Drug and Alcohol Dependence 117 (2011) 102–110

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Drug and Alcohol Dependence

journal homepage: www.elsevier.com/locate/drugalcdep

Review

Involvement of the endocannabinoid system in alcohol dependence:

The biochemical, behavioral and genetic evidence
Amaia M. Erdozain a,b , Luis F. Callado a,b,∗
a
Department of Pharmacology, University of the Basque Country, 48940 Leioa, Bizkaia, Spain
b
Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Spain

a r t i c l e i n f o a b s t r a c t

Article history: Background: Recent advances in the understanding of alcohol dependence suggest that the endocannabi-
Received 19 October 2010 noid system (ECS) plays a key role in the neurobiological mechanisms underlying this pathology.
Received in revised form 7 February 2011 Methods: The aim of the present review is to show the currently available biochemical, behavioral and
Accepted 14 February 2011
genetic evidence on the involvement of the ECS in alcohol dependence.
Available online 16 March 2011
Discussion: Firstly, biochemical studies have shown that both chronic and acute administration of ethanol
produce alterations in different elements of this neurotransmission system. Secondly, the pharmacologi-
Keywords:
cal and genetic manipulation of the ECS in rodents result in altered ethanol-related behavior. Furthermore,
Alcohol dependence
Cannabinoids
rodent strains with different preference for ethanol differ in their ECS state. Also, genetic studies have
Endocannabinoid system described that particular polymorphisms in the genes coding for some elements of this system are associ-
Receptors ated with some phenotypes of alcohol dependence. Finally, the possible efficacy of cannabinoid receptor
Rimonabant blockers 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
1.1. The endocannabinoid system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
1.2. The endocannabinoid system and addiction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
2. Biochemical evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
3. Behavioral evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
3.1. Pharmacological modulation of the endocannabinoid system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
3.2. Genetic modulation of the endocannabinoid system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
4. Genetic evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
5. Rimonabant for the treatment of alcohol dependence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Role of funding source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

1. Introduction morbidity, mortality and disability is very important. According to

the World Health Organization in 2000 alcohol dependence was
Alcohol dependence is a costly and socially devastating illness. responsible for 4% of global disease, constituting only a slightly
Ethanol produces a complex and multidimensional effect on health lower level than that caused by smoking (4.1%) and hypertension
and the global burden related to alcohol consumption in terms of (4.4%) (World Health Organization, 2004).
Ethanol is toxic to most body tissues, producing changes on
the cardiovascular system, digestive system, central nervous sys-
∗ Corresponding author at: Department of Pharmacology, University of the Basque tem, peripheral nerves, muscle–skeletal system and the fetus. In
Country, Leioa, Bizkaia, Spain. Tel.: +34 94 6012762; fax: +34 94 6013220. the central nervous system ethanol generates a deep depression of
E-mail address: lf.callado@ehu.es (L.F. Callado). neuronal 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
 A.M. Erdozain, L.F. Callado / Drug and Alcohol Dependence 117 (2011) 102–110
throughout the human brain (Wang et al., 2000). Nowadays, it is
well known that ethanol affects most of the neurochemical and
endocrine systems (Diamond and Gordon, 1997), among which is
the 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 process
of the development, tolerance, withdrawal, craving and relapse of
alcohol dependence. For this purpose we have performed a com-
puterized search of the literature on PubMed and Scopus in order
to obtain biochemical, behavioral or genetic evidence. A secondary
search was done by hand searching the reference lists from primary
studies 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 their
endogenous ligands, enzymes involved in the biosynthesis and
degradation of these ligands and putative membrane transport
proteins. So far, two cannabinoid receptors have been identified
and cloned. The CB1 receptor (Matsuda et al., 1990) is mainly
expressed in the brain, where it plays an important role in the
regulation of neurotransmitter release (Wilson and Nicoll, 2002),
and it is, indeed, the most abundant G-protein-coupled receptor
in the mammalian brain. Conversely, the CB2 receptor (Munro
et al., 1993) is mainly located in peripheral tissues, at the high-
est levels in the immune system, such as spleen, tonsil, thymus
and lymphoid tissues (Galiegue et al., 1995). The endocannabinoids
are polyinsaturated fatty acids derived compounds, which share
some pharmacologic properties of 9 -THC. To date, anandamide
(N-araquidonoylethanolamine, AEA) and 2-arachidonoylglycerol
(2-AG) are the most thoroughly studied endocannabinoids, which
are produced on demand and released in the extracellular environ-
ment, where they can bind and activate CB1 and CB2 cannabinoid
receptors among others. Their hydrolysis, resulting in the termina-
tion of endocannabinoid signaling, is an intracellular event carried
out 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, FAAH
immunoreactivity 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 the
neuroanatomical distribution of the ECS and some of the central
effects 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 on
higher cognitive and motor functions, memory, hypothermia and
nociception (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 of
abuse, including alcohol, has thoroughly been documented (Koob
and Volkow, 2010). In this way, the ECS seems to be involved
in the regulation of this reward pathway. Thus, CB1 cannabinoid
receptors are present in different regions of the mesocorticolim-
bic circuitry and are involved in the neurobiological mechanisms
underlying the addictive processes of drugs of abuse (Maldonado
et al., 2006). It has been described that following depolarization
dopaminergic neurons in the ventral tegmental area (VTA) release
endocannabinoids that act as a regulatory feedback mechanism
(Lupica and Riegel, 2005). However, since dopaminergic neurons
do not appear to express CB1 receptor (Herkenham et al., 1991;
103
Matsuda et al., 1993), this regulatory mechanism is likely to be
indirect.
Additional data supporting the involvement of the ECS in the
reward pathway arises from in vivo microdialysis studies. Firstly,
acute alcohol, and nicotine, induced increase of dopamine levels in
the nucleus accumbens is significantly reduced by the CB1 receptor
antagonist rimonabant (Cohen et al., 2002). Similarly, in CB1 recep-
tor knockout mice acute ethanol administration did not produce
any dopamine release in the same region (Hungund et al., 2003).
These studies confirm that the dopamine release induced by some
drugs of abuse is CB1 receptor dependent.
Nonetheless, the ECS is not only involved in the rewarding
properties of alcohol, but also in the motivation for drug seek-
ing, the individual vulnerability for developing a dependence, the
acquisition of ethanol self-administering behavior, the develop-
ment of the withdrawal syndrome, etc. There is strong evidence
of the involvement of the ECS in alcohol dependence. In this
article biochemical, behavioral and genetic evidence of this impli-
cation will be reviewed. Firstly, biochemical studies have shown
that both chronic and acute administration of ethanol produce
alterations in different elements of the endocannabinoid system.
Secondly, the pharmacological and genetic manipulation of this
system in rodents result in altered ethanol-related behavior. Fur-
ther, rodent strains with different preference for ethanol differ in
their ECS state. Finally, genetic studies have described that partic-
ular polymorphisms in the genes coding for some elements of this
neurotransmission system are associated with some phenotypes of
alcohol dependence.
2. Biochemical evidence
Several studies have demonstrated that chronic exposure to
ethanol modulates some elements of the ECS both in vitro and in
vivo (Table 1). The first in vitro study reported a concentration- and
time-dependent increase in anandamide formation after chronic
exposure of neuroblastoma SK-N-SH strain cells to ethanol. This
increase in the endocannabinoid formation was inhibited by the
CB1 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 neuron
cultures to ethanol, being similarly inhibited by rimonabant and
pertussis toxin (Basavarajappa et al., 2000).
The modulation of the endocannabinoid content by chronic
ethanol has also been observed in rodent brain, although the results
are not consistent among all the studies. Thus, anandamide lev-
els were increased in the limbic forebrain while decreased in the
midbrain of ethanol-treated rats (Gonzalez et al., 2002a, 2004).
Another study corroborated this increase of anandamide in mice
cerebral 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 microdialysis
study reported increased dialysate levels of this endocannabi-
noid in the nucleus accumbens of ethanol self-administering rats,
with no concomitant change in anandamide concentrations (Caille
et al., 2007). However, other studies observed either a decrease
(Gonzalez et al., 2002a) or no change (Gonzalez et al., 2004) in
2-AG levels in rat midbrain. Nevertheless, these endocannabinoid
levels modulated by chronic ethanol administration are further
affected by alcohol-deprivation and relapse to alcohol consumption
(Gonzalez et al., 2004).
Cannabinoid CB1 receptor mRNA, density and functionality have
also been reported to be altered by chronic exposure to ethanol. The
first study indicated a significant down-regulation of the CB1 recep-
tor density in whole mouse brain chronically exposed to ethanol,
104 A.M. Erdozain, L.F. Callado / Drug and Alcohol Dependence 117 (2011) 102–110

Table 1
Biochemical evidence about the involvement of the endocannabinoid system in alcohol dependence.

Type of study Results Reference

In vitro studies
Chronic ethanol increased AEA formation in SK-N-SH cells Basavarajappa and Hungund (1999)
ECBs
Chronic ethanol increased 2-AG formation in cerebellar granule neurons Basavarajappa et al. (2000)

Animal brain studies

Chronic ethanol decreased AEA and 2-AG contents in the midbrain and increased AEA content Gonzalez et al. (2002a)
ECBs in the limbic forebrain
Levels of ECBs underwent significant changes in reward-related rat brain areas during Gonzalez et al. (2004)
alcoholization, deprivation and relapse
Ethanol self- administration increased 2-AG levels, without altering AEA levels, in rat nucleus Caille et al. (2007)
accumbens (in vivo microdialysis)
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 rat Gonzalez et al. (2002b)
CB1R
brain regions examined
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)

Chronic ethanol reduced CB1 receptor binding and functionality in several mouse brain Vinod et al. (2006)
CB1R & ECBs
regions; increased AEA levels and reduced FAAH activity in cortex
Chronic intermittent ethanol withdrawal transiently down-regulated rat hippocampal CB1Rs, Mitrirattanakul et al. (2007)
followed by long-term up-regulation, with increased EBCs
Human brain studies
CB1R & ECBs Elevated CB1R density and functionality and ECBs in the prefrontal cortex of alcoholic suicide Vinod et al. (2005)
victims compared to non-suicidal alcoholics
CB1R & FAAH Decreased CB1R density and functionality and FAAH activity in the ventral striatum of Vinod et al. (2010)
non-suicidal alcoholics compared to controls
ECBs Decreased AEA in nucleus accumbens and frontal cortex of Cloninger type 1 alcoholics Lehtonen et al. (2010)

Acute alcohol
Reduced ECB levels in different rat brain regions Rubio et al. (2007)
ECBs 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 Rubio et al. (2009)
increased in any of the regions analyzed

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 and
functionality observed in particular brain regions of mice follow-
ing the same ethanol exposure (Vinod et al., 2006). Similarly, CB1
receptor mRNA expression was found to be decreased in several
rat brain regions after chronic ethanol consumption (Ortiz et al.,
2004a). Nonetheless, another study did not observe any changes
after chronic exposure to ethanol either in CB1 receptor binding
or mRNA levels in rat brain (Gonzalez et al., 2002b). In regard to
the neuromodulation that exerts the CB1 receptor over neurotrans-
mitter synthesis and release, ethanol also produces a functional
desensitization of CB1 receptors in rats, modulating the synthesis
of 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 of
these receptors followed by a long-term up-regulation, with con-
comitant increased endocannabinoid levels, has been described in
rat hippocampus after chronic intermittent ethanol withdrawal
(Mitrirattanakul et al., 2007). Similarly, a recovery of the decreased
CB1 receptor binding and functionality has also been reported after
withdrawal (Vinod et al., 2006).
On the whole, the biochemical studies performed in rodents that
chronically consume ethanol suggest a down-regulation of brain
CB1 receptors, which is likely to be the consequence of high endo-
cannabinoid levels observed in these animals. The reason for this
high endocannabinoid content is still unknown and might be either
a higher synthesis rate, a lower degradation rate or even a decreased
re-uptake of the synaptic endocannabinoids.
In recent years some studies have been performed in postmortem
human brain of subjects that had previously been diagnosed of alco-
hol dependence. A hyperactivity of the endocannabinoid signaling
has been reported in the prefrontal cortex of suicidal alcoholic sub-
jects compared to alcoholic subjects dying of other causes than
suicide. These suicidal alcoholic subjects presented higher CB1
receptor density and functionality, as well as higher anandamide
and 2-AG levels. The authors suggested that this endocannabinoid
hyperactivity in the prefrontal cortex could be a potential factor
for suicide in alcohol dependence (Vinod et al., 2005). Recently,
the same group has described decreased CB1 receptor binding
and functionality in the ventral striatum of non-suicidal alcoholic
subjects compared to controls. However, these parameters were
elevated in the suicidal alcoholics when compared to non-suicidal
alcoholic subjects (Vinod et al., 2010). In regard to the differences
between Cloninger type 1 and 2 alcoholics, decreased anandamide
levels were observed in the nucleus accumbens and frontal cortex
in type 1 alcoholics (Lehtonen et al., 2010). Thus, studies performed
in 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 different
brain regions after short-term exposure to ethanol (Rubio et al.,
2007). Similarly anandamide content was also found reduced in
several rat brain regions, which was not dependent on FAAH or N-
acyltransferase activity (Ferrer et al., 2007). An in vivo microdialysis
study confirmed this reduction of dyalisate anandamide levels in
the 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-term
exposure to ethanol, since a decrease in the receptor mRNA and pro-
 A.M. Erdozain, L.F. Callado / Drug and Alcohol Dependence 117 (2011) 102–110 105

Table 2
Behavioral evidence about the involvement of the endocannabinoid system in alcohol dependence, after pharmacological manipulation of the system.

Modulation Results Reference

Agonists
CP55,940 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)
Surinabant (SR147778)
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 of Cippitelli et al. (2005)
ethanol-seeking in both alcohol-preferring and non-preferring rats
Reduced ethanol self-administration in alcohol-preferring rats when Hansson et al. (2007)
administered systemically or locally into the prefrontal cortex
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 in De Bruin et al. (2011)
rats
FAAH inhibitors
Increased ethanol self-administration in rats Hansson et al. (2007)
URB597 Increased ethanol preference and consumption in mice Blednov et al. (2007)
Increased ethanol preference in mice Vinod et al. (2008)

Anandamide transporter inhibitors

AM404 Reduced ethanol self-administration in rats Cippitelli et al. (2007)

tein levels has been reported in particular rat brain regions (Oliva
et al., 2008; Rubio et al., 2009).
In summary, chronic or acute alcohol administration seem to
produce different effects in the ECS. These differences may be due
to the adaptative responses produced by the chronic exposure to
ethanol.
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 cannabinoid
agonists, antagonists, FAAH inhibitors or anandamide transporter
inhibitors, alters the ethanol-related behavior in rodents (summa-
rized in Table 2). These findings further support the involvement of
the 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 55,212-2
increased alcohol relapse during a period of alcohol deprivation in
rats (Alen et al., 2008).
Conversely, the administration of the cannabinoid antago-
nists rimonabant (SR141716) and surinabant (SR147778) reduces
alcohol-related behavior and consumption. The first research work
to study this issue showed that rimonabant inhibited ethanol intake
in selectively bred alcohol-preferring mice tested under the two-
bottle choice paradigm (Arnone et al., 1997). This phenomenon
was later confirmed in different strains of alcohol-preferring rats
(Colombo et al., 1998; Dyr et al., 2008), as well as in non-preferring
mice (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 allows
studying the motivation of the animal to consume alcohol. Thus,
rimonabant reduced motivation for alcohol in a dose-dependent
manner both in non-preferring (Gallate and McGregor, 1999) and
preferring rats (Colombo et al., 2004). Similarly, operant ethanol
self-administration was reduced by rimonabant in rats tested under
different procedures (Freedland et al., 2001; Rodriguez de Fonseca
et al., 1999; Caille et al., 2007; Cippitelli et al., 2005; Hansson et al.,
2007). Further, rimonabant prevented the acquisition of ethanol
drinking behavior in alcohol-preferring rats (Serra et al., 2001).
In order to study the withdrawal and relapse in animals, two
experimental procedures have been developed. Firstly, the alcohol
deprivation effect consists in the temporary increase in volun-
tary ethanol intake after a period of alcohol withdrawal. Indeed,
rimonabant completely abolished the alcohol deprivation effect
in alcohol-preferring rats (Serra et al., 2002). The second pro-
cedure assesses the reinstatement of alcohol-seeking behavior
induced by a specific stimulus which was previously associated
with alcohol availability. In the same way, rimonabant reduced
conditioned reinstatement of ethanol-seeking behavior both in
alcohol-preferring and non-preferring rats (Cippitelli et al., 2005).
Similar results to those obtained with rimonabant have
been further observed with another cannabinoid antagonist that
was more recently synthesized, the surinabant or SR1417778
(Rinaldi-Carmona et al., 2004). A first study indicated that under
the two-bottled choice paradigm surinabant dose-dependently
decreased ethanol consumption in mice (Rinaldi-Carmona et al.,
2004). Likewise, surinabant reduced ethanol intake and preference
in alcohol-preferring rats (Gessa et al., 2005) and non-preferring
rats that had been chronically alcoholized (Lallemand and De Witte,
2006). Also, this cannabinoid antagonist dose-dependently reduced
the acquisition of ethanol drinking in alcohol-preferring rats and
106 A.M. Erdozain, L.F. Callado / Drug and Alcohol Dependence 117 (2011) 102–110
Table 3
Behavioral evidence about the involvement of the endocannabinoid system in alcohol dependence, after genetic manipulation of the system.
Modulation Results
CB1R knockout mice
Decreased ethanol intake and absence of alcohol-induced DA release in N accumbens
Absence of ethanol withdrawal symptoms and stress-stimulated ethanol drinking
Decreased ethanol intake and preference in young mice
Decreased ethanol intake
Decreased ethanol self-administration and increased alcohol sensitivity and withdrawal
Decreased ethanol-induced conditioned place preference
Decreased ethanol preference; increased blood ethanol concentration at high ethanol doses
Decreased ethanol intake and preference; absence of conditioned place preference
FAAH knockout mice
Increased ethanol intake and preference; decreased sensitivity in female FAAH−/− mice
Increased ethanol intake and preference; no differences in the ethanol-induced place preference, sensitivity or withdrawal
Increased ethanol preference; decreased ethanol sensitivity and withdrawal convulsions
suppressed the alcohol deprivation effect after a period of alcohol
abstinence (Gessa et al., 2005).
Recently, it has been reported that another CB1 cannabinoid
receptor antagonist, SLV330, was effective in reducing ethanol self-
administration and reinstatement of ethanol seeking in rats (De
Bruin et al., 2011).
The indirect activation of the ECS has also been studied in regard
to alcohol dependence. Thus, FAAH inhibitor URB597 administra-
tion increased operant ethanol self-administration in rats (Hansson
et 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, the
acute 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 the
ECS in particular situations relies on the genetic modulation of the
system. 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 markedly
reduced voluntary alcohol consumption in a two-bottle choice
paradigm (Hungund et al., 2003; Poncelet et al., 2003), which was
concomitant with a complete lack of ethanol-induced dopamine
release in the nucleus accumbens (Hungund et al., 2003). A similar
study observed this decrease in ethanol intake, but only in young
mice that did not express CB1 receptors, since old mice showed
similar preference for ethanol regardless of their genetic back-
ground (Wang et al., 2003). The reduction in ethanol consumption
was further confirmed in an operant self-administration paradigm
(Naassila et al., 2004; Thanos et al., 2005). Also, these animals show
a reduced ethanol-induced conditioned place preference, which
further 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 there
are discrepancies in the results. Racz et al. (2003) reported that
ethanol withdrawal symptoms were completely absent in these
genetically modified mice, while Naassila et al. (2004) observed an
increase in alcohol sensitivity and withdrawal induced convulsions.
Additionally, ethanol induced higher blood ethanol concentrations
in CB1 knockout mice, while decreasing ethanol preference. This
fact revealed that CB1 receptors might also be involved in ethanol
absorption and distribution, particularly after administration of
high ethanol doses (Lallemand and De Witte, 2005).
Reference
Hungund et al. (2003)
Racz et al. (2003)
Wang et al. (2003)
Poncelet et al. (2003)
Naassila et al. (2004)
Houchi et al. (2005)
Lallemand and De Witte (2005)
Thanos et al. (2005)
Basavarajappa et al. (2006)
Blednov et al. (2007)
Vinod et al. (2008)
Secondly, the ethanol-related behavior has also been studied
in FAAH knockout mice, which indeed have higher levels of endo-
cannabinoids compared to their wild type mates. All the studies
agree that these mice show increased intake and preference for
ethanol (Basavarajappa et al., 2006; Blednov et al., 2007; Vinod
et al., 2008), although one of them only observed this phenomena
in female mice, suggesting a sex-link mechanism (Basavarajappa
et al., 2006). Also, FAAH knockout mice displayed lower sensitivity
to acute effects of ethanol, and a reduction in ethanol withdrawal
convulsions (Vinod et al., 2008). However, Blednov et al. (2007) did
not observe any difference in ethanol sensitivity or withdrawal.
The findings of this section show that the stimulation of the ECS
increases ethanol consumption, whereas a blockade of the system
reduces ethanol administration. These effects may be due to a possi-
ble mechanism through which the endocannabinoids may enhance
the reinforcing properties of ethanol.
4. Genetic evidence
Alcohol dependence is a heterogeneous complex disorder with a
multiple 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 associated
with genes involved in metabolism of ethanol and also several
genes of different neurotransmission systems that underlie alcohol
dependence.
In this context, genetic evidence about the involvement of
the ECS in alcohol dependence has risen from the study of
rodent strains that differ in their preference for ethanol (summa-
rized in Table 4). First of all, differences in CB1 receptor density
and functionality have been reported within animals with dif-
ferent preference for ethanol. On one hand, alcohol-preferring
mice showed decreased CB1 receptor density, but higher affin-
ity and functionality than alcohol-avoiding mice (Hungund and
Basavarajappa, 2000; Basavarajappa and Hungund, 2001). On the
other hand, both decreased CB1 receptor gene expression and
functionality have been observed in different brain areas of alcohol-
preferring rats (Ortiz et al., 2004b). Moreover, in the prefrontal
cortex of alcohol-preferring rats, not only has a decrease in CB1
receptor binding and functionality been reported, but a decrease
in 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 the
ECS could be associated with some phenotypes of alcohol depen-
dence (Table 4). However, although a great number of genetic
 A.M. Erdozain, L.F. Callado / Drug and Alcohol Dependence 117 (2011) 102–110 107

Table 4
Genetic evidence about the involvement of the endocannabinoid system in drug addiction and alcohol dependence.

Study Results Reference

Rodent strains with different alcohol preference

Decreased CB1R density but higher affinity in alcohol-preferring mice Hungund and Basavarajappa (2000)
CB1R 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 and Hansson et al. (2007)
activity; increased 2-AG, in the prefrontal cortex of alcohol-preferring rats
CB1R polymorphisms
Association with severe alcohol withdrawal syndrome Schmidt et al. (2002)
rs1049353 No significant association with different alcoholism-related phenotypes, Preuss et al. (2003)
including severe alcohol withdrawal syndrome
This meta-analysis showed no significant association with alcohol Benyamina et al. (2011)
dependence;
rs6454674 Association with alcohol dependence Zuo et al. (2007)
rs2023239 Association with CB1R density, alcohol-elicited brain activation, subjective Hutchison et al. (2008)
reward when consuming alcohol and positive outcomes with
pharmacotherapy that targets the dopaminergic system
Association with alcoholic patients who suffered from attention deficit/ Ponce et al. (2003)
AAT repeat
hyperactivity disorder during childhood
microsatellite
No significant association with alcohol dependence; although significant with Comings et al. (1997)
cocaine, amphetamine and cannabis dependence
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
Association with concomitant street drug and problem drug/alcohol use Sipe et al. (2002)
rs324420 Association with multiple different drug addictions Flanagan et al. (2006)
Association with reward-related ventral striatum reactivity Hariri et al. (2009)

CB2R polymorphisms
rs2501432 Association with alcoholism in a Japanese population Ishiguro et al. (2007)

MAGL polymorphisms
No 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 significant
association. First of all, a particular single nucleotide polymor-
phism (SNP) of the CB1 receptor, the rs1049353, was associated
with severe alcohol withdrawal syndrome (Schmidt et al., 2002).
Nonetheless, another study that assessed the same particular
variation failed to confirm the association with different alcohol
dependence-related phenotypes, including severe withdrawal syn-
drome (Preuss et al., 2003). Further, a nine-allele microsatellite
polymorphism of the CB1 receptor, containing repeats of the single
trinucleotide AAT, has been associated with childhood attention
deficit/hyperactivity disorder in alcoholic patients (Ponce et al.,
2003). For this microsatellite repeat, again, another study did not
find an association with alcohol dependence, although it showed
an association with other drug dependences and with intravenous
drug use (Comings et al., 1997). In the same context, a recent
meta-analysis did not observe any significant association of either
rs1049353 or AAT repeat polymorphisms with alcohol dependence
(Benyamina et al., 2011). Similarly, another study failed to find an
association 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 of
CB1 receptor variants with alcohol dependence. Thus a highly sig-
nificant difference was observed in a particular haplotype of three
different SNPs in Japanese alcoholics compared to controls (Zhang
et al., 2004). Also, risk for alcohol dependence was significantly
associated 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 the
prefrontal cortex of alcoholic patients, greater alcohol-elicited
brain 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, the
rs324420, which produces a mutant enzyme with reduced cellu-
lar stability and therefore reduced expression and activity (Chiang
et al., 2004), has also been studied in regard to alcohol and other
drug use, with positive outcomes. The first study observed an asso-
ciation of this SNP with concomitant street drug use and problem
drug/alcohol use (Sipe et al., 2002). Another study confirmed this
association in a group composed of subjects with different drug
addictions, including alcohol dependence (Flanagan et al., 2006).
Recently, an imaging study has reported that the A carriers for
this polymorphism have increased brain reactivity in the reward-
related ventral striatum, which highlights the importance of the
variability 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 in
alcohol dependence. A significant association of the CB2 recep-
tor polymorphism rs2501432 with alcohol dependence has been
reported in Japanese patients (Ishiguro et al., 2007). Also, 14 differ-
ent polymorphisms in the monoacylglycerol lipase (MAGL) gene
were studied in a Japanese alcoholic population, but none of the
markers showed a significant association (Iwasaki et al., 2007).
Many of the studies presented show genetic evidence about the
involvement of the ECS in alcohol dependence. However, the lack
of positive associations in some studies can be attributed to the
genetic differences between the populations of alcoholics and con-
trols studied. Therefore, further studies are required to verify the
tentative allelic and genotypic associations reported.
108 A.M. Erdozain, L.F. Callado / Drug and Alcohol Dependence 117 (2011) 102–110
5. Rimonabant for the treatment of alcohol dependence
As reviewed throughout the article, a great amount of evidence
suggests the involvement of the ECS in the neurobiology of alcohol
dependence. More precisely, CB1 receptor blockade by rimonabant
in rodents has been reported to decrease not only ethanol intake
and 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 II
clinical studies have been conducted in human alcoholics in order
to 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/day
in the prevention of alcohol relapse in alcoholic patients that had
recently been detoxified (Soyka et al., 2008). Treatment compliance
tended to be better in the group of patients receiving rimonabant
than in the control group (patients receiving placebo). Overall, the
clinical and biological safety and tolerability of rimonabant was
good, and the rate of adverse effects was similar in both groups. The
primary end point of the study was to assess the time to first drink
and the time to relapse to first heavy alcohol drinking. At the end
of 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% was
the relapse rate to heavy drinking. In regard to the secondary end
points of the study, there were no significant differences between
groups in cumulative abstinence duration in days, percentage of
drinking days and percentage of heavy drinking days, although
there was similarly a trend toward favorable effects of rimona-
bant. Authors suggest that the lack of improvement of rimonabant
over placebo might be explained, firstly, by a very high benefi-
cial response rate in the placebo group, and secondly, by the short
duration of the treatment.
The second clinical trial was a 3-week double-blind, placebo-
controlled study to evaluate the effect of rimonabant 20 mg/day
on alcohol consumption in nontreatment-seeking heavy alcohol
drinkers (George et al., 2010). First of all, after 1 week baseline, 18
participants received rimonabant and 21 received placebo during 2
weeks, 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 evaluated
in an alcohol self-administration paradigm in which after receiving
an oral priming dose of ethanol they had the option of consuming
up to eight alcohol drinks or receiving some money for each non-
consumed drink. The results show that rimonabant did not change
alcohol self-administration either.
Rimonabant was commercialized for the treatment of obesity.
Nevertheless, due to the incidence of severe adverse psychiatric
effects during the treatment (Christensen et al., 2007), its commer-
cialization has been recently discontinued (European Medicines
Agency, 2009).
6. Conclusion
In conclusion, several lines of evidence support the involvement
of the ECS in alcohol dependence. It is clear that this neurotransmis-
sion system is directly involved in the primary rewarding effects of
ethanol through different cellular mechanisms. Moreover, the ECS
seems to play an important role in the motivation to seek the drug
in alcoholics and in the relapse to drug-seeking in alcoholic patients
that had recently been detoxified. Further studies will be required
to clarify the specific mechanisms that mediate these effects. How-
ever, the ECS constitutes a clear therapeutic target for the treatment
of alcohol dependence. In this way, the development of new specific
cannabinoid antagonists could contribute to develop new pharma-
cological treatments to decrease not only ethanol intake, but also
the 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 de
Salud Carlos III, Centro de Investigación Biomédica en Red de Salud
Mental, CIBERSAM, Spain. AM Erdozain is recipient of a predoctoral
fellowship from the Basque Government. The funding sources had
no further role in the writing of the review, or in the decision to
submit the paper for publication.
Contributors
AM Erdozain performed the literature search and LF Callado
wrote the first draft of the manuscript. Both authors contributed
to and have approved the final manuscript.
Conflict of interest
There are no conflicts of interest.
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