Alphaxalone Activates a Cl⁻ Conductance Independent of GABA_A Receptors in Cultured Embryonic Human Dorsal Root Ganglion Neurons

Abstract

Whole cell and cell-attached patch-clamp techniques characterized the neurosteroid anesthetic alphaxalone's (5alpha-pregnane-3alpha-ol-11,20-dione) effects on GABAA receptors and on Cl- currents in cultured embryonic (5- to 8-wk old) human dorsal root ganglion neurons. Alphaxalone applied by pressure pulses from closely positioned micropipettes failed to potentiate the inward Cl- currents produced by application of GABA. In the absence of GABA, alphaxalone (0.1-5.0 microM) directly evoked inward currents in all dorsal root ganglion neurons voltage-clamped at negative membrane potentials. The amplitude of the current was directly proportional to the concentration of alphaxalone (Hill coefficient 1.3 +/- 0.15). The alphaxalone-induced whole cell current was carried largely by Cl- ions. Its reversal potential was close to the theoretical Cl- equilibrium potential, changing with a shift in the external Cl- concentration as predicted by the Nernst equation for Cl- ions. And because the alphaxalone-current was not suppressed by the competitive GABAA receptor antagonist bicuculline or by the channel blockers picrotoxin and t-butylbicyclophosphorothionate (TBPS; all at 100 microM), it did not appear to result from activation of GABAA receptors. In contrast to GABA-currents in the same neurons, the whole cell current-voltage curves produced in the presence of alphaxalone demonstrated strong inward rectification with nearly symmetrical bath and pipette Cl- concentrations. Fluctuation analysis of the membrane current variance produced by 1.0 microM alphaxalone showed that the power density spectra were best fitted to double Lorentzian functions. The elementary conductance for alphaxalone-activated Cl- channels determined by the relationship between mean amplitude of whole cell current and variance was 30 pS. Single-channel currents in cell-attached patches when the pipette solution contained 10 microM alphaxalone revealed a single conductance state with a chord conductance of approximately 29 pS. No subconductance states were seen. The current-voltage determinations for the single-channels activated by alphaxalone demonstrated a linear relationship. Mean open and shut times of single alphaxalone-activated channels were described by two exponential decay functions. Taken together, the results indicate that in embryonic human DRG neurons, micromolar concentrations of alphaxalone directly activate Cl- channels whose electrophysiological and pharmacological properties are distinct from those of Cl- channels associated with GABAA receptors.

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“…This suggests that, at high concentrations , alphaxalone directly activates the GABA A receptor/chloride channel complex in the absence of GABA, increasing the chloride conductance. Similar ndings were reported for cultured human embryonic dorsal root ganglion neurons (VALEYEV et al, 1999a;1999b). Barker and colleagues (BARKER et al, 1987) have shown in cultured rat spinal neurons that alphaxalone increases the chloride conductance by increasing the mean open time of the GABA activated channels, and directly activates the chloride conductance by stimulating long duration channel openings.…”

Long-term non-invasive interrogation of human dorsal root ganglion neuronal cultures on an integrated microfluidic multielectrode array platform

“…DRG, located along the spinal nerves, house the cell bodies of sensory neurons whose dendrites are located in the skin, muscles, tendons, joints and internal organs. [13][14][15][16][17][18][19][20][21][22][23][24][25][26] Studies of dissociated human dorsal root ganglion (hDRG) neurons have described changes in electrophysiology and ion channel function due to changing culture conditions, 23 exposure to viral proteins, 25 drugs, and chemicals. 5 Lack of access to primary human sensory neurons has hindered the understanding of the physiology of pain in humans; 6 most investigations have been performed on neurons retrieved from ganglionectomized chronic pain patients or fetal and autopsy tissues.…”

“…[7][8][9][10][11][12] In a few cases, viable human tissues have been dissociated into single neurons and utilized to characterize receptor function using traditional electrophysiological methods, such as patch clamping, to monitor alterations in the electrophysiological response. [13][14][15][16][17][18][19][20][21][22][23][24][25][26] Studies of dissociated human dorsal root ganglion (hDRG) neurons have described changes in electrophysiology and ion channel function due to changing culture conditions, 23 exposure to viral proteins, 25 drugs, and chemicals. 24,[26][27][28][29] While these techniques are very informative, they are restricted to individually interrogating single cells.…”