Ion channels and the ontogenesis of excitability

Patch-clamp techniques were used to study the expression of ionic currents in chick ciliary ganglion neurons dissociated acutely at various stages of embryonic development (stages 26, 30, 35 and 39). TTX-sensitive Na{dollar}sp+{dollar} currents (I{dollar}sb{lcub}rm Na{rcub}{dollar}), voltage-activated Ca{dollar}sp{lcub}2+{rcub}{dollar} currents (I{dollar}sb{lcub}rm Ca{rcub}{dollar}) and TEA-sensitive voltage-activated K{dollar}sp+{dollar} currents (I{dollar}sb{lcub}rm DR{rcub}{dollar}) were detectable at stage 26 and increased with amplitude and density with development. Transient TEA-resistant voltage-activated K{dollar}sp+{dollar} currents (I{dollar}sb{lcub}rm A{rcub}{dollar}) and Ca{dollar}sp{lcub}2+{rcub}{dollar}-activated K{dollar}sp+{dollar} currents (I{dollar}sb{lcub}rm K(Ca){rcub}{dollar}) were detectable only after stage 35. The densities of all currents (except I{dollar}sb{lcub}rm DR{rcub}{dollar}) increased dramatically when these neurons form synapses with their target tissues.; Isolation of these ciliary ganglion neurons from the in ovo environment at stage 35 and maintaining them in vitro for 4 days prevented the normal expression of I{dollar}sb{lcub}rm A{rcub}{dollar} and I{dollar}sb{lcub}rm K(Ca){rcub}{dollar} suggesting that the expression of these channels is regulated by extrinsic factors. Neurons were isolated from E9 chick embryos and maintained in vitro for 4 days under different conditions. I{dollar}sb{lcub}rm A{rcub}{dollar} expression is enhanced when the cells are co-cultured with striated myotubes but not when co-cultured with fibroblasts lysed myotubes, or when cultured as explants. Culturing these cells in the presence of aFGF enhances I{dollar}sb{lcub}rm A{rcub}{dollar} expression, but CNTF, bFGF, muscle-conditioned medium or High K{dollar}sp+{dollar} medium have no effect. Culture medium containing a soluble fraction of chick brain extract increases I{dollar}sb{lcub}rm A{rcub}{dollar} expression in these neurons. None of these culture conditions promote I{dollar}sb{lcub}rm K(Ca){rcub}{dollar} expression in ciliary ganglion neurons.; Surgical manipulations were used to remove the developing optic vesicle (OV), the middorsal region of the midbrain (MB) primordium, or both, all prior to the formation of the ciliary ganglion in chick embryos. I{dollar}sb{lcub}rm A{rcub}{dollar} and I{dollar}sb{lcub}rm K(Ca){rcub}{dollar} were recorded from acutely isolated neurons that developed in ovo in the absence of target tissue (OV{dollar}sp-{dollar}) or absence of preganglionic innervation (MB{dollar}sp-{dollar}). The activation and inactivation kinetics of I{dollar}sb{lcub}rm A{rcub}{dollar} were 2-3 times faster in OV{dollar}sp-{dollar} and OV{dollar}sp-{dollar}/MB{dollar}sp-{dollar} cells compared to OV{dollar}sp+{dollar} neurons. MB{dollar}sp-{dollar} cells expressed normal I{dollar}sb{lcub}rm A{rcub}{dollar}. I{dollar}sb{lcub}rm K(Ca){rcub}{dollar} was reduced by 90-100% in OV{dollar}sp-{dollar}, MB{dollar}sp-{dollar} and MB{dollar}sp-{dollar}/OV{dollar}sp-{dollar} neurons.