Name: a simple behavioral assay for testing visual function in xenopus laevis
Uploaded: 2014-06-12T15:00:00.000+00:00
Duration: 8 min 34 s
Description: Watch this Scientific Journal Video about A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis at JoVE.com

这项工作是由来自美国国立卫生研究院的赞助:EY015748，EY017964（MEZ），和EY019517（ASV）。这项工作还得到了防盲研究无限制补助金眼科部和中央纽约的狮子。我们还要感谢我们的动物技术员，马修丽尼，他无微不至的关怀动物和加强在该视频的明星。

动物护理 非洲爪蟾在这项研究中使用的蝌蚪生长，并提出处理根据实验动物的护理和使用经州医科大学IACUC和引导程序。 1，预行为设置:蝌蚪<ol><li>从市售来源获得受精爪蟾胚胎或体外受精的卵母细胞中，如前所述6。 </li><li>将囊胚神经板期胚胎在18˚C 60毫米并载0.1X孕产妇死亡率[10 X Marc的改良林格（MMR）解决方案股票（10毫米MgCl 2的培养皿中，20毫米氯化钾，20毫米氯化钙2; 50毫米肝素钠; 1 M氯化钠;用50微克/毫升庆大霉素抗生素调节至pH 7.5，高压灭菌，并存储在RT）]。改变0.1X孕产妇死亡率每一天，并移除任何死胚。 </li><li>一旦胚胎达到Nieuwkoop和法贝尔阶段〜七月27日至30日，第切换é胚胎成0.1X孕产妇死亡率无抗生素，使肠道细菌的生长。 </li><li>长出蝌蚪，直到他们到达饲养阶段（阶段45）和移动为100毫米培养皿充满0.1X孕产妇死亡率。喂它们荨麻粉上清液隔日了一个星期，改变0.1X孕产妇死亡率的干预一天。 </li><li>在100mm的培养皿中一周后，将动物以半加仑罐填充有水蛙[0.5 g / L的瞬间海洋和2mM磷酸氢二钠（的Na 2 HPO 4:分子量141.96），pH为6.8。放他们，如同在步骤2.4，但换水，每周只有2-3次水时是阴天。维持动物在12小时light/12暗光周期与灯光早上6点打开。 </li><li>将半加仑的油箱与测试对象在一个白色的表面像一个实验室工作台底垫，至少有12小时的O / N，在测试之前。 </li><li>在阶段45到50的测试蝌蚪在下面的步骤3所述。 </li></ol> 2，预行为设置:技术设备吨<ol><li>设置在实验室的行为分析的区域一边。它应该是在一个安静的，与标准荧光灯低流量区域。 </li><li>制备的测试槽的动物。 嵌套，半加仑的测试坦克是由两部分组成:一个内罐，将举行水和蝌蚪;和外罐的视觉刺激。 <ol><li>为内槽，使在外侧角部与Sharpie笔，5厘米从底部的小标记;这是吃水线（ 图1A，绿色虚线）。 </li><li>另外，对于内槽，填充在储罐中的d​​ivets（在角部和中心）的惰性化合物，类似的Sylgard弹性体。注:蝌蚪会在这些领域徘徊，如果他们不填写。 </li><li>对于外槽，盖1罐用黑色导电胶带，另一半用白色纸巾图1B的外侧的恰好一半。 </li></ol></li><li>将坦克上的接种转盘。 </li><li>安装摄像头/三脚架，这样它上面的测试坦克。调整相机以允许测试区域的可视化， 如图1C所示 。 </li><li>将摄像头连接到电脑与安装了5个QuickTime播放器软件。打开相机电源。在QuickTime Player，下文件，选择"新建动画拍摄。"行为分析安装程序将在计算机上看到的时候了。 </li><li>悬垂一个轻量级的棉布在整个安装，以减少外部线索，可能会影响蝌蚪的行为，以及，降低水的表面上反射光。 </li><li>确保在35-50之间cd /米2的布措施的亮度。 </li></ol> 3，行为分析<ol><li>填写内部测试坦克5cm的水痕在步骤1.2.1与青蛙的水制成。 </li><li>用一个小网，轻轻将动物移入内部测试箱。 </li><li>开放的问答计算机，并使用外罐为指导uickTime软件，请确保相机能够以可视化并记录试验区。 </li><li>写在一张纸上，并记录在相机的动物名称，日期和时间。 </li><li>将测试箱插入外箱与箱的黑色侧向右图2。 </li><li>开始安排的罐后，立即记录该电影，并设置计时器，持续2分钟。 </li><li>当计时器的蜂鸣声，取出内测试箱，旋转外槽180°，然后将外槽内的内槽试验。启动定时器。注:黑方现在应该在另一边。 </li><li>总共十次重复步骤3.7，八个次。使用图2为指导，检查了每个试验。 </li><li>重复的行为分析上的两个独立的日子。 </li></ol> 4，视网膜干切断<ol><li>将动物在0.02％三卡因，直到他们反应迟钝到尾巴捏了＃3镊子。 </li><li>熔体1％琼脂糖在0.1X孕产妇死亡率，然后添加了60毫米的培养皿。一旦冷却，使一个小的矩形草皮在琼脂糖和蝌蚪加至用少量0.02％三卡因的草皮，从而使动物被部分地浸没在液体中。 </li><li>刺穿皮肤的眼用25号针头的背侧区域后方成45度角，而对支撑在另一侧镊子的动物。 </li><li>注意不要剪断静脉，它位于靠近视神经，小心伸入带＃5镊子孔，剪断视神经和翻转出来的方式。如果出现严重出血，由于不小心剪断动脉，然后迅速将动物在2％三卡因安乐死。注意:2％三卡因溶液可引起麻木的人类。这个解决方案应该带手套处理。 </li><li>恢复的动物在手术后，将动物在100毫米的皮氏培养皿用0.7X MMR和50微克/毫升庆大霉素20分钟。 NEXT，蝌蚪转移到污水箱中的青蛙水。让动物休息的O / N。 </li><li>第二天早上，测试如上所述（步骤3.1-3.9）的视功能。 </li></ol> 5。分析结果<ol><li>后的试验完成后，测量时间的蝌蚪停留在罐的黑色侧的量。 <ol><li>手动查看使用软件所显示的时间，并允许频繁暂停的视频。看视频显示时间。写下两分钟的审判，这是当内罐成功巢外罐内部，平方鉴于摄像头的开始。 </li><li>使用的蝌蚪的眼睛的位置来定义该罐的侧面将动物游泳。定义跨越从水箱的一侧，以仅当两个眼睛已经越过了黑/白线对其他。 </li></ol></li><li>写下每个间隔（以秒为单位）的开始和结束该动物花费在黑边。暂停和重新风的视频，以确保准确性。总计每次试验中的秒。 </li><li>输入这些数字进入附加行为工作表。工作表将计算的时间，该动物减去花费在坦克从总120秒试验和除以以秒为单位的总时间的黑边所花费的时间上的白边的比[（120 - 总秒数花在黑边）/ 120秒]。工作表的平均值这一比例在所有10项试验。 </li><li>利用学生的T检验，配对，双尾分布，以确定是否天的试验是显著（P≤0.05）。 </li></ol>

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The authors have nothing to disclose.

我们在这里报告，可以很容易地进行下一个星期由受过训练的最低限度实验室人员一个简单的视觉引导行为分析。而其他检测需要专门的设备和专业技术在动物行为，此法可快速测试，以确定视觉功能。另一个行为分析中，视觉回避试验中，已开发，以确定如何在顶盖有助于视觉感知在爪 10。该测定法测量空间的调谐和对比敏感度响应于移动与稳定的背景，因此，可以测量比简单的检测更专门的基于视觉的行为报告在这里。一种自动化系统的行为已有报道，可以测量所花费的时间上的槽8的一侧上的量。也有市售软件（ 如任何迷宫软件）已被用来测量时间斑马鱼的花费在黑暗或明亮的环境中9的量。在一个SSAY我们在这里报告并不需要购买这些类型的昂贵的设备或软件的，而是依赖于日常的试剂和设备来测量视觉功能。 在视觉引导法是在一个动物进行，以测试其视觉功能。当几个蝌蚪的行为分析结果进行比较彼此，我们观察到在治疗组动物之间差异无统计学意义。我们测试了连续六天3蝌蚪，也测试了另外五个动物连续三天，每天运行十次。在任一种情况下，我们发现其中未处理的或单一干切断组无统计学显著变化（P = 0.2067），但得到的统计学显著变化时这些动物相比，双干切断组（P = 0.002）。这是使用Prism 6.0 c的软件用Tukey多重比较试验（n = 8蝌蚪）运行的双因子ANOVA统计学检验测定。有最大之前的数据点数量的手术，我们建议您使用六天的行为分析。如果结果需要更快的与组织的退变少得，我们建议您使用三天的系统。 此法测试一个动物在同一时间。我们进行了试点研究，看看如果两个蝌蚪或多个可以同时测量。要确定是否有在一个坦克2蝌蚪影响了他们的偏爱罐的白边，我们分开，然后测试每个蝌蚪在一起。由于图像拍摄的坦克上面，如果蝌蚪游高于或低于对方的任何时间，我们发现很难区分它们。如果我们使用不同大小的蝌蚪，我们观察到较大的一个，会影响其中较小的一个游。未来的技术可以发展到基因标记蝌蚪，这将可以更好地跟踪相同大小的多个动物。 在几十tadpo的执行这个实验LES，我们观察到一些蝌蚪首选的白边比别人更一致。蝌蚪，其行为连续两天变化超过10％，我们将采取再次手术前第三天测试。这可能是因为另一个观察:我们注意到，把动物在白色背景上也做了区别在他们的偏爱罐的白边。有这额外的一天可能有助于调节一些蝌蚪更喜欢坦克的白边。此法的局限性是它只能测试视力功能。我们观察到的可变性可能是由于其它发育缺陷，除了降低了视觉功能或失明。出于这个原因，动物，表明在手术前不到60％的偏好并没有进一步测试。 我们还测试了年龄范围预变质蝌蚪。我们发现，蝌蚪年龄小于45级是非常小的，他们的眼睛很难用我们的，我们来跟踪BCAM。较旧的蝌蚪（阶段51 - 55岁以上）更心烦意乱;虽然他们最终花更多的时间在坦克的白边，他们似乎需要更长的时间来游泳。这些年长的动物均较大，因此，更易于跟踪，但需要在手术之前测试如上所述。与此相反，大多数蝌蚪在阶段45到50表现为预期与这些不同年龄的动物的话最好在将来的研究中使用。 昼夜光线的变化已被证明会影响视觉导向的行为反应在爪蟾 2。与此相一致的观察，我们注意到，同一种动物，迅速游到试验罐的白边在上午进行的试验，花了更长的时间在下午的行为测试。在进行了一些"动物相同/不同的时间&#39;试验后，我们确定了行为分析的最佳时间是从早上6点到下午1点。 我们还使用了数字handhELD摄像机通过火线低光照条件下，5来测试动物的连接。夜间或暗视觉这允许测试，其中动物使用其杆光感受器看到的。条件是通过简单地找到执行视觉导引测定所需的动物最少量的光的改变。这里介绍的实验测试白昼视觉，其中该动物大多采用其锥光感受器看到的。通过结合这些方法，这两种类型的视野，暗适应和明，可以进行测试。 我们使用此测定法，以与从多能干细胞过度本质表示眼睛字段转录因子和外在因素，头蛋白生长异位眼测试动物的视觉功能。从这些组织所产生的眼睛功能4,11。头蛋白也扮演前中枢神经系统发育12的作用，但使用这种视觉导引法与动物帽TR结合ansplant法，它在眼睛发育的作用被揭露。影响早期胚胎的形成，但其他外在因素还没有被证明有在眼睛发育的作用可能在诱导多能干动物帽细胞4,11视网膜前体的形成。使用这个行为分析，未来的研究可以判断这些新生成的眼睛的视觉功能。

非洲爪蟾已被用来作为一种模式生物来研究眼的形成。眼睛快速发展，成长到成熟，在不到一个星期的那些对视觉发育和功能的影响测试基因或通路。来测试视觉功能，视动和optomotor反应已被用于在斑马鱼和爪蟾蝌蚪，分别1,2。由于非洲爪蟾蝌蚪的眼睛比斑马鱼相对较小，此法需要使用专门的设备和训练有素的人员来检测在非洲爪蟾的微妙尾巴翻转和眼动行为。在非洲爪蟾的更健壮的行为是偏爱使用白色背景，在此3所述游泳在一个罐中。当放置在一个半黑/半白罐蝌蚪，预变质蝌蚪迅速游到水箱的白边。我们以前使用此测定法来确定是否多能干细胞衍生的眼睛的功能4。这里，我们报告了详细的版本该测定的，它可以被用来测试premetamorphic 爪蟾蝌蚪的视觉功能。 此法比optomotor响应试验简单，因为它只需要安装数码摄像机，数码相机的软件和标准的设备在大多数非洲爪蟾的实验室发现。此外，记录的响应并不需要特别的训练来统计结果。我们的代表性的结果表明，在相同组的蝌蚪，已经经历双视网膜干切断，游随机周围的罐中。我们还包括来自代表蝌蚪的行为分析结果，显示一只眼睛可以为视觉反应测试。工作表已经被包括在内，以便在试验过程中获得的号码可以被插入和分析。此表可用于确定测试的蝌蚪是否有一个视觉响应。

<table><tbody><tr><td>1/2 Gallon Flex-Tank with Cover</td><td>eNasco</td><td>SB19271M</td><td>Size: 5-3/8&quot; x 7&quot; x 3-3/4&quot;</td></tr><tr><td>Black electrical tape</td><td></td><td></td><td></td></tr><tr><td>White tissue paper</td><td></td><td></td><td></td></tr><tr><td>Large inoculating turntable</td><td>VWR</td><td>50809-022</td><td>Size: Dia 114.3 x&amp;nbsp; H 76.2 mm (4 1/2&amp;nbsp; x&amp;nbsp; 3&quot;)</td></tr><tr><td>Durasorb underpad</td><td>VWR</td><td>82004-836</td><td>Size: 43.2 x 60.1 cm (17 x 24&quot;)</td></tr><tr><td>Kimwipe</td><td>Krackeler Scientific, Inc.</td><td>1945-34155-CS</td><td></td></tr><tr><td>Standard tripod</td><td></td><td>Various</td><td></td></tr><tr><td>iSight camera or webcam</td><td>Apple</td><td>M8817LL/A</td><td>Good for larger tadpoles but small ones are difficult to see</td></tr><tr><td>Portable computer</td><td>Apple/PC</td><td>Various</td><td>We used a 13&quot; MacBook, 2 GHz Intel Core 2 Duo running MacOSX Lion 10.7.5</td></tr><tr><td>MiniDV handycam camcorder</td><td>SONY</td><td>DCR-HC42</td><td>Connected by firewire to the computer with a 6-conductor and 4-conductor alpha FireWire 400&amp;nbsp;</td></tr><tr><td>Handycam station</td><td>SONY</td><td>DCRA-C121</td><td>This can be used for connecting firewire to camera</td></tr><tr><td>QuickTime Player software</td><td>Quicktime</td><td>Version 10.1</td><td></td></tr><tr><td>26 G Needle (5/8&quot; length)</td><td>VWR</td><td>BD305115</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11295-10</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Disposables</td><td></td><td></td><td></td></tr><tr><td>Gentamicin sulfate [50 mg/ml]</td><td>Fisher Scientific</td><td>17-528Z</td><td>Stored at RT</td></tr><tr><td>Sylgard 184 silicone elastomer</td><td>Fisher Scientific</td><td>NC9644388</td><td></td></tr><tr><td>Instant ocean</td><td>Doctors Foster and Smith</td><td>CD-116528</td><td>Stock solution = 100 g/L stored at RT</td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma Aldrich</td><td>S0876</td><td>Stock solution = 0.4 M stored at RT</td></tr><tr><td>&lt;em&gt;In vitro&lt;/em&gt; fertilized embryos</td><td>eNasco</td><td>LM00490MX</td><td>100 embryos/unit</td></tr></tbody></table>

a simple behavioral assay for testing visual function in xenopus laevis

在青蛙的蝌蚪视功能测量， 非洲爪蟾 ，允许筛选的盲目性在活的动物。在视动反应是已经在测试的所有脊椎动物中观察到一个基于视觉的，反身的行为。蝌蚪眼睛小所以尾部翻转响应作为替代措施，这需要训练有素的技术人员来记录微妙的响应。我们开发了一种替代行为分析基础上的事实，蝌蚪喜欢当放置在一个用黑色和白色的边槽上槽的白边游泳。这里提出的分析是，创建一个容易测量的响应一种廉价，简单的替代方法。该装置由一个三脚架，摄像头和嵌套测试的坦克，在大多数非洲爪蟾实验室一应俱全。本文包括一个电影展示蝌蚪的行为，前后切断视神经后。为了测试一只眼睛的功能，我们还包括下列国家代表蝌蚪在每只眼睛的视网膜进行干切断连续数天的sentative结果。未来的研究可以制定此法的自动版本测试许多蝌蚪的视力一次。

以前的报告显示，premetamorphic 爪蟾蝌蚪喜欢游泳，黑色/ ​​白色背心的白边，并呼吁该试验中，背景色彩偏好分析3，我们为了测试的两种蝌蚪眼在视功能改变了这一试验不到一个星期。以这种方式，他们的眼睛可以被收集用于组织学检查。 我们在这里显示了如何响应是由于视觉线索。在电影1中，在左面板和右面板的蝌蚪是相同的动物。在左边是手术前和右侧的动物，切断两者视神经后的动物。我们打​​旋水以表明没有被添加到从黑边驱赶动物，或将其吸引到白色的一面。在电影中，我们提供的蝌蚪在底角的示意图，它显示了视神经的状态为每组动物。在电影1，正常，U正操作的蝌蚪背景颜色响应，在不到30秒的测试箱​​，而盲蝌蚪游漫无目的地周围的罐中。动画的速度增加，而实际时间记录在右上角的角落。 为了测试一只眼睛的视力，我们已经有来自动物的视神经被切断了一只眼睛，并在我们的实验测试的结果。在另一只眼睛的视神经被切断后两天的测试，并在随后的日子里的视觉引导行为再次测试。在图3中 ，曲线示出了时间的蝌蚪选择游泳上水箱的黑色侧的量。这是衡量连续六个天图3A。如上所述，右视神经切断后行为上2天并在左边进行测试，在4天的行为测试后的蝌蚪优选罐的白边随机地在时间图3C的50％。请注意，一个进制在过去的6个试验在1天至4花在罐的白边更多的时间。花在了黑边的时间更甚至在5日和6，当动物是双目失明。从每两天的结果取平均值在一起，并以图表的形式图3C呈现。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51726/51726fig1highres.jpg" width="500" /> 图1实验装置的视觉引导行为分析（一）内部测试罐保持透明，并标记来指示水位（绿色虚线在拐角处行）（B）外箱测试是覆盖在黑色对另一半和白棉纸绝缘胶带（C）蝌蚪是使用一个摄像头放置在三脚架上并连接到便携式计算机可视化。测试箱休整上的转盘接种，以便试验之间易于车削， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51726/51726fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51726/51726fig2highres.jpg" width="500" /> 图2原理图如何视觉引导行为实验进行的矩形表示测试箱，呈现出每次试验怎么半白/黑一半面积的变化。这可以在行为分析中用来标记的试验次数。右边的测试箱是一个特写两分法在蝌蚪和它的预期行为。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51726/51726fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 欣页="总是"&gt; <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51726/51726fig3highres.jpg" width="500" /> 图3。代表实验清点的时间百分比在测试箱的白边蝌蚪游（A）秒的金额用在坦克的黑边蝌蚪被测量并放置在一个表（B ）A中的表被转换成所花费的时间槽的白色侧的百分比。这是通过减去总试验时间（120秒）花在了黑边的秒和120秒除以完成。这是未经处理的（U）蝌蚪，有一个单一的干切断（SA），然后双干切断（DA）进行了测试。（C）该图显示了时间的平均百分比在白方（白棒）和黑边花（黑条）。用于制作图形的表如下所示。g3highres.jpg"目标="_blank"&gt;请点击这里查看该图的放大版本。 电影1。在白色背景优先检测。蝌蚪短视和盲目的动物在左面板和右面板的行为反应是相同的，但在右边的那些经历了双重的视网膜干切断。在底角的示意图表明视神经是否完好或损坏的（蓝条）。在右上角的计时器显示的时间。这部电影是加快以方便查看。一旦在空白区域，注意蝌蚪游如何接近白色和黑色和反向之间的边界。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51726/51726_Viczian_Movie1.mov" target="_blank">请点击此处观看该视频。</a> <table border="0" cellpadding="0" cellspacing="0" width="616"><tbody><tr height="22"><td height="22" style="height:22px;width:88px;"></td><td style="width:88px;">大Ÿ1 </td><td style="width:88px;"> 2天</td><td style="width:88px;"> 3天</td><td style="width:88px;"> 4天</td><td style="width:88px;"> 5天</td><td style="width:88px;"> 6天</td></tr><tr height="22"><td height="22" style="height:22px;">审判1 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr><tr height="21"><td height="21" style="height:21px;">试验2 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr><tr height="21"><td height="21" style="height:21px;">试用3 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr><tr height="21"><td height="21" style="height:21px;">试用4 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr><tr height="21"><td height="21" style="height:21px;">试用5 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 &lt;/ TD&gt; <td> 0 </td></tr><tr height="21"><td height="21" style="height:21px;">试用6 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr><tr height="21"><td height="21" style="height:21px;">试用7 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr><tr height="21"><td height="21" style="height:21px;">试验8 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr><tr height="21"><td height="21" style="height:21px;">试验9 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr><tr height="21"><td height="21" style="height:21px;">试用10 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td colspan="3">的时间比花在白边</td><td></td><td></td><td></td></tr><tr height="21"><td height="21" style="height:21px;">比1 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td></tr><tr height="21"><td height="21" style="height:21px;">比2 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td></tr><tr height="21"><td height="21" style="height:21px;">比3 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td></tr><tr height="21"><td height="21" style="height:21px;">比4 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td></tr><tr height="21"><td height="21" style="height:21px;">比5 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td></tr><tr height="21"><td height="21" style="height:21px;">比6 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td></tr><tr height="21"><td height="21" style="height:21px;">比7 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td></tr><tr height="21"><td height="21" style="height:21px;">比8 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td></tr><tr height="21"><td height="21" style="height:21px;">比9 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td></tr><tr height="22"><td height="22" style="height:22px;">比10 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td></tr><tr height="25"><td height="25" style="height:25px;">平均</td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td><td> 1.00 </td> &lt;TD&gt; 1.00 </td></tr><tr height="22"><td height="22" style="height:22px;"></td><td>第1天</td><td> 2天</td><td> 3天</td><td> 4天</td><td> 5天</td><td> 6天</td></tr><tr height="22"><td height="22" style="height:22px;">审判1 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 69 </td><td> 69 </td></tr><tr height="21"><td height="21" style="height:21px;">试验2 </td><td> 0 </td><td> 3 </td><td> 0 </td><td> 88 </td><td> 68 </td><td> 58 </td></tr><tr height="21"><td height="21" style="height:21px;">试用3 </td><td> 7 </td><td> 0 </td><td> 28 </td><td> 0 </td><td> 58 </td><td> 47 </td></tr><tr height="21"><td height="21" style="height:21px;">试用4 </td><td> 0 </td><td> 0 </td><td> 11 </td><td> 0 </td><td> 59 </td><td> 72 </td></tr><tr height="21"><td height="21" style="height:21px;">试用5 </td><td> 0 </td><td> 0 </td><td> 7 </td><td> 22 </td><td> 57 </td><td> 57 </td></tr><tr height="21"><td height="21" style="height:21px;">试用6 </td><td> 1 </td><td> 98 </td><td> 4 </td><td> 56 </td><td> 69 </td><td> 53 </td></tr><tr height="21"><td height="21" style="height:21px;">试用7 </td><td> 0 </td><td> 13 </td><td> 33 </td><td> 24 </td><td> 44 </td><td> 57 </td></tr><tr height="21"><td height="21" style="height:21px;">试验8 </td><td> 0 </td><td> 113 </td><td> 38 </td><td> 24 </td><td> 36 </td><td> 64 </td></tr><tr height="21"><td height="21" style="height:21px;">试验9 </td><td> 0 </td><td> 0 </td><td> 4 </td><td> 43 </td><td> 60 </td><td> 57 </td></tr><tr height="21"><td height="21" style="height:21px;">试用10 </td><td> 1 </td><td> 21 </td><td> 0 </td><td> 32 </td><td> 63 </td><td> 80 </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td colspan="2">的时间比花在白边</td<html> &gt; <td></td><td></td><td></td><td></td></tr><tr height="21"><td height="21" style="height:21px;">比1 </td><td> 100％ </td><td> 100％ </td><td> 100％ </td><td> 100％ </td><td> 43％ </td><td> 43％ </td></tr><tr height="21"><td height="21" style="height:21px;">比2 </td><td> 100％ </td><td> 98％ </td><td> 100％ </td><td> 27％ </td><td> 43％ </td><td> 52％ </td></tr><tr height="21"><td height="21" style="height:21px;">比3 </td><td> 94％ </td><td> 100％ </td><td> 77％ </td><td> 100％ </td><td> 52％ </td><td> 61％ </td></tr><tr height="21"><td height="21" style="height:21px;">比4 </td><td> 100％ </td><td> 100％ </td><td> 91％ </td><td> 100％ </td><td> 51％ </td><td> 40％ </td></tr><tr height="21"><td height="21" style="height:21px;">比5 </td><td> 100％ </td><td> 100％ </td><td> 94％ </td><td> 82％ </td><td> 53％ </td><td> 53％ </td></tr><tr height="21">高度:21px;"&gt;比6 </td><td> 99％ </td><td> 18％ </td><td> 97％ </td><td> 53％ </td><td> 43％ </td><td> 56％ </td></tr><tr height="21"><td height="21" style="height:21px;">比7 </td><td> 100％ </td><td> 89％ </td><td> 73％ </td><td> 80％ </td><td> 63％ </td><td> 53％ </td></tr><tr height="21"><td height="21" style="height:21px;">比8 </td><td> 100％ </td><td> 6％ </td><td> 68％ </td><td> 80％ </td><td> 70％ </td><td> 47％ </td></tr><tr height="21"><td height="21" style="height:21px;">比9 </td><td> 100％ </td><td> 100％ </td><td> 97％ </td><td> 64％ </td><td> 50％ </td><td> 53％ </td></tr><tr height="22"><td height="22" style="height:22px;">比10 </td><td> 99％ </td><td> 83％ </td><td> 100％ </td><td> 73％ </td><td> 48％ </td><td> 33％ </td></tr><tr height="24"><td height="24" style="height:24px;">平均</td><td> 99％ </td><td> 79％ </td><td> 90％ </td><td> 76％ </td><td> 51％ </td><td> 49％ </td></tr></tbody></table> 工作表1。白色背景偏好分析工作表。这个表有内置的，这样它可以用于输入数字和符结果的计算。

Watch this Scientific Journal Video about A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis at JoVE.com

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

非洲爪蟾蝌蚪喜欢上一个黑/白罐的白边游泳。此行为是由他们的愿景指导。基于这种情况，我们提出了一个简单的实验来测试蝌蚪的视觉功能。

一个简单的行为分析在检测视觉功能<em&gt;非洲爪蟾</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

a-simple-behavioral-assay-for-testing-visual-function-xenopus

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

9.8K 次观看.  Center for Vision Research, SUNY Eye Institute, Upstate Medical University.  非洲爪蟾蝌蚪喜欢上一个黑/白罐的白边游泳。此行为是由他们的愿景指导。基于这种情况，我们提出了一个简单的实验来测试蝌蚪的视觉功能。 

视频: A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

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 ...

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  ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

发育生物学

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

组织测定动物第移植（ACT）的含量非洲爪蟾</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

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.

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 ...

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

活的嗅觉途径的功能评价

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

光学相干层析成像和光运动响应在小鼠和大鼠中的结构和功能视觉系统读数

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Zebrafish Avoidance and Thigmotaxis Assay: A High-Throughput Method to Examine Larval Behavior in Response to Aversive Visual Stimulus

Source: Richendrfer, H., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/50622/" target="_blank">Automated High-throughput Behavioral Analyses in Zebrafish Larvae</a>. &#160;J. Vis. Exp.&#160;(2013)
The video describes the avoidance and thigmotaxis assay of zebrafish larvae. The video examines the behavior of zebrafish larvae in response to visual aversive stimulus.

斑马鱼回避和触动性测定:一种检查幼虫对厌恶视觉刺激的反应行为的高通量方法

Source: Richendrfer, H., et al. Automated High-throughput Behavioral Analyses in Zebrafish Larvae. &#160;J. Vis. Exp.&#160;(2013)
The video describes the ...

一个简单的行为分析在检测视觉功能

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