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.     

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.     

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.     

“Disjunction” of Frog Neuromuscular Synapses by Treatment with Proteolytic Enzymes

VERTEBRATE skeletal muscle fibres are surrounded by the ectolemma or basement membrane, a thin sheath of filamentous material. At the neuromuscular junction, the ectolemma occupies the synaptic cleft and is continuous with a similar material which surrounds the axon terminal and its Schwann cell covering¹ (Fig. I A). Changes in the ectolemma of atrophic muscles have been observed^{2, 3}, but little is known about the structure and function of this material. The study described here demonstrates that certain proteolytic enzymes selectively digest the ectolemmal sheath and that concomitantly the motor nerve terminals and their associated Schwann cells dissociate from the muscle fibres.     

Efficacité de la mémoire associative inhérente à la potentiation post-tétanique

An associative memory is modeled in networks of cells that are assumed to have the short-term plasticity of the neuromuscular junction of the frog. The data relating synaptic transmission efficiency and stimulation frequency for post-tetanic potentiation of the neuromuscular junction are represented by polynomial expansions. Simulation of storage and retrieval demonstrates that functional associative memory is feasible based on this particular synaptic plasticity. Retrieval reaches a maximum efficiency at a delay of three minutes after storage and is lost after about 9 min. The signal to noise ratio of the retrieved pattern drops steadily as additional associations are stored in memory but retrieval appears to be possible with up to four stored associations. Although the data are derived from synapses not normally proposed as a basis for memory functions, the results here will generalize to other synaptic junctions located more centrally that have similar characteristics. This simulation technique allows the efficiency of associative memory based on various types of synaptic plasticity to be evaluated.     

Neuronal experience modifies synaptic long-term facilitation

In a crayfish phasic neuromuscular junction, we have demonstrated low-frequency depression (LFD), high-frequency depression (HFD), and long-term facilitation (LTF) in response to different regimens of stimulation. Chronic stimulation of the phasic axon supplying the closer muscle of the claw in Procambarus clarkii resulted in diminished expression of HFD and LTF. Conversely, when impulse production in the phasic motoneuron was reduced by claw immobilization, both HFD and LTF were enhanced. LFD was insensitive to these manipulations. These results provide further evidence for long-term adaptation of the phasic neuromuscular junction to ongoing levels of impulse activity and illustrate the importance of a neuron's past history for synaptic plasticity. The ability of the neuron to adjust its short-term plasticity in response to altered experience constitutes an adaptive response that could be of general significance.     

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.     

Perisynaptic Schwann Cells at the Neuromuscular Synapse: Adaptable,Multitasking Glial Cells

The neuromuscular junction (NMJ) is engineered to be a highly reliable synapse to carry the control of the motor commands of the nervous system over the muscles. Its development, organization, and synaptic properties are highly structured and regulated to support such reliability and efficacy. Yet, the NMJ is also highly plastic, able to react to injury and adapt to changes. This balance between structural stability and synaptic efficacy on one hand and structural plasticity and repair on another hand is made possible by the intricate regulation of perisynaptic Schwann cells, glial cells at this synapse. They regulate both the efficacy and structural plasticity of the NMJ in a dynamic, bidirectional manner owing to their ability to decode synaptic transmission and by their interactions via trophic-related factors.The vertebrate neuromuscular junction (NMJ), arguably the best characterized synapse in the peripheral nervous system (PNS), is composed of three closely associated cellular components: the presynaptic nerve terminal, the postsynaptic specialization, and nonmyelinating Schwann cells. These synapse-associated glial cells are called perisynaptic Schwann cells (PSCs), or terminal Schwann cells (see reviews by Todd and Robitaille 2006; Feng and Ko 2007; Griffin and Thompson 2008; Sugiura and Lin 2011). Multiple roles of PSCs have gained great appreciation since the 1990s and, along with the novel roles of astrocytes in central synapses, have led to the concept of the “tripartite” synapse (Araque et al. 1999, 2014; Volterra et al. 2002; Auld and Robitaille 2003; Kettenmann and Ransom 2013).Thus, to fully understand synaptic formation and function, it is critical to also consider the active and essential roles of synapse-associated glial cells. We will discuss evidence supporting the existence of a synapse–glia–synapse regulatory loop that helps maintain and restore synaptic efficacy at the NMJ. We will also explore the multiple functions that PSCs exert, functions that are adapted to a given situation at the NMJ (e.g., synapse formation, stability, and reinnervation). This will highlight the great adaptability and plasticity of the morphological and functional properties of PSCs.In this review, we will focus on the multiple roles PSCs play in synaptic formation, maintenance, remodeling, and regeneration, as well as synaptic function and plasticity. Based on the evidence presented, we propose a model in which PSCs, through specific receptor activation, play a prominent role in a continuum of synaptic efficacy, stability, and plasticity at the NMJ. These synaptic-regulated functions allow PSCs to orchestrate the stability and plasticity of the NMJ and, hence, are important for maintaining and adapting synaptic efficacy.     

The agrin gene codes for a family of basal lamina proteins that differ in function and distribution.

We isolated two cDNAs that encode isoforms of agrin, the basal lamina protein that mediates the motor neuron-induced aggregation of acetylcholine receptors on muscle fibers at the neuromuscular junction. Both proteins are the result of alternative splicing of the product of the agrin gene, but unlike agrin, they are inactive in standard acetylcholine receptor aggregation assays. They lack one (agrin-related protein 1) or two (agrin-related protein 2) regions in agrin that are required for its activity. Expression studies provide evidence that both proteins are present in the nervous system and muscle and that, in muscle, myofibers and Schwann cells synthesize the agrin-related proteins while the axon terminals of motor neurons are the sole source of agrin.     

Cytokines,growth factors and sprouting at the neuromuscular junction

The formation of neuronal sprouts, either from synaptic terminals or nearby nodes of Ranvier, is a widely known form of plasticity of motoneurons. Sprouts form in response to several stimuli, but most notably in partially denervated or paralyzed muscle. In search of the cellular or molecular basis of this phenomenon, several largely parallel lines of investigation have been pursued. Strong evidence is presented that at least four cytokines or growth factors may be involved in motoneuron sprouting, each of which uses a distinctive signaling pathway. Three of the different proposed sprouting molecules: neuroleukin, insulin-like growth factor, and neural cell adhesion molecules can be viewed as muscle-derived retrograde signaling molecules of roughly equal potency to induce motoneurons to sprout. A fourth molecule, ciliary neurotrophic factor (CNTF) is likely to form an essential anterograde signal, from Schwann cells to muscle fibers, that ultimately produces sprouting. Other cytokines and growth factors such a neurotrophins or GDNF family members are discussed, but their role in motoneuron sprouting is less clear. These cytokines and growth factors could represent redundant mechanisms for self-repair of the neuromuscular junction or they could interact at different levels of their cellular pathways.     

In vivo observations of terminal Schwann cells at normal, denervated, and reinnervated mouse neuromuscular junctions

We found a low-molecular-mass, fluorescent dye, Calcein blue am ester (CB), that labels terminal Schwann cells at neuromuscular junctions in vivo without damaging them. This dye was used to follow terminal Schwann cells at neuromuscular junctions in the mouse sternomastoid muscle over periods of days to months. Terminal Schwann cell bodies and processes were stable in their spatial distribution over these intervals, with processes that in most junctions were precisely aligned with motor nerve terminal branches. Three days after nerve cut, the extensive processes elaborated by terminal Schwann cells in denervated muscle were labeled by CB. The number and length of CB-labeled terminal Schwann cell processes decreased between 3 days and 1 month after denervation, suggesting that terminal Schwann cell processes are only transiently maintained in the absence of innervation. During reinnervation after nerve crush, however, terminal Schwann cell processes were extended in advance of axon sprouts, and these processes persisted until reinnervation was completed. By viewing the same junctions twice during reinnervation, we directly observed that axon sprouts used existing Schwann cell processes and chains of cell bodies as substrates for outgrowth. Thus, CB can be used to monitor the dynamic behavior of terminal Schwann cells, whose interactions with motor axons and their terminals are important for junction homeostasis and repair.     

Activation of Matrix Metalloproteinase-3 is altered at the frog neuromuscular junction following changes in synaptic activity

The extracellular matrix surrounding the neuromuscular junction is a highly specialized and dynamic structure. Matrix Metalloproteinases are enzymes that sculpt the extracellular matrix. Since synaptic activity is critical to the structure and function of this synapse, we investigated whether changes in synaptic activity levels could alter the activity of Matrix Metalloproteinases at the neuromuscular junction. In particular, we focused on Matrix Metalloproteinase 3 (MMP3), since antibodies to MMP3 recognize molecules at the frog neuromuscular junction, and MMP3 cleaves a number of synaptic basal lamina molecules, including agrin. Here we show that the fluorogenic compound (M2300) can be used to perform in vivo proteolytic imaging of the frog neuromuscular junction to directly measure the activity state of MMP3. Application of this compound reveals that active MMP3 is concentrated at the normal frog neuromuscular junction, and is tightly associated with the terminal Schwann cell. Blocking presynaptic activity via denervation, or TTX nerve blockade, results in a decreased level of active MMP3 at the neuromuscular junction. The loss of active MMP3 at the neuromuscular junction in denervated muscles can result from decreased activation of pro-MMP3, or it could result from increased inhibition of MMP3. These results support the hypothesis that changes in synaptic activity can alter the level of active MMP3 at the neuromuscular junction.     

Integration site-dependent expression of a transgene reveals specialized features of cells associated with neuromuscular junctions

After skeletal muscle is denervated, fibroblasts near neuromuscular junctions proliferate more than fibroblasts distant from synaptic sites, and they accumulate adhesive molecules such as tenascin (Gatchalian, C. L., M. Schachner, and J. R. Sanes. 1989. J. Cell Biol. 108:1873-1890). This response could reflect signals that arise perisynaptically after denervation, preexisting differences between perisynaptic and extrasynaptic fibroblasts, or both. Here, we describe a line of transgenic mice in which patterns of transgene expression provide direct evidence for differences between perisynaptic and extrasynaptic fibroblasts in normal muscle. Transgenic mice were generated using regulatory elements from a major histocompatibility complex (MHC) class I gene linked to the Escherichia coli beta-galactosidase (lacZ) gene. Expression of lacZ was detected histochemically. In each of eight lines, lacZ was detected in different subsets of cells, none of which included lymphocytes. In contrast, endogenous MHC is expressed in most tissues and at high levels in lymphocytes. Thus, the MHC gene sequences appeared inactive in the transgene, and lacZ expression was apparently controlled by genomic regulatory elements that were specific for the insertion site. In one line, cells close to the neuromuscular junction were lacZ positive in embryonic and young postnatal mice. Electron microscopy identified these cells as fibroblasts and Schwann cells associated with motor nerve terminals, as well as endoneurial fibroblasts, perineurial cells, and Schwann cells in the distal branches of motor nerves. No intramuscular cells greater than 200 microns from synaptic sites were lacZ positive. These results indicate that there are molecular differences between perisynaptic and extrasynaptic fibroblasts even in normal muscle and that diverse perisynaptic cell types share a specific pattern of gene expression.     

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.     

Modulation of Synaptic Vesicle Exocytosis in Muscle-Dependent Long-Term Depression at the Amphibian Neuromuscular Junction

We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry.     

Axon branch removal at developing synapses by axosome shedding

In many parts of the developing nervous system, the number of axonal inputs to each postsynaptic cell is dramatically reduced. This synapse elimination has been extensively studied at the neuromuscular junction, but how axons are lost is unknown. Here, we combine time-lapse imaging of fluorescently labeled axons and serial electron microscopy to show that axons at neuromuscular junctions are removed by an unusual cellular mechanism. As axons disappear, they shed numerous membrane bound remnants. These "axosomes" contain a high density of synaptic organelles and are formed by engulfment of axon tips by Schwann cells. After this engulfment, the axosome's contents mix with the cytoplasm of the glial cell. Axosome shedding might underlie other forms of axon loss and may provide a pathway for interactions between axons and glia.     

The Cytoplasmic Domain of Rat Synaptotagmin I Enhances Synaptic Transmission

Synaptotagmin, an integral membrane protein of synaptic vesicles, functions as a calcium sensor in the temporal control of neurotransmitter release. Although synaptotagmin facilitates lipid membrane fusion in biochemical experiments, overexpression of synaptotagmin inhibits neurotransmission. A facilitatory effect of synaptotagmin on synaptic transmission was never observed. To determine whether synaptotagmin may accelerate synaptic transmission in vivo, we injected the cytoplasmic domain of rat synaptotagmin I (CD-syt) into crayfish motor axons and tested the effect of CD-syt on synaptic response. We confirmed that CD-syt accelerates neuromuscular transmission. The injected preparation had larger synaptic potentials with shorter rise time. Experiments with varying calcium concentrations showed that CD-syt increased the maximum synaptic response of the neuromuscular synapses. Further tests on short-term plasticity of neuromuscular synapses revealed that CD-syt increases the release probability of the release-ready vesicles.     

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.