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 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-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 Cl– homeostasis.
Significance Statement The Cl– concentration in central neurons is critical to normal synaptic function and if not properly regulated may contribute to epilepsy, chronic pain and other pathologies. Here, we introduce a novel method to quantify the resting Cl– conductance of the neuronal membrane and show how this conductance contributes to the recovery of Cl– concentration after a high Cl– load. We also clarify that ion channels of the GABAA-receptor type underlie a major fraction of the resting Cl– conductance and show that an endogenous neurosteroid may quicken recovery of Cl– concentration via effects of spontaneously released neurotransmitter on the GABAA-receptors. The findings are important for understanding the neuronal Cl– homeostasis, critical to normal brain function.
- chloride Homeostasis
- GABA-a Receptor
- Miniature Postsynaptic Current
- Resting Chloride Conductance
Authors report no conflict of interest.
Authors contributions: TY, MD and SJ contributed to the study design. TY made the electrophysiological recordings and SJ made the computations. TY, MD and SJ contributed to analysis and interpretation. TY, MD and SJ contributed to writing the paper.
This work was supported by the Swedish Research Council (grant no 22292), by Gunvor och Josef Anérs Stiftelse and by Umeå University Medical Faculty (Insamlingsstiftelsen, Karin och Harald Silvanders fond, and Leila och Bertil Ehrengrens fond).