Steady growth cone currents revealed by a novel circularly vibrating probe: A possible mechanism underlying neurite growth

Abstract

The rate and direction of neurite growth have been shown in a number of studies to be determined by the distribution of adhesive sites on the growth cone. Recent evidence showing that the application of extrinsic electric fields can redistribute membrane molecules and alter both the rate and direction of neurite growth have raised the question whether endogenous electric fields might be produced by steady currents in growth cones. To investigate this question, we have devised a novel circularly vibrating microprobe capable of measuring current densities in the range of 5 nA/cm2 (near the theorectical limit of sensitivity), with a spatial resolution of 2 micron. The design of this device and the development of a novel algorithm for computing current vectors on-line is described. Using this probe we have found that cultured goldfish retinal ganglion cell growth cones generate steady inward currents at their tips. The measured currents, in the range of 10-100 nA/cm2, appear to flow into the filopodia at their tips and back outward near the junctures of the filopodia and the growth cone. The currents appear to be produced only during active growth. Ion substitution experiments support the conclusion that the majority of this current is carried by Ca2+ ions, which we postulate flow through a population of activated voltage-sensitive Ca2+ channels located on the filopodial tips. Calculation of the transmembrane current density (4 X 10(-6) nA/cm2) leads to an estimate of channel density (10 channels/micron2) in close agreement with the measured density of Ca2+ channels in other systems. The assumption that calcium channel proteins are conveyed to nerve terminals by active transport, whereas sodium channel proteins are conveyed passively by a slower somatofugal diffusion process [Strichartz et al, 1984], would explain why developing neurons tend to display Ca2+-sensitive electrogenesis at their growing tips, and Na+-sensitive action potentials later in development. In order to gain some insight into the possible role of these steady growth currents, we estimated the membrane depolarization and axial voltage gradient they produce. It is likely that the currents produce sufficient membrane depolarization (approximately equal to 4 mV) to cause autogenous activation of ion channel permeabilities. Similarly, the axial voltage gradient (approximately equal to 4 mV/cm) would be expected to move intracytoplasmic vesicles by electrophoresis at a rate (20-40 microns/hr) very close to that at which the filopodia are observed to grow.(ABSTRACT TRUNCATED AT 400 WORDS)

Digital imaging of free calcium changes and of spatial gradients in growing processes in single, mammalian central nervous system cells.

“…1) but almost never near the 2.0 level unless the cells were stimulated by high K or other means. The observations on growing cells reported are in agreement with studies using electrical techniques in other preparations that have shown a calcium influx into growing tips by means of either Ca spikes (3-5) or a mechanism that carries steady calcium current measurable by external vibrating probe electrodes (2). It was noted in the vibrating probe studies that currents were not generated by stationary growth cones, only those in an active growth state.…”

“…Elevated calcium levels, by analogy to other motile and secretory systems, could allow the local activation of actinomyosin systems to stretch or shape a growing region or promote the insertion of new membrane, as in vesicular secretion, and thereby produce outgrowth. Recent electrophysiological data indicate that transmembrane Ca transport activity exists in growth cones (1)(2)(3)(4)(5)(6), suggesting the possibility of elevated Ca2" concentration. The present study has employed fluorescent Ca indicators (7)(8)(9)(10) and a cooled, charge-coupled device (CCD) camera of the type used in astronomy over the past several years (11,12) to image free Ca2+ levels in individual cultured CNS cells from rat embryo.…”

Intracellular calcium levels do not change during contact-mediated collapse of chick DRG growth cone structure

“…Finally, it must be pointed out that these veils are extremely thin, and the corresponding fluorescence signals are weak and therefore may be subject to error. Several studies have localized calcium channels and calcium currents to the veil regions and to the base of filopodia (Anglister et al, 1982;Freeman et al, 1985;O'Lague et al, 1985;Lipscombe et al, 1988;Silver et al, 1990). Therefore, one might expect to find higher levels of free calcium near these calcium entry zones.…”

Application of conductive polymers, scaffolds and electrical stimulation for nerve tissue engineering

“…Patel et al (1982) suggested three possible ways by which electrical stimulation could act directly on a neuron, including the redistribution of cytoplasmic materials, the activation of growth-controlling transport processes across the plasma membrane due to change in cell membrane potential, and the electrophoretic accumulation of surface molecules responsible for neurite growth or cell-substratum adhesion. Changes in ionic currents around the growing fibre tips induced by electric fields have been suggested by Freeman et al (1985) as one possible mechanism through which electrical stimulation can affect nerve cells. Sisken et al (1989) suggested that electrical stimulation affects protein synthesis in transected sciatic nerve segments and stimulates neurite outgrowth in vitro.…”