Fig. 1: Nts-positive neurons in the LS were activated during hedonic feeding.

A The LS was activated during the intake of high-sucrose food without an energy deficit. Left panel: Food intake measured in 2 h for control mice (ad libitum access to standard chow, n = 10) and hedonic feeding mice (intermittent access to high-sucrose food along with ad libitum access to standard chow, n = 10). Mann–Whitney U test. ****P < 0.0001. Middle panel: Number of c-fos⁺ neurons in the LS of control (n = 10) and hedonic feeding mice (n = 10). Mann–Whitney U test. ****P < 0.0001. Right panel: Representative images of c-fos immunostaining in the LS of control and hedonic-feeding mice. Scale bar: 100 μm. B The LS showed preferential activation when mice consumed high-sucrose food (HSF) compared with standard chow after fasting. Left panel: Food intake measured in 2 h for standard chow (n = 8) or high-sucrose food (n = 8) after an overnight fast. Mann–Whitney U test. ****P < 0.0001. Middle panel: Number of c-fos⁺ neurons in the LS of the fasted, fasted + chow and fasted + HSF groups. One-way ANOVA (F_{(2, 21)} = 6.558, P < 0.01) followed by Tukey’s post hoc test. ns, no significant difference, *P < 0.05, and **P < 0.01. Right panel: Representative images of c-fos immunostaining in the LS of the fasted, fasted + chow and fasted + HSF groups. Scale bar: 100 μm. C Representative images of double in situ hybridization experiments for c-fos (green) and Nts (red). Scale bar: 100 μm. D Representative images of double in situ hybridization experiments for c-fos (green) and Penk (red). Scale bar: 100 μm. E Representative images of double in situ hybridization experiments for c-fos (green) and Crhr2 (red). Scale bar: 100 μm. F Percentages of Penk⁺, Crhr2⁺ and Nts⁺ cells among the c-fos⁺ population: Penk⁺/c-fos⁺ = 19.7 ± 2.5%, Crhr2⁺/c-fos⁺ = 13.5 ± 1.3%, and Nts⁺/c-fos⁺ = 57.6 ± 2.5%. One-way ANOVA (F_(2,14) = 43.71, P < 0.01) followed by Tukey’s post hoc test. ***P < 0.001 and ****P < 0.0001. Means ± s.e.m. G Representative images of double in situ hybridization experiments for Nts (red) and vGAT (green). Scale bar: 100 μm. H Percentage of Nts⁺ cells in the vGAT⁺ population and percentage of vGAT⁺ cells in the Nts⁺ population: (Nts⁺ & vGAT⁺)/vGAT⁺ = 21.3 ± 3.8%, (Nts⁺ & vGAT⁺)/Nts⁺ = 100%, and (Nts⁺ & vGluT2⁺)/Nts⁺ = 1.38 ± 0.5%.