Fig. 3: Npy1R⁺ neurons in PVH respond to simultaneous neuronal input from AgRP and POMC neurons.

a, Representative images (a) and quantification of the percentage of Mc4r, Pdyn, Glp1r and Npy1r expressing-neurons in PVH area that colocalize with Fos mRNA in CNO-treated mice for 1 h (b). *P = 0.0442 AgRP-Gq versus POMC-Gi in MC4R⁺ analysis, *P = 0.0402 control versus AgRP-Gq;POMC-Gi in Npy1R⁺ analysis. c, Schematic of experimental protocol for unbiased whole-brain Fos analysis. d, Representative images from Fos volumetric comparison (n = 4 mice per group; Methods) between CNO-treated control versus AgRP-Gq mice, showing coronal, sagittal and transcranial sections. Insert shows a coronal section at the PVH area, with increased Fos signal in the thalamus (orange) area and decreased Fos signal in the periventricular area (blue). e, Representative coronal images of the Fos volumetric comparisons between AgRP-Gq versus AgRP-Gq;POMC-Gi mice (upper) and POMC-Gi versus AgRP-Gq;POMC-Gi group (lower). f, Detailed image of Fos volumetric analysis at the coronal DVC area for each previous comparison. g, Representative images of medial (mNTS) and posterior NTS/AP (pNTS) areas from the DVC complex. h, Quantification of Fos protein expression in the DVC area from CNO-treated mice for 1 h. **P = 0.005 AgRP-Gq;POMC-Gi versus rest of groups in the mNTS area, *P = 0.0385 AgRP-Gq;POMC-Gi versus control and AgRP-Gq group in the pNTS area; and *P = 0.0362 AgRP-Gq;POMC-Gi versus control in the AP area. i, Quantification of cells expressing Fos mRNA in pNTS and AP areas from CNO-treated mice for 1 h. *P = 0.0184 AgRP-Gq;POMC-Gi versus AgRP-Gq and POMC-Gi groups, ***P = 0.0004 AgRP-Gq;POMC-Gi versus control. j, Representative FISH images of the pNTS area showing the colocalization of Slc17a6 (blue) and Slc32a1 (red) markers in Fos⁺ (yellow) neurons. k, Quantification of Fos⁺ neurons that co-express Slc17a6, Slc32a1 or none of these genes in the pNTS area. *P = 0.0141 AgRP-Gq;POMC-Gi versus control and AgRP-Gq for Slc17a6, *P = 0.059 AgRP-Gq;POMC-Gi versus rest of the groups for Slc32a1. Data are the average ± s.e.m. of the percentage of positive cells per hemisection of PVH area in b; and the mean ± s.e.m. of total positive cells per hemisection of NTS area in h, i and k. Biological replicates are: b, n = 6 mice per group for MC4R⁺ and Npy1R⁺, and 5 for Pdyn⁺ and Glp1R⁺ analysis; c–e, n = 4 mice per group; h, n = 5 control, 5 AgRP-Gq, 5 POMC-Gi and 6 AgRP-Gq;POMC-Gi mice; i and k, n = 4 control, 4 AgRP-Gq, 4 POMC-Gi and 5 AgRP-Gq;POMC-Gi mice. Box plots indicate the median ± minimum/maximum values and include data points of individual mice. Statistical significance was determined by one-way ANOVA followed by Tukey’s test for b, and two-way ANOVA followed by Sidak’s test for h, and Tukey’s test for i and k. White scale bar in a, g and j , 100 µm. Black scale bar in d–f, 1,500 µm. Figure 3c created with BioRender.com. IHC, immunohistochemistry; LSFM, light sheet fluorescence microscopy.

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