Live Imaging of Regenerating Lamprey Spinal Axons

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Abstract
The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Although the growing tips of developing axons in lamprey have not been described, in all species studied, growth cones are complex in shape, consisting of a lamellipodium and filopodia, rich in F-actin and lacking neurofilaments (NF). By contrast, static immunohistochemical and electron microscopic observations of fixed tissue suggested that the tips of regenerating lamprey spinal axons are simple in shape, densely packed with NF, but contain very little F-actin. Thus, it has been proposed that regeneration of axons in the CNS of mature animals is not based on the canonical pulling mechanism of growth cones but involves an internal protrusive force, perhaps generated by the transport and assembly of NF. To eliminate the possibility that these histological features are due to fixation artifact, fluorescently labeled regenerating axon tips were imaged live. Methods. Spinal cords were transected, and after 0 to 10 weeks, the CNS was isolated in lamprey Ringerat5°Cto12°Candthelargereticulospinalaxons were microinjected with fluorescent tracers. The proximal axon tips were imaged with a fluorescence dissecting microscope repeatedly over 2 to 5 days and photographed with confocal microscopy. Experiments were also performed through a dorsal incision in the living animal. Axon tips were microinjected as above or retrogradely labeled with tracer applied to the transection site and photographed through the fluorescence dissecting scope or with two-photon microscopy. The spinal cords were then fixed and processed for wholemount NF immunohistochemistry. Results. The living axon tips were simple in shape, not significantly different from those in fixed spinal cords, and filled with NF. In isolated CNS preparations, very little axon retraction and no regeneration was observed. In the living animal, rapid retraction, up to 3 mm/day, was seen during the 1st few days posttransection. At more than 2 weeks posttransection, some fibers showed regeneration of up to 35 μm/day. Conclusions. 1) The tips of regenerating lamprey axons are simple in shape and filled with NF. 2) Both axon retraction and axon extension are active processes, requiring factors present in the living animal that are missing in the isolated CNS.