Fig. 2: The LH contains molecularly distinct Penk-expressing neurons.

a Coronal diagrams depicting the region analyzed (squared in red; top) and confocal images showing the Penk expression in the multiple hypothalamic subregions of Penk-Cre × Ai14 mice (bottom), replicated independently with similar results in 4 mice. Scale bars, 50 μm. fx, fornix; 3 v, third ventricle. b Quantification of tdTomato-positive cells in the LH, Arc, PVN, VMH, and DMH (n = 4 Penk-Cre × Ai14 mice). Dashed line indicates the normalized level of tdTomato-positive cells in the LH. c–h, Representative images of RNA in situ hybridization for Pmch (c), Hcrt (e), and Lepr (g) with Penk in the LH. Arrowheads represent colocalization, replicated independently with similar results in 2 mice. Scale bars, 25 μm. Pie charts indicate % of LH^Penk neurons colocalizing with Pmch (d; n = 274 cells from two mice), Hcrt (f; n = 236 cells from two mice), or Lepr (h; n = 226 cells from two mice). A large fraction of LH^Penk neurons does not express Pmch, Hcrt, or Lepr. i, j Representative images of RNA in situ hybridization for VGAT and vGlut2 with Penk in the LH (i). Red, yellow, and white arrowheads represent colocalization of Penk with vGlut2, VGAT, and all respectively, replicated independently with similar results in 3 mice. Scale bars, 25 μm. Pie charts indicate % of LH^Penk neurons colocalizing with vGlut2, VGAT and all (j; n = 528 cells from three mice). k, l Representative images of RNA in situ hybridization for Penk and Cre in the LH using Penk-Cre mice and its high magnification image (squared in white dashed line) (k). Arrowheads represent colocalization. Scale bars, 20 μm. Quantification of the % overlap of Cre+ cells as a fraction of Penk+ cells and Penk+ cells as a fraction of Cre+ cells (l). (n = 20 images). n.s not significant. Data are presented as mean ± SEM.