Secondary analysis of scRNA-Seq results
Count and meta data were downloaded from GSE93374 and count data were normalized to counts per million (CPM). These data were filtered for cells in the ARH30. The count data were then joined to the meta data by cell ID, genes including SK1-4 and POMC were selected, and were exported to an Excel file. Further filtering was applied through Excel using metadata or expression data. We extracted the expression profiles of SK1-3 and POMC in neurons only. We calculated the number of SK neurons that express either only individual SK, both SK and POMC, both SK and AgRP, or all SK and POMC and AgRP, which resulted in four subpopulations of SK neurons: SK+/POMC-/AgRP-, SK+/POMC+/AgRP-, SK+/POMC-/AgRP+ and SK+/ POMC+/AgRP+. Meanwhile, we calculated the number of POMC neurons with or without the expression of each SK. We also compared the expression of SK3 in male and female POMC neurons.
Care of all animals and procedures was approved by Baylor College of Medicine Institutional Animal Care and Use Committee. Mice were housed in a temperature-controlled room at 22–24 0C using a 12-h light, 12-h dark cycle. Regular chow (5V5R, PicoLab) and water were provided ad libitum.
Tamoxifen-inducible POMC-CreERT2 transgenic allele31 was crossed with Rosa26-LSL-tdTomato (Jackson Laboratory, #007905) or with SK3lox/lox mice 29,32 (Jackson Laboratory, #019083) to generate Rosa26-LSL-tdTomato/POMC-CreERT2, SK3 lox/lox/POMC-CreERT2 (referred as pomc-SK3 KO) and SK3lox/lox (referred as controls) littermates. These mice received tamoxifen (Sigma, T5648) treatment at 11 weeks of age (0.2 g/kg, i.p.), which induced Cre-recombinase activity specifically in mature POMC neurons. Further, ERα-ZsGreen mouse strain33,34 was crossed with Rosa26-LSL-tdTomato/POMC-CreERT2 to generate POMC-CreERT2/Rosa26-LSL-tdTomato/ERα-ZsGreen for the electrophysiology recording of ERα-positive POMC neurons (POMC/ERα) in the ARH.
Validation of SK3lox/lox recombination in POMC cells
Control and pomc-SK3 KO mice (after tamoxifen inductions) were anesthetized with inhaled isoflurane and euthanized. The pituitary, the ARH and the nucleus of the solitary tract (NTS) were collected. Genomic DNAs were extracted using the REDExtract-N-Amp Tissue PCR Kit (# XNATS; Sigma-Aldrich, St Louis, MO), followed by PCR amplification of the floxed or recombined SK3 alleles. Primer for floxed SK3: Forward 5’-AGG AGA GGG CTG ATT CTC AAG-3’ and Reverse 5’-GTA TCG GTG ACT GCT TCA TCC-3’; Primer for recombinant band of the floxed SK3: Forward 5’-CTT CCC ATA TAA CAG TGT CAG-3’ and Reverse 5’-GTA TCG GTG ACT GCT TCA TCC-3’.
Serum corticosterone measurements
For the measurements of resting corticosterone, mice were rapidly anesthetized and decapitated at 9:00 am and the trunk blood was collected; to measure stress levels of corticosterone, mice were restrained for 60 min with a plastic restraint cone, and blood was collected from the tail vein. Plasma was obtained by centrifugation and assayed using a corticosterone ELIAS kit (EIACORT, Life Technologies Corporation, Grand Island, NY).
The body weight and the food intake were measured every 4 days. These mice were then put into TSE PhenoMaster metabolic cages at 5-6 months of age for 7 days to measure feeding and energy expenditure. Energy expenditure was analyzed using CalR analysis35 with body weight as a covariate. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed at 7 months of age. In GTT, overnight-fasted mice received intraperitoneal (i.p.) injections of D-glucose (1 mg/g body weight), and tail blood glucose was measured using a true-test glucometer immediately before and 15, 30, 60 and 120 min after injections. In ITT, 2 h-fasted mice received i.p. injections of 1 mU/g human insulin (Humulin R; Eli Lilly Corp., Indianapolis, IN), and blood glucose values were measured immediately before and 15, 30, 60 and 120 min after injections.
To label POMC neurons with tdTomato or ERα-ZsGreen for electrophysiological recordings, we generated double transgenic mouse POMC-CreERT2/Rosa26-LSL-tdTomato or triple transgenic mouse POMC-CreERT2/Rosa26-LSL-tdTomato/ERα-ZsGreen as described above. Four weeks after tamoxifen injection, mice were deeply anesthetized with isoflurane and transcardially perfused with a modified ice-cold sucrose-based cutting solution (pH 7.3) containing 10 mM NaCl, 25 mM NaHCO3, 195 mM Sucrose, 5 mM Glucose, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM Na-Pyruvate, 0.5 mM CaCl2, and 7 mM MgCl2, bubbled continuously with 95% O2 and 5% CO234. The mice were then decapitated, and the entire brain was removed and immediately submerged in the cutting solution. Slices (250 µm) were cut with a Microm HM 650V vibratome (Thermo Scientific). Three to four brain slices containing the ARH were obtained for each animal (bregma −2.54 mm to −1.46 mm; interaural 1.74 mm to 2.34 mm). The slices were recovered for 1 h at 34°C and then maintained at room temperature in artificial cerebrospinal fluid (aCSF, pH 7.3) containing 126 mM NaCl, 2.5 mM KCl, 2.4 mM CaCl2, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 5.0 mM glucose, and 21.4 mM NaHCO3) saturated with 95% O2 and 5% CO2 before recording. Slices were transferred to a recording chamber and allowed to equilibrate for at least 10 min before recording. The slices were superfused at 34°C in oxygenated aCSF at a flow rate of 1.8-2 ml/min.
Whole-cell patch clamp recordings were performed in POMC neurons in the ARH visually identified by an upright micro-scope (Eclipse FN-1; Nikon) equipped with IR-DIC optics (×40 NIR;Nikon). Signals were processed using Multiclamp 700B amplifier (Axon Instruments), sampled using Digidata 1440A, and analyzed offline on a PC with pCLAMP 10.3 (Axon Instruments). The brain slices containing ARH POMC neurons were bathed in oxygenated aCSF (32°C–34°C) at a flow rate of approximately 2 ml/min13,36. Patch pipettes with resistances of 3 to 5 MΩ were filled with solution containing: 126 mM K gluconate, 10mM NaCl, 10 mM EGTA, 1 mM MgCl2, 2 mM Na-ATP, and 0.1 mMMg-GTP (adjusted to pH 7.3 with KOH)13,29. Voltage clamp was used to record SK-like current in POMC neurons, as described before 13,29,37.
The minimal sample size was predetermined by the nature of the experiments and previous experience. For physiological readouts (body weight, food intake and GTT), 6-14 mice per group were included. For electrophysiological studies, 22-32 neurons from 3 different mice in each genotype or condition were included. For ELISA, 3-5 mice were included. The data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism to evaluate normal distribution and variations within and among groups. Methods of statistical analyses were chosen based on the design of each experiment and are indicated in figure legends. P < 0.05 was considered to be statistically significant.
Care of all animals and procedures conformed to the Guide for Care and Use of Laboratory Animals of the US National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine.