Transpinal and Transcortical Stimulation Alter Corticospinal Excitability and Increase Spinal Output

The objective of this study was to assess changes in corticospinal excitability and spinal output following noninvasive transpinal and transcortical stimulation in humans. The size of the motor evoked potentials (MEPs), induced by transcranial magnetic stimulation (TMS) and recorded from the right plantar flexor and extensor muscles, was assessed following transcutaneous electric stimulation of the spine (tsESS) over the thoracolumbar region at conditioning-test (C-T) intervals that ranged from negative 50 to positive 50 ms. The size of the transpinal evoked potentials (TEPs), induced by tsESS and recorded from the right and left plantar flexor and extensor muscles, was assessed following TMS over the left primary motor cortex at 0.7 and at 1.1× MEP resting threshold at C-T intervals that ranged from negative 50 to positive 50 ms. The recruitment curves of MEPs and TEPs had a similar shape, and statistically significant differences between the sigmoid function parameters of MEPs and TEPs were not found. Anodal tsESS resulted in early MEP depression followed by long-latency MEP facilitation of both ankle plantar flexors and extensors. TEPs of ankle plantar flexors and extensors were increased regardless TMS intensity level. Subthreshold and suprathreshold TMS induced short-latency TEP facilitation that was larger in the TEPs ipsilateral to TMS. Noninvasive transpinal stimulation affected ipsilateral and contralateral actions of corticospinal neurons, while corticocortical and corticospinal descending volleys increased TEPs in both limbs. Transpinal and transcortical stimulation is a noninvasive neuromodulation method that alters corticospinal excitability and increases motor output of multiple spinal segments in humans.     

The relationships among the severity of spinal cord injury,motor and somatosensory evoked potentials and spinal cord blood flow

To characterize the changes in axonal function in the motor and somatosensory tracts of the cord after spinal cord injury (SCI) and to correlate these changes with spinal cord blood flow (SCBF), the relationships among the severity of SCI, motor and somatosensory evoked potentials (MEPs and SSEPs) and SCBF were examined. Fifteen rats received a 1.5 g (n = 5), 20 g (n = 5) or 56 g (n = 5) clip compression injury of the cord at C8. SCBF at the injury site was measured by the hydrogen clearance technique 35 min before and 30 min after SCI. Concomitantly MEPs from the cord at T10 (MEP-C) and from the sciatic nerve (MEP-N) and SSEPs were recorded.A linear relationship (r = −0.89, P < 0.002) was found between the severity of SCI and the reduction in SCBF at the injury site. Linear discriminant analysis revealed that both the MEP (P < 0.0001) and SSEP (P < 0.003) were significantly related to the severity of SCI. Furthermore, the amplitude of the MEP (r = 0.65, P < 0.0001) and SSEP (r = 0.58, P < 0.0011) was significantly correlated with the posttraumatic SCBF. Multiple regression revealed that both the severity of cord injury and the degree of posttraumatic ischemia were significantly related to axonal dysfunction after SCI. While the MEP was more sensitive to injury than the SSEP, the SSEP more accurately distinguished between mild and moderate severities of cord injury.Axonal conduction in the motor and somatosensory tracts of the cord was significantly correlated with the reduction in posttraumatic SCBF and, therefore, these data provide quantitative evidence linking posttraumatic ischemia to axonal dysfunction following acute cord injury. Furthermore, this study validates the hypothesis that the combined recording of MEPs and SSEPs is an accurate technique to assess the physiological integrity of the cord after injury.     

Integrity of developing spinal motor columns is regulated by neural crest derivatives at motor exit points

Spinal motor neurons must extend their axons into the periphery through motor exit points (MEPs), but their cell bodies remain within spinal motor columns. It is not known how this partitioning is established in development. We show here that motor neuron somata are confined to the CNS by interactions with a neural crest subpopulation, boundary cap (BC) cells that prefigure the sites of spinal MEPs. Elimination of BC cells by surgical or targeted genetic ablation does not perturb motor axon outgrowth but results in motor neuron somata migrating out of the spinal cord by translocating along their axons. Heterologous neural crest grafts in crest-ablated embryos stop motor neuron emigration. Thus, before the formation of a mature transitional zone at the MEP, BC cells maintain a cell-tight boundary that allows motor axons to cross but blocks neuron migration.     

Axon kinematics change during growth and development

The microkinematic response of axons to mechanical stretch was examined in the developing chick embryo spinal cord during a period of rapid growth and myelination. Spinal cords were isolated at different days of embryonic (E) development post-fertilization (E12, E14, E16, and E18) and stretched 0%, 5%, 10%, 15%, and 20%, respectively. During this period, the spinal cord grew approximately 55% in length, and white matter tracts were myelinated significantly. The spinal cords were fixed with paraformaldehyde at the stretched length, sectioned, stained immunohistochemically for neurofilament proteins, and imaged with epifluorescence microscopy. Axons in unstretched spinal cords were undulated, or tortuous, to varying degrees, and appeared to straighten with stretch. The degree of tortuosity (ratio of the segment's pathlength to its end-to-end length) was quantified in each spinal cord by tracing several hundred randomly selected axons. The change in tortuosity distributions with stretch indicated that axons switched from non-affine, uncoupled behavior at low stretch levels to affine, coupled behavior at high stretch levels, which was consistent with previous reports of axon behavior in the adult guinea pig optic nerve (Bain, Shreiber, and Meaney, J. Biomech. Eng., 125(6), pp. 798-804). A mathematical model previously proposed by Bain et al. was applied to quantify the transition in kinematic behavior. The results indicated that significant percentages of axons demonstrated purely non-affine behavior at each stage, but that this percentage decreased from 64% at E12 to 30% at E18. The decrease correlated negatively to increases in both length and myelination with development, but the change in axon kinematics could not be explained by stretch applied during physical growth of the spinal cord. The relationship between tissue-level and axonal-level deformation changes with development, which can have important implications in the response to physiological forces experienced during growth and trauma.     

Contact-Mediated Inhibition Between Oligodendrocyte Progenitor Cells and Motor Exit Point Glia Establishes the Spinal Cord Transition Zone

Rapid conduction of action potentials along motor axons requires that oligodendrocytes and Schwann cells myelinate distinct central and peripheral nervous system (CNS and PNS) domains along the same axon. Despite the importance of this arrangement for nervous system function, the mechanisms that establish and maintain this precise glial segregation at the motor exit point (MEP) transition zone are unknown. Using in vivo time-lapse imaging in zebrafish, we observed that prior to myelination, oligodendrocyte progenitor cells (OPCs) extend processes into the periphery via the MEP and immediately upon contact with spinal motor root glia retract back into the spinal cord. Characterization of the peripheral cell responsible for repelling OPC processes revealed that it was a novel, CNS-derived population of glia we propose calling MEP glia. Ablation of MEP glia resulted in the absence of myelinating glia along spinal motor root axons and an immediate breach of the MEP by OPCs. Taken together, our results identify a novel population of CNS-derived peripheral glia located at the MEP that selectively restrict the migration of OPCs into the periphery via contact-mediated inhibition.     

Amyloid precursor protein at node of Ranvier modulates nodal formation

Amyloid precursor protein (APP), commonly associated with Alzheimer disease, is upregulated and distributes evenly along the injured axons, and therefore, also known as a marker of demyelinating axonal injury and axonal degeneration. However, the physiological distribution and function of APP along myelinated axons was unknown. We report that APP aggregates at nodes of Ranvier (NOR) in the myelinated central nervous system (CNS) axons but not in the peripheral nervous system (PNS). At CNS NORs, APP expression co-localizes with tenascin-R and is flanked by juxtaparanodal potassium channel expression demonstrating that APP localized to NOR. In APP-knockout (KO) mice, nodal length is significantly increased, while sodium channels are still clustered at NORs. Moreover, APP KO and APP-overexpressing transgenic (APP TG) mice exhibited a decreased and an increased thickness of myelin in spinal cords, respectively, although the changes are limited in comparison to their littermate WT mice. The thickness of myelin in APP KO sciatic nerve also increased in comparison to that in WT mice. Our observations indicate that APP acts as a novel component at CNS NORs, modulating nodal formation and has minor effects in promoting myelination.     

Amyloid precursor protein at node of Ranvier modulates nodal formation

Amyloid precursor protein (APP), commonly associated with Alzheimer disease, is upregulated and distributes evenly along the injured axons, and therefore, also known as a marker of demyelinating axonal injury and axonal degeneration. However, the physiological distribution and function of APP along myelinated axons was unknown. We report that APP aggregates at nodes of Ranvier (NOR) in the myelinated central nervous system (CNS) axons but not in the peripheral nervous system (PNS). At CNS NORs, APP expression co-localizes with tenascin-R and is flanked by juxtaparanodal potassium channel expression demonstrating that APP localized to NOR. In APP-knockout (KO) mice, nodal length is significantly increased, while sodium channels are still clustered at NORs. Moreover, APP KO and APP-overexpressing transgenic (APP TG) mice exhibited a decreased and an increased thickness of myelin in spinal cords, respectively, although the changes are limited in comparison to their littermate WT mice. The thickness of myelin in APP KO sciatic nerve also increased in comparison to that in WT mice. Our observations indicate that APP acts as a novel component at CNS NORs, modulating nodal formation and has minor effects in promoting myelination.     

Experimental Study of Low Dose Ultrashortwave Promoting Nerve Regeneration after Acellular Nerve Allografts Repairing the Sciatic Nerve Gap of Rats

Objectives To observe the effect of ultrashortwave (USW) therapy on nerve regeneration after acellular nerve allografts(ANA) repairing the sciatic nerve gap of rats and discuss its acting mechanisms. Methods Sixteen Wistar rats weighing 180–220 g were randomly divided into four groups with four rats in each group: normal control group; acellular group (ANA, treated by hypotonic-chemical detergent, was applied for bridging a 10 mm-long sciatic nerve defect); USW group (After 24 h of ANA repairing the sciatic nerve gap, low dose USW was administrated for 7 min, once a day, 20 times a course of treatment, three courses of treatment in all); and autografts group. 12 weeks after operation, a series of examinations was performed, including electrophysiological methods, the restoring rate of tibialis anterior muscle wet weight, histopathological observation (myelinated nerve number, myelin sheath thickness, and axon diameter), vascular endothelial growth factor (VEGF) mRNA expression of spinal cord, and muscle at injury site, and analyzed statistically. Results Compared to acellular nerve allografts alone, USW therapy can increase nerve conductive velocity, the restoring rate of tibialis anterior muscle wet weight, myelinated nerve number, axon diameter, VEGF mRNA expression of spinal cord, and muscle at injury site, the difference is significant. There were no differences between USW group and autografts group except myelin sheath thickness. Conclusions USW therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats, the upregulation of VEGF mRNA expression of spinal cord and muscle may play an important role.     

Startle stimuli exert opposite effects on human cortical and spinal motor system excitability in leg muscles

Increased excitability of the spinal motor system has been observed after loud and unexpected acoustic stimuli (AS) preceding H-reflexes. The paradigm has been proposed as an electrophysiological marker of reticulospinal tract activity in humans. The brainstem reticular formation also maintains dense anatomical interconnections with the cortical motor system. When a startling AS is delivered, prior to transcranial magnetic stimulation (TMS), the AS produces a suppression of motor evoked potential (MEP) amplitude in hand and arm muscles of healthy subjects. Here we analyzed the conditioning effect of a startling AS on MEP amplitude evoked by TMS to the primary motor leg area. Ten healthy volunteers participated in two experiments that used a conditioning-test paradigm. In the first experiment, a startling AS preceded a suprathreshold transcranial test stimulus. The interstimulus interval (ISI) varied between 20 to 160 ms. When given alone, the test stimulus evoked a MEP amplitude of approximately 0.5 mV in the slightly preinervated soleus muscle (SOL). In the second experiment, the startling AS was used to condition the size of the H-reflex in SOL muscle. Mean MEP amplitude was calculated for each ISI. The conditioning AS suppressed MEP amplitude at ISIs of 30-80 ms. By contrast, H-reflex amplitude was augmented at ISIs of 100-200 ms. In conclusions, acoustic stimulation exerts opposite and ISI-specific effects on the amplitude of MEPs and H-reflex in the SOL muscle, indicating different mechanism of auditory-to-motor interactions at cortical and spinal level of motor system.     

SEDIMENTATION ANALYSIS OF RIBONUCLEIC ACID EXTRACTED FROM ISOLATED MAUTHNER NERVE FIBRE COMPONENTS

Abstract— Segments of spinal cords from goldfish or from carp were incubated in vitro in the presence of RNA precursors for varying periods of time. Mauthner nerve fibres were isolated from the fresh unfixed tissue, or, for the separate analyses of axon and myelin sheath, from the fixed spinal cord.
The myelinated Mauthner fibre isolated from the incubated spinal cord showed RNA synthesis. A considerable part of the material sedimented at 4 S , but part of the nucleic acid was recovered at higher sedimentation values, up to 30 S. Newly synthesized RNA was extracted from the isolated myelin sheath as well as from the axon. Isolated myelinated Mauthner fibres were also incubated in vitro with RNA precursors. In this case incorporation occurred exclusively in material sedimenting at 4 S or lower. The turnover rate for RNA from the fibre was of a higher order than that of the bulk of RNA from the spinal cord. The findings of RNA synthesis in these tissue components lacking nuclei could possibly be explained as owing to mitochondria.
Studies by electron microscopy demonstrated the extent of purity of the isolated components and it was found that contamination was so small as to make it unlikely that the RNA investigated originated in contaminating tissue.     

Morphometric Analysis of Normal, Mutant, and Transgenic CNS: Correlation of Myelin Basic Protein Expression to Myelinogenesis

The neurological mutant mice shiverer (shi) and myelin deficient (shimld) lack a functional gene for the myelin basic proteins (MBP), have virtually no myelin in their CNS, shiver, seize, and die early. Mutant mice homozygous for an MBP transgene have MBP mRNA and MBP in net amounts approximately 25% of normal, have compact myelin, do not shiver or seize, and live normal life spans. We bred mice with various combinations of the normal, transgenic, shi, and shimld genes to produce mice that expressed MBP mRNA at levels of 0, 5, 12.5, 17.5, 50, 62.5, and 100% of normal. The CNS of these mice were analyzed for MBP content, tissue localization of MBP, degree of myelination, axon size, and myelin thickness. MBP protein content correlated with predicted MBP gene expression. Immunocytochemical staining localized MBP to white matter in normal and transgenic shi mice with an intensity of staining comparable to the degree of MBP gene expression. An increase in the percentage of myelinated axons and the thickness of myelin correlated with increased gene expression up to 50% of normal. The percentage of myelinated axons and myelin thickness remained constant at expression levels greater than 50%. The presence of axons loosely wrapped with oligodendrocytic membrane in mice expressing lower amounts of MBP mRNA and protein suggested that the oligodendroglia produced sufficient MBP to elicit axon wrapping but not enough to form compact myelin. Mean axon circumference of myelinated axons was greater than axon circumference of unmyelinated axons at each level of gene expression, further evidence that oligodendroglial cells preferentially myelinate axons of larger caliber.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Axonal Membrane Protein Caspr, a Homologue of Neurexin IV, Is a Component of the Septate-like Paranodal Junctions That Assemble during Myelination

We have investigated the potential role of contactin and contactin-associated protein (Caspr) in the axonal–glial interactions of myelination. In the nervous system, contactin is expressed by neurons, oligodendrocytes, and their progenitors, but not by Schwann cells. Expression of Caspr, a homologue of Neurexin IV, is restricted to neurons. Both contactin and Caspr are uniformly expressed at high levels on the surface of unensheathed neurites and are downregulated during myelination in vitro and in vivo. Contactin is downregulated along the entire myelinated nerve fiber. In contrast, Caspr expression initially remains elevated along segments of neurites associated with nascent myelin sheaths. With further maturation, Caspr is downregulated in the internode and becomes strikingly concentrated in the paranodal regions of the axon, suggesting that it redistributes from the internode to these sites. Caspr expression is similarly restricted to the paranodes of mature myelinated axons in the peripheral and central nervous systems; it is more diffusely and persistently expressed in gray matter and on unmyelinated axons. Immunoelectron microscopy demonstrated that Caspr is localized to the septate-like junctions that form between axons and the paranodal loops of myelinating cells. Caspr is poorly extracted by nonionic detergents, suggesting that it is associated with the axon cytoskeleton at these junctions. These results indicate that contactin and Caspr function independently during myelination and that their expression is regulated by glial ensheathment. They strongly implicate Caspr as a major transmembrane component of the paranodal junctions, whose molecular composition has previously been unknown, and suggest its role in the reciprocal signaling between axons and glia.     

Development of the axon membrane during differentiation of myelinated fibres in spinal nerve roots

Previous studies by a number of workers have shown that the axon membrane in normal mature myelinated fibres is highly differentiated, with the nodal axolemma exhibiting characteristics different to those of the internodal axolemma. However, the development of this axolemmal heterogeneity has not been previously explored. In the present study we used cytochemical methods to examine the development of nodal axolemma during the differentiation of myelinated fibres in rat spinal roots. The staining properties characteristic of normal nodal membrane appear in the axon, at gaps between Schwann cells, before the development of mature compact myelin or well defined paranodal axon--Schwann cell specializations close to the region of nodal axolemmal differentiation. These results are consistent with the hypothesis that the axon membrane differentiates into nodal and internodal regions before, or early in the process of, myelination, and suggest that the differentiation of the axon membrane may provide a signal demarcating the region to be covered by the myelin-forming cell.     

Changes within maturing neurons limit axonal regeneration in the developing spinal cord

Embryonic birds and mammals display a remarkable ability to regenerate axons after spinal injury, but then lose this ability during a discrete developmental transition. To explain this transition, previous research has emphasized the emergence of myelin and other inhibitory factors in the environment of the spinal cord. However, research in other CNS tracts suggests an important role for neuron-intrinsic limitations to axon regeneration. Here we re-examine this issue quantitatively in the hindbrain-spinal projection of the embryonic chick. Using heterochronic cocultures we show that maturation of the spinal cord environment causes a 55% reduction in axon regeneration, while maturation of hindbrain neurons causes a 90% reduction. We further show that young neurons transplanted in vivo into older spinal cord can regenerate axons into myelinated white matter, while older axons regenerate poorly and have reduced growth cone motility on a variety of growth-permissive ligands in vitro, including laminin, L1, and N-cadherin. Finally, we use video analysis of living growth cones to directly document an age-dependent decline in the motility of brainstem axons. These data show that developmental changes in both the spinal cord environment and in brainstem neurons can reduce regeneration, but that the effect of the environment is only partial, while changes in neurons by themselves cause a nearly complete reduction in regeneration. We conclude that maturational events within neurons are a primary cause for the failure of axon regeneration in the spinal cord.     

Hindshaker,a Novel Myelin Mutant Showing Hypomyelination Preferentially Affecting the Spinal Cord

Animals with spontaneous mutations affecting myelin formation have provided useful information about the genetic and cellular mechanisms regulating normal and abnormal myelination. In this paper we describe a novel murine mutation termed hindshaker (hsh) which is inherited in an automosal recessive manner. Affected mice are characterised by a variable tremor of the hind end which commences at about 2 weeks of age and largely disappears in animals older than 6 weeks. There is hypomyelination affecting predominantly the spinal cord, although the optic nerves and brain are involved to a much lesser degree. The defect of thinly myelinated and naked axons is maximal at 20 days of age and largely resolves with time so that in the adult most axons are myelinated. The myelin structure appears normal and immunostains for the major proteins. Although the distribution of oligodendrocytes in the spinal cord is similar to normal during the period of hypomyelination, there are fewer mature cells. The hsh mutation appears to delay the maturation of oligodenrocytes, particularly in the spinal cord. Additionally, there is a considerable variation in phenotypic expression and in penetrance when the mutation is expressed on different genetic backgrounds, suggesting the hsh locus is subject to the influence of modifying gene(s). Identification of the hsh gene should identify a factor important in the development of oligodendrocytes, particularly those in the spinal cord.     

Morphological and morphometric studies of early myelination in the cervical dorsal funiculus of the newborn mouse spinal cord

The early myelination of the dorsal funiculus at the level of the 4th cervical spinal cord was ultrastructurally studied in the one-day-old mouse. It was found that the fibers were mainly unmyelinated. However, some early myelinated fibers were scattered among unmyelinated fibers. In the initial stage of the myelination, the axon was partially contracted by a piece of cytoplasmic process of the oligodendroglial cell. The two lips of the oligodendroglial process then extended and converged, enwrapping the axon completely and forming the first contact point. With the anchorage of that contact point, the two lips of the process became elongated and enfolded by each other, and produced the internal and external tongues of the future myelin sheath. More contact points were formed at a regular interval by the regional fusion of the two external surface layers of the opposed cytoplasmic membranes of adjacent tongue processes. With the advanced bidirectional spiralization of the two tongue processes, many contact points were found between the adjacent lamellae of the concentrically arranged oligodendroglial process; simultaneously, the cleft between the neighboring contact points disappeared and formed the initial sites of the intraperiod line. During the early myelination, one single axon ensheathed concentrically by two different oligodendroglial processes as well as several axons enwrapped by a continuous spiral myelin sheath of one oligodendroglial cell were frequently observed. The cross-sectional areas of unmyelinated axons varied from 0.01 to 0.2 micron 2, with a median of 0.07 micron 2; whereas, that of promyelinated axons ranged from 0.09 to 1.4 micron 2, with a median at 0.61 micron 2. These data support the suggestion that the axon calibre is a critical factor for the initiation of central myelination.     

Nerve and muscle development in paralysé mutant mice

Nerve and muscle development was studied in paralysé mutant mice. The mutant phenotype is first recognizable 6-7 days after birth (PN 6-PN 7) as cessation of muscle growth and weakness and incoordination of movement. Mutant animals die between 2 and 3 weeks of age. Muscle fibers from paralysé mutants had a unimodal distribution of diameters and normal numbers and distributions of acetylcholine receptors. The only structural abnormality seen was a reduced extracellular space within muscle fascicles. Total muscle choline acetyltransferase activity was reduced compared with that of control muscles, indicating that synaptic terminal development was impaired. Light and electron microscopy showed that polyneuronal innervation was retained in mutant endplates, and the normal process of withdrawal of redundant innervation did not occur. The paralysé muscles reacted to experimental denervation with an increase in extrajunctional acetylcholine receptor numbers. Intramuscular axons failed to become myelinated in mutant animals, although sciatic nerve axons were myelinated with a normal myelin thickness/axon diameter ratio. Nodes of Ranvier were elongated and myelin lamellae in the paranodal regions were poorly fused. Sciatic nerves in mutant animals retained the neonatal unimodal distribution of axon diameters, whereas in control animals it became bimodal by 2 weeks of age. Our results are not consistent with a previous suggestion that paralysé mutant muscle endplates are progressively denervated. We conclude that the major expression of the paralysé mutant phenotype is an arrest in development of both nerve and muscle during the first week after birth. The paralysé mutant gene most likely is involved in the general support of development of many or all body tissues from 1 week of age. We found no regression of any aspect of differentiation, once achieved.     

Central nervous adaptations following 1 wk of wrist and hand immobilization.

Plastic neural changes have been documented in relation to different types of physical activity, but little is known about central nervous system plasticity accompanying reduced physical activity and immobilization. In the present study we investigated whether plastic neural changes occur in relation to 1 wk of immobilization of the nondominant wrist and hand and a corresponding period of recovery in 10 able-bodied volunteers. After immobilization, maximal voluntary contraction torque decreased and the variability of submaximal static contractions increased significantly without evidence of changes in muscle contractile properties. Hoffmann (H)-reflex amplitudes and the ratios of H-slope to M-slope increased significantly in flexor carpi radialis and abductor pollicis brevis at rest and during contraction without changes in corticospinal excitability, estimated from motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation. Corticomuscular coherence measures were derived from EEG and EMG obtained during static contractions. After immobilization, corticomuscular coherence in the 15- to 35-Hz range associated with maximum negative cumulant values at lags corresponding to MEP latencies decreased. One week after cast removal, all measurements returned to preimmobilization levels. The increased H-reflex amplitudes without changes in MEPs may suggest that presynaptic inhibition or postactivation depression of Ia afferents is reduced following immobilization. Reduced corticomuscular coherence may be caused by changes in afferent input at spinal and cortical levels or by changes in the descending drive from motor cortex. Further studies are needed to elucidate the mechanisms underlying the observed increased spinal excitability and reduced coupling between motor cortex and spinal motoneuronal activity following immobilization.     

Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and MAG) and their shared carbohydrate epitope and myelin basic protein in developing sciatic nerve

The cellular and subcellular localization of the neural cell adhesion molecules L1, N-CAM, and myelin-associated glycoprotein (MAG), their shared carbohydrate epitope L2/HNK-1, and the myelin basic protein (MBP) were studied by pre- and post-embedding immunoelectron microscopic labeling procedures in developing mouse sciatic nerve. L1 and N-CAM showed a similar staining pattern. Both were localized on small, non-myelinated, fasciculating axons and axons ensheathed by non- myelinating Schwann cells. Schwann cells were also positive for L1 and N-CAM in their non-myelinating state and at the onset of myelination, when the Schwann cell processes had turned approximately 1.5 loops. Thereafter, neither axon nor Schwann cell could be detected to express the L1 antigen, whereas N-CAM was found in the periaxonal area and, more weakly, in compact myelin of myelinated fibers. Compact myelin, Schmidt-Lanterman incisures, paranodal loops, and finger-like processes of Schwann cells at nodes of Ranvier were L1-negative. At the nodes of Ranvier, the axolemma was also always L1- and N-CAM-negative. The L2/HNK-1 carbohydrate epitope coincided in its cellular and subcellular localization most closely to that observed for L1. MAG appeared on Schwann cells at the time L1 expression ceased. MAG was then expressed at sites of axon-myelinating Schwann cell apposition and non-compacted loops of developing myelin. When compaction of myelin occurred, MAG remained present only at the axon-Schwann cell interface; Schmidt- Lanterman incisures, inner and outer mesaxons, and paranodal loops, but not at finger-like processes of Schwann cells at nodes of Ranvier or compacted myelin. All three adhesion molecules and the L2/HNK-1 epitope could be detected in a non-uniform staining pattern in basement membrane of Schwann cells and collagen fibrils of the endoneurium. MBP was detectable in compacted myelin, but not in Schmidt-Lanterman incisures, inner and outer mesaxon, paranodal loops, and finger-like processes at nodes of Ranvier, nor in the periaxonal regions of myelinated fibers, thus showing a complementary distribution to MAG. These studies show that axon-Schwann cell interactions are characterized by the sequential appearance of cell adhesion molecules and MBP apparently coordinated in time and space. From this sequence it may be deduced that L1 and N-CAM are involved in fasciculation, initial axon-Schwann cell interaction, and onset of myelination, with MAG to follow and MBP to appear only in compacted myelin. In contrast to L1, N- CAM may be further involved in the maintenance of compact myelin and axon-myelin apposition of larger diameter axons.