Journal of Neurocytology 25, 403-412 (1996) Clusters of axonal Na + channels remyelinating Schwann cells adjacent to SANJA D. NOVAKOVIC 1., THOMAS J. DEERINCK 2, S. ROCK. LEVINSON 3, PETER SHRAGER land MARK H. ELLISMAN 2 1 Department of Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA 2 Department of Neurosciences, University of California San Diego, La Jolla, CA 92093, USA 3 Department of Physiology, University of Colorado Health Sciences Center, Denver, CO 80262, USA Received 25 April 1995; revised 9 February 1996; accepted 8 March 1996 Summary Rat sciatic nerve fibres were demyelinated by injection of lysolecithin and examined at several stages as Schwann cells proliferated, adhered, and initiated remyelination. Immunoperoxidase EM has been used to follow the clustering of Na- channels that represents an early step in the formation of new nodes of Ranvier. At the peak of demyelination, 1 week post- injection, only isolated sites, suggestive of the original nodes, were labelled. As Schwann cells adhered and extended processes along the axons, regions of axonal Na t channel immunoreactivity were often found just beyond their leading edges. These channel aggregates were associated only with the axolemma and Na- channels were not detected on glial membranes. Sites with more than one cluster in close proximity and broadly labelled aggregates between Schwann cells suggested that new nodes of Ranvier formed as neighbouring Na + channel groups merged. Schwann cells thus seem to play a major role in ion channel distributions in the axolemma. In all of these stages Na + channel label was found primarily just outside the region of close contact between axon and Schwann cell. This suggests that Schwann cell adherence acts in part to exclude Na- channels, or that diffusible substances are involved and can act some distance from regions of direct contact. Introduction In the PNS repair processes following demyelination can be rapid and vigorous. Surviving Schwann cells are capable of extensive proliferation, and they adhere to fully demyelinated axons, elaborate myelin, and elongate. During this process new nodes of Ranvier are formed at short spacings within a single former internode. These new nodes have high densities of Na + channels and are functional in conduction at the earliest times at which they can be recognized in the light microscope (Shrager, 1988, 1989; Shrager & Rubinstein, 1990). The factors responsible for deter- mining the sites of nodes formed during remyelination are largely unknown. Observations of freeze-fracture replicas from Schwann cell deficient dystrophic mice revealed node-like patches without nearby Schwann cells, suggesting the possibility of axonal definition of such sites (Ellisman, 1979). Smith and colleagues (1982) then found new foci of inward current at very early stages of recovery in lysolecithin-demyelinated axons and this focused interest on determining the mechanisms through which Na § channels cluster. We have recently investigated several aspects of this process using immunofluorescence directed at an internal epitope of the Na + channel (Dugandzija- Novakovic et al., 1995). The results showed that Schwann cells may play a major role in promoting the aggregation of Na + channels. Specifically, it was found that within one day of Schwann cell adherence Na + channel clusters had formed near the edges of glial processes. Although an interaction between the axon and Schwann cell is clearly indicated for induction of channel clustering, the mechanism of this influence remains obscure. The previous work examined this interaction using light microscopy and, due to limitations in resolution, it was not possible to determine answers to some fundamental questions. Specifically, it could not be ascertained if the Na ~ channel aggregates were in the axolemma or in the Schwann cell membrane. Also, if in the axon, were these clusters at sites of close association of Schwann cell and axon surfaces, or were they just beyond the tip of the Schwann cell process? This last question bears *To whom correspondence should be addressed. 0300-4864/96 ( 1996 Chapman and Hall