使用 Vivo 单纤维记录和带附骨神经的固定背道根结条，以检查传导失败机制
1Department of Neurobiology, School of Basic Medicine,Fourth Military Medical University, 2Department of Toxicology, School of Public Health,ShanXi Medical University, 3Department of Psychology,Fourth Military Medical University, 4Department of Neurobiology, School of Basic Medical Sciences, Advanced Innovation Center for Human Brain Protection,Capital Medical University, 5Neuroscience Research Institute, Key Lab for Neuroscience, Ministry of Education/National Health Commission,Peking University, 6Department of Radiation Biology, Faculty of Preventive Medicine,Fourth Military Medical University
Summary
单纤维记录是一种有效的电生理技术，适用于中枢和周围神经系统。随着与附着的坐骨神经制备完整的DRG，检查传导失效的机制。这两种方案都有助于了解周围神经系统与疼痛的关系。
Abstract
单纤维记录是过去几十年中一种经典而有效的电生理技术，因为它在中枢和周围神经系统的神经纤维中有着特殊应用。这种方法特别适用于背根神经结膜（DRG），这是主要感觉神经元，表现出神经过程的伪单极结构。沿着斧子传递的动作电位的模式和特征在这些神经元中是可记录的。本研究使用体内单纤维记录来观察完全Freund的佐剂（CFA）处理大鼠的坐骨神经的传导失败。由于不能使用体内单纤维记录来研究底层机制，DRG神经元的贴片夹-记录在附加的坐骨神经的完整DRG的制剂上进行。这些记录揭示了在CFA处理的动物中，传导失败与DRG神经元后极化电位（AHP）的上升斜率之间的正相关关系。体内单纤维记录方案允许通过测量传导速度和监测某些疾病的神经纤维异常情况来对神经纤维进行分类。附加外周神经的完好 DRG 允许在大多数生理条件下观察 DRG 神经元的活动。最后，单纤维记录与完整DRG的电生理记录相结合是检查镇痛过程中传导失败作用的有效方法。
Introduction
沿神经纤维的信息的正常传输保证了神经系统的正常功能。神经系统的异常功能也反映在神经纤维的电信号传输中。例如，通过比较干预应用1前后神经传导速度的变化，可以对中枢脱骨髓病变的脱骨髓程度进行分类。很难在细胞内记录神经纤维，除了特殊制剂，如鱿鱼巨型斧子2。因此，电生理活性只能通过单纤维的细胞外记录进行记录。单纤维记录作为传统的电生理方法之一，其历史比其他技术要长。然而，尽管该方法应用广泛，但掌握这种方法的电生理学家较少。因此，需要详细介绍单光纤记录的标准协议，以便进行适当的应用。
虽然各种贴片夹技术主导了现代电生理学研究，但单纤维记录在记录神经纤维的活动方面仍然发挥着不可替代的作用，尤其是纤维以神经纤维的外在感。位于背根结节（DRG）的感官细胞体。这里使用单纤维记录的优点是，体内纤维记录提供了很长的观察时间，能够记录临床前模型中对自然刺激的反应，而不会干扰细胞内环境3,4.
近二十年来，越来越多的研究研究了神经纤维5沿线的复杂功能，传导失败，被定义为沿着斧子的神经脉冲传递不成功的状态，存在于许多不同的外围神经6，7。在我们的调查中，传导失败的存在是沿着C纤维8调制持续感知输入的一种内在的自我抑制机制。在高乳腺4、9的条件下，这种传导失败显著减弱。因此，针对传导失败所涉及的因素可能代表神经病痛的新疗法。为了观察传导失败，应根据单纤维记录的连续放电尖峰记录和分析点火模式。
为了彻底了解传导失效的机制，有必要根据子极性解剖特性，确定斧子的传输特性，或者更确切地说，确定DRG神经元的膜特性。以前在这一领域的许多研究都进行了分离的DRG神经元10，11，这可能是不可行的研究传导失败，由于两个障碍。首先，在解散过程中使用各种机械和化学方法来释放DRG神经元，这可能导致不健康的细胞或改变神经元的表型/特性，并混淆结果。其次，附着的外周神经基本消除，传导失效现象在这些准备中无法观察到。因此，对具有附加神经的完整DRG神经元的准备已经改进，以避免上述障碍。
Protocol
目前的议定书遵循了《美国公共卫生服务关于人道照料和使用实验室动物的政策指南》，第四军医大学动物实验伦理委员会批准了该议定书。 1. 动物 将24只斯普拉格-道利大鼠（4-8周大）分成两组。通过一组14只大鼠和另一组10只大鼠用盐水治疗，在平面内注射100μL的CFA，生成完整的Freund辅助（CFA）模型。注：所有动物都是从第四军医大学动物中心采集的。成年雄性?…
Representative Results
单光纤记录协议的结果取决于光纤解剖的质量。体内实验的动物必须在良好的状态，以保持神经躯干健康，便于解剖（见讨论部分的建议）。在许多情况下，在纤维上提供药物需要药物应用浴。图1说明了体内单纤维记录是如何操作的（图1A），并给出了一个经典记录，从CFA处理的动物的坐骨神经（图1B）。 以下实验调?…
Discussion
虽然最近的研究已经实现了DRG神经元在体内16的钙成像，从单个DRG受体进行体内贴片夹记录仍然极具挑战性。因此，在体内单纤维方法对疼痛场是持续重要的。本协议中的单光纤记录允许客观地观察传导失效现象，并且该技术与当前研究中开发的体外制剂相结合，可以检查临床前模型中的受体兴奋性变化。单光纤录制协议的三个步骤对于成功录制至关重要。首先，注意动物的麻?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金（31671089和81470060）和陕西省社会发展科学技术研究项目（2016SF-250）的资助。
Materials
| Instruments and software used in single fiber recording | |||
| Amplifier | Nihon kohden | MEZ-8201 | Amplification of the electrophysiological signals |
| Bioelectric amplifier monitor | ShangHai JiaLong Teaching instrument factory | SZF-1 | Monitor firing process via sound which is transformed from physiological discharge signal |
| Data acquisition and analysis system | CED | Spike-2 | Software for data acquisition and analysis |
| Electrode manipulator | Narishige | SM-21 | Contro the movement of the electrode as required |
| Hairspring tweezers | A.Dumont | 5# | Separate the single fiber |
| Isolator | Nihon kohden | SS-220J | |
| Memory oscilloscope | Nihon kohden | VC-9 | Display recorded discharge during |
| experiment | |||
| Stereomicroscope | ZEISS | SV-11 | Have clear observation when separate the local tissue and single fiber |
| Stimulator | Nihon kohden | SEZ-7203 | Delivery of the electrical stimuli |
| Von Frey Hair | Stoelting accompany | Delivery of the mechanical stimuli | |
| Water bath | Scientz biotechnology Co., Ltd. | SC-15 | Heating paroline to maintain at 37oC |
| Instruments and software used in patch clamp recording | |||
| Amplifier | Axon Instruments | Multiclamp 700B | Monitors the currents flowing through the recording electrode and also controls the stimuli by sending a signal to the electrode |
| Anti-vibration table | Optical Technology Co., Ltd. | Isolates the recording system from vibrations induced by the environment | |
| Camera | Olympus | TH4-200 | See the neurons in bright field; the controlling software allows to take pictures and do live camera image to monitor the approach of the electrode to the cell |
| Clampex | Axon | Clampex 9.2 | Software for data acquisition and delivery of stimuli |
| Clampfit | Axon | Clampfit 10.0 | Software for data analysis |
| Electrode puller | Sutter | P-97 | Prepare recording pipettes of about 2μm diameter with resistance about 5 to 8 MΩ |
| Glass pipette | Sutter | BF 150-75-10 | |
| Micromanipulator | Sutter | MP225 | Give a precise control of the microelectrode |
| Microscope | Olympus | BX51WI | Upright microcope equipped with epifluorescence for clearly observe the cells which would be patched |
| Origin | Origin lab | Origin 8 | Software for drawing picture |
| Perfusion Pump | BaoDing LanGe Co., Ltd. | BT100-1J | Perfusion of DRG in whole-cell patch clamp |
| Other instruments | |||
| Electronic balance | Sartorius | BS 124S | Weighing reagent |
| pH Modulator | Denver Instrument | UB7 | Adjust pH to 7.4 |
| Solutions/perfusion/chemicals | |||
| Calcium chloride | Sigma-Aldrich | C5670 | Extracellular solution |
| Chloralose | Shanghai Meryer Chemical Technology Co., Ltd. | M07752 | Mixed solution for Anesthesia |
| Collagenase | Sigma-Aldrich | SLBQ1885V | Enzyme used for clearing the surface of DRG |
| D (+) Glucose | Sigma-Aldrich | G7528 | Extracellular solution |
| Liquid Paraffin | TianJin HongYan Reagent Co., Ltd. | Maintain fiber wetting | |
| Magnesium sulfate | Sigma-Aldrich | M7506 | Extracellular solution |
| Potassium chloride | Sigma-Aldrich | P3911 | Extracellular solution |
| Protease | Sigma-Aldrich | 62H0351 | Enzyme used for clearing the surface of DRG |
| Sodium bicarbonate | Sigma-Aldrich | S5671 | Extracellular solution |
| Sodium chloride | Sigma-Aldrich | S5886 | Extracellular solution |
| Sodium phosphate monobasic | Sigma-Aldrich | S0751 | Extracellular solution |
| Sucrose | Sigma-Aldrich | S0389 | Extracellular solution |
| Urethane | Sigma-Aldrich | U2500 | Mixed solution for Anesthesia |
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Cite This Article
Mao, H., Wang, X., Chen, W., Liu, F., Wan, Y., Hu, S., Xing, J. Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure. J. Vis. Exp. (150), e59234, doi:10.3791/59234 (2019).