These studies showed that quantitative variations in passive ethanol exposure are positively associated with increases in subsequent voluntary ethanol intake and S+ preference in two inbred mouse strains, B6 and D2. In three independent experiments, we replicated and extended the principal findings from a previous study that only included an all-or-none comparison of experimental (passive ethanol infusions) vs. control (passive water infusion) groups within each strain (Fidler et al., 2011
). Consistent with our previous study, the current studies showed that B6 Water mice self-infused more ethanol and were less averse to the S+ than D2 Water mice. Moreover, absolute intakes for each strain during the choice phase were similar to those in the previous IGAC study and similar to intakes measured in home-cage two-bottle choice drinking procedures (Belknap, Crabbe & Young, 1993
). The low intakes in D2 Water groups confirmed that switching from the oral to IG route of administration was not sufficient to increase voluntary ethanol intake in this strain, supporting previous suggestions that aversive post-absorptive effects of ethanol normally contribute to avoidance of oral ethanol by D2 mice (Cunningham, Gremel & Groblewski, 2009
; Fidler et al., 2011
Also consistent with the previous study, passive ethanol exposure increased IG ethanol intake and preference in both strains and eliminated the strain difference in S+ preference. Moreover, the enhancing effect of passive exposure was greater in D2 mice than in B6 mice during the first few days of ethanol self-infusion (i.e., no-choice phase) in all three experiments. Importantly, the present studies extended our previous findings by showing that the increases in intake and preference were positively correlated with cumulative passive ethanol dose (). The high voluntary ethanol intakes and S+ preferences in ethanol-exposed D2 mice are of particular interest in light of the low intakes and preferences commonly reported for this strain in home-cage two-bottle drinking procedures.
Correlations Between Cumulative Passive Ethanol Exposure and IGAC
Analyses of ethanol intake patterns in all three experiments were also consistent in showing that B6 mice had more bouts per day than D2 mice, and that passive ethanol exposure increased daily intakes by increasing number of bouts per day in both strains. Additionally, all three studies showed that D2 mice had larger ethanol bouts than B6 mice and that D2 mice self-infused a higher percentage of their total ethanol intake in large bouts (≥ 1 g/kg/bout) than B6 mice. Thus, these findings strongly support our previous characterization of B6 mice as “sippers” and D2 mice as “gulpers.”
Experiment 1 showed that exposure to a high cumulative dose of ethanol each day was not sufficient to increase later IGAC. Rather, the pattern of daily ethanol exposures was critical, with three larger infusions (3-inf group) producing a stronger effect than six (6-inf) or nine (9-inf) smaller infusions. Number of infusions had an especially pronounced impact on choice phase intake and S+ preference in D2 mice. The direction of the effect was contrary to expectations based on a previous periodicity study in which rats that had received six smaller IG infusions per day later drank more ethanol than rats that had received the same total dose per day in three larger infusions (Le Magnen et al., 1984
). Possible reasons for the discrepant outcomes include the use of different species, different dosing schedules and different routes of administration during self-administration testing. Although Le Magnen et al. attributed their outcome to a group difference in dependence, their study provided no independent evidence that dependence was greater in the six-infusion group than in the three-infusion group. Thus, it is possible that their group difference in ethanol drinking was unrelated to dependence or withdrawal. In contrast, Experiment 1 strongly supports the suggestion that ethanol dependence and withdrawal played a role in enhancing ethanol intake in the D2 3-inf group.
More generally, the combined data from Experiments 1–3 showed a strong positive quantitative relationship between physical withdrawal signs during the passive phase and the later increase in ethanol self-infusion in D2 mice, a relationship that was apparent in correlations based on group averages () as well as in correlations based on individual subject scores (Supplementary Table 4
). To our knowledge, no other model of dependence-induced intake has ever provided as comprehensive a demonstration of this relationship. Since B6 mice showed negligible signs of withdrawal across a wide range of ethanol dosing regimens, one might conclude that dependence and physical withdrawal played little or no role in mediating their later increases in IGAC. However, it is also possible that our single time point HIC assessment procedure did not provide a sufficiently sensitive index of withdrawal in B6 mice, perhaps because the assessment was not made at the time of their peak withdrawal. It is also possible that examination of a comlete HIC time course and calculation of area-under-the-curve (e.g., Crabbe, 1998
) or affective signs of withdrawal (Heilig et al., 2010
) would provide more sensitive predictors of later IGAC in B6 mice. These issues should be addressed in future IGAC studies and in other models in which B6 mice have shown dependence-induced increases in ethanol intake (e.g., oral self-administration after intermittent vapor exposure: Becker & Lopez, 2004
The positive relationship between withdrawal severity and subsequent IGAC in D2 mice is consistent with theories that attribute the increase in ethanol intake to negative reinforcement based on alleviation of withdrawal (Becker, 2008
; Koob & Le Moal, 2008
; Fidler et al., 2011
). In other words, ethanol-induced alleviation of more severe withdrawal would be expected to have a greater positive impact on subsequent increases in ethanol intake than alleviation of less severe withdrawal. It is important to distinguish the positive environmental
(phenotypic) correlation observed here from the previously described negative genetic
correlation between sensitivity to ethanol withdrawal and oral ethanol intake and preference (Metten et al., 1998
). The negative genetic correlation is based on observations of both selectively bred and inbred mouse strains that indicate higher oral ethanol consumption and preference in genotypes with lower genetic susceptibility to ethanol withdrawal (e.g., B6, HAP) than in genotypes with higher genetic susceptibility to ethanol withdrawal (e.g., D2, LAP). In contrast to the environmental correlation, demonstration of the genetic correlation does not require measurement of ethanol intake subsequent to induction of withdrawal in the same animals. Whereas a genetic correlation suggests possible overlap in the genes influencing the related phenotypes (Crabbe et al., 1990
), the environmental correlation suggests a possible causal relationship (i.e., withdrawal caused increased ethanol intake).
The positive environmental correlation observed here contrasts with the negative phenotypic correlation recently reported in HAP-2/LAP-2 mice (Fidler et al., 2011
), suggesting that the direction of this relationship varies across genotypes. Nevertheless, all four of the genotypes that have been studied in the IGAC procedure (B6, D2, HAP-2, LAP-2) have shown a positive relationship between withdrawal and later ethanol intake based on comparisons between experimental and control mice within each strain (i.e., all experimental groups showed more severe withdrawal and higher intake than the same genotype control groups). If one assumes that negative reinforcement (i.e., alleviation of withdrawal) underlies the positive environmental correlation based on the group differences, the negative phenotypic correlation observed in HAP-2/LAP-2 mice might be explained by detrimental effects of withdrawal-induced physical impairment on opportunities to experience the contingency between S+ licking and ethanol infusion. In other words, the overall impact of negative reinforcement on ethanol intake might have been reduced for the most highly impaired HAP-2/LAP-2 mice simply because they experienced withdrawal alleviation less often than less impaired mice. By this account, differences in the direction of the phenotypic correlation are explained by genetic differences in the ability of acute withdrawal to interfere with approach toward and licking of the S+ tube (i.e., HAP-2/LAP-2 mice are affected more than B6/D2 mice). Similar response interference mechanisms might contribute to the negative genetic correlation between oral ethanol intake and sensitivity to ethanol withdrawal. Additional studies that examine the genetic and phenotypic relationship between withdrawal severity and IGAC in other genotypes, including other replicates of the HAP/LAP selection, are needed to evaluate the generality of these findings.
Although emphasis has been placed on the potential predictive relationship between withdrawal and later ethanol intake, consideration must also be given to the significant positive correlations between intoxication and ethanol intake (). If one interprets the intoxication measure as a general index of ethanol sensitivity, the direction of this correlation appears contrary to what would be predicted on the basis of the literature suggesting that a low level of response to ethanol is a risk factor for alcohol dependence (Crabbe, Bell & Ehlers, 2010
). That literature would suggest that more highly intoxicated individuals (or genotypes) should be less
likely (not more likely) to have high ethanol intakes. Given that prediction and the general difficulty in finding intoxication measures in rodents that model low response to alcohol (Crabbe et al., 2010
), we believe that the correlations between intoxication and IGAC observed here are better interpreted as byproducts of the strong correlation between our intoxication measure and withdrawal severity. Indeed, the correlations based on group medians from all three studies were +0.61 (n = 12, p < .05) in B6 mice and +0.94 (n = 12, p < .0001) in D2 mice; correlations based on individual scores were +0.33 (n = 12, p < .001) in B6 mice and +0.48 (n = 12, p < .0001) in D2 mice. In other words, mice that showed higher intoxication scores tended to also show more severe withdrawal, which provides the motivational substrate for enhancing ethanol intake via negative reinforcement.
Although no tolerance assessments were made, it is reasonable to suggest that passive ethanol exposure induced tolerance to one or more ethanol effects in these studies. Furthermore, tolerance would likely be influenced by variations in cumulative ethanol dose or periodicity of ethanol exposure (Kalant, LeBlanc & Gibbins, 1971
). Thus, consideration must be given to the possibility that tolerance contributed to the effects of passive ethanol exposure on IGAC. For example, ethanol intake might be enhanced by development of tolerance to ethanol’s motor inhibitory effects. Alternatively, tolerance to ethanol’s post-absorptive aversive effects might interfere with development of conditioned taste aversion to S+ (Risinger & Cunningham, 1995
). However, although tolerance to ethanol’s inhibitory or aversive effects might explain increased ethanol intake or a weaker S+ aversion, tolerance cannot readily explain the development of an absolute S+ preference (i.e., preference ratio > 0.5) in the D2 10-day group or as previously reported for B6, HAP-2 and LAP-2 ethanol-treated mice (Fidler et al., 2011
). Tolerance to negative effects would be expected to reduce S+ aversion, presumably leading to indifference in the selection of S+ and S− (i.e, S+ preference ratio = 0.5). However, an additional mechanism, such as negative reinforcement via alleviation of withdrawal, is needed to explain S+ preference ratios above 0.5.
Previously, we suggested that the effects of passive ethanol exposure on IGAC might be jointly determined by tolerance-induced reduction in ethanol’s post-absorptive aversive effects and by negative reinforcement through alleviation of withdrawal (Fidler et al., 2011
). Tolerance to post-absorptive aversive effects might be especially important in allowing ethanol-avoiding genotypes (e.g., D2) to administer sufficient ethanol to experience negative reinforcement. On the basis of the literature and current data, we further hypothesize that the relative contributions of tolerance and negative reinforcement might vary across genotypes. For example, in light of previous studies showing that B6 mice develop greater tolerance than D2 mice to ethanol’s hypothermic (Crabbe et al., 1982
) and taste-aversion-inducing (Risinger & Cunningham, 1995
) effects, tolerance might be more importantly involved in enhancing IGAC in B6 mice than in D2 mice. Conversely, given data suggesting a stronger relationship between withdrawal severity and IGAC in D2 mice, negative reinforcement might play a more important role for D2 than for B6 mice. As noted earlier, it is also possible that D2 mice show greater negative reinforcement based on alleviation of acute physical signs of withdrawal whereas B6 mice are influenced more by alleviation of dependence-induced affective changes (e.g., anxiety) during protracted abstinence. Future studies should address these hypotheses in B6 and D2 mice as well as in other inbred and selectively bred genotypes.
When interpreting and comparing results from the IGAC model to findings from other models, it is important to note that dependence induced enhancement of ethanol intake has frequently been reported with delays of several days or weeks of abstinence between onset of withdrawal and the assessment of ethanol intake (e.g., Roberts et al., 2000
; Lopez & Becker, 2005
; Sommer et al., 2008
). Thus, in contrast to the present studies, those studies offered no opportunity for ethanol intake to alleviate the acute physical symptoms of withdrawal that wax and wane during the first 24–48 hrs after removal of ethanol. Consequently, it has been suggested that enhanced ethanol intake after such delays are mediated by alleviation of more protracted effects of withdrawal on negative affective states (e.g., Roberts et al., 2000
; Becker, 2008
; Heilig et al., 2010
). Currently, it is not known whether withdrawal-related increases in IGAC depend on access to ethanol and withdrawal alleviation during the initial acute phase of withdrawal. Ongoing studies in our laboratory are designed to assess this issue.
In summary, the present studies provide a more comprehensive analysis of the quantitative relationship between chronic ethanol exposure and later ethanol intake than any previous set of studies. Across all three studies, two variables were strong predictors of increased IGAC in both strains: cumulative ethanol dose and intoxication during passive exposure. However, a high cumulative dose was not sufficient to induce an increase in IGAC unless the intermittently administered unit doses were large enough to produce intoxication and withdrawal. In D2 mice, withdrawal during passive ethanol exposure was also a strong predictor of increased IGAC. However, B6 mice showed little withdrawal in these studies, precluding analysis of its potential role. Overall, these data support the hypothesis that dependence-induced increases in IG ethanol intake and S+ preference are jointly determined by two processes that might vary across genotypes: (a) tolerance to aversive post-absorptive ethanol effects, and (b) negative reinforcement (i.e., alleviation of withdrawal by self-administered ethanol).