Rat hippocampal neurons in culture: properties of GABA-activated Cl- ion conductance

Abstract

The actions of gamma-aminobutyric acid (GABA) on the membrane properties of rat hippocampal neurons maintained in dissociated cell culture were studied using intracellular recording techniques. All the neurons tested were responsive to GABA applied by pressure from micropipettes containing 10-20 microM GABA. The response consisted of a marked increase in conductance associated with a potential change. The inversion potential was sensitive to the Cl- ion gradient across the cells. It was about -60 mV when measured during recordings utilizing K acetate-filled microelectrodes, about -15 mV when measured during recordings with KCl-filled microelectrodes, and about +15 mV when measured with KCl electrodes in a medium containing low [Cl-]o. These results indicate that the membrane conductance evoked by GABA primarily involves Cl- ions. There were no apparent differences between the inversion potential of responses elicited at the level of the cell body and those evoked on processes. The two-electrode voltage-clamp technique was used to study the membrane mechanisms underlying these responses. GABA generated current responses that were associated with an increase in both conductance and membrane current variance. At a given potential both the conductance change and increase in variance were directly proportional to the amplitude of the current response. Spectral analysis of the membrane current variance evoked by GABA revealed that many of the computed spectra could be fitted by a single Lorentzian equation, suggesting that GABA activates two-state (open-closed) Cl- ion channels whose durations are exponentially distributed. The mean duration of these channel openings was estimated to be 22.9 +/- 2.1 ms, while the average conductance was estimated to be 19.8 +/- 2.7 pS in 13 cells. Large-amplitude GABA responses evoked at -70 mV frequently faded in amplitude, often by as much as 50%, with little or no change in the associated conductance. Since the membrane current variance decreased in direct relation to the fading current response, while estimates of channel lifetime did not change, the results suggest that the fading is due to a shift in the Cl- gradient. Responses to constant amounts of GABA evoked at different membrane potentials showed that the macroscopic conductance activated by GABA varied with membrane potential. Often 4-5 times more conductance was generated at depolarized (0 to +10 mV) relative to hyperpolarized potentials (-60 to -70 mV).(ABSTRACT TRUNCATED AT 400 WORDS)