Behavioural Pharmacology
The influence of various glutamate receptors antagonists on anxiety-like effect of ethanol withdrawal in a plus-maze test in rats

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Abstract

The aim of the present study was to determine whether various glutamate receptor antagonists could affect ethanol withdrawal-induced anxiety-like behavior measured in the elevated plus-maze test in rats. In our study, memantine (8 and 12 mg/kg), a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, did not show any effect on ethanol withdrawal anxiety. Acamprosate (NMDA and metabotropic glutamate5 (mGlu5) receptor antagonist), at a dose of 400 mg/kg showed anxiolytic-like effect, thus increasing the percent of time spent in open arms and open arms entries. Antagonists of group I mGlu receptors, such as MTEP ([(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine, mGlu5 receptor) or EMQMCM (3-ethyl-2-methyl-quinolin-6-yl-(4-methoxy-cyclohexyl)-methanone methanesulfonate, mGlu1 receptor), caused similar effects to acamprosate. In contrast to acamprosate and MTEP, EMQMCM (5 mg/kg) elevated the ethanol withdrawal-induced decrease in locomotion. When given alone to the saline-treated group, EMQMCM indicated anxiolytic-like effect. Our results imply a crucial role of mGlu5 receptor in an anxiety-like effect of ethanol withdrawal because MTEP (a selective mGlu5 receptor antagonist) and acamprosate (which also indirectly inhibits mGlu5 receptor) attenuated ethanol withdrawal anxiety-like behavior without influence on ethanol withdrawal hypolocomotion and did not show any effect in the saline-treated groups. However, difference in anxiolytic-like potency between both these group I mGlu receptors antagonists may be due to the recent experimental design. Therefore, taking into account a positive correlation between ethanol withdrawal-induced anxiety and relapse to ethanol drinking, our results suggest that mGlu receptor antagonists of group I (similarly to acamprosate) could prevent relapse to drinking and, therefore they might be useful in therapy of alcoholism.

Introduction

Although the spectrum of physiological and somatic signs of withdrawal vary widely across different classes of abuse drugs (see Koob and Le Moal, 2005), withdrawal of almost all additive drugs (including ethanol) induce an increase in negative emotional response, such as feelings of anxiety, restlessness, hyperirritability and depressed mood/dysphoria. It has been argued that such negative emotional components of the withdrawal, mainly anxiety, may be of greater motivational relevance than these of somatic signs in maintaining drug seeking behavior and compulsive drug use (Schulteis et al., 1995, Weiss et al., 2001, Koob and Le Moal, 2001, Schulteis and Liu, 2006). Growing data indicate that an increase in glutamatergic neurotransmission may contribute to anxiety expressed during withdrawal from chronic ethanol (Dahchour and De Witte, 2003, De Witte et al., 2003, Läck et al., 2007).

The role of ionotropic glutamate receptors, such as N-methyl-d-aspartate (NMDA) receptors in ethanol's effects has been well documented. Much evidence indicates that (1) acute ethanol administration reduces flux of calcium ions through the NMDA-stimulated cyclic GMP accumulation (Hoffman et al., 1989, Lovinger et al., 1989); (2) chronic ethanol treatment increases ligand binding to NMDA receptors in brain (Michaelis et al., 1978, Snell et al., 1993) and increases the number of glutamate receptors (Hoffman, 1995, Hoffman et al., 1995); (3) acute ethanol withdrawal increases NMDA-type glutamate receptor activity and it appears to contribute to such withdrawal-related effects as emotional disturbances (e.g. craving, dysphoria) and motor signs (e.g. tremor, seizures etc.) (Grant et al., 1990, Nagy et al., 2005).

NMDA receptor antagonists block some of ethanol's effects. For example, noncompetitive NMDA receptor antagonist, dizocilpine (5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine) inhibits reinforcing effects of ethanol in sensitization (Broadbent and Weitemier, 1999, Camarini et al., 2000) and conditioned place preference (Biala and Kotlinska, 1999) suggesting involvement of the NMDA receptor in ethanol rewarding effects. Furthermore, NMDA receptor antagonists block ethanol withdrawal seizures (Grant et al., 1990, Kotlinska et al., 2004).

The metabotropic glutamate (mGlu) receptor subtype 1 (mGlu1 receptor) and subtype 5 (mGlu5 receptor) belong to the group I family of mGlu receptors. Growing evidence indicates that the group I of mGlu receptors plays an important role in regulation of effects of drugs of abuse (Kenny and Markou, 2004). The mGlu5 receptor has particularly been implicated in motivation to ethanol drinking because the antagonist of this type of receptor, 6-methyl-2-(phenylethynyl)pyridine (MPEP) reduced ethanol self-administration in animals (Schroeder et al., 2005, Hodge et al., 2006, Lominac et al., 2006) and relapse to ethanol seeking behavior (Schroeder et al., 2005). The role of mGlu1 receptor in reinforcing effect of ethanol is also documented but less clear (Schroeder et al., 2005, Lominac et al., 2006). There are very limited data about a role of group I mGlu receptor in ethanol withdrawal syndrome. Only Olive and Becker (2008) indicated that MPEP, a mGlu5 receptor antagonist, does not attenuate the handling-induced convulsions during repeated ethanol withdrawal in mice.

Anxiety-like effect has been observed in laboratory animals following withdrawal from chronic ethanol exposure (File, 1994, File and Andrews, 1991, File et al., 1993, Gatch et al., 1999, Overstreet et al., 2002, Valdez et al., 2002) and also is one of the withdrawal signs in alcoholics (Koob et al., 1998). Affective withdrawal signs from alcohol may be a risk to return to ethanol use, since depressed mood and anxiety associated with protracted abstinence were positively correlated with relapse (Hershon, 1977, Mossberg et al., 1985, De Soto et al., 1989, Annis et al., 1998, Miller and Harris, 2000, Willinger et al., 2002, Kushner et al., 2005).

Acamprosate (calcium acetyl homotaurinate) is commonly used in the treatment of alcohol addiction as an anticraving drug (Littleton, 1995) that prevents relapse to drinking (Lhuintre et al., 1985, Lhuintre et al., 1990). Although the mechanism of action of this drug is unclear, data indicate that acamprosate alters glutamatergic transmission via a weak antagonism of NMDA receptor (Popp and Lovinger, 2000, Rammes et al., 2001, Mayer et al., 2002) and by an indirect blockade of the mGlu5 receptor (Harris et al., 2002). In animal studies, acamprosate decreased ethanol craving in rats (Spanagel et al., 1996), the expression of ethanol-induced sensitization to hyperlocomotor effect of ethanol (Kotlinska et al., 2006) and the handling-induced convulsions during alcohol withdrawal in mice (Farook et al., 2008). Memantine (1-amino-3,5-dimethyl-adamantan), is an noncompetitive NMDA receptor antagonist with very few side effects (Parsons et al., 2007), which in clinical studies attenuated cue-induced alcohol craving in recovering alcohol dependent patients (Krupitsky et al., 2007b) but did not reduce craving associated with alcohol consumption (Bisaga and Evans, 2004). Moreover, it did not reduce drinking in a recent treatment study on alcoholics (Evans et al., 2007). However, memantine suppresses alcohol withdrawal symptoms in humans (Krupitsky et al., 2007a). Both of these substances, acamprosate and memantine were used in our study to examine their influence on the anxiety-like effect of ethanol withdrawal in rats. Furthermore, because group I mGlu receptors play a role in regulation of motivation to ethanol drinking (Schroeder et al., 2005, Lominac et al., 2006), two antagonists i.e. EMQMCM (3-ethyl-2-methyl-quinolin-6-yl-(4-methoxy-cyclohexyl)-methanone methanesulfonate), an antagonist of mGlu1 receptor and MTEP ([(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine), an antagonist of mGlu5 receptor were also used. The aim of this study was to compare the influence of group I mGlu receptors antagonists with those of acamprosate and memantine on the ethanol withdrawal-induced anxiety-like effect measured in the plus-maze test in rats.

Section snippets

Animals

Male Wistar rats (HZL, Warsaw, Poland, 200–250 g) were used in all experiments. The animals were housed under standard laboratory conditions (22 °C, 12:12 light–dark cycle) in groups of four rats per cage. The animals were allowed a period of 7 days for acclimation before the start of the experiments with access to standard food (Bacutil, Motycz, Poland) and water ad libitum. All procedures were performed in agreement with ethical regulations and were approved by the local Ethics Committee.

Anxiety-like effect of ethanol withdrawal

At

Experiment 1. Influence of acamprosate on anxiety-like effect of ethanol withdrawal measured in the plus-maze test in rats

One-way analysis of variance ANOVA revealed significant differences between groups on the percent time spent by rats in the open arms of the plus-maze [F(5, 44) = 5.828, P < 0.001]. As shown in Fig. 1A, ethanol withdrawal reduced the percent of time spent by rats in the open arms on the 10th day of experiment (P < 0.05). Acute acamprosate treatment at the dose of 400 mg/kg counteracted this deficit (P < 0.01). Acamprosate alone (200, 400 mg/kg) did not change the percent of time spent by control

Discussion

Our experiments indicate that withdrawal of ethanol on day 10 of the experiment, after 9 days of ethanol administration (2 g/kg, i.p. once daily plus 4% w/v ethanol solution) induced an anxiety-like effect in rats in the elevated plus-maze test. These symptoms were observed as a decrease in percent time spent in the open arms, percent open arms entries and a decrease in locomotion measured as the closed arms entries. Although a decrease in locomotion during ethanol withdrawal could result in

Acknowledgments

Authors acknowledge Prof. W. Danysz (Preclinical R and D, Merz Pharmaceuticals, Frankfurt/Main, Germany) for the generous gift of MTEP, EMQMCM and memantine. This work was supported by the International Centre for Genetic Engineering and Biotechnology (ICGEB, Trieste, Italy) grant No. CRP/POL05-02.

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