Distribution of Na+ and K+ Currents in Soma, Axons and Growth Cones of Leech Retzius Neurones in Culture

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Abstract
Leech Retzius neurones were isolated by a new technique which allowed investigation of macroscopic currents over the surface of the cell body and the axons using loose patch-clamp. The distribution of ion current densities was measured for neurones that had just been removed from the CNS, and for cultured cells in which neurite outgrowth had begun. To standardize the mapping procedure, the same patch electrode was used at various sites along the neurone. Immediately after isolation of the cell, rapidly activating and inactivating Na+ currents were recorded from distal segments of the axons, but not from the soma or the proximal segment. Na+ currents were isolated by using patch electrodes containing tetraethylammonium (TEA+) and 4-aminopyridine (4-AP) to block K+ channels and Cd2+ to block calcium channels. Na+ currents in all regions of the neurone where they could be recorded were similar in their voltage dependence and kinetics. The Na+ current density was highest at the broken tips of the axon stumps. Neurites began to extend from the broken axon tips approximately 30min after isolation. Newly grown processes showed a high density of Na+ currents at their growth cones. After 2 days in culture the current densities became more uniformly distributed and Na+ currents could then be recorded in the soma and proximal axon segments. In agreement with earlier studies made with conventional two-electrode voltage-clamp, three principal K+ currents were detected in Retzius cells: a rapidly activating and inactivatingA-type current blocked by 4-AP (IA); a more slowly activating and inactivating delayed K+ current blocked by TEA+ (IK1); and a Ca2+-activated K+ current (IC). Immediately after isolation of the Retzius cell, both rapid A-type and slow delayed K+ currents were distributed more uniformly than Na+ currents over the soma and axons. In their voltage sensitivities and kinetics, these two K+ currents were markedly different from each other; their characteristics were, however, constant in different regions of the cell. Ca2+ currents were too small to be measured directly during depolarizing pulses. However, tail currents were large enough to demonstrate the presence of Ca2+ channels in the proximal segment of the axon and in the soma; the currents were not sufficiently large to resolve their spatial distribution. It is concluded that ion channels are present in newly grown membranes and that the density of Na+ channels is highest in the tips of distal axon stumps from which outgrowth begins. By contrast, K+ currents are distributed more uniformly along the neurone.