使用共聚焦显微镜以高时间分辨率记录小鼠神经肌肉接头中的钙瞬变
1Laboratory of Biophysics of Synaptic Processes,Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, 2Department of Radiophotonics and Microwave Technologies,Kazan National Research Technical University named after A.N. Tupolev, 3Department of Normal Physiology,Kazan State Medical University
Summary
该方案描述了通过切割神经将荧光钙染料加载到小鼠运动神经末梢的方法。此外，还提出了一种使用共聚焦显微镜记录周围神经末梢快速钙瞬变的独特方法。
Abstract
突触前钙水平的估计是研究突触传递的关键任务，因为钙进入突触前细胞会触发导致神经递质释放的级联事件。此外，突触前钙水平的变化介导了许多细胞内蛋白的活性，并在突触可塑性中起重要作用。研究钙信号传导对于寻找治疗神经退行性疾病的方法也很重要。神经肌肉接头是研究突触可塑性的合适模型，因为它只有一种类型的神经递质。本文描述了通过切割的神经束将钙敏感染料加载到小鼠运动神经末梢的方法。该方法允许估计与细胞内钙变化相关的所有参数，例如基础钙水平和钙瞬变。由于钙从细胞外部流入神经末梢及其与钙敏感染料的结合/解除结合发生在几毫秒的范围内，因此需要一个快速的成像系统来记录这些事件。事实上，高速相机通常用于记录快速钙变化，但它们的图像分辨率参数较低。这里介绍的用于记录钙瞬态的方案允许共聚焦显微镜提供极好的时空分辨率。
Introduction
在可兴奋细胞中测量快速钙波的问题是研究中枢和周围神经系统信号传输的最重要和最具挑战性的方面之一。钙离子在触发神经递质释放、突触可塑性和调节各种细胞内蛋白的活性方面起着重要作用1，2，3，4，5。研究钙信号传导对于寻找治疗神经退行性疾病的方法也很重要6。为了测量钙水平的变化，通常使用荧光钙敏感染料，并分析其荧光水平的变化7，8，9。
钙染料加载到细胞中可以通过不同的方式实现。主要使用细胞渗透性染料10，11。然而，在这种情况下，不仅难以控制细胞内染料的浓度，而且也很难选择目标细胞进行上样。该方法不适用于研究周围神经末梢，因为染料进入突触后细胞。相反，细胞不透透的染料更适合这种制剂。在这种情况下，染料通过显微注射或通过贴片移液管12，13，14递送到细胞。还有一种通过神经残端加载的方法。后一种方法最适合于神经肌肉接头制剂15，16，17，18，19，20。它允许仅对感兴趣的细胞进行染色。尽管该方法不能准确评估靶细胞中染料的浓度，但可以通过比较溶液中静止细胞的荧光水平与已知浓度的钙21来近似估计浓度。在这项研究中，提出了应用于哺乳动物突触的该方法的修改。
在动作电位的去极化阶段钙进入是一个快速的过程，特别是在神经肌肉接头;因此，对于其注册，需要适当的设备1.最近使用电压敏感荧光染料的一项研究表明，小鼠外周突触中动作电位的持续时间约为300μs22。在青蛙的外围突触中使用钙敏感染料评估钙瞬变的持续时间更长：上升时间约为2-6毫秒，衰减时间约为30-90毫秒，具体取决于使用的钙染料23，24。为了在荧光染料的帮助下测量快速过程，通常使用CCD或CMOS相机，以及快速灵敏的CCD矩阵。然而，这些相机具有低分辨率的缺点，受限于矩阵25、26、27、28的敏感元件的大小。最快的相机具有足够的灵敏度来记录响应细胞低频刺激的动作电位和钙瞬变，扫描频率为2，000 Hz，矩阵尺寸为80 x 8029。为了获得具有更高空间分辨率的信号，使用共聚焦显微镜，特别是当需要评估信号中的一些体积变化时30，31，32。但应该记住，共聚焦显微镜在线扫描模式下具有很高的扫描速度，但在构建空间图像时，记录快速过程的速度仍然存在显着限制33。有基于旋转Nipkow盘（狭缝扫描显微镜）的共聚焦显微镜和具有更高扫描速度的多点阵列扫描仪。同时，它们在共聚焦图像滤波（带有Nipkow盘的显微镜的针孔串扰）方面不如经典共聚焦显微镜32，34，35。具有共振扫描的共聚焦成像还可以提供高时间测量所需的高时空分辨率36。然而，考虑到当使用共振扫描仪时，以高扫描速度配准弱荧光响应需要高灵敏度的检测器，例如混合检测器36。
本文提出了一种提高激光扫描共聚焦显微镜（LSCM）记录的信号的时间分辨率，同时保持空间分辨率37的方法。目前的方法是前面描述的方法的进一步发展，并转移到LSCM平台38，39，40。这种方法不需要改变显微镜硬件，并且基于一种算法的应用，用于记录周期性诱发的荧光信号，具有相对于刺激时刻的时间偏移。
Protocol
对来自小鼠BALB / C（20-23g，2-3个月大）的分离神经肌肉制剂进行实验41。 实验程序按照喀山联邦大学和喀山医科大学的实验动物使用指南进行，符合NIH实验动物护理和使用指南。实验方案符合欧洲共同体理事会指令86/609/EEC的要求，并得到喀山医科大学伦理委员会的批准。 1. 林格氏和归档解决方案的准备 通过混合以下成分来制备哺?…
Representative Results
根据所提出的技术用染料加载制剂后，位于神经残端附近的大多数突触具有足够的荧光水平（见 图2）。用染料加载制剂并应用所述配准和图像处理方法后，获得具有所需空间和时间分辨率的钙瞬变（见 图4）。钙瞬时已通过所提出的方法回收（见 图3）。 还分析了恢复信号的幅度和时间参数。平均数据如 <str…
Discussion
本文介绍了通过神经残端将Ca2+敏感染料加载到小鼠神经末梢以及使用共聚焦显微镜记录快速钙瞬变的方法。由于实施了这种加载方法，位于神经残端附近的大多数突触具有足够的荧光水平，以记录钙进入神经末梢以响应运动神经的低频刺激。
与之前提出的通过残肢加载钙染料的方案不同，该协议设计用于哺乳动物突触。以前用于冷血动物制剂的方案需要过夜孵育<sup …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作的荧光研究在俄罗斯科学基金会赠款（项目编号19-15-00329）的资助下进行。该方法是在政府为RAS АААААА-А18-118022790083-9的喀山科学中心分配的资助下开发的。该研究是使用联邦研究中心“RAS喀山科学中心”的设备开发的。作者要感谢Victor I. Ilyin博士对这份手稿的批判性阅读。
Materials
| Capillary Glass | Clark Electromedical instruments, UK | GC150-10 | |
| Confocal and multiphoton microscope system Leica TCS SP5 MP | Leica Microsystems , Heidelberg, Germany | ||
| Flaming/Brown Micropipette Puller P 97 | Sutter Instrument, USA | P-97 | |
| Flow regulator | KD Medical GmbH Hospital Products, Germany | KD REG | Disposable infusion set with Flow regulator |
| HEPES | Sigma-Aldrich, USA | H0887 | 100mL |
| Illumination system Leica CLS 150X | Leica Microsystems, Germany | ||
| ImageJ | National Institutes of Health, USA | http://rsb.info.nih.gov/ij/download.html | |
| Las AF software | Leica Microsystems, Heidelberg, Germany | ||
| Las X software | Leica Microsystems, Heidelberg, Germany | https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/ | |
| Magnetic Holder with Suction Tubing | BIOSCIENCE TOOLS, USA | MTH-S | |
| Microspin FV 2400 | Biosan, Latvia | BS-010201-AAA | |
| Minutien Pins | Fine science tools, Canada | 26002-20 | |
| Multi-spin MSC 3000 | Biosan, Latvia | BS-010205-AAN | |
| Oregon Green 488 BAPTA-1 pentapotassium salt | Molecular Probes, USA | O6806 | 500 μg |
| Pipette | Biohit, Russia | 720210 | 0.5-10 µL |
| Pipette tip | Biohit, Russia | 781349 | 10 µL |
| Plasticine | local producer | ||
| Single-use hypodermic needles | Bbraun | 100 Sterican | 0.4×40 mm |
| Spreadsheet program | Microsoft, USA | Microsoft Office Excel | |
| Stereomicroscope, Leica M80 | Leica Microsystems , Germany | ||
| Suction electrode | Kazakov A. SIMPLE SUCTION ELECTRODE FOR ELECTRIC STIMULATION OF BIOLOGICAL OBJECTS / A. Kazakov, M. Alexandrov, N. V. Zhilyakov et al. // International research journal. - 2015. – No. 9 (40) Part 3. – P. 13-16. | http://research-journal.org/biology/prostoj-vsasyvayushhij-elektrod-dlya-elektricheskoj-stimulyacii-biologicheskix-obektov/ | |
| Sylgard 184 elastomer | Dow Corning, USA | ||
| Syringe | local producer | 0.5 mL | |
| Syringe | local producer | 60 mL |
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Citazione di questo articolo
Zhilyakov, N. V., Arkhipov, A. Y., Khaziev, E. F., Mukhamedyarov, M. A., Samigullin, D. V. Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy. J. Vis. Exp. (178), e63308, doi:10.3791/63308 (2021).