
release by directly acting on the nicotinic receptors located on
the mesolimbic dopaminergic neurons which lead to locomotor
hyperactivity or hypoactivity depending on dose and animal strain
[9,69]. Behavioral sensitization is thought to be produced by incremental neuroadaptations of neural systems due to repeated use
of abused drugs [69]. Nicotine has been shown to increase the
release of agmatine from adrenal chromafn cells [47,62] which
is implicated in drug addiction [2,60] and several effects of psychostimulants [60,71]. The brain regions (amygdala, VTA and NAc)

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N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161174

Fig. 7. Effects of 2 -adrenoceptor agonist, clonidine alone (A) and in combination with agmatine (B) on nicotine (NIC) induced locomotor activity during 7-day development
phase. Mice were pretreated with aCSF (2 l/mouse, icv) or clonidine (0.1 and 0.2 g/mouse, icv) or clonidine (0.1 g/mouse, icv) in combination with non-effective dose
of agmatine (20 g/mouse, icv) 30 min before rst daily injections of saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) for 7 consecutive days. Each point represents the
mean locomotor counts SEM (n = 7). # P < 0.01, ## P < 0.001 vs. aCSFSAL. *P < 0.01, **P < 0.001 vs. aCSFNIC (days 17) (Two-way ANOVA followed by Bonferroni multiple
comparison test).

N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161174

169

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

Fig. 9. Effects of 2 -adrenoceptor agonist, clonidine alone and in combination with agmatine on nicotine (NIC) induced behavioral sensitization. Mice were pretreated
with saline (SAL) (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) twice daily for 7 consecutive days and injected with aCSF (NIC/aCSFNIC) or clonidine (0.1 and 0.2 g/mouse, icv)
(NIC/clonidineNIC) or clonidine (0.1 g/mouse, icv) in combination with non-effective dose of agmatine (20 g/mouse, icv) (NIC/Clonidine + AGMNIC) 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 + aCSFSAL) (n = 7).
#
P < 0.001 vs. SAL/aCSF + aCSFSAL, *P < 0.001 vs. NIC/aCSF + aCSFNIC, $ P < 0.001 vs. NIC/clonidine + aCSFNIC, @ P < 0.001 vs. NIC/aCSF + AGMNIC (One-way ANOVA followed
by NewmanKeuls test).

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N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161174

Fig. 10. Effects of 2 -adrenoceptor antagonists, yohimbine (Fig. 9A) and idazoxan (Fig. 9B) on the inuence of agmatine (AGM) on nicotine (NIC) induced locomotor activity
during 7-day development phase. Mice were pretreated with saline (SAL) (1 ml/kg, ip) or yohimbine (5 mg/kg, ip) or idazoxan (0.4 mg/kg, ip) before aCSF (2 l/mouse, icv)
or AGM (40 g/mouse, icv) 30 min before rst daily injections of SAL (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) for 7 consecutive days. Each point represents the mean locomotor
counts SEM (n = 7). # P < 0.001 vs. SAL + aCSFSAL, ! P < 0.05, !! P < 0.01, !!! P < 0.001 vs. SAL + aCSFNIC (days 17) *P < 0.05, **P < 0.01, ***P < 0.001 vs. SAL + AGMNIC (Two-way
ANOVA followed by Bonferroni multiple comparison test).

N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161174

171

Fig. 11. Effects of 2 -adrenoceptor antagonists, yohimbine (Fig. 9A) and idazoxan (Fig. 9B) on the inuence of agmatine (AGM) on locomotor activity in response to nicotine
(NIC) challenge on day 11. Mice were pretreated with saline (SAL) (1 ml/kg, ip) or yohimbine (5 mg/kg, ip) or idazoxan (0.4 mg/kg, ip) before aCSF (2 l/mouse, icv) or AGM
(40 g/mouse, icv) 30 min before injections of SAL (1 ml/kg, sc) or NIC (0.4 mg/kg, sc) during 7-day development phase and tested with SAL (1 ml/kg, sc) or NIC (0.4 mg/kg,
sc) challenge on day 11. Each column represents the mean locomotor counts SEM (n = 7). # P < 0.001 vs. SAL + aCSF/SALSAL, $ P < 0.001 vs. SAL + aCSF/NICNIC. *P < 0.001 vs.
SAL + AGM/NICNIC (One-way ANOVA followed by NewmanKeuls test).

involved in drug addiction has extensive distribution of agmatine,

its transporters and enzymes participated in its degradation and
biosynthesis [48]. Therefore we examined whether agmatine plays
a crucial role in locomotor response to the acute or chronic repeated
administration of nicotine in mice.
This study investigated the effect of exogenously (icv) injected
agmatine and the agents reported to increase endogenous brain
agmatine content on nicotine induced behavioral sensitization.
Administration of agmatine precursor, l-arginine or inhibition
of metabolic pathways might result in augmentation of endogenous agmatine levels in brain. In the present study, we used
diamine oxidase (DAO) inhibitor, aminoguanidine or agmatinase
inhibitor, arcaine and arginase inhibitor, DFMO to block the agmatine metabolism leading to increased agmatine levels in brain
[18,31,46,56,60]. Agmatine is extensively metabolized peripherally in liver, kidney and has very short biological half life [15].
On the other hand, its half life in spinal cord and brain is 1218 h
[49]. Therefore to avoid peripheral metabolism and to attain sufcient concentration in brain, agmatine and its modulators were
injected by icv route daily during development phase (days 17)
or during nicotine withdrawal phase (days 810). We found that
neither agmatine nor its modulators displayed any effect on spontaneous motor activity on day 1 (acute response) when administered
30 min before rst dose of nicotine. On the other hand, daily pretreatments (days 17) with these drugs signicantly attenuated the
development of nicotine induced locomotor sensitization. Interestingly none of these drugs by themselves at any dose level caused
any change in basal locomotor activity in saline treated animals.
Thus their effects on behavioral sensitization can not be attributed
to locomotor suppression or sedation. It is noteworthy that drugs

employed in the present investigation in addition to their effect

on agmatine [64] also possess other activities like NMDA antagonism (arcaine), NOS inhibition (aminoguanidine) as well as NO
precursor (l-arginine) activity. Nonetheless, the involvement of
imidazoline receptors and other biological targets of agmatine
like NMDA and NOS cannot be ruled out and warrant further
investigation. Aminoguanidine, a DAO inhibitor [31] that inhibits
degradation of agmatine to guanidinebutanoic acid, is also a selective inducible NOS inhibitor [57]. Based on these ndings it seems
plausible that rather than acute dose of agmatine, its daily pretreatment for chronic period may be required to block nicotine effect.
Thus although premature, it can be anticipated that a reduction
in agmatinergic tone in NAc shell may contribute to behavioral
sensitization to nicotine. However further behavioral studies using
immunoneutralization or selective drugs that reduce the agmatine
formation may help to clarify its role in nicotine sensitization.
Our ndings that agmatine did not block acute nicotine effects
in mice are consistent with the results of Zaniewska et al. [76] in
rats. This is the only study that investigated the effect of agmatine
(ip) on nicotine evoked behavioral responses in rats. Their pharmacological analysis indicated lack of effect of agmatine on locomotor,
sensitizing or subjective effects but displayed inhibition of conditioned hyperlocomotion induced by nicotine. In contrast, our data
shows blockade of development of nicotine sensitization. One possible explanation for these discrepancies could be due to different
doses, route, treatment schedule and animal strain. Additional evidence supporting our data is that agmatine attenuates ethanol
as well as morphine withdrawal [3,27,67], decreases cocaine and
fentanyl self-administration [36] and expression of morphine sensitization [71] in experimental animals.

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N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161174

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

Furthermore, in the present study icv injections of agmatine or

its modulators during nicotine withdrawal period (days 8, 9, 10)
resulted in a pronounced block in locomotor response to nicotine challenge on day 11. This suggests that exogenous agmatine
or increasing endogenous agmatine in brain by different means
inhibits the consolidation or incubation of sensitization. However,
acute dose of agmatine (4080 g/mouse, icv) given before nicotine challenge on day 11 (data not shown) does not counteract
the expression of nicotine sensitization. This agrees with previous
agmatine studies on nicotine sensitization [76]. Although the psychoactive actions of nicotine are mediated centrally though direct
interaction with nicotinic receptors (nAchRs) [9], some data exists
that implies the role for the other neurotransmitter systems like
glutamatergic and adrenergic systems [55,57]. Earlier studies have
suggested that NMDARs are key receptors in expression as well
as development of behavioral sensitization and that NO is also
involved in development but not in expression of nicotine sensitization [57,66]. The diverse central effects of agmatine also are
associated with its ability to bind to 2 -adrenoceptors and imidazoline binding sites, inhibition of NOS activity and blockade of NMDA
receptors or nicotinic receptors [3,13,30,35,44,74]. The fact that
agmatine acts on so many different biochemical processes, neurotransmitters or receptors makes it difcult to determine which
of its many effects are critical to suppress nicotine induced sensitization. Thus suppression of nicotine induced sensitization by
agmatine could also be due to its ability to block NMDA or nicotinic
receptors or inhibition of enzyme NOS.
Several lines of experimental evidence indicated the involvement of noradrenergic receptors in psychostimulant induced
behavioral effects including sensitization [16,59,70]. Systemic
administration of the 2 -adrenergic receptor agonists clonidine,
lofexidine and guanabenz attenuated stress-induced reinstatement

of cocaine-seeking behavior [11] and footshock-induced reinstatement of nicotine-seeking behavior in rats [79]. Moreover, chronic
administration of nicotine increases the density of 2 -adrenergic
binding sites in some brain regions [73]. Interestingly, several
studies have identied correlation between agmatinergic neurons
and 2 -adrenergic receptors in many brain regions [24,37]. Like
clonidine, agmatine has been shown to bind 2 -adrenoceptors
[63,75,80]. Agmatine alters the ring rate of locus ceruleus neurons
in vivo [43,52] and induces 2 -adrenoceptors dependant antinociception [39]. The potentiating effect of agmatine on morphine
induced analgesia is mediated by 2 -adrenoceptors [51,75]. In
view of these ndings, we studied the possible involvement of 2 adrenergic receptors in agmatine induced attenuation of nicotine
sensitization.
The present work showed that chronic administration of 2 adrenoceptors agonist, clonidine during development and nicotine
free period signicantly blocked the nicotine induced increase in
locomotor counts. Moreover, clonidine also augmented the suppression of nicotine sensitization by agmatine in the doses that
were ineffective when administered alone. Thus, our nding is
in line with reports of agmatine/clonidine synergism [77] and
strengthens the notion that several biological as well as pharmacological effects of agmatine are closely linked to 2 -adrenergic
receptors activation [63,75,80]. In fact clonidine has been reported
to reduce morphine [8] cocaine [21] and d-amphetamine [68]
induced sensitization. It may be recalled that, clonidine is clinically used for smoking cessation and attenuation of nicotine
withdrawal symptoms [6,14]. Involvement of 2 -adrenergic receptors in attenuation of nicotine sensitization by agmatine is further
supported by the fact that agmatine effect was completely abolished by selective 2 -adrenergic receptor antagonists, yohimbine
and idazoxan, a mixed antagonist of 2 /imidazoline I2 recep-

N.R. Kotagale et al. / Behavioural Brain Research 213 (2010) 161174

tor. This is consistent with previous reports that 2 -adrenergic

receptor antagonists attenuated several effects of agmatine in
drug addiction [63,75]. Thus the results clearly indicate that the
inhibitory effects of agmatine on nicotine sensitization are mediated through 2 -adrenoceptors. Alternatively the effects observed
following administration of agmatine and clonidine could be mediated by acting on entirely different systems. It is difcult for any
decision on this matter unless explored whether ability of NMDA
or nicotinic receptors antagonist to block nicotine induced sensitization could be reversed by 2 -adrenoceptor antagonists. Our
results suggest that endogenous brain agmatine and its resultant
interaction with 2 -adrenergic receptors may play an important
role in nicotine induced sensitization. Moreover, 2 -adrenoceptors
agonist, clonidine decreases the dopamine overow by psychostimulants [20]. It is important to note that midbrain regions like VTA
and NAc involved in sensitization expresses abundant agmatine
immunoreactivity [41]. Agmatine also diminishes dopaminergic
neurotransmission by reducing the levels of dopamine in VTA
evoked by morphine withdrawal [71]. Thus, it is reasonable to
assume that attenuation of nicotine induced locomotor sensitization by agmatine is associated with 2 -adrenergic receptor
activation. However, it needs critical investigation whether 2 adrenergic receptor activation and inhibition of dopaminergic
transmission by agmatine are directly related.
In conclusion, attenuation of nicotine induced locomotor sensitization by repeated agmatine administration seems to be mediated
via its interaction with 2 -adrenoceptors. However, whether prolong exposure to nicotine alters or lowers brain agmatine level and
is subsequently responsible for nicotine sensitization is not clear
at present. Although it is premature to conclude that endogenous
agmatine regulates behavior in nicotine addiction, the ndings
from this study suggest that agmatine or the agents augmenting endogenous agmatine levels may have therapeutic potential
in nicotine abuse.

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