Figure 2.

Orexin-dependent reversal of the effects of social defeat by calorie restriction. A, Mice from Figure 1C were killed 1 d after testing social interaction, and prepro-orexin mRNA levels were determined by quantitative PCR (n = 7 per group). Two-way ANOVA reports a main effect for social defeat (F_(1,24) = 9.6, p = 0.005) but no effect for diet (F_(1,24) = 0.22, p = 0.64) or interaction (F_(1,24) = 0.22, p = 0.64). Bonferroni's post hoc test analysis demonstrated no effect for calorie restriction in either group. Mice were subjected to social defeat and calorie restriction as in Figure 1C. On day 21, mice were tested for social interaction and then perfused with paraformaldehyde 1 h later. Tissue sections were processed for immunohistochemistry for orexin and c-Fos. (**p < 0.01.) B, Concentration of orexin-positive neurons in the lateral hypothalamus at bregma −1.8 mm. Two-way ANOVA reports a main effect for social defeat (F_(1,8) = 8.46, *p = 0.02) but no effect for diet (F_(1,8) = 0.03, p = 0.86) or interaction (F_(1,8) = 0.03, p = 0.86). Bonferroni's post hoc test analysis demonstrated no effect for calorie restriction in either group (p > 0.05). C, Percent of orexin-positive cells expressing c-Fos. Two-way ANOVA reports a main effect for social defeat (F_(1,8) = 92.89, p < 0.0001) and for calorie restriction (F_(1,8) = 150.89, p < 0.0001) with no significant interaction (F_(1,8) = 0.32, p = 0.57). Bonferroni's post hoc test analysis demonstrated a significant effect for calorie restriction in both groups (***p < 0.001). Planned a posteriori analysis demonstrated that social defeat causes a significant decrease in the percentage of orexin cells expressing c-Fos (***p < 0.001). D, Number of orexin cells expressing c-Fos plotted against social interaction scores. Best fit line calculated by linear regression (R² = 0.6440). E, Representative photomicrographs of double-labeled orexin (red) and c-Fos (blue). Examples of double-labeled cells marked with red arrows. All error bars presented as SEM.