药品监督管理局多个斑马鱼的新方法（<em>斑马鱼</em>）和退出的量化
Summary
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.
Abstract
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.
Introduction
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>。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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors have nothing to disclose.
Materials
| Three Shelf benchtop housing system | Aquatic Habitats | N/A | http://aquatichabitats.com/research-systems/z-hab-system/ |
| 1.5L Spawning tank w/400 micron baffle | Aquatic Habitats | N/A | http://aquatichabitats.com/consumables/tanks-lids-and-baffles/info/nursery-tank-and-baffles/ |
| Pure Grain Ethanol | Luxco, INC | N/A | http://www.luxco.com/public/brands/brands.asp?brandid=21 |
| Ethovision XT Motion tracking software | Noldus Information Technology | http://www.noldus.com/animal-behavior-research/products/ethovision-xt | |
| Pipette | Eppendorf Canada | http://www.eppendorf.ca/int/index.php?l=211&pb=e396f9e705e754a3&action=products&contentid=1&catalognode=9477. | |
| Light/Dark Arena | Custom | Construct as per procedure description. 9.5cm wide, 9.5cm deep, 55cm long |
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