The authors have nothing to disclose.

所有的程序和行为测试正在协议号12年6月11日，这是符合加拿大理事会的管理和使用实验动物的动物护理的指导方针批准MacEwan大学的动物研究伦理委员会。 1.准备配料罐，解决方案和管理计划<ol><li>得到给药时间表，使得给予动物在相同的环境，并在一天的同一时间，以避免时间或偏压任何的困惑视觉刺激。 </li><li>获得尽可能多的相同的，1.5L的，清晰的聚丙烯产卵所必需的组尺寸的数量的坦克。使用基团的每罐8鱼，其允许2组鱼的要每天稍后在过程中进行测试（参见步骤3）。使用1保持槽和每组一个配料罐（组2×总数）。 <ol><li>将400微米产卵插入所有保温水箱。 ˚F患病与生境的水或反渗透水在适当的温度坦克（斑马鱼，25-28°C），它与温度一致鱼通常安置在。 注意:可以有一些药物和缓冲栖息地水的化学组分之间不希望的化学相互作用。在这种情况下，使用缓冲的最小或没有水族馆的盐用于药物给药反渗透水，以及用于对照组。 </li><li>确保坦克处于中立的环境，以避免给药过程中调节鱼的外部视觉刺激。 </li></ol></li><li>制备药液。混在产卵箱的药物与人居水适量。通过组合3毫升的高品位的乙醇（95％非变性乙醇）与1497毫升的水制备的0.2％的乙醇溶液。通过组合21毫升乙醇与1479毫升的水制备的1.4％的乙醇溶液。 </li></ol> 2.组网鱼和EThanol行政程序<ol><li>从它们的栖息地仔细坦克净鱼转移到含有产卵插入适当的储存罐。理想的情况是，房子在插入产卵鱼，以消除扣除干脆。 </li><li>所有的鱼在各自的储水槽，轻轻抬起产卵插入了贮水箱，并将其放入相应的药液罐（ 图1A）。 <ol><li>根据需要记录的给药时间。用30分钟，在此处描述的过程的乙醇溶液。 </li><li>如果可能的话，有助理协助各实验组的传输同步进行的药液，以确保精确的计量时间。另外，转移一组的时间和跟踪个别群体的计量时间（ 图1A）。 </li></ol></li><li>在必要的给药期结束时，从乙醇溶液小心地提起产卵刀片去掉鱼出的药液，并把轻轻回贮水箱。 </li><li>轻轻地网鱼在暂养箱，把它们放回它们的栖息地，直到下一个预定的时间给药，或将产卵插回储存罐，消除网。 </li><li>作为动物壳体设备，房屋，用作贮水箱在同一水箱和产卵插入动物的参数范围内以前，如果可能的提及。这将消除管理过程中完全扣除。 </li></ol> 3.行为测试<ol><li> 55厘米长9.5厘米深的有白色防水地板（ 图1B）获得光/暗的舞台宽9.5厘米。贴上白色和黑色防水不反光的纸张使用魔术贴竞技场的内壁，其中一半盖在白色的半盖在黑色的舞台上。填写竞技场5厘米的栖息地水的深度在25-28℃的温度。 MAINT艾因在整个测试这个温度。 </li><li>通过构造一个白色的三面外壳为竞技场可以位于Minimalize外部视觉刺激。确保在测试区域具有扩散顶灯灯光不会导致在水面上的反射，但是足够亮的运动跟踪软件，或交-hoc手册量化从视频图像。 </li><li>将舞台上的外壳，并设置行为跟踪软件的记录和运动分析参数。将测试时间为5-15分钟，这取决于所研究的问题。 注意:在这里，我们用了5分钟。 </li><li>运输组鱼进行测试，以研究区的生境坦克，并放置在舞台上外壳的外面。适应的鱼10分钟。 </li><li>轻轻净一个从在光/暗竞技场的中心相应的组和地方鱼，一定要松开鱼，当它被定位成平行于所述舞台的长轴AVOID偏置鱼光或暗区。 </li><li>开始的动物被释放后立即记录的行为。观看任何软件问题跟踪鱼或鱼跳跃或冻结。旋转舞台上的180°后一半的受试者进行了测试，以防止任何的困惑，由于导致从竞技场的一端朝向机箱的开口端的偏见。 </li><li>经过庭审已经结束了，轻轻地网，从舞台上拆下来的鱼保持罐或栖息地的坦克。 </li></ol> 4.分析<ol><li>检查时间花在光与暗区。对于每个组，每个鱼，得到在光与暗区所花费的相对时间，并使用一个样本t检验进行分析（;从总试验时间（分）150秒差或Wilcoxon符号秩检验对非参数数据）的以确定群体显著喜欢一个区域比其他。 </li><li>为了比较偏好，计算prefe减去花费在从在黑暗区中花费的时间的光带和比较各组之间的差异的时间伦斯索引。 叔测试可以用来比较两个组。比较多组同方差的利用Tukey的HSD post hoc检验的单向分析在必要时（或Kruskal-Wallis检验与唐恩的多重比较事后检验用于非参数数据）。 </li><li>比较速度，区转换，曲折的数目，以及各组不动。使用方差利用Tukey的HSD post hoc检验单向分析在必要时（或Kruskal-Wallis检验与唐恩的多重比较事后检验用于非参数数据）。 </li></ol>

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作者承认约书亚盖洛普对使用其用于图1的拍摄设备。这项工作是由自然科学和工程研究理事会（NSERC），加拿大发现金（以TJH）的支持。

涉及药品监督管理局在斑马鱼以往的研究仅仅依靠扣除鱼从家里罐运送他们向药液12,16。网并不总是一致的，往往需要较长的时间比由于斑马鱼，其具有显著个体差异的逃避反应预期。传统的转印方法虽然有用，但也能够在通过减少总时间的鱼花的水以外的量，以及减少在动物之间的传输时间的变化量的改善。除了提高实用性，该方法为研究人员剂量大组鱼的同时在对于时间的精确量的解决方案的能力。先前的方法需要两种给药单个的鱼，或试图同时净整鱼的基团。前者是缓慢和降低吞吐量，而后者是一种笨拙和困难的任务。此外，运动追踪系统将c一个评估的多个鱼的行为一次可以从该方法的精确度中受益。鱼可以直接从产卵插入到浅水舞台，这将确保几乎同时暴露在舞台上所有的鱼被转移。 利用这种新方法生产的显著效果上三组斑马鱼超过21天的时间来实现时。控制鱼没有收到任何酒精，每天中度鱼，每天收到一次少量的酒精，以及每周 - 狂欢的鱼，每周接受了大剂量的酒精，一旦超过21天的期限。在光/暗任务，给药过程没有改变斑马鱼的正常偏爱对照条件下的暗区。然而，2天后酒精戒断的，斑马鱼在日常中度组优选的光区的舞台，逆转偏好。统计分析证实了在区域偏好一个显著的变化。这是可能的计量程序，可以重新产生了乙醇寻求行为由于条件性位置偏爱（CPP），用鱼求乙醇（一种食欲刺激物），并更喜欢白色区域，由于其相对相似性的明亮的给药区域。最近的研究显示，斑马鱼的地方都服用乙醇17,18 CPP可发生在斑马鱼和地区。每周狂欢鱼没有表现出偏爱后2天乙醇，尽管大约占该发现的神经机制证据不足，则可能是不频繁的，但大剂量的酒精可损伤大脑，从而尽可能地把鱼无法区段之间进行区分。 这个计量过程赋予研究者的另一个优点是它的普遍性。如所描述的，该过程产生清晰的和有用的结果，在斑马鱼。但是，该过程并不限于仅用于斑马鱼。关于该过程的约束条件相关的大小和定量给料罐，动物和TESTI的限制纳克装置，以及对数据进行分析的能力。随着使用移动跟踪软件，最近的研究已经在整个审讯调查许多额外的变量。白避免习惯，越过广场，运动习惯，延迟到白区，不稳定游动，thigmotaxis（靠近舞台的墙壁所花费的时间），以及风险评估也可以量化14。也可以考虑使用此过程在其他模式生物，如金鱼，先前已经被用于研究耐受和戒断19的作用。研究人员希望能调查的其它药理学试剂或环境毒素的影响，使用其它的行为测定法，或其他海洋或淡水动物，也可以利用这个基本方法。 使用这个协议是简单而有效的。但是，如果结果并不如预期，考虑故障排除的运动跟踪系统，以确保正确的数据集合。 Trackin克应该是整个审判顺利和准确。跟踪系统应当记录鱼的身体中心的运动在所有时间。跟踪任何跳跃或不稳定的运动点，由于视觉假象，如光线反射的水波纹，或不当的跟踪设置，可能混淆的结果。在结果有些差异可能来自于赛场上的设计还是环境剂量小的差异。可以肯定的是复制的，竞技场应描述，特别是对于使用白色地板最大化在斑马鱼中的跟踪效率。与其他鱼类，颜色较淡，深色地板应使用运动跟踪，以帮助。类似地，在一种环境，而不受任何视觉刺激可能可能偏压鱼给药的鱼是非常重要的。任何不同的颜色，图案或色调是感知到的鱼可能会混淆的结果。中性，浅色的背景相似，在住房制度的建议。&lt;/ P&gt; 尽管这是可行的，以根据需要修改为其它种类的鱼的该方法中，结果可能会在固有的光/暗偏好20-21而言会有所不同。同样重要的是要注意，即使这里描述的方法仍导致一些应力。虽然网是显著降低，该过程仍然涉及的时间，其中的鱼必须是选自水，可能诱发的应激反应在很短的时间。拉姆齐和他的同事22发现，斑马鱼网纹皮质醇了至少两倍于自己的身体的水平比那些没有被捕。因为皮质醇是连接于应力11的一种激素，这可能改变被测量的行为反应。而我们的方法减少了网，它并不能完全消除。但是，它为研究人员提供了机会，以消除电网的发病率，如果他们通过住房斑马鱼在产卵插入的渴望。未来的研究应调查是否使用产卵inser的吨鱼的传输可以降低相对于网应激反应。此外，这是极为重要的，以保持精确的给药次数与组之间传送倍。改变给药时间或较大的差异时花出去的水可能会改变结果。为了减少可能的生理作用，这也是关键性白天的类似倍剂量的鱼。在目前的实验鱼配料和上午10点至下午2点进行测试。此过程的准确，可靠的复制依赖于重复精度的协议。

斑马鱼（ 斑马鱼）是一个小的硬骨鱼类物种原产于印度是一个有用的模式生物行为1和医学研究2,3。斑马鱼也常用于各种药理物质的检测，以表征其对行为的影响。不同剂量和给药时间表已经被用于研究斑马鱼的，如兴奋剂4，抗焦虑药5和乙醇6-8化合物给药后的行为。 我们的实验室已调查的充分验证的光/暗检测9,20，通常也被称为scototaxic测定不同乙醇给药时间表对斑马鱼焦虑和运动的影响。开发乙醇给药的新方法，以提高效率为重复，日常管理过的很长一段时间（21天）6 </sUP&gt;。以前使用的方法是实用的，但是，我们试图开发一种方法，该方法减少了网，而其相关联的时间成本，并允许感兴趣的大量鱼的药物的同时，精确地定时给药。在传统的使用研究乙醇中，斑马鱼网状，并从一个罐转移到另一个含有乙醇和水10-12相应的混合物。虽然这种方法是被广泛接受的，扣除斑马鱼可能增加的可变性中引入和从药液去除鱼所需要的时间。因此，精确的曝光于所关注的化合物可能会发生变化对涉及重复给药的实验过程。一种方法是减少错误的变性运输时间所产生的来源是这样希望的。用我们的方法，我们能够同时移动所有的鱼，导致鱼的每一个相同的给药时间。以下乙醇曝光（此处描述的），斑马鱼可以在任何numbe被测试- [R行为测定法，包括那些评估焦虑。使用新的方法给药鱼群体已经超越精确复制和学科之间和整个鱼类群体规范计量能力的实际用途。新软件的出现，允许多个鱼的跟踪立刻可以看到研究人员利用我们的方法，以确保在他们的实验中可复制性和准确性。考虑到广泛使用斑马鱼作为模式生物进行行为神经科学，这种方法会提高效率和实用性于未来的药理研究。 在本范例中，重复给药方案被采用，大约有反映人类饮用的时间表。鱼被随机分成3组:对照组，每天中度或每周-狂欢。给药方案是在持续21天，选择，因为它显著在以前的研究中超过7曝光时间。控制鱼获得零ALCOHOL，每天中度鱼获得0.2％的酒精每天一次，以及每周 - 狂欢的鱼，每周收到1.4％的酒精一次。亮/暗任务，使用后第2天停药的评估焦虑。这是一个相对简单的测试来管理其使用的矩形舞台，其中在一侧的壁是白色并且在另一侧是暗9。成年斑马鱼更喜欢稳健的控制条件6,9,13下舞台的黑暗的一面。增加焦虑操作性定义为显著更多的时间在黑暗中度过区，降低焦虑可以当鱼花比较多的时间在光区花假设。随着运动追踪软件，其他的信息变量也可以量化，包括平均速度，不动，蜿蜒和过渡区的14。 在我们实验室开发的给药方法可以应用到其中的水溶性化合物被施用到一个或多个zebrafi任何研究SH。许多其他药剂可从该方法中受益，目前正在斑马鱼试验。常用的测试化合物包括尼古丁，利眠宁，丁螺环酮，和东莨菪碱，它们溶解在一种类似的方式，以乙醇;通过混合化学适量的水中。因此，该过程的一般范围更广，而不是仅限于乙醇。另外，用药物为多天的给药后，光/暗任务只有一个的许多行为测试，可以被采用。给药后或抽出时，可使用的其他流行的测定包括新颖槽潜水试验15和社会行为的测试，例如变浅16。下面的过程将概述的重复传送的鱼或鱼个体的群体到含有目标化合物的药理学方案的有效方法。此外，测试焦虑与光照/黑暗试验过程在鱼谁在戒断暴露于醇施用长时间表后组进行说明。

<table><tbody><tr><td>Three shelf benchtop housing system</td><td><a href="http://aquatichabitats.com/research-systems/z-hab-system/" target="_blank"><img src="/img/new_window.png" /> Aquatic Habitats</a></td><td>N/A</td><td></td></tr><tr><td>1.5 L Spawning tank w/400 &amp;mu;m baffle</td><td><a href="http://aquatichabitats.com/consumables/tanks-lids-and-baffles/info/nursery-tank-and-baffles/" target="_blank"><img src="/img/new_window.png" /> Aquatic Habitats</a></td><td>N/A</td><td></td></tr><tr><td>Pure Grain Ethanol</td><td><a href="http://www.luxco.com/brands/vodka-neutral-spirits-neutral-spirits-everclear" target="_blank"><img src="/img/new_window.png" /> Luxco, INC</a></td><td>N/A</td><td></td></tr><tr><td>Ethovision XT Motion tracking software</td><td><a href="http://www.noldus.com/animal-behavior-research/products/ethovision-xt" target="_blank"><img src="/img/new_window.png" /> Noldus Information Technology</a></td><td></td><td></td></tr><tr><td>Pipette</td><td><a href="http://eshop.eppendorf.ca/categories/1" target="_blank"><img src="/img/new_window.png" /> Eppendorf Canada</a></td><td></td><td></td></tr><tr><td>Light/Dark Arena</td><td>Custom</td><td></td><td>Construct as per procedure description. 9.5 cm wide, 9.5 cm deep, 55 cm long.</td></tr></tbody></table>

a novel method of drug administration to multiple zebrafish (danio rerio) and the quantification of withdrawal

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

为了保持精度和控制在药理研究与斑马鱼是很重要的时候乙醇给药的持续时间一致和准确，如上所述。我们的过程可以增加给药过程的便利性和吞吐量。对任一每周-狂欢或每日中度时间表乙醇的给药导致改变的焦虑程度，与光照/黑暗试验测定，与对照组相比。当最后一次给药后两天测试，斑马鱼，对照组（谁没有收到任何酒精，但还是被转移到配料罐）中显示的行为预期的模式，与对照鱼的消费显著更多的时间在舞台上的阴暗面（ 图2A）在其他研究中6,9,13类似天真斑马鱼。鱼每周狂欢组比较无偏好无论是浅色或深色区域中的任务时，他们最后的乙醇处理后测试了两种天（ 图2B </stroNG&gt;）。有趣的是，鱼在日常中度组显著的首选舞台（ 图2C）的光明面;相反，对照组。优先索引指示控制和日常中度组（ 图2D）之间的显著差异。有跨组（ 表1）中的游泳速度没有显著差异，或不动的，因此，这种作用并不是由于马达赤字在鱼。 <img alt="图1" src="/files/ftp_upload/51851/51851fig1highres.jpg" /> 图1:传送程序和光明/黑暗竞技场 ，这些照片说明了过户手续（一）首先，鱼都坐落在转移罐（左）。配料含有乙醇罐被描绘（右）。（BC）从转印槽的乙醇溶液移动的鱼需要researcher解除产卵卡壳从转移箱，进入乙醇溶液罐。（D）光/暗的舞台如图所示测量宽9.5厘米9.5厘米深，55厘米长。白色地板的情况下，伴随着上半竞技场（左）和在其另一半白墙的黑色壁，产生明暗区域。 <img alt="图2" src="/files/ftp_upload/51851/51851fig2display.jpg" /> 图2:代表性的结果和乙醇给药过程对三组斑马鱼的2天后停药trackplots。 （A）在整个5分钟亮/暗审单控制斑马鱼的运动路径的代表trackplot。下面是表示为热图，它是斑马鱼的运动的整个试验的着色表示的基础上，在鱼的每个像素表示的位置所花费的时间相同的斑马鱼trackplot。（B）的甲repres从周线，狂欢组单斑马鱼在整个5分钟光/暗任务entative trackplot。下面是来自同一斑马鱼的热图（C）的一个单一的每日中度斑马鱼的运动的整个5分钟光照/黑暗试验的代表性trackplot。下面是来自同一斑马鱼的热图。（D）中的优先级索引，计算各组中减去时间中的光带从时间中暗区工作的时间。负数表示偏爱的暗区。正数表示偏爱浅色区域。结果表明，在对照和日常中度组2天停药* P &lt;0.05（单向ANOVA）之间的偏好一个显著差异。请注意，还有为暗一个显著偏好于对照组，P &lt;0.05（1样本t检验 ，从0差），并在日常中度组为光的显著偏好，磷&lt;0.05（1样品&lt;EM&gt; t检验，从0差）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51851/51851fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr height="21"><td height="43" rowspan="2" style="height:42px;width:125px;"></td><td style="width:89px;"> 速度 </td><td style="width:100px;"> 动 </td></tr><tr height="22"><td height="22" style="height:22px;width:89px;"> （厘米/秒）的 </td><td style="width:100px;"> （秒） </td></tr><tr height="42"><td height="42" style="height:43px;width:125px;"></td><td style="width:89px;"> 2天WD </td><td style="width:100px;"> 2天WD </td></tr><tr height="22"><td height="22" style="height:22px;width:125px;">对照组（n = 13） </td><td style="width:89px;"> 9.1±0.6 </td><td style="width:100px;"> 1.9±0.6 </td></tr><tr height="22"><td height="22" style="height:22px;width:125px;">狂欢（N = 14） </td><td style="width:89px;"> 9.8±0.5 </td><td style="width:100px;"> 0.8±0.2 </td></tr><tr height="22"><td height="22" style="height:22px;width:125px;">慢性（N = 15） </td><td style="width:89px;"> 10.3±0.5 </td><td style="width:100px;"> 2.5±1.0 </td></tr></tbody></table> 表1:亮/暗测试期间的平均流速和固定性 。平均游泳速度（厘米/秒），并在2天后停药的代表性组斑马鱼的固定性（秒）（平均值±SEM）。在这里，被发现在任何速​​度或不动无显著差异。使用与贺尔康等人的许可。，（2013）。

Watch this Scientific Journal Video about A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal at JoVE.com

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

药品监督管理局多个斑马鱼的新方法（<em&gt;斑马鱼</em&gt;）和退出的量化

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

a-novel-method-drug-administration-to-multiple-zebrafish-danio-rerio

Nanyang Technological University

Research

JoVE Journal

Neuroscience

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

12.1K 次观看.  MacEwan University. A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

视频: A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

科研

癌症研究

Behavioral Approaches to Studying Innate Stress in Zebrafish

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of stress-related diseases and psychiatric disorders, yet further studies into the genetic and neuronal regulation of stress are still required to develop better therapeutics. The zebrafish provides a powerful genetic model to investigate the neural underpinnings of stress, as there exists a large collection of mutant and transgenic lines. Moreover, pharmacology can easily be applied to zebrafish, as most drugs can be added directly to water. We describe here the use of the 'novel tank test' as a method to study innate stress responses in zebrafish, and demonstrate how potential anxiolytic drugs can be validated using the assay. The method can easily be coupled with zebrafish lines harboring genetic mutations, or those in which transgenic approaches for manipulating precise neural circuits are used. The assay can also be used in other fish models. Together, the described protocol should facilitate the adoption of this simple assay to other laboratories.

This manuscript describes a simple method to measure stress behaviorally in adult zebrafish. The approach takes advantage of the innate tendency that zebrafish prefer the bottom half of a tank when in a stressful state. We also describe methods for coupling the assay with pharmacology.

斑马鱼内生应激研究的行为方法

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of ...

行为学

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

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 measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

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 ...

神经科学

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

成年斑马鱼的优化静脉注射

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging成年斑马鱼

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

生物学

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

利用斑马鱼胚胎对化合物毒性的快速评价

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

医学

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

利用斑马鱼对肠道转运时间进行高通量测量

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

优化腹腔注射用于成年斑马鱼化合物递送

An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as cardiomyopathy. Intraperitoneal (IP) injection is an important method that delivers compounds to the body for either testing therapeutic effects or generating disease models such as doxorubicin-induced cardiomyopathy (DIC). Currently, there are two methods of IP injection. Both&#160;methods have limitations when handling toxic compounds such as doxorubicin, which result in side effects manifesting as severe damage to the body shape and fish death. While these shortcomings could be overcome by extensive investigator training, a new IP injection method that has minimal side effects is desirable. Here, a unique IP injection method that is able to handle toxic compounds is reported. Consistently reduced cardiac function can result without incurring significant fish death. The technique can be easily mastered by researchers who have minimal experience with adult zebrafish.

作者聚焦: 斑马鱼心脏毒性模型的新型腹腔内注射方法

A new intraperitoneal (IP) injection method in adult zebrafish is described. When handling toxic compounds such as doxorubicin, this procedure is more effective than the two previously reported IP methods. The technique is designed to be easily adopted by researchers with limited experience in the zebrafish model.

一种成年斑马鱼腹腔注射技术，可最大限度地减少身体损伤和相关死亡率

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as ...

药品监督管理局多个斑马鱼的新方法（

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此视频中的章节

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

斑马鱼内生应激研究的行为方法

在斑马鱼幼虫的高通量自动化的行为分析

成年斑马鱼的优化静脉注射

Gavaging成年斑马鱼

利用斑马鱼胚胎对化合物毒性的快速评价

利用斑马鱼对肠道转运时间进行高通量测量

一种成年斑马鱼腹腔注射技术，可最大限度地减少身体损伤和相关死亡率

一种成年斑马鱼腹腔注射技术，可最大限度地减少身体损伤和相关死亡率

一种成年斑马鱼腹腔注射技术，可最大限度地减少身体损伤和相关死亡率