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Olfactory ensheathing cells (OEC), which normally associate closely with but do not myelinate axons in situ, myelinate axons in the adult mammalian spinal cord. They are of clinical interest as candidate cells for autologous transplantation but the ability of OEC to myelinate axons in vitro has been controversial. To clarify this issue, we isolated OEC from olfactory bulbs (OB) of juvenile and adult rats expressing GFP and analyzed their ability to myelinate axons. Using a well-defined assay for myelination of dorsal root ganglia (DRG) axons in culture, we found that OEC from juvenile pups associated with and then myelinated DRG axons. OEC assembled into bundles with the axons by 1week and required more than a week before myelination on axons was detected. In contrast, rat Schwann cells did not bundle axons and they formed P0(+) and MBP(+) myelin segments after as little as 1week. Most of the OEC in culture exhibited staining for calponin, a marker that was not found on Schwann cells in culture, whereas in both OEC and Schwann cell populations nearly all cells were positive for p75NTR and GFAP. These results confirm previous reports showing only subtle immunological differences between Schwann cells and OEC. Besides differences in the rate of myelination, we detected two additional functional differences in the interactions of OEC and Schwann cells with DRG axons. First, the diameter of OEC generated myelin was greater than for Schwann cell myelin on DRG axons. Second, OEC but not Schwann cells myelinated DRG axons in the absence of vitamin C. OEC isolated from adult OB were also found to bundle and myelinate DRG axons but the latter occurred only after incubation times of at least 3weeks. The results indicate that adult OEC require longer incubation times than juvenile OEC to myelinate axons and suggest that patterns of myelination by OEC and Schwann cells are distinguishable at least on axons in vitro. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair., (Copyright © 2010 Elsevier Inc. All rights reserved.)

NrCAM is a neural cell adhesion molecule of the L1 family that has been linked to autism spectrum disorders, a disease spectrum in which abnormal thalamocortical connectivity may contribute to visual processing defects. Here we show that NrCAM interaction with neuropilin-2 (Npn-2) is critical for semaphorin 3F (Sema3F)-induced guidance of thalamocortical axon subpopulations at the ventral telencephalon (VTe), an intermediate target for thalamic axon sorting. Genetic deletion of NrCAM or Npn-2 caused contingents of embryonic thalamic axons to misproject caudally in the VTe. The resultant thalamocortical map of NrCAM-null mutants showed striking mistargeting of motor and somatosensory thalamic axon contingents to the primary visual cortex, but retinogeniculate targeting and segregation were normal. NrCAM formed a molecular complex with Npn-2 in brain and neural cells, and was required for Sema3F-induced growth cone collapse in thalamic neuron cultures, consistent with a vital function for NrCAM in Sema3F-induced axon repulsion. NrCAM-null mice displayed reduced responses to visual evoked potentials recorded from layer IV in the binocular zone of primary visual cortex (V1), particularly when evoked from the ipsilateral eye, indicating abnormal visual acuity and ocularity. These results demonstrate that NrCAM is required for normal maturation of cortical visual acuity, and suggest that the aberrant projection of thalamic motor and somatosensory axons to the visual cortex in NrCAM-null mutant mice impairs cortical functions.

Saltatory conduction requires high-density accumulation of Na(+) channels at the nodes of Ranvier. Nodal Na(+) channel clustering in the peripheral nervous system is regulated by myelinating Schwann cells through unknown mechanisms. During development, Na(+) channels are first clustered at heminodes that border each myelin segment, and later in the mature nodes that are formed by the fusion of two heminodes. Here, we show that initial clustering of Na(+) channels at heminodes requires glial NrCAM and gliomedin, as well as their axonal receptor neurofascin 186 (NF186). We further demonstrate that heminodal clustering coincides with a second, paranodal junction (PNJ)-dependent mechanism that allows Na(+) channels to accumulate at mature nodes by restricting their distribution between two growing myelin internodes. We propose that Schwann cells assemble the nodes of Ranvier by capturing Na(+) channels at heminodes and by constraining their distribution to the nodal gap. Together, these two cooperating mechanisms ensure fast and efficient conduction in myelinated nerves.

Chondroitin sulfate proteoglycans (CSPGs) inhibit axonal growth, and treatment with chondroitinase ABC promotes axonal regeneration in some models of central nervous system (CNS) injury. The aims of this study were (1) to compare the spatiotemporal appearance of CSPG expression between spinal cord contusion and hemisection models, and (2) to evaluate chondroitinase treatment effects on axonal regrowth in the two injury models. After hemisection, CSPG-immunoreactivity (IR) in the injury site rose to peak levels at 18 days but then decreased dramatically by 49 days; in contrast, CSPG-IR remained high for at least 49 days after contusion. After hemisection, many anterogradely labeled corticospinal tract (CST) axons remained close to CSPG-rich lesion sites, but after contusion, most CST axons retracted by approximately 1 mm rostral from the rostral-most CSPG-rich cyst. Intraspinal injection of chondroitinase at 0, 1, 2, and 4 weeks following injury dramatically reduced CSPG-IR in both injury models within 4 days, and CSPG-IR remained low for at least 3 weeks. After the chondroitinase treatment, many axons grew around the lesion site in hemisected spinal cords but not in contused spinal cords. We propose that improved axonal growth in hemisected spinal cords is due to decreased inhibition resulting from degradation of CSPGs located adjacent to severed CST axons. However, in spinal cord contusions, retracted CST axons fail to grow across gliotic regions that surround CSPG-rich injury sites despite efficient degradation with chondroitinase, suggesting that other inhibitors of axonal growth persist in the gliotic regions.

Axon initial segments (AISs) and nodes of Ranvier are sites of action potential generation and propagation, respectively. Both domains are enriched in sodium channels complexed with adhesion molecules (neurofascin [NF] 186 and NrCAM) and cytoskeletal proteins (ankyrin G and betaIV spectrin). We show that the AIS and peripheral nervous system (PNS) nodes both require ankyrin G but assemble by distinct mechanisms. The AIS is intrinsically specified; it forms independent of NF186, which is targeted to this site via intracellular interactions that require ankyrin G. In contrast, NF186 is targeted to the node, and independently cleared from the internode, by interactions of its ectodomain with myelinating Schwann cells. NF186 is critical for and initiates PNS node assembly by recruiting ankyrin G, which is required for the localization of sodium channels and the entire nodal complex. Thus, initial segments assemble from the inside out driven by the intrinsic accumulation of ankyrin G, whereas PNS nodes assemble from the outside in, specified by Schwann cells, which direct the NF186-dependent recruitment of ankyrin G.

Severed axons of the inferior colliculus (IC) commissure can regenerate across a lesion in organotypic cultures from postnatal day (P) 6 gerbils, but this regenerative capacity is lost by P12 (Hafidi et al. [ 1995] J Neurosci 15:1298-1307, [1999] J Neurobiol 41:267-280). In the present study, we examined the mechanisms underlying this age-dependent failure of axons to regenerate. In P6-P12 heterochronic cultures, the P12 axons failed to cross the lesion site and project to the contralateral P6 IC lobe. In contrast, axons originating from the P6 lobe could regenerate through the lesion and invade the contralateral P12 IC lobe. To determine whether this age-dependent change in regenerative capacity can develop in organotypic cultures, IC slices with an intact commissure were obtained from P6 animals, grown in vitro for 6 days, and then lesioned at the commissure. In these slices, axon regeneration failure was similar to that observed in normal P12 tissue. Several in vitro treatments enhanced axon regeneration: removal of the entire midline region, inhibition of protein synthesis at the lesion site, and exposure to ABC chondroitinase. Furthermore, when the injured commissural axons were provided with a carpet of C6-R cells (a radial glia-like cell line), significantly more axons projected to the contralateral lobe of the IC. Taken together, these results suggest that the maturation of nonneuronal cells within the lesion site lead to failed axon regeneration in mature animals, and show that ameliorative strategies can be evaluated in vitro., (Copyright 2004 Wiley-Liss, Inc.)

Specialized cells at the midline of the central nervous system have been implicated in controlling axon projections in both invertebrates and vertebrates. To address the requirement for ventral midline cells in providing cues to commissural axons in mice, we have analyzed Gli2 mouse mutants, which lack specifically the floor plate and immediately adjacent interneurons. We show that a Dbx1 enhancer drives tau-lacZ expression in a subpopulation of commissural axons and, using a reporter line generated from this construct, as well as DiI tracing, we find that commissural axons projected to the ventral midline in Gli2(-/-) embryos. Netrin1 mRNA expression was detected in Gli2(-/-) embryos and, although much weaker than in wild-type embryos, was found in a dorsally decreasing gradient. This result demonstrates that while the floor plate can serve as a source of long-range cues for C-axons in vitro, it is not required in vivo for the guidance of commissural axons to the ventral midline in the mouse spinal cord. After reaching the ventral midline, most commissural axons remained clustered in Gli2(-/-) embryos, although some were able to extend longitudinally. Interestingly, some of the longitudinally projecting axons in Gli2(-/-) embryos extended caudally and others rostrally at the ventral midline, in contrast to normal embryos in which virtually all commissural axons turn rostrally after crossing the midline. This finding indicates a critical role for ventral midline cells in regulating the rostral polarity choice made by commissural axons after they cross the midline. In addition, we provide evidence that interactions between commissural axons and floor plate cells are required to modulate the localization of Nr-CAM and TAG-1 proteins on axons at the midline. Finally, we show that the floor plate is not required for the early trajectory of motoneurons or axons of the posterior commissure, whose projections are directed away from the ventral midline in both WT and Gli2(-/-) embryos, although they are less well organized in Gli2(-/-)mutants.

Nr-CAM is a neuronal cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily that has been implicated as a ligand for another CAM, axonin-1, in guidance of commissural axons across the floor plate in the spinal cord. Nr-CAM also serves as a neuronal receptor for several other cell surface molecules, but its role as a ligand in neurite outgrowth is poorly understood. We studied this problem using a chimeric Fc-fusion protein of the extracellular region of Nr-CAM (Nr-Fc) and investigated potential neuronal receptors in the developing peripheral nervous system. A recombinant Nr-CAM-Fc fusion protein, containing all six Ig domains and the first two fibronectin type III repeats of the extracellular region of Nr-CAM, retains cellular and molecular binding activities of the native protein. Injection of Nr-Fc into the central canal of the developing chick spinal cord in ovo resulted in guidance errors for commissural axons in the vicinity of the floor plate. This effect is similar to that resulting from treatment with antibodies against axonin-1, confirming that axonin-1/Nr-CAM interactions are important for guidance of commissural axons through a spatially and temporally restricted Nr-CAM positive domain in the ventral spinal cord. When tested as a substrate, Nr-Fc induced robust neurite outgrowth from dorsal root ganglion and sympathetic ganglion neurons, but it was not effective for tectal and forebrain neurons. The peripheral but not the central neurons expressed high levels of axonin-1 both in vitro and in vivo. Moreover, antibodies against axonin-1 inhibited Nr-Fc-induced neurite outgrowth, indicating that axonin-1 is a neuronal receptor for Nr-CAM on these peripheral ganglion neurons. The results demonstrate a role for Nr-CAM as a ligand in axon growth by a mechanism involving axonin-1 as a neuronal receptor and suggest that dynamic changes in Nr-CAM expression can modulate axonal growth and guidance during development., (Copyright 1999 Academic Press.)

Receptor-type protein tyrosine phosphatase beta (RPTP beta) is expressed in the developing nervous system and contains a carbonic anhydrase (CAH) domain as well as a fibronectin type III repeat in its extracellular domain. Fusion proteins containing these domains were used to search for ligands of RPTP beta. The CAH domain bound specifically to a 140 kDa protein expressed on the surface of neuronal cells. Expression cloning in COS7 cells revealed that this protein is contactin, a GPI membrane-anchored neuronal cell recognition molecule. The CAH domain of RPTP beta induced cell adhesion and neurite growth of primary tectal neurons, and differentiation of neuroblastoma cells. These responses were blocked by antibodies against contactin, demonstrating that contactin is a neuronal receptor for RPTP beta. These experiments show that an individual domain of RPTP beta acts as a functional ligand for the neuronal receptor contactin. The interaction between contactin and RPTP beta may generate unidirectional or bidirectional signals during neural development.

To elucidate the role of neuron-glia cell adhesion molecule (Ng-CAM) in axonal pathway formation of avian spinal interneurons, we have examined the ultrastructural expression of Ng-CAM in the developing spinal cord, by using a preembedding immunocytochemical method. Ng-CAM immunoreactivity was punctate and was restricted to cell surfaces. In accordance with our previous light microscopic observations (Shiga et al., '90), the earliest developing spinal interneurons were Ng-CAM-positive on their cell bodies, axons, and growth cones. Axons and growth cones that were either fasciculated or in contact with each other strongly expressed Ng-CAM, thus indicating the possible involvement of Ng-CAM in fasciculation of axons and in the contact guidance of growth cones along preexisting axons. By using higher resolution immunoelectron microscopy, the present study has also revealed new information on the subcellular localization of Ng-CAM on developing spinal interneurons, neuroepithelial cells, and floor plate cells. Although Ng-CAM immunoreactivity was prominent on both axons and growth cones, these structures were Ng-CAM-negative when they contacted the basal lamina around the spinal cord. By contrast, Ng-CAM was detectable on the surface of both neuroepithelial cells and floor plate cells only when they made contact with the Ng-CAM-positive axons and growth cones of interneurons. These results suggest that the subcellular distribution of Ng-CAM is regulated differentially, depending on the apposing cell surfaces, and that such differential and developmentally regulated expression may contribute to the elongation, fasciculation, and guidance of spinal axons.