Regulation of Intracellular Chloride by Cotransporters in Developing Lateral Superior Olive Neurons

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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−]imeasurement. Responses to glycine changed from depolarization to hyperpolarization during the second week after birth, resulting from [Cl−]idecrease. Furosemide equally altered the [Cl−]iof 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−]ielevation 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−]iat 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−]iand 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.