Contribution of Resting Conductance, GABA(A)-Receptor Mediated Miniature Synaptic Currents and Neurosteroid to Chloride Homeostasis in Central Neurons

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.