Stereotypic morphology of glutamatergic synapses on identified muscle cells of Drosophila larvae

The distribution and morphology of glutamatergic synapses on Drosophila bodywall muscle fibers were examined at the single-synapse level using immunocytochemistry and electrophysiology. We find that glutamate-immunoreactive motor endings innervate the entire larval bodywall musculature, with each muscle fiber receiving at least one glutamatergic ending. The innervation is initiated at stereotyped locations on each muscle fiber from where moderately branched varicose nerve processes project over the internally facing muscle surface. Individual muscle fibers have distinct stereotypic patterns of nerve endings that occupy characteristic regions on the cell surface. The muscle-specific branching pattern of motor endings is reiterated by segmentally homologous fibers. Two morphological types of innervating nerve processes can be distinguished by their bouton size distributions: (1) Type I processes, which have localized branching and a broad size distribution of relatively large varicosities ranging up to 8 microns (mean diameter, 3.1 +/- 1.6 microns; +/- SD, n = 521), and (2) thinner Type II processes, which have a narrower distribution of small varicosities with a mean diameter of only 1.4 +/- 0.6 microns (+/- SD, n = 214). Immunoelectron microscopy with peroxidase-labeled second antibody demonstrates that the varicosities are surrounded by a subsynaptic reticulum, that they contain immunoreactive vesicles of about 30-50 nm, and thus probably represent synaptic release sites. By iontophoretic application of glutamate we mapped the responsive sites on the muscle surface and found an excellent correspondence between transmitter sensitivity and the patterns of endings as described by immunocytochemistry. In contrast to our finding of numerous glutamate iontophoresis-sensitive sites, we did not detect any aspartate-responsive muscles. These data provide strong new evidence for glutamate being an endogenous transmitter at the Drosophila larval neuromuscular junction.     

Development of postsynaptic-like specializations of the neuromuscular synapse in the absence of motor nerve

It was previously reported that the acetylcholine receptor clusters and acetylcholinesterase appear on embryonic superior oblique muscle cells developing in vivo without motor nerve contacts. The objective of this study was to examine whether some other components of neuromuscular junction also form on muscle cells developing in vivo in the absence of motor neurons. In the present study, postsynaptic specializations such as junctional folds, postsynaptic density and basal lamina were studied in normal and aneural muscles. The superior oblique muscle of duck embryos was made aneural by permanent destruction of trochlear motor neurons by cauterizing midbrain on embryonic day 7; 3 days before the motor neurons normally project their axons into the muscle. Normal and aneural muscles from embryonic days 10 to 25 were processed for electron microscopy. The results indicate that morphological specializations such as junction-like folds, postsynaptic-like density, and basal lamina also develop in the absence of motor neuron contacts. Whether the differentiation of specialized synaptic basal lamina is dependent on the presence of motor neurons was examined by utilizing a monoclonal antibody against heparan sulfate proteoglycan. Immunohistochemical studies indicate that specialized synaptic basal lamina differentiates in the absence of motor neurons. Thus, the mechanism of development of postsynaptic components of neuromuscular junction in this muscle is not dependent on motor neuron contacts. These results also suggest that the postsynaptic cell plays a more active role in synapse formation than previously realized. The results are discussed in relation to the control of synapse numbers by the postsynaptic cell.     

Development of the spinal nerves in the lamprey: II. Outflows from the spinal cord

During a series of studies on the development of spinal nerves in the tail of larval lampreys (13 mm, 26 days), Lampetra japonica, we observed outflows of cytoplasmic processes (cytoplasmic outflow) or whole cell bodies (cellular outflow) from the neural tube or from the cord. Three types were distinguished according to their site of exit from the surface of the neuraxis. The dorsal outflow (DO) is the cellular outflow seen on the midsagittal surface of the dorsal wall of the caudalmost region of the neural tube, just rostral or caudal to the opening in the dorsal tube wall. It is hypothesized that the cells from the dorsal neural tube become polymorphous cells scattered in the extramedullar space. The dorsolateral outflow (DLO) is the cytoplasmic outflow emerging from the dorsolateral aspect of the spinal cord at the intermyotome level. DLO fibers are non-myelinated fibers arising from the dorsolateral tract (DLT) and, after piercing the basal lamina and the glia limitans, run in the myosepta laterally to spread along the deep surface of the dermis. DLO fibers terminate in two different ways: those of one group pierce the dermis and the basal lamina to end as intraepidermal free endings that contain aggregates of clear vesicles, and those of the other group form varicosities that lie within depressions on the lateral cell surface of myotomes. DLO fibers in the extramedullary space characteristically lack any sheaths, including the basal lamina. The ventrolateral outflow (VLO) represents primitive ventral roots consisting of both the cytoplasmic and the cellular outflows: the former shows the axonal outgrowth from primitive somatomotor neurons, and the latter is represented by elongated cells derived from the glia limitans, extending along the axonal processes to form the Schwann cell sheath around the proximal portion of the ventral root axons. The basal lamina covering the cord does not extend along the VLO fibers. The developing ventral root contains axons at different stages of differentiation. Some axons end on the medial surface of the myotome at the midsegmental level to form neuromuscular junctions. However, axonal processes of primitive motoneurons form close contacts with muscle cells by means of small cytoplasmic projections that contain no synaptic vesicles and lack the basal lamina interposed between them. The proposed sequence of development, based on stages in caudal to rostral sections, is the DO, the DLO, and then the VLO.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMDA receptors at the endplate of rat skeletal muscles: precise postsynaptic localization

In this study we demonstrate expression of the N-methyl-D-aspartate receptor NR1 subunit in the rat neuromuscular junction of skeletal muscles of different functional types (extensor digitorum longus, soleus, and diaphragm muscles) using fluorescence immunocytochemistry. Electron microscopic immunocytochemistry has shown that the NR1 subunit is localized solely on the sarcolemma in the depths of the postsynaptic folds. These findings suggest participation of the glutamatergic signaling system in functioning of the adult mammalian neuromuscular junction.     

Developmental and neural regulation of a subsarcolemmal component of the rat neuromuscular junction.

We have generated a monoclonal antibody, designated mAb 3G2, which reacts with a subsarcolemmal component of the neuromuscular junction in adult rats. mAb 3G2 immunoreactivity lies beneath and between the ACh receptor-rich synaptic gutters, around the sole plate nuclei, and at/near sarcomeric Z-disks in the vicinity of the synapse. Localization of mAb 3G2 immunoreactivity to neuromuscular junctions begins postnatally and gradually increases to adult levels. The establishment of this synaptic localization is neurally regulated, as neonatal denervation prevents its occurrence. In adults, denervation results in a loss of synaptic immunoreactivity that returns upon reinnervation. The antigen is also found at the myotendinous junction; its localization here is innervation independent. mAb 3G2 recognizes a 41 kDa protein on immunoblots of extracts of newborn muscle. Based on its distribution within muscle fibers, its developmental and neural regulation, and its molecular weight, the protein recognized by mAb 3G2 can be distinguished from other known postsynaptic proteins. Its neural dependence and developmental regulation suggest that it may participate in synaptic stabilization, perhaps as the intracellular component in a chain of proteins that serve to tether the nerve terminal to the perijunctional region of the muscle fiber.     

Drosophila olfactory local interneurons and projection neurons derive from a common neuroblast lineage specified by the empty spiracles gene

Background

The skeletal neuromuscular junction is a useful model for elucidating mechanisms that regulate synaptogenesis. Developmentally important intercellular interactions at the neuromuscular junction are mediated by the synaptic portion of a basal lamina that completely ensheaths each muscle fiber. Basal laminas in general are composed of four main types of glycosylated proteins: laminins, collagens IV, heparan sulfate proteoglycans and nidogens (entactins). The portion of the muscle fiber basal lamina that passes between the motor nerve terminal and postsynaptic membrane has been shown to bear distinct isoforms of the first three of these. For laminins and collagens IV, the proteins are deposited by the muscle; a synaptic proteoglycan, z-agrin, is deposited by the nerve. In each case, the synaptic isoform plays key roles in organizing the neuromuscular junction. Here, we analyze the fourth family, composed of nidogen-1 and -2.

Results

In adult muscle, nidogen-1 is present throughout muscle fiber basal lamina, while nidogen-2 is concentrated at synapses. Nidogen-2 is initially present throughout muscle basal lamina, but is lost from extrasynaptic regions during the first three postnatal weeks. Neuromuscular junctions in mutant mice lacking nidogen-2 appear normal at birth, but become topologically abnormal as they mature. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting the phenotype is not secondary to a general defect in the integrity of synaptic basal lamina. Further genetic studies suggest that synaptic localization of each of the four families of synaptic basal lamina components is independent of the other three.

Conclusion

All four core components of the basal lamina have synaptically enriched isoforms. Together, they form a highly specialized synaptic cleft material. Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction.     

Identification of the neuropeptide transmitter proctolin in Drosophila larvae: characterization of muscle fiber-specific neuromuscular endings

The cellular localization of the peptide neurotransmitter proctolin was determined for larvae of the fruitfly Drosophila melanogaster. Proctolin was recovered from the CNS, hindgut, and segmental bodywall using reverse-phase HPLC, and characterized by bioassay, immunoassay, and enzymatic analysis. A small, stereotyped population of proctolin-immunoreactive neurons was found in the larval CNS. Several of the identified neurons may be efferents. In the periphery, proctolin-immunoreactive neuromuscular endings were identified on both visceral and skeletal muscle fibers. On the hindgut, the neuropeptide is associated with endings on intrinsic circular muscle fibers. We propose that the hindgut muscle fibers are innervated by central neurons homologous to previously described proctolinergic efferents of grasshoppers. The segmental bodywall innervation consists of a pattern of segment-specific junctions on several singly identifiable muscle fibers. While it is generally accepted that Drosophila muscle fibers are innervated by glutamatergic motoneurons, our data indicate that a specialized subset of muscle fibers are also innervated by peptidergic efferents.     

A minigene of neural agrin encoding the laminin‐binding and acetylcholine receptor‐aggregating domains is sufficient to induce postsynaptic differentiation in muscle fibres

The extracellular matrix molecule agrin is both necessary and sufficient for inducing the formation of postsynaptic specializations at the neuromuscular junction (NMJ). At the mature NMJ, agrin is stably incorporated in synaptic basal lamina. The postsynapse‐inducing activity of chick agrin, as assayed by its capability of causing aggregation of acetylcholine receptors (AChRs) on cultured muscle cells, maps to a 21 kDa, C‐terminal domain. Binding of chick agrin to muscle basal lamina is mediated by the laminins and maps to a 25 kDa, N‐terminal fragment of agrin. Here we show that an expression construct encoding a 'mini'‐agrin, in which the laminin‐binding fragment was fused to the AChR‐clustering domain, is sufficient to induce postsynaptic differentiation in vivo when injected into non‐synaptic sites of rat soleus muscle. As shown for ectopic postsynaptic differentiation induced by full‐length neural agrin, myonuclei underneath the ectopic sites expressed the gene for the AChR ε‐subunit. Altogether, our data show that a 'mini'‐agrin construct encoding only a small fraction of the entire agrin protein is sufficient to induce postsynapse‐like structures that are reminiscent of those induced by full‐length neural agrin or innervation by motor neurons.     

Novel antigens at the neuromuscular junction

Three novel components of neuromuscular junctions have been identified by use of monoclonal antibodies (McAb) against glycoproteins obtained from a mouse neuroblastoma X human dorsal root ganglion cell hybrid line. Antigen distribution was assessed by fluorescent immunohistochemistry on frozen sections of human intercostal muscle counterstained with labeled alpha-bungarotoxin to identify neuromuscular junctions. Antigen SOS 6 stained exclusively in the neuromuscular junction, whereas antigens SOS 5 and SOS 13 were highly enriched in the junction but also stained extrasynaptic regions. These antigens can be distinguished from previously described components of the neuromuscular junction by their molecular weights, insensitivity to collagenase treatment, and solubility in 0.1% Triton X-100. Indirect evidence suggests that these species-specific antigens are located in the postsynaptic muscle membrane, but location in the junctional basal lamina or subsarcolemmal region cannot be excluded.     

Neuromuscular junction disassembly and muscle fatigue in mice lacking neurotrophin-4.

Neurotrophin-4 (NT-4) is produced by slow muscle fibers in an activity-dependent manner and promotes growth and remodeling of adult motorneuron innervation. However, both muscle fibers and motor neurons express NT-4 receptors, suggesting bidirectional NT-4 signaling at the neuromuscular junction. Mice lacking NT-4 displayed enlarged and fragmented neuromuscular junctions with disassembled postsynaptic acetylcholine receptor (AChR) clusters, reduced AChR binding, and acetylcholinesterase activity. Electromyographic responses, posttetanic potentiation, and action potential amplitude were also significantly reduced in muscle fibers from NT-4 knock-out mice. Slow-twitch soleus muscles from these mice fatigued twice as rapidly as those from wild-type mice during repeated tetanic stimulation. Thus, muscle-derived NT-4 is required for maintenance of postsynaptic AChR regions, normal muscular electrophysiological responses, and resistance to muscle fatigue. This neurotrophin may therefore be a key component of an activity-dependent feedback mechanism regulating maintenance of neuromuscular connections and muscular performance.     

Development of the spinal nerves of the larval lamprey: IV. Spinal nerve roots of 21-mm larval and adult lampreys, with special reference to the relation of meninges with the root sheath and the perineurium

Spinal nerve roots of 21-mm larval and adult lampreys were electron microscopically studied. In 21-mm larval lampreys, each ventral and dorsal rootlet contains axons of various diameters enclosed together as groups in individual troughs of a Schwann cell cytoplasm, lying in direct contact with one another, and is further ensheathed entirely by a basal lamina. Dorsal roots possess visceral axons, while ventral roots lack them. In adult lampreys the ventral and dorsal roots possess individual sheaths for larger somatic axons, each being surrounded by a single Schwann cell and the basal lamina and separated from one another by a considerable amount of connective tissue. Visceral fibers are present in both the dorsal and ventral roots of adult lampreys. They aggregate to form fascicles that lie among somatic axons, being separated from them. Two layers of the meningeal tissue invaginate to form a root sheath around the distal portion of individual dorsal and ventral roots of 21-mm larval lampreys. In adult lampreys the sheath is similarly formed but extends over most of the dorsal and ventral roots. The perineurium is not developed in 21-mm larval lampreys, but is present and ensheaths only the proximal portion of spinal nerve trunks outside the meninges in adult lampreys: it is completely absent along most of the length of peripheral nerves. In both larval and adult lampreys, the outer cell layer of the root sheath is open-ended near the middle of nerve roots with respect to the extramedullary connective tissue space. Similar loosening of the cellular barrier is seen along blood vessels. Thus, the outer meningeal fibrous layer is directly continuous with the extra medullary connective tissue space by way of the inner fibrous layer of the root sheath.     

Miller Fisher syndrome: immunofluorescence and immunoelectron microscopic localization of IgG at the mouse neuromuscular junction

In vitro electrophysiological experiments have demonstrated that IgG antibodies from patients with Miller Fisher syndrome (MFS) impair neuromuscular transmission by a fast and completely reversible combined pre- and postsynaptic blockade. In this study we investigated the cellular and subcellular binding sites of IgG from four MFS patients at the mouse hemidiaphragm by immunofluorescence and immunoelectron microscopy. IgG from all patients produced significant immunostaining at the neuromuscular junction, whereas sera from healthy volunteers or from patients with other neurological diseases did not stain neuromuscular junction. Immunoelectron microscopy revealed that, when living hemidiaphragms were incubated with IgG from MFS patients, labeling was found on both pre- and postsynaptic membranes of the neuromuscular junction, whereas terminal Schwann cells and the basal lamina covering the synaptic membranes were not labeled. These findings demonstrate that IgG from MFS patients binds to synaptic membranes of the neuromuscular junction where it might interfere with the function of both the pre- and postsynaptic activities.     

The membrane morphology of the neuromuscular junction, sarcolemma, sarcoplasmic reticulum and transverse tubule system in murine muscular dystrophy studied by freeze-fracture electron microscopy

‘Dystrophic’ mice of the 129/ReJ-dy strain have a genetic defect in Schwann cell proliferation and neuromuscular junction formation. The presynaptic membrane specialization associated with vesicle fusion and acetylcholine release, as well as the postsynaptic membrane specializations associated with acetylcholine receptivity, appear normal in these animals when visualized with freeze-fracture techniques. However, there is a reduction in the infolding of the postsynaptic membrane, which forms the secondary synaptic cleft at the motor endplate and is the site of acetylcholinesterase activity. The orthogonal arrays of the non-junctional sarcolemma are found on dystrophic muscles, but at lower than normal densities. These observations are made on muscle fibers in which the membrane molecular organization of the sarcoplasmic reticulum and transverse tubule systems appear normal. Several possible linkages between the deficit in myelination and the altered synaptic morphology are discussed in the context of neuromuscular interaction.     

Ultrastructure and innervation of regenerated intrafusal muscle fibres in heterochronous isografts of the fast rat muscle

Extensor digitorum longus (EDL) muscles from 2- to 28-day-old rats were grafted into EDL muscles of adult inbred recipients (n = 8). At 1–6 months after the operation, experimental muscles were excised and the ultrastructure and innervation of regenerated muscle spindles was examined. Regenerated muscle spindles (n = 36) in isografted EDL muscles contained 4.3 ± 0.2 (mean ± SEM) encapsulated muscle fibres. These “intrafusal” muscle fibres lacked nuclear bag and nuclear chain accumulations, which are characteristic of normal muscle spindles; thus, they rather resembled thin encapsulated extrafusal muscle fibres. In the same sample, myelinated axons were found in 33 (92%) muscle spindles, but no sensory terminals were found. These findings demonstrate that regenerated spindles in isografted EDL muscles were not reinnervated by spindle-specific sensory axons, but exclusively by motor axons. Typical intracapsular motor endplates (MEPs) were found in one third of regenerated spindles examined. Their motor terminals contained accumulated mitochondria and synaptic vesicles. As is characteristic for MEPs, axolemma and sarcolemma were separated by a synaptic cleft about 60 nm wide that contained a basal lamina. The underlying sarcolemma formed either small infoldings or none at all, and the subsynaptic area contained only small subsarcolemmal accumulations of mitochondria. It is apparent that the structures described here as “regenerated muscle spindles” do not perform their normal physiological function as stretch receptors because they lack the sensory innervation. The present results show that regeneration and reinnervation in heterochronous isografts corresponds to that previously described in autotransplanted free muscle grafts. The results also show that, during muscle spindle regeneration, intrafusal satellite cells develop into extrafusal-like muscle fibres, apparently due to their motor innervation. Received: 15 June 1999 / Revised, accepted: 20 December 1999     

Neuromuscular junction development in the cutaneous pectoris muscle of Rana catesbeiana

Synaptic specializations were studied in the developing cutaneous pectoris muscle of Rana catesbeiana tadpoles and froglets to correlate nerve terminal morphology (by light and electron microscopy), accumulation of acetylcholine receptors, and the ability of the muscle to contract following nerve stimulation. This correlated approach was used to determine the developmental timing and possible causal relationship of events in nerve and muscle maturation at the neuromuscular junction. Initially, the cutaneous pectoris nerve trunk was present in the undifferentiated presumptive cutaneous pectoris mesenchyme, prior to muscle maturation. At stage XII when the muscle was first able to contract weakly in response to nerve stimulation, the motor nerve terminal endings were simple bulbous enlargements associated with diffuse subneural aggregations of acetylcholine receptors (indicated by diffuse speckles of rhodamine alpha-bungarotoxin fluorescence). Before stage XII no rhodamine alpha-bungarotoxin fluorescence was present anywhere in the muscle. The first stage in the organization of acetylcholine receptors at the neuromuscular junction was the accumulation of diffuse speckles of fluorescence beneath the terminal enlargements. This was followed by the clustering of receptors into small polygonal areas at each synaptic site, and finally the organization of receptors into parallel linear rows. Presumably this final stage was associated with formation of junctional folds. By stage XV the synapses were multiply innervated and had developed acetylcholinesterase activity. The general nerve terminal morphology and pattern of accumulation of acetylcholine receptors at cutaneous pectoris neuromuscular junctions were similar to those of the adult throughout metamorphic climax except that they still contained more than one motor axon. After metamorphic climax, elimination of multiple innervation occurred.     

Differential targeting of components of the dystrophin complex to the postsynaptic membrane

Accumulating evidence points to the participation of dystroglycan in the clustering of nicotinic acetylcholine receptors at the neuromuscular junction [Côtéet al.. (1999) Nature Genet., 3 , 338–342]. Dystroglycan is part of a multimolecular complex, either associated with dystrophin (the dystrophin‐associated protein complex) at the sarcolemma or with utrophin (the utrophin‐associated protein complex) at the neuromuscular junction. Understanding the assembly of this complex at the developing synapse led us to investigate, in Torpedo electrocyte, the intracellular routing and the targeting of several of its components, including dystroglycan, syntrophin, dystrophin and dystrobrevin. We previously demonstrated that acetylcholine receptors and rapsyn, the 43‐kDa receptor‐associated protein at the synapse, are cotargeted to the postsynaptic membrane via the exocytic pathway [Marchand et al.. (2000) J. Neurosci., 20 , 521–528]. Using cell fractionation, immunopurification and immuno‐electron microscope techniques, we show that β‐dystroglycan, an integral glycoprotein that constitutes the core of the dystrophin‐associated protein complex localized at the innervated membrane, is transported together with acetylcholine receptor and rapsyn in post‐Golgi vesicles en route to the postsynaptic membrane. Syntrophin, a peripheral cytoplasmic protein of the complex, associates initially with these exocytic vesicles. Conversely, dystrophin and dystrobrevin were absent from these post‐Golgi vesicles and associate directly with the postsynaptic membrane. This study provides the first evidence for a separate targeting of the various components of the dystrophin‐associated protein complex and a step‐by‐step assembly at the postsynaptic membrane.     

Distinct localization of collagen Q and PRiMA forms of acetylcholinesterase at the neuromuscular junction

Acetylcholinesterase (AChE) terminates the action of acetylcholine at cholinergic synapses thereby preventing rebinding of acetylcholine to nicotinic postsynaptic receptors at the neuromuscular junction. Here we show that AChE is not localized close to these receptors on the postsynaptic surface, but is instead clustered along the presynaptic membrane and deep in the postsynaptic folds. Because AChE is anchored by ColQ in the basal lamina and is linked to the plasma membrane by a transmembrane subunit (PRiMA), we used a genetic approach to evaluate the respective contribution of each anchoring oligomer. By visualization and quantification of AChE in mouse strains devoid of ColQ, PRiMA or AChE, specifically in the muscle, we found that along the nerve terminus the vast majority of AChE is anchored by ColQ that is only produced by the muscle, whereas very minor amounts of AChE are anchored by PRiMA that is produced by motoneurons. In its synaptic location, AChE is therefore positioned to scavenge ACh that effluxes from the nerve by non-quantal release. AChE-PRiMA, produced by the muscle, is diffusely distributed along the muscle in extrajunctional regions.     

Structure and physiology of developing neuromuscular synapses in culture

The structure and function of developing neuromuscular synapses in culture have been investigated. We used neuromuscular junctions formed by coculturing dissociated muscle cells and dissociated neurons obtained from Xenopus embryos. After recording nerve-evoked endplate potentials (e.p.p.s) and spontaneously occurring miniature endplate potentials (m.e.p.p.s) from a given junction, the same specimen was investigated for electron-microscopic histology. We surveyed almost the total area of the junctional region by making serial sections. Even in preparations cocultured for only a short time (4-11 hr), both e.p.p.s and m.e.p.p.s could be obtained. The junctional region of these early synapses revealed a simple structure. The presynaptic terminals contained smooth-surfaced clear vesicles, but there were no presynaptic specializations such as active zones. The width of the synaptic cleft was variable, with predominance of narrow regions (10-30 nm), and there was no basal lamina inside the cleft. When the coculture time was 1 d or longer, the junctional area started to show structural features resembling a mature neuromuscular synapse. In the presynaptic terminal there were active zones, consisting of the presynaptic density and an accumulation of vesicles near the density. In many junctions, the postsynaptic membrane showed densities and thickenings, with a widened synaptic cleft, that contained basal lamina. It is known that growth cones, prior to making neuromuscular junctions, can release the transmitter substance with a very long latency if stimulated repetitively. In contrast, e.p.p.s with short latencies can be evoked by single stimuli soon after the growth cones attach to muscle cells. However, our data did not reveal any structural changes to account for such functional changes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Terminal Schwann cell structure is altered in diaphragm of mdx mice

The diaphragm muscle of the mdx mouse is a model system of Duchenne muscular dystrophy, since it completely lacks dystrophin and shows severe fiber necrosis and loss of specific muscle force by 4-6 weeks of age. Changes in neuromuscular junction structure also become apparent around 4 weeks including postsynaptic acetylcholine receptor declustering, loss of postsynaptic junctional folds, abnormally complex presynaptic nerve terminals, and muscle fiber denervation. Normally, terminal Schwann cells (TSCs) cap both nerve terminals and acetylcholine receptors at the neuromuscular junction, and play a crucial role in regeneration of motor axons following muscle denervation by guiding axons to grow from innervated junctions to nearby denervated junctions. However, their role in restoring innervation in dystrophic muscle is unknown. We now show that TSCs fail to cap fully the neuromuscular junction in dystrophic muscle; TSCs extend processes, but the organization of these extensions is abnormal. TSC processes of dystrophic muscle do not form bridges from denervated fibers to nearby innervated endplates, but appear to be directed away from these endplates. Adequate signaling for TSC reactivity is present, since significant muscle fiber denervation and acetylcholine receptor declustering are present. Thus, significant structural denervation is present in the diaphragm of mdx mice and the ability of TSCs to form bridges between adjacent endplates to guide reinnervation of muscle fibers is impaired, possibly attenuating the ability of dystrophic muscle to recover from denervation and ultimately leading to muscle weakness.