Immunolocalization of the Na(+)-Ca2+ exchanger in mammalian myelinated axons

Previous studies on the pathophysiology of white matter anoxic injury have revealed that the Na(+)-Ca2+ exchanger is an important mediator of Ca2+ overload. To date, however, the localization of this key Ca2+ transporter in myelinated axons has not been demonstrated. The present study uses immunofluorescence labeling with a monoclonal antibody (R3F1) to the canine cardiac type I Na(+)-Ca2+ exchanger to localize exchanger protein to rat peripheral and central myelinated axons. The indirect immunofluorescence labeling technique was used to study paraformaldehyde fixed frozen cryostat sections of sciatic nerve, optic nerve and spinal cord. Examination of sciatic nerve sections with both conventional and confocal microscopy revealed a staining pattern which suggested both a glial and axonal localization of the exchanger. In the rat optic nerve, positive label was associated with cell bodies and their processes, likely glia, and with numerous finer processes arranged in parallel, running longitudinally. These finer processes likely represent axonal profiles. A similar staining pattern was observed in lateral and dorsal columns from spinal cord. Immunoelectron microscopy of dorsal root axons revealed gold particles associated with the paranodal and internodal myelin, in the axoplasm, and close to the nodal/paranodal axon membrane. The high density of Na(+)-Ca2+ exchanger demonstrated in central and peripheral myelinated mammalian axons supports the importance of this transporter in Ca2+ regulation in these tissues.