In vivo imaging of evoked calcium responses indicates the intrinsic axonal regenerative capacity of zebrafish.

Calcium is an important messenger in the neuronal system, but its specific role in axonal regeneration has not been fully investigated. To clarify it, we constructed a noninvasive in vivo calcium-imaging model of zebrafish Mauthner cells and monitored subcellular calcium dynamics during axonal regeneration. Using the calcium indicator GCamp6f, we observed that the regenerative length correlated with the peak amplitude of the evoked calcium response before axotomy, which suggested that the evoked calcium response might serve as a useful indicator of evoked neuronal activity and axonal regenerative capacity. To investigate this possibility, we overexpressed an inward rectifying potassium channel protein, Kir2.1a, to decrease the Mauthner neuronal activity and found that the inhibition of the calcium response correlated with decreased axonal regeneration. In contrast, treatment of pentylenetetrazol and knockout of the sodium voltage-gated channel α subunit 1 gene increased the calcium response and thus enhanced axonal regeneration. Our results therefore increased the understanding of the correlation between the neural activity and the vertebrate axonal regeneration.-Chen, M., Huang, R.-C., Yang, L.-Q., Ren, D.-L., Hu, B. In vivo imaging of evoked calcium responses indicates the intrinsic axonal regenerative capacity of zebrafish.

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 24h after laser axotomy. After that, the proximal axons initiated a robust regeneration and many of the M-cell axons almost regenerated fully at 4days 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.