Elsevier

Neuropharmacology

Volume 54, Issue 6, May 2008, Pages 976-988
Neuropharmacology

CB1 receptor blockade reduces the anxiogenic-like response and ameliorates the neurochemical imbalances associated with alcohol withdrawal in rats

https://doi.org/10.1016/j.neuropharm.2008.02.005Get rights and content

Abstract

There is strong evidence that blocking CB1 receptors may reduce alcohol intake in alcohol-dependent individuals. However, there is still limited evidence that CB1 receptor antagonists may also be beneficial in the attenuation of alcohol withdrawal syndrome, even though alcohol withdrawal appears to be milder in CB1 receptor knockout mice. Here we have examined whether the CB1 receptor antagonist rimonabant (SR141716) can alleviate the behavioral symptoms and revert the neurochemical imbalance elicited by a 3-h interruption of chronic alcohol exposure (7.2% in the drinking water for 10 days) in male Wistar rats. Administration of rimonabant attenuated the strong anxiogenic traits of the animals that developed when regular alcohol intake was interrupted. This may reflect the correction of the GABA/glutamate imbalances developed by the animals that received rimonabant in various brain regions involved in emotional (e.g. prefrontal cortex) and motor (e.g. caudate-putamen and globus pallidus) responses. In addition, rimonabant also affected the dopamine deficits generated by alcohol abstinence in the amygdala and ventral-tegmental area, albeit to a lesser extent. However, this antagonist was unable to correct the impairment caused by alcohol abstinence in serotonin and neuropeptide Y. The endocannabinoid activity in the brain of alcohol-abstinent rats indicated that the behavioral and neurochemical improvements caused by rimonabant were not related to the attenuation of a possible increase in this activity generated by alcohol withdrawal. Conversely, the density of CB1 receptors was reduced in alcohol-abstinent animals (e.g. globus pallidus, substantia nigra), as were the levels of endocannabinoids and related N-acylethanolamines (e.g. amygdala, caudate-putamen). Thus, rimonabant possibly enhances an endogenous response generated by interrupting the regular use of alcohol. In summary, rimonabant might attenuate withdrawal symptoms associated with alcohol abstinence, an effect that was presumably due to the normalization of GABA and glutamate, and to a lesser extent, dopamine transmission in emotion- and motor-related areas.

Introduction

The endogenous cannabinoid system serves as a messenger system that is active in different brain processes, as well as in the periphery. The different elements in this pathway have been identified over the last 20 years (reviewed in Mackie and Stella, 2006) and are formed by: (i) endogenous ligands mostly derived from fatty acids; (ii) the enzymes involved in their metabolism; (iii) membrane receptors and in particular two G protein-coupled receptors called CB1 and CB2; and (iv) an inactivation mechanism that terminates the biological signal mediated by endocannabinoids (Mackie and Stella, 2006). Recent studies have postulated that this system might be involved in physiological processes that are altered in addictive states, which is consistent with the identification of endocannabinoid ligands, enzymes and receptors in addiction-related areas, and in particular the CB1 receptor (reviewed in Parolaro et al., 2005, Maldonado et al., 2006). This association would explain the relationship of this system with genetic vulnerability, reinforcement, drug-seeking behavior, dependence, relapse and other phenomena characteristic of different types of habit-forming drugs (Parolaro et al., 2005, Maldonado et al., 2006). Alcohol is one of the habit-forming drugs that interacts with the cannabinoid system (for review, see Hungund et al., 2002, Colombo et al., 2005). Genetic studies have shown a greater frequency in the occurrence of CB1 receptor polymorphisms in subpopulations of alcoholic patients that suffer severe withdrawal (Schmidt et al., 2002) or that have antecedents of childhood attention deficit/hyperactivity (Ponce et al., 2003). A second polymorphism affects the endocannabinoid degrading enzyme, fatty acid amide hydrolase (FAAH), and this has also been studied in populations of alcoholics (Sipe et al., 2002). These genetic studies support the idea that individuals bearing polymorphisms in proteins that regulate cannabinoid activity might have a higher risk of becoming addicted to alcohol. This phenomenon is probably associated with the changes in CB1 receptor function or in the activity of FAAH due to these genetic variants (Uhl et al., 2006). Indeed, this is the case in laboratory animals where the differences in alcohol preference between distinct strains of mice (Hungund and Basavarajappa, 2000, Basavarajappa and Hungund, 2001) or rats (Ortiz et al., 2004a) have been associated with differences in CB1 receptor gene expression, number and/or function in specific brain structures. In addition, genetic ablation of key proteins for cannabinoid signaling in mice (e.g. CB1 receptor or FAAH enzyme) produced marked alterations in alcohol preference, intake and/or withdrawal signs (Hungund et al., 2003, Racz et al., 2003, Basavarajappa et al., 2006, Blednov et al., 2007).

Biochemical studies have also demonstrated the effects of alcohol exposure on endocannabinoid levels and on their CB1 receptors in cultured nerve cells (Basavarajappa et al., 2000, Basavarajappa et al., 2003, Basavarajappa and Hungund, 1999b) and laboratory animals (Basavarajappa and Hungund, 1999a, González et al., 2002a, González et al., 2002b, González et al., 2004, Ortiz et al., 2004b, Vinod et al., 2006, Ferrer et al., 2007, Rubio et al., 2007). In general, these studies support the notion that homeostatic mechanisms linked to the development of tolerance and dependence to alcohol involve an elevation of endocannabinoid levels in certain brain structures. However, this increase was sometimes accompanied by a compensatory downregulation/desensitization of CB1 receptors. Indeed, FAAH-deficient mice, which have increased levels of anandamide, showed a preference for alcohol and voluntarily consumed more alcohol than wild-type animals (Basavarajappa et al., 2006, Blednov et al., 2007). Moreover, the selective CB1 receptor antagonist rimonabant significantly reduced the preference for alcohol, craving and/or intake in different species or strains of rodents (Arnone et al., 1997, Colombo et al., 1998, Colombo et al., 2004, Lallemand et al., 2001, Freedland et al., 2001, Serra et al., 2001, Wang et al., 2003, González et al., 2004). The influence of the cannabinoid system in these events is also evident in CB1 receptor knockout mice, which consume less alcohol in self-administration paradigms and have a reduced preference for alcohol (Hungund et al., 2003, Wang et al., 2003, Racz et al., 2003, Naassila et al., 2004).

Such biochemical and pharmacological experiments have more recently been extended to conditions of alcohol deprivation (withdrawal) and of relapse after chronic alcohol exposure, periods in which endocannabinoid levels are also notably altered (González et al., 2004, Vinod et al., 2006). This fact may explain why in addition to its effects on alcohol preference and/or intake, rimonabant also blocked the effects of alcohol deprivation which indicates it may also possess anti-relapse properties (Serra et al., 2002). Significantly, CB1 receptor-deficient mice appear to suffer no signs of withdrawal after discontinuation of alcohol when compared to wild-type animals (Racz et al., 2003), despite the higher blood levels of alcohol reached after chronic alcohol exposure in these animals than in wild-type mice (Lallemand and De Witte, 2005). The alleviation of alcohol withdrawal symptoms in the absence of the CB1 receptor would support the idea that blocking CB1 receptors might also be beneficial to treat alcohol abstinence. Thus, we tested the hypothesis that CB1 receptor antagonists may serve to attenuate withdrawal symptoms in alcohol-abstinent animals by evaluating the capability of rimonabant to improve the neurochemical imbalance and to reduce behavioral symptoms elicited by the interruption of chronic alcohol exposure in male Wistar rats. It should be noted that rimonabant (SR141716), a compound developed by Sanofi-Aventis, is the most representative CB1 receptor antagonist. This compound is currently being clinically tested for its potential application in obesity and tobacco dependence, and it is under preclinical evaluation for its potential use in alcohol dependence and other pathological conditions (see Muccioli and Lambert, 2005, for review). Animals were subjected to chronic alcohol exposure for 10 days, which was followed by a short period of alcohol deprivation. This short deprivation period represents an important difference with respect to earlier studies that were conducted much longer after alcohol removal (González et al., 2004). We chose this 3-h limit to assess the pharmacology and biochemistry after the discontinuation of alcohol exposure because preliminary time-course experiments indicated that this was a critical time-point for the appearance of withdrawal symptoms in rats (e.g. hyperactivity, anxiety-like responses), which mimic similar responses in humans (Becker, 2000, De Witte et al., 2003). Given that alcohol deprivation after a period of chronic alcohol exposure may be stressful for the animals and that this might in turn influence their behavioral and neurochemical responses during alcohol abstinence, we also compared the plasma corticosterone levels between alcohol-abstinent and control animals. We first tested the effects of rimonabant on the enhanced anxiety response elicited by alcohol withdrawal. Subsequently, we examined its effects on several neurochemical and molecular imbalances provoked by discontinuing alcohol exposure, particularly in regions of the brain directly or indirectly related to withdrawal responses, such as the basal ganglia (e.g. caudate-putamen, globus pallidus and substantia nigra), several limbic structures (e.g. nucleus accumbens, prefrontal cortex, amygdala, ventral-tegmental area) and the hypothalamus (De Witte et al., 2003, for review). Finally, we analyzed the changes that alcohol withdrawal produced in the number of CB1 receptors in brain structures related to motor activity, appetite and stress/emotion. Likewise, the levels of their endogenous ligands, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA, anandamide), as well as related N-acylethanolamines were also assessed.

Section snippets

Animals

Male Wistar rats were housed in a room with a controlled 12 h photoperiod (08:00–20:00 h darkness) and at a temperature of 23 ± 1 °C. The rats had free access to standard food and water and were used at 3 months of age, weighing between 250 and 300 g. Experiments were always conducted 3 h after the onset of the dark phase and under red light. We selected this time because it corresponds to a period in which the activity of animals is maximal. Two different series of experiments were carried out and the

Withdrawal symptoms elicited by alcohol abstinence in rats and amelioration of these symptoms with rimonabant

After a prolonged period of chronic alcohol exposure, discontinuation of alcohol exposure for 3 h elicits a series of withdrawal symptoms similar to those reported for alcohol abstinence in humans (De Witte et al., 2003, Valdez and Koob, 2004, for review). Thus, rats exhibited marked hyperactivity as measured in a computerized actimeter. In these animals the ambulation (distance traveled in cm) increased from 1597.5 (±87.5) in controls (n = 5) to 1867.7 (±88.1) in alcohol-abstinent animals (n = 5, p <

Discussion

Rimonabant and other CB1 receptor antagonists are clinically promising molecules to treat alcohol addiction, since they can reduce the preference, intake and craving for alcohol in laboratory animals, and especially in animals with a strong preference for alcohol (see references in Section 1, and Colombo et al., 2005, for a recent review). These observations are concordant with the alcohol self-administering behavior exhibited by mice lacking the CB1 receptor (Hungund et al., 2003, Wang et al.,

Acknowledgements

This work was supported by grants to MR, RDM, JFR and JAR from the “Comunidad de Madrid” (S-SAL-0261/2006), CIBERNED (CB06/05/0089) and the “Plan Nacional sobre Drogas” (PR153/03-12279) and from the US National Institutes of Health/National Institute on Drug Abuse (DA16825, DA018224), to JMW from the Linda and Jack Gill Center for Biomolecular Science, Indiana University, and the MetaCyt grant to Indiana University from the Lilly Foundation Inc. Indianapolis, IN. The authors are indebted to

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