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

筆者らの研究室で仕事に支えられたスタンパーリアラヴェッロ ローマ、テレソン (付与なし。GGP14066)。

All the animal experiments were approved by the ethics committee of Sapienza University of Rome-Unit of Histology and Medical Embryology and were performed in accordance with the current version of the Italian Law on the Protection of Animals.
1. Experimental set-up
<ol>
	<li>Set-up the experimental system composed of 1 actuator/transducer, 2 stimulators, 1 in-vitro muscle apparatus, 1 preparatory tissue bath, 1 suction electrode, 1 digital oscilloscope, 1 stereomicroscope, 1 cold l...

<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. 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上記実験のプロトコルは、測定、差別または筋肉に直接に発生した任意の機能変化間接的神経筋接合部のレベルでの理想的な方法を提供します。この手法は NMJ 機能の間接測定に基づいているので、かどうか瑕疵は形態学的変化または生化学的変化に関係を確立する使用できません。対照的に、任意の形態学的または生化学的変化が神経伝達シグナルの機能を減らしたかどうかを決定するの?...

神経筋接合部 (NMJ) は、筋線維の運動終板と運動ニューロンの軸索の端末間の接続によって形成される化学シナプスです。略記は、筋肉と神経との間の通信が高齢化や筋萎縮性側索硬化症 (ALS) が発生すると、障害に重要な役割を再生する示されています。筋肉や神経は、双方向の方法1,2通信、筋肉奇形とは切り離して NMJ 欠陥を測定することができるという、physiopathological の相互作用に新たな洞察があります。確かに、この機能の評価は、形態や生化学的変化を減らす神経伝達機能を伝達するかどうかを評価するために役立つことがあります。
神経刺激と同じ筋に、膜の直接刺激によって誘発される応答による筋収縮反応の比較は NMJ 機能の間接測定として提案されています。確かに、膜刺激パスによって神経伝達シグナル、以来 2 つの収縮応答の違いと思われる略記で変更します。このアプローチをラット3,4,5,6、7、広く提案されており、またマウス モデル8,9,10、11,12に関する情報を収集するために使用します。
ここでは、私たちは消費税し、2 つの筋の準備、すなわちヒラメ坐骨とダイヤフラム隔の準備をテストする手順の詳細に説明します。特注を使用してソフトウェア、考案した NMJ と筋肉の両方の機能を特徴付けるいくつかの測定を組み合わせた連続的なテスト プロトコルそれにより筋肉の別々 に NMJ 損傷の包括的な評価を降伏します。特に、収縮力と筋の動態、直接力周波数曲線, 神経刺激、発砲とテタヌス周波数と intratetanic 疲労7の両方の神経伝達障害13プロトコルを測定します。

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measuring neuromuscular junction functionality

神経筋接合部 (NMJ) 機能は運動ニューロンと筋肉の間の通信障害、加齢、筋萎縮性側索硬化症 (ALS) などの病気を勉強して極めて重要な役割を果たしています。ここで述べる NMJ 機能を電気刺激の 2 つのタイプを組み合わせることで測定するために使用することができます実験的プロトコル: 筋膜刺激と神経を通して刺激を直接します。これらの 2 つの異なる刺激に対する筋反応の比較は、筋肉の機能低下につながる略記で潜在的な変化の機能レベルで定義する助けることができます。
Ex vivo準備はよく統制された研究に適しています。ここで筋肉と NMJ 機能ヒラメ坐骨神経の準備のため、ダイヤフラム横隔神経の準備のためのいくつかのパラメーターを測定するための集中的なプロトコルについて述べる。プロトコルは約 60 分を持続し、カスタムメイドによる間断なく行われている収縮動態特性、筋肉や神経の刺激の力周波数の関係と 2 つパラメーター NMJ 機能に固有すなわち神経伝達障害と intratetanic の疲労を測定するソフトウェア。この方法論は、SOD1G93Aトランスジェニック マウス、普遍的突然変異体の抗酸化酵素スーパーオキシドジスムターゼ 1 (SOD1) を染色 ALS の実験モデルを用いたヒラメ筋と横隔膜筋の準備で損害を検出する使用されました。

我々 が説明されたプロトコルでは、いくつかの神経筋疾患や老化サルコペニア機能神経について説明します。かどうか (その場合は、どのレベルで) は、このプロトコルを使用することができます筋肉の変化がそれ自体筋肉または神経筋伝達で発生する選択の変更によるものです。下図のデータは、我々 グループの18、20疾患の末期...

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

Measuring Neuromuscular Junction Functionality

神経筋接合部 (NMJ) の機能評価は、筋肉と神経の間の通信に不可欠な情報を提供できます。ここで使用して 2 つの異なる筋神経剤、すなわちヒラメ坐骨とダイヤフラム隔 NMJ と筋肉の両方の機能を総合的に評価するためのプロトコルについて述べる。

神経筋接合部機能を測定

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

The overall goal of this procedure is to study neuromuscular junction functionality by means of an ex vivo experimental approach. This is accomplished by stimulating the muscle nerve preparation in two ways, directly on the muscle membrane and through the nerve. Since membrane stimulation bypasses neuro transmission signaling, any differences between the two contractile responses can be considered as an indirect measurement of neuromuscular junction functionality.Here we present this procedure in soleus sciatic nerve preparation. The muscle is dissected together with its nerve and placed into a perfused tissue bath. It is fixed to a force and length controller and a platinum pair of electrodes are placed parallel to the muscle.A glass suction electrode is then moved close to the cut end of the nerve. A comprehensive testing protocol is then applied to thoroughly evaluate both neuromuscular junction and muscle functionality. The functional connection between muscle and nerve is conclusion for both part and two surviving faction.In the first region of the level of each of the two tissue communicate that these neuromuscular junction, which normally displays a pressure like morphology. Nevertheless in several pathological conditions the functionality interplayed between muscle and nerve is severely compromised and neuromuscular junction lose the complex morphologic organization. The overall goal of our procedure is to study neuromuscular junction functionality by using and ex vivo experimental approach.This is accomplished by stimulating the muscle nerve preparation in two ways. One by directly stimulating the muscle membrane and the other by stimulating the nerve and analyze the muscle properties. Turn on the circulation water bath and adjust the temperature to 30 degrees celsius.Fill the bath with the Krebs-Ringer solution. Allow O point four bar of gas mixture to flow through the oxitube and into the bath. Turn on the actuator transducer and the two pulse stimulators.Set the current values to 300 million pair for the membrane stimulation and to five million pair for the nerve stimulation. After sacrificing the mouse by cervical dislocation, remove the skin from the legs. Now cut the achilles tendon and while tightly clamping the tendon, pull the gastrocnemius muscle and the soleus together upwards.Once the proximal tendon of the soleus is exposed cut the entire calf above it and quickly place the sample in the preparatory tissue bath located under the stereo microscope. Using a pair of forceps, tightly clamp the proximal tendon of the soleus and gently pull it to expose the sciatic innervation. Once the innervation is exposed remove the surrounding tissues to reveal about five millimeters of the nerve.Then use a fine pair of scissors to carefully cut the nerve. Complete the muscle, nerve excision by cutting the achilles tendon to separate the soleus from the gastrocnemius. Now the muscle, nerve preparation is ready to be mounted onto the testing apparatus.Create a slip knot at the end of the nylon thread and tighten it around the achilles tendon. Clamp the proximal tendon within the fixed clamp and tie the nylon wire around the lever arm of the force transducer. Allow the muscle to equilibrate in the solution.To determine the initial optimal length, stimulate the muscle with a series of single pulses while gently changing the creep load value. The optimal length is obtained when the twitch force is maximum. Place the suction electrodes near the muscle and pull the nerve in.Then, while gently altering the pulse current value, stimulate the muscle with a series of single pulses. The twitch force generated by the muscle when stimulated through the nerve should be equal to the values measured when stimulating it on the membrane. Once the optimal current value is determined, push the nerve out of the electrode and deliver a few current pulses.If the amount of previously selected current is excessive, the current pulses delivered through the suction electrode elicit the muscle contraction by conducting current through the bath. Using a home-made software we devised an automated testing protocol for the study of soleus neuromuscular junction functionality. The protocol lasts about 65 minutes and is made up of four different parts.In the first part, the muscle is stimulated with four single pulses. Two delivered directly and two through the nerve. Time to peak, half relaxation time, maximum value of force derivative and twitch force are then measured from the twitch responses.In the second part, the muscle is stimulated with a series of pulse trains ranging from 20 hertz to 80 hertz, which is the tetanic frequency. To compute the force, frequency curves for both nerve and direct stimulations. In the third and fourth parts of the protocol the muscle is subjected to two fatigue paradigms to measure neuro transmission failure and intra tetanic fatigue.During these fatigue paradigms, the muscle is continuously stimulated with one pulse train delivered on the membrane followed by 14 pulse trains delivered through the nerve. The entire sequence is repeated 20 times. The first paradigm is delivered at a firing frequency of 35 hertz.The second at the tetanic frequency of 80 hertz. Neuro transmission failure is believed to play an important role in the development of fatigue as it is related to the external block of action potential propagation, decreased transmitter release and decreased and plate excitability of junction fatigue ability. Another aspect of neuromuscular junction fatigue ability is clearly expressed by intratetanic fatigue which is an estimate of the muscle's ability to maintain force during a single tetanic contraction and reflects high frequency fatigue.At the end of the protocol, net muscle length and weight are measured using an analog caliper and a precision scale to compute the muscle cross sectional area. Studies on the SOD1 transgenic mouse model of amyotrophic lateral sclerosis have highlighted the potential of this methodology. In fact, the transgenic soleus muscles yield a reduced contractile response for both the force derivative and tetanic force when directly stimulated and an even greater reduction when stimulated through the nerve.As regards to tetanic force for example, these experiments have shown that muscle contractility accounts for 25%of the damage while a further 45%is related to defects in neuro transmission. Another interesting point is the absence of any difference in control muscles when stimulated directly or indirectly. This finding proves that the methodology does not induce any technical artifacts since the neuromuscular junction is expected to be fully functional in control animals.As regards intratetanic fatigue, the results have shown significantly lower values in transgenic soleus muscles than in their control counterparts. Interestingly, transgenic soleus muscle is significantly damaged by repetitive stimulation, which means that neuromuscular junction functionality can be evaluated for a maximum stimulation time of eight minutes. After eight minutes, the transgenic muscle returns to an almost null value of force when stimulated.After watching the video you should have got understanding how to measure neuromuscular junction functionality and soleus muscle of mouse. Given that this technique based on indirect measurement of neuromuscular junction functionality, it does not allow to be made where reported defects are related to morphologic or biochemical changes. On the other hand, this approach represents an essential way to assess whether these aggressions affect at user functionality of the neuro transmission signal.Finally, the protocol proposal can be easily adopted to measure neuromuscular junction functionality of the diaphragm, another muscle often involved in pathological diseases.

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Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

で、アクティブなゾーンの解剖とイメージングショウジョウバエ神経筋接合部

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

神経科学

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

シーケンシャル神経筋接合部におけるシングルシュワン細胞の輪郭を描くのにフォトブリーチング

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

全マウント神経筋接合解析のための トランス対アブドミン筋 の解剖

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

の検出<em&gt;その場で</emで&gt;タンパク質 - タンパク質複合体<em&gt;ショウジョウバエ</em近接ライゲーションアッセイの使用&gt;幼虫神経筋接合部

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

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

神経筋接合部：測定シナプスサイズ、断片化および共焦点蛍光顕微鏡を用いてシナプスタンパク質の密度の変化

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

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

経皮的電気神経刺激を使用した神経筋機能の評価

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

なぜ定量事項：での表現型のキャラクタリゼーション<em&gt;ショウジョウバエ</em&gt;幼虫の神経筋接合部

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

カエルの神経筋終末へのカルシウム色素の神経切片への積載：カエル神経筋接合部におけるカルシウムの一時的な位置合わせ

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

高スループットとのマルチパラメトリック定量化のための2個のアルゴリズム<em&gt;ショウジョウバエ</em&gt;神経筋接合部形態

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

ショウジョウバエ幼虫神経筋接合部のシナプス電流の焦点 Macropatch 録音

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...