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. 2020 Jul 30;11:3794. doi: 10.1038/s41467-020-17578-7

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© The Author(s) 2020

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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Fig. 3 — Sim1-Cre mice (8–10 weeks old) received injections of AAV-FLEX-GFP or AAV-EF1a-FLEX-EGFP-P2A-mNachBac vectors to bilateral PVH and used for studies. a, b Expression of EGFP in the PVH after the NachBac virus expression (a, left panels), and representative expression of c-Fos expression in the PVH after overnight fast alone (top panels) or overnight fast with 2 h refeeding (bottom panels) in NachBac mice (a, right panels) and control (b). c Comparison of number of neurons with c-Fos expression in the PVH between fast and fast–refeeding in control and NachBac mice (two-way ANOVA, n = 15/each, F(1, 56) = 385.3, ***p < 0.0001 Control fasting vs refeeding and p = 0.7212 NachBac fasting vs refeeding). At least three mice with five sections containing the PVH were used for counting the number of c-Fos in the PVH. Arrows point to the PVH proper. d–h Weekly body weight (d, two-way ANOVA, n = 8 for GFP and 7 for NachBac, F(8, 117) = 4.252, ***p < 0.0001, body weight at 8 weeks after injection between Control and NachBac), weekly net body gain (e, two-way ANOVA, n = 8 for GFP and 7 for NachBac, F(8, 171) = 7.256, ***p < 0.0001, body weight at 8 weeks after injection between Control and NachBac) were followed, real time traces showing energy expenditure (f), comparison in the difference of energy expenditure between day and night (g, unpaired two-tailed Student’s t-test, n = 8 for GFP and 7 for NachBac, t = 3.836, d.f. = 13, **p = 0.0021), and differences between day and night periods within groups (h, two-way ANOVA, n = 8 for GFP and 7 for NachBac, F(3, 26) = 9.314, *p = 0.0488 control GFP day vs night and p = 0.8657, NachBac day vs night). i, j Real time traces showing feeding (i) and comparison in the difference of feeding between day and night (j, two-way ANOVA, n = 8 for GFP and 7 for NachBac, F(3, 26) = 9.257, ***p = 0.0008 Control GFP day vs night, and *p = 0.0458 NachBac day vs night). j–l Daily feeding (k) and energy expenditure (l, unpaired two-tailed Student’s t-test, n = 8 for GFP and 7 for NachBac, t = 3.240, d.f. = 13, **p = 0.0064) were compared between groups. PVH paraventricular hypothalamus, 3V the third ventricle. Scale bar = 200 µM. All data presented as mean ± SEM. Source data are provided as a Source Data file.