我们感谢肖恩Pelkowski的协助优化的行为检测。这项工作是由美国国家儿童健康和人类发展研究所，R01 HD060647和全国环境卫生科学研究所，F32 ES021342和R03 ES017755支持。

1。斑马鱼胚胎，并提高幼虫的收集<ol><li>高硼硅玻璃菜与“假”的草（由绿色纱）（ 图1）应在黎明和插入的坦克，离开了两个小时，以收集斑马鱼的胚胎。的玻璃培养皿中的胚胎应浇在一个手持式过滤器中，并用去离子水漂洗。然后应胚胎生长在蛋水。的卵水中含有60毫克/升的去离子水和0.25 mg / L的亚甲基蓝的，这是用来作为防霉剂​​的即时海上活动。 </li><li>根据个别实验假说，胚胎可以立即或在特定的发展阶段，使用毒物或药物治疗。的有毒物质和药品通常是溶解在DMSO中（在1,000倍的浓度），应进行进一步的稀释，在蛋水介质直接。 </li><li>在胚胎和幼虫的治疗药品或毒物，幼虫和胚胎可以被安置在深的陪替氏培养皿中在每50毫升的密度为约50-60幼虫，直到7单丝旦（蛋水溶液应该改变至少每隔一天，以避免真菌/细菌生长的行为分析死胚）。 </li></ol> 2。准备模具行为分析<ol><li>专门开发的塑料模具测量11.7厘米×7.6厘米×5毫米，在内部构建定制。模具需要创建车道用琼脂糖倒入单井Thermo Scientific的塑料板。单一孔板测量12.4厘米×8.1厘米×1.2厘米。 </li><li>的模具包含5个车道，在它的两侧是有角度的，在60℃。在模具中的各泳道与碱上面的顶部，这是最宽的，而底部的宽度为14毫米的18毫米3.5毫米高。有4毫米的车道在模具之间的间隙（ 图2）。 </li><li>为了准备车道融化的琼脂糖倒入50毫升（0.8％琼脂糖去离子D水与60 mg / L的即时海洋）在一个单一的孔板。然后，模具应被放置在非常缓慢的顶部的液体琼脂糖，以消除任何气泡的形成，并可以被删除时琼脂糖冷却（这需要大约45分钟）。 </li><li>琼脂糖车道应不早于前一天运行行为实验（以避免干燥的琼脂糖）的泳道，可在室温下保存在碗碟上的盖大约36小时。琼脂糖车道应该只被用于一项实验中，然后应被丢弃。 </li></ol> 3。图像捕捉<ol><li>可放置最多20个幼虫，每个泳道中的板。通常5幼虫每泳道使用，以便最准确的跟踪游泳速度，并减少每次实验所需要的幼虫数。可填充有蛋水带或不带取决于实验的药物或毒物的泳道。然而，泳道不应为f酶联免疫吸附试验（ELISA），直到它们被放置在成像机柜的所有的方式，这将防止溢出。为了保持一致性，幼虫应该有一个驯化时间的10分钟后，它们被放置在琼脂糖模具定位在笔记本电脑的屏幕的顶部。高效移动幼虫从培养皿琼脂糖车道的，将有助于减少幼虫的压力。这是最简单的幼虫被安置在浅水坦克或培养皿。 </li><li>成像柜包括佳能数码相机使用时间推移摄影和一台笔记本电脑。摄像机应放置在机柜的顶部，旨在实现了15.6英寸屏幕的笔记本电脑屏幕朝上（ 图3），应放在机柜的底部。四大板块应手工定位的笔记本电脑屏幕上直接。此时车道可以封顶蛋水或化学处理，以便它是级别的顶部车道（以消除阴影的边缘上的升麻醉中的图像）。 </li><li>使用PowerPoint演示文稿的厌恶刺激的幼虫。在过去移动红球，斑马鱼幼虫在6或12多孔板15,17,18。当前的PowerPoint开始的15分钟，然后15分钟的移动板的上半部分（ 图4）上的红色条的空白白色背景。为了消除，幼虫过热，最好是购买一台笔记本电脑，屏幕温度不超过28°C。为了避免琼脂糖车道内的液体的蒸发，最大成像时间应保持在低于1小时。 </li><li>数码相机应该被编程为延时摄影，拍照每6秒每实验共300张图片。然而，频率和长度可以调整，根据实验和行为量化。在过去的扩展成像时间被雇用，每幅图像之间使用较长的时间间隔。 CAMER一个可以设置在较低的分辨率进行成像以视频速度（每秒30帧）。虽然较低的分辨率限制到一个单一的多孔板，录音录像适合成像快速游泳赛事15。 </li></ol> 4。图像分析<ol><li> ImageJ的图像应打开并使用内部专门写宏。宏自动分割可以被删除，使红色的颜色的颜色通道，减去背景，应用一个阈值，并确定通过颗粒分析幼虫。五车道幼虫分析是最当前的宏观Zebrafish_macro25k。使用提示宏设置的图像数量，颜色要减去，图像的阈值，等等。 </li><li>所有的图像后运行通过ImageJ的宏，将显示一个结果文件将包含X，Y坐标个别幼虫为每个影像随着图像数量和车道数量。 </LI&gt; <li>成果文件应当以Excel格式保存，并根据的空白背景与移动栏背景以及数排序。应使用Excel模板，有方程内置自动确定井安置幼虫，幼虫之间的距离，移动速度，其余金额。最当前的Excel模板25ib创建在Creton实验室，这是可根据要求提供。图应该建立到Excel表显示各治疗组，治疗组和对照组之间的比较用t检验。进一步的统计分析，可以采用SPSS。 </li></ol>

<ol>
	<li>Gerlai, R., Lahav, M., Guo, S., Rosenthal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drinks+like+a+fish:+zebra+fish+(Danio+rerio)+as+a+behavior+genetic+model+to+study+alcohol+effects.">Drinks like a fish: zebra fish (Danio rerio) as a behavior genetic model to study alcohol effects.</a> Pharmacol. Biochem. Behav. 67, 773-782 (2000).</li><li>Selderslaghs, I. W. T., Hooyberghs, J., De Coen, W., Witters, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotor+activity+in+zebrafish+embryos:+A+new+method+to+assess+developmental+neurotoxicity.">Locomotor activity in zebrafish embryos: A new method to assess developmental neurotoxicity.</a> Neurotoxicol. 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Teratol. 33, 742-751 (2011).</li><li>Stewart, A., Wu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+modulation+of+anxiety-like+phenotypes+in+adult+zebrafish+behavioral+models.">Pharmacological modulation of anxiety-like phenotypes in adult zebrafish behavioral models.</a> Prog. Neuropsychopharmacol. Biol. Psychiatry. 35, 1421-1431 (2011).</li><li>Eddins, D., Cerutti, D., Williams, P., Linney, E., Levin, E. 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(20), e923 (2008).</li><li>Pelkowski, S., Kapoor, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+high-throughput+imaging+system+for+automated+analyses+of+avoidance+behavior+in+zebrafish+larvae.">A novel high-throughput imaging system for automated analyses of avoidance behavior in zebrafish larvae.</a> Behav. Brain Res. 223, 135-144 (2011).</li><li>Richendrfer, H., Pelkowski, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+developmental+toxicity+by+automated+analyses+of+behavior+in+zebrafish+larvae.">Assessment of developmental toxicity by automated analyses of behavior in zebrafish larvae.</a> Unpublished observations. , (2012).</li><li>Richendrfer, H., Pelkowski, S., Colwill, R. M., Creton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+edge:+pharmacological+evidence+for+anxiety-related+behavior+in+zebrafish+larvae.">On the edge: pharmacological evidence for anxiety-related behavior in zebrafish larvae.</a> Behav. Brain Res. 228, 99-106 (2012).</li><li>Richendrfer, H. A., Pelkowski, S., Colwill, R., Creton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+sub-chronic+exposure+to+chlorpyrifos+reduces+anxiety-related+behavior+in+zebrafish+larvae.">Developmental sub-chronic exposure to chlorpyrifos reduces anxiety-related behavior in zebrafish larvae.</a> Neurotoxicol. Teratol. , (2012).</li><li>Egan, R. J., Bergner, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+behavioral+and+physiological+phenotypes+of+stress+and+anxiety+in+zebrafish.">Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish.</a> Behav. Brain Res. 205, 38-44 (2009).</li><li>Colwill, R., Creton, R., Kalluef, A., Stewart, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish protocols for neurobehavioral research. 66, (2012).</li></ol>

作者宣称，他们有没有竞争经济利益。

虽然我们不断地改善我们的新型行为检测，它一直是有用的检测在斑马鱼幼虫15的的避免和thigmotaxis行为。许多试验已经进行优化的化验结果，如所用颜色刺激，理想的数字幼虫每车道，长度和行为检测。以前，我们用多孔板（6孔或12孔）15,17,18。然而，最近我们已经创建了新型车道模具的幼虫，让我们聚集了大量行为的措施，在一个单一的含量16（ 图5）创建一个更大的游动空间。其他修改包括显示的PowerPoint演示的变化（改变检测运动或长度）的大小（我们也有更狭窄的小巷模具）车道。 目前，这种高通量的自动化系统是独一无二的，它能够测量大范围的行为zebrafish幼虫，如速度，回避，接近其他幼虫和多车道板thigmotaxis的，在相同的时间。结果可以得到快速，在成像时，可以分析大量的幼虫。该系统是既便宜构建和快速，易于设置。这个系统的一个局限是无法评估的3-D运动在斑马鱼幼虫。跟踪成年斑马鱼的自动化系统，具有3-D的能力，并能识别范围更广的行为，例如向上或向下运动内的水柱10,19。另一个限制是，目前我们的成像系统未优化视频速度高通量分析。视频速度成像是可以将相机设置为较低的分辨率15时，但是这限制了分析，以一个单一的板块。 在使用新创建的“车道”的方法，精确地检测几个部分需要执行。当幼虫放置在t他车道，这是至关重要的，以确保液体水平很浅，直至将板定位在笔记本电脑的屏幕的顶部。如果车道得太满了液体，幼虫会逃逸到该板的外周。此外，当插入到琼脂糖模具，必须采取降低模具非常缓慢。如果模具的插入速度太快，气泡将形成在琼脂糖中，将确定的图像J宏作为额外的幼虫。据表示，如果琼脂糖车道甚至有几个气泡，最好是结交新朋友。 在未来，我们希望优化我们的行为的检测，以分析其他复杂的行为，如在斑马鱼幼虫的学习和研究如何学习可能会受到暴露在早期发展中的有毒物质和药品。我们目前正在检测分析学习行为，即行为的结果可能有助于确定哪些脑区，可能是有用的在开发过程中受到某些毒物或药物。自动检测已开发出用于测量学习行为的斑马鱼幼虫20和这些实验可能是可修正通过使用强大的回避反应在多车道板的高通量筛选。 我们建议，在今后的研究中，这种行为可以用来检测测试了大量的药品和有毒物质的发展的影响。此类研究提供了丰富的信息，对特定的风险因素，有助于制定出更好的怀孕妇女和儿童的健康和安全指引。

斑马鱼的遗传，发育和行为科学的1-4成为一种流行的模式。他们从他们的绒毛膜孵化2-3天受精后（DPF），充分发展4-5 DPF功能的器官，并表现出了大量的行为由7 DPF 5,6。斑马鱼幼虫非常适合高通量分析，因为其体积小7,8。软件是市售9-14斑马鱼幼虫和成虫的行为自动分析。然而，这种软件可以是昂贵的，具有有限的选择斑马鱼幼虫多孔板测量复杂的行为。 我们创建了一个新的高吞吐量的成像系统，是廉价的设置和可以量化7单丝旦斑马鱼幼虫15,16中的一些不同的行为。该系统使我们能够快速，高效地测试后胚胎暴露了一些微妙的行为异常药品和环境毒物16-18。 该系统的建成使用木制柜，房子在机柜顶部的一台数码相机。相机朝下柜被放置在一台笔记本电脑，屏幕朝上15的底部。时间推移成像是用来捕捉幼虫放置在小巷里。幼虫可以安装在四个多井或者多车道板被定位在笔记本电脑的屏幕的顶部。我们使用PowerPoint演示文稿作为一个厌恶刺激，幼虫回应移开，以避免向边缘（thigmotaxis）15,17和游泳。 ImageJ的图像导入自动化的宏用来分裂的颜色通道，减去背景和应用的阈值，以确定个别幼虫。坐标都列出了每个幼虫在每个图像，并可以插入一个Excel文件，我们用它来避免和thigmota量化红双喜的行为，鱼鱼类距离，游泳速度，而其余16量。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		 </tr>
		 <tr>
			<td>Instant Ocean</td>
			<td>That Pet Place</td>
			<td>198262</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Agarose</td>
			<td>Fisher</td>
			<td>BP1356-100</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Methylene Blue</td>
			<td>That Pet Place</td>
			<td>214325</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>One well plates</td>
			<td>Fisher</td>
			<td>12-565-493</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Digital camera</td>
			<td>Canon </td>
			<td>EOS Rebel T1i</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaging Cabinets</td>
			<td>WoodCraft Towers</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Laptops</td>
			<td>Acer Aspire</td>
			<td>Any is good as long as it has a 15.6 in. LCD screen with 1366 x 768 pixel resolution and a brightness of 220 cd/m2.</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Camera Lens</td>
			<td>Canon</td>
			<td>EF-S 55 - 250 mm f/4.0 - 5.6 IS zoom lens</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Plastic diffuser</td>
			<td>Pendaflex </td>
			<td>52345</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Software</td>
		 </tr>
		 <tr>
			<td>PowerPoint 2010</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td><a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a></td>
		 </tr>
		 <tr>
			<td>Excel 2010</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Statistical software</td>
			<td>SPSS 20</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

automated high-throughput behavioral analyses in zebrafish larvae

我们已经创建了一个新的高通量成像系统行为的分析，在7日龄的斑马鱼幼虫在多车道板。该系统测量自发行为和厌恶刺激的反应，这是显示PowerPoint演示文稿的幼虫通过 。记录的图像分析与ImageJ的宏，它自动将颜色通道，减去背景，并应用一个阈值，以确定个别幼虫放置在小巷里。然后，我们可以到一个Excel工作表导入坐标量化游泳速度，优先车道的边缘或侧面，休息行为，thigmotaxis，幼虫之间的距离，和回避行为。微妙的变化，行为很容易被发现使用我们的系统，它可用于接触环境毒物或药物后的行为分析。

在我们较早的检测，使用反弹球厌恶刺激，野生型幼虫，未经处理的响应通过游泳在阱（回避行为）对的阱（行为thigmotaxis）15的边缘移动的球。我们后来证实在这个实验thigmotaxis行为是衡量焦虑相关行为的斑马鱼幼虫17。有显着差异的幼虫运动，远离球和偏好的边缘时相比，空白背景。这些行为也被证实在我们的新的检测使用移动的红色条和更强大的16。此外，我们现在可以品尝包括游泳速度，其余井的一端或侧面的偏爱，和鱼之间的距离（ 图5）在一个单一的检测数量较大的行为。控制幼虫生长在蛋水优先增加菜和车道的边缘FTER它们都带有一个厌恶刺激（移动红酒吧）。当幼虫生长在含有1微克/毫升DMSO的溶剂，通常用为1,000倍的储备溶液以溶解各种药品和有毒物质的蛋水时，得到类似的结果。 幼虫用鸡蛋水和DMSO（对照）和有不同浓度的非有机食品中常见的有机磷农药，代表性的结果如图5所示。所示的结果是从一个实验中的采样。然而，当重复的，结果表明游泳速度和thigmotaxis行为的改变而低浓度的有机磷农药，它模仿人类的食物消费水平18。 <img alt="图1" src="/files/ftp_upload/50622/50622fig1.jpg" /> 图1。收集盘。玻璃耐热菜是用来收集电子邮件mbryos从成人鱼缸。从高硼硅菜盖被切断，插入塑料电网和绿色纱缝上塑料电网。这将创建一个模仿自然的环境氛围的成年斑马鱼养殖。 <img alt="图2" fo:content-width="/files/ftp_upload/50622/50622fig2highres.jpg" fo:width="6in" src="/files/ftp_upload/50622/50622fig2.jpg" /> 图2。塑料模具和琼脂糖车道。 A）的模具如左图所示。 0.8％琼脂糖倒入一孔板，模具被缓慢插入，然后除去琼脂糖冷却时，B）在右边显示板琼脂糖的塑料模具中创建的车道。 <img alt="图3" src="/files/ftp_upload/50622/50622fig3.jpg" /> 图3。成像柜成像柜speciallŸ内置在我们的实验室和用于高通量行为分析。一个15万像素的数码相机被安装到机柜的顶部朝下，以收集时间的推移幼虫的递板放置一台笔记本电脑的屏幕上的图像。板和屏幕之间的是一个塑料的扩压器，用于在收集到的图像，以防止波纹图案。 <img alt="图4" fo:content-width="/files/ftp_upload/50622/50622fig4highres.jpg" fo:width="4.5in" src="/files/ftp_upload/50622/50622fig4.jpg" /> 图4。空白背景和PowerPoint厌恶的刺激 ， 这是当前PowerPoint，用于唤起在斑马鱼幼虫的行为变化。它提供了强大的A）空白的背景和B）移动红杠之间的行为差异。 <img alt="图5" fo:content-width="/files/ftp_upload/50622/50622fig5highres.jpg" fo:width="5.5in" src="/files/ftp_upload/50622/50622fig5.jpg" /> 图5。定量高通量检测的行为，例如，从我们的行为，我们使用Excel工作表的x，y坐标的幼虫内检测量化的行为。白条幼虫暴露到一个空白的背景，红柱红动条的幼虫暴露在PowerPoint中显示数据显示数据。这些图表显示测量结果，可以得到从行为分析）幼虫占车道，B）幼虫百分比的车道上，C）幼虫上的车道边缘的百分比，D）之间的距离鱼（毫米），E），游泳速度的幼虫（毫米/分钟），F）占其余的幼虫。在所示的曲线图，数据是从treatm耳鼻喉科的幼虫用DMSO控制和几种农药的浓度从0.001到0.1微米（发现在人类饮食中常见的水平）。 <a href="https://www.jove.com/files/ftp_upload/50622/50622fig5large.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Automated High-throughput Behavioral Analyses in Zebrafish Larvae at JoVE.com

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

本实验室开发了一种新的高通量自动化的成像系统，是有用的，用于检测的几个不同的行为，在7天龄的斑马鱼幼虫。该系统可用于检测幼虫后已暴露于环境的有毒物质或药品的行为的微妙的变化，。

在斑马鱼幼虫的高通量自动化的行为分析

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

automated-high-throughput-behavioral-analyses-in-zebrafish-larvae

Research

JoVE Journal

Neuroscience

15.1K 次观看.  Brown University. 本实验室开发了一种新的高通量自动化的成像系统，是有用的，用于检测的几个不同的行为，在7天龄的斑马鱼幼虫。该系统可用于检测幼虫后已暴露于环境的有毒物质或药品的行为的微妙的变化，。

视频: Automated High-throughput Behavioral Analyses in Zebrafish Larvae

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

科研

神经科学

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to a certain degree, many animals have to be analyzed, to obtain statistically significant data. This in turn requires a computer assisted automated quantification of locomotion patterns. To obtain high contrast images of almost translucent and small moving objects, a novel imaging technique based on frustrated total internal reflection called FIM was developed. In this setup, animals are only illuminated with infrared light at the very specific position of contact with the underlying crawling surface. This methodology results in very high contrast images. Subsequently, these high contrast images are processed using established contour tracking algorithms. Based on this, we developed the FIMTrack software, which serves to extract a number of features needed to quantitatively describe a large variety of locomotion characteristics. During the development of this software package, we focused our efforts on an open source architecture allowing the easy addition of further modules. The program operates platform independent and is accompanied by an intuitive GUI guiding the user through data analysis. All locomotion parameter values are given in form of csv files allowing further data analyses. In addition, a Results Viewer integrated into the tracking software provides the opportunity to interactively review and adjust the output, as might be needed during stimulus integration. The power of FIM and FIMTrack is demonstrated by studying the locomotion of Drosophila larvae.

FIM is a novel, cost effective imaging system designed to track small moving objects such as C. elegans, planaria or Drosophila larvae. The accompanying FIMTrack program is designed to deliver fast and efficient data analysis. Together, these tools allow high-throughput analysis of behavioral traits.

FIM成像和FIMtrack：两个新的工具允许高吞吐量和成本效益分析步态

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

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 ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene.

斑马鱼癫痫模型中的行为和生理分析

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research ...

Using Zebrafish Larvae to Study the Pathological Consequences of Hemorrhagic Stroke

Despite being the most severe subtype of stroke with high global mortality, there is no specific treatment for patients with intracerebral hemorrhage (ICH). Modelling ICH pre-clinically has proven difficult, and current rodent models poorly recapitulate the spontaneous nature of human ICH. Therefore, there is an urgent requirement for alternative pre-clinical methodologies for study of disease mechanisms in ICH and for potential drug discovery.
The use of zebrafish represents an increasingly popular approach for translational research, primarily due to a number of advantages they possess over mammalian models of disease, including prolific reproduction rates and larval transparency allowing for live imaging. Other groups have established that zebrafish larvae can exhibit spontaneous ICH following genetic or chemical disruption of cerebrovascular development. The aim of this methodology is to utilize such models to study the pathological consequences of brain hemorrhage, in the context of pre-clinical ICH research. By using live imaging and motility assays, brain damage, neuroinflammation and locomotor function following ICH can be assessed and quantified.
This study shows that key pathological consequences of brain hemorrhage in humans are conserved in zebrafish larvae highlighting the model organism as a valuable in vivo&#160;system for pre-clinical investigation of ICH. The aim of this methodology is to enable the pre-clinical stroke community to employ the zebrafish larval model as an alternative complementary model system to rodents.

Here we present a protocol to quantify brain injury, locomotor deficits and neuroinflammation following bleeding in the brain in zebrafish larvae, in the context of human intracerebral hemorrhage (ICH).

斑马鱼幼虫研究出血性脑卒中的病理后果

Despite being the most severe subtype of stroke with high global mortality, there is no specific treatment for patients with intracerebral hemorrhage ...

Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish

Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This alternate immersion protocol is a noninvasive, step-wise method of inducing hyperglycemia for up to eight weeks. Adult zebrafish are alternately exposed to sugar (glucose) and water for 24 hours each. The zebrafish begin treatment in a 1% glucose solution for 2 weeks, then a 2% solution for 2 weeks, and finally a 3% solution for the remaining 4 weeks. Compared to water-treated (stress) and mannitol-treated (osmotic) controls, glucose-treated zebrafish have significantly higher blood sugar levels. The glucose-treated zebrafish show blood sugar levels of 3-times that of controls, suggesting that after both four and eight weeks hyperglycemia can be achieved. Sustained hyperglycemia was associated with increased Glial Fibrillary Acidic Protein (GFAP) and increased nuclear factor Kappa B (NF-kB)&#160;levels in retina and decreased physiological responses, as well as cognitive deficits suggesting this protocol can be used to model disease complications.

This protocol noninvasively induces hyperglycemia in zebrafish for up to 8 weeks. Using this protocol, an in-depth study of the adverse effects of hyperglycemia can be made.

交替浸入葡萄糖中，在斑马鱼中产生长期血糖超血症

Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

生态HIV脑感染的老鼠模型

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae

Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between these features may be interrogated by controlling the phase transition of a disease-associated protein in a vulnerable cell type, although this experimental approach has been limited so far. Here, we describe a protocol to induce phase transition of the RNA/DNA-binding protein TDP-43 in spinal motor neurons of zebrafish larvae for modeling cytoplasmic aggregation of TDP-43 occurring in degenerating motor neurons in amyotrophic lateral sclerosis (ALS). We describe a bacterial artificial chromosome (BAC)-based genetic method to deliver an optogenetic TDP-43 variant selectively to spinal motor neurons of zebrafish. The high translucency of zebrafish larvae allows for the phase transition of the optogenetic TDP-43 in the spinal motor neurons by a simple external illumination using a light-emitting diode (LED) against unrestrained fish. We also present a basic workflow of live imaging of the zebrafish spinal motor neurons and image analysis with freely available Fiji/ImageJ software to characterize responses of the optogenetic TDP-43 to the light illumination. This protocol enables the characterization of TDP-43 phase transition and aggregate formation in an ALS-vulnerable cellular environment, which should facilitate an investigation of its cellular and behavioral consequences.

We describe a protocol to induce phase transition of TAR DNA-binding protein 43 (TDP-43) by light in the spinal motor neurons using zebrafish as a model.

TDP-43在斑马鱼幼虫脊髓运动神经元中的光遗传学相变

Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between ...

体内研究 斑马鱼幼虫的全脑成像

In Vivo Whole-Brain Imaging of Zebrafish Larvae Using Three-Dimensional Fluorescence Microscopy

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the cellular resolution. Here, we provide an optimized protocol for performing whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy, including sample preparation and immobilization, sample embedding, image acquisition, and visualization after imaging. The current protocol enables in vivo imaging of the structure and neuronal activity of a larval zebrafish brain at a cellular resolution for over 1 h using confocal microscopy and custom-designed fluorescence microscopy. The critical steps in the protocol are also discussed, including sample mounting and positioning, preventing bubble formation and dust in the agarose gel, and avoiding motion in images caused by incomplete solidification of the agarose gel and paralyzation of the fish. The protocol has been validated and confirmed in multiple settings. This protocol can be easily adapted for imaging other organs of a larval zebrafish.

作者聚焦：使用三维荧光显微镜对斑马鱼幼虫进行体内全脑成像  

Presented here is a protocol for in vivo whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy. The experimental procedure includes sample preparation, image acquisition, and visualization.

体内 使用三维荧光显微镜对斑马鱼幼虫进行全脑成像

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the ...