Regulation of intracellular chloride by cotransporters in developing lateral superior olive neurons

Abstract

The regulatory mechanisms of intracellular Cl- concentration ([Cl- ](i)) were investigated in the lateral superior olive (LSO) neurons of various developmental stages by taking advantage of gramicidin perforated patch recording mode, which enables neuronal [Cl-](i) measurement. Responses to glycine changed from depolarization to hyperpolarization during the second week after birth, resulting from [Cl-](i) decrease. Furosemide equally altered the [Cl-](i) of both immature and mature LSO neurons, indicating substantial contributions of furosemide-sensitive intracellular Cl- regulators; i.e., K+-Cl- cotransporter (KCC) and Na+-K+-Cl- cotransporter (NKCC), throughout this early development. Increase of extracellular K+ concentration and replacement of intracellular K+ with Cs+ resulted in [Cl-](i) elevation at postnatal days 13-15 (P13-P15), but not at P0-P2, indicating that the mechanism of neuronal Cl- extrusion is sensitive to both furosemide and K+-gradient and poorly developed in immature LSO neurons. In addition, removal of extracellular Na+ decreased [Cl-](i) at P0-P2, suggesting the existence of extracellular Na+-dependent and furosemide-sensitive Cl- accumulation in immature LSO neurons. These data show clearly that developmental changes of Cl- cotransporters alter [Cl-](i) and are responsible for the switch from the neonatal Cl- efflux to the mature Cl- influx in LSO neurons. Such maturational changes in Cl- cotransporters might have the important functional roles for glycinergic and GABAergic synaptic transmission and the broader implications for LSO and auditory development.