Article Figures & Data
Figures
- Figure 1.
Orexin signaling mediates the antidepressant-like effect of acute calorie restriction. A, Forced swim test results for female orexin null mice and wild-type littermates fed ad libitum (n = 6 wild-type and 10 null) or calorie restricted (n = 5 wild-type and 6 null). For latency, two-way ANOVA reports a significant interaction (F(1,23) = 5.31, p = 0.03) and significant effects for genotype (F(1,23) = 12.63, p = 0.002) and for diet (F(1,23) = 5.35, p = 0.03). For immobility, two-way ANOVA reports a nonsignificant interaction (F(1,23) = 3.58, p = 0.07) with significant effects for genotype (F(1,23) = 13.90, p = 0.001) and for diet (F(1,23) = 5.10, p = 0.03). Bonferroni's post hoc test analysis demonstrated a significant effect for latency and immobility (*p < 0.05) only in wild-type mice. B, Forced swim test results for male orexin null mice and wild-type littermates fed ad libitum (n = 5 wild-type and 7 null) or calorie restricted (n = 5 wild-type and 5 null). For latency, two-way ANOVA reports a significant interaction (F(1,18) = 6.59, p = 0.02) and nonsignificant effect for genotype (F(1,18) = 3.37, p = 0.08) but a significant effect for diet (F(1,18) = 7.80, p = 0.01). For immobility, two-way ANOVA reports a nonsignificant interaction (F(1,18) = 1.07, p = 0.32) and nonsignificant effects for genotype (F(1,18) = 1.02, p = 0.33) and for diet (F(1,18) = 2.41, p = 0.14). Bonferroni's post hoc test analysis demonstrated a significant effect for latency (*p < 0.05) only in wild-type mice. Eight-week-old C57BL/6 male mice were individually housed and subjected to either social defeat daily for 10 d or daily handling (Control). After the social defeat paradigm, mice received either ad libitum feeding or calorie restriction for 10 d. On the following day (day 21 overall), social interaction was measured by comparing time spent interacting with an unfamiliar mouse with an empty target. C, Interaction time presented as a percentage of target absent (n = 28 per group for ad libitum and 9 per group for calorie restriction). Two-way ANOVA showed a main effect for diet (F(1,70) = 38.97, p < 0.0001) and for social defeat (F(1,70) = 4.34, p = 0.04) with no interaction (F(1,70) = 0.95, p = 0.33). 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 social interaction in ad libitum-fed animals (**p < 0.01). D, Calorie restriction increases time spent in the interaction zone in wild-type mice, but not orexin null mice, after social defeat (n = 5 per group). Two-way ANOVA demonstrates a significant effect for genotype (F(1,16) = 14.54, p = 0.002), no effect for diet (F(1,16) = 1.90, p = 0.19), and a significant interaction (F(1,16) = 6.35, p = 0.02). Bonferroni's post hoc test analysis demonstrated a significant effect for calorie restriction in wild-type mice only (*p < 0.05). All error bars presented as SEM.
- 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.
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