Brain slice preparation and whole-cell patch-clamp recordings

Acute brain slices were prepared as described previously (Gao et al. 2012; Gao et al. 2017; Jiang et al. 2013; Molinas et al. 2019; Rezai-Zadeh et al. 2014). Briefly, under isoflurane anesthesia mice were decapitated, the brains were removed and immersed in ice-cold oxygenated artificial cerebrospinal fluid (aCSF) containing the following (in mM): 124 NaCl, 26 NaHCO₃, 1.4 NaH₂PO₄, 11 glucose, 3 KCl, 1.3 MgCl, 1.5 CaCl₂, pH: 7.3–7.4. Coronal brain slices containing the hypothalamus (300 μm) were prepared with a vibrating microtome (Leica VT 1200S). The slices were transferred to a holding chamber containing aCSF (for ~1 hour) before recordings.

Whole-cell patch-clamp recordings were performed at 34–36 °C from PVN neurons identified under a 40x water-immersion objective (N.A.=0.8). Epifluorescence was used to identify PRV-containing neurons and infrared illumination and differential interference contrast optics (IR-DIC) to target specific cells. The recording electrodes (2–5 MΩ) were filled with a solution containing the following (in mM): 130 K⁺ or Cs⁺-gluconate, 10 HEPES, 5 EGTA, 1 NaCl, 1 MgCl₂, 1 CaCl₂, 3 KOH or CsOH, 2–3 Mg-ATP, 0.2 % biocytin, pH 7.3–7.4. Liquid junction potential was corrected before determining membrane potential. The resting membrane potential was examined at rest in current-clamp mode.

To determine the excitability of iBAT-related PVN neurons, recordings were conducted in current clamp mode. During a step protocol, the recorded neurons were hyperpolarized to ~ −90 mV and then depolarizing current steps (duration of 1s) were applied to reveal the firing activity of iBAT-related PVN neurons before and after application of leptin (300 nM). The effect of bath application of leptin on firing rate was assessed 5 min after leptin reached the chamber.

Spontaneous excitatory postsynaptic currents (sEPSCs) were examined at a holding potential of −60 mV. Spontaneous inhibitory postsynaptic current (sIPSC) or evoked inhibitory postsynaptic current (eIPSCs) were examined at a holding potential of −10 mV. This experimental condition allowed us to exclude excitatory glutamatergic transmission without using glutamate receptor antagonists which are known to decrease the presynaptic release of GABA (Boychuk and Smith 2016; Xu and Smith 2015). Tetrodotoxin (TTX, 1 μM) was bath applied to record miniature excitatory and inhibitory post-synaptic currents (mEPSCs, mIPSCs). Electrophysiological signals were recorded using an Axoclamp 700B amplifier (Molecular Devices) and acquired by pClamp 10 software (Molecular Devices). Synaptic currents were analyzed offline using pClamp 10 and MiniAnalysis (Synaptosoft). Murine leptin (300 nM, PeproTech Inc) was dissolved in aCSF and bath applied in specific experiments.

For light-stimulation of channel rhodopsin (ChR2)-positive fibers a wide-spectrum UV light was used. The light was focused on the back aperture of the microscope, producing a wide-field exposure around the recorded cell. The light output was controlled by a programmable pulse stimulator, Master-8 (AMPI Co., Israel) and pClamp 10 software. The glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM, Tocris Bioscience) and D-(−)-2-amino-5-phosphonopentanoic acid (AP5, 50 μM; Tocris Bioscience), the GABA_A receptor antagonist bicuculline methiodide (30 μM; Tocris Bioscience), and 4-aminopyridine (4-AP, 100 μM, Tocris Bioscience) were included in the aCSF and bath applied for specific experiments. After recordings the brain slices were fixed in 4 % paraformaldehyde in 0.15 M sodium phosphate buffer overnight at 4 °C. After several rinses in 0.01 M phosphate-buffered saline (PBS), slices were immersed in AMCA Avidin D (1:200, Vector Labs) in 0.01M PBS containing 1 % Triton X-100 (Acros organics) for 72 h at 4 °C to visualize the recorded neurons.

Continuous recordings of EPSCs and IPSCs were conducted before and after drug application. The effects of drugs across the neuron groups were analyzed using paired t-test. The effects of drugs on PSC frequency and amplitude were analyzed within individual cells using the Kolmogorov-Smirnov (K-S) test by comparing 2 min epochs before and after drug application. Neurons were grouped (decrease, increase, or no change) based on K-S test. Significance was set at p<0.05 and numbers are reported as mean ± standard error (SEM).