神经微电路急性脑片电生理和形态特征采用配对膜片钳记录
Summary
Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.
Abstract
的膜片钳记录，从急性脑切片制剂与同时细胞内生物胞素填充两个（或多个）耦合突触神经元（配对记录）的组合允许它们的结构和功能特性的相关分析。用这种方法，可以通过它们的形态和电生理反应模式来识别和表征前和突触后神经元。配对录音允许研究这些神经元之间的连接模式，以及化学和电突触传递的属性。在这里，我们得到，以获得可靠的配对记录连同神经元形态学的最佳恢复所需的步骤的一步一步的描述。我们将描述如何对经由化学突触或间隙连接连接的神经元在大脑切片制剂被识别。我们将阐述如何神经元被重建，以获得dendrit自己的3D形态IC和轴突域以及如何突触联系的识别和本地化。我们还将讨论配对记录技术的警告和限制，尤其是脑片的制备过程中与树突和轴突截断相关，因为这些强烈地影响连接的估计。因为配对记录方法的多功能性。然而，它会留在在大脑中的神经元鉴定微电路表征突触传递的不同方面的宝贵工具。
Introduction
2突触耦合的神经元之间的神经元微电路是在大脑中的大型网络的积木并且突触信息处理的基本单位。一个先决条件，例如神经元微电路的特征是知道的形态和两个前和突触后神经元的伙伴的功能特性，突触连接（S）和它的结构和功能的机制的类型。然而，在突触连接的许多研究中的至少一个在微电路中的神经元的是不是很好的特点。这导致从通常的突触连接研究中使用的相对非特异性的刺激协议。因此，突触前神经元的结构和功能特性要么根本不或仅到一个相当小的范围内确定的（ 即，标记蛋白质的表达等 ）。在组合配对录音与细胞内染色用标志sUCH作为生物胞素，neurobiotin或荧光染料更适合用于研究小神经微电路。这种技术允许一个调查的同时一个形态鉴定突触连接的许多结构和功能参数。
所谓的“单一”突触两个神经元之间的联系进行了研究使用急性切片准备两种皮层和皮层下大脑区域1-10。最初，尖锐的微电极在这些实验中使用;之后，膜片钳记录是采用为了获得突触信号记录具有较低噪声电平以及一个改进的时间分辨率。
一个显著的技术进步是利用红外微分干涉对比（IR-DIC）的光学11-14，微观技术，显著改善神经细胞的知名度和识别的脑片，使之成为可能吨Ø获得录音从视觉上确定的突触连接15-17。在一般情况下，一对录音进行急性切片的筹备工作;只有极少数的出版物可从报告突触连接的神经元在体内 18-20录音。
配对记录的最重要的优点是，一个功能表征可以与同时在光学和电子显微镜水平形态解析被组合（ 例如见。，7,16,21）。组织化学处理后，将突触连接的神经元对的树突和轴突的形态被追踪。随后，有可能量化的形态特征，如长度，空间密度，取向，分支图案等 ，然后这些参数可以为特定的突触连接的一个目标分类提供了基础。此外，与此相反，以用于研究神经元connecti大多数其它技术VITY，配对录音还允许对单一的突触连接突触联系的标识。这可以直接使用光学和电子显微镜16,21-27的组合或使用树突棘的钙成像28,29来完成。但是，与后者的方法只兴奋性但不抑制连接，可以研究，因为它需要通过突触后受体通道的钙内流。
除了 突触传递的在确定的神经元微电路配对的记录进行详细的分析还允许的突触可塑性规则30,31的研究或-与激动剂/拮抗剂组合的应用-突触传递由神经递质如乙酰胆碱32和腺苷调制33。
Protocol
所有的实验过程进行了按照欧盟指令保护动物，德国动物福利法（Tierschutzgesetz）和欧洲实验动物科学协会联合会的指导原则。 1.建立了电与配对记录开始之前，一个电设置具有待建。简述怎么这么的建立组装下面给出： 安装一个防振台在其上的显微镜，操纵器和所有其他设备将被放置。 注：振动或任何其它类型的运动需要从突触连接的记录…
Representative Results
配对录音是选择的方法进行了深入的表征形态识别的单向或双向的突触连接，以及间隙连接（电）连接（ 图1）。在体感桶皮质层4配对记录的一个例子示于图1A。两个单向兴奋性和抑制性突触连接可被表征（ 图1B中的C）。此外成对录音允许来自双向突触连接， 即 ，记录，从连接，其中两个神经元中的一对是前和突触后彼此。这示于图1D，<…
Discussion
从突触耦合兴奋性和/或抑制性神经元成对录音是神经元微电路的研究中非常通用的方法。不仅这种方法允许一个来估计神经类型之间的突触连接，但也允许确定连接和前和突触后神经元的形态的功能特性。此外，激动剂和/或拮抗剂可以很容易地应用到神经元片的准备工作。这允许人们研究神经调的影响突触传递32的属性或一个深入表征使用定义酉突触连接的，例如，突触释放16,17,38,39…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank all members of ‘Function of Neuronal Microcircuits’ Group at Institute of Neuroscience and Medicine, INM-2, Research Centre Jülich and the ‘Function of Cortical Microcircuits’ Group in the Dept. of Psychiatry, Psychotherapy and Psychosomatics, Medical School, JARA, RWTH Aachen University for fruitful discussions. This work was supported by the DFG research group on Barrel Cortex Function (BaCoFun).
Materials
| Name | Company | Catalog Number | Comments |
| Amplifier | HEKA | EPC 10 USB Triple | with 2-3 preamplifiers |
| Microscope | Olympus | BX51WI | with 2 camera ports and a 4× objective, a 40× water-immersion objective |
| Camera | TILL Photonics | VX55 | infrared CCD camera |
| Workstation | Luigs & Neumann | Infrapatch 240 | with a motorized x-y stage and a motorized focus axis for the microscope |
| Micromanipulator | Luigs & Neumann | SM-5 | x-y-z manipulators for 2-3 preamplifiers |
| Faraday cage | Luigs & Neumann | ||
| Anti-vibration table | Newport Spectra-Physics | ||
| Patchmaster | HEKA | ||
| Microtome | Microm International | HM650V | |
| Micropipette puller | HEKA | Sutter P-97 | |
| Neurolucida system | Microbrightfield | with Neurolucida and Neuroexplorer softwares |
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Cite This Article
Qi, G., Radnikow, G., Feldmeyer, D. Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings. J. Vis. Exp. (95), e52358, doi:10.3791/52358 (2015).