斜脊髓切片用于神经前根刺激的制备
Summary
我们显示如何在年轻小鼠制备脊髓倾斜切片。该制剂允许前根的刺激。
Abstract
从脊髓切片电生理记录已经被证明是调查范围广泛的问题，对网络性能的有价值的技术，从细胞。我们展示如何准备年轻小鼠（P2 – P11）脊髓可行的斜片。在此制备方法中，运动神经元保留它们的轴突从脊髓的腹侧根部出来。这些轴突的刺激引起背传播动作电位侵入脊髓中运动神经元胞体和令人兴奋的运动神经元的抵押品。逆向动作电位记录是表征运动神经元的身份，这超过了其他的识别方法的直接，明确的和优雅的方式。此外，刺激运动神经元络是一种简单而可靠的方式来激发运动神经元的脊髓内抵押品目标，如其它运动神经元或Renshaw细胞。在这个协议中，我们提出从运动神经元胞体以及伦肖细胞激发逆向录音，腹侧根刺激所致。
Introduction
从历史上看，使用锋利的电极运动神经元录音被体内的大型动物，如猫或大鼠1或对小鼠2孤立全脊髓进行。视觉指导下，要达到的膜片钳技术在80年代的出现，要求直接进入体细胞运动神经元的密封需要。因此，脊髓切片准备一直以来随手20世纪90年代3月初实现。然而，早期的切片准备往往没有允许前根的刺激。据我们所知，只有两个研究报道在横切片腹侧根成功刺激，从小鼠4,5-得到无。
在这篇文章中，我们提出了一种技术来实现新生小鼠的存活脊髓切片（P2 – P11），其中运动神经元池保留其神经前根轴突出发。发泄拉尔根刺激引发逆向动作电位放回池中运动神经元从相同的前根离开的肉体。这也激发了运动神经元抵押的目标，其他的运动神经元6-10和伦肖细胞11-13。因为只有运动神经元发送它们的轴突沿着前根中，我们使用逆向动作电位的记录作为一个简单的和明确的方式来physiologicaly确定运动神经元10。
除了使用潜在的非包容性或误导性的电生理和形态准则确认身份的运动神经元，近期对脊髓运动神经元的研究还依赖于繁琐和耗时的事后染色16。这种识别通常只有在记录细胞的样品进行的。其他标识的策略依赖于它的运动神经元表达内源性荧光鼠标线<sup> 17-19。然而，当标志物的表达仍是变量，如果这项研究已经要求使用的转基因小鼠行中使用基因编码的标记可能难以在年轻的时候。另外，逆向动作电位的录音可以从常规细胞记录的发生进行的所有鼠标。在猫，大鼠和小鼠完整脊髓的准备工作实验者，都可靠地使用，因为1950年的1,2,20,21等识别技术。在最佳条件下，我们能够从几乎所有记录的运动神经元的诱发逆向动作电位。
此外，前根刺激可以用于可靠地激发其它运动神经元22,23或它们的靶标。在伦肖细胞10,24,25。我们在这里提出在逆向动作电位记录从运动神经元胞体的形式腹侧根刺激的应用，以及伦肖细胞的激发。
Protocol
实验是按照欧盟指令（86/609 / CEE和2010-63-UE）和法国的立法进行，由巴黎第五大学伦理委员会的批准。 1.脊髓切片准备每天准备了以下解决方案或提前一天。如果保持过夜，用95％ 的 O 2和5％CO 2的气泡并保持冷藏在密闭瓶。 制备低钠离子的人工脑脊液（ACSF）：3毫米氯化钾，1mM的的NaH 2 PO 4，230 mM的蔗…
Representative Results
采用逆向动作电位运动神经元身份确认 细胞靶向 运动神经元在腹角（在红色可见于图2C）中找到。从形成前根轴突束，至多直至完全捆绑分散一开始看到的大细胞（SOMA长轴线，高于20微米）。实现使用3初始电阻的电极至4MΩ圆形健康寻找细胞?…
Discussion
脊髓倾斜切片是重要的，因为它允许在一个可靠的，全面和具体的方式的单个椎骨段运动神经元池和伦肖细胞单方面刺激。此外，它允许一个快速的，优雅的和无歧义的识别记录的运动神经元。下一步，我们将突出相比其他片制备方法这一技术的优势，然后我们将强调出最常见的陷阱，以避免在执行此过程。
使用横切片大多数研究的基础上固有的电性能3,14,15,29鉴定?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢马林·曼努埃尔和Olivia高盛-Szwajkajzer他们在拍摄照片的帮助。作者还感谢阿琼Masukar和托拜厄斯·博克校对稿件。金融支持是由国民法新社提供的倒拉RECHERCHE（HYPER-MND，ANR-2010-汪曾祺，1429至1401年），美国国立卫生研究院，NINDS（R01NS077863）时，蒂埃里Latran基金会（OHEX项目），法国协会肌病（授权编号16026）和Target ALS是表示感谢。菲利克斯乐华是一个“成功承接博士”，从巴黎高等师范学校，卡尚的收件人。
Materials
| Na-kynurenate | ABCAM | ab120256 | dissolves better then other brands |
| KCl | Sigma | P3911 | |
| NaH2PO4 | Sigma | P5655 | |
| sucrose | Sigma | S9378 | |
| NaHCO3 | Sigma | S6014 | |
| CaCl2 | G Biosciences | R040 | |
| MgCl2 | Quality Biological | 351-033-721 | |
| glucose | Sigma | G5767 | |
| ascorbic acid | Sigma | A5960 | |
| Na-pyruvate | Sigma | P2250 | |
| K-gluconate | Sigma | P1847 | |
| EGTA | Sigma | E3889 | |
| HEPES | Sigma | H4034 | |
| NaCl | Sigma | S9888 | |
| Agar | Sigma | A9799 | |
| QX-314 | Alomone | Q150 | |
| Mg-ATP | Sigma | A9187 | |
| CsOH | Sigma | 232041 | |
| Na-GTP | Sigma | 51120 | |
| gluconic acid | Sigma | G1951 | |
| Cesium hydroxide solution | Sigma | 232041 | |
| KOH | Sigma | P5958 | |
| Vannas Spring Scissors – 2.5mm | FST | 15000-08 | only use for cutting the dura, might get damaged if cutting bones |
| Stimulator | A-M Systems | Isolated Pulse Stimulator Model 2100 | |
| Vibratome | Campden | Vibrating Microtome 7000 – Model 7000smz-2 | |
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Citer Cet Article
Leroy, F., Lamotte d’Incamps, B. The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation. J. Vis. Exp. (116), e54525, doi:10.3791/54525 (2016).