Pentobarbital-activated Cl(-) channels in cultured adult and embryonic human DRG neurons.

Developmental differences in pentobarbital-activated Cl(-) currents were studied in adult and embryonic human dorsal root ganglia (DRG) neurons using whole-cell patch-clamp recordings. Pentobarbital-induced Cl(-) conductance was significantly greater in adult DRGs (28.4 pS) than in embryonic cells (19.1 pS). Fluctuation analysis of the spectral density plots of Cl(-) channel activation by pentobarbital showed age differences in the length and number of open time constants (adult cells, tau(1), tau(2), tau(3) were 224, 8. 4, 1.5 ms, respectively; embryonic cells tau(1) and tau (2) were 165 and 26.3 ms, respectively). The different kinetic properties of human adult and embryonic DRG Cl(-) channels opened by pentobarbital may reflect the presence of different subunits in the two populations of neurons.

GABA-Induced Cl- current in cultured embryonic human dorsal root ganglion neurons.

gamma-Aminobutyric acid (GABA)-activated channels in embryonic (5-8 wk old) human dorsal root ganglion (DRG) neurons in dissociated culture were characterized by whole cell and single-channel techniques. All DRG neurons when held at negative holding membrane potentials displayed inward current to micromolar concentrations of GABA applied by pressure pulses from closely positioned micropipettes. The current was directly proportional to the concentration of GABA (EC50, 111 microM; Hill coefficient, 1.7). DRG neurons also responded to micromolar concentrations of pentobarbital and alphaxalone but not to cis-4-aminocrotonic acid (CACA), glycine, or taurine. Baclofen (100 microM) affected neither the holding currents nor K+ conductance (when patch pipettes were filled with 130 mM KCl) caused by depolarizing pulses. Whole cell GABA-currents were blocked by bicuculline, picrotoxin, and t-butylbicyclophosphorothionate (TBPS; all at 100 microM). The reversal potential of whole cell GABA-currents was close to the theoretical Cl- equilibrium potential, shifting with changes in intracellular Cl- concentration in a manner expected for Cl--selective channels. The whole cell I-V curve for GABA-induced currents demonstrated slight outward rectification with nearly symmetrical outside and inside Cl- concentrations. Spectral analysis of GABA-induced membrane current fluctuations showed that the kinetic components were best fitted by a triple Lorentzian function. The apparent elementary conductance for GABA-activated Cl- channels determined from the power spectra was 22.6 pS. Single-channel recordings from cell-attached patches with pipettes containing 10 microM GABA indicated that GABA-activated channels have a main and a subconductance level with values of 30 and 19 pS, respectively. Mean open and closed times of the channel were characterized by two or three exponential decay functions, suggesting two or three open channel states and two closed states. Single channels showed a lack of rectification. The actions of GABA on cultured human embryonic DRG neurons are mediated through the activation of GABAA receptors with properties corresponding to those found in the CNS of human and other mammalian species but differing from those of cultured human adult DRG neurons.

Alphaxalone activates a Cl- conductance independent of GABAA receptors in cultured embryonic human dorsal root ganglion neurons.

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.

Embryonic rat hippocampal neurons and GABAA receptor subunit-transfected non-neuronal cells release GABA tonically.

We used patch-clamp recording techniques to investigate the contribution of GABA to baseline membrane properties in cultured embryonic rat hippocampal neurons. Almost all of the neurons recorded with Cl--filled pipettes and clamped at negative potentials exhibited baselines that were noticeably noisy, with microscopic fluctuations superimposed on the macroscopic holding current. A gentle steam of saline applied to the neuronal surface rapidly and reversibly reduced the baseline current and fluctuations, both of which were completely eliminated by bicuculline. Fluctuation analysis showed that the variance in the baseline current signal was exponentially distributed with estimated kinetics comparable to those activated by submicromolar concentrations of exogenous GABA. The kinetics of Cl- channels activated by endogenous GABA displayed a potential sensitivity comparable to those activated by exogenous GABA. Non-neuronal cells stably transfected with alpha1 and gamma2 GABAA receptor subunits exhibited little baseline current variance when recorded with Cl--filled pipettes. Addition of micromolar GABA to the extracellular saline or to the pipette solution induced a saline- and bicuculline-sensitive baseline current signal comparable to that recorded in hippocampal neurons. Thus, both intra- and extracellular sources of GABA could contribute to the baseline properties recorded in these cultured neurons.

Pharmacologically novel GABA receptor in human dorsal root ganglion neurons.

1. Whole cell voltage-clamp studies of gamma-aminobutyric acid (GABA) receptors were performed on large (> 80 microns) cultured human dorsal root ganglion (DRG) neurons. 2. GABA and pentobarbital sodium when applied in micromolar concentrations evoked inward Cl- currents in DRG neurons voltage clamped at negative membrane potentials. 3. Diazepam (10 microM) and pentobarbital (10 microM) upmodulated the GABA current by approximately 149 and 168%, respectively. 4. The GABA currents in human DRG cells were unaffected by the classical GABA antagonists picrotoxin and bicuclline (100 microM). In contrast, the GABA responses evoked in adult rat DRG cells cultured in an identical manner were inhibited by both antagonists. The glycine receptor antagonist strychnine (100 microM) did not alter GABA currents in human DRG cells. 5. Human DRG cells did not respond to glycine (10-100 microM) or taurine (10-100 microM). The GABAB agonist baclofen had no effect on the holding current when patch pipettes were filled with 130 mM KCl. The GABAB antagonists saclofen applied either alone or with GABA was without effect. 6. The differences between the GABA receptors described here and GABA receptors in other species may reflect the presence of receptor subunits unique to human DRG cells.

Depolarizing GABA-activated Cl- channels in embryonic rat spinal and olfactory bulb cells.

1. We have compared the electrical properties of the Cl- channels activated by GABA in cells acutely dissociated from embryonic (E) spinal cord (SC) and olfactory bulb (OB) regions at E15 using different configurations of the patch-recording technique. By in situ analysis these cells express GABAA receptor mRNAs encoding a common set of subunits (alpha 2, beta 2, and beta 3). SC cells also express alpha 3, alpha 5 and gamma 2s transcripts. 2. Whole-cell recordings revealed current responses to GABA (0.5 microM to 1 mM) in 242 out of 294 cells. In both SC and OB cells, currents evoked by 2 microM GABA could be potentiated by diazepam (DZP) in a dose-dependent manner with an EC50 of approximately 50 nM in both SC and OB. The maximal effect was approximately 300%. Both SC and OB cells exhibited GABA-activated currents that were only partially sensitive to zinc even at high micromolar concentrations. The effect of DZP and the relatively modest sensitivity to zinc suggest the presence of gamma subunits in both preparations. 3. Spectral analysis of current responses in twenty-six cells showed that power spectra could be fitted by three exponential components (tau 1-3) in the cells of both areas. The tau of the longest-lasting component (tau 3) was significantly different in the cells of the two areas: approximately 50 ms in OB and 80-100 ms in SC. No statistically significant differences in the average inferred unitary conductance between the two cell types could be resolved. 4. Single-channel properties were examined directly using the cell-attached configuration. GABA-activated channels could be recorded in only 89 out of well-sealed 984 patches and most of them exhibited multiple channel activity. The mean open time in the response to 10 microM GABA was significantly shorter in OB cells (12 ms) compared to SC cells (25 ms) while the average conductance values were not significantly different between the two cell types. 5. On average, Cl- channels reversed polarity when the on-cell patch pipette potential was approximately -30 mV. Thus, in these embryonic neurons, micromolar GABA activates Cl- channels, which, when open, effectively depolarize cells by approximately 30 mV. 6. Cl- channels activated by GABA are open longer in embryonic SC cells than in OB cells. This statistically significant difference in native GABAA receptor Cl- channel properties correlates with, and may be related to differences in subunit mRNA expression.

Pharmacological properties of fetal rat hippocampal GABAA receptors.

Cells were cultured from embryonic (E) day 17 and 19 rat hippocampal tissues for one or more days in serum-supplemented growth medium. Intracellular recordings in the whole-cell configuration were made at room temperature. GABAA receptor agonists were applied by pressure pulses from closely positioned pipettes. Most of the cells recorded under these conditions responded to one or another ligand. When the equilibrium potential for Cl- was set near 0 mV, current responses to GABA and other GABAA receptor agonists reversed polarity near 0 mV, suggesting a dominant role for Cl- ions in the response. Many cells also responded to the general anesthetics (-)pentobarbital and alfaxalone (3 alpha-hydroxy-5 alpha-pregnane-11,20-dione). Micromolar concentrations of these drugs, like the transmitter GABA, elicited membrane current responses that reversed near 0 mV. Sequential exposure of individual neurons to both anesthetics revealed sensitivity to one but not the other agent as well as some responding to both. These results indicate: (1) the GABAA receptor function emerges pre-natally in rat hippocampal neurons; (2) barbiturates can have several sites for binding to in GABAA receptor complexes.

Cl- channels are randomly activated by continuous GABA secretion in cultured embryonic rat hippocampal neurons.

Throughout the adult vertebrate central nervous system (CNS) gamma-aminobutyric acid (GABA) mediates transient Cl- conductances commonly identified as fast, Cl(-)-dependent inhibitory synaptic signals [Prog. Neurobiol., 36 (1991) 35-92]. In the rat hippocampus Cl(-)-dependent excitatory transients mediated by GABA emerge during the first postnatal week superimposed on a steady-state baseline that is also Cl(-)- and GABA-dependent [Int. J. Dev. Neurosci., 8 (1990) 481-490]. Here we report that many embryonic rat hippocampal neurons cultured for hours to days exhibit random fluctuations in Cl- channel activity that are mediated by continuous secretion of GABA in the absence of transients. Thus, GABA is broadcast tonically before it is released transiently.

Characterization of the gamma-aminobutyric acidA receptor-channel complex composed of alpha 1 beta 2 and alpha 1 beta 3 subunits from rat brain.

The cloned alpha 1, beta 2 and beta 3 subunits of the gamma-aminobutyric acid (GABA)A receptor-channel complex from rat brain were coexpressed as alpha beta complexes in cultured Chinese hamster ovary cells. Electrophysiological characterization of alpha 1 beta 2 and alpha 1 beta 3 receptor subunit arrangements was performed utilizing patch electrodes in the whole-cell recording configuration. The reversal potential of the current activated by either GABA or muscimol corresponded to that expected for Cl- ions and was dependent on the Cl- gradient. The dose response to GABA for activation of Cl- currents by either subunit combination displayed similar potencies. Currents were partially blocked by the reversible antagonist bicuculline. (-)Pentobarbital was ineffective by itself, but potentiated responses to GABA. The steroid alphaxalone (3 alpha-hydroxy 5 alpha-pregnane 11,20-dione) produced just-detectable inward currents, but did not potentiate GABA-activated currents. Diazepam was completely ineffective. The kinetics and conductance of the Cl- ion channels were inferred from spectral analysis of agonist-induced current fluctuations. Both kinetics and conductance were dependent on agonist structure.

Inhibitions of the GABA-induced currents of rat neurons by the alkaloid isocoryne from the plant Corydalis pseudoadunca.

A novel substance, isocoryne, affecting the GABA receptor complex, was isolated from the Corydalis pseudoadunca plant. The inhibitory effect of isocoryne on GABA-activated transmembrane current of single neurons isolated from trigeminal ganglia of rats was tested using concentration-clamp, voltage-clamp and intracellular perfusion techniques. Isocoryne, like other phthalide isoquinoline alkaloids with a 1S,9R isomeric form, produced an inhibitory effect on GABA-activated currents with a Ki = 10(-6) M.