Walston Steven T, Chang Yao-Chuan, Weiland James D, Chow Robert H
Department of Biomedical Engineering, University of Southern California, Los Angeles, California.
USC Roski Eye Institute, University of Southern California, Los Angeles, California.
J Neurophysiol. 2017 Nov 1;118(5):2763-2769. doi: 10.1152/jn.00578.2017. Epub 2017 Aug 30.
Patch clamp recordings of neurons in the inner nuclear layer of the retina are difficult to conduct in a whole mount retina preparation because surrounding neurons block the path of the patch pipette. Vertical slice preparations or dissociated retinal cells provide access to bipolar cells at the cost of severing the lateral connection between neurons. We have developed a technique to remove photoreceptors from the rodent retina that exposes inner nuclear layer neurons, allowing access for patch clamp recording. Repeated application to and removal of filter paper from the photoreceptor side of an isolated retina effectively and efficiently removes photoreceptor cells and, in degenerate retina, hypertrophied Müller cell end feet. Live-dead assays applied to neurons remaining after photoreceptor removal demonstrated mostly viable cells. Patch clamp recordings from bipolar cells reveal responses similar to those recorded in traditional slice and dissociated cell preparations. An advantage of the photoreceptor peel technique is that it exposes inner retinal neurons in a whole mount retina preparation for investigation of signal processing. A disadvantage is that photoreceptor removal alters input to remaining retinal neurons. The technique may be useful for investigations of extracellular electrical stimulation, photoreceptor DNA analysis, and nonpharmacological removal of light input. This study reports a method for removing photoreceptors from rodent whole mount retina while preserving the architecture of the inner retina. The method enables easier access to the inner retina for studies of neural processing, such as by patch clamp recording.
在全层视网膜标本中，对视网膜内核层的神经元进行膜片钳记录很困难，因为周围的神经元会阻碍膜片吸管的路径。垂直切片标本或分离的视网膜细胞虽能接触到双极细胞，但代价是切断了神经元之间的横向连接。我们开发了一种从啮齿动物视网膜中去除光感受器的技术，该技术可暴露内核层神经元，便于进行膜片钳记录。将滤纸反复贴在分离视网膜的光感受器一侧并取下，能有效且高效地去除光感受器细胞，在退化的视网膜中还能去除肥大的米勒细胞终足。对去除光感受器后剩余的神经元进行活死检测，结果显示大部分细胞是存活的。从双极细胞进行的膜片钳记录揭示了与传统切片和分离细胞标本中记录到的类似反应。光感受器剥离技术的一个优点是，它能在全层视网膜标本中暴露视网膜内层神经元，用于研究信号处理。缺点是去除光感受器会改变剩余视网膜神经元的输入。该技术可能有助于细胞外电刺激研究、光感受器DNA分析以及光输入的非药物性去除。本研究报告了一种从啮齿动物全层视网膜中去除光感受器同时保留视网膜内层结构的方法。该方法能更方便地接触到视网膜内层，用于神经处理研究，如通过膜片钳记录。
