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 GABAA 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 similar to 6% of the total resting conductance. A major fraction of this resting Cl- conductance is picrotoxin (PTX)-sensitive and likely due to open GABAA receptors, but ClC-2 channels do not contribute. The results also show that when the decay of GABAA 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 Clhomeostasis.
Originally published in manuscript form