Wang Guangfu, Wyskiel Daniel R, Yang Weiguo, Wang Yiqing, Milbern Lana C, Lalanne Txomin, Jiang Xiaolong, Shen Ying, Sun Qian-Quan, Zhu J Julius
Department of Pharmacology, University of Virginia, Charlottesville, Virginia, USA.
Department of Zoology and Physiology, University of Wyoming, Laramie, Wyoming, USA.
Nat Protoc. 2015 Mar;10(3):397-412. doi: 10.1038/nprot.2015.019. Epub 2015 Feb 5.
Deciphering neuronal circuitry is central to understanding brain function and dysfunction, yet it remains a daunting task. To facilitate the dissection of neuronal circuits, a process requiring functional analysis of synaptic connections and morphological identification of interconnected neurons, we present here a method for stable simultaneous octuple patch-clamp recordings. This method allows physiological analysis of synaptic interconnections among 4-8 simultaneously recorded neurons and/or 10-30 sequentially recorded neurons, and it allows anatomical identification of >85% of recorded interneurons and >99% of recorded principal neurons. We describe how to apply the method to rodent tissue slices; however, it can be used on other model organisms. We also describe the latest refinements and optimizations of mechanics, electronics, optics and software programs that are central to the realization of a combined single- and two-photon microscopy-based, optogenetics- and imaging-assisted, stable, simultaneous quadruple-viguple patch-clamp recording system. Setting up the system, from the beginning of instrument assembly and software installation to full operation, can be completed in 3-4 d.
解析神经回路对于理解大脑功能及功能障碍至关重要，但这仍是一项艰巨的任务。为便于剖析神经回路（这一过程需要对突触连接进行功能分析以及对相互连接的神经元进行形态学鉴定），我们在此介绍一种稳定同步八通道膜片钳记录方法。该方法可对4至8个同时记录的神经元和/或10至30个顺序记录的神经元之间的突触互连进行生理分析，还能对超过85%的记录中间神经元和超过99%的记录主神经元进行解剖学鉴定。我们描述了如何将该方法应用于啮齿动物组织切片；然而，它也可用于其他模式生物。我们还描述了对实现基于单光子和双光子显微镜、光遗传学和成像辅助的稳定同步四通道至八通道膜片钳记录系统至关重要的机械、电子、光学和软件程序的最新改进与优化。从仪器组装和软件安装开始到全面运行，搭建该系统可在3至4天内完成。
