Whole-cell patch-clamp recordings were made in transverse brainstem slices (300–400 μm) containing the NTS from rats or mice (21–60 days old). Under deep anaesthesia (sodium pentobarbital; 100 mg kg−1, i.p.) or halothane inhalation, animals were decapitated and the brainstem removed and immersed in ice-cold (0–4°C), oxygenated (95% O2–5% CO2) artificial cerebrospinal fluid (ACSF) containing (mm): 124 NaCl, 3 KCl, 26 NaHCO3, 1.4 NaH2PO4, 11 glucose, 1.3 CaCl2, and 1.3 MgCl2, pH 7.3–7.4, with an osmolality of 290–305 mosmol kg−1. The brainstem then was mounted on a glass stage and transverse slices were cut with a vibratome. The slices were then transferred to a submersion-type recording chamber mounted on a fixed-stage platform under an upright microscope (Olympus BX50WI, Melville, NY, USA).
After an equilibration period of 1–2 h, whole-cell patch-clamp recordings were obtained from neurones in the NTS using patch pipettes with open tip resistance of 2–5 MΩ. Seal resistance was 1–5 GΩ and series resistance was < 24 MΩ, uncompensated. Patch pipettes were filled with (mm): 130–140 potassium or caesium gluconate, 1 NaCl, 5 EGTA, 10 Hepes, 1 MgCl2, 1 CaCl2, 3 KOH or CsOH, 2–4 ATP; biocytin, 0.2%; pH 7.2. Added to the ACSF for specific experiments were: tetrodotoxin (TTX; 2 μm; Sigma, St Louis, MO, USA or Alomone Laboratories, Jerusalem, Israel), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μm; Sigma), bicuculline methiodide (30 μm; Sigma), picrotoxin (PTX; 50–100 μm; Sigma), dl-2-amino-5-phosphono-valeric acid (APV; 50 μm; Sigma), tolbutamide (200 μm; Sigma), wortmannin (100–200 nm; Alomone Laboratories), and leptin (10 nm to 1 μm; Sigma; or PeproTech, Rocky Hill, NJ, USA). Wortmannin was dissolved in dimethylsulphoxide (DMSO) as a 5-mm stock solution and added to ACSF to obtain a final DMSO concentration of < 0.01%. Tolbutamide was dissolved in 100% ethanol, with the final ethanol concentration in ACSF of < 0.5%. Leptin was reconstituted in 15 mm HCl, pH normalized with NaOH. For all solvents, vehicle alone at the final concentration was without effect in separate recordings from NTS neurones. All other drugs were dissolved directly in the ACSF. Recording pipettes were pulled from borosilicate glass capillaries of 1.65 mm outer diameter and 0.45 mm wall thickness (Garner Glass Co., Claremont, CA, USA). Electrophysiological signals were recorded using an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA), low-pass filtered at 5 kHz, digitized at 88 kHz (Neuro-corder, Cygnus Technology, Delaware Water Gap, PA, USA), stored on videotape, and analysed off-line on a PC with pCLAMP programs (Axon Instruments) or Mini-analysis (Synaptosoft, Decatur, GA, USA). Recordings were performed under visual control in a recording chamber on an upright, fixed-stage microscope equipped with infrared differential interference contrast (IR-DIC) and epifluorescence to identify non-labelled and EGFP-labelled cells (Olympus BX50WI). Epifluorescence was briefly used to target fluorescent cells, at which time the light source was switched to IR-DIC to obtain the whole-cell recording. Once in the whole-cell configuration, cells were voltage clamped for 5 min near resting membrane potential (determined by temporarily removing the voltage clamp) to allow equilibration of recording pipette contents with the intracellular milieu. Synaptic currents were examined at holding potentials positive and negative to resting membrane potential (−30 to 0 mV for IPSCs and −60 to −80 mV for EPSCs). Current-clamp (i.e. voltage) recordings were performed at resting membrane potential. Input resistance was assessed by measuring voltage deflection at the end of the response to injected rectangular current pulses (100–500 ms of ±10–50 pA).