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

ABSTRACT: 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.

SUBMITTER: Zhang CL

PROVIDER: S-EPMC3364104 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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

Zhang Chuan-Li CL Rodenkirch Lance L Schultz Justin R JR Chiu Shing Yan SY

Journal of neuroscience methods 20120331 1

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 t ...[more]

PMID: 22484559

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Project description:Advances in brain connectomics set the need for detailed knowledge of functional properties of myelinated and non-myelinated (if present) axons in specific white matter pathways. The corpus callosum (CC), a major white matter structure interconnecting brain hemispheres, is extensively used for studying CNS axonal function. Unlike another widely used CNS white matter preparation, the optic nerve where all axons are myelinated, the CC contains also a large population of non-myelinated axons, making it particularly useful for studying both types of axons. Electrophysiological studies of optic nerve use suction electrodes on nerve ends to stimulate and record compound action potentials (CAPs) that adequately represent its axonal population, whereas CC studies use microelectrodes (MEs), recording from a limited area within the CC. Here we introduce a novel robust isolated "whole" CC preparation comparable to optic nerve. Unlike ME recordings where the CC CAP peaks representing myelinated and non-myelinated axons vary broadly in size, "whole" CC CAPs show stable reproducible ratios of these two main peaks, and also reveal a third peak, suggesting a distinct group of smaller caliber non-myelinated axons. We provide detailed characterization of "whole" CC CAPs and conduction velocities of myelinated and non-myelinated axons along the rostro-caudal axis of CC body and show advantages of this preparation for comparing axonal function in wild type and dysmyelinated shiverer mice, studying the effects of temperature dependence, bath-applied drugs and ischemia modeled by oxygen-glucose deprivation. Due to the isolation from gray matter, our approach allows for studying CC axonal function without possible "contamination" by reverberating signals from gray matter. Our analysis of "whole" CC CAPs revealed higher complexity of myelinated and non-myelinated axonal populations, not noticed earlier. This preparation may have a broad range of applications as a robust model for studying myelinated and non-myelinated axons of the CNS in various experimental models.

Project description:BACKGROUND:Small-diameter, myelinated axons are selectively susceptible to dysfunction in several inflammatory PNS and CNS diseases, resulting in pain and degeneration, but the mechanism is not known. METHODS:We used in vivo confocal microscopy to compare the effects of inflammation in experimental autoimmune neuritis (EAN), a model of Guillain-Barré syndrome (GBS), on mitochondrial function and transport in large- and small-diameter axons. We have compared mitochondrial function and transport in vivo in (i) healthy axons, (ii) axons affected by experimental autoimmune neuritis, and (iii) axons in which mitochondria were focally damaged by laser induced photo-toxicity. RESULTS:Mitochondria affected by inflammation or laser damage became depolarized, fragmented, and immobile. Importantly, the loss of functional mitochondria was accompanied by an increase in the number of mitochondria transported towards, and into, the damaged area, perhaps compensating for loss of ATP and allowing buffering of the likely excessive Ca2+ concentration. In large-diameter axons, healthy mitochondria were found to move into the damaged area bypassing the dysfunctional mitochondria, re-populating the damaged segment of the axon. However, in small-diameter axons, the depolarized mitochondria appeared to "plug" the axon, obstructing, sometimes completely, the incoming (mainly anterograde) transport of mitochondria. Over time (~?2 h), the transported, functional mitochondria accumulated at the obstruction, and the distal part of the small-diameter axons became depleted of functional mitochondria. CONCLUSIONS:The data show that neuroinflammation, in common with photo-toxic damage, induces depolarization and fragmentation of axonal mitochondria, which remain immobile at the site of damage. The damaged, immobile mitochondria can "plug" myelinated, small-diameter axons so that successful mitochondrial transport is prevented, depleting the distal axon of functioning mitochondria. Our observations may explain the selective vulnerability of small-diameter axons to dysfunction and degeneration in a number of neurodegenerative and neuroinflammatory disorders.

Dataset Information

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

Publications

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

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