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 et al., 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.

In addition to assessments of lever press responding for alcohol, we also measured numbers of head entries into the solution access area during maintenance and reinstatement tests. Head entries showed stronger statistical effects of ivermectin compared to active lever presses in both the maintenance and reinstatement tests. Rats emitted about twice as many head entries than earned reinforcers during the maintenance tests. This can be seen by comparing data on earned reinforcers to head entries in Table 1. Although it is obviously necessary for the animal to protrude its head into this access area to obtain the solution delivery, during the reinstatement tests when no solution is available for delivery, the ratio of head entries to active lever presses was even greater. Rats emit about three times as many head entries as lever presses (i.e., compare Figs. 3 and ​and4).4). A head entry can be considered a conditioned appetitive response because it had been associated with the solution delivery and, like lever pressing in the absence of reinforcer delivery, may be a measure of “craving”.

A reduction in craving should assist maintaining abstinence from alcohol in alcohol dependent persons. The challenge for treating alcohol dependence is to prevent relapse to drinking or, if drinking is initiated, attenuate the potential escalation into excessive drinking. Ivermectin may assist treatment for alcohol dependence on both fronts. Craving or head entries under conditions of alcohol delivery or no alcohol delivery is decreased by ivermectin. Ivermectin likely would decrease the chances of escalating alcohol drinking once initiated. This statement is based on the fact that ivermectin reduced responding under the progressive ratio condition in which the response requirement increases over the session.

This study shows support for the approach of testing a pharmacological agent that was chosen based on information gleaned from genetic studies. The idea to test the effects of ivermectin on behaviors supported by alcohol was based on the following. First, the P2rx4 gene was identified as a candidate gene related to alcohol drinking in recombinant inbred rats (Tabakoff et al., 2009) and this gene is differentially expressed in selectively bred P and NP rats (Kimpel et al., 2007). Both studies showed that higher levels of alcohol consumption were associated with lower levels of the gene. The P2rx4 gene codes for the P2X₄ receptor, part of the purinergic system of ion channels activated by ATP (Koles et al., 2007; North, 1996). When activated, P2X receptors exert excitatory effects in various neural regions. Alcohol interacts with the P2X₄ receptor system by inhibiting its excitatory effects (Asatryan et al., 2010; Davies et al., 2005; Li et al., 2000; Xiao et al., 2008). This system can be enhanced in the presence of ivermectin because it acts as a specific positive allosteric modulator of P2X₄ receptors (Khakh et al., 1999; Priel et al., 2004). For example, deletion of the P2rx4 gene in mice reduces long-term potentiation (LTP) in hippocampal Schaffer collateral synapses and ivermectin enhances LTP in wild-type but not in P2rx4 knock-out mice (Sim et al., 2006). Ivermectin also increases the amplitude and frequency of spontaneous P2X-mediated EPSCs in cortical slices (Lalo et al., 2007). Thus, it is possible that ivermectin altered, in part, the behavioral effects of alcohol seen in the present study through P2X₄ mechanisms. However, other actions of ivermectin have been reported, such as effects on GABA, glycine, and nicotinic systems (Adelsberger et al., 2000; Dawson et al., 2000; Hugel et al., 2000; Krause et al., 1998; Shan et al., 2001). Any of these systems may have contributed to the results reported herein either directly or indirectly (Haile et al., 2008; Vengeliene et al., 2008; Xiao et al., 2008).

One possible scenario to explain the effects of ivermectin on alcohol self-administration behavior is as follows. Under typical conditions, activation of P2X₄ exerts excitatory effects on the inhibitory GABAergic neurons in the ventral tegmental area (VTA). GABAergic neurons inhibit dopamine (DA) cells in nucleus accumbens (NAc) and DA levels in NAc are linked to the rewarding effects of various drugs of abuse (DiChiara et al., 1988; Haile et al., 2008). Alcohol inhibits GABAergic transmission to DA neurons in this region through purinergic mechanisms and this could reflect one way in which alcohol intake enhances DA levels in NAc. That is, alcohol would cause a decrease in P2X₄ receptor activity that would lead to a disinhibition of DA neurons through a reduction in GABA interneuron inhibition. However, ivermectin, a positive allosteric modulator of P2X₄ receptors, enhances the excitatory P2X₄ system and this would cause a reduction in DA neural activity due to an increase in GABA inhibition. Presumably, an indirect alcohol-induced increase in NAc DA levels could be counteracted by ivermectin via the P2X₄ mechanism although effects mediated through other systems likely contribute as well.

Several lines of evidence suggest that ivermectin crosses the blood-brain barrier to affect CNS processes. Systemic ivermectin administration causes gross motoric problems including ataxia, staggering, and loss of righting in rats among other signs suggesting involvement of the CNS (Dadarkar et al., 2007). Further support for the ability of ivermectin to cross the blood-brain barrier and affect the CNS is shown by its ability to protect against the convulsant effects of pentylenetetrazole, lidocaine, or strychnine, reverse picrotoxin-induced anxiety in elevated plus maze, and decrease spontaneous locomotor activity when administered systemically as seen in the present study and in previous reports (Dawson et al., 2000; Spinosa et al., 2002; Trailovic et al., 2007).

There are limitations in the present study. The ability of alcohol exposure to reinstate extinguishing responding on the lever previously associated with alcohol delivery is thought to be a model of relapse to drinking after abstinence in alcoholics due to its face validity. While this drug-induced reinstatement procedure in animals has been a popular tool for researchers in addiction (Le et al., 2002; Shaham et al., 2003), its construct and predictive validities as an animal model of relapse have not been confirmed (Katz et al., 2003; Koob et al., 2009). Further, the reinstatement procedure in animals necessarily utilizes active extinction (i.e., the contingency between lever pressing and solution delivery is eliminated) whereas for humans, abstinence is not typically an active process of extinguishing a behavior. The specificity of the effect of ivermectin on behavior maintained by alcohol vs another reinforcer is perhaps limited. P2 receptors have been implicated in motivated behaviors such as feeding although such effects are most likely due to P2Y receptor mediation and not P2X receptors (Kittner et al., 2001; Krugel et al., 2004). Although interest in role of the purinergic system in the CNS is growing, the literature about this system and behavior is limited and much of it relates to pain (Abbracchio et al., 2009). Another limitation is the lack of dose response function for ivermectin. Although in many cases, the ivermectin dose effects were significant, examination of the data in the figures and tables suggest that this likely reflect that assessments made under the vehicle condition differed from those made under any of the three doses of ivermectin tested (1 – 10 mg/kg). These doses of ivermectin are below the LD₅₀ of 18.2 mg/kg and outside its lower 95% confidence interval which is 15.5 mg/kg (Trailovic et al., 2007). Thus, the maximum dose used in the present study is about as high as can be used to avoid lethality or gross toxic effects.