Muscle Endplate Research Articles

Prevention of autoimmune attack by targeting specific T-cell receptors in a severe combined immunodeficiency mouse model of myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor. We have previously demonstrated a selection bias of CD4+ T cells expressing the Vbeta5.1 T-cell receptor gene in the thymus of HLA-DR3 patients with MG. To evaluate the pathogenicity of these cells, severe combined immunodeficiency mice engrafted with MG thymic lymphocytes were treated with anti-Vbeta5.1 antibody. Signs of pathogenicity (eg, acetylcholine receptor loss and complement deposits at the muscle end plates of chimeric mice) were prevented in anti-Vbeta5.1-treated severe combined immunodeficiency chimeras. Pathogenicity was mediated by autoantibodies against acetylcholine receptor. Thymic cells depleted of Vbeta5.1-positive cells in vitro before cell transfer were nonpathogenic, indicating that Vbeta5.1-positive cells are involved in the production of pathogenic autoantibodies. Acetylcholine receptor loss was prevented by Vbeta5.1 targeting in HLA-DR3 patients only, demonstrating specificity for HLA-DR3-peptide complexes. The action of the anti-Vbeta5.1 antibody involved both the in vivo depletion of Vbeta5.1-expressing cells and an increase in the interferon-gamma/interleukin-4 ratio, pointing to an immune deviation-based mechanism. This demonstration that a selective and specific T-helper cell population is involved in controlling pathogenic autoantibodies in MG holds promise for the treatment of MG.

Epibatidine: high potency and broad spectrum activity on neuronal and neuromuscular nicotinic acetylcholine receptors.

The activity of epibatidine at neuronal and neuromuscular nicotinic acetylcholine receptors was investigated in several in situ and in vitro systems and compared with the activity of nicotine and suxamethonium. Activation of ganglionic nicotinic receptors by epibatidine was shown in the guinea-pig ileum (contraction mediated by the cholinergic neurons of the ileum) and in pithed and atropinized rats (rise in blood pressure). Epibatidine also activated nicotinic receptors at the peripheral terminals of afferent C-fibres (rabbit ear) and in the brain (antidiuresis in rats). The agonistic effects of epibatidine were followed by long-lasting receptor desensitization. No antinociceptive effect of epibatidine was seen in rats at a dose free of motor impairment. On muscle end plate nicotinic receptors of the rat diaphragm (not responding to depolarizing agents by contraction), epibatidine was equipotent with suxamethonium in causing neuromuscular inhibition. On an extraocular muscle of the rabbit (responding to depolarizing agents by contraction) epibatidine in vitro and in situ caused a contraction at a 100-fold lower dose than suxamethonium. The Straub tail reaction in mice to epibatidine could be attributed to the sustained stimulation of motor end plate receptors of the "slow contracting" type of muscle fibres by epibatidine. In conclusion epibatidine was the most potent agonist on all neuronal and neuromuscular nicotinic receptors examined.

Metabolic Stabilization of Muscle Nicotinic Acetylcholine Receptor by Rapsyn

Although the metabolic half-life of muscle endplate acetylcholine receptor (AChR) changes during development and after denervation in the adult, little is known about the molecular mechanisms that influence receptor stability. We have investigated the effect on AChR turnover of its interaction with rapsyn, a 43 kDa peripheral membrane protein that is closely associated with the AChR in muscle cells and is required for its clustering at endplates. Both in transfected COS cells and in cultured myotubes from rapsyn-negative and rapsyn-positive mice, we have found that the presence of rapsyn slows the turnover of AChRs by as much as twofold. The effect was similar for both embryonic (alpha2betadeltagamma) and adult (alpha2betadeltaepsilon) AChRs and for AChRs whose beta subunit lacked a putative tyrosine phosphorylation site. Neither colchicine nor cytochalasin D altered AChR turnover or prevented the rapsyn effect. Mutant rapsyn proteins whose N-terminal myristoylation signal was eliminated, or whose C terminus or zinc-finger domains were deleted, failed to change the rate of receptor turnover. Each of these mutations affects the association of the AChR with rapsyn, suggesting that AChR stability is altered by interaction between the two proteins. Our results suggest that, in addition to its role in AChR clustering, rapsyn also functions to metabolically stabilize the AChR.

Fiber atrophy, but not changes in acetylcholine receptor expression, contributes to the muscle dysfunction after immobilization.

Muscle weakness associated with critical illness can be due to the illness itself, immobilization associated with it, and/or to concomitant use of drugs that affect neuromuscular transmission. This study investigated the contribution of immobilization per se to the muscle dysfunction, as well as the associated morphologic and biochemical changes. Prospective, laboratory study. Hospital research laboratory. Adult, male, Sprague-Dawley rats, weighing 200 to 250 g, were randomly allocated to three experimental groups, depending on the duration (7, 14, or 28 days) of limb immobilization (n = 9 to 11 per group) or sham immobilization (n = 5 to 6 per group). Chronic, unilateral immobilization (disuse) of the tibialis cranialis muscle was produced by fixing the knee and ankle joints at 90 degrees flexion. The contralateral unimmobilized leg and a separate group of sham-immobilized legs served as controls. After 7, 14, or 28 days of disuse of the tibialis muscles, the peak isometric twitch (Pt) and tetanic (Po) tensions, as well as fatigability during 5 secs of nerve stimulation at 50, 100, and 150 Hz, were measured simultaneously in situ in the immobilized group and in its contralateral control, and in the sham-immobilized group and in its contralateral control. Muscle fiber and endplate morphologies were determined by histochemical methods; membrane acetylcholine receptors (AChRs) were determined by 125I alpha-bungarotoxin assay; and the level of expression of AChR subunit transcripts was determined by reverse transcriptase-polymerase chain reaction. Immobilization reduced Pt, Po, fatigability, muscle mass, and fiber cross-sectional area (p<.001 vs. controls), but did not decrease tension per unit muscle mass, fiber oxidative capacity, or motor endplate size. Muscle mass correlated with fiber cross-sectional area. Changes in fiber cross-sectional area accounted for 23% and 46% (p< or =.043) of the variability in Pt and Po, respectively. Pt and Po correlated poorly with total AChR protein and expression of epsilon- and gamma-subunit messenger RNA. To the extent that the immobilization model simulates the disuse-induced muscle dysfunction of critical illness, the results suggest that disuse per se may contribute to the muscle weakness, and that the muscle weakness is explained, almost exclusively, by the fiber atrophy and not by the qualitative or quantitative changes in AChR expression.

Desensitizing function of calcium mobilized by the postsynaptic neuronal-type nicotinic acetylcholine receptors at the neuromuscular junction

Neuronal-type nicotinic acetylcholine receptors (N-nAChR) are co-localized with muscle-type (M-)nAChR in the postjunctional endplate membrane of adult skeletal muscle fibers. The postsynaptic desensitizing functions of the N-nAChR at the neuromuscular junction and at single skeletal muscle cells have been investigated using aequorin luminescence and fluorescence confocal imaging. A biphasic elevation of local intracellular Ca2+ is elicited by prolonged nicotinic action at the mouse muscle endplates. The contractile fast and non-contractile slow Ca2+ components are operated by postsynaptic M- and colocalized N-type nAChR, respectively. We have named the latter slow one RAMIC (receptor-activity modulating intracellular Ca2+). The N-nAChR are activated by nicotine and choline, and RAMIC are antagonized by methyllycaconitine and dihydro-beta-erythroidine. Neuromuscular functions may be regulated by a dual nAChR system to maintain the normal postsynaptic excitability. Certain N-nAChR may be also endowed with the same functional role in the central nervous system.

Distribution of myenteric NO neurons along the guinea-pig esophagus

Intrinsic nitrergic (NO) neurons of the guinea-pig esophagus were histologically studied to elucidate the physiological significance of the myenteric plexus located in the esophageal striated muscle and smooth muscle of the lower esophageal sphincter. Double staining for PGP 9.5 immunohistochemistry and NADPH-diaphorase histochemistry, which depicts whole neuronal elements and nitrergic NO neurons, respectively, revealed that the plexus had different network patterns along the entire course of the esophagus, and that NADPH-diaphorase positive neurons made up on average 69% of the total number of myenteric neurons. Motor endplates of the esophageal striated muscles that were stained by acetylcholinesterase histochemistry, were often observed in association with NADPH-diaphorase positive varicose fibers that were traced to the myenteric ganglia, though their direct continuity with the neuronal cell bodies could not be ascertained. We conclude that the myenteric NADPH-diaphorase positive neurons in the guinea-pig esophagus contribute to the innervation of the striated muscles as well as the smooth muscles of the lower esophageal sphincter.

Calcitonin gene-related peptide potentiates nicotinic acetylcholine receptor-operated slow Ca2+ mobilization at mouse muscle endplates.

1. The involvement of calcitonin gene-related peptide (CGRP) in the non-contractile slow Ca2+ mobilization induced by prolonged nicotinic stimulation was investigated by measurement of [Ca2+], levels in mouse single muscle cells (flexor digitorum brevis; FDB) loaded with a Ca2+ indicator fluo-3 using confocal laser scanning microscopy. 2. CGRP (3-30 nM) potentiated acetylcholine (ACh, 1 microM)-elicited slow Ca2+ mobilization in a concentration-dependent manner. 3. The potentiation by CGRP of the slow Ca2+ component was greatly depressed by a competitive nicotinic antagonist (+)-tubocurarine (5 microM). The Ca2+ channel blocker nitrendipine (1 microM) affected neither ACh responses nor the CGRP potentiation. 4. The slow Ca2+ component was completely abolished by reducing [Ca2+]0 from 2.5 to 0.25 mM whereas the fast component was not affected. The CGRP-induced potentiation of slow Ca2+ signal was also depressed by decreasing [Ca2+]0. 5. Isoproterenol (30 microM) and 8-bromo-adenosine 3',5'-cyclic monophosphate (1 mM) potentiated the ACh-elicited slow Ca2+ response. The potentiation by CGRP of the slow Ca2+ component was completely abolished by a protein kinase-A inhibitor H-89 (1 microM). 6. These findings indicate that CGRP potentiates the nicotinic ACh receptor-operated slow Ca2+ signal via the activation of protein kinase-A system at the skeletal muscle endplates.

Eyelid Movement During Complete Neuromuscular Block

Eyelid Movement During Complete Neuromuscular Block

Acetylcholine sensitivity of biphasic Ca2+ mobilization induced by nicotinic receptor activation at the mouse skeletal muscle endplate

1. Acetylcholine (ACh) was locally applied onto the endplate region in a mouse phrenic nerve-diaphragm muscle preparation to measure intracellular free calcium ([Ca2+]i) entry through nicotinic ACh receptors (AChRs) by use of Ca2+-aequorin luminescence. 2. ACh (0.1-3 mM, 20 microl) elicited biphasic elevation of [Ca2+]i (fast and slow Ca2+ mobilization) in muscle cells. The peak amplitude of the slow Ca2+ mobilization (not accompanied by twitch tension) was concentration-dependently increased by ACh, whereas that of the fast component (accompanied by twitch tension) reached a maximum response at a lower concentration (0.1 mM) of applied ACh. 3. A pulse of nicotinic agonists, (-)-nicotine (10 mM) and 1,1-dimethyl-4-phenyl-piperazinium (10 mM), but not a muscarinic agonist pilocarpine (10 mM), also elicited a biphasic Ca2+ signal. 4. Even though ACh release from motor nerve endings was blocked by botulinum toxin (5 microg, bolus i.p. before isolation of the tissue), the generation of both a fast and slow Ca2+ component caused by ACh application was observed. 5. These results strongly suggest that ACh locally applied onto the endplate region of skeletal muscle induces a slow Ca2+ signal reflecting Ca2+ entry through a postsynaptic nicotinic AChR, which has a low sensitivity to transmitter ACh.

Calcium in ischemic cell death.

This review article deals with the role of calcium in ischemic cell death. A calcium-related mechanism was proposed more than two decades ago to explain cell necrosis incurred in cardiac ischemia and muscular dystrophy. In fact, an excitotoxic hypothesis was advanced to explain the acetylcholine-related death of muscle end plates. A similar hypothesis was proposed to explain selective neuronal damage in the brain in ischemia, hypoglycemic coma, and status epilepticus. The original concepts encompass the hypothesis that cell damage in ischemia-reperfusion is due to enhanced activity of phospholipases and proteases, leading to release of free fatty acids and their breakdown products and to degradation of cytoskeletal proteins. It is equally clear that a coupling exists between influx of calcium into cells and their production of reactive oxygen species, such as .O2, H2O2, and .OH. Recent results have underscored the role of calcium in ischemic cell death. A coupling has been demonstrated among glutamate release, calcium influx, and enhanced production of reactive metabolites such as .O2-, .OH, and nitric oxide. It has become equally clear that the combination of .O2- and nitric oxide can yield peroxynitrate, a metabolite with potentially devastating effects. The mitochondria have again come into the focus of interest. This is because certain conditions, notably mitochondrial calcium accumulation and oxidative stress, can trigger the assembly (opening) of a high-conductance pore in the inner mitochondrial membrane. The mitochondrial permeability transition (MPT) pore leads to a collapse of the electrochemical potential for H+, thereby arresting ATP production and triggering production of reactive oxygen species. The occurrence of an MPT in vivo is suggested by the dramatic anti-ischemic effect of cyclosporin A, a virtually specific blocker of the MPT in vitro in transient forebrain ischemia. However, cyclosporin A has limited effect on the cell damage incurred as a result of 2 hours of focal cerebral ischemia, suggesting that factors other than MPT play a role. It is discussed whether this could reflect the operation of phospholipase A2 activity and degradation of the lipid skeleton of the inner mitochondrial membrane. Calcium is one of the triggers involved in ischemic cell death, whatever the mechanism.

The effect of aging on the morphological nerve changes during muscle reinnervation after nerve crush

The extent of the muscle endplate reinnervation that followed crush injury of the sciatic nerve was compared between young adult (4 and 5 months old) and aged (24 months old) animals. The time course of regeneration in the muscular nerve bundle, its ramification, and the nerve terminal was immunohistochemically estimated using an antibody against the neuron specific enolase (NSE), a neuronal marker. During early phases of regeneration (7, 21 and 28 days post-crush) in the young adult animal, there were tortuosity, vacuolation and/or unfasciculation in the nerve bundle and its ramification, along with immature nerve terminals and multiple innervation. Following a subsequent advancement in reinnervation to the denervated motor endplates, the adult type of single motor innervation was common on the day 56. The old muscles basically followed the course of reversible axotomy alike the young adult ones. The age difference accounted for as fol-lows: a reduced rate of reinnervation as indicated by a greater frequency of abnormal nerve bundles and immature nerve terminals at 28 days and 56 days post-crush, as well as unusual pathways or striking tortuosity represented by the NSE-labeled processes between day 7 and 56; late in the reinnervation period, abnormal regeneration characterized by damage of the nerve bundle, and poorly developed terminal architec-tures. These results suggest that despite the capability of the nerve from the old animals to extend its process, re-establishment of normal single motor innervation is reduced due to some age-related deficits, which may be related to the impaired Schwann cell-axon interactions.

Ultrastructural changes in motor endplates of the lumbrical muscles of rats induced by a microsomal Ca2+ ATPase inhibitor, 2,5-di(tert-butyl)-1,4-hydroquinone.

Female Wistar rats were treated orally for 5 days with 80 mg/kg body weight of 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), a microsomal Ca2+ ATPase inhibitor. Motor endplates of the lumbrical muscles were examined by light and electron microscopy. There was a decrease in body weight in the treated rats from the first day after administration, and toxic signs appeared after the third day, such as adoption of a prone position, salivation, lacrymation, and an abnormal gait and/or muscle weakness. No remarkable macroscopic or light microscopic changes were noted in the lumbrical muscles as well as other peripheral nerves of hind legs of the treated rats killed 1 day after the last DTBHQ treatment. Ultrastructurally, neurotoxicity characterized by loss of synaptic vesicles and mitochondria in the motor endplates, and by destruction of the motor terminals was detected in the lumbrical muscles of the treated rats. These results strongly indicate that DTBHQ targets the motor endplates in the rat lumbrical muscles and suggest that the resultant damage is responsible for the appearance of neurological signs, such as an abnormal gait and loss of muscle control.

O-20-287 - Reconstructive operations of brachial plexus injury and the experimental model of their management

Thirty five patients with brachial plexus injury have been operated on. Results were very good in the case of supraganglionic lesions in 15 patients. Fair and good results were obtained in 72% of Is patients with neurotization procedure of musculocutaneous and axillary nerves. Transfer of latissimus dorsi muscle instead of the biceps muscle was successful in another two cases. In the experimental animals the reinnervation of the biceps muscles was obtained by application of both cellular and acellular nerve grafts into the spinal cord and distally sutured with musculocutaneous nerve stump. Behavioural, electrophysiological as well as morphological methods proved the reconnection of the spinal motor neurons with the muscle end plates.

Neuronal nicotinic receptor operates slow Ca2+ mobilization at mouse muscle endplate

The contribution to neuromuscular functions by neuronal nicotinic acetylcholine receptor (nAChR) expressed at skeletal muscle endplate was investigated using intracellular Ca2+ measurements. A neuronal nAChR blocker, methyllycaconitine (MLA), depressed non-contractile Ca2+ mobilization without affecting muscle nAChR activity in nerve-stimulated mouse diaphragm muscle, after cholinesterase inhibition. Confocal imaging demonstrates that the MLA-sensitive Ca2+ mobilization also occurred at the endplate in single flexor digitorum brevis muscle cells as the slow component of two-phasic Ca2+ elevation after the prolonged nicotinic stimulation. A monoclonal antibody to α1 subunit of muscle nAChR depressed the fast but not the slow component. Thus, muscle neuronal-nAChR can induce the localized rise of Ca2+ at the postjunctional sites.

Autoimmunity to Ryanodine Receptor and Titin in Myasthenia Gravis is Associated with GM Allotypes

Myasthenia gravis (MG) is mediated by autoantibodies against the acetylcholine receptor at the muscle endplate. Some MG patients have in addition antibodies (Ab) to the skeletal muscle proteins ryanodine receptor (RyR) and titin. We have examined GM and KM allotypes, RyR and titin Ab in 44 MG patients (37 thymoma patients and 7 non-thymoma, late-onset patients) and 292 non-MG controls to see if GM/KM allotypes associate with differences in autoantibody production. All patients had titin Ab, and 15 thymoma patients had also RyR Ab. The phenotype GM 1, 2, 3 23 5, 21 was significantly increased in the patients with titin Ab compared with the non- MG controls (X2 = 4.93, p < 0.05). Thymoma patients with RyR Ab had a higher frequency of the GM 3 23 5 phenotype compared with RyR Ab negative patients and controls (X2 = 7.1, p < 0.05). KM allotypes did not differ between RyR Ab positive or titin Ab positive patients and controls. GM phenotypes may thus be associated with an autoimmune response against the muscle proteins titin and RyR in MG patients.

246 CGRP potentiates slow Ca 2+ mobilization via neuronal nicotinic receptor at mouse muscle endplates

The aim of this study was to investigate the mechanism for the reversal effect of NF449 (a suramin analogue) on the neuromuscular block induced by d-tubocurarine (d-TC).Nerve-stimulated muscle contractions and end-plate potentials were performed in mouse phrenic nerve-diaphragm preparations. Acetylcholine (ACh)-induced muscle contractions were performed in the chick biventer cervicis preparations. Presynaptic nerve terminal waveform recordings were performed in mouse triangularis sterni preparations.Amongst the suramin analogues in this study, only the NF449 and suramin were able to reverse the blockade effect produced by d-TC on nerve-stimulated muscle contractions. Each of these suramin analogues (NF007, NF023, NF279 and NF449) alone has no significant effect on the amplitude of nerve-stimulated muscle contractions. NF449 and suramin also showed the antagonising effects on the inhibition of end-plate potentials induced by d-TC. Furthermore, pre-treatment with NF449 can antagonise the inhibition of d-TC in ACh-induced contractions of chick biventer cervicis muscle. NF449 produced a greater rightward shift of the dose–response inhibition curve for d-TC than did suramin. Because other purinergic 2X (P2X) receptor antagonists, NF023 and NF279, do not have the reverse effects on the neuromuscular blockade of d-TC, the effect of NF449 seems irrelevant to inhibition of P2X receptors.These data suggest that NF449 was able to compete with the binding of d-TC on the nicotinic ACh receptors, and the effect of NF449 was more potent than suramin in reducing the inhibition of d-TC. The structure of NF449 may provide useful information for designing potent antidotes against neuromuscular toxins.

CGRP potentiates slow Ca mobilization via neuronal nicotinic receptor at mouse muscle endplates

CGRP potentiates slow Ca mobilization via neuronal nicotinic receptor at mouse muscle endplates

Nitric oxide synthase and cyclic GMP-dependent protein kinase concentrated at the neuromuscular endplate

Nitric oxide mediates diverse functions in development and physiology of vertebrate skeletal muscle. Neuronal type nitric oxide synthase-μ is enriched in fast-twitch fibers and binds to syntrophin, a component of the sarcolemmal dystrophin glycoprotein complex. Here, we show that cyclic GMP-dependent protein kinase type I, a primary effector for nitric oxide, occurs selectively at the neuromuscular junction, in mice and rats, and both neuronal type nitric oxide synthase-μ and cyclic GMP-dependent protein kinase type I remain at skeletal muscle endplates at least two weeks following muscle denervation. Expression of neuronal type nitric oxide synthase-μ and cyclic GMP-dependent protein kinase type I are up-regulated following fusion of cultured primary myotubes. Interestingly, the highest levels of neuronal type nitric oxide synthase-μ in muscle are found complexed with dystrophin at the sarcolemma of intrafusal fibers in muscle spindles.Localization of neuronal type nitric oxide synthase-μ and cyclic GMP-dependent protein kinase type I at the neuromuscular junction suggests functions for nitric oxide and cyclic GMP in the regulation of synaptic actions of intra- and extrafusal muscle fibers.

Galanin-containing nerve terminals that are involved in a dual innervation of the striated muscles of the rat esophagus

Galanin (GAL)-immunoreactive axon terminals on motor endplates of the esophageal striated muscles were demonstrated in mice, guinea-pigs and rats. The GAL-terminals innervated 33% of AChE-reactive motor endplates in mice and 6% of those in guinea-pigs. Double immunostaining revealed that separate GAL- and CGRP-positive terminals were localized within the same motor endplates in mice and rats. The GAL and CGRP terminals had different morphologies. No CGRP-immunoreactivity was found on motor endplates of the guinea-pig esophagus. Double immunostaining in rats showed that 68% of motor endplates with CGRP-nerve terminals were also supplied by GAL-nerve terminals, suggesting that the majority of esophageal striated muscles receive a dual innervation of GAL- and CGRP/ACh-containing terminals. By immuno-electronmicroscopy in the rat esophagus, GAL-immunoreactivity was found in a small type of nerve terminals that possessed many large cored vesicles (90–130 nm) with intense immunoreaction. Larger GAL-negative nerve terminals with a cluster of small clear vesicle (40–50 nm), which seemed to be ACh-containing nerve terminals, were adjacent to a depression or slight protrusion of the sarcolemma and well-developed folds in the muscle fibers. At the motor endplates, the GAL-positive terminals made a synaptic contact via basement membrane with the sarcolemma of the muscle fibers, which was characterized by post-synaptic intense electron density. In most of all situations, in which the GAL-positive terminals and GAL-negative or -positive terminals were adjacent to each other and were also apposed to the striated muscles, the terminals were separated by attenuated sheet- or tongue-like cytoplasmic processes that appeared to originate from Schwann cells. Thus, the GAL-nerve terminals seem to provide a direct innervation of the striated muscle fibers rather than innervating the ACh-containing motor nerve terminals adjacent to the GAL-terminals.

Galanin-containing nerve terminals that are involved in a dual innervation of the striated muscles of the rat esophagus

Galanin (GAL)-immunoreactive axon terminals on motor endplates of the esophageal striated muscles were demonstrated in mice, guinea-pigs and rats. The GAL-terminals innervated 33% of AChE-reactive motor endplates in mice and 6% of those in guinea-pigs. Double immunostaining revealed that separate GAL- and CGRP-positive terminals were localized within the same motor endplates in mice and rats. The GAL and CGRP terminals had different morphologies. No CGRP-immunoreactivity was found on motor endplates of the guinea-pig esophagus. Double immunostaining in rats showed that 68% of motor endplates with CGRP-nerve terminals were also supplied by GAL-nerve terminals, suggesting that the majority of esophageal striated muscles receive a dual innervation of GAL- and CGRP/ACh-containing terminals. By immuno-electronmicroscopy in the rat esophagus, GAL-immunoreactivity was found in a small type of nerve terminals that possessed many large cored vesicles (90–130 nm) with intense immunoreaction. Larger GAL-negative nerve terminals with a cluster of small clear vesicle (40–50 nm), which seemed to be ACh-containing nerve terminals, were adjacent to a depression or slight protrusion of the sarcolemma and well-developed folds in the muscle fibers. At the motor endplates, the GAL-positive terminals made a synaptic contact via basement membrane with the sarcolemma of the muscle fibers, which was characterized by post-synaptic intense electron density. In most of all situations, in which the GAL-positive terminals and GAL-negative or -positive terminals were adjacent to each other and were also apposed to the striated muscles, the terminals were separated by attenuated sheet- or tongue-like cytoplasmic processes that appeared to originate from Schwann cells. Thus, the GAL-nerve terminals seem to provide a direct innervation of the striated muscle fibers rather than innervating the ACh-containing motor nerve terminals adjacent to the GAL-terminals.