Cytoskeletal and Morphological Alterations Underlying Axonal Sprouting after Localized Transection of Cortical Neuron AxonsIn Vitro

We examined the cytoskeletal dynamics that characterize neurite sprouting after axonal injury to cortical neurons maintained in culture for several weeks and compared these with initial neurite development. Cultured neocortical neurons, derived from embryonic day 18 rats, were examined at 3 d in vitro (DIV) and at various time points after axotomy at 21 DIV. The postinjury neuritic response was highly dynamic, progressing through an initial phase of retraction, followed by substantial axonal sprouting within 4–6 hr. Postinjury sprouts were motile and slender with expanded growth cone-like end structures. Microtubule markers were localized to sprout shafts and the proximal regions of putative growth cones and filamentous actin was distributed throughout growth cones, whereas neurofilament proteins were restricted to sprout shafts. A similar distribution of cytoskeletal proteins was present in developing neurites at 3 DIV. Exposure of developing and mature, injured cultures to the microtubule stabilizing agent taxol (10 μg/ml) caused growth inhibition, process distension, the transformation of growth cones into bulbous structures, and abnormal neurite directionality. Microtubule and neurofilament segregation occurred after taxol exposure in developing neurites and postinjury sprouts. Exposure to the microtubule destabilizing agent nocodazole (100 μg/ml) resulted in substantial morphological alteration of developing neurons and inhibited neurite growth and postinjury axonal sprouting. Our results indicate that the axons of cortical neurons have an intrinsic ability to sprout after transection, and similar cytoskeletal dynamics underlie neurite development and postinjury axonal sprouting.