Fig. 6: Activation of vlPAGPenk neurons rescues binge-like eating habits of ELT mice.
a,b, Schematics showing the strategy for rabies-mediated retrograde tracing of monosynaptic inputs to the vlPAGPenk neurons. c–f, Confocal image showing starter cells in the vlPAGPenk neurons (c); scale bar, 250 μm. Green, expressing eGFP (d), red, expressing mRuby2 (e), yellow, expressing both eGFP and mRuby2 (f), replicated independently with similar results in 3 mice. Scale bars, 50 μm. g–i, Representative images of LH neurons sending inputs to vlPAGPenk neurons with mRNA labeling for Lepr (g), Hcrt (h) and Pmch (i), replicated independently with similar results in 2 mice. White arrows represent colocalization. Scale bars, 20 μm. j, Quantification of LH inputs expressing Lepr, Hcrt, Pmch, Nts and Cartpt to vlPAGPenk neurons. Box plots display the median (center) and the 2.5th to 97.5th percentiles of the distribution (bounds) with whiskers extending from minimum to maximum values (n = 284 cells from two Penk-Cre mice). k, Schematic depicting the injection of an AAV expressing a Cre-dependent Kir2.1 into the vlPAG of control Penk-Cre mice, replicated independently with similar results in 5 mice. Scale bar, 500 μm. l,m, Cumulative body weight gain during 4 d of ad libitum access to either NC or HFD (n = 5 mice per group). In l, two-way ANOVA (F(1,16) = 13.580, P = 0.002) was followed by Fisher’s LSD post hoc test for multiple comparisons; ***P < 0.001 compared with HFD-fed mice expressing DIO-eGFP or NC-fed mice expressing DIO-Kir2.1; in m, two-way RM ANOVA (F(3,24) = 41.954, P < 0.001) was followed by Bonferroni post hoc test for multiple comparisons; *P = 0.02 and ***P < 0.001, compared with DIO-eGFP at the respective day. n, Schematic for the chemogenetic manipulation of vlPAGPenk neurons. Confocal image showing DREADDs-expressing vlPAGPenk, replicated independently with similar results in 5 mice. Scale bar, 250 μm. o, 2.5 h Re-HFD consumption in control mice with or without CNO (n = 4, 6 and 5 mice for each group). Two-way RM ANOVA (F(6,20) = 22.438, P < 0.001) was followed by Bonferroni post hoc test for multiple comparisons; *P = 0.011 for mCherry versus hM4D; ***P < 0.001 for mCherry versus hM3D; †††P < 0.001 and †P = 0.022 for hM3D versus hM4D at the respective Re-HFD cycles. p, 2.5 h Re-HFD consumption in ELT mice with or without CNO (n = 5, 6 mice per group). Two-way RM ANOVA (F(2,18) = 23.197, P < 0.001) was followed by Bonferroni post hoc test for multiple comparisons; ***P < 0.001 compared with DIO-mCherry at the respective Re-HFD cycles. Data are the mean ± s.e.m.
