Calcium action potentials were extracellularly recorded from growth cones of differentiated N1E-115 neuroblastoma cells maintained in monolayer cultures. Extracellular recordings along the neurites suggest that voltage-activated Ca2+ channels are less abundant in the processes than in the growth cones. In order to investigate if Ca2+ entry into the growth cone plays a role in the regulation of neurite growth, we studied the morphological changes induced by experimental conditions which permit calcium entry. Cells were depolarized either by 30 mM potassium (for 10-60 min) or by stimulating the soma (for 20-120 min) with an intracellular electrode. Morphological changes in individual cells were followed by means of time-lapse video recordings. In more than 60% of the experiments, steady-state potassium depolarization induced a pronounced increase of 20-120% in the area of the growth cone. This was frequently associated with neurite elongation. However, such changes could not be detected in the presence of Cd2+ concentrations which block the Ca2+ channels. Similar results were obtained in the presence of 2 μM of the Ca2+ ionophore A-23187 or when the cells were repetitively stimulated (0.2 Hz) in a medium containing 10-6 M TTX and 15 mM TEA. Local microapplication, directly onto single growth cones, of a depolarizing solution containing 5 mM Ca2+ also led to similar observations. Scanning electron microscopy indicated that the depolarized growth cone membranes were flattened and contained markedly more rounded protuberances relative to control cultures. Our results indirectly suggest that Ca2+ entry might be a trigger in the process of neurite elongation.
Bibliographical noteFunding Information:
We thank Dr. M. E. Spira for critical comments on the manuscript. Supported by grants from the U. S.-Israel Binational Science Foundation, USPHS NS-14716, and from the Paralyzed Veterans of America, Technology and Research Foundation.