Neuropharmacology of alcohol addiction

Glutamate

The effect of glutamate to alter alcohol-induced LORR was studied in mice. Mice were injected with a high, hypnotic dose of alcohol (4 g kg⁻¹), which caused them to lose the righting reflex. After mice regained their righting reflex, they were immediately injected intracerebroventricularly with glutamate, which induced an instant return to the LORR within 60 s (^{Ferko, 1994}). These data indicate that glutamate can enhance the central depressant action of alcohol. In line with this link between glutamate and LORR, it has been demonstrated that in healthy individuals with a family history of alcoholism, the response to the glutamate/NMDA receptor (NMDAR) antagonist ketamine was altered, as opposed to the response of those with no such family history (^{Petrakis et al., 2004}). Similar findings were obtained in mice selectively bred for increased sensitivity to alcohol; these mice were more sensitive to ketamine treatment (^{Meyer and Phillips, 2003}), suggesting that altered NMDAR function contributes to the altered response to an acute alcohol challenge. This finding, however, is not surprising, given the fact that the NMDAR is a primary target of alcohol's action.

NMDAR antagonists are capable of preventing the acquisition of alcohol-induced conditioned place preference in rats (^{Biala and Kotlinska, 1999}). Furthermore, alcohol self-administration in a free-choice operant task (water vs 10% alcohol) was attenuated by microinjection into the NAC of 2-amino-5-phosphopentanoic acid—a competitive NMDAR antagonist (^{Rassnick et al., 1992}). These results suggest that glutamate neurotransmission in the NAC may modulate alcohol self-administration and its reinforcing effects.

The glutamatergic system in turn is tightly linked to the nitric oxide (NO) pathway. Thus, stimulation of NMDARs leads to a calcium influx, and the binding of calcium to calmodulin activates neuronal NO synthase, which produces NO. The close link between the glutamatergic/NMDA receptor system and NO production implies that the neuronal NO synthase gene is also involved in the modulation of acute effects of alcohol. In fact, several pharmacological studies and studies using neuronal NO synthase knockout mice show that NO signalling also modulates the duration of LORR induced by alcohol and alcohol reinforcement (^{Spanagel et al., 2002}).

GABA

Highly alcohol-sensitive and less alcohol-sensitive rat lines have marked line differences in GABA-mediated events, and these are correlated with the sedative effects of alcohol. Using different GABA_A receptor subunit knockout mouse models for the measurement of alcohol sensitivity provides the currently strongest evidence for a role of α1 subunits in alcohol-induced LORR (^{Boehm et al., 2006}). Polymorphisms in GABA_A receptor subunits have also been linked to alcohol response in humans (^{Dick et al., 2006}).

Interestingly, Sardinian alcohol non-preferring rats, selected for their low alcohol preference and consumption, carry a point mutation (R100Q) in the gene coding for the GABA_A receptor α6 subunit. Rats carrying this mutation voluntarily drank less alcohol than did normal rats during the acquisition phase, but this difference was lost during the maintenance phase (^{Sanna et al., 2004}). However, in a recent study by ^{Botta et al. (2007)} it has been demonstrated that the α6-R100Q GABA_A receptor subunit polymorphism did not change the acute alcohol sensitivity of extrasynaptic receptors. Furthermore, α6 subunit knockout mice did not differ in the sensitivity to the hypnotic effects of alcohol as measured by LORR (^{Homanics et al., 1997}). Activation of the GABA_B receptor was shown to suppress acquisition of alcohol-drinking behaviour in rats (^{Colombo et al., 2002a}; ^{Carai et al., 2005}).

nAChR/glycine

It has been shown that alcohol-induced stimulation of the mesolimbic DA system involves central nACh as well as strychnine-sensitive glycine receptors (^{Ericson et al., 1998}; ^{Söderpalm et al., 2000}; ^{Molander et al., 2005}), which suggests the possible involvement of these receptors in the initiation phase of alcohol consumption. Thus, alcohol-induced DA overflow within the NAC is mediated by nAChR located in the VTA as mecamylamine infused into this area can antagonize this effect and can reduce voluntary alcohol drinking (^{Ericson et al., 1998}, ²⁰⁰³).

DA/5-HT

Both neurotransmitters play, if at all, only a minor role in mediating sensitivity to high doses of alcohol. Thus, various manipulations of these systems did not produce consistent alterations in LORR measurements. In contrast, both systems are crucial for the initiation of alcohol reinforcement. Following the key publication by ^{Gessa et al. (1985)}, which showed that low doses of alcohol produce a dose-dependent increase in the firing rate of DAergic neurons in the VTA, it was later consistently shown that alcohol stimulates DA transmission in the mesolimbic pathway (^{Di Chiara and Imperato, 1988}). With respect to 5-HT, it was shown that alcohol potentiates the action of this neurotransmitter via the 5-HT₃ receptor (^{Lovinger and Zhou, 1994}), and it is suggested that inborn DAergic and/or 5-HTergic dysfunction might be of importance for the initial alcohol preference. For instance, low levels of DA and 5-HT were seen in different alcohol-preferring P, high-alcohol-drinking (HAD) and alcohol-preferring Fawn-Hooded rat lines as compared to their alcohol non-preferring counterpart lines (^{McBride and Li, 1998}). In agreement, 5-HT₃ receptor antagonists were shown to suppress the acquisition of voluntary alcohol consumption in alcohol-preferring P rats (^{Rodd-Henricks et al., 2000}).

Cannabinoids

There is only little evidence that CBs influence the initial sensitivity to hypnotic doses of alcohol. However, alcohol reinforcement processes are modulated by this system, and it is suggested that alcohol reward is dependent on CB1 receptor activity. Thus, CB1 receptors in alcohol-avoiding DBA/2 mice exhibit a lower efficacy than CB1 receptors in alcohol-preferring C57BL/6 mice (^{Hungund and Basavarajappa, 2000}). Genetically selected alcohol-preferring msP or alcohol-preferring AA (Alko alcohol) rat lines show specific differences in the organization of the brain endocannabinoid system in a number of brain regions when compared with unselected Wistars or alcohol-avoiding Alko non-alcohol (ANA) rat lines (^{Cippitelli et al., 2005}; ^{Hansson et al., 2007}) and CB1 receptor antagonism has been reported to specifically suppress acquisition of alcohol-drinking behaviour in rodents (^{Colombo et al., 2005}). More importantly, CB1 receptor knockout mice display reduced alcohol-induced conditioned place preference and self-administration (^{Wang et al., 2003}; ^{Thanos et al., 2005a}). Furthermore, the study from ^{Wang et al. (2003)} showed that there was an age-dependent decline in ethanol preference and intake in the wild-type but not CB1 knockout mice, which is consistent with the idea that reward-dependent mechanisms become less important with age and that a decrease in activity within the endogenous CB system might correlate with these events. Furthermore, the human CB1 receptor, which is encoded by the CNR1 gene, may play a role in the development of alcoholism as it has recently been reported by ^{Zuo et al. (2007)} in a large case–control sample.

Opioids

Endogenous opioids are crucial for mediating alcohol-induced reward. The opioid receptor antagonist naltrexone reverses alcohol-induced DA release in the NAC in rats, and suppression of operant alcohol-reinforced behaviour by naltrexone is associated with attenuation of the alcohol-induced increase in dialysate DA levels in the NAC (^{Gonzales and Weiss, 1998}). These findings not only show that alcohol reinforcement depends on the activity of endogenous opioid systems but also confirm that DA output in the NAC is associated with this reinforcement process (^{Herz, 1997}). Furthermore, alcohol-preferring AA rats show lower opioidergic activity in areas involved in alcohol reinforcement (^{Nylander et al., 1994}), and many other studies have also reported innate differences in opioid systems in other alcohol-preferring and -avoiding lines of animals, such as AA vs ANA, and P vs alcohol non-preferring rats, or C57BL/6 vs DBA/2 mice (^{Herz, 1997}; ^{Jamensky and Gianoulakis, 1997}; ^{McBride and Li, 1998}). In line with the conclusion that endogenous opioid systems are crucial in mediating alcohol reward, naltrexone was shown to slow down the acquisition of alcohol drinking in mice (^{Phillips et al., 1997}).

CRF/NPY

Innate hyperactivity of the CRF system and hypoactivity of the neuropeptide Y (NPY) system are known to promote alcohol consumption. Thus, high-alcohol-preferring msP rats show an anxious-like phenotype and higher density of CRF1 receptor in several limbic areas related to alcohol drinking motivation (^{Heilig and Koob, 2007}). ^{Thiele et al. (1998)} demonstrated that NPY-deficient mice show increased alcohol consumption compared with wild-type mice, and are also less sensitive to the hypnotic effects of alcohol, as shown by their more rapid recovery from LORR. In contrast, transgenic mice that overexpress an NPY gene in neurons have a lower preference for alcohol and are more sensitive than controls to the hypnotic effects of this drug. These data are direct evidence that alcohol consumption and resistance are inversely related to NPY levels in the brain (^{Thiele et al., 1998}). The alcohol-preferring P rats and non-selected C57BL/6J mice show low constitutive levels of NPY, which has been suggested to contribute to their known high level of alcohol consumption (^{Hwang et al., 2004}; ^{Hayes et al., 2005}). In summary, transgenic work with NPY and null-mutant mice, as well as studies with alcohol-preferring animals, has implicated NPY in the control of alcohol consumption, suggesting that genetic variations of the NPY gene may also contribute to the heritability of alcoholism. In fact, in a recent study an association of specific variants of the NPY gene and the onset of alcoholism has been shown (^{Mottagui-Tabar et al., 2005}). The same applies for the CRF system, as a link between the stress system and vulnerability to alcohol drinking has been recently documented in humans—polymorphisms of the genes encoding CRF1 receptor have been associated with alcohol consumption (^{Treutlein et al., 2006}).

In summary, the inborn differences in the activity of several neurotransmitter systems are one important reason why some individuals initiate alcohol drinking and others do not. This might as well explain the initiation of alcohol use early in life. The presence/absence of one or several alternative alleles of particular genes therefore results in an alcohol-sensitive phenotype that in the appropriate environment may lead to the initiation of alcohol drinking because the response of these individuals to appetitive properties of this drug will be much greater.

Glutamate

Adaptive responses such as changes in the number and/or affinity of synaptic glutamate receptors or their subunits will occur to counterbalance the acute inhibitory effect of alcohol on NMDAR function and glutamate release. However, the outcome of pharmacological studies using NMDAR antagonists with regard to alcohol self-administration is inconclusive, showing that different NMDAR antagonists can reduce or have no effect on alcohol intake (^{Shelton and Balster, 1997}; ^{Bienkowski et al., 1999}). In addition, NR2A subunit deletion in mice does not affect voluntary alcohol intake (^{Boyce-Rustay and Holmes, 2006}).

The application of the AMPA (DL-α-amino-3-hydroxy-5-methylsoxazole-4-propionate)/kainate receptor antagonist GYKI 52468 did not alter operant responding for alcohol (^{Stephens and Brown, 1999}). Experiments with knockout mice also do not suggest the involvement of these receptors in the maintenance of alcohol drinking, as both GluR1 and GluR3 deletions did not have any effect on either home cage alcohol drinking or operant self-administration (^{Cowen et al., 2003}; ^{Sanchis-Segura et al., 2006}).

Similarly, no changes in operant alcohol self-administration by alcohol-preferring P rats were found by use of a metabotropic glutamate receptor1 (mGluR1) antagonist (^{Schroeder et al., 2005a}). Neither agonists nor antagonists acting at mGluR2/3 receptors had an effect on maintenance responding for alcohol under operant conditions in P rats (^{Schroeder et al., 2005a}; ^{Rodd et al., 2006}). No difference in alcohol consumption was seen in mGluR4 knockout mice (^{Blednov et al., 2004}). In contrast, mGluR5 antagonists were capable of reducing alcohol-reinforced responding in mice (^{Hodge et al., 2006}) and alcohol-preferring P and alcohol-preferring Fawn-Hooded rats (^{Cowen et al., 2005}; ^{Schroeder et al., 2005a}).

GABA

Chronic alcohol consumption is associated with a decrease in the brain GABA_A receptor density and brain region-specific up- or downregulation of α and β subunit gene expression (^{Golovko et al., 2002}). Knockout mice lacking different GABA_A receptor subunits were examined in several alcohol-related paradigms and it was shown that α1, α2, α5 and δ subunit deletion leads to lower alcohol consumption (^{Mihalek et al., 2001}; ^{Boehm et al., 2004}; ^{Crabbe et al., 2006}; ^{June et al., 2007}). The critical determinant of alcohol's actions on GABA_A receptors seems to be protein kinase C (PKC) activity. Thus, mice lacking PKCɛ showed increased sensitivity of GABA_A receptors to allosteric modulation by alcohol (^{Hodge et al., 1999}). In contrast, GABA_A receptors are less sensitive to alcohol's effects in mice lacking PKCγ (^{Harris et al., 1995}). Accordingly, PKCɛ knockout mice are known to consume less alcohol (^{Hodge et al., 1999}), whereas PKCγ knockouts show increased voluntary alcohol consumption (^{Bowers and Wehner, 2001}).

Pharmacological manipulations of GABA_A receptors were studied in alcohol-preferring rat lines. Thus, negative allosteric modulators of the GABA_A receptor were shown to reduce alcohol consumption in several alcohol-preferring rat lines (^{Rassnick et al., 1993a}; ^{Wegelius et al., 1994}). Additionally, antagonism of GABA_A receptors within the VTA (^{Nowak et al., 1998}) or increasing the activity of those receptors in NAC (^{June et al., 1998}) suppressed alcohol consumption in alcohol-preferring P rats, suggesting the particular importance of these nuclei in the maintenance of alcohol drinking.

Metabotropic GABA_B receptors might also play a crucial role in controlling the levels of chronic alcohol intake. Thus, the GABA_B agonist baclofen suppressed voluntary alcohol consumption in alcohol-preferring sP rats. However, repeated use of baclofen might lead to the development of tolerance (^{Colombo et al., 2000}; ^{Carai et al., 2005}). Interestingly, co-administration of a positive allosteric modulator of the metabotropic GABA_B receptor such as CGP7930 (3-(3′,5′-di-tert-butyl-4′-hydroxy)phenyl-2,2-dimethylpropanol) enhances baclofen's potency and reduces the development of tolerance (^{Adams and Lawrence, 2007}).

nAChR/glycine

Cholinesterase inhibitors were shown to reduce alcohol intake and preference in alcohol-preferring AA rats (^{Doetkotte et al., 2005}). It remains, however, to be established what subunit composition of the nAChRs is most important in this respect. To date, it is known that α4β2 and α7 subtypes of nAChRs are not playing an important role in alcohol consumption (^{Lê et al., 2000}; ^{Ericson et al., 2003}), whereas antagonism of α3β2 and β3 subunits of the nAChR were capable of reducing voluntary alcohol consumption in both rats and mice (^{Larsson et al., 2004}; ^{Jerlhag et al., 2006b}).

Modulation of the activity of the glycinergic system might lead to reduced alcohol self-administration. Thus, ^{Molander et al. (2007)} recently showed that the glycine reuptake inhibitor Org25935—acting specifically on the glycine transporter 1 (GlyT1)—decreases alcohol intake and preference in male Wistar rats by increasing extracellular glycine levels that primarily activate inhibitory strychnine-sensitive glycine receptors.

Channels

^{Katsura et al. (2006)} demonstrated an increased expression of the α1C, α1D and α2/δ1 subunits of L-type voltage-sensitive calcium channels in the mouse cerebral cortex following chronic alcohol exposure. Accordingly, the Ca²⁺ channel antagonist verapamil was able to reduce alcohol intake in alcohol consuming monkeys (^{Rezvani et al., 1991}). Knockout mice models have shown that both N-type Ca²⁺ and GIRK2 channels might also have a certain role in the maintenance of alcohol consumption (^{Blednov et al., 2001}; ^{Newton et al., 2004}). Reduced alcohol consumption in humans was achieved by use of anticonvulsants, which are known to exert their effects via multiple mechanisms of action; however, one common feature of these compounds is the inhibition of voltage-gated Na⁺ and Ca²⁺ channels (^{Johnson, 2004}; ^{Johnson et al., 2004}).

DA/5-HT

Despite the fact that alcohol-induced DA release within the NAC is critically involved in the initiation of alcohol reinforcement processes (^{Di Chiara and Imperato, 1988}; ^{Spanagel and Weiss, 1999}), 6-hydroxy DA-induced lesion of the mesolimbic tract failed to alter voluntary self-administration in rats, suggesting a less important role of DA in maintaining alcohol consumption (^{Rassnick et al., 1993b}). However, postsynaptic changes in DA receptor signalling seem to be involved in the maintenance of voluntary alcohol intake as DA D1 and D2 receptor knockout mice show alterations in alcohol consumption (^{Crabbe et al., 2006}) and D1, D2 and D3 receptor agonists and antagonists were capable of modulating alcohol consumption in common stock rats (^{Pfeffer and Samson, 1988}; ^{Russell et al., 1996}; ^{Cohen et al., 1998}) and alcohol-preferring rats as well (^{Dyr et al., 1993}; ^{McBride and Li, 1998}; ^{Thanos et al., 2005b}).

Knockout mouse models and pharmacological manipulations of various components of the 5-HT system indicate a modulatory role of 5-HT in voluntary alcohol consumption. Thus, deletion of 5-HT transporters (^{Kelaï et al., 2003}) or overexpression of 5-HT₃ receptors (^{Engel et al., 1998}) leads to a reduction in alcohol self-administration as compared with control mice. Deletion of 5-HT_1B receptors was shown to increase alcohol intake (^{Crabbe et al., 1996}; but see also ^{Crabbe et al., 2006}). Pharmacological manipulations of 5-HT system activity revealed that administration of serotonin reuptake inhibitors, 5-HT₁, 5-HT₂ and 5-HT₃ agonists as well as antagonists were capable of reducing alcohol consumption in common stock as well as alcohol-preferring animals (^{Zhou et al., 1998}; ^{Lê and Funk, 2005}; ^{Ciccocioppo et al., 2006}; ^{Overstreet et al., 2006}).

Cannabinoids

Endocannabinoid transmission is acutely inhibited by alcohol (^{Ferrer et al., 2007}) and thus, becomes hyperactive during chronic alcohol administration, as revealed by the increase in the levels of endocannabinoids and the downregulation of CB1 receptors (^{Bilbao and Rodriguez de Fonseca, 2008}). Thus, chronic alcohol administration was found to be associated with an increased formation of the endocannabinoids anandamide and 2-AG (^{Basavarajappa and Hungund, 1999}; ^{Basavarajappa et al., 2000}). Most of the behavioural studies have focused on the pharmacological manipulation of the CB1 receptor showing that agonists increase (^{Colombo et al., 2002b}), whereas antagonists decrease alcohol intake and preference (^{Arnone et al., 1997}; ^{Gessa et al., 2005}). In agreement, CB1 receptor knockout mice were shown to consume less alcohol in most of the studies (^{Crabbe et al., 2006}).

Opioids

Opioid μ and κ receptor knockout mice were shown to exhibit lower alcohol self-administration, suggesting the significance of these receptors in the maintenance of alcohol consumption (^{Roberts et al., 2000}; ^{Kovacs et al., 2005}), whereas δ knockouts displayed greater alcohol preference and intake (^{Roberts et al., 2001}). In accordance, selective antagonists acting at either μ or κ opioid receptors were able to reduce alcohol consumption, which supports the hypothesis that following chronic alcohol intake the endorphin/μ opioid receptor system as well as the dynorphin/κ opioid receptor system is dysregulated (^{McBride and Li, 1998}; ^{Walker and Koob, 2008}).

CRF/NPY

A number of pharmacological studies using antagonists for the CRF1 receptor have demonstrated that activity of this receptor is responsible for excessive alcohol self-administration in non-selected rodents (^{Chu et al., 2007}; ^{Funk et al., 2007}) as well as in high-alcohol-preferring msP rats (^{Hansson et al., 2006}; ^{Gehlert et al., 2007}). In line with these findings, CRF1 receptor-deficient mice do not show any increase in self-administration following an induction of dependence (^{Chu et al., 2007}). It is, however, important to emphasize that CRF1 receptor antagonism is only efficient in animal models with excessive alcohol consumption but has only little effect on the maintenance of moderate voluntary alcohol consumption in the home cage. A possible reason is that long-term upregulation of CRF1 receptors is observed in the amygdala only following chronic and excessive alcohol drinking (^{Heilig and Koob, 2007}; ^{Sommer et al., 2008}). In this context, George Koob and Le Moal propose that changes in the activity of CRF system maintain hedonic stability in an allostatic state, as opposed to a homoeostatic state (^{Koob, 2003}; ^{Koob and Le Moal, 2006}) and thereby contribute to excessive alcohol consumption.

The NPY system is also believed to be involved in the maintenance of alcohol self-administration as administration of NPY antagonists have been shown to decrease alcohol self-administration in both genetically selected and non-selected alcohol-preferring lines (^{Gilpin et al., 2003}; ^{Schroeder et al., 2005b}) as well as in animals with a history of dependence (^{Rimondini et al., 2005}).

In summary, continuous alcohol use affects a wide range of brain neurotransmitter/neuromodulator systems, leading to adaptive, long-lasting changes. Thus, it is probably not possible to determine which of these systems contributes the most to the development of alcohol dependence, as it is likely to be interplay between different brain systems that is responsible for such a pathological process, rather than a specific action of alcohol exclusively on one of the systems. Some of those changes, however, cause the transition between controlled and compulsive alcohol use. This occurs only in a limited proportion of about 10–15% of alcohol users and is the result of initial genetic differences between individuals and environmental factors at critical phases of the life course.

Glutamate

Adaptive changes of glutamatergic systems may cause hyperexcitability of the CNS during withdrawal or conditioned withdrawal and further possibly represent one mechanism causing alcohol craving (^{Littleton, 2000}; ^{Gass and Olive, 2008}). However, NMDA receptors seem to play a minor role in conditioned alcohol-seeking responses (^{Bäckström and Hyytia, 2004}; ^{Bachteler et al., 2005}), whereas non-NMDA ionotropic glutamate receptors, especially AMPA receptors, are involved in this behaviour (^{Bäckström and Hyytia, 2004}; ^{Sanchis-Segura et al., 2006}). The use of AMPA receptor subunit GluRC knockout mice revealed an exceptional role of this subunit in cue-induced alcohol-seeking behaviour (^{Sanchis-Segura et al., 2006}). Not only ionotropic AMPA receptors but also mGluRs might be involved in alcohol-seeking behaviour. In particular, mGluR5 receptor antagonists are effective in the reinstatement model (^{Bäckström et al., 2004}). Activation of group II mGluRs attenuates both stress- and cue-induced reinstatement behaviour (^{Zhao et al., 2006}). However, another study using agonists acting at mGluR2/3 and mGluR8 receptors showed that significant effects of treatment on cue-induced reinstatement occurred only at doses that also decreased spontaneous locomotor activity (^{Bäckström and Hyytiä, 2005}).

GABA

Priming injections of the GABA_A receptor-positive modulator allopregnanolone dose-dependently reinstated extinguished responding for alcohol (^{Nie and Janak, 2003}), suggesting GABAergic control over alcohol seeking. The GABA_A receptor agonist muscimol administered directly into median raphe nucleus was also able to reinstate alcohol seeking (^{Lê et al., 2008}), implying an important role of this nucleus in this behaviour. Baclofen, an agonist acting at the GABA_B receptor, was shown to reduce the motivation to consume alcohol in rats (^{Colombo et al., 2003b}), suggesting a possible role of this receptor in alcohol craving as well. There are also indications that this drug might be efficient in reducing alcohol craving in humans (^{Addolorato et al., 2002}, ^2005a).

nAChR

Priming injections of nicotine reinstate alcohol seeking after a drug-free period in rats (^{Lê et al., 2003}, ^2006a), demonstrating an additional event that can induce craving. In fact, alcohol seeking and nicotine seeking are tightly associated most likely through their common action on nAChR. What is more, nAChR in the VTA mediate the DA-activating and -reinforcing properties of alcohol cues (^{Löf et al., 2007}).

Channels

The anticonvulsant drug lamotrigine, which was shown to inhibit Na⁺ channel activity and in turn reduce glutamate release, was found to significantly decrease cue-induced reinstatement of alcohol-seeking behaviour in rats (^{Vengeliene et al., 2007}).

DA/5-HT

D1, D2 and D3 DA receptor antagonists were shown to reduce cue-induced reinstatement of alcohol-seeking behaviour (^{Liu and Weiss, 2002b}; ^{Vengeliene et al., 2006}). The exposure of abstinent individuals to environmental cues previously associated with alcohol is known to induce mesolimbic DA release (^{Löf et al., 2007}), suggesting an important role of augmented extracellular DA levels in cue-induced alcohol craving/seeking.

Administration of a 5-HT_1A agonist, known to decrease 5-HT release, into median raphe nucleus reinstated alcohol seeking (^{Lê and Funk, 2005}), whereas systemic injections of 5-HT uptake blockers as well as 5-HT₃ antagonists attenuated intermittent foot-shock stress-induced reinstatement of alcohol seeking in rats (^{Lê et al., 1999}, ^2006b; ^{Lê and Funk, 2005}).

Cannabinoids

Acute administration of an anandamide reuptake inhibitor AM404 was incapable of altering cue-induced reinstatement of alcohol-seeking behaviour (^{Cippitelli et al., 2007}). In contrast, acute administration of a CB1 receptor antagonist has been shown to suppress this behaviour (^{Cippitelli et al., 2005}), suggesting a possible role of this particular receptor in cue-elicited craving for alcohol.

Opioids

Unselective opioid receptor antagonists such as naltrexone are efficient in reducing cue-induced responding of alcohol seeking (^{Katner et al., 1999}; ^{Hyytiä, 2005}) as well as priming-induced alcohol-seeking behaviour (^{Lê et al., 1999}). However, stress-induced reinstatement is not affected by this treatment (^{Lê et al., 1999}). Furthermore, selective antagonism of μ or δ opioid receptors inhibited alcohol-seeking behaviour elicited by drug-related environmental stimuli (^{Ciccocioppo et al., 2002}). A recent positron emission tomography study is in line with these preclinical findings as by means of the selective μ opioid receptor ligand carfentanil, a correlation of craving and μ opioid receptor availability in alcohol-dependent subjects could be detected (^{Heinz et al., 2005}).

CRF

Cue- and stress-induced reinstatement has been found to be blocked by CRF1 receptor antagonists (^{Lê et al., 2000}; ^{Gehlert et al., 2007}; ^{Marinelli et al., 2007}). Administration of a CRF1 receptor antagonist into median raphe nucleus blocked stress-induced reinstatement (^{Lê et al., 2002}), suggesting an interaction between CRF and 5-HT systems within this nucleus. In addition, the CRF system within the central nucleus of the amygdala is also believed to mediate stress-induced reinstatement of alcohol-seeking behaviour (^{Funk et al., 2003}). Antagonism of the CRF1 receptor was also shown to reduce reinstatement of alcohol seeking induced by the α2-adrenoreceptor antagonist yohimbine (^{Marinelli et al., 2007}).

In summary, exposure to environmental cues or stressors activates the mesolimbic DAergic system, which, together with a specific memory for alcohol, will induce an alcohol-seeking response. However, the fact that reinstatement of alcohol seeking can be initiated by at least four different events namely conditioned cues, priming, stress and nicotine shows that neurochemically and nueroanatomically distinct pathways can induce such a response, which is in accordance with the multi-pathway model of alcohol craving proposed by ^{Verheul et al. (1999)}. Given these findings, it is very unlikely that a specific pharmacological intervention will lead to overall blunted craving responses in alcohol-dependent patients.

Glutamate

One major hypothesis in the field of alcohol research proposes that the glutamatergic system is critically involved in addictive behaviour especially in relapse (^{Tsai and Coyle, 1998}; ^{Gass and Olive, 2008}). The clinically used drug acamprosate, known to reduce a hyper-glutamatergic system (^{Spanagel et al., 2005}), was capable of reducing the ADE in Wistar rats under home cage and operant conditions (^{Spanagel et al., 1996}; ^{Hölter et al., 1997}; ^{Heyser et al., 1998}). Furthermore, the study by ^{Hölter et al. (2000a)} has demonstrated that chronic treatment with a non-competitive NMDA channel antagonist selectively abolished the increased alcohol intake during the ADE. Similarly, reduction of relapse-like alcohol drinking after a deprivation phase occurred after administration of competitive and non-competitive antagonists acting at specific binding sites of the NMDA receptor (^{Vengeliene et al., 2005}). Reduced relapse-like drinking was also shown with use of an AMPA/kainate antagonist, whereas AMPA subunit deletion studies suggest a crucial role of the GluRC subunit in the expression of ADE (^{Cowen et al., 2003}; ^{Sanchis-Segura et al., 2006}). Antagonism of mGluR5 was also capable of reducing relapse-like alcohol self-administration in Wistar rats (^{Bäckström et al., 2004}) as well as of reducing repeated ADEs in alcohol-preferring P rats (^{Schroeder et al., 2005a}). And finally, agonists acting at mGluR2/3 receptors were found to be effective in reducing relapse-like alcohol self-administration (^{Rodd et al., 2006}). In conclusion, a decrease in glutamatergic activity and signalling in general seems to be sufficient to reduce relapse behaviour.

GABA

Administration of both the GABA_A agonist diazepam and antagonist flumazenil decreased post-deprivation alcohol consumption, whereas the inhibition of the GABA transporter by tiagabine resulted in an increase of the ADE (^{Schmitt et al., 2002}). GABA_B agonists were shown to decrease the expression of an ADE in alcohol-preferring sP rats (^{Colombo et al., 2003a}). In general, these findings suggest that the GABAergic system is involved in the expression of relapse-like drinking behaviour.

nAChR/glycine

A recent clinical study by ^{Mann et al. (2006)} showed that relapsed patients treated with the cholinesterase inhibitor galantamine consumed less alcohol per drinking day than patients treated with placebo.

The glycine reuptake inhibitor Org25935 significantly reduced alcohol intake after introduction of an alcohol deprivation period in rats (^{Molander et al., 2007}).

Channels

Administration of the anticonvulsant drug lamotrigine completely abolished the expression of an ADE in Wistar rats (^{Vengeliene et al., 2007}).

DA/5-HT

Free-choice alcohol drinking by alcohol-preferring P rats alters DA and 5-HT neurotransmission in the NAC during a deprivation phase. In the absence of alcohol, extracellular DA levels were enhanced for at least 2 weeks, suggesting that changes in monoaminergic systems may be in part responsible for increased alcohol drinking observed during the ADE (^{Thielen et al., 2004}).

Following long-term alcohol consumption with repeated deprivation phases, alcohol-preferring iP rats exhibited increased binding sites for D1 and D2 receptors in NAC, dorsal striatum and subamygdala regions (^{Sari et al., 2006}). Furthermore, an upregulation of DA D3 receptors was found in the dorsal striatum in alcohol-preferring HAD and P rats undergoing a similar procedure (^{Vengeliene et al., 2006}). This implies that the nigrostriatal DAergic pathway might mediate relapse-like drinking behaviour. Indeed, alcohol and other drugs of abuse produce strong habits that might involve the nigrostriatal pathway known to mediate those behaviours (^{Dickinson et al., 2002}; ^{Gerdeman et al., 2003}; ^{Everitt and Robbins, 2005}; ^{Spanagel and Heilig, 2005}). Thus, the observed upregulation of striatal D3 receptors after long-term home cage alcohol exposure might not be related to the alcohol intake per se but rather to the stimulus–response habit (^{Vengeliene et al., 2006}).

Administration of haloperidol, a DA antagonist with selectivity for D2-like receptors, led to a suppression of an ADE in mice (^{Salimov et al., 2000}). In addition, a highly selective D3 receptor antagonist caused a dose-dependent reduction of relapse-like drinking in the ADE model in Wistar rats (^{Vengeliene et al., 2006}).

5-HT₃ receptor antagonists, known to block alcohol-stimulated DA release in the mesolimbic system, were also able to suppress relapse-like drinking in alcohol-preferring P rats (^{Rodd-Henricks et al., 2000}).

Cannabinoids/opioids

^{González et al. (2004)} showed that the levels of endocannabinoids underwent significant changes in reward-related areas during relapse with lowest values in this phase. Thus, the induction of compensatory mechanisms, such as an upregulation of the CB1 receptor and a decrease in endocannabinoid levels within the endocannabinoidergic system, might be a determinant for relapse behaviour. In line with this suggestion is the observation that following alcohol consumption with repeated deprivation phases the CB1 receptor displays a long-lasting upregulation (^{Rimondini et al., 2002}). In fact the administration of rimonabant—an antagonist acting at the CB1 receptor—has been shown to suppress the ADE in alcohol-preferring sP rats (^{Gessa et al., 2005}).

Reduction of an ADE could also be achieved by the use of unselective opioid receptor antagonists (^{Hölter and Spanagel, 1999}; ^{Mormede et al., 2004}). In contrast, the initial increase in alcohol intake seen after a period of deprivation in high-alcohol-preferring rat lines was enhanced by administration of morphine (^{Mormede et al., 2004}). Similar results were obtained with the highly selective κ opioid receptor agonist CI-977 (enadoline). Thus, in long-term alcohol-experienced rats, a strong increase in their alcohol intake during expression of an ADE as a result of CI-977 treatment was seen (^{Hölter et al., 2000b}). Several studies in alcohol-dependent patients have linked specific genetic variations within opioidergic genes with alcoholism. ^{Xuei et al. (2006)}, for example, demonstrated an association of the κ opioid system with alcohol dependence.

NPY

Intracerebroventricular infusion of NPY has been shown to reduce alcohol intake after a deprivation phase (^{Gilpin et al., 2003}), whereas overexpression of NPY in the amygdala was shown to blunt the increase in alcohol intake following repeated deprivation phases (^{Thorsell et al., 2007}). This suggests an important role of this system in relapse to alcohol use.

In summary, the ADE model has been used in the last decade to map the neurochemical substrates underlying relapse-like drinking behaviour. Most evidence comes from pharmacological intervention studies on different glutamate receptors. Findings from these preclinical studies can be directly transferred to the human situation and in fact several new compounds, which have been characterized in the ADE model, are currently undergoing clinical testing for prevention of relapse behaviour (^{Spanagel and Kiefer, 2008}).