Distribution of N-CAM in synaptic and extrasynaptic portions of developing and adult skeletal muscle

Previous studies of denervated and cultured muscle have shown that the expression of the neural cell adhesion molecule (N-CAM) in muscle is regulated by the muscle's state of innervation and that N-CAM might mediate some developmentally important nerve-muscle interactions. As a first step in learning whether N-CAM might regulate or be regulated by nerve-muscle interactions during normal development, we have used light and electron microscopic immunohistochemical methods to study its distribution in embryonic, perinatal, and adult rat muscle. In embryonic muscle, N-CAM is uniformly present on the surface of myotubes and in intramuscular nerves; N-CAM is also present on myoblasts, both in vivo and in cultures of embryonic muscle. N-CAM is lost from the nerves as myelination proceeds, and from myotubes as they mature. The loss of N-CAM from extrasynaptic portions of the myotube is a complex process, comprising a rapid rearrangement as secondary myotubes form, a phase of decline late in embryogenesis, a transient reappearance perinatally, and a more gradual disappearance during the first two postnatal weeks. Throughout embryonic and perinatal life, N-CAM is present at similar levels in synaptic and extrasynaptic regions of the myotube surface. However, N-CAM becomes concentrated in synaptic regions postnatally: it is present in postsynaptic and perisynaptic areas of the muscle fiber, both on the surface and intracellularly (in T-tubules), but undetectable in portions of muscle fibers distant from synapses. In addition, N-CAM is present on the surfaces of motor nerve terminals and of Schwann cells that cap nerve terminals, but absent from myelinated portions of motor axons and from myelinating Schwann cells. Thus, in the adult, N-CAM is present in synaptic but not extrasynaptic portions of all three cell types that comprise the neuromuscular junction. The times and places at which N-CAM appears are consistent with its playing several distinct roles in myogenesis, synaptogenesis, and synaptic maintenance, including alignment of secondary along primary myotubes, early interactions of axons with myotubes, and adhesion of Schwann cells to nerve terminals.     

Neuron-glia interactions: the roles of Schwann cells in neuromuscular synapse formation and function

The NMJ (neuromuscular junction) serves as the ultimate output of the motor neurons. The NMJ is composed of a presynaptic nerve terminal, a postsynaptic muscle and perisynaptic glial cells. Emerging evidence has also demonstrated an existence of perisynaptic fibroblast-like cells at the NMJ. In this review, we discuss the importance of Schwann cells, the glial component of the NMJ, in the formation and function of the NMJ. During development, Schwann cells are closely associated with presynaptic nerve terminals and are required for the maintenance of the developing NMJ. After the establishment of the NMJ, Schwann cells actively modulate synaptic activity. Schwann cells also play critical roles in regeneration of the NMJ after nerve injury. Thus, Schwann cells are indispensable for formation and function of the NMJ. Further examination of the interplay among Schwann cells, the nerve and the muscle will provide insights into a better understanding of mechanisms underlying neuromuscular synapse formation and function.     

Localization and characterization of nitric oxide synthase at the frog neuromuscular junction

Summary. The frog neuromuscular junction is sensitive to nitric oxide (NO), since exogenously applied NO reduces the release of transmitter by presynaptic terminals and the size of ATP-induced Ca²⁺ responses in perisynaptic Schwann cells. This study aimed at determining whether an NO synthase (NOS) is present at the neuromuscular junction, notably in perisynaptic Schwann cells, the glial cells at this synapse. The NADPH-diaphorase (NADPH-d) histochemical technique revealed the presence of NOS in cell bodies and presumed processes of perisynaptic Schwann cells. Incubation with NOS inhibitors, N^G-nitro-L-arginine methyl ester or N^G-monomethyl-L-arginine-acetate, abolished the NADPH-d staining. Moreover, L-arginine, the precursor of NO, impeded the blockade by NOS inhibitors, establishing the NOS specificity of NADPH-d staining in frog tissue. The pattern of labelling with a polyclonal antibody against the neuronal form of NOS was similar to the NADPH-d staining, also suggesting the presence of a neuronal NOS in perisynaptic Schwann cells. Using electron microscopy, the NOS immunostaining was found at the membrane and occasionally in the cytoplasm of perisynaptic Schwann cells and was not detected in the nerve terminal or muscle. There was no enzymatic or immunocytochemical labelling of NOS 6 days after denervation. It is concluded that NOS is present in frog perisynaptic Schwann cells. The presence of this endogenous NOS suggests that NO may act as a diffusible glial messenger to modulate synaptic activity and synapse formation at the neuromuscular junction.     

Mechanisms controlling axonal sprouting at the neuromuscular junction

This review considers the relative roles of sprouting stimuli, perisynaptic Schwann cells and neuromuscular activity in axonal sprouting at the neuromuscular junction in partially denervated muscles. A number of sprouting stimuli, including insulin-like growth factor II, which are generated from inactive muscle fibers in partially denervated and paralyzed skeletal muscles, has been considered. There is also evidence that perisynaptic Schwann cells induce and guide axonal sprouting in adult partially denervated muscles. Excessive neuromuscular activity significantly reduces bridging of perisynaptic Schwann cell processes between innervated and denervated endplates and thereby inhibits axonal sprouting in partially denervated adult muscles. Elimination of neuromuscular activity is also detrimental to sprouting in these muscles, suggesting that calcium influx into the nerve is crucial for axonal sprouting. The role of neuromuscular activity in axonal sprouting will be considered critically in the context of the roles of sprouting stimuli and perisynaptic Schwann cells in the process of axonal sprouting.     

Overexpression of the CT GalNAc transferase in skeletal muscle alters myofiber growth, neuromuscular structure, and laminin expression.

Carbohydrates have been shown to mediate or modulate a number of important events in the development of the nervous system; however, there is little evidence that they participate directly in the development of synapses. One carbohydrate structure that is likely to be important in synaptic development of the neuromuscular junction is the CT carbohydrate antigen [GalNAcbeta1,4[NeuAcalpha2,3]Galbeta1(-3GalNAc or -4GlcNAc)]. The synaptic localization of the CT antigen is due to the presence of the terminal beta1,4 GalNAc linkage, and such linkages are localized to the neuromuscular junction in many species. Here we show that an enzyme that can create the synaptic CT structure, the CT GalNAc transferase, is also confined to the neuromuscular junction in mice. Using transgenic mice, we show that overexpression of the CT GalNAc transferase in extrasynaptic regions in skeletal myofibers caused as much as a 60% reduction in the diameter of adult myofibers and an order of magnitude increase in satellite cells. Neuromuscular junctions of transgenic mice had severely reduced numbers of secondary folds, Schwann cell processes were present in the synaptic cleft, and secondary folds were often misaligned with active zones. In addition, multiple presynaptic specializations occurred on individual myofibers. In addition, some normally synaptic proteins, including laminin alpha4, laminin alpha5, utrophin, and NCAM, were expressed along extrasynaptic regions of myofibers. One of the muscle proteins that displayed increased glycosylation with the CT antigen in the transgenic mice was alpha-dystroglycan. These experiments provide the first in vivo evidence that a synaptic carbohydrate antigen has important roles in the development of the neuromuscular synapse and suggest that the CT antigen is involved in controlling the expression of synaptic molecules.     

Antibodies that bind specifically to synaptic sites on muscle fiber basal lamina

Basal lamina (BL) ensheathes each skeletal muscle fiber and passes through the synaptic cleft at the neuromuscular junction. Synaptic portions of the BL are known to play important roles in the formation, function, and maintenance of the neuromuscular junction. Here we demonstrate molecular differences between synaptic and extrasynaptic BL. We obtained antisera to immunogens that might be derived from or share determinants with muscle fiber BL, and used immunohistochemical techniques to study the binding of antibodies to rat skeletal muscle. Four antisera contained antibodies that distinguished synaptic from extrasynaptic portions of the muscle fiber's surface. They were anti- anterior lens capsule, anti-acetylcholinesterase, anti-lens capsule collagen, and anti-muscle basement membrane collagen; the last two sera were selective only after antibodies binding to extrasynaptic areas had been removed by adsorption with connective tissue from endplate-free regions of muscle. Synaptic antigens revealed by each of the four sera were present on the external cell surface and persisted after removal of nerve terminal. Schwann cell, and postsynaptic plasma membrane. Thus, the antigens are contained in or connected to BL of the synaptic cleft. Details of staining patterns, differential susceptibility of antigens to proteolysis, and adsorption experiments showed that the antibodies define at least three different determinants that are present in synaptic but not extrasynaptic BL.     

Activity-dependent switch from synapse formation to synapse elimination during development of neuromuscular junctions

The embryonic development of neuromuscular junctions consists of two successive epochs, an early period marked by exuberant synapse formation and a later period marked by synapse elimination. In the frog muscles we have studied, myogenesis is protracted and overlaps the periods of synapse formation and elimination. Thus, the formative and regressive events of synaptic development do not occur in synchrony across different fibers in the muscle. We propose that local activity orchestrates a shift from synaptogenesis to synapse elimination at the level of single muscle fibers. We also present evidence that perisynaptic Schwann cells and the expression of ion channels in the sarcolemma play important roles in the development of neuromuscular junctions. Questions for future study are outlined.     

Fibroblasts that proliferate near denervated synaptic sites in skeletal muscle synthesize the adhesive molecules tenascin(J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan

Four adhesive molecules, tenascin(J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan, accumulate in interstitial spaces near synaptic sites after denervation of rat skeletal muscle (Sanes, J. R., M. Schachner, and J. Covault. 1986. J. Cell Biol. 102:420-431). We have now asked which cells synthesize these molecules, and how this synthesis is regulated. Electron microscopy revealed that mononucleated cells selectively accumulate in perisynaptic interstitial spaces beginning 2 d after denervation. These cells were identified as fibroblasts by ultrastructural and immunohistochemical criteria; [3H]thymidine autoradiography revealed that their accumulation results from local proliferation. Electron microscopic immunohistochemistry demonstrated that N-CAM is associated with the surface of the fibroblasts, while tenascin(J1) is associated with collagen fibers that abut fibroblasts. Using immunofluorescence and immunoprecipitation methods, we found that fibroblasts isolated from perisynaptic regions of denervated muscle synthesize N-CAM, tenascin(J1), fibronectin, and a heparan sulfate proteoglycan in vitro. Thus, fibroblasts that selectively proliferate in interstitial spaces near synaptic sites are likely to be the cellular source of the interstitial deposits of adhesive molecules in denervated muscle. To elucidate factors that might regulate the accumulation of these molecules in vivo, we analyzed the expression of tenascin(J1) and fibronectin by cultured fibroblasts. Fibroblasts from synapse-free regions of denervated muscle, as well as skin, lung, and 3T3 fibroblasts accumulate high levels of tenascin(J1) and fibronectin in culture, showing that perisynaptic fibroblasts are not unique in this regard. However, when they are first placed in culture, fibroblasts from denervated muscle bear more tenascin(J1) than fibroblasts from innervated muscle, indicating that expression of this molecule by fibroblasts is regulated by the muscle's state of innervation; this difference is no longer apparent after a few days in culture. In 3T3 cells, accumulation of tenascin(J1) is high in proliferating cultures, depressed in confluent cultures, and reactivated in cells stimulated to proliferate by replating at low density or by wounding a confluent monolayer. Thus, synthesis of tenascin(J1) is regulated in parallel with mitotic activity. In contrast, levels of fibronectin, which increase less dramatically after denervation in vivo, are similar in fibroblasts from innervated and denervated muscle and in proliferating and quiescent 3T3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

A Schwann cell matrix component of neuromuscular junctions and peripheral nerves

Molecules localized to the synapse are potential contributors to processes unique to this specialized region, such as synapse formation and maintenance and synaptic transmission. We used an immunohistochemical strategy to uncover such molecules by generating antibodies that selectively stain synaptic regions and then using the antibodies to analyse their antigens. In this study, we utilized a monoclonal antibody, mAb 6D7, to identify and characterize an antigen concentrated at frog neuromuscular junctions and in peripheral nerves. In adult muscle, immunoelectron microscopy indicates that the antigen is located in the extracellular matrix around perisynaptic Schwann cells at the neuromuscular junction and in association with myelinated and nonmyelinated axons in peripheral nerves. The maintenance of the mAb 6D7 epitope is innervation-dependent but is muscle-independent; it disappears from the synaptic region within 2 weeks after denervation, but persists after muscle damage when the nerve is left intact. mAb 6D7 immunolabelling is also detected at the neuromuscular junction in developing tadpoles. Biochemical analyses of nerve extracts indicate that mAb 6D7 recognizes a glycoprotein of 127 kDa with both N- and O-linked carbohydrate moieties. Taken together, the results suggest that the antigen recognized by mAb 6D7 may be a novel component of the synaptic extracellular matrix overlying the terminal Schwann cell. The innervation-sensitivity of the epitope at the neuromuscular junction suggests a function in the interactions between nerves and Schwann cells.     

Transmitter release increases intracellular calcium in perisynaptic Schwann cells in situ.

Glial cells isolated from the nervous system are sensitive to neurotransmitters and may therefore be involved in synaptic transmission. The sensitivity of individual perisynaptic Schwann cells to activity of a single synapse was investigated, in situ, at the frog neuromuscular junction by monitoring changes in intracellular Ca2+ in the Schwann cells. Motor nerve stimulation induced an increase in intracellular Ca2+ in these Schwann cells; this increase was greatly reduced when transmitter release was blocked. Furthermore, local application of the cotransmitters acetylcholine and ATP evoked Ca2+ responses even in the absence of extracellular Ca2+. Successive trains of nerve stimuli or applications of transmitters resulted in progressively smaller Ca2+ responses. We conclude that transmitter released during synaptic activity can evoke release of intracellular Ca2+ in perisynaptic Schwann cells. This Ca2+ signal may play a role in the maintenance or modulation of a synapse. These data show that synaptic transmission involves three cellular components with both postsynaptic and glial components responding to transmitter secretion.     

Modulation of neurotransmission by reciprocal synapse-glial interactions at the neuromuscular junction

Perisynaptic Schwann cells are glial cells that are closely associated with pre- and postsynaptic elements of the neuromuscular junction. Recent evidence shows that these cells detect and modulate neurotransmission in an activity-dependent fashion. Through G-protein signalling and Ca²⁺ released from internal stores they can decrease or increase neurotransmitter release, respectively. Thus, they help to establish the level of neurotransmission associated with activity dependent short-term synaptic plasticity. We discuss evidence implicating perisynaptic Schwann cells as being active partners in neurotransmission at the neuromuscular junction, with emphasis on the modulation of short-term plasticity and potential implications for long-term changes.     

Motor unit abnormalities in Dystonia musculorum mice

Dystonia musculorum (dt) is a mouse inherited sensory neuropathy caused by mutations in the dystonin gene. While the primary pathology lies in the sensory neurons of dt mice, the overt movement disorder suggests motor neurons may also be affected. Here, we report on the contribution of motor neurons to the pathology in dt(27J) mice. Phenotypic dt(27J) mice display reduced alpha motor neuron cell number and eccentric alpha motor nuclei in the ventral horn of the lumbar L1 spinal cord region. A dramatic reduction in the total number of motor axons in the ventral root of postnatal day 15 dt(27J) mice was also evident. Moreover, analysis of the trigeminal nerve of the brainstem showed a 2.4 fold increase in number of degenerating neurons coupled with a decrease in motor neuron number relative to wild type. Aberrant phosphorylation of neurofilaments in the perikaryon region and axonal swellings within the pre-synaptic terminal region of motor neurons were observed. Furthermore, neuromuscular junction staining of dt(27J) mouse extensor digitorum longus and tibialis anterior muscle fibers showed immature endplates and a significant decrease in axon branching compared to wild type littermates. Muscle atrophy was also observed in dt(27J) muscle. Ultrastructure analysis revealed amyelinated motor axons in the ventral root of the spinal nerve, suggesting a possible defect in Schwann cells. Finally, behavioral analysis identified defective motor function in dt(27J) mice. This study reveals neuromuscular defects that likely contribute to the dt(27J) pathology and identifies a critical role for dystonin outside of sensory neurons.