Tracing Central Nervous System Axon Regeneration in Xenopus.

Axonal tracing allows visualizing connectivity between neurons, providing useful information about structure, neuronal location, and function of the nervous system. Identifying regenerating axons and their neuron cell bodies present the particular challenges of labeling the projections of interest while unambiguously demonstrating regrowth of those axons that have been damaged. In the developing brain, an additional labeling challenge arises, as new connections are being made throughout the duration of an experiment. Various strategies have been used to label regenerating axons, including transgenic animals expressing neuron-specific fluorescent proteins, and application of a single labeling molecule after axotomy and regeneration. However, the single label approach is limited in its application to the developing brain, primarily because it leads to the conclusion that every axon that is labeled has regenerated. Double-labeling overcomes these obstacles by identifying regenerating cells as those that are labeled with two different tracing molecules. Moreover, the use of dextran amines, which are only taken up by injured axons and transported retrogradely, provides further confidence of labeling regenerating axons and neuron cell bodies. The procedure described herein provides a straightforward method for using fluorescently labeled dextran amines to identify regenerating supraspinal neurons in Xenopus, but can be applied to other areas of the central and peripheral nervous system as well.