Action potential conduction velocity increases dramatically during early development as axons become myelinated. Integral to this process is the clustering of voltage-gated Na+ (Nav) channels at regularly spaced gaps in the myelin sheath called nodes of Ranvier. We show here that some aspects of peripheral node of Ranvier formation are distinct from node formation in the CNS. For example, at CNS nodes, Nav1.2 channels are detected first, but are then replaced by Nav1.6. Similarly, during remyelination in the CNS, Nav1.2 channels are detected at newly forming nodes. By contrast, the earliest Nav-channel clusters detected during developmental myelination in the PNS have Nav1.6. Further, during PNS remyelination, Nav1.6 is detected at new nodes. Finally, we show that accumulation of the cell adhesion molecule neurofascin always precedes Nav channel clustering in the PNS. In most cases axonal neurofascin (NF-186) accumulates first, but occasionally paranodal neurofascin is detected first. We suggest there is heterogeneity in the events leading to Nav channel clustering, indicating that multiple mechanisms might contribute to node of Ranvier formation in the PNS.

Various animal models are available for studying human multiple sclerosis (MS). Most of them model the initial phase of MS, including the immune-triggered attack of the myelin membrane and/or oligodendrocytes and, occasionally, demonstrate the remission and relapsing phases. However, few mimic the late chronic demyelinating phase. Overexpression of the proteolipid protein gene (Plp) causes a unique demyelinating disorder in mice in which normal-appearing myelin forms early in life and chronic demyelination occurs later. We found that remyelination is severely affected in this late demyelinating phase, but is not caused by deprivation of oligodendrocyte progenitors expressing PDGF receptor alpha (PDGFRα) and Olig2, which are present at an even higher number in the demyelinated white matter of the mutants than in wild-type controls. Furthermore, mature oligodendrocytes containing PLP were observed, but failed to remyelinate. The ability of oligodendrocytes from older transgenic animals to produce a myelin membrane-like structure was not impaired when cultured in vitro, which indicates that the lack of remyelination is not simply caused by changes in the intrinsic properties of the oligodendrocytes. Glial activation also occurred much earlier than active demyelination in mutant mice. Thus, in addition to intrinsic mechanisms, extrinsic mechanisms might also have an important role in defects of remyelination. These features are also observed in patients at a late stage of MS, leading to chronic demyelinating lesions. Thus, this mouse model partly mimics the late stage of MS and can be used to study the cause of inhibition of remyelination.

The extension and directionality of neurite outgrowth are key to achieving successful target connections during both CNS development and during the re-establishment of connections lost after neural trauma. The degree of axonal elongation depends, in large part, on the spatial arrangement of astrocytic processes rich in growth-promoting proteins. Because astrocytes in culture align their processes on exposure to an electrical field of physiological strength, we sought to determine the extent to which aligned astrocytes affect neurite outgrowth. To this end, dorsal root ganglia cells were seeded onto cultured rat astrocytes that were pre-aligned by exposure to an electric field of physiological strength (500 mV mm−1). Using confocal microscopy and digital image analysis, we found that neurite outgrowth at 24 hours and at 48 hours is enhanced significantly and directed consistently along the aligned astrocyte processes. Moreover, this directed neurite outgrowth is maintained when grown on fixed, aligned astrocytes. Collectively, these results indicate that endogenous electric fields present within the developing CNS might act to align astrocyte processes, which can promote and direct neurite growth. Furthermore, these results demonstrate a simple method to produce an aligned cellular substrate, which might be used to direct regenerating neurites.

Live-cell imaging of glioblastoma U373 and U87 cells transfected with actin cytoskeleton markers has been used to study the re-arrangements that are associated with migration in two- and three-dimensional matrices and in brain tissue. In collagen gels and in brain slices, both cell types developed neuronal-like processes with ruffling membranes and filopodia. Blebbing cells were also observed, but these were mainly immobile. The retraction of trailing cell processes in a tissue environment was associated with the transient development and contraction of bundles of axial stress fibers. The inhibition of Rho-kinase caused glioblastoma cells in brain slices to become immobile and develop neurite-like processes at random, which indicates the requirement of Rho signaling and contractility for migration. Actin stress fibers were also observed in glioblastoma cells injected into the brains of living mice. Thus, invading glioblastoma cells use neurite-like extensions to penetrate between neuronal fibers and contractile actin bundles for traction of the cell body.

Neurostatin, a modified ganglioside in mammalian brain, is highly immunogenic. Fusion of spleen cells of Balb/C mice immunized with bovine neurostatin with SP2O myeloma cells, led to hybridoma producers of anti-neurostatin antibodies. Two, monoclonal, IgG secreters were selected that recognized the inhibitor in blots and tissue of cow, rat and pig. The lack of species specificity indicated that the molecular structure of the inhibitory epitope is similar in these mammals. That epitope was also present in other gangliosides of the b series, defining a new regulatory system of glial-cell proliferation and apoptosis. The antibodies further revealed the neuronal origin of neurostatin.

We provide both molecular and pharmacological evidence that the metabotropic, purinergic, P2Y6, P2Y12 and P2Y13 receptors and the ionotropic P2X4 receptor contribute strongly to the rapid calcium response caused by ATP and its analogues in mouse microglia. Real-time PCR demonstrates that the most prevalent P2 receptor in microglia is P2Y6 followed, in order, by P2X4, P2Y12, and P2X7 = P2Y13. Only very small quantities of mRNA for P2Y1, P2Y2, P2Y4, P2Y14, P2X3 and P2X5 were found. Dose-response curves of the rapid calcium response gave a potency order of: 2MeSADP>ADP=UDP=IDP=UTP>ATP>BzATP, whereas A2P4 had little effect. Pertussis toxin partially blocked responses to 2MeSADP, ADP and UDP. The P2X4 antagonist suramin, but not PPADS, significantly blocked responses to ATP. These data indicate that P2Y6, P2Y12, P2Y13 and P2X receptors mediate much of the rapid calcium responses and shape changes in microglia to low concentrations of ATP, presumably at least partly because ATP is rapidly hydrolyzed to ADP. Expression of P2Y6, P2Y12 and P2Y13 receptors appears to be largely glial in the brain, so that peripheral immune cells and CNS microglia share these receptors. Thus, purinergic, metabotropic, P2Y6, P2Y12, P2Y13 and P2X4 receptors might share a role in the activation and recruitment of microglia in the brain and spinal cord by widely varying stimuli that cause the release of ATP, including infection, injury and degeneration in the CNS, and peripheral tissue injury and inflammation which is signaled via nerve signaling to the spinal cord.

Early regeneration of injured peripheral nerves involves a series of events that are important in the success of eventual reconnection. In many nerve injuries, such as transections with gaps, axons and Schwann cells (SCs) penetrate into new microenvironments de novo, not involving zones of Wallerian degeneration. We studied unexplored axon–SC interactions by sampling of newly forming connections through a silicone conduit across transected rat sciatic peripheral nerve gaps. Axon and SC participation in bridge formation was addressed by light microscopy, electron microscopy and by double-labeling immunohistochemistry, including confocal imaging, and several, less appreciated aspects of early regrowth were identified. There are limitations to early and widespread regeneration of axons and SCs into bridges initially formed from connective tissue and blood vessels. Regrowth is ‘staggered’ such that only a small percentage of parent axons sampled the early bridge. There is an intimate, almost invariable relationship between SCs and extension of axons, which challenges the concept that axons lead and SCs follow. ‘Naked’ axons were infrequent and limited in scope. Axons did not seek out and adhere to vascular laminin but intimately followed laminin deposits associated with apposed SCs. Growth cones identified by labeling of β III tubulin, PGP 9.5 and GAP43/B50 were complex, implying a pause in their regrowth, and were most prominent at the proximal stump–regenerative bridge interface. There is surprising and substantial hostility to local regrowth of axons into newly forming peripheral nerve bridges. Early axon outgrowth, associated with apposed Schwann cell processes, is highly constrained even when not exposed to adjacent myelin and products of Wallerian degeneration.