Shift from depolarizing to hyperpolarizing glycine action in rat auditory neurones is due to age-dependent Cl- regulation

1. The inhibitory neurotransmitter glycine can elicit depolarizing response s in immature neurones. We investigated the changes in glycine responses an d their ionic mechanism in developing neurones of the rat lateral superior olive (LSO), an auditory brainstem nucleus involved in sound localization. 2. Whole-cell and gramicidin perforated-patch recordings were performed fro m visually identified LSO neurones in brain slices and glycine was pressure applied for 3-100 ms to the soma. Glycine-evoked currents were reversibly blocked by strychnine. They were mostly monophasic, but biphasic responses occurred in similar to 30% of P8-11 neurones in perforated patch recordings . 3. In whole-cell recordings from P2-11 neurones, the reversal potential of glycine-evoked currents (E-Gly) was determined by the transmembranous Cl- g radient and corresponded closely to the Nernst potential for Cl-, regardles s of age. This indicates that Cl- is the principle ion permeating glycine r eceptors, but is also consistent with a low relative (10-20%) permeability for HCO3-. The Cl- gradient also determined the polarity and amplitude of g lycine-evoked membrane potential changes. 4. Leaving the native intracellular [Cl-] undisturbed with gramicidin perfo rated-patch recordings, we found a highly significant, age-dependent change of E-Gly from -46.8 +/- 1.8 mV (P1-4, n = 28) to -67.6 +/- 3.3 mV (P5-8, n = 10) to -82.2 +/- 4.1 mV (P9-11, n = 18). The majority of P1-4 neurones w ere depolarized by glycine (similar to 80 %) and spikes were evoked in simi lar to 30 %. In contrast, P9-11 neurones were hyperpolarized. 5. In perforated-patch recordings, E-Gly was influenced by the voltage prot ocol and the glycine application interval; it could be shifted in the posit ive and negative direction. For a given application interval, these shifts were always larger in P1-4 than in P8-11 neurones, pointing to less effecti ve Cl- regulation mechanisms in younger neurones. 6. Furosemide (frusemide), a blocker of cation-Cl- cotransporters, reversib ly shifted E-Gly in the negative direction in P2-4 neurones, yet in the pos itive direction in P8-10 neurones, suggesting the blockade of net inward an d net outward Cl- transporters, respectively. 7. Taken together, age-dependent changes in active Cl- regulation are likel y to cause the developmental shift from depolarizing to hyperpolarizing gly cine responses. A high intracellular [Cl-] is generated in neonatal LSO neu rones which decreases during maturation.