Fig. 5: Synergistic effect of the 5-HT₃R and GABA_AR-α5 signaling within the MC4R^dBNST neurons on HFD-induced mental deficiency and obesity.

a Expression level of Htr1a-3a and Htr2c in the dBNST of WT mice treated with LFD or HFD. B Co-localization of MC4R neurons with both GABA_AR-α5 and 5-HT₃A signaling within the dBNST. Inset in (b) is a magnified view showing colocalized neurons with arrow heads. Scale bar in (b), 150 μm. scale bar in inset of (b), 50 μm. Error bars represent mean ± SEM. n = 9 per group; *p < 0.05; two-way ANOVA and followed by Bonferroni comparisons test. c Examples of in vivo fluorescence traces in GNS or vehicle-treated Mc4r^Cre mice with AAV9-FLEX-GCaMP6f into the dBNST followed with a chronic treatment of HFD or LFD (left). Right, Heat map of color-coded changes of fluorescence intensity in the mice as described in (C). d The number of fluorescence transients detected in MC4R^dBNST neurons. Error bars represent mean ± SEM. n = 10 per group; *p < 0.05, LFD + Veh vs HFD + Veh; ^#p < 0.05, HFD + Veh vs HFD + GNS; one-way ANOVA and followed by Tukey comparisons test. e–i The EPM (e, f), FST (g), TST (h), and MBT (i) test by the mice as described in (c). Error bars represent mean ± SEM. n = 7–9 per group; *p < 0.05, LFD + Vehicle vs HFD + Vehicle; ^#p < 0.05, HFD + Vehicle vs HFD + GNS; ^‡p > 0.05, LFD + Vehicle vs HFD + GNS. one-way ANOVA and followed by Tukey comparisons test. j, k OFT test by Mc4r^Cre::Rosa26^Cas9 mice with a bilateral injection of either vehicle (Vehicle), AAV9- FLEX-Gabra5^sgRNA-tdTomato (Gabra5^sgRNA), or AAV9-FLEX-Gabra5^sgRNA-tdTomato and AAV9-FLEX-Htr3a^shRNA-EYFP (Gabra5^sgRNA + Htr3a^shRNA) into the dBNST. Error bars represent mean ±  SEM. n = 6–8 per group; *p < 0.05, Vehicle vs Gabra5^sgRNA; ^#p < 0.05, Gabra5^sgRNA vs Gabra5^sgRNA + Htr3a^shRNA; ^‡p > 0.05, Vehicle vs Gabra5^sgRNA + Htr3a^shRNA; one-way ANOVA and followed by Tukey comparisons test. L Body weight (left) and daily food intake (right) in LFD or HFD-treated Mc4r^Cre::Rosa26^Cas9 mice with injection of AAV9-FLEX-Htr3a^shRNA-EYFP or combined AAV9-FLEX-Gabra5^sgRNA-tdTomato and AAV9-FLEX-Htr3a^shRNA-EYFP (Gabra5^sgRNA + Htr3a^shRNA) into the dBNST. Error bars represent mean ±  SEM. n = 9 per group; *p < 0.05, HFD vs HFD + Htr3a^shRNA; two-way ANOVA and followed by Bonferroni comparisons test. m Combined intake of HFD and LFD in a food preference test following the assay in the mice described in (l). Error bars represent mean ±  SEM. n = 9 per group; *p < 0.05, LFD vs Htr3a^shRNA and HFD vs Htr3a^shRNA in the intake of LFD; ^#p < 0.05 LFD vs Htr3a^shRNA and HFD vs Htr3a^shRNA in the intake of HFD. Two-way ANOVA and followed by Bonferroni comparisons test. n Macronutrient composition of daily food intake by the mice described in (l).