Fig. 6: Combination of zonisamide and granisetron alleviates HFD-induced mental dysfunctions and rescues obesity.

a Examples of in vivo fluorescence traces in ad-lib fed Mcr4^Cre mice with AAV9-FLEX-GCaMP6f into the dBNST followed with a chronic treatment of LFD or HFD or HFD combined with injection of ZNS into the dBNST. b Heat map of color-coded changes of fluorescence intensity in the mice as described in (a). c The numbers of fluorescence transients detected in MC4R^dBNST neurons of the mice as described in (a). d Daily food intake by WT mice with the chronic treatment of LFD or HFD or HFD combined with injection of ZNS into the dBNST. Error bars represent mean ± SEM. n = 7–9 per group; *p < 0.05, LFD vs HFD; ^#p > 0.05, HFD + ZNS vs LFD. One-way ANOVA and followed by Tukey comparisons test. d, e OFT (d) and MBT (e) assays by the mice as described in (a). f Diagram showing in vivo fiber photometry analysis of Mc4r^dBNST neurons in response to Gabra5-KO and ZNS. g Examples of in vivo fluorescence traces in ad-lib fed Mc4r^Cre::Rosa26^Cas9 mice with bilateral injection of either vehicle, AAV9-FLEX-Gabra5^sgRNA-tdTomato (Gabra5^sgRNA) into the dBNST, followed by microinjection of vehicle or ZNS into the dBNST. h Heat map of color-coded changes of fluorescence intensity in the mice as described in (g). i The numbers of fluorescence transients detected from the MC4R^dBNST neurons in mice as described in (g). j, k EPM test of the mice described in (g). Error bars represent mean ± SEM. n = 7–9 per group; *p < 0.05, Vehicle vs Gabra5^sgRNA; ^#p < 0.05, Vehicle vs ZNS; ^‡p > 0.05, Gabra5^sgRNA vs Gabra5^sgRNA + ZNS. One-way ANOVA and followed by Tukey comparisons test. l–q EPM (l, m), OFT(n), TST(o), FST(p), and MBT (q) assays in WT mice with i.p. or i.c.v injection of both ZNS and GNS into dBNST by chronic HFD feeding. r, s Body weight (r) and food intake (s) in mice with the chronic treatment of LFD or HFD or HFD combined with i.p. injection of ZNS and GNS into the dBNST or HFD combined with microinjection of ZNS and GNS into the dBNST during a 7-week treatment of HFD. Error bars represent mean ± SEM. n = 7 per group; *p < 0.05, HFD vs HFD + ZNS + GNS (i.p.); ^#p < 0.05, HFD vs HFD + ZNS + GNS (BNST); two-way ANOVA and followed by Bonferroni comparisons test. t Combined intake of LFD and HFD in mice as described in (r) and (s) during the last 7 days of the 7-week treatment of HFD. Error bars represent mean ± SEM. n = 7 per group; *p < 0.05, LFD vs HFD + ZNS + GNS (i.p.), HFD vs HFD + ZNS + GNS (BNST) and HFD + ZNS + GNS (i.p.) vs HFD + ZNS + GNS (BNST) in the intake of LFD; ^#p < 0.05, LFD vs HFD + ZNS + GNS (i.p.), HFD vs HFD + ZNS + GNS (BNST) and HFD + ZNS + GNS (i.p.) vs HFD + ZNS + GNS (BNST) in the intake of HFD. Two-way ANOVA and followed by Bonferroni comparisons test. u Macronutrient composition of food intake by the mice as described in (r). v Schematic diagram showing that a cocktail therapy of zonisamide and granisetron by action through the GABA_AR-α5 and 5-HT₃R signaling, respectively, within the MC4R^dBNST neurons effectively eliminate obesity and mental disorders comorbidity.