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Ethanol familiarity and naltrexone treatment affect ethanol responses in rats
Alcohol 37 (2005) 167–172
Ethanol familiarity and naltrexone treatment affect ethanol responses in rats Stephen W. Kiefer*, Katherine G. Hill, Daniel L. Coonfield, Frank M. Ferraro III Department of Psychology, Kansas State University, 492 Bluemont Hall, Manhattan, KS 66506-5302, USA Received 17 June 2005; received in revised form 22 September 2005; accepted 22 September 2005
Abstract In the present study, the effects of ethanol familiarity on the ability of naltrexone to alter ethanol palatability and consumption were examined. One group of rats was allowed continuous access to 10% vol/vol ethanol and water for 3 weeks. A second group received only water. At the end of this time, the groups were further subdivided and injected with either 3 mg/kg naltrexone or saline (total of four groups; n 5 11–13 per group) before ethanol taste reactivity tests with 10% vol/vol ethanol and ethanol consumption tests. Results showed that naltrexone effectively decreased ingestive responding and increased aversive responding. Further, rats familiar with alcohol made more ingestive responses to 10% vol/vol ethanol. A significant interaction of drug treatment and familiarity was found in the data for aversive responses: naltrexone treatment produced more aversive responses in ethanol-familiar rats, whereas saline treatment resulted in fewer aversive responses in rats familiar with ethanol. Naltrexone treatment clearly reduced consumption of 10% vol/vol ethanol, although its effects were attenuated somewhat by ethanol familiarity. The present data indicate that both alcohol familiarity and naltrexone treatment affect ethanol reactivity and ethanol consumption in outbred rats. The interaction of naltrexone treatment and ethanol familiarity only for aversive reactivity and the lack of such an interaction for the consumption measures suggests that the mechanisms underlying ethanol reactivity and ethanol consumption may be dissociable at the neural level. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Naltrexone; Taste reactivity; Rat; Palatability; Alcohol; Intake
1. Introduction Rats that are given acute treatments of naltrexone display a reliable change in response to the taste of ethanol and in the consumption of ethanol during short-term access tests (Ferraro et al., 2002; Hill & Kiefer, 1997). Specifically, ethanol becomes less palatable to rats as evidenced by a reliable increase in aversive responses accompanied by a decrease in ingestive responding (although the latter has occasionally failed to achieve standard levels of statistical significance; see Coonfield et al., 2002). As noted, acute treatment with naltrexone reliably reduces consumption of 10% vol/vol ethanol, an effect that may be partially mediated by the alterations in ethanol palatability that are revealed by the taste reactivity tests. Naltrexone is a nonspecific antagonist of endogenous opioid receptors (Herz, 1997; Oswald & Wand, 2004) and is currently being used in human clinical populations as a supplemental treatment (in addition to psychotherapy) for alcohol abuse and alcoholism (O’Malley et al., 1992; Volpicelli et al., 1992).
* Corresponding author. Tel.: 11-785-532-6850; fax: 11-785-5325401. E-mail address: [email protected] (S.W. Kiefer). 0741-8329/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2006.03.004
One factor that has been shown to influence the response of rats to a taste solution is whether that taste is novel or familiar. In an early article, Revusky and Bedarf (1967) demonstrated that rats associated illness with a novel taste much more readily than they associated illness with a familiar taste. This effect has been demonstrated many times since the original publication (Elkins, 1973; Kiefer & Braun, 1977; Kalat & Rozin, 1973) and the explanation has an intuitive appeal: it would be adaptive in an evolutionary sense to associate a bout of illness with a novel taste rather than one that was familiar and that had not produced adverse effects in previous exposures. Ethanol exposure or familiarity also alters a rat’s response to the taste of ethanol and consumption. For example, rats exposed to ethanol prenatally consume more ethanol as infants (Chotro & Arias, 2003; Domı´nguez et al., 1998) and also display increased palatability to the taste of ethanol (Arias & Chotro, 2005). Our examination of naltrexone’s effects on ethanol reactivity and consumption involved extensive ethanol familiarization procedures (Coonfield et al., 2002; Hill & Kiefer, 1997). Rats typically were given 2–3 weeks of continuous access to 10% vol/vol ethanol and water, thus providing extensive familiarization with the taste and the postingestive effects of ethanol. In the present experiment, it was
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determined if the familiarity of ethanol was an important component for the eventual effects of naltrexone treatment. To test this issue, rats familiar with the taste of ethanol were compared to rats for which ethanol was a novel stimulus, both in the taste reactivity paradigm and in a situation where rats were allowed to consume ethanol during periods of restricted fluid access.
2. Materials and methods 2.1. Subjects Forty-nine male Long Evans rats (Harlan, Indianapolis, IN) served as subjects. The rats were approximately 100 days old at the start of the experiment. The animals were individually housed and maintained on a 12-h light–dark cycle (lights on at 0700 h) in a room with an ambient temperature of 21 C. Food and water were available ad libitum throughout the major part of the study; the exception was during the ethanol consumption phase when rats were on restricted access to fluid. All procedures in the present study followed the guidelines developed by the National Institutes of Health and were examined and approved by the Institutional Animal Care and Use Committee at Kansas State University. 2.2. Drug Naltrexone hydrochloride (Sigma) was dissolved in 0.9% saline to make a 3-mg/cc solution; injections were calculated at 1.0 cc/kg body weight. All injections were intraperitoneal. 2.3. Procedure After a 2-week period of adjustment to the laboratory environment, each rat was implanted with an intraoral fistula. Rats were anesthetized with a combination 80 mg/kg ketamine, 0.05 mg/kg acepromazine solution. Subjects were then implanted with a 5-cm piece of 90-gauge polyethylene tubing, which was inserted lateral to the first maxillary molar using a curved needle. After insertion, the fistula was threaded subcutaneously along the cheek, exited at the top of the scalp, and secured with a Teflon washer. Rats were given Bicillin (0.2 cc, i.m.) postoperatively to prevent or minimize infection. After 1 week of postoperative recovery, rats were divided into two groups based on mean body weight. One group of rats was randomly selected to receive 10% vol/ vol ethanol (prepared from 95% ethanol diluted with distilled water) in one bottle and distilled water in a second bottle. The remaining group of rats was given access to two bottles of distilled water. All rats had access to the two-bottle situation for a period of 3 weeks. Bottle positions were switched daily, and consumption measures were taken every 48 h. Glass bottles (250 ml) with rubber stoppers and stainless steel drinking tubes were used.
At the end of ethanol familiarization, the rats were ranked, based on either ethanol consumption (grams per kilogram body weight) or water consumption, matched, and then assigned to one of four groups. One group (n 5 13) was familiarized with ethanol during the twobottle tests and injected with 3 mg/kg naltrexone before taste reactivity tests with 10% vol/vol ethanol. The remaining rats familiarized with ethanol (n 5 12) were injected with physiological saline prior to the taste reactivity tests. Similarly, the rats given only water during the two-bottle tests were assigned to either a naltrexone group (n 5 11) or a saline group (n 5 13). Treatment, either naltrexone or saline, was randomly determined for the groups. All rats were habituated to the taste reactivity chamber and then tested for reactivity to distilled water to provide experience with the procedures. The taste reactivity chamber (a Plexiglas cylinder 4 cm high and 9 cm in diameter) rested on a glass base placed above a mirror so that the animal’s ventral side could be recorded by a video camera focused on the mirror. A piece of polyethylene tubing was connected to a 10-cc glass barrel syringe from which solutions were delivered using an infusion pump. For a single trial, the pump delivered 1 ml of fluid (rate 5 1 ml/min) directly into the rat’s mouth. After the water trial, rats were tested for taste reactivity to 10% vol/vol ethanol over the next 4 days, with one trial per day. Injections of either naltrexone or saline were given exactly 30 min prior to the taste reactivity test. Taste reactivity data collected via videotape were analyzed frame by frame to quantify the different orofacial responses. Consistent with previous research conducted in this laboratory and previous work by Grill and Norgren (1978), responses were quantified as either ingestive or aversive. Ingestive responses consisted of mouth movements, tongue protrusions, and lateral tongue protrusions; aversive responses consisted of gapes, drips, head shakes, forelimb flails, fluid expulsions, and chin rubs. All videotaped trials were scored without the rater knowing the experimental condition of the individual rats. After reactivity data were collected, the animals were put on a schedule of restricted fluid access to determine the effect of ethanol familiarity and naltrexone treatment on ethanol consumption. The rats were habituated for 6 days to a restricted fluid schedule in which the animals had access to water at 1300 h for 30 min. For these trials, 50-ml polypropylene centrifuge tubes fitted with one-hole rubber stoppers and stainless steel drinking tubes were used. Water bottles (250 ml glass) were then put back on the home cages from 1700 to 0800 h to ensure adequate hydration. This particular schedule produced a very mild level of fluid deprivation (5-h deprivation) prior to the daily testing sessions. After 6 days, water was replaced during the 30-min session by 10% vol/vol ethanol. Exactly 10 min before ethanol access, rats were injected with either naltrexone or saline, consistent with the treatment received
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during reactivity tests. Rats received four consecutive days of drug treatment and ethanol access. 2.4. Data analysis The present design was a mixed factorial plan with drug treatment as one factor and ethanol familiarity as a second between-groups variable. Data from the taste reactivity and consumption tests were analyzed with a 2 3 2 3 4 mixed analysis of variance (ANOVA) (Familiarization 3 Drug Treatment 3 Day) with repeated measures on the last factor. Taste reactivity responses were pooled into total ingestive and total aversive responses for analysis. A standardized measure of effect size, partial eta squared (h2p), is provided in the results for each significant outcome (Kirk, 1982). Partial eta squared provides a way to evaluate the magnitude of the experimental treatment effect. A large treatment effect is indicated by an effect size of approximately 0.15 (Cohen, 1977). Separate analyses were run for the ingestive and the aversive responses using the General Linear Models procedure for repeated measures (SPSS). An additional 2 3 2 3 4 mixed ANOVA (Familiarization 3 Drug Treatment 3 Day) was used to analyze the ethanol consumption data. When overall differences exceeded the P ! .05 criterion, post hoc tests were used to compare treatment groups. A standard level of P ! .05 was also used for these comparisons. 3. Results 3.1. Ethanol familiarization During the 3-week period when half the rats had free access to 10% vol/vol ethanol and water, there was a large range of consumption levels. Ethanol consumption for the last 48-h period ranged from 5.5 to 64.4 g. When the data were converted to g EtOH/kg body weight, the range of ethanol consumption was from 1.08 to 21.88 g. Considering all 25 rats given access to 10% vol/vol ethanol, the mean amount of ethanol consumed was 33.8 g (6S.E.M. 5 3.17) per 48-h period (7.4 g EtOH/kg body weight 6 0.96 S.E.M.). These rats also consumed a mean of 32.8 g (6S.E.M. 5 3.49) of distilled water over the same 48-h period. Rats given access to two bottles of distilled water consumed a mean total of 58.4 g (6S.E.M. 5 3.0) of water, an amount roughly equivalent to the total amount consumed by rats given access to ethanol and water. Average body weights for each group, which were matched when the groups were formed (Familiar-Saline, 289 [69.7 S.E.M.] g; Novel-Saline, 308 [68.3 S.E.M.] g; Familiar-Naltrexone, 295 [66.3 S.E.M.] g; Novel-Naltrexone, 290 (66.1 S.E.M.) g), remained essentially equal during ethanol familiarization and through the remainder of the experiment. Body weights at the end of the experiment were not significantly different for the four groups (Familiar-Saline, 384 [616.0 S.E.M.] g; Novel-Saline, 419 [68.3 S.E.M.] g; Familiar-Naltrexone, 405 [67.2 S.E.M.] g; Novel-Naltrexone, 396 [69.9 S.E.M.] g).
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3.2. Taste reactivity The results from the taste reactivity tests, which were done after the animals had been given 3 weeks access to either ethanol and water or water alone, are shown in Figs. 1 and 2. To simplify data presentation, the Day component was collapsed as this factor was not a significant main effect in any of the analyses (nor did it interact significantly with the other factors). As can be seen in Fig. 1, the EthanolSaline rats consistently made the largest number of ingestive responses during testing. Naltrexone treatment significantly reduced ingestive responding [F(1, 45) 5 5.43, P !.05, h2p 5 0.11]. Familiarity was also a significant effect as rats preexposed to ethanol made significantly more ingestive responses overall [F(1, 45) 5 7.98, P !.05, h2p 5 0.15]. Drug treatment and ethanol familiarity did not interact in a statistically significant manner. Aversive responding by all four groups is shown in Fig. 2 (again, the Day factor was not a significant main effect so the data are collapsed across the four trials). Naltrexone treatment was quite effective for eliciting aversive responses [F(1, 45) 5 14.58, P !.05, h2p 5 0.24]. Although ethanol familiarity was not a significant main effect, there was a significant interaction between drug treatment and ethanol familiarity [F(1, 45) 5 7.11, P !.05, h2p 5 0.14]. In naltrexone-treated rats, ethanol familiarity resulted in higher levels of aversive responding than that produced by rats for which ethanol was novel. An opposite result was found for saline-treated rats: ethanol familiarity resulted in a decreased level of aversive responding compared to that of the novel condition. Visual examination of the data clearly shows that naltrexone was effective for eliciting aversive responses in rats, particularly in rats for which ethanol was a familiar solution. For comparison purposes, the data from the water trials before and after the ethanol reactivity tests are presented in Table 1. In general, rats perceived distilled water as relatively aversive. Responding to water was similar to that seen in the ethanol reactivity shown by the saline-treated
Fig. 1. Mean number of ingestive responses made to 10% vol/vol ethanol by rats in each of the four groups. Rats were familiarized with either ethanol or water; drug treatment consisted of either 3 mg/kg naltrexone or physiological saline. Rats were tested for taste reactivity to ethanol exactly 30 min after injection. Because the Day effect was not statistically significant, the data are shown for the Familiarization and Drug Treatment factors only.
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Fig. 2. Mean number of aversive responses to 10% vol/vol ethanol made by the four groups of rats. Format and explanation is the same as that described for Fig. 1.
rats. No significant group differences or testing effects were found in the water responding. 3.3. Ethanol consumption When taste reactivity testing was completed, rats were tested for consumption of 10% vol/vol ethanol after they were injected with either naltrexone or saline, the results of which are shown in Fig. 3. Statistical analysis revealed a significant effect of drug treatment [F(1, 45) 5 60.51, P !.05, h2p 5 0.56] in addition to a significant effect of ethanol familiarity [F(1, 45) 5 6.71, P !.05, h2p 5 0.19]. In general, rats treated with naltrexone consumed significantly less ethanol than rats treated with saline, replicating earlier reports. Rats familiar with ethanol consumed significantly more than rats for which ethanol was novel. The interaction between drug treatment and ethanol familiarity was not statistically significant.
4. Discussion This experiment revealed that, with one significant exception, familiarity with ethanol and acute treatment with naltrexone produced independent effects on ethanol taste reactivity and ethanol consumption in outbred rats. Rats that had ethanol experience produced more ingestive Table 1 Mean (6S.E.M.) ingestive and aversive responses to distilled water by the four groups Water trial Pre
Post
Ingestive responding Familiar-Saline 58.2 Novel-Saline 46.7 Familiar-Naltrexone 47.0 Novel-Naltrexone 82.73
(613.6) (65.2) (67.6) (611.5)
37.8 43.3 33.3 56.5
(66.9) (616.2) (69.9) (611.1)
Aversive responding Familiar-Saline Novel-Saline Familiar-Naltrexone Novel-Naltrexone
(63.9) (62.4) (63.9) (65.0)
15.2 17.0 15.3 11.7
(64.3) (67.8) (67.0) (66.3)
15.2 18.9 18.9 17.6
Fig. 3. Mean amount of 10% vol/vol alcohol consumed by rats during the 30-min access period. Rats were mildly fluid deprived, injected with the appropriate dosage of naltrexone, and 10 min later given half an hour of access to ethanol. The data are presented showing only the Familiarization and Drug Treatment factors as the Day effect was statistically nonsignificant.
responses than rats not familiar with the taste of ethanol. These data confirm earlier findings from the present laboratory that had shown that palatability shifted to a more ingestive response pattern of response with ethanol experience both in outbred rats (Kiefer et al., 1994) and in rats genetically selected for high ethanol consumption (BadiaElder & Kiefer, 1999; Bice & Kiefer, 1990; Kiefer et al., 1995). Ethanol familiarity also was a significant main effect in the consumption data where higher levels of ethanol consumption were found in rats familiar with 10% vol/vol ethanol (the ethanol novel groups had limited experience with the taste of ethanol during the reactivity tests; specifically, these rats were presented with 1 ml a day for 4 days). All rats were tested for ethanol taste reactivity first, thus potentially confounding the consumption tests. For example, it is possible that naltrexone would have carryover effects such that the consumption data might be compromised. However, the present results and prior work have generally indicated that acute treatment with naltrexone does not affect future testing. In the present experiment, the Day factor was not a significant effect in any of the dependent measures nor did it interact with the drug treatment, indicating that there were no consistent changes in responding over repeated testing. Also, in other reports (Coonfield et al., 2004; Hill & Kiefer, 1997), it has been consistently shown that, during acute treatments with naltrexone, ethanol consumption was significantly decreased but immediately returned to control levels the next day when treatment was halted. The results of acute naltrexone treatment on ethanol taste reactivity and ethanol consumption were also consistent with data published earlier (Coonfield et al., 2002; Hill & Kiefer, 1997). Rats treated with acute naltrexone produced significantly fewer ingestive responses and consumed significantly lower levels of ethanol than saline-treated rats. It has been suggested (Hill & Kiefer, 1997) that the reduced levels of consumption seen following naltrexone treatment are probably related to the decreased palatability of ethanol
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that is produced by this drug. It should be noted, however, that within the two groups treated with naltrexone, ethanol familiarity did attenuate somewhat the effectiveness of the drug (Fig. 3 shows that naltrexone treatment virtually eliminated ethanol consumption but rats familiar with ethanol still consumed ethanol at a somewhat higher level). Chotro and Arias (2003) noted that the opioid antagonist, naloxone, effectively reduced ethanol consumption in rats that were also preexposed to ethanol prenatally, further implicating the opioid system in ethanol responsivity. The results of aversive responding revealed the single significant interaction between ethanol familiarity and naltrexone treatment. Ethanol familiarity increased the effectiveness of naltrexone to elicit aversive responses to 10% vol/vol ethanol. Ethanol familiarity in saline-treated rats resulted in significantly lower levels of aversive responding than that seen in the saline-treated rats for which ethanol was novel. These latter results would probably be predicted by the fact that, with control saline injections, rats familiar with ethanol made more ingestive responses and later consumed more ethanol than their counterparts that experienced ethanol for essentially the first time. The fact that ethanol familiarity increased the ability of naltrexone to elicit aversive responses has clear implications for the use of naltrexone in the treatment of those individuals who chronically abuse alcohol. These individuals are obviously quite familiar with all the sensory aspects of ethanol solutions, and familiarity would generally result in a more positive palatability response to ethanol and higher ethanol consumption (conclusions reinforced by examination of the saline-treated rats in the present experiment). The addition of naltrexone treatment had the general effect of reducing both ethanol palatability and consumption; the most significant outcome was that naltrexone was particularly effective for increasing the aversiveness of the ethanol solution in rats familiar with ethanol. The clinical implications for individuals who abuse alcohol appear to be that naltrexone should be considered a viable treatment for discouraging alcohol consumption because of the drug’s ability to render the taste of alcohol as unpalatable. The clear interaction between naltrexone treatment and ethanol familiarity in the aversive response data but not in the other measures (ingestive responding and consumption) suggests that there are probably different neural mechanisms that underlie these specific behaviors. For example, it was suggested earlier that taste reactivity responses identified as ingestive and aversive may be dissociable (Berridge & Grill, 1985). In the cited experiment, sweet/bitter solutions were used to elicit mixed reactivity responses and it was found that one could independently manipulate one of the stimuli (e.g., sucrose) and affect just one of the response types (increasing the sucrose concentration increased ingestive responding but did not alter aversive responding). In the present experiment, the result that ethanol familiarity interacted with naltrexone treatment in the aversive responses but that these two variables acted
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independently on ingestive responding supports the dissociability of these taste reactivity response categories. Taste reactivity to and consumption of a solution generally are consonant. Sweet solutions produce virtually all ingestive-type responses in rats and are avidly consumed in a common drinking situation. Conversely, bitter substances elicit a great deal of aversive responding in rats and are avoided in a common drinking situation. Dissociation of reactivity and consumption was demonstrated in a study by Pelchat et al. (1983) where rats were trained to avoid drinking a sucrose solution by pairing it with one of three negative outcomes: gastrointestinal distress induced by LiCl treatment, shock, or lactose treatment. In all three cases, rats learned to avoid drinking the sucrose solution. It was only in the illness treatment, however, that rats also displayed a palatability shift for the sucrose. Taste reactivity responses to sucrose shifted to aversive following the taste–illness pairing. Thus, rats can be trained to avoid drinking a solution that still elicits positive taste reactivity responses. Demonstration of the converse, drinking a strongly unpalatable solution, has not been reported in the literature to the authors’ knowledge, but anecdotal evidence of humans consuming foul-tasting medicine would seem to be a good example. In a sense, the present data resulted in a dissociation between taste reactivity and consumption as ethanol familiarity attenuated naltrexone’s ability to reduce ingestive responding to ethanol and ethanol consumption, but familiarity facilitated aversive responding following naltrexone treatment. Separating the factors that influence alcohol use and abuse is obviously a difficult task given the number and the complexity of variables. Ethanol familiarity represents one significant factor. Pharmacological treatments that alter the behavioral response to ethanol provide yet another factor. With each of these two factors, understanding of the underlying neural and biochemical mechanisms will ultimately be extremely valuable in the attempts to treat or control abuse of alcohol. It is the interactions between factors, however, that will probably prove to be the most crucial to complete understanding. In the present article, some initial insight into the complex interactions between ethanol familiarity and naltrexone treatment is presented, which may serve as a guide for elucidating underlying neural and biochemical (e.g., opioid) mechanisms in the brain. Acknowledgments The authors thank Paula Schneider for assistance with the data analysis. This work was supported by grant AA11867 from the National Institute on Alcohol Abuse and Alcoholism. References Arias, C., & Chotro, G. (2005). Increased preference for ethanol in the infant rat after prenatal ethanol exposure, expressed on intake and taste reactivity tests. Alcohol Clin Exp Res 29, 337–346.
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Badia-Elder, N. B., & Kiefer, S. W. (1999). Taste reactivity in alcoholpreferring AA and alcohol-avoiding ANA rats. Alcohol 18, 159–163. Berridge, K. C., & Grill, H. J. (1985). Isohedonic tastes support a twodimensional hypothesis of palatability. Appetite 5, 221–231. Bice, P. J., & Kiefer, S. W. (1990). Taste reactivity in alcohol preferring and nonpreferring rats. Alcohol Clin Exp Res 14, 721–727. Chotro, M. G., & Arias, C. (2003). Prenatal exposure to ethanol increases ethanol consumption: a conditioned response? Alcohol 30, 19–28. Cohen, J. (1977). Statistical Power Analysis for the Behavioral Sciences, 2nd ed., Hillsdale, NJ: Lawrence Erlbaum Associates. Coonfield, D. L., Hill, K. G., Kaczmarek, H. J., Ferraro, F. M. III, & Kiefer, S. W. (2002). Low doses of naltrexone reduce palatability and consumption of ethanol in outbred male rats. Alcohol 26, 43–47. Coonfield, D. L., Kiefer, S. W., Ferrro, F. M. III, & Sinclair, J. D. (2004). Ethanol palatability and consumption by high ethanol-drinking rats: manipulation of the opioid system with naltrexone. Behav Neurosci 118, 1089–1096. Domı´nguez, H. D., Lo´pez, M. F., & Molina, J. C. (1998). Neonatal responsiveness to alcohol odor and infant alcohol intake as a function of alcohol experience during late gestation. Alcohol 16, 109–117. Elkins, R. L. (1973). Attenuation of drug-induced bait shyness to a palatable solution as an increasing function of its availability prior to conditioning. Behav Biol 9, 221–226. Ferraro, F. M. III, Hill, K. G., Kaczmarek, H. J., Coonfield, D. L., & Kiefer, S. W. (2002). Naltrexone modifies the palatability of basic tastes and alcohol in outbred male rats. Alcohol 27, 107–114. Grill, H. J., & Norgren, R. (1978). The taste reactivity test. I. Mimetic responses to gustatory stimuli in neurologically normal rats. Brain Res 143, 263–279. Herz, A. (1997). Endogenous opioid systems and alcohol addiction. Psychopharmacology 129, 99–111.
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Ethanol familiarity and naltrexone treatment affect ethanol responses in rats Stephen W. Kiefer*, Katherine G. Hill, Daniel L. Coonfield, Frank M. Ferraro III Department of Psychology, Kansas State University, 492 Bluemont Hall, Manhattan, KS 66506-5302, USA Received 17 June 2005; received in revised form 22 September 2005; accepted 22 September 2005
Abstract In the present study, the effects of ethanol familiarity on the ability of naltrexone to alter ethanol palatability and consumption were examined. One group of rats was allowed continuous access to 10% vol/vol ethanol and water for 3 weeks. A second group received only water. At the end of this time, the groups were further subdivided and injected with either 3 mg/kg naltrexone or saline (total of four groups; n 5 11–13 per group) before ethanol taste reactivity tests with 10% vol/vol ethanol and ethanol consumption tests. Results showed that naltrexone effectively decreased ingestive responding and increased aversive responding. Further, rats familiar with alcohol made more ingestive responses to 10% vol/vol ethanol. A significant interaction of drug treatment and familiarity was found in the data for aversive responses: naltrexone treatment produced more aversive responses in ethanol-familiar rats, whereas saline treatment resulted in fewer aversive responses in rats familiar with ethanol. Naltrexone treatment clearly reduced consumption of 10% vol/vol ethanol, although its effects were attenuated somewhat by ethanol familiarity. The present data indicate that both alcohol familiarity and naltrexone treatment affect ethanol reactivity and ethanol consumption in outbred rats. The interaction of naltrexone treatment and ethanol familiarity only for aversive reactivity and the lack of such an interaction for the consumption measures suggests that the mechanisms underlying ethanol reactivity and ethanol consumption may be dissociable at the neural level. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Naltrexone; Taste reactivity; Rat; Palatability; Alcohol; Intake
1. Introduction Rats that are given acute treatments of naltrexone display a reliable change in response to the taste of ethanol and in the consumption of ethanol during short-term access tests (Ferraro et al., 2002; Hill & Kiefer, 1997). Specifically, ethanol becomes less palatable to rats as evidenced by a reliable increase in aversive responses accompanied by a decrease in ingestive responding (although the latter has occasionally failed to achieve standard levels of statistical significance; see Coonfield et al., 2002). As noted, acute treatment with naltrexone reliably reduces consumption of 10% vol/vol ethanol, an effect that may be partially mediated by the alterations in ethanol palatability that are revealed by the taste reactivity tests. Naltrexone is a nonspecific antagonist of endogenous opioid receptors (Herz, 1997; Oswald & Wand, 2004) and is currently being used in human clinical populations as a supplemental treatment (in addition to psychotherapy) for alcohol abuse and alcoholism (O’Malley et al., 1992; Volpicelli et al., 1992).
* Corresponding author. Tel.: 11-785-532-6850; fax: 11-785-5325401. E-mail address: [email protected] (S.W. Kiefer). 0741-8329/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2006.03.004
One factor that has been shown to influence the response of rats to a taste solution is whether that taste is novel or familiar. In an early article, Revusky and Bedarf (1967) demonstrated that rats associated illness with a novel taste much more readily than they associated illness with a familiar taste. This effect has been demonstrated many times since the original publication (Elkins, 1973; Kiefer & Braun, 1977; Kalat & Rozin, 1973) and the explanation has an intuitive appeal: it would be adaptive in an evolutionary sense to associate a bout of illness with a novel taste rather than one that was familiar and that had not produced adverse effects in previous exposures. Ethanol exposure or familiarity also alters a rat’s response to the taste of ethanol and consumption. For example, rats exposed to ethanol prenatally consume more ethanol as infants (Chotro & Arias, 2003; Domı´nguez et al., 1998) and also display increased palatability to the taste of ethanol (Arias & Chotro, 2005). Our examination of naltrexone’s effects on ethanol reactivity and consumption involved extensive ethanol familiarization procedures (Coonfield et al., 2002; Hill & Kiefer, 1997). Rats typically were given 2–3 weeks of continuous access to 10% vol/vol ethanol and water, thus providing extensive familiarization with the taste and the postingestive effects of ethanol. In the present experiment, it was
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determined if the familiarity of ethanol was an important component for the eventual effects of naltrexone treatment. To test this issue, rats familiar with the taste of ethanol were compared to rats for which ethanol was a novel stimulus, both in the taste reactivity paradigm and in a situation where rats were allowed to consume ethanol during periods of restricted fluid access.
2. Materials and methods 2.1. Subjects Forty-nine male Long Evans rats (Harlan, Indianapolis, IN) served as subjects. The rats were approximately 100 days old at the start of the experiment. The animals were individually housed and maintained on a 12-h light–dark cycle (lights on at 0700 h) in a room with an ambient temperature of 21 C. Food and water were available ad libitum throughout the major part of the study; the exception was during the ethanol consumption phase when rats were on restricted access to fluid. All procedures in the present study followed the guidelines developed by the National Institutes of Health and were examined and approved by the Institutional Animal Care and Use Committee at Kansas State University. 2.2. Drug Naltrexone hydrochloride (Sigma) was dissolved in 0.9% saline to make a 3-mg/cc solution; injections were calculated at 1.0 cc/kg body weight. All injections were intraperitoneal. 2.3. Procedure After a 2-week period of adjustment to the laboratory environment, each rat was implanted with an intraoral fistula. Rats were anesthetized with a combination 80 mg/kg ketamine, 0.05 mg/kg acepromazine solution. Subjects were then implanted with a 5-cm piece of 90-gauge polyethylene tubing, which was inserted lateral to the first maxillary molar using a curved needle. After insertion, the fistula was threaded subcutaneously along the cheek, exited at the top of the scalp, and secured with a Teflon washer. Rats were given Bicillin (0.2 cc, i.m.) postoperatively to prevent or minimize infection. After 1 week of postoperative recovery, rats were divided into two groups based on mean body weight. One group of rats was randomly selected to receive 10% vol/ vol ethanol (prepared from 95% ethanol diluted with distilled water) in one bottle and distilled water in a second bottle. The remaining group of rats was given access to two bottles of distilled water. All rats had access to the two-bottle situation for a period of 3 weeks. Bottle positions were switched daily, and consumption measures were taken every 48 h. Glass bottles (250 ml) with rubber stoppers and stainless steel drinking tubes were used.
At the end of ethanol familiarization, the rats were ranked, based on either ethanol consumption (grams per kilogram body weight) or water consumption, matched, and then assigned to one of four groups. One group (n 5 13) was familiarized with ethanol during the twobottle tests and injected with 3 mg/kg naltrexone before taste reactivity tests with 10% vol/vol ethanol. The remaining rats familiarized with ethanol (n 5 12) were injected with physiological saline prior to the taste reactivity tests. Similarly, the rats given only water during the two-bottle tests were assigned to either a naltrexone group (n 5 11) or a saline group (n 5 13). Treatment, either naltrexone or saline, was randomly determined for the groups. All rats were habituated to the taste reactivity chamber and then tested for reactivity to distilled water to provide experience with the procedures. The taste reactivity chamber (a Plexiglas cylinder 4 cm high and 9 cm in diameter) rested on a glass base placed above a mirror so that the animal’s ventral side could be recorded by a video camera focused on the mirror. A piece of polyethylene tubing was connected to a 10-cc glass barrel syringe from which solutions were delivered using an infusion pump. For a single trial, the pump delivered 1 ml of fluid (rate 5 1 ml/min) directly into the rat’s mouth. After the water trial, rats were tested for taste reactivity to 10% vol/vol ethanol over the next 4 days, with one trial per day. Injections of either naltrexone or saline were given exactly 30 min prior to the taste reactivity test. Taste reactivity data collected via videotape were analyzed frame by frame to quantify the different orofacial responses. Consistent with previous research conducted in this laboratory and previous work by Grill and Norgren (1978), responses were quantified as either ingestive or aversive. Ingestive responses consisted of mouth movements, tongue protrusions, and lateral tongue protrusions; aversive responses consisted of gapes, drips, head shakes, forelimb flails, fluid expulsions, and chin rubs. All videotaped trials were scored without the rater knowing the experimental condition of the individual rats. After reactivity data were collected, the animals were put on a schedule of restricted fluid access to determine the effect of ethanol familiarity and naltrexone treatment on ethanol consumption. The rats were habituated for 6 days to a restricted fluid schedule in which the animals had access to water at 1300 h for 30 min. For these trials, 50-ml polypropylene centrifuge tubes fitted with one-hole rubber stoppers and stainless steel drinking tubes were used. Water bottles (250 ml glass) were then put back on the home cages from 1700 to 0800 h to ensure adequate hydration. This particular schedule produced a very mild level of fluid deprivation (5-h deprivation) prior to the daily testing sessions. After 6 days, water was replaced during the 30-min session by 10% vol/vol ethanol. Exactly 10 min before ethanol access, rats were injected with either naltrexone or saline, consistent with the treatment received
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during reactivity tests. Rats received four consecutive days of drug treatment and ethanol access. 2.4. Data analysis The present design was a mixed factorial plan with drug treatment as one factor and ethanol familiarity as a second between-groups variable. Data from the taste reactivity and consumption tests were analyzed with a 2 3 2 3 4 mixed analysis of variance (ANOVA) (Familiarization 3 Drug Treatment 3 Day) with repeated measures on the last factor. Taste reactivity responses were pooled into total ingestive and total aversive responses for analysis. A standardized measure of effect size, partial eta squared (h2p), is provided in the results for each significant outcome (Kirk, 1982). Partial eta squared provides a way to evaluate the magnitude of the experimental treatment effect. A large treatment effect is indicated by an effect size of approximately 0.15 (Cohen, 1977). Separate analyses were run for the ingestive and the aversive responses using the General Linear Models procedure for repeated measures (SPSS). An additional 2 3 2 3 4 mixed ANOVA (Familiarization 3 Drug Treatment 3 Day) was used to analyze the ethanol consumption data. When overall differences exceeded the P ! .05 criterion, post hoc tests were used to compare treatment groups. A standard level of P ! .05 was also used for these comparisons. 3. Results 3.1. Ethanol familiarization During the 3-week period when half the rats had free access to 10% vol/vol ethanol and water, there was a large range of consumption levels. Ethanol consumption for the last 48-h period ranged from 5.5 to 64.4 g. When the data were converted to g EtOH/kg body weight, the range of ethanol consumption was from 1.08 to 21.88 g. Considering all 25 rats given access to 10% vol/vol ethanol, the mean amount of ethanol consumed was 33.8 g (6S.E.M. 5 3.17) per 48-h period (7.4 g EtOH/kg body weight 6 0.96 S.E.M.). These rats also consumed a mean of 32.8 g (6S.E.M. 5 3.49) of distilled water over the same 48-h period. Rats given access to two bottles of distilled water consumed a mean total of 58.4 g (6S.E.M. 5 3.0) of water, an amount roughly equivalent to the total amount consumed by rats given access to ethanol and water. Average body weights for each group, which were matched when the groups were formed (Familiar-Saline, 289 [69.7 S.E.M.] g; Novel-Saline, 308 [68.3 S.E.M.] g; Familiar-Naltrexone, 295 [66.3 S.E.M.] g; Novel-Naltrexone, 290 (66.1 S.E.M.) g), remained essentially equal during ethanol familiarization and through the remainder of the experiment. Body weights at the end of the experiment were not significantly different for the four groups (Familiar-Saline, 384 [616.0 S.E.M.] g; Novel-Saline, 419 [68.3 S.E.M.] g; Familiar-Naltrexone, 405 [67.2 S.E.M.] g; Novel-Naltrexone, 396 [69.9 S.E.M.] g).
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3.2. Taste reactivity The results from the taste reactivity tests, which were done after the animals had been given 3 weeks access to either ethanol and water or water alone, are shown in Figs. 1 and 2. To simplify data presentation, the Day component was collapsed as this factor was not a significant main effect in any of the analyses (nor did it interact significantly with the other factors). As can be seen in Fig. 1, the EthanolSaline rats consistently made the largest number of ingestive responses during testing. Naltrexone treatment significantly reduced ingestive responding [F(1, 45) 5 5.43, P !.05, h2p 5 0.11]. Familiarity was also a significant effect as rats preexposed to ethanol made significantly more ingestive responses overall [F(1, 45) 5 7.98, P !.05, h2p 5 0.15]. Drug treatment and ethanol familiarity did not interact in a statistically significant manner. Aversive responding by all four groups is shown in Fig. 2 (again, the Day factor was not a significant main effect so the data are collapsed across the four trials). Naltrexone treatment was quite effective for eliciting aversive responses [F(1, 45) 5 14.58, P !.05, h2p 5 0.24]. Although ethanol familiarity was not a significant main effect, there was a significant interaction between drug treatment and ethanol familiarity [F(1, 45) 5 7.11, P !.05, h2p 5 0.14]. In naltrexone-treated rats, ethanol familiarity resulted in higher levels of aversive responding than that produced by rats for which ethanol was novel. An opposite result was found for saline-treated rats: ethanol familiarity resulted in a decreased level of aversive responding compared to that of the novel condition. Visual examination of the data clearly shows that naltrexone was effective for eliciting aversive responses in rats, particularly in rats for which ethanol was a familiar solution. For comparison purposes, the data from the water trials before and after the ethanol reactivity tests are presented in Table 1. In general, rats perceived distilled water as relatively aversive. Responding to water was similar to that seen in the ethanol reactivity shown by the saline-treated
Fig. 1. Mean number of ingestive responses made to 10% vol/vol ethanol by rats in each of the four groups. Rats were familiarized with either ethanol or water; drug treatment consisted of either 3 mg/kg naltrexone or physiological saline. Rats were tested for taste reactivity to ethanol exactly 30 min after injection. Because the Day effect was not statistically significant, the data are shown for the Familiarization and Drug Treatment factors only.
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Fig. 2. Mean number of aversive responses to 10% vol/vol ethanol made by the four groups of rats. Format and explanation is the same as that described for Fig. 1.
rats. No significant group differences or testing effects were found in the water responding. 3.3. Ethanol consumption When taste reactivity testing was completed, rats were tested for consumption of 10% vol/vol ethanol after they were injected with either naltrexone or saline, the results of which are shown in Fig. 3. Statistical analysis revealed a significant effect of drug treatment [F(1, 45) 5 60.51, P !.05, h2p 5 0.56] in addition to a significant effect of ethanol familiarity [F(1, 45) 5 6.71, P !.05, h2p 5 0.19]. In general, rats treated with naltrexone consumed significantly less ethanol than rats treated with saline, replicating earlier reports. Rats familiar with ethanol consumed significantly more than rats for which ethanol was novel. The interaction between drug treatment and ethanol familiarity was not statistically significant.
4. Discussion This experiment revealed that, with one significant exception, familiarity with ethanol and acute treatment with naltrexone produced independent effects on ethanol taste reactivity and ethanol consumption in outbred rats. Rats that had ethanol experience produced more ingestive Table 1 Mean (6S.E.M.) ingestive and aversive responses to distilled water by the four groups Water trial Pre
Post
Ingestive responding Familiar-Saline 58.2 Novel-Saline 46.7 Familiar-Naltrexone 47.0 Novel-Naltrexone 82.73
(613.6) (65.2) (67.6) (611.5)
37.8 43.3 33.3 56.5
(66.9) (616.2) (69.9) (611.1)
Aversive responding Familiar-Saline Novel-Saline Familiar-Naltrexone Novel-Naltrexone
(63.9) (62.4) (63.9) (65.0)
15.2 17.0 15.3 11.7
(64.3) (67.8) (67.0) (66.3)
15.2 18.9 18.9 17.6
Fig. 3. Mean amount of 10% vol/vol alcohol consumed by rats during the 30-min access period. Rats were mildly fluid deprived, injected with the appropriate dosage of naltrexone, and 10 min later given half an hour of access to ethanol. The data are presented showing only the Familiarization and Drug Treatment factors as the Day effect was statistically nonsignificant.
responses than rats not familiar with the taste of ethanol. These data confirm earlier findings from the present laboratory that had shown that palatability shifted to a more ingestive response pattern of response with ethanol experience both in outbred rats (Kiefer et al., 1994) and in rats genetically selected for high ethanol consumption (BadiaElder & Kiefer, 1999; Bice & Kiefer, 1990; Kiefer et al., 1995). Ethanol familiarity also was a significant main effect in the consumption data where higher levels of ethanol consumption were found in rats familiar with 10% vol/vol ethanol (the ethanol novel groups had limited experience with the taste of ethanol during the reactivity tests; specifically, these rats were presented with 1 ml a day for 4 days). All rats were tested for ethanol taste reactivity first, thus potentially confounding the consumption tests. For example, it is possible that naltrexone would have carryover effects such that the consumption data might be compromised. However, the present results and prior work have generally indicated that acute treatment with naltrexone does not affect future testing. In the present experiment, the Day factor was not a significant effect in any of the dependent measures nor did it interact with the drug treatment, indicating that there were no consistent changes in responding over repeated testing. Also, in other reports (Coonfield et al., 2004; Hill & Kiefer, 1997), it has been consistently shown that, during acute treatments with naltrexone, ethanol consumption was significantly decreased but immediately returned to control levels the next day when treatment was halted. The results of acute naltrexone treatment on ethanol taste reactivity and ethanol consumption were also consistent with data published earlier (Coonfield et al., 2002; Hill & Kiefer, 1997). Rats treated with acute naltrexone produced significantly fewer ingestive responses and consumed significantly lower levels of ethanol than saline-treated rats. It has been suggested (Hill & Kiefer, 1997) that the reduced levels of consumption seen following naltrexone treatment are probably related to the decreased palatability of ethanol
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that is produced by this drug. It should be noted, however, that within the two groups treated with naltrexone, ethanol familiarity did attenuate somewhat the effectiveness of the drug (Fig. 3 shows that naltrexone treatment virtually eliminated ethanol consumption but rats familiar with ethanol still consumed ethanol at a somewhat higher level). Chotro and Arias (2003) noted that the opioid antagonist, naloxone, effectively reduced ethanol consumption in rats that were also preexposed to ethanol prenatally, further implicating the opioid system in ethanol responsivity. The results of aversive responding revealed the single significant interaction between ethanol familiarity and naltrexone treatment. Ethanol familiarity increased the effectiveness of naltrexone to elicit aversive responses to 10% vol/vol ethanol. Ethanol familiarity in saline-treated rats resulted in significantly lower levels of aversive responding than that seen in the saline-treated rats for which ethanol was novel. These latter results would probably be predicted by the fact that, with control saline injections, rats familiar with ethanol made more ingestive responses and later consumed more ethanol than their counterparts that experienced ethanol for essentially the first time. The fact that ethanol familiarity increased the ability of naltrexone to elicit aversive responses has clear implications for the use of naltrexone in the treatment of those individuals who chronically abuse alcohol. These individuals are obviously quite familiar with all the sensory aspects of ethanol solutions, and familiarity would generally result in a more positive palatability response to ethanol and higher ethanol consumption (conclusions reinforced by examination of the saline-treated rats in the present experiment). The addition of naltrexone treatment had the general effect of reducing both ethanol palatability and consumption; the most significant outcome was that naltrexone was particularly effective for increasing the aversiveness of the ethanol solution in rats familiar with ethanol. The clinical implications for individuals who abuse alcohol appear to be that naltrexone should be considered a viable treatment for discouraging alcohol consumption because of the drug’s ability to render the taste of alcohol as unpalatable. The clear interaction between naltrexone treatment and ethanol familiarity in the aversive response data but not in the other measures (ingestive responding and consumption) suggests that there are probably different neural mechanisms that underlie these specific behaviors. For example, it was suggested earlier that taste reactivity responses identified as ingestive and aversive may be dissociable (Berridge & Grill, 1985). In the cited experiment, sweet/bitter solutions were used to elicit mixed reactivity responses and it was found that one could independently manipulate one of the stimuli (e.g., sucrose) and affect just one of the response types (increasing the sucrose concentration increased ingestive responding but did not alter aversive responding). In the present experiment, the result that ethanol familiarity interacted with naltrexone treatment in the aversive responses but that these two variables acted
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independently on ingestive responding supports the dissociability of these taste reactivity response categories. Taste reactivity to and consumption of a solution generally are consonant. Sweet solutions produce virtually all ingestive-type responses in rats and are avidly consumed in a common drinking situation. Conversely, bitter substances elicit a great deal of aversive responding in rats and are avoided in a common drinking situation. Dissociation of reactivity and consumption was demonstrated in a study by Pelchat et al. (1983) where rats were trained to avoid drinking a sucrose solution by pairing it with one of three negative outcomes: gastrointestinal distress induced by LiCl treatment, shock, or lactose treatment. In all three cases, rats learned to avoid drinking the sucrose solution. It was only in the illness treatment, however, that rats also displayed a palatability shift for the sucrose. Taste reactivity responses to sucrose shifted to aversive following the taste–illness pairing. Thus, rats can be trained to avoid drinking a solution that still elicits positive taste reactivity responses. Demonstration of the converse, drinking a strongly unpalatable solution, has not been reported in the literature to the authors’ knowledge, but anecdotal evidence of humans consuming foul-tasting medicine would seem to be a good example. In a sense, the present data resulted in a dissociation between taste reactivity and consumption as ethanol familiarity attenuated naltrexone’s ability to reduce ingestive responding to ethanol and ethanol consumption, but familiarity facilitated aversive responding following naltrexone treatment. Separating the factors that influence alcohol use and abuse is obviously a difficult task given the number and the complexity of variables. Ethanol familiarity represents one significant factor. Pharmacological treatments that alter the behavioral response to ethanol provide yet another factor. With each of these two factors, understanding of the underlying neural and biochemical mechanisms will ultimately be extremely valuable in the attempts to treat or control abuse of alcohol. It is the interactions between factors, however, that will probably prove to be the most crucial to complete understanding. In the present article, some initial insight into the complex interactions between ethanol familiarity and naltrexone treatment is presented, which may serve as a guide for elucidating underlying neural and biochemical (e.g., opioid) mechanisms in the brain. Acknowledgments The authors thank Paula Schneider for assistance with the data analysis. This work was supported by grant AA11867 from the National Institute on Alcohol Abuse and Alcoholism. References Arias, C., & Chotro, G. (2005). Increased preference for ethanol in the infant rat after prenatal ethanol exposure, expressed on intake and taste reactivity tests. Alcohol Clin Exp Res 29, 337–346.
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