PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Pharmacol Biochem Behav. Author manuscript; available in PMC Jun 1, 2012.
Published in final edited form as:
PMCID: PMC3081972
NIHMSID: NIHMS282026
Pharmacologically Targeting the P2rx4 Gene on Maintenance And Reinstatement of Alcohol Self-Administration In Rats
Therese A Kosten, Ph.D.
Therese A Kosten, Baylor College of Medicine, Michael E Debakey Veterans Administration Medical Center, Houston, TX 77030;
Corresponding author: Therese A Kosten, Ph.D., Menninger Department of Psychiatry and Behavioral Sciences, 2002 Holcombe Blvd, Houston, TX 77030, 713-794-7637 (voice), 713-794-7240 (fax), tkosten/at/bcm.edu
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 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 as a potential treatment agent for alcohol dependence. Caution is warranted due to modest specifity on behavior reinforced by alcohol, some reduction in general activity levels, and the lack of dose-response effects.
Keywords: ivermectin, purinergic system, P2X4 receptors, relapse, operant conditioning
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 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.
P2X4 receptors show excellent specificity to the allosteric modulator effects of ivermectin (Khakh et al., 1999; Priel et al., 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 et al., 2002; Richardson et al., 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.
2.1 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 Care” (NIH publication No.85-23, revised 1996).
2.2 Alcohol dependence induction procedure
In our previous reinstatement study (Vosler et al., 2001), the levels of reinstated active lever press responding were not very high (about 9 presses). We reasoned that we might be able to increase this level of responding by exposing the rats to alcohol prior to initial operant self-administration training. Rats (n=38) were initially exposed to a modified “drinking in the dark” procedure (Bell et al., 2009). In this procedure, rats were maintained on a multiple scheduled access to alcohol solution (5% w/v) concurrently with water. Initially, two bottles, one per solution type, were available 24-hr a day during Week 1, 16-hr a day during Week 2, 8-hr per day during Week 3, and 4-hr a day during Week 4 on Monday - Friday. Drinking periods of 8-hr or less occurred during the dark phase only and most of the 16-hr drinking period occurred during this phase. Thereafter, access was limited to three, 1-hr periods per day each separated by a 2-hr non-access break on Monday - Friday. Rats were provided ad libitum access to water on weekends. These schedules were in effect for 6-wks after which rats were returned to ad libitum water access and began training for operant self-administration. Note that several weeks of self-administration training were conducted prior to test sessions and thus rats would not be considered alcohol dependent or in withdrawal when tests were performed.
The 24 rats that drank the greatest proportion of alcohol out of the total intake during the last week of the drinking in the dark procedure were entered into the experiments. All rats drank more than half of their daily fluid intake as alcohol with mean (± S.E.M.) of 75 ± 3%. Half of these rats were assigned to the alcohol responding group (EtOH) and half to the sucrose responding group (Suc). Assignments were made so that groups did not differ in alcohol preference. One rat from the EtOH group failed to show stable lever press behavior and was not entered into either study. Some rats from each group were run in both the maintenance and reinstatement experiments and some rats were run in only one experiment.
2.3 Operant self-administration training
Sessions were conducted in standard operant chambers housed within sound-attenuating cubicles equipped with fans (Coulbourn Instruments, Whitehall, PA). Each chamber was outfitted with a house light on one side of the cage and two levers on the opposite wall. Above each lever was a triple cue light and in between the levers was an access area through which a dipper could protrude. The dipper was immersed in a solution reservoir and could be activated to present 0.1 ml of solution through the access area within the chamber. This access area was equipped with a light and with infrared sensors that were used to detect head entries into the area. Another infrared detector was mounted on the ceiling and used to tabulate the animal’s body movements. Tabulation of all data as well as programming of stimulus parameters was performed with a Coulbourn Instruments based hardware and software (Graphic State Notation) system.
Illumination of the house light signaled the beginning of the sessions that were 30-min in length. Rats were initially trained to press either lever and this would activate the dipper that was immersed within a reservoir of 10% sucrose (w/v) solution. Depression of a lever also illuminated the triple cue light above the lever and the light within the dipper access area for the duration of the dipper presentation. As training progressed, several parameters were changed gradually until test conditions were met. These included decreasing the duration of the dipper presentation from 15-sec to 3-sec to 1-sec and decreasing the concentration of the sucrose solution for the Suc group to 5% and then 3%. The concentration of sucrose for the EtOH group was also decreased as alcohol was added using a modified sucrose fading procedure (Samson et al., 1998; Vosler et al., 2001). The solution modification procedure for the EtOH group began at 0% EtOH/10% sucrose and proceeded to 5% EtOH/10% Sucrose, then to 10% EtOH/5% sucrose, then to 10%EtOH/3% sucrose, and finally to 10% ethanol/2% sucrose. Thus, lever presses were ultimately reinforced by 10% ethanol/2% sucrose (w/v) for the EtOH group and by 3% sucrose (w/v) in the Suc group. These two solutions at these concentrations support equivalent levels of operant responding under the conditions used in this study (Czachowski et al., 1999; Samson et al., 1998; Vosler et al., 2001). Finally, the response requirements were altered so only one lever was active and the fixed ratio was raised to FR2. Number of presses on the other lever (inactive) were tabulated but had no programmed consequences.
2.4 Drugs
Ivermectin (22,23-Dihydroavermectin B1; Sigma Chemicals, St. Louis, MO) was prepared in isotonic saline and a concentration of 1 mg/ml and administered IP at one of three doses (1, 3, or 10 mg/kg) 30-min prior to sessions. Alcohol (ethyl alcohol, 109 Proof, organic grain alcohol, USP grade; Pharmco-Aaper, Brookfield, CT) was diluted with water and sucrose (EMD Chemicals, Whitehouse Station, NJ) at various concentrations as described above.
2.5. Experiment 1: Maintenance of operant self-administration
2.5.1 Groups
Rats were assigned to either the alcohol responding responding group (EtOH; n=9) or to the sucrose responding group (Suc; n=11).
2.5.2 Progressive ratio tests
Once operant responding for the assigned solution was stable under the FR2 schedule of reinforcement (<20% variability of active lever presses emitted over 2 days), test sessions using a progressive ratio (PR) schedule were initiated twice per week with training sessions continuing on alternate days. The response requirements proceeded using the following schedule: 1, 1, 2, 2, 3, 3, 4, 4, 5, 5, 7, 7, 9, 9, 11, 11,13, 13, 15, 15, 18, 18, 21, 21, 24, 24, etc based on Walker & Koob (Walker et al., 2007). That is, to earn each successive reinforcer, the rat was required to respond at each level in sequence. The length of the test session was increased to 3-hr. All other stimulus parameters were not altered. Rats were injected (IP) with ivermectin or vehicle (isotonic saline) 30-min prior to the initiation of test sessions. Ivermectin doses were presented in a non-systematic order and were each tested twice. The means of the data collected on the two test sessions per dose were used in the analyses.
2.5.3 Data analysis
Measures assessed included the final ratio requirement completed as well as numbers of reinforcers earned, head entries into the dipper access area, and body movements made during the session. Data were analyzed with 2 X 4 ANOVA representing the between group factor of Group (EtOH vs. Suc) with repeated measures on Dose. Significance was set at P=0.05 and trends towards significance at P=0.10. Data are presented as mean ± standard error of the mean (S.E.M.).
2.6 Experiment 2: Reinstatement of operant responding
2.6.1 Groups
Rats were assigned to either the alcohol responding responding group (EtOH; n=9) or to the sucrose responding group (Suc; n=7).
2.6.2 Reinstatement tests
Once lever press response levels were stable under the FR2 schedule of reinforcement (<20% variability of active lever presses emitted over 2 days), extinction sessions were begun. In these sessions, no solutions were available for delivery although all other stimulus parameters remained constant. Lever press responding decreased over sessions to minimal levels. Once a rat emitted 5 or fewer active lever presses for two consecutive days, a reinstatement session was performed based on Bienkowski et al (Bienkowski et al., 2000). First, 15 non-contingent solution (alcohol or sucrose depending upon group assignment) presentations were made. The house light was illuminated for 12-sec during which the dipper delivered the solution to which the animal was trained for 8-sec followed by a 4-sec timeout. . Next, a 30-min session in which lever press responding was not followed by dipper presentations commenced. All other stimulus parameters were the same as in training sessions. Rats were injected (IP) with ivermectin or vehicle (isotonic saline) 30-min prior to the initiation of test sessions. Ivermectin doses were presented in a non-systematic order and were each tested twice. The means of the data collected on the two test sessions per dose were used in the analyses.
2.6.3 Data analysis
Measures assessed included numbers of active and inactive lever presses, head entries into dipper area, and body movements made during the session. Data were analyzed with 2 X 4 ANOVA representing the between group factor of Group (EtOH vs. Suc) with repeated measures on Dose. Significance was set at P=0.05 and trends towards significance at P=0.10. Data are presented as mean ± standard error of the mean (S.E.M.).
3.1 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-hr 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 Fig. 1. This statement is supported by the significant Group effect, F(1,18)=5.19; P<0.05, although the Group X Dose effect failed to reach significance (P>0.10).
Figure 1
Figure 1
Ivermectin attenuates the level of work rats will engender to obtain solution reinforcers in the maintenance study conducted under a progressive ratio (PR) schedule. Mean (± S.E.M.) last ratio requirement completed over the 3-hr progressive ratio (more ...)
Data on active lever presses emitted during the ivermectin test sessions are presented in Table 1. Ivermectin decreased active lever press responses as supported by the significant Dose effect, F(3,54)=2.76; P=0.05. The EtOH group tended to show lower levels of active lever press responding compared to the Suc group as supported by the trends towards significance for the Group effect, F(1,18)=3.74; P<0.07. The Group X Dose interaction was not significant (P>0.10). Numbers of inactive lever presses were minimal (means across Group and Dose were less than 4) and did not differ across doses or between groups (P’s>0.10; data not shown).
Table 1
Table 1
Numbers of active lever presses, earned reinforcers, and head entries into dipper access area shown during maintenance test sessions conducted under a progressive ratio schedule are presented by ivermectin dose and solution reinforcer group.
Numbers of earned reinforcers decreased significantly with ivermectin pretreatment and were lower in the EtOH compared to the Suc group as seen in Table 1. These conclusions are supported by the significant Dose, F(3,54)=3.02; P<0.05; and Group F(1,18)=4.77; P<0.05, effects although the interaction failed to reach significance (P>0.10). The number of head entries made into the dipper access area were decreased significantly by ivermectin administration, a statement supported by the significant Dose effect, F(3,54)=5.09; P<0.005. These data are also presented in Table 1. The Group effect and its interaction with Dose were not significant (P’s>0.10) for head entries. Finally, ivermectin decreased the number of body movements made during the sessions in rats of both groups. This is supported by the significant Dose effect, F(3,54)=8.61; P<0.001 and these data are presented in Fig. 2. The Group effect and its interaction with Dose were not significant (P’s>0.10).
Figure 2
Figure 2
Ivermectin decreases general activity levels during the maintenance of operant self-administration study. Mean (± S.E.M.) number of body movements recorded during the 3-hr tests are shown by ivermectin dose. Each data point is based on the mean (more ...)
3.2. Experiment 2: Reinstatement of operant responding
The average numbers of active lever presses emitted during the two FR2 training trials prior to implementing extinction sessions did not differ between groups (EtOH: 68.9 ± 9.0; Suc: 66.3 ± 8.4; p>0.10). On the last extinction session prior to the first reinstatement, the mean (± S.E.M.) number of active lever presses did not differ between groups (EtOH: 2.2 ±0.6; Suc: 3.3 ± 0.5; p<0.10) and were significantly less than the number of active lever presses emitted during the first reinstatement session, F(1,16)=11.19; P<0.005.
The effect of ivermectin on reinstatement of operant responding for alcohol or sucrose induced by solution presentation was assessed. Data on active lever presses emitted during the reinstatement sessions are shown in Fig. 3. As seen in Fig. 3, ivermectin tended to decrease responding as supported by the trend towards significance of the Dose effect, F(3,42)= 2.43; P<0.08. The Suc group tended to show lower levels of active lever press responding compared to the EtOH group as supported by the trends towards significance for the Group effect, F(1,14)=3.18; P<0.10. The Group X Dose interaction was not significant (P>0.10).
Figure 3
Figure 3
Ivermectin tends to decrease reinstatement of extinguished lever press responding. Mean (± S.E.M) number of active lever presses are shown for the EtOH (closed symbols) and Suc (open symbols) groups by ivermectin dose. Each data point is based (more ...)
Ivermectin significantly decreased numbers of head entries made into the dipper access area as shown in Fig. 4. This statement is supported by the significant Dose effect, F(3,42)=3.34; P<0.05. The Group effect and its interaction with Dose were not significant (P’s>0.10). There was no effect of ivermectin on numbers of inactive lever presses emitted or on numbers of body movements made during sessions (P’s>0.10). These data are presented in Table 2.
Figure 4
Figure 4
Ivermectin decreases conditioned approach during reinstatement test sessions. Mean (± S.E.M) number of head entries into the dipper access area are shown for the EtOH group (closed symbols) and Suc group (open symbols) by ivermectin dose test (more ...)
Table 2
Table 2
Numbers of inactive lever presses emitted and body movements made during reinstatement test sessions are presented by ivermectin dose and solution reinforcer group.
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 the Group X Dose interaction was not significant, data shown in Fig. 3 suggest that most of the trend towards significance of the ivermectin dose effect was carried by the Suc group. In contrast, examination of the data from the maintenance study shown in Fig. 1 and Table 1 suggests that most of the significance of the ivermectin dose effect was carried by the EtOH group. And, although the Group X Dose interaction failed to reach significance, the Group effect was significant for the maintenance study. Ivermectin administration did decrease general activity levels (e.g., number of body movements) under maintenance conditions although this effect was not seen during reinstatement tests. The differential effects of ivermectin on movements between the two session types likely reflect the different session lengths (3-hr for PR tests vs. 30-min for reinstatement tests). Nonetheless, the data are consistent with the notion that pharmacological manipulations of P2X4 receptor activity affect behaviors maintained by alcohol or sucrose reinforcement.
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.
Ivermectin is used in humans to treat lymphatic filariasis and onchocerciasis, two parasite-induced diseases found mostly in Africa (WHO, 1997). It is generally considered safe with few side effects and is acceptable to patients (Pacqui et al., 1991). Thus, ivermectin could be considered as a potential pharmacological treatment for alcohol dependence. Interestingly, an interaction between alcohol and ivermectin in humans has been described. Based on clinical reports of increased cases of ataxia and postural hypotension among individuals who ingested alcohol along with ivermectin, a study of plasma ivermectin levels was conducted (Shu et al., 2000). The group administered alcohol along with ivermectin showed significantly higher plasma ivermectin concentrations up to 4 hr after administration compared to the group given ivermectin with water. Thus, if ivermectin is tested in alcohol dependent individuals, caution is needed in dose considerations. Another caution about the potential use of ivermectin to treat alcoholism is its limited specificity to alcohol.
Acknowledgements
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.
Footnotes
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
  • Abbracchio MP, Burnstock G, Verkhratsky A, Zimmermann H. Purinergic signalling in the nervous system: an overview. Trends in Neurosciences. 2009;32:19–29. [PubMed]
  • Adelsberger H, Lepier A, Dudel J. Activation of rat recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptor by the insecticide ivermectin. European Journal of Pharmacology. 2000;394:163–170. [PubMed]
  • Anton RF. Pharmacologic appoaches to the management of alcoholism. Journal of Clinical Psychiatry. 2001;62:11–17. [PubMed]
  • Asatryan L, Popova M, Perkins D, Trudell JR, Alkana RL, Davies DL. Ivermectin antagonizes ethanol inhibition in purinergic P2X4 receptors. Journal of Pharmacology and Experimental Therapeutics. 2010;334:720–728. [PubMed]
  • Bell RL, McBride WJ. Drinking in the dark - Multiple scheduled access (DID-MSA) 2009 http://www.scripps.edu/cnad/inia/modelratdrinkingindark.pdf.
  • Bienkowski P, Koros E, Kostowski W, Bogucka-Bonikowska A. Reinstatement of ethanol seeking in rats: behavioral analysis. Pharmacology Biochemistry and Behavior. 2000;66 [PubMed]
  • Campbell WC. Ivermectin and Abamectin Springer Verlag. New York: 1989.
  • Czachowski CL, Samson HH, Dening CE. Independent ethanol-and sucrose-maintained responding on a multiple schedule of reinforcement. Alcohol: Clinical and Experimental Research. 1999;23:398–403. [PubMed]
  • Dadarkar SS, Deore MD, Gatne MM. Comparative evaluation of acute toxicity of ivermectin by two methods afer single subcutaneous administration in rats. Regulatory Toxicology and Pharmacology. 2007;47:257–260. [PubMed]
  • Davies DL, Machu TK, Guo Y, Alkana RL. Ethanol sensitivity in ATP-gated P2X receptors in subunit dependent. Alcohol: Clinical and Experimental Research. 2005;27:743–755. [PubMed]
  • Dawson GR, Wafford KA, Smith A, Marshall GR, Bayley PJ, Schaeffer JM, Meinke PT, McKernen RM. Anticonvulsant and adverse effects of avermectin analogs in mice are mediated through the g-aminobutyric acidA receptor. Journal of Pharmacology and Experimental Therapeutics. 2000;295:1051–1060. [PubMed]
  • DiChiara G, Imperato A. Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proceedings of the National Academy of Sciences USA. 1988;85:5274–5278. [PubMed]
  • Haile CN, Kosten TA, Kosten TR. Pharmacogenetic treatments for drug addiction: alcohol and opiates. American Journal of Drug and Alcohol Abuse. 2008;34:355–381. [PubMed]
  • Hodos W. Progessive ratio as a measure of reward. Science. 1961;134:943–944. [PubMed]
  • Hugel S, Schlichter R. Presynaptic P2X receptors facilitate inhibitory GABAergic transmission between cultured rat spinal cord dorsal horn neurons. Journal of Neuroscience. 2000;20:2121–2130. [PubMed]
  • Katz JL, Higgins ST. The validity of the reinstatement model of craving and relapse to drug use. Psychopharmacology. 2003;168:21–30. [PubMed]
  • Khakh BS, Proctor WR, Dunwiddie TV, Labarca C, Lester HA. Allosteric control of gating and kinetics at P2X4 receptor channels. Journal of Neuroscience. 1999;19:7289–7299. [PubMed]
  • Kimpel MW, Strother WN, McClintick JN, Carr LG, Liang T, Edenberg HJ, McBride WJ. Functional gene expression differences between inbred alcohol-preferring and -non-preferring rats in five brain regions. Alcohol. 2007;41:95–132. [PMC free article] [PubMed]
  • Kittner H, Krugel U, Hoffmann E, Illes P. Stimulation of P2Y receptors enhances feeding behaviour in rats. Naunyn-Schmiedeberg's Archives of Pharmacology. 2001;363:R33.
  • Koles L, Furst S, Illes P. Purine ionotropic (P2X) receptors. Current Pharmaceutical Design. 2007;13:2368–2384. [PubMed]
  • Koob GF, Lloyd GK, Mason BJ. Development of pharmacotherapies for drug addiction: a Rosetta Stone approach. Nature Reviews Drug Discovery. 2009;8:500–515. [PMC free article] [PubMed]
  • Krause RM, Buisson B, Bertrand S, Corringer PJ, Galzi JL, Changeux JP, Bertrand D. Ivermectin: a positive allosteric effector of the alpha7 neuronal nicotinic acetylcholine receptor. Molecular Pharmacology. 1998;53:283–294. [PubMed]
  • Krugel U, Spies O, Regenthal R, Kittner H. P2 receptors are involved in the mediation of motivation-related behavior. Purinergic Signalling. 2004;1:21–29. [PMC free article] [PubMed]
  • Lalo U, Verkhratsky A, Pankratov Y. Ivermectin potentiates ATP-induced ion currents in cortical neurones: evidence for functional expression of P2X4 receptors? Neuroscience Letters. 2007;421:158–162. [PubMed]
  • Le AD, Shaham Y. Neurobiology of relapse to alcohol in rats. Pharmacology & Therapeutics. 2002;94:137–156. [PubMed]
  • Le KT, Villeneuve P, Ramjaun AR, McPherson PS, Beaudet A, Seguela P. Sensory presynaptic and widespread somatodendritic immunolocalization of central ionotropic P2X ATP receptors. Neuroscience. 1998;83:177–190. [PubMed]
  • Li C, Xiong K, Weight FF. Ethanol inhibition of adenosine 5'-triphosphate-activated current in freshly isolated adult rat hippocampal CA1 neurons. Neuroscience Letters. 2000;295:77–80. [PubMed]
  • Lumeng L, Waller MB, McBride WJ, Li TK. Different sensitivities to ethanol in alcohol-preferring and -nonpreferring rats. Pharmacology Biochemistry and Behavior. 1982;16:125–130. [PubMed]
  • McBride WJ, Li TK. Animal models of alcoholism: neurobiology of high alcohol-drinking behavior in rodents. Critical Reviews in Neurobiology. 1998;12:339–369. [PubMed]
  • NCBI. Ivermectin, Pub Med Health. Vol. 2010. Bethesda, MD: U.S. National Libary of Medicine; 2008.
  • North RA. Families of ion channels with two hydrophobic segments. Current Opinion in Cell Biology. 1996;8:474–483. [PubMed]
  • Pacqui M, Munoz B, Greene BM, Taylor HR. Community-based treatment of onchocerciasis with ivermectin: safety, efficacy, and acceptability of yearly treatment. Journal of Infectious Disease. 1991;163:381–385. [PubMed]
  • Priel A, Silberberg SD. Mechanism of ivermectin facilitation of human P2X4 receptor channels. Journal of General Physiology. 2004;123:281–293. [PMC free article] [PubMed]
  • Regier RA, Farmer ME, Rae DS, Locke BZ, Keith SJ, Judd LL, Goodwin FK. Comorbidity of mental disorders with alcohol and other drug abuse: results from the epidemiologic catchment area (ECA) study. Journal of the American Medical Association. 1990;264:2511–2518. [PubMed]
  • Richardson NR, Roberts DC. Progressive ratio schedules in drug self-administration studies in rats: a method to evaluate reinforcing efficacy. Journal of Neuroscience Methods. 1996;66:1–11. [PubMed]
  • Samson H, Slawecki C, Sharpt A, Chappell A. Appetitive and consummatory behaviors in the control of ethanol consumption: a measure of ethanol seeking behavior. Alcoholism: Clinical and Experimental Research. 1998;22:1783–1787. [PubMed]
  • Schuckit MA, Smith TL, Danko GP, Kramer J, Godinez J, Bucholz KK, Nurnberger JI, Hesselbrock V. Prospective evaluation of the four DSM-IV criteria for alcohol abuse in a large population. American Journal of Psychiatry. 2005;162:350–360. [PubMed]
  • Shaham Y, Shalev U, Lu L, DeWit H. The reinstatement model of drug relapse: history, methodology and major findings. Psychopharmacology. 2003;168:3–20. [PubMed]
  • Shan Q, Haddrill JL, Lynch JW. Ivermectin, an unconventional agonist of the glycine receptor chloride channel. Journal of Biological Chemistry. 2001;276:12556–12564. [PubMed]
  • Shu EN, Onwujekwe EO, Okonkwo PO. Do alcoholic beverages enhance availability of ivermectin? European Journal of Clinical Pharmacology. 2000;56:437–438. [PubMed]
  • Sim JA, Chaumont S, Jo J, Ulmann L, Young MT, Cho K, Buell G, North RA, Rassendren F. Altered hippocampal synaptic potentiation in P2X4 knock-out mice. Journal of Neuroscience. 2006;26:9006–9009. [PubMed]
  • Spinosa HS, Stilck SRAN, Bernardi MM. Possible anxiolytic effects of ivermectin in rats. Veterinary Research Communications. 2002;26:309–321. [PubMed]
  • Swift RM. Drug therapy for alcohol dependence. New England Journal of Medicine. 1999;340:1482–1490. [PubMed]
  • Tabakoff B, Saba L, Printz M, Flodman P, Hodgkinson C, Goldman D, Koob G, Richardson HN, Kechris K, Bell RL, Hubner N, Heinig M, Pravenec M, Mangion J, Legault L, Dongier M, Conigave KM, Whitfield JB, Saunders J, Grant B, Hoffman PL. WHO/ISBRA_Study_on_State_and_Trait_Markers_of_Alcoholism. Genetical genomic determinants of alcohol consumption in rats and humans. BMC Biology. 2009;7:70. [PMC free article] [PubMed]
  • Trailovic SM, Varagic VM. The effect of ivermectin on convulsions in rats produced by lidocaine and strychnine. Veterinary Research Communications. 2007;31:863–872. [PubMed]
  • Vengeliene V, Molander A, Spanagel R. Neuropharmacology of alcohol addiction. British Journal of Pharmacology. 2008;154:299–315. [PubMed]
  • Vosler PS, Bombace JC, Kosten TA. A discriminative, two-lever test of dizocilpine's ability to reinstate ethanol-seeking behavior. Life Sciences. 2001;69:591–598. [PubMed]
  • Walker BM, Koob GF. The g-aminobutyric acid-b receptor agonist baclofen attenuates responding for ethanol in ethanol-dependent rats. Alcoholism: Clinical and Experimental Research. 2007;31:11–18. [PMC free article] [PubMed]
  • Weight FF, Li C, Peoples RW. Alcohol action on membrane ion channels gated by extracellular ATP (P2X receptors) Neurochemistry International. 1999;35:143–152. [PubMed]
  • WHO. Thirteenth Programme Report. Geneva: World Health Organization; 1997. Tropical disease research, progress 1995–1996.
  • Xiao C, Zhou C, Li K, Davies DL, Ye JH. Purinergic Type 2 receptors at GABAergic synapses on ventral tegmental area dopamine neurons are targets for ethanol action. Journal of Pharmacology and Experimental Therapeutics. 2008;327:196–205. [PMC free article] [PubMed]