Drugs for Relapse Prevention of Alcoholism (Milestones in Drug Therapy)

Editors: Spanagel, Rainer; Mann, Karl F.

Title: Drugs for Relapse Prevention of Alcoholism, 1st Edition

Copyright 2005 Springer

> Table of Contents > Serotonergic compounds: preclinical data

Serotonergic compounds: preclinical data

Anh Dzung L ^{1, 2, 3}

Douglas R. Funk¹

¹ Department of Neuroscience, Center for Addiction and Mental Health, Toronto, Ontario, Canada M5S 2S1

² Departments of Pharmacology and ³Psychiatry, University of Toronto, Toronto, Ontario, Canada M5S 1A8

Introduction

Evidence for a role of the brain serotonergic system in alcohol dependence was first reported in 1968 by Myers and Veale [1]. Depletion of brain serotonin by treatment with parachlorophenylalanine (pCPA), a serotonin synthesis inhibitor, produced a strong and long-lasting suppression of alcohol consumption in rats. Since that time, the involvement of brain 5-HT in alcohol dependence has been studied extensively. Over the years, a variety of neurobiological approaches have been used to investigate the role of 5-HT in alcohol self-administration. Early work was dominated by studies examining the effects of lesioning the central serotonergic system by the administration of selective neurotoxins such as 5,6- and 5, 7- dihydroxytryptamine (DHT) to destroy 5-HT terminals [2]. These studies were followed by the extensive examination of the effects of raising 5-HT levels with serotonin precursors such as tryptophan or 5-hydroxytryptophan [3] or with selective serotonin reuptake inhibitors (SSRIs) [4]. The vast literature on the effects of 5-HT manipulations on alcohol consumption in experimental animals has been a subject of many excellent reviews [5, 6 and 7]. In general, it can be said that although there are some inconsistencies in the literature, the preclinical data generated over the last three decades strongly suggest that the brain 5-HT system plays a significant role in alcohol consumption and that manipulations that augment 5-HT function reduce alcohol consumption [5, 6 and 7].

Over the last few years many advances have been made in understanding the potential mechanisms underlying the role of 5-HT receptors in alcohol consumption. The 5-HT₁, 5-HT₂ and 5-HT₃ receptors and associated subtypes have received the most attention. To a great extent, these advances can be attributed to the development of new agents that have high selectivity for these receptor types and subtypes as well as the availability of transgenic mice with selective deletion of these receptors. The involvement of these 5-HT receptor types and subtypes in alcohol consumption will be the focus of this chapter.

Relapse is the major clinical problem in the treatment of alcohol abuse, as up to one-half of alcoholics relapse shortly after detoxification [7]. Most preclinical

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In this chapter we will first examine the recent preclinical literature on the involvement of 5-HT₁, 5-HT₂, and 5-HT₃ receptor types or subtypes on alcohol consumption. We will then examine existing studies concerning the involvement of the 5-HT system in relapse to alcohol.

5-HT receptor types and subtypes in alcohol consumption

Our understanding of the 5-HT receptor systems has progressed considerably over the last decade. There are seven families of 5-HT receptors, and at least 14 distinct receptor subtypes have been characterized, the majority of which appear to have functional roles in the central nervous system (CNS) [9]. Of these various receptors, the functional involvement of 5-HT₁, 5-HT₂ and 5-HT₃ subtypes in alcohol intake have been examined in detail due to the availability of selective ligands.

5-HT_1A

The 5-HT_1A receptors occur at high densities in 5-HT cell body areas, particularly in the dorsal and the median raphe nuclei where they function as inhibitory autoreceptors. They are also found in various forebrain areas known to be involved in the effects of abused drugs, including the frontal cortex, amygdala, hippocampus and septum where they function as postsynaptic receptors [9, 10]. This distribution is consistent with a role for this receptor in mediating the effects of 5-HT on cortical and limbic structures and also in modulating the activity of 5-HT neurons. Stimulation of 5-HT_1A receptors produces complex changes in the activity of 5-HT systems, depending on the doses of drugs employed or where they are administered. For example, peripheral administration of low doses of 8-hydroxy-2-di-n-propylamino tetralin (8-OH-DPAT), a prototype 5-HT_1A receptor agonist, or its central injection directly into the median and dorsal raphe, activates 5-HT_1A autoreceptors resulting in decreases in 5-HT cell firing and 5-HT release in terminal regions [10].

Evidence for the involvement of 5-HT_1A receptors in the regulation of alcohol drinking derives from two experimental strategies: a) evaluation of 5-HT

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Treatment with a range of chemically diverse 5-HT_1A receptor agonists such as 8-OH-DPAT, ipsapirone or gepirone has been shown to reduce alcohol intake in a variety of experimental procedures used to assess alcohol consumption [12, 13, 14, 15, 16, 17, 18, 19, 20 and 21]. The most common 5-HT_1A receptor agonist, 8-OH-DPAT, in doses ranging from 0.125 to 2.5 mg/kg has been shown to suppress alcohol intake in various animal models of alcohol consumption including two-bottle choice drinking or operant self-administration [12, 13 and 14, 18, 19] in different strains of rats including those selectively bred for high and low alcohol consumption. Low doses of 8-OH-DPAT (0.03 to 0.06 mg/kg), however, have been shown to stimulate alcohol intake in a limited access drinking procedure when access to alcohol solution was restricted to 60 min per day [13, 14]. Such stimulation of alcohol consumption induced by low doses of 8-OH-DPAT has been attributed to the selective stimulation of 5-HT_1A autoreceptors located in the raphe nuclei, as direct infusions of 8-OH-DPAT into the dorsal or median raphe also enhance alcohol intake [12]. The stimulation of alcohol intake induced by 8-OH-DPAT in the dorsal raphe is more selective to ethanol as it did not increase water intake, whereas infusions of 8-OH-DPAT into the median raphe increase both water and alcohol intake. Direct infusion of 8-OH-DPAT into the dorsal raphe, however, has been shown to reduce alcohol intake in the 8 h period following infusion [15]. The discrepancy in these findings might be related to a number of factors ranging from the strain of rat used, the nature of the alcohol drinking procedure and most importantly, the doses of 8-OH-DPAT employed. The effects of 0.1 to 2.5 g 8-OH-DPAT injected into the dorsal raphe were used to assess alcohol intake in Wistar rats during a 40 min period in the Tomkins et al. study [12], whereas alcohol intake in AA rats selectively bred for high alcohol intake was measured following infusion of 10 g into the dorsal raphe in the Schreiber et al. study [15].

The highly selective 5-HT_1A receptor antagonist WAY 100635 in doses ranging from 0.05 to 0.5 mg/kg has been shown to suppress alcohol intake in female P rats [16]. The suppression of alcohol intake, however, was not dose-dependent, and the effect was not seen at a higher dose of 1 mg/kg. Tomkins and co-workers [17] also found that the 1 mg/kg dose of WAY 100635 did not alter alcohol self-administration in Wistar rats. It is possible that the absence of the effect of WAY 100635 at the high dose might be due to the loss of its specificity at the pre-synaptic 5-HT_1A autoreceptor. 8-OH-DPAT or ipsapirone, at doses which reduced operant alcohol self-administration, have also been shown to suppress responding for water or saccharin [18, 19], suggesting that

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The involvement of post-synaptic 5-HT_1A receptors in alcohol consumption is unclear. Direct infusions of 5-HT_1A agonists to the raphe as well as to other brain structures such as dorsal and ventral hippocampus, amygdala, septum and striatum have been used to dissociate the contribution of pre- and post-synaptic 5-HT_1A receptors to their anxiolytic and antidepressant actions [10]. While it is clear that pre-synaptic 5-HT_1A receptors can modulate alcohol intake, the nature of the involvement of post-synaptic 5-HT_1A receptors in alcohol consumption remains to be determined. To our knowledge, there are no reports in the literature of studies specifically designed to examine the effect of direct infusions of 5-HT_1A receptor ligands in limbic brain structures on alcohol intake. Such studies are necessary to clarify the role of post-synaptic 5-HT_1A receptors in the regulation of alcohol consumption.

5-HT_1B

5-HT_1B receptor binding is localized primarily to sites in the basal ganglia, including the ventral pallidum, globus pallidus, substantia nigra and in the limbic system, most notably the dorsal subiculum of the hippocampus [22]. The mRNA for the receptor, on the other hand, is found in the cingulate and entorhinal cortex, nucleus accumbens, striatum, subthalamic nucleus, hippocampus, raphe nuclei and cerebellum [23]. Lesion and electron microscopic studies have localized the 5-HT_1B receptors to axons and terminals [23, 24], so it is likely that the mismatch between receptor binding and mRNA reflects the fact that the receptors are transported away from the cell body after synthesis. The localization of the 5-HT_1B receptors to terminals is consistent with the results of in vitro studies showing that the release of a variety of transmitters is modulated by 5-HT_1B receptors, including gamma aminobutyric acid (GABA), substance P, glutamate and acetylcholine [25, 26, 27 and 28]. 5-HT_1B receptors also regulate the release of 5-HT from the terminals of the midbrain raphe projection neurons in the forebrain [29].

A number of approaches provide converging evidence that the 5-HT_1B receptor is linked to alcohol dependence. Studies examining the effects of 5-HT_1B receptor agonists point to the involvement of 5-HT_1B receptors in alcohol preference and intake in laboratory animals. Administration of the mixed 5-HT_1B/2C agonist mCPP was reported to reduce both alcohol and water intake in a two-bottle, limited access design [30]. In a more recent study, done using a similar design, mCPP reduced alcohol, water and food intake [31]. These

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Genetic studies have also provided evidence for the involvement of 5-HT_1B receptors in alcoholism, although there are some inconsistent findings. In humans, variations in the gene coding for the 5-HT_1B receptor have been linked to alcoholism in a subset of alcoholics with antisocial behavioral traits [33]. In mice, a mapping study showed that a locus associated with alcohol preference was found at the precise location of the 5-HT_1B receptor gene [34]. The results of studies on alcohol intake in mice with targeted gene knockouts of the 5-HT_1B receptor are controversial. In an initial study, 5-HT_1B-/- mice were shown to have greater preference for and spontaneous consumption of alcohol than wild type controls [35]. The same group later failed to replicate this finding [36]. Other studies have also found that 5-HT_1B-deficient mice do not differ in these parameters [37]. At present, the reasons for these inconsistencies are not clear.

5-HT_2A

5-HT_2A receptors are expressed throughout the rat CNS including several regions involved in alcohol- or drug-related behaviors. Studies using receptor autoradiography, immunohistochemistry and in situ hybridization have demonstrated that 5-HT_2A receptors are expressed at high levels in the frontal cortex, nucleus accumbens, septum, amygdalar nuclei, hippocampus, hypothalamus, ventral tegmental area (VTA) and raphe nuclei [38, 39]. Administration of agonists active at the 5-HT_2A receptor such as 2,5-dimethoxy-4-iodoamphetamine (DOI) [40, 41] have been shown to reduce alcohol intake using a 24 h access procedure. Maurel et al. [40] replicated their earlier finding in showing that the 5-HT_2A agonist DOI significantly and selectively reduced operant responding for alcohol. Interestingly, antagonists such as amperozide, FG 5974, MDL 100,907, mianserin and ritanserin also reduce alcohol intake [31, 40, 41 and 42]. A likely reason for this discrepancy is that the drugs have affinity for other subtypes of 5-HT receptors.

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Downregulation of 5-HT_2A receptors in discrete brain areas with microinjections of antisense have been shown to have significant effects on alcohol intake. Blakley et al. [43] found that chronic infusion of 5-HT_2A antisense via osmotic minipumps intracerebroventricularly, or directly into the central amygdala (CeA) significantly reduced alcohol preference and intake in a 24 h access design. Antisense treatment of the prefrontal cortex (PFC), on the other hand, significantly increased alcohol preference and intake. The specificity of these effects is called into question, as changes in saccharin preference in the same direction as the effects on alcohol were observed. Antisense application to the hippocampus or dorsal raphe nucleus did not affect either saccharin or alcohol preference or intake.

5-HT_2C

The 5-HT_2C receptor is expressed in a number of brain areas related to drug reinforcement including the frontal cortex, septum, hippocampus and amygdala [39, 44]. There are few studies examining the effects of 5-HT_2C selective drugs on alcohol preference or consumption. Tomkins et al. [17] showed that the 5-HT_2C agonist Ro60-0175 reduced operant responding for alcohol, and that this effect was blocked by the selective 5-HT_2C antagonist SB242,084. The antagonist also blocked the reduction of responding for alcohol induced by the 5-HT releaser dexfenfluramine. SB242,084 alone increased responding for alcohol. These compounds also had effects on blood alcohol levels, with the agonist decreasing and the antagonist increasing alcohol concentration.

5-HT₃

Of the 5-HT receptors, the 5-HT₃ receptor is the only one linked to an iongated channel. 5-HT₃ receptor binding is found at low levels throughout cortical and subcortical structures as revealed by autoradiographic mapping techniques [45, 46]. In situ hybridization and immunohistochemical analysis reveal that 5-HT₃ receptors are expressed in a large proportion of GABA neurons that co-express cholycystokinin immunoreactivity [45]. 5-HT₃ receptors have been shown to regulate the activity of the mesolimbic dopamine system at the level of the VTA as well as the nucleus accumbens [47, 48]. 5-HT₃ receptors have been shown to be involved in many actions of alcohol [46] and currently drugs acting at the 5-HT₃ receptors are targeted as potential pharmacotherapeutic agents for the treatment of alcohol abuse [7].

Preclinical studies examining the effects of various 5-HT₃ receptor antagonists on alcohol consumption have revealed a conflicting pattern. A number of studies have found that administration of 5-HT₃ receptor antagonists such as MDL 72222, ondansetron, zacopride and tropisetron reduce alcohol consumption in rats in a two-bottle choice paradigm [49, 50, 51, 52, 53 and 54]. The reduction of alcohol

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The effects of 5-HT₃ receptor agonists on alcohol consumption have also been investigated. Intraventricular or intra-accumbens injection of the 5-HT₃ receptor agonist 2-methyl-5-HT or m-chlorophenylbiguanide (mCPBG) reduced alcohol consumption without affecting food or water intake in rats [57]. Transgenic mice over-expressing the 5-HT₃ receptors also show reduced alcohol consumption compared to controls [58]. The 5-HT₃ transgenic mice have also been shown to be more sensitive to the effects of ethanol compared to controls and it has been suggested that such differences might account for their low alcohol consumption [59]. Whether the suppression of alcohol intake induced by central administration of 5-HT₃ receptor agonists is due to an enhancement of sensitivity to the effects of ethanol remains to be determined.

5-HT₃ receptor antagonists have been shown to attenuate or block the stimulation of dopamine (DA) release in the nucleus accumbens or somatodendritic DA release in the VTA induced by ethanol [11, 46]. The antagonism of ethanol-stimulated mesolimbic DA activity by 5-HT₃ receptor antagonists has been suggested as the primary mechanism underlying their attenuation of alcohol intake [7, 11, 46]. In support of this, it was observed that in 6-OHDA lesioned rats, the suppression of alcohol intake induced by tropisetron was markedly reduced [60]. A recent study by Rodd-Henricks et al. [61] demonstrated, furthermore, that intracranial self-administration of ethanol in the posterior VTA by Wistar rats can be blocked by co-administration of a 5-HT₃ receptor antagonist.

5-HT and relapse to alcohol

As mentioned earlier, most of the neurobiological investigation into the mechanisms underlying alcohol dependence has focused on the neurochemical mechanisms underlying alcohol reinforcement with little attention being paid to those underlying relapse [8]. Knowledge of the mechanisms underlying drug reinforcement, however, might not be sufficient to explain why individuals are still vulnerable to relapse after a prolonged period of abstinence [62].

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Animal models of relapse to alcohol

Currently there are two animal models that have been developed to study relapse to alcohol in experimental animals [8]. The first model utilizes the alcohol deprivation effect (ADE), which is described as the temporary increase in alcohol consumption following a short period of abstinence [63]. The magnitude of the ADE effects increases with repeated cycles of deprivation [64, 65]. The increase in alcohol-taking after abstinence and the increase in the intensity of ADE after repeated cycles of deprivation parallel the effects of repeated deprivation seen on the priming effects of alcohol in humans [8]. The second model that has been used to assess relapse to alcohol is the reinstatement procedure [8]. In this model, animals are trained to lever press for alcohol, the operant response is extinguished and the effects of acute exposure to alcohol [66, 67 and 68], cues previously associated with alcohol [69] or stressors [67, 68] on the reinstatement of the lever pressing response are examined.

While these two models have been used quite extensively over the last several years to evaluate the effects of various compounds, particularly those acting at opioidergic or corticotrophin-releasing factor receptors [8] on relapse to alcohol, little work has been conducted with serotonergic agents. To our knowledge there has been only one study that examined the effects of 5-HT on relapse to alcohol using the ADE model. Rodd-Henricks et al. [65] found that the 5-HT₃ receptor antagonists MDL 7222 or tropisetron can suppress ADE. The doses of these compounds required to suppress ADE, however, were much higher than those required to reduce baseline alcohol consumption. We have also investigated the effects of serotonergic agents on relapse to alcohol using the reinstatement procedure. In our early studies [68, 69], we have shown that treatment with fluoxetine can attenuate reinstatement of alcohol seeking induced by exposure to foot shock stress, but not by that induced by priming with alcohol. Recently, we have found that paroxetine, another serotonin re-uptake inhibitor, or the 5-HT₃ receptor antagonist tropisetron can also attenuate the reinstatement of alcohol seeking induced by foot shock stress [70].

Conclusions

Our understanding of the role of the serotonergic systems in alcohol dependence has advanced greatly over the last several years. While the availability of

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While little work has been carried out examining the involvement of 5-HT systems in relapse, the existing data suggest that the 5-HT mechanisms mediating relapse and alcohol consumption can be dissociated, as evidenced by the differential dose requirement [65] or the fact that they have differential effects on the reinstatement of ethanol-seeking induced by stressors and priming [68].

Acknowledgements

Supported by grant AA13108 from the NIAAA to Dr. A.D. L .

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