In Vivo Imaging Of Mauthner Axon Regeneration,Remyelination And Synapses Re-establishment After Laser Axotomy In Zebrafish Larvae

In mammals,central nervous system(CNS)axons are unable to regenerate after injury,resulting in permanent impairments of movements and sensations.In contrast,many severed axons in the zebrafish CNS possess a great capacity to regrow precisely and functionally to re-innervate their target sites.Zebrafish is an excellent model to study 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 axon regenerates poorly after mechanical spinal cord injury.Inconsistent with this result,however,we have found that Mauthner cell possesses a great capacity for axon regeneration after two-photon laser ablation.By using ZEISS LSM 710 two-photon microscope,we performed precise unilateral axotomy of GFP labeled Mauthner cell in the Tol-056 enhancer trap line larvae.Our results showed that distal axons almost degenerated completely at 24 hours after laser axotomy.After that,the proximal axons initiated a robust regeneration and many of the Mauthner cell axons almost regenerated fully at 4 days post axotomy.Furthermore,we also visualized that regenerated axons were remyelinated when we severed red fluorescent dye labeled Mauthner cell in the Tg(mbp:EGFP-CAAX)line larvae.To the best of our knowledge,this is the first in vivo characterization of remyelination of the regenerated axons in zebrafish CNS.Moreover,by single Mauthner 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.Thus,we provided the first in vivo observation of synapses re-establishment of regenerating axon in zebrafish CNS.In addition,we further demonstrated that microtubule-disrupting drug nocodazole administration could completely abolish this regeneration capacity.These results together suggested that in vivo time-lapse imaging of Mauthner cell axon laser injury may provide a powerful analytical model for understanding the underlying cellular and molecular mechanisms of the CNS axon regeneration.