In vivo Imaging of Mitochondrial Transport in Single-Axon Regeneration of Zebrafish Mauthner Cells

Abstract

Mitochondrial transport is essential for neuronal function, but the evidence of connections between mitochondrial transport and axon regeneration in the central nervous system (CNS) of living vertebrates remains limited. Here, we developed a novel model to explore mitochondrial transport in a single Mauthner axon (M axon) of zebrafish with non-invasive in vivo imaging. To confirm the feasibility of using this model, we treated labeled zebrafish with nocodazole and demonstrated that it could disrupt mitochondrial transport. We also used two-photon laser axotomy to precisely axotomize M axons and simultaneously recorded their regeneration and the process of mitochondrial transport in living zebrafish larvae. The findings showed that the injured axons with stronger regenerative capability maintain greater mitochondrial motility. Furthermore, to stimulate axon regeneration, treatment with dibutyryl cyclic adenosine monophosphate (db-cAMP) could also augment mitochondrial motility. Taken together, our results provide new evidence that mitochondrial motility is positively correlated with axon regeneration in the living vertebrate CNS. This promising model will be useful for further studies on the interaction between axon regeneration and mitochondrial dynamics, using various genetic and pharmacological techniques.

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

“…As for the details of the molecular mechanism underlying this process, we did not mention them in this study. However, we speculated that mitochondria may play an important role in this activity-dependent axonal regeneration (20,47,62). Mitochondria are the main source of cellular energy, whereas axonal regeneration is a process costing huge amounts of energy.…”

“…However, our previous work has suggested that M cells have robust axonal regeneration after 2photon axotomy, indicating that M cells are a good model for the in vivo imaging of the axonal regeneration at a single-cell resolution. An additional benefit is that these large cells are clearly and easily viewed in vivo (19)(20)(21). By combining the single-cell electroporation technology, the use of noninvasive techniques to intervene and image M-cell axonal regeneration in vivo provided us the possibility of studying the unclear precise cellular mechanism of regeneration in CNS.…”

MicroRNA-133b Negatively Regulates Zebrafish Single Mauthner-Cell Axon Regeneration through Targeting tppp3 in Vivo

“…A Zeiss microscope (LSM710, Germany) was used to ablate the M-cell axons over cloacal pores. We normally set the 800 nm two-photon laser at an intensity of 12–15% to damage axon over ~1.5 s (Xu et al, 2017 ).…”

“…In vivo imaging of single-axon regeneration in intact vertebrate is a powerful approach to gain mechanistic insights into this process (Kerschensteiner et al, 2005 ; Canty et al, 2013 ; Lorenzana et al, 2015 ; Xu et al, 2017 ). Although, previous studies have established miRNAs as crucial regulators in regenerative processes, little is known regarding their role in a single neuron during regeneration.…”

“…Using two-photo axotomy, a technology that can precisely injure a single axon (O'Brien et al, 2009 ; Canty et al, 2013 ; Xu et al, 2017 ), we have demonstrated that Mauthner-cells, a hindbrain neuronal type with large soma and long axons projecting toward the spinal cord, have the capacity to regenerate (Xu et al, 2017 ). In this study, we examined the role of miR-133b in M-cell regeneration.…”