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.

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.