Figure 3. Effects of HDAC2 siRNA infusion into the CeA on anxiety-like behaviors and alcohol and sucrose intake in P rats.

A. The light/dark box (LDB) exploration test showed that HDAC2 siRNA infusion attenuated anxiety-like behaviors of P rats in comparison to vehicle- or control siRNA-infused rats. The percentage of time spent in the light and dark compartments was significantly different among the treatment groups (F_{2, 36} = 36.3, p<0.001). HDAC2 siRNA infused rats spent more time in the light compartment and less time in the dark compartment than vehicle- and control siRNA-infused rats. Values are represented as the mean ± SEM of the percentage of time spent in each compartment averaged from 5–17 P rats per group. *Significantly different from control groups (p<0.001; one-way ANOVA followed by post hoc analysis by Tukey’s test).

B. The elevated plus maze (EPM) exploration test also showed that HDAC2 siRNA infusion into the CeA resulted in a reduction in the anxiety-like behaviors of P rats. P rat performance on the EPM test was significantly different between the treatment groups in percentage of open arm entries (F_2,21 = 46.7, p< 0.001) and the percentage of time spent in the open arms (F_2,21 = 52.2, p < 0.001). P rats infused with HDAC2 siRNA showed a higher percentage of open arm entries and time spent in the open arms than those infused with vehicle or control siRNA. The number of total arm entries does not differ significantly between the groups, suggesting that there are no effects of HDAC2 siRNA infusion on the general activity of P rats. Values represent the mean ± SEM of the percentage of open arm entries, percentage of time spent in the open arm, and number of total arm entries from 8 rats per group. *Significantly different from control groups (p<0.001; one-way ANOVA followed by post hoc analysis by Tukey’s test).

C. Voluntary ethanol consumption as measured by the two-bottle free choice paradigm was reduced by infusion of HDAC2 siRNA, but not vehicle, into the CeA of P rats. Analysis by two-way repeated measures ANOVA identified a significant difference in the amount of ethanol consumed between the treatment groups overall and daily, as indicated by the group × day interaction (Group: F_1,120 = 71.9, p<0.001; Group × Day: F_12,120 = 13.8, p<0.001). P rats were given access to water and 7% ethanol followed by water and 9% ethanol. Following the sixth day of ethanol access, P rats received infusion of vehicle or HDAC2 siRNA and consumption of water and 9% ethanol were monitored for several days. Total fluid intake did not significantly differ between the groups. Values are represented as the mean ± SEM (6 P rats per group) of the ethanol consumption (g/kg/day) or total fluid intake (mL) plotted daily. *Significantly different between the groups (p<0.01–0.001; post hoc analysis of the group by day interaction by Tukey’s test).

D. Voluntary sucrose consumption as measured by the two-bottle free choice paradigm was unaltered by the infusion of HDAC2 siRNA and vehicle into the CeA of P rats. Analysis by two-way repeated measures ANOVA indicated no significant differences between groups. P rats were given access to water and sucrose solution as described in the methods section. Following the third day of 4% sucrose intake, P rats received infusion of vehicle or HDAC2 siRNA and consumption of water and sucrose solution were monitored for several days. Total fluid intake did not significantly differ between the groups. Values are represented as the mean ± SEM (5 P rats per group) of the sucrose intake (g/kg/day) or total fluid intake (mL) plotted daily.