Differential expression pattern of chloride transporters NCC, NKCC2, KCC1, KCC3, KCC4, and AE3 in the developing rat auditory brainstem

Abstract

During development of inhibitory synapses, the action of the two neurotransmitters GABA and glycine shifts from depolarizing to hyperpolarizing. The shift is due to an age-dependent regulation of the intracellular free chloride concentration ([Cl(-)](i)) in postsynaptic neurons. A model system to study this maturation process is a glycinergic projection in the mammalian auditory brainstem. It is formed in the superior olivary complex (SOC) by neurons of the medial nucleus of the trapezoid body, whose axons terminate in the lateral superior olive (LSO). LSO neurons of perinatal rats and mice are depolarized upon glycine application, whereas older cells (>postnatal day (P) 8) are hyperpolarized. Here we examined the expression of six secondary active chloride transporter genes ( NCC, NKCC2, KCC1, KCC3, KCC4, and AE3) in the rat SOC to unravel the molecular mechanisms underlying this change. RT-PCR analysis demonstrated brainstem expression of KCC1, KCC3, KCC4, and AE3, but not of NCC and NKCC2. RNA in situ hybridization showed that only AE3 is highly expressed both at P3 (high [Cl(-)](i)) and P12 (low [Cl(-)](i)) in LSO neurons. KCC1 and KCC4 are weakly expressed in LSO neurons at P3 and P12, respectively. This study completes the expression analysis of all known chloride transporters sensitive to loop diuretic drugs in the SOC and demonstrates differences in the maturation between hippocampal and brainstem inhibitory synapses.