High Alcohol-Preferring Mice Show Reaction to Loss of Ethanol Reward Following Repeated Binge Drinking

Abstract

Background: Beyond yielding high blood ethanol (EtOH) concentrations (BECs), binge-drinking models allow examination of drinking patterns which may be associated with EtOH's rewarding effects, including front-loading and consummatory successive negative contrast (cSNC), a decrease in intake when only water is available to subjects expecting EtOH. The goals of the current study were to broaden our understanding of these reward-related behaviors during binge EtOH access in high alcohol-preferring (HAP) replicate lines (HAP2 and HAP3) of mice selectively bred to prefer alcohol. We hypothesized that both lines would show evidence of front-loading during binge EtOH access and that we would find a cSNC effect in groups where EtOH was replaced with water, as these results have been shown previously in HAP1 mice.

Methods: HAP replicate 2 and replicate 3 female and male mice were given 2 hours of EtOH or water access in the home cage for 15 consecutive days using "drinking in the dark" (DID) procedures. Mice received the same fluid (either 20% unsweetened EtOH or water) for the first 14 days. However, on the 15th day, half of the mice from these 2 groups were provided with the opposite assigned fluid (EtOH groups received water and vice versa). Intake was measured in 1-minute bins using specialized sipper tubes, which allowed within-session analyses of binge-drinking patterns.

Results: EtOH front-loading was observed in both replicates. HAP3 mice displayed front-loading on the first day of EtOH access, whereas front-loading developed following alcohol experience in HAP2 mice, which may suggest differences in initial sensitivity to EtOH reward. Consummatory SNC, which manifests as lower water intake in mice expecting EtOH as compared to mice expecting water, was observed in both replicates.

Conclusions: These findings increase confidence that defined changes in home cage consummatory behavior are driven by the incentive value of EtOH. The presence of cSNC across HAP replicates indicates that this reaction to loss of reward is genetically mediated, which suggests that there is a biological mechanism that might be targeted.

Keywords: Binge Drinking; Consummatory Successive Negative Contrast; Ethanol Intake; Front-Loading; High Alcohol-Preferring Mice; Rate of Alcohol.

Figure 1.. Mean Daily Intake and Distance Travelled Over Two Weeks.

Intake: In both replicates, the water group consumed more total fluid each day during DID than the EtOH group, [post-hoc analysis following up on a main effect of group; p < 0.05]. We also note that EtOH intake in both replicates was reliable such that all animals readily consumed pharmacologically relevant (binge) levels of EtOH every day. Error bars are sometimes obscured by the plot symbols (A, B). Distance: In HAP2 mice, a day*group interaction is driven by initial greater locomotor activity in the EtOH group rapidly declining at a much faster rate than the water group whose activity stabilizes around day 3 (C). In HAP3, no significant effects were observed, indicating similar levels of activity between EtOH and water consuming animals in this replicate (D). Multiple comparison tests were conducted wherein distance travelled on each day within a given fluid group (EtOH or Water) was compared to day 1. Results indicate significant differences in the HAP2 replicate only, where days which differed from day 1 are indicated with *. These results indicate the potential of EtOH-induced locomotor sedation in HAP2 mice only.

Figure 2.. Assessment of Front-Loading.

Intake patterns (displayed as means of central moving averages ± SEM) for each replicate were compared on day 1 (A and B) versus day 14 (C and D). Except for day 1 in HAP2 mice, the EtOH groups always had a significantly higher proportion of intake within the first 15 minutes (front-loading) than the water group (E and F). * indicates significant group differences as indicated by Sidak’s post-hoc testing which were conducted following a main effect of group in both replicates, see Results. # Indicates percentage of intake significantly differed from the 12.5% frontloading threshold, indicating front-loading in EtOH groups with the exception of HAP2 day 1.

Figure 3.. Assessment of Front-Loading Over Days.

Analyses considering the percentage of intake within the first 15-minutes of each two-hour DID session over the two-week period indicated a main effect of group in both replicates, where the EtOH group front-loaded more than the water group. * indicates days where percent intake within the first 15 minutes between groups significantly differed as determined by Sidak’s post-hoc testing, black lines indicate days where the EtOH group’s first 15 minute intake differed from the described 12.5% frontloading threshold (always significantly higher), gray lines indicate days where the water group’s first 15 minute intake significantly differed from this threshold (always significantly lower).

Figure 4.. Day 15 EtOH Intake Time course.

In mice experiencing EtOH for the first time (WE), we observed different drinking patterns between the replicates. In HAP2 (A), WE mice do not front-load EtOH at the same level as mice who have a 2-week drinking history (EE). In HAP3 (B), an opposite pattern was observed, where WE mice front-load significantly more than EE.

Figure 5.. Assessment of cSNC.

WW mice drink significantly more than EW mice on day 15, a cSNC effect that is seen across both HAP replicates. No significant differences in intake exist between EE and WE groups in either replicate (no evidence for postive contrast).

Figure 6.. Day 15 Water Intake Time course.

In both replicates we observe a cSNC effect which is further demonstrated by prolonged differences in water intake patterns, where mice with an EtOH history (EW) consumed approximately half as much water during the early portion of the session as mice without any previous EtOH experience (WW).