Endocannabinoid system in the development of tolerance to alcohol
Balapal S. Basavarajappa1&2 1Division of Analytical Psychopharmacology, New York State Psychiatric Institute, New York, NY. 2Nathan Kline Institute for Psychiatric Research, Orangeburg, NY, USA
The author presents an integrative review of current research examining the evidence that the endocannabinoid system plays an important role in the development of tolerance to alcohol. The identification of cannabinoid receptor (CB1 receptor), which was activated by the major psychoactive component of marijuana, (9-tetrahydrocannabinol ((9-THC), led to discovery of endocannabinoids. So far, arachidonylethanolamide (AEA), 2-arachidonylglycerol (2-AG), 2-arachidonylglycerol ether (noladin ether), and virodhamine have been isolated from both nervous and peripheral tissues. Both AEA and 2-AG have been shown to mimic the pharmacological and behavioral effects of (9-THC. Accumulated evidence suggests that various aspects of alcoholism including tolerance development to alcohol and alcohol intake are regulated by endocannabinoid system. AEA uptake, mRNA for CB1 receptors, CB1 receptors and its signal transduction are down regulated by chronic alcohol treatment. The observed down-regulation of CB1 receptor function results from the persistent stimulation of the receptors by AEA and 2-AG, the synthesis of which has been shown to be increased by chronic alcohol treatment. This enhanced formation of endocannabinoids may subsequently influence the synaptic plasticity. It was found that the CB1 receptor antagonist SR141716A (Rimonabant) or deletion of CB1 receptor gene has been shown to reduce voluntary alcohol intake. CB1 knockout mice also exhibited increased alcohol sensitivity, withdrawal and reduced conditioned place preference in rodents. Similarly, activation of the CB1 receptor system promoted alcohol craving. The current knowledge taken together clearly suggests a role for the endocannabinoid system in the development of alcoholism. (Klinik und Forschung 2005;11:16-19) Key words: Chronic Alcohol, Endocannabinoids, Alcohol Tolerance, Anandamide, 2-Arachidonylglycerol.
Alcohol dependence is usually accompanied by tolerance to the intoxicating effects of alcohol and by withdrawal symptoms including tremors and confusion when consumption of alcohol ceases. Although important advances have been made in recent years in understanding the mechanisms underlying the development of tolerance to and dependence on alcohol, the exact mechanisms are still elusive. The endocannabinoid system is constituted of cannabinoid receptors, endogenous cannabinoids, and the molecules involved in the inactivation of endocannabinoids (uptake and degradation enzyme known as fatty acid amide hydrolase, FAAH). Cannabinoid receptors belong to the large family of seven transmembrane-spanning G-protein-coupled receptors (GPCRs). There are two types of cannabinoid receptors, CB1 and CB2, defined by their unique localization. The CB1 receptor is widely distributed in several brain regions, with high-density in the cortex, hippocampus, basal ganglia, and cerebellum. Both CB1 and CB2 receptors have been characterized and cloned. The functional response of the CB1 and CB2 receptors is coupled through Gi/Go proteins, negatively to adenylate cyclase and N- and P/Q-type Ca2+ channels. They are positively coupled to A-type and inwardly rectifying K+ channels and mitogen- activated protein kinases .
In 1992, Devane and coworkers discovered the existence of endogenous cannabinoid substance anandamide (AEA) in mammalian brain. Since then three other endocannabinoids such as 2-arachidonylglycerol (2-AG), 2-arachidonylglycerol ether (noladin ether), and virodhamine have been identified. Unlike classical neurotransmitters and neuropeptides, AEA and 2-AG are not stored in intracellular compartments, but are produced on demand by receptor-stimulated cleavage of lipid precursors and released from neurons immediately afterwards .
Alcohol-induced brain cannabinoids
In the last eight years, several lines of evidence support the involvement of endocannabinoid system in the pharmacological actions of alcohol and in alcohol-drinking behavior . It was demonstrated that chronic alcohol exposure leads to activation of specific PLA2, a key enzyme involved in the formation of endocannabinoids in neuronal cells and brain [4,8]. Later it was found that the exposure of SK-N-SH cells or cerebellar granular neurons (CGNs) to chronic alcohol resulted in increased accumulation of AEA [5,10] and 2-AG (Table 1) . In these studies, it was demonstrated that the AEA and 2-AG synthesis increased with increasing duration of alcohol exposure, peaking at 72 h with 100mM alcohol, the experimental condition known to cause cellular tolerance and dependence to alcohol in neurons. These adaptive changes were further increased by the Ca2+-ionophore or ionomycin and inhibited by pertussis toxin (which selectively inactivates G-protein) and the CB1 receptor antagonist rimonabant, which is also shown to inhibit alcohol drinking in rodents [1,15,21,22,44]. Chronic alcohol treatment of Swiss-Webster male mice led to a significant increase in the brain levels of AEA and a significant decrease in N-ArPE, an immediate precursor for AEA synthesis, compared to control brains . A recent study also demonstrated that chronic alcohol exposure of rats caused a decrease in the content of both AEA and 2-AG in the midbrain, while AEA content increased in the limbic forebrain, a key area for the reinforcing properties of habit-forming drugs including alcohol . Although, the mechanism by which chronic alcohol exposure leads to selective increase in the levels of AEA and 2- AG remains to be established, these observations point to the possible involvement of endocannabinoids in alcohol-induced neuroadaptive changes in the brain. These neuroadaptive changes may influence the neuronal plasticity and may be responsible for the activation of the reward system.
AEA signaling at the cannabinoid CB1 receptors is terminated through an uptake mechanism that transports AEA into the cell, where it subsequently undergoes rapid degradation by FAAH [13,18,29,39]. Co-localization of both FAAH and CB1 receptors in the brain may point to a possible role of FAAH in AEA signaling and uptake . Thus, chronic alcohol-induced increases in extracellular AEA could result in a decrease in AEA influx, an increase in AEA efflux from the cell, and/ or altered intracellular metabolism . In a recent study, it was found that chronic exposure to alcohol leads to an increase in extracellular AEA by inhibiting the uptake of AEA. This effect was independent of the CB1 receptor since CB1 receptor knockout mice have normal uptake activity . After prolonged exposure to alcohol, cells become tolerant to the effects such that AEA uptake is no longer inhibited by acute alcohol . Chronic alcohol did not show any direct inhibition of FAAH activity in these neurons. These observations suggest that alcohol-induced inhibition of AEA uptake may, in part, be responsible for alcohol-induced increase in extracellular AEA.
Alcohol-induced regulation of cannabinoid receptors
Further, it was found that chronic alcohol not only increases endocannabinoids in neuronal cells and brain but also impairs the CB1 receptor function in the brain [3,6]. The results of this study indicate that the chronic alcohol exposure decreased the number of CB1 receptors and inhibited the ability of the CB1 receptor agonist to stimulate GTP(S binding in mice, possibly through sustained higher levels of endocannabinoids [5,9,31]. The observed down-regulation of the CB1 receptor signaling system by chronic alcohol may also result from over-stimulation of receptors through increased synthesis of the endogenous CB1 receptor agonist (AEA and or 2-AG). These observations are consistent with the recent data which indicate that forced consumption of high levels of alcohol significantly decreases CB1 receptor gene expression in the caudate-putamen (Cpu), ventromedial nucleus of the hypothalamus (VMN), and CA1 and CA2 fields of the hippocampus . It was shown that chronic alcohol exposure of rats was not effective in altering either CB1 receptor binding or mRNA levels in brain regions . The reason for this inconsistency is not known. These observations strongly support the participation of the endocannabinoid system in mediating some of the pharmacological and behavioral effects of alcohol, and the CB1 receptor may thus constitute an important target for therapeutic intervention in alcohol-related behaviors.
Further studies to examine whether the chronic alcohol-mediated down-regulation of brain CB1 receptors has any functional effect on CB1 receptor-activated G-proteins revealed that the net CB1 receptor agonist (CP-55, 940)-stimulated [35S] GTP(S binding was reduced significantly in chronic alcohol-exposed mice without any significant changes in the G-protein affinity . These results suggest that the observed down-regulation of CB1 receptors by chronic alcohol has a dramatic effect on desensitization of cannabinoid-activated signal transduction similar to that observed for (9-THC or other cannabinoids.
Cannabinoid receptors and voluntary alcohol consumption
Several studies have shown the inhibition of voluntary alcohol intake by CB1 receptor antagonist rimonabant in rodents. Rimonabant has been shown to decrease voluntary alcohol intake in alcohol-preferring C57BL/6 mice , in Sardinian alcohol-preferring (sP) rats, in alcohol self-administering Long Evans rats , and in alcohol-preferring congenic B6.Cb4i5-(/13C/Vad and B6.Cb4i5-(14/Vad mouse strains . Further, acute administration of the CB1 receptor agonist CP55, 940 increased the motivation to consume alcohol in Wistar rats and this effect was prevented by pretreatment with rimonabant [22,23]. An acute dose of rimonabant abolished the alcohol deprivation effect (i.e., the temporary increase in alcohol intake after a period of alcohol withdrawal) in sP rats . Acute administration of CB1 receptor agonists WIN 55,212-2 and CP-55, 940 significantly stimulated voluntary alcohol consumption in alcohol-preferring sP rats and this was prevented by rimonabant . Recently another CB1 receptor blocker SR147778 significantly decreased ethanol consumption in both rats and mice . In alcohol-experienced sP rats SR-147778 reduced the alcohol intake. When alcohol-experienced sP rats are deprived of alcohol for 15 days, there is a large intake of alcohol on reintroduction of alcohol, and this response was almost abolished by treatment with SR-147778 . These observations indicate that the endogenous cannabinoid system may play a critical role in the appetitive value of alcohol in these experimental models of alcoholism. Further studies to address the mechanism by which CB1 receptor blocked by rimonabant or SR147778 reduce the voluntary alcohol consumption needs further investigation. Since rimonabant blocked alcohol-evoked dopamine release in the shell of the NAc following alcohol administration  and CB1 receptor knockout mice lack acute alcohol-induced increase in dopamine in NAc dialysates in C57BL/6  suggest the role of dopaminergic system that projects from the ventral tegmental area (VTA) of the midbrain to the nucleus accumbens (NAc), and to other forebrain sites including the dorsal striatum, is the major substrate of reward and reinforcement produced by alcohol [19,33-35,43,49,50]. It is well established that cannabinoids activate dopaminergic neurons in the VTA [19,24,43,47,49,50], resulting in the release of dopamine in the NAc . CB1 receptor knockout mice displayed increased dopamine D2 receptors in striatum . In addition, pretreatment with rimonabant enhanced the hyperactivity exhibited by administration of D2-like receptor agonist , suggesting that existence of functional interaction between endocannabinoid system and dopaminergic system. It should be to noted that CB1 receptors are not localized on dopamine cell bodies or on their nerve terminals [28,36]. It is therefore unlikely that observed blockade of alcohol-induced dopamine release by rimonabant may involve afferent pathways to the VTA. This action may also explain the reducing effects of rimonabant on alcohol self-administration by indirectly blocking the activation of the mesolimbic dopaminergic transmission .
The adaptive changes noted in the endocannabinoid system after chronic alcohol treatment may be important for the development of alcohol-seeking behavior and further research is required to establish this phenomenon (Fig. 1). The further evidence for the participation of the endocannabinoid system in alcohol drinking behavior is derived from the observed differences in CB1 receptor function in two genetic strains of alcohol-preferring C57BL/6 and alcohol-avoiding DBA/2 mice. It was found that DBA/2 mice have significantly lower functional CB1 receptors than C57BL/6 mice , suggesting the CB1 receptor mediated signaling mechanism in regulating voluntary alcohol consumption. Thus genetically determined differences in the activities of distinct components of the endocannabinoid system under basal conditions or in response to alcohol exposure may exist between alcohol-preferring and alcohol-avoiding animals and may be partially responsible for the differences in their voluntary alcohol intake.
This hypothesis was further examined using genetically modified CB1 receptor knockout mice. The CB1 receptor expression could be detected in regions that influence several key functions including memory, cognition, mood, motor coordination, sensation, and autonomic function. Several studies have also shown the inhibition of voluntary alcohol intake in CB1 receptor knockout mice. It has been shown that young CB1 receptor wild-type mice showed a significantly higher alcohol preference and voluntary alcohol intake compared to their CB1 knockout littermates . Further, rimonabant has been shown to reduce voluntary alcohol intake in CB1 receptor wild-type but not in knockout mice . Similarly, administration of rimonabant significantly reduced alcohol and sucrose intake in C57BL/6x129/Ola mice and had no effect in CB1 receptor knockout C57BL/6x129/Ola mice . Recent study also provides unequivocal evidence for the participation of CB1 receptor in the regulation of voluntary alcohol consumption and some of the acute intoxicating effects caused by administration of alcohol . Alcohol consumption and preference are decreased, whereas alcohol sensitivity and withdrawal severity are increased in CB1 knockout mice . CB1 receptor knockout mice showed an increase in alcohol withdrawal-induced convulsions suggesting that alcohol consumption is also inversely related to alcohol withdrawal severity. Alcohol produced similar reduction in body temperature in CB1 knockout and wild-type mice . The motor coordination on rotarod was reduced in both CB1 knockout and wild-type mice . In another study, CB1 knockout mice (CD1 back ground) were more sensitive to the hypothermic and sedative/hypnotic effects of alcohol than wild-type mice . CB1 knockout mice displayed a significant decrease in locomotor activity following injection of alcohol . Surprisingly, it was shown recently that alcohol tolerance was not affected by the CB1 receptor deletion in C57BL/6 mice and acute alcohol effects such as hypothermia and motor-incoordination were shown not to be mediated by CB1 receptors . However, alcohol withdrawal symptoms observed in CB1 receptor wild-type mice were not observed in CB1 receptor knockout mice . Currently the reason for this discrepancy is not clear. In recent study, an increased endogenous brain level of AEA and related fatty acid amides and an endocannabinoid tone was shown in FAAH knockout (FAAH/) mice . Using these mice, recent study (preliminary data) suggests that female mice exhibited increased alcohol intake and less sensitive to acute alcohol effects . These data taken together indicate that the endocannabinoid system could be important for alcoholism including alcohol drinking behavior and alcohol tolerance. These findings are significant for the development of potential therapeutic strategies for the treatment of alcoholism.
Over the past several years, remarkable advances have been made in our understanding of the role of the endocannabinoid system in the development of alcohol tolerance and alcohol-drinking behavior (Fig. 1). These studies have provided strong evidence that the endocannabinoid system serve as an attractive therapeutic target for the treatment of alcohol tolerance and alcohol-related disorders. However, further studies are necessary to unfold the exact mechanism by which alcohol exerts its pharmacological and behavioral effects through the endocannabinoid system. These results suggest that the cannabinoid antagonist, rimonabant or SR147778, may be useful as a potential therapeutic agents for not only obesity but also for alcoholism.
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Correspondence address: Balapal S. Basavarajappa, Ph.D. Division of Analytical Psychopharmacology Nathan Kline Institute for Psychiatric Research, 140 Old Orangeburg Rd, Orangeburg, NY-10962, Tel: 845-398-3234; Fax, 845-398-5451, Basavaraj@nki.rfmh.org.