Contribution of Resting Conductance, GABA_A-Receptor Mediated Miniature Synaptic Currents and Neurosteroid to Chloride Homeostasis in Central Neurons

Abstract

Maintenance of a low intraneuronal Cl^- concentration, [Cl^-]_i, is critical for inhibition in the CNS. Here, the contribution of passive, conductive Cl^- flux to recovery of [Cl^-]_i after a high load was analyzed in mature central neurons from rat. A novel method for quantifying the resting Cl^- conductance, important for [Cl^-]_i recovery, was developed and the possible contribution of GABA_A and glycine receptors and of ClC-2 channels to this conductance was analyzed. The hypothesis that spontaneous, action potential-independent release of GABA is important for [Cl^-]_i recovery was tested. [Cl^-]_i was examined by gramicidin-perforated patch recordings in medial preoptic neurons. Cells were loaded with Cl^- by combining GABA or glycine application with a depolarized voltage, and the time course of [Cl^-]_i was followed by measurements of the Cl^- equilibrium potential_, as obtained from the current recorded during voltage ramps combined with GABA or glycine application. The results show that passive Cl^- flux contributes significantly, in the same order of magnitude as does K⁺-Cl^- cotransporter 2 (KCC2), to [Cl^-]_i recovery and that Cl^- conductance accounts for ∼ 6% of the total resting conductance. A major fraction of this resting Cl^- conductance is picrotoxin (PTX)-sensitive and likely due to open GABA_A receptors, but ClC-2 channels do not contribute. The results also show that when the decay of GABA_A receptor-mediated miniature postsynaptic currents (minis) is slowed by the neurosteroid allopregnanolone, such minis may significantly quicken [Cl^-]_i recovery, suggesting a possible steroid-regulated role for minis in the control of Cl^- homeostasis.