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. 2022 Dec 12;26(1):79–91. doi: 10.1038/s41593-022-01208-0

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 5 — (a) Representative images of Cre (green) and Lepr mRNA (red) expression in the LH of wild-type (top) or Lepr-Cre mice (bottom), replicated independently with similar results in four mice. Scale bars, 20 μm. (b) Representative images of Lepr (red) and VGAT mRNA (white) in the LH. Scale bars, 20 μm. Pie charts indicate percentage of LH^Lepr cells either colocalizing or non-colocalizing with probes to VGAT (n = 225 cells from three mice). (c) Lepr expression in the LH of Lepr-Cre x Ai14 mice along the rostral-caudal axis, replicated independently with similar results in three mice. Scale bars, 50 μm. (d) Representative image of the LH of Lepr-Cre x Ai14 mice (tdTom, red), with either Mch or Hcrt immunostaining (green), replicated independently with similar results in three mice. Scale bars, 50 μm. (e) Representative images showing tdTom (red) and c-fos immunoreactivity (green) in the LH of HFD-primed Lepr-Cre x Ai14 mice following exposure to either NC or Re-HFD. White arrowheads indicate the colocalization, replicated independently with similar results in five mice. Scale bars, 50 μm. (f) Quantification of the proportion of c-fos-positive cells among tdTom-expressing LH^Lepr neurons in response to NC or Re-HFD (n = 5 mice per group). Two-tailed unpaired t-test, t8 = −5.793, ***p = 0.000408. (g) Sample traces of neuronal firing in response to 150 pA current injections (500 ms) in LH^Lepr neurons of control Lepr-Cre mice expressing DIO-EmGFP (control: DIO-EmGFP), control Lepr-Cre mice expressing DIO-Lepr shRNA (control: DIO-Lepr shRNA) and ELT Lepr-Cre mice expressing DIO-EmGFP (ELT: DIO-EmGFP). (h) Action potential firing rate in response to increasing current injection steps in LH^Lepr neurons (n = 8 cells from three control: DIO-EmGFP mice, n = 9 cells from three control: DIO-Lepr shRNA mice, and n = 10 cells from three ELT: DIO-EmGFP mice). Two-way RM ANOVA (F(30,360) = 3.237, p < 0.001) was followed by Bonferroni post hoc test for multiple comparisons; at 100 pA, **p = 0.008 for control: DIO-EmGFP versus control: DIO-Lepr shRNA, ***p < 0.001 for control: DIO-EmGFP versus ELT: DIO-EmGFP; at 120 pA, **p = 0.001 for control: DIO-EmGFP versus control: DIO-Lepr shRNA, ***p < 0.001 for control: DIO-EmGFP versus ELT: DIO-EmGFP; at 140 pA, **p = 0.002 for control: DIO-EmGFP versus control: DIO-Lepr shRNA, ***p < 0.001 for control: DIO-EmGFP versus ELT: DIO-EmGFP; at 160 pA, **p = 0.004 for control: DIO-EmGFP versus control: DIO-Lepr shRNA, ***p < 0.001 for control: DIO-EmGFP versus ELT: DIO-EmGFP; at 180 pA, *p = 0.011 for control: DIO-EmGFP versus control: DIO-Lepr shRNA, **p = 0.003 for control: DIO-EmGFP versus ELT: DIO-EmGFP; at 200 pA, *p = 0.024 for control: DIO-EmGFP versus ELT: DIO-EmGFP; at 220 pA, *p = 0.049 for control: DIO-EmGFP versus ELT: DIO-EmGFP. (i) The rheobase measurements for LH^Lepr neurons from (h) (n = 8 cells from three control: DIO-EmGFP mice, n = 9 cells from three control: DIO-Lepr shRNA mice and n = 10 cells from three ELT: DIO-EmGFP mice). One-way ANOVA (F(2, 24) = 6.664, p = 0.005) was followed by Fisher LSD post hoc test for multiple comparisons; **p = 0.002, **p = 0.009 compared with control: DIO-EmGFP mice. Data are presented as mean ± SEM.