Fig. 4: AgRP neurons are required for the enhanced saccharin intake during calorie restriction.

a Representative photomicrographs of coronal section of hypothalamic arcuate nucleus (ARH) of wild-type (WT) or AgRP-Cre mice stereotaxically injected with pAAV8-hSyn-DIO-hM3D(Gq)-mCherry (red). Mice were perfused after CNO treatment and brain sections were subjected to a fluorescent immunohistochemistry against c-Fos (green). b Bar graphs display saccharin intake for 4 h after vehicle or CNO treatment 30 min before access to saccharin solution in AgRP-Gq mice in a crossover protocol (n = 5 mice per group, paired t-test, p = 0.029, Cohen’s d = 1.21). c Displays body weight change of CR AgRP-Gi mice treated with vehicle or CNO (n = 11 and 9 mice per group, two-way ANOVA, p-time x treatment = 0.413; p-treatment = 0.515; p-time < 0.001, Cohen’s f = 3.96). d Shows saccharin intake of CR AgRP-Gi mice that were treated with vehicle or CNO before saccharin exposure (n = 9 and 8 mice per group; two-way ANOVA: p-time x treatment = 0.703; p-treatment = 0.263; p-time < 0.001; Cohen’s f = 1.183. Dunnett’s post hoc tests: days 1, 2, 3, and 4 vs. day 0 in the vehicle-treated group: p = 0.203, 0.030, 0.006, and 0.036; in the CNO-treated group: p = 0.994, 0.999, 0.042, and 0.010. Vehicle- vs. CNO-treated groups: t-test on day 1, p = 0.165; day 2, p = 0.003, Cohen’s d = 1.55; day 3; p = 0.028, Cohen’s d = 1.02; and day 4, p = 0.0.466). e Bar graph displaying the latency to eat in vehicle- or CNO-treated AgRP-Gi mice (n = 4 and 5 mice per group; two-way ANOVA: p-time × treatment = 0.008, Cohen’s f = 0.861; Sidak’s post hoc test: vehicle- vs. CNO-treated AgRP-Gi mice on day 2, p = 0.004). f Bar graph displaying 1-hour food intake after saccharin exposure in vehicle- or CNO-treated AgRP-Gi mice (n = 7 and 6 mice per group; t-test: day 1, p = 0.065; day 2, p = 0.027, Cohen’s d = 1.46; day 3, p = 0.0.282; and day 4, p = 0.276). g Representative photomicrographs of coronal section of ARH of AgRP-Gi mice treated with vehicle or CNO subjected to chromogenic immunohistochemistry against c-Fos (cyan) and fluorescent immunohistochemistry against AgRP (red). Colocalization is shown with white arrowheads. h Representative photomicrographs of coronal section of paraventricular hypothalamic nucleus (PVH) of AgRP-Gi mice treated with vehicle or CNO subjected to chromogenic immunohistochemistry against c-Fos (brown signal, blue arrowheads). Scale Bar: 50 µm low magnification and 25 µm high magnification. Data represent the mean ± SEM, with error bars representing the SEM. *, p < 0.05; †, p < 0.1 vs. different treatment; a, p < 0.05 vs day 0 same treatment.