This work was supported by a Howard Hughes Medical Institute Education Grant and an internal grant from the Saint Joseph's University chapter of Sigma Xi. We would also like to thank the three anonymous reviewers who helped strengthen the protocol and interpretations.

为住房，保健和斑马鱼的研究以下方法已经批准圣约瑟夫大学IACUC。 1.斑马鱼的居所和照顾<ol><li>获取主题斑马鱼从本地供应商，还是从野外捕获的人群。请注意，住房斑马鱼是受IACUC的指导方针和额外的许可证都需要住房野生捕捞种群。 </li><li>根据性别鉴定和独立的斑马鱼。另外，请跳过此步骤或根据研究假设，分离不同的方法进行更换。 注:性别特征是难以确定在斑马鱼，尤其是在女性。参照本指引执行10或有用的在线手册来区分性别。 </li><li>随机进一步分离种群成焦鱼的适当比例对刺激鱼。注:焦鱼考试科目随后通过一系列behavio的拉尔测定，并刺激鱼是将占据侵略和浅水实验刺激室的个人。 注意:传统上，1和5之间每焦鱼刺激鱼在浅水测定法和1:1的刺激:用于在侵略测定14,17焦鱼比。然而，研究人员需要专门设计时考虑的假设研究（ 即，应该刺激鱼保持不变，保持在焦鱼行为的一致性，还是应该刺激鱼被每个检测之间旋转，以确保适当的随机化）。 </li><li>严格的12时12分光/暗周期下存储在斑马鱼房架所有的鱼（27.5-28.5℃）。日采食所有重点和激励鱼糜食品和每周一次的丰年虾，对非研究天。 </li><li>确保所有测试ZT1和ZT5（时间授时）之间进行，以避免昼夜节律的影响26。 </li></ol> 2.随机化和坦克安装<ol><li>镭ndomize所有个人先验行为检测的特定序列。设置使用在线随机序列发生器27或从统计软件包（R）的一个随机序列发生器一致随机化方案。 <ol><li>标签1每个检测到3，并保持这个任务是一致的。分配一个随机序列将每个独立的，并根据行为分析标签映射序列。例如，对于总共三个试验（1映射到变浅，2映射到胆识和3映射到侵略），一个给定的个体可以具有&#39;2,3,1&#39;的一个随机序列，这将要求它们执行特定行为检测指定的顺序排列。 </li></ol></li><li>设立在实验室试验罐（胆识，侵略和浅水）在尽量减少外界不良刺激的可能性，并确保有足够的空间用于视频摄像机捕捉整个坦克的地方。确保摄像机已enoug^ h清晰查看罐的标签，以及能够区分焦点鱼和安装在这个位置的摄像机的微妙行为。 </li><li>记录与保持在与指定的箱的全视图，以尽量减少在行为得分作为差分录音的结果意外差别相同位置的相机中的所有行为试验。 注意:避免将坦克重型交通区域和门的动作和声音会影响斑马鱼的行为。 </li><li>建立独特的JWatcher协议24得分每个行为的检测。请参阅JWatcher手册，了解如何建立联络主文件分配行为，以键盘和焦点分析​​文件，总结收集到的数据。 注:JWatcher是行为记分软件，允许可靠和可重复的定制行为得分24。 </li><li>定义标记方案来跟踪日期，鱼数量，并为所有的行为实验测定编号（ 如:"F5-B-123115"暗示鱼5，胆识检测，12月31日日 ，2015年），并在标签视图和摄像机的焦点罐用胶带之外。保持并记录在实验室笔记本为快速参考和未来的质量控制信息。 </li><li>设置将所有测定之间使用以及直接在个人从壳体齿条移除之后的驯化槽。 注:驯化槽和终端舱是小的水族馆砾石英寸（40厘米×20厘米×25厘米），并且接近于壳体机架的过滤器。 </li><li>该实验全部完成后，转移个人到一个单独的终端坦克将作为所有使用焦距个人最终目的地坦克。使用槽是大到足以容纳一组个体的生命（ 例如25加仑）的其余部分。 注:保持一致和相似的水的温度和条件ACROS这是非常重要的一切都坦克。这包括住房，驯化，和所有的测试坦克。 </li></ol> 3.开展侵略分析<ol><li>进行侵略测定在一个19升矩形槽测量30厘米×15厘米×10厘米。装在围绕侵略坦克三面外不透明隔板槽，留，以观看前面，以减少无用的外界刺激。分与外部标记罐分成四个相等的矩形象限并永久固定的反射镜（用硅酮填缝）左下象限内，在22.5°倾斜形成对罐的最左侧一个直角三角形。 </li><li>确保水在驯化槽和侵略罐中的温度在壳体机架2℃。要做到这一点，有新鲜的，老化水预热至27.5-28.5℃，准备加入到侵略坦克。请注意，以改变水在每个测定之间，以消除无端是重要嗅觉线索。 </li><li>转移，通过拔罐，焦点鱼从壳体机架到一个单独的驯化槽那里将适应环境10分钟。 注:重要的是要通过降低处理不当引入到鱼的刺激。传输个人如前所述27。 <ol><li>具体地，手动和小心地将一个小的，透明的塑料杯到感兴趣罐。而不破坏鱼不亚于通过使用净转移，慢慢鱼舀进杯子。 </li></ol></li><li>确保视频摄像机准备通过确认试验侵略罐（见步骤3.1）是焦点，并立即个体转移到侵略罐，在设备上按记录之前记录。通过拔罐从顺应坦克侵略罐转移焦点鱼。 注:记录鱼进入坦克侵略跟踪条目的确切时间之前是很重要的。 </li><li>记录汇聚分裂国家罐10分钟，30秒，然后杯从侵略罐放回驯化槽的焦点的鱼。 </li><li>在观看录制，等待30秒的鱼已经被引入到罐后的进球行为之前，一个额外的适应期。 </li><li>使用用于攻击行为限定的JWatcher评分系统，量化为10分钟以下行为:邻近镜花费时间，镜像方法，飞镖的数量，试图叮咬（在反射镜的方向张口）的数目，和横向显示的号码数（背鳍，胸鳍，臀鳍和尾鳍勃起）9。 </li><li>分数单侵略试验至少两次，直到检测与迭代之间的一致性高得分。 </li></ol> 4.开展大胆试验<ol><li>在开展小1.5升梯形水箱大胆试验，​​测量15厘米高x26.5厘米顶的X22.5厘米底×6厘米宽。环绕四面不透明的纸ØF中的坦克，但留下暴露观察前方。一个单一的，薄片胶带外部的放置在罐从罐的底部划界的顶部。 </li><li>确保水在大胆罐中的温度在壳体机架2℃（参见步骤3.2） </li><li>从壳体机架驯化槽转移焦点鱼（参见步骤3.3），并让适应环境10分钟。 </li><li>确保摄像机准备拍摄大胆试验，​​一旦准备转移焦点鱼，按记录设备。从驯化罐转移焦点鱼的胆量槽和录制了8分30秒的。 </li><li>当观看记录，允许另外的30秒的适应期的鱼被引入到得分前油箱之后。 </li><li>使用JWatcher软件，量化大胆行为8分钟。具体来说，量化以下行为:近表面的时间，延迟进入TRA的上部，数nsitions进入上部，和29不稳定的运动（飞镖）的数目。 </li><li>分数单大胆试验至少两次，直到检测与迭代之间的一致性高得分。 </li></ol> 5.开展了浅水分析<ol><li>进行浅水实验中有三个隔间用硅胶密封填缝两个玻璃隔板分成一个76升罐（所产生的三个室由两个刺激区和一个联络室）。在外部，标记指定每个刺激区&#39;玻璃分隔的6.35厘米双重偏好的区域，表明两种车身长度30。应用不透明的分区外部周围的浅水坦克的三面，但留下暴露观察前方。 注:此首选项区可进行定制，以测试不同大小的物种的特定假设。 </li><li>根据研究的性质，选择刺激滩涂适应环境至少12小时beforÈ测试并保留在浅水罐在整个实验过程中。 注:刺激滩涂是用来评估焦点鱼在这些浅滩的存在或不存在的行为鱼个体的集合。不同的刺激浅滩组合可以选择测试多个不同的浅水假设。可以肯定的刺激浅滩各自刺激区域之间旋转，以避免侧偏置，并且也使其适应测试前至少12小时。 </li><li>如前所述转移焦点鱼入驯化罐（见步骤3.3） </li><li>传送，通过从中间驯化槽在浅水罐中央室拔罐，焦点鱼。 </li><li>允许10分钟的现场得分的观察期前的10分钟适应期。另外，记录浅水试验，然后再使用JWatcher得分。鱼被确定为变浅时，他们在腔室中的优先区域内住房目标浅滩。 </li><li>量化在中心容积花各人喜好区和时间花费的时间。 </li><li>作为额外的描述计量，监测"浅水的实力"（SOS）。其中大小描述如何经常单个鱼群分配-1和+1之间的值。 </li><li>计算由浅水区的面积除以所花费的时间变浅的比率和减去的时间的比例花费不变浅和非浅水区的面积。放置此数量超过由每个区域的体积单独分开的每个区所花费的时间的总和。关于该计算的详细的信息可以在先前的研究中14中找到。 </li></ol> 6，数据质量控制<ol><li>分析使用传统的数据分析工具31-32的数据。 </li><li>测试使用非参数列文的测试的所有行为方差齐性。如果一个行为是不均匀的执行日志变换33。 </li><li>确认没有顺序的影响（ 即，随机化为了不影响个人的行为）对同质数据34使用秩和检验方差分析。 </li><li>确认有与侧固定系数35 SOS的指数创建单变量一般线性模型无副作用偏见的浅水检测。 注意:如果有一点与数据没有问题，下游分析可以进行。如果有问题，调和额外的转换，具有生物合理的解释合理，和/或增加样本量。 </li></ol> 7.分析数据<ol><li>计算双尾Spearman秩相关性36分开内的胆略和侵略检测所有行为。 </li><li>计算组内相关系数（ICC），为与行为分析稠度进行了一个以上的添双向混合模型E对于一个给定的个人36。在这一背景下，国际刑事法院是在给定的实验乖鱼如何衡量持续超过一次的测量。 </li><li>由中心和拉平所有行为预处理魄力和侵略行为，以输入准备一个主成分分析（PCA）25。 注:由中心和拉平预处理测量是一个重要的步骤。如果没有可变更多的次数多，会出现过度影响提取成分的解释这个预处理步骤的行为。居中和拉平的过程中会删除此偏差。 </li><li>执行与相关矩阵PCA一，以确定是否魄力和侵略是作为行为综合症联系在了一起。在&gt; 0.6诠释每个组件显著的行为负荷。见2010 Budaev 24报告PCA的行为学研究的综合指南。一个碎石图将知识性上eigenve构建函数应保持解释。 </li><li>使用统计软件包如R或SPSS 31,32，提取得分为每个单独的每个组件。该分数代表多少给定的个人的行为可以由特定的组件进行说明。 </li><li>计算因素回归分数和不冻36的强度之间的Spearman等级相关。 注:行为综合症被视为存在，如果魄力和侵略行为加载相同的组件上，或者，如果有显著可解释的负载任何组件与浅水的实力相关。另外，还要注意浅水的实力不包含在PCA，以允许更多复杂的行为quantifications（胆略和侵略）更好的解释。该PCA将正交崩溃魄力和侵略特性引入特征向量，最大限度的变化说明，并非常适用于这些复杂的测量量。解读所产生的特征向量和回归我们的浅水测量强度对他们每个人给出了每个复杂的行为（胆略和侵略）涉及到一个更明确的二进制浅水测量一个更加直观的解释（ 即鱼是不冻或不不冻）。 </li></ol>

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The authors (GW and SPM) have no competing financial interests or conflicts of interest.

该协议将确定是否有在魄力，侵略一致协会，和在斑马鱼变浅的行为。如果有一致的关联的任何这些行为之间的给定人群中，然后一个行为综合症存在。通过研究人口的自然行为综合症，研究人员可以有它的行为的动态的一个更完整的理解，种群结构，以及可能的进化史3。此外，操纵影响这些行为综合征，像在药理学6，毒理学7，行为遗传学8,9的环境中，和内分?...

有文献的从一个给定的群体调查个别动物内不同的行为之间的关联越来越多。这些协会被称为行为综合征，并测量通常包括气魄，侵略，探索行为和社交1-5。行为综​​合征是直接和间接原因有价值。直接，行为症状的知识，可以提供进化理论，人口结构和人口动态3的更完整视图。间接的，对于行为的关联知识可能通知量化的行为，如药理6，7毒理学，行为遗传学8,9，和内分泌10个字段。因为这些直接和间接的利益，行为症状的知识的增加是常用的模式生物，如斑马鱼特别有价值。使用斑马鱼的研究发现我呐多种学科，包括行为综合征11-13的分析。为了推进行为综合症的研究知识，因为其他学科也使用行为三围为假设检验，性能可靠，简洁的描述都需要有效的分析和解释，标准化的协议将有利于物种的学习间的比较。我们的协议被开发用于测量实验室饲养的斑马鱼14人口的气魄侵略，浅水行为综合症。然而，该协议的基础上（跟踪单个鱼，确保适当的随机化，以及适当的分析）可以很容易地修改各种替代行为的措施。此外，气魄，侵略，或浅水测定可单独针对不同的假设进行测试运行。因此，尽管我们的目标是描述如何进行行为综合症的研究和成功的人，微升协议埃维尔行为测量，此过程的每个方面都可以单独存在。 对行为综合征的文献跨越几个分类组，从节肢动物对人类4和，以测量一个行为综合症，至少两个行为背景必须量化。不幸的是，在用于量化横跨行为的轴线的行为测量测定法往往很少的一致性。例如，在鱼，粗体可能使用T-迷宫试验，旷场试验中，或引入一种新的或外国的刺激15的测量。在鱼的侵略研究可能涉及对子互动，视频刺激试验，或粘土模型试验12,16,17。同样，变浅的行为，这通常涉及shoalmate偏爱的测量分析，可以在不同类型的罐进行的，用不同的方法来确定缔合时间21-23。在这个协议中的overa的特定子集LL行为分析的剧目呈现。具体而言，该协议提出了一个方法来跟踪通过气魄，侵略和浅水测定个人，有利于个体内比较，以确定比较是否在整个人口中的所有个体一致的方式。我们在以前的研究中14,18执行该协议与斑马鱼和慈鲷罪犯（Amatitlania nigrofasciata），它可以与任何同样大小的淡水鱼工作。 魄力测定法中，有一个水平行划定在水箱的上部和下部相等大小的区域一个1.5升梯形罐进行。量化行为包括跃迁受水箱的上部和下部，在各部分，飞镖的数量所花费的时间之间的受试鱼的数目，并进入上部的延迟。侵略测定在一个19L的矩形箱，其包括执行一个3英寸×5英寸的镜面倾斜约22°，位于罐19的左下角。量化行为包括由目标鱼与镜20相互作用花费的总时间量，具有特定侵略性指标沿-试验鱼和其反射之间叮咬的数目，横向显示，飞镖。对于这些具体指标，叮咬定义为走向张开嘴镜子快速弓步，横向显示被定义为横向，胸鳍，臀鳍和背鳍的在镜子的方向燃烧，和飞镖任何不稳定的运动，是不朝镜子执导。最后，变浅测定量化测试鱼类中的三腔槽的中心腔室中的行为。该储箱的侧室要么空，或者含有鱼的"目标浅滩"，并且测试鱼花费每侧室接近的时间被测量21-23。单个综合得分称为STREN变浅的GTH（SOS），计算每个单独的测试鱼类，具体到刺激，并且可以在下游分析14一起使用。所有行为是由一个单一的浏览器，或使用被称为JWatcher 24自由行为量化软件多个观看者得分。 测试行为综合症的存在主要是一个统计的努力，并且在2010年Budeav所提出，建议遵循的准则25，具体而言，建议在中心和范数据执行主分量分析（PCA），其中输入是一个人的行为与多种行为测量 （ 即气魄和侵略）测定的载体。在PCA上的相关矩阵进行的，减少了行为测量的维数，因此，提取，解释大部分变化的最重要的知识。然后将提取的部件可以被解释浅上高的因子载荷为感兴趣和回归分数个体行为编辑可以提取用于说明组件的基础上，每一个人。然后，这些回归的分数可以比在SOS测量等各种非行为的测量，如鱼的大小或性别。 这个工作流程是在该胆识和浅水14之间存在特定的性别行为综合征被发现斑马鱼的行为综合征的研究中得到落实。在这种情况下，更大胆的斑马鱼男性更可能具有较大的，更积极的物种（D. albolineatus）相关联，但是这种关联在雌性丢失。该工作流还在其一个行为综合症未发现的，潜在地表明该物种的行为可塑性少年囚犯鲷亲属（Amatitlania nigrofasciata）18的研究来实现。因此，下面的协议，提出瓦特第i个在研究个人层面的行为综合症的框架划定的三个具体分析（气魄，侵略和浅水）性质的目标。

<table><tbody><tr><td>Zebrafish Rack System</td><td>Aquaneering Inc</td><td>Cat. # ZS550</td><td></td></tr><tr><td>Pet Valu Tropical Fish Food, 224.0 g</td><td>Pet Valu</td><td>Cat. # 31700</td><td></td></tr><tr><td>Premium Grade Brine Shrimp Eggs, 16 oz</td><td>Brine Shrimp Direct</td><td></td><td></td></tr><tr><td>1.5 L Trapezoidal Tank</td><td>Pentair Aquatic Ecosystems</td><td>Cat. # itsts-a</td><td></td></tr><tr><td>19 L rectangular tank</td><td>That Fish Place</td><td>211932</td><td></td></tr><tr><td>76 L rectangular tank</td><td>That Fish Place</td><td>212180</td><td></td></tr><tr><td>Hitachi KP-D20A CCD Camera</td><td>Prescott&#039;s, Inc.</td><td></td><td></td></tr><tr><td>Nikon AF Nikkor 35-105 mm f/305~4.5s MACRO lens</td><td>Nikon Corporation</td><td></td><td></td></tr><tr><td>ArtMinds Square Mirror, Value Pack 3&quot; x 3&quot;</td><td>Michaels</td><td>Cat. # 10334162</td><td></td></tr><tr><td>Jwatcher</td><td></td><td></td><td></td></tr><tr><td>SPSS Statistics Base</td><td>IBM</td><td></td><td></td></tr><tr><td>R</td><td>The R Foundation</td><td></td><td></td></tr></tbody></table>

boldness, aggression, and shoaling assays for zebrafish behavioral syndromes

在具体的行为在不同的上下文交互一种行为综合征的存在。斑马鱼已经测试对象中最近的研究和标准化的协议，以确保正确的分析和解释是重要的。在我们以前的研究中，我们通过监测了一系列的1.5升水箱的梯形行为（近地表的时间，潜伏期过渡，过渡的数量和飞镖）的测量气魄。同样，我们在一个长方形19升油箱量化侵​​略观察叮咬，横向显示，飞镖和时间近一个倾斜的镜子。通过将一个76升罐投入三分，我们还研究了浅水偏好。变浅检测是一个高度可定制的试验和可以为特定的假设进行调整。然而，对于该测定的协议也必须是标准化的，但是定制不够灵活。在以前的研究中，末端腔要么空的，包含5或10的斑马鱼，或5闪电（鱼丹）（D. albolineatus）。在FOL哞哞叫的手稿，我们提出了一个详细的协议，并伴随该协议，该协议将允许的行为综合症实验复制的成功应用的代表性数据。

根据研究的性质，和所用的具体协议，几种不同的结果在一个行为综合征实验是可能的。下面的表格和数字，其中指出，改编自我们以前的杂志上发表的研究行为流程14和斑马鱼杂志17。当提案（如上所述）将其整体，两组结果，"测定法相关&#39;内，并进行"测定法相关之间，"预期（ 图1）。 所述第...

Watch this Scientific Journal Video about Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes at JoVE.com

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

气魄，侵略和浅水测定斑马鱼行为综合征

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is ...

boldness-aggression-shoaling-assays-for-zebrafish-behavioral

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.2K 次观看.  Saint Joseph’s University. This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

视频: Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

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

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

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

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

斑马鱼的响应行为的检测，以测量光强的变化

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

科研

生物学

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

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

We present a behavioral chamber designed to assess cognitive performance. We provide data showing that once acquired, zebrafish remember the task 8 weeks later. We also show that hyperglycemic zebrafish have altered cognitive performance, indicating that this paradigm is applicable to studies assessing cognition and memory.

以斑马鱼为模型系统的三室选择行为任务

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or ...

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social defects.

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, ...

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 an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

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

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

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

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

行为的：评估成年斑马鱼神经毒性作用的测试电池

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

作者聚焦：成年斑马鱼环境和药物诱导的行为变化评估 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.