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PubMed2003

Structural alterations at the neuromuscular junctions of matrix metalloproteinase 3 null mutant mice.

Matrix metalloproteinases are important regulators of extracellular matrix molecules and cell-cell signaling. Antibodies to matrix metalloproteinase 3 (MMP3) recognize molecules at the frog neuromuscular junction, and MMP3 can remove agrin from synaptic basal lamina (VanSaun & Werle, 2000). To gain insight into the possible roles of MMP3 at the neuromuscular junction, detailed observations were made on the structure and function of the neuromuscular junctions in MMP3 null mutant mice. Striking differences were found in the appearance of the postsynaptic apparatus of MMP3 null mutant mice. Endplates had an increased volume of AChR stained regions within the endplate structure, leaving only small regions devoid of AChRs. Individual postsynaptic gutters were wider, containing prominent lines that represent the AChRs concentrated at the tops of the junctional folds. Electron microscopy revealed a dramatic increase in the number and size of the junctional folds, in addition to ectopically located junctional folds. Electrophysiological recordings revealed no change in quantal content or MEPP frequency, but there was an increase in MEPP rise time in a subset of endplates. No differences were observed in the rate or extent of developmental synapse elimination. In vitro cleavage experiments revealed that MMP3 directly cleaves agrin. Increased agrin immunofluorescence was observed at the neuromuscular junctions of MMP3 null mutant mice. These results provide strong evidence that MMP3 is involved in the control of synaptic structure at the neuromuscular junction and they support the hypothesis that MMP3 is involved in the regulation of agrin at the neuromuscular junction.

Agrin↗

PubMed2003

Ca2+ channels and synaptic transmission at the adult, neonatal, and P/Q-type deficient neuromuscular junction.

Different types of voltage-activated Ca(2+) channels have been established based on their molecular structure and pharmacological and biophysical properties. One of them, the P/Q-type, is the main channel involved in nerve-evoked neurotransmitter release at neuromuscular junctions and the immunological target in Eaton-Lambert Syndrome. At adult neuromuscular junctions, L- and N-type Ca(2+) channels become involved in transmitter release only under certain experimental or pathological conditions. In contrast, at neonatal rat neuromuscular junctions, nerve-evoked synaptic transmission depends jointly on both N- and P/Q-type channels. Synaptic transmission at neuromuscular junctions of the ataxic P/Q-type Ca(2+) channel knockout mice is also dependent on two different types of channels, N- and R-type. At both neonatal and P/Q knockout junctions, the K(+)-evoked increase in miniature endplate potential frequency was not affected by N-type channel blockers, but strongly reduced by both P/Q- and R-type channel blockers. These differences could be accounted for by a differential location of the channels at the release site, being either P/Q- or R-type Ca(2+) channels located closer to the release site than N-type Ca(2+) channels. Thus, Ca(2+) channels may be recruited to mediate neurotransmitter release where P/Q-type channels seem to be the most suited type of Ca(2+) channel to mediate exocytosis at neuromuscular junctions.

Adult↗

PubMed1995

A computer program for the study of synaptic transmission at the neuromuscular junction.

The study of synaptic biopotentials at the neuromuscular junction has proven to be an invaluable tool in understanding the pre- and post-synaptic mechanisms underlying chemical synaptic transmission and exploring the pathogenesis of neuromuscular disorders. Computer technology has become an important part of these experiments as it increases accuracy and automates the process, allowing more data to be collected and quantitatively analysed. This paper presents a computer program for IBM PC compatible computers capable of recording and analysing the spontaneously occurring miniature end-plate potentials (MEPPs), neurally evoked end-plate potentials (EPPs) and muscle fiber action potentials (APs). The program performs its basic analysis immediately following the sampling of biopotentials from a muscle fiber, thus providing the user with immediate feedback on the status of the experiment; 45 parameters of neuromuscular transmission are computed on-line and displayed on the monitor along with the signal-averaged waveforms. After a series of fibers has been recorded, the program will compile the data into summary tables. The program is designed to provide an experimenter with an easy-to-use, comprehensive method of performing electrophysiological measurements and analysis at the neuromuscular junction.

Action Potentials↗

PubMed1994

Factors causing different properties at neuromuscular junctions in fast and slow rat skeletal muscles.

Neuromuscular junctions on fast and slow skeletal muscle fibers have different properties. Possible reasons for these differences were examined in adult rat soleus (SOL) muscle fibers reinnervated at new ectopic or old denervated sites by fast fibular (FIB) or slow SOL motoneurons. FIB motoneurons formed large ectopic junctions with a high density of nerve terminal varicosities (fast appearance), whereas SOL motoneurons formed small ectopic junctions with a low density of varicosities (slow appearance). Both FIB and SOL motoneurons formed small junctions with a slow appearance when reinnervating old SOL endplates. FIB nerves innervating ectopic sites and SOL nerves reinnervating old sites had the same appearance whether they contacted the SOL fibers alone (single innervation) or together (dual innervation). Continuous stimulation of the FIB nerve at 10 Hz for 3-4 months reduced the size of ectopic FIB and intact extensor digitorum longus (EDL) junctions and caused a modest reduction in density of terminal varicosities in EDL. Junction size and muscle fiber diameter were positively correlated, but the slope describing this relation was steeper for FIB junctions than for SOL junctions. It is concluded that in the present system (1) motoneuron type and not muscle fiber type determines the fast or slow character of the neuromuscular junction. (2) denervated endplates of one type place stable and severe constraints on the termination pattern of reinnervating axons of another type, (3) the appearance of fast EDL junctions undergoes a modest fast to slow transformation when exposed to long-term slow pattern stimulation, and (4) not only the size of the muscle fibers, but also the type and firing pattern of the motoneurons and the spatial constraints at preformed endplates influence the relation between junction size and muscle fiber diameter.

Animals↗

PubMed1989

Quantitative investigation of the neuromuscular junction of rat skeletal muscle fibres after double innervation.

In normal (untreated) rats the mean length ratio of postsynaptic to presynaptic membrane was 2.7 +/- 0.8 for neuromuscular junctions of slow-twitch soleus muscle fibres and 4.2 +/- 1.0 for neuromuscular junctions of fast-twitch extensor digitorum longus muscle fibres; this difference was significant (P less than 0.001). After experimental double innervation by fast and slow muscle nerves for four months, the ratio was (1) 2.9 +/- 0.8 for the original slow-twitch fibre end-plate and 2.8 +/- 0.8 for the newly established one, both not significantly different from that of the normal slow-twitch fibres; and (2) 2.2 +/- 0.5 for the original fast-twitch fibre end-plate and 2.2 +/- 0.7 for the newly established one, both significantly smaller than that of the normal fast-twitch fibres (P less than 0.001). This means that the double innervated slow-twitch muscle fibres retained their original neuromuscular junction type, whereas the doubly-innervated fast-twitch muscle fibres underwent a dramatic transformation of their neuromuscular junction from the fast-muscle to the slow-muscle type. In both doubly innervated fibres, the ultrastructural characteristics of neuromuscular junctions, whether altered or not, were identical at both end-plate regions.

Animals↗

PubMed1986

Effects of phorbol ester on spontaneous transmitter release at frog neuromuscular junction.

Spontaneous transmitter release was studied at frog neuromuscular junctions exposed to the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), a specific activator of protein kinase C (PrkC). TPA at concentrations between 10(-7) and 10(-6) M induced a dose-dependent increase in miniature end-plate potential frequency. This frequency increase was enhanced by raising [Ca]o, diminished by lowering the temperature of the bath and virtually abolished in Ca2+ -free Ringer. The TPA effect was only poorly reversible after washing. TPA was ineffective in increasing spontaneous release of transmitter at phosphatidylcholine-pretreated neuromuscular junctions. It is suggested that PrkC might play a role in neuromuscular transmission processes.

Animals↗

PubMedDate not supplied

The postsynaptic submembrane machinery at the neuromuscular junction: requirement for rapsyn and the utrophin/dystrophin-associated complex.

Neuromuscular synapse formation is brought about by a complex bi-directional exchange of information between the innervating motor neuron and its target skeletal muscle fiber. Agrin, a heparin sulfate proteoglycan, is released from the motor nerve terminal to activate its muscle-specific kinase (MuSK) receptor that leads to a second messenger cascade requiring rapsyn to ultimately bring about AChR clustering in the muscle membrane. Rapsyn performs many functions in skeletal muscle. First, rapsyn and AChRs co-target to the postsynatic apparatus. Second, rapsyn may self associate to stabilize and promote AChR clustering. Third, rapsyn is essential for AChR cluster formation. Fourth, rapsyn is required to transduce the agrin-evoked MuSK phosphorylation signal to AChRs. Finally, rapsyn links AChRs to the utrophin-associated complex, which appears to be required for AChR stabilization as well as maturation of the neuromuscular junction. Proteins within the utrophin-associated complex such as alpha-dystrobrevin and alpha-syntrophin are also important for signaling events that affect neuromuscular synapse stability and function. Here we review our current understanding of the role of the postsynaptic-submembrane machinery involving rapsyn and the utrophin-associated complex at the neuromuscular synapse. In addition we briefly review how these studies of the neuromuscular junction relate to GABAergic and glycinergic synapses in the CNS.

Animals↗

PubMed1993

In vivo observations of pre- and postsynaptic changes during the transition from multiple to single innervation at developing neuromuscular junctions.

Synaptic rearrangements in developing muscle were studied by visualizing individual neuromuscular junctions in the sternomastoid muscle of living neonatal mice as they underwent the transition from multiple to single innervation. Vital staining of ACh receptors (AChRs) with rhodamine-conjugated alpha-bungarotoxin showed that while junctions were still multiply innervated (usually by two motor axons), regions of the postsynaptic membrane within each junction became depleted of receptors. Usually, several small postsynaptic areas lost AChRs in succession. In these areas, AChRs already in the membrane rapidly disappeared compared to a low level of receptor turnover elsewhere in the junction. Moreover, there was no evidence of new AChRs being inserted into these areas. Within each postsynaptic area undergoing AChR depletion, the intensity of receptor staining decreased gradually over 1-2 d. In some junctions, it appeared that AChRs were migrating away from areas being depleted of receptors. The depletion of AChRs from some sites in combination with the spreading apart of the entire receptor-rich area due to muscle fiber growth accounts for the transformation from plaque-like to branched receptor distributions at developing neuromuscular junctions. Vital staining of presynaptic motor nerve terminals at junctions whose postsynaptic AChRs were also stained showed that motor nerve terminals were lost from the same areas that were depleted of receptors postsynaptically. Postsynaptic areas began to be depleted of AChRs before there was any obvious loss of membrane or intracellular staining in the overlying nerve terminal. Only when a single innervating axon remained at a junction did loss of motor nerve terminals and underlying AChRs largely cease. That former synaptic areas could at later times be identified as uninnervated regions within a junction indicates that synapse elimination during development leaves an indelible mark on synaptic structure. These observations suggest that the withdrawal of a motor axon from a neuromuscular junction occurs as a consequence of the stepwise elimination of all of its synapses with that muscle fiber. These results also suggest that an important aspect of synaptic competition leading to axon withdrawal is the precocious loss of AChRs beneath the nerve terminals of the axon that will be eliminated. A similar early loss of AChRs beneath one axon's synapses has been shown to occur during synapse elimination in reinnervated adult muscle (Rich and Lichtman, 1989a).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

[Histochemical study of acetylcholine receptors at the neuromuscular junctions of the cat posterior cricoarytenoid muscle using erabutoxin B].

Acetylcholine receptors (AchRs) at the neuromuscular junctions of the cat posterior cricoarytenoid muscle (PCA muscle) were demonstrated by using erabutoxin b (Eb) which has a curare-like action. Eb is one of the short-chain neurotoxins which is obtained from Laticauda-semifasciata. At the light microscopic level, the localization of the neuromuscular junctions was detected on the muscle fiber by rhodamine-labeled Eb (TMR-Eb) under a fluorescein microscopy. For the electron microscopy horseradish peroxidase-labeled Eb (HRP-Eb) was used. After incubation with HRP-Eb conjugate the tissue was fixed in a mixture of paraformaldehyde and glutaraldehyde, then embedded in Epon. The reaction products were largely restricted to the postsynaptic membrane at the neuromuscular junction. Acetylcholinesterase (AchE) activity was also demonstrated electron microscopically by Karnovsky's and Lewis' methods respectively. The reaction products were localized at the subneural apparatus of the neuromuscular junction. Both these results were compared.

Animals↗

PubMed1984

The location of neuromuscular junctions on regenerating adult mouse muscle in culture.

The location of neuromuscular junctions which form in vitro between regenerating adult mouse muscle fibres and sections of embryonic mouse spinal cord was examined. The position of the original motor end-plates on the explanted muscle fibres was determined by using either rhodamine-labelled alpha-bungarotoxin (R alpha BT) binding to the acetylcholine receptors, or by stains to demonstrate acetylcholinesterase (AChE) also located at the end-plate. In this culture system, the explanted muscle fibres degenerate and regenerate to form new myotubes which develop cross-striations and contractions. The location of the newly-formed neuromuscular junctions in these mature cultures was then demonstrated using R alpha BT-binding to acetylcholine receptors, silver impregnation and cholinesterase techniques. Less than half the new neuromuscular junctions were at the original end-plate areas indicating that, at least in this system, junctions can form at sites other than those of the original end-plate.

Acetylcholinesterase↗

PubMed1979

Glutamate potential : differences from the excitatory junctional potential revealed by diltiazem and concanavalin A in crayfish neuromuscular junction.

1. The effect of diltiazem and concanavalin A (Con A) on the crayfish neuromuscular junction was investigated in order to compare the action of L-glutamate with that of the excitatory transmitter. 2. When diltiazem (0.3 nM) was added to the perfusion fluid, the iontophoretic glutamate potential was reduced to about half, whereas the amplitude of excitatory junctional potentials (EJPs) increased by about two times. 3. Dose-response curves of L-glutamate suggested that diltiazem acted in a non-competitive manner. The decrease in amplitude of the glutamate potential caused by diltiazem was not due to the acceleration of desensitization of the glutamate receptor. 4. The increase in amplitude of EJPs caused by diltiazem was due to the increase in membrane resistance. The quantal content and size of extracellular EJPs were not affected by diltiazem. 5. In normal saline, bath application of glutamate decreased the amplitude of both glutamate potentials and EJPs because of desensitization of the glutamate receptor. The decrease in amplitude of the glutamate potential was completely prevented by previous application of Con A (10(-6) M). On the other hand, Con A had no influence on the decrease in amplitude of EJPs. 6. Some possible explanations of these pharmacological differences between glutamate potentials and EJPs revealed by diltiazem and Con A are considered.

Animals↗

PubMed1961

Observations on the fine structure and cytochemistry of mouse and human intercostal neuromuscular junctions.

The fine structure of the mouse and human intercostal muscle neuromuscular junction was studied after brief fixation in a new formol-sucrose fixative. This primary formalin fixation was followed by brief postosmication in buffered 1 per cent osmium tetroxide. Muscle blocks were embedded in methacrylate or Epon 812 epoxy resin. Marked similarities between mouse and human motor end-plates were observed. Neuromuscular junctions from both mouse and human intercostal muscle showed synaptic vesicles, primary and secondary synaptic clefts, and layered differentiation of the amorphous surface material (ASM) present on the surface of the Schwann cell plasma membrane and on the muscle surface membrane in the region of the neuromuscular junction. An attempt to stain the ASM with lead was unsuccessful. Observations on thick and thin plastic-embedded sections stained by PAS after diastase digestion showed that the ASM within the subneural apparatus is PAS positive. Alcian blue stained the endoneurium and perineurium of peripheral nerve bundles and portions of the end-plates. The similarity of the PAS-positive ASM to other basement membranes described in other sites is discussed and its possible physiologic significance within the subsynaptic apparatus is considered.

Animals↗

PubMedDate not supplied

Localization of choline acetyltransferase at neuromuscular junctions.

Choline acetyltransferase (ChAc) was localized at the neuromuscular junctions of rabbit diaphragm with immunohistochemical staining, which produced a brown color, in combination with histochemical staining for cholinesterase, which produced a blue color, in the same section. The innervations of nerve terminals at the neuromuscular junctions were comparable when a silver impregnation was substituted for the immunohistochemical staining of ChAc.

Animals↗

PubMed1994

In vivo imaging shows loss of synaptic sites from neuromuscular junctions in a model of myasthenia gravis.

We examined the pre- and postsynaptic elements of the neuromuscular junction during immune attack on the postsynaptic acetylcholine receptors (AChRs) in a model of myasthenia gravis (MG). We followed, in the sternomastoid muscle of living mice, the staining of nerve terminals and postsynaptic AChRs at individual neuromuscular junctions in situ for up to 16 days after exposure to a monoclonal anti-AChR antibody. Several exposures to this antibody over 6 days led to spotty loss of AChR staining 1 to 3 days later within individual neuromuscular junctions. In addition, we observed loss of motor nerve terminal staining at presynaptic sites opposed to postsynaptic regions that had lost AChRs. Sites that lost pre- and postsynaptic staining were often immediately adjacent to other junctional regions that maintained a high density of AChRs and still stained presynaptically. Ultimately, the loss of synaptic sites resulted in neuromuscular junctions that appeared to be abnormally fragmented. To determine whether junctions recovered from the immune attack, we followed some antibody-treated muscle fibers for an additional 8 days without further exposure to antibody. Signs of recovery were evident because some of the synaptic regions that had previously lost AChRs subsequently regained them. But these junctions still remained fragmented both pre- and postsynaptically. These findings suggest that the postsynaptic membrane is affected in a highly local way by the immune attack on AChRs occurring in MG. One consequence of this attack is a long-term loss of not only postsynaptic components but also the overlying nerve terminals.

Animals↗

PubMed2000

Ectopic expression of NCAM in skeletal muscle of transgenic mice results in terminal sprouting at the neuromuscular junction and altered structure but not function.

The neuromuscular system provides an excellent model for the analysis of molecular interactions involved in the development and plasticity of synaptic contacts. The neural cell adhesion molecule (NCAM) is believed to be involved in the development and plasticity of the neuromuscular junction, in particular the axonal sprouting response observed in paralyzed and denervated muscle. In order to explore the role of myofiber NCAM in modulating the differentiation of motor neurons, we generated transgenic mice expressing a GPI-anchored NCAM isoform that is normally found in developing and denervated muscle, under the control of a skeletal muscle-specific promoter. This results in the constitutive expression of NCAM at postnatal ages, a time when the endogenous mouse NCAM is absent from the myofiber. We found that a significant number of neuromuscular junctions in adult transgenic animals displayed terminal sprouting (>20%) reminiscent of that elicited in response to cessation of neuromuscular activity. Additionally, a significant increase in the size and complexity of neuromuscular synapses as a result of extensive intraterminal sprouting was detected. Electrophysiological studies, however, revealed no significant alterations of neuromuscular transmission at this highly efficient synapse. Sprouting in response to paralysis or following nerve crush was also significantly enhanced in transgenic animals. These results suggest that in this ectopic expression model NCAM can directly modulate synaptic structure and motor neuron-muscle interactions. The results contrast with knockout experiments of the NCAM gene, where very limited changes in the neuromuscular system were observed.

Acetylcholinesterase↗

PubMed2001

Miller Fisher syndrome: immunofluorescence and immunoelectron microscopic localization of IgG at the mouse neuromuscular junction.

In vitro electrophysiological experiments have demonstrated that IgG antibodies from patients with Miller Fisher syndrome (MFS) impair neuromuscular transmission by a fast and completely reversible combined pre- and postsynaptic blockade. In this study we investigated the cellular and subcellular binding sites of IgG from four MFS patients at the mouse hemidiaphragm by immunofluorescence and immunoelectron microscopy. IgG from all patients produced significant immunostaining at the neuromuscular junction, whereas sera from healthy volunteers or from patients with other neurological diseases did not stain neuromuscular junction. Immunoelectron microscopy revealed that, when living hemidiaphragms were incubated with IgG from MFS patients, labeling was found on both pre- and postsynaptic membranes of the neuromuscular junction, whereas terminal Schwann cells and the basal lamina covering the synaptic membranes were not labeled. These findings demonstrate that IgG from MFS patients binds to synaptic membranes of the neuromuscular junction where it might interfere with the function of both the pre- and postsynaptic activities.

Animals↗

PubMed2003

Neurophysiology of the neuromuscular junction: overview.

This manuscript reviews the physiology of neuromuscular transmission with emphasis on four features: (1) the structure of the neuromuscular junction, (2) release of vesicles of acetylcholine from the nerve terminal, (3) the roles of postsynaptic Na(+) channels and acetylcholine receptors in converting the chemical signal from the nerve terminal into a propagated action potential on the muscle fiber, and (4) the safety factor for neuromuscular transmission. It also introduces new information about how the neuromuscular junction maintains physiological integrity during muscle fiber stretch and contraction.

Acetylcholine↗

PubMed1993

Ultrastructural analysis of sympathetic neuromuscular junctions on mesenteric veins of the guinea pig.

This study reports on the detailed ultrastructure of sympathetic postganglionic varicose axon terminals on mesenteric veins leading from the ileum of the guinea pig and in particular the structural arrangement of the varicosities with venous smooth muscle cells. The response to nerve stimulation in veins has a long time course and it has been suggested that this reflects a wide separation between the site of transmitter release and the receptors on the effector cell membrane. The aim of this study was to determine the distance between individual sympathetic varicosities and smooth muscle cells in mesenteric veins. Fluorescent histochemical preparations of the sympathetic innervation of the different branches of mesenteric veins indicate the branching network of varicose axons around the vessel to be relatively dense. Electron micrographs show the innervation to be confined to the adventitia close to the medio-adventitial border and to be predominantly catecholaminergic. A serial section ultrastructural analysis of the relationship of the varicosities with the outer smooth muscle cells showed that almost all (98%) of the exposed axon varicosities in the adventitia formed neuromuscular junctions. Three-dimensional reconstructions from serial sections of individual varicosities have shown that the junctions have structural specialisations identical to neuromuscular junctions described on arterial vessels and similar to those found at skeletal neuromuscular junctions. The density of neuromuscular junctions on the veins was found to be similar to that on the corresponding artery in the same animal. We suggest that in veins, noradrenaline is released focally at neuromuscular junctions.

Animals↗