在跨脊柱直流刺激期间类型识别大鼠脊柱腺瘤的 Vivo 细胞内记录
Summary
该协议描述了大鼠腰椎瘤的体内细胞内记录，同时具有跨脊柱直电流刺激。该方法使我们能够测量膜特性，并记录脊髓的腺体或阴极化之前、期间和之后有节奏的分子发射。
Abstract
体内脊髓腺细胞内记录为确定完整脊柱网络中的细胞电生理特征提供了”黄金标准”，与传统的体外或细胞外记录技术相比具有显著优势。体内细胞内记录的优点是，这种方法可以在神经系统完全成熟的成年动物身上进行，因此许多观察到的生理机制可以转化为实际应用。在本文中，我们描述了这个程序与外部应用的恒定电流刺激相结合，它模仿脊柱神经元网络内发生的极化过程。跨脊柱直流刺激（tsDCS）是一种创新的方法，越来越多地用作各种神经损伤后康复和运动的神经调节干预。tsDCS对神经系统的影响仍然不为人所知，其行动背后的生理机制在很大程度上未知。tsDCS 与细胞内记录同时应用使我们能够直接观察莫托龙膜特性的变化和节律激发的特征，以应对脊柱神经元网络的极化，这对理解 tsDCS 操作至关重要。此外，当提出的协议包括识别内侧肌肉及其功能（柔性与拉伸器）以及生理类型（快速与缓慢）的motoneuron时，它提供了一个机会，有选择地研究tsDCS对已识别的脊柱回路成分的影响，这些成分似乎受到极化的影响不同。所介绍的程序侧重于细胞内记录和刺激的手术准备，重点是实现制备稳定性和结果可重复性所必需的步骤。讨论了阴极或阴极 tsDCS 应用方法的细节，同时关注实际和安全问题。
Introduction
跨脊柱直流刺激（tsDCS）作为一种有效的方法，在健康和疾病1，2，3中改变2脊柱回路兴奋性， 正在得到认可。1在这项技术中，一个恒定电流在位于选定脊髓段上方的有源电极之间传递，参考电极位于呼吸口或更红润的 4。几项研究已经证实，tsDCS可用于管理某些病理状况，如神经病变疼痛5，痉挛6，脊髓损伤7或促进康复8。研究人员认为，tsDCS会唤起细胞内和细胞膜外空间之间离子分布的改变，根据当前方向,9、10、11，,10这可促进或抑制神经元活动。然而，直到最近，还缺乏对莫托龙这种影响的直接证实。
在这里，我们描述了一个详细的方案，用于在体内进行麻醉大鼠腰椎莫酮的电位记录，同时应用 tsDCS，以观察莫托内隆膜的变化和燃烧特性，以应对脊髓神经网络的腺癌或阴极化。细胞内记录打开神经元属性的几个区域调查，不适用于以前使用的细胞外技术9,9，12。例如，可以精确测量由 tsDCS 诱导的莫托龙膜膜电压响应，以指示尖峰生成电压阈值，或分析作用电位参数。此外，该技术还使我们能够确定莫托龙无源膜特性，如输入电阻，并观察细胞内刺激电流与分子节律发射频率之间的关系。基于功能识别神经（即向屈曲器或挤出器提供发源的神经）的抗浪漫性识别，使我们能够进一步识别内向运动单元的类型（快与慢），从而有机会测试极化是否以不同的方式影响成熟的脊柱神经元系统的单个元素。由于在录音前进行了广泛的手术，并且对录音的稳定性和可靠性要求很高，这项技术极具挑战性，但允许对一个motoneuron的电生理特性进行直接和长期的评估：在tsDCS应用之前、期间和之后，这对于确定其急性作用和持续效应至关重要。由于motoneuron直接激活外混肌纤维14，并参与肌肉收缩的反馈控制，并发展力量15，16任何观察到15,的tsDCS对运动单元或肌肉收缩特性的影响可能与motoneuron兴奋性或燃烧特性的调制有关。
Protocol
与本议定书有关的所有程序都已被有关当局（例如地方道德委员会）接受，并遵守关于动物福利和管理的国家和国际规则。 注：参与手术的每个参与者必须接受基本外科手术方面的训练，并且必须拥有进行动物实验的有效许可证。 1. 麻醉和预谋 麻醉大鼠与内丙酮注射五巴比妥钠（初始剂量60毫克+千克-1 为6个月大的雄性威斯塔大鼠体重400\…
Representative Results
当确保细胞穿透条件稳定时，可以在细胞内记录的基础上计算作用电位参数和若干膜特性。 图1A 提供了细胞内刺激引起的典型正交作用潜力，它符合数据包含的所有标准（至少为-50 mV的静膜电位，峰值振幅高于50 mV，具有正过冲）。可测量动作电位参数，如尖峰振幅、后白化幅度或后白化半衰减时间（AHP-HDT）。大鼠莫托龙中后一个参数的值是区分快速和慢莫托龙（AHP-HDT > 20…
Discussion
如果执行正确，所述方案的手术部分应在大约三小时内完成。在手术过程中，应特别注意保持动物的生理状况，特别是体温和麻醉深度。除了明显的伦理考虑外，缺乏适当的麻醉可能导致神经解剖或拉明切除术期间肢体过度运动，并导致制剂损伤或实验终止。在用微电子穿透脊髓之前使动物瘫痪后，监测麻醉深度和心率以及应用基于动物体重和肺活量的适当通气参数至关重要。任何偏离所需生理?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家科学中心第2017/25/B/NZ7/00373号赠款的支持。作者们要表彰汉娜·德日马尼亚-塞利乔夫斯卡和沃齐米尔兹·姆罗奇斯基的作品，他们都为收集和分析本文件介绍的结果做出了贡献。
Materials
| Durgs and solutions | – | – | – |
| Atropinum sulfuricum | Polfa Warszawa | – | – |
| Glucose | Merck | 346351 | – |
| NaHCO3 | Merck | 106329 | – |
| Pancuronium Jelfa | PharmaSwiss/Valeant | – | Neuromuscular blocker |
| Pentobarbital sodium | Biowet Puławy Sp. z o.o | – | Main anesthetic agent |
| Pottasium citrate | Chempur | 6100-05-06 | – |
| Tetraspan | Braun | – | HES solution |
| Surgical equipment | – | – | – |
| 21 Blade | FST | 10021-00 | Scalpel blade |
| Cauterizer | FST | 18010-00 | – |
| Chest Tubes | Mila | CT1215 | – |
| Dumont #4 Forceps | FST | 11241-30 | Muscle forceps |
| Dumont #5 Forceps | FST | 11254-20 | Dura forceps |
| Dumont #5F Forceps | FST | 11255-20 | Nerve forceps |
| Dumont #5SF Forceps | FST | 11252-00 | Pia forceps |
| Forceps | FST | 11008-13 | Blunt forceps |
| Forceps | FST | 11053-10 | Skin forceps |
| Hemostat | FST | 13013-14 | – |
| Rongeur | FST | 16021-14 | For laminectomy |
| Scissors | FST | 15000-08 | Vein scissors |
| Scissors | FST | 15002-08 | Dura scissors |
| Scissors | FST | 14184-09 | For trachea cut |
| Scissors | FST | 104075-11 | Muscle scissors |
| Scissors | FST | 14002-13 | Skin scissors |
| Tracheal tube | – | – | Custom made |
| Vein catheter | Vygon | 1261.201 | – |
| Vessel cannulation forceps | FST | 18403-11 | – |
| Vessel clamp | FST | 18320-11 | For vein clamping |
| Vessel Dilating Probe | FST | 10160-13 | For vein dissection |
| Sugrgical materials | – | – | – |
| Gel foam | Pfizer | GTIN 00300090315085 | Hemostatic agent |
| Silk suture 4.0 | FST | 18020-40 | – |
| Silk suture 6.0 | FST | 18020-60 | – |
| Equipment | – | – | – |
| Axoclamp 2B | Molecular devices | discontinued | Intracellular amplifier/ new model Axoclamp 900A |
| CapStar-100 End-tidal CO2 Monitor | CWE | 11-10000 | Gas analyzer |
| Grass S-88 | A-M Systems | discontinued | Constant current stimulator |
| Homeothermic Blanket Systems with Flexible Probe | Harvard Apparatus | 507222F | Heating system |
| ISO-DAM8A | WPI | 74020 | Extracellular amplifier |
| Microdrive | – | – | Custom made/replacement IVM/Scientifica |
| P-1000 Microelectrode puller | Sutter Instruments | P-1000 | Microelectrode puller |
| SAR-830/AP Small Animal Ventilator | CWE | 12-02100 | Respirator |
| Support frame | – | – | Custom made/replacement lab standard base 51601/Stoelting |
| Spinal clamps | – | – | Custom made/replacement Rat spinal adaptor 51695/Stoelting |
| TP-1 DC stimulator | WiNUE | – | tsDCS stimulator |
| Miscellaneous | – | – | – |
| 1B150-4 glass capillaries | WPI | 1B150-4 | For microelectrodes production |
| Cotton wool | – | – | – |
| flexible tubing | – | – | For respirator and CO2 analyzer connection |
| MicroFil | WPI | MF28G67-5 | For filling micropipettes |
| Silver wire | – | – | For nerve electrodes |
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Diesen Artikel zitieren
Bączyk, M., Krutki, P. In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation. J. Vis. Exp. (159), e61439, doi:10.3791/61439 (2020).