Ethanol Agm

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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 b s t r a c ta 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)

Present study investigated the role of agmatine in ethanol-induced anxiolysis and withdrawal anxiety usingelevated plus maze (EPM) test in rats. The anxiolytic-like effect of ethanol was potentiated by pretreatment

with imidazoline I1/I2 receptor agonist agmatine (10–20 mg/kg, i.p.), imidazoline I1 receptor agonists, moxonidine

(0.25 mg/kg, i.p.) andclonidine (0.015 mg/kg, i.p.),imidazoline I2 receptoragonist, 2-BFI (5 mg/kg, i.p.)as well as by

thedrugsknownto increase endogenous agmatine levelsin brain viz., L -arginine, an agmatine biosyntheticprecursor

(100 µg/rat, i.c.v.), ornithine decarboxylase inhibitor, DFMO (125 µg/rat, i.c.v.), diamine oxidase inhibitor,

aminoguanidine (65 µg/rat, i.c.v.) and agmatinase inhibitor, arcaine (50 µg/rat, i.c.v.). Conversely, prior administra-

tionofI1 receptor antagonist,efaroxan(1 mg/kg,i.p.),I2 receptor antagonist,idazoxan(0.25 mg/kg,i.p.) andarginine

decarboxylaseinhibitor, D-arginine (100 µg/rat,i.c.v.) blocked theanxiolytic-like effectof ethanol.Moreover, ethanol

withdrawal anxiety was markedly attenuated by agmatine (10–20 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 µg/rat, i.c.v.), DFMO (125 µg/rat, i.c.v.),

aminoguanidine (65 µg/rat, i.c.v.) and arcaine (50 µg/rat, i.c.v.). The anti-anxiety effect of agmatine in ethanol-

withdrawn rats was completely blocked by efaroxan (1 mg/kg, i.p.) and idazoxan (0.25 mg/kg, i.p.).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 throughmultiple

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 hasbeen implicated in the

process of drug addiction (Aricioglu-Kartal and Uzbay, 1997; Li et al.,

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 neurotransmitterin 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),

European Journal of Pharmacology 637 (2010) 89–101

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

E-mail address: [email protected] (C.T. Chopde).

0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.ejphar.2010.03.058

Contents lists available at ScienceDirect

European Journal of Pharmacology

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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 neurotrans-

mitters with multireceptorial af nity and diverse physiological func-

tions. 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 af nity to imidazoline

and α2-adrenoceptors (Li et al., 1994; Regunathan and Reis, 1996; Reisand 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 brainsupportsthis correlation (De

Vos et al., 1994; Raasch et al., 1995). Further, several reports have

demonstrated the effect of imidazoline receptor ligands like agmatine,

harmane andβ-carboline on ethanol actions, intakeand 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 recep-

tors in ethanol-induced anxiolysis and withdrawal anxiety in rats

using an elevated plus maze (EPM) paradigm.

2. Materials and methods

2.1. Animals

Adult healthySprague Dawley rats(230–250 g) were housed four per

cage (640× 410× 250 mm height) or individually after intracerebroven-

tricular (i.c.v.) cannulation, under controlled conditions (25±2 °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 hydro-

chloride, moxonidine hydrochloride, DL -α-diuoromethyl ornithine

hydrochloride (DFMO), aminoguanidine hemisulfate, arcaine sulfate,

efaroxan hydrochloride, idazoxan hydrochloride and L -arginine mono-

hydrochloride (Sigma-Aldrich Co., USA.), 2-(2-benzofuranyl)-2-imidazo-

line 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 µl/rat) route to alter the levels of brain

agmatine and avoid peripheral effects. For intracerebroventricular (i.c.v.)

administration of drugs, dilutions were madewith articial cerebrospinal

uid(aCSF) of following composition 0.2 M NaCl,0.02 M NaH2CO3, 2 m M

KCl, 0.5 mM KH2PO4, 1.2 mM CaCl2, 1.8 mM MgCl2, 0.5 mMNa2SO4, 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).

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

lateralventricle. 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). Theguidecannulae were thenxed to the skull withdental 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 μ 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 1min

after each injection to promote diffusion of drugs before being slowly

withdrawn to prevent backow.

2.4. Anxiety test (elevated plus maze)

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 eachother withan 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 ef ciency 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 µl of blood was

mixed with 160 µl perchloric acid (3%) and stored at 4 °C until analysis.

Ethanol was quantied by thealcoholdehydrogenase assay(Zapataet al.,

2006). Briey, 20 µl clear aliquot was incubated in duplicate in 1 ml of 0.5 M Tris–Cl buffer (pH 8.8) containing 5.5 µg/ml of alcohol dehydro-

genase and 1.5 mM β-nicotinamide adenine dinucleotide (β-NAD) for

40 min at room temperature. Reduction of β-NAD to β-NADH was

measured by reading the absorbance at 340 nm. The ethanol concentra-

tion 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, 1–2.5 g/kg, i.p.),

agmatine (10–80 mg/kg, i.p.) or saline 30 min before being subjected to

EPM test. For combination studies, agmatine (10–

20 mg/kg, i.p.) was

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

and subjected to EPM test as mentioned earlier.

To assesswhether agmatine hasany 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 dehydroge-

nase 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 µl/rat,

i.c.v.), agmatine precursor, L -arginine (100 µg/rat, i.c.v.), an ornithine

decarboxylase inhibitor, DFMO (125 µg/rat, i.c.v.), a diamine oxidase

inhibitor, aminoguanidine (65 µg/rat, i.c.v.), or an agmatinase inhibitor,

arcaine (50 µg/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 arereported to signicantlyenhance thebrain agmatine contentat

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 µg) 10 min prior to anxiolytic dose of ethanol

(2 g/kg, i.p.) and subsequently subjected to EPM test.

2.7. In uence of imidazoline receptor agonists and antagonists on

ethanol-induced anxiolysis

In view of high af nity of endogenous agmatine towards imidazo-

line 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, imidazo-

line I1/ α2 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.) before10 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 I1receptor antagonist, efaroxan (1 mg/kg, i.p.) or I2/ α2 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 allowadaptationto

the novel food. From 8th day onwards, half of thesubjects 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).

Control ratswere pair fedwithan isocaloricliquiddietcontainingsucrose

as a caloric substitute to ethanol. At 24 h post ethanol withdrawal, the

pair-fedgroups as well as ethanol fed groups were subjected to EPMtest.

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 bodyweightof each animal was monitored

daily (0900 h) for all groups. Each rat daily consumed 13.6±2.3 g/kg

ethanol and body weight were not signicantly deviated from pair-fed

animals.On 21st dayof experiment,the mean bodyweight of pair-fed and

ethanol fed animals was 238± 2.3 g and 245± 3.2 g respectively.

2.9. Effect of drugs on ethanol withdrawal anxiety

These protocolsweredesigned to assessthe effectof 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 =6–8) received one of the following drugs, agmatine

(5–20 mg/kg, i.p.), DFMO (125 μ g/rat, i.c.v.), aminoguanidine (65 μ g/rat,

i.c.v.), arcaine (50 μ g/rat, i.c.v.), L -arginine(100 μ 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 μ l/rat,i.c.v.) or saline(5 ml/kg,i.p.). These doseswere selectedon

the basis of acute studies.

To determine blood ethanol concentration, the blood samples

were collected from pair-fed control, ethanol fed and in ethanol-

withdrawn 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/ α2 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 placementwas veried histologically for distribution of ink

in theventricles.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 statisticallyanalyzed by one wayanalysis of

variance (ANOVA) with repeated measures on drug treatmentsfollowed by post hoc Dunnett's test. The data obtained from ethanol-

withdrawn and pair-fed rats were compared by unpaired t -test. The

acute combination protocols and treatment of drugs in ethanol-

withdrawn rats were analyzed by either unpaired ‘t’ test or one way

repeated-measures ANOVA and individual means were compared

either by Dunnett's or Newman–Keuls post hoc test. A value of P b0.05

was considered signicant.

3. Results

3.1. Agmatine potentiated anxiolytic effect of ethanol in EPM

As shown in Fig. 1, acute injections of ethanol (1.5–2 g/kg, i.p.) and

agmatine (40–

80 mg/kg, i.p.) showed signicant effect on percent open-

91B.G. Taksande et al. / European Journal of Pharmacology 637 (2010) 89–101


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arm time (% OAT) [ethanol — F (4, 39)=38.55, P b0.001; agmatine — F

(4, 39)=32.89, P b0.001] and percent open-arm entries (% OAE)

[ethanol — F (4, 39)=15.70, P b0.001; agmatine — F (4, 39)=4.947,

P b0.01] as compared to respective control. Post hoc analysis showed

signicant increase in % OAT [ethanol — 1.5 g/kg (P b0.001) and 2 g/kg

(P b0.001); agmatine — 40 mg/kg (P b0.001), 80 mg/kg (P b0.001)] and

% OAE [ethanol — 1.5 g/kg (P b0.05), 2 g/kg (P b0.01); agmatine —40 mg/kg (P b0.05),80 mg/kg (P b0.05)]but didnot affected closed-arm

entries in these doses. Though their lower doses i.e. 1 g/kg, ethanol and

10–20 mg/kg, agmatine were found to be ineffective, the higher dose of

ethanol (2.5 g/kg) however signicantlyreduced thepercent time spent

(P b0.05) and entries into open-arm (P b0.01) as well as the number of

closed-arm entries (P b0.001). 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 b0.01; % OAE — F (2, 17)=7.287, P b0.01] with

ineffective dose of ethanol (1 g/kg i.p.). The post hoc analysis showed

that agmatine (10 and 20 mg/kg; P b0.05 and P b0.01 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 μ g/rat, i.c.v.), DFMO (125 μ g/rat, i.c.v.), aminogua-

nidine (65 μ g/rat, i.c.v.) and arcaine (50 μ g/rat, i.c.v.) showed signicant

effect on % OAT (One way ANOVA — F (9, 69)=11.95, P b0.001) and %

OAE(One wayANOVA— F (9,69)= 2.745,P b0.01)produced by ethanol

(1 g/kg). Post hoc Newman–Kuels comparisons showed signicant

potentiation of anxiolytic effect of ethanol by L -arginine (% OAT —

P b0.001; % OAE — P b0.05), DFMO (% OAT — P b0.01; % OAE — P b0.05),

aminoguanidine (% OAT — P b0.001; % OAE — P b0.05) and arcaine

(%OAT— P b0.001;% OAE—P b0.05). However, these treatmentshad 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.

Fig. 1. Effect of (A) ethanol (1–2.5 g/kg, i.p.), (B) agmatine (10–80 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 (10–20 mg/kg, i.p.) and 30 min later injected with

ethanol (1 g/kg, i.p.).Thirty minthereafter, individual ratwas placed in the centerof thearmto explore theEPMfor 5 min. Each barrepresents mean±S.E.M.( n =6–8). *, #, $P b0.05,

**, $$P b0.01, # #, ***P b0.001 vs respective control (One way repeated measure ANOVA followed by Dunnett's test).



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

ethanol in EPM

Pretreatment of animals with arginine decarboxylase inhibitor, D-

arginine (100 μ g/rat, i.c.v.) signicantly blocked anxiolytic effect of

ethanol (8% w/vin saline, 2 g/kg, i.p.) as evident from decreased% OAT

[Unpaired ‘t’ test, t =2.942, df =10, P b0.05] as well as % OAE

[Unpaired ‘t’ test, t =5.345, df =10, P b0.001] as compared to ethanol

(2 g/kg, i.p.) treated group.However,this treatmenthad no signicant

effect on total number of closed-arm entries. The administration of D-

arginine 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.

3.4. Imidazoline receptor agonists enhanced while antagonist blocked

anxiolytic effect of ethanol and agmatine

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 b0.001; % OAE —

F (7, 47)=12.16, P b0.001] 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,

Fig. 2. Effect of enhanced brain agmatine contenton 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 µg/rat), aminoguanidine (65 µg/rat), arcaine (50 µg/rat), L -arginine (100 µg/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 mean±S.E.M. (n=7) *P b0.05, **P b0.01, ***P b0.001 vs respective

control (One way ANOVA post hoc Newman–Kuels test).

Fig. 3. Effect of D-arginine on anxiolytic effect of ethanol (2 g/kg, i.p.) in EPM showing

(A) % time spentin open arm, (B)% open-arm entries and(C) closed-arm entries. Rats were

injected with the D-arginine (8% w/v in saline; 100 μ 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

wasplacedin thecenterof thearmto exploretheEPMfor 5 min. Eachbarrepresents mean±

S.E.M. (n =6). *P b0.05, **P b0.001 vs control group (Unpaired ‘t’ test).



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P b0.001]. Post hoc Newman–Kuels comparisons indicated the signi-

cant potentiation of anxiolytic effect of ethanol and agmatine by

moxonidine [ethanol (% OAT — P b0.001, % OAE — P b0.001); agmatine

(% OAT — P b0.01, % OAE — P b0.001)], clonidine [ethanol (% OAT —

P b0.001, % OAE — P b0.001); agmatine (% OAT — P b0.001, % OAE —P b0.001)] and 2-BFI [ethanol (% OAT — P b0.001, % OAE — P b0.001);

agmatine (% OAT — P b0.001, % OAE — P b0.001)]. 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 b0.001; % OAE — F (5, 35)=4.309,

P b0.01] and agmatine (40 mg/kg, i.p.) [% OAT — F (5, 35)=18.92,

P b0.001; % OAE — F (5, 35)=4.848, P b0.01] in EPM test (Fig. 5). Post

hoc Newman–Kuelscomparison demonstrated the signicant effectof

efaroxan (1 mg/kg, i.p.) [ethanol (% OAT— P b0.001,% OAE— P b0.01);

agmatine (% OAT — P b

0.001, % OAE — P b

0.01)] and idazoxan

(0.25 mg/kg, i.p.) [ethanol (% OAT — P b0.001, % OAE — P b0.01);

agmatine (% OAT— P b0.001, %OAE— P b0.01)]on theEPM 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 blockedby pretreatment of ratswith imidazoline

receptor antagonists [One way ANOVA — % OAT — F (5, 35)=10.48,

P b0.001; % OAE — F (5, 35)=13.99, P b0.05]. Post hoc Newman–Kuels

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.)

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 spentin open arm, (B)% open-armentriesand (C)the numberof 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 b0.01, $$, *P b0.001 vs respective control group (One way

ANOVA post hoc Newman–Kuels test).

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

wereinjected withthe 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 b0.01,**, $$P b0.001vs respective

control group (One way ANOVA post hoc Newman–Kuels test).



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[% OAT — P b0.01; % OAE — P b0.01]. 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

Chronicexposure to ethanolfor 21 daysand itsabruptdiscontinuation

produced marked anxiety at 24 h. Withdrawal from chronic ethanol

exposure led to decrease in % OAT [unpaired ‘t’ test; t =3.822, df =10,

P b0.01], % OAE [unpaired ‘t’ test; t =6.859, df =10, P b0.001] and total

number of closed-arm entries [unpaired ‘t’ test; t =3.721, df =10,

P b0.01] as compared to pair-fed animals (Fig. 7). Acute administration

of agmatine (10–20 mg/kg, i.p.) [% OAT — F (3, 23)=5.202, P b0.01;

% OAE — F (3, 23)=13.18, P b0.001] attenuated ethanol withdrawal-

induced anxiety. Post hoc comparisons indicated signicant effect of 10

(% OAT — P b0.05; % OAE — P b0.001) as well as 20 mg/kg (% OAT —

P b0.01; % OAE — P b0.001) 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.) andethanol (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 b0.05, **P b0.01,***P b0.001 vs respective control (Oneway ANOVA post hoc

Newman–Kuels test).

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

blockadeby agmatine(5–20 mg/kg, i.p.).Rats weregivenfree access to ethanolthrough

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 b0.01, **P b0.001 vs control (Unpaired ‘t’

test), # P b0.05, ## P b0.01, ###P b0.001 vs ethanol-withdrawn group (One way ANOVA

post hoc Dunnett's test).



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Similarly, L -arginine, aminoguanidine and arcaine showed signif-

icant effecton ethanol withdrawal anxiety [One wayANOVA; % OAT—

F (4, 29)=9.228, P b0.001; % OAE — F (4, 29)=14.37, P b0.001].

Post hoc Dunnett's comparisons showed signicant reduction in

ethanol withdrawal anxiety by L -arginine (100 μ g/rat, i.c.v.) (% OAT —

P b0.01; % OAE — P b0.001), DFMO (125 μ g/rat, i.c.v.) (% OAT — P b0.01;

% OAE — P b0.001), aminoguanidine (65 μ g/rat, i.c.v.) (% OAT — P b0.001;

% OAE — P b0.001) and arcaine (50 μ g/rat, i.c.v.) (% OAT — P b0.001;

% OAE —

P b

0.001). The total number of closed-arm entries remainedunaffected (Fig. 8).

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

receptor agonistsviz.,moxonidine (0.25 mg/kg,i.p.),clonidine(0.015 mg/

kg, i.p.), 2-BFI (5 mg/kg, i.p.) signicantly ameliorated ethanol with-

drawal anxiety [One way ANOVA; % OAT — F (3, 23)=8.508, P b0.001;

% OAE — F (3, 23)=4.959, P b0.01]. Post hoc comparison demonstrated

the signicant effect of moxonidine (OAT — P b0.01; OAE — P b0.05) and

clonidine (% OAT — P b0.01; % OAE — P b0.05), 2-BFI (% OAT — P b0.001;

% OAE — P b0.01) on the EPM behavior of ethanol-withdrawn rats.

However, total number of closed-arm entries remained unaffected.

Fig. 8. Effect of increased brain agmatine content on 24 h of ethanol withdrawal-

induced anxiety. Rats were injected i.c.v. with DFMO (125 µg/rat) or aminoguanidine

(65 µg/rat) or arcaine (50 µg/rat) or L -arginine (100 µg/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 b0.01, **P b0.001

vs ethanol-withdrawn rats (One way ANOVA post hoc Dunnett's test).

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 inopen arms, (B) % open-arm entries and (C) the closed-arm entries. Each bar represents

the mean±S.E.M. (n =6). *P b0.05, **P b0.01, ***P b0.001 vs ethanol-withdrawn rats

(One way ANOVA post hoc Dunnett's test).



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As shown in Fig. 10, the effect of agmatine (20 mg/kg, i.p.) on ethanol

withdrawal anxietywas completely abolished by efaroxan(1 mg/kg, i.p.)

[% OAT — F (3, 23)= 9.967, P b0.001; % OAE — F (3, 23)= 8.105,

P b0.001] or idazoxan (0.25 mg/kg, i.p.) [% OAT — F (3, 23)= 10.82,

P b0.001; % OAE— F (3,23)= 11.82,P b0.001].Post hoc Dunnett's analysis

indicated signicant effect of efaroxan (% OAT — P b0.01; % OAE —

P b0.01) and idazoxan (% OAT — P b0.01; % OAE — P b0.01) on the

anxiolytic effect of agmatine in ethanol-withdrawn rats. Administration

of efaroxan or idazoxan to ethanol-withdrawn rats did not evoke anyresponse in EPM test.

3.7. Blood ethanol concentration

As shown in Table 1, blood ethanol levels were 92.5± 2.4, 177.6±3.7

and 5.3± 1.3 mg/dl (mean±S.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

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 ofothers (Durcanand Lister, 1988; Belzung andBerton, 1997;

Bilkei-Gorzo et al., 1998; LaBuda and Fuchs, 2002; LaBuda and Hale,

2000) acute administration of ethanol (1.5–2 g/kg) produced a dose-

dependent anti-anxiety effect in the EPM test as evident from increased

percent time spent and entries into open arms without any change inclosed-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 in-

coordination 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

(Lavinskyet al., 2003; Gong et al., 2006) agmatine (40and 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 ofethanol inEPM 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 thepresenceof

agents like L -arginine, DFMO, aminoguanidine and arcaine modulat-

ing 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 L -

ornithine decarboxylase and arginase enzyme (Slotkin et al., 1982;

Selamnia et al., 1998) and subsequentlystimulates L-ADC (Hernandez

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 ratswere injectedwith 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-armentries and (C) the closed-arm entries. Eachbar represents the mean±

S.E.M. (n =6). *P b0.01, **P b0.001 vs ethanol-withdrawn rats (One way ANOVA post

hoc Newman–

Kuels test).

Table 1

Blood ethanol concentration (mg/dl).

Treatment groups Blood ethanol concentration

(BEC) (mg/dl)

Acute study

Saline 0.89 ±0.27

Ethanol (2 g/kg) 92.5 ±2.4

Ethanol (1 g/kg) 56.8 ±4.7

Agmatine (10 mg/kg)+ethanol (1 g/kg) 53.2±3.9

Chronic studyChronic ethanol fed group 177.6 ±3.7

Control etha nol withdrawal group 5.3 ± 1.3

Agmatine (40 mg/kg, ip) treated ethanol

withdrawal group

4.7±3.5

Bloodethanolconcentrationin ratsfollowingacute treatmentof 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 beforeand afterwithdrawaltreated witheither

saline or agmatine 30 min before 24 h withdrawal. Data expressed as mean± S.E.M.

(n =6).



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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 previousstudy 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 adminis-

tered acutely together with ethanol exhibited synergistic anxiolytic

effect. It may be due to additive accumulation of agmatine

concentration in brain suf cient 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 bindimidazoline 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, etcthat areinvolved inregulationof anxiety(Otake et al.,1998;

Davis, 1997; Koob, 2003). Major depression, opioid addiction, neurode-

generative 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 involvementof 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/ α2receptor 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 imidazo-

line 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 (Gruppet 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 dif cult

to rule out the involvement of additional α2 adrenoreceptors since

agmatine displays af nity for both I1/I2 and α2 adrenoreceptors.

Polyamines play variety of roles in CNS development (Slotkin and

Bartolome, 1986) and increased polyamine expression in certain brain

areas hasbeen reportedduringethanol withdrawal (Davidsonand Wilce,

1998; Gibsonet al., 2003). Since agmatineis a polyamineprecursor andis

reported to alleviate behaviors associated with ethanol withdrawal

syndrome (Uzbay et al., 2000), we extended the present study to

withdrawal anxiety. It was found thataveragedaily ethanol consumption

of individual rat was about 13.6±2.3 g/kg and attains adequate blood

ethanol levels (177.6±3.7 mg/dl) as measured just before the ethanol

withdrawal. Severalreportssuggest thattheconsumptionof 9–15 g/kgof

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 signi

cant reduction inpercentage 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 ethanolwithdrawalanxietytest counteractedanxiety 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 neuro-

toxicityin rat hippocampal slice cultures through signicantreduction 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 (Davidsonand Wilce,1998). Theynot onlypotentiate

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 Regu-

nathan, 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 processesincluding 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.



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Thepresent study alsodemonstrated 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 demonstratedthe complex interactionbetween imidazoline

receptors system and ethanol. Chronic ethanol administration tends to

counteract the hypotensive effect of imidazoline I1 receptor agonistsclonidine 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 I2receptor 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

β-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

depressionassociated with ethanol withdrawalin 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 α2−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 agmatinein 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.

References

Adell, A., Myers, R.D., 1994. Increased alcohol intake in low alcohol drinking rats afterchronic infusion of the beta-carboline harman into the hippocampus. Pharmacol.Biochem. Behav. 49, 949–953.

Aricioglu, F., Means, A., Regunathan, S., 2004. Effect of agmatine on the development of morphine dependence in rats: potential role of cAMP system. Eur. J. Pharmacol. 504,191–197.

Aricioglu-Kartal, F., Uzbay, I.T., 1997. Inhibitory effect of agmatine on naloxone-precipitatedabstinence syndrome in morphine dependent rats. Life Sci. 61, 1775–1781.

Askalany, A.R., Yamakura, T., Petrenko, A.B., Kohno, T., Sakimura, K., Baba, H., 2005.Effect of agmatine on heteromeric N-methyl-D-aspartate receptor channels.Neurosci. Res. 52, 387–392.

Baldwin, H.A., Rassnick, S., Rivier, J., Koob, G.F., Britton, K.T., 1991. CRF antagonistreverses the “anxiogenic” response to ethanol withdrawal in the rat. Psychophar-macology 103, 227–232.

Bedford, A., McIver, D., Foulds,, 1978. “general instability” and “psychopathy”16PFscales and their relationship to psychiatric moodstate. J. Clin.Psychol. 34, 417–418.

Belzung, C., Berton, F., 1997. Further pharmacological validation of the BALB/cneophobia in the free exploratory paradigm as an animal model of trait anxiety.Behav. Pharmacol. 8, 541–548.

Bence, A.K., Worthen, D.R., Stables, J.P., Crooks, P.A., 2003. An in vivo evaluation of theantiseizure activity and acute neurotoxicity of agmatine. Pharmacol. Biochem.Behav. 74, 771–775.

Bertoglio, L.J., Carobrez, A.P., 2002. Prior maze experience required to alter midazolameffects in ratssubmitted to the elevatedplus-maze. Pharmacol. Biochem. Behav. 72,449–455.

Bilkei-Gorzo, A., Gyertyan, I., Levay, G., 1998. mCPP-induced anxiety in the light-darkbox in rats— a new method for screening anxiolytic activity. Psychopharmacology(Berl.) 136, 291–298.

Chandler, L.J., Sutton, G., Norwood, D., Sumners, C., Crews, F.T., 1997. Chronic ethanolincreases N-methyl-D-aspartate-stimulated nitric oxide formation but not receptordensity in cultured cortical neurons. Mol. Pharmacol. 51, 733–740.

Costa, E.T., Ferreira, V.M., Valenzuela, C.F., 2003. Evidence that nitric oxide regulates theacute effects of ethanol on rat N-methyl-D-aspartate receptors in vitro. Neurosci.

Lett. 343, 41–

44.Dalvi, A., Rodgers, R.J., 1996. GABAergic inuences on plus-maze behaviour in mice.Psychopharmacology 128, 380–397.

Dandekar, M.P., Singru, P.S., Kokare, D.M., Lechan, R.M., Thim, L., Clausen, J.T., Subhedar,N.K., 2008. Importance of cocaine- and amphetamine-regulated transcript peptidein the central nucleus of amygdala in anxiogenic responses induced by ethanolwithdrawal. Neuropsychopharmacology 33, 1127–1136.

Davidson, M., Wilce, P., 1998. Chronic ethanol treatment leads to increased ornithinedecarboxylaseactivity: implications fora roleof polyamines in ethanoldependenceand withdrawal. Alcohol Clin. Exp. Res. 22, 1205–1211.

Davis, M., 1997. Neurobiology of fear responses: therole of theamygdala.J. NeuropsychiatryClin. Neurosci. 9, 382–402.

De Vos, H., Bricca, G., De Keyser, J., De Backer, J.P., Bousquet, P., Vauquelin, G., 1994.Imidazoline receptors, non-adrenergic idazoxan binding sites and alpha 2-adrenoceptors in the human central nervous system. Neuroscience 59, 589–598.

Dobrydnjov, I., Axelsson, K., Berggren, L., Samarütel, J., Holmström, B., 2004. Intrathecaland oral clonidine as prophylaxis for postoperative alcohol withdrawal syndrome:a randomized double-blinded study. Anesth. Analg. 98, 738–744.

Durcan, M.J., Lister, R.G., 1988. Time course of ethanol's effects on locomotor activity,

exploration and anxiety in mice. Psychopharmacology (Berl.) 96, 67–72.Durcan, M.J., Lister, R.G., Linnoila, M., 1989. Behavioral effects of alpha 2 adrenoceptor

antagonists and their interactions with ethanol in tests of locomotion, explorationand anxiety in mice. Psychopharmacology (Berl.) 97, 189–193.

El-Mas, M.M., Abdel-Rahman, A.A., 2001. Effect of long-term ethanol feeding onbrainstem alpha(2)-receptor binding in Wistar–Kyoto and spontaneously hyper-tensive rats. Brain Res. 900, 324–328.

Fadda, F., Mosca, E., Colombo, G., Gessa, G.L., 1989. Effect of spontaneous ingestion of ethanol on brain dopamine metabolism. Life Sci. 44, 281–287.

Feng, Y., LeBlanc, M.H., Regunathan, S., 2005. Agmatine reduces extracellular glutamateduring pentylenetetrazole-induced seizures in rat brain: a potential mechanism forthe anticonvulsive effects. Neurosci. Lett. 390, 129–133.

Ferreira, V.M.M., Valenzuela, C.F., Morato, G.S., 1999. Role of nitric oxide-dependentpathways in ethanol induced anxiolytic effects in rats. Alcohol Clin. Exp. Res. 23,1898–1904.

Galea, E., Regunathan, S., Eliopoulos, V., Feinstein, D.L., Reis, D.J., 1996. Inhibition of mammalian nitric oxide synthases by agmatine, an endogenous polyamine formedby decarboxylation of arginine. Biochem. J. 316, 247–249.

Garcia-Sevilla, J.A., Escriba, P.V., Ozaita, A., Walzer, C., Guimon, J., 1998. Density of imidazoline receptors in platelets of euthymic patients with bipolar affectivedisorder and in brains of lithium-treated rats. Biol. Psychiatry. 43, 616–618.

Garthwaite, J., Garthwaite, G., Palmer, R.M., Moncada, S., 1989. NMDA receptoractivation induces nitric oxide synthesis from arginine in rat brain slices. Eur. J.Pharmacol. 172, 413–416.

Gatch, M.B., Wallis, C.J., Lal, H., 2000. Effects of ritanserin on ethanol withdrawal-induced anxiety in rats. Alcohol. 21, 11–17.

Gibson, D.A., Harris, B.R., Rogers, D.T., Littleton, J.M., 2002. Radioligand binding studiesreveal agmatine is a more selective antagonist for a polyamine-site on the NMDAreceptor than arcaine or ifenprodil. Brain Res. 952, 71–77.

Gibson, D.A., Harris, B.R., Prendergast, M.A., Hart, S.R., Blanchard II, J.A., Holley, R.C.,Pedigo, N.W., Littleton, J.M., 2003. Polyamines contribute to ethanol withdrawal-induced neurotoxicity in rat hippocampal slice cultures through interactions withthe NMDA receptor. Alcohol Clin. Exp. Res. 27, 1099–1106.

Gong, Z.H., Li, Y.F., Zhao, N., Yang, H.J., Su, R.B., Luo, Z.P., Li, J., 2006. Anxiolytic effect of agmatine in rats and mice. Eur. J. Pharmacol. 550, 112–116.

Grupp,L.A.,Lau,V., Harding, S.,1997.Thereduction inalcoholintake bythe 5-HT1Aagonist8-OH DPAT and its attenuation by the alpha 2 adrenergic antagonist idazoxan

correlates with blood glucose levels. Psychopharmacology (Berl.) 133, 172–

178.Halaris, A., Plietz, J., 2007. Agmatine: metabolic pathway and spectrum of activity in

brain. CNS Drugs 21, 885–900.Hao, Y.J., Kitashiba, H., Honda, C., Nada, K., Moriguchi, T., 2005. Expression of arginine

decarboxylase and ornithine decarboxylase genes in apple cells and stressedshoots. J. Exp. Botany 56, 1105–1115.

Hernandez, S., Schwarcz de Tarlovsky, S., 1999. Arginine decarboxylase in Trypanosomacruzi, characteristics and kinetic properties. Cell. Mol. Biol. (Noisy-le-grand) 45,383–391.

Hirani, K., Sharma, A.N., Jain, N.S., Ugale, R.R., Chopde, C.T., 2005. Evaluation of GABAergic neuroactive steroid 3alpha-hydroxy-5alpha-pregnane-20-one as aneurobiological substrate for the anti-anxiety effect of ethanol in rats. Psycho-pharmacology (Berl.) 180, 267–278.

Huang, M.J., Regunathan, S., Botta, M., Lee, K., McClendon Yi, G.B., Pedersen, M.L.,Berkowitz, D.B., Wang, G., Travagli, M., Piletz, J.E., 2003. Structure–activity analysis of guanidine group in agmatine for brain agmatinase. Ann. N. Y. Acad. Sci. 1009, 52–63.

Isome, M., Lortie, M.J., Murakami, Y., Parisi, E., Matsufuji, S., Satriano, J., 2007. Theantiproliferative effects of agmatine correlate with the rate of cellular proliferation.Am. J. Physiol. Cell Physiol. 293, C705–C711.



12/13

Itzhak, Y., 2008. Role of the NMDA receptor and nitric oxide in memory reconsolidationof cocaine-induced conditioned place preference in mice. Ann.N. Y. Acad. Sci. 1139,350–357.

Jung, M.W., Qin, Y., Lee, D., Mook-Jung, I., 2000. Relationship among discharges of neighboring neurons in the rat prefrontal cortex during spatial working memorytasks. J. Neurosci. 20, 6166–6172.

Kokare, D.M., Chopde, C.T., Subhedar, N.K., 2006. Participation of α-melanocytestimulating hormone in ethanol-induced anxiolysis and withdrawal anxiety inrats. Neuropharmacology 51, 536–545.

Koob, G.F., 2000. Neurobiology of addiction. Toward the development of new therapies.Ann. N. Y. Acad. Sci. 909, 170–185.

Koob, G.F., 2003. Alcoholism:allostasisand beyond. Alcohol Clin. Exp.Res. 27, 232–

243.LaBuda, C.J., Fuchs, P.N., 2002. Catecholamine depletion by reserpine blocks theanxiolytic actions of ethanol in the rat. Alcohol 26, 55–59.

LaBuda, C.J., Hale, R.L., 2000. Anxiety in mice following acute aspartame and ethanolexposure. Alcohol 20, 69–74.

Lal, H., Prather, P.L., Rezazadeh, S.M., 1991. Anxiogenic behavior in rats during acuteand protracted ethanol withdrawal: reversal by buspirone. Alcohol 8, 467–471.

Lal, H., Prather, P.L., Rezazadeh, S.M., 1993. Potential role of 5HT1C and/or 5HT2receptors in the mianserin-induced prevention of anxiogenic behaviors occurringduring ethanol withdrawal. Alcohol Clin. Exp. Res. 17, 411–417.

Lavinsky, D., Arteni, N.S., Netto, C.A., 2003. Agmatine induces anxiolysis in the elevatedplus maze task in adult rats. Behav. Brain Res. 141, 19–24.

Lee, W.T., Hong, S., Yoon, S.H., Kim, J.H., Park, K.A., Seong, G.J., Lee, J.E., 2009. Neuro-protective effects of agmatine on oxygen-glucose deprived primary-culturedastrocytesand nuclear translocation of nuclear factor-kappaB. BrainRes. 1281,64–70.

Lewis, B., Wellmann, K.A., Barron, S., 2007. Agmatine reduces balance decits in a ratmodel of third trimester binge-like ethanol exposure. Pharmacol. Biochem. Behav.88, 114–121.

Li, G., Regunathan, S., Barrow, C.J., Eshraghi, J., Cooper, R., Reis, D.J., 1994. Agmatine: an

endogenous clonidine-displacing substance in the brain. Science 263, 966–969.Li, J., Li, X., Pei, G., Qin, B.Y., 1999. Correlation between inhibitions of morphine

withdrawal and nitric-oxide synthase by agmatine. Zhongguo Yao Li Xue Bao. 20,375–380.

Linnoila,M., Eckardt,M., Durcan, M., Lister, R., Martin, P., 1987. Interactions of serotoninwith ethanol: clinical and animal studies. Psychopharmacol. Bull. 23, 452–457.

Littleton, J.M., Lovinger, D., Liljequist, S., Ticku, R., Matsumoto, I., Barron, S., 2001.Role of polyamines and NMDA receptors in ethanol dependence and withdrawal. AlcoholClin. Exp. Res. 25, 132S–136S.

Liu, P., Bergin, D.H., 2009. Differential effects of i.c.v. microinfusion of agmatine onspatial working and reference memory in the rat. Neuroscience 159, 951–961.

Liu, P., Rushaidhi, M., Collie, N.D., Leong, M.T., Zhang, H., 2008. Behavioral effects of intracerebroventricular microinfusion of agmatine in adult rats. Behav. Neurosci.122, 557–569.

Loring, R.H., 1990. Agmatine acts as an antagonist of neuronal nicotinic receptors. Br. J.Pharmacol. 99, 207–211.

Lovinger, D.M., 1997. Serotonin's role in alcohol's effects on the brain. Alcohol HealthRes. World 2, 114–120.

Lu, G., Su, R.B., Li, J., Qin, B.Y., 2003. Modulation by a-diuoromethyl-ornithine andaminoguanidineof painthreshold,morphine analgesia and tolerance. Eur. J. Pharmacol.478, 139–144.

Mao, L., Abdel-Rahman, A.A., 1996. Synergistic behavioral interaction between ethanoland clonidine in rats: role of alpha-2 adrenoceptors. J. Pharmacol. Exp. Ther. 279,443–449.

Mello, C.F., Rubin, M.A., Sultana, R., Barron, S., Littleton, J.M., Buttereld, D.A., 2007.Diuoromethylornithine decreases long-lasting protein oxidation induced byneonatal ethanol exposure in the hippocampus of adolescent rats. Alcohol Clin.Exp. Res. 31, 887–894.

Mondavio, M., Ghiazza, G., 1989. Use of clonidine in the prevention of alcoholwithdrawal syndrome. Minerva Med. 80, 1233–1235.

Morgana, A.D., Campbellb, U.C., Fonsa, R.D., Carrolla, M.E., 2002. Effects of agmatine onthe escalation of intravenous cocaine and fentanyl self-administration in rats.Pharmacol. Biochem. Behav. 72, 873–880.

Navarro, E., Alonso, S.J., Martin, F.A., Castellano, M.A., 2005. Toxicological andpharmacological effects of D-arginine. Basic Clin. Pharmacol. Toxicol. 97, 149–154.

Nunes-de-Souza,R.L., Canto-de-Souza,A., da-Costa, M.,Fornari, R.V.,Graeff, F.G., Pela,I.R., 2000. Anxiety-induced antinociception in mice: effectsof systemicand intra-

amygdala administration of 8-OH-DPAT and midazolam. Psychopharmacology150, 300–310.

Onal, A., Soykan, N., 2001. Agmatine produces antinociception in tonic pain in mice.Pharmacol. Biochem. Behav. 69, 93–97.

Onal, A., Delen, Y., Ulker, S., Soykan, N., 2003. Agmatine attenuates neuropathic pain inrats: possible mediation of nitric oxide and noradrenergic activity in the brainstemand cerebellum. Life Sci. 73, 413–428.

Opitz, K., 1990. The effect of clonidine and related substances on voluntary ethanolconsumption in rats. Drug Alcohol Depend. 25, 43–48.

Otake, K., Ruggiero, D.A.,Regunathan,S., Wang, H., Milner, T.A.,Reis, D.J.,1998. Regionallocalization of agmatine in the rat brain: an immunocytochemical study. Brain Res.787, 1–14.

Pandey, S.C., Zhang, D., Mittal, N., Nayyar, D., 1999. Potential role of the genetranscription factor cyclic AMP-responsive element binding protein in ethanolwithdrawal-related anxiety. J. Pharmacol. Exp. Ther. 288, 866–878.

Parale, M.P., Kulkarni, S.K., 1986. Studies with alpha 2-adrenoceptor agonists andalcohol abstinence syndrome in rats. Psychopharmacology (Berl.) 88, 237–239.

Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinates. Academic Press,London.

Pellow, S., File, S.E., 1986. Anxiolytic and anxiogenic drug effects on exploratory activityin an elevated plus-maze: a novel test of anxiety in the rat. Pharmacol. Biochem.Behav. 24, 525–529.

Polivy, J., Herman, C.P., 1976. Effects of alcohol on eating behavior: inuence of moodand perceived intoxication. J. Abnormal Psychology 85, 601–606.

Prendergast, M.A., Harris, B.R., Blanchard II, J.A., Mayer, S., Gibson, D.A., Littleton, J.M.,2000. In vitro effects of ethanol withdrawal and spermidine on viability of hippocampus from male and female rat. Alcohol Clin. Exp. Res. 24, 1855–1861.

Raasch, W., Regunathan, S., Li, G., Reis, D.J., 1995. Agmatine, the bacterial amine, iswidely distributed in mammalian tissues. Life Sci. 56, 2319–2330.

Raasch, W., Schafer, U., Chun, J., Dominiak, P., 2001. Biological signicance of agmatine,

an endogenous ligand at imidazoline binding sites. Br. J. Pharmacol. 133, 755–

780.Regunathan, S., 2006. Agmatine: biological role and therapeutic potential in morphineanalgesia and dependence. AAPS J. 8, E479–E484.

Regunathan, S., Reis, D.J., 1996. Imidazoline receptors and their endogenous ligands.Ann. Rev. Pharmacol. Toxicol. 36, 511–544.

Reis, D.J., Regunathan, S., 1998. Agmatine: an endogenous ligand at imidazolinereceptors may be a novel neurotransmitter in brain. J. Auton. Nerv. Sys. 72, 80–85.

Reis, D.J., Regunathan, S., 2000. Is agmatine a novel neurotransmitter in brain? TrendsPharmacol. Sci. 21, 187–193.

Rezazadeh, S.M., Prather, P.L., Lal, H., 1993. Sensitization to 5-HT1C receptor agonist inrats observed following withdrawal from chronic ethanol. Alcohol 10, 281–283.

Roberts, J.C., Grocholski, B.M., Kitto, K.F., Fairbanks, C.A., 2005. Pharmacodynamic andpharmacokinetic studies of agmatine after spinal administration in the mouse.

J. Pharmacol. Exp. Ther. 314, 1226–1233.Roelofs, S.M., 1985. Hyperventilation, anxiety, craving for alcohol: a subacute alcohol

withdrawal syndrome. Alcohol 2, 501–505.Rommelspacher, H., Schmidt, L.G., May, T., 1991. Plasma norharman (beta-carboline)

levels are elevated in chronic alcoholics. Alcohol Clin. Exp. Res. 15, 553–559.Rommelspacher, H., Dufeu, P., Schmidt, L.G., 1996. Harman and norharman in

alcoholism: correlations with psychopathology and long-term changes. AlcoholClin. Exp. Res. 20, 3–8.

Rosenfeld, H.J., Roberts, J., 1976. Arginine decarboxylase from a Pseudomonas species. J. Bacteriol. 125, 601–607.

Satriano, J., Schwartz, D., Ishizuka, S., Lortie, M.J., Thomson, S.C., Gabbai, F., Kelly, C.J.,Blantz, R.C., 2001. Suppression of inducible nitric oxide generation by agmatinealdehyde: benecial effects in sepsis. Cell. Physiol. 188, 313–320.

Selamnia, M., Mayeur, C., Robert, V., Blachicr, F., 1998. Alpha-diuoromethylornithine(DFMO) as a potent arginase activity inhibitor in human colon carcinoma cells.Biochem. Pharmacol. 55, 1241–1245.

Sessa, A., Perin, A., 1997. Ethanol and polyamine metabolism: physiologic andpathologic implications: a review. Alcohol Clin. Exp. Res. 21, 318–325.

Sessa, A., Desiderio, M.A., Perin, A., 1987. Ethanol and polyamine metabolism in adultand fetal tissues: possible implication in fetus damage. Adv. Alcohol Subst. Abuse 6,73–85.

Sharma, A.N., Chopde, C.T., Hirani, K., Kokare, D.M.,Ugale, R.R., 2007.Chronic progesteronetreatment augments while dehydroepiandrosterone sulphate prevents tolerance toethanol anxiolysis and withdrawal anxiety in rats. Eur. J. Pharmacol. 567, 211 –222.

Slotkin, T.A., Bartolome, J., 1986. Role of ornithine decarboxylase and the polyamines innervous system development: a review. Brain Res. Bull. 17, 307–320.

Slotkin, T.A., Seidler, F.J., Trepanier, P.A., Whitmore, W.L., Lerea, L., Barnes, G.A., Weigel,S.J., Bartolome, J., 1982. Ornithine decarboxylase and polyamines in tissues of theneonatal rat: effects of alpha-diuoromethylornithine, a specic, irreversibleinhibitor of ornithine decarboxylase. J. Pharmacol. Exp. Ther. 222, 741–745.

Spies, C.D., Rommelspacher, H., Winkler, T., Müller, C., Brummer, G., Funk, T., Berger, G.,Fell, M., Blum, S., Specht, M., Hannemann, L., Schaffartzik, W., 1996. Beta-carbolinesin chronic alcoholics following trauma. Addict. Biol. 1, 93–103.

Su, R.B., Li, J., Qin, B.Y., 2003. Effect of L -arginine and anti-L -arginine decarboxylaseantibody on morphine analgesia and tolerance. Chin. J. Drug Depend. 12, 97–101.

Su, R.B., Wang, W.P., Lu, X.Q., Wu, N., Liu, Z.M., Li, J., 2009. Agmatine blocks acquisitionand re-acquisition of intravenous morphine self-administration in rats. Pharmacol.Biochem. Behav. 92, 676–682.

Taksande, B.G., Kotagale, N.R., Tripathi, S.J., Ugale, R.R., Chopde, C.T., 2009. Antidepres-sant like effect of selective serotonin reuptake inhibitors involve modulation of imidazoline receptors by agmatine. Neuropharmacology 57, 415–424.

Utkan, T., Ulak,G., Yildiran, H.G.,Yardimoglu, M., Gacar, M.N., 2000. Investigationon themechanism involved in the effects of agmatine on ethanol-induced gastric mucosal

injury in rats. Life Sciences 66, 1705–

1711.Uzbay, I.T., Erden, B.F., 2003. Attenuation of ethanol withdrawal signs by high doses of

L -arginine in rats. Alcohol 38, 213–218.Uzbay,I.T., Kayaalp, S.O., 1995. A modied liquid dietof chronic ethanol administration:

validation by ethanol withdrawal syndrome in rats. Pharmacol. Res. 31, 37–42.Uzbay, I.T., Akarsu, E.S., Kayaalp, S.O., 1994. Effects of bromocriptine and haloperidol on

ethanol withdrawal syndrome in rats. Pharmacol. Biochem. Behav. 49, 969–974.Uzbay, I.T., Erden, B.F., Tapanyigit, E.E., Kayaalp, S.O., 1997. Nitric oxide synthase

inhibition attenuates signs of ethanol withdrawal in rats. Life Sci. 61, 2197–2209.Uzbay, I.T., Yeşilyurt, O., Celik, T., Ergün, H., Işimer, A., 2000. Effects of agmatine on

ethanol withdrawal syndrome in rats. Behav. Brain Res. 107, 153–159.Valenzuela, C.F., 1997. Alcohol and neurotransmitter interactions. Alcohol Health Res.

World 21, 141–148.Vandergriff, J., Kallman, M.J., Rasmussen, K., 2000. Moxonidine, a selective imidazoline-

1 receptor agonist, suppresses the effects of ethanol withdrawal on the acousticstartle response in rats. Biol. Psychiatry 47, 874–879.

Yang, X.C., Reis, D.J., 1999. Agmatine selectively blocks the N-methyl-D-aspartatesubclass of glutamate receptor channels in rat hippocampal neurons. J. Pharmacol.Exp. Ther. 288, 544–549.



13/13

Zaniewska, M., McCreary, A.C., Sezer, G., Przegaliński, E., Filip, M., 2008. Effects of agmatine on nicotine-evoked behavioral responses in rats. Pharmacol. Rep. 60,645–654.

Zapata, A., Gonzales, R.A., Shippenberg, T.S., 2006. Repeated ethanol intoxication inducesbehavioral sensitization in the absence of a sensitized accumbens dopamine responsein C57BL/6J and DBA/2J Mice. Neuropsychopharmacol 31, 396–405.

Zeidan, M.P., Zomkowski, A.D.E., Rosa, A.O., Rodrigues, A.L.S., Gabilan, N.H., 2007.Evidence for imidazoline receptors involvement in the agmatine antidepressant-like effect in the forced swimming test. Eur. J. Pharmacol. 565, 125–131.

Zomkowski, A.D., Hammes, L., Lin, J., Calixto, J.B., Santos, A.R., Rodrigues, A.L., 2002.Agmatine produces antidepressant-like effects in two models of depression in mice.Neuroreport 13, 387–391.


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