Basal lamina directs acetylcholinesterase accumulation at synaptic sites in regenerating muscle

In skeletal muscles that have been damaged in ways which spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fibers are characterized by junctional folds and accumulations of acetylcholine receptors and acetylcholinesterase (AChE). The formation of junctional folds and the accumulation of acetylcholine receptors is known to be directed by components of the synaptic portion of the myofiber basal lamina. The aim of this study was to determine whether or not the synaptic basal lamina contains molecules that direct the accumulation of AChE. We crushed frog muscles in a way that caused disintegration and phagocytosis of all cells at the neuromuscular junction, and at the same time, we irreversibly blocked AChE activity. New muscle fibers were allowed to regenerate within the basal lamina sheaths of the original muscle fibers but reinnervation of the muscles was deliberately prevented. We then stained for AChE activity and searched the surface of the new muscle fibers for deposits of enzyme they had produced. Despite the absence of innervation, AChE preferentially accumulated at points where the plasma membrane of the new muscle fibers was apposed to the regions of the basal lamina that had occupied the synaptic cleft at the neuromuscular junctions. We therefore conclude that molecules stably attached to the synaptic portion of myofiber basal lamina direct the accumulation of AChE at the original synaptic sites in regenerating muscle. Additional studies revealed that the AChE was solubilized by collagenase and that it remained adherent to basal lamina sheaths after degeneration of the new myofibers, indicating that it had become incorporated into the basal lamina, as at normal neuromuscular junctions.     

Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites

Axons regenerate to reinnervate denervated skeletal muscle fibers precisely at original synaptic sites, and they differentiate into nerve terminals where they contact muscle fibers. The aim of this study was to determine the location of factors that influence the growth and differentiation of the regenerating axons. We damaged and denervated frog muscles, causing myofibers and nerve terminals to degenerate, and then irradiated the animals to prevent regeneration of myofibers. The sheath of basal lamina (BL) that surrounds each myofiber survives these treatments, and original synaptic sites on BL can be recognized by several histological criteria after nerve terminals and muscle cells have been completely removed. Axons regenerate into the region of damage within 2 wk. They contact surviving BL almost exclusively at original synaptic sites; thus, factors that guide the axon's growth are present at synaptic sites and stably maintained outside of the myofiber. Portions of axons that contact the BL acquire active zones and accumulations of synaptic vesicles; thus by morphological criteria they differentiate into nerve terminals even though their postsynaptic targets, the myofibers, are absent. Within the terminals, the synaptic organelles line up opposite periodic specializations in the myofiber's BL, demonstrating that components associated with the BL play a role in organizing the differentiation of the nerve terminal.     

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.     

Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve

We examined the role of nerve terminals in organizing acetylcholine receptors on regenerating skeletal-muscle fibers. When muscle fibers are damaged, they degenerate and are phagocytized, but their basal lamina sheaths survive. New myofibers form within the original basal lamina sheaths, and they become innervated precisely at the original synaptic sites on the sheaths. After denervating and damaging muscle, we allowed myofibers to regenerate but deliberately prevented reinnervation. The distribution of acetylcholine receptors on regenerating myofibers was determined by histological methods, using [125I] alpha-bungarotoxin or horseradish peroxidase-alpha-bungarotoxin; original synaptic sites on the basal lamina sheaths were marked by cholinesterase stain. By one month after damage to the muscle, the new myofibers have accumulations of acetylcholine receptors that are selectively localized to the original synaptic sites. The density of the receptors at these sites is the same as at normal neuromuscular junctions. Folds in the myofiber surface resembling junctional folds at normal neuromuscular junctions also occur at original synaptic sites in the absence of nerve terminals. Our results demonstrate that the biochemical and structural organization of the subsynaptic membrane in regenerating muscle is directed by structures that remain at synaptic sites after removal of the nerve.     

Globular and asymmetric acetylcholinesterase in the synaptic basal lamina of skeletal muscle

The aim of this study was to characterize the molecular forms of acetylcholinesterase (AChE) associated with the synaptic basal lamina at the neuromuscular junction. The observations were made on the neuromuscular junctions of cutaneous pectoris muscles of frog, Rana pipiens, which are similar to junctions of most other vertebrates including mammals, but are especially convenient for experimentation. By measuring relative AChE activity in junctional and extrajunctional regions of muscles after selective inactivation of extracellular AChE with echothiophate, or of intracellular AChE with DFP and 2-PAM, we found that > 66% of the total AChE activity in the muscle was junction- specific, and that > 50% of the junction-specific AChE was on the cell surface. More than 80% of the cell surface AChE was solubilized in high ionic strength detergent-free buffer, indicating that most, if not all, was a component of the synaptic basal lamina. Sedimentation analysis of that fraction indicated that while asymmetric forms (A12, A8) were abundant, globular forms sedimenting at 4-6 S (G1 and G2), composed > 50% of the AChE. It was also found that when muscles were damaged in various ways that caused degeneration of axons and muscle fibers but left intact the basal lamina sheaths, the small globular forms persisted at the synaptic site for weeks after phagocytosis of cellular components; under certain damage conditions, the proportion of globular to asymmetric forms in the vacated basal lamina sheaths was as in normal junctions. While the asymmetric forms required high ionic strength for solubilization, the extracellular globular AChE could be extracted from the junctional regions of normal and damaged muscles by isotonic buffer. Some of the globular AChE appeared to be amphiphilic when examined in detergents, suggesting that it may form hydrophobic interactions, but most was non-amphiphilic consistent with the possibility that it forms weak electrostatic interactions. We conclude that the major form of AChE in frog synaptic basal lamina is globular and that its mode of association with the basal lamina differs from that of the asymmetric forms.     

Synapse-specific expression of acetylcholine receptor genes and their products at original synaptic sites in rat soleus muscle fibres regenerating in the absence of innervation.

To test the hypothesis that synaptic basal lamina can induce synapse-specific expression of acetylcholine receptor (AChR) genes, we examined the levels mRNA for the alpha- and epsilon-subunits of the AChR in regenerating rat soleus muscles up to 17 days of regeneration. Following destruction of all muscle fibres and their nuclei by exposure to venom of the Australian tiger snake, new fibres regenerated within the original basal lamina sheaths. Northern blots showed that original mRNA was lost during degeneration. Early in regeneration, both alpha- and epsilon-subunit mRNAs were present throughout the muscle fibres but in situ hybridization showed them to be concentrated primarily at original synaptic sites, even when the nerve was absent during regeneration. A similar concentration was seen in denervated regenerating muscles kept active by electrical stimulation and in muscles frozen 41-44 hours after venom injection to destroy all cells in the synaptic region of the muscle. Acetylcholine-gated ion channels with properties similar to those at normal neuromuscular junctions were concentrated at original synaptic sites on denervated stimulated muscles. Taken together, these findings provide strong evidence that factors that induce the synapse-specific expression of AChR genes are stably bound to synaptic basal lamina.     

Development of basal lamina in synaptic and extrasynaptic portions of embryonic rat muscle

Each vertebrate skeletal muscle fiber is ensheathed by a basal lamina (BL) which passes through the synaptic cleft of the neuromuscular junction. In the adult, the synaptic portion of the BL is both functionally and chemically specialized. We have used an immunofluorescence method to compare the development of synaptic and extrasynaptic portions of BL in embryonic rat intercostal muscles. Immunohistochemical staining of adult muscle fibers with monoclonal and serum antibodies defines "synaptic" antigens (including acetylcholinesterase) that are concentrated in synaptic BL, "extrasynaptic" antigens that are concentrated in extrasynaptic regions, and "shared" antigens (including collagen IV, fibronectin, laminin, and a heparan sulfate proteoglycan) that are present in both synaptic and extrasynaptic BL ( Sanes and Chiu , 1983). Synapses appear on newly formed myotubes on embryonic Day 14 (E14; birth is on E22 ). Patches of BL that contain shared and extrasynaptic antigens are present on myotube surfaces by E15, and BL forms a continuous sheath by E17. Shared antigens are present at but not confined to synaptic areas by E15. Two synaptic antigens appear in synaptic areas a day later, and are not detectable extrasynaptically . At least one extrasynaptic antigen is present at immature synapses, and lost or masked by E19 . Thus synaptic BL is not assembled as a unit; rather, components are added, lost, or modified as synaptogenesis proceeds.     

Aggregates of acetylcholine receptors are associated with plaques of a basal lamina heparan sulfate proteoglycan on the surface of skeletal muscle fibers

Hybridoma techniques have been used to generate monoclonal antibodies to an antigen concentrated in the basal lamina at the Xenopus laevis neuromuscular junction. The antibodies selectively precipitate a high molecular weight heparan sulfate proteoglycan from conditioned medium of muscle cultures grown in the presence of [35S]methionine or [35S]sulfate. Electron microscope autoradiography of adult X. laevis muscle fibers exposed to 125I-labeled antibody confirms that the antigen is localized within the basal lamina of skeletal muscle fibers and is concentrated at least fivefold within the specialized basal lamina at the neuromuscular junction. Fluorescence immunocytochemical experiments suggest that a similar proteoglycan is also present in other basement membranes, including those associated with blood vessels, myelinated axons, nerve sheath, and notochord. During development in culture, the surface of embryonic muscle cells displays a conspicuously non-uniform distribution of this basal lamina proteoglycan, consisting of large areas with a low antigen site-density and a variety of discrete plaques and fibrils. Clusters of acetylcholine receptors that form on muscle cells cultured without nerve are invariably associated with adjacent, congruent plaques containing basal lamina proteoglycan. This is also true for clusters of junctional receptors formed during synaptogenesis in vitro. This correlation indicates that the spatial organization of receptor and proteoglycan is coordinately regulated, and suggests that interactions between these two species may contribute to the localization of acetylcholine receptors at the neuromuscular junction.     

The clustering of acetylcholine receptors and formation of neuromuscular junctions in regenerating mammalian muscle grafts

The present investigation was undertaken to study the relationship between acetylcholine receptor (AchR) clustering and endplate formation within regenerating skeletal muscle grafts. Silver staining of nerves was combined with rhodamine-alpha-bungarotoxin labeling of AchR clusters in heterotopic grafts of the rat soleus muscle. Two major graft procedures were used: whole muscle grafts and grafts which lacked the zone of original motor endplates (MEP-less grafts). These categories were subdivided into standard grafts, where subsequent innervation was allowed, and noninnervated grafts, which were experimentally deprived of innervation. Grafting brought about the death and removal of muscle fibers, followed by regeneration of myotubes within surviving basal lamina sheaths. A transient population of small extra-junctional AchR clusters spontaneously appears shortly after myotube formation in all four muscle graft types. Early myotubes of whole muscle grafts (both innervated and standard grafts, prior to the time of innervation) also develop presumptive secondary synaptic clefts and large, organized aggregations of AchRs at original synaptic sites. At later times, nerves regenerating into standard whole muscle and MEP-less grafts lead to the formation of numerous ectopic endplates. In whole muscle grafts, endplates may also form at original synaptic sites. Functional graft innervation is achieved in whole muscle and MEP-less grafts as early as 20 days postgrafting. The results of this study support the existence of still-unknown factors associated with the original synaptic site which can direct postsynaptic differentiation independent of innervation. They also demonstrate that functional endplates may form in mammalian muscle grafts at both original synaptic sites and ectopic locations, thus indicating that the zone of original synaptic sites is not necessary for the establishment of numerous functional and morphologically well-differentiated endplates.     

Agrin and acetylcholine receptors are removed from abandoned synaptic sites at reinnervated frog neuromuscular junctions

Changes in the distribution of agrin and acetylcholine receptors (AChRs) were examined during reinnervation and following permanent denervation as a means of understanding mechanisms controlling the distribution of these molecules. Following nerve damage in the peripheral nervous system, regenerating nerve terminals preferentially return to previous synaptic sites leading to the restoration of synaptic activity. However, not all portions of original synaptic sites are reoccupied: Some of the synaptic sites are abandoned by both the nerve terminal and the Schwann cell. Abandoned synaptic sites contain agrin, AChRs, and acetylcholinesterase (AChE) without an overlying nerve terminal or Schwann cell providing a unique location to observe changes in the distribution of these synapse-specific molecules. The distribution of anti-agrin and AChR staining at abandoned synaptic sites was altered during the process of reinnervation, changing from a dense, wide distribution to a punctate, pale pattern, and finally becoming entirely absent. Agrin and AChRs were removed from abandoned synaptic sites in reinnervated frog neuromuscular junctions, while in contralateral muscles which were permanently denervated, anti-agrin and AChR staining remained at abandoned synaptic sites. Decreasing synaptic activity during reinnervation delayed the removal of agrin and AChRs from abandoned synaptic sites. Altogether, these results support the hypothesis that synaptic activity controls a cellular mechanism that directs the removal of agrin from synaptic basal lamina and the loss of agrin leads to the dispersal of AChRs. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 999–1018, 1997     

Induction of a specialized muscle basal lamina at chimaeric synapses in culture

To identify mechanisms that regulate the formation of the neuromuscular junction, we examined the cellular origin of a heparan sulfate proteoglycan (HSPG) that becomes highly concentrated within the synaptic cleft during the initial deposition of the junctional basal lamina. Using cultured nerve and muscle cells from anuran and urodele embryos, we prepared species-chimaeric synapses that displayed spontaneous cholinergic potentials, and eventually developed organized accumulations of acetylcholine receptors and HSPG at the sites of nerve-muscle contact. To determine the cellular origin of synaptic HSPG molecules, these chimaeric junctions were stained with both species-specific and cross-reactive monoclonal antibodies, labeled with contrasting fluorochromes. Our results demonstrate that synaptic HSPG is derived almost exclusively from muscle. Since it has already been shown that muscle cells can assemble virtually all of the known constituents of the junctional basal lamina into organized surface accumulations, without any input from nerve cells, we consider the possibility that the specialized synaptic basal lamina may be generated primarily by the myofibre, in response to another 'inductive' positional signal at the site of nerve-muscle contact.     

Sodium channels aggregate at former synaptic sites in innervated and denervated regenerating muscles

The role of innervation in the establishment and regulation of the synaptic density of voltage-activated Na channels (NaChs) was investigated at regenerating neuromuscular junctions. Rat muscles were induced to degenerate after injection of the Australian tiger snake toxin, notexin. The loose-patch voltage clamp technique was used to measure the density and distribution of NaChs on muscle fibers regenerating with or without innervation. In either case, new myofibers formed within the original basal lamina sheaths, and, NaChs became concentrated at regenerating endplates nearly as soon as they formed. The subsequent increase in synaptic NaCh density followed a time course similar to postnatal muscles. Neuromuscular endplates regenerating after denervation, with no nerve terminals present, had NaCh densities not significantly different from endplates regenerating in the presence of nerve terminals. The results show that the nerve terminal is not required for the development of an enriched NaCh density at regenerating neuromuscular synapses and implicate Schwann cells or basal lamina as the origin of the signal for NaCh aggregation. In contrast, the change in expression from the immature to the mature form of the NaCh isoform that normally accompanies development occurred only partially on muscles regenerating in the absence of innervation. This aspect of NaCh regulation is thus dependent upon innervation.     

Activity-dependent accumulation of basal lamina by cultured rat myotubes

Myoblasts from 20-day rat embryos fuse and differentiate in culture to form spontaneously active myotubes. The myotubes acquire an extracellular matrix that includes a patchy basal lamina (BL) and a layer of fibrils that runs among and above the cells. Several antibodies that bind to muscle fiber basement membrane in vivo were used to study the organization of the extracellular matrix and the effect of muscle activity on the accumulation of its components. Light and electron microscopic immunohistochemical methods showed that the composition and organization of myotube BL in vitro resemble those seen in vivo. Antibodies that bind to both synaptic and extrasynaptic muscle fiber BL, in vivo stain the entire myotube BL in vitro, while antisera that bind preferentially to synaptic BL in vivo stain small patches of myotube BL, which are usually associated with regions rich in acetylcholine receptors. The effects of activity on accumulation of BL were studied by comparing control myotubes to myotubes paralyzed with tetrodotoxin or lidocaine. Immunohistochemical and 125I-antibody binding experiments with three antisera that stain the entire BL showed that paralyzed myotubes accumulate less BL than active myotubes. The effects of activity and inactivity are reversible: new BL forms if toxin is removed from cultures and BL is lost if active myotubes are paralyzed. Thus, accumulation of BL by myotubes is dependent, at least in part, on activity. In contrast, the number of patches stained by synapse-specific BL antibodies is increased in inactive cultures. Thus, immunologically distinguishable components of BL are differentially affected by activity.     

Nerve-induced remodeling of muscle basal lamina during synaptogenesis

To identify mechanisms that regulate the deposition of the junctional basal lamina during synaptogenesis, immunocytochemical experiments were carried out on cultured nerve and muscle cells derived from Xenopus laevis embryos. In some experiments successive observations were made on individual muscle cells after pulse-labeling with a fluorescent monoclonal antibody specific for a basal lamina proteoglycan. In others, old and new proteoglycan molecules were differentially labeled with antibody conjugated to contrasting fluorochromes. These observations revealed that surface deposits of antibody-labeled proteoglycan remain morphologically stable for several days on developing muscle cells. Over the same period, however, new sites of proteoglycan accumulation formed that contained primarily those antigenic sites recently exposed at the cell surface. When muscle cells became innervated by cholinergic neurites, new proteoglycan accumulations were induced at the developing neuromuscular junctions, and these too were composed almost exclusively of recently deposited antigen. In older muscle cultures, where many cells possessed relatively high background concentrations of antigen over their surfaces, developing neuromuscular junctions initially showed a markedly reduced proteoglycan site-density compared with the adjacent, extrajunctional muscle surface. Much of this perineural region eventually became filled with dense, nerve induced proteoglycan plaques at later stages of synapse development. Motoneurons thus appear to have two, superficially paradoxical effects on muscle basal lamina organization. They first cause the removal of any existing, extrajunctional proteoglycan from the path of cell contact, and then induce the deposition of dense plaques of recently synthesized proteoglycan within the developing junctional basal lamina. This observation suggests that the proteolytic enzyme systems that have already been implicated in tissue remodeling may also contribute to the inductive interaction between nerve and muscle cells during synaptogenesis.     

Muscle-derived agrin in cultured myotubes: expression in the basal lamina and at induced acetylcholine receptor clusters.

The synaptic basal lamina (SBL) directs key aspects of the differentiation of regenerating neuromuscular junctions. A range of experiments indicate that agrin or a closely related molecule is stably associated with the SBL and participates in inducing the formation of the postsynaptic apparatus after damage to adult muscle. The selective concentration of agrin-related molecules in the SBL suggests that agrin is secreted locally by cellular components of the nerve-muscle synapse. In vivo studies on aneural embryonic muscle indicate that the muscle cell is one source of the agrin-like molecules in the SBL. Here we have used cultured chick muscle cells to study the expression of agrin-related molecules in the absence of innervation. Immunofluorescence and immunoelectron microscopy show that myogenic cells in culture express agrin-related molecules on their surfaces, and that at least a subset of these molecules are associated with the basal lamina. Moreover, in short term cultures agrin-like molecules accumulate on the surfaces of myogenic cells grown in unsupplemented basal media. We quantified the expression of agrin-like molecules on the cell surface using a solid-phase radioimmune assay. The expression of these molecules is relatively low during the first 6 days of culture and increases fourfold during the second week. The stimulation of the expression of agrin-related molecules in these long-term cultures requires the presence of chick embryo extract or fetal calf serum. We also characterized the expression of muscle-derived agrin-like molecules at clusters of AChR. These agrin-related molecules are not consistently colocalized at spontaneous AChR aggregates; however, they are selectively concentrated at greater than or equal to 90% of the AChR clusters that are induced by Torpedo agrin. These data, together with previous results from in vivo developmental experiments indicate that the agrin-like molecules in the synaptic basal lamina are derived at least in part from the muscle cell. In addition, the expression of agrin-like molecules can be regulated by soluble factors present in CEE and FBS. Finally, the selective localization of these molecules at induced AChR clusters, taken together with their localization in the basal lamina, suggests that agrin-like molecules secreted by the muscle cell play an important role in the formation and/or the stabilization of the postsynaptic apparatus.     

Acetylcholinesterase from the motor nerve terminal accumulates on the synaptic basal lamina of the myofiber

Acetylcholinesterase (AChE) in skeletal muscle is concentrated at neuromuscular junctions, where it is found in the synaptic cleft between muscle and nerve, associated with the synaptic portion of the myofiber basal lamina. This raises the question of whether the synaptic enzyme is produced by muscle, nerve, or both. Studies on denervated and regenerating muscles have shown that myofibers can produce synaptic AChE, and that the motor nerve may play an indirect role, inducing myofibers to produce synaptic AChE. The aim of this study was to determine whether some of the AChE which is known to be made and transported by the motor nerve contributes directly to AChE in the synaptic cleft. Frog muscles were surgically damaged in a way that caused degeneration and permanent removal of all myofibers from their basal lamina sheaths. Concomitantly, AChE activity was irreversibly blocked. Motor axons remained intact, and their terminals persisted at almost all the synaptic sites on the basal lamina in the absence of myofibers. 1 mo after the operation, the innervated sheaths were stained for AChE activity. Despite the absence of myofibers, new AChE appeared in an arborized pattern, characteristic of neuromuscular junctions, and its reaction product was concentrated adjacent to the nerve terminals, obscuring synaptic basal lamina. AChE activity did not appear in the absence of nerve terminals. We concluded therefore, that the newly formed AChE at the synaptic sites had been produced by the persisting axon terminals, indicating that the motor nerve is capable of producing some of the synaptic AChE at neuromuscular junctions. The newly formed AChE remained adherent to basal lamina sheaths after degeneration of the terminals, and was solubilized by collagenase, indicating that the AChE provided by nerve had become incorporated into the basal lamina as at normal neuromuscular junctions.