“Warming yang and invigorating qi” acupuncture alters acetylcholine receptor expression in the neuromuscular junction of rats with experimental autoimmune myasthenia gravis

Myasthenia gravis is an autoimmune disorder in which antibodies have been shown to form against the nicotinic acetylcholine nicotinic postsynaptic receptors located at the neuromuscular junction.＂Warming yang and invigorating qi＂ acupuncture treatment has been shown to reduce serum inflammatory cytokine expression and increase transforming growth factor beta expression in rats with experimental autoimmune myasthenia gravis.However,few studies have addressed the effects of this type of acupuncture on the acetylcholine receptors at the neuromuscular junction.Here,we used confocal laser scanning microscopy to examine the area and density of immunoreactivity for an antibody to the nicotinic acetylcholine receptor at the neuromuscular junction in the phrenic nerve of rats with experimental autoimmune myasthenia gravis following ＂warming yang and invigorating qi＂ acupuncture therapy.Needles were inserted at acupressure points Shousanli（LI10）,Zusanli（ST36）,Pishu（BL20）,and Shenshu（BL23） once daily for 7 consecutive days.The treatment was repeated after 1 day of rest.We found that area and the integrated optical density of the immunoreactivity for the acetylcholine receptor at the neuromuscular junction of the phrenic nerve was significantly increased following acupuncture treatment.This outcome of the acupuncture therapy was similar to that of the cholinesterase inhibitor pyridostigmine bromide.These findings suggest that ＂warming yang and invigorating qi＂ acupuncture treatment increases acetylcholine receptor expression at the neuromuscular junction in a rat model of autoimmune myasthenia gravis.

Active zone stability:insights from fly neuromuscular junction

The presynaptic active zone is a dynamic structure that orchestrates regulated release of neurotrans- mitters. Developmental and aging processes, and changes in neuronal network activity can all modulate the number, size and composition of active zone and thereby synaptic efficacy. However, very little is known about the mechanism that controls the structural stability of active zone. By study- ing a model synapse, the Drosophila neuromuscular iunction, our recent work shed light on how two scaffolding proteins at the active zone regulate active zone stability by promoting a localized dephos- phorylation event at the nerve terminal. Here we discuss the major insights from our findings and their implications for future research.

The Drosophila adult neuromuscular junction as a model for unravelling amyloid peptide influence on synapse dynamics

Amyloid peptide（Aβ）oligomers are considered one of the primary causal factors for the synaptic loss characteristic of Alzheimer’s disease（AD）（Karran and De Strooper,2016）.However,Aβis generated in normal brains and accumulates at synaptic sites,which raises the question whether Aβplays a physiological role in synapses.

EFFECTS OF MANGANESE ON NEUROMUSCULAR JUNCTION TRANSMISSION

The effects of manganese on neuromuscular junction transmission were studied in the isolated rat phrenic nerve diaphragm preparation, chick biventer cervicis nerve-muscle preparation and toad rectus abdominis muscle preparation. It wa found that manganese could adversely andconcentration-dependently inhibit muscular contraction response developed by indirect stimulation or direct stimulation. The IC50 of indirect and direct stimulation were 0.28 mmol/L and 10 mmol/Lrespectively. The time required to inhibit 50% of the contraction by indirect stimulation was 4.3±1. 0 min. The inhibitory action could be antagonized partially by high Ca2+. Manganese could reduce the seusitivity of the chick biventer cervicis muscle to acetylcholine, and shift the dose-respond curves foracetylcholine to the right in a nonparallel manner with a pD2' value of 2. 62. The result suggested that manganese acted on pre- and post-synaptic sites.

Lesson from the neuromuscular junction:role of pattern and timing of nerve activity in synaptic development

The anatomical plan of adult muscle innervation is relatively simple： a given muscle comprises several motor units, each constituted by one motor neuron and the muscle fibers that it innervates; moreover, every muscle fiber is innervated by only one axonal terminal. In other words, motor units have separate, although intermingled, territories of inner- vation （Figure 1D）. In striking contrast, the anatomical organization is different at birth, when every muscle fiber is innervated by several nerve terminals belonging to different motor neurons, a condition known as ＂polyneuronal inner- vation＂, with the consequence that motor units have larger and overlapped territories of innervation （Figure 1A） （Tapia and Lichtman, 2012）. Soon after birth, redundant nerve ter- minals are progressively eliminated in a couple of weeks in rodents, and muscle fibers acquire their mature mononeu- ronal innervation. The same process occurs again in the adult muscle during reinnervation after nerve damage, when a transient period of polyneuronal innervation involves a good fraction of the fibers （Rich and Lichtman, 1989;

Remnant neuromuscular junctions in denervated muscles contribute to functional recovery in delayed peripheral nerve repair

Schwann cell proliferation in peripheral nerve injury(PNI)enhances axonal regeneration compared to central nerve injury.However,even in PNI,long-term nerve damage without repair induces degeneration of neuromuscular junctions(NMJs),and muscle atrophy results in irreversible dysfunction.The peripheral regeneration of motor axons depends on the duration of skeletal muscle denervation.To overcome this difficulty in nerve regeneration,detailed mechanisms should be determined for not only Schwann cells but also NMJ degeneration after PNI and regeneration after nerve repair.Here,we examined motor axon denervation in the tibialis anterior muscle after peroneal nerve transection in thy1-YFP mice and regeneration with nerve reconstruction using allografts.The number of NMJs in the tibialis anterior muscle was maintained up to 4 weeks and then decreased at 6 weeks after injury.In contrast,the number of Schwann cells showed a stepwise decline and then reached a plateau at 6 weeks after injury.For regeneration,we reconstructed the degenerated nerve with an allograft at 4 and 6 weeks after injury,and evaluated functional and histological outcomes for 10 to 12 weeks after grafting.A higher number of pretzel-shaped NMJs in the tibialis anterior muscle and better functional recovery were observed in mice with a 4-week delay in surgery than in those with a 6-week delay.Nerve repair within 4 weeks after PNI is necessary for successful recovery in mice.Prevention of synaptic acetylcholine receptor degeneration may play a key role in peripheral nerve regeneration.All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University on 5 July 2017,30 March 2018,and 15 May 2019(A2017-311C,A2018-297A,and A2019-248A),respectively.

The postsynaptic effect of taxol on rat neuromuscular junction

Taxol was used as a tool reagent and the function of the microtubules (MTs) beneath thepostsynaptic membrane was studied in an isolated non-uniformly stretched muscle preparation of ratdiaphragm.After exposure to taxol (20 μmol/L,10min),the amplitude of acetylcholine potential ofinnervated muscle endplate was decreased by 30%,but the lime course of AChP and membrane po-tential remained unchanged.The results indicate that taxol can inhibit the responsiveness ofpostsynapfic membrane.It is therefore suggested that the site of action of taxol indudng inhibitionof postsynapfic responsiveness in neuromuscular junction may be the microtubules beneath thepostsynapfic membrane.

Ethylene thiourea exposure induces neurobehavioral toxicity in zebrafish by disrupting axon growth and neuromuscular junctions

Ethylene thiourea(ETU)converted from ethylene bisdithiocarbamate(EBDC)fungicides has aroused great concern because of its prevalence and harmful effects.Although ETU-induced neurotoxicity has been reported,the potential mechanisms remain unclear.This study provided insights into its neurotoxic effects at environmentally relevant concentrations in zebrafish.Our findings showed that embryonic exposure to ETU decreased the hatch rate and delayed somite development.Furthermore,ETU treatment significantly reduced the dark velocity in the locomotion assay.The upregulated tendency of the mitogen-activated protein kinases(MAPK)pathway(mknk1,atf4,mapkapk3)screened by transcriptome analysis implied motor neuron degeneration,which was validated by subsequent morphological observation,as axon length and branches were truncated in the 62.5μg/L ETU group.However,although the rescue experiment with a p38 MAPK inhibitor(SB203580)successfully ameliorated axon degeneration,it failed to reverse the locomotion behaviors.Further exploration of transcriptome data revealed the varied expression of presynaptic scaffold protein-related genes(pcloa,pclob,bsna),whose downregulation might impair the neuromuscular junction(NMJ).Therefore,we reasonably suspected that ETU-induced neurobehavioral deficits might result from the combined effects of the MAPK pathway and presynaptic proteins.Considering this,we highlighted the necessity to take precautions and early interventions for susceptible ETU-exposed populations.

Tissue optical clearing imaging for structural changes of neuromuscular junctions after mice ischemic stroke[Invited]

Ischemic stroke causes long-term disability and results in motor impairments.Such impairments are associated with structural changes in the neuromuscular junction(NMJ),including detailed morphology and three-dimensional(3D)distribution.However,previous studies only explored morphological changes of individual NMJs after stroke,which limits the understanding of their role in post-stroke motor impairment.Here,we examine 3D distributions and detailed morphology of NMJs in entire mouse muscles after unilateral and bilateral strokes induced by photothrombosis.The results show that 3D distributions and numbers of NMJs do not change after stroke,and severe unilateral stroke causes similar levels of NMJ fragmentation and area enlargement to bilateral stroke.This research provides structural data,deepening the understanding of neuromuscular pathophysiology after stroke.

C9orf72 poly-GA proteins impair neuromuscular transmission

Amyotrophic lateral sclerosis(ALS) is a devastating motoneuron disease,in which lower motoneurons lose control of skeletal muscles.Degeneration of neuromuscular junctions(NMJs) occurs at the initial stage of ALS.Dipeptide repeat proteins(DPRs) from G4C2repeat-associated non-ATG(RAN) translation are known to cause C9orf72-associated ALS(C9-ALS).However,DPR inclusion burdens are weakly correlated with neurodegenerative areas in C9-ALS patients,indicating that DPRs may exert cell non-autonomous effects,in addition to the known intracellular pathological mechanisms.Here,we report that poly-GA,the most abundant form of DPR in C9-ALS,is released from cells.Local administration of poly-GA proteins in peripheral synaptic regions causes muscle weakness and impaired neuromuscular transmission in vivo.The NMJ structure cannot be maintained,as evidenced by the fragmentation of postsynaptic acetylcholine receptor(AChR) clusters and distortion of presynaptic nerve terminals.Mechanistic study demonstrated that extracellular poly-GA sequesters soluble Agrin ligands and inhibits Agrin-MuSK signaling.Our findings provide a novel cell non-autonomous mechanism by which poly-GA impairs NMJs in C9-ALS.Thus,targeting NMJs could be an early therapeutic intervention for C9-ALS.

Atrial Septal Defect, Neuromuscular Junction and Skeletal Abnormalities in Spinal Muscular Atrophy Type Ⅲ

Spinal muscular atrophy （SMA） is a genetic disorder which is clinically characterized by progressive muscle weakness and atrophy and is associated with the degeneration of spinal and lowers bulbar motor neurons. SMAis the most common genetic cause of infant mortality, and seems to be present in general populations. The clinical spectrum of SMA ranges from early infant death to normal adult life with only mild weakness. Approximately 81.2–95.0% of cases of SMA resulted from homozygous deletion of survival of motor neuron 1 （SMN1） and 5.0% were compound heterozygous patients.[1] SMA might manifest not only the dysfunction of pure motor neurons but also abnormalities in neuromuscular junction （NMJ）, osteoporotic bone formation, cardiac abnormalities, and vascular defects.[2] These phenomena have been described in severe SMA （Type I, II） patients and in mouse models while data from SMA Type III individuals are not available. Patients with SMA Type III demonstrate progressive proximal weakness affecting the legs more severely than the arms, and might ultimately end up in the wheelchair. Herein, we report one patient with SMA Type III manifesting an atrial septal defect （ASD）, NMJ defect, short stature, and thick toes.

TDP-43 dysregulation and neuromuscular junction disruption in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis(ALS)is a disease characterized by upper and lower motor neuron(MN)loss with a signature feature of cytoplasmic aggregates containing TDP-43,which are detected in nearly all patients.Mutations in the gene that encodes TDP-43(TARBDP)are known to result in both familial and sporadic ALS.In ALS,disruption of neuro-muscular junctions(NMJs)constitutes a critical event in disease pathogenesis,leading to denervation atrophy,motor impairments and disability.Morphological defects and impaired synaptic transmission at NMJs have been reported in several TDP-43 animal models and in vitro,linking TDP-43 dysregulation to the loss of NMJ integrity in ALS.Through the lens of the dying-back and dying-forward hypotheses of ALS,this review discusses the roles of TDP-43 related to synaptic function,with a focus on the potential molecular mechanisms occurring within MNs,skeletal muscles and glial cells that may contribute to NMJ disruption in ALS.

Human neuromuscular junction three-dimensional organoid models and the insight in motor disorders

The neuromuscular junction(NMJ),a peripheral synaptic connection between motoneurons and skeletal muscle fibers,controls movement.Dysregulation of NMJs has been implicated in various motor disorders.Because of their large size and easy accessibility,NMJs have been extensively investigated in the neuroscience field and have greatly contributed to our understanding of the fundamental principles of synapses in the central nervous system.Researchers have tried multiple ways to develop models to recreate NMJs.Rapid progress in the research and development of tissue-like organoids has made it possible to produce human NMJ three-dimensional(3D)models in vitro,providing an additional powerful strategy to study NMJs.Here,we introduce the most recent advances of human embryonic stem cell-or induced pluripotent stem cell-derived organoids to model 3D NMJs.

Protective effect of Sijunzi decoction on neuromuscular junction ultrastructure in autoimmune myasthenia gravis rats

OBJECTⅣE: To investigate the protective role of Sijunzi decoction in neuromuscular junction(NMJ)and muscle cell mitochondria ultrastructure; as well as its effects on the amount of adenosine triphosphate(ATP) and the activities of mitochondrial respiratory chain complexes I, Ⅱ, Ⅲ, and Ⅳ in autoimmune myasthenia gravis rats.METHODS: An experimental autoimmune myasthenia gravis(EAMG) rat model was established by inoculating rats with acetylcholine receptors extracted from Torpedo. Rats were divided into three groups: model, prednisone, and Sijunzi decoction, and were fed physiological saline, prednisone, or Sijunzi decoction, respectively. NMJ and muscle cell mitochondria ultrastructure were observed by transmission electron microscope. The amount of ATP was assessed by high performance liquid chromatography. The activities ofmitochondrial respiratory chain complexes I, Ⅱ, Ⅲ,and Ⅳ was determined using the Clark oxygen electrode method.RESULTS: In the model group, there were sparse muscle fibers, with decreased mitochondria, and sparse, diffluent, or absent NMJ folds. After intervention with Sijunzi decoction, the above pathology changes were improved: muscle fiber structure was clear and complete; the mitochondria count was higher; and the NMJ structure was close to normal. Gastrocnemius muscle mitochondria in the model group produced significantly less ATP than those in the prednisone group(P<0.01). Conversely, the ATP of Sijunzi decoction group was significantly higher than prednisone group(P<0.01). The activities of gastrocnemius muscle mitochondrial respiratory chain complexes I, Ⅱ, Ⅲ, and Ⅳ in both the prednisone and Sijunzi decoction groups was dramatically higher compared with the model group(P<0.05). The activities of complexes I and Ⅲ in the Sijunzi decoction group were significantly higher than those in the prednisone group(P<0.05), but there was no obvious difference in complex Ⅱ or Ⅳ activities between the two groups(P>0.05).CONCLUSION: Sijunzi decoction improved pathological changes in muscle mitochondria and NMJ,enhanced the amount of ATP in gastrocnemius muscle mitochondria, and improved the activities of respiratory chain complexes I, Ⅱ, Ⅲ, and Ⅳ(especially I and Ⅲ) of the EAMG rats.

RESISTANCE OF THE NEUROMUSCULAR JUNCTION OF THE SNAKE TO THE BLOCKING ACTION OF BOTULINUM TOXIN TYPE A

As the most potent natural poison, botulinum toxin (BoTX) blocks the cholinergic synaptic transmission in skeletal muscles selectively by interfering in the acetylcholine release from motor nerve terminals and results in the death of the animals due to the paralysis

Skeletal muscle as a molecular and cellular biomarker of disease progression in amyotrophic lateral sclerosis:a narrative review

Amyotrophic lateral sclerosis is a fatal multisystemic neurodegenerative disease with motor neurons being a primary target.Although progressive weakness is a hallmark feature of amyotrophic lateral sclerosis,there is considerable heterogeneity,including clinical presentation,progression,and the underlying triggers for disease initiation.Based on longitudinal studies with families harboring amyotrophic lateral sclerosis-associated gene mutations,it has become apparent that overt disease is preceded by a prodromal phase,possibly in years,where compensatory mechanisms delay symptom onset.Since 85-90%of amyotrophic lateral sclerosis is sporadic,there is a strong need for identifying biomarkers that can detect this prodromal phase as motor neurons have limited capacity for regeneration.Current Food and Drug Administration-approved therapies work by slowing the degenerative process and are most effective early in the disease.Skeletal muscle,including the neuromuscular junction,manifests abnormalities at the earliest stages of the disease,before motor neuron loss,making it a promising source for identifying biomarkers of the prodromal phase.The accessibility of muscle through biopsy provides a lens into the distal motor system at earlier stages and in real time.The advent of“omics”technology has led to the identification of numerous dysregulated molecules in amyotrophic lateral sclerosis muscle,ranging from coding and non-coding RNAs to proteins and metabolites.This technology has opened the door for identifying biomarkers of disease activity and providing insight into disease mechanisms.A major challenge is correlating the myriad of dysregulated molecules with clinical or histological progression and understanding their relevance to presymptomatic phases of disease.There are two major goals of this review.The first is to summarize some of the biomarkers identified in human amyotrophic lateral sclerosis muscle that have a clinicopathological correlation with disease activity,evidence of a similar dysregulation in the SOD1G93A mouse during presymptomatic stages,and evidence of progressive change during disease progression.The second goal is to review the molecular pathways these biomarkers reflect and their potential role in mitigating or promoting disease progression,and as such,their potential as therapeutic targets in amyotrophic lateral sclerosis.

Three-dimensional in vitro models of neuromuscular tissue

Skeletal muscle is a dynamic tissue in which homeostasis and function are guaranteed by a very defined three-dimensional organization of myofibers in respect to other nonmuscular components,including the extracellular matrix and the nervous network.In particular,communication between myofibers and the nervous system is essential for the overall correct development and function of the skeletal muscle.A wide range of chronic,acute and genetic-based human pathologies that lead to the alteration of muscle function are associated with modified preservation of the fine interaction between motor neurons and myofibers at the neuromuscular junction.Recent advancements in the development of in vitro models for human skeletal muscle have shown that three-dimensionality and integration of multiple cell types are both key parameters required to unveil pathophysiological relevant phenotypes.Here,we describe recent achievement reached in skeletal muscle modeling which used biomaterials for the generation of three-dimensional constructs of myotubes integrated with motor neurons.

果蝇Neuroligin 4以非依赖于Neurexin方式调控突触的发育

目的探究果蝇突触黏附分子神经连接蛋白-4(Drosophila neuroligin 4,DNlg4)对幼虫神经肌肉突触发育的调控是否依赖于经典的neuroligin(Nlg)突触前配体分子轴突蛋白(Drosophila neurexin,DNrx)。方法主要利用免疫荧光染色和激光共聚焦显微观测技术,对包括野生型、dnlg4突变体、dnrx突变体和dnlg4,dnrx双突变体等不同基因型果蝇三龄幼虫的神经肌肉接头(neuromuscular junction,NMJ)的形态进行观察,测量其突触分枝总长度并计数突触终扣(bouton)数目。以此作为NMJ发育状态参数,通过比较不同基因型果蝇的NMJ发育缺陷程度,确定DNlg4和DNrx在调控神经肌肉突触发育中的关系。结果DNrx缺失会导致较DNlg4缺失更严重的NMJ发育缺陷;缺失DNlg4会进一步加重因DNrx缺失而导致的NMJ发育缺陷;运动神经元表达外源的DNlg4可部分挽救DNrx缺失造成的NMJ发育缺陷;未检测到两类分子存在在体的相互结合。结论DNlg4与DNrx对果蝇幼虫神经肌肉突触发育的正向调控具有协同效应,DNlg4以非依赖于DNrx的方式调控神经肌肉突触的发育。

七氟烷或丙泊酚复合瑞芬太尼麻醉在重症肌无力患者胸腺切除术中的应用

目的探讨七氟烷或丙泊酚复合瑞芬太尼麻醉在重症肌无力患者经胸骨正中切口胸腺扩大切除术中的应用,并比较其效果。方法搜集2008年7月-2011年6月于解放军309医院麻醉科行胸骨正中切口胸腺扩大切除术的重症肌无力患者156例,分为2组,其中应用靶控静脉输注丙泊酚和瑞芬太尼麻醉(P组)80例,应用吸入七氟烷复合静脉输注瑞芬太尼麻醉患者(S组)76例,均不加用肌松药。采用4个成串刺激(TOF)检测神经肌肉传递功能。以手术开始(切皮)前作为0时点,每30min记录1次各规定时间点的血流动力学变化,以及基础状态下与拔管后的血气分析及呼吸频率等指标,直至麻醉结束,同时记录麻醉时间、手术时间、清醒时间、麻醉恢复室(PACU)停留时间、拔管时间、输入液体及瑞芬太尼总量。结果两组患者在不使用肌松药的情况下均顺利完成手术,S组手术开始后各时间点TOF值均明显低于P组(P<0.05)。手术后,P组pH值和PaCO2较S组显著升高(P<0.05),PaO2则显著降低(P<0.05)。在患者清醒时间、拔管时间、PACU停留时间及瑞芬太尼用量的比较上,S组明显低于P组(P<0.05)。两组患者在血流动力学及脑电双频指数(BIS)、手术时间、麻醉时间、输入液体总量上差异无统计学意义(P>0.05)。结论重症肌无力患者接受胸骨正中切口胸腺扩大切除术时,在不使用肌松药的情况下,应用七氟烷或丙泊酚复合瑞芬太尼麻醉安全有效。七氟烷复合瑞芬太尼对神经肌肉传递功能的抑制更具有优势,对术后呼吸功能的影响更小。

神经—骨骼肌的继发性兴奋——“循经感传”机制研究(Ⅰ)

方法 :用离体的坐骨神经—腓肠肌组织和后肢的坐骨神经及其分支、腓肠肌、趾长伸肌—胫前肌。结果 :刺激坐骨神经引起的腓肠肌纤维兴奋过程中的去极化电位形成的综合电流能刺激穿越该肌的神经 ,引起该神经支配的肌肉继发性兴奋反应。结论 :由于外周神经中还包含有传入的感觉神经 ,即可能引起该神经感受野出现继发性的感觉 。