提耳伸肌压钳条件下哺乳动物神经肌肉传输的检查准备
Summary
本文所描述的协议使用鼠标提耳肌 (拉尔) 肌肉记录自发性和神经诱发突触后电位 (电流钳) 和电流 (电压钳) 在神经肌肉连接。使用该技术可以为正常和疾病条件下突触传递机制提供关键的见解。
Abstract
本协议描述了一种在电流钳和电压钳条件下记录神经肌肉结突触传递的技术。使用肛提耳肌 (拉尔) 的前体制备, 是因为它是一种薄肌肉, 可在马达终板上为微电极掷镖师掷提供易于可视化的神经肌肉连接。该方法允许记录自发微型终板电位和电流 (mEPPs 和 mEPCs), 神经诱发终板电位和电流 (无害性和 EPCs), 以及电机终板的膜性能。从该方法得到的结果包括量子含量 (QC), 囊泡释放部位的数量 (n), 囊泡释放的可能性 (p相对), 突触促进和抑郁症, 以及肌肉膜时间常数 (τm) 和输入电阻。这种技术应用于人类疾病的小鼠模型可以突出疾病状态中的关键病理, 并有助于确定新的治疗策略。通过完全压紧单个突触, 该方法提供了目前可用的突触传输的最详细的分析之一。
Introduction
研究神经肌肉连接处的突触传递, 可以洞察神经和骨骼肌肉系统之间的动态关系, 是检查突触生理学的绝佳模型。提肛耳肌 (拉尔) 是一种薄肌肉, 允许神经肌肉连接容易形象化。以前的报告描述了使用拉尔检查突触药物和毒素的便利性, 并描绘了拉尔12的骨骼肌纤维类型特征。许多研究使用了拉尔检查神经肌肉生理学3,4,5,6,7,8。对于电生理, 能够轻松地观察拉尔神经肌肉连接允许准确地放置电极在电机终板, 并大大减少空间钳位问题记录突触传输。现有的肌肉膜性能的钳位记录, 如膜时间常数 (τm) 和输入电阻 (R in) 是容易获得的.此外, 这些特性可以用用于记录神经肌肉传输的相同肌肉纤维来测量, 从而直接将突触功能与肌肉膜的特性进行比较。对这些数据的分析可以为许多神经肌肉疾病的物理机制和改变活动状态提供关键的洞察力。
这里描述的技术的一个关键方面是使用电压钳的突触录音, 这是不受非非线性遇到的电流钳和独立的肌肉膜的性质。使用电压钳, 而不是电流钳检查神经肌肉传输的优点是通过二十世纪五十年代9的开创性努力建立的。在电流钳下, 在振幅超过 10-15 mV 的无害产品不是 mEPP 振幅9的线性乘积。例如, 如果平均 mEPP 是 1 mv, 5 mv 的 EPP 可以假设是 5 mEPPs 的产品 (QC 5);然而, 40 mV 的 EPP 将是超过 40 mEPPs 的产品。这种非线性在更大的无害性发生, 因为 EPP 的驱动力, 这是不同的膜电位和平衡电位的乙酰胆碱受体 (10 mV), 大大减少在大型无害性。在电压钳实验中, 由于肌肉膜电位没有改变, 所以在压夹试验中避免了这个问题。缺点是, 在技术上比电流钳记录更难完成电压钳的实验。考虑到这一点, McLachlan 和马丁开发了一个简单的数学校正, 它在非线性10的当前钳型录音中占了不可用的。更正工作良好11,12,13, 但重要的是, 假设肌肉膜的性能没有中断。
肌肉膜的性质是特别重要的考虑, 如果研究条件或疾病状态, 扰乱肌肉。例如, R6/2 基因模型的骨骼肌从亨廷顿氏病是 hyperexcitable 由于逐步减少休息氯和钾电流14,15。结果, mEPPs 和无害的 R6/2 骨骼肌肉被放大。当然, 其他因素可以改变 mEPPs 和无害环境。与亨廷顿病小鼠 (R6/1) 的不同模型工作发现了无害性的变化, 似乎与诱捕蛋白8有关。为了评估导致神经肌肉传导改变的机制, 用电压钳消除改变肌肉膜性能的效果将是有益的。在最近的一项研究中, R6/2 神经肌肉传输的研究在电流和电压钳条件下使用的技术描述。通过将两个电极放在板板16的长度常数内, 电机终板的整体被压紧, 误差小于1%。结果表明, 电压钳和校正的电流钳记录产生了 R6/2 肌神经肌肉传递的对比测量。这突出表明, 如果肌肉膜的性能已经改变, 并显示获得独立于肌肉膜性能的电压钳记录的好处, 则可能很难纠正非非线性的无害化。本文提出的协议是检查影响突触传递和突触后膜性能的条件或疾病状态的理想选择。
Protocol
Representative Results
Discussion
这里描述的是制备和使用的小鼠拉尔肌肉测量神经肌肉传输在电流或电压钳条件下。解剖拉尔有几个重要的要点要考虑。在电极掷镖师掷中清除肌肉辅助组织中多余的结缔组织, 因为电极在定位掷镖师掷时可能会感染结缔组织。然而, 只有去除结缔组织, 可以很容易地被带走, 以限制伤害的机会, 肌肉。神经的隔离应该小心地进行, 因为它非常微妙。为了避免神经损伤, 将一些周围的组织连接到神经?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢马克 m. 富先生和丹尼尔. 米兰达的社论评论, 艾哈迈德. Khedraki 帮助建立了这项技术, 莱特州立大学提供财政支持 (启动基金到 A.A.V.)。
Materials
| Olympus Compound Microscope | Olympus | BX51WI | |
| 10x Objective | Olympus | UMPLFLN10XW | |
| 40x Objective | Olympus | LUMPLFLN40XW | |
| Borosilicate Glass | Sutter Instruments | BF150-86-7.5 | |
| CCD Camera | Santa Barbara Instruments Group | ST-7XMEI | |
| Axoclamp 900A Amplifier | Molecular Devices | 2500‐0179 | |
| Mater-9 Pulse Generator | AMPI | ||
| Iso-flex Stimulus Isolator | AMPI | ||
| pCLAMP 10 Data Acquisition and Analysis Software | Molecular Devices | 1-2500-0180 | |
| Concentric Bipolar Electrode | FHC | CBDSH75 | |
| Ball-joint Manipulator | Narishige | ||
| Non-metalic Syringes 34 Gauge | World Precision Instruments | MF34G-5 | |
| Nikon Stereomicroscope | Nikon | SMZ800N | |
| No. 5 Forceps | Fine Science Tools | ||
| Spring Scissors | Fine Science Tools | 15006-09 | |
| No. 2 Forceps | Roboz | RS-5Q41 | |
| Microdissecting Scissors | Roboz | RS-5912SC | |
| Sylgard 184 Silicone Elastomer Kit | Dow Corning | 2404019862 | |
| Hair Removal Cream | Nair | ||
| Grass SD9 Stimulator | Grass Medical | ||
| Model P-1000 Micropipette Puller | Sutter Instruments | P-1000 | |
| Axon Digidata 1550 Low-noise Data Acuisition System | Molecular Devices | ||
| Low Pass Bessell Filter | Warner Instrument Corp. | LPF-8 | |
| Left-handed Micromanipulator | Siskiyou Corp. | MX1641/45DL | |
| Right-handed Micromanipulator | Siskiyou Corp. | MX1641/45DR | |
| Single Motion Controler | Siskiyou Corp. | MC100e | |
| Crossed Roller Micromanipulator | Siskiyou Corp. | MX1641R | This was added to the Z-axis of the Left and Right-handed micromanipulators to allow the z axis to be motorized. This custom set-up is cheaper and less bulky than buying a 4-axis motorized micromanipulator. It also allows us to control both micromanipulators with one controller |
| All chemicals were orded from Fisher except, | |||
| BTS | Toronto Research Chemicals | B315190 | |
| CTX | Alomone Labs | C-270 | |
| 4-Di-2-Asp | Molecular Probes | Molecular probes is no longer a company. Now ordered through Fisher | |
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