Inducible deletion of endothelial cell Efnb2 delays capillary regeneration and attenuates myofibre reinnervation following myotoxin injury in mice.

Acute injury of skeletal muscle disrupts myofibres, microvessels and motor innervation. Myofibre regeneration is well characterized, however its relationship with the regeneration of microvessels and motor nerves is undefined. Endothelial cell (EC) ephrin-B2 (Efnb2) is required for angiogenesis during embryonic development and promotes neurovascular regeneration in the adult. We hypothesized that, following acute injury to skeletal muscle, loss of EC Efnb2 would impair microvascular regeneration and the recovery of neuromuscular junction (NMJ) integrity. Mice (aged 3-6 months) were bred for EC-specific conditional knockout (CKO) of Efnb2 following tamoxifen injection with non-injected CKO mice as controls (CON). The gluteus maximus, tibialis anterior or extensor digitorum longus muscle was then injured with local injection of BaCl2. Intravascular staining with wheat germ agglutinin revealed diminished capillary area in the gluteus maximus of CKO vs. CON at 5 days post-injury (dpi); both recovered to uninjured (0 dpi) level by 10 dpi. At 0 dpi, tibialis anterior isometric force of CKO was less than CON. At 10 dpi, isometric force was reduced by half in both groups. During intermittent contractions (75 Hz, 330 ms s-1, 120 s), isometric force fell during indirect (sciatic nerve) stimulation whereas force was maintained during direct (electrical field) stimulation of myofibres. Neuromuscular transmission failure correlated with perturbed presynaptic (terminal Schwann cells) and postsynaptic (nicotinic acetylcholine receptors) NMJ morphology in CKO. Resident satellite cell number on extensor digitorum longus myofibres did not differ between groups. Following acute injury of skeletal muscle, loss of Efnb2 in ECs delays capillary regeneration and attenuates recovery of NMJ structure and function. KEY POINTS: The relationship between microvascular regeneration and motor nerve regeneration following skeletal muscle injury is undefined. Expression of Efnb2 in endothelial cells (ECs) is essential to vascular development and promotes neurovascular regeneration in the adult. To test the hypothesis that EfnB2 in ECs is required for microvascular regeneration and myofibre reinnervation, we induced conditional knockout of Efnb2 in ECs of mice. Acute injury was then induced by BaCl2 injection into gluteus maximus, tibialis anterior or extensor digitorum longus (EDL) muscle. Capillary regeneration was reduced at 5 days post-injury (dpi) in gluteus maximus of conditional knockout vs. controls; at 10 dpi, neither differed from uninjured. Nerve stimulation revealed neuromuscular transmission failure in tibialis anterior with perturbed neuromuscular junction structure. Resident satellite cell number on EDL myofibres did not differ between groups. Conditional knockout of EC Efnb2 delays capillary regeneration and attenuates recovery of neuromuscular junction structure and function.

Effects of membrane cholesterol-targeting chemicals on skeletal muscle contractions evoked by direct and indirect stimulation.

Cholesterol is one of the major components of plasma membrane, where its distribution is nonhomogeneous and it participates in lipid raft formation. In skeletal muscle cholesterol and lipid rafts seem to be important for excitation-contraction coupling and for neuromuscular transmission, involving cholesterol-rich synaptic vesicles. In the present study, nerve and muscle stimulation-evoked contractions were recorded to assess the role of cholesterol in contractile function of mouse diaphragm. Exposure to cholesterol oxidase (0.2 U/ml) and cholesterol-depleting agent methyl-ß-cyclodextrin (1 mM) did not affect markedly contractile responses to both direct and indirect stimulation at low and high frequency. However, methyl-ß-cyclodextrin at high concentration (10 mM) strongly decreased the force of both single and tetanus contractions induced by phrenic nerve stimulation. This decline in contractile function was more profoundly expressed when methyl-ß-cyclodextrin application was combined with phrenic nerve activation. At the same time, 10 mM methyl-ß-cyclodextrin had no effect on contractions upon direct muscle stimulation at low and high frequency. Thus, strong cholesterol depletion suppresses contractile function mainly due to disturbance of the neuromuscular communication, whereas muscle fiber contractility remains resistant to decline.

Acute effect of Δ-9-tetrahydrocannabinol on neuromuscular transmission and locomotive behaviors in larval zebrafish.

Given the increasing trend of cannabis use for recreational and therapeutic purposes, a comprehensive examination of cannabis effects is warranted. The principal psychoactive constituent of cannabis, Δ-9-tetrahydrocannabinol (THC), is a potent disrupter of neurodevelopment. Nevertheless, the impact of acute exposure to THC on developing motor systems is not well-investigated. In this study, using a neurophysiological whole cell patch clamp approach we demonstrated that a 30-min exposure to THC can alter spontaneous synaptic activities at neuromuscular junctions of 5-day post-fertilized zebrafish. An increased frequency of synaptic activity and altered decay kinetic properties were documented in the THC-treated larvae. Locomotive behaviors, including swimming activity rate and C-start escape response to sound were also affected by THC. Although the THC-treated larvae displayed hyperactivity of their basal swimming levels, their escape response rate to sound stimuli was reduced. These findings suggest that acute exposure to THC can disrupt neuromuscular transmission and locomotor-driven responses in developing zebrafish.NEW & NOTEWORTHY Acute exposure to THC alters motor neuron-muscle communication and motor behaviors in developing zebrafish. Our neurophysiology data indicated that the properties of spontaneous synaptic activity at neuromuscular junctions, such as decay component of acetylcholine receptors and frequency of synaptic events, were affected by a 30-min exposure to THC. Hyperactivity and reduced responsiveness to the sound stimulus were also observed in the THC-treated larvae. Exposure to THC during early developing stages may induce motor dysfunction.

Single fiber electromyography and measuring jitter with concentric needle electrodes.

This monograph contains descriptions of the single fiber electromyography (SFEMG) method and of the more recently implemented method of recording jitter with concentric needle electrodes (CNEs). SFEMG records action potentials from single muscle fibers (SFAPs), which permits measuring fiber density (FD), a sensitive measure of reinnervation, and jitter, a sensitive measure of abnormal neuromuscular transmission (NMT). With voluntary activation, jitter is measured between two SFAPs with acceptable amplitude and rise time. With activation by axon stimulation, jitter is measured between the stimulus and individual SFAPs. Pitfalls due to unstable triggers and inconstant firing rates during voluntary activation and subliminal stimulation during axon stimulation should be identified and avoided. In CNE recordings, spikes with shoulders or rising phases that are not parallel are produced by summation of SFAPS; these should be excluded and reference values for CNE jitter should be used. CNE and SFEMG have similar and very high sensitivity in detecting increased jitter, as in myasthenia gravis and other myasthenic conditions. However, jitter is also seen in ongoing reinnervation and some myopathic conditions. With SFEMG, these can be identified by increased FD; however, FD cannot be measured with CNE, and conventional electromyography should be performed in muscles with increased jitter to detect neurogenic or myogenic abnormalities. Jitter is abnormal after injections of botulinum toxin, even in muscles remote from the injection site, and can persist for 6 mo or more. This can complicate the detection or exclusion of abnormal NMT.

Single fiber EMG and measuring jitter with concentric needle electrodes.

This monograph contains descriptions of the single-fiber electromyography (SFEMG) method and of the more recently implemented method of recording jitter with concentric needle electrodes (CNE). SFEMG records action potentials from single muscle fibers (SFAPs), which permits measuring fiber density (FD), a sensitive measure of reinnervation, and jitter, a sensitive measure of abnormal neuromuscular transmission (NMT). With voluntary activation, jitter is measured between two SFAPs with acceptable amplitude and rise time. With activation by axon stimulation, jitter is measured between the stimulus and individual SFAPs. Pitfalls due to unstable triggers and inconstant firing rates during voluntary activation and subliminal stimulation during axon stimulation should be identified and avoided. In CNE recordings, spikes with shoulders or rising phases that are not parallel are produced by summation of SFAPS; these should be excluded and reference values for CNE jitter should be used. CNE and SFEMG have similar and very high sensitivity in detecting increased jitter, as in myasthenia gravis and other myasthenic conditions. However, jitter is also seen in ongoing reinnervation and some myopathic conditions. With SFEMG, these can be identified by increased FD; however, FD cannot be measured with CNE, and conventional EMG should be performed in muscles with increased jitter to detect neurogenic or myogenic abnormalities. Jitter is abnormal after injections of botulinum toxin, even in muscles remote from the injection site, and can persist for 6 mo or more. This can complicate the detection or exclusion of abnormal NMT.

The interest of 100 versus 200 Hz tetanic stimulations to quantify low levels of residual neuromuscular blockade with mechanomyography: a pilot study.

A more sensitive method than the train-of-four ratio seems required to detect low levels of residual neuromuscular blockade before tracheal extubation. The goal of the study was to determine the potential benefit of 5 s of 100 versus 200 Hz tetanic stimulation to quantify the residual block with mechanomyography in anesthetised patients. Twenty informed and consenting 18- to 80-year-old patients undergoing nose surgery were included. On the left hand, neuromuscular transmission was continuously monitored by acceleromyography. On the right side, a new mecanomyographic device (Isometric Thumb Force©) recorded the force of thumb adduction (N) developed during 5 s of 100- and 200 Hz tetanic stimulations of the ulnar nerve at three consecutive times: baseline before inducing the neuromuscular blockade, at the time of contralateral train-of-four ratio 0.9 recovery, and 3 min after additional sugammadex reversal. Tetanic Fade Ratios (TFR = F residual/F max) were compared between 100 and 200 Hz stimulations using Student's t test. At the time of TOF ratio 0.9 recovery, both 100 and 200 Hz TFR were significantly decreased compared to baseline (0.61 and 0.16 on average, respectively, p < 0.0001). The 200 Hz TFR was significantly lower than the 100 Hz TFR (p < 0.0001). There were no differences between baseline and post-reversal TFR. The 200 Hz TFR has the potential to better describe low levels of residual neuromuscular blockade than the TOF ratio and 100 Hz TFR and would benefit from further investigations. Retrospectively registered in the Australian and New Zealand Clinical Trials Registry ACTRN12619000273189.

TrkB signaling contributes to transdiaphragmatic pressure generation in aged mice.

Ventilatory deficits are common in old age and may result from neuromuscular dysfunction. Signaling via the tropomyosin-related kinase receptor B (TrkB) regulates neuromuscular transmission and, in young mice, is important for the generation of transdiaphragmatic pressure (Pdi). Loss of TrkB signaling worsened neuromuscular transmission failure and reduced maximal Pdi, and these effects are similar to those observed in old age. Administration of TrkB agonists such as 7,8-dihydroxyflavone (7,8-DHF) improves neuromuscular transmission in young and old mice (18 mo; 75% survival). We hypothesized that TrkB signaling contributes to Pdi generation in old mice, particularly during maximal force behaviors. Old male and female TrkBF616A mice, with a mutation that induces 1NMPP1-mediated TrkB kinase inhibition, were randomly assigned to systemic treatment with vehicle, 7,8-DHF, or 1NMPP1 1 h before experiments. Pdi was measured during eupneic breathing (room air), hypoxia-hypercapnia (10% O2/5% CO2), tracheal occlusion, spontaneous deep breaths ("sighs"), and bilateral phrenic nerve stimulation (Pdimax). There were no differences in the Pdi amplitude across treatments during ventilatory behaviors (eupnea, hypoxia-hypercapnia, occlusion, or sigh). As expected, Pdi increased from eupnea and hypoxia-hypercapnia (∼7 cm H2O) to occlusion and sighs (∼25 cm H2O), with no differences across treatments. Pdimax was ∼50 cm H2O in the vehicle and 7,8-DHF groups and ∼40 cm H2O in the 1NMPP1 group (F8,74 = 2; P = 0.02). Our results indicate that TrkB signaling is necessary for generating maximal forces by the diaphragm muscle in old mice and are consistent with aging effects of TrkB signaling on neuromuscular transmission.NEW & NOTEWORTHY TrkB signaling is necessary for generating maximal forces by the diaphragm muscle. In 19- to 21-mo-old TrkBF616A mice susceptible to 1NMPP1-induced inhibition of TrkB kinase activity, maximal Pdi generated by bilateral phrenic nerve stimulation was ∼20% lower after 1NMPP1 compared with vehicle-treated mice. Treatment with the TrkB agonist 7,8-dihydroxyflavone did not affect Pdi generation when compared with age-matched mice. Inhibition of TrkB kinase activity did not affect the forces generated during lower force behaviors in old age.

Methocarbamol blocks muscular Nav 1.4 channels and decreases isometric force of mouse muscles.

BACKGROUND: The muscle relaxant methocarbamol is widely used for the treatment of muscle spasms and pain syndromes. To elucidate molecular mechanisms of its action, we studied its influence on neuromuscular transmission, on isometric muscle force, and on voltage-gated Na+ channels. METHODS: Neuromuscular transmission was investigated in murine diaphragm-phrenic nerve preparations and muscle force studied on mouse soleus muscles. Nav 1.4 channels and Nav 1.7 channels were functionally expressed in eukaryotic cell lines. RESULTS: Methocarbamol, at 2 mM, decreased the decay of endplate currents, slowed the decay of endplate potentials and reduced tetanic force of soleus muscles. The drug reversibly inhibited current flow through muscular Nav 1.4 channels, while neuronal Nav 1.7 channels were unaffected. CONCLUSIONS: The study provides evidence for peripheral actions of methocarbamol on skeletal muscle. Muscular Na+ channels are a molecular target of methocarbamol. Since Nav 1.7 currents were unaffected, methocarbamol is unlikely to exert its analgesic effect by directly blocking Nav 1.7 channels.

Alterations in fast-twitch muscle membrane conductance regulation do not explain decreased muscle function of SOD1G93A rats.

INTRODUCTION/AIMS: Both neuromuscular junction (NMJ) dysfunction and altered electrophysiological properties of muscle fibers have been reported in amyotrophic lateral sclerosis (ALS) patients. ALS-related preclinical studies typically use rodent SOD1G93A overexpression models, but translation to the human disease has been challenged. The present work explored NMJ function and cellular electrophysiological properties of muscles fibers in SOD1G93A overexpression rats. METHODS: Longitudinal studies of compound muscle action potentials (CMAPs) were performed in SOD1G93A rats. Cellular studies were performed to evaluate electrophysiological properties of muscle fibers, including the resting membrane conductance (Gm ) and its regulation during prolonged action potential (AP) firing. RESULTS: SOD1G93A rats showed a substantial loss of gastrocnemius CMAP amplitude (35.8 mV, P < .001) and a minor increase in CMAP decrement (8.5%, P = .002) at 25 weeks. In addition, SOD1G93A EDL muscle fibers showed a lower baseline Gm (wild-type, 1325 µS/cm2 ; SOD1G93A , 1137 µS/cm2 ; P < .001) and minor alterations in Gm regulation during repeated firing of APs as compared with wild-type rats. DISCUSSION: The current data suggest that loss of CMAP amplitude is largely explained by defects in either lower motor neuron or skeletal muscle with only minor indications of a role for neuromuscular transmission defects in SOD1G93A rats. Electrophysiological properties of muscle fibers were not markedly affected, and an elevated Gm , as has been reported in motor neuron disease (MND) patients, was not replicated in SOD1G93A muscles. Collectively, the neuromuscular pathology of SOD1G93A rats appears to differ from that of ALS/MND patients with respect to neuromuscular transmission defects and electrophysiological properties of muscle fibers.

Postganglionic sympathetic neurons, but not locus coeruleus optostimulation, activates neuromuscular transmission in the adult mouse in vivo.

Recent work demonstrated that sympathetic neurons innervate the skeletal muscle near the neuromuscular junction (NMJ), and muscle sympathectomy and sympathomimetic agents strongly influence motoneuron synaptic vesicle release ex vivo. Moreover, reports attest that the pontine nucleus locus coeruleus (LC) projects to preganglionic sympathetic neurons and regulates human mobility and skeletal muscle physiology. Thus, we hypothesized that peripheral and central sympathetic neurons projecting directly or indirectly to the skeletal muscle regulate NMJ transmission. The aim of this study was to define the specific neuronal groups in the peripheral and central nervous systems that account for such regulation in adult mice in vivo by using optogenetics and NMJ transmission recordings in 3-5-month-old, male and female ChR2(H134R/EYFP)/TH-Cre mice. After detecting ChR2(H134R)/EYFP fluorescence in the paravertebral ganglia and LC neurons, we tested whether optostimulating the plantar nerve near the lumbricalis muscle or LC neurons effectively modulates motor nerve terminal synaptic vesicle release in living mice. Nerve optostimulation increased motor synaptic vesicle release in vitro and in vivo, while the presynaptic adrenoceptor blockers propranolol (ß1/ß2) and atenolol (ß1) prevented this outcome. The effect is primarily presynaptic since miniature end-plate potential (MEPP) kinetics remained statistically unmodified after stimulation. In contrast, optostimulation of LC neurons did not regulate NMJ transmission. In summary, we conclude that postganglionic sympathetic neurons, but not LC neurons, increased NMJ transmission by acting on presynaptic ß1-adrenergic receptors in vivo.

Appraisal of a novel pedagogical approach to demonstrating neuromuscular transmission to medical students.

Although traditional didactic lecturing is the principal instructional approach used in numerous medical schools, this method has several limitations. Experiential learning approaches place students in the center of the learning process and creates a positive and supportive classroom learning environment. In this article, we propose an active pedagogical approach for teaching a basic physiology concept, namely, neuromuscular transmission (NMT), and we evaluate the effectiveness of this approach among undergraduate medical students. One-hundred forty-two undergraduate medical students were involved in this study (cohort A, n = 62; cohort B, n = 80). Cohort A received a didactic lecture on NMT and subsequently tested their knowledge of the topic via test 1, after which their perceptions of didactic lectures were recorded. The students were then asked to participate in a touch-and-go (TAG) active-learning session regarding NMT, after which their learning was subsequently tested by taking test 2. Their perceptions about the TAG game were also recorded. Students in cohort B were first administered test 1 regarding NMT. Then, they were asked to attend a didactic lecture blended with a TAG active-learning session, after which test 2 was administered, and their perceptions were recorded. Both the didactic lecture and active learning sessions facilitated student learning regarding the concepts involved in NMT. A comparison of the lecture followed by the game used in cohort A with the game blended lecture used in cohort B revealed no significant differences. However, both formats created a positive influence on students' interest in learning the concept, and the students preferred active learning over didactic sessions alone.

Clinical features of LRP4/agrin-antibody-positive myasthenia gravis: A multicenter study.

INTRODUCTION: Our aim in this study was to identify the prevalence and clinical characteristics of LRP4/agrin-antibody-positive double-seronegative myasthenia gravis (DNMG). METHODS: DNMG patients at 16 sites in the United States were tested for LRP4 and agrin antibodies, and the clinical data were collected. RESULTS: Of 181 DNMG patients, 27 (14.9%) were positive for either low-density lipoprotein receptor-related protein 4 (LRP4) or agrin antibodies. Twenty-three DNMG patients (12.7%) were positive for both antibodies. More antibody-positive patients presented with generalized symptoms (69%) compared with antibody-negative patients (43%) (P ≤ .02). Antibody-positive patients' maximum classification on the Myasthenia Gravis Foundation of America (MGFA) scale was significantly higher than that for antibody-negative patients (P ≤ .005). Seventy percent of antibody-positive patients were classified as MGFA class III, IV, or V compared with 39% of antibody-negative patients. Most LRP4- and agrin-antibody-positive patients (24 of 27, 89%) developed generalized myathenia gravis (MG), but with standard MG treatment 81.5% (22 of 27) improved to MGFA class I or II during a mean follow-up of 11 years. DISCUSSION: Antibody-positive patients had more severe clinical disease than antibody-negative patients. Most DNMG patients responded to standard therapy regardless of antibody status.

Sympathomimetics regulate neuromuscular junction transmission through TRPV1, P/Q- and N-type Ca2+ channels.

Increasing evidence indicates that, first, the sympathetic nervous system interacts extensively with both vasculature and skeletal muscle fibers near neuromuscular junctions (NMJs) and, second, its neurotransmitter, noradrenaline, influences myofiber molecular composition and function and motor innervation. Since sympathomimetic agents have been reported to improve NMJ transmission, we examined whether two in clinical use, salbutamol and clenbuterol, affect the motor axon terminal via extracellular Ca2+ and molecular targets, such as TRPV1 and P/Q- and N-type voltage-activated Ca2+ channels. Electrophysiological recordings in ex-vivo preparations of peroneal nerves and lumbricalis muscles from young adult mice focused on spontaneous miniature end-plate potentials and singly and repetitively evoked end-plate potentials. Adding one dose of salbutamol or clenbuterol to the nerve/muscle preparation or repeatedly administering salbutamol to a mouse for 4 weeks increased spontaneous and evoked synaptic vesicle release but induced a steep decline in EPP amplitude in response to repetitive nerve stimulation. These effects were mediated primarily by ω-agatoxin IVA-sensitive P/Q-type and secondarily by ω-conotoxin GVIA-sensitive N-type Ca2+ channels. Presynaptic arvanil-sensitive TRPV1 channels seem to regulate Ca2+ at the motor neuron terminal at rest, while putative presynaptic ß-adrenergic receptors may mediate sympathomimetic and catecholamine effects on presynaptic Ca2+ channels during NMJ activation.

Philips Intellivue NMT module: precision and performance improvements to meet the clinical requirements of neuromuscular block management.

The variability or inaccuracy of acceleromyographic measurements could interfere with the interpretation of the train-of-four (TOF) ratio during neuromuscular block (NMB) recovery. This study evaluated the precision and performance of the Philips Intellivue NMT module (NMT) before (part 1) and after (part 2) several technical upgrades (i.e., firmware upgrade, new cable, and hand adapter) that were recently available. Two cohorts of 30 patients who were scheduled to undergo rhino/septoplasty under general anesthesia were included in the study. TOF ratios were recorded simultaneously every 15 s on both hands with the NMT and a TOF-Watch SX installed inside a SL TOF-Tube (TWX). Before rocuronium was administered and once final responses were stabilized, the average of the four successive measurements that determined the baselines and repeatability coefficients were compared using a z test. Simultaneous measurements were recorded at different NMB stages: onset, depth of NMB after intubation, when TWX recovered TOF count 2, TOF ratios 0.5 and 0.9, and when NMT recovered TOF ratio 0.9. The results were compared using a Student t test; p < 0.05 was considered significant. The NMT repeatability coefficients obtained in part 1 were significantly higher than with the TWX, they were significantly lower in part 2. Initially, the NMT significantly overestimated NMB recovery at every stage. Conversely, in the second part of the study, no difference reached statistical significance. With the recent upgrades and the new hand adapter, the NMT provided similar results compared with the TWX, Their implementation should be recommended in clinical practice.

Placement of TOF-Cuff® on the lower leg for neuromuscular and blood pressure monitoring during anesthetic induction for shoulder surgeries.

PURPOSE: The aim of this study was to compare two devices for neuromuscular monitoring during anesthetic induction. TOF-Cuff® was installed on the lower leg stimulating the tibial nerve, while the more conventional TOF-Scan® was installed over the ulnar nerve at the wrist. Methods Twenty adult patients were enrolled in this prospective, controlled study. Train-of-four (TOF) was recorded every 15 s until TOF ratio of 0%. Mean arterial blood pressure (MAP) was assessed with TOF-Cuff® and with standard anesthesia monitoring from the brachial artery. MAP was measured before and after anesthetic induction. Time to TOF ratio = 0% was compared with one-sample t test and Bland-Altman plots. Results Patients received 0.53 ± 0.09 mg atracurium per kg body weight intravenously. Mean time to TOF ratio = 0% was 150.8 s (± 43.7) for TOF-Scan®, and 174.4 s (± 42.7) for TOF-Cuff® (p = 0.1356). Bias was - 15.9 (95% confidence interval - 37.5 to 5.6) with 95% limits of agreement of - 95.2 to 63.3. Twenty-five percent of the patients had a technical issue with a TOF-Cuff® measurement. For MAP, mean difference was 1.4 (95% confidence interval - 2.4 to 5.2) with 95% limits of agreement of - 22.7 to 25.5. Conclusion The time from administration of a common dose of atracurium to a TOF ratio of 0% assessed with TOF-Cuff® stimulating the tibial nerve compared to TOF-Scan® stimulating the ulnar nerve showed large limits of agreement in Bland-Altman analysis. There was a high failure rate with TOF-Cuff® measurements on the lower leg.

Diaphragm neuromuscular transmission failure in aged rats.

In aging Fischer 344 rats, phrenic motor neuron loss, neuromuscular junction abnormalities, and diaphragm muscle (DIAm) sarcopenia are present by 24 mo of age, with larger fast-twitch fatigue-intermediate (type FInt) and fast-twitch fatigable (type FF) motor units particularly vulnerable. We hypothesize that in old rats, DIAm neuromuscular transmission deficits are specific to type FInt and/or FF units. In phrenic nerve/DIAm preparations from rats at 6 and 24 mo of age, the phrenic nerve was supramaximally stimulated at 10, 40, or 75 Hz. Every 15 s, the DIAm was directly stimulated, and the difference in forces evoked by nerve and muscle stimulation was used to estimate neuromuscular transmission failure. Neuromuscular transmission failure in the DIAm was observed at each stimulation frequency. In the initial stimulus trains, the forces evoked by phrenic nerve stimulation at 40 and 75 Hz were significantly less than those evoked by direct muscle stimulation, and this difference was markedly greater in 24-mo-old rats. During repetitive nerve stimulation, neuromuscular transmission failure at 40 and 75 Hz worsened to a greater extent in 24-mo-old rats compared with younger animals. Because type IIx and/or IIb DIAm fibers (type FInt and/or FF motor units) display greater susceptibility to neuromuscular transmission failure at higher frequencies of stimulation, these data suggest that the age-related loss of larger phrenic motor neurons impacts nerve conduction to muscle at higher frequencies and may contribute to DIAm sarcopenia in old rats. NEW & NOTEWORTHY Diaphragm muscle (DIAm) sarcopenia, phrenic motor neuron loss, and perturbations of neuromuscular junctions (NMJs) are well described in aged rodents and selectively affect FInt and FF motor units. Less attention has been paid to the motor unit-specific aspects of nerve-muscle conduction. In old rats, increased neuromuscular transmission failure occurred at stimulation frequencies where FInt and FF motor units exhibit conduction failures, along with decreased apposition of pre- and postsynaptic domains of DIAm NMJs of these units.

Optogenetic analysis of neuromuscular transmission in the colon of ChAT-ChR2-YFP BAC transgenic mice.

Propulsion of luminal content along the gut requires coordinated contractions and relaxations of gastrointestinal smooth muscles controlled by the enteric nervous system. Activation of excitatory motor neurons (EMNs) causes muscle contractions, whereas inhibitory motor neuron (IMN) activation causes muscle relaxation. EMNs release acetylcholine (ACh), which acts at muscarinic receptors on smooth muscle cells and adjacent interstitial cells of Cajal, causing excitatory junction potentials (EJPs). IMNs release ATP (or another purine) and nitric oxide to cause inhibitory junction potentials (IJPs) and muscle relaxation. We used commercially available choline acetyltransferase (ChAT)-channelrhodopsin-2 (ChR2)-yellow fluorescent protein (YFP) bacterial artificial chromosome (BAC) transgenic mice, which express ChR2 in cholinergic neurons, to study cholinergic neuromuscular transmission in the colon. Intracellular microelectrodes were used to record IJPs and EJPs from circular muscle cells. We used blue light stimulation (BLS, 470 nm, 20 mW/mm2) and electrical field stimulation (EFS) to activate myenteric neurons. EFS evoked IJPs only, whereas BLS evoked EJPs and IJPs. Mecamylamine (10 µM, nicotinic cholinergic receptor antagonist) reduced BLS-evoked IJPs by 50% but had no effect on electrically evoked IJPs. MRS 2179 (10 µM, a P2Y1 receptor antagonist) blocked BLS-evoked IJPs. MRS 2179 and Nω-nitro-l-arginine (100 µM, nitric oxide synthase inhibitor) isolated the EJP, which was blocked by scopolamine (1 µM, muscarinic ACh receptor antagonist). Immunohistochemistry revealed ChAT expression in ~88% of enhanced YFP (eYFP)-expressing neurons, whereas 12% of eYFP neurons expressed nitric oxide synthase. These data show that cholinergic interneurons synapse with EMNs and IMNs to cause contraction and relaxation of colonic smooth muscle.NEW & NOTEWORTHY Electrical stimulation of interganglionic connectives has been used widely to study synaptic transmission in the enteric nervous system. However, electrical stimulation will activate many types of neurons and nerve fibers, which complicates data interpretation. Optogenetic activation of enteric neurons using genetically modified mice expressing channelrhodopsin-2 in cholinergic neurons offers a new approach that provides more specificity for nerve stimulation when studying myenteric plexus nerve circuitry.

Trouble at the junction: When myopathy and myasthenia overlap.

Although myopathies and neuromuscular junction disorders are typically distinct, their coexistence has been reported in several inherited and acquired conditions. Affected individuals have variable clinical phenotypes but typically display both a decrement on repetitive nerve stimulation and myopathic findings on muscle biopsy. Inherited causes include myopathies related to mutations in BIN1, DES, DNM2, GMPPB, MTM1, or PLEC and congenital myasthenic syndromes due to mutations in ALG2, ALG14, COL13A1, DOK7, DPAGT1, or GFPT1. Additionally, a decrement due to muscle fiber inexcitability is observed in certain myotonic disorders. The identification of a defect of neuromuscular transmission in an inherited myopathy may assist in establishing a molecular diagnosis and in selecting patients who would benefit from pharmacological correction of this defect. Acquired cases meanwhile stem from the co-occurrence of myasthenia gravis or Lambert-Eaton myasthenic syndrome with an immune-mediated myopathy, which may be due to paraneoplastic disorders or exposure to immune checkpoint inhibitors.

Neuromuscular transmission defects in myopathies: Rare but worth searching for.

INTRODUCTION: Decremental responses in repetitive nerve stimulation have been reported in a few hereditary myopathies. We examined the frequency of decrement in a cohort of myopathy patients. METHODS: We reviewed all patients referred for myopathy who underwent repetitive nerve stimulation between January 2007 and May 2017. We included patients with decrement (>10%) and either a pathological or molecular diagnosis of myopathy. RESULTS: Among 157 patients with myopathies, 4 patients had decrement (2 hydroxychloroquine-associated vacuolar myopathy, 1 centronuclear myopathy, and 1 distal myopathy). One hydroxychloroquine-associated vacuolar myopathy patient also had inflammatory myopathy. Pyridostigmine improved weakness in the centronuclear myopathy patient, but not in the distal myopathy patient. No patient with an acquired myopathy received pyridostigmine. CONCLUSIONS: Despite the rare occurrence of decrement in myopathy, its presence may urge consideration of pharmacological intervention. Muscle Nerve 59:475-478, 2019.

Cholesterol and the Safety Factor for Neuromuscular Transmission.

A present review is devoted to the analysis of literature data and results of own research. Skeletal muscle neuromuscular junction is specialized to trigger the striated muscle fiber contraction in response to motor neuron activity. The safety factor at the neuromuscular junction strongly depends on a variety of pre- and postsynaptic factors. The review focuses on the crucial role of membrane cholesterol to maintain a high efficiency of neuromuscular transmission. Cholesterol metabolism in the neuromuscular junction, its role in the synaptic vesicle cycle and neurotransmitter release, endplate electrogenesis, as well as contribution of cholesterol to the synaptogenesis, synaptic integrity, and motor disorders are discussed.