Phylogenetic polymorphism on lectin binding to junctional and non-junctional basal lamina at the vertebrate neuromuscular junction

Summary The histochemical binding of seven fluoreceinated lectins was comparatively studied in muscular tissue from twenty three different animal species including mamalians, amphibians, avians and fishes. Special interest was taken in the exploration of the differential lectin-binding properties at the neuromuscular synapse. Binding to synaptic sites was demonstrated using lectins that recognizes N-acetylgalactosamine and among of them, Dolichus biflorus agglutinin (DBA), was the most specific. Nevertheless, DBA fails to stain endplates in the muscle from most of the avians and the fishes (including the Torpedo electric organ) indicating that a polymorphic distribution of glycoconjugates exist at the vertebrate neuromuscular junction. Other lectins such as Concanavalin A (ConA) or Wheat germ agglutinin (WGA), share a similar staining properties in all animals that we examined making an intense label over the complete muscle surface. Although the species-related polymorphism on lectin binding does not reveal a clear relationship with the evolutionary tree, they give an evidence on the chemical heterogeneity of molecules specifically concentrated at the neuromuscular junction.     

Simultaneous labelling of basal lamina components and acetylcholinesterase at the neuromuscular junction

Summary A double labelling technique has been developed which permits the concomitant localization of basal lamina constituents together with acetylcholinesterase in mouse skeletal muscles. First, using the protein A-gold technique, type IV collagen and laminin were revealed on basal laminae ensheathing skeletal muscle fibres. The immunolabelling for both proteins was higher in synaptic than extrasynaptic regions. At synaptic sites the anti-type IV collagen immunolabelling exhibited an asymmetry; it was more intense on the portion of basal lamina closest to the postsynaptic membrane, whereas the anti-laminin immunolabelling was more uniformly distributed. It was also observed that the laminin immunoreactivity associated with Schwann and perineural cells was higher than that of skeletal muscle fibres. Secondly, the two basal lamina antigens were revealed simultaneously with another synaptic protein, acetylcholinesterase, using a refined cytochemical technique prior to the immunolabelling. The cytochemical reaction, which facilitates the location of endplates, did not alter the immunolabelling pattern. This double labelling procedure permits ready comparison of the distributions of type IV collagen and laminin with that of acetylcholinesterase, and may prove to be a useful approach in studies on synaptic components in developing and diseased muscle.     

N-CAM at the vertebrate neuromuscular junction

We have detected the neural cell adhesion molecule, N-CAM, at nerve-muscle contacts in the developing and adult mouse diaphragm. Whereas we found N-CAM staining with fluorescent antibodies consistently to overlap with the pattern of alpha-bungarotoxin staining at nerve-muscle contacts both during development and in the adult, we observed N-CAM staining on the surfaces of developing myofibers and at much lower levels on adult myofibers. Consistent with its function, N-CAM was also detected on axons and axon terminals. Immunoblotting experiments with anti-N-CAM antibodies on detergent extracts of embryonic (E) diaphragm muscle revealed a polydisperse polysialylated N-CAM polypeptide, which in the adult (A) was converted to a discrete form of Mr 140,000; this change, called E-to-A conversion, was previously found to occur in different neural tissues at different rates. The Mr 140,000 component was not recognized by monoclonal antibody anti-N-CAM No. 5, which specifically recognizes antigenic determinants associated with N-linked oligosaccharide determinants on N-CAM from neural tissue. The relative concentration of the Mr 140,000 component prepared from diaphragm muscle increased during fetal development and then decreased sharply to reach adult values. Nevertheless, expression of N-CAM in muscle could be induced after denervation: one week after the sciatic nerve was severed, the relative amount of N-CAM increased dramatically as detected by immunoblots of extracts of whole muscle. Immunofluorescent staining confirmed that there was an increase in N-CAM, both in the cell and at the cell surface; at the same time, however, staining at the motor endplate was diminished. Our findings indicate that, in muscle, in addition to chemical modulation, cell-surface modulation of N-CAM occurs both in amount and distribution during embryogenesis and in response to denervation.     

Receptors to agglutinin fromDolichus biflorus (DBA) at the synaptic basal lamina of rat neuromuscular junction

Summary The binding of agglutinin fromDolichus biflorus (DBA) and other lectins (Concanavalin A, agglutinin from wheat germ and lectin fromBandeiraea simplicifolid) to synaptic and extrasynaptic portions of the basal lamina of muscle fibers, was studied with histochemical methods. In rat muscle, DBA-binding is specifically detected at the basal lamina of neuromuscular junction. However, long-term (6 months) denervated end-plate in adult rat muscle failed to bind DBA. During normal development, synaptic DBA receptors appear later than acetylcholine receptors or acetylcholinesterase at the rat neuromuscular junction. Generalized DBA-binding to motor end-plates is first visualized in 3-day-old rats, but section of sciatic nerve in 1-day-old rats prevents the appearence of synaptic DBA-binding on the leg end-plates. It is suggested, therefore, that the synaptic DBA receptors could be related to the postnatal stabilization of rat neuromuscular synapses.     

Assembly and regulation of acetylcholinesterase at the vertebrate neuromuscular junction

The collagen-tailed form of acetylcholinesterase (ColQ-AChE) is the major if not unique form of the enzyme associated with the neuromuscular junction (NMJ). This enzyme form consists of catalytic and non-catalytic subunits encoded by separate genes, assembled as three enzymatic tetramers attached to the three-stranded collagen-like tail (ColQ). This synaptic form of the enzyme is tightly attached to the basal lamina associated with the glycosaminoglycan perlecan. Fasciculin-2 is a snake toxin that binds tightly to AChE. Localization of junctional AChE on frozen sections of muscle with fluorescent Fasciculin-2 shows that the labeled toxin dissociates with a half-life of about 36h. The fluorescent toxin can subsequently be taken up by the muscle fibers by endocytosis giving the appearance of enzyme recycling. Newly synthesized AChE molecules undergo a lengthy series of processing events before final transport to the cell surface and association with the synaptic basal lamina. Following co-translational glycosylation the catalytic subunit polypeptide chain interacts with several molecular chaperones, glycosidases and glycosyltransferases to produce a catalytically active enzyme that can subsequently bind to one of two non-catalytic subunits. These molecular chaperones can be rate limiting steps in the assembly process. Treatment of muscle cells with a synthetic peptide containing the PRAD attachment sequence and a KDEL retention signal results in a large increase in assembled and exportable AChE, providing an additional level of post-translational control. Finally, we have found that Pumilio2, a member of the PUF family of RNA-binding proteins, is highly concentrated at the vertebrate neuromuscular junction where it plays an important role in regulating AChE translation through binding to a highly conserved NANOS response element in the 3'-UTR. Together, these studies define several new levels of AChE regulation in electrically excitable cells.     

ARIA is concentrated in the synaptic basal lamina of the developing chick neuromuscular junction

ARIA is a member of a family of polypeptide growth and differentiation factors that also includes glial growth factor (GGF), neu differentiation factor, and heregulin. ARIA mRNA is expressed in all cholinergic neurons of the central nervous systems of rats and chicks, including spinal cord motor neurons. In vitro, ARIA elevates the rate of acetylcholine receptor incorporation into the plasma membrane of primary cultures of chick myotubes. To study whether ARIA may regulate the synthesis of junctional synaptic acetylcholine receptors in chick embryos, we have developed riboprobes and polyclonal antibody reagents that recognize isoforms of ARIA that include an amino-terminal immunoglobulin C2 domain and examined the expression and distribution of ARIA in motor neurons and at the neuromuscular junction. We detected significant ARIA mRNA expression in motor neurons as early as embryonic day 5, around the time that motor axons are making initial synaptic contacts with their target muscle cells. In older embryos and postnatal animals, we found ARIA protein concentrated in the synaptic cleft at neuromuscular junctions, consistent with transport down motor axons and release at nerve terminals. At high resolution using immunoelectron microscopy, we detected ARIA immunoreactivity exclusively in the synaptic basal lamina in a pattern consistent with binding to synapse specific components on the presynaptic side of the basal lamina. These results support a role for ARIA as a trophic factor released by motor neuron terminals that may regulate the formation of mature neuromuscular synapses.     

Mitochondrial clustering at the vertebrate neuromuscular junction during presynaptic differentiation

During vertebrate neuromuscular junction (NMJ) development, presynaptic motor axons differentiate into nerve termini enriched in synaptic vesicles (SVs). At the nerve terminal, mitochondria are also concentrated, but how mitochondria become localized at these specialized domains is poorly understood. This process was studied in cultured Xenopus spinal neurons with mitochondrion-specific probe MitoTracker and SV markers. In nerve-muscle cocultures, mitochondria were concentrated stably at sites where neurites and muscle cells formed NMJs, and mitochondria coclustered with SVs where neurites were focally stimulated by beads coated with growth factors. Labeling with a mitochondrial membrane potential-dependent probe JC-1 revealed that these synaptic mitochondria were with higher membrane potential than the extrasynaptic ones. At early stages of bead-stimulation, actin-based protrusions and microtubule fragmentation were observed in neurites at bead contact sites, suggesting the involvement of cytoskeletal dynamics and rearrangement during presynaptic differentiation. Treating the cultures with an actin polymerization blocker, latrunculin A (Ltn A), almost completely abolished the formation of actin-based protrusions and partially inhibited bead-induced mitochondrial and SV clustering, whereas the microtubule disrupting agent nocodazole was ineffective in inhibiting the clustering of mitochondria and SVs. Lastly, in contrast to Ltn A, which blocked bead-induced clustering of both mitochondria and SVs, the ser/thr phosphatase inhibitor okadaic acid inhibited SV clustering but not mitochondrial clustering. These results suggest that at developing NMJs, synaptogenic stimuli induce the clustering of mitochondria together with SVs at presynaptic terminals in an actin cytoskeleton-dependent manner and involving different intracellular signaling molecules.     

Neurotrophic control of 16S acetylcholinesterase at the vertebrate neuromuscular junction

Endplate 16S acetylcholinesterase (16S-AChE) from rat anterior gracilis muscle was assessed, 6 hr to 10 days after denervation, by velocity sedimentation analysis on linear sucrose gradients. The innervating obturator nerve was transected either close (1-2 mm, short stump) or far (35-40 mm, long stump) from the muscle. In both instances, the activity of 16S-AChE gradually decreased and reached approximately the same level (10%-20% of control) by 6 days after denervation. However, enzymatic decay started considerably earlier in short stump (12-24 hr) as compared to long stump (4-5 days) preparations, i.e., the time of onset of 16S-AChE loss depended on the length of nerve that remained attached to the muscle. Whether this result extended to other AChE molecular forms (10S, 4S) in muscle endplates could not be determined because, in contrast to 16S-AChE, these forms were also detected in red blood cells (4S) and plasma (10S). Only small amounts of 16S-AChE were found in intact obturator nerves (1/100 of that in gracilis endplate regions). Thus a faster depletion of enzyme from shorter nerve stumps after axotomy could not entirely account for the substantial effect of nerve stump length on 16S-AChE. Since muscle contraction ceases immediately following nerve transection, regardless of nerve stump length, the results can be ascribed to the lack of some neural influence other than nerve-evoked muscle activity. The present findings are consistent with the view that maintenance of 16S-AChE at neuromuscular junctions primarily depends on regulatory substances which are conveyed by axonal transport and released from nerve terminals.     

Distribution and turnover rate of acetylcholine receptors throughout the junction folds at a vertebrate neuromuscular junction

The distribution and turnover rate of acetylcholine receptors labeled with 125I-alpha-bungarotoxin were examined in innervated mouse sternomastoid muscle by electron microscope autoradiography using the "mask" analysis procedure. We compared the total population of receptors with receptors newly inserted at the junction 2 d after inactivation with nonradioactive alpha-bungarotoxin, both at the top (thickened) region of the postjunctional folds (pjm) and the nonthickened bottom folds. We found that the receptor site density was approximately 10 times greater on the thickened pjm than on the nonthickened bottom folds for both total and newly inserted receptors. This ratio does not change significantly during a 6-d period after labeling the new receptors. Furthermore, calculated values for turnover time of receptors show that both total and newly inserted receptors at both regions of the junctional folds have half-lives for degradation within the range given in the literature for slow junctional receptors. These data exclude a simple migration model whereby receptors are preferentially inserted in the nonthickened region of the junctional folds and then migrate into the thickened membrane at a rate equal to the turnover rate of the receptors.     

Neurotrophic control of 16S acetylcholinesterase at the vertebrate neuromuscular junction.

Endplate 16S acetylcholinesterase (16S-AChE) from rat anterior gracilis muscle was assessed, 6 hr to 10 days after denervation, by velocity sedimentation analysis on linear sucrose gradients. The innervating obturator nerve was transected either close (1--2 mm, short stump) or far (35--40 mm, long stump) from the muscle. In both instances, the activity of 16S-AChE gradually decreased and reached approximately the same level (10%--20% of control) by 6 days after denervation. However, enzymatic decay started considerably earlier in short stump (12--24 hr) as compared to long stump (4--5 days preparations, i.e., the time of onset of 16S-AChE loss depended on the length of nerve that remained attached to the muscle. Whether this result extended to other AChE molecular forms (10S, 4S) in muscle endplates could not be determined because, in contrast to 16S-AChE, these forms were also detected in red blood cells (4S) and plasma (10S). Only small amounts of 16S-AChE were found in intact obturator nerves (1/100 of that in gracilis endplate regions). Thus a faster depletion of enzyme from shorter nerve stumps after axotomy could not entirely account for the substantial effect of nerve stump length on 16S-AChE. Since muscle contraction ceases immediately following nerve transection, regardless of nerve stump length, the results can be ascribed to the lack of some neural influence other than nerve-evoked muscle activity. The present findings are consistent with the view that maintenance of 16SAChE at neuromuscular junctions primarily depends on regulatory substances which are conveyed by axonal transport and released from nerve terminals.     

Acetylcholine receptor aggregation parallels the deposition of a basal lamina proteoglycan during development of the neuromuscular junction

To determine the time course of synaptic differentiation, we made successive observations on identified, nerve-contacted muscle cells developing in culture. The cultures had either been stained with fluorescent alpha-bungarotoxin, or were maintained in the presence of a fluorescent monoclonal antibody. These probes are directed at acetylcholine receptors (AChR) and a basal lamina proteoglycan, substances that show nearly congruent surface organizations at the adult neuromuscular junction. In other experiments individual muscle cells developing in culture were selected at different stages of AChR accumulation and examined in the electron microscope after serial sectioning along the entire path of nerve-muscle contact. The results indicate that the nerve-induced formation of AChR aggregates and adjacent plaques of proteoglycan is closely coupled throughout early stages of synapse formation. Developing junctional accumulations of AChR and proteoglycan appeared and grew progressively, throughout a perineural zone that extended along the muscle surface for several micrometers on either side of the nerve process. Unlike junctional AChR accumulations, which disappeared within a day of denervation, both junctional and extrajunctional proteoglycan deposits were stable in size and morphology. Junctional proteoglycan deposits appeared to correspond to discrete ultrastructural plaques of basal lamina, which were initially separated by broad expanses of lamina-free muscle surface. The extent of this basal lamina, and a corresponding thickening of the postsynaptic membrane, also increased during the accumulation of AChR and proteoglycan along the path of nerve contact. Presynaptic differentiation of synaptic vesicle clusters became detectable at the developing neuromuscular junction only after the formation of postsynaptic plaques containing both AChR and proteoglycan. It is concluded that motor nerves induce a gradual formation and growth of AChR aggregates and stable basal lamina proteoglycan deposits on the muscle surface during development of the neuromuscular junction.     

Structural determinants of the reliability of synaptic transmission at the vertebrate neuromuscular junction

Brain Cell Biology - The reliability of neuromuscular transmission depends on the size and molecular organization of the neuromuscular junction. Comparative studies show that the quantal release...     

Amphetamine inhibition of alpha-bungarotoxin binding at the mouse neuromuscular junction.

Amphetamine inhibited neuromuscular transmission and prevented the irreversible blockade produced by α-bungarotoxin (α-BGT) in the isolated mouse phrenic nerve-diaphragm preparation. Similarly, amphetamine (1.35 × 10^?4 to 3 × 10^?3M) inhibited the binding of ¹²⁵I-α-BGT to mouse hemidiaphragms in a concentration-dependent manner; (+)-amphetamine was found to be twice as potent as its (-)-isomer with respect to inhibition of ¹²⁵I-α-BGT binding. It is suggested that amphetamine binds to the nicotinic, cholinergic receptor of skeletal muscle and may produce weakness and paralysis in amphetamine overdosage.     

The voltage-dependent sodium channel is co-localized with the acetylcholine receptor at the vertebrate neuromuscular junction

Isolated motor endplates from mouse intercostal muscles can be obtained after subcellular fractionation. On these motor endplates, localization of the nicotinic receptor and of the voltage-dependent Na+ channel coincides as demonstrated by double labeling with rhodamine alpha-bungarotoxin and a specific anti-Na+ channel monoclonal antibody. High density of Na+ channel at the motor endplate is confirmed by the enrichment in TTX binding sites as compared to the crude homogenate. In contrast isolated motor endplates are almost completely devoid of Ca2+ channel antagonist binding sites.     

Wnt4 participates in the formation of vertebrate neuromuscular junction

Neuromuscular junction (NMJ) formation requires the highly coordinated communication of several reciprocal signaling processes between motoneurons and their muscle targets. Identification of the early, spatially restricted cues in target recognition at the NMJ is still poorly documented, especially in mammals. Wnt signaling is one of the key pathways regulating synaptic connectivity. Here, we report that Wnt4 contributes to the formation of vertebrate NMJ in vivo. Results from a microarray screen and quantitative RT-PCR demonstrate that Wnt4 expression is regulated during muscle cell differentiation in vitro and muscle development in vivo, being highly expressed when the first synaptic contacts are formed and subsequently downregulated. Analysis of the mouse Wnt4−/− NMJ phenotype reveals profound innervation defects including motor axons overgrowing and bypassing AChR aggregates with 30% of AChR clusters being unapposed by nerve terminals. In addition, loss of Wnt4 function results in a 35% decrease of the number of prepatterned AChR clusters while Wnt4 overexpression in cultured myotubes increases the number of AChR clusters demonstrating that Wnt4 directly affects postsynaptic differentiation. In contrast, muscle structure and the localization of several synaptic proteins including acetylcholinesterase, MuSK and rapsyn are not perturbed in the Wnt4 mutant. Finally, we identify MuSK as a Wnt4 receptor. Wnt4 not only interacts with MuSK ectodomain but also mediates MuSK activation. Taken together our data reveal a new role for Wnt4 in mammalian NMJ formation that could be mediated by MuSK, a key receptor in synaptogenesis.     

MAGI-1c: a synaptic MAGUK interacting with muSK at the vertebrate neuromuscular junction

The muscle-specific receptor tyrosine kinase (MuSK) forms part of a receptor complex, activated by nerve-derived agrin, that orchestrates the differentiation of the neuromuscular junction (NMJ). The molecular events linking MuSK activation with postsynaptic differentiation are not fully understood. In an attempt to identify partners and/or effectors of MuSK, cross-linking and immunopurification experiments were performed in purified postsynaptic membranes from the Torpedo electrocyte, a model system for the NMJ. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis was conducted on both cross-link products, and on the major peptide coimmunopurified with MuSK; this analysis identified a polypeptide corresponding to the COOH-terminal fragment of membrane-associated guanylate kinase (MAGUK) with inverted domain organization (MAGI)-1c. A bona fide MAGI-1c (150 kD) was detected by Western blotting in the postsynaptic membrane of Torpedo electrocytes, and in a high molecular mass cross-link product of MuSK. Immunofluorescence experiments showed that MAGI-1c is localized specifically at the adult rat NMJ, but is absent from agrin-induced acetylcholine receptor clusters in myotubes in vitro. In the central nervous system, MAGUKs play a primary role as scaffolding proteins that organize cytoskeletal signaling complexes at excitatory synapses. Our data suggest that a protein from the MAGUK family is involved in the MuSK signaling pathway at the vertebrate NMJ.     

Reversibility and mode of action of Black Widow spider venom on the vertebrate neuromuscular junction

Black widow spider venom (BWSV) stimulates transmitter release and depletes synaptic vesicles from muscles bathed in a sodium free medium containing 1 mM EGTA. However, frog neuromuscular junctions treated with BWSV in glucosamine Ringer's and post-treated with antivenin recover normal function. This suggests that probably the permanent block of neuromuscular transmission is due to changes in permeability of the nerve ending plasma membrane to cations such as Na+. When BWSV is applied in a medium lacking divalent cations and containing 1 mM EGTA, in most of the cases no effect is observed. We found that this inhibition can be overcome in three ways: (a) by adding divalent cations to the medium; (b) by increasing the tonicity of the medium with sucrose; (c) by raising the temperature of the medium. These results suggest that the lack of divalent cations influences the membrane fluidity. Moreover, in view of the report by Yahara and Kakimoto-Sameshima (1977. Proc. Natl. Acad. Sci. U.S.A. 74:4511--4515) that hypertonic media induce capping of surface receptors in lymphocytes and thymocytes, we think that these data further support the hypothesis that BWSV stimulates release by a dual mode of action; namely, it increases the nerve ending permeability to cations and also stimulates release directly via a process of redistribution of membrane components, a process which may also inhibit vesicle recycling.     

Activity and synapse elimination at the neuromuscular junction

The neuromuscular junction undergoes a loss of synaptic connections during early development. This loss converts the innervation of each muscle fiber from polyneuronal to single. During this change the number of motor neurons remains constant but the number of muscle fibers innervated by each motor neuron is reduced. Evidence indicates that a local competition among the inputs on each muscle fiber determines which inputs are eliminated. The role of synapse elimination in the development of neuromuscular circuits, other than ensuring a single innervation of each fiber, is unclear. Most evidence suggests that the elimination plays little or no role in correcting for errant connections. Rather, it seems that connections are initially highly specific, in terms of both which motor neurons connect to which muscles and which neurons connect to which particular fibers within these muscles. A number of attempts have been made to determine the importance of neuromuscular activity during early development for this rearrangement of synaptic connections. Experiments reducing neuromuscular activity by muscle tenotomy, deafferentation and spinal cord section, block of nerve impulse conduction with tetrodotoxin, and the use of postsynaptic and presynaptic blocking agents have all shown that normal activity is required for normal synapse elimination. Most experiments in which complete muscle paralysis has been achieved show that activity may be essential for the occurrence of synapse elimination. Furthermore, experiments in which neuromuscular activity has been augmented by external stimulation show that synapse elimination is accelerated. A plausible hypothesis to explain the activity dependence of neuromuscular synapse elimination is that a neuromuscular trophic agent is produced by the muscle fibers and that this production is controlled by muscle-fiber activity. The terminals on each fiber compete for the substance produced by that fiber. Inactive fibers produce large quantities of this substance; on the other hand, muscle activity suppresses the level of synthesis of this agent to the point where only a single synaptic terminal can be maintained. Inactive muscle fibers would be expected to be able to maintain more nerve terminals. The attractiveness of this scheme is that it provides a simple feedback mechanism to ensure that each fiber retains a single effective input.     

Desensitization and recovery at the frog neuromuscular junction

The time course of carbachol-induced desensitization onset and recovery of sensitivity after desenitization have been compared at the frog neuromuscular junction. The activation-desensitization sequence was determined from input conductance measurements using potassium-depolarized muscle preparations. Both desensitization onset and recovery from desensitization could be adequately described by single time constant expressions, with tauonset being considerably shorter than taurecovery. In nine experiments, tauonset was 13+/-1.3 s and taurecovery was 424+/-51 s with 1 mM carbachol. Elevating the external calcium or carbachol concentration accelerated desensitization onset without changing the recovery of sensitivity after equilibrium desensitization. Desensitization onset was accelerated by a prior activation-desensitization sequence to an extent determined by the recovery interval that followed the initial carbachol application. The time course of return of tauonset was closely parallel to, but slower than the time course of recovery of sensitivity. These results are consistent with a cyclic model in which intracellular calcium is a factor controlling the rate of development of desensitization.