Whole-cell patch-clamp recording of T-type Ca2+ channel current on dissociated DRG neurons

Whole-cell patch-clamp recording on dissociated DRG neurons was performed, as described previously.³⁹ In brief, the DRG (L3, L4, and L5) were rapidly harvested from the isoflurane-anesthetized animals and were incubated in 0.01% blendzyme 2 (Roche Diagnostics, Madison, WI) for 30 min followed by incubation in 0.25% trypsin and 0.125% DNase for 30 min, both dissolved in DMEM/F12 with glutaMAX (ThermoFisher). After exposure to 0.1% trypsin inhibitor and centrifugation, the pellet was gently triturated in culture medium containing Neural basal media A (ThermoFisher) plus 0.5 µm glutamine. Dissociated cells were plated onto 5% laminin-coated glass coverslips (ThermoFisher) and maintained at 37°C in humidified 95% air and 5% CO₂ for 2 h and were studied no later than 6–8 h after harvest.

Electrophysiological recordings on the DRG neurons were performed in a blind manner where the electrophysiologist was not aware of the treatment (MIA or saline). Voltage-induced currents flowing through Ca²⁺ channels were recorded using an extracellular solution containing (in mM): 2 BaCl₂, 4-aminopyridine 1, 10 HEPES, 140 tetraethylammonium chloride (TEACl), pH of 7.4, with an osmolarity of 300 mOsm. To selectively record T-type low-voltage activated (LVA) calcium currents (I_Ca), neurons were preincubated in a Tyrode’s solution with 0.2 µM ω-conotoxin GVIA, 0.2 µM nisoldipine, and 0.2 µM ω-conotoxin MVIIC for at least 30 min. ω-conotoxin GVIA irreversibly blocks N-type I_Ca, and ω-conotoxin MVIIC irreversibly blocks P-/Q-type I_Ca. The concentrations used were saturating in preliminary experiments.³⁹ Any residual high-voltage activated (HVA) I_Ca following incubation of HVA calcium channel blockers was eliminated by using fluoride in the internal pipette solution.^39,40 The fluoride (F⁻)-based internal solution, which was used in all experiments examining LVA I_Ca, contained (in mM): 135 tetra-methyl ammonium hydroxide (TMA-OH), 10 EGTA, 40 HEPES, and 2 MgCl₂, adjusted to pH 7.2 with hydrofluoric acid. A selective and reversible T-type Ca²⁺ channel blocker, TTA-P2 (3, 5-dichloro-N-[1–(2,2-dimethyl-tetrahydropyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide, Alomone Labs, Jerusalem, Israel),⁴⁰ was used to confirm the T-type I_Ca.³⁹ Patch pipettes, ranging from 2–4MΩ resistance, were formed from borosilicate glass (King Precision Glass Co., Claremont, CA) and fire polished. Recordings were made with an Axopatch 700B amplifier (Molecular Devices, Downingtown, PA). Signals were filtered at 2 kHz and sampled at10 kHz with a Digidata 1440 A digitizer and pClamp10 software (Molecular Devices). Series resistance (5–10 MΩ) was monitored before and after the recordings, and data were discarded if the resistance changed by 20%. After achieving the whole cell recording, capacitance and series resistance were compensated accordingly. Leak currents were digitally subtracted using a P/4 leak subtraction protocol. Voltage protocols consisted of 400-ms square-wave commands from a holding potential of −90 mV for LVA to +60 mV in 10mV increments with 5 s intervals between steps. The peak T-current was measured after subtracted from the current at the end of the depolarizing test potential to avoid contamination with residual HVA currents.⁴⁰ Measured inward current was normalized by membrane capacitance, which results in a T-channel I_Ca density corrected for cell size (pA/pF). To determine the current–voltage (I–V) relationship of voltage-dependent activation, the peak I_Ca densities during each voltage command step were fitted to a smooth curve with a Boltzmann equation: I = G_max(V−E_rev)/[(1+exp[(V−V₅₀)/k))], which provided the maximum conductance (G_max). Normalized activation curves were fitted with a Boltzmann equation G/G_max = 1/(1+exp(V₅₀−Vm)/k), where G was calculated as follows: G = I/(Vm−E_rev). The steady-state inactivation curves were fitted with I/Imax = 1/(1+exp(V₅₀−Vm)/k). In all of the equations, V₅₀ denotes the half-activation and half inactivation potentials, Vm is the membrane potential, Erev is the inversion potential, k is the slope factor, G is the conductance, and I is the current at a given Vm; G_max and I_max are the maximum conductance and current, respectively.