Complex Correlations Between Desmin Content, Myofiber Types, and Innervation Patterns in the Human Extraocular Muscles.

Purpose: To investigate whether the distribution of intermediate filament protein desmin is related to the different patterns of innervation in the human extraocular muscles (EOMs). Methods: EOM samples were analyzed with immunohistochemistry using antibodies against desmin, vimentin, different myosin heavy chain (MyHC) isoforms, and fetal and adult acetylcholine receptor (AChR) subunits. Neuromuscular junctions (NMJs) were identified with α-bungarotoxin or with antibodies against neurofilament and synaptophysin. Results: Desmin was present in the vast majority of myofibers, but it was weakly present or absent in a limited area in the close vicinity of the single en plaque NMJs in less than half of these myofibers. Desmin was either present or lacking in MyHCsto/I myofibers displaying multiple en grappe endings but present in MyHCsto/I myofibers receiving spiral nerve endings. In MyHCeom myofibers displaying multiterminal en plaque endings, desmin was either present or absent irrespective of AChR subunits or EOM layer. Vimentin did not substitute for the lack of desmin. Conclusions: The results indicate that the human EOMs have a more complex cytoskeletal organization than other muscles and suggest additional signalling mechanisms from the NMJs to the myofibers.

NMDA receptors at the endplate of rat skeletal muscles: precise postsynaptic localization.

In this study we demonstrate expression of the N-methyl-D-aspartate receptor NR1 subunit in the rat neuromuscular junction of skeletal muscles of different functional types (extensor digitorum longus, soleus, and diaphragm muscles) using fluorescence immunocytochemistry. Electron microscopic immunocytochemistry has shown that the NR1 subunit is localized solely on the sarcolemma in the depths of the postsynaptic folds. These findings suggest participation of the glutamatergic signaling system in functioning of the adult mammalian neuromuscular junction.

[Histochemical findings of and fine structural changes in motor endplates in diseases with neuromuscular transmission abnormalities].

We herein review the histochemical findings and fine structural changes of motor endplates associated with diseases causing neuromuscular transmission abnormalities. In anti-acetylcholine receptor (AChR) antibody-positive myasthenia gravis (MG), type 2 fiber atrophy is observed, and the motor endplates show a reduction in the nerve terminal area, simplification of the postsynaptic membrane, decreased number of acetylcholine receptors, and deposition of immune complexes. In anti-MuSK antibody-positive MG, the fine structure shows a decrease in the postsynaptic membrane length, but the secondary synaptic cleft is preserved. There is no decrease in the number of AChRs, and there are no deposits of immune complexes at the motor endplates. Patients with Lambert-Eaton myasthenic syndrome show type 2 fiber atrophy, their motor endplates show a decrease in both the mean postsynaptic area and postsynaptic membrane length in the brachial biceps muscle. Congenital myasthenic syndrome with episodic apnea is characterized only by small-sized synaptic vesicles; the postsynaptic area is preserved. In subjects with congenital myasthenic syndrome with acetylcholinesterase deficiency, quantitative electron microscopy reveals a significant decrease in the nerve terminal size and presynaptic membrane length; further, the Schwann cell processes extend into the primary synaptic cleft, and partially or completely occlude the presynaptic membrane. The postsynaptic folds are degenerated, and associated with pinocytotic vesicles and labyrinthine membranous networks. Patients with slow-channel congenital myasthenia syndrome show type 1 fiber predominance, and their junctional folds are typically degenerated with widened synaptic space and loss of AChRs. Patients with AChR deficiency syndrome caused by recessive mutations in AChR subunits also show type 1 fiber predominance, and while most junctional folds are normal, some are simplified and have smaller than normal endplates. Rapsin and MuSK mutations cause type 1 fiber predominance, and the small postsynaptic area is associated with AChR decrease.

The differential effects of pathway- versus target-derived glial cell line-derived neurotrophic factor on peripheral nerve regeneration.

OBJECT: Glial cell line-derived neurotrophic factor (GDNF) has potent survival effects on central and peripheral nerve populations. The authors examined the differential effects of GDNF following either a sciatic nerve crush injury in mice that overexpressed GDNF in the central or peripheral nervous systems (glial fibrillary acidic protein [GFAP]-GDNF) or in the muscle target (Myo-GDNF). METHODS: Adult mice (GFAP-GDNF, Myo-GDNF, or wild-type [WT] animals) underwent sciatic nerve crush and were evaluated using histomorphometry and muscle force and power testing. Uninjured WT animals served as controls. RESULTS: In the sciatic nerve crush, the Myo-GDNF mice demonstrated a higher number of nerve fibers, fiber density, and nerve percentage (p < 0.05) at 2 weeks. The early regenerative response did not result in superlative functional recovery. At 3 weeks, GFAP-GDNF animals exhibit fewer nerve fibers, decreased fiber width, and decreased nerve percentage compared with WT and Myo-GDNF mice (p < 0.05). By 6 weeks, there were no significant differences between groups. CONCLUSIONS: Peripheral delivery of GDNF resulted in earlier regeneration following sciatic nerve crush injuries than that with central GDNF delivery. Treatment with neurotrophic factors such as GDNF may offer new possibilities for the treatment of peripheral nerve injury.

Defective neuromuscular synapses in mice lacking amyloid precursor protein (APP) and APP-Like protein 2.

Biochemical and genetic studies place the amyloid precursor protein (APP) at the center stage of Alzheimer's disease (AD) pathogenesis. Although mutations in the APP gene lead to dominant inheritance of familial AD, the normal function of APP remains elusive. Here, we report that the APP family of proteins plays an essential role in the development of neuromuscular synapses. Mice deficient in APP and its homolog APP-like protein 2 (APLP2) exhibit aberrant apposition of presynaptic marker proteins with postsynaptic acetylcholine receptors and excessive nerve terminal sprouting. The number of synaptic vesicles at presynaptic terminals is dramatically reduced. These structural abnormalities are accompanied by defective neurotransmitter release and a high incidence of synaptic failure. Our results identify APP/APLP2 as key regulators of structure and function of developing neuromuscular synapses.

Enteric co-innervation of motor endplates in the esophagus: state of the art ten years after.

The existence of a distinct ganglionated myenteric plexus between the two layers of the striated tunica muscularis of the mammalian esophagus represented an enigma for quite a while. Although an enteric co-innervation of vagally innervated motor endplates in the esophagus has been repeatedly suggested, it was not possible until recently to demonstrate this dual innervation. Ten years ago, we were able to demonstrate that motor endplates in the rat esophagus receive a dual innervation from both vagal nerve fibers originating in the brain stem and from varicose enteric nerve fibers originating in the myenteric plexus. Since then, a considerable amount of data could be raised on enteric co-innervation and its occurrence in a variety of species, including humans, its neurochemistry, spatial relationships on motor endplates, ontogeny, and possible roles during esophageal peristalsis. These data underline the significance of this newly discovered innervation component, although its function is still largely unknown. The aim of this review is to summarize current knowledge about enteric co-innervation of esophageal striated muscle and to provide some hints as to its functional significance.

Are MuSK antibodies the primary cause of myasthenic symptoms?

OBJECTIVE: To investigate the morphologic, electrophysiologic, and molecular correlates of muscle-specific tyrosine kinase-seropositive [MuSK(+)] myasthenia gravis (MG). BACKGROUND: Anti-MuSK antibodies are detected in some of acetylcholine receptor-seronegative [AChR(-)] patients with MG with prominent facial, bulbar, and respiratory muscle involvement. The morphologic and electrophysiologic correlates of MuSK(+) MG have not been investigated to date. METHODS: Immunohistochemistry, electron microscopy, and in vitro electrophysiology studies were performed on an intercostal muscle specimen of a patient with MuSK(+) MG and in control subjects. MUSK was directly sequenced, and the nucleotide changes were traced with allele-specific PCR in control subjects. RESULTS: A man aged 34 years has had facial weakness since childhood and progressive bulbar and respiratory muscle weakness and intermittent diplopia since age 21 years. He has thin temporalis and masseter muscles, a high-arched palate, and an atrophic tongue. EMG shows a 36% decrement in facial muscles. His mother has similar facial features. His endplates (EPs) show no AChR or MuSK deficiency, but the amplitudes of the miniature EP potentials and currents are reduced to 35% and 55% of normal, respectively. EP ultrastructure is well preserved, but some junctional folds immunostain faintly for immunoglobulin G. Mutation analysis of MUSK reveals one rare and two common DNA polymorphisms. CONCLUSIONS: 1) The circulating anti-muscle-specific tyrosine kinase antibodies caused neither muscle-specific tyrosine kinase nor acetylcholine receptor deficiency at the endplates; 2) the reduced intercostal miniature endplate potential and current amplitudes were not accounted for by acetylcholine receptor deficiency; 3) the faint immunoglobulin G deposits at the endplates may or may not represent anti-muscle-specific tyrosine kinase antibodies; and 4) the anti-muscle-specific tyrosine kinase antibodies may not be the primary cause of myasthenic symptoms in this patient.

Vesicular glutamate transporter 1 immunoreactivity in motor endplates of striated esophageal but not skeletal muscles in the mouse.

Glutamate, the major excitatory transmitter in the central nervous system, has been speculated for years to influence mammalian motor endplates but trials to identify glutamatergic motor terminals failed because specific markers were not available. Recently, antibodies to vesicular glutamate transporters (VGLUTs) opened new possibilities for further morphological investigations. We detected VGLUT1 immunoreactivity (-ir), but not VGLUT2-ir and VGLUT3-ir, respectively, in many motor nerve terminals in motor endplates of the mouse esophagus as identified by alpha-bungarotoxin or colocalization of VGLUT1 with choline acetyltransferase. These findings suggest that glutamate is co-stored with acetylcholine in esophageal neuromuscular junctions. Surprisingly, we found neither VGLUT1-ir nor VGLUT2-ir or VGLUT3-ir in neuromuscular junctions of somitic and branchiogenic skeletal muscles. This may reflect differences in functional properties and the embryonic origin between skeletal and esophageal striated muscle fibers.

The spatiotemporal relationship among Schwann cells, axons and postsynaptic acetylcholine receptor regions during muscle reinnervation in aged rats.

To morphologically define the aging-related features during muscle reinnervation the spatiotemporal relationships among the major components of the neuromuscular junctions (NMJs) were investigated. A total of 64 rats, 30 adults (4 months old) and 34 aged adults (24 months old), were used. Between 1 and 12 weeks after sciatic nerve-crushing injury, cryosections of skeletal muscle were single or double labeled for S100, a marker of Schwann cells (SCs), for protein gene product 9.5, a neuronal marker, and for alpha-bungarotoxin (alpha-BT), a marker of the acetylcholine receptor site (AChR site), and then observed by confocal laser microscopy. The most obvious age changes were noted: (1) the regenerating SCs and axons were delayed in their arrival at the NMJ, (2) the dimensions of terminal SCs and AChR sites displayed a drastic and long-lasting drop (for terminal SCs, during 1-8 weeks; for AChR sites, during 1-12 weeks); (3) the degree of spatial overlap between AChR sites and terminal SCs was markedly low until 8 weeks post-crush; (4) damage and poor formation in the SCs, terminal axons and AChR sites, together with poor process extension from the terminal SC or terminal axon, were pronounced; (5) persistent aberrant changes, such as multiple innervation and terminal axon sprouting, together with poorly formed collateral innervation, nerve bundles, and NMJs, more frequently occurred in the later reinnervation period. Thus, with aging, regeneration is impaired during the period in which regenerating SC strands and axons extend into NMJs and the subsequent establishment of nerve-muscle contact is in progress. A complex set of morphological abnormalities between or among the TSCs, terminal axons, and AChR sites may be important in slowing of regeneration and reinnervation in aged motor endplates.

Ciliary neurotrophic factor is required for motoneuron sprouting.

We used mutant mice that lack the gene for ciliary neurotrophic factor (CNTF) to test the hypothesis that it is an endogenous sprouting factor. Fibers in the lateral gastrocnemius muscle were either partially denervated by transection of one of the branches of its nerve or paralyzed by intramuscular injection of botulinum toxin. This results in a significant sprouting response at the terminals of intact motoneurons in normal animals. We did not detect sprouting produced by either stimulus in mice lacking CNTF. When exogenous CNTF was administered to CNTF knockout mice following partial muscle denervation, they mounted a typical sprouting response. Thus CNTF is a critical factor in the process of sprout formation after both partial denervation injury and neuromuscular paralysis. It may function as part of a cellular compensatory mechanism after neuronal injury.

Development of neuron-neuron synapses.

Our understanding of neuronal synapse development has advanced in recent years. The development of glycinergic synapses appears to depend on gephyrin and glycine receptor activity. Molecular characterization of the structure and development of glutamatergic synapses is in progress, but the underlying mechanisms remain unclear. Activity-dependent mechanisms and specific molecules that regulate the morphological development of dendritic spines have recently been identified.

Neurogenic calcitonin gene-related peptide: a neurotrophic factor in the maintenance of acetylcholinesterase molecular forms in adult skeletal muscles.

This work addresses the role of calcitonin gene-related peptide (CGRP) in the physiological maintenance of acetylcholinesterase (AChE) molecular forms in motor endplate regions of adult Sprague-Dawley rat fast-twitch anterior gracilis muscles. Results show that: (a) CGRP is present in obturator nerve motor neurons which supply the gracilis muscle, as well as in the corresponding motor endplate regions where high levels of both AChE activity and acetylcholine receptors (AChRs) are detected; (b) endplate-associated CGRP declines with muscle denervation several hours before any changes in AChE forms are detected; (c) a single subcutaneous injection of CGRP reversibly reduces the activities of all AChE forms in endplate regions of normally innervated and otherwise untreated gracilis muscles; and (d) similar treatment with hCGRP(8-37), a potent and selective CGRP antagonist, produces the opposite effects, i.e., it reversibly elevates the activities of all AChE forms. These and other findings indicate that CGRP and hCGRP(8-37) influence the mechanism(s) by which AChE forms are maintained in intact adult gracilis muscles. Indeed, the findings lend strong support to the hypothesis that nerve-derived CGRP plays a key role in the trophic regulation of AChE forms at the neuromuscular junction.

Myasthenia gravis, thymoma, intestinal pseudo-obstruction, and neuronal nicotinic acetylcholine receptor antibody.

Intestinal pseudo-obstruction occurs rarely in patients with myasthenia gravis (MG) and thymoma. The etiology of the intestinal pseudo-obstruction remains to be elucidated, although an autoimmune mechanism is postulated. We present the first report of neuronal nicotinic acetylcholine receptor (AChR)-specific antibody in a patient with seropositive MG, malignant thymoma, and intestinal pseudo-obstruction. This finding provides evidence that intestinal pseudo-obstruction associated with thymoma and possibly other neoplasms may be related to antibodies against the neuronal nicotinic receptors at autonomic ganglia.

Distinct structures and functions of related pre- and postsynaptic carbohydrates at the mammalian neuromuscular junction.

Carbohydrates that terminate in beta-linked N-acetylgalactosamine (betaGalNAc) residues are concentrated in the postsynaptic apparatus of the skeletal neuromuscular junction and have been implicated in the differentiation of the postsynaptic membrane. We now report that distinct synapse-specific betaGalNAc-containing carbohydrates are associated with motor nerve terminals. Two monoclonal antibodies that recognize distinct betaGalNAc-containing epitopes, CT1 and CT2, both stain synaptic sites on skeletal muscle fibers. However, CT1 selectively stains nerve terminal, whereas CT2 selectively stains the postsynaptic apparatus. Likewise, CT1 and CT2 selectively stain motoneuron-like and muscle cell lines, respectively. Using the cell lines, we identify distinct CT1- and CT2-reactive glycolipids and glycoproteins. Finally, we show that GalNAc modulates the adhesion of motoneuron-like cells to recombinant fragments of a synaptic cleft component, laminin beta2. Together, these results show that pre- as well as postsynaptic membranes bear and are affected by distinct but related synapse-specific carbohydrates.

Endurance training increases acetylcholine receptor quantity at neuromuscular junctions of adult rat skeletal muscle.

The aim of the study was to test the hypothesis that a 16 week endurance training program would alter the abundance of endplate-associated nicotinic acetylcholine receptors (nAChR) in various rat skeletal muscles. We found a 20% increase in endplate-specific [125I]alpha-bungarotoxin binding in several muscles of trained rats, accompanied by equal susceptibility of toxin binding to the inhibitory effect of D-tubocurarine in sedentary and trained muscles. We conclude that the neuromuscular junction adaptations that occur with increased chronic activation include an increase in nAChR number. Results of experiments designed to determine nAChR turnover also suggest that this effect is mediated by an alteration in the receptor's metabolic state. The potential implications and mechanisms of this adaptation are discussed.

Schwann cells proliferate at rat neuromuscular junctions during development and regeneration.

Terminal Schwann cells (TSCs) cover neuromuscular junctions and are important in the repair and maintenance of these synapses. We have examined how these cells are generated at developing junctions and how their number is regulated during repair of nerve injury. At birth, approximately half of the junctions in rat soleus and extensor digitorum longus muscles have one TSC soma. Somata are absent from the remainder, although Schwann cell (SC) processes arising from somata along the preterminal axon cover almost all of these synapses. By 2 months of age, junctions have gained an additional two to three TSCs. Most of this gain occurs during the first 2 postnatal weeks and largely precedes the expansion of endplate size. Although the initial addition is caused by cell migration, mitotic labeling shows extensive division of TSCs at junctions. A slower addition of TSCs occurs in adult muscles, and TSC number in the adult is correlated with endplate size. During repair of nerve injury, TSC number is regulated by a combination of signals from motor neurons and denervated tissue. As shown previously (Connor et al., 1987), denervation of adult muscles did not, in itself, cause TSC mitosis. However, TSCs became mitotic during reinnervation. Partial denervation induced division of TSCs at innervated but not denervated endplates. A disproportionate number of these mitotic cells were found at endplates contacted by TSC processes extended from nearby denervated endplates, contacts known to promote nerve sprouting. These results show an association between TSC mitotic activity and alterations in synaptic structure during development, sprouting, and reinnervation.

Neurocalcin-immunopositive nerve terminals in the muscle spindle, Golgi tendon organ and motor endplate.

The present study revealed the immunohistochemical distribution of neurocalcin, a three EF-hand calcium-binding protein, in the rat muscles and tendons. In the muscle spindles, annulospiral endings, which made spirals around the intrafusal muscles, showed intense neurocalcin-immunoreactivity. In the Golgi tendon organs, immunopositive thick nerve fibers entered the collagenous fibers resulting in the projection of many swelling terminals. In all examined muscles, nerve terminals in the motor endplates showed neurocalcin-immunoreactivity associated with the membranes of synaptic vesicles and mitochondria. These findings suggest that neurocalcin is distributed and regulates calcium signaling in both afferent and efferent nerve terminals in the muscles and tendons.

Polyneural innervation in the psoas muscle of the developing rat.

Polyneural innervation was studied in the psoas muscle in developing rats from P4 till P25 and at adult age, with the combined silver-acetylcholinesterase technique. Nerve endings were counted, and end-plates were measured. These data were compared with such data in the human. The end of polyneural innervation in the rat (around P20) and in the human (around 12 weeks postterm age) in both cases coincides with a transformation in motor behavior and postural control. The rat's psoas muscle at early stages is less heavily innervated than this muscle in the human. Up to three axons per motor end-plate were counted at P4, but in the human up to five axons at 25 weeks of post menstrual age. This difference might be related to the lower percentage of type I muscle fibers in the rat.

Nonvagal origin of galanin-containing nerve terminals innervating striated muscle fibers of the rat esophagus.

We investigated the origin of galanin-positive nerve fibers on motor endplates in rat esophagus using anterograde 1,1'-dioleyl-3,3,3', 3'-tetramethylindocarbocyanine methane sulfonate (DiI) tracing from the nucleus ambiguus combined with galanin immunocytochemistry and calcitonin gene-related peptide immunocytochemistry. To demonstrate spatial relationships of galanin-positive nerve fibers to vagal and enteric nerve fibers on motor endplates, we combined galanin immunocytochemistry with calcitonin gene-related peptide immunostaining for labeling of vagal terminals, and vasoactive intestinal peptide immunoreactivity and NADPH-diaphorase histochemistry for demonstration of enteric nerve fibers. Within fine varicose nerve fibers, galanin was colocalized with vasoactive intestinal peptide and NADPH-diaphorase to a high degree and turned out to be completely separated from calcitonin gene-related peptide-positive or anterogradely DiI-labeled vagal motor terminals. These results indicate that the enteric nervous system is the most important and possibly the only source of galanin-positive nerve terminals on motor endplates in rat esophagus. Galanin may be, in addition to nitric oxide and vasoactive intestinal peptide, a mediator of the enteric coinnervation of striated muscle in this organ.

Age-related remodeling of neuromuscular junctions on type-identified diaphragm fibers.

Previous studies have reported fiber-type differences in the morphological adaptations of neuromuscular junctions (NMJs) to aging by comparing limb muscles consisting of predominantly type I or II fibers. A confounding factor in these studies is age-related change in activity, which may differ between muscles. In the present study, we assessed age-related changes of the NMJ in type-identified fibers of the rat diaphragm muscle, which maintains consistent inspiratory-related activation throughout life. In 6- and 24-month-old rats, a fluorescent triple-labeling technique was used to visualize phrenic axons, presynaptic nerve terminals, and postsynaptic acetylcholine receptors (end-plates) on type-identified fibers. The NMJs were then imaged using three-dimensional (3D) confocal microscopy. On type IIx and IIb fibers, nerve terminal and end-plate 2D planar and 3D surface areas expanded, and the number of nerve terminal and end-plate branches increased, indicating fragmentation of the NMJ with aging. On the other hand, NMJs on type I and IIa fibers displayed little adaptation. These morphological adaptations may be geared toward maintaining the efficacy of inspiratory-related activity of the diaphragm muscle, but may affect the functional reserve of the aging diaphragm.