Reversed scototaxis during withdrawal after daily-moderate, but not weekly-binge, administration of ethanol in zebrafish

Abstract

Alcohol abuse can lead to severe psychological and physiological damage. Little is known, however, about the relative impact of a small, daily dose of alcohol (daily-moderate schedule) versus a large, once per week dose (weekly-binge schedule). In this study, we examined the effect of each of these schedules on behavioural measures of anxiety in zebrafish (Danio rerio). Adult wild-type zebrafish were administered either 0.2% ethanol on a daily-moderate schedule or 1.4% ethanol on a weekly-binge schedule for a period of 21 days, and then tested for scototaxis (preference for darkness) during withdrawal. Compared to a control group with no alcohol exposure, the daily-moderate group spent significantly more time on the light side of the arena (indicative of decreased anxiety) on day two of withdrawal, but not day 9 of withdrawal. The weekly-binge group was not significantly different from the control group on either day of withdrawal and showed no preference for either the light or dark zones. Our results indicate that even a small dose of alcohol on a daily basis can cause significant, though reversible, changes in behaviour.

Figure 1. Scototaxis arena and ethanol administration schedule used with control, weekly-binge, and daily-moderate groups.

(A) The scototaxis arena measured 9.5 cm wide, 9.5 cm deep, and 55 cm long, with black walls covering one half of the arena, white walls covering the other half, and white flooring throughout; water depth was maintained at 5 cm. (B) Ethanol administration and testing took place over a 30 day period. The daily-moderate group (•) received ethanol (0.2%) each day for 21 days of the exposure period, whereas the weekly-binge group (○) received ethanol (1.4%) only on the 7th, 14th and 21st days. All groups, including control, underwent the same daily procedures with the exception of ethanol administration. All animals were tested (T) on days 23 (2^nd day of withdrawal) and 30 (9^th day of withdrawal).

Figure 2. Naive preference for light/dark zones with and without a black floor.

(A) When a black floor was used, zebrafish spent more time in the dark zone (186.9±11.16 sec, n = 20) compared to the light zone (113.1±11.16 sec, n = 20). (B) With a white floor, the fish also spent more time in the dark zone (207.8±15.37 sec, n = 12) compared to the light zone (92.22±15.37 sec, n = 12). ***p<0.001, paired t-test.

Figure 3. Daily-moderate ethanol exposure reverses scototactic preference on day two of withdrawal.

(A) i: A plot of a single control zebrafish as it moved throughout the arena during the five minute trial. ii: Heatmap (pseudocolour representation of zebrafish movement) of the same fish throughout the five minute trial. iii: Total time spent in the light and dark zones for the five minute trial. Control fish preferred the dark zone (paired t-test, p = 0.0442, n = 13). (B) i–ii: Same as in (A) but for a representative fish in the weekly-binge group. iii: Weekly-binge fish did not have a significant preference for either zone (paired t-test, p = 0.3325, n = 14). (C) i–ii: Same as in (A) but for a representative fish in the daily-moderate group. iii: Daily-moderate fish spent significantly more time in the light zone (paired t-test, p = 0.0366, n = 15). (D) Preference index for all groups. There was a significant difference in preference between daily-moderate and control groups (One way ANOVA, F (2,39) = 4.32, p = 0.0202 with post-hoc Tukey test). *p<0.05. (E) The number of transitions between the dark and light zones for each group for the duration of the 5 minute trials. No significant differences in zone transitions were observed across all groups (One way ANOVA, F (2, 34) = 1.266, p = 0.2949).

Figure 4. Dark preference and zone transitions after nine days of withdrawal.

(A) The preference index was calculated as in Figure 3. No group showed a significant preference for light versus dark. There were no significant differences between groups (One way ANOVA, F (2, 39) p = 0.5487; n = 15 for control, n = 12 for weekly-binge, n = 15 for daily-moderate). (B) The number of zone transitions were calculated as in Figure 3. There were no significant differences in number of zone transitions between groups nine days after ethanol exposure. (One way ANOVA, F (2,39) = 1.592, p = 0.2164; n = 15 for control, n = 12 for weekly-binge, n = 15 for daily-moderate).