Membraneless condensates by Rapsn phase separation as a platform for neuromuscular junction formation.

Our daily life depends on muscle contraction, a process that is controlled by the neuromuscular junction (NMJ). However, the mechanisms of NMJ assembly remain unclear. Here we show that Rapsn, a protein critical for NMJ formation, undergoes liquid-liquid phase separation (LLPS) and condensates into liquid-like assemblies. Such assemblies can recruit acetylcholine receptors (AChRs), cytoskeletal proteins, and signaling proteins for postsynaptic differentiation. Rapsn LLPS requires multivalent binding of tetratricopeptide repeats (TPRs) and is increased by Musk signaling. The capacity of Rapsn to condensate and co-condensate with interaction proteins is compromised by mutations of congenital myasthenic syndromes (CMSs). NMJ formation is impaired in mutant mice carrying a CMS-associated, LLPS-deficient mutation. These results reveal a critical role of Rapsn LLPS in forming a synaptic semi-membraneless compartment for NMJ formation.

Adrenaline Facilitates Synaptic Transmission by Synchronizing Release of Acetylcholine Quanta from Motor Nerve Endings.

The long history of studies on the effect of catecholamines on synaptic transmission does not answer the main question about the mechanism of their action on quantal release in the neuromuscular junction. Currently, interest in catecholamines has increased not only because of their widespread use in the clinic for the treatment of cardiovascular and pulmonary diseases but also because of recent data on their possible use for the treatment of certain neurodegenerative diseases, muscle weakness and amyotrophic sclerosis. Nevertheless, the effects and mechanisms of catecholamines on acetylcholine release remain unclear. We investigated the action of noradrenaline and adrenaline on the spontaneous and evoked quantal secretion of acetylcholine in the neuromuscular junction of the rat soleus muscle. Noradrenaline (10 µM) did not change the spontaneous acetylcholine quantal release, the number of released quanta after nerve stimulation, or the timing of the quantal secretion. However, adrenaline at the same concentration increased spontaneous secretion by 40%, increased evoked acetylcholine quantal release by 62%, and synchronized secretion. These effects differ from those previously described by us in the synapses of the frog cutaneous pectoris muscle and mouse diaphragm. This indicates specificity in catecholamine action that depends on the functional type of muscle and the need to take the targeted type of muscle into account in clinical practice.

Remodeling of the skeletal muscle and postsynaptic component after short-term joint immobilization and aquatic training.

Joint immobilization is commonly used as a conservative treatment for osteoarticular and musculotendinous traumas. However, joint immobilization might elicit degenerative effects on the neuromuscular system and muscle atrophy. For this reason, the choice of strategies that mitigate these effects is essential in the post-immobilization period. Therefore, this study aimed to investigate the impact of aquatic training on the morphology of muscle fibers and motor endplates of the gastrocnemius muscle in the post-immobilization period. Male Wistar rats (90 days old) were divided into groups: Sedentary: no procedure; Immobilization: joint immobilization protocol (10 days); Immobilization/non-training: joint immobilization protocol (10 days) followed by four weeks without exercise intervention; Immobilization/training: joint immobilization protocol (10 days) and post-immobilization aquatic training (4 weeks). After the procedures, we quantified the cross-sectional area (CSA), volume and numerical density of different myofibers types, and total and stained area and perimeter of the motor endplate. We demonstrate the following main results: (a) short-term joint immobilization resulted in myofibers atrophy; however, we verified a small change in the postsynaptic component; (b) the period of inactivity after immobilization caused severe changes in the motor endplate (lower stained area, stained perimeter, total area, and total perimeter) and maintenance of muscle atrophy due to immobilization; (c) the prescription of post-immobilization exercise proved to be effective in restoring muscle morphology and inducing plasticity in the motor endplate. We conclude that short-term joint immobilization (10 days) results in atrophy type I and II myofibers, in addition to a decline in the total perimeter of the motor endplate. Besides, the post-immobilization period appears to be decisive in muscle and postsynaptic remodeling. Thus, aquatic training is effective in stimulating adjustments associated with muscle hypertrophy and plasticity of the motor endplate during the post-immobilization period.

Herbal complex 'Buyang Huanwu Tang' improves motor endplate function of denervated-dependent skeletal muscle atrophy in rat.

Denervated-dependent skeletal muscle atrophy is a disease induced by skeletal muscle associated peripheral neuro-disconnection. Its specific molecular mechanisms remain unknown. The treating for denervated-dependent skeletal muscle atrophy is applied with an herbal complex Buyang Huanwu Tang used in traditional Chinese medicine and subjected to the established denervated-dependent skeletal muscle atrophy in rat models, and the therapeutic effects and associated mechanisms were evaluated in the pathogenesis of denervated-dependent skeletal muscle atrophy. Denervated-dependent skeletal muscle atrophy in rats was established and randomly divided into eight groups, including Normal control, Model, Positive control, Model + Buyang Huanwu Tang, Model + astragalus extracts, Model + Buyang Huanwu Tang-astragalus, Buyang Huanwu Tang + LY294002, and astragalus extract + LY294002 group. Hematoxylin-eosin staining and quantitative RT-PCR (qRT-PCR) assay were used to examine the inflammatory response of muscle tissues. Quantitative RT-PCR and Western blotting assay were utilized to analyze mRNA and protein expression. Immunohistochemistry assay was used to detect molecule expression in anterior cervical muscle tissues. Motor endplate activity was examined using the wholemount acetylcholinesterase staining method. The wet mass ratio of anterior cervical muscle was measured. The results indicated that Buyang Huanwu Tang treatment significantly alleviated inflammatory response, enhanced acetylcholinesterase activity, and motor endplate functions, and promoted wet mass of anterior cervical muscle compared to denervated-dependent skeletal muscle atrophy rat models (P < 0.05). Buyang Huanwu Tang regulated molecules of PI3K/PKB/GSK3ß/FOXO1 signaling pathway. Buyang Huanwu Tang significantly reduced muscle atrophy F-box protein, MuFR-1, Bax and caspase 9 expression, significantly enhanced Bcl-2 expression, and remarkably increased element-binding protein and vascular endothelial growth factor levels, compared to Model group (P < 0.05). Buyang Huanwu Tang suppressed caspase 9 and caspase 3 activity and associated apoptosis. Moreover, PI3K specific blocker, LY294002, significantly inhibited the effects of Buyang Huanwu Tang on the above molecule expression (P < 0.05). In conclusion, Buyang Huanwu Tang improved motor endplate functions of denervated-dependent skeletal muscle atrophy rat model through suppressing mitochondria-mediated apoptosis and activating PI3K/PKB/FOXO1 signaling pathway.

Nicotinic α7 receptor-induced adenosine release from perisynaptic Schwann cells controls acetylcholine spillover from motor endplates.

Acetylcholine (ACh) spillover from motor endplates occurs after neuronal firing bursts being potentiated by cholinesterase inhibitors (e.g., neostigmine). Nicotinic α7 receptors (α7nAChR) on perisynaptic Schwann cells (PSCs) can control ACh spillover by unknown mechanisms. We hypothesized that adenosine might be the gliotransmitter underlying PSCs-nerve terminal communication. Rat isolated hemidiaphragm preparations were used to measure (1) the outflow of [3 H]ACh, (2) real-time transmitter exocytosis by video-microscopy with the FM4-64 fluorescent dye, and (3) skeletal muscle contractions during high-frequency (50 Hz) nerve stimulation bursts in the presence of a selective α7nAChR agonist, PNU 282987, or upon inhibition of cholinesterase activity with neostigmine. To confirm our prediction that α7nAChR-mediated effects require direct activation of PSCs, we used fluorescence video-microscopy in the real-time mode to measure PNU 282987-induced [Ca2+ ]i transients from Fluo-4 NW loaded PSCs in non-stimulated preparations. The α7nAChR agonist, PNU 282987, decreased nerve-evoked diaphragm tetanic contractions. PNU 282987-induced inhibition was mimicked by neostigmine and results from the reduction of ACh exocytosis measured as decreases in [3 H]ACh release and FM4-64 fluorescent dye unloading. Methyllycaconitine blockage of α7nAChR and the fluoroacetate gliotoxin both prevented inhibition of nerve-evoked ACh release and PSCs [Ca2+ ]i transients triggered by PNU 282987 and neostigmine. Adenosine deamination, inhibition of the ENT1 nucleoside outflow, and blockage of A1 receptors prevented PNU 282987-induced inhibition of transmitter release. Data suggest that α7nAChR controls tetanic-induced ACh spillover from the neuromuscular synapse by promoting adenosine outflow from PSCs via ENT1 transporters and retrograde activation of presynaptic A1 inhibitory receptors.

Opposed Actions of PKA Isozymes (RI and RII) and PKC Isoforms (cPKCßI and nPKCε) in Neuromuscular Developmental Synapse Elimination.

BACKGROUND: During neuromuscular junction (NMJ) development, synapses are produced in excess. By sensing the activity-dependent release of ACh, adenosine, and neurotrophins, presynaptic receptors prompt axonal competition and loss of the unnecessary axons. The receptor action is mediated by synergistic and antagonistic relations when they couple to downstream kinases (mainly protein kinases A and C (PKA and PKC)), which phosphorylate targets involved in axonal disconnection. Here, we directly investigated the involvement of PKA subunits and PKC isoforms in synapse elimination. METHODS: Selective PKA and PKC peptide modulators were applied daily to the Levator auris longus (LAL) muscle surface of P5-P8 transgenic B6.Cg-Tg (Thy1-YFP) 16 Jrs/J (and also C57BL/6J) mice, and the number of axons and the postsynaptic receptor cluster morphology were evaluated in P9 NMJ. RESULTS: PKA (PKA-I and PKA-II isozymes) acts at the pre- and postsynaptic sites to delay both axonal elimination and nAChR cluster differentiation, PKC activity promotes both axonal loss (a cPKCßI and nPKCε isoform action), and postsynaptic nAChR cluster maturation (a possible role for PKCθ). Moreover, PKC-induced changes in axon number indirectly influence postsynaptic maturation. CONCLUSIONS: PKC and PKA have opposed actions, which suggests that changes in the balance of these kinases may play a major role in the mechanism of developmental synapse elimination.

Interaction of Axonal Chondrolectin with Collagen XIXa1 Is Necessary for Precise Neuromuscular Junction Formation.

Chondrolectin (Chodl) is needed for motor axon extension in zebrafish and is dysregulated in mouse models of spinal muscular atrophy (SMA). However, the mechanistic basis of Chodl function is not known. Here, we use Chodl-deficient zebrafish and mouse mutants to show that the absence of Chodl leads to anatomical and functional defects of the neuromuscular synapse. In zebrafish, the growth of an identified motor axon beyond an "en passant" synapse and later axon branching from synaptic points are impaired, leading to functional deficits. Mechanistically, motor-neuron-autonomous Chodl function depends on its intracellular domain and on binding muscle-derived collagen XIXa1 by its extracellular C-type lectin domain. Our data support evolutionarily conserved roles of Chodl in synaptogenesis and provide evidence for a "synapse-first" scenario of motor axon growth in zebrafish.

Nicotinic acetylcholine receptor at vertebrate motor endplates: Endocytosis, recycling, and degradation.

At vertebrate motor endplates, the conversion of nerve impulses into muscle contraction is initiated by binding of acetylcholine to its nicotinic receptor (nAChR) at the postsynapse. Efficiency and safety of this process are dependent on proper localization, density, and molecular composition of the receptors. To warrant this, intricate machineries regulating the turnover of nAChR are in place. They control and execute the processes of i) expression, ii) delivery to the postsynaptic membrane, iii) clustering at the plasma membrane, iv) endocytic retrieval, v) activity-dependent recycling, and vi) degradation of nAChR. Concentrating on aspects iv-vi, this review addresses the current status of techniques, concepts, and open questions on endocytosis, recycling, and degradation of nAChR. A picture is emerging, that shows connections between executing machineries and their regulators. The first group includes the actin cytoskeleton, myosin motor proteins, Rab G-proteins, and the autophagic cascade. The second group features protein kinases A and C, Cdk5, and CaMKII as well as other components like the E3-ligase MuRF1 and the membrane shaping regulator, SH3GLB1. Recent studies have started to shed light onto nerve inputs that appear to master the tuning of the postsynaptic protein trafficking apparatus and the expression of critical components for nAChR turnover.

BDNF is a mediator of glycolytic fiber-type specification in mouse skeletal muscle.

Brain-derived neurotrophic factor (BDNF) influences the differentiation, plasticity, and survival of central neurons and likewise, affects the development of the neuromuscular system. Besides its neuronal origin, BDNF is also a member of the myokine family. However, the role of skeletal muscle-derived BDNF in regulating neuromuscular physiology in vivo remains unclear. Using gain- and loss-of-function animal models, we show that muscle-specific ablation of BDNF shifts the proportion of muscle fibers from type IIB to IIX, concomitant with elevated slow muscle-type gene expression. Furthermore, BDNF deletion reduces motor end plate volume without affecting neuromuscular junction (NMJ) integrity. These morphological changes are associated with slow muscle function and a greater resistance to contraction-induced fatigue. Conversely, BDNF overexpression promotes a fast muscle-type gene program and elevates glycolytic fiber number. These findings indicate that BDNF is required for fiber-type specification and provide insights into its potential modulation as a therapeutic target in muscle diseases.

Motor Endplate-Anatomical, Functional, and Molecular Concepts in the Historical Perspective.

By mediating voluntary muscle movement, vertebrate neuromuscular junctions (NMJ) play an extraordinarily important role in physiology. While the significance of the nerve-muscle connectivity was already conceived almost 2000 years back, the precise cell and molecular biology of the NMJ have been revealed in a series of fascinating research activities that started around 180 years ago and that continues. In all this time, NMJ research has led to fundamentally new concepts of cell biology, and has triggered groundbreaking advancements in technologies. This review tries to sketch major lines of thought and concepts on NMJ in their historical perspective, in particular with respect to anatomy, function, and molecular components. Furthermore, along these lines, it emphasizes the mutual benefit between science and technology, where one drives the other. Finally, we speculate on potential major future directions for studies on NMJ in these fields.

Regional variations in the number distribution of intrinsic myenteric neurons and coinnervated motor endplates on the striated muscles in the rat esophagus.

The roles of intrinsic neurons and the significance of the coinnervated striated muscles in the esophagus are unclear. We examined the number distribution of intrinsic neurons and coinnervated motor endplates on the striated muscles in the rat esophagus using immunohistochemistry to investigate whether these neurons and coinnervated striated muscles may be relevant to the local control of esophageal motility. The number of PGP9.5-positive neurons was higher in the cervical esophagus (segment 1) and gradually decreased toward the aboral, with a moderate increase in the abdominal (segment 5). This pattern was similar to that of NOS-positive neurons, while the number of ChAT-positive neurons decreased toward the aboral, but it was not significantly different among segments 3 to 5. The number of ChAT-positive motor endplates increased toward the aboral, with the highest number in segment 5. The proportion of coinnervated motor endplates was approximately 80% in segments 1 to 4, but approximately 66% in segment 5. NPY-IR was localized in some nerve terminals among the smooth muscles of the muscularis mucosa and some NOS- or ChAT-positive esophageal intrinsic neurons. ENK-8-IR was found in some NOS- or ChAT-positive intrinsic neurons, and nerve terminals surrounding intrinsic neurons in the esophagus, but not in motor neurons at the NA or DMV. This study suggests that regional variations in the number of intrinsic neurons and coinnervated striated muscles in the rat esophagus may be involved in local regulations of esophageal motility, and that the rat esophageal intrinsic neurons may contain, at least, motor neurons and interneurons.

Cycles of myofiber degeneration and regeneration lead to remodeling of the neuromuscular junction in two mammalian models of Duchenne muscular dystrophy.

Mice lacking the sarcolemmal protein dystrophin, designated mdx, have been widely used as a model of Duchenne muscular dystrophy. Dystrophic mdx mice as they mature develop notable morphological abnormalities to their neuromuscular junctions, the peripheral cholinergic synapses responsible for activating muscle fibers. Most obviously the acetylcholine receptor aggregates are fragmented into small non-continuous, islands. This contrasts with wild type mice whose acetylcholine receptor aggregates are continuous and pretzel-shaped in appearance. We show here that these abnormalities in mdx mice are also present in a canine model of Duchenne muscular dystrophy and provide additional evidence to support the hypothesis that NMJ remodeling occurs due to myofiber degeneration and regeneration. Using a method to investigate synaptic AChR replacement, we show that neuromuscular junction remodeling in mdx animals is caused by muscle fiber degeneration and regeneration at the synaptic site and is mimicked by deliberate myofiber injury in wild type mice. Importantly, the innervating motor axon plays a crucial role in directing the remodeling of the neuromuscular junction in dystrophy, as has been recorded in aging and deliberate muscle fiber injury in wild type mice. The remodeling occurs repetitively through the life of the animal and the changes in junctions become greater with age.

Dysfunction of Neuromuscular Synapses in the Genetic Model of Alzheimer's Disease.

The function of synaptic transmission and presynaptic vesicular cycle in the neuromuscular synapses of the diaphragm was studied in transgenic APP/PS1 mice (Alzheimer's disease model). The decrease in the quantal content of end-plate potential, intense depression of the amplitude of terminal plate potentials under conditions of lasting high frequency stimulation (50 Hz), a drastic prolongation of the synaptic vesicle recycling time in APP/PS1 mice in comparison with wild type mice were detected. Manifest dysfunction of the neuromuscular synapses, caused by disordered neurosecretion and recycling of the synaptic vesicles in the presynaptic nerve endings, was detected in the Alzheimer's disease model on transgenic APP/PS1 mice. The study supplemented the notions on the pathogenesis of Alzheimer's disease as a systemic disease, while the detected phenomena could just partially explain the development of motor disorders in this disease.

Neuromuscular Junction Morphology and Gene Dysregulation in the Wobbler Model of Spinal Neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset neuromuscular disease for which there is currently no effective treatment. The progression of ALS includes loss of motor neurons controlling the voluntary muscles, with much of this loss occurring at the neuromuscular junction. In an effort to better understand changes at the neuromuscular junction, we utilized the wobbler mouse model of motor neuron loss. We examined biceps and end plate morphologies and monitored selected factors involved in end plate function. Structural volumes were determined from 3D reconstructions that were generated for the end plates. Wobbler mice exhibited size reductions of both the muscle fibers and the end plates within the biceps, and we found that the end plate volumes were the most sensitive indicator of the degeneration. Concurrently, we found increases in calcitonin gene-related peptide (CGRP) and its receptor in wobbler biceps and spinal cord. We also found increases in gene expression of two acetylcholine receptors within the wobbler biceps, which may be a result of altered CGRP/CALCRL (calcitonin receptor-like receptor) expression.

TGF-ß/SMAD signaling inhibits intermittent cyclic mechanical tension-induced degeneration of endplate chondrocytes by regulating the miR-455-5p/RUNX2 axis.

A mechanical stimulation plays a pivotal role in maintaining normal cartilage function. Our objective was to reveal the mechanism of action of the tension-sensitive molecule miR-455-5p in the degeneration of endplate chondrocytes and to identify whether the transforming growth factor beta (TGF-ß)/SMAD signaling pathway has a regulatory effect on it. The expression profiles of members of the TGF-ß/SMAD pathway, miR-455-5p, and RUNX2 were determined by microRNA microarray analysis, reverse transcription quantitative polymerase chain reaction, luciferase reporter assay, and Western blot analysis. Intermittent cyclic mechanical tension (ICMT) induced the degeneration of endplate chondrocytes without affecting their viability. The tension-sensitive molecule miR-455-5p specifically bound to RUNX2, a gene involved in the degeneration of endplate chondrocytes. Activation of the TGF-ß/SMAD signaling pathway upregulated miR-455-5p expression and thus inhibited RUNX2 levels. Therefore, the TGF-ß/SMAD signaling pathway inhibits the ICMT-induced degeneration of endplate chondrocytes by regulating the miR-455-5p/RUNX2 axis.

Ryanodine- and CaMKII-dependent release of endogenous CGRP induces an increase in acetylcholine quantal size in neuromuscular junctions of mice.

OBJECTIVE: The aim of this study was to identify the mechanism responsible for an increase in miniature endplate potentials (MEPPs) amplitude, induced by ryanodine as an agonist of ryanodine receptors in mouse motor nerve terminals. METHODS: Using intracellular microelectrode recordings of MEPPs and evoked endplate potentials (EPPs), the changes in spontaneous and evoked acetylcholine release in motor synapses of mouse diaphragm neuromuscular preparations were studied. RESULTS: Ryanodine (0.1 µM) increased both the amplitudes of MEPPs and EPPs to a similar extent (up to 130% compared to control). The ryanodine effect was prevented by blockage of receptors of calcitonin gene-related peptide (CGRP) by a truncated peptide CGRP8-37 . Endogenous CGRP is stored in large dense-core vesicles in motor nerve terminals and may be released as a co-transmitter. The ryanodine-induced increase in MEPPs amplitude may be fully prevented by inhibition of vesicular acetylcholine transporter by vesamicol or by blocking the activity of protein kinase A with H-89, suggesting that endogenous CGRP is released in response to the activation of ryanodine receptors. Activation of CGRP receptors can, in turn, upregulate the loading of acetylcholine into synaptic vesicles, which will increase the quantal size. This new feature of endogenous CGRP activity looks similar to recently described action of exogenous CGRP in motor synapses of mice. The ryanodine effect was prevented by inhibitors of Ca/Calmodulin-dependent kinase II (CaMKII) KN-62 or KN-93. Inhibition of CaMKII did not prevent the increase in MEPPs amplitude, which was caused by exogenous CGRP. CONCLUSIONS: We propose that the activity of presynaptic CaMKII is necessary for the ryanodine-stimulated release of endogenous CGRP from motor nerve terminals, but CaMKII does not participate in signaling downstream the activation of CGRP-receptors followed by quantal size increase.

Adenosine Promotes Endplate nAChR Channel Activity in Adult Mouse Skeletal Muscle Fibers via Low Affinity P1 Receptors.

Adenosine is a powerful modulator of skeletal neuromuscular transmission, operating via inhibitory or facilitatory purinergic-type P1 receptors. To date, studies have been focused mainly on the effect of adenosine on presynaptic P1 receptors controlling transmitter release. In this study, using two-microelectrode voltage-clamp and single-channel patch-clamp recording techniques, we have explored potential postsynaptic targets of adenosine and their modulatory effect on nicotinic acetylcholine receptor (nAChR)-mediated synaptic responses in adult mouse skeletal muscle fibers in vitro. In the whole-mount neuromuscular junction (NMJ) preparation, adenosine (100 µM) significantly reduced the frequency of the miniature endplate currents (MEPCs) and slowed their rising and decay time. Consistent with a postsynaptic site of action, adenosine and the potent P1 receptor agonist NECA significantly increased the open probability, the frequency and the open time of single nAChR channels, recorded at the endplate region. Using specific ligands for the P1 receptor subtypes, we found that the low-affinity P1 receptor subtype A2B was responsible for mediating the effects of adenosine on the nAChR channel openings. Our data suggest that at the adult mammalian NMJ, adenosine acts not only presynaptically to modulate acetylcholine transmitter release, but also at the postsynaptic level, to enhance the activity of nAChRs. Our findings open a new scenario in understanding of purinergic regulation of nAChR activity at the mammalian endplate region.

Changes in Myosin Heavy Chain Isoforms Along the Length of Orbital Fibers in Rabbit Extraocular Muscle.

Purpose: Extraocular muscles express 10 myosin heavy chain (MyHC) isoforms that cater for a wide range of contractile speeds. We aim to characterize the variations in MyHC expression along the length of singly (SIFs) and multiply innervated fibers (MIFs) in the orbital layer of rabbit superior rectus muscle. Methods: Monospecific antibodies to nine MyHCs, including an anti-slow-tonic antibody characterized here were used to immunohistochemically map variations in MyHC distribution in serial sections along the muscle's full length. Results: The fastest MyHC, EO, is expressed at the endplate zone (EPZ) of SIFs, flanked proximally and distally by segments expressing the slower 2A, with or without embryonic MyHC. MIFs with constant diameter express α-cardiac MyHC at the EPZ, flanked by segments co-expressing α-cardiac/embryonic and possibly slow-tonic MyHCs. MIFs with varying diameter also express α-cardiac MyHC at the EPZ in their thin, central region, flanked by thin segments co-expressing α-cardiac/embryonic MyHCs, with long proximal and distal extensions of larger diameter that co-express embryonic/slow-tonic and α-cardiac or ß/slow MyHCs. Conclusions: Orbital fiber types express multiple MyHCs, with faster ones in SIFs, slower ones in MIFs, but all have fast EPZs and slower end segments. We hypothesize that these unique MyHC distributions enable these fibers to relax in two kinetically distinct phases while acting in an antagonistic manner during a saccade: the fast phases facilitate acceleration of eyeball rotation during agonist contraction, while the slow phases help its deceleration toward the visual target, thereby linearizing the saccade. These properties also facilitate pulley movements to implement Listing's law.

A Novel Type of Multiterminal Motor Endplate in Human Extraocular Muscles.

Purpose: To investigate the relation between type of motor endplate, acetylcholine receptor (AChR) subunit composition, and fiber types in human extraocular muscles (EOMs). Methods: EOM samples collected from subjects aged 34 to 82 years were serially sectioned and processed for immunohistochemistry, with specific antibodies against different myosin heavy chain (MyHC) isoforms, neurofilament, synaptophysin, and adult epsilon (ε) and fetal gamma (γ) AChR subunits as well as α-bungarotoxin. Results: A novel type of motor endplate consisting of large, multiterminal en plaque endings was found in human EOMs, in addition to the previously well-described single en plaque and multiple en grappe endplates. Such novel endplates were abundant but exclusively observed in myofibers lacking MyHC slow and fast IIa but containing MyHC extraocular (MyHCeom), isoforms. Multiple en grappe endings were found only in myofibers containing MyHC slow-tonic isoform and contained fetal γ AChR subunit. Adult ε and fetal γ AChR subunits, alone or combined, were found in the multiterminal endplates. Distinct AChR subunits were present in adjacent motor endplates of a given myofiber containing MyHCeom. Conclusions: Human EOMs have a more complex innervation pattern than previously described, comprising also a novel type of multiterminal motor endplate present in myofibers containing MyHCeom. The heterogeneity in AChR subunit composition in a given myofiber suggests the possible presence of polyneuronal innervation in human EOMs.

Depressed Synaptic Transmission and Reduced Vesicle Release Sites in Huntington's Disease Neuromuscular Junctions.

Huntington's disease (HD) is a progressive and fatal degenerative disorder that results in debilitating cognitive and motor dysfunction. Most HD studies have focused on degeneration of the CNS. We previously discovered that skeletal muscle from transgenic R6/2 HD mice is hyperexcitable due to decreased chloride and potassium conductances. The progressive and early onset of these defects suggest a primary myopathy in HD. In this study, we examined the relationship between neuromuscular transmission and skeletal muscle hyperexcitability. We used an ex vivo preparation of the levator auris longus muscle from male and female late-stage R6/2 mice and age-matched wild-type controls. Immunostaining of the synapses and molecular analyses revealed no evidence of denervation. Physiologically, we recorded spontaneous miniature endplate currents (mEPCs) and nerve-evoked EPCs (eEPCs) under voltage-clamp, which, unlike current-clamp records, were independent of the changes in muscle membrane properties. We found a reduction in the number of vesicles released per action potential (quantal content) in R6/2 muscle, which analysis of eEPC variance and morphology indicate is caused by a reduction in the number of vesicle release sites (n) rather than a change in the probability of release (prel). Furthermore, analysis of high-frequency stimulation trains suggests an impairment in vesicle mobilization. The depressed neuromuscular transmission in R6/2 muscle may help compensate for the muscle hyperexcitability and contribute to motor impersistence.SIGNIFICANCE STATEMENT Recent evidence indicates that Huntington's disease (HD) is a multisystem disorder. Our examination of neuromuscular transmission in this study reveals defects in the motor nerve terminal that may compensate for the muscle hyperexcitability in HD. The technique we used eliminates the effects of the altered muscle membrane properties on synaptic currents and thus provides hitherto the most detailed analysis of synaptic transmission in HD. Clinically, the striking depression of neurotransmission we found may help explain the motor impersistence in HD patients. Therapies that target the highly accessible peripheral nerve and muscle system provide a promising new avenue to lessen the debilitating motor symptoms of HD.