End-plate Potentials Research Articles

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.

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.

Adrenergic receptors control frequency-dependent switching of the exocytosis mode between “full-collapse” and “kiss-and-run” in murine motor nerve terminal

AimsNeurotransmitter release from the synaptic vesicles can occur through two modes of exocytosis: “full-collapse” or “kiss-and-run”. Here we investigated how increasing the nerve activity and pharmacological stimulation of adrenoceptors can influence the mode of exocytosis in the motor nerve terminal. MethodsRecording of endplate potentials with intracellular microelectrodes was used to estimate acetylcholine release. A fluorescent dye FM1–43 and its quenching with sulforhodamine 101 were utilized to visualize synaptic vesicle recycling. Key findingsAn increase in the frequency of stimulation led to a decrease in the rate of FM1–43 unloading despite the higher number of quanta released. High frequency activity promoted neurotransmitter release via the kiss-and-run mechanism. This was confirmed by experiments utilizing (I) FM1–43 dye quencher, that is able to pass into the synaptic vesicle via fusion pore, and (II) loading of FM1–43 by compensatory endocytosis. Noradrenaline and specific α2-adrenoreceptors agonist, dexmedetomidine, controlled the mode of synaptic vesicle recycling at high frequency activity. Their applications favored neurotransmitter release via full-collapse exocytosis rather than the kiss-and-run pathway. SignificanceAt the diaphragm neuromuscular junctions, neuronal commands are translated into contractions necessary for respiration. During stress, an increase in discharge rate of the phrenic nerve shifts the exocytosis from the full-collapse to the kiss-and-run mode. The stress-related molecule, noradrenaline, restricts neurotransmitter release in response to a high frequency activity, and prevents the shift in the mode of exocytosis through α2-adrenoceptor activation. This may be a component of the mechanism that limits overstimulation of the respiratory system during stress.

Antagonistic postsynaptic and presynaptic actions of cyclohexanol on neuromuscular synaptic transmission and function.

Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Some survivors develop a severe, acute or delayed myasthenic syndrome. In animal models, similar myasthenia has been associated with increasing plasma concentration of one insecticide solvent metabolite, cyclohexanol. We investigated possible mechanisms using voltage and current recordings from mouse neuromuscular junctions (NMJs) and transfected human cell lines. Cyclohexanol (10-25mM) reduced endplate potential (EPP) amplitudes by 10-40% and enhanced depression during repetitive (2-20Hz) stimulation by up to 60%. EPP decay was prolonged more than twofold. Miniature EPPs were attenuated by more than 50%. Cyclohexanol inhibited whole-cell currents recorded from CN21 cells expressing human postjunctional acetylcholine receptors (hnAChR) with an IC50 of 3.74mM. Cyclohexanol (10-20mM) also caused prolonged episodes of reduced-current, multi-channel bursting in outside-out patch recordings from hnAChRs expressed in transfected HEK293T cells, reducing charge transfer by more than 50%. Molecular modelling indicated cyclohexanol binding (-6kcal/mol) to a previously identified alcohol binding site on nicotinic AChR α-subunits. Cyclohexanol also increased quantal content of evoked transmitter release by ∼50%. In perineurial recordings, cyclohexanol selectively inhibited presynaptic K+ currents. Modelling indicated cyclohexanol binding (-3.8kcal/mol) to voltage-sensitive K+ channels at the same site as tetraethylammonium (TEA). TEA (10mM) blocked K+ channels more effectively than cyclohexanol but EPPs were more prolonged in 20mM cyclohexanol. The results explain the pattern of neuromuscular dysfunction following ingestion of organophosphorus insecticides containing cyclohexanol precursors and suggest that cyclohexanol may facilitate investigation of mechanisms regulating synaptic strength at NMJs. KEY POINTS: Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Survivors may develop a severe myasthenic syndrome or paralysis, associated with increased plasma levels of cyclohexanol, an insecticide solvent metabolite. Analysis of synaptic transmission at neuromuscular junctions in isolated mouse skeletal muscle, using isometric tension recording and microelectrode recording of endplate voltages and currents, showed that cyclohexanol reduced postsynaptic sensitivity to acetylcholine neurotransmitter (reduced quantal size) while simultaneously enhancing evoked transmitter release (increased quantal content). Patch recording from transfected cell lines, together with molecular modelling, indicated that cyclohexanol causes selective, allosteric antagonism of postsynaptic nicotinic acetylcholine receptors and block of presynaptic K+ -channel function. The data provide insight into the cellular and molecular mechanisms of neuromuscular weakness following intentional ingestion of agricultural organophosphorus insecticides. Our findings also extend understanding of the effects of alcohols on synaptic transmission and homeostatic synaptic function.

Homeostatic plasticity induced by increased acetylcholine release at the mouse neuromuscular junction

At the neuromuscular junction (NMJ), changes to the size of the postsynaptic potential induce homeostatic compensation. At the Drosophila NMJ, increased glutamate release causes a compensatory decrease in quantal content, but it is unknown if this mechanism operates at the cholinergic mammalian NMJ. We addressed this question by recording endplate potentials (EPP) and muscle contraction in 3-month and 24-month ChAT-ChR2-EYFP mice that overexpress vesicular acetylcholine transporter and release more acetylcholine per vesicle. At 3 months, the quantal content of EPPs from ChAT-ChR2-EYFP mice were not different from WT controls, however tetanic depression was greater, and quantal size during high-frequency stimulation and the size of the readily releasable pool (RRP) were decreased. At 24 months of age, quantal content was reduced in ChAT-ChR2-EYFP mice, which normalized synaptic depression despite smaller RRP. The effect of pancuronium on indirect evoked muscle twitch was not different between groups. These results indicate that an increase in the amount of acetylcholine per vesicle induces two distinct age-dependent homeostatic mechanisms compensating excessive acetylcholine release.

Confocal Endomicroscopy of Neuromuscular Junctions Stained with Physiologically Inert Protein Fragments of Tetanus Toxin.

Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066–1315 (emGFP-1066:TetC), 1093–1315 (emGFP-1093:TetC) and 1109–1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.

The Safety Factor for Neuromuscular Transmission: Effects of Dimethylsulphoxide, Cannabinoids and Synaptic Homeostasis.

BackgroundIn myasthenia gravis, impaired postsynaptic sensitivity to acetylcholine results in failure of neuromuscular transmission and fatiguing muscle weakness.ObjectiveDevelop an ex vivo muscle contraction assay to test cannabinoids and other substances that might act on the myasthenic neuromuscular junction to restore control of the muscle.MethodsTubocurarine was added to an ex vivo, mouse phrenic nerve-hemidiaphragm muscle preparation to reduce acetylcholine sensitivity. This produced a myasthenia-like decrement in twitch force during a train of 10 nerve impulses (3 / sec). Endplate potential (EPP) recordings were used to confirm and extend the findings.ResultsSurprisingly, addition to the bath of dimethylsulphoxide (DMSO), at concentrations as low as 0.1%(v/v), partially reversed the decrement in nerve-evoked force. Intracellular electrophysiology, conducted in the presence of tubocurarine, showed that DMSO increased the amplitudes of both the spontaneous miniature EPP (MEPP) and the (nerve-evoked) EPP. In the absence of tubocurarine (synaptic potentials at physiological levels), an adaptive fall in quantal content negated the DMSO-induced rise in EPP amplitude. The effects of cannabinoid receptor agonists (solubilized with DMSO) in the contraction assay do not support their further exploration as useful therapeutic agents for myasthenia gravis. CP 55,940 (a dual agonist for cannabinoid receptor types 1 and 2) reversed the beneficial effects of DMSO.Conclusions:We demonstrate a powerful effect of DMSO upon quantal amplitude that might mislead pharmacological studies of synaptic function wherever DMSO is used as a drug vehicle. Our results also show that compounds targeting impaired neuromuscular transmission should be tested under myasthenic-like conditions, so as to avoid confounding effects of synaptic homeostasis.

A comparison of acute mouse hindlimb injuries between tourniquet- and femoral artery ligation-induced ischemia-reperfusion

The tourniquet or femoral artery ligation is widely used to stop extremity hemorrhage or create a bloodless operating field in the combat scenario and civilian setting. However, these procedures with subsequent reperfusion also induce ischemia-reperfusion (IR) injuries. To fully evaluate animal models of limb IR injuries, we compared tourniquet- and femoral artery ligation-induced IR injuries in the hindlimb of mice. In C57/BL6 mice, 3 h of unilateral hindlimb ischemia was induced by placement of a rubber band at the hip joint or a surgical ligation of the femoral artery. The tourniquet or femoral artery ligation was then released, allowing for 24 h of reperfusion. Compared to the femoral artery ligation/IR, the tourniquet/IR induced more severe skeletal muscle damage, including muscle necrosis and interruption of muscle fibers. There was no gastrocnemius muscle contraction in tourniquet/IR, while femoral artery ligation/IR markedly weakened gastrocnemius muscle contraction. Motor nerve terminals disappeared, and endplate potentials (EPPs) were undetectable in tourniquet/IR, whereas femoral artery ligation/IR only induced mild impairment of motor nerve terminals and decreased the amplitude of EPPs. Additionally, western blot data showed that proinflammatory cytokine levels (IL-1β and TNF-α) were higher in the tourniquet/IR than that in femoral artery ligation/IR. Moreover, tourniquet/IR caused significant tissue edema and dilation of lymphatic vessels in the hindlimb, compared to femoral artery ligation/IR. The above data demonstrated that tourniquet/IR-induced acute hindlimb injuries are more severe than those induced by femoral artery ligation/IR. This suggests that future investigators should determine which hindlimb IR model (tourniquet/IR or femoral artery ligation/IR) is optimal depending on the purpose of their study.

Clinical Efficacy and Safety of Eculizumab for Treating Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune disease primarily mediated by acetylcholine receptor antibodies (AChR-Ab), cellular immune dependence, and complement system involvement. Since the AChR on the postsynaptic membrane is destroyed by an immune attack, sufficient endplate potential cannot be generated, resulting in the development of a synaptic transmission disorder at the neuromuscular junction and in muscle weakness. The role of the complement system in MG has been demonstrated in animal models and clinical tests, and it has been determined that complement inhibition in patients with MG can prevent disease induction and reverse its progression. Eculizumab is a humanized monoclonal antibody that inhibits the cleavage of complement protein C5 and prevents autoimmune damage; additionally, it has received subsequent approval by the Federal Drug Administration of the United States for MG treatment. However, various concerns regarding the use of eculizumab persist. In this review, we have discussed the treatment time, cost effectiveness, long-term efficacy, and tolerability of eculizumab for MG treatment. We have also summarized historical information and have presented perspectives on this new therapeutic modality.

Characterization of LRP4/Agrin Antibodies From a Patient With Myasthenia Gravis.

To determine whether human anti-LRP4/agrin antibodies are pathogenic in mice and to investigate underpinning pathogenic mechanisms. Immunoglobulin (Ig) was purified from a patient with myasthenia gravis (MG) with anti-LRP4/agrin antibodies and transferred to mice. Mice were characterized for body weight, muscle strength, twitch and tetanic force, neuromuscular junction (NMJ) functions including compound muscle action potential (CMAP) and endplate potentials, and NMJ structure. Effects of the antibodies on agrin-elicited muscle-specific tyrosine kinase (MuSK) activation and AChR clustering were studied and the epitopes of these antibodies were identified. Patient Ig-injected mice had MG symptoms, including weight loss and muscle weakness. Decreased CMAPs, reduced twitch and tetanus force, compromised neuromuscular transmission, and NMJ fragmentation and distortion were detected in patient Ig-injected mice. Patient Ig inhibited agrin-elicited MuSK activation and AChR clustering. The patient Ig recognized the β3 domain of LRP4 and the C-terminus of agrin and reduced agrin-enhanced LRP4-MuSK interaction. Anti-LRP4/agrin antibodies in the patient with MG is pathogenic. It impairs the NMJ by interrupting agrin-dependent LRP4-MuSK interaction.

Metformin Increases Sarcolemma Integrity and Ameliorates Neuromuscular Deficits in a Murine Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease characterized by progressive muscle weakness and wasting. Stimulation of AMP-activated protein kinase (AMPK) has been demonstrated to increase muscle function and protect muscle against damage in dystrophic mice. Metformin is a widely used anti-hyperglycemic drug and has been shown to be an indirect activator of AMPK. Based on these findings, we sought to determine the effects of metformin on neuromuscular deficits in mdx murine model of DMD. In this study, we found metformin treatment increased muscle strength accompanied by elevated twitch and tetanic force of tibialis anterior (TA) muscle in mdx mice. Immunofluorescence and electron microscopy analysis of metformin-treated mdx muscles revealed an improvement in muscle fiber membrane integrity. Electrophysiological studies showed the amplitude of miniature endplate potentials (mEPP) was increased in treated mice, indicating metformin also improved neuromuscular transmission of the mdx mice. Analysis of mRNA and protein levels from muscles of treated mice showed an upregulation of AMPK phosphorylation and dystrophin-glycoprotein complex protein expression. In conclusion, metformin can indeed improve muscle function and diminish neuromuscular deficits in mdx mice, suggesting its potential use as a therapeutic drug in DMD patients.

The Different Responses to Femoral Artery Ligation‐induced Ischemia between Balb/c and C57BL/6 mice

Peripheral arterial disease(PAD) is a common circulatory problem in lower extremities, and the murine experimental model is used to reproduce human PAD. Here, the left femoral artery was blocked by ligation to reduce blood flow to the limb of Balb/c and C57BL/6 mice, and the functional and morphological changes of gastrocnemius muscle were compared between Balb/c and C57BL/6 mice. It was found that Balb/c mice display serious skeletal muscle injuries after 6 weeks of the artery ligation, including irregular myofibers with more adipose-liked tissue (17.77 ± 0.88) compared to C57BL/6 mice (0.27% ± 0.07). The pretzel-like nicotinic acetylcholine receptor (nAChR) clusters were fragmented by femoral artery ligation-induced ischemia in the gastrocnemius muscle from Balb/c mice (8.8 ± 1.3, compared to 2.5 ± 0.7 in C57BL/6). There is no difference in end plate potentials (EPPs) and skeletal muscle contraction between Balb/c (29.19 ± 2.25 mV and 77.1 ± 2.65 g) and C57BL/6 mice (29.2 mV ± 1.42 mV and 78.83 ± 2.47 g). After 6 weeks of the femoral artery ligation, EPPs and skeletal muscle contraction are smaller in Balb/c mice (8.9 ± 1.6 mV and 38.6 ± 7.3 g), compared to C57BL/6 mice (19.9 ± 2.0 mV and 71.9 ± 3.2 g). These results confirmed that pathophysiological alterations in the neuromuscular junction and skeletal muscle of Balb/c mice are kept a long period after the femoral artery ligation, while neuromuscular junction and skeletal muscle function is rapidly recovered in C57BL/6 mice with the femoral artery ligation. This study suggests that femoral artery ligation-induced ischemia in Balb/c mouse is an appropriate experimental model to reproduce PAD.

Взаимодействие механизмов угнетения квантового выделения ацетилхолина при активации ваниллоидных (TRPV1) и пуриновых рецепторов в нервно-мышечном синапсе мыши

The main goal of the study was to analyze the relationship between the signaling pathways of the regulation of acetylcholine (ACh) quantal release in the peripheral synapse triggered by the activation of vanilloid (TRPV1) and purine receptors. In electrophysiological experiments carried out at the neuromuscular synapses of the m. Levator Auris Longus, it was found that the frequency of miniature endplate potentials (mEPPs) and the quantal content of endplate potentials (EPPs) decrease in the presence of TRPV1 agonist capsaicin. This effect was completely reversed by SB366791, a specific competitive antagonist of TRPV1 receptors. ATP, like capsaicin, decreased the frequency of mEPPs and the EPP quantal content. Against the background of the TRPV1 antagonist, the inhibitory effect of ATP on ACh secretion was realized in full. At the same time, against the background of TRPV1 channels activation by capsaicin, the effect of ATP on both spontaneous and evoked ACh release was absent. It was suggested that the mechanisms of action of ATP and capsaicin may be associated with a change in Ca2+ entry into the nerve ending. To test this hypothesis, experiments were carried out to assess the changes in the presynaptic calcium level (Ca2+ transient) using a fluorescent calcium dye upon nerve stimulation. The amplitude of the calcium transient did not change either with the application of ATP or with the addition of capsaicin. Thus, in the neuromuscular synapse of mammals, along with purinergic pathway of ACh secretion regulation, there is also a mechanism of neurosecretion modulation mediated by the activation of TRPV1 channels. The triggering of these mechanisms leads to the suppression of the processes of both spontaneous and evoked release of ACh quanta from the motor nerve endings. It was demonstrated that both pathways of regulation are not accompanied by a change in the Ca2+ transient, but have a common link of regulation of the quantal neurosecretion.

Effects of inhibition of glycogen synthase kinase 3p on neuromuscular synaptic transmission in transgenic mice with model of alzheimer's disease

Alzheimer's disease is the most common neurodegenera-tive disease. Synaptic dysfunction plays important role in pathogenesis of Alzheimer's disease. Recently, glycogen synthase kinase 3 has been considered as a potential target for therapy of Alzheimer's disease, since the activity of this enzyme, which regulates many cellular and synaptic functions, is impaired in this pathology. In this paper, we studied the effects of inhibition of glycogen synthase kinase 3p on the parameters of the peripheral (neuromuscular) synapse functioning in the Alzheimer's disease model in APP/PS1 transgenic mice. In electrophysiological experiments on the diaphragm of APP/PS1 of mice, application of the glycogen synthase kinase 3p inhibitor AR-A014418 (1 pM) did not cause significant changes in the parameters of spontaneous and evoked neurotransmitter release, as well as in the dynamics of the amplitude of the end-plate potentials during high-frequency stimulation. In fluorescent experiments using the dye FM 1-43, it was found that the application of AR-A014418 (1 pM) does not significantly change the intensity of endocytosis, but causes moderate enhancement of exocytosis of synaptic vesicles during high-frequency stimulation. Enhancement of synaptic vesicle exocytosis due to inhibition of glycogen synthase kinase 3p may alleviate neuromuscular synaptic dysfunction in APP/PS1 mice. The obtained results can be used in studies aimed to development of therapeutic strategies for Alzheimer's disease.

The Neuromuscular Junction in Health and Disease: Molecular Mechanisms Governing Synaptic Formation and Homeostasis.

The neuromuscular junction (NMJ) is a highly specialized synapse between a motor neuron nerve terminal and its muscle fiber that are responsible for converting electrical impulses generated by the motor neuron into electrical activity in the muscle fibers. On arrival of the motor nerve action potential, calcium enters the presynaptic terminal, which leads to the release of the neurotransmitter acetylcholine (ACh). ACh crosses the synaptic gap and binds to ACh receptors (AChRs) tightly clustered on the surface of the muscle fiber; this leads to the endplate potential which initiates the muscle action potential that results in muscle contraction. This is a simplified version of the events in neuromuscular transmission that take place within milliseconds, and are dependent on a tiny but highly structured NMJ. Much of this review is devoted to describing in more detail the development, maturation, maintenance and regeneration of the NMJ, but first we describe briefly the most important molecules involved and the conditions that affect their numbers and function. Most important clinically worldwide, are myasthenia gravis (MG), the Lambert-Eaton myasthenic syndrome (LEMS) and congenital myasthenic syndromes (CMS), each of which causes specific molecular defects. In addition, we mention the neurotoxins from bacteria, snakes and many other species that interfere with neuromuscular transmission and cause potentially fatal diseases, but have also provided useful probes for investigating neuromuscular transmission. There are also changes in NMJ structure and function in motor neuron disease, spinal muscle atrophy and sarcopenia that are likely to be secondary but might provide treatment targets. The NMJ is one of the best studied and most disease-prone synapses in the nervous system and it is amenable to in vivo and ex vivo investigation and to systemic therapies that can help restore normal function.

Review on Muscle Movement Recording System Using EMG

Electromyography is a neurophysiological technique for examining the electrical activity of skeletal muscles. It occurred from the muscle membrane potential. In a healthy muscle at rest, the spontaneous physiological activity can be a record by End plate potential, insertion activity. EMG recording of the activity of selective cranial nerve muscles is currently included in the intraoperative set during surgical manipulation of the brainstem. When during the process, weak muscles can be detected and treat those muscles to regain their function. EMG is also used to diagnose neuromuscular performance. Its gain is less and frequency response may be broader. Using the EMG technique to record the potential of muscle is an easy way to generate the resting potential of muscle.

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

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. 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. 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. 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.

Lack of Desmin in Mice Causes Structural and Functional Disorders of Neuromuscular Junctions.

Desmin, the major intermediate filament (IF) protein in muscle cells, interlinks neighboring myofibrils and connects the whole myofibrillar apparatus to myonuclei, mitochondria, and the sarcolemma. However, desmin is also known to be enriched at postsynaptic membranes of neuromuscular junctions (NMJs). The pivotal role of the desmin IF cytoskeletal network is underscored by the fact that over 120 mutations of the human DES gene cause hereditary and sporadic myopathies and cardiomyopathies. A subgroup of human desminopathies comprises autosomal recessive cases resulting in the complete abolition of desmin protein. In these patients, who display a more severe phenotype than the autosomal dominant cases, it has been reported that some individuals also suffer from a myasthenic syndrome in addition to the classical occurrence of myopathy and cardiomyopathy. Since further studies on the NMJ pathology are hampered by the lack of available human striated muscle biopsy specimens, we exploited homozygous desmin knock-out mice which closely mirror the striated muscle pathology of human patients lacking desmin protein. Here, we report on the impact of the lack of desmin on the structure and function of NMJs and the transcription of genes coding for postsynaptic proteins. Desmin knock-out mice display a fragmentation of NMJs in soleus, but not in the extensor digitorum longus muscle. Moreover, soleus muscle fibers show larger NMJs. Further, transcription levels of acetylcholine receptor (AChR) genes are increased in muscles from desmin knock-out mice, especially of the AChRγ subunit, which is known as a marker of muscle fiber regeneration. Electrophysiological recordings depicted a pathological decrement of nerve-dependent endplate potentials and an increased rise time of the nerve-independent miniature endplate potentials. The latter appears related to the fragmentation of NMJs in desmin knockout mice. Our study highlights the essential role of desmin for the structural and functional integrity of mammalian NMJs.

Effects of a Fat-Rich Diet on the Spontaneous Release of Acetylcholine in the Neuromuscular Junction of Mice.

Western societies are facing a clear increase in the rate of obesity and overweight which are responsible for musculoskeletal pain. Some of the substances described in the environment of myofascial trigger points (MTrPs) are the same as those found in the skeletal muscle of obese people, such as cytokines. Furthermore, elevated neuromuscular neurotransmission has been associated with MTrPs. The main objective of this study is to assess whether obesity or overweight may be a facilitator of myofascial pain. The experiments were performed on male Swiss mice. One experimental group was given a typical “cafeteria” diet and another group a commercial high-fat diet for six weeks. Intramuscular adipocytes were assessed with Sudan III. The functional study was performed with electromyographic recording to determine the plaque noise and intracellular recording of miniature endplate potentials (MEPPs). The intake of a cafeteria diet showed the presence of more adipocytes in muscle tissue, but not with the fat-supplemented diet. Both experimental groups showed an increase in the plaque noise and an increase in the frequency of MEPPs that lasted several weeks after interrupting diets. In summary, the supply of a hypercaloric diet for six weeks in mice increases spontaneous neurotransmission, thus facilitating the development of MTrPs.

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 (β1/β2) and atenolol (β1) 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 β1-adrenergic receptors in vivo.