测量神经肌肉接头功能
Summary
（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。
Abstract
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。
Introduction
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。
Protocol
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 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…
Representative Results
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…
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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)。
Materials
| Dual-Mode Lever System | Aurora Scientific Inc. | 300B | actuator/transducer |
| High-Power Bi-Phase Stimulator | Aurora Scientific Inc. | 701B | pulse stimulator (nerve) |
| High-Power Bi-Phase Stimulator | Aurora Scientific Inc. | 701C | pulse stimulator (muscle) |
| In vitro Muscle Apparatus | Aurora Scientific Inc. | 800A | |
| Preparatory tissue bath | Radnoti | 158400 | |
| Monopolar Suction Electrode | A-M Systems | 573000 | with a home-made reference |
| Oscilloscope | Tektronix | TDS2014 | |
| Stereomicroscope | Nikon | SMZ 800 | |
| Cold light illuminator | Photonic Optics | PL 3000 | |
| Acquisition board | National Instruments | NI PCIe-6353 | |
| Connector block | National Instruments | NI 2110 | |
| Personal computer | AMD Phenom II x4 970 | Processor 3.50 Ghz with Windows 7 | |
| LabView 2012 software | National Instruments | ||
| Krebs-Ringer Bicarbonate Buffer | Sigma-Aldrich | K4002 | physiological buffer |
| Sodium bicarbonate | Sigma-Aldrich | S5761 | |
| Calcium chloride CaCl2 | Sigma-Aldrich | C4901 | anhydrous, powder, ≥97% |
| Potassium dihydrogen phosphate KH₂PO₄ | AnalaR | 7778-77-0 | |
| Magnesium sulphate MgSO₄ | AnalaR | 7487-88-9 | |
| Buffer HEPES | Sigma-Aldrich | H3375 | ≥99.5% (titration) |
| Dishes 60mm x 15mm | Falcon | 353004 | Polystyrene |
| Silicone | Sylgard | 184 Silicone | Elastomer Kit 0.5Kg. |
| Thermostat | Dennerle | DigitalDuomat 1200 | |
| Pump | Newa Mini | MN 606 | for aquarium |
| Heat resistance Thermocable | Lucky Reptile | 61403-1 | 50/60Hz 50W |
| Bucket | any 10 liters | Polypropylene | |
| O2 + 5%CO2 | siad | Mix gas | |
| #5 Forceps | Fine Science Tools | 11252-20 | 2 items |
| Spring Scissors – 8 mm Blades | Fine Science Tools | 15024-10 | nerve excision |
| Sharp Scissors | Fine Science Tools | 14059-11 | muscle removal |
| Delicate Scissors | Wagner | 02.06.32 | external of the animal |
| Student Scalpel Handle #3 | Fine Science Tools | 91003-12 | |
| Scalpel Blades #10 | Fine Science Tools | 10010-00 | |
| Scalpel Blades #11 | Fine Science Tools | 10011-00 | |
| nylon wire Ø0.16 mm | any |
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Tags
Keywords:
Neuromuscular Junction FunctionalityEx Vivo Experimental ApproachMuscle Nerve PreparationMembrane StimulationNerve StimulationContractile ResponsesSoleus Sciatic Nerve PreparationForce And Length ControllerPlatinum ElectrodesGlass Suction ElectrodeTesting ProtocolMuscle FunctionalityPathological ConditionsKrebs-Ringer SolutionGas MixtureActuator TransducerPulse StimulatorsMuscle MembraneNerve StimulationSacrificingCervical DislocationGastrocnemius MuscleSoleus MuscleProximal Tendon
Citar este artigo
Rizzuto, E., Pisu, S., Nicoletti, C., Del Prete, Z., Musarò, A. Measuring Neuromuscular Junction Functionality. J. Vis. Exp. (126), e55227, doi:10.3791/55227 (2017).