Mechanisms of stationary voltage fluctuation in the neuromuscular junction endplate and corresponding denoising paradigms.

The neuromuscular junction (NMJ) has an elaborate anatomy to ensure agile and accurate signal transmission. Based on our formerly obtained expressions of the thermal and conductance induced voltage fluctuations, in this paper, the mechanisms underlying the conductance-induced voltage fluctuation are characterized from two aspects: the scaling laws with respect to either of the two system-size factors, the number of receptors or the membrane area; and the "seesaw effect" with respect to the intensive parameter, the concentration of acetylcholine. According to these mechanisms, several aspects of the NMJ anatomy are explained from a denoising perspective. Finally, the power spectra of the two types of voltage fluctuations are characterized by their specific scaling laws, based on which we explain why the endplate noise has the low-frequency property that is described by the term "seashell sound".

Electromyography of the muscle spindle.

In needle electromyography, there are two spontaneous waveforms, miniature end plate potentials and "end plate spikes", appearing usually together. Miniature end plate potentials are local, non-propagating postsynaptic waves, caused by spontaneous exocytosis of acetylcholine in the neuromuscular junction. The prevailing hypothesis states that "end plate spikes" are propagated postsynaptic action potentials of muscle fibers, caused by presynaptic irritation of the motor nerve or nerve terminal. Using several small concentric needle electrodes in parallel with the muscle fibers, most "end plate spikes" are strictly local or propagating for 2-4 mm. At the end plate zone, there are miniature end plate potentials without "end plate spikes". Local "end plate spikes" are junctional potentials of intrafusal gamma neuromuscular junctions of the nuclear bag fibers, and propagated "end plate spikes" are potentials of nuclear chain muscle fibers of muscle spindles. Miniature end plate potentials without "end plate spikes" at the end plate zone derive from alpha neuromuscular junctions. These findings contrast with the prevailing hypothesis. The history of observations and different hypotheses of the origin of end plate spikes are described.

Mechanisms of the end-plate potential noise and its implication for the neuromuscular junction anatomy.

We construct a compact model to mimic the membrane voltage response to the concentration of acetylcholine ([ACh]) which is mediated by the stochastic gating of acetylcholine (ACh) receptors. The patterns of the voltage depolarization against [ACh] as well as the accompanying voltage noises are presented. The mechanism of the voltage fluctuation that caused by the stochastic gating of receptors is explained. We consider that our results explain the frequently observed "end-plate (potential) noise" in physiology and electromyographic literature. These results, together with the requirements of evolution pressure on the motor units, explain reasonably the anatomical structure of the neuromuscular junction.

Laryngeal Reconstruction Using Tissue-Engineered Implants in Pigs: A Pilot Study.

OBJECTIVE/HYPOTHESIS: There are currently no treatments available that restore dynamic laryngeal function after hemilaryngectomy. We have shown that dynamic function can be restored post hemilaryngectomy in a rat model. Here, we report in a first of its kind, proof of concept study that this previously published technique is scalable to a porcine model. STUDY DESIGN: Animal study. METHODS: Muscle and fat biopsies were taken from three Yucatan minipigs. Muscle progenitor cells (MPCs) and adipose stem cells (ASCs) were isolated and cultured for 3 weeks. The minipigs underwent a left laterovertical partial laryngectomy sparing the left arytenoid cartilage and transecting the recurrent laryngeal nerve. Each layer was replaced with a tissue-engineered implant: 1) an acellular mucosal layer composed of densified Type I oligomeric collagen, 2) a skeletal muscle layer composed of autologous MPCs and aligned oligomeric collagen differentiated and induced to express motor endplates (MEE), and 3) a cartilage layer composed of autologous ASCs and densified oligomeric collagen differentiated to cartilage. Healing was monitored at 2 and 4 weeks post-op, and at the 8 week study endpoint. RESULTS: Animals demonstrated appropriate weight gain, no aspiration events, and audible phonation. Video laryngoscopy showed progressive healing with vascularization and re-epithelialization present at 4 weeks. On histology, there was no immune reaction to the implants and there was complete integration into host tissue with nerve and vascular ingrowth. CONCLUSIONS: This pilot study represents a first in which a transmural vertical partial laryngectomy was performed and successfully repaired with a customized, autologous stem cell-derived multi-layered tissue-engineered implant. LEVEL OF EVIDENCE: NA Laryngoscope, 131:2277-2284, 2021.

Effects of Vocal Training on Thyroarytenoid Muscle Neuromuscular Junctions and Myofibers in Young and Older Rats.

The purpose of this investigation was to determine the effects of vocal training on neuromuscular junction (NMJ) morphology and muscle fiber size and composition in the thyroarytenoid muscle, the primary muscle in the vocal fold, in younger (9-month) and older (24-month) Fischer 344 × Brown Norway male rats. Over 4 or 8 weeks of vocal training, rats of both ages progressively increased their daily number of ultrasonic vocalizations (USVs) through operant conditioning and were then compared to an untrained control group. Neuromuscular junction morphology and myofiber size and composition were measured from the thyroarytenoid muscle. Acoustic analysis of USVs before and after training quantified the functional effect of training. Both 4- and 8-week training resulted in less NMJ motor endplate dispersion in the lateral portion of the thyroarytenoid muscle in rats of both ages. Vocal training and age had no significant effects on laryngeal myofiber size or type. Vocal training resulted in a greater number of USVs with longer duration and increased intensity. This study demonstrated that vocal training induces laryngeal NMJ morphology and acoustic changes. The lack of significant effects of vocal training on muscle fiber type and size suggests vocal training significantly improves neuromuscular efficiency but does not significantly influence muscle strength changes.

Evidence That the Central Nervous System Can Induce a Modification at the Neuromuscular Junction That Contributes to the Maintenance of a Behavioral Response.

Neurons within the spinal cord are sensitive to environmental relations and can bring about a behavioral modification without input from the brain. For example, rats that have undergone a thoracic (T2) transection can learn to maintain a hind leg in a flexed position to minimize exposure to a noxious electrical stimulation (shock). Inactivating neurons within the spinal cord with lidocaine, or cutting communication between the spinal cord and the periphery (sciatic transection), eliminates the capacity to learn, which implies that it depends on spinal neurons. Here we show that these manipulations have no effect on the maintenance of the learned response, which implicates a peripheral process. EMG showed that learning augments the muscular response evoked by motoneuron output and that this effect survives a sciatic transection. Quantitative fluorescent imaging revealed that training brings about an increase in the area and intensity of ACh receptor labeling at the neuromuscular junction (NMJ). It is hypothesized that efferent motoneuron output, in conjunction with electrical stimulation of the tibialis anterior muscle, strengthens the connection at the NMJ in a Hebbian manner. Supporting this, paired stimulation of the efferent nerve and tibialis anterior generated an increase in flexion duration and augmented the evoked electrical response without input from the spinal cord. Evidence is presented that glutamatergic signaling contributes to plasticity at the NMJ. Labeling for vesicular glutamate transporter is evident at the motor endplate. Intramuscular application of an NMDAR antagonist blocked the acquisition/maintenance of the learned response and the strengthening of the evoked electrical response.SIGNIFICANCE STATEMENT The neuromuscular junction (NMJ) is designed to faithfully elicit a muscular contraction in response to neural input. From this perspective, encoding environmental relations (learning) and the maintenance of a behavioral modification over time (memory) are assumed to reflect only modifications upstream from the NMJ, within the CNS. The current results challenge this view. Rats were trained to maintain a hind leg in a flexed position to avoid noxious stimulation. As expected, treatments that inhibit activity within the CNS, or disrupt peripheral communication, prevented learning. These manipulations did not affect the maintenance of the acquired response. The results imply that a peripheral modification at the NMJ contributes to the maintenance of the learned response.

Examination of the human motor endplate after brachial plexus injury with two-photon microscopy.

INTRODUCTION: After traumatic nerve injury, neuromuscular junction remodeling plays a key role in determining functional outcomes. Immunohistochemical analyses of denervated muscle biopsies may provide valuable prognostic data regarding clinical outcomes to supplement electrodiagnostic studies. METHODS: We performed biopsies on nonfunctioning deltoid muscles in two patients after gunshot wounds and visualized the neuromuscular junctions using two-photon microscopy with immunohistochemistry. RESULTS: Although the nerves in both patients showed evidence of acute Wallerian degeneration, some of the motor endplates were intact but exhibited significantly decreased surface area and volume. Both patients exhibited substantial recovery of motor function over several weeks postinjury. DISCUSSION: Two-photon microscopic assessment of neuromuscular junction integrity and motor endplate morphometry in muscle biopsies provided evidence of partial sparing of muscle innervation. This finding supported the clinical judgment that eventual recovery would occur. With further study, this technique may help to guide operative decisionmaking after traumatic nerve injuries.

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.

Effect of Pulsed and Continuous Ultrasound Therapy on the Degree of Collateral Axonal Branching at the Lesion Site, Polyinnervation of Motor End Plates, and Recovery of Motor Function after Facial Nerve Reconstruction.

The aim of the present study is to test whether ultrasound therapy of muscles denervated by nerve injury would improve the quality of their reinnervation by reduction of the collateral axonal branching at the lesion site and poly-innervation degree at the neuromuscular junctions. After transection and suture of the buccal branch of the facial nerve, pulsed or continuous type of ultrasound therapy was applied to the paralyzed whisker pad muscles of rats in the course of 2 months. Instead of reduction, we found a significant increase in the collateral axonal branching after continuous ultrasound therapy when compared to the branching determined after pulsed or sham ultrasound therapy. Both types of ultrasound therapy also failed to reduce the proportion of polyinnervated end plates in the reinnervated facial muscles. Accordingly, continuous ultrasound therapy failed to restore any parameter of the motor performance of the vibrissal hairs. Application of pulsed ultrasound therapy promoted slight improvements of the functional parameters angular velocity and acceleration. The inhomogeneous structural and functional results achieved after both types of ultrasound therapy let us conclude that further studies are required to evaluate its effects on peripheral nerve regeneration. Anat Rec, 302:1314-1324, 2019. © 2019 Wiley Periodicals, Inc.

Spatial Distribution of Motor Endplates and its Adaptive Change in Skeletal Muscle.

Motor endplates (MEPs) are the important interfaces between peripheral nerves and muscle fibers. Investigation of the spatial distribution of MEPs could help us better understand neuromuscular functional activities and improve the diagnosis and therapy of related diseases. Methods: Fluorescent α-bungarotoxin was injected to label the motor endplates in whole-mount skeletal muscles, and tissue optical clearing combined with light-sheet microscopy was used to investigate the spatial distribution of MEPs and in-muscle nerve branches in different skeletal muscles in wild-type and transgenic fluorescent mice. Electrophysiology was used to determine the relationship between the spatial distribution of MEPs and muscle function. Results: The exact three-dimensional distribution of MEPs in whole skeletal muscles was first obtained. We found that the MEPs in the muscle were distributed in an organized pattern of lamella clusters, with no MEPs outside the lamella zone. Each MEP lamella was innervated by one independent in-muscle nerve branch and mediated an independent muscle subgroup contraction. Additionally, the MEPs changed along the lamella clusters after denervation and regained the initial pattern after reinnervation. The integrity and spatial distribution of MEPs could reflect the functional state of muscles. The signal absence of a certain MEP lamella could suggest a problem in certain part of the muscle. Conclusions: The MEP lamella clusters might be the basis of neuromuscular function, and the spatial distribution of MEPs could serve as a testbed for evaluating the functional status of muscle and the therapeutic targeting map related to MEPs.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions.

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (τm) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

A presynaptic congenital myasthenic syndrome attributed to a homozygous sequence variant in LAMA5.

We report a severe defect of neuromuscular transmission in a consanguineous patient with a homozygous variant in the laminin α5 subunit gene (LAMA5). The variant c.8046C > T (p.Arg2659Trp) is rare and has a predicted deleterious effect. The affected individual, who also carries a rare homozygous sequence variant in LAMA1, had normal cognitive function, but magnetic resonance brain imaging showed mild volume loss and periventricular T2 prolongation. Repetitive nerve stimulation at 2 Hz showed 50% decrement of compound muscle action potential amplitudes but 250% facilitation immediately after exercise, similar to that seen in Lambert-Eaton myasthenic syndrome. Endplate studies demonstrated a profound reduction of the endplate potential quantal content but normal amplitudes of miniature endplate potentials. Electron microscopy showed endplates with increased postsynaptic folding that were denuded or only partially occupied by small nerve terminals. Expression studies revealed that p.Arg2659Trp caused decreased binding of laminin α5 to SV2A and impaired laminin-521 cell adhesion and cell projection support in primary neuronal cultures. In summary, this report describing severe neuromuscular transmission failure in a patient with a LAMA5 mutation expands the list of phenotypes associated with defects in genes encoding α-laminins.

Nerve growth factor and basic fibroblast growth factor promote reinnervation by nerve-muscle-endplate grafting.

INTRODUCTION: This study was designed to test whether exogenous application of nerve growth factor (NGF) and basic fibroblast growth factor (FGF-2) to muscles reinnervated with nerve-muscle-endplate band grafting (NMEG) could promote specific outcomes. METHODS: The right sternomastoid muscle in adult rats was experimentally denervated and immediately reinnervated by implanting an NMEG pedicle from the ipsilateral sternohyoid muscle. A fibrin sealant containing NGF and FGF-2 was focally applied to the implantation site. Maximal tetanic force, muscle weight, regenerated axons, and motor endplates were analyzed 3 months after treatment. RESULTS: Mean tetanic force, wet muscle weight, and number of regenerated axons in the treated muscles were 91%, 92%, and 84% of the contralateral controls, respectively. The majority of endplates (86%) in the treated muscles were reinnervated by regenerated axons. DISCUSSION: Focal administration of NGF and FGF-2 promotes efficacy of the NMEG technique. Muscle Nerve 57: 449-459, 2018.

Extracorporeal shock wave treatment can selectively destroy end plates in neuromuscular junctions.

INTRODUCTION: This study assesses the effect of radial extracorporeal shock wave (rESW) exposure on neuromuscular transmission and neuromuscular junction (NMJ) morphology. METHODS: We applied 2,000 rESWs at 0.18 mJ/mm2 and a frequency of 15 Hz to the right calf of male rats, measured the compound muscle action potential (CMAP), and examined NMJ morphology using electron microscopy. Left calf muscles were used as controls. RESULTS: rESW exposure significantly reduced CMAP amplitude without delayed latency in exposed muscles compared with controls. All rESW-exposed muscles exhibited NMJs with irregular end plates. Mean interjunctional fold interval was significantly increased compared with controls. However, axon terminals and muscle fibers surrounding NMJs with irregular end plates were unchanged. DISCUSSION: This localized destruction of end plates may be caused by differences in acoustic impedance induced by the density of acetylcholine receptors. These results provide a possible mechanism for the effectiveness of rESW treatment for spasticity and dystonia. Muscle Nerve 57: 466-472, 2018.

Divergent effects of resistance training and anabolic steroid on the postsynaptic region of different skeletal muscles of aged rats.

This study aimed to analyze the effects of resistance training associated with testosterone administration in the neuromuscular junction (NMJ) postsynaptic region of different skeletal muscle types of aged rats. Wistar rats were divided into: SEI - 20-months-old control, SEF - 24-months-old control, T - 20-months-old with testosterone, S - 20-months-old resistance trained and ST - 20-months-old with resistance training associated with testosterone propionate. All groups were submitted to familiarization and maximum load carrying testing (MLCT). The MLCT was applied before and after the resistance training (RT) period. RT (6-8×/session with progressive loads of 50 to 100%, 3×/week and 120s interval) was performed in ladder climbing for 15weeks. The administration of testosterone propionate was performed 2×/week (10mg/kg/body weight). After euthanize, soleus and plantaris muscles were removed and prepared for histochemistry and cytofluorescence. T, S and ST significantly increased their maximum carrying load capacity compared to SEI and SEF (p<0.05). For soleus postsynaptic region, ST had lower total and stained area than SEF (p<0.05). For plantaris, the postsynaptic component of T was statistically larger than SEI (p<0.05). For soleus histochemistry, T, S and ST groups showed the same magnitude of type I myofibers hypertrophy, thus statistically different from SEI and SEF (p<0.05). The cross-sectional area of the type IIa myofibers of the ST was larger than SEF (p<0.05). The volume density of type I myofibers show to be lower in ST than SEI (p<0.05). As for type IIa myofibers, ST increased Vv [type IIa] compared to SEI and SEF (p<0.05). For plantaris, T significantly hypertrophied type I myofibers compared to SEI and SEF (p<0.05). S and ST demonstrated significant increases of type I myofibers compared to SEI and SEF (p<0.05). As for type IIx myofibers, both S and ST showed myofibers larger than SEI (p<0.05). However, only the ST had significant difference compared to SEF (p<0.05). In conclusion, both therapies, alone or combined, have little effect on the morphology of the NMJ postsynaptic region of distinct muscles. Moreover, the three therapies are potentially stimulating for strength gains and muscle hypertrophy.

Effects of Titanium Mesh Cage End Structures on the Compressive Load at the Endplate Interface: A Cadaveric Biomechanical Study.

BACKGROUND This study aimed to evaluate whether obliquely angled and ring-shaped titanium mesh cage (TMC) end structures can improve the compressive load on the endplate interface in anterior cervical corpectomy and fusion (ACCF). MATERIAL AND METHODS A total of 23 volunteers underwent cervical lateral x-ray. The oblique angle of the superior endplate was measured, which was used to construct the gradient of the TMC end. Forty-two fresh cadaveric vertebral bodies were harvested and randomly distributed among four TMC groups with different ends. The baseline indicators of bone mineral density and anteroposterior and transverse dimensions were recorded. The superior endplate was placed at an angle of 12° when performing uniaxial compression testing. The maximum loads of the four TMCs were assessed. RESULTS There were no significant differences among the groups regarding the baseline indicators. The conventional TMC had the lowest maximum load (1362.3±221.78 N, p<0.05), whereas the TMC with an obliquely end ring had the highest maximum load (2095.82±285.64 N, p<0.05). The maximum loads of the TMCs with oblique footprints and flat end ring were much higher than that of the conventional TMC (p<0.05) but significantly lower than that of the TMC with the obliquely end ring (p<0.05), with average values of 1806.91±246.98 N and 1725.3±213.33 N, respectively. CONCLUSIONS Both the ring shape and oblique angle of the TMC end contributed to an increase in compressive force and are advocated for use in TMC structure optimization to decrease the incidence of TMC subsidence in ACCF.

Muscarinic cholinoreceptors (M1-, M2-, M3- and M4-type) modulate the acetylcholine secretion in the frog neuromuscular junction.

Muscarinic cholinoreceptors regulate the neurosecretion process in vertebrate neuromuscular junctions. The diversity of muscarinic effects on acetylcholine (ACh) secretion may be attributed to the different muscarinic subtypes involved in this process. In the present study, the location of five muscarinic receptor subtypes (M1, M2, M3, M4 and M5) on the motor nerve terminals of frog cutaneous pectoris muscle was shown using specific polyclonal antibodies. The modulatory roles of these receptors were investigated via assessment of the effects of muscarine and specific muscarinic antagonists on the quantal content of endplate currents (EPCs) and the time course of secretion, which was estimated from the distribution of "real" synaptic delays of EPCs recorded in a low Ca2+/high Mg2+ solution. The agonist muscarine decreased the EPC quantal content and synchronized the release process. The depressing action of muscarine on the EPC quantal content was abolished only by pretreatment of the preparation with the M3 blockers 4-DAMP (1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide) and J 104129 fumarate ((αR)-α-Cyclopentyl-α-hydroxy-N-[1-(4-methyl-3-pentenyl)-4-piperidinyl]benzeneacetamide fumarate). Moreover, antagonists of the M1, M2, M3 and M4 receptors per se diminished the intensity of secretion, which suggests a putative up-regulation of the release by endogenous ACh.

Loss of laminin-α4 results in pre- and postsynaptic modifications at the neuromuscular junction.

Synaptic basal lamina such as laminin-421 (α4ß2γ1) mediate differentiation of the neuromuscular junction (NMJ). Laminins interact with their pre- or postsynaptic receptors to provide stability and alignment of the pre- to postsynaptic specializations. Knockout of the laminin-α4 gene (lama4) does not alter gross NMJ morphogenesis. However, mice deficient in laminin-α4 (lama4-/- mice) display disruptions in the alignment of the active zones and postsynaptic folds at the NMJ, although the physiological consequences of this loss have not been examined. The present study investigated the differences in neurotransmission during the early development and maturation of the NMJ in lama4-/- and wild-type mice. Lama4-/- NMJs demonstrated a decrease in miniature end-plate potential (EPP) frequency and increased amplitude of miniature EPPs and evoked EPPs. Binomial parameters analysis of neurotransmitter release revealed a decrease in quantal release, the result of a decrease in the number of active release sites, but not in the probability of transmitter release. Lama4-/- NMJs displayed higher levels of synaptic depression under high-frequency stimulation and altered facilitation, suggesting compromised delivery of synaptic vesicles. This idea is supported by our molecular investigations of lama4-/- NMJs, where we see altered distribution of Bassoon, a molecular component of active zones, presumably resulting from perturbed neurotransmission.-Chand, K. K., Lee, K. M., Lavidis, N. A., Noakes, P. G. Loss of laminin-α4 results in pre- and postsynaptic modifications at the neuromuscular junction.

Preservation of neuromuscular function in symptomatic SOD1-G93A mice by peripheral infusion of methylene blue.

In mutant superoxide dismutase 1 (SOD1) mouse models of familial amyotrophic lateral sclerosis (fALS) some of the earliest signs of morphological and functional damage occur in the motor nerve terminals that innervate fast limb muscles. This study tested whether localized peripheral application of a protective drug could effectively preserve neuromuscular junctions in late-stage disease. Methylene blue (MB), which has mitochondria-protective properties, was infused via an osmotic pump into the anterior muscle compartment of one hind limb of late pre- symptomatic SOD1-G93A mice for ≥3weeks. When mice reached end-stage disease, peak twitch and tetanic contractions evoked by stimulation of the muscle nerve were measured in two anterior compartment muscles (tibialis anterior [TA] and extensor digitorum longus [EDL], both predominantly fast muscles). With 400µM MB in the infusion reservoir, muscles on the MB-infused side exhibited on average a ~100% increase in nerve-evoked contractile force compared to muscles on the contralateral non-infused side (p<0.01 for both twitch and tetanus in EDL and TA). Pairwise comparisons of endplate innervation also revealed a beneficial effect of MB infusion, with an average of 65% of endplates innervated in infused EDL, compared to only 35% on the non-infused side (p<0.01). Results suggested that MB's protective effects required an extracellular [MB] of ~1µM, were initiated peripherally (no evidence of retrograde transport into the spinal cord), and involved MB's reduced form. Thus peripherally-initiated actions of MB can help preserve neuromuscular structure and function in SOD1-G93A mice, even at late stages of disease.