在斑马鱼幼虫的视觉反应的视网膜电图分析
Summary
We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.
Abstract
电图（ERG）是确定视网膜功能的无创性的方法电。通过放置在角膜的表面上的电极的方法，电活性响应于光而产生可以测量并且用于评估视网膜细胞在体内的活性。这个手稿描述了使用所述的ERG的测定视觉功能在斑马鱼。斑马鱼一直被用作模型脊椎动物发育由于易于基因抑制由吗啉代寡核苷酸和药理操纵。 5-10 DPF，只有视锥细胞的功能是在幼虫视网膜。因此，斑马鱼，不像其它动物，是对于锥形视觉功能的体内研究的强有力的模型系统。该协议使用标准麻醉，显微操作，并且是常见的执行斑马鱼的研究实验室立体显微镜协议。概述的方法使用标准的电EQuipment和低光相机的放置记录微电极引导到幼虫角膜。最后，我们将演示如何市售ERG刺激/录像机最初设计用于小鼠可以很容易地适合与斑马鱼中使用。幼虫斑马鱼的ERG提供在动物中测定锥体视觉功能的一个极好的方法，已经由吗啉代寡核苷酸注射改性以及诸如锌指核酸酶（ZFN）较新的基因组工程方法，转录因子样效应核酸酶（TALENS），和群集定期相互间隔短回文重复序列（CRISPR）/ Cas9，所有这些都大大地提高了效率和基因在斑马鱼的靶向效力。此外，我们采取的药理学试剂穿透斑马鱼幼体来评估向光响应的分子成分的能力的优势。这个协议概述了可以被修改并用来研究一个安装各种实验目标。
Introduction
电图（ERG）是已被广泛地应用于临床，用于确定在人类视网膜的功能的非侵入性的电生理学方法。响应于光激励的电活动是通过将记录电极对角膜的外表面测量的。刺激范例和响应波形的特性限定的视网膜神经细胞贡献的响应。该方法已被改编为与一些动物模型包括小鼠和斑马鱼中使用。典型的脊椎动物的ERG响应具有四个主要组成部分：一个波，这是从光感受器细胞活性衍生的角膜负电位; b-波，从接通双极细胞衍生的角膜正电位;的d波，角膜正电位解释为OFF双极细胞的活性;而C波，其中b波后出现几秒钟，反映了米勒的神经胶质细胞和RET活动伊纳勒色素上皮1-4。了解ERG分析的历史和原则在人类和动物模型的附加 引用了网上购书，Webvision，犹他州和文本的大学，如原则和愿景4,5临床电生理实践。
斑马鱼（斑马鱼）长期以来被青睐的一种模式脊椎动物发展，由于其快速成熟和透明性，其允许器官系统，行为分析的非侵入性的形态学分析和正向和反向遗传筛选（对于综述，参见Fadool和道林6）。斑马鱼幼体是高度适合于遗传学和药理学处理，其中，加上它们的高繁殖力时，使它们的优异动物模型，用于高通量生物分析。锥体在幼虫斑马鱼棒较高比率 – 大约1：1的比小鼠（〜3％浓S） -使其成为锥功能7-9的研究特别有用。
在脊椎动物的视网膜，锥杆10前发展。有趣的是，斑马鱼锥体可操作早在4单丝旦，允许锥体的选择性电生理分析在该阶段6中，11,12。与此相反，在杆ERG响应11和21旦13之间出现。因此，斑马鱼幼体在4-7 DPF功能作为全视网膜锥。然而，4-7单丝旦幼虫天然明视视网膜电响应由b波为主。 （+） – – 2-氨基-4-膦酰基丁酸（L-AP4），激动剂为代谢型谷氨酸（mGluR6）受体由上表达双极细胞，有效地阻止了生成的药理学试剂，如L-应用b-波和揭示了分离的锥形质量受体电位，（以下简称“a波”），14-17。
在这里，我们描述了一个简单而reliabl使用市售ERG设备设计用于与已被改编为与斑马鱼幼虫用鼠标E法进行ERG分析。该系统可用于对不同的遗传背景，以及那些具有药理剂处理，以帮助研究人员在信号通路有助于视觉灵敏度和光适应16的识别的斑马鱼幼虫。在这个协议中所列的实验程序将引导侦查员在使用ERG分析来解答各种有关视觉的生物学问题，并表现出灵活的ERG安装施工。
Protocol
Representative Results
Discussion
在这个协议中的幼虫斑马鱼的ERG记录一个简单的过程详细说明。这个程序允许视觉function.There的快速和全面的分析是整个应该牢记的程序几个关键步骤。斑马鱼幼虫应该是健康的实验前，以避免死亡时潜在的药物治疗，并确保在ERG录制长时间的生活。此外，重要的是，在实验中使用的幼虫是密切年龄匹配。这是由于视网膜（锥体亚型即 ，差动发展），同时也幼虫的整体形态和生理的快速发?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank members of the UNC Zebrafish Aquaculture facility for maintenance of the zebrafish. We would also like to thank Diagnosys, LLC for assistance with the setup of the ERG apparatus. Additional thanks go to Dr. Portia McCoy and the laboratory of Dr. Ben Philpot for assistance with electrophysiological methods. We also wish to thank Lizzy Griffiths for her illustration of a larval zebrafish. This work was supported by National Institutes of Health awards F32 EY022279 (to J.D.C) and R21 EY019758 (to E.R.W).
Materials
| Name of the Material/Equipment | Company | Catalog Number | Comments/ Description (optional) |
| Faraday cage | 80/20 Inc | custom | Custom designed aluminum "Industrial Erector Set" for Cage framework |
| PVA sponge | Amazon | B000ZOWG1C | Provides a soft, moist platform for placement of zebrafish larvae |
| 150 ml Sterile Filter systems | Corning | 431154 | Filtering solutions to prevent small articulates from blocking micropipettes |
| Espion E2 | Diagnosys, LLC | contact | Modular electrophysiology system capable of generating visual stimuli for any stimulator and digital recording and analysis of responses using propietary software, more information at http://www.diagnosysllc.com |
| Colordome | Diagnosys, LLC | contact | Light stimulator with RGB LED and Xenon light sources for Ganzfeld ERG, more information at http://www.diagnosysllc.com |
| Micromanipulator | Drummond | 3-000-024-R | Holding and positioning the recording microelectrode |
| Magnetic ring stand | Drummond | 3-000-025-MB | Holding and positioning of the camera and refrence electrode |
| Lead extensions | Grass Technologies | F-LX | Spare female to male 1.5 mm lead cables for connecting electrodes |
| Male Pin to Female SAFELEAD Adaptor | Grass Technologies | DF-215/10 | Connecting 2 mm pins to 1.5 headboard pins |
| Window screen frame (metal) and spline | Lowes or Home Depot | various | For attaching copper mesh to Faraday cage framework |
| Steriflip 50 ml filters | Millipore | SCGP00525 | Filtering solutions to prevent small articulates from blocking micropipettes |
| BNC adaptor | Monoprice | 4127 | Connecting camera to BNC cable |
| BNC cable | Monoprice | 626 | Connecting camera to video adaptor |
| Camera lens | Navitar | 1582232 | Visualizing the positioning of the recording microelectrode onto the larval cornea |
| Camera coupler | Navitar | 1501149 | Visualizing the positioning of the recording microelectrode onto the larval cornea |
| Luna BNC to VGA + HDMI Converter | Sewell | SW-29297-PRO | BNC to VGA adaptor allowing camera image to project on computer monitor |
| APB | Sigma | A1910 | mGluR6 agonist, blocks b-wave allowing analysis of the isolated cone mass receptor potential |
| Borosilicate glass | Sutter | BF-150-86-10 | Fire- polished borosilicate glass (metling temperature = 821°C) with filament and dimensions of 1.5mm x 0.86 mm (outer diameter by inner diameter) |
| P97 Flaming/Brown puller | Sutter | P97 | For pulling glass micropipettes |
| Sorbothane sheet | Thorlabs | SB12A | Synthetic viscoelastic urethane polymer, placed under Passive Isolation Mounts and ERG platform to absorb shock and prevent slipping, can be cut to size |
| Breadboard | Thorlabs | B2436F | Vibration isolation platfrom for ERG stimulator and zebrafish specimen |
| Passive Isolation Mounts | Thorlabs | PWA074 | Provides vibration isolation to breadboard |
| Copper mesh | TWP | 022X022C0150W36T | To line Faraday Cage |
| Pipette pump | VWR | 53502-233 | Used with Pasteur pipettes to carefully transfer zebrafish larvae |
| Pasteur pipettes | VWR | 14672-608 | Used with Pipette pump to carefully transfer zebrafish larvae |
| Camera | Watec | WAT-902B | Visualizing the positioning of the recording microelectrode onto the larval cornea |
| Tricaine (MS-222) | Western Chemical | Tricaine-S | Pharmaceutical-grade anesthetic, |
| Micro-fil | WPI | MF28G-5 | Filling microelectrode holder and microelectrode glass |
| Microelectrode holder | WPI | MEH2SW15 | Holds glass microelectrode, connects to ERG equipment |
| Reference Electrode | WPI | DRIREF-5SH | Carefully break off last centimeter of casing to drain electrolyte and expose sintered Ag/AgCl pellet electrode |
| Reference Electrode (alternative) | WPI | EP1 | Alternative to DRIREF-5SH. Ag/AgCl electrode that must be wired/soldered to connecting lead |
| Low-noise cable for Microelectrode holder | WPI | 13620 | Connecting recording microelctrode holder to adaptor/headboard |
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