Neural adaption in midbrain GABAergic cells contributes to high-fat diet–induced obesity

Figures

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  Fig. 1.. Long-term activation of vlPAG GABAergic cells reverses DIO. (A) Schematic and representative images. Scale bar, 100 μm. (B) Experimental timeline and group schematic. (C to F) hM3Dq-expression and mCherry-expression DIO mice were treated with CNO (0.25 mg/kg) intraperitoneally each day for 2 weeks. Two-way repeated-measures ANOVA was used (hM3Dq: n = 7 mice; mCherry: n = 7 mice) for body weight (*P < 0.05), fat mass (*P < 0.05), lean mass (P = 0.4843), and fat rate (*P < 0.05). Holm-Sidak post hoc test was used to determine time point when hM3Dq group is significantly different from control group. (G to J) hM3Dq expression and mCherry expression DIO mice were treated with CNO at 5:00 p.m. and then placed in metabolic cages for 24 hours. Energy expenditure and respiratory exchange ratios (RER) from each group of mice are shown. Bar graphs (H and J) represent averages of dark (8:00 p.m. to 8:00 a.m.) and light cycles. Shaded parts represent the dark cycle (mCherry: n = 12 hours averaged from seven mice; hM3Dq: n = 12 hours averaged from six mice; unpaired t test). (K) Representative H&E staining of the inguinal white adipose tissue (iWAT) from CNO treatment hM3Dq expression and…

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  Fig. 2.. Calcium imaging of vlPAG GABAergic cells during refeeding of normal chow or high-fat chow. (A) Schematic for expressing GCaMP6m. Scale bar, 100 μm. (B) Experimental timeline and group schematic. (C) Amplitude of Ca2+ signaling in vlPAG GABAergic neurons described in (C) to (N) (test 1 group I: n = 7 mice, test 1 group II: n = 6 mice, test 2 group I: n = 7 mice, test 2 group II: n = 6 mice, test 3 group I: n = 7 mice, test 3 group II: n = 6 mice). Data are represented as mean ± SEM, and two-way repeated-measures ANOVA (*P < 0.05) followed by the Holm-Sidak post hoc test was used (P = 0.5746 for test 1, P = 0.7859 for test 2, and ****P < 0.0001 for test3). (D to F) Sample recording trace (D), heatmaps (E), and mean GCaMP6m signal (F) of all mice (group I and group II) aligned to the initiation and termination of feeding corresponding to (D) (n = 13 mice) in vlPAG GABAergic neurons in response to normal chow stimuli in test 1. (G to I) Sample recording trace (G), heatmaps (H), and mean GCaMP6m signal of all mice (I) showing the Ca2+ signals in vlPAG GABAergic neurons in response to first time high-fat chow exposure. (J to L) Sample recording trace (J), heatmaps (K), and mean GCaMP6m…

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  Fig. 3.. GABAergic synaptic transmission and intrinsic properties of vlPAG GABAergic cells were altered in DIO mice. (A) Diagram showing virus injection (left) and electrophysiological recording (right). Scale bar, 100 μm. (B) Representative traces of mIPSCs recorded in vlPAG GABAergic neurons from mice that had undergone 7 weeks of NFD or HFD. (C) Average mIPSC frequency from the two groups (NFD: n = 15 cells from three mice, HFD 7 W: n = 21 cells from three mice, ***P < 0.001, Mann-Whitney test). (D) Average mIPSC amplitude from the two groups (P = 0.247, unpaired t test). (E) Representative traces of spontaneous action potential (AP) firing. (F) Spontaneous AP firing rate of vlPAG GABAergic neurons from mice that had undergone 7 W NFD or HFD (NFD: n = 43 of 53 cells from eight mice, HFD 7 W: n = 43 of 52 cells from nine mice; *P < 0.05, Mann-Whitney test). (G) Representative traces of single AP. Dot lines represent −40 mV. (H to L) Activation threshold (H, P = 0.74, unpaired t test), AP duration (I, P = 0.063), AHP amplitude (J, *P < 0.05, unpaired t test), resting potential (K, **P < 0.01, Mann-Whitney test), and AP threshold (L, **P < 0.01, Mann-Whitney test) of vlPAG GABAergic…

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  Fig. 4.. Transcriptional expression changes in vlPAG GABAergic cells in DIO mice. (A) Schematic representation of the workflow. (B) UMAP visualization of 14438 cells (7625 cells for NFD control group and 6813 cells for DIO group). (C) UMAP visualization of eight transcriptionally distinct clusters expressing canonical markers. Related markers see fig. S11. (D) Unsupervised clustering of vlPAG GABA neuronal cell types represented in a UMAP plot (n = 3606 cells). Different cell type clusters are color coded. Related markers see fig. S11. (E) Violin plot showing normalized expression of Cacna2d1 for vlPAGGABAergic neuronal cell types in NFD control and DIO groups; gaba1: ****P = 9.0 × 10−14 (< 0.0001), gaba2: P = 0.9, gaba3: P = 0.1938, gaba4: P = 0.6042, gaba5: P = 0.3396, gaba6: P = 0.8732, gaba7: P = 0.5349, Wilcoxon. (F) RNA FISH costaining of Vgat and Cacna2d1 mRNA in vlPAG area. Scale bars, 50 μm. (G) Proportion of Vgat-positive neurons expressing Cacna2d1 (NFD ctr: n = 3900 cells from 27 slides of four mice, DIO: n = 3369 cells from 27 slides of four mice; ****P < 0.0001, unpaired t test). (H) Experimental timeline and schematic for over-expressing Cacna2d1. (I to L) Body weight (g),…

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  Fig. 5.. Overexpression Cacna2d1 rescued synaptic transmission and neural activity of vlPAG GABAergic neurons in DIO mice. (A) Experimental timeline and schematic. (B) Representative traces of mIPSCs recorded in vlPAGGABAergic neurons from DIO mice with CACNA2D1 overexpression or eGFP-expression. (C) Average mIPSC frequency from the three groups (NFD: 15 cells from three mice, DIO ctr: 30 cells from three mice, DIO rescue: 21 cells from three mice; *P < 0.05, ***P < 0.001, Kruskal-Wallis with Dunn’s multiple test). (D) Average mIPSC amplitude from three groups (NFD: 15 cells from three mice, DIO ctr: 30 cells from three mice, DIO rescue: 21 cells from three mice; P = 0.088, one-way ANOVA). (E) Experimental timeline and schematic for over-expressing Cacna2d1 and GCaMP6m. Scale bar, 100 μm. (F and G) Sample recording traces showing calcium signaling in response to high-fat chow stimuli in control group (F) and rescue group (G) that underwent 7 weeks HFD. Light-blue shaded parts represent feeding event. (H) Amplitude of Ca2+ signaling in different groups (DIO rescue group: n = 6 mice, DIO control group: n = 6 mice, NFD control group: n = 6 mice; data for NFD control group was the same from…