INTERACTIONS BETWEEN CANNABINOID AND OPIOID RECEPTOR SYSTEMS IN THE MEDIATION OF ETHANOL EFFECTS

Alcohol & Alcoholism Vol. 40, No. 1, pp. 25–34, 2005 doi:10.1093/alcalc/agh112Advance Access publication 18 November 2004

SPECIAL ISSUE ARTICLE

INTERACTIONS BETWEEN CANNABINOID AND OPIOID RECEPTOR SYSTEMS INTHE MEDIATION OF ETHANOL EFFECTS

JORGE MANZANARES*, SERGIO ORTIZ, JOSÉ M. OLIVA, SANDRA PÉREZ-RIAL and TOMÁS PALOMO

Servicio de Psiquiatría y Centro de Investigación, Hospital Universitario 12 de Octubre, Avda. Córdoba s/n, 28041 Madrid, Spain

(Received 16 September 2004; first review notified 2 September 2004; in revised form 14 September 2004; accepted 16 September 2004)

Abstract — Over the past few years, advances in the investigation of the neurochemical circuits involved in the development andtreatment of alcohol dependence have identified peptides and receptors as potential key targets in the treatment of problems related toalcohol consumption. The endogenous opioid system is modified by alcohol intake in areas of the brain related to reward systems, anddifferential basal levels of opioid gene expression are found in rodents with a high preference for ethanol. This suggests a greatervulnerability to alcohol consumption in relation to differences in genetic background. Further evidence of the involvement of opioidpeptides in alcohol dependence is the ability of the opioid antagonist naltrexone to reduce alcohol intake in animal models ofdependence and in alcohol-dependent patients. Abundant evidence indicates that the activation of cannabinoid receptors stimulates therelease of opioid peptides, therefore the cannabinoid receptor antagonists may presumably alter opioid peptide release, thus facilitatingthe reduction of ethanol consumption. However, little is known about the effects of ethanol on the endogenous cannabinoid system, thevulnerability of cannabinoid receptors to alcohol intake or their neurochemical implications in reducing consumption of alcohol. Inthis paper, we review the role of opioid and cannabinoid receptor systems, their vulnerability to alcohol intake and the developmentof dependence, and the targeting of these systems in the treatment of alcoholism.

Alcohol & Alcoholism Vol. 40, No. 1 © Medical Council on Alcohol 2005; all rights reserved

*Author to whom correspondence should be addressed: Edificio Materno-Infantil, Planta 6, 613-A, Hospital Universitario 12 de Octubre, Avda.Cordoba s/n, 28041 Madrid, Spain. Tel.: 34 91 390 8252; Fax: 34 91 3908538; E-mail: [email protected]

25

INTRODUCTION

Many reports suggest the involvement of the endogenousopioid system as a relevant part of the neurobiologicalmechanisms that are functionally involved in the reinforce-ment of drinking behaviour and development of alcoholdependence. In particular, findings showing that alcohol-induced dopamine release can be blocked by opioidantagonists implicate opioidergic activity as an intermediaryin the process (Benjamin et al., 1993; Gonzales and Weiss,1998). Available data suggest that alcohol increases opioidneurotransmission and that this activation is part of themechanism responsible for the reinforcing effects of alcohol(Gianoulakis, 1996; Ulm et al., 1995). Furthermore, a numberof studies suggest that alterations in gene expression of opioidpeptides may determine the vulnerability to alcohol depend-ence (Oswald and Wand, 2004) and the therapeutic efficacy ofanticraving drugs.

A summary of facts relating alcohol intake, developmentof alcohol dependence and genetic vulnerability to alcoholwith endogenous opioid and cannabinoid receptor systems aredepicted in Fig. 1. As shown, excessive alcohol consumptionis a complex, multifactorial problem that includes not only thealteration of neurochemical elements in the brain but also anumber of psychosocial conditions that may possibly favourthe development of problems related to alcohol intake, whichhas been observed in dependent subjects. From theseconditions (left part of Fig. 1), several personality traits suchas high impulsivity, low self-esteem and sensation-seeking

behaviour may contribute greatly to enhance vulnerability toalcohol dependence. In addition, a number of psychiatricdisorders (phobias, attention-deficit hyperactive disorder oraffective disorders) may also modify the effects of alcohol,enhancing its reinforcing properties and leading towards theprogression to alcohol dependence. Although psychologicaltraits and psychiatric conditions play a major role in thedevelopment, treatment and relapse of alcoholism, thefindings of neurochemical alterations reported in a number ofpreclinical studies have allowed the identification of potentialtargets involved in the development, treatment andreinstatement of alcohol seeking behaviour.

It is well known that alcohol intake increases the release ofopioid peptides and subsequently increases gene expressionin mesencephalic brain areas related to reinforcement andreward, such as the nucleus accumbens or the ventral part ofcaudate-putamen. In these regions, alcohol-induced opioidrelease stimulates dopamine neurons by acting directly onthe nucleus accumbens and by disinhibiting GABA mesen-cephalic neurons projecting into the dopamine tegmental area(Spanagel and Weiss, 1999). Alterations in dopamine neuronsin the terminals (nucleus accumbens, cortex) and/or cellbodies (ventral tegmental area) of mesolimbic and mesocorticalsystems lead to loss of a neurochemical homeostatic control,contributing to the development of relapse and facilitating theprogression to alcohol dependence. Differences in opioidfunctional activity have also been associated with a distinctvulnerability to drugs of abuse in animal models of drugdependence. Indeed, the examination of basal opioid geneexpression has revealed that lower opioid activity in the areasinvolved in motivation and reward of rats that were selectedfor their high preference for alcohol appears to be relatedto increased vulnerability to alcohol dependence (Nylanderet al., 1994). In preclinical studies, blockade of opioid

26 J. MANZANARES et al.

receptors with the non-selective receptor antagonistnaltrexone decreases alcohol intake in alcohol-preferring ratsand reduces the alcohol-induced increase in opioidergicactivity (Cowen and Lawrence, 2001), thereby inhibiting, atleast in part, the reinforcing properties of alcohol.

In recent years, several reports have suggested a closeinteraction between the endogenous cannabinoid system andalcohol consumption. It has been suggested that ethanol, usingneuronal cells, increases the release of the endogenouscannabinoid ligands arachidonylethanolamide (AEA) and 2-arachidonylglycerol (2-AG) (Basavarajappa and Hungund,1999a; Basavarajappa et al., 2000, 2003). The increase inligand release may continuously activate the receptor,potentially down-regulating the cannabinoid CB1 receptor,after chronic ethanol administration, as shown recently(Basavarajappa et al., 1998; Basavarajappa and Hungund,1999b; Ortiz et al., 2004a). On the other hand, although itremains to be clarified, the administration of low doses ofcannabinoid agonists enhances ethanol intake and this effectappears to be dependent upon the conditions of theexperimental paradigm (Gallate and McGregor, 1999; Colomboet al., 2002). Conversely, several authors suggest that theadministration of cannabinoid receptor antagonists reducesethanol intake in rodents (Arnone et al., 1997; Colombo et al.,1998). Several mechanisms may participate in reducingethanol intake in rats treated with the cannabinoid antagonist.First, as shown in Fig. 1, the blockade of cannabinoid CB1receptors may impede the increase in opioid release inducedby ethanol. Second, cannabinoid receptor antagonists mayreduce the ethanol-induced increase in mesencephalicdopamine neurons or block the disinhibition of GABAergicneurons (for review see Piomelli, 2003) that, in turn, results

in the activation of dopamine neurons. This representshypothetically distinct mechanisms by which the use ofcannabinoid antagonists may be effective in reducing ethanolintake.

Taking into account that the administration of cannabinoidreceptor agonists enhances endogenous opioid activity(Corchero et al., 1997a,b; Manzanares et al., 1999), differ-ences in endogenous cannabinoid and opioid function maysuggest a distinct vulnerability to ethanol consumption and/ordependence. Therefore, it is tempting to speculate that animalswith low opioid expression and more vulnerability to ethanolmay have impaired cannabinoid receptor function in keyregions of the brain related to motivation and reward.

In the present review, we examined some of the effects ofethanol on opioid and cannabinoid receptors systems in thebrain in relation to chronic consumption, genetic vulnerabilitytowards high preference for ethanol and the pharmacologicalresponse to opioid and cannabinoid receptor antagonists inrelation to ethanol consumption.

ROLE OF OPIOID SYSTEM ON ETHANOL INTAKE,VULNERABILITY AND DEPENDENCE

Opioid receptors

Consumption of ethanol in animal models of ethanoldependence modifies the expression and function of opioidreceptors. However, in several reports a great variability hasbeen detected that appears to depend on the method usedto induce ethanol dependence, the region examined and/or thestrain of rats or mice used (Gianoulakis, 2001; Oswald andWand, 2004). For instance, prolonged (30 days) ethanol

ETHANOL

OPIOIDS

NALTREXONE

⊗

⊗ VULNERABILITY

DOPAMINE

MESENCEPHALIC

TEGMENTAL AREA

GABA

MESENCEPHALIC

+

-

NUCLEUS ACCUMBENS

PSYCHOSOCIAL

CONDITIONS

+CANNABINOIDS

+ ?

⊗

CB1 ANTAGONIST

⊗

Personality (impulsivity, low self-

esteem, search of new sensations)

Psychiatric Disorders (phobia,

ADHD, affective disorders)

LOSS OF CONTROL

REINFORCEMENT

BEHAVIOUR

PROGRESS TO DEPENDENCE RELAPSE

+

¿VULNERABILITY?

Fig. 1. Effects of ethanol on opioid and cannabinoid receptor systems. Role of psychological traits in the development of alcohol dependence.

CANNABINOID AND OPIOID SYSTEMS AND ALCOHOLISM 27

consumption increases the binding of mu-opioid receptors inthe caudate putamen of Sardinian rats (Fadda et al., 1999) butdownregulates these receptors in the nucleus accumbens andcaudate-putamen of Wistar rats (Turchan et al., 1999).Similarly, Chen and Lawrence (2000) have shown that chronic(50 days) voluntary ethanol intake inhibits DAMGO-stimulated [35S]GTPγ binding in the nucleus accumbens,caudate-putamen, and lateral septum in Fawn-Hoodedethanol-preferring rats. In contrast, Sim-Selley et al. (2002)have reported that mu-opioid-stimulated [35S]GTPγS bindingwas lower in the prefrontal cortex of brains from ethanol self-administering Long Evans rats, although voluntary ethanolintake showed no effect on mu-opioid function in the cingulatecortex, caudate-putamen, nucleus accumbens, amygdala,hypothalamus, thalamus, and locus coeruleus compared withsucrose self-administering rats. These results taken togethersuggest that alterations in the mu-opioid receptor may berelated to the duration and level of ethanol consumption,the regions of the brain examined and pattern of ethanolintake (Table 1).

The development of gene knockout technique has furtherstrengthened the evidence supporting the key role of opioidreceptors in ethanol dependence. Indeed, Roberts et al. (2000)have reported that mice deficient in mu-opioid receptor donot self-administer ethanol. Similarly, delta-opioid receptorknockout mice displayed a greater preference for ethanol andconsumed more ethanol than wild-type mice, suggesting thata decrease in delta-receptor activity may be associated withincreased ethanol-intake behaviour (Roberts et al., 2001).

Opioid peptides

It is well established that ethanol induces the release of opioidpeptides, which interact with brain nuclei closely involvedin reward and positive reinforcement systems (Jamensky andGianoulakis, 1997; Koob et al., 1998). Acute ethanoladministration increases endorphin and enkephalin geneexpression in discrete regions of the brain and increases therelease of these peptides in the brain and pituitary of rodents(Schulz et al., 1980; Gianoulakis and Barcomb, 1987; Pateland Pohorecky, 1989; Anwer and Soliman, 1995; Li et al.,1998; Rasmussen et al., 1998; Oliva et al., 2002a, 2003a)(Table 2).

On the other hand, prolonged ethanol administration maydecrease the release of endogenous opioid peptides. Forinstance, chronic ethanol administration decreased pro-opiomelanocortin gene expression in the forebrain (Olivaet al., 2002b; Rasmussen et al., 2002) and in the pituitarygland (Patel and Pohorecky, 1989; Oliva et al., 2002b) of ratsand β-endorphin release in cultured hypothalamic neurons(Boyadjieva and Sarkar, 1994). However, Cowen and

Lawrence (2001) have shown that ethanol consumptionincreases preproenkephalin mRNA in central and intercalatednuclei of the amygdala but decreases preproenkephalin mRNAin the nucleus accumbens and olfactory tubercle. Theseauthors suggest that alterations in opioid peptide releaseand/or gene expression are dependent on the regions of thebrain examined and the duration of ethanol consumption(Table 2).

The role of opioid peptide release and opioid gene expressionhas also been investigated in relation to increased vulnerabilityto ethanol dependence. Several studies have shown that basalopioid activity differs between ethanol-preferring and non-preferring strains of rodents. Selectively bred AA (ethanol-preferring) rats present a higher hypothalamic basalpro-opiomelanocortin gene expression compared with ANA(ethanol-avoiding) rats (Marinelli et al., 2000) and moresensitivity to ethanol consumption in selectively bred, ethanol-preferring P rats compared with ethanol-avoiding NP rats(Krishnan-Sarin et al., 1998) as well as in ethanol-preferringC57BL/6 mice compared with ethanol non-preferring DBA/2mice (Jamensky and Gianoulakis, 1999).

Met-enkephalin and Leu-enkephalin peptide levels werelower in the nucleus accumbens of AA compared with ANArats (Nylander et al., 1994), whereas a more intenseproenkephalin expression was reported in the prefrontal cortexof AA compared with ANA rats (Marinelli et al., 2000).Despite a number of studies that examined the basal functionalactivity of opioid receptors in selectively bred rats with a highpreference for ethanol consumption, the results remaininconclusive. It has been shown that the density of opioidreceptors in various regions of the brain is lower, higher orsimilar in ethanol-preferring compared with non-preferringrats (McBride et al., 1998; Marinelli et al., 2000).

Our laboratory has determined opioid functional activity innaïve ethanol-preferring Fawn-Hooded rats and ethanol non-preferring Wistar rats. The Fawn-Hooded strain of rat shows ahigh preference for ethanol intake (10% v/v) in a two-bottlefree-choice situation (Rezvani et al., 1990; Ortiz et al., 2004b)that may be related, at least in part, to decreased brain opioidfunction (Cowen et al., 1998; Rezvani et al., 2002). Indeed, asdepicted in Fig. 2, we have observed lower mu-opioid receptor-stimulated [35S]GTPγS-binding autoradiography in thecaudate-putamen and cingulate cortex, lower proenkephalingene expression in the caudate-putamen and nucleusaccumbens, and lower pro-opiomelanocortin gene expression inthe arcuate nucleus of Fawn-Hooded compared with Wistarrats. Therefore, the results of this study, in agreement withprevious reports, strongly support the hypothesis that the basalfunctional activity of the opioid system plays a critical role inthe vulnerability to ethanol intake.

Table 1. DAMGO-stimulated [35S]GTPγ binding in different brain regions of several rat strains after chronic ethanol consumption

[35S]GTPγ EthanolAnimals Brain regions binding administration References

Sardinian CPu, Acc ↑ Two-bottle choice paradigm Fadda et al., (1999)Wistar CPu, Acc ↓ Two-bottle choice paradigm Turchan et al., (1999)Fawn-Hooded CPu, Acc, Lateral Septum ↓ Two-bottle choice paradigm Chen and Lawrence, (2000)Long Evans PF cortex Cg, CPu, Acc, ↓ Ethanol self-administration Sim-Selley et al., (2002)

Ce, hyp, thalamus, LC = Two-bottle choice paradigm

28 J. MANZANARES et al.

Table 2. Acute and chronic effects of ethanol on opioid release and gene expression in various brain and pituitary regions of rodents

Acute Chronic

POMC ↑ Oliva et al. (2002b) ↓ Rasmussen et al. (2002)(AL) (Forebrain)

↓ ↓ Oliva et al. (2002b)(Arc, IL) (Arc, IL, AL)

PENK ↑ ↑(CPu, Acc, Tu, Oliva et al. (2002a) (Ce, IM) Cowen and Lawrence (2001)

Pir, Ce, Me, ↓VMN, PVA) (Acc, Tu)

↓(Acc, Ce)

β-endorphin ↑ Li et al. (1998) ↓ Patel and Pohorecky (1989);(Hyp, septum, Anwer and Soliman (Pituitary) Schulz et al. (1980)

forebrain) (1995)=

(PFx) Patel and Pohorecky ↓(1989) (Cultured

↑ hyp. Boyadjieva and(pituitary) Rasmussen et al. (1998) neurons) Sarkar (1994)

↑ ↓Met-enkephalin (CPu) (CPu, Schulz et al. (1980)

= Schulz et al. (1980) medulla)(Hyp, forebrain =

medulla) (Forebrain)

POMC, pro-opiomelanocortin; PENK, proenkephalin; CPu, caudate-putamen; Acc, nucleus accumbens; Tu, olfactory tubercle; Pir, piriform cortex; Ce,central amygdala; Me, medial amygdaloid nucleus; IM, intercalated amygdaloid nucleus; VMN, ventromedial nucleus; PVA, paraventricular nucleus;Hyp, hypothalamus; Arc, arcuate nucleus; IL, intermediate lobe of the pituitary; AL, anterior lobe of pituitary; PFx, prefrontal cortex.

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Fig. 2. Proenkephalin gene expression in the caudate-putamen (CPu), pro-opiomelanocortin gene expression in the arcuate nucleus (ARC), andDAMGO-stimulated [35S]GTPγ binding in CPu and cingulate cortex (Ccg) of 10 Fawn-Hooded and Wistar rats. Both strains of rats received food andwater ad libitum. Gene expression was measured by in situ hybridization histochemistry, using synthetic oligonucleotide probes complementary toproenkephalin (PENK) or pro-opiomelanocortin (POMC) genes labelled with 35S using terminal deoxytransferase as described previously (Young et al.,1986; Oliva et al., 2003). [35S]GTPγ binding in CPu and cingulate cortex (Ccg) was carried out following the method described by Sim-Selley et al.(2002). Symbols represent the means and vertical lines set the ± SEM in 10 rats of both strains. *Values from proenkephalin, pro-opiomelanocortin geneexpression and DAMGO-stimulated [35S]GTPγ binding that are significantly different (P < 0.05, Student’s t-test) in the comparison of both strains of rats.

Representative autoradiograms of coronal brain sections at the level of CPu, Ccg and ARC in Fawn-Hooded and Wistar rats. Bar represents 1 mm.

CANNABINOID AND OPIOID SYSTEMS AND ALCOHOLISM 29

Regulation of ethanol intake by opioid receptor agonists

The precise mechanisms underlying the reinforcing propertiesof ethanol remain to be determined. Nevertheless,experimental results suggest that the endogenous opioidsystem plays a major role in the consumption of ethanol inanimals. Indeed, pharmacological manipulations of opioidreceptors affect the rewarding properties of ethanol,acquisition and maintenance of its consumption. Adminis-tration of small doses of morphine plus ethanol showed astrong conditioned place preference compared with morphineor ethanol administration alone (Marglin et al., 1988).Furthermore, the potentiation of ethanol consumption inducedby intracerebroventricular (Linseman and Harding, 1990)or systemic (Reid and Hunter, 1984; Hubbell et al., 1991,1987; Stromberg et al., 1997a,b) administration of doses ofmorphine is due to its interaction with opioid receptors, whichmodulate the ethanol rewarding effects.

ROLE OF THE CANNABINOID SYSTEM ONETHANOL INTAKE, VULNERABILITY AND

ADDICTION

Effects of ethanol intake on the cannabinoid system

The behavioural and neurochemical effects of marihuanaalong with ethanol have interested a great number of scientistsfor many years. However, not until recently have researchersbeen able to describe an endocannabinoid system in the braincomposed of cannabinoid CB1 receptors and endogenousligands. Similarly, the necessary pharmacological tools(cannabinoid receptor agonists and antagonists) to explore therole of this system towards the development and treatmentof ethanol dependence became available only recently. Thepioneering studies in this precise area were the elegantinvestigations conducted by Arnone et al. (1997), whodescribed for the first time the inhibition of ethanol intake bythe cannabinoid receptor antagonist SR 141716A. The resultsof this study were extended and confirmed by other authors(Colombo et al., 1998; Freedland et al., 2001), strengthening

the involvement of the cannabinoid CB1 receptor in thepharmacotherapy of ethanol dependence. The fact that theblockade of cannabinoid CB1 receptor modulates ethanolintake led other investigators to examine the effects of ethanolon the endogenous cannabinoid system. The initial studies,which were a considerable achievement in this line ofresearch, were the investigations carried out by Basavarajappaand Hungund (1999a,b). These authors pointed out for the firsttime that chronic ethanol administration downregulatedcannabinoid receptors in synaptic plasma membranes of themouse brain (Basavarajappa et al., 1998) and increased thelevels of the endogenous cannabimimetic compoundanandamide and its precursor N-arachidonoylphos-phatidylethanolamine in SK-N-SH cells (Basavarajappa andHungund, 1999a). In cerebellar granule neurons, chronicethanol exposure induced a significant increase in the levels ofthe endocannabinoid 2-AG (Basavarajappa et al., 2000).Confirming these studies in our laboratory (Ortiz et al., 2004a),we recently reported that chronic (52 days) forced con-sumption of high levels of ethanol (average ethanol intake =5.8 ± 0.1 g of ethanol/kg/day) significantly decreased thecannabinoid CB1 receptor gene expression in rat brain areasof the rat brain such as the caudate-putamen, ventromedialnucleus of the hypothalamus, and certain fields of thehippocampus (CA1, CA2) (Fig. 3). These results provide addi-tional support to the hypothesis that prolonged ethanol intakeincreases brain endocannabinoid ligands (Basavarajappa andHungund, 1999a; Basavarajappa et al., 2000), which in turndecrease the CB1 receptor gene expression and function(Basavarajappa et al., 1998; Basavarajappa and Hungund,1999b; Ortiz et al., 2004a).

Role of the cannabinoid system in vulnerability toethanol intake

Coinciding evidence suggests that the CB1 receptor signallingsystem may play an important role in modulating ethanol-reinforcing effects and in the preference for ethanol intakebehaviour. It has been demonstrated that ethanol-preferringC57BL/6J mice have a significantly lower level of CB1

CB

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Fig. 3. Cannabinoid CB1 receptor gene expression in CPu, Ccg, hippocampal areas (CA1, CA2, CA3 and DG) and ventromedial nucleus (VMN)in control and forced-ethanol groups of rats. Rats had unlimited access to only a solution of ethanol (10% v/v) containing saccharin (0.25% w/v) or towater containing the same saccharin solution for a total period of 52 days. Columns represent the means and vertical lines + SEM of CB1 mRNA levelsin 8 rats; *values from cannabinoid CB1 receptor gene expression levels in CPu, Ccg, hippocampal areas and VMN from the forced-ethanol group that differ significantly (P < 0.05) from the control group. (Reproduced from Ortiz et al., 2004a from Alcohol and Alcoholism by permission of

Oxford University Press).

30 J. MANZANARES et al.

receptors and higher affinity for [3H]CP-55,940 than ethanol-avoiding DBA/2 mice, which do not readily consume ethanol(Hungund and Basavarajappa, 2000). Furthermore, the higherlevels of CB1 receptors found in DBA/2 mice are less stronglycoupled to G-proteins than in C57BL/6J mice, as shown bythe CB1 receptor agonist (WIN-55,212–2 or HU-210 or CP-55,940)-stimulated [35S]GTPγS-binding assay (Basavarajappaand Hungund, 2001).

Taking into account the idea that lower cannabinoid CB1receptor function may be associated with increasedvulnerability to high ethanol preference and consumption, werecently compared the activity of cannabinoid CB1 receptor(WIN-55,212-stimulated [35S]GTPγS-binding autoradio-graphy, and gene expression) in ethanol-preferring Fawn-Hooded and ethanol non-preferring Wistar rats under naïveconditions (Ortiz et al., 2004b). The results of this studyconfirmed the presence of lower cannabinoid CB1 receptor-stimulated [35S]GTPγS binding in cingulate cortex, caudate-putamen, nucleus accumbens, ventromedial hypothalamicnucleus, amygdaloid area, and certain fields of thehippocampus in Fawn-Hooded compared with Wistar rats(Fig. 4). The notion is further supported by the fact that thecannabinoid CB1 receptor gene expression was also lowerin the cingulate cortex, caudate-putamen, ventromedialhypothalamic nucleus and CA3 area of hippocampus in Fawn-Hooded compared with Wistar rats (Ortiz et al., 2004b).

The development of mice deficient in CB1 receptor gene hasalso been critical in demonstrating that the endocannabinoidsignalling acting on CB1 receptors is involved in ethanolpreference. It has been shown that these CB1�/� miceexhibited dramatically reduced voluntary ethanol consump-tion (Hungund et al., 2003; Poncelet et al., 2003; Wang et al.,2003; Naassila et al., 2004).

Regulation of ethanol intake by cannabinoid receptoragonists

The fact that the exposure and intake of ethanol may inducealterations in the function and activity of the endocannabinoidsystem supports the notion that pharmacologicalmanipulations of the cannabinoid CB1 system may affectethanol consumption. Earlier studies by McMillan andSnodgrass (1991) showed that acute administration of ∆9-tetrahydrocannabinol (THC) reduced ethanol intake in ratsby 5 and 7%. However, these authors also reported thatchronic administration of THC initially decreased ethanolintake and, when tolerance occurred, ethanol consumptionincreased, even during THC withdrawal. The hypothesis,which suggests that the activation of cannabinoid CB1 receptorstimulates ethanol intake, was further confirmed by the studiesof Gallate and McGregor (1999) and Colombo et al. (2002).These authors showed that acute administration of thecannabinoid CB1 receptor agonists CP-55,940 or WIN-55,212

WIN

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Fig. 4. WIN-55,212–2-stimulated [35S]GTPγ binding in CPu, Ccg, nucleus accumbens (Acc), VMN, amygdala (AMG), CA1, CA2, CA3 and DG ofhippocampus and substantia nigra pars reticulata (SNr) of 10 Fawn-Hooded and Wistar rats. Symbols represent the means (arbitrary units of O.D) andvertical lines ± SEM in 10 rats of both strains. *Values from WIN-55,212–2-stimulated [35S]GTPγ binding receptor levels that differ significantly (P <0.05, Student’s t-test) in both strains of rats. Representative autoradiograms of coronal brain sections of WIN-55,212–2-stimulated [35S]GTPγ bindingin CPu, Cg, Acc, VMN, AMG, areas of hippocampus and SNr from Fawn-Hooded and Wistar rats. Bar represents 1 mm. (Reproduced from

Ortiz et al., 2004b from Alcohol and Alcoholism by permission of Oxford University Press).

CANNABINOID AND OPIOID SYSTEMS AND ALCOHOLISM 31

promoted voluntary ethanol intake in Sardinian ethanol-preferring (sP) rats (Colombo et al., 2002) and increased thebreakpoints for beer (Gallate and McGregor, 1999). In bothstudies, the increase in ethanol intake induced by CP-55,940or WIN-55,212 was prevented by cannabinoid or opioidreceptor antagonists, strongly suggesting the participation ofboth systems in this process.

ROLE OF CANNABINOID AND OPIOIDANTAGONISTS IN THE TREATMENT OF

ETHANOL DEPENDENCE

A variety of rewarding stimuli including ethanol, enhance theactivity of the endogenous opioid system (Gianoulakis, 1996).It has been hypothesized that this increase in opioid activitymay explain, at least in part, the reward response producedby ethanol. Therefore, the blockade of central opioid receptorsusing selective and non-selective opioid antagonists maymodulate the positive reinforcing properties of ethanol (Weisset al., 1990; Franck et al., 1998) and prove effective inreducing ethanol consumption. Indeed, naltrexone, whichattenuates ethanol consumption in laboratory animals(Volpicelli et al., 1986; Cowen and Lawrence, 2001; Olivaet al., 2003b) and in humans (Volpicelli et al., 1992; Kranzleret al., 1997), is a non-selective opioid antagonist approved forthis clinical indication in Europe and United States.

The mechanisms by which the administration of naltrexonereduces ethanol intake have not been examined precisely.Recent reports suggest that naltrexone would render as‘normal’, the endogenous opioid activity modified previouslyby prolonged ethanol consumption. In agreement, someresearchers have shown that the administration of naltrexonereversed the alterations in opioid gene expression (Cowen andLawrence, 2001; Oliva et al., 2003b) and opioid receptordensities (Overstreet et al., 1999) induced by ethanol intake.Preliminary results from our laboratory using the two-bottle-choice preference model revealed that administration ofnaltrexone reduced ethanol consumption by �50% and thatthis decrease was accompanied by a reduction inproenkephalin gene expression (elevated previously byprolonged ethanol consumption) in the caudate-putamen,core and shell parts of the nucleus accumbens, and olfactorytubercle (Oliva et al., 2003b).

The administration of cannabinoid receptor antagonists alsoreduces ethanol intake in a wide variety of experimentalparadigms. The administration of CB1 receptor-selectiveantagonists such as SR-141716A, SR-147778 or AM-251reduced voluntary ethanol consumption under the home-cagetwo-bottle regimen or self administration procedures inethanol-consuming rats and mice (Arnone et al., 1997;Colombo et al., 1998; Freedland et al., 2001; Lallemand et al.,2001; Rinaldi-Carmona et al., 2004), blocked acquisitionof ethanol-drinking behaviour in rats (Serra et al., 2001),decreased the motivation to consume ethanol in rats (Gallateand McGregor, 1999) and completely abolished the ethanoldeprivation effect (Serra et al., 2002). However, little is knownof the neurochemical mechanisms involved in this action.Since the increase of ethanol consumption induced by thecannabinoid receptor agonists CP-55,940 or WIN-55,212can be blocked by administration of either naltrexone or

SR-141716A, and cannabinoid receptor agonists increase theendogenous opioid function (for review, see Manzanares et al.,1999), it is tempting to speculate that the reduction of ethanolintake produced by administration of cannabinoid receptorantagonists may be related, as suggested previously for opioidreceptor antagonists, to the ‘normalization’ of opioid peptidesor opioid receptor functional activity altered by prolongedconsumption of ethanol. Recent studies carried out in ourlaboratory revealed that the reduction of ethanol intakeinduced by the cannabinoid receptor antagonist AM-251 wasassociated with a lower decrease of DAMGO-stimulated[35S]GTPγ binding in the caudate-putamen and pro-opiomelanocortin gene expression in the anterior lobe of thepituitary gland compared with the reduction produced byvehicle-ethanol treated rats (Ortiz et al., unpublished results).That is, administration of AM-251 tended to normalise mu-opioid receptor binding, altered previously by continuousexposure to ethanol.

The neurochemical mechanisms involved in the reductionof ethanol intake induced by either opioid or cannabinoidreceptor antagonists may not be exclusively related toalterations in opioid functional activity, and still remain tobe determined. Nevertheless, the fact that both opioid andcannabinoid antagonists tend to ‘normalize’ opioid functiondisrupted by ethanol intake suggests a potential synergisticaction between both antagonists to reduce the consumption ofethanol. Indeed, Gallate et al. (2004) reported recently thata combined low-dose treatment with opioid and cannabinoidreceptor antagonists synergistically reduces the motivation toconsume ethanol in rats. The mechanisms responsible forthis synergistic action are still unknown and a number ofpreclinical studies are needed to clarify the precise nature ofthe interaction induced by blockade of both opioid andcannabinoid CB1 receptors. Nevertheless, although theseresults should be interpreted with caution, they may have animportant impact in the treatment of problems related toethanol in clinical practice. Further double-blind, placebo-controlled studies should be carried out to evaluate theoutcome of combined treatment in ethanol-dependent patients.

CONCLUSIONS

The pharmacological treatment of ethanol dependence is achallenge in our society. The search for the key elementsinvolved in the genetic differences between individuals thatmake them more vulnerable to ethanol dependence and theidentification of targets for the design of new, potentiallyuseful drugs to reduce ethanol intake and craving and toprevent relapse are an ongoing concern of scientists workingin this field. It is now clear that opioid transmission in thebrain is important in the development of ethanol dependenceand possibly in dependence induced by other drugs of abuse.The best evidence of this idea is the good response of anumber of ethanol-dependent patients (throughout the world)to the opioid antagonist naltrexone. The fact that cannabinoidagonists increase the release of opioids and also increaseethanol intake in preclinical studies has stimulated researchon the role played by the endocannabinoid system in ethanoldependence. To date, it appears that endogenous cannabinoidligands, cannabinoid CB1 receptor function and gene

32 J. MANZANARES et al.

expression are altered by ethanol consumption. Evidence frompreclinical studies strongly suggests that the administrationof cannabinoid receptor antagonists reduces ethanol intake.Furthermore, a synergistic action to decrease ethanol consump-tion has been reported between opioid and cannabinoidreceptor antagonists. Although the future design andapplication of drugs that modulate cannabinoid receptors toimprove problems related to ethanol need much moreinvestigation, the preclinical studies discussed in this review,point to the cannabinoid receptor system as an importantpotential target that should be seriously considered in futurestudies of the pharmacological treatment of alcoholism.

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