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

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Abstract
The actions of 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 .mu.M 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 potassium 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. The membrane conductance evoked by GABA primarily involves Cl-. GABA generated current responses that were associated with an increase in both conductance and membrane current variance. At a given potential, 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 2-state (open-closed) Cl- 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 [picoSiemens] 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. The fading is due to a shift in the Cl- gradient. Responses to constant amounts of GABA evoked at different membrane potentials showed the macroscopic conductance activated by GABA varied with membrane potential. Often 4-5 times more conductance was generated at depolarized (0 to +10 mV) than at hyperpolarized potentials (-60 to -70 mV). Changes in the kinetics rather than the conductance of the microscopic channels could account for the observed voltage sensitivity of the GABA response. Glycine and muscimol also generated Cl--dependent current responses that were associated with an increase in membrane current variance. Both activate ion channels with about the same conductance as that activated by GABA, but with significantly longer durations. Cl- channels activated by GABA have relatively constant properties in neurons cultured from spinal and supraspinal regions, while those activated by glycine do not.

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