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).

动物保养和实验方案由北卡罗莱纳大学教堂山分校的机构动物护理和使用委员会批准，并符合实验动物福利NIH办公室和协会的评估和实验动物保健国际资格认证的所有要求。 注:要获得幼虫ERG分析，公布的协议标准的斑马鱼的饲养和维护被雇用18。幼虫通过自然繁殖获得，住在一个14小时光照/ 10小时黑暗周期。该协议已优化幼虫5-7天受精后（DPF），但也最好能在老鱼的小修改的程序进行。在这里，使用野生型斑马鱼幼虫的TL应变5 DPF。 1，微管生产<ol><li>用拉1.5×0.86毫米（外径由内径）若干微量火抛光硼硅玻璃毛细管丝（熔融温度821°C）和P-97火焰山/布朗微量拉马装有箱热灯丝。使用该程序用于制作表1所述微量。 </li><li>请与相应的刻度标尺，在显微镜下每个微管，以确保提示是10-15微米，直径有一个平滑的顶部开口（ 即无锯齿边缘）。 </li><li>小心保存微量防止尖损伤和接触灰尘。存储选项包括培养皿实验室胶带，泡棉内衬盒，或市售的微量储存容器。 注意:其他微量拉拔器及玻璃毛细管可以使用，只要微量的正确直径和高品质的尖端得以实现。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 压力 </td><td> 热 </td><td> 拉 </td><td> 速度 </tD&gt; <td> 时间 </td></tr><tr><td> 500 </td><td> 560 </td><td> - </td><td>三十</td><td> 200 </td></tr><tr><td> 500 </td><td> 450 </td><td> - </td><td>三十</td><td> 200 </td></tr><tr><td> 500 </td><td> 410 </td><td> 55 </td><td> 40 </td><td> 200 </td></tr></tbody></table> 表1:程序用于使用P-97燃烧的/棕色微量拉马微量的制备配有框热灯丝微量移液器使用1.5×1.0 平方毫米（外径由内径）火抛光的硼硅玻璃毛细管长丝制成。 （熔融温度，821℃）。 2.缓冲准备<ol><li>使用过滤，充氧金鱼林格氏缓冲器19中的微电极的毛细管，并以饱和的聚乙烯醇（PVA）海绵在其上的幼虫被放置于实验。此外，使用E3<html>胚胎培养基或Hank氏平衡盐溶液。 </li><li>制备10倍金鱼林格氏溶液，如表2中所述，调整至pH 7.8，并消毒用0.22微米的过滤器，并存储10倍的库存在4℃。 </li><li>通过稀释10倍林格氏液为1x用去离子蒸馏水创建在实验当天的工作溶液。过滤器采用0.22微米的过滤系统。含氧化合物通过用95％氧气鼓泡4/10％CO 2的气体10分钟。帽紧紧事后以确保该溶液保持氧化。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>氯化钠</td><td>的1.25M </td></tr><tr><td>氯化钾</td><td> 26毫米</td></tr><tr><td> 氯化钙 </td><td> 25毫米</td></tr><tr><td> 氯化镁 </td><td> 10毫</td></tr><tr><td>葡萄糖</td><td> 100毫米</td></tr><tr><td> HEPES </td><td> 100毫米</td></tr></tbody></table>jove_content"&gt; 表2:10X金鱼林格氏溶液的制备。 3.平台电图<ol><li>执行ERG实验上的防振台法拉第笼内部，以改善信噪比。安装一个定制的钢平台使用六角螺母的防振动台。放置一个可移动的塑料平台，在桌子上的光源下，粘弹性聚氨酯聚合物吸震底部。 </li><li>定位相机与磁化的立场，在可移动的塑料平台，旨在下来。定位显微（其将保持在记录微电极）与第二磁化支架于可动塑料平台的右侧。确保相机和显微不会被其他设备的运动受到干扰，而它们不会阻挡从所述光源照明。 </li><li>将相机连接到视频监控和定位它来查看的眼睛幼虫用于放置电极，在适当的位置。 </li><li>确保安装正确地与铜线接地。检查噪声，放置在记录微电极的参考电极和尖端在35毫米培养皿中填充有林格氏液。检查设置的电噪声水平用示波器或ERG的装置的内置功能。噪音水平应不超过±10μV从基线。 </li></ol> 4.海绵准备<ol><li>切一小矩形的干PVA海绵将紧贴在35毫米培养皿。海绵的厚度不应大于盘的深度。使用一个工具刀，用干净的刀片进行切割。 </li><li>进行额外切成海绵容纳参比电极（无论是一个浅切口长度在海绵上的底部或竖直切过的小端1蝶式）。 </li><li>使用耐化学性的标记物标记上的海绵（其中幼虫将被放置），可以被用于定位所述相机的小圆点。 </li><li>浸泡PVA海绵林格氏液，直至饱和。取出并迅速涂抹在纸巾上的2-3倍。放置海绵在一个干净的35mm平皿。 </li><li>定位含有该塑料平台上的海绵，使得该标记可被相机可视培养皿。 </li></ol> 5.电极的制备注:斑马鱼设置包括在与林格氏溶液饱和PVA海绵和与角膜接触的记录电极接触的参考电极。参考电极由一个银/氯化银颗粒。记录电极是一个拉玻璃微量吸管由含Ag丝微电极保持器填充有林格氏溶液和保持。 <ol><li>通过5分钟（记录MICR浸泡在6-9％次氯酸钠（漂白剂），氯化电极oelectrode线）或15分钟（参考电极）。空气干燥上的Kimwipe 5分钟。 <ol><li>根据剪裁的样式在步骤4.2制成，将银/氯化银沉淀的参考电极的成（用于垂直蝶形切）或下（对于浅切纵向在底部）的海绵。附加参比电极引线到记录系统。 </li><li>或者，如果设置ERG具有空间限制或有从Ag / AgCl电极特别强光伏工件，通过琼脂盐桥连接的基准电极到海绵移动电极离开光路。 </li></ol></li><li>附上〜40厘米适当尺寸的管道，以5ml的非鲁尔锁注射器。充满林格氏液的注射器。微持有人持有的压力端口通常附带的适配器，以适应管与1/16内径"3/32"，1/8"或5/32"。 </li><li>填写1毫升非鲁尔锁注射器林格氏溶液和，使用Micro-FIL，小心地填充微电极保持器。防止气泡的形成。 </li><li>附上5毫升注射器与管的微电极保持器的压力端口，并用它来确保微电极保持器充满林格氏液。使用微FIL和1-ml注射器填充有林格氏液，从尖端填充微量的玻璃，并确保没有气泡存在。 </li><li>装上玻璃微到微电极夹，小心翼翼地保持电极丝直。一旦保护，​​使用5号毫升注射器的仔细强制通过微林格氏液，直至溶液小钱是尖端可见。的压力的注射器（当没有施加到角膜）偶发的应用将防止形成气泡，以及遮挡由于灰尘或盐的积累，在微量移液器尖端。 <ol><li>如果解决方案出来作为流，更换克姑娘微量，由于顶端开口太大或损坏。 </li></ol></li><li>小心地在显微记录微电极，并连接引线到记录系统。 </li></ol> 6.电图分析注:由于幼虫视网膜锥支配地位，高品质的ERG结果可以当根据间接白光低水平执行的准备用于记录获得的（&lt;1勒克斯）或具有短周期更高强度（&lt;1分钟）（ ≤250勒克司）工作灯。暗适应短时间的前记录仍然是必需的（见步骤6.7）。然而，实验可以在昏暗的红色或红外光使用红外感光摄像头进行。所有实验均在过滤灭菌（0.22微米）系统水从所述UNC斑马鱼养殖设施进行，但可使用的替代胚胎介质。 <ol><li>切纸巾方格测量大约1厘米2。 </li><li>如果测量孤立锥体质量受体电位，孵育3-5幼虫与500微米（±）-2-氨基-4- phosphonobutyric酸（APB）系统水5分钟。 注意:虽然APB是活动（L）的外消旋混合物和非活性（R） - 形式的AP4的，这是因为有效的为L-AP4和更便宜。 </li><li>麻醉3-5幼虫在系统水用0.02％（重量/体积）三卡因直至反应迟钝，约1-2分钟。 </li><li>使用巴斯德吸管和吸管泵在使用最小照度（≤250勒克司&lt;1分钟）解剖体视个人仔细的幼虫转移到纸巾广场。检查每个幼虫的位置，并选择一个候选者是背侧与未被遮挡的眼睛。 <ol><li>延长录音（&gt; 30分钟），由玻璃体内达到，但用细驼毛刷刷不包括头3％甲基纤维素保持幼虫湿润。 </li></ol></li><li>使用镊子，转移纸巾SQUARË与幼虫到潮湿的PVA海绵。 <ol><li>扩展记录（&gt; 30分钟），通过在含有蒸馏水的侧臂烧瓶一个airstone鼓泡气体申请水饱和的100％ 的 O 2气体的连续流过的幼虫。定位管道离开烧瓶的侧臂，传达附近幼虫的头部的加湿氧气。 注意:步骤6.4.1和6.5.1步骤将延长鱼16的寿命。 </li></ol></li><li>下最小照度，使用显微和照相机在鼻和眼的尾端部之间的中点来定位微电极前端轻轻压在角膜的背极限。 注意:电极末端到角膜的远末端区域的错位可导致极性相反ERG波形。 </li><li>允许幼虫到暗适应5-10分钟。 </li><li>使用阿瓦伊记录测试闪光响应从LED光源或光刺激提供的光拉布勒刺激和录音设备。调整协议参数，如闪光强度，闪长，闪颜色，背景亮度和颜色过滤器设置，以适应实验。 </li><li>当与试验结束时，根据AVMA / IACUC准则安乐死幼虫。 </li></ol>

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没有利益冲突的声明。

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展。例如，7旦上皮蒸腾效率后减小使之更难以保持鱼活着。另一个重要因素是在记录和参考电极的质量。拉玻璃毛细管时要防止粗犷吨要小心IPS。微量microforge可以采用火抛光拉电极和帮助，以提高信号采集。另外，保持的Ag / AgCl电极的质量是非常重要的。使用后，必须将它们用蒸馏水冲洗并空气立即干燥。而变暗的电极的外表面的可发生的时间和不影响性能，玷污（黄色至深红色）或应避免进站电极。避免用手直接处理电极，作为蛋白质的污染可以电极的行为产生不利影响。最后，应该指出的是，银/氯化银电极是感光，并可能导致闪工件如果没有保护。然而，我们发现这些文物是最小的（见图例图1和图2），不要用我们的幼虫斑马鱼的反应，以刺激闪烁测量干扰。可替代地，盐桥可以用来经由琼脂SA连接的Ag / AgCl电极的海绵LT桥将电极从光路。尽管盐桥中使用的直流（DC）的记录，以稳定的电极电位23，ERG记录利用交流电（AC）。因此，在本系统中的盐桥的唯一目的是，如果在特定的设置有空间限制或特别强光伏工件。 ERG具有超过其它技术的几个优点来测量视功能。的主要优点是，它是一个在体内的记录。的缺点是，特定的细胞的活性，必须从而不是直接测得的波形来推断，作为将是光感受器和其他细胞在视网膜的抽吸电极或膜片钳记录的情况。通常情况下，抽吸电极的录音，膜片钳记录和整个视网膜尔格具有药理学试剂可以容易地引入细胞来定义分子元件的优点响应，而这是在使用哺乳动物模型体内更加困难。斑马鱼幼体对这些药剂的通透性克服了这个缺点。 暗适应幼虫在没有药理学试剂分析产生的ERG的波形由b波为主，已观察到由多个其它基团2，9,24 -26的结果。我们证明具备通过服用药理学试剂穿透幼虫斑马鱼的能力的优势，以隔离锥体质量受体电位。幼虫与APB处理显示b-波的废除，使"一个波"，以可视化14-17。波形具有优良信噪比分别通过平均3次扫描获得的。变化的响应波形有时提到的，但Makhankov 等 2所报道，从同一动物中重复测量的方差较小比看到个人之间。因此，该方差可能是生物变异，而不是在该技术可变性的结果。 我们还利用ERG分析来检验使用双闪光灯范例，类似于发表的结果中的背景照明9，16的存在和不存在锥形恢复体内 。这是通过测定幼虫视网膜到不同的ISI连续闪烁反应的能力来完成的。当与药理学试剂或基因组工程策略，例如组合用作TALENS淘汰通路目标，斑马鱼是功能强大的系统为锥形适应和视力恢复的分子力学的体内研究。 我们的过程和设备可以很容易地适应于隔离检查幼虫的眼睛，老年斑马鱼或其他脊椎动物27,28。事实上，ERG刺激和记录系统我们聘请了主要设计用于在临床上使用，然后修改了实验小鼠。为适应斑马鱼幼体平台非常简单，并且很容易在其他实验室进行低成本复制。相似的配置，包括经典电设备可以为最小的成本来构造，并已被证明提供可靠的结果16。总体而言，这种技术是非常有益的研究人员在研究视功能的机制。

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。

<table><tbody><tr><td>Faraday cage</td><td>80/20 Inc</td><td>custom</td><td>Custom designed aluminum &quot;Industrial Erector Set&quot; for Cage framework</td></tr><tr><td>PVA sponge</td><td>Amazon</td><td>B000ZOWG1C</td><td>Provides a soft, moist platform for placement of zebrafish larvae</td></tr><tr><td>150 ml Sterile Filter systems</td><td>Corning</td><td>431154</td><td>Filtering solutions to prevent small articulates from blocking micropipettes</td></tr><tr><td>Espion E2</td><td>Diagnosys, LLC</td><td>contact</td><td>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</td></tr><tr><td>Colordome</td><td>Diagnosys, LLC</td><td>contact</td><td>Light stimulator with RGB LED and Xenon light sources for Ganzfeld ERG, more information at http://www.diagnosysllc.com</td></tr><tr><td>Micromanipulator</td><td>Drummond</td><td>3-000-024-R</td><td>Holding and positioning the recording microelectrode</td></tr><tr><td>Magnetic ring stand</td><td>Drummond</td><td>3-000-025-MB</td><td>Holding and positioning of the camera and refrence electrode</td></tr><tr><td>Lead extensions</td><td>Grass Technologies</td><td>F-LX</td><td>Spare female to male 1.5 mm lead cables for connecting electrodes</td></tr><tr><td>Male Pin to Female SAFELEAD Adaptor</td><td>Grass Technologies</td><td>DF-215/10</td><td>Connecting 2 mm pins to 1.5 headboard pins</td></tr><tr><td>Window screen frame (metal) and spline</td><td>Lowes or Home Depot</td><td>various</td><td>For attaching copper mesh to Faraday cage framework</td></tr><tr><td>Steriflip 50 ml filters</td><td>Millipore</td><td>SCGP00525</td><td>Filtering solutions to prevent small articulates from blocking micropipettes</td></tr><tr><td>BNC adaptor</td><td>Monoprice</td><td>4127</td><td>Connecting camera to BNC cable</td></tr><tr><td>BNC cable</td><td>Monoprice</td><td>626</td><td>Connecting camera to video adaptor</td></tr><tr><td>Camera lens</td><td>Navitar</td><td>1582232</td><td>Visualizing the positioning of the recording microelectrode onto the larval cornea</td></tr><tr><td>Camera coupler</td><td>Navitar</td><td>1501149</td><td>Visualizing the positioning of the recording microelectrode onto the larval cornea</td></tr><tr><td>Luna BNC to VGA + HDMI Converter</td><td>Sewell</td><td>SW-29297-PRO</td><td>BNC to VGA adaptor allowing camera image to project on computer monitor</td></tr><tr><td>APB</td><td>Sigma</td><td>A1910</td><td>mGluR6 agonist, blocks b-wave allowing analysis of the isolated cone mass receptor potential</td></tr><tr><td>Borosilicate glass</td><td>Sutter</td><td>BF-150-86-10</td><td>Fire- polished borosilicate glass (metling temperature = 821&amp;deg;C) with filament and dimensions of 1.5mm x 0.86 mm (outer diameter by inner diameter)&amp;nbsp;</td></tr><tr><td>P97 Flaming/Brown puller</td><td>Sutter</td><td>P97</td><td>For pulling glass micropipettes</td></tr><tr><td>Sorbothane sheet</td><td>Thorlabs</td><td>SB12A</td><td>Synthetic viscoelastic urethane polymer, placed under Passive Isolation Mounts and ERG platform to absorb shock and prevent slipping, can be cut to size</td></tr><tr><td>Breadboard</td><td>Thorlabs</td><td>B2436F</td><td>Vibration isolation platfrom for ERG stimulator and zebrafish specimen</td></tr><tr><td>Passive Isolation Mounts</td><td>Thorlabs</td><td>PWA074</td><td>Provides vibration isolation to breadboard</td></tr><tr><td>Copper mesh</td><td>TWP</td><td>022X022C0150W36T</td><td>To line Faraday Cage</td></tr><tr><td>Pipette pump</td><td>VWR</td><td>53502-233</td><td>Used with Pasteur pipettes to carefully transfer zebrafish larvae</td></tr><tr><td>Pasteur pipettes</td><td>VWR</td><td>14672-608</td><td>Used with Pipette pump to carefully transfer zebrafish larvae</td></tr><tr><td>Camera</td><td>Watec</td><td>WAT-902B</td><td>Visualizing the positioning of the recording microelectrode onto the larval cornea</td></tr><tr><td>Tricaine (MS-222)</td><td>Western Chemical</td><td>Tricaine-S</td><td>Pharmaceutical-grade anesthetic,</td></tr><tr><td>Micro-fil</td><td>WPI</td><td>MF28G-5</td><td>Filling microelectrode holder and microelectrode glass</td></tr><tr><td>Microelectrode holder</td><td>WPI</td><td>MEH2SW15</td><td>Holds glass microelectrode, connects to ERG equipment</td></tr><tr><td>Reference Electrode</td><td>WPI</td><td>DRIREF-5SH</td><td>Carefully break off last centimeter of casing to drain electrolyte and expose sintered Ag/AgCl pellet electrode</td></tr><tr><td>Reference Electrode (alternative)</td><td>WPI</td><td>EP1</td><td>Alternative to DRIREF-5SH. Ag/AgCl electrode that must be wired/soldered to connecting lead</td></tr><tr><td>Low-noise cable for Microelectrode holder</td><td>WPI</td><td>13620</td><td>Connecting recording microelctrode holder to adaptor/headboard</td></tr></tbody></table>

electroretinogram analysis of the visual response in zebrafish larvae

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。

通常情况下，尔格记录从斑马鱼幼虫在5旦，由于大量的研究发表的ERG记录在这个阶段9，16,20。使用白色LED的光的20毫秒刺激没有背景照明的暗适应的条件下，测定幼虫的响应。我们利用选自由Ganzfeld光刺激器和计算机控制器/记录器的市售的E​​RG系统。刺激器采用了严格控制的专有脉冲宽度调制（PWM）系统，以控制两者的背景和闪光刺激的亮度。使用专有的全差动放大器与硬件抗混叠滤波器驱动一个16位模拟 - 数字转换器（ADC）的反应被记录。刺激和反应录音被通过根据制造商的方案包含在设备的专用软件控制。我们的设备被编程为利用1kHz的采样速率，数字贝塞尔CASCADE过滤器设置为0.312赫兹和300赫兹和60赫兹陷波滤波器去除多余噪声之间的带通。在暗适应幼虫的b波的增加幅度的增加的光强度（ 图1）。在一个波通常遮盖的b波，并且不能可靠地检测到。 b-波可阻断孵育与代谢型谷氨酸受体（mGluR6）激动剂幼虫，4-膦酰基丁酸（APB）。这允许锥形质量受体电位（或"一个波"）被检测到。这种"一个波"响应的增加的幅度与光的强度增大（ 图2）。 附加范例可以用于测试超越基本视觉功能和灵敏度视觉参数。通过使用双闪光灯范例，一种可以测量锥光响应的，从最初的刺激（ 图3A）中恢复的能力。随着刺激间隔（ISI）的增加，第二响应的幅度增加，这表明从最初的刺激（ 图3B）的恢复。提出了APB隔离"一个波"的痕迹有三种扫描平均，符合斑马鱼幼体尔格利用类似的刺激范式2，9,16发表的报告。 <img alt="图1" src="/files/ftp_upload/52662/52662fig1.jpg" /> 图1:典型的ERG记录在5 DPF幼虫斑马鱼 。暗适应条件下，所获得的强度系列。鱼与强度等于1，10，25，50 125，250，500，1250，2500和5000坎德拉/ m 2的暴露于LED的白色光为20毫秒的持续时间。的光刺激的发作被表示为虚线垂直线。负电位的波（垂直箭头）难以分辨，而正潜在b-波（角度箭头）是波形的主峰。一个小的光伏伪影可以观察到发病的一个波前轻微阳性偏转。插图，平均b波反应振幅随光线已经使用了中落桨式（21），22贴合强度。误差棒表示SEM。 <img alt="图2" src="/files/ftp_upload/52662/52662fig2.jpg" /> 图2:从在5旦暗适应条件下获得的幼虫斑马强度系列记录，APB-分离锥质量受体电位 。刺激是20毫秒的LED白光与强度等于1，10，25，50 125，250，500，1250，2500和5000烛光/米2。光刺激的发作被表示为虚线垂直线。所述分离锥质量受体电位（"a波"）是波形（箭头）的主导因素。小光伏伪影可以观察到发病锥体响应的前一个次要正偏转。插图，平均反应振幅随光线已经使用了中落桨式贴合强度。误差棒表示SEM。 <img alt="图3" src="/files/ftp_upload/52662/52662fig3.jpg" /> 图3:利用双闪光灯范例锥质量受体电位记录 5旦的APB处理幼虫进行的白色光（LED光源）2 20毫秒闪烁，各为1000坎德拉/ m 2的强度（A）的与刺激间隔（ISI）等于2秒到2连续闪烁的响应。光曝光的标志是垂直虚线。第二响应的幅度比所述第一反应的低，表明不完全反应的恢复。（B）中的最大分离的锥形质量受体POTEN的比率TiAl基第二个刺激，对于每个ISI最初刺激的反应。最佳拟合非线性回归分析得到了应用。由于ISI的增加，响应第二刺激增长幅度相对于响应初始刺激表明感光灵敏度的渐进复苏。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52662/52662fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Electroretinogram Analysis of the Visual Response in Zebrafish Larvae at JoVE.com

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

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.

在斑马鱼幼虫的视觉反应的视网膜电图分析

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

electroretinogram-analysis-of-the-visual-response-in-zebrafish-larvae

Nanyang Technological University

Research

JoVE Journal

Neuroscience

15.5K 次观看.  University of North Carolina at Chapel Hill. 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.

视频: Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

斑马鱼的响应行为的检测，以测量光强的变化

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

科研

生物学

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

VisioTracker，创新的自动化到动眼神经分析方法

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

神经科学

The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish

Zebrafish are a proven model for vision research, however many of the earlier methods generally focused on larval fish or demonstrated a simple response. More recently adult visual behavior in zebrafish has become of interest, but methods to measure specific responses are new coming. To address this gap, we set out to develop a methodology to repeatedly and accurately utilize the optokinetic response (OKR) to measure visual acuity in adult zebrafish. Here we show that the adult zebrafish's visual acuity can be measured, including both binocular and monocular acuities. Because the fish is not harmed during the procedure, the visual acuity can be measured and compared over short or long periods of time. The visual acuity measurements described here can also be done quickly allowing for high throughput and for additional visual procedures if desired. This type of analysis is conducive to drug intervention studies or investigations of disease progression.

Zebrafish have been a valuable tool for many research areas. Here we demonstrate a method to elicit a visual response and calculate a functional visual acuity in the adult zebrafish.

在视动反应在斑马鱼视力的定量测量

Zebrafish are a proven model for vision research, however many of the  earlier methods generally focused on larval fish or demonstrated a simple  ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Electroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For functional measurements of visual performance, electroretinography (ERG) is an ideal non-invasive method, which has been well established in higher vertebrate species. This approach is increasingly being used for examining the visual function in zebrafish, including during the early developmental larval stages. However, the most commonly used recording electrode for larval zebrafish ERG to date is the glass micropipette electrode, which requires specialized equipment for its manufacture, presenting a challenge for laboratories with limited resources. Here, we present a larval zebrafish ERG protocol using a cone-shaped sponge-tip electrode. The novel electrode is easier to manufacture and handle, more economical, and less likely to damage the larval eye than the glass micropipette. Like previously published ERG methods, the current protocol can assess outer retinal function through photoreceptor and bipolar cell responses, the a- and b-wave, respectively. The protocol can clearly illustrate the refinement of visual function throughout the early development of zebrafish larvae, supporting the utility, sensitivity, and reliability of the novel electrode. The simplified electrode is particularly useful when establishing a new ERG system or modifying existing small-animal ERG apparatus for zebrafish measurement, aiding researchers in the visual neurosciences to use the zebrafish model organism.

Here, we present a protocol that simplifies the measurement of light evoked electroretinogram responses from larval zebrafish. A novel cone-shaped sponge-tip electrode can help to make the study of visual development in larval zebrafish using the electroretinogram ERG easier to achieve with reliable outcomes and lower cost.

一种新型锥形海绵尖电极在幼虫斑马鱼中的电录记录

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For ...

发育生物学

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

活斑马鱼幼虫的眼睛去除以检查视觉系统的神经支配依赖性生长和发育

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Photomotor Response Assay: A Method to Measure the Behavioral Response of Larval Zebrafish to a Sudden Change in Lighting Condition

Source: Steele, W. B., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/57938/" target="_blank">Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine</a>.&#160;J. Vis. Exp.&#160;(2018).
This video describes the photomotor response assay of zebrafish and fathead minnow larvae using an automated tracking software. The protocol measures the behavioral response of the larval fish following a sudden change in lighting condition.

光运动反应测定:一种测量斑马鱼幼鱼对照明条件突然变化的行为反应的方法

Source: Steele, W. B., et al. Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant ...

JoVE Encyclopedia of Experiments

蓝斑马（斑马鱼）

幼虫

Analyzing the Prey-Evoked Neuronal Activity in a Transgenic Zebrafish Larva

Source: Muto, A. et al.,&#160; <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57485">Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae.</a> J. Vis. Exp. (2018)
This video demonstrates the method for studying visual prey detection in transgenic zebrafish larvae using calcium indicator-based fluoresce imaging with their neurons expressing fluorescent calcium indicator complexes.

分析转基因斑马鱼幼虫中猎物诱发的神经元活动

1. Ablation of a Subpopulation of Neurons Using a Two-photon Laser Microscope
NOTE: If users plan on performing Ca imaging following ablation, use the ...