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From the DEPARTMENT OF EXPERIMENTAL MEDICINE AND PUBLIC HEALTH,
University of Camerino, Italy
Role of the endocannabinoid system in the control
of alcohol abuse mechanisms
Andrea Cippitelli
Supervisors:
-
Prof. Roberto Ciccocioppo
Prof. Fernando Rodriguez de Fonseca
Camerino 2006
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List of Papers
This thesis is based on the papers listed below:
1. Rodriguez de fonseca F., Del Arco I., Bermudez-Silva FJ., Bilbao A., Cippitelli
A., Navarro M. The endocannabinoid system: physiology and pharmacology.
Alcohol Alcohol. Jan-Feb; 40 (1): 2-14, 2005. Review.
2. Cippitelli A., Bilbao A., Hansson A.C., Del Arco I., Sommer W., Heilig M.,
Massi M., Bermudez-Silva F.J., Navarro M., Ciccocioppo R., de Fonseca F.R.;
The European TARGALC Consortium. Cannabinoid CB1 receptor antagonism
reduces conditioned reinstatement of ethanol-seeking behavior in rats. Eur J
Neurosci. Apr; 21(8): 2243-51, 2005.
3. Pavon F.J., Bilbao A., Hernández-Folgado L., Cippitelli A., Jagerovic N.,
Abellán G., Rodríguez-Franco M.I., Serrano A., Macias M., Gómez R., Navarro
M., Goya, P. and Rodríguez de Fonseca F. Pharmacological evaluation of the
novel in vivo cannabinoid receptor antagonist 5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-3-hexyl-1H-1,2,4-triazole – LH 21 –on food intake: evidence for
a peripheral site of action. Manuscript submitted to second revision at
Neuropharmacology.
4. Cippitelli A., Bilbao A., Navarro M.,Gorriti M.A., Massi M., Pomelli D.,
Ciccocioppo R. and Rodríguez de Fonseca F.. Selective reduction of ethanol selfadministration by the Anandamide trasport inhibitor AM 404. Manuscript
submitted to second revision at Eur J Neurosci.
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Table of contents
I. INTRODUCTION
Cannabis history and use
Endocannabinoid system
Cannabinoids and alcohol links
Animal and experimental models
Aim and contents
References
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II. THE ENDOCANNABINOID SYSTEM: PHYSIOLOGY AND PHARMACOLOGY
Abstract
Introduction
Biochemistry of the endogenous cannabinoid system
Functional neuroanatomy of the endogenous cannabinoid system
Pharmacology of the endogenous cannabinoid system
Physiology of the endogenous cannabinoid system
A practical approach: role for the endocannabinoid system in alcoholism
Conclusion
References
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III. CANNABINOID CB1 RECEPTOR ANTAGONISM REDUCES CONDITIONED
REINSTATEMENT OF ETHANOL-SEEKING BEHAVIOUR IN RATS
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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IV. PHARMACOLOGICAL EVALUATION OF THE NOVEL IN VIVO CANNABINOID
RECEPTOR ANTAGONIST 5-(4-CHLOROPHENYL)-1-(2,4-DICHLOROPHENYL)-3HEXYL-1H-1,2,4-TRIAZOLE – LH 21 – ON FOOD INTAKE: EVIDENCE FOR A
PERIPHERAL SITE OF ACTION
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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V. SELECTIVE REDUCTION OF ETHANOL SELF-ADMINISTRATION BY THE
ANANDAMIDE TRANSPORT INHIBITOR AM 404
Abstract
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Introduction
Materials and Methods
Results
Discussion
References
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VI. PPAR-α AGONISTS MODULATE ALCOHOL CRAVING AND RELAPSE THROUGH
A PERIPHERAL MECHANISM
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Introduction
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Materials and Methods
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Results
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Discussion
102
References
104
VII. GENERAL DISCUSSION
References
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109
ACKNOWLEDGEMENTS
110
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I
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I.INTRODUCTION
Cannabis history and use
The medical and recreational use of Cannabis sativa derivatives has been practised for thousands of
years and nowadays marijuana is the most commonly used illicit drug in the United States and in
many other countries throughout the world. Their use in USA has reached the pick at the end of
seventies and 4-5% of the general population and 15-20 % of high school seniors and college
students used marijuana at least once a month in 1994 (Chalsma and Boyum,1994). Above all, the
use between adolescents represent a big medical and social problem. However the consumers belong
to an heterogeneous population for age and gender. The term marijuana, which is Mexican, refers to
a particular form of cannabis that derives mainly from the leaves of the hemp plant growing in hot
and dry climates but also in more temperate zones which has been harvested for thousands of years
because of its usefulness in production of hemp for ropes and textiles, but also for its psychotropic
effects and the multitude of therapeutic indications ascribed to it. Several different cannabis
formulations are used, including hashish which consists of the psychoactive sticky resin pressed into
blocks, and bhang, a liquid distillate used in India.
The earliest archeological evidence of cannabis use dates back 10000 years. Between 2700 and
2000 BC, cannabis was used in Cina to treat rheumatic pains and other conditions (Adams and
Martins, 1996) and in India cannabis use played an important role in religion. In 1000 AD hashish
was known in the most part of arab world. The Cannabis plant attracted the attenction of European
scientists when Napoleon’s troops brought back from Egypt intriguing accounts of its psychotropic
activity. We now now that cannabis effects in humans include disruption of short-term memory,
cognitive impairments, enhanced body awareness, incoordination, sleepiness, reflex tachycardia,
hypothermia and mood alterations with euphoria or dysphoria depending on prior experience of the
user, mood state at the time of onset, drug dose and route of administration (Pertwee, 1988).
Figure 1. Timeline of Cannabis use from ancient world to the present (Childers and Breivogel, 1998).
In 1964 the main psychoactive component of cannabis and its chemical structure was isolated by
Gaoni & Mechoulam. The compound was a dibenzopyrane derivative, Δ9-tetrahydrocannabinol (Δ9THC) present in yellow resin that covers the leaves and flower clusters of the ripe female plant. In
this period intensive research on the molecular mode of action of cannabis sativa preparations
started and over the last fifteen years, huge advances in our knowledge of the physiology and
pharmacology of the cannabinoid system have taken place. Some effects of cannabinoids may be
therapeutically useful and currently nabilone, a structural analogue of Δ9-THC, is used in parts of
the USA and the UK for treatment of chemotherapy-induced nausea and vomit. Dronabinol, Δ9THC itself, is used as an appetite stimulator in AIDS patients in the USA (House of Lords, 1998)
and to reduce intraocular pressure in patients with glaucoma. In their hearing in 1998, the Science
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and Technology Committee of the UK House of Lords concluded that the large amount of anecdotal
evidence as to the therapeutic efficacy of cannabis in multiple sclerosis and chronic pain conditions
warranted ‘‘as a matter of urgency’’ investigation in proper clinical trials (House of Lords, 1998).
Clinical trials of standardized cannabis extracts are now being undertaken for these indications
(Wade et al., 2003; Zajicek et al., 2003; Berman et al., 2004), and a new drug based on cannabis
extracts (Sativex A) has recently been approved in Canada as adjunctive treatment for the
symptomatic relief of neuropathic pain associated with multiple sclerosis. Lastly the cannabinoid 1
receptor antagonist rimonabant is in phase III of clinical trials for the treatment of obesity and as an
aid to smoking cessation (Cleland et al., 2004; Van Gaal et al., 2005). Nonetheless, the usefulness as
therapeutic agents of such extracts, of Δ9-THC itself, or of synthetic compounds with the same
pharmacological actions as Δ9-THC, is greatly hampered by their psychotropic effects (Pryce &
Baker, 2005) and by their abuse potential. A major goal for many researchers is to find new
approaches to harness the therapeutic properties of these cannabinoids without producing unwanted
effects.
Endocannabinoid system
In the 1970’s and early 1980’s, it was generally assumed that the psychotropic effects of cannabis
terpenoid derivative Δ9-THC were dued by its hydrophobic nature that might act by influencing
membrane fluidity, rather than combining with a specific receptor. However, by the mid 1980’s,
several groups had shown that cannabinoid activity was highly stereospecific (Razdan, 1986) which
led to the search for a specific receptor and its endogenous mediators. The first‘‘hard’’ evidence for
receptors was the finding that 9-THC inhibited adenylyl cyclase activity in neuroblastoma cell
membranes (Howlett, 1984), followed by radioligand binding studies using the synthetic
cannabinoid agonist CP55,940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3hydroxypropyl)cyclohexanol) (Devane et al., 1988). Shortly after, the cannabinoid1 receptor was
cloned from rat brain (Matsuda et al., 1990). At the present, two cannabinoid receptors types have
been determined. Both receptor types are members of the seven transmembrane G-protein-coupled
receptor superfamily, with 44% and were named CB1 and CB2. CB1 is centrally located and
mediates the ‘‘high’’ produced by smoked cannabis (Huestis et al., 2001). Their distribution in the
brain is consistent with the ability of cannabinoids to alter pain perception, affect motor function,
impair cognition and memory, and stimulate feeding (Hohmann and Herkenham, 2000; Herkenham
et al., 1990; Matsuda et al., 1993; Freund et al., 2003). Those actions are effectively inhibited by the
CB1 receptor antagonist SR141716A (Rinaldi-Carmona et al., 1994), and are abolished in CB1 null
mutants (Lutz, 2002). CB2 is mainly a peripheral receptor and was cloned from human
promyelocytic leukaemia HL-60 cells (Munro et al.. 1993). The identification of cannabinoid
receptors led to the intriguing speculation that an endogenous cannabinoid-like substance might
exist. In 1992, the first endogenous compound to exert THC-like activity, the lipid
arachidonylethanolamide, was extracted from pig brain and named anandamide, from the Sanskrit
word for bliss, ananda (Devane et al., 1992). Anandamide has been shown to mimic in vivo
pharmacological and behavioural effects of Δ9-THC such as motor depression in an open field,
catalepsy on an elevated ring, analgesia on a hot plate, as well as hypothermia. Three years later, 2arachidonoylglycerol, a monoacylglycerol involved as an intermediate in a variety of signalling
pathways, was reported to interact with cannabinoid receptors (Mechoulam et al., 1995; Sugiura et
al., 1995) and following this, other endogenous compounds also may bind cannabinoid receptors,
including dihomo-g-linolenoylethanolamide and docosatetraenoylethanolamide (Hanus et al., 1993),
2-arachidonoylglyceryl ether (noladin ether, Hanus et al., 2001), O-arachidonoyl-ethanolamine
(virhodamine; Porter et al., 2002), N-arachidonoyl-dopamine (NADA; Bisogno et al., 2000) were
discovered (Figure 2).
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Figure 2. Timeline. Advances in research on the mechanisms of cannabinoid actions (Childers and Breivogel, 1998).
Recently, a compound named oleylethanolamide with similar structure of anandamide and thought
to activate cannabinoid receptor was described to show affinity for a subtype of peroxisome
proliferator-activated receptor, PPAR-α (Fu et al., 2003) and regulate feeding and body weight
through peripheral mechanisms (Rodriguez de Fonseca et al., 2001). Anandamide and
oleylethanolamide belong to the fatty acid ethanolamide (FAE) family of lipid mediators and are
released on demand by stimulated neurons and rapidly eliminated through hydrolysis catalyzed by
the enzyme fatty acid amide hydrolase. Anandamide, that meets all key criteria of an endogenous
cannabinoid substance, is inactivated by a previous step process consisting of carrier-mediated
transport that reuptake inter-synaptic endocannabinoid. Cannabinoid receptor activation results
mainly in an inhibition of cAMP formation and inhibition of Ca2+ influx as well as in the activation
of inwardly rectifying potassium conductance and A currents. These actions are relevant to the role
of cannabinoids as modulators of neurotransmitter release (Schlicker and Kathmann, 2001) and
short-term synaptic plasticity (Wilson and Nicoll, 2001). Recent physiological, pharmacological and
high-resolution anatomical studies provided evidence that the major physiological effect of
cannabinoids is the regulation of neurotransmitter release via activation of presynaptic CB1
receptors located on distinct types of axon terminals throughout the brain. Subsequent discoveries
shed light on the functional consequences of this localization by demonstrating the involvement of
endocannabinoids in retrograde signalling at GABAergic and glutamatergic synapses, as well as
modulators of postsynaptic transmission, interacting with other classical neurotransmitter including
dopamine. Thus, endogenous cannabinoid system may play an important role in many physiological
processes: pain perception, motor impairments, cognitive processes, memory, anxiety and other
psychiatric disorders including drug addiction.
Cannabinoids and alcohol links
Our laboratory has been working from several years on drug addiction reaching a good international
tradition particularly in the study alcoholism. Alcohol use is a major cause of morbidity and
mortality. Recent data indicate that it accounts for approximately 85000 deaths/ year in the US only,
making it the number three externally modifiable cause of mortality (Mokdad et al., 2004). Genetic
susceptibility factors interact with the environment to account for a considerable heritability in
alcohol use disorders (Enoch and Goldman, 2001). Identification of heritable susceptibility factors
offers a promise of improved and ultimately individualized pharmacological treatment in this
disorder, a promise which is in part beginning to be realized (Oslin et al., 2003). A large body of
evidence suggests functional interactions between the effects of cannabis and ethanol. First, the
ability of this small molecule to interact with numerous physiologic systems. The initial effects of
ethanol currently are believed to result primarily from facilitation of GABA A receptors and
inhibition of NMDA glutamate receptors transmission, respectively. Second, cannabis and alcohol
share some similar behavioural profiles: at low doses both produce stimulation of locomotor activity
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and at high doses both produce sedation, although obviously the dose levels at which these effects
take place are much different. Lastly, both activate the same reward pathways and CB1 receptor
plays an important role in regulating its positive reinforcing properties. It is well established that the
mesolimbic DA system plays a central role in reward circuitry and is a common route in the
reinforcement produced by most drugs of abuse. Indeed, alcohol administration enhances DA levels
in the nucleus accumbens (NAc). Furthermore, Tanda et al. (1997) and Gessa et al. (1998) have
shown that cannabinoids can cause the release of DA in the NAc by activating DA-containing
neurons in the ventral tegmental area, from which the mesoaccumbal DA-mediated pathway
originates. Thus, cannabinoids and alcohol lead to a common effect of central importance
(Mechoulam and Parker, 2003). These results strongly support the notion that the release of DA in
the NAc is a general biochemical consequence of several drugs of abuse; many, if not all, drugs
with rewarding properties act directly or indirectly through mechanisms that involve DA at the level
of the NAc. These effects most probably stem from the role of this system in the control of
motivational properties of natural rewards, such as food and sex (Wise and Rompre, 1989).
Exogenously administered cannabis and ethanol activate these natural reward circuits (Figure 3).
Figure 3. Diagram of the brain reward circuitry of the mammalian (laboratory rat) brain, indicating sites of action (on
the basis of the best presently available evidence) of various drugs, including cannabinoids, that enhance brain reward
and reward-related behaviours. ABN, anterior bed nuclei of the medial forebrain bundle; Acb, nucleus accumbens,
AMYG, amygdala; BNST, bed nucleus of the stria terminalis; BSR, brain-stimulation reward; CRF, corticotropin
releasing factor; DA, dopamine; DYN, dynorphin; END, endorphin; ENK, enkephalin; FCX, frontal cortex; GABA,
gamma-aminobutyric acid; GLU, glutamate; HIPP, hippocampus; 5HT, 5-hydroxytryptamine (serotonin); HYPOTHAL,
hypothalamus; LAT-TEG, lateral tegmental noradrenergic cell groups; LC, locus coeruleus; NE, norepinephrine
(noradrenaline); OFT, olfactory tubercle; OPIOID, endogenous opioid; PAG, periaqueductal grey matter; Raphe´,
Raphe´ nuclei of the brain stem; RETIC, reticular formation of the brain stem; VP, ventral pallidum; VTA, ventral
tegmental area (Gardner, 2005).
There is also considerable interest in the role of gradual adaptations in the mesolimbic DA system in
the development of addiction to psychoactive drugs and relapse to their use following withdrawal
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(Robinson and Berrige, 2003). Furthermore behavioural evidences show that SR141716A inhibits
ethanol intake in the ethanol-consuming C57Black/6 mice (Arnone et al., 1997) and Sardinian
alcohol-preferring (sP) rats (Serra et al., 2001). Voluntary ethanol intake has also been shown to be
enhanced by cannabinoid receptor agonists in ethanol-preferring sP rats (Colombo et al., 2002).
Indeed, using beer (which rats drink readily without prompting) as the test solution, Gallate et al.
reported that the cannabinoid receptor agonist CP55940 dose-dependently increased responding for
beer, an effect that was reversed by SR141716A. An operant self-administration study has shown
that SR-141716A reduces operant responding in Wistar rats that were made ethanol dependent by
14-day exposure to ethanol-vapour chambers, but not in rats that are not ethanol dependent
(Rodriguez de Fonseca et al., 1999).Cross-tolerance between ethanol and Δ9-THC has been
documented (Newman et al., 1972; Hungund and Basavarajappa, 2000). Decreased central CB1
receptor density (Basavarajappa et al., 1998) and receptor functionality (Basavarajappa and
Hungund, 1999) have been observed in mice chronically exposed to ethanol, providing further
evidence for a close link between ethanol and the endocannabinoid system.
Animal and experimental models
As evidenced by its widespread compulsive use, alcohol is a reinforcing and addictive substance.
Ethanol and other drugs of abuse reinforcement can be demonstrated in animal studies involving
oral and other routes of self-administration in which an experimental subject is located in a box
where has to carry out a task to receive the reinforcing drug. The great majority of selfadministration studies with rats have used simple fixed ratio (FR) schedules of reinforcement. The
FR 1 schedule is useful for exploring patterns of rate of drug intake and can be used effectively for
preliminary screening of drugs with abuse liability (Arnold and Roberts, 1997). In the case of
alcohol and in our experiments FR 1 schedule in which the animal each time pressing a lever
receives a reward is used (Figure 4).
Figure 4. Self-administration cage (left) and frontal panel of the box (right). When the animal press the circled lever
earns a rewarding liquid such as alcohol.
This experimental paradigm provide information on the motivational value of the addictive drugs.
However, a better predictor of the reinforcing value of the stimulus consist on a break point measure
under a progressive ratio (PR) operant schedule. Here, the response requirements to earn the drug
reward escalate after the delivery of each reinforcement until the animal is no longer motivated to
press the lever.
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Alcoholism is a chronic relapsing disorder and recurrent resumption of alcohol abuse after
detoxification and abstinence is one of the principal characteristics of dependence on alcohol
(American Psychiatric Association, 1994; O’Brien et al., 1990, 1998; O’Brien and McLellan, 1996).
The scientific approach to find a remedy for alcoholics is moved to develop new compounds able to
affect not only the amount of the drug ingested but also to decrease the compulsive use and seeking
of the drug. Various types of stimuli can increase relapse in drug addicts; a small dose recalling the
drug use, stress and drug-paired environmental stimuli appear to be the most important factors (Le
et al., 1998; Katner et al., 1999; Monti et al., 1999; Martin-Fardon et al., 2000; Rohsenow et al.,
2000; Ciccocioppo et al., 2001). One of recently developed experimental models of relapse to
alcohol-seeking and use is the cue-induced reinstatement paradigm. It consists in reinstating learned
responses evoked by environmental stimuli that have become associated with the subjective actions
of ethanol by means of classical conditioning after a period of extinction that resemble withdrawal
in human subjects.
Animal models based on selective breeding for excessive ethanol drinking have demonstrated a
utility for identifying and validating novel alcoholism treatment targets (McBride and Li, 1998).
The alcohol-preferring AA (Alko Alcohol) and the alcohol-avoiding ANA (Alko Non-Alcohol) rat
lines are among the best-established selection-based models, and have been bi-directionally bred for
high and low alcohol consumption, respectively, for over ninety generations (Sinclair et al., 1989).
In addition to the normal line of Wistar rats, in this study we use an alternative strain of genetically
selected alcohol-preferring rats. They are bred in our department of Experimental Medicine and
Public Health of the University of Camerino (Marche, Italy) for more of fifty generations from
Sardinian alcohol-preferring rats of the 13th generation, provided by the Department of
Neurosciences of the University of Cagliari (Colombo, 1997; Gessa et al., 1991).They are referred
to as marchigian-sardinian alcohol-preferring (msP) rats.
Aim and contents
The aim of this study is to elucidate the physiology and pharmacology of endogenous cannabinoid
system (Rodriguez de Fonseca et al., 2005) and to extend our knowledge on the effects of
cannabinoid drugs on alcohol reward and relapse to alcohol use. For this purpose, rodent models of
ethanol self-administration and reinstatement to alcohol-seeking behaviour will be used. To this
extent, the endocannabinoid system will be farmacologically manipulated using cannabinoid CB1
receptor agonists and antagonists. In the set of experiment we use a selective CB1 receptor
antagonist/inverse agonist SR 141716A to functionally validate previous in situ hybridization data
showing an over-expression of mRNA encoding for CB1 receptor of alcohol-preferring line of rats
compared with mRNA levels of normal Wistars (Cippitelli et al., 2005). This result is of scientific
relevance because indicates a direct correlation between genetic predisposition to consume ethanol
and an increased density of cannabinoid receptor in brain areas associated with addiction. In a
second study, we report the effects of a new synthetic cannabinoid receptor antagonist that results
ineffective in modifying ethanol self-administration but reduces feeding through a peripheral
mechanism because it is not able to cross blood, brain barrier. Thus 5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-3-hexyl-1H-1,2,4- triazole , named LH 21 could be an interesting compound for the
treatment of eating disorders and obesity because devoid of the potential side effects derived from
central blockade of cannabinoid CB1 receptors (Pavon et al., second revision Neuropharmacology).
Then, we report data about experiments realized using cannabinoid agonists. Despite several
bibliographic evidences reporting an increased ethanol consumption following treatment with CB1
receptor agonists we show an inhibition of operant responding for ethanol, modulating the
endocannabinoid tone and increasing inter-synaptic levels of anandamide using indirect cannabinoid
agonist such as AM 404 or directly activating neuronal CB1 receptors with ACEA and WIN 5521211
2 (Cippitelli et al., second revision Eur J Neurosci). These findings could be the first evidence that
increase in endocannabinoid signalling reduces the propensity to drink alcohol. Lastly we report
experimental evidence of a new receptor system involved in alcohol addiction. Oleylethanolamide, a
non-cannabinoid endogenous lipid with anorexic properties (Rodriguez de Fonseca et al., 2001),
binding its nuclear cognate receptor peroxisome proliferator-activated receptor-α (PPAR-α) (Fu et
al., 2003) modulates alcohol self-administration and play an important role in vulnerability to
alcohol abuse.
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References
Adams I.B., Martin B.R. (1996). Cannabis: pharmacology and toxicology in animals and humans. Addiction 91, 19851614.
American Psychiatric Association (1994). Diagnostic and Statistical Manual of Mental Disorders. 4 th ed. American
Psychiatric Association, Washington D.C,
Arnold J.M. and Roberts D.C.S. (1997). A critique of Fixed and Progressive Ratio Schedules Used to Examine the
Neural Substrates of Drug Reinforcement. Pharmacol Biochem Behav 57(3), 441-447.
Arnone M., Maruani, J. Chaperon F., Thiebot M.H., Poncelet M., Soubrie P. & Le Fur G. (1997). Selective inhibition of
sucrose and ethanol intake by SR 141716, an antagonist of central cannabinoid (CB1) receptors. Psychopharmacology,
132, 104–106.
Basavarajappa BS, Hungund BL (1999). Chronic ethanol increases the cannabinoid receptor agonist anandamide and its
precursor N-arachidonoylphosphatidylethanolamine in SK-N-SH cells. J Neurochem 72:522–528
Basavarajappa BS, Cooper TB, Hungund BL (1998). Chronic ethanol administration down-regulates cannabinoid
receptors in mouse brain synaptic plasma membrane. Brain Res 793:212–218
Bisogno T., Melck D., Bobrov MY., Gretskaya NM, Bezuglov VV., De Petrocellis L., Di Marzo V. (2000). N-acyldopamines: novel synthetic CB(1) cannabinoid-receptor ligands and inhibitors of anandamide inactivation with
cannabimimetic activity in vitro and in vivo. Biochem J. Nov 1; 351 Pt 3:817-24.
Berman J. S., Symonds C. & Birch R. (2004). Efficacy of two cannabis based medicinal extracts for relief of central
neuropathic pain from brachial plexus avulsion: results of a randomised controlled trial. Pain, 112, 299–306.
Chalsma A.L., Boyum D. (1994). Marijuana Situation Assessment.Washington, D.C., Office of National Drug Control
Policy.
Childers S.R. and Breivogel C.S. (1998). Cannabis and endogenous cannabinoid system. Drug
Dependence 51: 173-187.
and Alcohol
Ciccocioppo R., Sanna P.P., Weiss F. (2001). Cocaine-predictive stimulus induces drug-seeking behavior and neural
activation in limbic brain regions after multiple months of abstinence: reversal by D1 antagonists. ProcNatlAcad
SciUSA 98:1976–1981
Cippitelli A., Bilbao A., Hansson A.C., Del Arco I., Sommer W., Heilig M., Massi M., Bermudez-Silva F.J., Navarro
M., Ciccocioppo R., de Fonseca F.R.; The European TARGALC Consortium. Cannabinoid CB1 receptor antagonism
reduces conditioned reinstatement of ethanol-seeking behavior in rats. (2005).Eur J Neurosci. Apr;21(8):2243-51.
Cippitelli A., Bilbao A., Navarro M.,Gorriti M.A., Massi M., Pomelli D., Ciccocioppo R. and Rodríguez de Fonseca F.
Selective reduction of ethanol self-administration by the Anandamide trasport inhibitor AM 404. Eur J Neurosci. 2006
(second revision)
Cleland J. G. F., Ghosh J., Freemantle N., Kaye G. C., Nasir M., Clark A. L. & Coletta A. P. (2004). Clinical trials
update and cumulative meta-analyses from the American College of Cardiology: WATCH, SCD-HeFT, DINAMIT,
CASINO, INSPIRE, STRATUS-US, RIO-Lipids and cardiac resynchronisation therapy in heart failure. Eur. J. Heart
Failure, 6, 501–508.
Colombo G. (1997). Ethanol drinking behaviour in Sardinian alcohol-preferring rats. Alcohol.Alcohol 32: 443-453.
Colombo G., Serra S., Brunetti G., Gomez R., Melis S., Vacca G., Carai M.M. & Gessa G.L. (2002). Stimulation of
voluntary ethanol intake by cannabinoid receptor agonists in ethanol-preferring sP rats. Psychopharmacology, 159, 181–
187.
Devane W. A., Dysarz F. A. 3rd, Johnson M., Melvin L. S. and Howlett A. C. (1988). Determination and
characterization of a cannabinoid receptor in rat brain. Molecular Pharmacology 34, 605–613.
13
Devane W.A., Hanus L., Breuer A., Pertwee R.G., Stevenson L.A., Griffin G., Gibson D., Mandelbaum A., Etinger
A. and Mechoulam R. (1992). Isolation and structure of a brain constituent that binds to the cannabinoid receptor.
Science. 258, 1946-1949.
Enoch M.A., Goldman D. (2001).. The genetics of alcoholism and alcohol abuse. Curr Psychiatr Rep 3: 144–151.
Freund T. F., Katona I. & Piomelli D.(2003). Role of endogenous cannabinoids in synaptic signaling. Physiol. Rev., 83,
1017–1066 (review).
Fur J., Gaetani S., Oveisi F., Lo Verme J., Serrano A., Rodriguez de Fonseca F., Rosengarth A., Luecke H., Di Giacomo
B., Tarzia G., and Piomelli D. (2003). Oleylethanolamide regulates feeding and body weight through activation of the
nuclear receptor PPAR-alpha. Nature 425, 90–93.
Gallate J.E., Saharov T., Mallet P.E., McGregor I.S. (1999). Increased motivation for beer in rats following
administration of a cannabinoid CB1 receptor agonist. Eur. J. Pharmacol. 370, 233–240
Gaoni Y., Mechoulam R. (1964). Isolation, structure and partial synthesis of an active constituent of hashish. J. Am.
Chem. Soc. 86, 1646-164.
Gardner E.L. (2005). Endocannabinoid signalling system and brain reward: emphasis on dopamine. Pharmacol Biochem
Behav 81: 263-284.
Gessa G.L., Colombo G., Fadda F. (1991). Rat lines genetically selected for difference in voluntary ethanol
consumption, in Current Practices and Future Developments in the Pharmacotherapy of Mental Disorders (Meltzer
HY, Nerozzi D eds), pp 193-200. Excerpta Medica, Amsterdam
Gessa G.L., Melis M., Muntoni A.L., Diana M. (1998). Cannabinoids activate mesolimbic dopamine neurons by an
action on cannabinoid CB1 receptors. Eur J Pharmacol;341:39–44.
Hanus L., Abu-Lafi S., Fride E., Breuer A., Vogel Z., Shalev D.E., Kustanovich I., and Mechoulam R. (2001). 2Arachidonyl glyceryl ether, an endogenous agonist of them cannabinoid CB1 receptor. Proc Natl Acad Sci USA
98:3662–3665.
Hanus L., Gopher A., Almog S., and Mechoulam R. (1993). Two new unsaturated fatty acid ethanolamides in brain that
bind to the cannabinoid receptor. J Med Chem 36:3032–3034.
Herkenham M., Lynn A.B., Little M.D., Johnson M.R., Melvin L.S., de Costa B.R., and Rice K.C. (1990). Cannabinoid
receptor localization in brain. Proc Natl Acad Sci USA 87:1932–1936.
Hohmann A.G., Herkenham M. (2000). Localization of cannabinoid CB(1) receptor mRNA in neuronal subpopulations
of rat striatum: a double-label in situ hybridization study. Synapse 37: 71–80.
House of Lords, The United Kingdom Parliament (1998). [http:// www.parliament.the-stationery office .co.uk/pa/ ld
199798/ldselect/ ldsctech/151/15101.htm]
Howlett A.C. (1984). Inhibition of neuroblastoma adenylyl ciclase by cannabinoids and nantradol compounds.Life Sci.
35, 1803-1810.
Huestis M.A., Gorelick D.A., Heishman S.J., Preston K.L., Nelson R.A., Moolchan E.T., and Frank R.A. (2001).
Blockade of effects of smoked marijuana by the CBl-selective cannabinoid receptor antagonist SR141716. Arch Gen
Psychiat 58:322–328.
Hungund B.L., Basavarajappa B.S. (2000). Are anandamide and cannabinoid receptors involved in ethanol tolerance? A
review of the evidence. Alcohol Alcohol 35:126–133
Katner S.N., Magalong J.G., Weiss F. (1999). Reinstatement of alcoholseeking behavior by drug-associated
discriminative stimuli after prolonged extinction in the rat. Neuropsychopharmacology 20: 471–479
Le A.D., Quan B., Juzytch W., Fletcher P.J., Joharchi N., Shaham Y.(1998). Reinstatement of alcohol-seeking by
priming injections of alcohol and exposure to stress in rats. Psychopharmacology 135:169–174
Lutz B. (2002). Molecular biology of cannabinoid receptors. Prostag Leukot Essent Fatty Acids 66: 123–142.
14
Martin-Fardon R., Ciccocioppo R., Massi M., Weiss F. (2000). Nociceptin prevents stress-induced ethanol- but not
cocaine-seeking behavior in rats. Neuroreport 11:1939–1943
Matsuda L.A., Bonner T.I., Lolait S.J. (1993). Localization of cannabinoid receptor mRNA in rat brain. J Comp Neurol
327: 535–550.
Matsuda L.A., Lolait S.J., Brownstein M. J., Young A. C. and Bonner T. I. (1990). Structure of a cannabinoid receptor
and functional expression of the cloned cDNA. Nature 346, 561–564.
McBride W.J., Li T.K. (1998). Animal models of alcoholism: neurobiology of high alcohol-drinking behavior in
rodents. Crit Rev Neurobiol 12: 339–369.
Mechoulam R and Parker L. (2003). Cannabis and alcohol: a close friendship. Trends in Pharmacological sciences 24(6)
266-268.
Mechoulam R., Ben-Shabat S., Hanus L., Ligumsky M., Kaminski N.E., Schatz A.R., Gopher A., Almog S., Martin
B.R., Compton D.R., et al. (1995). Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to
cannabinoid receptors. Biochem Pharmacol 50:83–90.
Mokdad A.H., Marks J.S., Stroup D.F., Gerberding J.L. (2004). Actual causes of death in the United States, 2000.
JAMA 291: 1238–1245.
Monti P.M., Rohsenow D.J., Hutchison K.E., Swift R.M., Mueller T.I., Colby S.M., Brown R.A., Gulliver S.B., Gordon
A., Abrams D.B. (1999) Naltrexone’s effect on cue-elicited craving among alcoholics in treatment. Alcohol Clin Exp
Res 23:1386–1394
Munro S., Thomas K.L., and Abu-Shaar M. (1993). Molecular characterization of a peripheral receptor for
cannabinoids. Nature (Lond) 365:61–65.
Newman L.M., Lutz M.P., Gould M.H., Domino E.F. (1972). Δ 9- Tetrahydrocannabinol and ethyl alcohol: evidence for
cross-tolerance in the rat. Science 175:1022–1023
O’Brien C.P., Childress A.R., Ehrman R., Robbins S.J. (1998). Conditioning factors in drug abuse: can they explain
compulsion? J. Psychopharmacol. 12:15-22
O’Brien C.P., Childress A.R., McLellan A.T., Ehrman R. (1990). Integrating systematic cue exposure with standard
treatment in recovering drug dependent patients. Addict Behav 15:355-365
O’Brien C.P., McLellan A.T. (1996). Myths about the treatment of addiction. Lancet 347:237-240
Oslin D.W., Berrettini W., Kranzler H.R., Pettinati H., Gelernter J., Volpicelli J.R. et al (2003). A functional
polymorphism of the mu-opioid receptor gene is associated with naltrexone response in alcohol-dependent patients.
Neuropsychopharmacology 28: 1546–1552.
Pavon F.J., Bilbao A., Hernández-Folgado L., Cippitelli A., Jagerovic N., Abellán G., Rodríguez-Franco M.I., Serrano
A., Macias M., Gómez R., Navarro M., Goya P. and Rodríguez de Fonseca F. Pharmacological evaluation of the novel
in vivo cannabinoid receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,4-triazole – LH 21 –
on food intake: evidence for a peripheral site of action. Neuropharmacology 2006 (submitted)
Pertwee R.G. (1988). The central pharmacology of psychotropic cannabinoids. Pharmacol. Therapeut. 36, 189-261
Porter A. C., Sauer J. M., Knierman M. D. et al. (2002). Characterization of a novel endocannabinoid, virodhamine,
with antagonist activity at the CB1 receptor. Journal of Pharmacology and Experimental Therapeutics 301, 1020–1024.
Pryce G. & Baker D. (2005). Emerging properties of cannabinoid medicines in management of multiple sclerosis.
Trends Neurosci., 28, 272–276 (review).
Razdan R.K. (1986). Structure-activity relationships in cannabinoids. Pharmacol Rev 38:75-149
Rinaldi-Carmona M., Barth F., Heaulme M., Shire D., Calandra B., Congy C., Martinez S., Maruani J., Neliat G., Caput
D., Ferrara P., Soubrie´ P., Breliere J.C., and Le Fur G. (1994) SR141716A, a potent and selective antagonist of the
brain cannabinoid receptor. FEBS Lett 350:240–244.
15
Robinson T.E. and Berridge K.C. (2003) Addiction. Annu. Rev. Psychol. 54, 25–53
Rodriguez de fonseca F., Del Arco I., Bermudez-Silva F.J., Bilbao A., Cippitelli A., Navarro M. (2005). The
endocannabinoid system: physiology and pharmacology. Alcohol Alcohol. Jan-Feb;40 (1):2-14. Epub 2004 Nov 18.
Review.
Rodriguez D.F., Navarro M., Gomez R., Escuredo L., Nava F., Fu J. et al (2001). An anorexic lipid mediator regulated
by feeding. Nature 414: 209–212.
Rodriguez de Fonseca F., Roberts A.J., Bilbao A., Koob G.F., Navarro M. (1999). Cannabinoid receptor antagonist SR
141716A decreases operant ethanol self administration in rats exposed to ethanol-vapor chambers. Zhongguo Yao Li
Xue Bao 20: 1109–1114
Rohsenow D.J., Monti P.M., Hutchison K.E., Swift R.M., Colby S.M., Kaplan G.B. (2000). Naltrexone’s effects on
reactivity to alcohol cues among alcoholic men. J Abnorm Psychol 109:738–742
Schlicker E., Kathmann M. (2001). Modulation of transmitter release via presynaptic cannabinoid receptors.
Trends Pharmacol Sci. Nov; 22(11):565-72. Review.
Serra S., Carai M.A., Brunetti G., Gomez R., Melis S., Vacca G., Colombo G., Gessa G.L. (2001). The cannabinoid
receptor antagonist SR 141716A prevents acquisition of drinking behavior in alcohol-preferring rats. Eur J Pharmacol
430:369–371
Sinclair J.D., Le A.D., Kiianmaa K. (1989). The AA and ANA rat lines, selected for differences in voluntary alcohol
consumption. Experientia 45: 798–805.
Sugiura T., Kondo S., Sukagawa A., Nakane S., Shinoda A., Itoh K., Yamashita A., and Waku K. (1995). 2Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res Commun
215:89–97.
Tanda G, Pontieri FE, Di Chiara G. (1997). Cannabinoid and heroin activation of mesolimbic dopamine transmission by
a common mu1 opioid receptor mechanism. Science; 276:2048–50.
Van Gaal L. F., Rissanen A. M., Scheen A. J., Ziegler O. & Rössner S., for the RIO-Europe Study Group (2005). Effects
of the cannabinoid-1 receptor blocker rimonabant on weight reduction and cardiovascular risk factors in overweight
patients: 1-year experience from the RIO-Europe study. Lancet, 365, 1389–1397.
Wade D. T., Robson P., House H., Makela P. & Aram J. (2003). A preliminary controlled study to determine whether
whole-plant cannabis extracts can improve intractable neurogenic symptoms. Clin. Rehab., 17, 21–29.
Wilson R.I. and Nicoll R.A. (2001). Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses.
Nature (Lond) 410:588–592.
Wise R.A. and Rompre P.P. (1989). Brain dopamine and reward. Annu. Rev. Psychol. 40, 191–225
Zajicek J., Fox P., Sanders H., Wright D., Vickery J., Nunn A. & Thompson A. on behalf of the UK MS Research
Group (2003) Cannabinoids for treatment of spasticity and other symptoms related to multiple sclerosis (CAMS study):
multicentre randomised placebo- controlled trial. Lancet, 362, 1517–1526.
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II
17
II. THE ENDOCANNABINOID SYSTEM:
PHYSIOLOGY AND PHARMACOLOGY
Abstract
The endogenous cannabinoid system is an ubiquitous lipid signalling system that appeared early in
evolution and which has important regulatory functions throughout the body in all vertebrates. The
main endocannabinoids (endogenous cannabis-like substances) are small molecules derived from
arachidonic acid, anandamide (arachidonoylethanolamide) and 2-arachidonoylglycerol. They bind
to a family of G-protein-coupled receptors, of which the cannabinoid CB1 receptor is densely
distributed in areas of the brain related to motor control, cognition, emotional responses, motivated
behaviour and homeostasis. Outside the brain, the endocannabinoid system is one of the crucial
modulators of the autonomic nervous system, the immune system and microcirculation.
Endocannabinoids are released upon demand from lipid precursors in a receptor-dependent manner
and serve as retrograde signalling messengers in GABAergic and glutamatergic synapses, as well as
modulators of postsynaptic transmission, interacting with other neurotransmitters, including
dopamine. Endocannabinoids are transported into cells by a specific uptake system and degraded by
two well-characterized enzymes, the fatty acid amide hydrolase and the monoacylglycerol lipase.
Recent pharmacological advances have led to the synthesis of cannabinoid receptor agonists and
antagonists, anandamide uptake blockers and potent, selective inhibitors of endocannabinoid
degradation. These new tools have enabled the study of the physiological roles played by the
endocannabinoids and have opened up new strategies in the treatment of pain, obesity, neurological
diseases including multiple sclerosis, emotional disturbances such as anxiety and other psychiatric
disorders including drug addiction. Recent advances have specifically linked the endogenous
cannabinoid system to alcoholism, and cannabinoid receptor antagonism now emerges as a
promising therapeutic alternative for alcohol dependence and relapse.
Introduction
Twenty-four years of pharmacological research separate the identification of the main psychoactive
constituent of Cannabis sativa preparations, (-)-∆9-tetrahydrocannabinol (THC) (Gaoni and
Mechoulam, 1964; Mechoulam, 1970) from the characterization (Devane et al., 1988; Herkenham et
al., 1991) and molecular cloning (Matsuda et al., 1990) of its cellular target, the cannabinoid CB1
receptor (CB1). The extensive research on the structure and activity of the natural constituents of
Cannabis (termed cannabinoids) and the development of synthetic compounds with high potency
and stereoselectivity have led to the identification of the main physiological functions that are
modulated by this new class of drugs (Howlett et al., 1990). The discovery of the cannabinoid
receptor and the availability of highly selective and potent cannabimimetics led to the rapid
identification of a family of lipid transmitters that serve as natural ligands for the CB1 receptor:
arachidonoylethanolamide (AEA), named anandamide from the Sanskrit ‘internal bliss’ (Devane et
al., 1992) and 2-arachidonoylglycerol (2-AG) (Mechoulam et al., 1995; Sugiura et al., 1995). The
pharmacological properties of the endocannabinoids were found to be very similar to those of the
synthetic cannabimimetics. The subsequent description of a complex biochemical pathway for the
synthesis, release (Di Marzo et al., 1994; Cadas et al., 1996), transport (Beltramo et al., 1997) and
degradation (Cravatt et al., 1996) of endocannabinoids completed the scaffold of a new signalling
system termed the ‘endocannabinoid system’. Since the discovery of anandamide, more than 3500
18
scientific reports have comprehensively explored the main aspects of the endocannabinoid system.
This system now appears as a relevant modulator of physiological functions not only in the central
nervous system but also in the autonomic nervous system, the endocrine network, the immune
system, the gastrointestinal tract, the reproductive system and in microcirculation (Di Marzo et al.,
1998; Table 1).
The present review gives a general perspective of the endogenous cannabinoid system, including the
main pharmacological advances in the development of drugs capable of modulating their dynamics.
The review focuses on the role of endocannabinoids as modulators of reward circuits and motivated
behaviour that are relevant for drug addiction, including alcoholism. In light of the extensive
research over the past 12 years, several specialized reviews wherein the reader will find a more
profound analysis of the role played by the endocannabinoid system in selected physiological
functions are shown in table 1.
Topic
References
Biochemistry and molecular
biology
Signalling
Anatomy and development
Physiology
Pharmacology
Addiction
Matsuda, 1997; Felder and Glass, 1998; Giuffrida et al., 2001; Piomelli, 2003
Schlicker and Kathmann, 2001; Wilson and Nicoll, 2002; Freund et al., 2003
Breivogel and Childers, 1998; Fernandez-Ruiz et al., 2000; Elphick and Egertova, 2001
Di Marzo et al., 1998; De Petrocellis et al., 2004
Howlett et al., 2002
Maldonado, 2002; Maldonado and Rodríguez de Fonseca, 2002; Tanda and Goldberg, 2003;
Martin et al., 2004
Calignano et al., 2000; Pertwee, 2001
Rodríguez de Fonseca et al., 1998; Chaperon and Thiebot, 1999; Castellano et al., 2003
Izzo et al., 2001
Cabral, 2001
Kunos et al., 2002; Randall et al., 2002; Hiley and Ford, 2004
Piomelli et al., 2000; Cravatt and Lichtman, 2003; Guzman, 2003; Smith et al., 2004
Pain
Behaviour
Gastrointestinal system
Immune system
Cardiovascular system
Therapeutic applications
Sice the discovery of anandamide in 1992,over 3500 pubblications have reported new data on the biological role of the
endogenous cannabinoid system
Table 1. A selection of reviews and reports that explore in depth the main aspects of endocannabinoids and their
receptors.
Biochemistry of the endogenous cannabinoid system
Endocannabinoids. When discovered, the endocannabinoids were found to be derivatives of
arachidonic acid, which resembled other lipid transmitters (eicosanoids such as prostaglandins or
leukotrienes). Additional studies revealed the existence of other structure-related lipid messengers
including palmitylethanolamide or oleoylethanolamide, which are not active at cannabinoid
receptors. These messengers will not be included in this review, although they serve important
physiological functions in inflammation, pain control, feeding behaviour and lipid metabolism
(Calignano et al. 1998; Rodríguez de Fonseca et al., 2001; Fu et al., 2003; Piomelli, 2003).
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Endocannabinoids are derivatives of arachidonic acid conjugated with ethanolamine or glycerol.
Figure 1 depicts the chemical structure of four endocannabinoids, anandamide, 2arachidonoylglycerol (2-AG), the ester of arachidonic acid and ethanolamine; virodhamine which
resembles anandamide (Porter et al., 2002), and the 2-arachidonyl glyceryl ether noladin, an
analogue of 2-AG (Hanus et al., 2001).
Figure 1. Cannabinoid receptor agonists. Left, the structure of four arachidonic acid derivatives that have been
identified as endogenous ligands for both the cannabinoid CB1 and CB2 receptors. Right, the structure of ∆9
tetrahydrocannabinol (THC), the main cannabinoid receptor agonist present in Cannabis preparations and that of the
aminoalkylindole WIN-55,2122, a synthetic cannabinoid receptor agonist active at CB1 and CB2 receptors.
All these endocannabinoids have been found in the brain, plasma and peripheral tissues, although
the relevance of noladin has been questioned recently (Oka et al., 2003) because its concentration in
the brain is too low for this compound to act as an endogenous cannabinoid receptor ligand. In the
brain, the concentration of anandamide is 200-fold lower than that of 2-AG (Sugiura et al., 1995;
Stella et al., 1997). The monoglyceride 2-AG is a metabolic intermediate in lipid metabolism
whereas anandamide is the product of the cleavage of a membrane phospholipid. However, after
depolarization or receptor stimulation (e.g. dopamine D2 receptor-mediated), the concentration of
anandamide can rise up to 5–12 fold in a time-limited fashion (Giuffrida et al., 1999; Stella and
Piomelli, 2001; Kim et al., 2002).
Synthesis and release. Different pathways are involved in the synthesis and release of anandamide
and 2-AG. Figure 2 shows the dynamics of formation and degradation of anandamide. Anandamide
is formed by the cleavage of phospholipid precursor, the N-arachidonoyl-phosphatidylethanolamine
(NAPE). The precursor is synthesized by the enzyme N-acyltransferase (NAT), which catalyses the
transfer of arachidonic acid from phosphatidylcholine to the head group of
phosphatidylethanolamine. This enzyme requires the presence of Ca2+ and is regulated by cAMP,
which enhances the activity of NAT by phosphorylation mediated through the cAMP-dependent
activity of protein kinase A (Cadas et al., 1996; Piomelli, 2003). The release of anandamide from
NAPE is catalysed by a specific phospholipase D (PLD), which has been cloned recently (Okamoto
et al., 2004). This enzyme has no homology with the known PLD enzymes and is classified as a
member of the zinc metallohydrolase family. Its presence is highest in the brain, kidneys and testis.
The activity of PLD is regulated by depolarization or by activation of the ionotropic glutamate Nmethyl-D-Aspartate (NMDA) receptors or nicotinic α7 neuronal receptors (Stella and Piomelli,
2001; Piomelli, 2003) or stimulation of the metabotropic receptors of major neurotransmitters
20
including dopamine, glutamate and acetylcholine (Giuffrida et al., 1999; Varma et al., 2001; Kim et
al., 2002). The synthesis and release of 2-AG is different from that of anandamide. Because 2-AG
is a monoglyceride, its formation is closely associated with the metabolism of triacylglycerol,
mainly by the receptor-dependent activation of phosphatidylinositol- specific phospholipase C
(PLC). The standard model proposes that activation of metabotropic receptors coupled to the PLC
and diacylglycerol (DG) lipase pathway will systematically lead to increases in 2-AG production
(Stella et al., 1997; Piomelli, 2003). Cloning of the enzyme 1,2- diacylglycerol lipase (Bisogno et
al., 2003) has confirmed this hypothesis, as well as the contribution of ionotropic purinergic
receptors such as P2XT, which boosts 2-AG formation (Witting et al., 2004). Although 2-AG
formation is dependent on Ca2+, its regulation is independent of anandamide synthesis and release.
Once anandamide and 2-AG are formed, they target the CB1 receptors in the same cell where they
were formed, via diffusion within the plasmalemma, or they can be released to the extracellular
fluid where they reach distant targets (i.e. presynaptic terminals) with the apparent help of protein
carriers such as lipocalins or albumin (Piomelli, 2003).
Figure 2. Overview of the biochemical pathways for synthesis, degradation and cellular actions of the endogenous
cannabinoid anandamide. Anandamide is released from a membrane lipid precursor (N-arachidonoylphosphatidylethanolamine, NAPE) by the action of a specific phospholipase D (PLD) activated by depolarization or Gprotein-coupled receptor (GPCR) stimulation. NAPE biosynthesis is catalysed by a membrane enzyme, Nacyltransferase (NAT) activated by calcium (Ca2+) and cAMP. Anandamide acts as a retrograde messenger at
presynaptic cannabinoid receptors (CB1), where it regulates neurotransmitter release (NT) through its second
transduction systems [mainly Ca2+ incorporated through voltage-gated calcium channels (VGCC) or glutamate NMDA
(N-methyl-D-aspartate) receptors]. Anandamide also acts as a neuromodulator of major transmitter systems, including
dopamine, at postsynaptic cells, where it regulates excitability and synaptic plasticity through its modulation of
potassium (K+) channels, and the regulation of a broad spectrum of protein kinases (PK) including protein kinase A and
mitogen-activated protein kinases (MAPK). Anandamide action is terminated through a two-step process, which
includes, first, its cellular uptake through a specific anandamide transporter (AT) and second, degradation by enzymatic
cleavage to arachidonic acid (AA) and ethanolamide by the membrane-bound enzyme fatty acid amidohydrolase
(FAAH).
21
Uptake and degradation. Endocannabinoid signalling is terminated by a two-step process that
includes transport into cells and hydrolysis by two specific enzymatic systems. Both steps exert a
tight control of endocannabinoid levels in tissues, rapidly eliminating these signalling molecules.
Endocannabinoid uptake is mediated by a transporter (Beltramo et al., 1997), which is widely
distributed throughout the brain (Giuffrida et al., 2001). The transporter is an elusive molecule
which works in a manner that is similar to other lipid carriers: it facilitates the uptake of both
anandamide and 2-AG in an energy-independent fashion (Beltramo et al., 1997). The anandamide
transporter is saturable, displays substrate specificity and can be blocked by specific drugs such as
AM 404 (Figure 4). A major issue of debate has been the potential coupling of endocannabinoid
transport and degradation: it is possible that the energy for the uptake process is obtained by its
coupling to the enzymatic hydrolysis of anandamide. However, a recent report seems to confirm that
transport and degradation are independent processes (Fegley et al., 2004). The degradation of
endocannabinoids is performed by two specific enzymatic systems: the fatty acid amide hydrolase
(FAAH) (Cravatt et al., 1996) and the monoacylglyceride lipase (MAGL) (Dinh et al., 2002).
FAAH is a membrane enzyme that belongs to the serine–hydrolase family. FAAH is widely
distributed throughout the body, with high concentrations in the brain and liver. FAAH can degrade
many fatty acid amides, including acylethanolamides such as anandamide and the sleep factor
oleamide. Although FAAH can inactivate 2-AG, the main enzyme responsible for the inactivation
of this monoglyceride is MAGL (Dinh et al., 2002). This enzyme is also a serine hydrolase and its
distribution in the nerve terminals of specific brain neurons has been determined recently (Gulyas et
al., 2004).
Receptors: Two major cannabinoid receptors have been cloned, both of which belong to the
superfamily of G-protein coupled receptors. The first receptor described was named the CB1
receptor and it is mainly located in the terminals of nerve cells (central and peripheral neurons and
glial cells), the reproductive system (i.e. testis), some glandular systems and the microcirculation
(Devane et al., 1988; Howlett et al., 1990; Herkenham et al., 1991; Wagner et al., 1997; Batkai et
al., 2001). The CB2 cannabinoid receptor was found initially in multiple lymphoid organs with the
highest expression detected in B lymphocytes, moderate expression in monocytes and
polymorphonuclear neutrophils and the lowest expression in T lymphocytes, although subsequent
studies identified it in microglial cells as well (Munro et al., 1993; Galiègue et al., 1995; Piomelli,
2003). An interesting aspect of cannabinoid receptors is their expression during development of the
brain, where they control cell differentiation (Rueda et al., 2002), and their presence in tumour cells
derived from glial cells and the main epithelia (Galve-Roperh et al., 2000; Sanchez et al., 2001;
Casanova et al., 2003). Pharmacological studies revealed the existence of other endocannabinoid
targets including the vanilloid receptor (Zygmunt et al., 1999) and at least two non-CB1 non-CB2
‘CB-like’ receptors, one in the vascular bed and the other in glutamatergic axon terminals (Hajos et
al., 2001; Howlett et al., 2002; Kunos et al., 2002). The existence of these and other putative
cannabinoid receptors, and their role in endocannabinoid physiology can be clarified only after their
molecular characterization. Cannabinoid receptors, especially the CB1 receptor, display unique
properties. The most relevant property is their preservation throughout evolution: e.g. human, rat
and mouse CB1 receptors have 97–99% amino acid sequence identity. The preservation of this
ancient signalling system in vertebrates and several invertebrate phyla reflects the important
functions played by the endocannabinoids in cell and system physiology. A second remarkable
characteristic of the CB1 receptors is their high expression in the brain. The CB1 receptor is the
most abundant G-protein-coupled receptor, with densities 10–50 fold above those of classical
transmitters such as dopamine or opioid receptors (Howlett et al., 1990; Herkenham et al., 1991).
Another important characteristic is the low efficiency of CB1 receptor coupling to its transduction
system: e.g. when compared with opioid receptors, CB1 receptors are 7-fold less efficient in their
ability to couple to G proteins (Breivogel et al., 1998; Felder and Glass, 1998; Manzanares et al.,
1999). Both cannabinoid receptors are coupled to similar transduction systems. Cannabinoid
receptor activation was initially reported to inhibit cAMP formation through its coupling to Gi
22
proteins (Devane et al., 1988; Howlett et al., 1990), resulting in a decrease of the protein kinase Adependent phosphorylation processes as well. However, additional studies found that the
cannabinoid receptors were also coupled to ion channels through the Golf protein, resulting in the
inhibition of Ca2+ influx through N (Mackie and Hille, 1992), P/Q (Twitchell et al., 1997) and L
(Gebremedhin et al., 1999) type calcium channels, as well as the activation of inwardly rectifying
potassium conductance and A currents (Mackie et al., 1995; Childers and Deadwyler, 1996). These
actions are relevant to the role of cannabinoids as modulators of neurotransmitter release (Schlicker
and Kathmann, 2001) and short-term synaptic plasticity (Wilson and Nicoll, 2001), as discussed
below. Further research also described the coupling of CB1 and CB2 receptors to the mitogenactivated protein kinase cascade, to the phosphatidylinositol 3-kinase, to the focal adhesion kinase,
to ceramide signalling and to nitric oxide production (Derkinderen et al., 1996; Bouaboula et al.,
1997; Molina- Holgado et al., 1997; Galve-Roperh, 2000; Howlett et al., 2002). Finally, recent
studies revealed that under certain conditions, the CB1 receptors can stimulate formation of cAMP
by coupling to the Gs protein (Felder et al., 1998).
Endocannabinoids exhibit different binding properties and intrinsic activity at CB1 and CB2
receptors. Anandamide behaves as a partial agonist at both CB1 and CB2 receptors, but has higher
affinity for the CB1 receptor (Hillard et al., 1999; Howlett et al., 2002). The intrinsic activity of
anandamide at CB1 receptors is 4–30 fold higher than at CB2 receptors. However, 2-AG is a
complete agonist at both CB1 and CB2 receptors and it exhibits less affinity than anandamide for
both CB1 and CB2 receptors (Stella et al., 1997; Howlett et al., 2002).
Functional neuroanatomy of the endogenous cannabinoid system
As described above, the endogenous cannabinoid system is widely distributed throughout the body.
In the peripheral tissues the localization of the elements of the endogenous cannabinoid system
reflects the distribution of the cell types where they are located (e.g. B lymphocytes in spleen and
lymph nodes). However, in the nervous system the distribution is much more complex and
structured, and clearly reflects the importance of this system in synaptic transmission. In some
regions, such as the hippocampus, there is a complementary distribution of cannabinoid receptors,
endocannabinoid transporters and degradation enzymes. However, in other areas of the brain, for
instance the thalamus, there are discrepancies (i.e. transport activity and MAGL expression in the
absence of a relevant presence of the CB1 receptors) in its distribution, which reflects the gaps in
our knowledge of the composition of the endocannabinoid system.
Receptors. From the early work of Herkenham et al. (1991) it was clear that the CB1 receptor
distribution was unique among G-protein-coupled receptors, not only because of the very high
densities of cannabinoid binding sites but also because of the dynamics of CB1 receptor synthesis
and transport. Binding studies and in situ hybridization analysis showed that the cannabinoid
receptors are synthesized in somata and the protein transported to axon terminals (Herkenham et al.,
1991; Matsuda et al., 1993). The phenotype of the CB1 receptor-expressing neurons corresponds
mainly to GABAergic neurons including cholecystokinin-containing neocortical, amygdalar and
hippocampal neurons and dynorphin- and substance P-expressing medium spiny neurons of the
outflow nuclei of basal ganglia (Tsou et al., 1999; Julian et al., 2003). Several glutamatergic and
cholinergic telencephalic and cerebellar neurons also express the CB1 receptors (Piomelli, 2003). In
the peripheral nervous system, the CB1 receptors are located in sensory neurons of the dorsal root
ganglia. Figure 3 shows how the CB1 receptors are synthesized in medium spiny neurons of the
caudate-putamen and the protein transported to the axon terminals in the globus pallidus and
substantia nigra. The dense presence of CB1 binding sites in the cerebellum, hippocampus, striatum,
globus pallidum and substantia nigra clearly reflects this biological characteristic of CB1 receptors.
23
Figure 3. Imaging cannabinoid CB1 receptor in circuits of the rat brain reward system. Cannabinoid receptors are
mainly located at presynaptic axon terminals. In the basal ganglia, CB1 receptor mRNA expression (panels A and B) is
located mainly in GABAergic projecting neurons of the caudateputamen (Cpu), but not in the target nuclei, the globus
pallidus or the substantia nigra (GP and SN). However, the protein is mainly detected by immunohistochemistry (panels
C and D) in the axon terminals innervating both outflow nuclei of the basal ganglia. Panel E shows the dense presence
of CB1 receptors in the substantia nigra and ventral tegmental area (VTA) as mapped by CB1 receptor agoniststimulated GTP-γ-S incorporation. In these areas, CB1 receptors are not located in dopaminergic neurons (Panel F):
confocal imaging using specific antibodies against CB1 receptors (green) and tyrosine hydroxilase (red) shows the
compartmentalization of CB1 receptors in GABAergic afferents to the substantia nigra pars reticulata (SNr), whereas
dopaminergic cells are restricted to the pars compacta (SNc). The segregation of CB1 receptors and catecholaminergic
transmission is also observed in the hippocampus-dentate gyrus (Hpc-DG, panel G).
Enzymes. Fatty acid amide hydrolase is present in large principal neurons, such as the pyramidal
cells of the cerebral cortex, the pyramidal cells of the hippocampus, the Purkinje cells of the
cerebellar cortex and the mitral cells of the olfactory bulb.
Immunocytochemical analysis of these brain regions revealed a complementary pattern of FAAH
and CB1 expression with CB1 immunoreactivity occurring in fibres surrounding FAAHimmunoreactive cell bodies and/or dendrites (Egertova et al., 2003). This complementary
distribution suggests that FAAH closely controls the duration of cannabinoid effects, although there
are sites where this association does not occur, such as the outflow nuclei of basal ganglia.
Monoglyceride lipase is located mainly in the hippocampus, cortex, cerebellum and anterior
thalamus, with moderate expression in the extended amygdala including the shell of the nucleus
accumbens (Dinh et al., 2002). Comparison of the distribution of FAAH and MAGL at the cellular
level shows that FAAH is primarily a postsynaptic enzyme, whereas MAGL is presynaptic. The
spatial segregation of the two enzymes suggests that anandamide and 2-AG signalling may subserve
24
functional roles that also involve spatial segregation, raising a controversy with respect to the nature
and function of the retrograde endocannabinoid signal (Gulyas et al., 2004).
Transporter. The distribution of the anandamide transporter has been only partially characterized
because the transporter has not been cloned. The distribution of transport activity is highest in areas
expressing CB1 receptors, such as the hippocampus, the amygdala, the striatum and the
somatosensory, motor and limbic areas of the cortex. Transport activity is also present in areas with
low expression of the CB1 receptor, such as the thalamus and the hypothalamus (Beltramo et al.,
1997; Giuffrida et al., 2001).
Pharmacology of the endogenous cannabinoid system
During the last twenty years, and especially after the discovery of the CB1 receptor and
anandamide, an intense research effort has yielded numerous series of drugs that interact with most
of the main elements of the endogenous cannabinoid system. Today we have drugs that bind to the
CB1 receptor as agonists or antagonists, drugs that block the endocannabinoid transport and drugs
that inhibit the activity of FAAH. We lack specific NAT, PLD, sn1-DAGL and MAGL inhibitors.
Both in vitro and in vivo bioassays have been used to evaluate the activity of the new compounds.
Prior to the availability of radioligand cannabinoid receptors, in vitro assays included the inhibition
of forskolin-stimulated cAMP production and the inhibition of electrically evoked contractions of
isolated smooth muscle preparations. Smooth muscle preparations most often used for the bioassay
of cannabinoids are the mouse-isolated vas deferens and the myenteric plexus-longitudinal muscle
preparation from the guinea pig small intestine. These bioassays, which are particularly sensitive,
rely on the ability of cannabinoid receptor agonists to act via the CB1 receptors to inhibit
electrically evoked contractions. In vivo bioassays include behavioural tests for analgesia and
locomotion. A cluster of four effects (analgesia, hypothermia, immobility and catalepsy) in mice
constituting the ‘mouse tetrad’, is classically considered as a signature of cannabimimetic activity.
The recent availability of mouse knockouts for the cannabinoid receptors and FAAH (Ledent et al.,
1999; Cravatt et al., 2001) has facilitated these studies, offering a reliable model in the search for
selective compounds.
What is the logic of a cannabinoid approach to pharmacotherapeutics? Cannabinoid receptor
agonists may be designed to mimic the signalling processes mediated by anandamide and 2-AG,
mainly in pathological situations where a boost in cannabinoid receptor stimulation might be
needed. Cannabinoid receptor antagonism might be the approach selected in conditions with
enhanced endocannabinoid signalling. Transport inhibition and inhibition of degradation are more
sophisticated approaches, both oriented towards magnifying the tonic actions of endocannabinoids.
A rational use of these therapeutic strategies requires the identification and evaluation of the
functional status of endocannabinoid signalling in reference disorders. Thus, a deficit of anandamide
signalling during conditions of stress might be counteracted by the blockade of anandamide
degradation (Kathuria et al., 2003). As a summary of cannabinoid pharmacology, Table 2 shows the
reference compound for each molecular target, indicating Ki in the case of ligand–receptor
interaction or IC50 in the case of enzymatic inhibitors.
Cannabinoid receptor agonists. According to the International Union of Pharmacology (reviewed in
Howlett et al., 2002), cannabinoid agonists can be divided into classical cannabinoids, non-classical
cannabinoids, aminoalkylindoles and eicosanoids. New series of compounds have been recently
described, including diarylether sulfonylesters (Mauler et al., 2002) and pyrrole derivatives (Tarzia
et al., 2003b).
Classical cannabinoids are tricyclic dibenzopyran derivatives that are either compounds occurring
naturally in the plant Cannabis sativa, or synthetic analogues of these compounds. The most
representative forms are ∆9-THC (Figure 1), a partial agonist at both the CB1 and CB2 receptors
and the main psychoactive constituent of Cannabis, along with 11-hydroxy-∆8-THC-dimethylheptyl
25
(HU-210), a synthetic compound that displays the highest potency at the CB1 receptor (Howlett et
al., 2002). Classical cannabinoids are usually CB1/CB2 agonists, although changes in the THC
molecule have led to the synthesis of selective CB2 receptor agonists such as HU-308 (Hanus et al.,
1999).
Non-classical cannabinoids are synthetic THC analogues that lack the dihydropyran ring. The most
representative form is the Pfizer compound CP-55 940, a potent and complete agonist at both the
CB1 and CB2 receptors, which was used to characterize the CB1 receptor for the first time (Devane
et al., 1988; Herkenham et al., 1991).
Aminoalkylindoles were the first non-cannabinoid molecules that displayed cannabimimetic activity
(Pacheco et al., 1991). R-(+)-WIN-55,212–2 (Figure 1) is the most representative form, and it
behaves as a complete agonist at both the CB1 and CB2 receptors, with higher intrinsic activity at
the CB2 receptor.
Eicosanoids are the prototypic endocannabinoids (Figure 1), of which anandamide (a partial agonist
at both the cannabinoid receptors) and 2-AG (a complete agonist at both the CB1 and CB2
receptors) are the most representative compounds. Based on the structure of anandamide, minor
chemical changes have led to the development of the first generation of CB1-selective agonists, of
which R(+)- methanandamide and arachidonyl-2’-chloroethylamiden (ACEA) (Table 2) are the
most representative forms (Hillard et al., 1999).
Name
ACEA
SR141716A
HU-308
SR 144528
UCM 707
OL-135
URB 597
Target
CB1
CB1
CB2
CB2
AT
FAAH
FAAH
Action
Agonist
Antagonist
Agonist
Antagonist
Blocker
Inhibitor (reversible)
FAAH
Inhibitor
(irreversible)
Ki/IC50 (nM)
1.4
5.6
22.7
0.60
800
2.1
4.6
Reference
Hillard et al., 1999
Rinaldi-Carmona et al., 1994
Hanus et al., 1999
Rinaldi-Carmona et al., 1998
Lopez-Rodriguez et al., 2001
Lichtman et al., 2004
Kathuria et al., 2003
Table 2. Targeting the endogenous cannabinoid system: synthetic drugs of reference for cannabinoid CB1 and CB2
receptors, anandamide transporter (AT) and endocannabinoid degradation enzyme, fatty acid amidohydrolase (FAAH)
Cannabinoid receptor antagonists. Several series of compounds have been developed as CB1
receptor antagonists. The most representative are diarylpyrazoles, substituted benzofuranes,
aminoalkylindoles and triazole derivatives.
Diarylpyrazoles include both the first CB1 receptor antagonist synthesized (SR 141716A, RinaldiCarmona et al., 1994) and the first CB2 receptor antagonist (SR 144528). They were synthesized by
Sanofi and are considered the reference antagonists. However, they are not neutral antagonists since
they display significant inverse agonist properties. Modification of the SR 141716A molecule has
yielded other CB1 receptor antagonists with improved properties, including SR 147778 and AM 281
(Howlett et al., 2002; Rinaldi- Carmona et al., 2004). Diarylpyrazoles are orally active and
are currently under clinical trials for the treatment of obesity.
Substituted benzofuranes include LY 320135, a CB1 receptor antagonist with affinity at serotonin
and muscarinic receptors (Felder et al., 1998).
Aminoalkylindoles include a CB2 receptor antagonist, AM 630, which also displays activity as a
low-affinity partial CB1 agonist (Howlett et al., 2002).
26
Triazole derivatives include LH-21 (Jagerovic et al., 2004), an in vivo CB1 antagonist with a
paradoxic low affinity in vitro for CB1 receptors and devoid of inverse agonist properties.
Uptake blockers. Based on the structure of anandamide, a series of eicosanoid derivatives that have
the ability to block anandamide transport have been synthesized. The molecular structures of the
three prototypical uptake blockers are depicted in Figure 4.
Figure 4. Structure of three anandamide uptake blockers. UCM 707 is the compound with the highest affinity at the
anandamide transporter. AM 404 was the first blocker designed and has been extensively described. Both molecules,
however, had a significant impact on the activity of the fatty acid amidohydrolase (FAAH), the enzyme that degrades
anandamide. AM 1172 is a recently described compound without inhibitory action at FAAH, which has been used to
demonstrate the independence of anandamide transport and degradation processes.
The first and best studied transport inhibitor is AM 404 (Beltramo et al., 1997). The administration
of AM 404 results in the accumulation of anandamide and potentiates the effects of exogenously
administered anandamide. The compound AM 404 can be degraded by FAAH and behaves as an
agonist of vanilloid receptors. A second series of compounds is represented by UCM 707, which
displays a higher affinity at the transporter than AM 404 (Lopez-Rodríguez et al., 2001; De Lago et
al., 2002). A latest addition is AM 1172, a FAAH-resistant transport inhibitor that allows the study
of anandamide uptake processes without interference in FAAH activity (Fegley et al., 2004).
However the IC50 of AM 1172 (2000 nM) is lower than that reported for UCM 707 (800 nM).
Inhibitors of fatty acid amide hydrolase. As in the case of the cannabinoid receptors, different lines
of research have led to the discovery of chemically heterogeneous FAAH inhibitors. The earlier
inhibitors described consisted of reversible electrophilic carbonyl inhibitors (trifluoromethyl
ketones, alphaketo esters and amides, and aldehydes) or irreversible inhibitors (sulfonyl fluorides
and fluorophosphonates) incorporated into the fatty acid structures. Based on the structure of
alphatrifluoromethyl ketones a series of potent inhibitors were developed. Of these, alpha-keto N4oxazolopyridine provides inhibitors that are 102–103 times more potent than the corresponding
trifluoromethyl ketones (Boger et al., 2000). A recent series of alpha heterocycles has been shown to
possess very high potency and selectivity to reversibly inhibit FAAH activity in vivo and in vitro.
The most potent of these new compounds is OL-135, which exhibits IC50 in the low nanomolar
range (Lichtman et al., 2004). A different strategy has been selected by the group of Piomelli et al.,
27
who have developed exceptionally potent irreversible FAAH inhibitors, which exhibit a promising
anxiolytic profile (Kathuria et al., 2003; Tarzia et al., 2003a). These new classes of inhibitors are
carbamate derivatives capable of directly interacting with the serine nucleophile of FAAH.
However, these new inhibitors, although extremely potent, are not selective because they may
potentially inactivate other serine hydrolases such as heart triacylglycerol hydrolase (Lichtman et
al., 2004).
Physiology of the endogenous cannabinoid system
The ubiquitous presence of the endogenous cannabinoid system correlates with its role as a
modulator of multiple physiological processes. A comprehensive analysis of all the functions of the
endocannabinoids is beyond the scope of the present review. The reader will find an extensive list of
recent reviews that explore the physiological relevance of the endogenous cannabinoid system, as
depicted in Table 1. In this section, we focus on the cellular and system physiological events
mediated by endocannabinoids that are relevant to our understanding of the contribution of the
endogenous cannabinoid system in alcoholism.
Cellular physiology. As described in the section on biochemistry of the endogenous cannabinoid
system, endocannabinoids are released upon demand after cellular depolarization or receptor
stimulation in a calcium-dependent manner. Once produced, they act on the cannabinoid receptors
located in the cells surrounding the site of production. This property indicates that endocannabinoids
are local mediators similar to the autacoids (e.g. prostaglandins). In the CNS, the highly organized
distribution of endocannabinoid signalling elements in GABAergic and glutamatergic synapses and
their preservation throughout evolution suggests a pivotal role in synaptic transmission. Because of
the inhibitory effects on adenylyl cyclase, the activation of K+ currents and the inhibition of Ca2+
entry into cells, the net effect of the CB1 receptor stimulation is a local hyperpolarization that leads
to the general inhibitory effects described. If endocannabinoids act postsynaptically they will
counteract the activatory inputs entering the postsynaptic cells. This mechanism has been proposed
for postsynaptic interactions with dopaminergic transmission (Felder et al., 1998; Rodríguez de
Fonseca et al., 1998; Giuffrida et al., 1999). Despite its importance, this effect is secondary to the
important presynaptic actions whose existence is supported by two facts: (i) the concentration of the
CB1 receptors in presynaptic terminals and (ii) the well-documented inhibitory effects of the CB1
receptor agonists on the release of GABA, glutamate, acetylcholine and noradrenaline (Schlicker
and Kathmann, 2001; Piomelli, 2003). This inhibitory effect has been demonstrated for
neuropeptides such as corticotrophinreleasing factor and cholecystokinin as well (Rodríguez de
Fonseca et al., 1997; Beinfeld and Connolly, 2001). Presynaptic inhibition of neurotransmitter
release is associated with the inhibitory action of endocannabinoids on Ca2+ presynaptic calcium
channels via the activation of CB1 receptors. Presynaptic inhibition of transmitter release by
endocannabinoids may adopt two different forms of short-term synaptic plasticity, depending on the
involvement of GABA or glutamate transmission, respectively: depolarization-induced suppression
of inhibition (DSI) and depolarization-induced suppression of excitation (DSE) (Wilson and Nicoll,
2002; Diana and Marty, 2004). Both forms of synaptic plasticity involve the initial activation of a
postsynaptic large projecting neuron (pyramidal or Purkinje cells) that sends a retrograde messenger
to a presynaptic GABA terminal (DSI) or a presynaptic glutamate terminal (DSE), inducing a
transient suppression of either the presynaptic inhibitory or the presynaptic excitatory input. The
contribution of endocannabinoids to these forms of short term synaptic plasticity has been described
in the hippocampus (Wilson and Nicoll, 2001; Wilson et al., 2001) and the cerebellum (Diana et al.,
2002). The nature of the endocannabinoid system acting as a retrograde messenger is still unknown.
The role of endocannabinoid-induced DSI or DSE seems to be the coordination of neural networks
within the hippocampus and the cerebellum that are involved in relevant physiological processes,
such as memory or motor coordination.
28
Additional forms of endocannabinoid modulation of synaptic transmission involve the induction of
long-term synaptic plasticity, namely long-term potentiation (LTP) and long-term depression
(LTD). Both forms of synaptic plasticity involve long-term changes in the efficacy of synaptic
transmission in glutamatergic neurons, which have a major impact on consolidation and remodelling
of the synapsis. Activation of the cannabinoid receptors prevents the induction of LTP in the
hippocampal synapses (Stella et al., 1997) and a facilitation of LTD in the striatum (Gerdeman et
al., 2002) and the nucleus accumbens (Robbe et al., 2002). In the hippocampus, the
endocannabinoid messengers regulate a form of LTD that affects inhibitory GABAergic neurons
(Chevaleyre and Castillo, 2003).
Overall, endocannabinoids act as local messengers that adjust synaptic weight and contribute
significantly to the elimination of information flow through specific synapses in a wide range of
time frames. The fact that cannabinoid receptor stimulation has a major impact on second
messengers involved not only in synaptic remodelling (Derkinderen et al., 1996; Piomelli, 2003) but
also in neuronal differentiation (Rueda et al., 2002) and neuronal survival (Panikashvili et al., 2001;
Marsicano et al., 2003) indicates that the signalling system is a major homeostatic mechanism that
guarantees a fine adjustment of information processing in the brain and provides counter-regulatory
mechanisms aimed at preserving the structure and function of major brain circuits. Both processes
are relevant for homeostatic behaviour such as motivated behaviour (feeding, reproduction,
relaxation, sleep) and emotions, as well as for cognition, since learning and memory require
dynamic functional and morphologic changes in brain circuits. An experimental confirmation of this
hypothetical role of the endogenous cannabinoid system was the demonstration of its role in the
control of the extinction of aversive memories (Marsicano et al., 2002; Terranova et al., 1996).
System physiology. The cellular effects of endogenous cannabinoids have a profound impact on the
main physiological systems that control body functions (Table 1). Despite the peripheral modulation
of the immune system, vascular beds, reproductive organs, gastrointestinal motility and metabolism,
the endogenous cannabinoid system tightly regulates perception processes including nociception
(cannabinoids are potent analgetics, Martin and Litchman, 1998) and visual processing in the retina
(Straiker et al., 1999). Additional functions exerted by the endogenous cannabinoid system involve
the regulation of basal ganglia and cerebellar circuits, where it is involved in the modulation of
implicit learning of motor routines (Rodriguez de Fonseca et al., 1998).
Among the varied functions in which the endogenous cannabinoid system is engaged, the
homeostatic control of emotions and the regulation of motivated behaviour merit special attention
because of its impact on human diseases, including addiction. The endogenous cannabinoid system
controls the motivation for appetite stimuli, including food and drugs (Di Marzo et al., 1998, 2001;
Navarro et al., 2001; Gomez et al., 2002). The positive effects of endocannabinoids on motivation
seem to be mediated not only by the peripheral sensory systems in which cannabinoid receptors are
present (i.e. the promotion of feeding induced by cannabinoid CB1 receptor agonists, Gomez et al.,
2002), but also by the action of endocannabinoids on the reward system, a set of in-series circuits
that link the brain stem, the extended amygdala and the frontal executive cortex. The endogenous
cannabinoid system is widely distributed in the extended amygdala, a set of telencephalic nuclei
located in medial septal neurons, the nucleus accumbens shell and amygdalar complex, and are
involved in the control of motivated behaviour, conditioned responses and gating-associated
emotional responses. This hypothesis is supported by two facts: the inhibition of motivated
behaviour observed after administration of a cannabinoid antagonist (Colombo et al., 1998; Navarro
et al., 2001) and the reward deficits observed in the CB1 receptor knockout mice (Ledent et al.,
1999; Maldonado and Rodríguez de Fonseca, 2002; Sanchis-Segura et al., 2004). Research on the
neurobiological basis of endocannabinoid effects on motivated behaviour has focused on
endocannabinoid–dopamine interaction as well as on the role of the endocannabinoid system in
habit learning and conditioning. The extended amygdala is the target of the ascending
mesocorticolimbic projections of the ventral tegmental area (VTA) dopaminergic neurons, a subset
of mesencephalic neurons that display a consistent response to drugs of major abuse, which appear
29
to be a common substrate for the reward properties of drugs of dependence (Maldonado and
Rodríguez de Fonseca, 2002). Most drugs of dependence activate the VTA dopaminergic neurons,
as monitored by the dopamine release in terminal areas, especially in the nucleus accumbens and
prefrontal cortex, or by the firing rates of VTA dopaminergic neurons. THC and other CB1 receptor
agonists increase dopamine efflux in the nucleus accumbens and prefrontal cortex and increase the
dopaminergic cell firing in the VTA (for review see Gardner and Vorel, 1998). This effect is not
caused by the direct activation of dopaminergic neurons because they do not express CB1 receptors
(Julian et al., 2003). Although the effects of cannabinoid agonists on dopamine release in the
projecting areas (i.e. nucleus accumbens) can be blocked by the opioid antagonist naloxone, the
increase in VTA dopaminergic cell firing cannot be blocked. This discrepancy may suggest the
existence of a differential role for endogenous opioid systems as the modulators of cannabinoid
actions in dopamine cell bodies with respect to their axon terminals. Cannabinoid effects might also
involve glutamatergic and GABAergic inputs to the nucleus accumbens and VTA, because
presynaptic CB1 receptors regulate glutamate and GABA release in these areas, inducing LTD
(Schlicker and Kathmann, 2001; Robbe et al., 2002). In agreement with these actions of
cannabinoids in brain reward circuits, repeated cannabinoid exposure can induce behavioural
sensitization similar to that produced by other drugs of dependence. Chronic cannabinoid
administration also produces cross-sensitization to the locomotor effects of psychostimulants
(Maldonado and Rodríguez de Fonseca, 2002). Because endocannabinoids induce LTD in the
nucleus accumbens (which affect glutamatergic inputs coming from the prefrontal cortex), they
probably regulate the acquisition of habit learning and conditioned responses relevant to the
progressive loss of control that characterize drug addiction (Maldonado and Rodríguez de Fonseca,
2002). Interestingly, administration of a CB1 receptor antagonist blocks cue-induced reinstatement
to heroin and cocaine self-administration (De Vries et al., 2001, 2003). The importance of the
endogenous cannabinoid system in the control of motivated behaviour goes far beyond the control
of processing ongoing reward signals. The CB1 receptors are apparently involved in the control of
reward homeostasis (Sanchis-Segura et al., 2004). Moreover, when cannabinoid homeostatic
mechanisms are not adequate to restore the lost equilibrium in reward control derived from
continuous uncontrolled exposure to a reinforcer (e.g. opiates or alcohol), allostatic changes
involving CB1 receptors are set in motion to counteract the spiralling distress imposed on the
reward circuit. This has been demonstrated in rodents exposed to cycles of dependence–abstinence
to alcohol and morphine (Navarro et al., 2001; Rimondini et al., 2002). In this model, a history of
dependence is associated with a permanent up-regulation of the expression of CB1 receptors in
reward-related areas and with an enhanced sensitivity to reward disruption induced by cannabinoid
receptor antagonists (Rodríguez de Fonseca et al., 1999; Rimondini et al., 2002). Whether these
allostatic changes occur in other models of motivated behaviour (i.e. feeding) remains to be
determined. Cannabinoid receptors are not only associated with motivational disturbances, but also
related to emotional processing. A key station for the endocannabinoid regulation of emotions is the
amygdalar complex. Endocannabinoids are able to depress the release of glutamate and
corticotropinreleasing factor, reducing the amygdalar output and the activity of basolateral
inhibitory GABA projections to the central nucleus of the amygdala, thereby activating the
amygdalofugal pathway (Rodríguez de Fonseca et al., 1996, 1997; Navarro et al., 1997; Marsicano
et al., 2002; Piomelli, 2003). The final balance will lead to anxiety or anxiolysis, depending on the
rate of activation of descending projections of the central nucleus of the amygdala to the
hypothalamus (endocrine responses) and brainstem (behavioural and autonomic responses).
However, recent studies indicate that anxiolysis is the normal response to enhanced cannabinoid
transmission in the limbic system, as reflected by the phenotype of FAAH knockout mice and the
effects of FAAH inhibitors (Cravatt et al., 2003; Kathuria et al., 2003). The induction of anxiety by
cannabinoid receptor antagonists (Navarro et al., 1997) supports this notion as well.
30
A practical approach: role for the endocannabinoid system in alcoholism
The presence of the endogenous cannabinoid system in reward circuits and its role in motivational
and emotional homeostasis suggests that drugs which modulate cannabinoid signalling might serve
as therapeutic tools in drug addiction. In accordance with this rationale, the CB1 receptor
antagonists are able to modulate opioid self-administration in rodents (Navarro et al., 2001).
Extending this hypothesis, converging research lines have established a role for both anandamide
and the CB1 receptor in alcohol dependence (Hungund and Basaravajappa, 2000; Hungund et al.,
2002; Mechoulam and Parker, 2003). The administration of CB1 receptor agonists promotes alcohol
intake (Colombo et al., 2002), whereas the administration of a CB1 receptor antagonist decreases
alcohol self-administration, especially in animals with a history of alcohol dependence (Rodríguez
de Fonseca et al., 1999) or in alcohol-preferring rat lines (Colombo et al., 1998). Molecular studies
have shown that chronic alcohol administration is associated with an increased formation of both
anandamide and its membrane precursor NAPE (Basavarajappa and Hungund, 1999). Chronic
alcohol exposure also resulted in the stimulation of a second endocannabinoid, 2-AG
(Basavarajappa et al., 2000). Animal studies also revealed that chronic exposure to alcohol downregulated the CB1 receptors in the brain (Basavarajappa et al., 1998). Finally, a recent gene
screening study has identified the CB1 receptor as one of the genes whose expression is
permanently affected by serial cycles of alcohol dependence and withdrawal (Rimondini et al.,
2002). These data indicate a role for the endogenous cannabinoid system as a relevant contributor to
alcoholism. Human gene studies support this experimental hypothesis, since a linkage between
clinical forms of alcoholism and polymorphisms and/or mutations of the genes encoding either the
CB1 receptor (Comings et al., 1997; Schmidt et al., 2002) or the FAAH (Sipe et al., 2002), the
enzyme responsible for AEA inactivation (Cravatt et al., 1996), have been described. In the present
issue, the reader will find additional experimental approaches to the role of the endogenous
cannabinoid system in alcoholism.
Conclusion
Since the discovery of anandamide, the increasing information on the physiological roles played by
the endogenous cannabinoid system and its contribution to pathology have led to this signalling
system becoming more important in neurobiology. The intense pharmacological research based on
this information has yielded, in a very short time, potent, selective drugs targeting the endogenous
cannabinoid system that have opened up new avenues for the understanding and treatment of major
diseases including cancer, pain, neurodegeneration, anxiety and addiction. This is a very promising
starting point for a new age that takes over from the ancient use of Cannabis as a medicine. Now is
the time for clinical trials aimed at evaluating the efficacy of cannabinoid drugs in disorders lacking
effective therapeutic approaches, such as alcoholism.
31
References
Basavarajappa B. S., Cooper T. B. and Hungund B. L. (1998). Chronic ethanol administration down-regulates
cannabinoid receptors in mouse brain synaptic plasma membrane. Brain Research 793, 212–218.
Basavarajappa B. S. and Hungund B. L. (1999). Chronic ethanol increases the cannabinoid receptor agonist anandamide
and its precursor N-arachidonoylphosphatidyl-ethanolamine in SK-N-SH cells. Journal of Neurochemistry 72, 522–528.
Basavarajappa B. S., Saito M., Cooper T. B. and Hungund B. L. (2000). Stimulation of cannabinoid receptor agonist 2arachidonylglycerol by chronic ethanol and its modulation by specific neuromodulators in cerebellar granule neurons.
Biochimica et Biophysica Acta 1535, 78–86.
Batkai S., Jarai Z., Wagner J. A. et al. (2001). Endocannabinoids acting at vascular CB1 receptors mediate the
vasodilated state in advanced liver cirrhosis. Nature Medicine 7, 827–832.
Beltramo M., Stella N., Calignano A., Lin S. Y., Makriyannis A. and Piomelli D. (1997). Functional role of high-affinity
anandamide transport, as revealed by selective inhibition. Science 277, 1094–1097.
Beinfeld M. C. and Connolly K. (2001). Activation of CB1 cannabinoid receptors in rat hippocampal slices inhibits
potassiumevoked cholecystokinin release, a possible mechanism contributing to the spatial memory defects produced by
cannabinoids. Neuroscience Letters 301, 69–71.
Bisogno T., Howell F., Williams G. et al. (2003). Cloning of the first sn1-DAG lipases points to the spatial and temporal
regulation of endocannabinoid signalling in the brain. Journal of Cell Biology 163, 463–468.
Bisogno T., Melck D., Bobrov MY., Gretskaya NM, Bezuglov VV., De Petrocellis L., Di Marzo V. (2000). N-acyldopamines: novel synthetic CB(1) cannabinoid-receptor ligands and inhibitors of anandamide inactivation with
cannabimimetic activity in vitro and in vivo. Biochem J. Nov 1; 351 Pt 3:817-24.
Boger D. L., Sato H., Lerner A. E. et al. (2000). Exceptionally potent inhibitors of fatty acid amide hydrolase: the
enzyme responsible for degradation of endogenous oleamide and anandamide. Proceedings of the National Academy of
Sciences of the United States of America 97, 5044–5049.
Bouaboula M., Perrachon S., Milligan L. et al. (1997). A selective inverse agonist for central cannabinoid receptor
inhibits mitogenactivated protein kinase activation stimulated by insulin or insulinlike growth factor 1. Evidence for a
new model of receptor/ ligand interactions. Journal of Biological Chemistry 272, 22330–22339.
Breivogel C. S. and Childers S. R. (1998). The functional neuroanatomy of brain cannabinoid receptors. Neurobiology
of Disease 5, 417–431.
Breivogel C. S., Selley D. E. and Childers S. R. (1998). Cannabinoid receptor agonist efficacy for stimulating
[35S]GTPγS binding to rat cerebellar membranes correlates with agonist-induced decreases in GDP affinity. Journal of
Biological Chemistry 273, 16865–16873.
Cadas H., Gaillet S., Beltramo M., Venance, L. and Piomelli, D. (1996). Biosynthesis of an endogenous cannabinoid
precursor in neurons and its control by calcium and cAMP. Journal of Neuroscience 16, 3934–3942.
Cabral G. A. (2001). Marijuana and cannabinoids: effects on infections, immunity and AIDS. Journal of Cannabis
Therapeutics 1, 61–85.
Calignano A., La Rana G., Giuffrida A. and Piomelli D. (1998). Control of pain initiation by endogenous cannabinoids.
Nature 394, 277–281.
Calignano A., La Rana G., Loubet-Lescoulie P. and Piomelli D. (2000). A role for the endogenous cannabinoid system
in the peripheral control of pain initiation. Progress in Brain Research 129, 471–482.
Casanova M. L., Blazquez C., Martinez-Palacio J., Villanueva C., Fernandez-Acenero M. J., Huffman J. W., Jorcano J.
L. And Guzman M. (2003). Inhibition of skin tumor growth and angiogenesis in vivo by activation of cannabinoid
receptors. Journal of Clinical Investigation 111, 43–50.
32
Castellano C., Rossi-Arnaud C., Cestari V. and Costanzi M. (2003). Cannabinoids and memory: animal studies. Current
Drug Targets — CNS & Neurological Disorders 2, 389–402.
Chaperon F. and Thiébot M.-H. (1999). Behavioral effects of cannabinoid agents in animals. Critical Reviews in
Neurobiology 13, 243–281.
Chevaleyre V. and Castillo P. E. (2003). Heterosynaptic LTD of hippocampal GABAergic synapses: a novel role of
endocannabinoids in regulating excitability. Neuron 38, 461–472.
Childers S. R. and Deadwyler S. A. (1996). Role of cyclic AMP in the actions of cannabinoid receptors. Biochemical
Pharmacology 52, 819–827.
Colombo G., Agabio R., Fa M., Guano L., Lobina C., Loche A., Reali R. and Gessa G. L. (1998). Reduction of
voluntary ethanol intake in ethanol-preferring sP rats by the cannabinoid antagonist SR-141716. Alcohol and
Alcoholism 33, 126–130.
Colombo G., Serra S., Brunetti G., Gómez R., Melis S., Vacca G., Carai M. M., and Gessa G. L. (2002). Stimulation of
voluntary ethanol intake by cannabinoid receptor agonists in ethanolpreferring sP rats. Psychopharmacology 159, 181–
187.
Comings D. E., Muhleman D., Gade R., Johnson P., Verde R., Saucier, G., and MacMurray J. (1997). Cannabinoid
receptor gene (CNR1): association with i.v. drug use. Molecular Psychiatry 2, 161–168.
Cravatt B. F., Demarest K., Patricelli M. P., Bracey M. H., Giang D. K., Martin B. R. and Lichtman A. H. (2001).
Supersensitivity to anandamide and enhanced endogenous cannabinoid signalling in mice lacking fatty acid amide
hydrolase. Proceedings of the National Academy of Sciences of the United States America 98, 9371–9376.
Cravatt B. F., Giang D. K., Mayfield S. P., Boger D. L., Lerner R. A., and Gilula N. B. (1996). Molecular
characterization of an enzyme that degrades neuromodulatory fatty-acid amides. Nature 384, 83–87.
Cravatt B. F. and Lichtman A. H. (2003). Fatty acid amide hydrolase: an emerging therapeutic target in the
endocannabinoid system. Current Opinion in Chemical Biology 7, 469–475.
De Lago E., Fernandez-Ruiz J., Ortega-Gutierrez S., Viso A., Lopez-Rodriguez M. L. and Ramos J. A. (2002).
UCM707, a potent and selective inhibitor of endocannabinoid uptake, potentiates hypokinetic and antinociceptive
effects of anandamide. European Journal of Pharmacology 449, 99–103.
De Petrocellis L., Cascio M. G. and Di Marzo V. (2004). The endocannabinoid system: a general view and latest
additions. British Journal of Pharmacology 141, 765–774.
Derkinderen P., Toutant M., Burgaya F., Le Bert M., Siciliano J. C., De Franciscis V., Gelman M. and Girault J. A.
(1996). Regulation of a neuronal form of focal adhesion kinase by anandamide. Science 273, 1719–1722.
Devane W. A., Dysarz F. A. 3rd, Johnson M., Melvin L. S. and Howlett A. C. (1988). Determination and
characterization of a cannabinoid receptor in rat brain. Molecular Pharmacology 34, 605–613.
Devane W. A., Hanus L., Breuer A. et al. (1992). Isolation and structure of a brain constituent that binds to the
cannabinoid receptor. Science 258, 1946–1949.
De Vries T. J., Homberg J. R., Binnekade R., Raaso H. and Schoffelmeer A. N. (2003). Cannabinoid modulation of the
reinforcing and motivational properties of heroin and heroin associated cues in rats. Psychopharmacology 168, 164–169.
De Vries T. J., Shaham Y., Homberg J. R., Crombag H., Schuurman K., Dieben J., Vanderschuren L. J. and
Schoffelmeer A. N. (2001). A cannabinoid mechanism in relapse to cocaine seeking. Nature Medicine 7, 1151–1154.
Diana M. A., Levenes C., Mackie K. and Marty A. (2002). Short-term retrograde inhibition of GABAergic synaptic
currents in rat Purkinje cells is mediated by endogenous cannabinoids. Journal of Neuroscience 22, 200–208.
Diana M. A. and Marty A. (2004). Endocannabinoid-mediated shortterm synaptic plasticity: depolarization-induced
suppression of inhibition (DSI) and depolarization-induced suppression of excitation (DSE). British Journal of
Pharmacology 142, 9–19.
33
Di Marzo V., Fontana A., Cadas H., Schinelli S., Cimino G., Schwartz J. C. and Piomelli D. (1994). Formation and
inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372, 686–691.
Di Marzo V., Goparaju S. K., Wang L. et al. (2001). Leptin-regulated endocannabinoids are involved in maintaining
food intake. Nature 410, 822–825.
Di Marzo V., Melck D., Bisogno T. and De Petrocellis L. (1998). Endocannabinoids: endogenous cannabinoid receptor
ligands with neuromodulatory action. Trends in Neurosciences 21, 521–528.
Dinh T. P., Carpenter D., Leslie F. M., Freund T. F., Katona I., Sensi S. L., Kathuria S. and Piomelli D. (2002). Brain
monoglyceride lipase participating in endocannabinoid inactivation. Proceedings of the National Academy of Sciences
of the United States of America 99, 10819–10824.
Egertova M., Cravatt B. F. and Elphick M. R. (2003). Comparative analysis of fatty acid amide hydrolase and cb(1)
cannabinoid receptor expression in the mouse brain: evidence of a widespread role for fatty acid amide hydrolase in
regulation of endocannabinoid signaling. Neuroscience 119, 481–496.
Elphick M. R. and Egertova M. (2001). The neurobiology and evolution of cannabinoid signalling. Philosophical
Transactions of the Royal Society of London. Series B, Biological Sciences 356, 381–408.
Fegley D., Kathuria S., Mercier R., Li C., Goutopoulos A., Makriyannis A. and Piomelli D. (2004). Anandamide
transport is independent of fatty-acid amide hydrolase activity and is blocked by the hydrolysis-resistant inhibitor
AM1172. Proceedings of the National Academy of Sciences of the United States of America 101, 8756–8761.
Felder C.C. and Glass M. (1998).Cannabinoid receptors and their endogenous agonists. Annual Review of
Pharmacology and Toxicology 38, 179–200.
Felder C. C., Joyce K. E., Briley E. M. et al. (1998). LY320135, a novel cannabinoid CB1 receptor antagonist, unmasks
coupling of the CB1 receptor to stimulation of cAMP accumulation. Journal of Pharmacology and Experimental
Therapeutics 284, 291–297.
Fernandez-Ruiz J., Berrendero F., Hernandez M. L. and Ramos J. A. (2000). The endogenous cannabinoid system and
brain development. Trends in Neurosciences 23, 14–20.
Freund T. F., Katona I. and Piomelli D. (2003). Role of endogenous cannabinoids in synaptic signaling. Physiological
Reviews 83, 1017–1066.
Fu J., Gaetani S., Oveisi F. et al. (2003). Oleylethanolamide regulates feeding and body weight through activation of the
nuclear receptor PPAR-alpha. Nature 425, 90–93.
Galiègue S., Mary S., Marchand J., Dussossoy D., Carrière D., Carayon P., Bouaboula M., Shire D., Le Fur G. and
Casellas P. (1995). Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte
subpopulations. European Journal of Biochemistry 232, 54–61.
Galve-Roperh I., Sanchez C., Cortes M. L., del Pulgar T. G., Izquierdo M. and Guzman M.(2000). Anti-tumoral action
of cannabinoids: involvement of sustained ceramide accumulation and extracellular signal-regulated kinase activation.
Nature Medicine 6, 313–319.
Gaoni Y. and Mechoulam R. (1964). Isolation, structure and partial synthesis of an active constituent of hashish. Journal
of the American Chemical Society 86, 1646–1647.
Gardner E. L. and Vorel S. R. (1998). Cannabinoid transmission and reward-related events. Neurobiology of Disease 5,
502–533.
Gebremedhin D., Lange A. R., Campbell W. B., Hillard C. J. and Harder D. R. (1999). Cannabinoid CB1 receptor of cat
cerebral arterial muscle functions to inhibit L-type Ca2+ channel current. American Journal of Physiology 276, H2085–
2093.
Gerdeman G. L., Ronesi J. and Lovinger D. M. (2002). Postsynaptic endocannabinoid release is critical to long-term
depression in the striatum. Nature Neuroscience 5, 446–451.
34
Giuffrida A., Beltramo M. and Piomelli D. (2001). Mechanisms of endocannabinoid inactivation: biochemistry and
pharmacology. Journal of Pharmacology and Experimental Therapeutics 298, 7–14.
Giuffrida A., Parsons L. H., Kerr T. M., Rodriguez de Fonseca F., Navarro M. and Piomelli D. (1999). Dopamine
activation of endogenous cannabinoid signaling in dorsal striatum. Nature Neuroscience 2, 358–363.
Gomez R., Navarro M., Ferrer B., Trigo J.M., Bilbao A., Del Arco I., Cippitelli A., Nava F., Pomelli D., and Rodriguez
de Fonseca F. (2002). A peripheral mechanism for CB1 cannabinoid receptor-dependent modulation of feeding. Journal
of Neuroscience 22, 9612–9617.
Gulyas A. I., Cravatt B. F., Bracey M. H., Dinh T. P., Piomelli D., Boscia F. and Freund T. F. (2004). Segregation of
two endocannabinoid-hydrolyzing enzymes into pre- and postsynaptic compartments in the rat hippocampus,
cerebellum and amygdala. European Journal of Neuroscience 20, 441–458.
Guzman M. (2003). Cannabinoids: potential anticancer agents. Nature Reviews Cancer 3, 745–755.
Hajos N., Ledent C. and Freund T. F. (2001). Novel cannabinoid-sensitive receptor mediates inhibition of glutamatergic
synaptic transmission in the hippocampus. Neuroscience 106, 1–4.
Hanus L., Breuer A., Tchilibon et al. (1999). HU-308: a specific agonist for CB(2), a peripheral cannabinoid receptor.
Proceedings of the National Academy of Sciences of the United States of America 96, 14228–14233.
Hanus L., Abu-Lafi S., Fride E., Breuer A., Vogel Z., Shalev D. E., Kustanovich I. and Mechoulam R. (2001). 2arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB1 receptor. Proceedings of the National
Academy of Sciences of the United States of America 98, 3662–3665.
Herkenham M., Lynn A. B., Johnson M. R., Melvin L. S., de Costa B. R. and Rice K. C. (1991). Characterization and
localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. Journal of Neuroscience
11, 563–583.
Hiley C. R. and Ford W. R. (2004). Cannabinoid pharmacology in the cardiovascular system: potential protective
mechanisms through lipid signalling. Biological Reviews of the Cambridge Philosophical Society 79, 187–205.
Hillard C. J., Manna S., Greenberg M. J., Di Camelli R., Ross R. A., Stevenson L. A., Murphy V., Pertwee R. G. and
Campbell W. B. (1999). Synthesis and characterization of potent and selective agonists of the neuronal cannabinoid
receptor (CB1). Journal of Pharmacology and Experimental Therapeutics 289, 1427–1433.
Howlett A. C., Barth F., Bonner T. I. et al. (2002). International Union of Pharmacology. XXVII. Classification of
cannabinoid receptors. Pharmacological Reviews 54, 161–202.
Howlett A. C., Bidaut-Russell M., Devane W. A., Melvin L. S., Johnson M. R. and Herkenham M. (1990). The
cannabinoid receptor: biochemical, anatomical and behavioral characterization. Trends in Neurosciences 13, 420–423.
Hungund B. L. and Basavarajappa B. S. (2000). Are anandamide and cannabinoid receptors involved in ethanol
tolerance? A review of the evidence. Alcohol and Alcoholism 35, 126–133.
Hungund B. L., Basavarajappa B. S., Vadasz C., Kunos G., Rodriguez de Fonseca F., Colombo G., Serra S., Parsons L.
and Koob G. F. (2002). Ethanol, endocannabinoids, and the cannabinoidergic signaling system. Alcohol: Clinical and
Experimental Research 26, 565–574.
Izzo A. A., Mascolo N. and Capasso F. (2001). The gastrointestinal pharmacology of cannabinoids. Current Opinion in
Pharmacology 11, 597–603.
Jagerovic N., Hernández-Folgado L., Alkorta I. et al. (2004). Discovery of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3hexyl- 1h-1,2,4-triazole, a novel in vivo cannabinoid antagonist containing a 1,2,4-triazole motif. Journal of Medicinal
Chemistry 47, 2939–2942.
Julian M. D., Martin A. B., Cuellar B., Rodriguez de Fonseca F., Navarro M., Moratalla R. And Garcia-Segura L. M.
(2003). Neuroanatomical relationship between type 1 cannabinoid receptors and dopaminergic systems in the rat basal
ganglia. Neuroscience 119, 309–318.
35
Kathuria S., Gaetani S., Fegley D. et al. (2003). Modulation of anxiety through blockade of anandamide hydrolysis.
Nature Medicine 9, 76–81.
Kim J., Isokawa M., Ledent C. and Alger B. E. (2002). Activation of muscarinic acetylcholine receptors enhances the
release of endogenous cannabinoids in the hippocampus. Journal of Neuroscience 22, 10182–10191.
Kunos G., Batkai S., Offertaler L., Mo F., Liu J., Karcher J. and Harvey-White J. (2002). The quest for a vascular
endothelial cannabinoid receptor. Chemistry and Physics of Lipids 121, 45–56.
Ledent C., Valverde O., Cossu G. et al. (1999). Unresponsiveness to cannabinoids and reduced addictive effects of
opiates in CB1 receptor knockout mice. Science 283, 401–404.
Lichtman A. H., Leung D., Shelton C., Saghatelian A., Hardouin C., Boger D. and Cravatt B. F. (2004). Reversible
inhibitors of fatty acid amide hydrolase that promote analgesia: evidence for an unprecedented combination of potency
and selectivity. Journal of Pharmacology and Experimental Therapeutics. First published June 30, 2004,
doi.10.1124/jpet.104.069401.
Lopez-Rodriguez M. L., Viso A., Ortega-Gutierrez S., Lastres-Becker I., Gonzalez S., Fernandez-Ruiz J. and Ramos J.
A. (2001). Design, synthesis and biological evaluation of novel arachidonic acid derivatives as highly potent and
selective endocannabinoid transporter inhibitors. Journal of Medicinal Chemistry 44, 4505–4508.
Mackie K. and Hille B. (1992). Cannabinoids inhibit N-type calcium channels in neuroblastoma–glioma cells.
Proceedings of the National Academy of Sciences of the United States of America 89, 3825–3829.
Mackie K., Lai Y., Westenbroek R. and Mitchell R. (1995). Cannabinoids activate an inwardly rectifying potassium
conductance and inhibit Q-type calcium currents in AtT20 cells transfected with rat brain cannabinoid receptor. Journal
of Neuroscience 15, 6552–6561.
Maldonado R. (2002). Study of cannabinoid dependence in animals. Pharmacology and Therapeutics 95, 153–164.
Maldonado R. and Rodriguez de Fonseca F. (2002) Cannabinoid addiction: behavioral models and neural correlates.
Journal of Neuroscience 22, 3326–3321.
Manzanares J., Corchero J., Romero J., Fernandez-Ruiz J. J., Ramos J. A. and Fuentes J. A. (1999). Pharmacological
and biochemical interactions between opioids and cannabinoids. Trends in Pharmacological Sciences 20, 287–294.
Marsicano G., Goodenough S., Monory K. et al. (2003). CB1 cannabinoid receptors and on-demand defense against
excitotoxicity. Science 302, 84–88.
Marsicano G., Wotjak C. T., Azad S. C. et al. (2002). The endogenous cannabinoid system controls extinction of
aversive memories. Nature 418, 530–534.
Martin B. R. and Lichtman A. H. (1998). Cannabinoid transmission and pain perception. Neurobiology of Disease 5,
447–461.
Martin B. R., Sim-Selley L. J. and Selley D. E. (2004). Signaling pathways involved in the development of cannabinoid
tolerance. Trends in Pharmacological Sciences 25, 325–330.
Matsuda L. A. (1997). Molecular aspects of cannabinoid receptors. Critical Reviews in Neurobiology 11, 143–166.
Matsuda L. A., Bonner T. I. and Lolait S. J. (1993). Localization of cannabinoid receptor mRNA in rat brain. Journal
of Comparative Neurology 327, 535–550.
Matsuda L. A., Lolait S. J., Brownstein M. J., Young A. C. and Bonner T. I. (1990) Structure of a cannabinoid receptor
and functional expression of the cloned cDNA. Nature 346, 561–564.
Mauler F., Mittendorf J., Horvath E. and De Vry J. (2002). Characterization of the diarylether sulfonylester (-)-I-3- (2hydroxymethylindanyl-4-oxy)phenyl-4,4,4-trifluoro-1-sulfonate (BAY 38-7271) as a potent cannabinoid receptor
agonist with neuroprotective properties. Journal of Pharmacology and Experimental Therapeutics 302, 359–368.
Mechoulam R. (1970). Marihuana chemistry. Science 168, 1159–1166.
36
Mechoulam R., Ben-Shabat S., Hanus L. et al. (1995). Identification of an endogenous 2-monoglyceride, present in
canine gut, that binds to cannabinoid receptors. Biochemical Pharmacology 50, 83–90.
Mechoulam R. and Parker, L. (2003). Cannabis and alcohol — a close friendship. Trends in Pharmacological Sciences
24, 266–268.
Molina-Holgado F., Lledó A. and Guaza C. (1997) Anandamide suppresses nitric oxide and TNF-α responses to
Theiler’s virus or endotoxin in astrocytes. Neuroreport 8, 1929–1933.
Munro S., Thomas K. L. and Abu-Shaar M. (1993). Molecular characterization of a peripheral receptor for
cannabinoids. Nature 365, 61–65.
Navarro M., Carrera M. R., Fratta W. et al. (2001). Functional interaction between opioid and cannabinoid receptors in
drug selfadministration. Journal of Neuroscience 21, 5344–5350.
Navarro M., Hernandez E., Munoz R. M., del Arco I., Villanua M. A., Carrera M. R. and Rodríguez de Fonseca F.
(1997) Acute administration of the CB1 cannabinoid receptor antagonist SR 141716A induces anxiety-like responses in
the rat. Neuroreport 8, 491–496.
Oka S., Tsuchie A., Tokumura A., Muramatsu M., Suhara Y., Takayama H., Waku K. and Sugiura T. (2003). Etherlinked analogue of 2-arachidonoylglycerol (noladin ether) was not detected in the brains of various mammalian species.
Journal of Neurochemistry 85, 1374–1381.
Okamoto Y., Morishita J., Tsuboi K., Tonai T. and Ueda N. (2004). Molecular characterization of a phospholipase D
generating anandamide and its congeners. Journal of Biological Chemistry 279, 5298–5305.
Pacheco M., Childers S. R., Arnold R., Casiano F. and Ward S. J. (1991). Aminoalkylindoles: actions on specific Gprotein-linked receptors. Journal of Pharmacology and Experimental Therapeutics 257, 170–183.
Panikashvili D., Simeonidou C., Ben-Shabat S., Hanus L., Breuer A., Mechoulam R. and Shohami E. (2001). An
endogenous cannabinoid (2-AG) is neuroprotective after brain injury. Nature 413, 527–531.
Pertwee R. G. (2001). Cannabinoid receptors and pain. Progress in Neurobiology 63, 569–611.
Piomelli D. (2003). The molecular logic of endocannabinoid signalling. Nature Reviews Neuroscience 4, 873–884.
Piomelli D., Giuffrida A., Calignano A. and Rodríguez de Fonseca F. (2000). The endocannabinoid system as a target
for therapeutic drugs. Trends in Pharmacological Sciences 21, 218–224.
Porter A. C., Sauer J. M., Knierman M. D. et al. (2002). Characterization of a novel endocannabinoid, virodhamine,
with antagonist activity at the CB1 receptor. Journal of Pharmacology and Experimental Therapeutics 301, 1020–1024.
Randall M. D., Harris D., Kendall D. A. and Ralevic V. (2002). Cardiovascular effects of cannabinoids. Pharmacology
and Therapeutics 95, 191–202.
Rimondini R., Arlinde C., Sommer W. and Heilig M. (2002). Long-lasting increase in voluntary ethanol consumption
and transcriptional regulation in the rat brain after intermittent exposure to alcohol. The FASEB Journal 16, 27–35.
Rinaldi-Carmona M., Barth F., Congy C. et al. (2004). SR147778, a new potent and selective antagonist of the CB1
cannabinoid receptor. Biochemical and pharmacological characterization. Journal of Pharmacology and Experimental
Therapeutics 310, 905–914.
Rinaldi-Carmona M., Barth F., Héaulme M. et al. (1994). SR141716A, a potent and selective antagonist of the brain
cannabinoid receptor. FEBS Letters 350, 240–244.
Rinaldi-Carmona M., Barth F., Millan J. et al. (1998). SR 144528, the first potent and selective antagonist of the CB2
cannabinoid receptor. Journal of Pharmacology and Experimental Therapeutics 284, 644–650.
Robbe D., Kopf M., Remaury A., Bockaert J. and Manzoni O. J. (2002). Endogenous cannabinoids mediate long-term
synaptic depression in the nucleus accumbens. Proceedings of the National Academy of Sciences of the United States of
America 99, 8384–8348.
37
Rodríguez de Fonseca F., Carrera M. R., Navarro M., Koob G. F., and Weiss F. (1997). Activation of corticotropinreleasing factor in the limbic system during cannabinoid withdrawal. Science 276, 2050–2054.
Rodríguez de Fonseca F., Del Arco I., Martin-Calderon J. L., Gorriti M. A. and Navarro M. (1998). Role of the
endogenous cannabinoid system in the regulation of motor activity. Neurobiology of Disease 5, 483–501.
Rodríguez de Fonseca F., Navarro M., Gomez R., et al. (2001). An anorexic lipid mediator regulated by feeding. Nature
414, 209–212.
Rodríguez de Fonseca F., Roberts A. J., Bilbao A., Koob, G. F. and Navarro, M. (1999). Cannabinoid receptor
antagonist SR141716A decreases operant ethanol self administration in rats exposed to ethanol-vapor chambers.
Zhongguo Yao Li Xue Bao 20, 1109–1114.
Rodríguez de Fonseca F., Rubio P., Menzaghi F., Merlo-Pich E., Rivier J., Koob G. F. and Navarro M. (1996).
Corticotropinreleasing factor (CRF) antagonist [D-Phe12,Nle21,38,C alpha MeLeu37]CRF attenuates the acute actions
of the highly potent cannabinoid receptor agonist HU-210 on defensive–withdrawal behavior in rats. Journal of
Pharmacology and Experimental Therapeutics 276, 56–64.
Rueda D., Navarro B., Martinez-Serrano A., Guzman M., Galve-Roperh I. (2002). The endocannabinoid anandamide
inhibits neuronal progenitor cell differentiation through attenuation of the Rap1/B-Raf/ERK pathway. Journal of
Biological Chemistry 277, 46645–46650.
Sanchez C., de Ceballos M. L., Del Pulgar, T. G. et al. (2001). Inhibition of glioma growth in vivo by selective
activation of the CB(2) cannabinoid receptor. Cancer Research 61,5784–5789.
Sanchis-Segura C., Cline B. H., Marsicano G., Lutz B. and Spanagel R. (2004). Reduced sensitivity to reward in CB1
knockout mice. Psychopharmacology 2004 Apr 9.
Schlicker E. and Kathmann M. (2001). Modulation of transmitter release via presynaptic cannabinoid receptor. Trends
in Pharmacological Sciences 22, 565–572.
Schmidt L. G., Samochowiec J., Finckh U., Fiszer-Piosik E., Horodnicki J., Wendel B., Rommelspacher H. and Hoehe
M. R. (2002). Association of a CB1 cannabinoid receptor gene (CNR1) polymorphism with severe alcohol dependence.
Drug and Alcohol Dependence 65, 221–224.
Sipe J. C., Chiang K., Gerber A. L., Beutler E. and Cravatt B. F. (2002). A missense mutation in human fatty acid amide
hydrolase associated with problem drug use. Proceedings of the National Academy of Sciences of the United States of
America 99, 8394–8399.
Smith P. F. (2004). Medicinal cannabis extracts for the treatment of multiple sclerosis. Current Opinion in
Investigational Drugs 5, 727–730.
Stella, N. and Piomelli, D. (2001). Receptor-dependent formation of endogenous cannabinoids in cortical neurons.
European Journal of Pharmacology 425, 189–196.
Stella N., Schweitzer P. and Piomelli D. (1997). A second endogenous cannabinoid that modulates long-term
potentiation. Nature 388, 773–778.
Straiker A., Stella N., Piomelli D., Mackie K., Karten H. J. And Maguire G. (1999). Cannabinoid CB1 receptors and
ligands in vertebrate retina: localization and function of an endogenous signaling system. Proceedings of the National
Academy of Sciences of the United States of America 96, 14565–14570.
Sugiura T., Kondo S., Sukagawa A., Nakane S., Shinoda A., Itoh K., Yamashita A. and Waku K. (1995). 2Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochemistry Biophysics Research
Communications 215, 89–97.
Tanda G. and Goldberg S. R. (2003). Cannabinoids: reward, dependence, and underlying neurochemical mechanisms —
a review of recent preclinical data. Psychopharmacology 169, 115–134.
Tarzia G., Duranti A., Tontini A., Piersanti G., Mor M., Rivara S., Plazzi P. V., Park C., Kathuria S. and Piomelli, D.
(2003a). Design, synthesis, and structure-activity relationships of alkylcarbamic acid aryl esters, a new class of fatty
acid amide hydrolase inhibitors. Journal of Medicinal Chemistry 46, 2352–2360.
38
Tarzia G., Duranti A., Tontini A., Spadoni G., Mor M., Rivara S., Plazzi P. V., Kathuria S. and Piomelli D. (2003b).
Synthesis and structure–activity relationships of a series of pyrrole cannabinoid receptor agonists. Bioorganic and
Medicinal Chemistry 11, 3965–3973.
Terranova J. P., Storme J. J., Lafon N., Perio A., Rinaldi-Carmona M., Le Fur G. and Soubrié P. (1996). Improvement
of memory in rodents by the selective CB1 cannabinoid receptor antagonist, SR 141716. Psychopharmacology 126,
165–172.
Tsou K., Mackie K., Sanudo-Pena M. C. and Walker J. M. (1999). Cannabinoid CB1 receptors are localized primarily
on cholecystokinin-containing GABAergic interneurons in the rat hippocampal formation. Neuroscience 93, 969–975.
Twitchell W., Brown S. and Mackie K. (1997). Cannabinoids inhibit N- and P/Q-type calcium channels in cultured rat
hippocampal neurons. Journal of Neurophysiology 78, 43–50.
Varma N., Carlson G. C., Ledent C. and Alger B. E. (2001). Metabotropic glutamate receptors drive the
endocannabinoid system in hippocampus. Journal of Neuroscience 21, RC188.
Wagner J. A., Varga K., Ellis E. F., Rzigalinski B. A., Martin B. R. and Kunos G. (1997). Activation of peripheral CB1
cannabinoid receptors in haemorrhagic shock. Nature 390, 518–521.
Wilson R. I., Kunos G. and Nicoll R. A. (2001). Presynaptic specificity of endocannabinoid transnission in the
hippocampus. Neuron 31, 453–462.
Wilson R. I. and Nicoll R. A. (2001). Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses.
Nature 410, 588–592.
Wilson, R. I. and Nicoll, R. A. (2002). Endocannabinoid signaling in the brain. Science 296, 678–682.
Witting A., Walter L., Wacker J., Moller T. and Stella N. (2004). P2X7 receptors control 2-arachidonoylglycerol
production by microglial cells. Proceedings of the National Academy of Sciences of the United States of America 101,
3214–3219.
Zygmunt P. M., Petersson J., Andersson D. A., Chuang H., Sörgärd M., Di Marzo V., Julius D. and Högestätt E. D.
(1999). Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature 400, 452–457.
39
III
40
III. Cannabinoid CB1 receptor antagonism reduces
conditioned reinstatement of ethanol-seeking
behaviour in rats
Abstract
The endocannabinoid system is involved in a variety of effects of drugs of misuse, and blockade of
the cannabinoid CB1 receptor by selective antagonists elicits marked reductions in opioid and
alcohol self-administration. The present study was designed to extend our knowledge of the role of
the cannabinoid CB1 receptor in the modulation of alcohol misuse vulnerability in rats.
Accordingly, using non-selected Wistar rats and genetically selected Marchigian Sardinian alcoholpreferring (msP) rats, we investigated the effect of the CB1 antagonist SR141716A on operant
alcohol self-administration and on reinstatement of alcohol-seeking behaviour by environmental
conditioning factors. In addition, in situ hybridization studies in both strains were performed to
measure cannabinoid CB1 receptor mRNA in different brain areas of these animals. Results showed
that intraperitoneal administration of SR141716A (0.3, 1.0 and 3.0 mg ⁄ kg) markedly inhibits
ethanol self-administration and conditioned reinstatement of ethanol-seeking behaviour in both
strains of rats. ED50 analysis showed significantly higher sensitivity (P < 0.05) to the effect of
SR141716A in msP rats than in heterogeneous Wistar rats. In situ hybridization studies revealed
that, compared with Wistar rats, msP animals have consistently greater cannabinoid CB1 receptor
mRNA expression in a number of brain areas, including the frontoparietal cortex, caudate-putamen
and hippocampus (CA1 and dentate gyrus areas). In conclusion, we provide clear evidence that
blockade of CB1 receptors reduces both ethanol self-administration and conditioned reinstatement
of alcohol-seeking behaviour in rats. In addition, current pharmacological and neuroanatomical data
suggest that an altered function of the CB1 receptor system exists between genetically selected
alcohol-preferring msP rats and a heterogeneous animal population.
Introduction
Converging research lines have established a role for both the endogenous cannabinoid anandamide
(AEA) and the cannabinoid CB1 receptor in ethanol dependence (Hungund & Basavarajappa, 2000;
Gonzalez et al., 2002; Hungund et al., 2002). Whereas administration of cannabinoid CB1 receptor
agonists promotes ethanol intake (Colombo et al., 2002), administration of a cannabinoid CB1
receptor antagonist decreases ethanol intake and self-administration (Arnone et al., 1997; Freedland
et al., 2001), especially in animals with a history of ethanol dependence (Rodrıguez de Fonseca et
al., 1999) or in alcohol-preferring rat lines (Colombo et al., 1998; Gessa et al., 2005). Molecular
studies have shown that chronic ethanol administration is associated with an increased formation of
both AEA and its membrane precursor N-arachidonylphosphatidylethanolamine (Basavarajappa &
Hungund, 1999; Ortiz et al., 2004). Chronic alcohol exposure also results in stimulation of 2arachidonylglycerol, a second endocannabinoid (Basavarajappa et al., 2000). Animal studies have
revealed that chronic exposure to ethanol down-regulated cannabinoid CB1 receptors in the brain
(Basavarajappa et al., 1998). Finally, a recent gene screening study has identified the cannabinoid
CB1 receptor as one of the genes whose expression is permanently affected by serial cycles of
ethanol dependence and withdrawal (Rimondini et al., 2002). These data indicate a role for the
endogenous cannabinoid system as a relevant contributor to alcoholism in addition to other major
transmission systems (Weiss & Porrino, 2002). Human genetic studies support this experimental
hypothesis as a link between clinical forms of alcoholism and polymorphisms and ⁄ or mutations of
41
the genes encoding either the cannabinoid CB1 receptor (Comings et al., 1997; Schmidt et al., 2002)
or the fatty acid amidohydrolase (FAAH) (Sipe et al., 2002), the enzyme responsible for AEA
inactivation (Cravatt et al., 1996), have been described. Despite extensive information on the effects
of chronic exposure to alcohol, there is a lack of information on the effects of pharmacological
manipulation of the endogenous cannabinoid system on ethanol self-administration and relapse.
Moreover, we do not know whether a line of animals selected for high alcohol drinking displays
alterations in the functionality of the endogenous cannabinoid system when compared with a normal
rat population. In the present study we addressed this problem by analysing the effects of
cannabinoid receptor blockade on alcohol self-administration and cue-induced relapse in normal
Wistar rats and in the genetically selected Marchigian Sardinian alcohol-preferring (msP) rats.
Using quantitative in situ hybridization histochemistry we also studied the expression of
cannabinoid CB1 receptor mRNA on both strains of rats.
Materials and methods
Animals
One hundred and four male Wistar rats and 32 genetically selected msP rats were used. The msP
rats were bred in the Department of Pharmacological Sciences and Experimental Medicine of the
University of Camerino (Italy) for more than 42 generations from Sardinian alcohol-preferring rats
(sP) of 13th generation provided by the Department of Neurosciences of the University of Cagliari
(Agabio et al., 1996; Lobina et al., 1997; Colombo et al., 1998). All rats, weighing 175–225 g, were
housed in groups of two in a temperature- and humidity-controlled vivarium on a reverse 12-h
light/dark cycle (lights on 18:00–06:00 h). All training and experimental sessions were conducted
during the dark phase of the cycle. Standard National Institutes of Health laboratory rat chow and
water were available ad libitum in the home cage, except as noted in ‘Behavioural training and
testing procedures’. All procedures were conducted in adherence to the European Community
Council Directive and National Institutes of Health Guidelines for Care and Use of Laboratory
Animals.
Drugs
SR141716A (Sanofi-Synthelabo, Montpellier, France) was suspended with 2–3 drops of Tween 80
in saline as vehicle. It was administered intraperitoneally at doses of 0.3, 1 and 3 mg ⁄ kg.
Locomotion studies
Twenty-four male Wistar rats were used for this experiment. Photocell cages of 40 · 35 · 35 cm
were used to measure locomotor behaviour. Animals were placed in the cages for 6 h on the day
before testing for habituation. The following day the animals were placed in the cages again to
reacclimatize for a period of 5 min before drug injection. Animals (eight per group) were then
injected with either vehicle or SR141716A (0.3 or 3 mg ⁄ kg). The number of beam interruptions
were was during 10-min intervals, over a period of 120 min.
Food reinforcement
A separate group of eight Wistar rats were trained to lever-press for food (45-mg food pellet; BioServe, Frenchtown, NJ, USA) on a fixed ratio 1 schedule of reinforcement, and food restricted to 13
g chow per rat per day. Once stable responding was achieved, animals were trained to acquire a
fixed ratio 5, time out 2 min, schedule of reinforcement. They were kept on food restriction for the
rest of the experiment. When a stable baseline was achieved, they were used to study the effects of
acute administration of SR141716A (0, 0.3, 1 and 3 mg⁄ kg, i.p.). Animals received the cannabinoid
antagonist in a Latin square design fashion 30 min before the test session. Baseline sessions were
interposed between testing sessions to assess carryover effects.
42
Operant training for liquid reinforcers
Training and testing were conducted in standard operant chambers located in sound-attenuating,
ventilated environmental cubicles. Each chamber was equipped with a drinking reservoir (volume
capacity 0.10 ml) positioned 4 cm above the grid floor in the centre of the front panel of the
chamber, and two retractable levers were located 3 cm to the right and left of the drinking
receptacle. Auditory and visual stimuli were presented via a speaker and a light located on the front
panel. A microcomputer controlled the delivery of fluids, presentation of auditory and visual
stimuli, and recording of the behavioural data. Rats were trained to self-administer 10% (v/v)
ethanol (48 rats), 0.2% (w/v) saccharin (eight rats), 10% sucrose (eight rats) or water (eight rats) in
30-min daily sessions on a fixed ratio 1 schedule of reinforcement, where each response resulted in
delivery of 0.1 ml of fluid, as previously described (Weiss et al., 1993). Briefly, for the first 3 days
of training, water availability in the home cage was restricted to 2 h per day in order to facilitate
acquisition of operant responding for a liquid reinforcer. During this time, lever pressing reinforced
by 0.2% (w ⁄ v) saccharin or 10% sucrose solution was established. At this point water was made
freely available, and saccharin ⁄ sucrose self-administration training continued until animals reached
stable baseline responding. A separate subset of rats from the saccharin-trained group were then
trained to self-administer ethanol by using a modification of the sucrose-fading procedure (Samson,
1986) that used saccharin instead of sucrose (Weiss et al., 1993). During the first 6 days of this
ethanol initiation phase a 5% (w/v) ethanol solution containing 0.2% saccharin (w/v) was available
to the rats. Starting on day 7, the concentration of ethanol was gradually increased from 5.0% to
8.0% and finally to 10.0% (w/v), whereas the concentration of saccharin was correspondingly
decreased to 0%. At the beginning of the saccharin-fading procedure a second, inactive lever was
introduced. During all training and testing phases responses at this lever were recorded as a measure
of non-specific behavioural activation, but they had no programmed consequences.
Saccharin and sucrose self-administration: effect of SR141716A
Following completion of the saccharin or sucrose training Wistar rats were used to study the effects
of the CB1 receptor antagonist SR141716A (0.0, 0.3, 1.0 and 3.0 mg ⁄ kg) given 30 min prior to a
self-administration session. The experiment was conducted every fourth day using a Latin square
counterbalanced design. Responding at the inactive lever was recorded throughout the experiment to
monitor non-specific behavioural effects.
Ethanol self-administration: effect of SR141716A
Following completion of the saccharin fading procedure Wistar and msP rats (n= 8 per group) were
trained in sessions of 30 min per day to lever-press for 10% ethanol (0.1 ml per response) until
stable baseline of responding was reached. We studied the effect of the CB1 receptor antagonist
SR141716A (0.0, 0.3, 1.0 and 3.0 mg ⁄ kg) given 30 min prior to a self-administration session.
Experiments were conducted every fourth day using a Latin square counterbalanced design.
Responding at the inactive lever was recorded throughout the experiment to monitor non-specific
behavioural effects.
Reinstatement of ethanol-seeking behaviour: effect of SR141716A
Conditioning phase
At completion of the fading procedure, in 30-min daily sessions, animals were trained to
discriminate between 10% ethanol and water. Beginning with self-administration training at the
10% ethanol concentration, discriminative stimuli (SD) predictive of ethanol vs. water availability
were presented during the ethanol and water self-administration sessions, respectively. The
discriminative stimulus for ethanol consisted of the odour of an orange extract (S+) whereas water
availability (i.e. non-reward) was signalled by an anise extract (S–). The olfactory stimuli were
generated by placing 6–8 drops of the respective extract into the bedding of the operant chamber. In
43
addition, each lever-press resulting in delivery of ethanol was paired with illumination of the
chamber’s house light for 5 s (CS+). The corresponding cue during water sessions was a 5-s tone (70
dB) (CS–). Concurrently with the presentation of these stimuli, a 5-s time-out period was in effect,
during which responses were recorded but not reinforced. The olfactory stimuli serving as S+ or S–
for ethanol or water availability were introduced 1 min before extension of the levers and remained
present throughout the 30-min sessions. The bedding of the chamber was changed and bedding trays
were cleaned between sessions. During the first 3 days of the conditioning phase the rats were given
ethanol sessions only. Subsequently ethanol and water sessions were conducted in random order
across training days, with the constraint that all rats received a total of ten ethanol and ten water
sessions.
Extinction phase
After the last conditioning day, rats were subjected to 30-min extinction sessions for 15 consecutive
days. During this phase, sessions began by extension of the levers without presentation of the
discriminative stimuli. Responses at the lever activated the delivery mechanism but did not result in
the delivery of liquids or the presentation of the response-contingent cues (house light or tone).
Reinstatement testing
Reinstatement tests began the day after the last extinction session. This test lasted 30 min under
conditions identical to those during the conditioning phase, except that alcohol and water were not
made available. Half the animals were tested under the S+/CS+ condition on day 1 and under the S–
/CS– condition on day 2. The order of cue presentation was inverted for the remaining rats.
Reinstatement experiments were conducted every fourth day (on days 6, 10, 14) and SR141716A
was administered 30 min prior to the sessions. Responding at the inactive lever was constantly
recorded to monitor possible non-specific behavioural effects.
CB1 receptor mRNA in situ hybridization
Ethanol-naϊve msP and Wistar rats as well as 18-day ethanol experienced msP rats (two bottles, free
choice between 10% ethanol and water, unlimited access) were used (n = 8 per group). Rats were
killed between 13:00 and 16:00 h by decapitation, brains were quickly removed, snap frozen in -40
°C isopentane and stored at -70°C until use. Ten-micrometer brain sections were taken at Bregma
levels (i) +2.5 to +1.7 mm, (ii) -0.3 to -0.4 mm and (iii) -2.3 to -3.3 mm according to the atlas of
Paxinos & Watson (1997). The CB1 receptor riboprobe was generated by PCR and corresponds to
nucleotides 1232–1272 of the cDNA sequence [NM_012784 (Matsuda et al., 1990)]. Probe RNA
synthesis and in situ hybridization were as described in detail by Caberlotto et al. (2004). The
hybridized sections were exposed to Fuji BAS-5000 Phosphorimager plates. Phosphorimagergenerated digital images were analysed using AIS Image Analysis Software (Imaging Research Inc.,
St. Catharines, Ontario, Canada). Regions of interest were defined by anatomical landmarks as
described in Paxinos & Watson (1997). Based on the known radioactivity in the 14C standards,
image values were converted to values of nCi/g. Slides were then exposed for 1 month to Kodak
BioMax MR film (Eastman Kodak Company, UK).
Statistics
Statistical significance of behavioural studies was assessed by multifactorial ANOVA, using
phenotype (alcohol preference), treatment (vehicle ⁄ SR141716) and time points as the main factors.
Following a significant F-value, post hoc analysis (Student–Newman–Keuls) was performed. ED50
was calculated by nonlinear regression analysis using PRISM software. Cannabinoid CB1 receptor
in situ hybridization data from the 13 brain regions selected were analysed by individual t-tests
followed by Holm’s ranked Bonferroni’s correction for multiple comparisons.
44
Results
I. Ethanol self-administration: effect of SR141716A
Acquisition of ethanol self-administration was quicker in msP rats than in Wistar rats (phenotype ·
day effect, F1,23= 10.4, P < 0.01; data not shown). When injected with SR141716A 30 min prior to
the session, both strains exhibited a dose-dependent reduction in ethanol self-administration
(treatment effect, F3,60= 10.6, P < 0.001; Figure 1).
Figure 1. Administration of the cannabinoid CB1 receptor antagonist SR141716A reduces ethanol self-administration
in Wistar and msP rats, with higher potency in this alcohol-preferring strain. *P < 0.05, vs. vehicle-treated animals; n= 8
animals per group.
We observed clear differences in this response between phenotypes (F1,60= 5.2, P < 0.05). Post hoc
analysis revealed that SR141716A was more effective in msP rats than in Wistar rats, with a
significant reduction of responses for ethanol starting at doses of 1.0 mg ⁄ kg in the former, whereas
in Wistar rats the effect was significant only at the highest dose of 3.0 mg ⁄ kg (simple effects of
genotype at doses of 1.0 and 3.0 mg ⁄ kg, F1,60 =4.1, P < 0.05). The different sensitivity to the effect
of SR141716A was confirmed by the different ED50 measured in the two rat lines, 0.509 ± 0.25 for
msP and 1.033 ± 0.12 for Wistar rats. The number of responses at the inactive lever was very low
throughout the experiment and was not influenced by treatment with SR141716A (Wistar, vehicle:
0.12 ± 0.12; SR 0.3 mg ⁄ kg: 0.12 ± 0.12; SR 1 mg ⁄ kg: 0.5 ± 0.03; SR 3 mg ⁄ kg: 0.12 ± 0.12; msP,
vehicle: 2.2 ± 1.1; SR 0.3 mg ⁄ kg: 2 ± 0.8; SR 1 mg ⁄ kg: 4 ± 3; SR 3 mg ⁄ kg: 1.5 ± 0.8; F1,23= 2.81,
45
n.s.). In order to assess potential sedative effects derived of SR141716A administration, both
locomotor activity and operant behavior tests were performed in male Wistar rats.
II. Locomotor activity: effects of SR141716A
Analysis of locomotor activity patterns in male Wistar rats habituated to the open field revealed that
the administration of the cannabinoid receptor antagonist SR141716A (0, 0.3 and 3 mg ⁄ kg, i.p.) did
not modify either total locomotor activity (F2,20= 1.1, n.s.) or global activity pattern, i.e. timeassociated decrease in locomotor activity when measured for at least 2 h (Figure 2A).
III. Food, saccharin and sucrose reinforcement: effects of SR141716A
The acute administration of SR141716A (0, 0.3, 1 and 3 mg ⁄ kg) resulted in reinforcer-dependent
effects of this cannabinoid receptor antagonist on three different operant behaviour responses.
Whereas SR141716A did not affect operant responses for food (fixed ratio 5, time out 2 min, F3,30=
0.9, n.s.; Figure 2B), it decreased operant responses for a solution containing 0.2% saccharin at 3
mg ⁄ kg (F3,28= 14.1, P < 0.005; Fig. 2C) and for a solution of 10% sucrose (at doses of 1 and 3 mg ⁄
kg, F3,30 =12.7, P < 0.005; Fig. 2D). These results suggest that the effects of cannabinoid receptor
blockade on operant responses are not dependent on sedative effects or hypolocomotion, but on
selective deficits in reward evaluation.
IV. Reinstatement of ethanol-seeking behaviour: effect of SR141716A
MsP rats learned to discriminate water from ethanol better than Wistar rats (genotype · liquid · day
effect, F9,414 = 32, P < 0.001; Figure 3A). During the extinction period, we also observed phenotype
differences (F1,23 =27.5, P < 0.001; Figure 3B), revealing that extinction was achieved quicker in
Wistar than in msP rats (phenotype · time effect F1,322 = 1.9, P < 0.003). On the first reinstatement
test, under the S+/CS+ stimulus condition, responses at the active lever were significantly increased
over the last extinction day in both Wistar (F1,11 = 28.58, P < 0.01) and msP (F1,11 =13.64, P < 0.01)
rats (Figure 3C). In addition, ANOVA revealed significantly higher levels of responding in msP
than in Wistar rats (F1,22 =4.3, P < 0.05). In contrast, when tested under S–/CS– conditions rats failed
to increase responding over extinction and no differences between the two rat lines were observed
(Figure 3C). When the cannabinoid receptor antagonist was injected 30 min prior to cue
presentation, we observed a significant and dose-dependent reduction in lever pressings in both rat
strains (treatment effect, F3,64 = 14.6, P < 0.001; Figure 3D). Post hoc analysis revealed that msP
rats responded with a reduction in lever presses to ethanol associated cues at lower doses of
SR141716A (i.e. 1 mg/kg) than Wistar rats. Again, detailed analysis of the dose–response in each
strain revealed different ED50 for the effect of SR141716A (0.80 ± 0.13 in msP and 1.26 ± 0.15 in
Wistar rats, F1,58 = 5.96, P < 0.02). Responses at the inactive lever were very low during both the
discrimination and the extinction phases. Similarly, during the reinstatement phase, responding at
this level was almost undetectable and was not influenced by treatment with SR141716A.
46
Figure 2. (A) Acute administration of the cannabinoid CB1 receptor antagonist SR141716A to field-habituated rats
does not affect locomotor actitvity or (B) food reinforcement in food-restricted animals. However, SR141716A reduced
operant responses for both a solution of 0.2% saccharin at 3 mg ⁄ kg (C) and for a solution containing 10% sucrose at 1
and 3 mg ⁄ kg dose (D), suggesting a reinforcer-dependent modulation of reward by cannabinoid CB1 receptors. *P <
0.05, vs. vehicle-treated animals; n= 8–9 animals per group.
47
Figure 3. (A) Differences in the discrimination phase, (B) extinction and (C) cue-induced relapse between Wistar and
alcohol-preferring msP rats. *P < 0.05, msP vs. Wistar rats. (D) Administration of the cannabinoid CB1 receptor
antagonist SR141716A reduces cue-induced relapse to ethanol self-administration in Wistar and msP rats, with a higher
potency in this alcohol-preferring strain. *P < 0.05, vs. vehicle-treated animals; n= 8 animals per group. #P < 0.05, msP
vs. Wistar rats in S+ ⁄ CS+ condition.
48
V. CB1 mRNA levels in msP and Wistar rats
CB1 mRNA expression was assessed by in situ hybridization in a number of brain regions (Figure
4).
Figure 4. Schematic representations of the areas sampled for mRNA measurements (grey and black overlay) in a
coronal section from different Bregma levels according to the atlas of Paxinos and Watson (1997). Left: bright-field
photomicrographs from in situ hybridization film autoradiograms showing CB1 receptor expression levels in brain
regions of respective Bregma levels in msP rats. Frontal cortex (fr cx); cingulate cortex (cg cx); frontoparietal cortex (fp
cx); caudate putamen (CP) in Bregma level +2.2 mm (CP, +2.2), Bregma level -0.4 mm (CP, -0.4), mediolateral part of
CP in Bregma level -0.4 mm (ml CP, -0.4); central amygdaloid nucleus (CeA); basolateral amygdaloid nucleus (BLA);
dorsal hippocampal subregions (CA: cornus ammon areas, CA1 to CA4; dentate gyrus, DG). Scale bar, 1 mm. For
details of treatment, see Materials and methods.
Strong differences in CB1 mRNA levels between ethanol-naϊve msP and Wistar rats were found in
the rostral part of the caudate putamen (Cpu), the frontoparietal cortex (fpCx), and the CA1 and
CA4 subregions of the hippocampus (13– 26% lower in Wistar rats) as revealed by a Holm ⁄
Bonferroni corrected t-test (rostral Cpu: F1,14 = 24.0, corrected P < 0.001; fpCx: F1,13 = 19.6,
corrected P < 0.01; CA1: F1,14 = 18.9, corrected P < 0.01; CA4: F1,14 = 7.8, corrected P < 0.05;
Figure 5A).
In msP rats voluntary ethanol consumption over a period of 18 days (ethanol intake progressively
increased over this period and ranged from 4.93 ± 0.39 g ⁄ kg on day 1 to 8.36 ± 0.36 g ⁄ kg on day
18) resulted in a trend towards a general down-regulation of CB1 expression in the forebrain regions
analysed; however, this effect only reached significance in the rostral part of the caudate putamen
and in the pituitary (Pit) (about 27 and 39% down-regulation by ethanol, respectively), as
demonstrated by a Holm ⁄ Bonferroni corrected t-test (rostral Cpu: F1,14=18.0, corrected P < 0.01;
Pit: F1,14 = 11.0, corrected P < 0.05; Figure 5B, Table 1).
49
Figure 5. Relative CB1 receptor expression levels in different brain regions of ethanol-naϊve msP and Wistar rats (A),
and of the same ethanol- naϊve msP and 10-days ethanol-experienced msP rats (B). Data are given in percentage of msP
rats as means ± SEM, n = 7–8. The statistical analysis was performed by a t-test followed by Holm’s ranked
Bonferroni’s correction (*P < 0.05; **P < 0.01, vs. msP rats). Frontal cortex (fr cx); cingulate cortex (cg cx);
frontoparietal cortex (fp cx); caudate putamen (CP) in Bregma level +2.2 mm (CP, +2.2), Bregma level -0.4 mm (CP, 0.4), mediolateral part of CP in Bregma level -0.4 mm (ml CP, -0.4); central amygdaloid nucleus (CeA); basolateral
amygdaloid nucleus (BLA); dorsal hippocampal subregions (CA: cornus ammon areas, CA1 to CA4; dentate gyrus,
DG); pituitary, anterior (PIT (ant)); for details see Materials and methods.
Reached significance in the rostral part of the caudate putamen and in the pituitary (Pit) (about 27
and 39% down-regulation by ethanol, respectively), as demonstrated by a Holm ⁄ Bonferroni
corrected t-test (rostral Cpu: F1,14 = 18.0, corrected P < 0.01; Pit: F1,14 =11.0, corrected P < 0.05;
Figure 5B, Table 1).
50
Table 1. Data are expressed as density values (nCi ⁄ g, means ± SEM; n = 7–8). Frontal cortex (fr cx); cingulate cortex
(cg cx); frontoparietal cortex (fp cx); caudate putamen (CP) in Bregma level +2.2 mm (CP, +2.2), Bregma level -0.4
mm (CP, -0.4), mediolateral part of CP in Bregma level -0.4 mm (ml CP, -0.4); central amygdaloid nucleus (CeA);
basolateral amygdaloid nucleus (BLA); dorsal hippocampal subregions (CA: cornus ammon areas, CA1 to CA4; dentate
gyrus, DG); pituitary, anterior (PIT (ant)); for details see Material and methods.
Discussion
Two main findings emerge from the present study. First, the endogenous cannabinoid system,
specifically the cannabinoid CB1 receptor, is a relevant contributor to the neuroadaptions associated
with cue-induced relapse to ethanol self-administration behaviour in rats. Second, at least in a strain
of rats bred for its ethanol preference, msP rats, there is increased cannabinoid CB1 receptor mRNA
expression in brain areas relevant for the processing of reward and reward-associated behaviors.
Ethanol drinking appears to reduce CB1 gene expression, and this effect is most pronounced in the
rostral part of the caudate putamen, the region with the strongest difference in expression between
msP and Wistar rats. These changes are reversed by ethanol experience. These two findings extend
the potential use of cannabinoid receptor antagonist-based therapy for relapse to addictive drugs, to
ethanol, one of the two main drugs of misuse, as previously described for cocaine and heroin (De
Vries et al., 2001, 2003; Navarro et al., 2001; Fattore et al., 2003).
A possible concern in the interpretation of the present results is that SR141716A-induced inhibition
of ethanol responding could, at least in part, reflect non-specific behavioural inhibition caused by
the drug. Several factors, however, argue against this possibility. First, we observed that
SR141716A does not suppress locomotor activity in field-habituated animals, nor does it affect
operant responses for food under fixed ratio 5, time out 2 min, experimental conditions that
demanded a sustained attentive ⁄ motivational drive in the tested animals. Moreover, we showed that
administration of SR141716A although inhibiting responding at an ethanol paired lever did not
modify responding at the inactive lever. Secondly, in a previous study in which reinstatement
paradigms were used, SR141716A given at the same doses as employed here did not block operant
responding induced by foot-shock (De Vries et al., 2001). However, our results indicate that
SR141716A does affect operant responses for sweet solutions (both 10% sucrose and 0.2%
51
saccharin), although with different potencies, indicating the existence of a reduced reward
evaluation of these two liquid reinforcers. Overall, these findings are consistent with recent reports
that propose a relevant role for CB1 receptors in reward sensitivity (De Vry et al., 2004; SanchisSegura et al., 2004). A potential role of this kind of reinforcer on cannabinoid CB1 receptor
antagonist-induced suppression of operant responses may be proposed on the basis of the
differential effects of SR141716A on operant responses for food, ethanol, saccharin or sucrose
(Figures 1 and 2).
Our results also support both genetic reports in humans and behavioural findings in genetically
modified animals in which a relevant role for either the cannabinoid CB1 receptor or the
endocannabinoid-degradating enzyme FAAH was recently described (Schmidt et al., 2002; Sipe et
al., 2002; Hungund et al., 2003; Wang et al., 2003). The nature of the contribution of the
endogenous cannabinoid system to alcoholism is complex. Chronic exposure to ethanol not only
modifies the bioavailability of anandamide by modifying the synthesis of its membrane precursor, it
also affects the expression of cannabinoid receptors in neurons, indicating that the endogenous
cannabinoid system adapts to the presence of ethanol. Because of the multiple physiological roles of
the endocannabinoids in the regulation of reward, memory, emotional states and behavioural
outcome, it was predicted that pharmacological or genetic manipulations of the endogenous
cannabinoid system may lead to alterations in ethanol self-administration behaviour. Deletion of the
cannabinoid CB1 receptor in mice has been reported to reduce ethanol preference and consumption
(Wang et al., 2003; Hungund et al., 2003), while the administration of cannabinoid CB1 receptor
antagonists blocked operant responses for ethanol, as well as stress- and ethanol-deprivationinduced increases in alcohol self-administration (Freedland et al., 2001; Serra et al., 2002; Racz et
al., 2003). Moreover, the history of ethanol dependence seems to be crucial for ethanol-selfadministration (Rodriguez de Fonseca et al., 1999), a result supported by at least one genetic study
that indicates that cycles of ethanol exposure up-regulate both cannabinoid CB1 receptor genes and
signal transduction systems associated with CB1 receptor-mediated signalling (i.e. MAP-kinases,
Rimondini et al., 2002).
An interesting aspect of endacannabinoid–alcohol interactions is the possibility of the existence of a
genetic contribution of endogenouscannabinoid system-related genes to alcoholism. Although two
studies have linked cannabinoid CB1 receptors and FAAH enzyme to severe alcohol dependence
(Schmidt et al., 2002; Sipe et al., 2002), and we have evidence of endocannabinoid–alcohol intake
interaction in CB1 knockout mice (Hungund et al., 2003; Wang et al., 2003), we lacked an animal
model of genetic vulnerability to alcoholism in which a contribution of the endogenous cannabinoid
system might be established. Partial evidence in this direction is provided by the in situ
hybridization results of our studies to clarify the reasons for the different sensitivity to the effect of
SR141716A between non-selected Wistar and msP rats. These results, in fact, clearly demonstrated
that msP rats display distinct regional differences in CB1 receptor mRNA expression compared with
Wistar rats.
A thorough evaluation of the role of CB1 receptors as a genetic predisposing f actor to alcohol
misuse would have required a comparative study in which the non-preferring control line for msP
rats was included. This was beyond the scope of the present work because the study was merely
aimed at evaluating the pharmacology of SR14176A on alcohol-related behaviours (ethanol
drinking and relapse), which cannot be measured in alcohol-non-preferring controls. The in situ
hybridization study was, in fact, planned a posteriori, simply to understand whether the different
sensitivity to the effect of the SR14176A between msP and Wistar rats was linked to different CB1
receptor system functionality. The results of this experiment clearly point to the existence of a
linkage of variations in brain CB1 receptor expression and distribution with genetic predisposition
to alcohol misuse. This view is further supported by the observation that ethanol experience
(voluntary consumption for 18 days) appears to decrease CB1 expression. Notably, this effect was
more pronounced in the rostral caudate putamen, the region which is most different between
ethanol-naϊve msP and Wistar rats. The restriction of this effect to the most rostral part most likely
52
points to compartmentalization within the caudate putamen, and its functional importance for
ethanol dependence warrants further investigation. The changes observed are in line with those
described in other pharmacological models in which the stimulation of the CB1 receptor promotes
alcohol intake whereas its blockade decreases intake: msP rats may have more CB1 receptors than
Wistar rats, and ethanol seems to reverse these differences, in a similar way to that described for
alcohol-non-preferring rats (i.e. a decrease in the expression of CB1 receptors after repeated ethanol
exposure, Basavarajappa et al., 1998).
The differences in CB1 mRNA expression in msP rats might be associated with the wellcharacterized phenotype of msP rats, which acquire ethanol self-administration more easily, and
exhibit differences in extinction and cue-associated relapse. We observed that there is a greater
sensitivity of msP rats than Wistar rats to the effects of cannabinoid receptor blockade on both selfadministration and cue-associated relapse. The fact that hippocampal areas (CA1 and CA4)
displayed selective differences in cannabinoid CB1 receptor mRNA expression and respond to
ethanol exposure may account for the increased sensitivity to the inhibitory effects of SR141716A
on cueinduced relapse in msP, because cannabinoid receptors in the hippocampus are crucial for
normal memory processing. The recently described regulatory role of cannabinoid CB1 receptors on
glutamate release in the hippocampus (Schlicker & Kathmann, 2001; Wilson & Nicoll, 2001) might
contribute to the well-characterized effects of ethanol on hippocampal glutamate release and
signalling (Lovinger et al., 1989; Martin & Swartzwelder, 1992) and on glutamate-mediated
regulation of ethanol self-administration and relapse (Backstrom et al., 2004). However, the
existence of at least one additional, but as yet uncloned cannabinoid CB1-like receptor on these
glutamatergic terminals (Hajos et al., 2001) demands further research in order to clarify whether all
the effects observed can be attributed solely to the already cloned CB1 receptor, which was
analyzed in the present study.
Whatever the case, the present findings highlight the importance of considering a cannabinoid CB1
receptor antagonist-based therapy not only for actual alcohol consumption but also for contextinduced promotion of relapse to alcohol drinking, one of the major problems seen in alcoholism
therapy. The results also suggest that, at least in msP rats, an altered function of the CB1 receptor
system may be linked to genetic vulnerability to alcohol misuse.
53
References
Agabio R., Cortis G., Fadda F., Gessa G.L., Lobina C., Reali R. & Colombo G. (1996). Circadian drinking pattern of
Sardinian alcohol-preferring rats. Alcohol Alcohol., 31, 385–388.
Arnone M., Maruani J., Chaperon F., Thiebot M.H., Poncelet M., Soubrie P. & Le Fur G. (1997). Selective inhibition of
sucrose and ethanol intake by SR 141716, an antagonist of central cannabinoid (CB1) receptors. Psychopharmacology,
132, 104–106.
Backstrom P., Bachteler D., Koch S., Hyytia P. & Spanagel R. (2004). mGluR5 antagonist MPEP reduces ethanolseeking and relapse behaviour. Neuropsychopharmacology, 29, 921–928.
Basavarajappa B.S., Cooper T.B. & Hungund B.L. (1998). Chronic ethanol administration down-regulates cannabinoid
receptors in mouse brain synaptic plasma membrane. Brain Res., 793, 212–218.
Basavarajappa B.S. & Hungund B.L. (1999). Chronic ethanol increases the cannabinoid receptor agonist anandamide
and its precursor N-arachidonoylphosphatidyl- ethanolamine in SK-N-SH cells. J. Neurochem., 72, 522–528.
Basavarajappa B.S., Saito M., Cooper T.B. & Hungund B.L. (2000). Stimulation of cannabinoid receptor agonist 2arachidonylglycerol by chronic ethanol and its modulation by specific neuromodulators in cerebellar granule neurons.
Biochim. Biophys. Acta, 1535, 78–86.
Caberlotto L., Rimondini R., Hansson A., Eriksson S. & Heilig M. (2004). Corticotropin-releasing hormone (CRH)
mRNA expression in rat central amygdala in cannabinoid tolerance and withdrawal: evidence for an allostatic shift?
Neuropsychopharmacology, 29, 15–22.
Colombo G., Agabio R., Fa M., Guano L., Lobina C., Loche A., Reali R. & Gessa G.L. (1998). Reduction of voluntary
ethanol intake in ethanol-preferring sP rats by the cannabinoid antagonist SR-141716. Alcohol Alcohol., 33, 126–130.
Colombo G., Serra S., Brunetti G., Gomez R., Melis S., Vacca G., Carai M.M. & Gessa G.L. (2002). Stimulation of
voluntary ethanol intake by cannabinoid receptor agonists in ethanol-preferring sP rats. Psychopharmacology, 159, 181–
187.
Comings D.E., Muhleman D., Gade R., Johnson P., Verde R., Saucier G. & MacMurray J. (1997). Cannabinoid receptor
gene (CNR1): association with i.v. drug use. Mol. Psychiatry, 2, 161–168.
Cravatt B.F.,Giang D.K., Mayfield S.P., Boger D.L., Lerner R.A. & Gilula N.B. (1996). Molecular characterization of
an enzyme that degrades neuromodulatory fatty-acid amides. Nature, 384, 83–87.
De Vries T.J., Homberg J.R., Binnekade R., Raaso H. & Schoffelmeer A.N. (2003). Cannabinoid modulation of the
reinforcing and motivational properties of heroin and heroin-associated cues in rats. Psychopharmacology, 168, 164–
169.
De Vries T.J., Shaham Y., Homberg J.R., Crombag H., Schuurman K., Dieben J., Vanderschuren L.J. & Schoffelmeer
A.N. (2001). A cannabinoid mechanism in relapse to cocaine seeking. Nat. Med., 7, 1151–1154.
De Vry J., Schreie R., Eckel G. & Jentzsch K.R. (2004). Behavioral mechanisms underlying inhibition of foodmaintained responding by the cannabinoid receptor antagonist ⁄ inverse agonist SR 141716A. Eur. J. Pharmacol., 483,
55–63.
Fattore L., Spano M.S., Cossu G., Deiana S. & Fratta W. (2003). Cannabinoid mechanism in reinstatement of heroinseeking after a long period of abstinence in rats. Eur. J. Neurosci., 17, 1723–1726.
Freedland C.S., Sharpe A.L., Samson H.H. & Porrino L.J. (2001). Effects of SR141716A on ethanol and sucrose selfadministration. Alcohol. Clin. Exp. Res., 25, 277–282.
Gessa G.L., Serra S., Vacca G., Carai M.A.M. & Colombo G. (2005). Suppressing effect of the cannabinoid CB1
receptor antagonist SR147778 on alcohol intake and motivational properties of alcohol in alcohol-preferring sP rats.
Alcohol Alcohol., 40, 46–53.
54
Gonzalez S., Cascio M.G., Fernandez-Ruiz J., Fezza F., Di Marzo V. & Ramos J.A. (2002). Changes in
endocannabinoid contents in the brain of rats chronically exposed to nicotine, ethanol or cocaine. Brain Res., 954, 73–
81.
Hajos N., Ledent C. & Freund T.F. (2001) Novel cannabinoid-sensitive receptor mediates inhibition of glutamatergic
synaptic transmission in the hippocampus. Neuroscience, 106, 1–4.
Hungund B.L. & Basavarajappa B.S. (2000). Are anandamide and cannabinoid receptors involved in ethanol tolerance?
A review of the evidence. Alcohol Alcohol., 35, 126–133.
Hungund B.L., Basavarajappa B.S., Vadasz C., Kunos G., Rodriguez de Fonseca F., Colombo G., Serra S., Parsons L. &
Koob G.F. (2002). Ethanol, endocannabinoids, and the cannabinoidergic signaling system.Alcohol Clin. Exp. Res., 26,
565–574.
Hungund B.L., Szakall I., Adam A., Basavarajappa B.S. & Vadasz C. (2003). Cannabinoid CB1 receptor knockout mice
exhibit markedly reduced voluntary alcohol consumption and lack alcohol-induced dopamine release in the nucleus
accumbens. J. Neurochem., 84, 698–704.
Lobina C., Agabio R., Diaz G., Fa M., Fadda F., Gessa G.L., Reali R. & Colombo G. (1997). Constant absolute ethanol
intake by Sardinian alcohol-preferring rats independent of ethanol concentrations. Alcohol Alcohol., 32, 19–22.
Lovinger D.M., White G. & Weight F.F. (1989). Ethanol inhibits NMDAactivated ion current in hippocampal neurons.
Science, 243, 1721–1724.
Martin D. & Swartzwelder H.S. (1992). Ethanol inhibits release of excitatory aminoacids from slices of hippocampal
area CA1. Eur. J. Pharmacol., 219, 469–472.
Matsuda L.A., Lolait S.J., Brownstein M.J., Young A.C. & Bonner T.I. (1990). Structure of a cannabinoid receptor and
functional expression of the cloned cDNA. Nature, 346, 561–564.
Navarro M., Carrera M.R., Fratta W., Valverde O., Cossu G., Fattore L., Chowen J.A., Gomez R., del Arco I., Villanua
M.A., Maldonado R., Koob G.F. & Rodriguez de Fonseca F. (2001). Functional interaction between opioid and
cannabinoid receptors in drug self-administration. J. Neurosci., 21, 5344–5350.
Ortiz S., Oliva J.M., Perez-Rial S., Palomo T. & Manzanares J. (2004). Chronic ethanol consumption regulates
cannabinoid CB1 receptor gene expression in selected regions of rat brain. Alcohol Alcohol., 39, 88–92.
Paxinos G. & Watson C. (1997). The Rat Brain in Stereotaxic Coordinates. Compact 3 rd edn. Academic Press, San
Diego.
Racz I., Bilkei-Gorzo A., Toth Z.E., Michel K., Palkovits M. & Zimmer A. (2003). A critical role for the cannabinoid
CB1 receptors in alcohol dependence and stress-stimulated ethanol drinking. J. Neurosci., 23, 2453– 2458.
Rimondini R., Arlinde C., Sommer W. & Heilig M. (2002). Long-lasting increase in voluntary ethanol consumption and
transcriptional regulation in the rat brain after intermittent exposure to alcohol. FASEB J., 16, 27–35.
Rodriguez de Fonseca F., Roberts A.J., Bilbao A., Koob G.F. & Navarro M. (1999). Cannabinoid receptor antagonist
SR141716A decreases operant ethanol self administration in rats exposed to ethanol-vapor chambers. Zhongguo Yao Li
Xue Bao, 120, 1109–1114.
Samson H.H. (1986). Initiation of ethanol reinforcement using a sucrose-substitution procedure in food- and water-sated
rats. Alcohol Clin. Exp. Res., 10, 436–442.
Sanchis-Segura C., Cline B.H., Marsicano G., Lutz B. & Spanagel R. (2004). Reduced sensitivity to reward in CB1
knockout mice. Psychopharmacology, 176, 223–232.
Schlicker E. & Kathmann M. (2001). Modulation of transmitter release via presynaptic cannabinoid receptors. Trends
Pharmacol. Sci., 22, 565–572.
Schmidt L.G., Samochowiec J., Finckh U., Fiszer-Piosik E., Horodnicki J., Wendel B., Rommelspacher H. & Hoehe
M.R. (2002). Association of a CB1 cannabinoid receptor gene (CNR1) polymorphism with severe alcohol dependence.
Drug Alcohol Depend., 65, 221–224.
55
Serra S., Brunetti G., Pani M., Vacca G., Carai M.A., Gessa G.L. & Colombo G. (2002). Blockade by the cannabinoid
CB (1) receptor antagonist, SR 141716, of alcohol deprivation effect in alcohol-preferring rats. Eur. J. Pharmacol., 443,
95–97.
Sipe J.C., Chiang K., Gerber A.L., Beutler E. & Cravatt B.F. (2002). A missense mutation in human fatty acid amide
hydrolase associated with problem drug use. Proc. Natl Acad. Sci. USA, 99, 8394–8399.
Wang L., Liu J., Harvey-White J., Zimmer A. & Kunos G. (2003). Endocannabinoid signaling via cannabinoid receptor
1 is involved in ethanol preference and its age-dependent decline in mice. Proc. Natl Acad. Sci. USA, 100, 1393–1398.
Weiss F., Lorang M.T., Bloom F.E. & Koob G.F. (1993). Oral alcohol self-administration stimulates dopamine release
in the rat nucleus accumbens: genetic and motivational determinants. J. Pharmacol. Exp. Ther., 267, 250–258.
Weiss F. & Porrino L.J. (2002). Behavioral neurobiology of alcohol addiction: recent advances and challenges. J.
Neurosci., 22, 3332–3337.
Wilson R.I. & Nicoll R.A. (2001). Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses.
Nature, 410, 588–592.
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IV
57
IV. Pharmacological evaluation of the novel in vivo
cannabinoid receptor antagonist 5-(4chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H1,2,4-triazole – LH 21 – on food intake: evidence
for a peripheral site of action
Abstract
The present study evaluates the pharmacological profile of the new in vivo cannabinoid CB1
receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,4- triazole –LH-21–
on feeding behaviour and alcohol self-administration in rats, two behaviours inhibited by
cannabinoid CB1 receptor antagonists. Administration of LH-21 (0.03, 0.3 and 3 mg/kg) to fooddeprived rats resulted in a dose-dependent inhibition of feeding. This effect was not associated with
changes in locomotor activity or with anxiety-like behaviours. Additionally, after the administration
of LH-21 we did not observe the induction of complex motor behaviours such as grooming or
scratching sequences, usually observed after central cannabinoid receptor blockade. Sub-chronic
administration of LH-21 reduced food intake and body weight gain in obese Zucker rats. Finally,
LH-21 did not markedly reduce alcohol self-administration (30% reduction observed only at a high
dose of 10 mg/kg). This pharmacological pattern partially overlaps that of the reference cannabinoid
CB1 receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole3-carboxamide, SR141716A (0.3, 1 and 3 mg/kg). In vitro analysis of blood-brain barrier
permeability using a parallel artificial membrane permeation assay demonstrated that LH-21 has a
low blood-brain barrier permeation, suggesting a mostly peripheral action for this compound. This
was confirmed by the low potency of peripherally injected LH-21 to antagonize motor depression
induced by intracerebroventricular administration of the CB1 agonist CP55,940. The present data
suggest that LH-21 is a promising compound for the treatment of eating disorders and obesity, and
that it will be devoid of potential side effects derived from central blockade of cannabinoid CB1
receptors.
Introduction
Since the discovery of the existence of receptors for the psychoactive compounds of cannabis sativa
(Devane et al., 1988; Matsuda et al., 1990) and the isolation of their endogenous ligands,
anandamide (Devane et al., 1992) and 2-arachidonoylglycerol (Mechoulam et al., 1995; Sugiura et
al., 1995), more than 4000 scientific reports have explored in depth the main aspects of the so called
“endocannabinoid system”. This system emerges nowadays as a relevant modulator of physiological
functions not only in the central nervous system but also in the autonomic nervous system, the
endocrine network, the immune system, the gastrointestinal tract, the reproductive system and the
microcirculation (Piomelli et al., 2003; Rodríguez de Fonseca et al., 2005).
Pharmacological studies reveal that there are at least two types of cannabinoid receptors, CB1 and
CB2, and a wide range of CB1 and CB2 ligands with diverse chemical structures are now available
(Howlett et al., 2002). Several series of compounds have been developed as cannabinoid CB1
58
receptor antagonists. The most representative are diarylpyrazoles, substitute benzofuranes,
aminoalkylindoles and triazole derivatives (For review see Rodriguez de Fonseca et al., 2005).
Diarylpyrazoles include both the first CB1 receptor antagonist synthesized (SR 141716A, RinaldiCarmona et al., 1994) and the first CB2 receptor antagonist (SR144528). Both are considered the
reference antagonists. Modification of the SR141716A molecule has yielded other CB1 receptor
antagonists with improved properties including SR147778 and AM281 (Howlett et al., 2002;
Rinaldi-Carmona et al., 2004). However, diarylpyrazoles are not neutral antagonists since they
display significant inverse agonist properties. In this context we have recently reported a silent
cannabinoid antagonist derived from a 1,2,4-triazole which represents a novel entry in cannabinoid
chemistry (Figure 1, Jagerovic et al., 2004). 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl1H-1,2,4-triazole –LH-21– is an in vivo CB1 antagonist with a paradoxic low affinity in vitro for
CB1 receptors, and devoid of inverse agonist properties.
The interest to develop cannabinoid antagonists derives from the multiple functions in which the
endogenous cannabinoid system is engaged, some of which are relevant for major human diseases
(Piomelli et al., 2000 and 2003). The endogenous cannabinoid system controls motivation for
appetitive stimuli, including food and drugs (Arnone et al., 1997; Colombo et al., 1998; Gómez et
al., 2002; Navarro et al., 2001). The homeostatic control of motivated behaviours and the regulation
of emotions warrant special attention because of the impact on the health systems of entities such as
feeding disorders (including obesity), addiction (especially alcoholism and tobacco smoking) or
mood alterations such as anxiety and depression. Following this rationale, clinical trials of the
effects of the cannabinoid receptor antagonist SR141716A on obesity and tobacco smoking are
currently under way (Cleland et al., 2004; Fernandez and Allison, 2004). Because of the inverse
agonism properties of the cannabinoid antagonist SR141716A (Maclennan et al., 1998; Mato et al.,
2002) the evaluation of a neutral cannabinoid CB1 receptor antagonist such as LH-21 will help to
clarify whether the inhibitory effects on feeding or alcohol self-administration are derived from
receptor blockade and not from the intrinsic activity of these compounds.
In the present study we tested LH-21 on models of motor behaviour, anxiety, feeding and alcohol
self-administration. Because of the structure of the triazole motif of LH-21 that might reduce its
crossing through biological membranes, we evaluated its potential penetration through the bloodbrain barrier using a parallel artificial membrane permeation assay. This information is relevant
because the inhibition of food intake is mediated both centrally (Jamshidi and Taylor, 2001;
Kirkahm et al, 2002; Hanus et al, 2003;) and peripherally (Gomez et al., 2002) while the effects on
reward/addiction, mood and motor control are mediated through cannabinoid receptors located in
central circuits (Rodríguez de Fonseca et al., 2005).
Figure 1. Chemical structure of the new in vivo cannabinoid CB1 receptor antagonist 5-(4- Chlorophenyl)-1-(2,4dichlorophenyl)-3-hexyl-1H-1,2,4-triazole (LH-21).
59
Material and methods
Animals
All experiments were performed in male Wistar rats, weighing 175-225 g at the start of the
experiments, or in obese Zucker rats aged 12 to 16 weeks (Panlab, Balona). Animals were housed in
groups of two in a temperature and humidity controlled vivarium on a reverse 12-hr light/dark cycle
(on 6:00 PM; off 6 AM). All training and experimental sessions were conducted during the dark
phase of the cycle. Standard National Institutes of Health laboratory rat chow and water were
available ad libitum in the home cage, except as noted in “Behavioural Training and Testing
Procedures”. All the procedures were conducted in adherence with the European Community
Council Directive 86/609/EEC regulating animal research.
Surgery and intracerebroventricular administration of drugs
For intracerebroventricular injections, stainless steel guide cannulas aimed at the lateral ventricle
were implanted in the rats. The animals were anesthetized with equithesin and placed in a David
Kopf Instruments (Tujunga, CA) stereotaxic instrument with the incisor bar set at 5 mm above the
interaural line. A guide cannula (7 mm, 23 gauge) was secured to the skull by using two stainless
steel screws and dental cement and was closed with 30 gauge obturators (Gómez et al, 2002). The
implantation coordinates were 0.6 mm posterior to bregma, ±2.0 mm lateral, and 3.2 mm below the
surface of the skull. These coordinates placed the cannula 1 mm above the ventricle. After a 7 d
postsurgical recovery period, cannula patency was confirmed by gravity flow of isotonic saline
through an 8-mm-long, 30 gauge injector inserted within the guide to 1 mm beyond its tip. This
procedure allowed the animals to become familiar with the injection technique. For
intracerebroventricular administration, the obturator was removed from the guide cannula and an 8
mm injector (30 gauge stainless steel tubing) that was connected to 70 cm of calibrated
polyethylene-10 tubing was lowered into the ventricle. The tubing was then raised until flow began,
and 5 µl of a solution containing 10 micrograms of CP 55,940 was infused over a 30-60 sec period.
The injector was left in the guide cannula for an additional 30 sec and then removed. The stylet was
immediately replaced. Animals were tested 5 min after injections. The intracerebroventricular
cannula placements were evaluated after each experiment by dye injection. Only rats with proper
intracerebroventricular placements were included in the data analysis.
Drugs
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,4-triazole –LH-21– (Figure 1) was
synthesized in the laboratory as previously described (Jagerovic et al., 2004). N-Piperidino-5-(4chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3- carboxamide, SR141716A, was a gift
from Sanofi-Aventis, Montpellier, France. (-)-cis- 3[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]trans-4-(3-hydroxypropyl) cyclohexanol (CP 55,940) was obtained through Tocris Cookson
(Avonmouth, UK). The drugs were suspended with 2-3 drops of Tween 80 in saline as vehicle and
administered intraperitoneally (i.p.) at doses of 0.03, 0.3 and 3 mg/kg (LH-21) or 0.3, 1 and 3 mg/kg
(SR141716A).
Food intake studies
The acute effects of drugs on feeding behaviour were analyzed in animals deprived of food for 24 hr
and habituated to handling (Gomez et al., 2002). To habituate the animals, 72 hr before the testing
with drugs, animals were food-deprived for 24 hr. Then, the bedding material was removed from the
cage and a small can containing weighed food pellets was placed inside the cage for 4 hr and the
amount of food eaten registered. After the initial test, the animals were under a free-feeding period
of 48h. Then, the animals were food-deprived for 24 hr again, with access to water ad libitum.
Fifteen minutes before the start of the test drugs were administered i.p., the animals were returned to
their home cage, where a measured amount of food (usually 30-40 gm) and a bottle containing 250
60
ml of fresh water were placed again. Food pellets and food spillage were weighed at 30, 60 and 120
min after starting the test, and the amount of food eaten was recorded. At the end of the test, the
amount of water consumed was also measured.
For analyzing the sub-chronic (8 days of treatment) effects of LH-21 in an obesity model, freefeeding Zucker rats were daily injected with LH-21 (0.3 and 3 mg/kg) at the start of the dark period
of the light cycle. The amount of food eaten and the body weight was registered daily.
Open-field test
Motor behaviours in the open field were studied in an opaque open field (100 × 100 × 40 cm) as
described previously (Beltramo et al., 2000). The field was illuminated using a ceiling halogen lamp
regulated to yield 350 lux at the center of the field. Rats were habituated to the field for 10 min the
day before testing. On the experimental day, the animals were treated and 30 min later placed in the
centre of the field, and locomotor activity (number of lines crossed or distance travelled in cm) and
rearing and grooming behaviour (number of rearings and time spent grooming) were scored for 5
min at 5, 30, 60, and 120 min after drug injection. Scratching sequences, a behaviour elicited by
cannabinoid receptor antagonism (Navarro et al., 1997) were also monitored. Behaviour was scored
by trained observers who were unaware of the experimental conditions.
Elevated plus-maze
As previously described (Navarro et al., 1997), the elevated plus-maze was made of opaque plastic,
with two opposite open arms (45 × 10 cm) and two opposite closed arms of the same size and 50cm-high walls. The arms were connected by a central square (10 × 10 cm). The entire apparatus was
elevated 75 cm above a white floor and exposed to dim illumination (70 lux). Rats were randomly
placed in the central square of the maze, facing an open arm. The number of entries onto and time
spent on each arm were scored, through the use of a video monitor placed in an adjacent room, for
the first 5 min (an arm entry was defined as all four feet in the arm). At the end of the test, each rat
was returned to its home cage. Final data are expressed as percentage of time spent on the exposed
or the closed arms of the maze.
Operant training for liquid reinforcers
Training and testing were conducted in standard operant chambers located in sound-attenuating,
ventilated environmental cubicles. Each chamber was equipped with a drinking reservoir (volume
capacity: 0.10 ml) positioned 4 cm above the grid floor in the centre of the front panel of the
chamber, and two retractable levers were located 3 cm to the right and left of the drinking
receptacle. Auditory and visual stimuli were presented via a speaker and a light located on the front
panel. A microcomputer controlled the delivery of fluids, presentation of auditory and visual
stimuli, and recording of the behavioural data. Rats were trained to self-administer 10%
[volume/volume (vol./vol.)] ethanol in daily 30-min sessions on a fixed-ratio 1 schedule of
reinforcement, where each response resulted in delivery of 0.1 ml. Briefly for the first 3 days of
training, water availability in the home cage was restricted to 2hr/day in order to facilitate
acquisition of operant responding for a liquid reinforcer. During this time, lever pressing reinforced
by 0.2% (w/v) saccharin solution was established. At this point water was made freely available,
and saccharin self-administration training continued until animals reached stable baseline
responding. Rats were then trained to self-administer ethanol by using a modification of the sucrose
fading procedure (Samson, 1986) that used saccharin instead of sucrose (Weiss et al., 1993). During
the first 6 days of this ethanol initiation phase a 5% (w/v) ethanol solution containing 0.2%
saccharin (w/v) was available to the rats. Starting on day 7, the concentration of ethanol was
gradually increased from 5.0% to 8.0% and finally to 10.0% (w/v), whereas the concentration of
saccharin was correspondingly decreased to 0%. At the beginning of the saccharin-fading procedure
a second, but inactive lever was introduced. During all training and testing phases responses at this
61
lever, which had no programmed consequences, were recorded as a measure of non-specific
behavioural activation.
Ethanol Self-administration: Effect of LH-21 and SR141716A
Following completion of the saccharin fading procedure rats were trained in 30 min session/day to
lever-press for 10% ethanol (0.1 ml/response) until a stable baseline level of responding was
reached. The CB1 receptor antagonists LH-21 (0.03, 0.3, 3 and 10 mg/kg) or SR141716A (0.0, 0.3,
1.0 and 3.0 mg/kg) were administered i.p. 30 min prior to the self-administration session. The
experiment was conducted every fourth day using a Latin square counterbalanced design.
Responding at the inactive lever was recorded throughout the experiment to monitor non-specific
behavioural effects.
Prediction of the brain penetration of LH-21
Prediction of the brain penetration was performed using a parallel artificial membrane permeation
assay (PAMPA), in a similar manner as described previously (Di et al., 2003). Commercial drugs,
phosphate buffered saline solution at pH 7.4 (PBS), and dodecane were purchased from Sigma,
Aldrich, Acros, and Fluka. Ethanol was reagent grade from Merck. The Millex filter units (PVDF
membrane, diameter 25 mm, pore size 0.45 µm) were acquired from Millipore. The porcine brain
lipid (PBL) was obtained from Avanti Polar Lipids. The donor microplate was a 96-well filter plate
(PVDF membrane, pore size 0.45 µm) and the acceptor microplate was an indented 96-well plate,
both from Millipore. The acceptor 96-well microplate was filled with 170 µl of PBS: ethanol
(70:30) and the filter surface of the donor microplate was impregnated with 4 µl of PBL in dodecane
(20 mg ml-1). Compound LH-21 and commercial drugs of known CNS permeability were dissolved
in PBS : ethanol (70:30) at 1 mg ml-1, filtered through a Millex filter, and then added to the donor
wells (170 µl). The donor filter plate was carefully put on the acceptor plate to form a sandwich,
which was left undisturbed for 120 minutes at 25 ºC. After incubation, the donor plate was carefully
removed and the concentration of compounds in the acceptor wells was determined by UV
spectroscopy. Every sample was analyzed in four wells and the average of the runs is reported,
including quality control standards of known permeability to validate the analysis set.
Statistics
Statistical significance of behavioural studies was assessed by analysis of variance (ANOVA). All
the studies were performed in between subjects. Following a significant F value, post hoc analysis
(Student-Newman-Keuls) was performed to assess specific comparisons between dose groups.
62
Results
I. Effects of LH-21 on food intake
The administration of LH-21 (0.03, 0.3 and 3 mg/kg) resulted in dose-dependent reduction in
feeding behaviour of food-deprived animals (Figure 2). Doses of 0.3 and 3 mg/kg suppressed
feeding with a high degree of efficacy, an effect that lasted up to 4 h. This effect was significant
(p<0.05, Newman-Keuls) at all the time points tested for the 3 mg/kg dose.
Figure 2. Acute administration of the in vivo cannabinoid CB1 receptor antagonist LH-21 (0.03, 0.3 or 3 mg/kg) to
24-hr food-deprived rats resulted in dose dependent reduction in feeding behaviour. Data are means ± SEM of 8
determinations per group. (*) p<0.05, versus vehicle-treated animals.
II. Effects of sub-chronic administration of LH-21 on food intake on obese Zucker rats.
The daily administration of LH-21 for 8 days reduced food intake (Figure 3A) and body weight gain
(Figure 3B) in obese Zuker rats. This effect was only observed with the highest dose tested (3
mg/kg). As described previously in Zucker rats treated with the cannabinoid receptor antagonist
SR141716A (rimonabant), the effects on weight gain were more evident and prolonged than the
reduction on feeding, suggesting the induction of metabolic adaptations (Vickers et al, 2003).
63
Figure 3. Subchronic (8 days) administration of the in vivo cannabinoid CB1 receptor antagonist LH-21 (0.3 or 3
mg/kg) reduced relative food intake (g/kg of body weight) and relative weight gain (g/g of body weight at day 1). Data
are means ± SEM of 8 determinations per group. (*) p<0.05, versus vehicle-treated animals.
64
III. Effect of LH-21 on ethanol self-administration.
We tested LH-21 on ethanol self-administration using a broad range of doses, since we have
recently observed that suppression of ethanol self-administration by SR141716A is a central effect
(Hansson et al., 2006). Using a Latin-square counterbalanced design we observed only a small
decrease in ethanol self-administration at the highest dose tested (10 mg/kg, Figure 4). This lack of
activity on ethanol self-administration of LH-21 contrasts with the high potency displayed in
suppressing feeding behaviour (Figures 2 and 3).
Figure 4. Acute intraperitoneal injection of LH-21 (0.03 to 10 mg/kg) 30 min before the testing procedure reduced
alcohol self-administration in male Wistar rats only at the highest dose tested (10 mg/kg). Data are means ± SEM of 810 determinations per group. (*) p<0.05, versus vehicle-treated animals.
IV. Open field: effects of LH-21 and SR141716A on locomotor activity and motor
behaviours.
LH-21 (3 and 10 mg/kg) did not affect locomotor activity (Figure 5A) nor did it induce complex
motor sequences (such as grooming behaviour or scratching sequences, Table 1) as it is typically
observed after the injection of the reference cannabinoid receptor antagonist/inverse agonist
SR141716A (3 mg/kg, Navarro et al., 1997). Although the highest dose of LH-21 (10 mg/kg)
apparently reduced locomotor activity during the first time interval of the open field test (5 min after
starting the trial), the difference was not statistically significant.
V. Effects of LH-21 on the elevated plus-maze.
The administration of the reference cannabinoid receptor antagonist SR141716A is associated with
decreased exploration on the elevated plus-maze (Navarro et al., 1997). This anxiety-like behaviour
65
was not observed when LH-21 (3 and 10 mg/kg) was administered to drug-naϊve animals (Figure
5B).
Figure 5. (A) Acute administration of LH-21 did not affect locomotor activity on the open field or (B) performance of
the animals in the elevated plus-maze, a standard anxiety test in rodents. Animals were injected i.p. 30 min before the
test. Data are means ± SEM of 8 determinations per group. (*)p<0.05, versus vehicle-treated animals.
66
Table 1 Effects of acute administration of the cannabinoid receptor antagonists SR141716A and LH-21 on behaviours
recorded in the open field. Data are means ± SEM of 8-10 determinations per group. (*)p<0.05, versus vehicle-treated
animals.
VI. Effects of SR141716A on feeding behaviour and ethanol self-administration
The i.p. administration of SR141716A (0.3, 1 and 3 mg/kg) to food-deprived animals resulted in a
dose-dependent reduction in food intake, which was constantly observed with the doses of 1 and 3
mg/kg (Figure 6A). With respect to ethanol self-administration, SR141716A was less potent
although a sustained inhibition of this behaviour was observed when the dose of 3 mg/kg was
injected into the animals 30 min prior to testing (Figure 6B)
Figure 6. (A)Acute administration of SR141716A reduced feeding in 24 hr food-deprived male Wistar rats. (B) Acute
administration of SR141716A reduced ethanol self-administration in Wistar rats at the dose of 3 mg/kg. Animals were
injected i.p. 30 min before the test. Data are means ± SEM of 8-10 determinations per group. (*) p<0.05, versus vehicle
treated animals.
67
VII. Prediction of the brain penetration of LH-21.
Because the different profile of LH-21 and SR141716A, the reference cannabinoid receptor
antagonist, we suspected that both drugs may have a differential permeability through the bloodbrain barrier. To this end we characterized this permeability in an in vitro model, and then we
studied it on an in vivo test. The in vitro permeability of LH-21 and ten commercial drugs through a
lipid extract from porcine brain was determined using a PAMPA test, and the results are gathered in
table 1. Assay validation was made comparing the experimental permeability with the reported
values of the commercial drugs (Di et al., 2003), which gave a good linear correlation (Figure 7).
Figure 7. Lineal correlation between experimental and reported permeability of ten commercial drugs using the
parallel artificial membrane permeation assay. Details of compounds and permeability values are described in Table 2.
From the straight line equation and taking into account the pattern determined by Di et al. (2003) for
blood-brain barrier (BBB) permeation prediction to classify compounds, we established the ranges
of this assay as follows:
Compounds of high BBB permeation: Pe (10-6 cm s-1) > 5.30
Compounds of low BBB permeation: Pe (10-6 cm s-1) < 3.10
As can be seen in table 2, the assay predicted the control compounds correctly and showed that
compound LH-21 had low blood-brain barrier permeation. Data obtained for the reference
cannabinoid receptor antagonist SR141716A revealed an experimental permeability above 3.77,
higher than that observed for LH-21, but intermediate between drugs with high or low blood-brain
barrier permeability. These results suggest that LH- 21 is not a good drug for targeting central
cannabinoid receptors.
68
a Taken from Di et al. (2003)
b Values are represented as the mean ± SEM.
Table 2. Prediction of the blood-brain barrier (BBB) penetration using a parallel artificial membrane permeation assay
(PAMPA). Experimental PAMPA results for tested compounds are compared with those described by Di et al., (2003).
Blood-bain barrier penetration was classified as positive (CNS+), intermediate (CNS +/-) or negative (CNS-),
depending on the value of experimental permeability: compounds of high BBB permeation display values of Pe (10-6
cm s-1) > 5.30 and compounds of low BBB permeation values of Pe (10-6 cm s-1) < 3.10.
VIII. Antagonism of central actions of the cannabinoid CB1 receptor agonist CP55,940
after peripheral administration of LH-21 or SR141716A.
In order to validate the in vitro permeability assay , we conducted an in vivo experiment on which
we tested whether LH-21 or SR141716A were able of antagonize the motor depression induced by
central administration (icv) of a cannabinoid receptor agonist (CP 55940). Either vehicle, LH-21
(0.3, 3 and 3 mg/kg) or SR141716A (3 mg/kg) were injected i.p. 30 min before the administration of
µg of CP 55,940 on the lateral ventricles of the rat. CP 55,940 dramatically reduced locomotion
(Figure 8A) and rearing activity (Figure 8B). Pre-treatment with SR141716A reversed this effect.
However LH-21 only exhibited a weak antagonistic activity on locomotion at the highest dose tested
(10 mg/kg) and was devoided of antagonistic activity on the rearing suppression induced by CP
55,940. These results indicate that LH-21 did not cross the blood brain barrier at a concentration
sufficient to reverse the acute actions of CP55,940.
69
Figure 8. Effects of peripheral (i.p.) administration of cannabinoid CB1 receptor antagonist LH- 21 (LH, 0.3., 3 and
10 mg/kg) or SR141716A (SR, 3 mg/kg) on the acute motor depression induced by central administration (icv) of CP
55,940 (10 µg in 5 µl) in male Wistar rats. SR141716A but not LH-21 antagonized the effects of CP, suggesting a better
brain penetration of SR141716A. (A) Cumulative locomotion score (distance travelled in cm) along a 2-hr testing. (B)
Cumulative number of rearings along a 2-hr testing. Animals were injected the antagonist i.p. 30 min before the
administration of CP 55,940. Testing started 5 min after CP injection. Data are means ± SEM of 8 determinations per
group. (*) p<0.05, versus vehicle-treated animals.
Discussion
Three major conclusions derive from the present study. First, LH-21, an in vivo neutral cannabinoid
CB1 receptor antagonist, has a different behavioural profile than the reference cannabinoid
antagonist / inverse agonist SR141716A, but retains its ability to reduce food intake and weight gain
in obese animals. Second, LH-21 has a poor penetrability through biological membranes, suggesting
that its effects are mainly produced through peripheral mechanisms (Table 2). Third, the high
efficacy of LH-21 as an inhibitor of feeding and the low efficacy of this compound in suppressing
ethanol self-administration are consistent with previous reports that suggest that while the inhibitory
actions of cannabinoid receptor antagonists on feeding have both peripheral and central
components, the effects on reward / drug self-administration are mediated via central cannabinoid
CB1 receptors (Maldonado and Rodríguez de Fonseca, 2002; Hansson et al., 2006). The lack of
central effects of LH-21 is further supported by the absence of two behavioural markers of brain
cannabinoid receptor blockade, the induction of anxiety-like responses in the elevated plus-maze
and the increase in complex motor sequences, such as grooming or scratching sequences (Navarro et
al., 1997 Rodríguez de Fonseca et al., 1997) (Table 1). Finally, peripheral administration of this
neutral antagonist at doses that reduces feeding and body weight gain in obese rats was unable of
preventing the acute motor depression induced by the central (icv) administration of a full
cannabinoid CB1 receptor agonist (CP 55,940).
70
Interestingly, although SR141716A has higher permeability through lipid membranes than LH-21,
its experimental permeability also suggests that it may have less penetration in the brain than
originally suspected, an observation which has recently been proposed after an initial clinical trial of
the effects of SR141716A in schizophrenics (Meltzer et al., 2004). In this study, the authors
discussed the lack of effects of this cannabinoid receptor antagonist in relation to the low dosage (20
mg, daily) and poor penetrability through the blood-brain barrier. Further support for this comes
from the observation of the different potency of SR141716A and LH-21 on food intake and ethanol
self-administration. While suppression of feeding induced by cannabinoid antagonists can be
mediated by CB1 receptors located in peripheral nerve terminals (Gómez et al., 2002) or in brain
areas with an open blood-brain barrier such as the hypothalamus (Jamshidi and Taylor, 2001; Cota
et al., 2003; Hanus et al, 2003), ethanol self-administration is regulated by central cannabinoid
receptors located in the ventral striatum and the prefrontal cortex (Maldonado and Rodriguez de
Fonseca, 2002; Rodriguez de Fonseca et al., 2005; Hansson et al., 2006). Both LH-21 and
SR141716A display more efficacy as feeding inhibitors than as suppressants of ethanol selfadministration. When compared, LH-21 and SR141716A have similar efficacies with respect to
feeding inhibition but SR141716A, which has better penetration through lipid bilayers, is at least
three times more potent when a central test such as inhibition of alcohol self-administration or
antagonism of central-cannabinoid agonist-induced motor depression (Figure 8) is used. Moreover,
the induction of grooming, a complex behaviour regulated by both central cannabinoid CB1 and
dopamine D1 receptors located in the ventral striatum (Rodríguez de Fonseca et al., 1998), was
observed after the administration of SR141716A but not after LH-21, again supporting the different
route of access of the two compounds to the central nervous system. Following this rationale, and as
a last example, LH-21 failed to inhibit exploration in the elevated plus-maze. Anxiety and anxietylike behaviours are adaptive responses regulated by limbic circuits, especially those involving the
amygdalar complex. Endocannabinoids and cannabinoid receptors modulate anxiety-like responses
in close association with the anxiogenic amygdalar neuropeptide corticotropin-releasing factor
(Caberlotto et al., 2004; Rodríguez de Fonseca et al., 1996 and 1997). The lack of effects of LH-21
on anxiety tests (elevated plus-maze as well as time spent in the centre of the open field, data not
shown) again suggest a lack of central actions. However, despite the suggestive set of experimental
data, an additional source of difference between both compounds may come from the inverse
agonist properties of SR141716A (For review see Pertwee, 2005), a pharmacological profile not
found in LH-21 (Jagerovic et al., 2004).
In conclusion, because of the poor penetration of LH-21 in the central nervous system and the
described role for peripheral cannabinoid CB1 receptors in the regulation of appetite, glucose
homeostasis and lipid metabolism, we believe that this drug can be considered as the first of a new
series of compounds designed to treat obesity and obesity associated disorders, in line with the
recent clinical observations in humans receiving the cannabinoid receptor antagonist Rimonabant
(Van Gaal et al., 2005), and which may avoid the potential side effects derived from central
blockade of cannabinoid receptors.
71
References
Arnone M., Maruani J., Chaperon F., Thiebot M.H., Poncelet M., Soubrie P., Le Fur, G., (1997). Selective inhibition of
sucrose and ethanol intake by SR 141716, an antagonist of central cannabinoid (CB1) receptors. Psychopharmacology
132, 104-106.
Beltramo M, Rodriguez de Fonseca F., Navarro M., Calignano A., Gorriti M.A., Grammatikopoulos G., Sadile A.G.,
Giuffrida A., Piomelli D. (2000). Reversal of dopamine D(2) receptor responses by an anandamide transport inhibitor. J
Neurosci. 20, 3401-3407.
Caberlotto L., Rimondini R., Hansson A., Eriksson S., Heilig M. (2004). Corticotropin-releasing hormone (CRH)
mRNA expression in rat central amygdala in cannabinoid tolerance and withdrawal: evidence for an allostatic shift?
Neuropsychopharmacology. 29, 15-22.
Cleland J.G., Ghosh J., Freemantle N., Kaye G.C., Nasir M., Clark A.L., Coletta A.P. (2004). Clinical trials update and
cumulative meta-analyses from the American College of Cardiology: WATCH, SCD-HeFT, DINAMIT, CASINO,
INSPIRE, STRATUS-US, RIO-Lipids and cardiac resynchronisation therapy in heart failure. Eur J Heart Fail. 6, 501508.
Colombo G., Agabio R., Fa M., Guano L., Lobina C., Loche A., Reali R., Gessa G.L. (1998). Reduction of voluntary
ethanol intake in ethanol-preferring sP rats by the cannabinoid antagonist SR-141716. Alcohol Alcohol 33,126-130.
Cota D., Marsicano G., Tschop M., Grubler Y., Flachskamm C., Schubert M., Auer D., Yassouridis A., Thone-Reineke
C., Ortmann S., Tomassoni F., Cervino C., Nisoli E., Linthorst A.C., Pasquali R., Lutz B., Stalla G.K., Pagotto U.
(2003). The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral
lipogenesis. J Clin Invest. 112, 423-431.
Devane W.A., Dysarz F.A. 3rd, Johnson M.., Melvin L.S. and Howlett A.C. (1988). Determination and characterization
of a cannabinoid receptor in rat brain. Molecular Pharmacology. 34, 605-613.
Devane W.A., Hanus L., Breuer A., Pertwee R.G., Stevenson L.A., Griffin G., Gibson D., Mandelbaum A., Etinger A.
and Mechoulam R. (1992). Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science.
258, 1946-1949.
Di L., Kerns E.H., Fan K., McConnell O.J., Carter G.T. (2003). High throughput artificial membrane permeability assay
for blood-brain barrier. Eur. J. Med. Chem. 38, 223-232.
Fernandez J.R., Allison D.B. (2004). Rimonabant Sanofi-Synthelabo. Curr Opin Investig Drugs. 5, 430-435.
Gomez R., Navarro M., Ferrer B., Trigo J.M., Bilbao A., Del Arco I., Cippitelli A., Nava F., Piomelli D., Rodriguez de
Fonseca F. (2002). A peripheral mechanism for CB1 cannabinoid receptor-dependent modulation of feeding. J Neurosci.
22, 9612-7.
Hansson A.C., Bermúdez-Silva F.J., Malinen H., Hyytiä P., Sanchez-Vera I., Rimondini R., Rodriguez de Fonseca F.R.,
Kunos G., Sommer W., Heilig M. (2006). Genetic impairment of frontocortical endocannabinoid degradation and high
alcohol preference. Neuropsychopharmacology (in press).
Hanus L., Avraham Y., Ben-Shushan D., Zolotarev O., Berry E.M., Mechoulam R. (2003). Short-term fasting and
prolonged semistarvation have opposite effects on 2-AG levels in mouse brain. Brain Res. 983, 144-151.
Howlett A.C., Barth F., Bonner T.I., Cabral G., Casellas P., Devane W.A., Felder C.C., Herkenham M., Mackie K.,
Martin B.R., Mechoulam R. and Pertwee RG. (2002). International Union of Pharmacology. XXVII. Classification of
cannabinoid receptors. Pharmacological Reviews. 54, 161-202.
Jagerovic N., Hernandez-Folgado L., Alkorta I., Goya P., Navarro M., Serrano A., Rodriguez de Fonseca F., Dannert
M.T., Alsasua A., Suardiaz M., Pascual D., Martín M.I. (2004). Discovery of 5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-3-hexyl- 1h-1,2,4-triazole, a novel in vivo cannabinoid antagonist containing a 1,2,4-triazole motif. J
Med Chem. 47, 2939-242.
Jamshidi N., Taylor D. A. (2001). Anandamide administration into the ventromedial hypothalamus stimulates appetite
in rats. Br J Pharmacol. 134, 1151-15544.
72
Kirkham T.C., Williams C.M., Fezza F., Di Marzo V. (2002). Endocannabinoid levels in rat limbic forebrain and
hypothalamus in relation to fasting, feeding and satiation: stimulation of eating by 2-arachidonoyl glycerol. Br J
Pharmacol. 136, 550-5577.
MacLennan S.J., Reynen P.H., Kwan J., Bonhaus D.W. (1998). Evidence for inverse agonism of SR141716A at human
recombinant cannabinoid CB1 and CB2 receptors. Br J Pharmacol. 124, 619-22.
Maldonado R., Rodriguez de Fonseca F. (2002). Cannabinoid addiction: behavioral models and neural correlates. J
Neurosci. 22, 3326-3331.
Mato S., Pazos A., Valdizan E.M.. (2002). Cannabinoid receptor antagonism and inverse agonism in response to
SR141716A on cAMP production in human and rat brain. Eur J Pharmacol. 443, 43-46.
Matsuda L.A., Lolait S.J., Brownstein M.J., Young A.C., Bonner T.I. (1990). Structure of a cannabinoid receptor and
functional expression of the cloned cDNA. Nature 346, 561-564.
Mechoulam R., Ben-Shabat S., Hanus L., Ligumsky M., Kaminski N.E., Schatz A.R., Gopher A., Almog S., Martin
B.R., Compton DR, Pertwee R.G. GriffinG., Bayewithch M Barg J. and Vogel Z. (1995). Identification of an
endogenous 2- monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochemical Pharmacology.
50, 83-90.
Meltzer H.Y., Arvanitis L., Bauer D., Rein W. Meta-Trial Study Group. (2004). Placebo-controlled evaluation of four
novel compounds for the treatment of schizophrenia and schizoaffective disorder. Am J Psychiatry. 161, 975-984.
Navarro M., Carrera M.R., Fratta W., Valverde O., Cossu G., Fattore L., Chowen J.A., Gomez R., del Arco I., Villanua
M.A., Maldonado R., Koob G.F. and Rodriguez de Fonseca F. (2001). Functional interaction between opioid and
cannabinoid receptors in drug self-administration. J. Neurosci. 21, 5344-50.
Navarro M., Hernández E., Muñoz R.M., del Arco, I. Villanua M.A., Carrera M.R., Rodriguez de Fonseca F. (1997).
Acute administration of the CB1 cannabinoid receptor antagonist SR 141716A induces anxiety-like responses in the rat.
Neuroreport. 8, 491- 496.
Pertwee R.G. (2005). Inverse agonism and neutral antagonism at cannabinoid CB(1) receptors. Life Sci. 76,1307-24.
Piomelli D. (2003). The molecular logic of endocannabinoid signalling. Nat Rev Neurosci. 4, 873-84.
Piomelli D., Giuffrida A., Calignano A., Rodriguez de Fonseca F. (2000). The endocannabinoid system as a target for
therapeutic drugs.Trends Pharmacol Sci. 21, 218-24.
Rinaldi-Carmona M., Barth F., Congy C., Martinez S., Oustric D., Perio A., Poncelet M., Maruani J., Arnone M.,
Finance O., Soubrie P.and Le Fur G. (2004). SR147778, a new potent and selective antagonist of the CB1 cannabinoid
receptor. Biochemical and pharmacological characterization.. J Pharmacol Exp Ther. 310, 905- 914.
Rinaldi-Carmona M., Barth F., Héaulme M., Shire D., Calandra B., Congy C., Martinez S., Maruani J., Néliat G., Caput
D., Ferrara P., Soubrié Ph., Brelière J. C. and Le Fur G. (1994). SR141716A, a potent and selective antagonist of the
brain cannabinoid receptor. FEBS Letters 350, 240-244.
Rodriguez de Fonseca F., Del Arco I., Bermudez-Silva F.J., Bilbao A., Cippitelli A., Navarro M. (2005). The
endocannabinoid system: physiology and pharmacology. Alcohol Alcohol. 40, 2-14.
Rodriguez de Fonseca F., Del Arco I., Martin-Calderon J.L., Gorriti M.A., Navarro M. (1998). Role of the endogenous
cannabinoid system in the regulation of motor activity. Neurobiol Dis. 5, 483-501.
Rodriguez de Fonseca F., Carrera M.R., Navarro M., Koob G.F., Weiss F. (1997). Activation of corticotropin-releasing
factor in the limbic system during cannabinoid withdrawal. Science. 276, 2050-2054.
Rodriguez de Fonseca F., Roberts A.J., Bilbao A., Koob G.F., Navarro M. (1999). Cannabinoid receptor antagonist
SR141716A decreases operant ethanol self administration in rats exposed to ethanol-vapor chambers. Zhongguo Yao Li
Xue Bao. 120, 1109-14
Rodriguez de Fonseca F., Rubio P., Menzaghi F., Merlo-Pich E., Rivier J., Koob G.F., Navarro M. (1996).
Corticotropin-releasing factor (CRF) antagonist [Dphe12, Nle21,38,C alpha MeLeu37]CRF attenuates the acute actions
73
of the highly potent cannabinoid receptor agonist HU-210 on defensive-withdrawal behavior in rats. J Pharmacol Exp
Ther. 276, 56-64.
Samson H.H. (1986). Initiation of ethanol reinforcement using a sucrose-substitution procedure in food- and water-sated
rats. Alcohol Clin Exp Res. 10, 436-442
Sugiura T., Kondo S., Sukagawa A., Nakane S., Shinoda A., Itoh K., Yamashita A., Waku K. (1995). 2Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res Commun.
215, 89-97.
Van Gaal L.F., Rissanen A.M., Scheen A.J., Ziegler O., Rossner S.; RIO-Europe Study Group. (2005). Effects of the
cannabinoid-1 receptor blocker rimonabant on weight reduction and cardiovascular risk factors in overweight patients:
1-year experience from the RIO-Europe study. Lancet. 365, 1389-1397.
Vickers S.P., Webster L.J., Wyatt A., Dourish C.T., Kennett G.A. (2003). Preferential effects of the cannabinoid CB1
receptor antagonist, SR 141716, on food intake and body weight gain of obese (fa/fa) compared to lean Zucker rats.
Psychopharmacology (Berl). 167, 103-11.
Weiss F., Lorang M.T., Bloom F.E. & Koob G.F. (1993). Oral alcohol self-administration stimulates dopamine release
in the rat nucleus accumbens: genetic and motivational determinants. J Pharmacol Exp Ther. 267, 250-258.
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V
75
V. SELECTIVE REDUCTION OF ETHANOL SELFADMINISTRATION BY THE ANANDAMIDE
TRANSPORT INHIBITOR AM404
Abstract
The endocannabinoid system mediates the pharmacological actions of ethanol and genetic studies
link endocannabinoids and their receptors to alcoholism. The variable results obtained with CB1
receptor agonists with respect to alcohol consumption, make difficult the interpretation of this
contribution limiting the therapeutic potential of direct cannabinoid agonists for the treatment of
alcohol abuse. An alternative approach may be to develop drugs that amplify the effects of
endogenous cannabinoids by preventing their inactivation. In the present study we addressed this
hypothesis by studying the effects of the anandamide transport inhibitor, AM404 on ethanol selfadministration as well as in reinstatement of ethanol seeking behaviour. The results show that
AM404 significantly reduced ethanol self-administration in a dose-dependent manner, but failed to
modify reinstatement for lever pressing induced by the stimuli associated with ethanol. This effect
was not due to a motor depressant effect and was not related to a decrease in general motivational
state, since it was not effective with other rewarding substances such as lever pressing for a saccharin
solution and food intake in 24 h – food deprived rats. In addition AM404 failed to reduce break point
for ethanol using a progressive ratio schedule of reinforcement. The mechanism of action of AM404
seems to be not mediated by cannabinoid CB1 receptor antagonist SR141716A neither by vanilloid
VR1 receptor because it is not antagonized by the VR1 receptor antagonist capsazepine. However
cannabinoid agonist- induced suppression of ethanol self-administration may imply cannabinoid CB1
receptors since the CB1-selective agonist ACEA [N-(2- chloroethyl)-5Z,8Z,11Z,14Zeicosatetraenamide] as the potent synthetic cannabinoid agonist WIN55,212-2 also reduced ethanol
self-administration. These results indicate that drugs aimed to block anandamide transport may be
considered as an innovative approach to treat alcohol abuse.
Introduction
Cannabis and alcohol are two of the oldest abused drugs used by humans and together with nicotine,
they represent a relevant health problem because of the clinical problems derived of their abuse.
Their psychotropic effects are well known and recent research has shown that there is a close link
between cannabis and alcohol (Arnone et al., 1997; Basavarajappa and Hungund, 2002). The
endogenous cannabinoid system (a system of lipid ligands and receptors that are the target of natural
and synthetic cannabinoids, for review see Piomelli, 2003) has been shown to mediate some of the
pharmacological and behavioural aspects of alcohol (Basavarajappa and Hungund, 2002; Rodríguez
de Fonseca et al., 2005). Both, cannabinoids and alcohol activate the same reward pathways and the
cannabinoid CB1 receptor plays and important role in regulating the positive reinforcing effects of
alcohol, as well as relapse to alcohol–seeking behaviour (Cippitelli et al., 2005; Hungund et al.,
2003; Wang et al., 2003). Several studies have documented that endocannabinoid transmission
becomes hyperactive in reward-related areas during chronic ethanol administration, as revealed by an
increase in the levels of endocannabinoids and the induction of down-regulation of CB1 receptors
possibly by over-stimulation of receptors through increased synthesis of the endogenous CB1
76
receptor agonist (anandamide and 2-arachidonylglicerol) (Basavarajappa and Hungund, 2000).
Following this rationale, cannabinoid CB1 receptor knockout mice show reduced alcohol preference
and self-administration (Hungund et al., 2003; Naassila et al 2004; Poncelet et al, 2003; Wang et al.,
2003). However, the role of cannabinoids in alcohol and drug-induced reward modulation demands
further research because of contradictory reports concerning the ability of cannabinoid agonists and
antagonists in modulating drug self-administration and relapse. The discrepancies in results have
been interpreted as being due to differences in apparatus, experimental design and subjects used. To
date, there is not a consensus in the literature with regard to the ability of cannabinoids to increase or
decrease the rewarding properties of alcohol and other drugs of abuse (Carriero et al., 1998;
Colombo et al., 1998 and 2002; Cossu et al., 2001; Martellota et al., 1998; Martin et al., 2000;
Solinas et al., 2005; Soria et al., 2005; Vlachou et al., 2003). In the literature concerning to
reinforcing effects in the self-administration context, most behavioural studies of the CB1 employing
pharmacological manipulation have focused on the use of direct cannabinoid receptor ligands (either
agonists or antagonists). Examples of these contradictory findings show how the administration of
the synthetic cannabinoid agonist WIN55,212-2 increases ethanol consumption (Colombo et al,
2002) while it decreases cocaine intravenous self-administration in rats (Fattore et al, 1999) and the
reinforcing actions of cocaine (Vlachou et al., 2003); another study by Braida and Sala (2002)
confirmed that the combination of CP55,940 with MDMA reduced the number of drug-associated
lever pressings. On the other hand, cannabinoid antagonists have been shown to block the rewarding
properties of most of drugs of abuse. It has been shown to reduce self- administration and subjective
effects of THC in rats, monkeys and recently in humans (Tanda et al., 2000; Solinas et al., 2003;
Huestis et al., 2001), heroin self administration (De Vries et al., 2003; Solinas et al., 2003) as well as
morphine-induced conditioned place preference (CPP) (Chaperón et al., 1998; Martin et al., 2000),
nicotine self-administration (Cohen et al., 2002) and nicotine-induced CPP (Le Foll and Goldberg
2004).
The cannabinoid antagonist SR141716A can also decrease ethanol intake and preference (Arnone et
al., 1997; Colombo et al., 1998; Rodríguez de Fonseca et al., 1999; Rinaldi-Carmona et al., 2004;
Cippitelli et al., 2005), as well as ethanol-induced CPP (Houchi et al., 2005). At least in one study,
cannabinoid receptor antagonism produces a biphasic effect, with a transient increase in heroin selfadministration, followed by a profound inhibition of operant responding for the opiate (Navarro et
al., 2001). The wide distribution of cannabinoid receptors and its role as a modulator of synaptic
transmission makes difficult the interpretation of these findings, and limit the utility of direct
cannabinoid CB1 receptor ligands for the treatment of drug abuse. A pharmacological alternative that
might reduce these problems may be offered by anandamide reuptake inhibitors. These drugs have
been used in vivo in an effort to demonstrate their ability to inhibit cellular accumulation of
anandamide and thereby stimulate cannabimimetic signalling. The anandamide reuptake inhibitor
AM404 produces physiological effects similar to anandamide in vivo and potentiates the receptormediated effects of exogenously administered anandamide (Beltramo et al., 1997 and 2000;
Calignano et al., 1997). Although numerous studies have examined and compared the pharmacology
of cannabinoids agonists and antagonist in reinforcing effects of ethanol, there are no studies
addressing the effects of the anandamide transport blocker AM404 on ethanol-related behaviours
such as ethanol self-administration and relapse. Considering the role of endocannabinoid system in
ethanol intake and reinforcement, the aim of this study was to test the effect of cannabinoid agonists
including the anandamide reuptake blocker AM404 on ethanol self-administration and in cueinduced reinstatement paradigms and to evaluate a possible mechanism of action responsible for the
modulation of alcohol-related behaviours by endogenous cannabinoid system.
77
Material and Methods
Animals
Male Wistar rats weighting 175-225g. were housed in groups of two in a temperature and humidity
controlled vivarium on a reverse 12-hr light/dark cycle (lights on 6:00 PM; off 6 AM). All training
and experimental sessions were conducted during the dark phase of the cycle. Standard laboratory rat
chow and water were available ad libitum in the home cage, except as noted in “Behavioural
Training and Testing Procedures”. All experimental procedures met the guidelines for the care and
use of laboratory animals of the European Communities directive 86/609/EEC regulating animal
research.
Drugs
AM404 (N-(4-hydroxyphenyl) arachidonylethanolamide), ACEA (Biogen Tocris), WIN552122(Sigma) and capsazepine (Sigma) were mixed in a vehicle of DMSO, Tween 80 and distilled water
in a ratio of 10:10:80. SR141716A (Sanofy-Synthelabo, Montpellier, France) was suspended within
2-3 drops of Tween 80 in saline. All of drugs and were injected intraperitoneally at a volume of
1ml/kg throughout the experiment.
Locomotor studies
We studied the effects of AM404, ACEA and WIN55,212-2 on immobility and horizontal locomotor
activity in rats. Motor activity was studied in an open field (100x100cm) interfaced to a computer
(SMART, Panlab, Barcelona) that recorded activity automatically. Animals were placed in the arena
for 10 min the day before testing for habituation. On the experimental day, the animals were placed
in the centre of the testing chambers, and immobility (difference between sample time and time spent
moving) and horizontal activity (distance travelled by the animal in a given sample period) were
recorded at 5min intervals. This procedure was performed at 0, 30, 60 and 120 min after the injection
of either vehicle or AM404 (2.0, 10.0mg/kg), ACEA (0.2, 1.0, 2.0mg/kg), WIN55,212-2 (0.4, 2.0,
5.0mg/kg). All behavioural tests were conducted in a sound-isolated room, illuminated with an
indirect halogen light (125 lux). Testing arenas were cleaned with water between subject
performances.
Feeding experiments
For this experiment, we measured food intake in rats habituated to the experimental setting and
deprived of food for 24 h. We administered AM404 (0, 0.4, 2.0 and 10.0 mg/kg) 30min before food
presentation and food intake was measured 60 and 120’ after the drug injection.
Operant training for liquid reinforcers
Training and testing were conducted in standard operant chambers located in sound – attenuating,
ventilated environmental cubicles. Each chamber was equipped with a drinking reservoir (volume
capacity: 0.10 ml) positioned 4 cm. above the grid floor in the centre of the front panel of the
chamber, and a retractable lever, located 3 cm. to the right of the drinking receptacle. Auditory and
visual stimuli were presented via a speaker and a light located on the front panel. A microcomputer
controlled the delivery of fluids, presentation of auditory and visual stimuli, and recording of the
behavioural data. Rats were trained to self-administer 10% ethanol (v/v) or 0.2% (w/v) saccharin or
water in 30-min. daily sessions on a fixed-ratio 1 schedule of reinforcement, where each response
resulted in delivery of 0.1 ml of fluid as previously described (Weiss et al., 1993). Briefly, for the
first 3 days of training, water availability in the home cage was restricted to 2hr/day in order to
facilitate acquisition of operant responding for a liquid reinforcer. During this time, rats were
permitted to lever-press for a 0.2% (w/v) saccharin solution. At this point water was made freely
available, and saccharin self-administration training continued for another 3 days. The rats, then,
78
were trained to self-administer ethanol by using a modification of the sucrose-fading procedure
(Samson, 1986) that used saccharin instead of sucrose (Weiss et al., 1993). During the first 6 days of
training rats were allowed to lever-press for a 5.0% (w/v) ethanol solution containing 0.2% (w/v)
saccharin. Starting on day 7, the concentration of ethanol was gradually increased from 5.0% to 8.0%
and finally to 10.0% (w/v), while the concentration of saccharin was correspondingly decreased to
0%. At the beginning of the saccharin fading procedure a second but inactive lever was introduced.
During all training and testing phases responses at this lever were recorded as a measure of nonspecific behavioural activation, but they had no programmed consequences.
Ethanol Self-administration: Effects of ACEA, WIN55212-2 and AM404 alone or in combination
with SR141716A and capsazepine
Following completion of the saccharin fading procedure, rats were trained in 30 min session/day to
lever-press for 10% ethanol until stable baseline of responding was reached. Firstly, we performed
the doses-response effect of the selective CB1 agonist ACEA (0.0, 0.2, 1.0 and 2.0mg/kg), the potent
synthetic cannabinoid agonist WIN55,212-2 (0.0, 0.4, 2.0 and 5.0 mg/kg) the anandamide transport
inhibitor AM404 (0.0, 0.4, 2.0 and 10.0 mg/kg), and the vanilloid receptor VR1 competitive
antagonist capsazepine (0.0, 3.0 and 10.0 mg/kg) on ethanol self-administration. Drugs were given
intraperitoneally 30 min prior to self-administration session. Secondly, we pre-treated the animals
with either capsazepine (10 mg/kg) or with the selective CB1 antagonist SR141716A (0.3 mg/kg)
prior to the injection of AM404 (2mg/kg). Pre-treatments were performed 30 min prior to AM404
injection. The experiments were conducted every fourth day using a Latin square counterbalanced
design. Responding at the inactive lever was recorded throughout the experiment to monitor nonspecific behavioural effects.
Saccharin self administration: effect of ACEA, WIN55,212-2 and AM404
When a stable baseline response for 0.2 (w/v) saccharin solution was reached, rats were used to study
the effect of ACEA, WIN55,212-2 and AM404 (0.0, 0.4 and 2mg/kg) given i.p. 30 min prior to the
self-administration session. Sessions lasted 30 min and a 20” time-out was scheduled to keep the
baseline response to lower levels similar to ethanol responding. The experiment was conducted every
fourth day using a latin square counterbalanced design. Responding at the inactive lever was
recorded throughout the experiment to monitor non-specific behavioural effects.
Reinstatement of ethanol-seeking behaviour: effect of AM404
Conditioning Phase
At completion of the fading procedure, in 30 min daily sessions, animals were trained to discriminate
between 10% ethanol and water. Beginning with self-administration training at the 10% ethanol
concentration, discriminative stimuli (SD) predictive of ethanol versus water availability were
presented during the ethanol and water self-administration sessions, respectively. The discriminative
stimulus for ethanol consisted of the odour of an orange extract (S+) whereas water availability (i.e.
no reward) was signalled by an anise extract (S-). The olfactory stimuli were generated by depositing
six-eight drops of the respective extract into the bedding of the operant chamber. In addition, each
lever-press resulting in delivery of ethanol was paired with illumination of the chamber’s house light
for 5 sec (CS+). The corresponding cue during water sessions was a 5 second tone (70 dB) (CS -).
Concurrently with the presentation of these stimuli, a 5 sec. time-out period was in effect, during
which responses were recorded but not reinforced. The olfactory stimuli serving as S + or S- for
ethanol availability were introduced one minute before extension of the levers and remained present
throughout the 30min. sessions. The bedding of the chamber was changed and bedding trays were
cleaned between sessions. During the first three days of the conditioning phase the rats were given
ethanol sessions only. Subsequently ethanol and water sessions were conducted in random order
79
across training days, with the constraint that all rats received a total of 10 ethanol and 10 water
sessions.
Extinction Phase
After the last conditioning day, rats were subjected to 30min extinction sessions for 15 consecutive
days. During this phase sessions began by extension of the levers without presentation of the SD.
Responses at the lever activated the delivery mechanism but did not result in the delivery of liquids
or the presentation of the response-contingent cues (house light or tone).
Reinstatement Testing
Reinstatement tests began the day after the last extinction session. This test lasted 30min under
conditions identical to those during the conditioning phase, except that alcohol and water were not
made available. Sessions were initiated by the extension of both levers and presentation of either the
ethanol S+ or water S- paired stimuli. The respective SD remained present during the entire session
and responses at the previously active lever were followed by activation of delivery mechanism and
a 5sec. presentation of CS+ in the S+ condition or the CS- (tone) in the S- condition. Animals were
tested under the S+/CS+ condition on day 1 and under the S-/CS- condition on day 2. Subsequently,
reinstatement experiments were conducted every fourth day (on days 6, 10, 14). AM404 was
administered i.p. 30 min prior to the sessions at the doses of 0.0, 0.4 and 2.0 mg/kg. Responding at
the inactive lever was constantly recorded to monitor possible non-specific behavioural effects.
Progressive ratio schedule of reinforcement: effect of AM404
In this experiment, rats (n=8) were tested under a progressive ratio schedule of reinforcement to
measure the BP (the last ratio completed by the animals) for ethanol. For this purpose, animals were
first trained to self-administer 10% alcohol under a FR1 schedule of reinforcement (see above).
Following acquisition of a stable baseline of responding for 10% ethanol, rats were tested under the
PR condition, in which the response requirement (i.e. the number of lever responses or the ratio
required to receive one dose of 10% ethanol) was increased as follows. For each of the first four
ethanol deliveries, the ratio was increased by 1; for the next four deliveries the ratio was increased by
2; for all the following deliveries the ratio was increased by 4. Each ethanol-reinforced response
resulted in a 1.0-s illumination of the house light, while sessions were terminated when more than 30
min had elapsed since the last reinforced response (Ciccocioppo et al. 2004). Drug testing was
carried out once a week as follows. The PR baseline was established on days 1 and 2, while PR drug
testing took place on day 3. For the next 2 days, animals were placed in the chambers under FR1
condition to re-establish the ethanol self-administration baseline, while on days 6 and 7 they
remained confined to their home cages. AM 404 (0.4 and 2.0 mg/kg; i.p.) or its vehicle was given 30
min before the PR session. The experiment was repeated for the following 2 weeks, counterbalancing
the treatment.
Statistics
Statistical significance of was assessed by analysis of variance (ANOVA). Following a significant F
value, post hoc analysis (Student-Newman-Keuls) was performed to assess specific comparisons
between dose groups.
80
Results
I. Experiment 1: AM404 decreases ethanol self-administration but not break point for
ethanol or motivation for other reinforcers.
Pre-treatment with the anandamide transport inhibitor AM404 significantly reduced the operant
response for ethanol in a dose-dependent manner. Doses of 2.0 and 10.0 mg/kg resulted effective.
Effect of treatment F3,7 =9.237, p<0.01 (Figure 1A). However, when efficacy of AM404 on natural
reinforcers was tested, the results showed that operant responding for 0.2% (w/v) saccharin was not
modified at the range of doses of 0.4 and 2 mg/kg (Figure 1B). In addition, administration of
AM404 did not alter motivation for food intake in rats food-deprived for 24h (Figure 1C). When rats
were tested under PR conditions, break point for ethanol did not result changed by treatment with
AM404 (Figure 1D). These results suggest that the effects derived from an increased
endocannabinoid tone on the reinforcing properties of alcohol are selective for it and do not involve
the motivational value of ethanol, sweet rewards or food.
Figure 1. Acute administration of anandamide transport inhibitor AM404 (0.0, 0.4, 2.0, 10.0 mg/kg, i.p.) 30 min before
the testing procedure in (A) ethanol self-administration, (B)saccharin self-administration, (C) food intake, (D)
progressive ratio paradigm. AM404 reduced alcohol self-administration in male Wistar rats at the highest dose tested (2
and 10 mg/kg). Operant responding for a 0.2% (w/v) saccharin solution and break point for ethanol did not result
modified as well as feeding behaviour in 24-hr food-deprived rats. Data are means ± SEM of 8-10 determinations per
group. (*) p<0.05, versus vehicle-treated animals. (**) p<0.01, versus vehicle treated animals. Operant responding at the
inactive referred to experiments A, B, D did not result modified by the treatment.
81
II. Experiment 2: Effect of AM404 on locomotor activity
As previously reported (Gonzalez et al 1999; Beltramo et al, 2000; Giuffrida et al, 2000), systemic
administration of AM404 (10mg/kg) caused a decrease in motor activity, which was measured as a
decrease of number of crossings This effect was statistically significant at 30, 60,120 min following
AM404 administration. As figure 2 shows, AM404 reduces locomotor activity only at the highest
dose tested (10 mg/kg). Overall treatment effect is F2,24=3.635,p<0.05
FIGURE 2. Effect of AM404 on locomotor behaviour in the open field test. Systemic administration of AM404
(10mg/kg, i.p) caused decrease in motor activity, which was measured in reduction of number of crossings. This effect
was statistically significant at 30, 60 and 120 min following AM404 administration. Data are means ± SEM of 9
determinations per group. (*)p<0.05, versus vehicle-treated animals.
82
III. Experiment 3: Effect of AM404 on relapse to ethanol-seeking behaviour.
The efficacy to suppress not only operant responding for ethanol but also reinstatement to lever
presses elicited by contextual stimuli associated to ethanol was tested with AM404. As shown
previously (Giuffrida et al., 2000) and in the present study, we did not administered the highest dose
of AM404 (10mg/kg) because it resulted in a significant inhibition of locomotor activity. Once a
stable extinction baseline was observed, we induced relapse by presenting cues associated with
ethanol delivery along self-administration training. Ethanol-related contextual stimuli elicited
ethanol-seeking behaviour, since operant response induced by ethanol-associated stimuli was more
intense and significantly higher when compared with the last day of extinction F2,8=8.650, p<0.01
(Figure 3A). When AM404 was injected 30min prior to cue presentation, failed to alter responses for
ethanol-seeking behaviour (Figure 3B).
FIGURE 3. Effects of acute administration of AM404 on cue-induced relapse to ethanol-seeking behaviour. (A) Cueinduced reinstatement of lever pressing under S+/CS+ or S-/CS- conditions. Responses to the active lever were
significantly higher when compared to the last day of extinction. In contrast, under S -/CS- conditions, rats did not
enhance operant responding when compared to the last day of extinction. Data are means ± SEM of 8-10 determinations
per group. (*) p<0.01, versus extinction. (B) AM404 failed to alter the responses for ethanol-seeking behaviour.
Responses at the inactive lever were not modified throughout reinstatement test as well as by the treatment. Data are
means ± SEM of 8-10 determinations per group.
83
IV. Experiment 4: Effect of ACEA and WIN55,212-2 on ethanol self-administration.
As AM404, an indirect agonist of cannabinoid CB1 receptors increasing anandamide brain levels,
reduced ethanol self-administration (figure 1A), we tested also two synthetic cannabinoid agonists
such as ACEA (selective CB1 receptor agonist) and WIN55,212-2 (potent CB1/CB2 receptors
agonist). Both drugs were administered i.p. 30 min prior self-administration sessions and reduced
operant responding for ethanol in a dose-dependent way. ACEA resulted effective at the dose of
2mg/kg (effect of treatment F3,9 =4.033, p<0.05, figure 4A) while WIN55,212-2 suppressed ethanol
at the dose of 5 mg/kg (effect of treatment F3,7 =5.623, p<0.01, figure 4B).
Figure 4. Acute administration of (A) ACEA (0.0, 0.2, 1.0, 2.0 mg/kg, i.p.) and (B) WIN55,212-2 (0.0, 0.4, 2.0,
5.0mg/kg) 30 min before the testing procedure in ethanol self-administration paradigm. Both drug reduced operant
responding for ethanol in a dose-dependent manner. ACEA resulted statistically significant at the higher dose tested (2
mg/kg) while WIN55,212-2 showed effect at 5 mg/kg. In A responses at the inactive lever were not influenced by the
treatment. At the contrary in B, WIN55,212-2 induced significant reduction of operant responding at the inactive lever
(data not shown). Data are means ± SEM of 8-10 determinations per group. (*) p<0.05, versus vehicle-treated animals.
(**) p<0.01, versus vehicle treated animals.
84
V. Experiment 5: Effect of ACEA and WIN55,212 on self-administration of 0.2% (w/v)
saccharin solution.
Given the suppression of operant ethanol self-administration by all cannabinoids tested, we evaluated
the selectivity of this effect measuring the efficacy of ACEA and WIN55,212-2 on operant
responding for natural reinforcers. Data showed that ACEA failed to reduce self-administration for
0.2% (w/v) saccharin while WIN55,212-2 modified in a dose-dependent way not only ethanol (figure
4B) but also self-administration for sweet rewards (effect of treatment F3,5=40.550, p<0.01). Doses
of 2.0 and 5.0 mg/kg resulted statistically significant. We also measured the possible motor effects of
this compound on the open field test but despite a strong reduction of locomotor activity evoked by
WIN55,212-2, these data are not shown here. When open field test, following a challenge with
ACEA, was performed we did not observe changes in locomotor behaviour of treated rats respect to
vehicle group (data not shown).
Figure 5. (A) Effect of CB1 receptor selective agonist ACEA (n=8) and (B) of the potent cannabinoid unselective
agonist WIN55,212-2(n=6) on 0.2% (w/v) saccharin self-administration. Number of lever presses for saccharin did not
modified by ACEA but WIN reduced operant behaviour. In A responses at the inactive lever were not influenced by the
treatment. At the contrary in B, WIN55,212-2 induced significant reduction of operant responding at the inactive lever
(data not shown)Data are means ± SEM of 8-10 determinations per group. (*) p<0.05, versus vehicle-treated animals.
(**)p<0.01 versus vehicle-treated animals.
85
VI. Experiment 6: Effect of cannabinoid CB1 antagonist SR141716A and competitive
VR1 receptor antagonist capsazepine on AM404-inuced reduction of ethanol selfadministration.
In the extent to identify the mechanism of action of cannabinoid agonists in reducing operant
responding for ethanol, we considered the possibility that this effect might be induced by activation
of CB1 cannabinoid brain receptors or by stimulation of vanilloid VR1 receptors given the affinity of
anandamide for vanilloid receptors evidenced in several studies (Zygmunt et al., 1999; 2000; Smart
et al 2000). In this experiment one group of rats received a pre-treatment with the selective CB1
antagonist SR141716A (0.3 mg/kg) or its vehicle 30 min prior to AM404 (2mg/kg) or vehicle
administration. As expected and previously demonstrated in our laboratory (Cippitelli et al., 2005),
pre-treatment with the selective CB1 receptor antagonist SR141716A alone, suppressed ethanol
response, showing similar potency when comparing to AM404. AM404-induced suppression of
ethanol self-administration was not blocked by the selective CB1 antagonist (effect of treatment
F3,7=5.335, p<0.01, figure 6A). In a second experiment a group of animals was pre-treated with the
competitive VR1 antagonist capsazepine (10mg/kg) or vehicle administered 30 min prior to the
injection of AM404 (2mg/kg) in rats self-administering ethanol. The effect of AM404 was not
reversed by pre-treatment with the competitive vanilloid VR1 receptor antagonist capsazepine.
(effect of treatment F3,7=4.851, p<0.05, figure 6B). In order to identify the appropriate dose of
capsazepine to be used in association with AM404, a previous experiment evaluating the doseresponse effect was carried out. None of the doses tested (3, 10 mg/kg) resulted in a significant
alteration in ethanol responding (data not shown).
86
Figure 6. Effect of pre-tratment with (A) SR141716A (0.3 mg/kg) or (B) capsazepine (10mg/kg) on suppression of
ethanol self-administration induced by AM404. The effect of AM404 was not reversed by both antagonists. Data are
means± SEM of 8 determinations per group. (*)p< 0.05 versus vehicle treated animals. Operant responding at the
inactive lever was evaluated throughout the experiments but did not result modified by treatments.
87
Discussion
The major finding of the present study is the demonstration that the increase of endocannabinoid
tone is associated to a reduction of ethanol consumption in operant conditions. Administration of the
anandamide transporter inhibitor AM404 that enhances inter-synaptic endocannabinoid levels
reduces ethanol self-administration, but does not affect the relapse induced by contextual cues
associated to ethanol. AM404 was the first synthetic inhibitor of anandamide uptake (Beltramo et al.,
1997) and has been shown to potentiate many effects elicited by anandamide in vitro (Beltramo et
al., 1997) and in vivo (Beltramo et al., 1997; Calignano et al., 2000). Because of the inability of
AM404 to activate cannabinoid receptors (Beltramo et al., 1997; 2000), the effects of this drug were
suggested to result from the elevation of endogenous anandamide levels. AM404 is not able to
suppress responding for natural reinforcers, such the operant responding for saccharin or food intake
neither break point parameter of progressive ratio was modified suggesting that its effect is not
related to a decrease in a general motivational state. This suppressive effect of AM404 seems to be
independent of known anandamide-induced motor impairment, since the lowest effective dose tested
in reducing ethanol self-administration did not alter motor behaviour in the open field. The selective
CB1 receptor agonist ACEA and the potent agonist WIN55,212-2 reduced ethanol selfadministration. However, the selective CB1 receptor antagonist SR141716A did not prevent AM404
action. Anandamide has been described to act as a partial agonist at ligand-activated cation channel
vanilloid receptor VR1 (Di Marzo et al., 2001; Piomelli, 2001). However, in the present study, the
effect of AM404 was not reversed by pre-treatment with the competitive vanilloid VR1 receptor
antagonist capsazepine, indicating that the inhibitory action of AM404 is not mediated by VR1
stimulation.
We cannot exclude that the effect of AM404 on alcohol consumption could be associated to other
targets such as non-cloned CB1-like receptors (Hajos et al., 2001) or to CB2 receptors that recently
have been found to participate to some central effects of cannabinoid. For example, vomit inhibition
seems to be mediated by CB2 binding site located in brainstem areas, cerebellum and cortex (Van
Sickle et al., 2005).
Operant ethanol self-administration was markedly reduced by treatment with the anandamide
transport inhibitor AM404 but controversial data exist in literature concerning the ability of CB1
agonists to modulate ethanol intake (Colombo et al., 2002), as well as divergent results has been
reported on reward and reinforcement behaviours with others drugs of abuse. These contrasting
results could be attributed to differences in the pharmacological properties and dose range of the
compounds tested, to the strain of the animals used and to the methods followed. Our study is
consistent with others that have examined the actions of various cannabinoid agonist on rewarding
properties of other drugs. For example, it has been shown that WIN55212,2 decreases cocaine
intravenous self-administration in rats (Fattore et al., 1999) and the reinforcing actions of cocaine
(Vlachou et al, 2003); additional studies by Braida and Sala (2002) confirmed that the combination
of the CB1 agonist CP55,940 with MDMA reduced the number of drug-associated lever pressings.
These data together with the present results indicate that cannabinoids compounds such as AM404
may produce reinforcing effects that may substitute those induced by ethanol. Hence, the AM404induced suppressive effect on ethanol self-administration found in these experiments could be
explained as a lower motivation for maintaining a second reward, i.e. ethanol intake, because the
animal previously received a reward–stimulating drug. Alternatively the suppression of ethanol selfadministration could be due to a potentiation of the sedative effect ethanol following increase of the
endocannabinoid tone In addition, pharmacokinetic factors limiting alcohol absorption, metabolism
and excretion might have also influnced our results.
Present results also showed that acute administration of AM404 does not suppress the relapse
response for ethanol, following presentation of contextual cues previously associated to ethanol
consumption. The differential response to AM404 under active ethanol self-administration and
88
conditioned responding during reinstatement, where alcohol is not available, may underly the
possibility that the endocannabinoid system plays a different role in the regulation of these two
distinct mechanisms.
Chronic ethanol treatment has been shown to down-regulate CB1 receptors and related signal
transduction processes. This down-regulation of CB1 receptor function is due to persistent
stimulation of the receptors by anandamide and 2-arachidonylglicerole, the synthesis of which is
increased by chronic ethanol treatment. The enhanced formation of endocannabinoids may
subsequently influence the release of other neurotransmitters (Basavarajappa and Hungund, 2005).
On the other hand, González et al (2004) showed that the levels of endocannabinoids underwent
significant changes in reward-related areas during relapse, showing lowest values in this phase. The
levels of both anandamide and 2-AG were significantly reduced when rats were allowed to relapse to
alcohol use. Thus, the induction of compensatory mechanisms such as up-regulation of the
cannabinoid CB1 receptors and a decrease in the endocannabinoid levels in the status of
endocannabinoid system may be determinant in the actions of AM404 in relapse behaviour to
ethanol-seeking.
In conclusion, our results showing that AM404 administration reduced ethanol self-administration
confirm and give support to the hypothesis that the inhibition of the reuptake of endocannabinoids
can be useful to reduce ethanol intake in alcoholic non abstinent patients. Since it is well known that
the psychotropic effects of cannabinoids are an obstacle to the development of cannabinoid basedtherapy. The possibility of finding a drug that shares the therapeutic efficacy of cannabinoid but
avoids the potential for abuse may be useful to pass the adverse effects of exogenous cannabinoid
administration. For this reason, pharmacological targetting of anandamide transport might be a
promising therapeutic tool for alcoholic patients.
89
References
Arnone M., Maruani J., Chaperon F., Thiebot M.H., Poncelet M., Soubrie P. & Le Fur G. (1997). Selective inhibition of
sucrose and ethanol intake by SR 141716, an antagonist of central cannabinoid (CB1) receptors. Psychopharmacology
(Berl.) 132, 104-106.
Basavarajappa B.S. & Hungund B.L. (2002). Neuromodulatory role of the endocannabinoid signalling system in
alcoholism: and overview. Prostaglandins, Leukotrienes and Essential Fatty Acids; 66, 287-299.
Basavarajappa B.S. & Hungund B.L. (2005). Role of the endocannabinoid system in the development of tolerance to
alcohol. Alcohol alcohol 40(1): 15-24.
Basavarajappa B. S., Saito M., Cooper T. B. and Hungund B. L. (2000). Stimulation of cannabinoid receptor agonist 2arachidonylglycerol by chronic ethanol and its modulation by specific neuromodulators in cerebellar granule neurons.
Biochimica et Biophysica Acta 1535, 78–86.
Beltramo M., Rodríguez de Fonseca F., Navarro M., Calignano A., Gorriti M.A., Grammatikopoulos, G., Sadile, A.G.,
Giuffrida, A.& Piomelli, D. (2000). Reversal of dopamine D2 receptor responses by an anandamide transport inhibitor. J.
Neurosci. 20, 3401-3407.
Beltramo M., Stella N., Calignano A., Lyn S.Y., Makriyannis A. & Piomelli D. (1997). Functional role of high-affinity
anandamide transport, as revealed by selective inhibition. Science 277, 1094-1097.
Braida D. & Sala M. (2002). Role of the endocannabinoid system in MDMA intracerebral self-administration in rats. Br J
Pharmacol. 136, 1089-92.
Calignano A., La Rana G., Beltramo M., Makriyannis A. & Piomelli D. (1997). Potentiation of anandamide hypotension
by the transport inhibitor, AM404. Eur. J Pharmacol. 337, R1-2.
Calignano A., La Rana G., Loubet-Lescoulie P. & Piomelli D. (2000). A role for the endogenous cannabinoid system in
the peripheral control of pain initiation. Prog Brain Res. 129, 471-82.
Carriero D., Aberman J., Lin S.Y., Hill A., Makriyannis A. & Salamone J.D. (1998). A detailed characterization of the
effects of four cannabinoids agonists on operant lever pressing. Psychopharmacology (Berl.) 137, 147-156.
Chaperon F., Soubrie P., Puech A.J. & Thiebot M.H.. (1998). Involvement of central cannabinoid (CB1) receptors in the
establishment of place conditioning in rats. Psychopharmacology (Berl.) 135, 324-332.
Cippitelli A., Bilbao A., Hansson A.C., Del Arco I., Sommer W., Heilig M., Massi M., Bermudez-Silva F., Navarro M.,
Ciccocioppo R. & Rodríguez de Fonseca F. (2005). Cannabinoid receptor CB1 antagonism reduces conditioned
reinstatement of ethanol-seeking behavior in rats. Eur. J. Neurosci., 21, 2243-2251.
Ciccocioppo R., Economidou D., Fedeli A., Angeletti S., Weiss F., Heilig M., Massi M. (2004). Attenuation of ethanol
self-administration and of conditioned reinstatement of alcohol seeking behavior by the anti-opioid peptide nociceptin/
orphanin FQ in alcohol-preferring rats. Psychopharmacology 172:170–178.
Cohen C., Perrault G., Voltz C., Steinberg R. & Soubrie P. (2002). SR141716, a central cannabinoid (CB(1)) receptor
antagonist, blocks the motivational and dopaminereleasing effects of nicotine in rats. Behav Pharmacol. 13, 451-63.
Colombo G., Agabio R., Fa M., Guano L., Lobina C., Loche A., Reali R. & Gessa G.L.. (1998). Reduction of voluntary
ethanol intake in ethanol-preferring sP rats by the cannabinoid antagonist SR-141716. Alcohol Alcohol. 33, 126-130.
Colombo G., Serra S., Brunetti G., Gomez R., Melis S., Vacca G., Carai M.M., Gessa L. (2002). Stimulation of voluntary
ethanol intake by cannabinoid receptor agonists in ethanol-preferring sP rats. Psychopharmacology (Berl). 159, 181-7.
Cossu G., Ledent C., Fattore L., Imperato A., Bohme G.A., Parmentier M. & Fratta W. (2001). Cannabinoid CB1
receptor knockout mice fail to self-administer morphine but not other drugs of abuse. Behav Brain Res. 118, 61-65.
De Vries T.J., Homberg J.R., Binnekade R., Raaso H. & Schoffelmeer A.N. (2003). Cannabinoid modulation of the
reinforcing and motivational properties of heroin and heroin-associated cues in rats. Psychopharmacology (Berl.) 168,
164-169.
90
Di Marzo V., Bisogno T., De Petrocellis L., Brandi I., Jefferson R.G., Winckler R.L., Davis, J.B., Dasse, O., Mahadevan,
A., Razdan, R.K. & Martin BR. (2001). Higly selective CB1 cannabinoid receptor ligands and novel CB1/VR1 vanilloid
receptor “hybrid” ligands. Biochem Biophys Res Commun.; 281, 444-451.
Fattore L., Martellotta M.C., Cossu G., Mascia M.S. & Fratta W (1999). CB1 cannabinoid receptor agonist WIN 55,2122 decreases intravenous cocaine selfadministration in rats. Behav Brain Res. 104, 141-146.
Giuffrida A., Rodríguez de Fonseca F., Nava F., Loubet-Lescouliè P.& Piomelli D. (2000). Elevated circulating levels of
anandamide after administration of the transport inhibitor, AM404. Eur J. Pharmacol 408, 161-168.
Gonzalez S., Romero J., de Miguel R., Lastres-Becker I., Villanua M.A., Makriyannis A., Ramos J.A. & Fernandez-Ruiz
J.J. (1999). Extrapyramidal and neuroendocrine effects of AM404, an inhibitor of the carrier-mediated transport of
anandamide. Life Sci 65, 327-336.
Gonzalez S., Valenti M., de Miguel R., Fezza F., Fernandez-Ruiz J., Di Marzo V. & Ramos J.A. (2004). Changes in
endocannabinoid contents in reward-related brain regions of alcohol-exposed rats, and their possible relevance to alcohol
relapse. Br J Pharmacol. 143, 455-464.
Hajos N., Ledent C. & Freund T.F. (2001). Novel cannabinoid-sensitive receptor mediates inhibition of glutamatergic
synaptic transmission in the hippocampus. Neuroscience. 106, 1-4.
Houchi H., Babovic D., Pierrefiche O., Ledent C., Daoust M. & Naassila M.. (2005). CB1 receptor knockout mice
display reduced ethanol-induced conditioned place preference and increased striatal dopamine D2 receptors.
Neuropsychopharmacology. 30, 339-349.
Huestis M.A., Gorelick D.A., Heishman S.J., Preston K.L., Nelson R.A., Moolchan E.T. & Frank R.A. (2001). Blockade
of effects of smoked marijuana by the CB1- selective cannabinoid receptor antagonist SR141716. Arch Gen Psychiatry.
58, 322- 328.
Hungund B.L., Szakall I., Adam A., Basavarajappa B.S. & Vadasz C. (2003). Cannabinoid CB1 receptor knockout mice
exhibit markedly reduced voluntary alcohol consumption and lack alcohol-induced dopamine release in the nucleus
accumbens. J Neurochem. 84, 698-704.
Le Foll B. & Goldberg S. (2004). Rimonabant, a CB1 antagonist, blocks nicotineconditioned place preferences.
Neuroreport, 15, 2139-2143
Martellotta M.C., Cossu G., Fattore L., Gessa G.L. & Fratta W. (1998). Self-administration of the cannabinoid receptor
agonist WIN 55,212-2 in drug-naive mice. Neuroscience. 85, 327-330.
Martin M., Ledent C., Parmentier M., Maldonado R. & Valverde O. (2000). Cocaine, but not morphine, induces
conditioned place preference and sensitization to locomotor responses in CB1 knockout mice. Eur J Neurosci. 12, 40384046.
Naassila M., Pierrefiche O., Ledent C. & Daoust M. (2004). Decreased alcohol self-administration and increased alcohol
sensitivity and withdrawal in CB1 receptor knockout mice. Neuropharmacology. 46, 243-253.
Navarro M., Carrera M.R., Fratta W., Valverde O., Cossu G., Fattore L., Chowen J.A., Gomez R., del Arco I., Villanua
M.A., Maldonado R., Koob G.F., Rodriguez de Fonseca F. (2001). Functional interaction between opioid and
cannabinoid receptors in drug selfadministration. J Neurosci. 21, 5344-50.
Piomelli D. (2003). The molecular logic of endocannabinoid signalling. Nat Rev Neurosci. 4, 873-884.
Piomelli D. (2001). The ligand that came from within. Trends Pharmacol Sci. 22, 17- 19.
Poncelet M., Maruani J., Calassi R. & Soubrie P. (2003). Overeating, alcohol and sucrose consumption decrease in CB1
receptor deleted mice. Neurosci Lett. 343, 216- 218.
Rinaldi-Carmona M., Barth F., Congy C., Martinez S., Oustric D., Perio A., Poncelet M., Maruani J., Arnone M.,
Finance O., Soubrie P. & Le Fur G. (2004). SR147778 [5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-ethyl-N- (1piperidinyl)-1Hpyrazole- 3-carboxamide], a new potent and selective antagonist of the CB1 cannabinoid receptor:
biochemical and pharmacological characterization. J Pharmacol Exp Ther. 310, 905-914.
91
Rodriguez de Fonseca F., Del Arco I., Bermudez-Silva F.J., Bilbao A., Cippitelli A. & Navarro M. (2005). The
endocannabinoid system: physiology and pharmacology. Alcohol Alcohol. 40, 2-14.
Rodriguez de Fonseca F., Roberts A.J., Bilbao A., Koob G.F. & Navarro M. (1999). Cannabinoid receptor antagonist
SR141716A decreases operant ethanol self-administration in rats exposed to ethanol-vapor chambers. Zhongguo Yao Li
Xue Bao. 20, 1109-1114.
Samson H,H. (1986). Initiation of ethanol reinforcement using a sucrose-substitution procedure in food- and water-sated
rats. Alcohol Clin Exp Res.10, 436-442.
Smart D., Gunthorpe M.J., Jerman J.C., Nasir S., Gray J., Muir A.I., Chambers J.K., Randall A.D. & Davis J.B. (2000).
The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br J Pharmacol. 129, 227230.
Solinas M., Panlilio L.V., Antoniuo K., Pappas L.A. & Goldberg S.R. (2003). The cannabinoid CB1 antagonist NPiperidinyl - 5- (4-chlorophenyl) - 1- (2,4-dichlorophenyl) - 4-methylpyrazole -3-carboxamide / SR141716A)
differentially alters the reinforcing effects of heroin under continous reinforcement, fixed ratio, and progressive ratio
schedules of drug self-administration in rats. J. Pharmacol. Exp. Ther. 306, 93-102.
Solinas M., Panlilio L.V., Tanda G., Makriyannis A., Matthews S.A. & Goldberg SR. (2005). Cannabinoid Agonists but
not Inhibitors of Endogenous Cannabinoid Transport or Metabolism Enhance the Reinforcing Efficacy of Heroin in Rats.
Neuropsychopharmacology. [Epub ahead of print]
Soria G., Mandizábal V., Touriño C., Robledo P., Ledent C., Parmentier M., Maldonado R. & Valverde, O. (2005). Lack
of CB1 cannabinoid receptor impairs cocaine self-administration. Neuropsychopharmacology [Epub ahead of print]
Tanda G. & Punzar P. & Goldberg S.R. (2000). Self-administration behaviour is maintained by the psychoactive
ingredient of marijuana in squirrel monkeys. Nat Neurosci. 3, 1073-1074.
Van Sickle M.D., Duncan M., Kingsley P.J., Mouihate A., Urbani P., Mackie K., Stella N., Makriyannis A., Piomelli D.,
Davison J.S., Marnett L.J., Di Marzo V., Pittman Q.J., Patel K.D., Sharkey K.A. (2005). Identification and functional
characterization of brainstem cannabinoid CB2 receptors. Science 310, 329-332.
Vlachou S., Nomikos G.G. & Panagis G. (2003). WIN 55,212-2 decreases the reinforcing actions of cocaine through
CB1 cannabinoid receptor stimulation. Behav Brain Res 141, 215-222.
Wang L., Liu J., Harvey-White J., Zimmer A. & Kunos G. (2003). Endocannabinoid signaling via cannabinoid receptor 1
is involved in ethanol preference and its agedependent decline in mice. Proc Natl Acad Sci U S A. 100, 1393-8.
Weiss F., Lorang M.T., Bloom F.E. & Koob G.F. (1993). Oral alcohol self-administration stimulates dopamine release in
the rat nucleus accumbens: genetic and motivational determinants. J Pharmacol Exp Ther. 267, 250-258.
Zygmunt P.M., Chuang H., Movahed P., Julius D. & Hogestatt E.D. (2000). The anandamide transport inhibitor AM404
activates vanilloid receptors. Eur J Pharmacol. 396, 39-42.
Zygmunt P.M., Petersson J., Andersson D.A., Chuang H., Sorgard M., Di Marzo V., Julius D. & Hogestatt E.D. (1999).
Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature. 400, 452-457.
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VI
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VI. PPAR-α agonists modulate alcohol craving and
relapse through a peripheral machanism
Introduction
The endogenous cannabinoid anandamide (AEA) (Devane et al., 1992; Di Marzo et al., 1994), the
peripheral satiety factor oleylethanolamide (OEA) (Rodriguez de Fonseca et al., 2001; Fu et al.,
2003), and the analgesic and anti-inflammatory factor palmitoylethanolamide (PEA) (Kuehl et al.,
1957; Calignano et al., 1998; Jaggar et al., 1998) are all members of the fatty acid ethanolamide
(FAE) family of lipid mediators. FAEs are thought to be produced in a stimulus-dependent manner
by activation of N-acylphoshatidylethanolamine-specific phospholipase D (Okamoto et al., 2004)
and mainly degradated by intracellular hydrolysis catalyzed by the serine enzyme fatty acid amide
hydrolase (Di Marzo et al., 1994). AEA and OEA take part in a variety of biological functions,
including regulation of feeding. Anandamide causes overeating in rats because of its ability to
activate cannabinoid receptors (Berry and Mechoulam, 2002). The selective CB1 receptor
antagonist SR141716A (Rinaldi-Carmona et al., 1995) counteracts these effects and, when
administered alone, decreases standard chow intake and caloric consumption (i.e., sucrose or
ethanol intake), presumably by antagonizing the actions of endogenously released endocannabinoids
such as anandamide and 2-arachidonoylglycerol (Arnone et al., 1997; Colombo et al., 1998;
Simiand et al, 1998; Kirkham and Williams, 2001; Rowland et al., 2001). This action is of
therapeutic relevance: cannabinoid agonists such as Δ9-tetrahydrocannabinol are currently used to
alleviate anorexia and nausea in AIDS patients, whereas the CB1 antagonist rimonabant
(SR141716A) was recently found to be effective in latest age clinical trials for the treatment of
obesity (Berry and Mechoulam, 2002). Despite the existence of central mechanisms for regulation
of food intake by endocannabinoids, evidence indicates that peripheral mechanisms unexpectedly
mediated by CB1 receptors also exist (Gomez et al., 2002).In contrast to anandamide, the
monounsaturated FAE oleoylethanolamide decreases food intake and body weight gain through a
cannabinoid receptor-independent mechanism (Rodriguez de Fonseca et al., 2001, Gaetani et al.,
2003). Pharmacological and molecular biological experiments have demonstrated that these effects
result from the high affinity binding of OEA to, and consequent activation of, the nuclear receptor
PPAR-α (peroxisome proliferator-activated receptor-α) (Fu et al.,2003) which serves an essential
function in the regulation of lipid metabolism (Berger and Moller, 2002, Bocher et al., 2002).
In addition, a large body of evidence suggests functional interactions between the effects of
cannabinoids and ethanol and recently it has been shown that the selective CB1 antagonist SR
141716A is able to decrease ethanol self-administration and reinstatement to alcohol-seeking
behaviour in two different lines of rats such as genetically-selected alcohol- preferring rats (msP)
and unselected Wistars (Cippitelli et al, 2005). Due to the structural similarities between AEA and
OEA, to the important role of endogenous cannabinoid system in feeding behaviour and alcohol
addiction and to the anorexic properties of OEA, in this study we have been testing the role of
PPAR-α receptors in experimental models of alcohol abuse and relapse to alcohol-seeking that is
self-administration and cue-induced reinstatement paradigms. To this purpose, a pharmacological
manipulation of PPAR-α receptor using its natural ligand OEA and a second synthetic agonist WY
14643 was carried out.
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Materials and methods
Animals
Male Wistar rats (175-200 gm) were housed individually with food and water available ad libitum,
except when restriction was required. All animal procedures met the National Institutes of Health
guidelines for the care and use of laboratory animals and the European Communities directive
86/609/EEC regulating animal research.
Drugs
Capsaicin was purchased from Sigma (St. Louis, MO) and dissolved in 5% Tween 80, 5%
propyleneglycol, and 90% saline. Oleylethanolamide was synthesized in the laboratory (Giuffrida et
al., 2000) and WY 14643 was purchased from TOCRIS. Both drugs were dissolved in a vehicle
conteining 10%DMSO, 10%TWEEN 80 and 80% distilled water. WY 14643 for icv administration
was dissolved in 50:50 parts of DMSO-water N-piperidino-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methylpyrazole-3- carboxamide (SR141716A) was a gift from Sanofi Recherche
(Montpellier, France) and suspended with 2-3 drops of TWEEN 80 and saline.
Surgery and intracerebroventricular administration of WY 14643
For intracerebroventricular injections, stainless steel guide cannulas aimed at the lateral ventricle
were implanted in the rats. The animals were anesthetized with equithesin and placed in a David
Kopf Instruments (Tujunga, CA) stereotaxic instrument with the incisor bar set at 5 mm above the
interaural line. A guide cannula (7 mm, 23 gauge) was secured to the skull by using two stainless
steel screws and dental cement and was closed with 30 gauge obturators (Gómez et al, 2002). The
implantation coordinates were 0.6 mm posterior to bregma, ±2.0 mm lateral, and 3.2 mm below the
surface of the skull. These coordinates placed the cannula 1 mm above the ventricle. After a 7 d
postsurgical recovery period, cannula patency was confirmed by gravity flow of isotonic saline
through an 8-mm-long, 30 gauge injector inserted within the guide to 1 mm beyond its tip. This
procedure allowed the animals to become familiar with the injection technique. For
intracerebroventricular administration, the obturator was removed from the guide cannula and an 8
mm injector (30 gauge stainless steel tubing) that was connected to 70 cm of calibrated
polyethylene-10 tubing was lowered into the ventricle. The tubing was then raised until flow began,
and 1 µl of a solution containing 0, 1, 3 and 10 micrograms of WY 14643 was infused over a 30 sec
period. The injector was left in the guide cannula for an additional 30 sec and then removed. The
stylet was immediately replaced. Animals were tested 5 min after injections. The
intracerebroventricular cannula placements were evaluated after each experiment by dye injection.
Only rats with proper intracerebroventricular placements were included in the data analysis.
Deafferentation
Capsaicin was administered subcutaneously (12.5 mg/ml) (Kaneko et al., 1998) in rats anesthetized
with ethyl ether. The total dose of capsaicin (125 mg/kg) was divided into three injections (25
mg/kg in the morning and 50 mg/kg in the afternoon, and then 50 mg/kg on the next day). Control
rats received vehicle injections. Experiments were performed 10 d after capsaicin treatment in rats
that had lost the corneal chemosensory reflex (eye wiping for 1–3 min after application of 0.1%
ammonium hydroxide into one eye).
Operant training for liquid reinforcers
Training and testing were conducted in standard operant chambers located in sound-attenuating,
ventilated environmental cubicles. Each chamber was equipped with a drinking reservoir (volume
capacity 0.10 ml) positioned 4 cm above the grid floor in the centre of the front panel of the
chamber, and two retractable levers were located 3 cm to the right and left of the drinking
receptacle. Auditory and visual stimuli were presented via a speaker and a light located on the front
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panel. A microcomputer controlled the delivery of fluids, presentation of auditory and visual
stimuli, and recording of the behavioural data. Rats were trained to self-administer 10% (v/v)
ethanol (48 rats), 0.2% (w/v) saccharin (eight rats), 10% sucrose (eight rats) or water (eight rats) in
30-min daily sessions on a fixed ratio 1 schedule of reinforcement, where each response resulted in
delivery of 0.1 ml of fluid, as previously described (Weiss et al., 1993). Briefly, for the first 3 days
of training, water availability in the home cage was restricted to 2 h per day in order to facilitate
acquisition of operant responding for a liquid reinforcer. During this time, lever pressing reinforced
by 0.2% (w/v) saccharin solution was established. At this point water was made freely available,
and saccharin self-administration training continued until animals reached stable baseline
responding. A separate subset of rats from the saccharin-trained group were then trained to selfadminister ethanol by using a modification of the sucrose-fading procedure (Samson, 1986) that
used saccharin instead of sucrose (Weiss et al., 1993). During the first 6 days of this ethanol
initiation phase a 5% (w/v) ethanol solution containing 0.2% saccharin (w/v) was available to the
rats. Starting on day 7, the concentration of ethanol was gradually increased from 5.0% to 8.0% and
finally to 10.0% (w/v), whereas the concentration of saccharin was correspondingly decreased to
0%. At the beginning of the saccharin-fading procedure a second, inactive lever was introduced.
During all training and testing phases responses at this lever were recorded as a measure of nonspecific behavioural activation, but they had no programmed consequences.
Ethanol self-administration: effect of OEA and WY 14643
Following completion of the saccharin fading procedure Wistar rats (n= 8 per group) were trained in
sessions of 30 min per day to lever-press for 10% ethanol (0.1 ml per response) until stable baseline
of responding was reached. We studied the effect of PPAR-α receptor agonists OEA (0.0, 1.0, 5.0,
10.0 mg/kg) and WY 14643(0.0, 1.0, 5.0, 20.0, 40.0 mg/kg given i.p., 30 min prior to a 30 min selfadministration session. Experiments were conducted every fourth day using a Latin square
counterbalanced design. Responding at the inactive lever was recorded throughout the experiment to
monitor non-specific behavioural effects.
Saccharin self-administration: effect of OEA and WY 14643
Wistar rats (n= 8 per group) were trained in sessions of 30 min per day to lever-press for a saccharin
0,2% liquid solutions (0.1 ml per response) until stable baseline of responding was reached. We
studied the effect of PPAR-α receptor agonists OEA (0.0, 1.0, 5.0, 10.0 mg/kg) and WY 14643 (0.0,
1.0, 5.0, 20.0, 40.0 mg/kg) given i.p., 30 min prior to a 30 min self-administration session.
Experiments were conducted every fourth day using a Latin square counterbalanced design.
Responding at the inactive lever was recorded throughout the experiment to monitor non-specific
behavioural effects.
Reinstatement of ethanol-seeking behaviour: effect of OEA and WY
Conditioning phase
At completion of the fading procedure, in 30-min daily sessions, animals were trained to
discriminate between 10% ethanol and water. Beginning with self-administration training at the
10% ethanol concentration, discriminative stimuli (SD) predictive of ethanol vs. water availability
were presented during the ethanol and water self-administration sessions, respectively. The
discriminative stimulus for ethanol consisted of the odour of an orange extract (S +) whereas water
availability (i.e. non-reward) was signalled by an anise extract (S–). The olfactory stimuli were
generated by placing 6–8 drops of the respective extract into the bedding of the operant chamber. In
addition, each lever-press resulting in delivery of ethanol was paired with illumination of the
chamber’s house light for 5 s (CS+). The corresponding cue during water sessions was a 5-s tone (70
dB) (CS–). Concurrently with the presentation of these stimuli, a 5-s time-out period was in effect,
during which responses were recorded but not reinforced. The olfactory stimuli serving as S+ or S–
for ethanol or water availability were introduced 1 min before extension of the levers and remained
96
present throughout the 30-min sessions. The bedding of the chamber was changed and bedding trays
were cleaned between sessions. During the first 3 days of the conditioning phase the rats were given
ethanol sessions only. Subsequently ethanol and water sessions were conducted in random order
across training days, with the constraint that all rats received a total of ten ethanol and ten water
sessions.
Extinction phase
After the last conditioning day, rats were subjected to 30 min extinction sessions for 15 consecutive
days. During this phase, sessions began by extension of the levers without presentation of the
discriminative stimuli. Responses at the lever activated the delivery mechanism but did not result in
the delivery of liquids or the presentation of the response-contingent cues (house light or tone).
Reinstatement testing
Reinstatement tests began the day after the last extinction session. This test lasted 30 min under
conditions identical to those during the conditioning phase, except that alcohol and water were not
made available. Half the animals were tested under the S+ ⁄ CS+ condition on day 1 and under the S–
/CS– condition on day 2. The order of cue presentation was inverted for the remaining rats.
Reinstatement experiments were conducted every fourth day (on days 6, 10, 14) and OEA and WY
14643 were administered i.p. 30 min prior to the sessions. Responding at the inactive lever was
constantly recorded to monitor possible non-specific behavioural effects.
Open-field test
Motor behaviours in the open field were studied in an opaque open field (100 × 100 × 40 cm) as
described previously (Beltramo et al., 2000). The field was illuminated using a ceiling halogen lamp
regulated to yield 350 lux at the centre of the field. Rats were habituated to the field for 10 min for
two days before testing. The test was carried out following a Latin square counterbalanced design
on days 3, 6, 9. On the experimental days, the animals were treated and 30 min later placed in the
centre of the field, and locomotor activity (number of lines crossed or distance travelled and
immobility time) and rearing and grooming behaviour (number of rearings and time spent
grooming) were scored for 10 min after drug injection. Behaviour was scored by trained observers
who were unaware of the experimental conditions.
Statistics
Statistical significance of behavioural studies was assessed by analysis of variance (ANOVA). All
the studies were performed in counterbalanced within subject design. Following a significant F
value, post hoc analysis (Student-Newman-Keuls) was performed to assess specific comparisons
between dose groups.
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Results
I. Ethanol self-administration: effect of OEA and WY 14643
When OEA and WY 14643 were injected intraperitoneally 30 min prior to the session, both
suppressed ethanol self-administration; OEA resulted effective at a lower range of doses (5 and 20
mg/kg, Fig. 1A) compared with WY 14643 (20 and 40 mg/kg; Fig. 1B) (treatment effect for OEA
F3,7=5.053, p<0.01) (treatment effect for WY F4,7=2.587, p<0.05). The number of responses at the
inactive lever was evaluated throughout the experiment but it was not influenced by treatment (data
not shown).
Figure 1. A Effect of OEA (0, 1, 5, 20 mg/kg) and B WY 14643 (0, 1, 5, 20, 40 mg/kg) on 10% ethanol selfadministration. Treatment with OEA 30 min prior self-administration sessions reduces lever pressing for ethanol at the
doses of 5, 20 mg/kg, i.p.) (n=8). Treatment with WY 14643 30 min prior self-administration sessions reduces lever
pressing for ethanol at the doses of 20, 40 mg/kg, i.p.) (n=8). * p <0.05 different from vehicle group. **p<0.01 different
from vehicle group.
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II. Saccharin self-administration: effect of OEA and WY 14643
OEA and WY 14643 dose-dependently suppress 0.2% saccharin solution self-administration when
injected intraperitoneally 30 min prior to the session. Both drugs resulted effective at the same range
of doses (5 and 20 mg/kg) (treatment effect for OEA F3,7=28.559, p<0.01Fig 2A) (treatment effect
for WY F3,7=12.664, p<0.01;Fig 2B). The number of responses at the inactive lever was evaluated
throughout the experiment but it was not influenced by treatment (data not shown).
Figure 2. (A) Effect of OEA (0, 1, 5, 20 mg/kg) and (B) WY 14643 (0, 5, 20 mg/kg) on 0.2% saccharin selfadministration. Treatment with both compounds 30 min prior self-administration sessions reduces lever pressing for the
sweet reward at the doses of 5, 20 mg/kg, i.p.) (n=8 per group).**p<0.01 different from vehicle group
III. Effect of WY 14643 on locomotor activity
The synthetic PPAR-α agonist WY 14643, administered intraperitoneally at doses of 0, 5, 20 mg/kg,
30 min prior the performance in the open field lasting 10 min, did not modified locomotor
behaviour. Evaluation of number of crossings and immobility time did not result statistically
significant in treated rats compared to vehicle group. As reported previously OEA did not result
effective in reducing locomotor behaviour at the doses of 5 but the dose of 20 mg/kg was found
working in habituated animals (Rodriguez de Fonseca, 2001)
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Figure 3. Effect of WY 14643 (0, 5, 20 mg/kg) on locomotor activity in the open field test. Intraperitoneal treatment
30 min prior the performance lasting 10 min (n=10 per group) did not modify significatively number of crossings and
immobility time
IV. Effect of OEA, WY 14643 and SR 141716A on rats submitted to sensory
deafferentation
Treatment with neurotoxin capsaicin prevented OEA (5 mg/kg, i.p.) and WY 14643 (20 mg/kg, i.p.)
effect on reducing ethanol self-administration (Fig 4.A) but not that induced by the centrally acting
CB1 antagonist SR 141716A (3 mg/kg, i.p., Fig. 4B) indicating that sensory terminals had been
destroyed.**F1,6=18.946, p<0.01. Both experiment were conducted following a within subject
design. The number of responses at the inactive lever was evaluated throughout the experiment but
it was not influenced by treatments (data not shown).
V. Central administration of WY 14643
WY 14643 does not affect ethanol self-administration when administered icv at the doses of 1 and 3
µg/rat (Fig. 4C) supplying the confirmation of a peripheral effect of PPAR-α agonists on operant
lever-pressing for ethanol. The experiment was carried out following a within subject design. The
number of responses at the inactive lever was evaluated throughout the experiment but it was not
influenced by treatment (data not shown).
100
Figure. 4. Ethanol self-administration of rats submitted to deafferentation following a challenge of OEA, WY 14643
and SR141716A. (A) Rats treated with PPAR- α agonists (OEA 5 and WY 20 mg/kg, ip)(n=7 per group) continue to
press the lever as the vehicle group whereas (B) CB1 antagonist SR 141716A reduces operant responding for ethanol
(n=7).** p<0.01 compared with vehicle group. (C) Icv administration of WY 14643 (1, 3 µg/rat) on ethanol self –
administration. Response to the active lever was not modified in treated rats compared to vehicle group (n=9).
VI. Effect of OEA and WY 14643 on cue-induced reinstatement
When both PPAR-α agonists were injected intraperitoneally 30 min prior to the session resulted in a
suppression of cue-induced reinstatement of lever pressing. Effect of OEA and WY 14643 resulted
in a progressing decrement of response but in the case of OEA it was effective at a lower range of
doses (1, 5 and 20 mg/kg, Fig. 5A) when compared with WY 14643 effect (20 and 40 mg/kg; Fig.
5B)(treatment effect for OEA F3,8=12.014, p<0.01) (treatment effect for WY F3,7=5.407, p<0.01).
Both experiments were conducted following a Latin square counterbalanced design. The number of
responses at the inactive lever was evaluated throughout the experiment but it was not influenced by
treatment (data not shown).
101
Figure. 5. Effect of OEA (n=9) and WY 14643 (n=8) on cue-induced relapse to ethanol-seeking behaviour. (A) OEA
(1, 5, 20 mg/kg, i.p.) provokes a statistically significant reduction at all doses tested whereas (B) WY14643 (5, 20, 40
mg/kg) induces reduction of reinstatement at the higher doses of 20 and 40 mg/kg. *p<0.05 compared with vehicle
group; **p<0.01 compared with vehicle group
Discussion
Oleyletanolamide (OEA) is an endogenous lipid that belongs to fatty acid ethanolamide (FAE)
family of lipid mediators and contributes to the regulation of the food intake through activation of
nuclear receptor PPAR-α in peripheral sensory fibres. Then, peripheral inputs related to appetite
suppression recruit CNS structures involved in the control of satiety as the paraventricular
hypothalamic nucleus and nucleus of the solitary tract in brainstem (Rodriguez de Fonseca et al.,
2001; Fu et al., 2003). In this study we tested the effects of OEA and WY 14643, a synthetic PPARα agonist in ethanol self administration and in cue-induced reinstatement to ethanol-seeking
behaviour paradigms. Results show that both compounds affect operant responding for ethanol and
also stimuli-conditioned reinstatement of lever pressing. The endogenous ligand of PPAR-α
receptor OEA is more effective in reducing ethanol self-administration when compared to WY
14643 according to the major affinity of OEA for its cognate receptor. These results identify the role
of PPAR-α receptor in the regulation of the motivational and addictive behaviour. Similar results
have been found for the effect of OEA and WY14643 on cue-induced reinstatement paradigm. In
addition both compounds resulted effective in decreasing lever pressing a sweet reward liquid
consisting on 0.2% (w/v) saccharin solution. The effect of reducing operant responding for both
reinforcers tested is not due to a general sedation evoked by a selective activation of PPAR-α
102
receptors since when the open field test was carried out, locomotor and exploratory activities of
WY 14643 -treated rats did not result modified despite a dose-effect trend to decrease locomotor
behaviour was noticed. As reported previously, OEA did not result effective in reducing horizontal
activity at the doses of 5 but the dose of 20 mg/kg was found working in habituated animals
(Rodriguez de Fonseca, 2001). The reason of such especific effect could be found in the role of
PPAR-α receptor and OEA in the neural processes responsible for the regulation of consumption
and intake behaviours.
Despite PPARα receptors are localized not only in peripheral sensory terminals but also in CNS and
particularly in limbic system, the speculation that anandamide and oleoylethanolamide act in a
coordinated manner to control feeding responses through opposing actions on sensory nerve
terminals within the gut (Rodriguez de Fonseca et al., 2001) led to us to the hypothesis that OEA
might be able to reduce motivation for ethanol acting on peripheral sites. In order to find out the site
of action of OEA, we treated rats able to self-administer ethanol with the neurotoxin capsaicin to
induce deafferentation of the peripheral sensory fibres. The result that rats submitted to sensory
denervation and treated with OEA and WY 14643 continue to self-administer ethanol and the
treatment with SR141716A, selective antagonist of centrally located CB1 receptors results effective
in decreasing this task suggest that the effect of PPAR-α agonists is related to a peripheral
mechanism that not affect directly the central nervous system. In addition when
intracerebroventricular administration of WY 14643 was carried out, no changes in alcohol
consumption were seen according to the fact that the reduction of ethanol self-administration is dued
to a peripheral mechanism.
The effects of such PPARα receptor agonists on relapse to alcohol-seeking reflect the same results
obtained in self-administration paradigm. It means that OEA and WY 14643 are able to induce
satiety not only when the reinforcer is available but also when environmental stimuli are associated
to the reinforcer. Processes of learning and memory storage underlie this association and
conditioning. A recent study indicates the role of the nucleus of the solitary tract in the processes of
memory storage after its stimulation (Miyashita and Williams, 2003). In this sense, the stimulation
of the solitary tract nucleus induced by the ethanol might play a pivotal role in transmission of
signals processing the memory of emotional experiences from peripheral areas to the central
nervous system. OEA might to be inhibiting craving and relapse for alcohol acting at this level.
103
References
Arnone M., Maruani J., Chaperon F., Thiebot M.H., Poncelet M., Soubrie P., Le Fur G. (1997). Selective inhibition of
sucrose and ethanol intake by SR 141716, an antagonist of central cannabinoid (CB1) receptors. Psychopharmacology
132:104–106.
Beltramo M., Rodriguez de Fonseca F., Navarro M., Calignano A., Gorriti MA., Grammatikopoulus G., Sadile G.,
Giuffrida A., Piomelli D. (2000). Reversal of dopamine D2 receptor responses by an anandamide transport inhibitor. J
Neurosci 20:3401–3407.
Berger J., and Moller D. E. (2002). The mechanisms of action of PPARs. Annu. Rev. Med. 53, 409–435
Berry E. M., and Mechoulam R. (2002). Tetrahydrocannabinol and endocannabinoids in feeding and appetite.
Pharmacol. Ther. 95, 185–190
Bocher V., Chinetti G., Fruchart J. C., and Staels B. (2002). [Role of the peroxisome proliferator-activated receptors
(PPARS) in the regulation of lipids and inflammation control] J. Soc. Biol. 196, 47–52
Calignano A, La Rana G, Giuffrida A, and Piomelli D (1998). Control of pain initiation by endogenous cannabinoids.
Nature (Lond) 394:277–281.
Cippitelli A., Bilbao A., Hansson A.C., Del Arco I., Sommer W., Heilig M., Massi M., Bermudez-Silva F.J., Navarro
M., Ciccocioppo R., de Fonseca F.R.; (2005). The European TARGALC Consortium. Cannabinoid CB1 receptor
antagonism reduces conditioned reinstatement of ethanol-seeking behavior in rats. Eur J Neurosci. Apr; 21(8):2243-51.
Colombo G., Agabio R., Fa M., Guano L., Lobina C., Loche A., Reali R., Gessa G.L. (1998). Reduction of voluntary
ethanol intake in ethanol-preferring sP rats by the cannabinoid antagonist SR-141716. Alcohol Alcohol 33:126–130.
Devane W.A., Hanus L., Breuer A., Pertwee R.G., Stevenson L.A., Griffin G., Gibson D., Mandelbaum A., Etinger A.
and Mechoulam R. (1992). Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science.
258, 1946-1949.
Di Marzo V., Fontana A., Cadas H., Schinelli S., Cimino G., Schwartz J.C., and Piomelli D. (1994). Formation and
inactivation of endogenous cannabinoid anandamide in central neurons. Nature (Lond) 372:686–691.
Fu J., Gaetani S., Oveisi F., Lo Verme J., Serrano A., Rodriguez de Fonseca F., Rosengarth A., Luecke H., Di Giacomo
B., Tarzia G., and Piomelli D. (2003). Oleylethanolamide regulates feeding and body weight through activation of the
nuclear receptor PPAR-alpha. Nature 425, 90–93.
Gaetani S., Oveisi F., and Piomelli, D. Modulation of meal pattern in the rat by the anorexic lipid mediator
oleoylethanolamide. (2003). Neuropsychopharmacology 28, 1311–1316
Giuffrida A., Rodriguez de Fonseca F., Piomelli D. (2000) Quantification of bioactive acylethanolamides in rat plasma
by electrospray mass spectrometry. Anal Biochem 280:87–93
Gomez R., Navarro M., Ferrer B., Trigo J.M., Bilbao A., Del Arco I., Cippitelli A., Nava F., Pomelli D. and Rodriguez
de Fonseca F. (2002). A peripheral mechanism for CB1 cannabinoid receptor-dependent modulation of feeding. Journal
of Neuroscience 22, 9612–9617.
Jaggar S.I., Hasnie F.S., Sellaturay S., and Rice A.S. (1998). The anti-hyperalgesic actions of the cannabinoid
anandamide and the putative CB2 receptor agonist palmitoylethanolamide in visceral and somatic inflammatory pain.
Pain 76:189–199.
Kaneko H., Kaunitz J., Tache Y. (1998). Vagal mechanisms underlying gastric protection induced by chemical
activation of raphe pallidus in rats. Am J Physiol 275:G1056–G1062
Kirkham T.C., Williams C.M. (2001). Synergistic effects of opioid and cannabinoid antagonists on food intake.
Psychopharmacology 153:267– 270.
Kuehl F.A., Jacob T.A., Ganley O.H., Ormond R.E., and Meisinger M.A.P. (1957). The identification of N-(2hydroxyethyl) palmitamide as a naturally occurring anti-inflammatory agent. J Am Chem Soc 79:5577–5578.
104
Miyashita T., Williams C.L. Enhancement of noradrenergic neurotransmission in the nucleus of the solitary tract modulates
memory storage processes. (2003). Brain Research.Oct17; 987 (2), 164-175
Okamoto Y., Morishita J., Tsuboi K., Tonai T., and Ueda N. (2004). Molecular characterization of a phospholipase D
generating anandamide and its congeners. J Biol Chem 279:5298–5305.
Rinaldi-Carmona M., Barth F., Heaulme M., Alonso R., Shire D., Congy C., Soubrie P., Breliere J.C., Le Fur G. (1995).
Biochemical and pharmacological characterisation of SR141716A, the first potent and selective brain cannabinoid
receptor antagonist. Life Sci 56:1941–1947.
Rodriguez de Fonseca F., Navarro M., Gomez R., Escuredo L., Nava F., Fu J., Murillo-Rodriguez E., Giuffrida A.,
LoVerme J., Gaetani S., Kathuria S., Gall C., and Piomelli D. (2001). An anorexic lipid mediator regulated by feeding.
Nature 414, 209–212
Rowland N.E., Mukherjee M., Robertson K. (2001). Effects of the cannabinoid receptor antagonist SR 141716, alone
and in combination with dexfenfluramine or naloxone, on food intake in rats. Psychopharmacology 159:111–116.
Simiand J., Keane M., Keane P.E., Soubrie P. (1998). SR 141716, a CB1 cannabinoid receptor antagonist, selectively
reduces sweet food intake in marmoset. Behav Pharmacol 9:179–181.
Weiss F., Lorang M.T., Bloom F.E. & Koob G.F. (1993). Oral alcohol self-administration stimulates dopamine release
in the rat nucleus accumbens: genetic and motivational determinants. J. Pharmacol. Exp. Ther., 267, 250–258.
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VII
106
VII. GENERAL DISCUSSION
The results demonstrated that the endocannabinoid system plays a foundamental role in the
regulation of the motivational effects of ethanol. Manipulation of the system, in fact, resulted in a
marked inhibition of alcohol drinking as well as in a reduction of conditioned reinstatement of
alcohol-seeking. Moreover, as shown in Chapter 3, innate functional differences of the
endocannabinoid system may represent an important genetic trait increasing predisposition to
develop alcohol abuse. For instance we have demonstrated that at least in a strain of rats bred for its
ethanol preference, the msP line, there is increased cannabinoid CB1 receptor mRNA expression in
brain areas relevant for the processing of reward and reward-associated behaviors. Ethanol drinking
in these animals appears to reduce CB1 gene expression, and this effect is most pronounced in the
rostral part of the caudate putamen, the region with the strongest difference in expression between
msP and Wistar rats. These changes have functional relevance because, then, msP rats are more
sensitive to SR141716A-induced inhibition of ethanol drinking and reinstatement of alcohol
seeking. These results are in line with genetic reports in humans and behavioural findings in
genetically modified animals in which a relevant role for either the cannabinoid CB1 receptor or the
endocannabinoid-degradating enzyme FAAH was recently described (Schmidt et al., 2002; Sipe et
al., 2002; Hungund et al., 2003; Wang et al., 2003). For example, two studies have linked
cannabinoid CB1 receptors and FAAH enzyme to severe alcohol dependence (Schmidt et al., 2002;
Sipe et al., 2002), and we have evidence of endocannabinoid–alcohol intake interaction in CB1
knockout mice (Hungund et al., 2003; Wang et al., 2003).
What is apparently surprising is that, as shown in Chapter 3-5, ethanol drinking is significantly
reduced following blockade of the CB1 receptor by SR141716A but also following direct activation
of this receptors by the CB1 selective agonist ACEA or by modulation of the endogenous
endocannabinoid levels subsequent to treatment with the transported inhibitor AM404. This paradox
can be, however, explained if one consider that the motivation to ethanol drinking can be modulated
bidirectionally by blocking its rewarding effect but also by substitution of its pharmacological
actions. Based on the results of our work, we hypothesize that the blockade of the CB1 receptors
results in a marked inhibition of the motivation for ethanol. This explains also why SR141716A not
only reduced ethanol self-administration but also decreased reinstatement of alcohol-seeking
motivated by conditioning factors. The SR-141716A efficacy in controlling operant-reinforced
responding is not limited to ethanol, but also extends to sucrose and saccharin self-administration,
two natural reinforcers (Cippitelli et al., 2005) and interestingly only modestly reduces lever
pressing for a NaCl solution in Na-depleted animals (Economidou et al., 2006). Sodium depletion is
known to induce a strong motivational state that is largely controlled by homeostatic factors (Clark
and Ilene 2004). Under these circumstances, therefore, the hedonic nature of the stimulus plays only
a modest role in motivating animals’ consummatory responses. The failure of SR-141716A to
strongly reduce NaCl self-administration led to speculate that the endogenous cannabinoid system is
critically involved in the control of goal-directed behaviours motivated by stimuli with high hedonic
impact, regardless of whether they are pharmacological in nature (alcohol) or natural (saccharin or
sucrose). Conversely, the endocannabinoid system does not seem to play a major role in the control
of behaviours motivated by need-state conditions (i.e. Na-depletion) (Economidou et al., 2006). Our
results highlight the importance of considering a cannabinoid CB1 receptor antagonist-based
therapy not only for alcohol consumption but also for context-induced promotion of relapse to
alcohol drinking, one of the major problems seen in alcoholism therapy.
Of interest however, is also that treatments aimed at increasing cannabinoid neurotransmission
decreases ethanol consumption. However in this case, as shown for ACEA and AM404 drug
treatments result in a potent inhibition of alcohol drinking while conditioned reinstatement of
107
alcohol-seeking is only modestly affected if any. In addition, at appropriate doses these compounds
selectively reduces alcohol self-administration leaving unaltered operant responding for both food
and sucrose. Overall these results suggest that the increase of the cannabinoid system specifically
reduces the motivation to ethanol consumption by substitution mechanisms that cannot generalize to
natural rewards consumption and is relevant only if ethanol is consumed (i.e., relapse in the absence
of ethanol is not affected). The substitution hypothesis should also help to reconcile the present
results with already published data showing that activation of CB1 receptors increases alcohol
consumption. In fact Colombo et al. (2002) found an enhancement in voluntary ethanol intake in
ethanol-preferring sP rats following activation of CB1 receptors and Gallate et al. (1999) reported
that the cannabinoid receptor agonist CP55940 dose-dependently increased responding for beer, an
effect that was reversed by SR141716A. In these studies authors used a two bottle choice paradigm
to study ethanol consumption, a condition in which the motivational requirement to obtain ethanol is
lower to that needed when ethanol is operantly self-administered. Hence, if the increase of
endocannabinoid neurotransmission result in a partial substitution of motivational ethanol effects it
can reduce drug consumption when its availability is contingent to high motivational requirement
(i.e., operant self-administration); whereas under condition of freely access to alcohol, due to a low
level of motivational requirement it increases intake by “priming” for the effect of the drug.
Overall the results of the studies presented here provide strong rational for the involvement of the
endocannabinoid system in the regulation of alcohol drinking and relapse. This system should be
considered an important target for the development of novel pharmacotherapy for treatment of
alcoholism. At present, the best candidate for further development is SR141716A (Rimonabant®)
that is already in clinical Phase III for treatment of obesity and nicotine dependence.
108
References
Cippitelli A., Bilbao A., Hansson A.C., Del Arco I., Sommer W., Heilig M., Massi M., Bermudez-Silva F., Navarro M.,
Ciccocioppo R. & Rodríguez de Fonseca F. (2005). Cannabinoid receptor CB1 antagonism reduces conditioned
reinstatement of ethanol-seeking behavior in rats. Eur. J. Neurosci., 21, 2243-2251.
Cippitelli A., Bilbao A., Navarro M.,Gorriti M.A., Massi M., Pomelli D., Ciccocioppo R. and Rodríguez de Fonseca F.
Selective reduction of ethanol self-administration by the Anandamide trasport inhibitor AM 404. (Eur J Neurosci. 2006
second revision)
Clark J.J., Ilene B.L. (2004). Reciprocal cross-sensitization between amphetamine and salt appetite. Pharmacol Biochem
Behav 78: 691–698
Colombo G., Serra S., Brunetti G., Gomez R., Melis S., Vacca G., Carai M.M., Gessa L. (2002). Stimulation of voluntary
ethanol intake by cannabinoid receptor agonists in ethanol-preferring sP rats. Psychopharmacology (Berl). 159, 181-7.
Economidou D., Mattioli L., Cifani C., Perfumi M., Massi M., Cuomo V., Trabace L., Ciccocioppo R. (2006). Effect of
the cannabinoid CB1 receptor antagonist SR-141716A on ethanol self-administration and ethanol-seeking behaviour in
rats Psychopharmacology (Berl). Jan; 183(4):394-403.
Gallate J.E., Saharov T., Mallet P.E., McGregor I.S. (1999). Increased motivation for beer in rats following
administration of a cannabinoid CB1 receptor agonist. Eur. J. Pharmacol. 370, 233–240
Hungund B.L., Szakall I., Adam A., Basavarajappa B.S. & Vadasz C. (2003). Cannabinoid CB1 receptor knockout mice
exhibit markedly reduced voluntary alcohol consumption and lack alcohol-induced dopamine release in the nucleus
accumbens. J. Neurochem., 84, 698–704.
Schmidt L.G., Samochowiec J., Finckh U., Fiszer-Piosik E., Horodnicki J., Wendel B., Rommelspacher H. & Hoehe
M.R. (2002). Association of a CB1 cannabinoid receptor gene (CNR1) polymorphism with severe alcohol dependence.
Drug Alcohol Depend., 65, 221–224.
Sipe J.C., Chiang K., Gerber A.L., Beutler E. & Cravatt B.F. (2002). A missense mutation in human fatty acid amide
hydrolase associated with problem drug use. Proc. Natl Acad. Sci. USA, 99, 8394–8399.
Wang L., Liu J., Harvey-White J., Zimmer A. & Kunos G. (2003). Endocannabinoid signaling via cannabinoid receptor
1 is involved in ethanol preference and its age-dependent decline in mice. Proc. Natl Acad. Sci. USA, 100, 1393–1398.
109
ACKNOWLEDGEMENTS
This work has been started in Prof. Rodriguez de Fonseca’ s laboratory at the Department of
Psychobiology, Complutense University of Madrid, Spain and finished in Prof. Ciccocioppo’s
laboratory at the Department of Experimental Medicine and Public Health, University of Camerino.
I wish to express my gratitude to all the people that has helped me during this work. In particular I
would like to thank:
Prof. Roberto Ciccocioppo and Fernando Rodriguez de Fonseca, my supervisors, for giving me a
chance and for introducing me into scientific research.
Ainhoa Bilbao, for carrying out with me most of the experiments here presented.
All the guys working in Camerino’s group and in particular: Amalia Fedeli, Daina Economidou,
Nazzareno Cannella, Laura Soverchia, Massimo Ubaldi, Anbarasu Lourdusamy, Admir
Malaj, Alfredo Fiorelli and Marino Cucculelli.
And finally to Prof. Maurizio Massi, prof. Marina Perfumi, prof. Miguel Navarro, Luis Franco,
Santiago Climent, Anita Hansson, Wolfgang Sommer, Markus Heilig and specially to my
family.
110