involvement of the endocannabinoid system in alcohol dependence: the biochemical, behavioral and...
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Drug and Alcohol Dependence 117 (2011) 102–110
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Drug and Alcohol Dependence
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Review
Involvement of the endocannabinoid system in alcohol dependence:The biochemical, behavioral and genetic evidence
Amaia M. Erdozaina,b, Luis F. Calladoa,b,∗
a Department of Pharmacology, University of the Basque Country, 48940 Leioa, Bizkaia, Spainb Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Spain
a r t i c l e i n f o
Article history:Received 19 October 2010Received in revised form 7 February 2011Accepted 14 February 2011Available online 16 March 2011
Keywords:Alcohol dependenceCannabinoidsEndocannabinoid systemReceptorsRimonabant
a b s t r a c t
Background: Recent advances in the understanding of alcohol dependence suggest that the endocannabi-noid system (ECS) plays a key role in the neurobiological mechanisms underlying this pathology.Methods: The aim of the present review is to show the currently available biochemical, behavioral andgenetic evidence on the involvement of the ECS in alcohol dependence.Discussion: Firstly, biochemical studies have shown that both chronic and acute administration of ethanolproduce alterations in different elements of this neurotransmission system. Secondly, the pharmacologi-cal and genetic manipulation of the ECS in rodents result in altered ethanol-related behavior. Furthermore,rodent strains with different preference for ethanol differ in their ECS state. Also, genetic studies havedescribed that particular polymorphisms in the genes coding for some elements of this system are associ-ated with some phenotypes of alcohol dependence. Finally, the possible efficacy of cannabinoid receptorblockers in the prevention of relapse to alcohol has been tested in clinical trials.Conclusion: Altogether, these multiple lines of evidence suggest that the ECS is implicated in the devel-opment of alcohol abuse and dependence.
© 2011 Elsevier Ireland Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1021.1. The endocannabinoid system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1031.2. The endocannabinoid system and addiction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
2. Biochemical evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1033. Behavioral evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
3.1. Pharmacological modulation of the endocannabinoid system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053.2. Genetic modulation of the endocannabinoid system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
4. Genetic evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1065. Rimonabant for the treatment of alcohol dependence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1086. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Role of funding source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
1. Introduction
Alcohol dependence is a costly and socially devastating illness.Ethanol produces a complex and multidimensional effect on healthand the global burden related to alcohol consumption in terms of
∗ Corresponding author at: Department of Pharmacology, University of the BasqueCountry, Leioa, Bizkaia, Spain. Tel.: +34 94 6012762; fax: +34 94 6013220.
E-mail address: [email protected] (L.F. Callado).
morbidity, mortality and disability is very important. According tothe World Health Organization in 2000 alcohol dependence wasresponsible for 4% of global disease, constituting only a slightlylower level than that caused by smoking (4.1%) and hypertension(4.4%) (World Health Organization, 2004).
Ethanol is toxic to most body tissues, producing changes onthe cardiovascular system, digestive system, central nervous sys-tem, peripheral nerves, muscle–skeletal system and the fetus. Inthe central nervous system ethanol generates a deep depression ofneuronal functions, concomitantly decreasing glucose metabolism
0376-8716/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.drugalcdep.2011.02.003
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throughout the human brain (Wang et al., 2000). Nowadays, it iswell known that ethanol affects most of the neurochemical andendocrine systems (Diamond and Gordon, 1997), among which isthe endocannabinoid system (ECS).
The aim of the present review is to provide the most recent sci-entific evidence about the involvement of the ECS in the processof the development, tolerance, withdrawal, craving and relapse ofalcohol dependence. For this purpose we have performed a com-puterized search of the literature on PubMed and Scopus in orderto obtain biochemical, behavioral or genetic evidence. A secondarysearch was done by hand searching the reference lists from primarystudies and former reviews.
1.1. The endocannabinoid system
The ECS constitutes a newly discovered system of neuromodu-lation and comprises the cannabinoid receptors, along with theirendogenous ligands, enzymes involved in the biosynthesis anddegradation of these ligands and putative membrane transportproteins. So far, two cannabinoid receptors have been identifiedand cloned. The CB1 receptor (Matsuda et al., 1990) is mainlyexpressed in the brain, where it plays an important role in theregulation of neurotransmitter release (Wilson and Nicoll, 2002),and it is, indeed, the most abundant G-protein-coupled receptorin the mammalian brain. Conversely, the CB2 receptor (Munroet al., 1993) is mainly located in peripheral tissues, at the high-est levels in the immune system, such as spleen, tonsil, thymusand lymphoid tissues (Galiegue et al., 1995). The endocannabinoidsare polyinsaturated fatty acids derived compounds, which sharesome pharmacologic properties of �9-THC. To date, anandamide(N-araquidonoylethanolamine, AEA) and 2-arachidonoylglycerol(2-AG) are the most thoroughly studied endocannabinoids, whichare produced on demand and released in the extracellular environ-ment, where they can bind and activate CB1 and CB2 cannabinoidreceptors among others. Their hydrolysis, resulting in the termina-tion of endocannabinoid signaling, is an intracellular event carriedout by different enzymes, mainly the fatty acid amide hydrolase(FAAH) and monoacylglycerol lipase (Alexander and Kendall, 2009).FAAH is the main enzyme involved in AEA degradation, produc-ing arachidonic acid and ethanolamime. In neural tissues, FAAHimmunoreactivity is associated primarily with neurons, in a pat-tern extensively complementary to the expression of CB1 receptors(Tsou et al., 1998).
1.2. The endocannabinoid system and addiction
There is much evidence suggesting an association between theneuroanatomical distribution of the ECS and some of the centraleffects produced by ethanol. More precisely, CB1 receptor localiza-tion in the cerebral cortex, hippocampus, thalamus, basal ganglia,cerebellum and medulla is consistent with the effects of alcohol onhigher cognitive and motor functions, memory, hypothermia andnociception (Herkenham et al., 1991; Glass et al., 1997).
The crucial role of the mesocorticolimbic dopaminergic path-way in the reward and reinforcement produced by all drugs ofabuse, including alcohol, has thoroughly been documented (Kooband Volkow, 2010). In this way, the ECS seems to be involvedin the regulation of this reward pathway. Thus, CB1 cannabinoidreceptors are present in different regions of the mesocorticolim-bic circuitry and are involved in the neurobiological mechanismsunderlying the addictive processes of drugs of abuse (Maldonadoet al., 2006). It has been described that following depolarizationdopaminergic neurons in the ventral tegmental area (VTA) releaseendocannabinoids that act as a regulatory feedback mechanism(Lupica and Riegel, 2005). However, since dopaminergic neuronsdo not appear to express CB1 receptor (Herkenham et al., 1991;
Matsuda et al., 1993), this regulatory mechanism is likely to beindirect.
Additional data supporting the involvement of the ECS in thereward pathway arises from in vivo microdialysis studies. Firstly,acute alcohol, and nicotine, induced increase of dopamine levels inthe nucleus accumbens is significantly reduced by the CB1 receptorantagonist rimonabant (Cohen et al., 2002). Similarly, in CB1 recep-tor knockout mice acute ethanol administration did not produceany dopamine release in the same region (Hungund et al., 2003).These studies confirm that the dopamine release induced by somedrugs of abuse is CB1 receptor dependent.
Nonetheless, the ECS is not only involved in the rewardingproperties of alcohol, but also in the motivation for drug seek-ing, the individual vulnerability for developing a dependence, theacquisition of ethanol self-administering behavior, the develop-ment of the withdrawal syndrome, etc. There is strong evidenceof the involvement of the ECS in alcohol dependence. In thisarticle biochemical, behavioral and genetic evidence of this impli-cation will be reviewed. Firstly, biochemical studies have shownthat both chronic and acute administration of ethanol producealterations in different elements of the endocannabinoid system.Secondly, the pharmacological and genetic manipulation of thissystem in rodents result in altered ethanol-related behavior. Fur-ther, rodent strains with different preference for ethanol differ intheir ECS state. Finally, genetic studies have described that partic-ular polymorphisms in the genes coding for some elements of thisneurotransmission system are associated with some phenotypes ofalcohol dependence.
2. Biochemical evidence
Several studies have demonstrated that chronic exposure toethanol modulates some elements of the ECS both in vitro and invivo (Table 1). The first in vitro study reported a concentration- andtime-dependent increase in anandamide formation after chronicexposure of neuroblastoma SK-N-SH strain cells to ethanol. Thisincrease in the endocannabinoid formation was inhibited by theCB1 receptor antagonist rimonabant and pertussis toxin, sug-gesting a CB1 receptor and Gi/o protein mediated regulation(Basavarajappa and Hungund, 1999). Shortly thereafter, the stim-ulation of the other main endocannabinoid formation, the 2-AG,was reported after chronic exposure of cerebellar granule neuroncultures to ethanol, being similarly inhibited by rimonabant andpertussis toxin (Basavarajappa et al., 2000).
The modulation of the endocannabinoid content by chronicethanol has also been observed in rodent brain, although the resultsare not consistent among all the studies. Thus, anandamide lev-els were increased in the limbic forebrain while decreased in themidbrain of ethanol-treated rats (Gonzalez et al., 2002a, 2004).Another study corroborated this increase of anandamide in micecerebral cortex after chronic ethanol exposure, which was con-sistent with a concomitant significant reduction in FAAH activity(Vinod et al., 2006). In regard to 2-AG, an in vivo microdialysisstudy reported increased dialysate levels of this endocannabi-noid in the nucleus accumbens of ethanol self-administering rats,with no concomitant change in anandamide concentrations (Cailleet al., 2007). However, other studies observed either a decrease(Gonzalez et al., 2002a) or no change (Gonzalez et al., 2004) in2-AG levels in rat midbrain. Nevertheless, these endocannabinoidlevels modulated by chronic ethanol administration are furtheraffected by alcohol-deprivation and relapse to alcohol consumption(Gonzalez et al., 2004).
Cannabinoid CB1 receptor mRNA, density and functionality havealso been reported to be altered by chronic exposure to ethanol. Thefirst study indicated a significant down-regulation of the CB1 recep-tor density in whole mouse brain chronically exposed to ethanol,
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Table 1Biochemical evidence about the involvement of the endocannabinoid system in alcohol dependence.
Type of study Results Reference
In vitro studies
ECBsChronic ethanol increased AEA formation in SK-N-SH cells Basavarajappa and Hungund (1999)Chronic ethanol increased 2-AG formation in cerebellar granule neurons Basavarajappa et al. (2000)
Animal brain studies
ECBsChronic ethanol decreased AEA and 2-AG contents in the midbrain and increased AEA contentin the limbic forebrain
Gonzalez et al. (2002a)
Levels of ECBs underwent significant changes in reward-related rat brain areas duringalcoholization, deprivation and relapse
Gonzalez et al. (2004)
Ethanol self- administration increased 2-AG levels, without altering AEA levels, in rat nucleusaccumbens (in vivo microdialysis)
Caille et al. (2007)
CB1R
Chronic ethanol down-regulated CB1Rs in whole mouse brain membranes Basavarajappa et al. (1998)Chronic ethanol was usually ineffective in altering CB1R binding and mRNA levels in all ratbrain regions examined
Gonzalez et al. (2002b)
Chronic ethanol reduced CB1R mRNA expression in some rat brain regions Ortiz et al. (2004a)Ethanol desensitized CB1R modulating the synthesis of monoamines Moranta et al. (2006)
CB1R & ECBsChronic ethanol reduced CB1 receptor binding and functionality in several mouse brainregions; increased AEA levels and reduced FAAH activity in cortex
Vinod et al. (2006)
Chronic intermittent ethanol withdrawal transiently down-regulated rat hippocampal CB1Rs,followed by long-term up-regulation, with increased EBCs
Mitrirattanakul et al. (2007)
Human brain studiesCB1R & ECBs Elevated CB1R density and functionality and ECBs in the prefrontal cortex of alcoholic suicide
victims compared to non-suicidal alcoholicsVinod et al. (2005)
CB1R & FAAH Decreased CB1R density and functionality and FAAH activity in the ventral striatum ofnon-suicidal alcoholics compared to controls
Vinod et al. (2010)
ECBs Decreased AEA in nucleus accumbens and frontal cortex of Cloninger type 1 alcoholics Lehtonen et al. (2010)
Acute alcohol
ECBsReduced ECB levels in different rat brain regions Rubio et al. (2007)Reduced AEA in different rat brain regions, which was not dependent on FAAH or NAT activity Ferrer et al. (2007)Reduced AEA, increased 2-AG in rat nucleus accumbens (in vivo microdialysis) Alvarez-Jaimes et al. (2009)
CB1R Reduced CB1R mRNA in different rat brain regions Oliva et al. (2008)CB1R & FAAH Reduced CB1R levels in the rat amygdala and prefrontal cortex; FAAH activity was not
increased in any of the regions analyzedRubio et al. (2009)
ECBs: endocannabinoid levels. CB1R: CB1 receptor.
without any changes in the affinity (Basavarajappa et al., 1998).This finding agrees with a reduction in the CB1 receptor density andfunctionality observed in particular brain regions of mice follow-ing the same ethanol exposure (Vinod et al., 2006). Similarly, CB1receptor mRNA expression was found to be decreased in severalrat brain regions after chronic ethanol consumption (Ortiz et al.,2004a). Nonetheless, another study did not observe any changesafter chronic exposure to ethanol either in CB1 receptor bindingor mRNA levels in rat brain (Gonzalez et al., 2002b). In regard tothe neuromodulation that exerts the CB1 receptor over neurotrans-mitter synthesis and release, ethanol also produces a functionaldesensitization of CB1 receptors in rats, modulating the synthesisof brain monoamines (Moranta et al., 2006).
Some studies have focused on the effect of alcohol with-drawal on the CB1 receptors. A transient down-regulation ofthese receptors followed by a long-term up-regulation, with con-comitant increased endocannabinoid levels, has been described inrat hippocampus after chronic intermittent ethanol withdrawal(Mitrirattanakul et al., 2007). Similarly, a recovery of the decreasedCB1 receptor binding and functionality has also been reported afterwithdrawal (Vinod et al., 2006).
On the whole, the biochemical studies performed in rodents thatchronically consume ethanol suggest a down-regulation of brainCB1 receptors, which is likely to be the consequence of high endo-cannabinoid levels observed in these animals. The reason for thishigh endocannabinoid content is still unknown and might be eithera higher synthesis rate, a lower degradation rate or even a decreasedre-uptake of the synaptic endocannabinoids.
In recent years some studies have been performed in postmortemhuman brain of subjects that had previously been diagnosed of alco-
hol dependence. A hyperactivity of the endocannabinoid signalinghas been reported in the prefrontal cortex of suicidal alcoholic sub-jects compared to alcoholic subjects dying of other causes thansuicide. These suicidal alcoholic subjects presented higher CB1receptor density and functionality, as well as higher anandamideand 2-AG levels. The authors suggested that this endocannabinoidhyperactivity in the prefrontal cortex could be a potential factorfor suicide in alcohol dependence (Vinod et al., 2005). Recently,the same group has described decreased CB1 receptor bindingand functionality in the ventral striatum of non-suicidal alcoholicsubjects compared to controls. However, these parameters wereelevated in the suicidal alcoholics when compared to non-suicidalalcoholic subjects (Vinod et al., 2010). In regard to the differencesbetween Cloninger type 1 and 2 alcoholics, decreased anandamidelevels were observed in the nucleus accumbens and frontal cortexin type 1 alcoholics (Lehtonen et al., 2010). Thus, studies performedin human brain show contradictory results probably due to con-founding variables as suicide.
Several recent reports have shown that acute alcohol adminis-tration does also induce changes in the endocannabinoid system.Reduced levels of endocannabinoids were observed in differentbrain regions after short-term exposure to ethanol (Rubio et al.,2007). Similarly anandamide content was also found reduced inseveral rat brain regions, which was not dependent on FAAH or N-acyltransferase activity (Ferrer et al., 2007). An in vivo microdialysisstudy confirmed this reduction of dyalisate anandamide levels inthe nucleus accumbens after an acute ethanol injection, but con-versely observed an increase in 2-AG levels (Alvarez-Jaimes et al.,2009). Furthermore, CB1 receptors are also affected by short-termexposure to ethanol, since a decrease in the receptor mRNA and pro-
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Table 2Behavioral evidence about the involvement of the endocannabinoid system in alcohol dependence, after pharmacological manipulation of the system.
Modulation Results Reference
AgonistsCP55,940 Increased the motivation for consuming beer in rats Gallate et al. (1999)WIN 55,212-2 and CP55,940 Increased ethanol intake in alcohol preferring Sardinian rats Colombo et al. (2002)WIN 55,212-2 Increased alcohol relapse during a period of alcohol deprivation Alen et al. (2008)
Antagonists
Rimonabant (SR141716)
Reduced ethanol intake in alcohol-preferring mice Arnone et al. (1997)Reduced ethanol intake in alcohol-preferring rats Colombo et al. (1998) and Dyr et al. (2008)Reduced the motivation for consuming beer in rats Gallate and McGregor (1999)Reduced ethanol self-administration in alcohol dependent rats Rodriguez de Fonseca et al. (1999)Reduced ethanol preference after chronic alcoholization in rats Lallemand et al. (2001)Reduced ethanol seeking and consumption in rats Freedland et al. (2001)Prevented acquisition of drinking behavior in alcohol-preferring rats Serra et al. (2001)Abolished the alcohol deprivation effect in alcohol-preferring rats Serra et al. (2002)Abolished motivational properties of alcohol in alcohol-preferring rats Colombo et al. (2004)Reduced ethanol self-administration and conditioned reinstatement ofethanol-seeking in both alcohol-preferring and non-preferring rats
Cippitelli et al. (2005)
Reduced ethanol self-administration in alcohol-preferring rats whenadministered systemically or locally into the prefrontal cortex
Hansson et al. (2007)
Surinabant (SR147778)Reduced ethanol intake in mice and rats Rinaldi-Carmona et al. (2004)Reduced ethanol intake and motivational properties in alcohol-preferring rats Gessa et al. (2005)Reduced ethanol preference after chronic alcoholization in rats Lallemand and De Witte (2006)
SLV330 Reduced ethanol self-administration and reinstatement of ethanol seeking inrats
De Bruin et al. (2011)
FAAH inhibitors
URB597Increased ethanol self-administration in rats Hansson et al. (2007)Increased ethanol preference and consumption in mice Blednov et al. (2007)Increased ethanol preference in mice Vinod et al. (2008)
Anandamide transporter inhibitorsAM404 Reduced ethanol self-administration in rats Cippitelli et al. (2007)
tein levels has been reported in particular rat brain regions (Olivaet al., 2008; Rubio et al., 2009).
In summary, chronic or acute alcohol administration seem toproduce different effects in the ECS. These differences may be dueto the adaptative responses produced by the chronic exposure toethanol.
3. Behavioral evidence
3.1. Pharmacological modulation of the endocannabinoid system
A wide variety of reports have demonstrated that the pharmaco-logical modulation of the endocannabinoid system, by cannabinoidagonists, antagonists, FAAH inhibitors or anandamide transporterinhibitors, alters the ethanol-related behavior in rodents (summa-rized in Table 2). These findings further support the involvement ofthe ECS in neurobiology underlying alcohol dependence.
First of all, cannabinoid agonists WIN 55,212-2 and CP 55,940,have been shown to increase ethanol consumption and prefer-ence. They produced an augment in the motivation for beer in rats(Gallate et al., 1999), in addition to enhance ethanol intake in mice(Kelai et al., 2006) and in a selectively bred alcohol-preferring Sar-dinian rat strain (Colombo et al., 2002). Furthermore, WIN 55,212-2increased alcohol relapse during a period of alcohol deprivation inrats (Alen et al., 2008).
Conversely, the administration of the cannabinoid antago-nists rimonabant (SR141716) and surinabant (SR147778) reducesalcohol-related behavior and consumption. The first research workto study this issue showed that rimonabant inhibited ethanol intakein selectively bred alcohol-preferring mice tested under the two-bottle choice paradigm (Arnone et al., 1997). This phenomenonwas later confirmed in different strains of alcohol-preferring rats(Colombo et al., 1998; Dyr et al., 2008), as well as in non-preferringmice (Poncelet et al., 2003) and both non-preferring rats exposed
or not to pulmonary chronic alcoholization (Lallemand et al., 2001).In other type of studies animals are required to complete an oper-ant response in order to obtain access to alcohol, which allowsstudying the motivation of the animal to consume alcohol. Thus,rimonabant reduced motivation for alcohol in a dose-dependentmanner both in non-preferring (Gallate and McGregor, 1999) andpreferring rats (Colombo et al., 2004). Similarly, operant ethanolself-administration was reduced by rimonabant in rats tested underdifferent procedures (Freedland et al., 2001; Rodriguez de Fonsecaet al., 1999; Caille et al., 2007; Cippitelli et al., 2005; Hansson et al.,2007). Further, rimonabant prevented the acquisition of ethanoldrinking behavior in alcohol-preferring rats (Serra et al., 2001).
In order to study the withdrawal and relapse in animals, twoexperimental procedures have been developed. Firstly, the alcoholdeprivation effect consists in the temporary increase in volun-tary ethanol intake after a period of alcohol withdrawal. Indeed,rimonabant completely abolished the alcohol deprivation effectin alcohol-preferring rats (Serra et al., 2002). The second pro-cedure assesses the reinstatement of alcohol-seeking behaviorinduced by a specific stimulus which was previously associatedwith alcohol availability. In the same way, rimonabant reducedconditioned reinstatement of ethanol-seeking behavior both inalcohol-preferring and non-preferring rats (Cippitelli et al., 2005).
Similar results to those obtained with rimonabant havebeen further observed with another cannabinoid antagonist thatwas more recently synthesized, the surinabant or SR1417778(Rinaldi-Carmona et al., 2004). A first study indicated that underthe two-bottled choice paradigm surinabant dose-dependentlydecreased ethanol consumption in mice (Rinaldi-Carmona et al.,2004). Likewise, surinabant reduced ethanol intake and preferencein alcohol-preferring rats (Gessa et al., 2005) and non-preferringrats that had been chronically alcoholized (Lallemand and De Witte,2006). Also, this cannabinoid antagonist dose-dependently reducedthe acquisition of ethanol drinking in alcohol-preferring rats and
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Table 3Behavioral evidence about the involvement of the endocannabinoid system in alcohol dependence, after genetic manipulation of the system.
Modulation Results Reference
CB1R knockout miceDecreased ethanol intake and absence of alcohol-induced DA release in N accumbens Hungund et al. (2003)Absence of ethanol withdrawal symptoms and stress-stimulated ethanol drinking Racz et al. (2003)Decreased ethanol intake and preference in young mice Wang et al. (2003)Decreased ethanol intake Poncelet et al. (2003)Decreased ethanol self-administration and increased alcohol sensitivity and withdrawal Naassila et al. (2004)Decreased ethanol-induced conditioned place preference Houchi et al. (2005)Decreased ethanol preference; increased blood ethanol concentration at high ethanol doses Lallemand and De Witte (2005)Decreased ethanol intake and preference; absence of conditioned place preference Thanos et al. (2005)
FAAH knockout miceIncreased ethanol intake and preference; decreased sensitivity in female FAAH−/− mice Basavarajappa et al. (2006)Increased ethanol intake and preference; no differences in the ethanol-induced place preference, sensitivity or withdrawal Blednov et al. (2007)Increased ethanol preference; decreased ethanol sensitivity and withdrawal convulsions Vinod et al. (2008)
suppressed the alcohol deprivation effect after a period of alcoholabstinence (Gessa et al., 2005).
Recently, it has been reported that another CB1 cannabinoidreceptor antagonist, SLV330, was effective in reducing ethanol self-administration and reinstatement of ethanol seeking in rats (DeBruin et al., 2011).
The indirect activation of the ECS has also been studied in regardto alcohol dependence. Thus, FAAH inhibitor URB597 administra-tion increased operant ethanol self-administration in rats (Hanssonet al., 2007), and similarly produced an increase in ethanol prefer-ence and consumption in mice in a two-bottle choice paradigm(Blednov et al., 2007; Vinod et al., 2008). On the other hand, theacute administration of AM404, an inhibitor of the putative anan-damide transporter, reduced ethanol self-administration in rats(Cippitelli et al., 2007).
3.2. Genetic modulation of the endocannabinoid system
An additional approach to understand the involvement of theECS in particular situations relies on the genetic modulation of thesystem. So far, CB1 receptor and fatty acid amide hydrolase (FAAH)enzyme knockout mice have been studied (summarized in Table 3)and show altered ethanol-related behavior.
First of all, mice lacking CB1 receptor exhibited markedlyreduced voluntary alcohol consumption in a two-bottle choiceparadigm (Hungund et al., 2003; Poncelet et al., 2003), which wasconcomitant with a complete lack of ethanol-induced dopaminerelease in the nucleus accumbens (Hungund et al., 2003). A similarstudy observed this decrease in ethanol intake, but only in youngmice that did not express CB1 receptors, since old mice showedsimilar preference for ethanol regardless of their genetic back-ground (Wang et al., 2003). The reduction in ethanol consumptionwas further confirmed in an operant self-administration paradigm(Naassila et al., 2004; Thanos et al., 2005). Also, these animals showa reduced ethanol-induced conditioned place preference, whichfurther suggests the involvement of the ECS in alcohol depen-dence, beyond the rewarding properties of ethanol (Houchi et al.,2005; Thanos et al., 2005). Furthermore, ethanol withdrawal is sig-nificantly altered in CB1 receptor knockout mice, although thereare discrepancies in the results. Racz et al. (2003) reported thatethanol withdrawal symptoms were completely absent in thesegenetically modified mice, while Naassila et al. (2004) observed anincrease in alcohol sensitivity and withdrawal induced convulsions.Additionally, ethanol induced higher blood ethanol concentrationsin CB1 knockout mice, while decreasing ethanol preference. Thisfact revealed that CB1 receptors might also be involved in ethanolabsorption and distribution, particularly after administration ofhigh ethanol doses (Lallemand and De Witte, 2005).
Secondly, the ethanol-related behavior has also been studiedin FAAH knockout mice, which indeed have higher levels of endo-cannabinoids compared to their wild type mates. All the studiesagree that these mice show increased intake and preference forethanol (Basavarajappa et al., 2006; Blednov et al., 2007; Vinodet al., 2008), although one of them only observed this phenomenain female mice, suggesting a sex-link mechanism (Basavarajappaet al., 2006). Also, FAAH knockout mice displayed lower sensitivityto acute effects of ethanol, and a reduction in ethanol withdrawalconvulsions (Vinod et al., 2008). However, Blednov et al. (2007) didnot observe any difference in ethanol sensitivity or withdrawal.
The findings of this section show that the stimulation of the ECSincreases ethanol consumption, whereas a blockade of the systemreduces ethanol administration. These effects may be due to a possi-ble mechanism through which the endocannabinoids may enhancethe reinforcing properties of ethanol.
4. Genetic evidence
Alcohol dependence is a heterogeneous complex disorder with amultiple genetic background. Twin studies on alcohol dependence,one of the classical genetic approaches, have revealed a heritabil-ity of alcohol dependence of over 50% (between 40 and 70%)(Köhnke, 2008). Thus, vulnerability for excessive alcohol consump-tion or development of alcohol dependence has been associatedwith genes involved in metabolism of ethanol and also severalgenes of different neurotransmission systems that underlie alcoholdependence.
In this context, genetic evidence about the involvement ofthe ECS in alcohol dependence has risen from the study ofrodent strains that differ in their preference for ethanol (summa-rized in Table 4). First of all, differences in CB1 receptor densityand functionality have been reported within animals with dif-ferent preference for ethanol. On one hand, alcohol-preferringmice showed decreased CB1 receptor density, but higher affin-ity and functionality than alcohol-avoiding mice (Hungund andBasavarajappa, 2000; Basavarajappa and Hungund, 2001). On theother hand, both decreased CB1 receptor gene expression andfunctionality have been observed in different brain areas of alcohol-preferring rats (Ortiz et al., 2004b). Moreover, in the prefrontalcortex of alcohol-preferring rats, not only has a decrease in CB1receptor binding and functionality been reported, but a decreasein FAAH gene expression and activity has also been described,together with an increase in 2-AG levels (Hansson et al., 2007).
Genetic studies in humans have also described that particu-lar polymorphisms in the genes coding for some elements of theECS could be associated with some phenotypes of alcohol depen-dence (Table 4). However, although a great number of genetic
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Table 4Genetic evidence about the involvement of the endocannabinoid system in drug addiction and alcohol dependence.
Study Results Reference
Rodent strains with different alcohol preference
CB1RDecreased CB1R density but higher affinity in alcohol-preferring mice Hungund and Basavarajappa (2000)Increased CB1R functionality in alcohol-preferring mice Basavarajappa and Hungund (2001)Decreased CB1R mRNA and functionality in alcohol-preferring rats Ortiz et al. (2004b)
CB1R, FAAH & ECBs Decreased CB1R binding and functionality; decreased FAAH expression andactivity; increased 2-AG, in the prefrontal cortex of alcohol-preferring rats
Hansson et al. (2007)
CB1R polymorphisms
rs1049353
Association with severe alcohol withdrawal syndrome Schmidt et al. (2002)No significant association with different alcoholism-related phenotypes,including severe alcohol withdrawal syndrome
Preuss et al. (2003)
This meta-analysis showed no significant association with alcoholdependence;
Benyamina et al. (2011)
rs6454674 Association with alcohol dependence Zuo et al. (2007)rs2023239 Association with CB1R density, alcohol-elicited brain activation, subjective
reward when consuming alcohol and positive outcomes withpharmacotherapy that targets the dopaminergic system
Hutchison et al. (2008)
AAT repeatmicrosatellite
Association with alcoholic patients who suffered from attention deficit/hyperactivity disorder during childhood
Ponce et al. (2003)
No significant association with alcohol dependence; although significant withcocaine, amphetamine and cannabis dependence
Comings et al. (1997)
Significant association with illicit substance dependence Benyamina et al. (2011)
No significant association of different SNPs with alcohol dependence in a Japanese population Herman et al. (2006)Association of a three-SNP haplotype with alcohol dependence Zhang et al. (2004)
FAAH polymorphisms
rs324420Association with concomitant street drug and problem drug/alcohol use Sipe et al. (2002)Association with multiple different drug addictions Flanagan et al. (2006)Association with reward-related ventral striatum reactivity Hariri et al. (2009)
CB2R polymorphismsrs2501432 Association with alcoholism in a Japanese population Ishiguro et al. (2007)
MAGL polymorphismsNo significant association of any of the studied SNP of MAGL and FAAH with alcoholism in a Japanese population Iwasaki et al. (2007)
ECBs: endocannabinoid levels. CB1R: CB1 receptor.
studies have been performed, only a few have found a significantassociation. First of all, a particular single nucleotide polymor-phism (SNP) of the CB1 receptor, the rs1049353, was associatedwith severe alcohol withdrawal syndrome (Schmidt et al., 2002).Nonetheless, another study that assessed the same particularvariation failed to confirm the association with different alcoholdependence-related phenotypes, including severe withdrawal syn-drome (Preuss et al., 2003). Further, a nine-allele microsatellitepolymorphism of the CB1 receptor, containing repeats of the singletrinucleotide AAT, has been associated with childhood attentiondeficit/hyperactivity disorder in alcoholic patients (Ponce et al.,2003). For this microsatellite repeat, again, another study did notfind an association with alcohol dependence, although it showedan association with other drug dependences and with intravenousdrug use (Comings et al., 1997). In the same context, a recentmeta-analysis did not observe any significant association of eitherrs1049353 or AAT repeat polymorphisms with alcohol dependence(Benyamina et al., 2011). Similarly, another study failed to find anassociation of 4 different CB1 receptor SNPs with alcohol depen-dence in a Japanese population (Herman et al., 2006).
Three additional studies have provided positive association ofCB1 receptor variants with alcohol dependence. Thus a highly sig-nificant difference was observed in a particular haplotype of threedifferent SNPs in Japanese alcoholics compared to controls (Zhanget al., 2004). Also, risk for alcohol dependence was significantlyassociated with the rs6454674 single nucleotide polymorphism.Furthermore, recently, the C allele for the rs2023239 CB1 recep-tor SNP was associated with greater CB1 receptor density in theprefrontal cortex of alcoholic patients, greater alcohol-elicitedbrain activation, greater subjective reward when consum-
ing alcohol and more positive outcomes with pharmacother-apy that targets the dopaminergic mesocorticolimbic circuitry(Hutchison et al., 2008).
A single nucleotide polymorphism in the FAAH gene, thers324420, which produces a mutant enzyme with reduced cellu-lar stability and therefore reduced expression and activity (Chianget al., 2004), has also been studied in regard to alcohol and otherdrug use, with positive outcomes. The first study observed an asso-ciation of this SNP with concomitant street drug use and problemdrug/alcohol use (Sipe et al., 2002). Another study confirmed thisassociation in a group composed of subjects with different drugaddictions, including alcohol dependence (Flanagan et al., 2006).Recently, an imaging study has reported that the A carriers forthis polymorphism have increased brain reactivity in the reward-related ventral striatum, which highlights the importance of thevariability in the endocannabinoid signaling in behavioral pro-cesses related to addiction (Hariri et al., 2009).
To conclude, two other genes of the ECS have been studied inalcohol dependence. A significant association of the CB2 recep-tor polymorphism rs2501432 with alcohol dependence has beenreported in Japanese patients (Ishiguro et al., 2007). Also, 14 differ-ent polymorphisms in the monoacylglycerol lipase (MAGL) genewere studied in a Japanese alcoholic population, but none of themarkers showed a significant association (Iwasaki et al., 2007).
Many of the studies presented show genetic evidence about theinvolvement of the ECS in alcohol dependence. However, the lackof positive associations in some studies can be attributed to thegenetic differences between the populations of alcoholics and con-trols studied. Therefore, further studies are required to verify thetentative allelic and genotypic associations reported.
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5. Rimonabant for the treatment of alcohol dependence
As reviewed throughout the article, a great amount of evidencesuggests the involvement of the ECS in the neurobiology of alcoholdependence. More precisely, CB1 receptor blockade by rimonabantin rodents has been reported to decrease not only ethanol intakeand preference, but also the motivation for consuming the drug,along with the alcohol deprivation effect and the conditioned rein-statement of ethanol-seeking behavior. In this context, two Phase IIclinical studies have been conducted in human alcoholics in orderto study the possible utility of rimonabant in alcohol dependence.
The first clinical trial was a 12-week double-blind, placebo-controlled study to evaluate the efficacy of rimonabant 20 mg/dayin the prevention of alcohol relapse in alcoholic patients that hadrecently been detoxified (Soyka et al., 2008). Treatment compliancetended to be better in the group of patients receiving rimonabantthan in the control group (patients receiving placebo). Overall, theclinical and biological safety and tolerability of rimonabant wasgood, and the rate of adverse effects was similar in both groups. Theprimary end point of the study was to assess the time to first drinkand the time to relapse to first heavy alcohol drinking. At the endof the study there was a little favorable effect of rimonabant in pre-venting relapse, although it did not reach statistical signification:41.5% vs 47.7% was the general relapse rate, and 27.7% vs 35.6% wasthe relapse rate to heavy drinking. In regard to the secondary endpoints of the study, there were no significant differences betweengroups in cumulative abstinence duration in days, percentage ofdrinking days and percentage of heavy drinking days, althoughthere was similarly a trend toward favorable effects of rimona-bant. Authors suggest that the lack of improvement of rimonabantover placebo might be explained, firstly, by a very high benefi-cial response rate in the placebo group, and secondly, by the shortduration of the treatment.
The second clinical trial was a 3-week double-blind, placebo-controlled study to evaluate the effect of rimonabant 20 mg/dayon alcohol consumption in nontreatment-seeking heavy alcoholdrinkers (George et al., 2010). First of all, after 1 week baseline, 18participants received rimonabant and 21 received placebo during 2weeks, and they reported their daily alcohol consumption by tele-phone. Rimonabant was not shown to alter alcohol consumption.Secondly, after the 2 weeks treatment, all patients were evaluatedin an alcohol self-administration paradigm in which after receivingan oral priming dose of ethanol they had the option of consumingup to eight alcohol drinks or receiving some money for each non-consumed drink. The results show that rimonabant did not changealcohol self-administration either.
Rimonabant was commercialized for the treatment of obesity.Nevertheless, due to the incidence of severe adverse psychiatriceffects during the treatment (Christensen et al., 2007), its commer-cialization has been recently discontinued (European MedicinesAgency, 2009).
6. Conclusion
In conclusion, several lines of evidence support the involvementof the ECS in alcohol dependence. It is clear that this neurotransmis-sion system is directly involved in the primary rewarding effects ofethanol through different cellular mechanisms. Moreover, the ECSseems to play an important role in the motivation to seek the drugin alcoholics and in the relapse to drug-seeking in alcoholic patientsthat had recently been detoxified. Further studies will be requiredto clarify the specific mechanisms that mediate these effects. How-ever, the ECS constitutes a clear therapeutic target for the treatmentof alcohol dependence. In this way, the development of new specificcannabinoid antagonists could contribute to develop new pharma-
cological treatments to decrease not only ethanol intake, but alsothe motivation for consuming alcohol.
Role of funding source
Funding for this study was provided by Plan Nacional sobre Dro-gas (PI 20061045), Gobierno Vasco (IT-199-07), and the Instituto deSalud Carlos III, Centro de Investigación Biomédica en Red de SaludMental, CIBERSAM, Spain. AM Erdozain is recipient of a predoctoralfellowship from the Basque Government. The funding sources hadno further role in the writing of the review, or in the decision tosubmit the paper for publication.
Contributors
AM Erdozain performed the literature search and LF Calladowrote the first draft of the manuscript. Both authors contributedto and have approved the final manuscript.
Conflict of interest
There are no conflicts of interest.
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