A novel method to study the local mitochondrial fusion in myelinated axons in vivo.

Mitochondrial remodeling (replication, fission/fusion) is a dynamically regulated process with diverse functions in neurons. A myelinated axon is an extension from the cell soma of a fully differentiated neuron. Mitochondria, once synthesized in the cell body, enter the axon displaying robust trafficking and accumulation at nodes of Ranvier to match metabolic needs. This long-distance deployment of mitochondria to axons raises the issue of whether myelinated axons can function independently of the cell body to execute mitochondrial remodeling to match local demands. Mitochondrial fusion has been suggested to occur in axons in simple neuronal cultures in vitro. However, whether such events occur in vivo in an intact nervous system remains unanswered. Here we describe a novel technique which allows monitoring of mitochondrial fusion in intact sciatic nerve of frog (Xenopus laevis). Mitochondrial population was labeled by injecting two different MitoTracker dyes (Red and Green), spatially apart along sciatic nerves surgically and then allow to "meet"in vivo. At 24h post-surgery, the sciatic nerves were taken out for mitochondrial imaging at the half-way point. During the post-injection periods, the anterograde-directed Green mitochondria meet with the retrograde-directed Red mitochondria. If fusion occurs, the merged of Green and Red fluorophores in the same mitochondrion will produce a Yellow color in merged images. The labeled mitochondria were observed with a Nikon A1 confocal microscope. Our new mitochondrial imaging method opens an avenue to separately assess the role of local axonal mitochondrial fusion, independent of the cell body of nerve fibers.