Fig. 4. Inhibition of Npy1R^PVH neurons activates glutamatergic and GABAergic neurons at the NTS area and promotes food intake in male mice.

a, Schematic of AAV delivery into the PVH area of Npy1R-Cre^+/− mice. b, Representative image of AAV-Flex-tdTomato injection site into the PVH area (n = 3 independent replicates). c, Image of posterior NTS/AP area showing axonal projections from Npy1R^PVH-tdTomato⁺ neurons (n = 2 independent replicates). d, Representative FISH images of pNTS area showing the colocalization of Fos (yellow) and Slc17a6 (cyan) or Slc32a1 (red) markers in control-Gi^PVH and Npy1R-Gi^PVH mice treated with CNO for 1 h in the absence of food. e, Quantification of total Fos⁺ cells in the pNTS and AP areas in control-Gi^PVH (grey bars, n = 3 vehicle-treated and 6 CNO-treated mice) and Npy1R-Gi^PVH mice (orange bars, n = 5 vehicle-treated and 6 CNO-treated mice) after vehicle (open bars) or CNO injection (solid bars). **P = 0.022 and ****P < 0.001. f, Quantification of the total Fos⁺ cells that co-expressed Slc17a6 and Slc32a1 markers in pNTS area after vehicle or CNO injection. (n = 3 vehicle-treated and 6 CNO-treated control-Gi^PVH mice; and n = 5 vehicle-treated and 6 CNO-treated Npy1R-Gi^PVH mice). *P = 0.0164, **P = 0.0052 and ****P < 0.001. g, Cumulative food intake during 24 h in ad libitum-fed Npy1R-Gi^PVH mice treated with vehicle (open dots) and CNO (solid dots; n = 9 male mice), *P = 0.0253. h, Quantification of delta food intake (∆ = CNO − vehicle) at different intervals after vehicle or CNO injection in Npy1R-Gi^PVH mice (n = 9 male mice), *P = 0.013 and ****P < 0.001. i, Average of RER in light and dark cycles from Npy1R-Gi^PVH mice treated with vehicle or CNO (n = 9 male mice). j, Scheme of chronic CNO treatments in Npy1R-Gi^PVH mice. k, Cumulative food intake along 3 d in mice treated chronically with CNO or vehicle in drinking water (n = 10 male mice) **P = 0.0026. l, Percentage of body weight gain after 3 d chronic treatment with vehicle or CNO (n = 15 male mice), **P = 0.0016. m, Average of food intake during light and dark cycles along the 3-d chronic treatment with vehicle or CNO in drinking water (n = 10 male mice), **P = 0.0051, ****P < 0.001. n, Average respiratory coefficient (RER) from 3 d of chronic treatment during light and dark cycles (n = 10 male mice), ****P < 0.0001. Data are presented as the average ± s.e.m. for each biological replicate. Box plots indicate the median ± minimum/maximum and include data points of individual mice. Statistical significance was determined by a two-tailed paired t-test for l; two-way ANOVA followed by Tukey’s test for e, f and n; and Bonferroni’s test for g–m. Scale bar, 100 µm. Figure 4a,j created with BioRender.com. Data from female mice are displayed in Extended Data Fig. 6.

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