2.12 Whole-cell patch-clamp electrophysiology of cultured DRG neurons
Whole-cell patch-clamp techniques were utilized to examine the electrophysiological and pharmacological properties of high-voltage-activated (HVA) calcium currents in DRG neurons from adult naïve mice. After a 48–72-hr incubation period, cultured DRG neuron preparations were placed into a submersion-type recording chamber (RC-22; Warner Instruments, Hamden CT, USA), secured to an inverted microscope, and visualized with a bright-field imaging system (Eclipse TE2000-U; Nikon). Patch-clamp electrodes were constructed from single-filament borosilicate glass (1.5 mm outer diameter and 0.84 mm inner diameter; World Precision Instruments) with a microelectrode puller (P-1000; Sutter Instruments, Novato, CA). Electrode tip impedance ranged from 2 to 4 MΩ and formed seal resistances > 1 GΩ when filled with an internal recording solution composed of (in mM) 140 tetraethylammonium chloride, 10 EGTA, 1 MgCl2, 10 HEPES, 0.5 GTP, and 3 ATP (pH = 7.4 by 1 M N-methyl-D-glucamine [NMDG]; ~300 mOsm, adjusted with sucrose, measured by a Wescor Vapro 5600, ELITech Group). Cultured neuron preparations were maintained under constant gravity-driven perfusion of an oxygenated external solution consisting of (in mM) 130 NMDG chloride, 5 BaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH = 7.4 by HCl; ~310-315 mOsm adjusted with sucrose), delivered at a rate of 1–2 ml∙min-1 at room temperature. Tetraethylammonium chloride and NMDG chloride were added to each solution to block voltage-dependent K+conductance and Na+ conductance, respectively. Ba2+ was added to the external solution to function primarily as a preferential charge carrier through the HVA channels, but also as a background K+ conductance blocker. The junction potential between the internal and external solutions was not corrected for.
For patch-clamp recordings of HVA Ca2+(HVA-ICa) currents in small diameter (<20 µm diameter) DRG neurons, series resistance, if necessary was compensated and maintained at <20 MΩ approximately 1–2 min after the whole-cell configuration was established. The voltage protocol used to evoke HVA-ICa was modified from that of prior publications (Chen & Ikeda, 2004; Li et al., 2017). Briefly, neurons were held at -80 mV, and a 40 mV square wave voltage pulse was applied via the patch electrode (evoked to -40 mV) for 20 ms to activate low-voltage-activated (LVA) Ca2+ channels. The holding voltage was then set at -60 mV for 20 ms followed by a 50 mV voltage application delivered via the electrode (evoked to -10 mV) for 20 ms to evoke HVA Ca2+ channels. After we recorded baseline HVA-ICa for 1 min to assess the stability of each evoked current, we applied either DALDA or morphine (1 μM) to the neurons using a six-channel perfusion valve control system (VC-6; Warner Instruments) for a period of 2 min (time of full bath exchange) followed by a 5-min washout with the external solution. This HVA stimulation protocol was run every 10 s for a total of 8 min. Acquired recordings of HVA-ICa were filtered at 4 kHz with a -3 dB, 4-pole, lowpass Bessel filter, sampled at a rate of 20 kHz, and stored on a personal computer (Dell) using pClamp 9.2 and a digitizer (Digidata 1322A, Molecular Devices). Offline, currents were digitally filtered by using a lowpass Gaussian filter with a -3 dB cutoff set to 2 kHz (Clampfit software; pClamp 9.2, Molecular Devices).