Extended Data Fig. 4: Homogenous function of VMH^Pdyn neurons with different projections.

a, We searched a published dataset (https://mouse.digital-brain.cn/projectome/hy) and found 241 VMH Pdyn+ neurons. We manually picked 172 neurons whose somata located in the dorsomedial part of the VMH for further analyses. b, We clustered these 172 dmVMH Pdyn+ neurons into 3 projectome-defined subtypes using the algorithm previously described. (Left) The total axon projections of neurons in each subtype were plotted in a 3D mouse brain space. Colors were randomly assigned to each neuron. (Right) A summary of axon projection length (in μm) for each subtype, in each brain area. Each tick represents the axon projection length of a single neuron in each brain area in a heatmap fashion with the scale shown above. c, A dot plot showing the preferred target brain area (column) for each subtype (row). The dot circle’s size and color intensity indicate the percentage of neurons in each subtype that projected to the indicated brain area and the average projection length (in μm) in a heatmap fashion, respectively, with the scale on the right. d, Soma distribution of the 3 subtypes of neurons along the anterior-posterior (AP), dorsal-ventral (DV), and lateral-medial (ML) axis of the Allen mouse brain common coordinate frame (CCFv3). e, Schematics for laser stimulation of VMH^pdyn neurons→VLPO projections (left) and a representative brain slice of the fluorescent signals in VMH^pdyn neurons→VLPO projections expressing AAV-DIO-SSFO-eYFP (right). White lines indicate the boundaries of brain structures or optic fibers. Scale bar, 200 μm. f–h, Average traces of food intake (f), core (g) and BAT (h) temperature after optogenetic activation of VMH^pdyn neurons→VLPO projections. (f–h, N = 3 mice per group). i, Schematics for laser stimulation of VMH^pdyn → PAG projections (left) and a representative brain slice of the fluorescent signals in VMH^pdyn neurons →PAG projections expressing AAV-DIO-SSFO-eYFP (right). Scale bar, 200 μm. j–l, Average traces of food intake (j), core (k) and BAT (l) temperature after optogenetic activation of VMH^pdyn neurons →PAG projections (j–l, N = 3 mice per group). m, Schematics for laser stimulation of VMH^pdyn neurons →MEA projections (left) and a representative brain slice of the fluorescent signals in VMH^pdyn neurons →MEA projections expressing AAV-DIO-SSFO-eYFP (right). Scale bar, 200 μm. n–p, Average traces of food intake (n), core (o) and BAT (p) temperature after optogenetic activation of VMH^pdyn → MEA projections (n–p, N = 3 mice per group). Blue arrows and dotted lines indicate the time of 5 Hz 15-second laser stimulation. e–p, plots data from female mice. See Extended Data Fig. 5 for results from male mice. Shaded area of the graph(e–p) indicated time points that are statistically significant as compared to eYFP group. Data are represented as mean ± SEM. P values were determined by Two-way ANOVA followed by Sidak’s multiple-comparison tests in f–h, j–l, n–p.

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