LOCALIZED AND TRANSIENT ELEVATIONS OF INTRACELLULAR CA2+ INDUCE THE DEDIFFERENTIATION OF AXONAL SEGMENTS INTO GROWTH CONES

The formation of a growth cone at the tip of a severed axon is a key s tep in its successful regeneration, This process involves major struct ural and functional alterations in the formerly differentiated axonal segment. Here we examined the hypothesis that the large, localized, an d transient elevation in the free intracellular calcium concentration ([Ca2+](i)) that follows axotomy provides a signal sufficient to trigg er the dedifferentiation of the axonal segment into a growth cone. Rat iometric fluorescence microscopy and electron microscopy were used to study the relations among spatiotemporal changes in [Ca2+](i), growth cone formation, and ultrastructural alterations in axotomized and inta ct Aplysia californica neurons in culture. We report that, in neurons primed to grow, a growth cone forms within 10 min of axotomy near the tip of the transected axon. The nascent growth cone extends initially from a region in which peak intracellular Ca2+ concentrations of 300-5 00 mu M are recorded after axotomy. Similar [Ca2+](i) transients, prod uced in intact axons by focal applications of ionomycin, induce the fo rmation of ectopic growth cones and subsequent neuritogenesis. Electro n microscopy analysis reveals that the ultrastructural alterations ass ociated with axotomy and ionomycin-induced growth cone formation are p ractically identical. In both cases, growth cones extend from regions in which sharp transitions are observed between axoplasm with major ul trastructural alterations and axoplasm in which the ultrastructure is unaltered. These findings suggest that transient elevations of [Ca2+]( i) to 300-500 mu M, such as those caused by mechanical injury, may be sufficient to induce the transformation of differentiated axonal segme nts into growth cones.