To investigate a potential for cold exposure to modulate gene expressions for lipid lipolysis and thermogenesis in the hypothalamus, mice were exposed to a cold (4°C) chamber for 4–6 hr, a more physiologically relevant condition. Immediately after the cold challenge, mouse brains were acutely extracted and sectioned in ice-cold oxygenated artificial cerebrospinal fluids (ACSFs). Hypothalamic sections that, respectively, include the paraventricular nucleus of the hypothalamus (PVH), lateral hypothalamus (LH), dorsomedial hypothalamus (DMH), ventromedial hypothalamus (VMH), and arcuate nucleus (ARC) were transferred to ACSF-containing incubators. Micro-punches of these brain regions were subsequently made for gene assays of lipid metabolic markers.

To evaluate the effects of cold on the gene markers of lipid lipolysis, we measured the mRNA levels of two key lipolytic markers adipose triglyceride lipase (Pnpla2) and hormone-sensitive lipase (Lipe). We observed that cold-challenged male mice showed a significant increase in the gene expressions of the both of Pnpla2 (Figure 1A₁) and Lipe (Figure 1A₂) selectively in the PVH but not in other brain regions (Figure 1B–E). We also assayed the gene expressions of thermogenic marker uncoupling protein 2 (Ucp2) and additional thermogenic factors (Cidea, Prdm16). Cold did not significantly affect mRNA expressions of these thermogenic markers in all the examined regions in this study (Figure 1). Cold did not significantly affect gene expressions of these lipolytic and thermogenic markers in female mice (Figure 1—figure supplement 1). These results suggest that cold exposure (4–6 hr) could induce a rapid lipolytic activity to release fatty acids primarily in the PVH in males. Because cold did not significantly affect lipolytic markers in other regions and did not affect thermogenic markers, we next focused on studying cold-induced lipid mobilization and lipolysis in the PVH in male mice.

Figure 1 with 1 supplement see all

Download asset Open asset

Figure 1—source data 1

Lipid metabolic gene markes in different hypothalamic areas in male mice.

https://cdn.elifesciences.org/articles/98353/elife-98353-fig1-data1-v1.xlsx

Download elife-98353-fig1-data1-v1.xlsx

To verify the cold-induced effects on lipolytic gene expressions, we next stained the protein expressions of ATGL and HSL in PVH sections using antibodies against the ATGL and HSL, respectively. Matching with the gene expression results, cold-challenged mice showed significant increases in ATGL (Figure 2) and HSL (Figure 3) expressions in both neurons and astrocytes, respectively, compared to control mice.

Figure 2

Download asset Open asset

Figure 2—source data 1

Group data of ATGL expressing neurons and astrocyets.

https://cdn.elifesciences.org/articles/98353/elife-98353-fig2-data1-v1.xlsx

Download elife-98353-fig2-data1-v1.xlsx

Figure 3

Download asset Open asset

Figure 3—source data 1

Group data of HSL expressing neurons and astrocyets.

https://cdn.elifesciences.org/articles/98353/elife-98353-fig3-data1-v1.xlsx

Download elife-98353-fig3-data1-v1.xlsx

Moreover, we examined whether and how cold modified the phosphorylation of the HSL, a key enzyme in the regulation of lipid lipolysis. After the cold exposure of cohort groups of mice, micro-punches of PVH sections were collected and directly used for western blots of phosphorylated HSL (p-HSL), HSL, and actin. Cold significantly increased activation (S660) site phosphorylation of the HSL compared to controls (Figure 4).

Figure 4

Download asset Open asset

Figure 4—source data 1

Western blots of the HSL proteins in the PVH.

https://cdn.elifesciences.org/articles/98353/elife-98353-fig4-data1-v1.zip

Download elife-98353-fig4-data1-v1.zip

Figure 4—source data 2

Gels of western blots of HSL, p-HSL, and actin.

https://cdn.elifesciences.org/articles/98353/elife-98353-fig4-data2-v1.zip

Download elife-98353-fig4-data2-v1.zip

Figure 4—source data 3

Group data of western blots of HSL proteins in the PVH.

https://cdn.elifesciences.org/articles/98353/elife-98353-fig4-data3-v1.xlsx

Download elife-98353-fig4-data3-v1.xlsx

It is increasingly appreciated that lipid droplets (LDs), endoplasmic reticulum-derived intracellular neutral lipid storage dynamic organelles (Olzmann and Carvalho, 2019), are predominantly accumulated in adipose tissues and liver under normal physiological conditions (Murphy, 2001; Farese and Walther, 2009). Also, there is evidence to indicate that glial cells such as astrocytes and tanycytes in the brain accumulate LDs under metabolic and hypoxic stress (Geller et al., 2019; Smolič et al., 2021). A recent elegant study shows that hyperactive neurons release fatty acids from phospholipids to formulate LDs in the brain and activation of neurons promotes lipolysis by increasing cytoplasmic lipases (Ioannou et al., 2019). These findings suggest that increased cell activity and accumulated LDs probably contribute to the cold-induced increase in the lipolytic markers we observed in this study.

To probe the mechanism of the upregulation of lipolytic markers in the PVH, we evaluated the ability for cold to accumulate LDs. After the cold challenge, mouse brains were perfused, fixed, and sectioned. Hypothalamic sections containing PVH were stained using the BODIPY 493 (BD493), an LD probe which has been well developed and widely applied to measure LD number and area in fixed tissues and live cells, respectively (Spangenburg et al., 2011; Long et al., 2012; Liu et al., 2015; Geller et al., 2019; Ioannou et al., 2019; Smolič et al., 2021). Interestingly, we observed that a short-term (30 min to 1 hr) cold exposure induced an increase in the LD area in the PVH (Figure 5A–C). We verified LDs by using the cytoplasmic LD-binding protein perilipin-2 (Figure 5D–F). To define the cell type selectivity of LD accumulation induced by cold, we co-stained LDs with the BD493, astrocytes with S100b, and neurons with neuronal marker NSE, respectively, in PVH sections. We observed that cold exposure significantly increased the BD493-positive S100b-expressing astrocytes but not NSE-expressing neurons (Figure 5—figure supplement 1), consistent with the previous reports that LDs were primarily accumulated in glial cells (Bailey et al., 2015; Liu et al., 2015; Marschallinger et al., 2020; Smolič et al., 2021). However, a longer (4–6 hr) cold exposure reduced the number of LDs (Figure 5—figure supplement 2), which might be due to cold-induced liberations of fatty acids from the LDs to mitochondria for β-oxidation.

Figure 5 with 2 supplements see all

Download asset Open asset

Figure 5—source data 1

Group data of BD493 area in cold-challenged mice and control mice.

https://cdn.elifesciences.org/articles/98353/elife-98353-fig5-data1-v1.xlsx

Download elife-98353-fig5-data1-v1.xlsx

We next examined whether and how cold modified cell activities within the PVH. Mice were placed in a cold chamber before mouse brains were perfused and fixed. PVH sections (40 μm in thickness) were stained using anti-Fos antibodies. Mice subjected to the cold challenge showed increased expressions of the cell activity indicator Fos in the PVH as compared to control mice (Figure 6A, B).

Figure 6

Download asset Open asset

Figure 6—source data 1

Group data of Fos-positive cells in the PVH.

https://cdn.elifesciences.org/articles/98353/elife-98353-fig6-data1-v1.xlsx

Download elife-98353-fig6-data1-v1.xlsx

As lipid peroxidation is essential in activity-dependent accumulation of LDs (Ioannou et al., 2019), we next evaluated the capability for cold to induce lipid peroxidation. Fiber photometry has recently been applied to detect the levels of fluorescent biosensors in vivo by us (Chen et al., 2022) and others (Sun et al., 2018; Andersen et al., 2023). To achieve this goal, we took advantage of the BODIPY581/591 C11 (BD-C11) ratiometric lipid peroxidation sensor, coupled with in vivo time-lapse two-color photometry monitoring approach. We developed and validated this approach to simultaneously monitor both red and green signals in one brain region through a single implanted photometry fiber connected to a two-color photometry system, for the BD-C11 sensor as it shifts its fluorescence emission peak from 590 (red) and 510 (green) nm when the sensor is oxidized. A custom-made Optical fiber multiple Fluid injection Cannula (OmFC) implanted over PVH was used for both BD-C11 injection and photometry monitoring of the two signals in freely behaving mice. Intra-PVH injection of the BD-C11 sensor through the OmFC was performed 4 hr prior to placing the mice in a temperature-controlled chamber. Cold potently increased the BD-C11 ratio (green to red), indicating increased lipid peroxidation (Figure 7A). To verify this result, we treated mice with a lipid soluble antioxidant α-tocopherol (α-TP) to inhibit lipid peroxidation. Compared to the vehicle-treated mice (Figure 7B), prior inhibition of lipid peroxidation with the α-TP blunted the cold-induced effects on lipid peroxidation (Figure 7C). These results demonstrate that cold induces lipid peroxidation in the PVH and that our combined fiber photometry and BD-C11 approach can be reliably applied to evaluate the dynamics of brain lipid metabolism in a spatiotemporal manner.

Figure 7 with 1 supplement see all

Download asset Open asset

Figure 7—source data 1

Group data of photometry monitoring of lipid peroxidation in the PVH.

https://cdn.elifesciences.org/articles/98353/elife-98353-fig7-data1-v1.xlsx

Download elife-98353-fig7-data1-v1.xlsx

We next sought to examine whether cell activity is required to modulate lipid peroxidation. To examine whether cold alters cell activity, we transduced PVH cells with the Ca²⁺ indicator GCaMP_6f- and placed the PVH transduced and photometry fiber-implanted mice in a cold chamber (Figure 7—figure supplement 1A). Cold exposure increased the intensity of PVH GCaMP_6f signals (Figure 7—figure supplement 1B, C), consistent with the increased Fos expressions (Figure 6). To inhibit cell activity, we utilized the GABA_A receptor agonist muscimol (MUS) (Sanders and Shekhar, 1995; Barbalho et al., 2009) and glutamate receptor antagonist Kynurenic acid (KYN) (Yoshida et al., 2012). To verify the inhibitory effect of the combined two chemicals, we performed intra-PVH injection of both MUS and KYN in PVH GCaMP_6f-transduced and fiber-implanted mice. Thirty minutes post the MUS and KYN administration via intra-PVH injection was able to reduce cell activity, as indicated by the decreased intensity of GCaMP_6f signals, compared to vehicle treatment (Figure 7—figure supplement 1D, E).

To test whether cell inactivation could diminish cold-induced effects on lipid peroxidation, intra-PVH injections of MUS and KYN were performed 30 min before placing the previously BD-C11-injected mice in a cold chamber and performing photometry monitoring of BD-C11 signals. Administration of the MUS and KYN prevented the cold-induced increase in the BD-C11 ratio (Figure 7D), suggesting that cell activation is required for the cold-induced lipid peroxidation.

To evaluate lipid lipolysis in the PVH, we performed intra-PVH injections of the EnzCheck lipase substrate through an implanted OmFC. The lipase substrate shifts its fluorescence to emission peak 515 nm (green) in the presence of lipases, and we detected the green signals using our fiber photometry system in a temporal manner. Intra-PVH injection of EnzCheck lipase substrate was performed 4 hr before placing the mice in a temperature-controlled chamber. Cold induced a potent increase in the green signals generated from the lipase substrate, indicating increased lipolysis (Figure 8A, B). We injected mice with the pan-lipase inhibitor diethylumbelliferyl phosphate (DEUP) via intra-PVT injections to inhibit lipolysis. Cold-induced lipolysis was reduced with the treatment of DEUP compared to vehicle-treated mice (Figure 8C). These results are consistent with the cold-induced increase in lipolytic markers (Figures 1—4), further indicating that cold induces lipid lipolysis in the PVH by increasing cytoplastic lipases. To examine whether cell activity is required for the cold-induced lipolysis, in cohort groups of PVH-implanted mice which were treated with the lipase substrate via intra-PVH injections, we performed intra-PVH injections of the MUS and KYN 30 min before placing the mice in a cold chamber. Administration of MUS and KYN prevented the cold-induced conversion of the lipase substrate, indicating decreased lipolytic activity (Figure 8D). These findings suggest that cold induces a rapid cell activity-dependent lipid lipolytic effect.

Figure 8

Download asset Open asset

Figure 8—source data 1

Group data of photometry monitoring of lipid lipolysis in the PVH.

https://cdn.elifesciences.org/articles/98353/elife-98353-fig8-data1-v1.xlsx

Download elife-98353-fig8-data1-v1.xlsx

To verify our results of LDs detected in the fixed tissues (Figure 5), we next performed time-lapse photometry monitoring of dynamics of LDs in vivo in freely behaving mice. To achieve this goal, we implanted an OmFC cannula in PVH for LD marker BD493 (green) injections and monitoring in mice. Matching with the results collected from the fixed sections, cold induced an increase in the intensity of BD493 signals (Figure 9A, B). This result indicates that cold increases the formation and accumulation of LDs, which could be directly used for lipolysis by releasing fatty acids to mitochondria for β-oxidation. The lipid soluble antioxidant α-TP blocked the cold-induced LD accumulation (Figure 9C). To define a role of cell activity in modulating LDs, we performed intra-cannula injections of both MUS and KYN in BD493-injected mice in the PVH. MUS and KYN administration blunted the cold-induced accumulation of LDs, as cold failed to increase the BD493 signals in MUS- and KYN-treated mice compared to controls (Figure 9D).

Figure 9

Download asset Open asset

Figure 9—source data 1

Group data of photometry monitoring of LD dynamics in the PVH.

https://cdn.elifesciences.org/articles/98353/elife-98353-fig9-data1-v1.xlsx

Download elife-98353-fig9-data1-v1.xlsx

C57/BL6J wild-type mice (#000664, Jackson Laboratory) have been described previously and are available from The Jackson Laboratory. Both male and female mice (age 8–12 weeks) were used at the start of experiments. Mice were group-housed 3–5 mice per cage in humidity- and temperature (22–25°C)-controlled rooms on a 12-hr light:dark cycle (lights on from 8:00 a.m. to 8:00 p.m.) with ad libitum access to water and mouse regular chow (#5001, LabDiet). Mice were single-caged after they received viral transductions with or without guide or optic fiber cannula insertion until all the experimental procedures were finished.

All the chemicals were purchased from Sigma except where noted. For the experiments requiring intra-PVH injections, an injector with 1 mm extension beyond the custom-made OmFC (Doric Lens) implanted over the PVH was attached by polyethylene tubing to a Hamilton syringe. The injection was performed at a speed of 50 nl per min for 4 min using a matched fluid injector consisting of a 1.25-mm ferrule and a sleeve connector, and the injector was withdrawn 10 min after the final injection. Grip cement (DENTSPLY) was used to anchor the cannula to the skull, and a plug was inserted to keep the cannula from becoming clogged when the injector was not in place. Mice were then returned to the home cage for 1 week at least before the experiments. The amount for intra-PVH injection was 200 nl of vehicle or chemicals (in μM): 100 BODIPY 493/503 (D3922, Invitrogen), 100 BODIPY 581/591 C11 (D3861, Invitrogen), 1 MitoSOX Mitochondrial Superoxide Indicators (M36005, Invitrogen), 1 EnzChek Lipase Substrate 505/515 (E33955, Invitrogen), 100 DEUP (D7692, Sigma), and 500 α-Tocopherol (#258024, Sigma); or 200 nl of 250 pmol Muscimol (M1523, Sigma) and 100 mol Kynurenic acid (K3375, Sigma).

Following our previously documented protocols (Chen et al., 2022), mice were anesthetized with 3% isoflurane to induce the anesthesia and with 1.5–2.0% isoflurane to maintain anesthesia during the surgery and placed in a stereotaxic frame (Kopf Instruments). A small incision was made in the skin of the head, a small hole was drilled on the skull, and mice were implanted with a customer-made OmFC (Doric Lens) over the PVH (coordinates from bregma: AP –0.8 mm, 0.2 mm from midline, DV –4.0 mm). The cannula was fixed to the skull with stainless steel screws and dental cement. After the surgery, all mice received meloxicam (5 mg/kg) and continued to be housed individually. Two weeks after surgery, the mice were briefly anesthetized and inserted with the fluid injector (with 1 mm protrusion) into PVH. For intra-PVH injection, the injector was attached to a Hamilton syringe through a polyethylene tube and the mice received 200 nl of vehicle, chemicals as listed in the Pharmacology section, or viral vectors (AAV₅-CamKII-GCaMP_6f-WPRE-SV40, addgene#100834, titer, 2.3 × 10¹³ GC/ml), at a rate of 50 nl/min.

As described in our previously published studies (Qi and Yang, 2015; Zhang et al., 2020; Chen et al., 2022), acute brain slices that include hypothalamic PVH, LH, DMH, VMH, and ARC, respectively, were prepared. Briefly, mice were first placed in a cold chamber (4°C) for 30 min or 4–6 hr, respectively, as described in the text and figure legends. After the cold exposure, mice were deeply anesthetized with isoflurane and decapitated. The mouse brains were dissected rapidly and placed in ice-cold oxygenated (95% O₂ and 5% CO₂) solution containing the following (in mM): 110 choline chloride, 26.2 D-glucose, 2.5 KCl, 1.25 NaH₂PO₄, 2 CaCl₂, 7 MgSO₄, 11.6 Na-L-ascorbic acid, and 3.1 Na-pyruvate. Coronal brain slices (260 μm thick) were cut with a vibratome (Leica; VT 1200 S) and maintained in an incubation chamber containing ice-cold ACSFs (in mM): 119 NaCl, 25 NaHCO₃, 11 D-glucose, 2.5 KCl, 2.5 CaCl₂, 1.3 MgSO₄, and 1 NaH₂PO₄. Micro-punches of the PVH, LH, DMH, VMH, and ARC were performed using a punch (1.5 mm diameter; Stoelting#57403) or a pipette tip (1.5 mm diameter). Six micro-punches of each region were obtained from each mouse were, respectively, collected and immediately placed in 200 μl Trizol reagent (Invitrogen) and stored at –80°C until analysis.

Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instruction. Briefly, the collected tissues were lysed in 200 μl of Trizol reagent (Invitrogen) and 40 μl of chloroform was added into each tube and subsequently vortexed for 15 s. After incubation for 2 min, centrifuged at 12,000 × g for 10 min (4°C). The aqueous phase was transferred to fresh tube and equal volume of isopropanol was added. The tube was incubated for 20 min and centrifuged at 12,000 × g for 10 min (4°C). The RNA pellet was washed with 70% ethanol by vortex and centrifuged at 12,000 × g for 10 min (4°C). cDNA was synthesized by High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer’s instructions. Real-time qPCR was performed using QuantStudio 3 instruments (Applied Biosystems). The mixture for qPCR reaction was prepared in a final volume of 20 μl containing 1 μl cDNAs and 10 μl of LightCycler 480 SYBR Green I Master (Roche) in the presence of primers at 500 nM. The specific primer sequences used were the following:

The specific primer sequences used were the following:

• Pnpla2 forward, 5′-CCAACACCAGCATCCAGT-3′;

• Pnpla2 reverse, 5′-CAGCGGCAGAGTATAGGG-3′;

• Lipe forward, 5′-CGCCATAGACCCAGAGTT-3′

• Lipe reverse, 5′-TCCCGTAGGTCATAGGAGAT-3′;

• Cidea forward, 5′-TGCTCTTCTGTATCGCCCAGT-3′

• Cidea reverse, 5′-GCCGTGTTAAGGAATCTGCTG-3′;

• Prdm16 forward, 5′-CCACCAGCGAGGACTTCAC-3′

• Prdm16 reverse, 5′-GGAGGACTCTCGTAGCTCGAA-3′;

• Ucp2 forward, 5′-CAGAGCACTGTCGAAGCCTA-3′

• Ucp2 reverse, 5′-GTATCTTTGATGAGGTCATA-3′;

• Actb forward, 5′-GCTGTCCCTGTATGCCTCT-3′

• Actb reverse, 5′-GTCTTTACGGATGTCAACG-3′;.

The relative level of expression was calculated using the comparative 2^−ΔΔCT method.

Briefly, three excitation wavelengths were used: 560, 505/490, and 405 nm. Excitation lights were generated through fiber-coupled two connectorized LEDs (CLED_560 for 560 nm; CLED_505/490 for 505 or 490 nm; CLE_405 for 405 nm; Doric Lenses) driven by a four-channel LED driver (LEDD_4; Doric Lenses). The LEDD_4 was controlled by a fiber photometry console (Doric Lenses) connected to a computer. Excitation lights were passed through two fluorescence MiniCubes (iFMC6_IE(400-410)_E1(460-490)_F1(500-540)_E2(555-570)_F2(580-680)_S)-6 ports with two integrated photodetector heads. The single detector measures both signals within the fluorescence detection windows from 500 to 540 nm and 580 to 680 nm band. The combined excitation light was sent into a patch cord made of a 400-µm core, 0.48 NA, low-autofluorescence optical fiber (Doric Lenses). The patch cord was connected to the implanted OmFC consisting of a 1.25-mm diameter optic fiber via a sleeve (Doric Lenses; Zirconia Sleeve 1.25 mm with black cover; Sleeve_ZR_1.25-BK). Both green and red fluorescence signals were collected through the same patch cord and passed through the same Minicube and focused onto a Fluorescence Detector Head (FDH; Doric Lenses). The photometry experiments were run in a Lock-in mode, and the acquisition rate was set to 12.0 ksps*C controlled by Doric Neuroscience Studio software (Doric Lenses). The fiber photometry experiments were performed 5 min after connecting the optic fibers to the animals. We processed the signals using the Doric Neuroscience Studio software (V5.3.3.14) to calculate the normalized fluorescence variation of the images (ΔF/F), and averaged the signals at 1 s bins. To avoid or minimize bleaching over time, we performed patch cord photobleaching for 12 hr before each experiment, reduced the illumination power outputs as much as possible, and recorded the signals for 30 s every 5 min. We used a rotary joint for long-term photometry recordings in freely moving animals.

Mice were euthanized and transcardially perfused with 1× phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in phosphate buffer (PFA, pH 7.2). Mouse brains were removed and post-fixed in 4% PFA overnight. Fixed brains were transferred to 30% sucrose in PFA for cryoprotection for three days. Next, 30 μm coronal sections were cut in a freezing cryostat (Leica).

For immunofluorescence, slices were washed three times in 1× PBS for 10 min each and heat incubated with target antigen retrieval solution (Invitrogen) for 2 min at 95°C. And then, the slices were washed three times in 1× PBS for 10 min each, followed by permeabilization in 1% Triton X-100 solution in 1× PBS for 40 min at room temperature. After blocking for 1 hr at room temperature, the slices were incubated with a mouse Fos antibody (1:150, sc-166940 AF647, Santa Cruz Biotechnologies), rabbit ATGL (1:150, bs-3831R-BF488, Bioss), rabbit HSL (1:150, bs-3223R-BF488, Bioss), rabbit NSE (1:200, bs-10445R-BF594, Bioss), and mouse S100b (1:200, bsm-10832-BF594, Bioss), respectively, for overnight at 4°C in the dark. The slices were then rinsed three times in 0.1% Triton X-100 solution in 1× PBS for 10 min each. For LD staining, slices were washed three times in 1× PBS for 10 min each and then incubated with 1% Triton X-100 solution in 1× PBS for 40 min. After blocking for 1 hr, the slices were stained with 20 μg/ml BODIPY 493/503 (Invitrogen, D3922) for 4 hr at room temperature in the dark. After the incubation, the slices were rinsed three times in 0.1% Triton X-100 solution in 1× PBS for 10 min each.

For BODIPY 493/503 co-staining with perilipin-2, NSE, and S100b, respectively, after permeabilization in 1% Triton X-100 solution in 1× PBS for 40 min and blocking for 1 hr at room temperature, the slices were incubated with rabbit perilipin-2 (1:200, CL594-15294, Proteintech), rabbit NSE, and mouse S100b antibodies, respectively, for overnight at 4°C in the dark. The slices were then washed three times in 0.1% Triton X-100 solution in 1× PBS for 10 min and incubated with 20 μg/ml BODIPY 493/503 (Invitrogen, D3922) for 4 hr at room temperature in the dark and then washed three times in 0.1% Triton X-100 solution in 1× PBS for 10 min each time. The stained slices were dried and mounted with mounting medium (0100-20, Southern Biotech). Images were taken using the All-in-One Fluorescence Microscope (BZ-X800E Keyence) and analyzed using the BZ-X800 Analyzer (Keyence).

Acute coronal brain slices (260 μm thickness) were collected following the procedures as stated in the above. Six micro-punches of PVH sections from each mouse were made and immediately placed in 40 μl cold lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, 0.25% Na-deoxycholate, 1 mM EDTA, 1% NP-40, 1 mM PMSF (20 mM PMSF in 100% ethanol), and 1 protease inhibitor tablet (A32955, Thermo Scientific). The samples were homogenized in lysis buffer and centrifuged at 14,000 × rpm at 4°C for 20 min. The supernatants were transferred to a fresh tube and used for Bradford assay to quantify the protein concentration. Equal amounts of protein from each sample were denatured and boiled at 100°C for 5 min. Proteins were separated by electrophoresis in 10% SDS–PAGE gel and transferred to PVDF membrane. The membrane was blocked with Protein-Free blocking buffer (927-80001, LI-COR) for 1 hr and incubated overnight with relevant primary antibodies at 4°C, including the p-HSL (1:1000, PA5-64494, Invitrogen), HSL (1:1000, PA5-17196, Invitrogen), and β-actin (1:5000, MAB8929, R&D Systems). After washing in PBS-Tween 20 (PBST), the membranes were incubated with secondary antibodies for 1 hr and a subsequent PBST washing. Images were acquired using Odyssey Classic Imager (LI-COR). The band intensity of HSL and p-HSL was quantified using the ImageJ.

Animals were randomly assigned to experimental or control groups. Following post hoc histological conformation of viral transfections and cannula/fiber placements, all mice with inaccurate viral infections or cannula placements were excluded from behavior experiments. All experiments were repeated at least twice, and data represent the technical and biological replicates. Paired or unpaired Student’s t tests were used to analyze differences between two groups of the same or different mice when appropriate, respectively. One-way ANOVA with Tukey’s post hoc test was used to compare group data from more than two groups of mice. Two-way ANOVA was used to analyze data from more than two groups across various time points. All data were analyzed by using Prism 10.0 (GraphPad Software).

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank all the members of the Yang laboratory for discussion and critical comments on this study. We thank the Doric Lenses for helping us build and adjust the two-channel two-color photometry rig. For the genetically encoded calcium indicator GCaMP_6f plasmids, we thank Dr. James M Wilson for depositing the plasmid of pENN-AAV-CamKII-GCaMP_6f-WPRE-SV40. This work was supported by the NIH (R01 DK112759, R01 DK135717 to Y.Y.) and Einstein Research Foundation.

Experimental protocols were approved by the Institutional Animal Care and Use Committees at the Albert Einstein College and conducted following the U.S. National Institutes of Health guidelines for animal research. The experimental protocol (#00001306).

1. Sent for peer review: April 9, 2024
2. Preprint posted: April 17, 2024
3. Reviewed Preprint version 1: June 13, 2024
4. Reviewed Preprint version 2: December 16, 2024
5. Version of Record published: January 30, 2025

You can cite all versions using the DOI https://doi.org/10.7554/eLife.98353. This DOI represents all versions, and will always resolve to the latest one.

© 2024, Min et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.