Calcium currents in dissociated cochlear neurons from the chick embryo and their modification by neurotrophin-3

Abstract

Calcium entry through voltage-dependent channels play a critical role in neuronal development. Using patch-clamp techniques we have identified the components of the macroscopic Ca 2+ current in acutely-isolated chick cochlear ganglion neurons and analysed their functional expression throughout embryonic development. With Ba 2+ as a charge carrier, the currents exhibited two main components, both with a high activation threshold but differing in their inactivation kinetics. One component showed inactivation with a time constant around 100 ms (transient) whereas the other hardly inactivated (sustained). The currents were sensitive to ω-Conotoxin GVIA and dihydropyridines, blocked by 20 μM Cd 2+, but unaffected by ω-Agatoxin IVA. In a few cases, only with Ca 2+ as a charge carrier, an additional component with low activation threshold and fast inactivation (time constant of 20 ms), was observed. Currents were first detected at day 7 of embryonic development. Current density (amplitude/cell capacitance) increased through embryonic day 9, when early synaptic contacts are established, and decreased thereafter to lower steady values. The effect of neurotrophin-3, a neurotrophic factor required for survival and differentiation of cochlear ganglion neurons, was also examined. Neurons isolated at embryonic day 7 or day 11 and maintained two days in culture with 2 ng/ml neurotrophin-3 showed a substantial increase in Ca 2+ current density, particularly in the transient component. These findings indicate that the expression of neuronal Ca 2+ channels is predominant at the time of synapse formation between transducing hair cells and their primary afferents. Besides its effects on survival and neuritogenesis, neurotrophin-3 enhances the expression of Ca 2+ channels in cultured neurons. Taken together these results suggest that the functional expression of Ca 2+ channels is regulated during embryonic development of cochlear neurons by the release of neurotrophin-3 from the differentiating sensory epithelium of the cochlea.