Patch-clamp electrophysiology

Whole-cell voltage-clamp recordings were performed on cultured DRG neurons of C57BL/6, TRPV1^-/-, TRPA1^-/- and TRPV1/TRPA1 ^{= / =} mice. The standard extracellular solution contained the following in mM: 140 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES, and 10 glucose at pH 7.4 (adjusted by Tetramethylammonium hydroxide). Pipette solution contained in mM: 140 KCl, 2 MgCl₂, 5 EGTA, and 10 HEPES (pH = 7.4 by KOH). Solutions were bath applied using a gravity-driven polytetrafluoroethylene multibarrel perfusion system (custom-made). All recordings were performed at room temperature. Transmembrane ion currents were acquired using an Axopatch 200B amplifier (Molecular Devices). Data were low-pass filtered at 2 kHz and sampled at 5 kHz. Patch pipettes were pulled from thick-walled borosilicate glass capillaries (GB150F-8P, Science Products) and heat-polished to give a resistance of 1.5–3.0 MΩ. Unless otherwise noted, cells were held at a potential of -60 mV. pCLAMP 10.3 (Molecular Devices) was used for data acquisition, digitization and for further offline analysis.