Whole-cell patch-clamp recordings

TRPV1 currents were recorded using an Axopatch 200B ultra-low-noise patch-clamp amplifier (Axon Instruments, USA). DRG neurons were placed in an open recording bath filled with Ringer's solution (mM: 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose, pH 7.4, 21 °C). Glass electrodes (recording electrodes, 1.65 ± 0.05 μm outer diameter) were pulled with an electrode puller and filled with an internal solution of electrodes (mM: 140 KCl, 5 MgCl₂, 4 Na₂ATP, 0.3 Na₃GTP, 2.5 CaCl₂, 5 EGTA, and 10 HEPES, pH 7.2) with an electrode resistance of 2–4 MΩ before recording. In whole-cell mode, the current and voltage of neurons were maintained at 0 and less than − 45 mV for 5 min, respectively. Capsaicin (10 μM), Mustard oil (10 mM), Menthol (100 μM), ATP (10 μM), Ac2-26 (10 μM), Boc2 (100 μM) (the antagonist of FPR2), and saline (vehicle control) were applied to adjacent recording cells through a gravity-driven drug-loading perfusion system. All testing was performed at approximately 22 °C. The clamp voltage (Vh) was set to − 60 mV for all experiments, and capsaicin evoked currents were recorded in voltage clamp mode. Divide the whole-cell current by the cell capacitance to calculate the current density. Capsaicin evoked whole-cell currents were filtered and the signal sampling frequency was 1 kHz. Membrane potentials were expressed as absolute values (millivolts, mV), and TRPV1 currents were expressed as absolute values (nanoamperes, nA) or multiples of changes compared with basal values. To detect neuronal excitability, the rheobase was measured in current-clamp mode by input step currents (100 pA for 500 ms). The rheobase was reflected by the change in the basal value of the stimulus current intensity (normalized to the basal value 1) and expressed as a percentage.