These data show a time-dependent enhancement of ethanol intake during withdrawal in D2 mice, but not in B6 mice. More specifically, dependent D2 mice significantly increased ethanol intake (relative to non-dependent mice) when initial ethanol access began during the first 24 h of withdrawal, but not when initial access was delayed for 1, 3 or 5 days after withdrawal (Exp. 2). In contrast, dependent B6 mice showed consistently high ethanol intakes in all conditions, with little enhancement of intake relative to control mice, regardless of when initial access began (Exp. 2). When dependent D2 mice received initial ethanol access during the first 2 days of withdrawal, the enhancement persisted over a 5-day abstinence period (Exp. 3). Moreover, dependent D2 mice showed greater oral intake of ethanol that was paired with IG ethanol infusion during acute withdrawal (Exp. 4).
The time-dependent enhancement in D2 mice provides strong support for the potentially important role played by acute withdrawal relief in the development of excessive ethanol intake and alcoholism. One possible interpretation is that ethanol intake during the first 24 h of withdrawal established a learned preference for S+ based on negative reinforcement produced by alleviation of the aversive physical symptoms of acute withdrawal [2
] or by alleviation of a concurrent negative affective state such as anxiety [4
]. In the absence of data showing a graded relationship between acute withdrawal severity () and ethanol intake between 8-25 h post withdrawal, it is difficult to argue that these data uniquely support an interpretation based on alleviation of physical withdrawal symptoms. However, regardless of the underlying mechanism, these data show that ethanol access during the initial 24 h of withdrawal can play a critical role in determining subsequent ethanol intake.
Of particular interest, withdrawal-prone D2 mice showed a greater detrimental effect of delayed ethanol access on ethanol intake than withdrawal-resistant B6 mice, raising the possibility of a positive genetic correlation between sensitivity to withdrawal severity and sensitivity to the intake enhancing effect of ethanol access during acute withdrawal. However, this conclusion must be tempered by several considerations. Most critically, because B6 and D2 are inbred strains, they differ in many different phenotypes that may or may not be related to ethanol intake during withdrawal. It will be necessary to study a larger number of inbred strains (or mouse lines selectively bred for withdrawal sensitivity) in this procedure in order to determine whether the relationship observed here represents a true genetic correlation [25
]. Since non-dependent B6 mice self-infused ethanol at relatively high levels, another reason for cautiously interpreting the genetic differences is that lack of increased intake in dependent B6 mice could simply reflect a ceiling effect. Alternatively, the parameters of passive ethanol exposure might not have established a sufficient level of dependence in B6 mice [21
]. It is also possible that the predisposition to high drinking in B6 mice is completely unrelated to withdrawal sensitivity. Regardless of the interpretation, the present data underscore the importance of genotype in determining ethanol intake in both dependent and non-dependent mice.
A previous meta-analysis suggested a genetic correlation different from that seen here. That is, withdrawal-prone genotypes have generally been found to drink less ethanol than withdrawal-resistant genotypes [26
]. However, a critical difference between our studies and those included in the meta-analysis is that we examined ethanol intake in dependent mice given ethanol access during withdrawal. In contrast, studies included in the meta-analysis examined ethanol intake only in mice that never experienced withdrawal. Indeed, the difference between non-dependent B6 and D2 mice in Exp. 2 is quite consistent with the meta-analysis in showing higher ethanol intake in the withdrawal-resistant B6 strain than in the withdrawal-prone D2 strain. Our finding that ethanol access during acute withdrawal had a relatively greater impact on later ethanol intake in D2 mice than in B6 mice raises the possibility that the genetic relationship between withdrawal sensitivity and ethanol intake might be different when animals receive ethanol access during acute withdrawal.
The persistence of enhanced ethanol intake and preference across a 5-day abstinence period in D2 mice given initial ethanol access during acute withdrawal is consistent with the hypothesized development of a learned Pavlovian preference for S+ based on negative reinforcement during acute withdrawal. Alternatively, it could be explained by negative reinforcement of the instrumental licking response. In either case, the persistence of such learned responses over a period of abstinence could explain relapse to alcohol taking long after the physical symptoms of acute withdrawal have subsided. Since the magnitude of negative reinforcement diminishes after acute withdrawal has ended, learned responses would be expected to gradually extinguish, which might also explain the slow decrease in ethanol intake over the 5 days of choice testing ().
In addition to negative reinforcement, enhanced ethanol intake in D2 mice can be explained, at least in part, by tolerance to aversive post-absorptive ethanol effects that would otherwise produce conditioned taste aversion [20
]. Thus, the detrimental effect of delaying initial ethanol access during withdrawal might also reflect a temporal decay in tolerance to those effects. Future studies could address this possibility by directly examining the decay in tolerance to ethanol's aversive effect during the first 5 days of withdrawal to see whether the time courses for loss of tolerance and reduction in ethanol intake are similar. Another alternative to the negative reinforcement interpretation is that passive ethanol exposure enhanced ethanol's rewarding effects in a time-dependent manner in D2 mice. This possibility could be tested using conditioned place preference, although it would be difficult to distinguish experimentally between a transient sensitization-like enhancement of ethanol reward and a transient withdrawal-related increase in ethanol's negative reinforcing effect since both effects could explain increased place preference.
Although our studies have focused on negative reinforcement during acute withdrawal, these findings do not preclude possible contributions by other risk factors, including differences in sensitivity to ethanol's ability to alleviate negative affective states (e.g., anxiety, depression) that may persist during protracted abstinence [4
]. The possibility that genetically mediated individual differences in sensitivity to acute withdrawal or in ethanol's ability to alleviate acute withdrawal deserves greater attention in the study of the brain mechanisms underlying alcoholism, especially in light of data suggesting that adult children of alcoholics who have not yet developed alcoholism (but are more likely to become alcoholic) experience more severe acute withdrawal symptoms (“hangovers”) than children of non-alcoholics [27
]. Such findings raise the possibility that drinking during acute withdrawal might be more strongly reinforced in individuals who are at high risk for developing alcoholism. Finally, these data encourage greater consideration of treatment and relapse prevention pharmacotherapies that target processes influencing negative reinforcement during acute withdrawal.