Persistent activation of GABAA receptor/Cl- channels by astrocyte-derived GABA in cultured embryonic rat hippocampal neurons

Whole cell patch-clamp recordings using Cl--filled pipettes revealed more negative levels of baseline current and associated current variance in embryonic rat hippocampal neurons co-cultured on a monolayer of astrocytes than those cultured on poly-D-lysine. These effects were mimicked by culturing neurons on poly-D-lysine in astrocyte-conditioned medium (ACM). The baseline current and variance decreased immediately in all cells after either local perfusion with saline or exposure to bicuculline, an antagonist of GABA at GABA(A) receptor/Cl- channels. Baseline current and variance in all cells reached a nadir at similar to 0 mV, the calculated equilibrium potential for Cl-. Perfusion of ACM rapidly induced a sustained current in neurons, which also reversed polarity at similar to 0 mV. Bicuculline attenuated or eliminated the ACM-induced current at a concentration that completely blocked micromolar GABA-induced current. Quantitative analyses of spontaneously occurring fluctuations superimposed on the ACM-induced current revealed estimated unitary properties of the underlying channel activity similar to those calculated for GABA's activation of GABA(A) receptor/Cl- channels. Bicuculline-sensitive synaptic-like transients, which reversed at similar to 0 mV, were also detected in neurons cultured in ACM, and these were immediately eliminated along with the negative baseline current and superimposed current fluctuations by perfusion. Furthermore bicuculline-sensitive synaptic-like transients were rapidly and reversibly triggered when ACM was acutely applied. ACM induced an increase in cytoplasmic Ca2+ in cultured embryonic hippocampal neurons that was completely blocked by bicuculline and strychnine. We conclude that astrocytes release diffusible substances, most likely GABA, that persistently activate GABAA receptor/Cl- channels in co-cultured neurons.