European Journal of Pharmacology 637 (2010) 89101

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European Journal of Pharmacology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Behavioural Pharmacology

Agmatine, an endogenous imidazoline receptor ligand modulates ethanol anxiolysis

and withdrawal anxiety in rats
Brijesh G. Taksande, Nandkishor R. Kotagale, Mital R. Patel, Gajanan P. Shelkar,
Rajesh R. Ugale, Chandrabhan T. Chopde
Division of Neuroscience, Department of Pharmacology, Shrimati Kishoritai Bhoyar, College of Pharmacy, New Kamptee, Nagpur (M.S.), 441 002, India

a r t i c l e

i n f o

Article history:
Received 4 December 2009
Received in revised form 6 March 2010
Accepted 31 March 2010
Available online 13 April 2010
Keywords:
Agmatine
Imidazoline receptor
Ethanol
Withdrawal anxiety
EPM (elevated plus maze)

a b s t r a c t
Present study investigated the role of agmatine in ethanol-induced anxiolysis and withdrawal anxiety using
elevated plus maze (EPM) test in rats. The anxiolytic-like effect of ethanol was potentiated by pretreatment
with imidazoline I1/I2 receptor agonist agmatine (1020 mg/kg, i.p.), imidazoline I1 receptor agonists, moxonidine
(0.25 mg/kg, i.p.) and clonidine (0.015 mg/kg, i.p.), imidazoline I2 receptor agonist, 2-BFI (5 mg/kg, i.p.) as well as by
the drugs known to increase endogenous agmatine levels in brain viz., L-arginine, an agmatine biosynthetic precursor
(100 g/rat, i.c.v.), ornithine decarboxylase inhibitor, DFMO (125 g/rat, i.c.v.), diamine oxidase inhibitor,
aminoguanidine (65 g/rat, i.c.v.) and agmatinase inhibitor, arcaine (50 g/rat, i.c.v.). Conversely, prior administration of I1 receptor antagonist, efaroxan (1 mg/kg, i.p.), I2 receptor antagonist, idazoxan (0.25 mg/kg, i.p.) and arginine
decarboxylase inhibitor, D-arginine (100 g/rat, i.c.v.) blocked the anxiolytic-like effect of ethanol. Moreover, ethanol
withdrawal anxiety was markedly attenuated by agmatine (1020 mg/kg, i.p.), moxonidine (0.25 mg/kg, i.p.),
clonidine (0.015 mg/kg, i.p.), 2-BFI (5 mg/kg, i.p.), L-arginine (100 g/rat, i.c.v.), DFMO (125 g/rat, i.c.v.),
aminoguanidine (65 g/rat, i.c.v.) and arcaine (50 g/rat, i.c.v.). The anti-anxiety effect of agmatine in ethanolwithdrawn rats was completely blocked by efaroxan (1 mg/kg, i.p.) and idazoxan (0.25 mg/kg, i.p.). These results
suggest that agmatine and imidazoline receptor system may be implicated in ethanol-induced anxiolysis and
withdrawal anxiety and strongly support further investigation of agmatine in ethanol dependence mechanism.
The data also project agmatine as a potential therapeutic target in overcoming alcohol withdrawal symptoms such
as anxiety.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Ethanol reinforcement and subsequent addiction are the prominent
factors for its abuse in society. Acute ethanol exerts anxiolytic effect
(Polivy and Herman, 1976; Bedford et al., 1978), whereas its abrupt
cessation following prolonged consumption leads to withdrawal
anxiety (Lal et al., 1991; Rezazadeh et al., 1993; Pandey et al., 1999;
Gatch et al., 2000). Though ethanol withdrawal anxiety is one of the
leading causes for its reinstatement and dependence (Baldwin et al.,
1991; Koob, 2000, 2003; Roelofs, 1985), its neurochemical basis is not
clearly understood. Ethanol exerts its biological action through multiple
receptors, including ion channels like GABAA, NMDA, 5HT3 receptors,
certain peptides and neurosteroids (Lal et al., 1993; Linnoila et al., 1987;
Fadda et al., 1989; Dalvi and Rodgers, 1996; Lovinger, 1997; Valenzuela,
1997; LaBuda and Fuchs, 2002; Hirani et al., 2005; Kokare et al., 2006;
Sharma et al., 2007).
Recently, agmatine an endogenous amine has been implicated in the
process of drug addiction (Aricioglu-Kartal and Uzbay, 1997; Li et al.,

Corresponding author. Tel.: + 91 7109 288650; fax: + 91 7109 287094.

E-mail address: chopdect@hotmail.com (C.T. Chopde).
0014-2999/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2010.03.058

1999; Aricioglu et al., 2004; Su et al., 2009). Considerable evidence have

shown that agmatine attenuates ethanol as well as morphine
withdrawal symptoms (Aricioglu-Kartal and Uzbay, 1997; Li et al.,
1999; Uzbay et al., 2000). It decreases morphine, cocaine and fentanyl
self-administration (Morgana et al., 2002) and inhibits nicotine induced
conditioned hyperlocomotion (Zaniewska et al., 2008). Several brain
nuclei like ventral tegmental area (VTA), nucleus accumbens (NAc),
amygdala, etc., which are involved in important aspects of alcoholism
including reinforcement of ethanol and withdrawal anxiety expresses
high agmatine immunoreactivity (Otake et al., 1998; Reis and
Regunathan, 2000).
Agmatine is biosynthesized following decarboxylation of L-arginine
by arginine decarboxylase (ADC) in mammalian brain and other tissues
(Reis and Regunathan, 2000). It is considered as neurotransmitter in the
brain, being synthesized, stored in synaptic vesicles, accumulated by
uptake, released by depolarization and degraded by agmatinase into
polyamine putrescine (Reis and Regunathan, 2000; Halaris and Plietz,
2007). Agmatine is a pleiotropic molecule with many central and
peripheral functions. Its systemic administration exerts anxiolytic (Liu
et al., 2008; Lavinsky et al., 2003), antidepressant (Zomkowski et al.,
2002; Taksande et al., 2009), antinociceptive (Onal and Soykan, 2001;
Onal et al., 2003), anticonvulsive (Bence et al., 2003; Feng et al., 2005),

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antiinammatory (Satriano et al., 2001), antiproliferative (Isome et al.,

2007) and neuroprotective effects (Lee et al., 2009) and facilitates
memory (Liu and Bergin, 2009).
It is important to note that agmatine is one of the few neurotransmitters with multireceptorial afnity and diverse physiological functions. Agmatine antagonizes NMDA, nAch receptors, inhibits nitric oxide
synthase (NOS) (Yang and Reis, 1999; Loring, 1990; Galea et al., 1996;
Reis and Regunathan, 2000) and binds with high afnity to imidazoline
and 2-adrenoceptors (Li et al., 1994; Regunathan and Reis, 1996; Reis
and Regunathan, 1998). It is considered as an endogenous ligand for
imidazoline receptors (Reis and Regunathan, 1998; Raasch et al., 2001)
and its anatomical distribution within brain supports this correlation (De
Vos et al., 1994; Raasch et al., 1995). Further, several reports have
demonstrated the effect of imidazoline receptor ligands like agmatine,
harmane and -carboline on ethanol actions, intake and development of
dependence or withdrawal syndrome (Uzbay et al., 2000; Dobrydnjov
et al., 2004; Mao and Abdel-Rahman, 1996; Rommelspacher et al., 1991,
1996; Spies et al., 1996; Lewis et al., 2007).
Thus it can be intrigued whether agmatine and its interaction with
imidazoline receptor system endogenously play an active role to
inuence ethanol intake, its behavioral effects and subsequent
development of alcoholism. The present work constitutes an
evaluation of relationship between agmatine and imidazoline receptors in ethanol-induced anxiolysis and withdrawal anxiety in rats
using an elevated plus maze (EPM) paradigm.

2.3. Surgery
Rats were anesthetized with thiopentone sodium (60 mg/kg, i.p.)
(Abbott Pharmaceuticals Ltd., Mumbai) and a 22-gauge stainless steel
guide cannula (C313G/Spc, plastic UK) was stereotaxically (David Kopf
Instruments, CA, USA) implanted (Kokare et al., 2006) into the right
lateral ventricle. The surgical coordinates 0.8 mm posterior, +1.2 mm
lateral to midline and 3.5 mm ventral to bregma were used for i.c.v.
cannulation (Paxinos and Watson, 1998). The guide cannulae were then
xed to the skull with dental cement (DPI-RR cold cure, acrylic powder,
Dental Product of India, Mumbai) and secured in two stainless steel
screws. A 28-gauge stainless steel dummy cannula was used to occlude
the guide cannula when not in use. Following surgery, the rats were
placed individually in cage and allowed to recover at least for 7 days
before being tested in elevated plus maze (EPM). Rats were then
randomly assigned to different groups (n = 8) and habituated to the
testing environment by transferring to experimental room and twice
daily handling for 1 week. Drugs were injected (5 l/rat) into the right
ventricle over a one min period with a microliter syringe (Hamilton,
Reno, NV, USA) connected by PE-10 polyethylene tubing to a 28-gauge
internal cannula (C313I/Spc, plastic one, internal diameter 0.18 mm,
outer diameter 0.20 mm) that extended 0.5 mm beyond the guide
cannula. The internal cannula was held in a position for another 1 min
after each injection to promote diffusion of drugs before being slowly
withdrawn to prevent backow.
2.4. Anxiety test (elevated plus maze)

2. Materials and methods

2.1. Animals
Adult healthy Sprague Dawley rats (230250 g) were housed four per
cage (640410250 mm height) or individually after intracerebroventricular (i.c.v.) cannulation, under controlled conditions (252 C and
12 h light/dark cycle, light on at 0700 am) with free access to food and
water. All experimental procedures were approved by the Institutional
Animal Ethical Committee and executed in strict accordance with the
guidelines of Committee for the Purpose of Control and Supervision of
Experiments on Animals, Govt. of India.

2.2. Drugs
The following drugs were used: agmatine sulfate, clonidine hydrochloride, moxonidine hydrochloride, DL--diuoromethyl ornithine
hydrochloride (DFMO), aminoguanidine hemisulfate, arcaine sulfate,
efaroxan hydrochloride, idazoxan hydrochloride and L-arginine monohydrochloride (Sigma-Aldrich Co., USA.), 2-(2-benzofuranyl)-2-imidazoline hydrochloride (2-BFI) (Tocris Biosciences, UK) and ethanol (absolute
ethyl alcohol) (Merck chemicals, Mumbai, India). Ethanol was diluted or
drugs like agmatine, moxonidine, 2-BFI, efaroxan and idazoxan were
dissolved in saline (0.9%) and administered by intraperitoneal (i.p.) route.
For withdrawal studies ethanol was given in liquid diet (Novartis, India).
Drugs like L-arginine, DFMO, arcaine and aminoguanidine were injected
by intracerebroventricular (i.c.v., 5 l/rat) route to alter the levels of brain
agmatine and avoid peripheral effects. For intracerebroventricular (i.c.v.)
administration of drugs, dilutions were made with articial cerebrospinal
uid (aCSF) of following composition 0.2 M NaCl, 0.02 M NaH2CO3, 2 mM
KCl, 0.5 mM KH2PO4, 1.2 mM CaCl2, 1.8 mM MgCl2, 0.5 mM Na2SO4, and
5.8 mM D-glucose.
Doses and timing of the drug injections with respect to the
behavioral testing employed in the protocols were selected on the
basis of previous experiments in our lab and available literature
(Pandey et al, 1999; Utkan et al., 2000; Lavinsky et al., 2003; Roberts
et al., 2005; Kokare et al., 2006; Zeidan et al., 2007; Dandekar et al.,
2008; Taksande et al., 2009).

The EPM apparatus (Pellow and File, 1986) was made up of black
painted plexiglass and consisted of two open arms (50 10 cm) and
two closed arms (50 10 40 cm) facing each other with an open roof
and connected by a central platform (10 10 cm). The maze was
elevated 60 cm above the oor and illuminated by a 100-W lamp,
xed 2 m above the maze oor. Testing began by placing the rat
individually in the center square of the plus maze facing one of the
open arms. An entry was registered only when all four paws of the
animal were placed into an arm. The classic variables, time spent and
numbers of entries in each arm were recorded for the period of 5 min.
After each test, platform of the maze was wiped and cleaned with
damp cotton. All subjects were experimentally naive at the beginning
of each study and tested once only to avoid one-trial tolerance to
anxiolytic efciency of drugs (Bertoglio and Carobrez, 2002) in the
EPM test. All the behavioral tests were conducted during the light
cycle between 0900 and 1200 h by an observer unaware of the
treatment conditions.
2.5. Blood ethanol concentration
Blood sample was removed from rat tail vein. 40 l of blood was
mixed with 160 l perchloric acid (3%) and stored at 4 C until analysis.
Ethanol was quantied by the alcohol dehydrogenase assay (Zapata et al.,
2006). Briey, 20 l clear aliquot was incubated in duplicate in 1 ml of
0.5 M TrisCl buffer (pH 8.8) containing 5.5 g/ml of alcohol dehydrogenase and 1.5 mM -nicotinamide adenine dinucleotide (-NAD) for
40 min at room temperature. Reduction of -NAD to -NADH was
measured by reading the absorbance at 340 nm. The ethanol concentration was estimated using standard calibration curve.
2.6. Effect of agmatine and its modulators on ethanol-induced anxiolysis
In these experiments we investigated the effects of exogenously
administered agmatine or drugs that alter the brain agmatine levels on
ethanol-induced anxiolysis. Rats were randomly assigned to different
groups (n = 8) and received either ethanol (8% w/v, 12.5 g/kg, i.p.),
agmatine (1080 mg/kg, i.p.) or saline 30 min before being subjected to
EPM test. For combination studies, agmatine (1020 mg/kg, i.p.) was

B.G. Taksande et al. / European Journal of Pharmacology 637 (2010) 89101

administered 10 min before an ineffective dose of ethanol (1 g/kg, i.p.)

and subjected to EPM test as mentioned earlier.
To assess whether agmatine has any effects on ethanol metabolism
we determined its blood concentration in ethanol and agmatine plus
ethanol treated groups. Blood samples were collected from tail vein
immediately after EPM test and analyzed using alcohol dehydrogenase assay (Zapata et al., 2006).
Next we determined the inuence of inducers or inhibitors of
enzymes involved in agmatine biosynthesis on anxiolytic effect of
ethanol. The present investigation deals with the role of brain agmatine
in anxiolytic effect of ethanol and withdrawal-induced anxiety. Hence to
modulate the agmatine concentration within rat brain and avoid possible
peripheral effects, agmatine modulators were administered by i.c.v.
route. Separate groups of animals were treated with either aCSF (5 l/rat,
i.c.v.), agmatine precursor, L-arginine (100 g/rat, i.c.v.), an ornithine
decarboxylase inhibitor, DFMO (125 g/rat, i.c.v.), a diamine oxidase
inhibitor, aminoguanidine (65 g/rat, i.c.v.), or an agmatinase inhibitor,
arcaine (50 g/rat, i.c.v.), 10 min before an ineffective dose of ethanol
(1 g/kg, i.p.). Thirty min later each rat was subjected to EPM test. DFMO
inhibits conversion of L-arginine to ornithine and subsequently to
putrescine (Lu et al., 2003). Aminoguanidine and arcaine inhibit
metabolism of endogenous agmatine to guanido-butanoic acid and
putrescine respectively (Lu et al., 2003; Regunathan, 2006). All these
agents are reported to signicantly enhance the brain agmatine content at
the doses used here.
In separate experiment, the effect of arginine decarboxylase (ADC)
inhibitor, D-arginine that blocks the biosynthesis of agmatine
(Rosenfeld and Roberts, 1976; Hao et al., 2005) was examined on
ethanol-induced anxiolysis. Rats were intracranially injected with
aCSF or D-arginine (100 g) 10 min prior to anxiolytic dose of ethanol
(2 g/kg, i.p.) and subsequently subjected to EPM test.
2.7. Inuence of imidazoline receptor agonists and antagonists on
ethanol-induced anxiolysis
In view of high afnity of endogenous agmatine towards imidazoline receptors, we investigated the effect of imidazoline receptor
agonists and antagonists on ethanol and agmatine induced anxiolysis.
The doses are selected on the basis of our preliminary experiments
and reports (Zeidan et al., 2007; Taksande et al., 2009). These drugs at
the doses used here per se did not evoke any response in EPM.
Separate groups of rats were treated either with saline, imidazoline I1/2 receptor agonists moxonidine (0.25 mg/kg, i.p.), clonidine
(0.015 mg/kg, i.p.) or imidazoline I2 receptor agonist, 2-BFI (5 mg/kg,
i.p.) before 10 min of subeffective dose of ethanol (8% w/v; 1 g/kg, i.p.)
or agmatine (10 mg/kg, i.p.) and subjected to EPM test.
For the antagonism studies, rats were pretreated with imidazoline I1
receptor antagonist, efaroxan (1 mg/kg, i.p.) or I2/2 receptor antagonist,
idazoxan (0.25 mg/kg, i.p.) 10 min prior to anxiolytic dose of ethanol (8%
w/v; 2 g/kg, i.p.) or agmatine (40 mg/kg, i.p.) or their subeffective dose
combination and subjected to EPM test as described earlier.
2.8. Ethanol withdrawal anxiety
Briey, rats housed individually were given free access to balanced
liquid diet (Novartis India Ltd., Mumbai) for 7 days to allow adaptation to
the novel food. From 8th day onwards, half of the subjects received liquid
diet containing 2.4% ethanol for 3 days, which was increased to 4.8% for
the following 4 days and nally to 7.2% for 14 days i.e. total 21 days
(ethanol fed group) (Uzbay et al., 2000). The remaining animals
continued on the control liquid diet (pair-fed group). Fresh aliquot of
ethanol diet and/or control liquid diet was introduced in the cage each
morning at 0900 h. From day 22 (0900 h), pair-fed groups were
continued on the same diet, whereas to ethanol fed groups, ethanol
containing diet was discontinued and replaced with ethanol free liquid
diet until the termination of the experiment (ethanol withdrawal).

91

Control rats were pair fed with an isocaloric liquid diet containing sucrose
as a caloric substitute to ethanol. At 24 h post ethanol withdrawal, the
pair-fed groups as well as ethanol fed groups were subjected to EPM test.
This time point was selected based on our earlier studies (Kokare et al.,
2006; Taksande et al., 2009) carried out at different time intervals
including 0, 12, 24, 48 and 72 h. Post ethanol withdrawal showed peak
anxiety in EPM at 24 h. Moreover, 24 h post ethanol withdrawal time
point for peak anxiety is very well accepted in most of the alcohol
withdrawal studies (Dandekar et al., 2008; Pandey et al, 1999).
The diet consumption and body weight of each animal was monitored
daily (0900 h) for all groups. Each rat daily consumed 13.62.3 g/kg
ethanol and body weight were not signicantly deviated from pair-fed
animals. On 21st day of experiment, the mean body weight of pair-fed and
ethanol fed animals was 2382.3 g and 2453.2 g respectively.
2.9. Effect of drugs on ethanol withdrawal anxiety
These protocols were designed to assess the effect of various drugs on
ethanol withdrawal anxiety. At 24 h post withdrawal, i.e. on day 23
(0900 h) the animals showed maximum ethanol abstinence anxiety.
Ethanol-withdrawn group was pretreated with agmatine or its
modulators and ligands of imidazoline receptors 30 min prior to EPM
test. Animals (n = 68) received one of the following drugs, agmatine
(520 mg/kg, i.p.), DFMO (125 g/rat, i.c.v.), aminoguanidine (65 g/rat,
i.c.v.), arcaine (50 g/rat, i.c.v.), L-arginine (100 g/rat, i.c.v.), moxonidine
(0.25 mg/kg, i.p.), clonidine (0.015 mg/kg, i.p.), 2-BFI (5 mg/kg, i.p.),
aCSF (5 l/rat, i.c.v.) or saline (5 ml/kg, i.p.). These doses were selected on
the basis of acute studies.
To determine blood ethanol concentration, the blood samples
were collected from pair-fed control, ethanol fed and in ethanolwithdrawn animals at 0 and 24 h post withdrawal time point.
In this set of experiment we investigated whether agmatine
induced attenuation of ethanol withdrawal anxiety can be blocked by
imidazoline receptor antagonists. Imidazoline I1 receptor antagonist,
efaroxan (1 mg/kg, i.p.) or imidazoline I2/2 receptor antagonist,
idazoxan (0.25 mg/kg, i.p.) were injected to ethanol-withdrawn rats
10 min prior to anti-anxiety dose of agmatine (20 mg/kg, i.p.). After
30 min, all the animals were subjected to the EPM test.
2.10. Data analysis
At the end of the experiment, dilute India ink was injected by i.c.v.
route and the animals were euthanized by an overdose of pentobarbital
sodium (80 mg/kg, i.p.). Immediately, the brain of rat was dissected out
and cannula placement was veried histologically for distribution of ink
in the ventricles. The guide cannulae were found to be incorrectly placed
in some animals (20%) and these were excluded from the observations.
Data from only those animals with uniform distribution of ink in the
ventricles were considered for statistical analysis.
The results are presented as mean S.E.M. The effects of different
acute drug treatments were statistically analyzed by one way analysis of
variance (ANOVA) with repeated measures on drug treatments
followed by post hoc Dunnett's test. The data obtained from ethanolwithdrawn and pair-fed rats were compared by unpaired t-test. The
acute combination protocols and treatment of drugs in ethanolwithdrawn rats were analyzed by either unpaired t test or one way
repeated-measures ANOVA and individual means were compared
either by Dunnett's or NewmanKeuls post hoc test. A value of P b 0.05
was considered signicant.
3. Results
3.1. Agmatine potentiated anxiolytic effect of ethanol in EPM
As shown in Fig. 1, acute injections of ethanol (1.52 g/kg, i.p.) and
agmatine (4080 mg/kg, i.p.) showed signicant effect on percent open-

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B.G. Taksande et al. / European Journal of Pharmacology 637 (2010) 89101

Fig. 1. Effect of (A) ethanol (12.5 g/kg, i.p.), (B) agmatine (1080 mg/kg, i.p.) and (C) their combination on behavior of rat in the EPM test. Rats were injected 30 min before testing
with vehicle, increasing doses of ethanol or agmatine. Separate groups of rats were injected with ineffective doses of agmatine (1020 mg/kg, i.p.) and 30 min later injected with
ethanol (1 g/kg, i.p.). Thirty min thereafter, individual rat was placed in the center of the arm to explore the EPM for 5 min. Each bar represents mean S.E.M. (n = 68). *, #, $P b 0.05,
**, $$P b 0.01, # #, ***P b 0.001 vs respective control (One way repeated measure ANOVA followed by Dunnett's test).

arm time (% OAT) [ethanol F (4, 39) = 38.55, P b 0.001; agmatine F

(4, 39) = 32.89, P b 0.001] and percent open-arm entries (% OAE)
[ethanol F (4, 39) = 15.70, P b 0.001; agmatine F (4, 39) = 4.947,
P b 0.01] as compared to respective control. Post hoc analysis showed
signicant increase in % OAT [ethanol 1.5 g/kg (P b 0.001) and 2 g/kg
(P b 0.001); agmatine 40 mg/kg (P b 0.001), 80 mg/kg (P b 0.001)] and
% OAE [ethanol 1.5 g/kg (P b 0.05), 2 g/kg (P b 0.01); agmatine
40 mg/kg (P b 0.05), 80 mg/kg (P b 0.05)] but did not affected closed-arm
entries in these doses. Though their lower doses i.e. 1 g/kg, ethanol and
1020 mg/kg, agmatine were found to be ineffective, the higher dose of
ethanol (2.5 g/kg) however signicantly reduced the percent time spent
(P b 0.05) and entries into open-arm (P b 0.01) as well as the number of
closed-arm entries (P b 0.001). Based upon these results, ethanol 1 g/kg
and agmatine 10 mg/kg dose were selected as sub-threshold dose for
further experiments.
Pretreatment of agmatine revealed signicant interaction [% OAT
F (2, 17) = 7.210, P b 0.01; % OAE F (2, 17) = 7.287, P b 0.01] with
ineffective dose of ethanol (1 g/kg i.p.). The post hoc analysis showed
that agmatine (10 and 20 mg/kg; P b 0.05 and P b 0.01 resp.) enhanced
the % OAT and % OAE produced by ethanol (Fig. 1C). The total numbers
of closed-arm entries remained unchanged in all treatment groups.

Based upon these results, ethanol 1 g/kg and agmatine 10 mg/kg

dose were selected as sub-threshold dose for further experiments.

3.2. Enhanced brain agmatine content augmented anxiolytic effect of

ethanol in EPM
As depicted in Fig. 2, pretreatment with the agmatine modulators
viz., L-arginine (100 g/rat, i.c.v.), DFMO (125 g/rat, i.c.v.), aminoguanidine (65 g/rat, i.c.v.) and arcaine (50 g/rat, i.c.v.) showed signicant
effect on % OAT (One way ANOVA F (9, 69) = 11.95, P b 0.001) and %
OAE (One way ANOVA F (9, 69) = 2.745, P b 0.01) produced by ethanol
(1 g/kg). Post hoc NewmanKuels comparisons showed signicant
potentiation of anxiolytic effect of ethanol by L-arginine (% OAT
P b 0.001; % OAE P b 0.05), DFMO (% OAT P b 0.01; % OAE P b 0.05),
aminoguanidine (% OAT P b 0.001; % OAE P b 0.05) and arcaine
(% OAT P b 0.001; % OAE P b 0.05). However, these treatments had no
signicant effect on total number of closed-arm entries. Administration
of L-arginine or DFMO or aminoguanidine or arcaine per se had no effect
on time spent and entries into open or closed arm as compared to aCSF
treated rats.

B.G. Taksande et al. / European Journal of Pharmacology 637 (2010) 89101

93

Fig. 3. Effect of D-arginine on anxiolytic effect of ethanol (2 g/kg, i.p.) in EPM showing
(A) % time spent in open arm, (B) % open-arm entries and (C) closed-arm entries. Rats were
injected with the D-arginine (8% w/v in saline; 100 g/rat, i.c.v.) or vehicle. Thirty min after
rats were injected with ethanol (2 g/kg, i.p.) or saline and 30 min thereafter, individual rat
was placed in the center of the arm to explore the EPM for 5 min. Each bar represents mean
S.E.M. (n=6). *Pb 0.05, **Pb 0.001 vs control group (Unpaired t test).

Fig. 2. Effect of enhanced brain agmatine content on anxiolytic effect of ethanol (1 g/kg, i.p.)
recorded during exploration for 5 min in EPM showing (A) % time spent in open arm,
(B) % open-arm entries and (C) closed-arm entries. Rats were injected i.c.v. with the DFMO
(125 g/rat), aminoguanidine (65 g/rat), arcaine (50 g/rat), L-arginine (100 g/rat) or
vehicle. Thirty min after rats were injected with ethanol (1 g/kg, i.p.) or saline and 30 min
thereafter, individual rat was placed in the center of the arm to explore the EPM for 5 min.
Each bar represents meanS.E.M. (n=7) *Pb 0.05, **P b 0.01, ***Pb 0.001 vs respective
control (One way ANOVA post hoc NewmanKuels test).

effect on total number of closed-arm entries. The administration of Darginine alone to saline treated animals did not show any response in
EPM test at the dose used here. These results are shown in Fig. 3.

3.3. Agmatine synthesis inhibitor, D-arginine blocked anxiolytic effect of

ethanol in EPM

3.4. Imidazoline receptor agonists enhanced while antagonist blocked

anxiolytic effect of ethanol and agmatine

Pretreatment of animals with arginine decarboxylase inhibitor, Darginine (100 g/rat, i.c.v.) signicantly blocked anxiolytic effect of
ethanol (8% w/v in saline, 2 g/kg, i.p.) as evident from decreased % OAT
[Unpaired t test, t = 2.942, df = 10, P b 0.05] as well as % OAE
[Unpaired t test, t = 5.345, df = 10, P b 0.001] as compared to ethanol
(2 g/kg, i.p.) treated group. However, this treatment had no signicant

Fig. 4, shows that imidazoline receptor agonists viz., moxonidine

(0.25 mg/kg, i.p.), clonidine (0.015 mg/kg, i.p.) and 2-BFI (5 mg/kg, i.p.)
signicantly potentiated the effect of ethanol (8% w/v in saline, 1 g/
kg, i.p.) [One way ANOVA; % OAT F (7, 47) = 13.06, P b 0.001; % OAE
F (7, 47) = 12.16, P b 0.001] and agmatine (10 mg/kg, i.p.) [One way
ANOVA; % OAT F (7, 47) = 24.79, P b 0.001; % OAE F (7, 47) = 21.61,

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Fig. 4. Effect of imidazoline agonists on anxiolytic effect of ethanol (1 g/kg, i.p.) or

agmatine (10 mg/kg, i.p.) recorded during exploration for 5 min in the EPM showing
(A) % time spent in open arm, (B) % open-arm entries and (C) the number of closed-arm
entries. Rats were injected with moxonidine (0.25 mg/kg, i.p.), clonidine (0.015 mg/kg,
i.p.) or 2-BFI (5 mg/kg, i.p.) or vehicle. Thirty min after rats were injected with ethanol
(1 g/kg, i.p.) or agmatine (10 mg/kg, i.p.) or saline and 30 min thereafter, individual rat
was placed in the center of the arm to explore the EPM for 5 min. Each bar represents
mean S.E.M. (n = 6). $P b 0.01, $$, *P b 0.001 vs respective control group (One way
ANOVA post hoc NewmanKuels test).

P b 0.001]. Post hoc NewmanKuels comparisons indicated the signicant potentiation of anxiolytic effect of ethanol and agmatine by
moxonidine [ethanol (% OAT P b 0.001, % OAE P b 0.001); agmatine
(% OAT P b 0.01, % OAE P b 0.001)], clonidine [ethanol (% OAT
P b 0.001, % OAE P b 0.001); agmatine (% OAT P b 0.001, % OAE
P b 0.001)] and 2-BFI [ethanol (% OAT P b 0.001, % OAE P b 0.001);
agmatine (% OAT P b 0.001, % OAE P b 0.001)]. However, these
treatments had no signicant effect on total number of closed-arm
entries. Administration of ethanol, agmatine, moxonidine, clonidine or
2-BFI alone at the doses employed here did not evoke any response in
EPM test as compared to their respective control.
On the other hand, pretreatment with imidazoline receptor
antagonists signicantly blocked anxiolytic effect of ethanol (2 g/kg,
i.p.) [% OAT F (5, 35) = 18.18, P b 0.001; % OAE F (5, 35) = 4.309,
P b 0.01] and agmatine (40 mg/kg, i.p.) [% OAT F (5, 35) = 18.92,
P b 0.001; % OAE F (5, 35) = 4.848, P b 0.01] in EPM test (Fig. 5). Post
hoc NewmanKuels comparison demonstrated the signicant effect of
efaroxan (1 mg/kg, i.p.) [ethanol (% OAT P b 0.001, % OAE P b 0.01);
agmatine (% OAT P b 0.001, % OAE P b 0.01)] and idazoxan

Fig. 5. Effect of imidazoline antagonists on anxiolytic effect of ethanol (2 g/kg, i.p.)

or agmatine (40 mg/kg, i.p.) recorded during exploration for 5 min in EPM showing
(A) % time spent in open arm, (B) % open-arm entries and (C) closed-arm entries. Rats
were injected with the efaroxan (1 mg/kg, i.p.) or idazoxan (0.25 mg/kg, i.p.) or vehicle.
Thirty min after rats were injected with ethanol (1 g/kg, i.p.) or saline and 30 min
thereafter, individual rat was placed in the center of the arm to explore the EPM for
5 min. Each bar represents mean S.E.M. (n = 6). *, $P b 0.01, **, $$P b 0.001 vs respective
control group (One way ANOVA post hoc NewmanKuels test).

(0.25 mg/kg, i.p.) [ethanol (% OAT P b 0.001, % OAE P b 0.01);

agmatine (% OAT P b 0.001, %OAE P b 0.01)] on the EPM behavior of
ethanol or agmatine treated rats (Fig. 5). However, these treatments
had no signicant effect on total number of closed-arm entries.
Efaroxan (1 mg/kg, i.p.) or idazoxan (0.25 mg/kg, i.p.) alone did not
signicantly affect open-arm or closed-arm variables. These effects are
depicted in Fig. 5.
3.5. Imidazoline receptors antagonists attenuated synergistic anxiolytic
effect of ethanol and agmatine
The effect of efaroxan and idazoxan on anxiolysis produced
by coadministration of ethanol and agmatine are shown in Fig. 6.
Simultaneous administration of ethanol (1 g/kg, i.p.) and agmatine
(10 mg/kg, i.p.) at the doses that were ineffective themselves resulted
in signicant anxiolysis in EPM test. Synergistic effect of ethanol and
agmatine, however was blocked by pretreatment of rats with imidazoline
receptor antagonists [One way ANOVA % OAT F (5, 35)=10.48,
Pb 0.001; % OAE F (5, 35)=13.99, Pb 0.05]. Post hoc NewmanKuels
comparisons demonstrated signicant effect of efaroxan (1 mg/kg, i.p.)
[% OAT P b 0.001; % OAE P b 0.05] or idazoxan (0.25 mg/kg, i.p.)

B.G. Taksande et al. / European Journal of Pharmacology 637 (2010) 89101

95

Pb 0.01], % OAE [unpaired t test; t=6.859, df =10, Pb 0.001] and total

number of closed-arm entries [unpaired t test; t=3.721, df =10,
Pb 0.01] as compared to pair-fed animals (Fig. 7). Acute administration
of agmatine (1020 mg/kg, i.p.) [% OAT F (3, 23)=5.202, P b 0.01;
% OAE F (3, 23)=13.18, Pb 0.001] attenuated ethanol withdrawalinduced anxiety. Post hoc comparisons indicated signicant effect of 10
(% OAT P b 0.05; % OAE P b 0.001) as well as 20 mg/kg (% OAT
P b 0.01; % OAE P b 0.001) dose of agmatine. Its lower dose (5 mg/kg)
was found to be ineffective (Fig. 7).

Fig. 6. Effect of imidazoline antagonists on subeffective dose combination of agmatine

(10 mg/kg, i.p.) and ethanol (1 g/kg, i.p.) in EPM (5 min) showing (A) % time spent in
open arm, (B) % open-arm entries and (C) number of closed-arm entries. Rats were
injected with the efaroxan (1 mg/kg, i.p.) or idazoxan (0.25 mg/kg, i.p.) or vehicle.
Thirty min after rats were injected with combination of agmatine (10 mg/kg, i.p.) and
ethanol (1 g/kg, i.p.) or saline, and 30 min thereafter, individual rat was placed in the
center of the arm to explore the EPM for 5 min. Each bar represents mean S.E.M.
(n = 6). *P b 0.05, **P b 0.01, ***P b 0.001 vs respective control (One way ANOVA post hoc
NewmanKuels test).

[% OAT P b 0.01; % OAE P b 0.01]. However, these treatments had no

signicant effect on closed-arm entries.
3.6. Ethanol withdrawal anxiety ameliorated by agmatine, its modulators
and imidazoline receptor agonists
Chronic exposure to ethanol for 21 days and its abrupt discontinuation
produced marked anxiety at 24 h. Withdrawal from chronic ethanol
exposure led to decrease in % OAT [unpaired t test; t=3.822, df =10,

Fig. 7. Anxiogenic effects of withdrawal from chronic ethanol consumption and

blockade by agmatine (520 mg/kg, i.p.). Rats were given free access to ethanol through
liquid modied diet for of 21 days and from the 22nd day onwards they were deprived
of ethanol. After 24 h of withdrawal they were treated with agmatine and 30 min
thereafter the exploratory behavior was observed for 5 min in EPM test showing (A) %
time spent in open arms, (B) % open-arm entries and (C) the closed-arm entries. Each
bar represents the mean S.E.M. (n = 6). *P b 0.01, **P b 0.001 vs control (Unpaired t
test), #P b 0.05, ##P b 0.01, ###P b 0.001 vs ethanol-withdrawn group (One way ANOVA
post hoc Dunnett's test).

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B.G. Taksande et al. / European Journal of Pharmacology 637 (2010) 89101

Similarly, L-arginine, aminoguanidine and arcaine showed significant effect on ethanol withdrawal anxiety [One way ANOVA; % OAT
F (4, 29) = 9.228, P b 0.001; % OAE F (4, 29) = 14.37, P b 0.001].
Post hoc Dunnett's comparisons showed signicant reduction in
ethanol withdrawal anxiety by L-arginine (100 g/rat, i.c.v.) (% OAT
Pb 0.01; % OAE Pb 0.001), DFMO (125 g/rat, i.c.v.) (% OAT Pb 0.01;
% OAE Pb 0.001), aminoguanidine (65 g/rat, i.c.v.) (% OAT Pb 0.001;
% OAE Pb 0.001) and arcaine (50 g/rat, i.c.v.) (% OAT P b 0.001;
% OAE P b 0.001). The total number of closed-arm entries remained
unaffected (Fig. 8).

Fig. 9. Effect of imidazoline receptor agonists on 24 h of ethanol withdrawal. Rats were

injected with moxonidine (0.25 mg/kg, i.p.), clonidine (0.015 mg/kg, i.p.) or 2-BFI
(5 mg/kg, i.p.) or vehicle and 30 min thereafter rats were subjected to test. The
exploratory behavior was observed for 5 min in EPM test showing (A) % time spent in
open arms, (B) % open-arm entries and (C) the closed-arm entries. Each bar represents
the mean S.E.M. (n = 6). *P b 0.05, **P b 0.01, ***P b 0.001 vs ethanol-withdrawn rats
(One way ANOVA post hoc Dunnett's test).

Fig. 8. Effect of increased brain agmatine content on 24 h of ethanol withdrawalinduced anxiety. Rats were injected i.c.v. with DFMO (125 g/rat) or aminoguanidine
(65 g/rat) or arcaine (50 g/rat) or L-arginine (100 g/rat) or aCSF and 30 min
thereafter rats were subjected to test. The exploratory behavior was observed for 5 min
in EPM test showing (A) % time spent in open arms, (B) % open-arm entries and (C) the
closed-arm entries. Each bar represents the mean S.E.M. (n = 6). *P b 0.01, **P b 0.001
vs ethanol-withdrawn rats (One way ANOVA post hoc Dunnett's test).

As shown in Fig. 9, ethanol-withdrawn rats, treated with imidazoline

receptor agonists viz., moxonidine (0.25 mg/kg, i.p.), clonidine (0.015 mg/
kg, i.p.), 2-BFI (5 mg/kg, i.p.) signicantly ameliorated ethanol withdrawal anxiety [One way ANOVA; % OAT F (3, 23)=8.508, Pb 0.001;
% OAE F (3, 23)=4.959, Pb 0.01]. Post hoc comparison demonstrated
the signicant effect of moxonidine (OAT Pb 0.01; OAE Pb 0.05) and
clonidine (% OAT Pb 0.01; % OAE Pb 0.05), 2-BFI (% OAT Pb 0.001;
% OAE Pb 0.01) on the EPM behavior of ethanol-withdrawn rats.
However, total number of closed-arm entries remained unaffected.

B.G. Taksande et al. / European Journal of Pharmacology 637 (2010) 89101

As shown in Fig. 10, the effect of agmatine (20 mg/kg, i.p.) on ethanol
withdrawal anxiety was completely abolished by efaroxan (1 mg/kg, i.p.)
[% OAT F (3, 23)=9.967, Pb 0.001; % OAE F (3, 23)=8.105,
Pb 0.001] or idazoxan (0.25 mg/kg, i.p.) [% OAT F (3, 23)=10.82,
Pb 0.001; % OAE F (3, 23)=11.82, Pb 0.001]. Post hoc Dunnett's analysis
indicated signicant effect of efaroxan (% OAT Pb 0.01; % OAE
Pb 0.01) and idazoxan (% OAT P b 0.01; % OAE P b 0.01) on the
anxiolytic effect of agmatine in ethanol-withdrawn rats. Administration
of efaroxan or idazoxan to ethanol-withdrawn rats did not evoke any
response in EPM test.

97

Table 1
Blood ethanol concentration (mg/dl).
Treatment groups
Acute study
Saline
Ethanol (2 g/kg)
Ethanol (1 g/kg)
Agmatine (10 mg/kg) + ethanol (1 g/kg)
Chronic study
Chronic ethanol fed group
Control ethanol withdrawal group
Agmatine (40 mg/kg, ip) treated ethanol
withdrawal group

Blood ethanol concentration

(BEC) (mg/dl)
0.89 0.27
92.5 2.4
56.8 4.7
53.2 3.9
177.6 3.7
5.3 1.3
4.7 3.5

Blood ethanol concentration in rats following acute treatment of ethanol (1, 2 g/kg, ip) and
subeffective dose combination of ethanol (1 g/kg, ip) + agmatine (10 mg/kg, ip). In
chronic studies, separate group of rats received ethanol in liquid modied diet and blood
ethanol concentration was measured 24 h before and after withdrawal treated with either
saline or agmatine 30 min before 24 h withdrawal. Data expressed as mean S.E.M.
(n = 6).

3.7. Blood ethanol concentration

As shown in Table 1, blood ethanol levels were 92.52.4, 177.63.7
and 5.31.3 mg/dl (meanS.E.M.) after acute ethanol administration,
21 days ethanol exposure and at the time of 24 h of ethanol withdrawal,
respectively. No signicant differences in blood ethanol concentration
were observed between the ethanol and ethanol+agmatine treated
groups.
4. Discussion

Fig. 10. Effect of imidazoline receptor antagonists on anti-anxiety effect of agmatine in

24 h ethanol withdrawal rats. Rats were pretreated with the efaroxan (1 mg/kg, i.p.) or
idazoxan (0.25 mg/kg, i.p.) or vehicle. Thirty min after rats were injected with agmatine
(40 mg/kg, i.p.) or saline 30 min thereafter rats were subjected to test. The exploratory
behavior was observed for 5 min in EPM test showing (A) % time spent in open arms,
(B) % open-arm entries and (C) the closed-arm entries. Each bar represents the mean
S.E.M. (n = 6). *P b 0.01, **P b 0.001 vs ethanol-withdrawn rats (One way ANOVA post
hoc NewmanKuels test).

The present study demonstrated the effects of agmatine on acute

ethanol anxiolysis and withdrawal anxiety in rats using EPM test.
Consistent with our previous ndings (Hirani et al., 2005; Kokare et al.,
2006) and of others (Durcan and Lister, 1988; Belzung and Berton, 1997;
Bilkei-Gorzo et al., 1998; LaBuda and Fuchs, 2002; LaBuda and Hale,
2000) acute administration of ethanol (1.52 g/kg) produced a dosedependent anti-anxiety effect in the EPM test as evident from increased
percent time spent and entries into open arms without any change in
closed-arm entries. However, animals treated with higher dose (2.5 g/
kg, i.p.) of ethanol showed decrease in open as well as closed-arm
activity in the EPM test, indicating mild sedation or motor incoordination as previously reported (Hirani et al., 2005). Absolute
number of closed-arm entries is a more reliable measure for locomotion
in EPM paradigm (Nunes-de-Souza et al., 2000). As reported earlier
(Lavinsky et al., 2003; Gong et al., 2006) agmatine (40 and 80 mg/kg, i.p.)
exerted anxiolytic action in rats. The present work shows for the rst
time that agmatine in per se noneffective doses signicantly potentiated
anxiolytic effect of ethanol in EPM test. Blood ethanol analysis showed no
signicant differences in blood ethanol concentration between ethanol
and ethanol + agmatine treated rats, suggesting that the effects of
agmatine were not due to any obvious effects on ethanol metabolism
(Lewis et al., 2007).
We have also investigated the ethanol anxiolysis in the presence of
agents like L-arginine, DFMO, aminoguanidine and arcaine modulating endogenous brain agmatine levels. The greater agmatine levels in
brain may be accomplished by increased biosynthesis or decreased
degradation of this substance. Biosynthesis of agmatine by L-arginine
decarboxylase (L-ADC) depends upon the availability of L-arginine (Su
et al., 2003). L-arginine is also converted into ornithine and nitric
oxide (NO) by an enzyme arginase and nitric oxide synthase,
respectively (Reis and Regunathan, 2000). Ornithine subsequently
converted into putrescine by L-ornithine decarboxylase. Inhibition of
either metabolic pathway is reported to increase activity in other
metabolic pathways. Administration of DFMO, which inhibits both Lornithine decarboxylase and arginase enzyme (Slotkin et al., 1982;
Selamnia et al., 1998) and subsequently stimulates L-ADC (Hernandez

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B.G. Taksande et al. / European Journal of Pharmacology 637 (2010) 89101

and Schwarcz de Tarlovsky, 1999), might increase the availability of

agmatine in brain (Lu et al., 2003). Agmatine is metabolized to
putrescine and guanido-butanoic acid by enzyme agmatinase and
diamine oxidase respectively (Reis and Regunathan, 2000) and
inhibition of these enzymes results in augmentation of endogenous
agmatine (Regunathan, 2006; Lu et al., 2003; Huang et al., 2003). In
the present study we used diamine oxidase (DAO) inhibitor,
aminoguanidine (Lu et al., 2003) and agmatinase inhibitor, arcaine
(Huang et al., 2003; Regunathan, 2006). In fact, results of our previous
study clearly indicate that all these drugs substantially increase the
levels of agmatine in rat brain (Taksande et al., 2009). All these
manipulations potentiated the anxiolytic effect of ethanol in EPM.
Conversely, L-ADC inhibitor, D-arginine attenuated anxiolytic effect of
ethanol. D-arginine (isomer of L-arginine) is known to inhibit ADC in
plants and bacteria (Rosenfeld and Roberts, 1976; Hao et al., 2005)
and thereby blocks conversion of L-arginine to agmatine. It is
extensively used in studies on L-arginine/nitric oxide pathway in
mammals (Navarro et al., 2005). Thus, anxiolytic effect of ethanol in
EPM might be closely related with modulation of endogenous
agmatine levels in brain. The acute treatment of control rats with
either of these modulators alone was devoid of any effect in EPM test.
Thus, the agmatine accumulation in brain by these agents may not be
adequate to evoke anxiolytic effect per se. However, when administered acutely together with ethanol exhibited synergistic anxiolytic
effect. It may be due to additive accumulation of agmatine
concentration in brain sufcient to induce anxiolysis. More extensive
analytical studies are required to verify these ndings.
The central effects of agmatine are diverse and have been commonly
linked to its ability to bind imidazoline receptors. Brain regions regulating
endocrine and affective functions have abundant imidazoline binding
sites and colocalized with its endogenous ligands like agmatine (De Vos
et al., 1994; Raasch et al., 1995; Otake et al., 1998). Moreover, substantial
agmatine immunoreactivity has been detected in areas like VTA, NAc,
amygdala, etc that are involved in regulation of anxiety (Otake et al., 1998;
Davis, 1997; Koob, 2003). Major depression, opioid addiction, neurodegenerative diseases and glial tumors are associated with disturbances of
imidazoline receptors in the human brain (Garcia-sevilla et al., 1998).
Therefore, we investigated in the present study the possible involvement
of imidazoline receptors in ethanol or agmatine induced anxiolysis. The
anxiolytic effects of both agmatine and ethanol were augmented by
ineffective doses of moxonidine or clonidine, mixed imidazoline I1/2
receptor agonists or 2-BFI, imidazoline I2 receptor agonist. In contrast,
efaroxan (1 mg/kg, i.p.) and idazoxan (0.25 mg/kg, i.p.) an antagonist of
imidazoline I1 and imidazoline I2 receptor respectively counteracted the
anxiolytic effect of not only agmatine and ethanol alone but also the
synergistic interaction of their combination. The alone doses of imidazoline receptor agonists and antagonists used here were devoid of any
effect in EPM. This is in agreement with the reports that efaroxan and
idazoxan both attenuated several behavioral and autonomic actions of
chronic and acute ethanol administration (Grupp et al., 1997; El-Mas and
Abdel-Rahman, 2001). Although we have not carried out blood ethanol
concentration in the presence of imidazoline drugs, the chances of
pharmacokinetic interaction between ethanol and imidazoline agents
employed in the study cannot be ruled out. However, this issue clearly
merits further investigation.
Thus, our results suggest a critical role for endogenous brain
agmatine levels and its resultant interaction with imidazoline
receptors in ethanol-induced anxiolysis. However, it is not clear
whether this interaction of ethanol and imidazoline receptors is direct
or involves endogenous release of agmatine. Nonetheless it is difcult
to rule out the involvement of additional 2 adrenoreceptors since
agmatine displays afnity for both I1/I2 and 2 adrenoreceptors.
Polyamines play variety of roles in CNS development (Slotkin and
Bartolome, 1986) and increased polyamine expression in certain brain
areas has been reported during ethanol withdrawal (Davidson and Wilce,
1998; Gibson et al., 2003). Since agmatine is a polyamine precursor and is

reported to alleviate behaviors associated with ethanol withdrawal

syndrome (Uzbay et al., 2000), we extended the present study to
withdrawal anxiety. It was found that average daily ethanol consumption
of individual rat was about 13.62.3 g/kg and attains adequate blood
ethanol levels (177.6 3.7 mg/dl) as measured just before the ethanol
withdrawal. Several reports suggest that the consumption of 915 g/kg of
ethanol per day for more than 15 days leads to physical dependence in
rodents (Uzbay et al., 1994; Uzbay and Kayaalp, 1995). Our data shows
that termination of ethanol ingestion caused signicant reduction in
percentage of open-arm entries and open-arm time in EPM test
indicating anxiety like behavior. The closed-arm entries were also
markedly decreased. This is in agreement with the nding that
withdrawal of ethanol following chronic treatment generates anxiety
(Lal et al., 1991; Rezazadeh et al., 1993; Pandey et al., 1999; Gatch et al.,
2000), which is a measure of psychological dependence and almost
always associated with a decrease in general activity as measured by
closed or total arm entries (Lal et al., 1993; Pandey et al., 1999; Gatch
et al., 2000; Jung et al., 2000). In the present study, systemic injection of
agmatine and its enhancers like L-arginine, DFMO, aminoguanidine and
arcaine prior to ethanol withdrawal anxiety test counteracted anxiety like
behavior in ethanol-withdrawn rats. The doses of agmatine or its
modulators used here did not alter the behavior of animals per se. This is
in agreement with earlier nding of Uzbay et al (2000) demonstrating
dose-dependent effect of agmatine on ethanol withdrawal syndrome in
rats. It was shown that agmatine signicantly inhibited stereotyped
behaviors, wet dog shakes and tremors appeared during the ethanol
withdrawal. Attenuation of ethanol withdrawal syndrome in rats by high
doses of L-arginine (1000 mg/kg, i.p.) is reported to be associated with its
metabolite agmatine (Uzbay and Erden, 2003). DFMO, a polyamine
synthesis inhibitor decreases long lasting protein oxidation induced by
neonatal ethanol exposure in the hippocampus of adolescent rats (Mello
et al., 2007). Moreover, it inhibits ethanol withdrawal-induced neurotoxicity in rat hippocampal slice cultures through signicant reduction in
polyamines like putrescine, spermine and spermidine (Gibson et al.,
2003). The concentration of polyamines is positively correlated with the
severity of withdrawal-induced tremors and seizures in ethanol
dependent animals (Davidson and Wilce, 1998). They not only potentiate
ethanol withdrawal-induced cell death in vitro (Prendergast et al., 2000;
Gibson et al., 2003) but also are implicated in the pathogenesis of fatal
alcohol syndrome (Littleton et al., 2001; Sessa et al., 1987; Sessa and
Perin, 1997). It is important to note that agmatine competitively binds
with polyamine site of NMDA receptors through which polyamines
mediate its actions related with ethanol withdrawal (Gibson et al., 2002,
2003; Lewis et al., 2007).
Our data also shows that pretreatment with aminoguanidine also
reversed the ethanol withdrawal anxiety. Aminoguanidine augments
the endogenous agmatine levels by inhibiting enzyme DAO that
metabolizes agmatine into guanido-butanoic acid (Reis and Regunathan, 2000). Besides its interaction with imidazoline receptors the
elevation of agmatine levels promotes inhibition of NOS in brain.
Several NOS inhibitors like NG-nitroarginine-methyl-ester (L-NAME)
and 7-nitroindazole (7-NI) enhance the anxiolytic effect of alcohol
(Ferreira et al., 1999) and exhibit potent inhibitory effects on ethanol
withdrawal symptoms (Uzbay et al., 1997). This indicates that NO
production may also be critically involved in ethanol withdrawal
anxiety. The neuronal glutamate-NO pathway is known to modulate
many physiological processes including drug dependence (Costa et al.,
2003; Itzhak, 2008). NO is an intermediary in the actions of glutamate
and is known to form as a result of the NMDA receptor activation
(Garthwaite et al., 1989; Chandler et al., 1997). Since agmatine
selectively block NMDA receptor and NO formation (Yang and Reis,
1999; Askalany et al., 2005), it is likely that this property may have
some relevance to benecial effect of agmatine in ethanol withdrawal
anxiety. However, it should be veried whether NOS inhibition,
NMDA receptor inhibition and imidazoline receptors activation by
agmatine are directly related.

B.G. Taksande et al. / European Journal of Pharmacology 637 (2010) 89101

The present study also demonstrated reversal of ethanol withdrawal

anxiety by imidazoline I1 agonists, clonidine and moxonidine and
imidazoline I2 agonists, 2-BFI. This effect of imidazoline receptor
agonists on ethanol withdrawal anxiety is also supported by various
preclinical studies (Mao and Abdel-Rahman, 1996; Opitz, 1990; Parale
and Kulkarni, 1986; Vandergriff et al., 2000). Number of preclinical
studies has demonstrated the complex interaction between imidazoline
receptors system and ethanol. Chronic ethanol administration tends to
counteract the hypotensive effect of imidazoline I1 receptor agonists
clonidine and rilmenidine (El-Mas and Abdel-Rahman, 2001) which
was completely reversed by efaroxan, an imidazoline I1 receptor
antagonist. Clonidine, produced synergistic behavioral interaction
with ethanol, reduced alcohol intake in ethanol preferring rats and
alleviated alcohol abstinence syndrome in rats (Mondavio and Ghiazza,
1989; Mao and Abdel-Rahman, 1996; Opitz, 1990; Parale and Kulkarni,
1986). Moxonidine, an imidazoline I1 receptor agonist signicantly
attenuated the ethanol withdrawal-induced elevation of acoustic startle
response in rats (Vandergriff et al., 2000). Conversely, imidazoline I2
receptor antagonist idazoxan, stimulated ethanol intake in rats and
blocked locomotor stimulant, anxiolytic and hypothermic effect of acute
ethanol administration (Durcan et al., 1989; Grupp et al., 1997). In
addition, imidazoline receptors endogenous ligands like harmane and
-carboline act on brain reward system and increase voluntary ethanol
drinking in animals (Adell and Myers, 1994). In fact, uctuation in the
blood levels of harmane was directly correlated with anxiety and
depression associated with ethanol withdrawal in rats (Rommelspacher
et al., 1996). More importantly, agmatine itself has inhibited behavioral
symptoms during the ethanol withdrawal syndrome in rats (Uzbay
et al., 2000). These ndings suggest that agmatine and its interaction
with imidazoline receptors may mediate some effects of ethanol.
However, apart from imidazoline receptors, the role of other biological
targets of agmatine like nitric oxide synthase, NMDA and 2
adrenoreceptors needs further investigation.
In conclusion, this study demonstrated that agmatine not only
potentiate ethanol-induced anxiolysis but also block the withdrawal
anxiety. We also showed similar inuence of imidazoline receptor
agonists or drugs augmenting endogenous agmatine levels on above
ethanol-induced behavioral parameters. Anti-anxiety effect of agmatine
in ethanol withdrawal rats was blocked by imidazoline antagonists.
Taken together it is reasonable to conclude that acute and chronic
behavioral effects of ethanol may be directly linked to endogenous
agmatine levels and its interaction with imidazoline receptors. These
ndings strongly support further investigation of agmatine in ethanol
dependence mechanism. The data indirectly project agmatine as a
potential therapeutic target in overcoming alcohol abuse associated
problem such as anxiety.
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