Live Imaging of Axonal Dynamics After Laser Axotomy of Peripheral Neurons in Zebrafish

Axon severing results in diverse outcomes, including successful regeneration and reestablishment of function, failure to regenerate, or neuronal cell death. Experimentally injuring an axon makes it possible to study degeneration of the distal stump that was detached from the cell body and document the successive steps of regeneration. Precise injury reduces damage to the environment surrounding an axon, and thereby the involvement of extrinsic processes, such as scarring or inflammation, enabling researchers to isolate the role that intrinsic factors play in regeneration. Several methods have been used to sever axons, each with advantages and disadvantages. This chapter describes using a laser on a two-photon microscope to cut individual axons of touch-sensing neurons in zebrafish larvae, and live confocal imaging to monitor its regeneration, a method that provides exceptional resolution.