How do myelinated axons signal to the nuclei of cells that enwrap them? The cell bodies of oligodendrocytes and Schwann cells are segregated from axons by multiple layers of bimolecular lipid leaflet and myelin proteins. Conventional signal transduction strategies would seem inadequate to the challenge without special adaptations. Wallerian degeneration provides a model to study axon-to-Schwann cell signaling in the context of nerve injury. We show a hitherto undetected rapid, but transient, activation of the receptor tyrosine kinase erbB2 in myelinating Schwann cells after sciatic nerve axotomy. Deconvolving microscopy using phosphorylation state-specific antibodies shows that erbB2 activation emanates from within the microvilli of Schwann cells, in direct contact with the axons they enwrap. To define the functional role of this transient activation, we used a small molecule antagonist of erbB2 activation (PKI166). The response of myelinating Schwann cells to axotomy is inhibited by PKI166 in vivo . Using...

Action potentials (APs) are electric phenomena that are recorded both intracellularly and extracellularly. APs are usually initiated in the short segment of the axon called the axon initial segment (AIS). It was recently proposed that at the onset of an AP the soma and the AIS form a dipole. We study the extracellular signature [the extracellular AP (EAP)] generated by such a dipole. First, we demonstrate the formation of the dipole and its extracellular signature in detailed morphological models of a reconstructed pyramidal neuron. Then, we study the EAP waveform and its spatial dependence in models with axonal AP initiation and contrast it with the EAP obtained in models with somatic AP initiation. We show that in the models with axonal AP initiation the dipole forms between somatodendritic compartments and the AIS, and not between soma and dendrites as in the classical models. The soma–dendrites dipole is present only in models with somatic AP initiation. Our study has consequences for interpreting extr...

Although axon degeneration is believed to contribute significantly to the permanent functional deficits in diseases such as multiple sclerosis, the extreme variability in sizes and locations of lesions associated with autoimmune diseases has made it difficult to evaluate the effectiveness of interventions intended to prevent such axon loss. In this study we developed a strategy for assessing the degree of axon loss a murine EAE model based on the anatomical convergence of somatosensory sensory axons. EAE was induced in 8-10 week C57BL/6 mice by immunization with recombinant myelin oligodendrocyte glycoprotein (MOG) peptides in complete Freund’s adjuvant and pertussus toxin to enhance immunological responses (Suen et al 1997, J. Exp. Med. 186:1233-1240). Four weeks after initial immunization, mice were perfused and spinal cords processed for standard plastic sectioning. Cross-sectional areas of dorsal columns were measured in 1 µm sections collected every 0.25 mm and axons were then counted at 5 predefined ...

Regeneration of axons in peripheral nerves is impeded by growth inhibitory molecules, including chondroitin sulfate, heparan sulfate, and keratan sulfate proteoglycans. We investigated whether specific enzymatic degradation of these molecules in the environment of regenerating peripheral axons would enhance their outgrowth. Transected common fibular (CF) nerves in Thy-1-YFP-H mice (Feng et al, Neuron, 28: 41-51, 2002) were repaired with 3-5 mm long grafts obtained from the CF nerves of wild-type littermates. The grafts were used to form a dark background against which regenerating axons containing yellow fluorescent protein (YFP) could be studied by fluorescence microscopy. The grafts also served as a vehicle for delivery of treatments at the time of repair with enzymes designed to degrade different proteoglycans in the territory of the regenerating axons. Lengths of individual reconstructed regenerating axon profiles were measured from images of optical sections through grafts treated with chondroitinase,...

Schwann cells (SCs) cover most of the surface of all axons in peripheral nerves. Axons and these glial cells are not only in intimate physical contact but also in constant and dynamic communication, each one influencing and regulating the development, function, and maintenance of the other. In

Preconditioning nerve injuries activate a pro-regenerative program that enhances axon regeneration for most classes of sensory neurons. However, nociceptive sensory neurons and central nervous system neurons regenerate poorly. In hopes of identifying novel mechanisms that promote regeneration, we screened for drugs that mimicked the preconditioning response and identified a nociceptive ligand that activates a preconditioning-like response to promote axon outgrowth. We show that activating the ion channel TRPV1 with capsaicin induces axon outgrowth of cultured dorsal root ganglion (DRG) sensory neurons, and that this effect is blocked in TRPV1 knockout neurons. Regeneration occurs only in NF200-negative nociceptive neurons, consistent with a cell-autonomous mechanism. Moreover, we identify a signaling pathway in which TRPV1 activation leads to calcium influx and protein kinase A (PKA) activation to induce a preconditioning-like response. Finally, capsaicin administration to the mouse sciatic nerve activates...

The hippocampal mossy fibers (MFs) travel within the stratum lucidum close to CA3 stratum pyramidale and form giant synapses with target neurons therein. Because of the difficulty to visualize the MFs, little is known about the mechanisms that guide the MFs to this restricted area. In the present study, we have attempted to solve this problem with an explant coculture system, using transgenic rats expressing green fluorescence protein (GFP(+) rats). When collagen gels were interposed between dentate gyrus (DG) slices prepared from postnatal day 6 (P6) GFP(+) rats and Ammon′s horn slices of wild-type littermates, GFP(+) MFs passed through the gels and reached CA3 stratum lucidum after 10 days in culture, which indicates that the MFs are guided by chemoattractants. However, surprisingly, the MFs could project normally to the host stratum lucidum even when Ammon′s slices were fixed with paraformaldehyde. These results imply that both diffusible and non-diffusible cues are involved in MF pathfinding. To invest...

How synaptic components are deposited at the right place at the right time is unknown. During transport of presynaptic precursors along the developing axon, vesicles pause frequently. Here we test the hypothesis that during synapse formation, delivery of presynaptic proteins to sites of contact is mechanistically similar to the stopping seen during axonal transport. We use primary cultures of postnatal rodent visual cortical neurons and time-lapse confocal imaging of fluorescently-tagged fusion proteins to test this hypothesis. We characterize the spatial distribution of pausing in the axon. Quantitative analysis of the probability of stopping and distributions of stop durations will be presented (n=1075). We then investigate the relationship between pausing and cycling of the transport vesicles with the plasma membrane. We find that synapsins regulate pausing (n=892). Finally, we determine whether stopping is regulated by calcium and phosphorylation pathways (n>5000). We then discuss our results in terms ...

Receptors for the Netrin family of axon guidance ligands in vertebrates include Deleted in Colorectal Cancer (DCC) and Neogenin. These receptors are members of a subgroup of the immunoglobulin superfamily of transmembrane proteins, and both possess four immunoglobulin-like domains, six fibronectin-type III repeats, a single transmembrane region and an intracellular region of ~300 amino acids. Although DCC and its invertebrate homologues have been shown to function in axon guidance as receptors of Netrin, a possible neural function of vertebrate Neogenin has not been elucidated. In the present study, we have examined the expression and role of Neogenin in the embryonic forebrain of Xenopus. In situ hybridisation on Stage 32 Xenopus forebrains revealed that Neogenin mRNA is specifically expressed in tissue surrounding the early axon scaffold. When Neogenin was overexpressed after blastomere injection of synthetic RNA transcripts, forebrains displayed abnormalities in the development of particular axon tracts...

Ephrins and Eph receptors have been implicated in a number of developmental processes including axon growth and guidance. This group of guidance molecules has been characterized as mainly inhibitory in nature, regulating the migration of growth cones through repulsive mechanisms. One of the best identified guidance roles for these molecules is their ability to regulate axonal crossing of the central nervous system (CNS) midline. While a great deal of information is known of a variety of guidance cues that regulate midline guidance, less is known of how ephrins and their receptors integrate these signals to regulate this process. Our studies have examined the role of B-class ephrins and Eph receptors in regulating developmental midline guidance of corpus callosum (CC) axons. Our results indicate that ephrins and Eph receptors are expressed in the developing fiber tract and extracellular environment at embryonic day (ED) 15, 16, 17, and 18, thus present to play a role in the guidance of these axons. Our prel...