Morphological properties and membrane channels of the growth cones induced in PC12 cells by nerve growth factor

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Abstract
Large growth cones were produced in vitro by nerve growth factor (NGF) treatment of multinucleate cells produced by chemical fusion of cells of the neuron‐like clone PC12. These endings were studied both at the light microscopic and ultrastructural levels. The activity of ionic channels at growth cones was recorded with intracellular microelectrodes, patch recording of single channels, and whole cone recording from mechanically isolated growth cones. Morphologically, these large growth cones were characterized by the presence of microspikes and filopodia, by the presence of actin demonstrated immunohistochemically, and by the presence of catecholamine fluorescence. At the ultrastructural level they contained a broad spectrum of organelles with a distribution characteristic of neuronal growth cones, including dense core vesicles, abundant smooth membrane cisternae, microtubules, and a filamentous network. The presence of channels capable of generating action potentials was revealed by intracellular microelectrode recording from the growth cone in the presence of locally applied tetraethylammonium (TEA). TEA appeared to block outward current channels that could effectively shunt inward current activated by depolarization. Action potentials elicited by depolarizing current in the presence of TEA could be blocked reversibly by Cd2+, a specific blocker of Ca channels. These action potentials were often followed by a long after‐hyperpolarization lasting hundreds of milliseconds. This after‐hyperpolarization was similar to that recorded in the cell body of PC12 cells where it appears to be mediated by Ca‐activated K current. Single channel recording from outside‐out excised patches of membrane from the growth cones perfused with KF revealed the presence of voltage sensitive Na channels, Ca‐activated K channels, and K channels resembling delayed rectifier K channels. Macroscopic currents recorded from mechanically isolated growth cones in the “whole cone” configuration showed rapid inward currents at potentials ≥ ‐40 mV, followed by delayed outward currents at more positive potentials, a finding providing additional evidence for the presence of Na and K channels in growth cones.