The role of conditioning factors in the formation of growth cones and neurites from the axon stump after axotomy.

Knowledge of the cellular events that underlie initiation of outgrowth is crucial to understanding regulation of development and regeneration of the nervous system. This study utilized a culture preparation in which growth cone formation could be studied independent of cellular responses to the presence of conditioning factors. Identified neurons were removed from the buccal ganglion of the mollusc, Helisoma trivolvis, and plated into defined culture medium. A large growth cone formed at the end of the attached axon stump. Although this axonal growth cone exhibited filopodial and lamellipodial activity, it did not advance across the substrate, suggesting that growth cone formation and motility were independent of the presence of conditioning factors. Axonal growth cones of identified neurons B19 and B5 exhibited differences in their morphological and behavioral properties. In response to the addition of conditioning factors, several new neurites extended from the periphery of the axonal growth cone. Extension of outgrowth from the axonal growth cone was accompanied by a redistribution of cytoskeletal elements in the axonal growth cone. Cytoskeletal staining revealed a loss of the peripheral actin filament network and microtubules were found to extend into the peripheral lamellipodium of the axonal growth cone, an area normally devoid of microtubule staining. Thus, these experiments indicate that growth cone formation is an intrinsic property of the distal axon stump and that neurite extension from this structure involves reorganization of the neuronal cytoskeleton.