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).