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.     

Perisynaptic Schwann cells at neuromuscular junctions revealed by a novel monoclonal antibody

This study aimed to generate a probe for perisynaptic Schwann cells (PSCs) to investigate the emerging role of these synapse-associated glial cells in the formation and maintenance of the neuromuscular junction (NMJ). We have obtained a novel monoclonal antibody, 2A12, which labels the external surface of PSC membranes at the frog NMJ. The antibody reveals PSC fine processes or “fingers” that are interposed between nerve terminal and muscle membrane, interdigitating with bands of acetylcholine receptors. This antibody also labels PSCs at the avian neuromuscular junction and recognizes a 200 kDa protein in Torpedo electric organs. In frog muscles, axotomy induces sprouting of PSC processes beyond clusters of acetylcholine receptors and acetylcholinesterase at denervated junctional branches. PSC branches often extend across several muscle fibers. At some junctions, PSC sprouts join the tips of neighboring branches. The average length of PSC sprouts is approximately 156 µ at 3-week denervated NMJs. PSC sprouting is accompanied by a significant increase in the number of Schwann cell bodies per NMJ. Following nerve regeneration, nerve terminals reinnervate the junction along the PSC processes. In vivo observations of normal frog muscles also show PSC processes longer than nerve terminals at some junctional branches. The results suggest that nerve injury induces profuse PSC sprouting that may play a role in guiding nerve terminal regeneration at frog NMJs. In addition, antibody 2A12 reveals the fine morphology of PSCs in relation to other synaptic elements and is a useful probe in elucidating the function of these synapse-associated glial cells in vivo.     

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.     

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.     

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.     

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.     

Motor function recovery: deciphering a regenerative niche at the neuromuscular synapse

The coordinated movement of many organisms relies on efficient nerve–muscle communication at the neuromuscular junction (NMJ), a peripheral synapse composed of a presynaptic motor axon terminal, a postsynaptic muscle specialization, and non-myelinating terminal Schwann cells. NMJ dysfunctions are caused by traumatic spinal cord or peripheral nerve injuries as well as by severe motor pathologies. Compared to the central nervous system, the peripheral nervous system displays remarkable regenerating abilities; however, this capacity is limited by the denervation time frame and depends on the establishment of permissive regenerative niches. At the injury site, detailed information is available regarding the cells, molecules, and mechanisms involved in nerve regeneration and repair. However, a regenerative niche at the final functional step of peripheral motor innervation, i.e. at the mature neuromuscular synapse, has not been deciphered. In this review, we integrate classic and recent evidence describing the cells and molecules that could orchestrate a dynamic ecosystem to accomplish successful NMJ regeneration. We propose that such a regenerative niche must ensure at least two fundamental steps for successful NMJ regeneration: the proper arrival of incoming regenerating axons to denervated postsynaptic muscle domains, and the resilience of those postsynaptic domains, in morphological and functional terms. We here describe and combine the main cellular and molecular responses involved in each of these steps as potential targets to help successful NMJ regeneration.     

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.     

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.     

Shp2 is dispensable in the formation and maintenance of the neuromuscular junction

SHP2, a protein tyrosine phosphatase with two SH2 domains, has been implicated in regulating acetylcholine receptor (AChR) gene expression and cluster formation in cultured muscle cells. To understand the role of SHP2 in neuromuscular junction (NMJ) formation in vivo, we generated mus cle-specific deficient mice by using a loxP/Cre strategy since Shp2 null mutation causes embryonic lethality. Shp2(floxed/floxed) mice were crossed with mice expressing the Cre gene under the control of the human skeletal alpha-actin (HSA) promoter. Expression of SHP2 was reduced or diminished specifically in skeletal muscles of the conditional knockout (CKO) mice. The mutant mice were viable and fertile, without apparent muscle defects. The mRNA of the AChR alpha subunit and AChR clusters in CKO mice were localized in a narrow central region surrounding the phrenic nerve primary branches, without apparent change in intensity. AChR clusters colocalized with markers of synaptic vesicles and Schwann cells, suggesting proper differentiation of presynaptic terminals and Schwann cells. In comparison with age-matched littermates, no apparent difference was observed in the size and length of AChR clusters in CKO mice. Both the frequency and amplitude of mEPPs in CKO mice were similar to those in controls, suggesting normal neurotransmission when SHP2 was deficient. These results suggest that Shp2 is not required for NMJ formation and/or maintenance.     

The function of p120 catenin in filopodial growth and synaptic vesicle clustering in neurons

At the developing neuromuscular junction (NMJ), physical contact between motor axons and muscle cells initiates presynaptic and postsynaptic differentiation. Using Xenopus nerve-muscle cocultures, we previously showed that innervating axons induced muscle filopodia (myopodia), which facilitated interactions between the synaptic partners and promoted NMJ formation. The myopodia were generated by nerve-released signals through muscle p120 catenin (p120ctn), a protein of the cadherin complex that modulates the activity of Rho GTPases. Because axons also extend filopodia that mediate early nerve-muscle interactions, here we test p120ctn's function in the assembly of these presynaptic processes. Overexpression of wild-type p120ctn in Xenopus spinal neurons leads to an increase in filopodial growth and synaptic vesicle (SV) clustering along axons, whereas the development of these specializations is inhibited following the expression of a p120ctn mutant lacking sequences important for regulating Rho GTPases. The p120ctn mutant also inhibits the induction of axonal filopodia and SV clusters by basic fibroblast growth factor, a muscle-derived molecule that triggers presynaptic differentiation. Of importance, introduction of the p120ctn mutant into neurons hinders NMJ formation, which is observed as a reduction in the accumulation of acetylcholine receptors at innervation sites in muscle. Our results suggest that p120ctn signaling in motor neurons promotes nerve-muscle interaction and NMJ assembly.     

The role of nitric oxide signaling in the formation of the neuromuscular junction

The formation of the vertebrate neuromuscular junction (NMJ) depends on the action of neural agrin on the muscle cell. The requirement for agrin and its receptor, muscle-specific kinase (MuSK), has been well established over the past 20 years. However, the signaling mechanisms through which agrin and MuSK cause synaptic differentiation are not well understood. New evidence from studies of muscle cells in culture and in embryos indicates that nitric oxide (NO) is an effector of agrin-induced postsynaptic differentiation at the NMJ. Cyclic GMP (cGMP) production by guanylate cyclase appears to be an important downstream step in this pathway. Nitric oxide and cGMP regulate the activity of several kinases, some of which may influence interaction of dystrophin and utrophin with the actin cytoskeleton to mediate or modulate postsynaptic differentiation in muscle cells. These signaling molecules could also play a role in retrograde signaling to influence differentiation of presynaptic nerve terminals.     

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.     

Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 +/- 0.06 ms in wild-type junctions and 5.4 ms +/- 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.     

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.     

Glia and Muscle Sculpt Neuromuscular Arbors by Engulfing Destabilized Synaptic Boutons and Shed Presynaptic Debris

Synapse remodeling is an extremely dynamic process, often regulated by neural activity. Here we show during activity-dependent synaptic growth at the Drosophila NMJ many immature synaptic boutons fail to form stable postsynaptic contacts, are selectively shed from the parent arbor, and degenerate or disappear from the neuromuscular junction (NMJ). Surprisingly, we also observe the widespread appearance of presynaptically derived “debris” during normal synaptic growth. The shedding of both immature boutons and presynaptic debris is enhanced by high-frequency stimulation of motorneurons, indicating that their formation is modulated by neural activity. Interestingly, we find that glia dynamically invade the NMJ and, working together with muscle cells, phagocytose shed presynaptic material. Suppressing engulfment activity in glia or muscle by disrupting the Draper/Ced-6 pathway results in a dramatic accumulation of presynaptic debris, and synaptic growth in turn is severely compromised. Thus actively growing NMJ arbors appear to constitutively generate an excessive number of immature boutons, eliminate those that are not stabilized through a shedding process, and normal synaptic expansion requires the continuous clearance of this material by both glia and muscle cells.     

β-Catenin gain of function in muscles impairs neuromuscular junction formation

Neuromuscular junction (NMJ) formation requires proper interaction between motoneurons and muscle cells. β-Catenin is required in muscle cells for NMJ formation. To understand underlying mechanisms, we investigated the effect of β-catenin gain of function (GOF) on NMJ development. In HSA-β-cat(flox(ex3)/+) mice, which express stable β-catenin specifically in muscles, motor nerve terminals became extensively defasciculated and arborized. Ectopic muscles were observed in the diaphragm and were innervated by ectopic phrenic nerve branches. Moreover, extensive outgrowth and branching of spinal axons were evident in the GOF mice. These results indicate that increased β-catenin in muscles alters presynaptic differentiation. Postsynaptically, AChR clusters in HSA-β-cat(flox(ex3)/+) diaphragms were distributed in a wider region, suggesting that muscle β-catenin GOF disrupted the signal that restricts AChR clustering to the middle region of muscle fibers. Expression of stable β-catenin in motoneurons, however, had no effect on NMJ formation. These observations provide additional genetic evidence that pre- and postsynaptic development of the NMJ requires an intricate balance of β-catenin activity in muscles.     

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.