脊髓切片 的离体 制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录
Summary
该协议描述了一种使用膜片钳研究运动神经元对脊髓刺激（SCS）的电反应的方法，具有高时空分辨率，这可以帮助研究人员提高分离脊髓和同时维持细胞活力的技能。
Abstract
脊髓刺激（SCS）可有效恢复脊髓损伤（SCI）后的运动功能。由于运动神经元是执行感觉运动行为的最终单位，因此直接研究SCS运动神经元的电反应可以帮助我们理解脊髓运动调节的底层逻辑。为了同时记录不同的刺激特征和细胞反应，膜片钳是在单细胞尺度上研究电生理特征的好方法。然而，实现这一目标仍然存在一些复杂的困难，包括维持细胞活力、快速将脊髓与骨结构分离，以及利用SCS成功诱导动作电位。在这里，我们提出了一个详细的方案，使用膜片钳以高时空分辨率研究运动神经元对SCS的电反应，这可以帮助研究人员提高分离脊髓和维持细胞活力的技能，以顺利研究SCS对运动神经元的电机制，避免不必要的试验和错误。
Introduction
脊髓刺激（SCS）可有效恢复脊髓损伤（SCI）后的运动功能。Andreas Rowald 等人报告说，SCS 在一天内实现了下肢运动和躯干功能1.探索SCS对运动恢复的生物学机制是制定更精确的SCS策略的关键和趋势研究领域。例如，Grégoire Courtine的团队证明，脊髓中的兴奋性Vsx2中间神经元和Hoxa10神经元是响应SCS的关键神经元，细胞特异性神经调控在SCI2后恢复大鼠行走能力是可行的。然而，很少有研究关注单细胞尺度上SCS的电机制。尽管众所周知，超阈值直流电刺激可以引发经典鱿鱼实验3,4,5中的动作电位（AP），但脉冲交变电刺激（如SCS）如何影响运动信号的产生仍不清楚。
鉴于脊髓内神经回路的复杂性，适当选择细胞群对于研究SCS的电机制非常重要。尽管 SCS 通过激活本体感受通路6 来恢复运动功能，但运动神经元是执行运动命令的最终单元，该命令来自整合本体感受信息传入输入7。因此，直接用SCS研究运动神经元的电特性可以帮助我们理解脊髓运动调节的底层逻辑。
众所周知，膜片钳是细胞电生理记录的金标准方法，具有极高的时空分辨率8。因此，本研究描述了一种使用膜片钳研究运动神经元对SCS的电反应的方法。与脑膜片钳9相比，脊髓膜片钳的难度更大，原因如下：（1）脊髓由体积小的椎管保护，需要非常精细的显微操作和严格的冰冷维护才能获得更好的细胞活力。（2）由于脊髓太细，无法固定在切割盘上，应将其浸入低熔点琼脂糖中，固化后进行修整。
因此，该方法在解剖脊髓和保持细胞活力的同时提供了技术细节，从而顺利地研究SCS对运动神经元的电机制，避免不必要的试验和错误。
Protocol
机构动物护理和使用委员会批准了所有动物实验，研究是按照相关的动物福利法规进行的。 1. 动物准备 动物饲养信息：在特定的无病原体环境中饲养雄性Sprague-Dawley大鼠（出生后10-14天，P10-P14）。注意：室温保持在20°C±2°C，湿度：50%-60%，光/暗循环12小时。动物可以自由获得食物和水。 逆行标记运动神经元：将氟金（FG）注射到双侧胫骨…
Representative Results
由于精细操作期间严格的低温维护（补充图1、补充图2和图1），细胞活力足以进行后续的电生理记录。为了尽可能地模拟临床场景，我们使用显微操作将 SCS 阴极和阳极分别放置在背中线和 DREZ 附近（图 2），这可以启动背角中的神经信号传播到运动柱背外侧区域的运动神经元。在这项研究中，我们使用FG定位直径为20-50μm的运动神经元。<st…
Discussion
SCS调制的运动信息最终收敛到运动神经元。因此，以运动神经元为研究靶点可以简化研究设计，更直接地揭示SCS的神经调控机制。为了同时记录不同的刺激特征和细胞反应，膜片钳是在单细胞尺度上研究电生理特征的好方法。然而，仍然存在一些困难，包括如何保持细胞活力，如何快速将脊髓与骨结构分离，以及如何利用SCS成功诱导APs。因此，本研究旨在帮助研究人员快速掌握基本的操作技能，?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
本研究由国家自然科学基金青年基金（52207254和82301657）和中国博士后科学基金（2022M711833）资助。
Materials
| Adenosine 5’-triphosphate magnesium salt | Sigma | A9187 | |
| Ascorbic Acid | Sigma | A4034 | |
| CaCl2·2H2O | Sigma | C5080 | |
| Choline Chloride | Sigma | C7527 | |
| Cover slide tweezers | VETUS | 36A-SA | Clip a slice |
| D-Glucose | Sigma | G8270 | |
| EGTA | Sigma | E4378 | |
| Fine scissors | RWD Life Science | S12006-10 | Cut the diaphragm |
| Fluorescence Light Source | Olympus | U-HGLGPS | |
| Fluoro-Gold | Fluorochrome | Fluorochrome | Label the motor neuron |
| Guanosine 5′-triphosphate sodium salt hydrate | Sigma | G8877 | |
| HEPES | Sigma | H3375 | |
| infrared CCD camera | Dage-MTI | IR-1000E | |
| KCl | Sigma | P5405 | |
| K-gluconate | Sigma | P1847 | |
| Low melting point agarose | Sigma | A9414 | |
| MgSO4·7H2O | Sigma | M2773 | |
| Micromanipulator | Sutter Instrument | MP-200 | |
| Micropipette puller | Sutter instrument | P1000 | |
| Micro-scissors | Jinzhong | wa1020 | Laminectomy |
| Microscope for anatomy | Olympus | SZX10 | |
| Microscope for ecletrophysiology | Olympus | BX51WI | |
| Micro-toothed tweezers | RWD Life Science | F11008-09 | Lift the cut vertebral body |
| NaCl | Sigma | S5886 | |
| NaH2PO4 | Sigma | S8282 | |
| NaHCO3 | Sigma | V900182 | |
| Na-Phosphocreatine | Sigma | P7936 | |
| Objective lens for ecletrophysiology | Olympus | LUMPLFLN60XW | working distance 2 mm |
| Osmometer | Advanced | FISKE 210 | |
| Patch-clamp amplifier | Axon | Multiclamp 700B | |
| Patch-clamp digitizer | Axon | Digidata 1550B | |
| pH meter | Mettler Toledo | FE28 | |
| Slice Anchor | Multichannel system | SHD-27H | |
| Spinal cord stimulatior | PINS | T901 | |
| Toothed tweezer | RWD Life Science | F13030-10 | Lift the xiphoid |
| Vibratome | Leica | VT1200S | |
| Wide band ultraviolet excitation filter | Olympus | U-MF2 |
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Citer Cet Article
Yao, Q., Luo, X., Liu, J., Li, L. The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation. J. Vis. Exp. (199), e65385, doi:10.3791/65385 (2023).