The Specificity of 130-kDa Leech Sensory Afferent Proteins Is Encoded by Their Carbohydrate Epitopes

From early development through adulthood in the leech, sensory afferents, glial cells, and connective tissue express different epitopes located on a group of 130-kDa glycoproteins. The sensory epitope [reactive with monoclonal antibody (mAb) Lan3-2] is shared by the peripheral sensory afferents of different sensory modalities. In contrast, three other immunocytochemically distinct epitopes (reactive with mAbs Laz2-369, Laz7-79, and Laz6-212) differentiate these sensory afferents according to their sensory modalities. The glial epitope (mAb Laz6-297) is expressed on all macroglial processes, and the connective tissue epitope (mAb Laz9-84) is located on connective tissue surrounding the CNS, as well as in the peripheral tissues. The hydrophilic-hydrophobic nature of the 130-kDa sensory afferent and glial proteins was determined by phase separation with Triton X-114 and hypoosmotic extraction. They behave as peripheral membrane proteins. Deglycosylation of 130-kDa glycoproteins with N-Glycanase or preincubation of their respective mAbs with alpha-methylmannoside showed that the sensory epitope contains mannose, whereas the modality epitopes are of an undefined carbohydrate character. Immunoprecipitation and a peptide mapping experiment confirmed the existence of four distinct sensory afferent epitopes. Previous studies provided evidence that the mannose-containing Lan3-2 epitope mediates normal sensory afferent growth in the synaptic neuropile. We, therefore, postulate that the carbohydrate epitopes on sensory afferent glycoproteins participate in synapse formation.     

Specific pathway selection by the early projections of individual peripheral sensory neurons in the embryonic medicinal leech

In leech, the central annulus of each midbody segment possesses seven pairs of sensilla, which are mixed clusters of primary peripheral sensory neurons that extend their axons into the CNS where they segregate into distinct fascicles. Pathway selection by individual afferent growth cones of sensillar neurons was examined by double labeling using intracellular dye-filling with anitobody labeling in early Hirudo medicinalis embryos. The monoclonal antibody Lan3–2 was used because sensillar neuronal tracts are specifically labeled by this antibody. Examining 68 individually filled neurons we found that sensillar neuron growth cones bifurcate within the CNS, that they project long filopodia capable to sampling the local environment, and that all of them appeared to choose a single particular CNS fascicle without apparent retraction or realignment of growth cones. Furthermore, each side of the bifurcating afferent growth cones always chose the same fascicle, implying a specific choice of a distinct labeled pathway. By dye-filling individual central neurons (P-cells), we show that there are centrally projecting axons present at the time sensillar afferents enter the ganglionic primordia and select a particular fascicle, and we confirm that at least the dorsal peripheral nerve is likely to be pioneered by central neurons, not by the peripheral afferent. In the sensillum studied here, we sound examples of sensory neurons extending axons into one of all the avilable fascicles. Thus, an individual embryonic sensillum possesses a heterogeneous population of afferents with respect to the central fascicle chosen. This is consistent with the idea that segregation into distinct axon fascicles may be based upon functional differences between individual afferent neurons. Our findings argue strongly in favor of specific pathway selection by afferents in this system and are consistent with previous suggestions that there exists a hierarchy of cues, including surface glycoconjugates that mediate navigation of the sensillar growth cones and the fasciculation of their axons. 1994 John Wiley & Sons, Inc.     

Tract formation and axon fasciculation of molecularly distinct peripheral neuron subpopulations during leech embryogenesis.

In leech, the central projections of peripheral sensory neurons segregate into specific axonal tracts, which are distinguished by differential expression of surface antigens recognized by the monoclonal antibodies Lan3-2 and Lan4-2. Lan3-2 recognizes an epitope expressed on axons that segregate into three distinct axon fascicles. In contrast, the Lan4-2-positive axons selectively project into only one of the Lan3-2-positive axon tracts. These observations provide evidence for a hierarchy of guidance cues mediating specific pathway formation in this system. Since the Lan3-2 antibody has been shown to perturb this process and since, as shown here, the Lan3-2 and Lan4-2 antigens are closely molecularly interrelated, these antibodies may help define molecules and epitopes mediating neuronal recognition and axonal guidance.     

Phylogenetic conservation of the cell-type-specific Lan3-2 glycoepitope in Caenorhabditis elegans

The biological function of a cell-type-specific glycosylation of an adhesion molecule belonging to the L1CAM immunoglobulin superfamily was previously determined in the nervous system of the embryonic leech, Hirudo medicinalis. The Lan3-2 glycoepitope is a surface marker of sensory afferent neurons and is required for their appropriate developmental collateral branching and synaptogenesis in the CNS. The chemical structure of the Lan3-2 glycoepitope consists of β-(1,4)-linked mannopyranose. Here, we show the conservation of the cell-type-specific expression of this mannose polymer in Caenorhabditis elegans. The Lan3-2 glycoepitope is expressed on the cell surface of a subset of dissociated embryonic neurons and, in the adult worm, by the pharyngeal motor neuron, M5, and the chemosensory afferents, the amphids. Additionally, the vulval epithelium expresses the Lan3-2 glycoepitope in late L4 larvae and in adult hermaphrodites. To investigate proteins carrying this restrictively expressed glycoepitope, worm extract was immunoaffinity purified with Lan3-2 monoclonal antibody and Western blotted. A polyclonal antibody reactive with the cytoplasmic tail of LAD-1/SAX-7, a C. elegans member of the L1CAM family, recognizes a 270 kDa protein band while Lan3-2 antibody also recognizes a 190 kDa glycoform, its putative Lan3-2 ectodomain. Thus, in C. elegans, as in leech, the Lan3-2 epitope is located on a L1CAM homologue. The cell-type-specific expression of the Lan3-2 glycoepitope shared by leech and C. elegans will be useful for understanding how cell-type-specific glycoepitopes mediate cell–cell interactions during development.     

Regeneration and asexual reproduction share common molecular changes: upregulation of a neural glycoepitope during morphallaxis in Lumbriculus

Neural morphallaxis is a regenerative process characterized by wide-spread anatomical and physiological changes in an adult nervous system. During segmental regeneration of the annelid worm, Lumbriculus variegatus, neural morphallaxis involved a reorganization of sensory, interneuronal, and motor systems as posterior fragments gained a more anterior body position. A monoclonal antibody, Lan 3-2, which labels a neural glyco-domain in the leech, was reactive in Lumbriculus. In the worm, this antibody labeled neural structures, particularly axonal tracts and giant fiber pathways of the central nervous system. A 60kDa protein, possessing a lumbriculid mannose-rich glycoepitope, was upregulated during neural morphallaxis, peaking in its expression at 3 weeks post-amputation. Peak upregulation of the Lan 3-2 epitope, or the protein possessing it, corresponded to the time of major neurobehavioral plasticity during regeneration. Analyses of asexually reproducing animals also revealed induction of the Lan 3-2 epitope. In this developmental context, Lan 3-2 epitope upregulation was also confined to segments expressing both changes in positional identity and neurobehavioral plasticity, but these molecular and behavioral changes occurred prior to body fragmentation. These results suggest that the lumbriculid Lan 3-2 glycoepitope and proteins that bear them have been co-opted for neural morphallactic programs, induced both in anticipation of reproductive fragmentation and in compensation for injury-induced fragmentation.     

In vivo dynamics of CNS sensory arbor formation: a time-lapse study in the embryonic leech

In the embryo of the leech Hirudo medicinalis, afferent projections of peripheral sensory neurons travel along common nerve tracts to the CNS, where they defasciculate, branch, and arborize into separate, modality-specific synaptic laminae. Previous studies have shown that this process requires, at least in part, the constitutive and then modality-specific glycosylations of tractin, a leech L1 homologue. We report here on the dynamics of growth of these projections as obtained by examining the morphology of single growing dye-filled sensory afferents as a function of time. Using 2-photon laser-scanning microscopy of the intact developing embryo, we obtained images of individual sensory projections at 3 to 30 min intervals, over several hours of growth, and at different stages of development. The time-lapse series of images revealed a highly dynamic and maturation-state-dependent pattern of growth. Upon entering the CNS, the growth cone-tipped primary axon sprouted numerous long filopodial processes, many of which appeared to undergo repeated cycles of extension and retraction. The growth cone was transformed into a sensory arbor through the formation of secondary branches that extended within the ganglionic neuropil along the anterior-posterior axis of the CNS. Numerous tertiary and quaternary processes grew from these branches and also displayed cycles of extension and retraction. The motility of these higher-order branches changed with age, with younger afferents displaying higher densities and greater motility than older, more mature sensory arbors. Finally, coincident with a reduction in higher order projections was the appearance of concavolar structures on the secondary processes. Rows of these indentations suggest the formation of presynaptic en-passant specializations accompanying the developmental onset of synapse formation.     

Netrin signal is produced in leech embryos by segmentally iterated sets of central neurons and longitudinal muscle cells

Abstract. Netrins are secreted molecules capable of attracting or repelling growing axons. They and their receptors, along with other netrin-interacting proteins, are widely conserved among animals from a broad range of phyla. We have raised and purified an antibody against a recently cloned leech netrin, which has allowed us to characterize embryonic netrin expression by cells in peripheral tissues and in the central nervous system. During early gangliogenesis, netrin expression was detected at particularly high levels in five bilateral pairs of central neurons. Towards the end of the period of axonal outgrowth, netrin expression was observed to be restricted to only six central neurons, comprising two bilateral pairs and two unpaired cells. A pair of netrin-producing central neurons, the bipolar cells, was identified by their expression of the antigen recognized by the monoclonal antibody Laz1-1. Double staining of sensory afferents from segmental sensilla with the monoclonal antibody Lan3-2 and the bipolar cells with the netrin antibody revealed that the terminals of these afferents grow up to the bipolar cells and turn anteriorly or posteriorly, without extending any further medially. Peripheral netrin expression was found to be restricted to longitudinal muscle cells in the ventral half of the body wall. Extracellular, secreted netrin was detected in a broad longitudinal stripe located symmetrically with respect to the ventral midline. The pattern of expression of netrin in leech embryos is consistent with observed expression patterns in other animals, suggesting that developmental netrin functions are conserved among all bilateral animals.     

In vivo dynamics of CNS sensory arbor formation: A time‐lapse study in the embryonic leech

In the embryo of the leech Hirudo medicinalis, afferent projections of peripheral sensory neurons travel along common nerve tracts to the CNS, where they defasciculate, branch, and arborize into separate, modality‐specific synaptic laminae. Previous studies have shown that this process requires, at least in part, the constitutive and then modality‐specific glycosylations of tractin, a leech L1 homologue. We report here on the dynamics of growth of these projections as obtained by examining the morphology of single growing dye‐filled sensory afferents as a function of time. Using 2‐photon laser‐scanning microscopy of the intact developing embryo, we obtained images of individual sensory projections at 3 to 30 min intervals, over several hours of growth, and at different stages of development. The time‐lapse series of images revealed a highly dynamic and maturation‐state‐dependent pattern of growth. Upon entering the CNS, the growth cone‐tipped primary axon sprouted numerous long filopodial processes, many of which appeared to undergo repeated cycles of extension and retraction. The growth cone was transformed into a sensory arbor through the formation of secondary branches that extended within the ganglionic neuropil along the anterior‐posterior axis of the CNS. Numerous tertiary and quaternary processes grew from these branches and also displayed cycles of extension and retraction. The motility of these higher‐order branches changed with age, with younger afferents displaying higher densities and greater motility than older, more mature sensory arbors. Finally, coincident with a reduction in higher order projections was the appearance of concavolar structures on the secondary processes. Rows of these indentations suggest the formation of presynaptic en‐passant specializations accompanying the developmental onset of synapse formation. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 41–53, 2003     

Ectopic CNS projections guide peripheral neuron axons along novel pathways in leech embryos

Previous studies have indicated that the formation of stereotyped segmental nerves in leech embryos depends on the interactions between CNS projections and ingrowing afferents from peripheral neurons. Especially, CNS-ablation experiments have suggested that CNS-derived guidance cues are required for the correct navigation of several groups of peripheral sensory neurons. In order to directly test this hypothesis we have performed transplantations of CNS ganglia into ectopic sites in segments from which the resident ganglia have been removed. We find that the transplanted ganglia extend numerous axons distributed roughly equally in all directions. When these CNS projections reach and make contact with peripheral sensory axons they are used as guides for peripheral neurons to grow toward and into the ectopic ganglia even when this means following novel pathways that cross the midline and/or segmental boundaries. The peripheral sensory axons turn and grow toward the ectopic ganglia only when in physical contact with CNS axons, suggesting that diffusible chemoattractants are not a factor. These results demonstrate that the guidance cues provided by ectopic CNS projections are both necessary and sufficient to steer peripheral sensory neuron axons into the CNS.     

Multiple strategies for directed growth cone extension and navigation of peripheral neurons

Leeches have a diverse constellation of peripheral neural elements that are challenged to extend growth cones in highly specific ways in a constantly changing embryonic environment. Two major systems are reviewed here. In one, peripheral afferents extend growth cones toward the central nervous system (CNS), forming common pathways, and then segregate into particular tracts within the CNS. A majority of these afferents depend on CNS-derived guidance cues and projections from the CNS to guide their way. However, not all of the nerves are established this way and at least one of the peripheral nerves is likely to be pioneered by sensillar sensory afferents. The distribution of particular antigens (such as the lan3–2 antigen) suggests the identity of molecules involved in homophilic adhesion along common pathways, whereas others (such as the lan4–2 and 3–6 antigens) are candidates for mediating specific pathway choices. In the second system, the myo-organizing Comb cell (C cell) projects multiple growth cones simultaneously along oblique trajectories not influenced by segmental or midline boundaries. Its parallel growth cones exhibit space-filling as well as directional growth and are guided by local cues to extend in discrete phases that are coordinated with the development of the environment. Both systems exhibit highly directed outgrowth orchestrated by a hierarchy of cues, establish patterns of neurites used to direct later migrating cells, and seem to be regulated temporally and spatially by interactions with the embryonic environment. These systems illustrate the strengths of examining neural development in vivo across several levels of analysis. © 1995 John Wiley & Sons, Inc.     

Development of wing sensory axons in the central nervous system of Drosophila during metamorphosis

The development of new, adult-specific axonal pathways in the central nervous system (CNS) of insects during metamorphosis is still largely uncharacterized. Here we used axonal labeling with DiI to describe the timing and pattern of growth of sensory axons originating in the wing of Drosophila as they establish their adult projection pattern in the CNS during pupal life. The wing of Drosophila carries a small number of readily identifiable sensory organs (sensilla) whose neurons are located in the periphery and whose axons travel along specific routes within the adult CNS. The neurons are born and undergo axonogenesis in a characteristic order. The order of axon arrival in the CNS appears to be the same as that of their development in the periphery. Within the CNS, the formation of four prominent axon bundles leading to distant termination sites is followed by the formation of a compact axon termination site near the point of wing nerve entry into the CNS. This sensillum-specific pattern persists into adulthood without discernible modification. We also find a small number of axons filled with DiI prior to the formation of the four permanent bundles. We have only been able to fill them for a few hours in early pupal life and therefore consider them to be transient. The bundles of wing sensory axons travel within tracts that contain other axons as well. Using immunocytochemistry, the tracts start to be histologically identifiable at around 12 h after pupariation (AP), and grow substantially as metamorphosis proceeds. Wing sensory neurons are found in the tracts by 18–20 h AP and the full adult pattern is established by 48 h AP. When sensory axons first enter the CNS, they fan out in the region where their appropriate tracts are located, but they do not wander extensively. They quickly form bundles that become increasingly compact over time. Calculations show that the rate of axon extension within the CNS varies from bundle to bundle and is equal to or greater than that of the same axons growing through wing tissue. © 1995 John Wiley & Sons, Inc.     

Sequential steps in axonal targeting are mediated by carbohydrate markers

Mannose and hybrid/complex-type oligosaccharides serve as markers for both the full set of peripheral sensory afferent neurons in the leech and also for disjoint subsets of these neurons. We have shown that these various surface carbohydrates play crucial roles in the multistep process by which afferents meet their synaptic parterns in the central nervous system (CNS). The carbohydrate marker common to all these afferents allows their projections (which are fasciculated as they enter the CNS) to disperse and search out target regions. Carbohydrate markers specific for subsets of these afferents subsequently allow each subset to consolidate the position of its projections in appropriate regions of the CNS where it contacts its synaptic partners. - 1995 John Wiley & Sons, Inc.     

Synaptic input from serial chordotonal organs onto segmentally homologous interneurons in the grasshopper Schistocerca gregaria

We investigated the synaptic inputs from the serially homologous pleural, tympanal and wing-hinge chordotonal organs onto a set of identified homologous interneurons (714, 539, 529) in the ventral nerve cord of the grasshopper Schistocerca gregaria. Cobalt backfills show that afferents from all chordotonal organs project into stereotypic tracts in the central nervous system in which intracellular staining reveals the interneurons to have dendritic arborizations. Neuron 714 was found to receive excitatory bilateral synaptic input from all the serial chordotonal organs tested, from the second thoracic segment down to the seventh abdominal segment. Neuron 531, by contrast, only receives input from the chordotonal afferents on the first abdominal segment; those on the axon side are excitatory, while those on the soma side are inhibitory. The pattern of chordotonal input onto neuron 529 is similar to that seen for neuron 714, with the exception that neuron 529 receives no input from the forewing chordotonal organs. The pattern of afferent connectivities onto neurons 714, 531 and 529 differs with respect to those afferents which synapse directly or indirectly with the respective neuron. The synaptic inputs demonstrate a segmental specialization in the chordotonal system and thereby offer an insight into information processing in a modular sensory system.     

Degeneration of primary afferent terminals following brachial plexus extensive avulsion injury in rats

Important breakthroughs in the understanding regeneration failure in an injured CNS have been made by studies of primary afferent neurons. Dorsal rhizotomy has provided an experimental model of brachial plexus (BP) avulsion. This is an injury in which the central branches of primary afferents are disrupted at their point of entry into the spinal cord, bringing motor and sensory dysfunction to the upper limbs. In the present work, the central axonal organization of primary afferents was examined in control (without lesion) adult Wistar rats and in rats subjected to a C3-T3 rhizotomy. Specific sensory axon subtypes were recognized by application of antibodies to the calcitonin gene-related peptide (CGRP), the P2X3 purinoreceptor, the low-affinity p75-neurotrophin receptor and the retrograde tracer cholera toxin subunit beta (TCbeta). Other subtypes weres labeled with the lectin Griffonia simplicifolia 1B4. Using immunohistochemistry and high resolution light microscopy, brachial plexus rhizotomy in adult rats has proven a reliable model for several neural deficits in humans. This lesion produced different degrees of terminal degeneration in the several types of primary afferents which define sub-populations of sensitive neurons. Between the C6 and C8 levels of the spinal cord,, deafferentation was partial for peptidergic GCRP-positive fibers, in contrast with elimination of non peptidergic and myelinated fibers. Dorsal rhizotomy has provided an adequate experimental model to study sensory alterations such as acute pain and allodynia as well as factors that affect regeneration into the CNS., Therefore, the differential deafferentation response must be considered inr the evaluation of therapies for nociception (pain) and regeneration for brachial plexus avulsion. The anatomical diffierences among the primary afferent subtypes also affect their roles in normal and damaged conditions.     

Peripheral glia direct axon guidance across the CNS/PNS transition zone.

CNS glia have integral roles in directing axon migration of both vertebrates and insects. In contrast, very little is known about the roles of PNS glia in axonal pathfinding. In vertebrates and Drosophila, anatomical evidence shows that peripheral glia prefigure the transition zones through which axons migrate into and out of the CNS. Therefore, peripheral glia could guide axons at the transition zone. We used the Drosophila model system to test this hypothesis by ablating peripheral glia early in embryonic neurodevelopment via targeted overexpression of cell death genes grim and ced-3. The effects of peripheral glial loss on sensory and motor neuron development were analyzed. Motor axons initially exit the CNS in abnormal patterns in the absence of peripheral glia. However, they must use other cues within the periphery to find their correct target muscles since early pathfinding errors are largely overcome. When peripheral glia are lost, sensory axons show disrupted migration as they travel centrally. This is not a result of motor neuron defects, as determined by motor/sensory double-labeling experiments. We conclude that peripheral glia prefigure the CNS/PNS transition zone and guide axons as they traverse this region.     

Sequential steps in synaptic targeting of sensory afferents are mediated by constitutive and developmentally regulated glycosylations of CAMs.

Sensory afferents in the leech are labeled with both constitutive and developmentally regulated glycosylations (markers) of their cell adhesion molecules (CAMs). Their constitutive mannose marker, recognized by Lan3-2 monoclonal antibody (mAb), mediates the formation of their diffuse central arbors. We show that, at the ultrastructural level, these arbors consist of large, loosely organized axons rich with filopodia and synaptic vesicles. Perturbing the mannose-specific adhesion of this first targeting step leads to a gain in cell-cell contact but a loss of filopodia and synaptic vesicles. During the second targeting step, galactose markers divide afferents into different subsets. We focus on the subset labeled by the marker recognized by Laz2-369 mAb. Initially, the galactose marker appears where afferents contact central neurons. Subsequently it spreads proximally and distally, covering the entire afferent surface. Afferents now gain cell-cell contact, with central neurons and self-similar afferents, but lose filopodia and synaptic vesicles. Extant synaptic vesicles prevail where afferents are apposed to central neurons. These neurons develop postsynaptic densities and en passant synapses are forming. Perturbing the galactose-specific adhesion of this second targeting step causes a loss of cell-cell contact but a gain in filopodia and synaptic vesicles, essentially returning afferents to the first targeting step. The transformation of afferent growth, progressing from mannose- to galactose-specific adhesion, is consistent with a change from cell-matrix to cell-cell adhesion. By performing opposing functions in a temporal sequence, constitutive and developmentally regulated glycosylations of CAMs collaborate in the synaptogenesis of afferents and the consolidation of self-similar afferents.     

Role of motoneuron-derived neurotrophin 3 in survival and axonal projection of sensory neurons during neural circuit formation

Sensory neurons possess the central and peripheral branches and they form unique spinal neural circuits with motoneurons during development. Peripheral branches of sensory axons fasciculate with the motor axons that extend toward the peripheral muscles from the central nervous system (CNS), whereas the central branches of proprioceptive sensory neurons directly innervate motoneurons. Although anatomically well documented, the molecular mechanism underlying sensory-motor interaction during neural circuit formation is not fully understood. To investigate the role of motoneuron on sensory neuron development, we analyzed sensory neuron phenotypes in the dorsal root ganglia (DRG) of Olig2 knockout (KO) mouse embryos, which lack motoneurons. We found an increased number of apoptotic cells in the DRG of Olig2 KO embryos at embryonic day (E) 10.5. Furthermore, abnormal axonal projections of sensory neurons were observed in both the peripheral branches at E10.5 and central branches at E15.5. To understand the motoneuron-derived factor that regulates sensory neuron development, we focused on neurotrophin 3 (Ntf3; NT-3), because Ntf3 and its receptors (Trk) are strongly expressed in motoneurons and sensory neurons, respectively. The significance of motoneuron-derived Ntf3 was analyzed using Ntf3 conditional knockout (cKO) embryos, in which we observed increased apoptosis and abnormal projection of the central branch innervating motoneuron, the phenotypes being apparently comparable with that of Olig2 KO embryos. Taken together, we show that the motoneuron is a functional source of Ntf3 and motoneuron-derived Ntf3 is an essential pre-target neurotrophin for survival and axonal projection of sensory neurons.     

Differential Glycosylation of Tractin and LeechCAM, Two Novel Ig Superfamily Members, Regulates Neurite Extension and Fascicle Formation

By immunoaffinity purification with the mAb Lan3-2, we have identified two novel Ig superfamily members, Tractin and LeechCAM. LeechCAM is an NCAM/FasII/ApCAM homologue, whereas Tractin is a cleaved protein with several unique features that include a PG/YG repeat domain that may be part of or interact with the extracellular matrix. Tractin and LeechCAM are widely expressed neural proteins that are differentially glycosylated in sets and subsets of peripheral sensory neurons that form specific fascicles in the central nervous system. In vivo antibody perturbation of the Lan3-2 glycoepitope demonstrates that it can selectively regulate extension of neurites and filopodia. Thus, these experiments provide evidence that differential glycosylation can confer functional diversity and specificity to widely expressed neural proteins.     

Antibody against myelin-associated inhibitor of neurite growth neutralizes nonpermissive substrate properties of CNS white matter

CNS white matter from higher vertebrates and cultured differentiated oligodendrocytes are nonpermissive substrates for neurite growth and fibroblast spreading. Membrane proteins of 35 kd and 250 kd with highly nonpermissive substrate properties could be extracted from CNS myelin fractions. Monoclonal antibodies were raised against these proteins: IN-1 and IN-2 bound both to the 35 kd and 250 kd inhibitors and to the surface to differentiated cultured oligodendrocytes. Adsorption of nonpermissive CNS myelin or nonpermissive oligodendrocytes with either antibody markedly improved their substrate properties. Optic nerve explants injected with IN-1 or IN-2 allowed axon ingrowth of cocultured sensory and sympathetic neurons. We conclude that the nonpermissive substrate properties of CNS white matter are due to these membrane proteins on the surface of differentiated oligodendrocytes and to their in vivo product, myelin.