Whole-cell patch clamp recordings from cultured DRG neurones

Standard whole-cell patch clamp experiments from small and medium sized (≤35 µm diameter) DRG neurones were performed at room temperature using an EPC-10 USB-double amplifier in combination with PATCHMASTER software (HEKA Electronics, Lambrecht, Germany). For recording of TTX-R Na⁺ currents the cells within 8 h after plating were continuously superfused with standard bath solution (in mM): 35 NaCl, 78 choline-Cl, 5 KCl, 30 TEA-Cl, 1.8 CaCl₂, 1 MgCl₂, 0.1 CdCl₂, 10 glucose, 10 HEPES, TTX 500 nM, 5 4-aminopyridine (4-AP). TEA and 4-AP were added to block K⁺ currents and CdCl₂ was added to block Ca²⁺ currents. The pH was set to 7.4 with HCl, the osmolarity was adjusted to 300 mOsm/kg with glucose. For recording of TTX-S Na⁺ currents we used cells from Na_V1.8^-/- mice because TTX-R Na_v1.8 currents can contaminate the recording of TTX-S currents. The standard bath solution was the same except that no TTX was added. Testing compounds were IL-17A-F, final bath concentrations 50 ng/ml. Patch pipettes were made by two step vertical PC-10 puller (Narishige, Tokyo, Japan) from borosilicate glass (Kimble Chase Gerresheimer, Santiago de Querétaro, Mexico) and filled with (internal) solution (in mM): 10 NaCl, 110 CsCl, 20 TEACl, 2.5 MgCl₂, 5 HEPES, 5 EGTA. The pH was adjusted to 7.0 with CsOH. Osmolarity was adjusted to 300 mOsm/kg with sucrose. Current signals were low-pass filtered at 1.1 kHz (4-pole Bessel) and 20 kHz (Bessel). For presentation, data were then digitalized at 500 Hz. Capacitive and leakage currents were subtracted digitally by the P/4 protocol. The data were analysed using the FITMASTER (HEKA Electronics, Lambrecht, Germany) and Origin 8.1 G (Microcal Software, Northampton, MA) software programs. Current densities were calculated by dividing the peak current (I_peak) evoked at each membrane potential (V_m) by the cell capacitance (C_m). The peak conductance (G) of Na⁺ currents at each potential was calculated from the corresponding peak current by using the equation G = I/(E − E_Rev) (E_Rev: reversal potential of Na⁺ current; I: peak current amplitude of Na⁺ current; E: membrane potential).

For determination of the steady-state inactivation a double-pulse protocol was used. Starting again from a holding potential of -80 mV the membrane was first depolarized for 500 ms in 5 mV steps in a range of −75 to + 10 mV, and then the membrane was depolarized to −5 mV for 100 ms.

Normalised peak conductance (G/G_max) and steady-state inactivation (I/I_max) were fitted with a Boltzmann function G/G_max (I/I_max) = [1 + exp((V_½ − V_m)/k)]⁻¹ where V_½ is the membrane potential generating half maximal activation or inactivation, V_m is the membrane potentials (or prepulse potential in steady-state inactivation experiments), and k is the slope of the function. For the display of I/V curves the average peak currents at each voltage test were used.

In some neurones we tested the excitability using current-clamp recordings. In these experiments the bath was perfused with HEPES solution (control; 118 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4). Test compounds were added with an application system. The recording pipettes contained 140 mM KCl, 10 mM NaCl, 1 mM MgCl₂, 0.5 mM CaCl₂, 2 mM Na₂-ATP, 5 mM EGTA, 10 mM HEPES, and 10 mM sucrose, pH 7.2. We included only neurones with a membrane potential less than −45 mV. To assess neuronal excitability, APs were elicited by current injection through the recording pipette. At the resting potential, current was applied at amplitudes from 0 pA in 25 pA steps (pulse duration 5 msec, interpulse interval 2 seconds) until an AP with the typical overshoot was elicited. This protocol was repeated every 2 minutes before application of IL-17A, and within 3–7 minutes after application of IL-17A to the bath. In addition, ramp current (0 pA to 3x threshold current) was applied for 500 ms, and the latency of the first AP and the numbers of elicited APs were measured before and during IL-17A application.