Brain region selective responses to cold.

Micro-punches of PVH, LH, DMH, VMH, and ARC were made from male mice exposed to a cold chamber for 4-6 h, which were directly used for RT-qPCR of the gene markers of lipolysis and thermogenesis. (A1-A5) Group data of the lipolytic marker ATGL (A1) and HSL (A2) as well as thermogenic marker Ucp2 (A3), Cidea (A4) and Prdm16 (A5) in the PVH. (B1-B5) Group data of the lipolytic marker ATGL (B1) and HSL (B2) as well as thermogenic marker Ucp2 (B3), Cidea (B4) and Prdm16 (B5) in the LH.

(C1-C5) Group data of the lipolytic marker ATGL (C1) and HSL (C2) as well as thermogenic marker Ucp2 (C3), Cidea (C4) and Prdm16 (C5) in the DMH. (D1-D5) Group data of the lipolytic marker ATGL (D1) and HSL (D2) as well as thermogenic marker Ucp2 (D3), Cidea (D4) and Prdm16 (D5) in the VMH. (E1-E5) Group data of the lipolytic marker ATGL (E1) and HSL (E2) as well as thermogenic marker Ucp2 (E3), Cidea (E4) and Prdm16 (E5) in the ARC. Data represent mean ± s.e.m. Student t tests were performed. *p<0.05. Each dot represents one animal in each group of all the panels.

Cold increases protein expressions of ATGL in neurons and astrocytes in PVH.

Control or cold (4-6)-challenged mice were perfused and fixed. Mouse brains were sectioned. ATGL, NSE, and S100b were stained using relevant antibodies respectively. (A) Representative images of ATGL (green), NSE (red), and ATGL/NSE overlay (yellow) signals in PVH sections from cold-challenged mouse. (B) Group data of relative ATGL/NSE overlay signals to total NSE signals in control and cold-challenged mice (n=5 each group). (C) Group data of relative ATGL/S100b overlay signals to total S100b signals in control and cold-challenged mice (n=5 each group). Data represent mean ± s.e.m. Student t tests were performed. ***p<0.001, *p<0.05. Scale bar, 100 μm for (A). PVH, paraventricular of hypothalamus; 3rd V, third ventricle.

Cold increases HSL protein expressions in both neurons and astrocytes in PVH.

Control or cold (4-6)-challenged mouse brains were sectioned. HSL, NSE, and S100b were respectively co-stained using relevant antibodies. (A) Representative images of HSL (green), NSE (red) and HSL/NSE overlay (yellow) signals in PVH sections from cold-challenged mouse. (B) Group data of relative HSL/NSE overlay signals to total NSE signals in control and cold-challenged mice (n=5 each group). (C) Group data of relative HSL/S100b overlay signals to total S100b signals in control and cold-challenged mice (n=5 each group). Data represent mean ± s.e.m. Student t tests were performed. ***p < 0.001, *p < 0.05. Scale bar, 100 μm for (A) PVH, paraventricular of hypothalamus; 3rd V, third ventricle.

Cold increases the level of phosphorylated HSL in PVH.

Micro-punches of PVH were collected in control and cold (4-6)-challenged mice, which were used for (A) western blots of p-HSL (Ser660), HSL, and β-action. (B) Group data of p-HSL fold change to HSL (n = 6 each group). Data represent mean ± s.e.m. Student t tests were performed. ** p < 0.01. Each dot represents one mouse.

Cold-induced LD accumulation.

Control or cold (30 min∼1 h)-challenged mice were perfused and fixed. Mouse brains were sectioned. LDs in the PVH were labelled using BODIPY493 (BD493). (A, B) Representative images of BD493 signals in PVH sections from one control (A) and one cold-challenged mouse (B). (C) Group data of relative BD493-labelled area in PVH in control and cold-challenged mice (n = 6 each group). (D-F) Sample images of BD493 (D) and perilipin2 (PLIN2) (E) and overlay (F) signals in the PVH. Data represent mean ± s.e.m. Student t tests were performed. ****p < 0.0001. Scale bars, 50 μm for (A, B), and 1 μm for (D-F). PVH, paraventricular of hypothalamus; 3rd V, third ventricle.

Cold increased Fos expressions in the PVH.

Control or cold-challenged mouse brains were perfused and fixed and sectioned. Fos in the PVH were labelled using anti-Fos antibodies. (A) Representative images of Fos signals in PVH from one control (top) and one cold-challenged mouse (bottom). (B) Group data of Fos-positive cells in PVH in control and cold-challenged mice (n = 5 each group). Data represent mean ± s.e.m. Student t tests were performed. **p<0.01. Scale bars, 100 μm for (A). 3rd V, third ventricle.

Cold-induced cell activity-dependent lipid peroxidation in the PVH.

Mice were injected with the lipid peroxidation indicator BD-C11 in the PVH via the implanted OmFC cannula 4 h before placing them in a temperature-controlled chamber. Two-color fiber photometry was applied for time-lapse monitoring of BD-C11 convertion via the optic fiber of the OmFC. (A) Representative traces of real-time photometry monitoring of red and green signals simultaneously. (B-D) Group data of relative BD-C11 ratio to baseline in mice treated with vehicle (B, n=6), α-TP (C, n= 4), and KYN+MUS (D, n=4). Data represent mean ± s.e.m. Student t tests were performed.

Cold-induced cell activity-dependent lipid lipolysis.

Mice were injected with the lipase substrate in the PVH via the implanted OmFC cannula 4 h before placing them in a temperature-controlled chamber. Two-color fiber photometry was used for time-lapse monitoring of lipase substrate convertion via the optic fiber of the OmFC. (A) Representative traces of real-time photometry monitoring of green signals. (B-D) Group data of lipase substrate convertion in mice treated with vehicle (B, n=4), α-TP (C, n=4), and KYN+MUS (D, n=4). Data represent mean ± s.e.m. Student t tests were performed.

Cold-induced cell activity-dependent LD accumulation.

Mice were injected with the LD marker BODIPY493 in the PVH via the OmFC cannula 4 h before placing them in a temperature-controlled chamber. Fiber photometry was used for time-lapse monitoring of BODIPY493 signals via the optic fiber of the OmFC. (A) Representative traces of real-time photometry monitoring of BODIPY493 signals. (B-D) Group data of BODIPY493 in mice treated with vehicle (B, n=4), α-TP (C, n= 4), and KYN+MUS (D, n=6). Data represent mean ± s.e.m. Student t tests were performed.