Fast step perfusion system

Whole cell recordings were performed on dissociated nodose ganglion neurons or transfected HEK293 cells using an inverted Olympus IX50 microscope and on NTS neurons contained in horizontal brainstem slices using an upright Nikon FN1 microscope. For HEK293 cell experiments, TRPV1-GFP+ cells were identified visually using 488 nm light illuminations prior to patching. Recording electrodes (2.8–3.8 MΩ) were filled with an intracellular solution containing (mM): 10 CsCl, 4 CsOH, 110 Cs-methanesulfonate, 11 EGTA, 1 CaCl₂, 2 MgCl₂, 10 HEPES, 2 MgATP, and 0.2 MgGTP. The intracellular solution was pH 7.4 and 296 mOsm. All cells were studied under voltage clamp conditions with an Axopatch 200A or MultiClamp 700A amplifier (Molecular Devices, Union City, CA, USA). Neurons were held at V_H = −60 mV using pipettes in whole cell patch configuration. Signals were filtered at 3 kHz and sampled at 30 kHz using p-Clamp software (version 10, Molecular Devices). Liquid junction potentials were not corrected. Extracellular solution (artificial cerebral spinal fluid, aCSF) was continuously perfused and specific drugs were either bath applied (slice) or locally applied using a fast-step perfusion system (Warner Instruments, Hamden, CT, USA).

Transfected Neuro2a cells were immersed in recording buffer (20 mM HEPES, 140 mM NaCl, 2.8 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose; pH 7.4) and subjected to whole-cell patch clamp with the aid of HEKA amplifier (HEKA Elektronik, Germany). Micropipettes were pulled on a PC-10 instrument (Narishige, Japan) from borosilicate glass capillaries (Harvard Apparatus, USA) and filled with internal buffer (140 mM CsCl, 6 mM CaCl2, 2 mM MgCl2, 2 mM MgATP, 0.4 mM NaGTP, 10 mM HEPES/CsOH, 20 mM BAPTA/KOH; pH 7.3). During a typical experiment, acetylcholine at concentrations from 1 to 100 μM was applied to a cell through a Fast-Step perfusion system (1 ml/min, Warner Instruments, US) along with PNU120596 (Tocris, UK) given at 10 μM. Electrophysiological recordings were performed at a holding potential of -40 mV. Currents were recorded through Patchmaster software (HEKA Elektronik, Germany) and analyzed using OriginPro 7.5 (OriginLab, USA). Current amplitudes were normalized to a maximal response for each cell.

Patch-clamp recordings were performed 48–72 h after the transfection using whole-cell scheme on EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany). External perfusion solution contained 140 mM NaCl, 2 mM CaCl2, 2.8 mM KCl, 4 mM MgCl2, 20 mM HEPES and 10 mM glucose,. Solution pH was adjusted to 7.4 (320–330 mOsm). The patch pipette contained 140 mM CsCl, 6 mM CaCl2,2mM MgCl2,2mM MgATP, 0.4 mM NaGTP, 10 mM HEPES/CsOH, 20 mM BAPTA/KOH with pH adjusted to 7.3. Pipettes were pulled from filament-supplied borosilicate glass capillaries (Harvard Apparatus Ltd., Holliston, MA, USA) using Narishige (Tokyo, Japan) pipette puller. Pipettes had resistances of 5–6 MOhm. Acetylcholine was delivered by FastStep perfusion system (Warner Instruments, Hamden, CT, USA).