In vivo imaging of Mauthner axon regeneration, remyelination and synapses re-establishment after laser axotomy in zebrafish larvae

Zebrafish is an excellent model to study central nervous system (CNS) axonal degeneration and regeneration since we can observe these processes in vivo and in real time in transparent larvae. Previous studies have shown that Mauthner cell (M-cell) axon regenerates poorly after mechanical spinal cord injury. Inconsistent with this result, however, we have found that M-cell possesses a great capacity for axon regeneration after two-photon laser ablation. By using ZEISS LSM 710 two-photon microscope, we performed specific unilateral axotomy of GFP labeled M-cells in the Tol-056 enhancer trap line larvae. Our results showed that distal axons almost degenerated completely at 24 h after laser axotomy. After that, the proximal axons initiated a robust regeneration and many of the M-cell axons almost regenerated fully at 4 days post axotomy. Furthermore, we also visualized that regenerated axons were remyelinated when we severed fluorescent dye labeled M-cells in the Tg (mbp:EGFP-CAAX) line larvae. Moreover, by single M-cell co-electroporation with Syp:EGFP and DsRed2 plasmids we observed synapses re-establishment in vivo during laser injury-induced axon re-extension which suggested re-innervation of denervated pathways. In addition, we further demonstrated that nocodazole administration could completely abolish this regeneration capacity. These results together suggested that in vivo time-lapse imaging of M-cell axon laser injury may provide a powerful analytical model for understanding the underlying cellular and molecular mechanisms of the CNS axon regeneration.