Chrispell Jared D, Rebrik Tatiana I, Weiss Ellen R
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill.
Department of Ophthalmology, Duke University.
J Vis Exp. 2015 Mar 16(97):52662. doi: 10.3791/52662.
The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.
视网膜电图（ERG）是一种用于测定视网膜功能的非侵入性电生理方法。通过将电极放置在角膜表面，可以测量对光产生的电活动，并用于评估体内视网膜细胞的活性。本手稿描述了使用ERG来测量斑马鱼的视觉功能。由于吗啉代寡核苷酸和药理学操作易于实现基因抑制，斑马鱼长期以来一直被用作脊椎动物发育的模型。在5至10日龄的仔鱼期，幼虫视网膜中只有视锥细胞具有功能。因此，与其他动物不同，斑马鱼是研究体内视锥细胞视觉功能的强大模型系统。本实验方案使用了在进行斑马鱼研究的实验室中常见的标准麻醉、显微操作和体视显微镜技术。所概述的方法利用标准的电生理设备和低光照相机，将记录微电极放置在幼虫角膜上。最后，我们展示了一种最初设计用于小鼠的市售ERG刺激器/记录器如何轻松地适配用于斑马鱼。斑马鱼幼虫的ERG为检测通过吗啉代寡核苷酸注射以及更新的基因组工程技术（如锌指核酸酶（ZFN）、转录激活样效应核酸酶（TALEN）和成簇规律间隔短回文重复序列（CRISPR）/Cas9）修饰的动物的视锥细胞视觉功能提供了一种出色的方法，所有这些技术都大大提高了斑马鱼基因靶向的效率和效力。此外，我们利用药物穿透斑马鱼幼虫的能力来评估对光反应有贡献的分子成分。本实验方案概述了一种可以由具有各种实验目标的研究人员进行修改和使用的设置。
