Name: measuring neuromuscular junction functionality
Uploaded: 2017-08-06T13:00:00.000+00:00
Duration: 10 min 40 s
Description: Watch this Scientific Journal Video about Measuring Neuromuscular Junction Functionality at JoVE.com

在作者的实验室的工作被支持基金会罗姆人和节目 （给予。GGP14066)。

<ol>
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E., Sawyer, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+and+failure+of+neurotransmission+in+the+diaphragm+of+mdx+mice.">Variability and failure of neurotransmission in the diaphragm of mdx mice.</a> Neuromuscular Disord. 16 (3), 168-177 (2006).</li><li>R&#246;der, I. V., Petersen, Y., Choi, K. R., Witzemann, V., Hammer, J. A., Rudolf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+myosin+Va+in+the+plasticity+of+the+vertebrate+neuromuscular+junction+in+vivo.">Role of myosin Va in the plasticity of the vertebrate neuromuscular junction in vivo.</a> PLoS ONE. 3 (12), e3871 (2008).</li><li>Chevessier, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+congenital+myasthenic+syndrome+due+to+MuSK+mutations+reveals+defects+in+structure+and+function+of+neuromuscular+junctions.">A mouse model for congenital myasthenic syndrome due to MuSK mutations reveals defects in structure and function of neuromuscular junctions.</a> Hum Mol Genet. 17 (22), 3577-3595 (2008).</li><li>Farchi, N., Soreq, H., Hochner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+acetylcholinesterase+overexpression+induces+multilevelled+aberrations+in+mouse+neuromuscular+physiology.">Chronic acetylcholinesterase overexpression induces multilevelled aberrations in mouse neuromuscular physiology.</a> J Physiol. 546 (1), 165-173 (2002).</li><li>Kuei, J. H., Shadmehr, R., Sieck, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+contribution+of+neurotransmission+failure+to+diaphragm+fatigue.">Relative contribution of neurotransmission failure to diaphragm fatigue.</a> J Appl Physiol (Bethesda, Md: 1985). 68 (1), 174-180 (1990).</li><li>Del Prete, Z., Musar&#242;, A., Rizzuto, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mechanical+properties,+including+isotonic+fatigue,+of+fast+and+slow+MLC/mIgf-1+transgenic+skeletal+muscle.">Measuring mechanical properties, including isotonic fatigue, of fast and slow MLC/mIgf-1 transgenic skeletal muscle.</a> Ann Biomed Eng. 36 (7), 1281-1290 (2008).</li><li>Lynch, G. S., Hinkle, R. T., Chamberlain, J. S., Brooks, S. V., Faulkner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+power+output+of+fast+and+slow+skeletal+muscles+from+mdx+mice+6-28+months+old.">Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old.</a> J Physiol. 535 (2), 591-600 (2001).</li><li>Brooks, S. V., Faulkner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+properties+of+skeletal+muscles+from+young,+adult+and+aged+mice.">Contractile properties of skeletal muscles from young, adult and aged mice.</a> J Physiol. 404, 71-82 (1988).</li><li>Lynch, G. S., Hinkle, R. T., Faulkner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+power+output+of+diaphragm+muscle+strips+from+mdx+and+control+mice+after+clenbuterol+treatment.">Force and power output of diaphragm muscle strips from mdx and control mice after clenbuterol treatment.</a> Neuromuscul Disord. 11 (2), 192-196 (2001).</li><li>Rizzuto, E., Pisu, S., Musar, A., Del Prete, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Neuromuscular+Junction+Functionality+in+the+SOD1+G93A+Animal+Model+of+Amyotrophic+Lateral+Sclerosis.">Measuring Neuromuscular Junction Functionality in the SOD1 G93A Animal Model of Amyotrophic Lateral Sclerosis.</a> Ann Biomed Eng . 43, (2015).</li><li>Soliani, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manuale di statistica per la ricerca e la professione statistica univariata e bivariata parametrica e non-parametrica per le discipline ambientali e biologiche. , (2004).</li><li>Gurney, M. E., Pu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+neuron+degeneration+in+mice+that+express+a+human+Cu,Zn+superoxide+dismutase+mutation.">Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.</a> Science (New York, N.Y). 264 (5166), 1772-1775 (1994).</li></ol>

作者没有透露。

上述实验协议提供一个理想的测量和识别任何功能的改变发生在肌肉中直接或间接在神经肌肉接头处一级途径。由于这项技术基于间接测量的诸多功能，它不能用于建立与形态学变化或与生化指标的变化，是否有关任何缺陷。与此相反的是，它提供确定是否任何形态或生化改建有减少神经递质信号功能的有效途径。然而，力测量完成后，肌肉可以从浴、 涂抹、 在最佳长度固定，，包围嵌入化合?...

神经肌肉接头 （诸多） 是由肌肉纤维运动终板和运动神经元轴突末端之间的连接形成化学突触。诸多已被证明至关重要的作用，当肌肉和神经之间的通信是受损，发生在老化或萎缩侧索硬化 (ALS)。肌肉和神经沟通双向方式1，2，能够衡量诸多缺陷分别从肌肉缺损可提供其病理相互作用的新见解。事实上，这个功能的评价可能有助于评估是否形态或生化改建减少神经传递信号的功能。
作为间接测量的诸多功能，提出了肌肉收缩反应诱发神经刺激和诱发其膜的直接刺激同一块肌肉的反应的比较。事实上，自膜刺激通过传递神经传递信号，任何两个收缩反应的差异可归因于诸多的变化。这种方法已经被广泛提出大鼠3，4，，56，7，并也用于在鼠标模型8，，910，，1112上收集信息。
在这里，我们描述详细的消费税和测试两种肌肉神经制剂，即的比目鱼坐骨神经和膈肌膈筹备工作的过程。使用定制软件，我们设计了一个连续的测试协议，结合测量的几个重要参数表征诸多和肌肉的功能，从而产生诸多损害综合评价分别从那肌肉。特别是，议定书 》 的措施抽搐力、 肌肉动力学、 直接力频率曲线和神经刺激，神经递质失败13射击和破伤风的频率，和 intratetanic 疲劳7。

<table><tbody><tr><td>Dual-Mode Lever System&amp;nbsp;</td><td>Aurora Scientific Inc.</td><td>300B</td><td>actuator/transducer</td></tr><tr><td>High-Power Bi-Phase Stimulator&amp;nbsp;</td><td>Aurora Scientific Inc.</td><td>701B</td><td>pulse stimulator (nerve)</td></tr><tr><td>High-Power Bi-Phase Stimulator&amp;nbsp;</td><td>Aurora Scientific Inc.</td><td>701C</td><td>pulse stimulator (muscle)</td></tr><tr><td>In vitro Muscle Apparatus&amp;nbsp;</td><td>Aurora Scientific Inc.</td><td>800A</td><td></td></tr><tr><td>Preparatory tissue bath</td><td>Radnoti</td><td>158400</td><td></td></tr><tr><td>Monopolar Suction Electrode</td><td>A-M Systems</td><td>573000</td><td>with a home-made reference&amp;nbsp;</td></tr><tr><td>Oscilloscope&amp;nbsp;</td><td>Tektronix</td><td>TDS2014</td><td></td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ 800</td><td></td></tr><tr><td>Cold light illuminator&amp;nbsp;</td><td>Photonic Optics</td><td>PL 3000</td><td></td></tr><tr><td>Acquisition board</td><td>National Instruments</td><td>NI PCIe-6353</td><td></td></tr><tr><td>Connector block</td><td>National Instruments</td><td>NI 2110</td><td></td></tr><tr><td>Personal computer</td><td>AMD Phenom II x4 970</td><td></td><td>Processor 3.50 Ghz with Windows 7</td></tr><tr><td>LabView 2012 software</td><td>National Instruments</td><td></td><td></td></tr><tr><td>Krebs-Ringer Bicarbonate Buffer&amp;nbsp;</td><td>Sigma-Aldrich</td><td>K4002&amp;nbsp;</td><td>physiological buffer</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761&amp;nbsp;</td><td></td></tr><tr><td>Calcium chloride CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C4901</td><td>anhydrous, powder, &amp;ge;97%</td></tr><tr><td>Buffer HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>&amp;ge;99.5% (titration)</td></tr><tr><td>Dishes 60mm x 15mm</td><td>Falcon</td><td>353004</td><td>Polystyrene</td></tr><tr><td>Silicone</td><td>Sylgard&amp;nbsp;</td><td>184 Silicone&amp;nbsp;</td><td>Elastomer Kit&amp;nbsp; 0.5Kg.</td></tr><tr><td>Thermostat</td><td>Dennerle</td><td>DigitalDuomat 1200</td><td></td></tr><tr><td>Pump</td><td>Newa Mini</td><td>MN 606</td><td>for aquarium</td></tr><tr><td>Heat resistance Thermocable</td><td>Lucky Reptile</td><td>61403-1</td><td>50/60Hz 50W</td></tr><tr><td>Bucket</td><td>any 10 liters</td><td></td><td>Polypropylene</td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt; + 5%CO2</td><td>siad</td><td></td><td>Mix gas</td></tr><tr><td>#5 Forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>11252-20</td><td>2 items</td></tr><tr><td>Spring Scissors - 8 mm Blades</td><td>Fine Science Tools</td><td>15024-10</td><td>nerve excision</td></tr><tr><td>Sharp Scissors&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>14059-11</td><td>muscle removal</td></tr><tr><td>Delicate Scissors</td><td>Wagner</td><td>02.06.32</td><td>external of the animal</td></tr><tr><td>Student Scalpel Handle #3</td><td>Fine Science Tools&amp;nbsp;</td><td>91003-12&amp;nbsp;</td><td></td></tr><tr><td>Scalpel Blades #10</td><td>Fine Science Tools&amp;nbsp;</td><td>10010-00</td><td></td></tr><tr><td>Scalpel Blades #11</td><td>Fine Science Tools&amp;nbsp;</td><td>10011-00</td><td></td></tr><tr><td>nylon wire &amp;Oslash;0.16 mm</td><td>any</td><td></td><td></td></tr></tbody></table>

measuring neuromuscular junction functionality

学习运动神经元和肌肉之间的通信受损，老化和萎缩侧索硬化 (ALS) 等疾病时，神经肌肉接头 （诸多） 功能发挥了关键作用。在这里，我们描述了一个实验性的协议，可以用于衡量诸多功能相结合的电刺激的两种类型: 直接肌肉膜刺激和通过神经刺激。对这些两不同刺激的肌肉反应的比较可以帮助定义，一级功能，在诸多导致肌肉功能下降的潜在变化。
Ex vivo筹备工作适合于严谨的研究。在这里，我们描述密集的协议来衡量的肌肉和诸多功能为比目鱼坐骨神经制备和膈肌膈神经编写几个参数。议定书 》 持续约 60 分钟，进行不间断地定制抽搐动力学性质、 力频率关系的肌肉和神经的刺激和两个参数特定于诸多功能，即神经递质失败和 intratetanic 疲劳的措施的软件。这种方法用于检测损害比目鱼肌和膈膜肌肉神经制剂中通过使用 SOD1G93A转基因小鼠，ALS 无所不在 overexpresses 突变体的抗氧化剂酶超氧化物歧化酶 1 (SOD1) 实验模型。

我们描述了议定书 》 提供了几种神经肌肉疾病或衰老骨骼肌功能失神经支配有关的信息。这个协议可以用于确定是否 （以及，如果是这样，在哪个层面） 肌肉变化是发生在本身的肌肉或神经肌肉传递的选择性变化。如下图所示的数据是工作的以前由我们组18，肌萎缩侧索硬化疾病20结束阶段 SOD1G93A转基因小鼠模型上进行的结果...

Watch this Scientific Journal Video about Measuring Neuromuscular Junction Functionality at JoVE.com

Measuring Neuromuscular Junction Functionality

（诸多） 神经肌肉接头功能评估可以提供有关肌肉和神经之间的通信的基本信息。在这里，我们描述一项议定书，全面评估的诸多和肌肉功能使用两种不同的肌肉神经制剂，即比目鱼坐骨神经和膈肌膈。

测量神经肌肉接头功能

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

measuring-neuromuscular-junction-functionality

Research

Education

JoVE Journal

JoVE Core

Anatomy and Physiology

Pharmacology

Neuroscience

Unit 1

Muscle Tissue

Skeletal Muscle Relaxants

SCIENTISTS IN ACTION

17.9K 次观看.  Sapienza University of Rome. （诸多） 神经肌肉接头功能评估可以提供有关肌肉和神经之间的通信的基本信息。在这里，我们描述一项议定书，全面评估的诸多和肌肉功能使用两种不同的肌肉神经制剂，即比目鱼坐骨神经和膈肌膈。

视频: Measuring Neuromuscular Junction Functionality

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.

从人类诱导多能干细胞诱导的体外神经肌肉结点

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

科研

发育生物学

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

神经肌肉接头处:测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

神经科学

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 (&#964;m) 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.

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

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

提耳伸肌压钳条件下哺乳动物神经肌肉传输的检查准备

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform&#39;s ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.

人类神经肌肉接头光遗传学模型的工程和表征

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

生物工程

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

单骨骼肌肉纤维解剖，用于全山神经肌肉结的免疫荧光和形态分析

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker &#945;-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.

We describe a method to generate human motor units in commercially available microfluidic devices by co-culturing human induced pluripotent stem cell-derived motor neurons with human primary mesoangioblast-derived myotubes resulting in the formation of functionally active neuromuscular junctions.

在微流体装置中生成具有功能性神经肌肉接头的人类运动单元

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle ...

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of three essential histological components: the pre-synaptic motor axon terminals, post-synaptic nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs). In order to demonstrate the morphological characteristics of NMJ, the rat medial gastrocnemius muscle was selected as the target-tissue and examined by using multiple fluorescent staining with various kinds of biomarkers, including neurofilament 200 (NF200) and vesicular acetylcholine transporter (VAChT) for the motor nerve fibers and their pre-synaptic terminals, alpha-bungarotoxin (&#945;-BTX) for the post-synaptic nicotinic AchRs, and S100 for the PSCs. In this study, staining was performed in two groups: in the first group, samples were stained with NF200, VAChT, and &#945;-BTX, and in the second group, samples were stained with NF200, &#945;-BTX, and S100. It was shown that both protocols can effectively demonstrate the detailed structure of NMJ. Using the confocal microscope, morphological characteristics of the pre-synaptic terminals, post-synaptic receptors, and PSC were seen, and their Z-stacks images were reconstructed in a three-dimensional pattern to further analyze the spatial correlation among the different labeling. From the perspective of methodology, these protocols provide a valuable reference for investigating the morphological characteristics of NMJ under physiological conditions, which may also be suitable to evaluate the pathological alteration of NMJ, such as peripheral nerve injury and regeneration.

The protocol shows a method to examine spatial correlation among the pre-synaptic terminals, post-synaptic receptors, and peri-synaptic Schwann cells in the rat medial gastrocnemius muscle using fluorescent immunohistochemistry with different biomarkers, namely, neurofilament 200, vesicular acetylcholine transporter, alpha-bungarotoxin, and S100.

可视化大鼠内侧腓肠肌神经肌肉接头的形态学特征

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of ...

<meta charset="utf8"></head><body>啮齿动物的 SFEMG:评估 NMJ 在健康和疾病中的功能

Stimulated Single Fiber Electromyography (SFEMG) for Assessing Neuromuscular Junction Transmission in Rodent Models

As the final connection between the nervous system and muscle, transmission at the neuromuscular junction (NMJ) is crucial for normal motor function. Single fiber electromyography (SFEMG) is a clinically relevant and sensitive technique that measures single muscle fiber action potential responses during voluntary contractions or nerve stimulations to assess NMJ transmission. The assessment and quantification of NMJ transmission involves two parameters: jitter and blocking. Jitter refers to the variability in timing (latency) between consecutive single-fiber action potentials (SFAPs). Blocking signifies the failure of NMJ transmission to initiate an SFAP response. Although SFEMG is a well-established and sensitive test in clinical settings, its application in preclinical research has been relatively infrequent. This report outlines the steps and criteria employed in performing stimulated SFEMG to quantify jitter and blocking in rodent models. This technique can be used in preclinical and clinical studies to gain insights into NMJ function in the context of health, aging, and disease.

<meta charset="utf8"></head><body>作者聚焦:刺激 SFEMG 在啮齿动物模型中的转化应用

In this study, we demonstrate a refined single fiber electromyography (SFEMG) protocol to allow in vivo measurement of neuromuscular junction (NMJ) transmission in rodent models. A step-by-step approach to the SFEMG technique is described to allow quantification of NMJ transmission variability and failure in rat gastrocnemius muscle.

刺激单纤维肌电图 （SFEMG） 用于评估啮齿动物模型中神经肌肉接头的传递

As the final connection between the nervous system and muscle, transmission at the neuromuscular junction (NMJ) is crucial for normal motor function. ...

测量神经肌肉接头功能

摘要

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此视频中的章节

从人类诱导多能干细胞诱导的体外神经肌肉结点

神经肌肉接头处:测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

提耳伸肌压钳条件下哺乳动物神经肌肉传输的检查准备

人类神经肌肉接头光遗传学模型的工程和表征

单骨骼肌肉纤维解剖，用于全山神经肌肉结的免疫荧光和形态分析

在微流体装置中生成具有功能性神经肌肉接头的人类运动单元

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

可视化大鼠内侧腓肠肌神经肌肉接头的形态学特征

刺激单纤维肌电图（SFEMG）用于评估啮齿动物模型中神经肌肉接头的传递

刺激单纤维肌电图（SFEMG）用于评估啮齿动物模型中神经肌肉接头的传递

测量神经肌肉接头功能

摘要

探索更多视频

此视频中的章节

从人类诱导多能干细胞诱导的体外神经肌肉结点

神经肌肉接头处:测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

提耳伸肌压钳条件下哺乳动物神经肌肉传输的检查准备

人类神经肌肉接头光遗传学模型的工程和表征

单骨骼肌肉纤维解剖，用于全山神经肌肉结的免疫荧光和形态分析

在微流体装置中生成具有功能性神经肌肉接头的人类运动单元

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

可视化大鼠内侧腓肠肌神经肌肉接头的形态学特征

刺激单纤维肌电图 （SFEMG） 用于评估啮齿动物模型中神经肌肉接头的传递

刺激单纤维肌电图 （SFEMG） 用于评估啮齿动物模型中神经肌肉接头的传递

刺激单纤维肌电图（SFEMG）用于评估啮齿动物模型中神经肌肉接头的传递

刺激单纤维肌电图（SFEMG）用于评估啮齿动物模型中神经肌肉接头的传递