Pharmacologically targeting the P2rx4 gene on maintenance and reinstatement of alcohol self-administration in rats

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Abstract

Genetic studies indicate that alcohol consumption associates with expression of the P2rx4 gene, a gene that codes for the P2X4 receptor. This receptor is a subtype in the purinergic system of ligand-gated ion channels that when activated exerts excitatory effects in CNS. P2X4 function is inhibited by alcohol and P2X4 receptors are modulated positively by the antiparasitic agent, ivermectin. Two experiments were performed to test the ability of ivermectin to alter the behavioral effects of alcohol in rats. After alcohol exposure was achieved via the “drinking in the dark” procedure, separate groups of Sprague–Dawley rats were trained to lever press for either alcohol (10% ethanol/2% sucrose) or sucrose (3%) solutions in operant chambers. Rats were tested for maintenance of operant self-administration under a progressive ratio condition (Experiment 1) and for reinstatement of extinguished responding induced by solution presentation (Experiment 2) after ivermectin (0; 1–10 mg/kg; IP) administration. Ivermectin decreased the amount of work that the animal performed to obtain reinforcers in the maintenance study, particularly in the group reinforced with alcohol, and tended to decrease reinstated lever press responding. Conditioned approach behavior (head entries) was significantly reduced by ivermectin in both experiments. Reduction in motor activity was seen during the longer maintenance sessions but not in the shorter reinstatement sessions. Results suggest some support for ivermectin-like drugs as potential treatment agents for alcohol dependence. Caution is warranted due to modest specificity on behavior reinforced by alcohol, some reduction in general activity levels, and the lack of dose–response effects.

Research Highlights

► Ivermectin attenuates responding for alcohol under a progressive ratio schedule. ► Ivermectin tends to diminish alcohol-induced reinstated responding. ► Ivermectin decreases conditioned approach behavior.

Introduction

Alcohol dependence is a serious problem in the United States affecting about 10–13% of the population (Regier et al., 1990). It is a chronic relapsing disorder in which the individual has difficulty controlling his or her drinking, develops tolerance to its effects, suffers from withdrawal symptoms, and typically has social, financial, or personal problems directly related to excessive drinking (Schuckit et al., 2005). Preventing relapse to drinking is a major challenge in alcohol abuse treatment. After obtaining abstinence, exposure to cues associated with drinking or to alcohol itself can promote the return to excessive drinking. Thus, an important treatment goal is to either attenuate the ability of alcohol or its cues to provoke the re-initiation of drinking or, once drinking has begun, to reduce the amount of drinking. Pharmacological treatments may help obtain this goal (Anton, 2001, Swift, 1999).

Part of the process of developing potential pharmacotherapies for addiction is the use of animal models of alcohol drinking, craving, and relapse. No one model can emulate the whole spectrum of addiction; rather, different procedures can be used to probe mechanisms of or test manipulations on specific phases of the addiction process (Koob et al., 2009). For example, maintenance of operant self-administration of alcohol models the binge–intoxication phase of drinking and the reinstatement of this behavior after extinction models the preoccupation–anticipation phase (Koob et al., 2009). Pharmacological agents to be tested in these models can be identified based on their ability to target genes that are associated with alcohol consumption or are differentially expressed between strains that vary in alcohol preference such as the alcohol-preferring (P) and alcohol-non-preferring (NP) rats. P and NP rats were selectively bred for divergence in alcohol preference under free-choice access conditions and are commonly used in alcohol research (Lumeng et al., 1982, McBride and Li, 1998).

One candidate to target pharmacologically is the P2rx4 gene. P rats show lower functional expression of this gene compared to NP rats (Kimpel et al., 2007). Further support is provided by a study that used a genetical genomic approach in conjunction with phenotypic analysis of alcohol drinking among 23 recombinant inbred rat strains (Tabakoff et al., 2009). In this study, the P2rx4 gene showed a strongly significant negative correlation with alcohol drinking. The P2rx4 gene codes for the P2X4 receptor, one of seven distinct P2X subtypes of the purinergic system (Koles et al., 2007, North, 1996). P2X receptors are ligand-gated ion channels that can be activated by extracellular adenosine 5′-triphosphate (ATP). When activated, P2X receptors exert excitatory actions at various regions of the central nervous system (Lalo et al., 2007, Le et al., 1998, Sim et al., 2006). P2X4 function is inhibited by alcohol (Asatryan et al., 2010, Davies et al., 2005, Li et al., 2000, Xiao et al., 2008) perhaps due to its ability to decrease its agonist affinity (Weight et al., 1999). This suggests that pharmacological agents that affect P2X4 receptor function may alter the behavioral effects of alcohol.

P2X4 receptors show excellent specificity to the allosteric modulator effects of ivermectin (Khakh et al., 1999, Priel and Silberberg, 2004), an antiparasitic drug used widely in veterinary medicine (Campbell, 1989). Ivermectin is also prescribed for humans as an antiparasitic agent (NCBI, 2008) and thus, if promising results are seen in animals, research can be readily translated into human populations. The present report describes the results of two experiments aimed at testing the ability of ivermectin to alter the behavioral effects of alcohol in rats. First, we examined the effects of ivermectin on maintenance of operant self-administration of alcohol in outbred, Sprague–Dawley rats. We utilized a progressive ratio (PR) procedure in which the response requirement increases over the session and this is thought to measure the motivation to work to obtain alcohol (or other reinforcer) delivery (Hodos, 1961, Le and Shaham, 2002, Richardson and Roberts, 1996). Next, we investigated the effects of ivermectin on reinstatement of extinguished operant responding induced by exposure to alcohol along with the cues associated with its delivery. To assess the specificity of the effects of ivermectin on maintenance and reinstatement of responding for alcohol, we performed parallel studies in rats trained to lever press for sucrose solution as we did previously (Vosler et al., 2001). In our prior study, levels of reinstated responding induced by alcohol were low. We attempted to increase response levels in rats in the present study by exposing them to alcohol prior to initial self-administration training although all tests were conducted several weeks after termination of the alcohol exposure procedure.

Section snippets

Subjects and setting

Adult (70–90 days at the start of the study), male Sprague–Dawley rats (Harlan Sprague–Dawley Inc., Indianapolis, IN) were housed individually in polypropylene cages in a temperature- and humidity-controlled room maintained on a reverse 12:12 light/dark cycle (lights on at 19:00). Food and water were available ad libitum except as noted under experimental procedures. Protocols were approved by the Institutional Animal Care and Use Committees and followed the “Principles of Laboratory Animal

Experiment 1: maintenance of operant self-administration

The effects of ivermectin on maintenance of operant responding for alcohol or sucrose were assessed under a progressive ratio (PR) schedule during 3-h test sessions. Data on last ratio completed under these conditions with ivermectin pretreatment are shown in Fig. 1. Ivermectin lowered the ratio the animal would complete as supported by the significant Dose effect, F(3,54) = 2.92; P < 0.05. Final ratios completed were lower in rats from the EtOH group compared to rats from the Suc group as seen in

Discussion

The results of the present study show that ivermectin decreased operant responding for alcohol and sucrose in rats. Ivermectin also tended to attenuate reinstatement of extinguished responding for alcohol and sucrose. The specificity of the effects of ivermectin to reduce the reinforcing effects of alcohol is modest. Reinstatement of lever press behavior originally reinforced by sucrose shows a tendency to be attenuated more by ivermectin than behavior originally reinforced by alcohol. Although

Acknowledgments

This research was supported by grant U01-AA013476. We greatly acknowledge the technical assistance provided by A. Aruffo, W. Huang, M. Kosten, P. O'Malley, M. Rengasamy, J. Taylor, W. Webb, and X. Xiang.

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