Reciprocal interactions between perisynaptic Schwann cells and regenerating nerve terminals at the frog neuromuscular junction

The perisynaptic Schwann cell (PSC) has gained recent attention with respect to its roles in synaptic function, remodeling, and regeneration at the vertebrate neuromuscular junction (NMJ). Here we test the hypothesis that, following nerve injury, processes extended by PSCs guide regenerating nerve terminals (NTs) in vivo, and that the extension of sprouts by PSCs is triggered by the arrival of regenerating NTs. Frog NMJs were double-stained with a fluorescent dye, FM4-64, for NTs, and fluorescein isothiocyanate (FITC)-tagged peanut agglutinin (PNA) for PSCs. Identified NMJs were imaged in vivo repeatedly for several months after nerve injury. PSCs sprouted profusely beginning 3-4 weeks after nerve transection and, as reinnervation progressed, regenerating NTs closely followed the preceding PSC sprouts, which could extend tens to hundreds of microns beyond the original synaptic site. The pattern of reinnervation was dictated by PSC sprouts, which could form novel routes joining neighboring junctions or develop into new myelinated axonal pathways. In contrast to mammals, profuse PSC sprouting in frog muscles was not seen in response to axotomy alone, and did not occur at chronically denervated NMJs. Instead, sprouting coincided with the arrival of regenerating NTs. Immunofluorescent staining revealed that in muscle undergoing reinnervation 4 weeks after axotomy, 91% of NMJs bore PSC sprouts, compared to only 6% of NMJs in muscle that was chronically denervated for 4 weeks. These results suggest that reciprocal interactions between regenerating NTs and PSCs govern the process of reinnervation at frog NMJs: regenerating NTs induce PSCs to sprout, and PSC sprouts, in turn, lead and guide the elaboration of NTs.     

The role of perisynaptic Schwann cells in development of neuromuscular junctions in the frog (Xenopus laevis)

Fluorescence microscopy was used to study the behavior of perisynaptic Schwann cells (PSCs) in relation to motor nerve terminals and postsynaptic clusters of acetylcholine receptors, during the development of the neuromuscular junction (NMJ) in the frog Xenopus laevis. Pectoral (supracoracoideus) muscles were labeled with monoclonal antibody 2A12 for Schwann cells, the dye FM4-64 for nerve terminals (NTs), alpha-bungarotoxin for acetylcholine receptors (AChRs), and Hoechst 33258 for cellular nuclei, in animals from tadpole stage 57 to fully grown adults. When muscle fibers first appeared in stage 57, NMJs consisted of tightly apposed NTs and AChRs and were only partially covered with PSCs or their processes. Within a few stages, PSCs fully occupied and overgrew the NMJs, extending fine sprouts between a few micrometers and hundreds of micrometers beyond the borders of the junction. Sprouts of PSCs were most abundant during the time when secondary myogenesis, synaptogenesis, and synaptic growth occurred at their highest rates. PSCs were recruited to NMJs during synaptic growth, at rates between 1.3 PSCs/100 microm junctional length early on and 0.4 PSCs/100 microm later. Shortly after metamorphosis, PSC sprouts disappeared and NMJs acquired the adult appearance, in which PSCs, NTs, and AChRs were mostly congruent. The results suggest that, although PSCs may not be required for initial nerve-muscle contacts, PSCs sprouts lead synaptic growth and play a role in the extension and maturation of developing NMJs.     

Roles of glial cells in the formation,function, and maintenance of the neuromuscular junction

Like other vertebrate synapses, the neuromuscular junction (NMJ) has glial cells that are closely associated with the pre- and post-synaptic components. These “perisynaptic Schwann cells” (PSCs) cover nerve terminals and are in close proximity to the synapse, yet their role at the NMJ has remained mysterious for decades. In this review we explore historical perspectives on PSCs and highlight key developments in recent years that have provided novel insight into PSC functions at the NMJ. First among these developments is the generation of specific antibody probes for PSCs. Using one such antibody and the principle of complement-mediated cell lysis, we have developed a novel technique to selectively ablate PSCs en masse from frog NMJs in vivo. Applying this approach, we have shown that PSCs are essential for the long-term maintenance of synaptic structure and function. In addition, PSCs are essential for the growth and maintenance of NMJs during development. Probes for PSCs also allow us to observe in vivo that processes extended by PSCs guide nerve terminals during synapse development, remodeling, and regeneration. PSCs may therefore dictate the pattern of innervation at the NMJ. Finally, PSCs may also induce postsynaptic acetylcholine receptor expression and aggregation. This wealth of recent findings about PSCs suggests that these synapse-associated glial cells are a more integral and essential component of the NMJ than previously appreciated. New approaches currently being applied at the NMJ may further support the emerging view that glial cells help make bigger, stronger, and more stable synapses.     

Glial cells maintain synaptic structure and function and promote development of the neuromuscular junction in vivo

To investigate the in vivo role of glial cells in synaptic function, maintenance, and development, we have developed an approach to selectively ablate perisynaptic Schwann cells (PSCs), the glial cells at the neuromuscular junction (NMJ), en masse from live frog muscles. In adults, following acute PSC ablation, synaptic structure and function were not altered. However, 1 week after PSC ablation, presynaptic function decreased by approximately half, while postsynaptic function was unchanged. Retraction of nerve terminals increased over 10-fold at PSC-ablated NMJs. Furthermore, nerve-evoked muscle twitch tension was reduced. In tadpoles, repeated in vivo observations revealed that PSC processes lead nerve terminal growth. In the absence of PSCs, growth and addition of synapses was dramatically reduced, and existing synapses underwent widespread retraction. Our findings provide in vivo evidence that glial cells maintain presynaptic structure and function at adult synapses and are vital for the growth and stability of developing synapses.     

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.     

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.     

Hypothalamic Sirt1 protects terminal Schwann cells and neuromuscular junctions from age‐related morphological changes

Neuromuscular decline occurs with aging. The neuromuscular junction (NMJ), the interface between motor nerve and muscle, also undergoes age‐related changes. Aging effects on the NMJ components—motor nerve terminal, acetylcholine receptors (AChRs), and nonmyelinating terminal Schwann cells (tSCs)—have not been comprehensively evaluated. Sirtuins delay mammalian aging and increase longevity. Increased hypothalamic Sirt1 expression results in more youthful physiology, but the relationship between NMJ morphology and hypothalamic Sirt1 was previously unknown. In wild‐type mice, all NMJ components showed age‐associated morphological changes with ~80% of NMJs displaying abnormalities by 17 months of age. Aged mice with brain‐specific Sirt1 overexpression (BRASTO) had more youthful NMJ morphologic features compared to controls with increased tSC numbers, increased NMJ innervation, and increased numbers of normal AChRs. Sympathetic NMJ innervation was increased in BRASTO mice. In contrast, hypothalamic‐specific Sirt1 knockdown led to tSC abnormalities, decreased tSC numbers, and more denervated endplates compared to controls. Our data suggest that hypothalamic Sirt1 functions to protect NMJs in skeletal muscle from age‐related changes via sympathetic innervation.     

Neuromuscular activity impairs axonal sprouting in partially denervated muscles by inhibiting bridge formation of perisynaptic Schwann cells

Following partial denervation of rat hindlimb muscle, terminal Schwann cells extend processes from denervated endplates to induce and guide sprouting from the remaining intact axons. Increased neuromuscular activity significantly reduces motor unit enlargement and sprouting during the acute phase of sprouting. These findings led to the hypothesis that increased neuromuscular activity perturbs formation of Schwann cell bridges and thereby reduces sprouting. Adult rat tibialis anterior (TA) muscles were extensively denervated by avulsion of L4 spinal root and were immediately subjected to normal caged activity or running exercise (8 h daily) for 3, 7, 14, 21, and 28 days. Combined silver/cholinesterase histochemical staining revealed that the progressive reinnervation of denervated endplates by sprouts over a 1 month period in the extensively partially denervated TA muscles was completely abolished by increased neuromuscular activity. Immunohistochemical staining and triple immunofluorescence revealed that the increased neuromuscular activity did not perturb the production of Schwann cell processes, but prevented bridging between Schwann cell processes at innervated and denervated endplates. Our findings suggest that failure of Schwann cell processes to bridge between endplates accounts, at least in part, for the inhibitory effect of increased neuromuscular activity on sprouting.     

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.     

The signaling pathways mediated by P2Y nucleotide receptors in the formation and maintenance of the skeletal neuromuscular junction

The motor neuron, the Schwann cell and the muscle cell are highly specialized at the vertebrate skeletal neuromuscular junction (NMJ). The muscle cell surface contains a high local density of acetylcholine (ACh) receptors (AChRs), acetylcholinesterase (AChE) and their interacting macromolecules at the NMJ, forming the postsynaptic specializations. During the early stages of development, the incoming nerve terminal induces the formation of these postsynaptic specializations; the nerve secretes agrin and neuregulin (NRG), which are known to aggregate existing AChRs and to increase the expression of AChR at the synaptic region, respectively. In addition, adenosine 5'-triphosphate (ATP) is stored at the motor nerve terminals and is coreleased with ACh during muscle contraction. Recent evidence suggests that ATP can play a role in forming and maintaining the postsynaptic specializations by activating its corresponding receptors. In particular, one of the nucleotide receptor subtypes, the P2Y(1) receptor, is specifically localized at the NMJs. The gene expression of AChR and AChE is upregulated after the activation of P2Y(1) receptors. Thus, the synaptic ATP together with agrin and NRG can act as a synapse-organizing factor to induce the expression of postsynaptic functional effectors.     

Strategic location of calcium channels at transmitter release sites of frog neuromuscular synapses

The localization of Ca2+ channels relative to the position of transmitter release sites was investigated at the frog neuromuscular junction (NMJ). Ca2+ channels were labeled with fluorescently tagged omega-conotoxin GVIA, an irreversible Ca2+ channel ligand, and observed with a confocal laser scanning microscope. The Ca2+ channel labeling almost perfectly matched that of acetylcholine receptors which were labeled with fluorescent alpha-bung-arotoxin. This indicates that groups of Ca2+ channels are localized exclusively at the active zones of the frog NMJ. Cross sections of NMJs showed that Ca2+ channels are clustered on the presynaptic membrane adjacent to the postsynaptic membrane.     

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.     

Developmental and adult-specific processes contribute to de novo neuromuscular regeneration in the lizard tail

Peripheral nerves exhibit robust regenerative capabilities in response to selective injury among amniotes, but the regeneration of entire muscle groups following volumetric muscle loss is limited in birds and mammals. In contrast, lizards possess the remarkable ability to regenerate extensive de novo muscle after tail loss. However, the mechanisms underlying reformation of the entire neuromuscular system in the regenerating lizard tail are not completely understood. We have tested whether the regeneration of the peripheral nerve and neuromuscular junctions (NMJs) recapitulate processes observed during normal neuromuscular development in the green anole, Anolis carolinensis. Our data confirm robust axonal outgrowth during early stages of tail regeneration and subsequent NMJ formation within weeks of autotomy. Interestingly, NMJs are overproduced as evidenced by a persistent increase in NMJ density 120 and 250 days post autotomy (DPA). Substantial Myelin Basic Protein (MBP) expression could also be detected along regenerating nerves indicating that the ability of Schwann cells to myelinate newly formed axons remained intact. Overall, our data suggest that the mechanism of de novo nerve and NMJ reformation parallel, in part, those observed during neuromuscular development. However, the prolonged increase in NMJ number and aberrant muscle differentiation hint at processes specific to the adult response. An examination of the coordinated exchange between peripheral nerves, Schwann cells, and newly synthesized muscle of the regenerating neuromuscular system may assist in the identification of candidate molecules that promote neuromuscular recovery in organisms incapable of a robust regenerative response.     

Assembly,plasticity and selective vulnerability to disease of mouse neuromuscular junctions

Although physiological differences among neuromuscular junctions (NMJs) have long been known, NMJs have usually been considered as one type of synapse, restricting their potential value as model systems to investigate mechanisms controlling synapse assembly and plasticity. Here we discuss recent evidence that skeletal muscles in the mouse can be subdivided into two previously unrecognized subtypes, designated FaSyn and DeSyn muscles. These muscles differ in the pattern of neuromuscular synaptogenesis during embryonic development. Differences between classes are intrinsic to the muscles, and manifest in the absence of innervation or agrin. The distinct rates of synaptogenesis in the periphery may influence processes of circuit maturation through retrograde signals. While NMJs on FaSyn and DeSyn muscles exhibit a comparable anatomical organization in postnatal mice, treatments that challenge synaptic stability result in nerve sprouting, NMJ remodeling, and ectopic synaptogenesis selectively on DeSyn muscles. This anatomical plasticity of NMJs diminishes greatly between 2 and 6 months postnatally. NMJs lacking this plasticity are lost selectively and very early on in mouse models of motoneuron disease, suggesting that disease-associated motoneuron dysfunction may fail to initiate maintenance processes at “non-plastic” NMJs. Transgenic mice overexpressing growth-promoting proteins in motoneurons exhibit greatly enhanced stimulus-induced sprouting restricted to DeSyn muscles, supporting the notion that anatomical plasticity at the NMJ is primarily controlled by processes in the postsynaptic muscle. The discovery that entire muscles in the mouse differ substantially in the anatomical plasticity of their synapses establishes NMJs as a uniquely advantageous experimental system to investigate mechanisms controlling synaptic rearrangements at defined synapses in vivo.     

Intense ultraterminal sprouting from motor nerves and ultrastructural aspects of the neuromuscular junction and non-junctional sarcolemma of the soleus (slow-twitch) muscle in motor endplate disease in the mouse

Motor end-plate disease (med) in the mouse is an hereditary defect of the neuromuscular system, with partial functional denervation and muscle inactivity in late stages of the disease. Motor end-plate disease is characterized by an intense ultraterminal sprouting of the motor nerves from swollen nerve terminal branches in the soleus muscle. At the ultrastructural level, the neuromuscular junctions extend to very wide territories, often outside the original motor end-plate, in regions where the nerve sprouts are in simple apposition to the muscle fiber, with no secondary synaptic folds. The nerve terminals are rich in neurofilaments and poor in synaptic vesicles.Freeze fracture analysis of the pre-synaptic and post-synaptic membrane specializations fails to reveal any important structural alteration which could suggest a defect in acetylcholine release or in muscle membrane excitability. However, the non-junctional sarcolemmal specializations (the so-called ‘square arrays’) arc found with a frequency slightly higher than in normal muscle.The nerve abnormalities at the neuromuscular junction may be either a consequence of muscle inactivity or the morphological expression of some primary nerve abnormality. Further studies of the soleus muscle at early stages of the disease may provide evidence in favor of either possibility.     

Terminal Schwann cells guide the reinnervation of muscle after nerve injury

Schwann cells and axons labeled by transgene-encoded, fluorescent proteins can be repeatedly imaged in living mice to observe the reinnervation of neuromuscular junctions. Axons typically return to denervated junctions by growing along Schwann cells contained in the old nerve sheaths or “Schwann cell tubes”. These axons then commonly “escape” the synaptic sites by growing along the Schwann cell processes extended during the period of denervation. These “escaped fibers” grow to innervate adjacent synaptic sites along Schwann cells bridging these sites. Within the synaptic site, Schwann cells, originally positioned above the synaptic site continue to cover the acetylcholine receptors (AChRs) immediately following denervation, but gradually vacate portions of this site. When regenerating axons return, they first deploy along the Schwann cells and ignore sites of AChRs vacated by Schwann cells. In many cases these vacated sites are never reinnervated and are ultimately lost. Following partial denervation, Schwann cells grow in an apparently tropic fashion from denervated to nearby innervated synaptic sites and serve as the substrates for nerve sprouting. These experiments show that Schwann cells provide pathways that stimulate axon growth and insure the rapid reinnervation of denervated or partially denervated muscles.     

Botulinum neurotoxins: from paralysis to recovery of functional neuromuscular transmission.

The neuromuscular junction is one of the most accessible mammalian synapses which offers a useful model to study long-term synaptic modifications occurring throughout life. It is also the natural target of botulinum neurotoxins (BoNTs) causing a selective blockade of the regulated exocytosis of acetylcholine thereby triggering a profound albeit transitory muscular paralysis. The scope of this review is to describe the principal steps implicated in botulinum toxin intoxication from the early events leading to a paralysis to the cellular response implementing an impressive synaptic remodelling culminating in the functional recovery of neuromuscular transmission. BoNT/A treatment promotes extensive sprouting emanating from intoxicated motor nerve terminals and the distal portion of motor axons. The current view is that sprouts have the ability to form functional synapses as they display a number of key proteins required for exocytosis: SNAP-25, VAMP/synaptobrevin, syntaxin-I, synaptotagmin-II, synaptophysin, and voltage-activated Na+, Ca2+ and Ca2+-activated K+ channels. Exo-endocytosis was demonstrated (using the styryl dye FM1-43) to occur only in the sprouts in vivo, at the time of functional recovery emphasising the direct role of nerve terminal outgrowth in implementing the restoration of functional neurotransmitter release (at a time when nerve stimulation again elicited muscle contraction). Interestingly, sprouts are only transitory since a second distinct phase of the rehabilitation process occurs with a return of synaptic activity to the original nerve terminals. This is accompanied by the elimination of the dispensable sprouts. The growth or elimination of these nerve processes appears to be strongly correlated with the level of synaptic activity at the parent terminal. The BoNT/A-induced extension and later removal of "functional" sprouts indicate their fundamental importance in the rehabilitation of paralysed endplates, a finding with ramifications for the vital process of nerve regeneration.