We would like to thank Chris Wreden and Michelle Bland for comments on the manuscript and for technical help.

1.幼虫的制备<ol><li>一个星期录制的行为之前，成立了一个跨（25处女和5男最小值）。保持在25℃，所有的十字架和后代。 注:培养条件的温度可以改变，但下面描述的时间线将需要进行调整，以帐户为发育速度的变化。 </li><li>录制时，在早晨的第一件事前5天，将交叉成一个集笼琼脂/果汁盖和酵母泥中的琼脂/果汁盖中央的DAB（0.5-1毫升）。 <ol><li>为了使集合笼，万佛洞成6盎司方形的聚乙烯瓶果蝇 。 </li><li>为了使琼脂/果汁瓶盖，在一个锥形瓶中混合18克用600毫升的水琼脂。在用200毫升的苹果汁单独的烧瓶中混合20克蔗糖。微波直到固体溶解。结合并搅拌，使混合物冷却至60℃。加20含10％甲基对羟基苯甲酸在95％的乙醇，并搅拌。添加〜7ml至35×10毫米的圆形培养皿的底部一半。允许琼脂/果汁巩固在室温1小时。保存在4℃。 </li><li>使酵母膏，按体积干酵母和水添加等份。保存在4℃。 </li></ol></li><li> 检查幼虫藏品的健康。 <ol><li>在早晨，记录前4天和3天，除去帽，并放置在笼子新鲜帽。每天在同一时间更换瓶盖拿到鸡蛋24小时收集和刚孵出的幼虫。 </li><li>每个帽取下后，请检查看跨多少鸡蛋铺设。 500-2,000鸡蛋之间的期望。调节成人的数量，如果需要的话。 注:几个鸡蛋应该已经孵化成幼虫，以及新孵幼虫会发现从酵母泥之遥。 </li><li>老化每个盖帽24小时。重新审视帽确定幼虫是否健康。 注意:大多数蛋应该孵化成幼虫，而幼虫应该已经爬进你们AST粘贴。身体不好的症状包括大量未孵化的卵，幼虫死者尸体从酵母泥远，幼虫腹部显著暗斑，表明免疫反应。如果幼虫是不健康的，改变笼，让新鲜帽/酵母泥，并检查基因型。 </li><li>废弃旧帽子。 </li></ol></li><li> 收集行为记录幼虫。 <ol><li>在记录之前既2和第1天，去除盖子和用新鲜帽替换。 </li><li>每个标签取下盖子，并保持在25℃。这将产生行为记录一个上演一系列的幼虫。幼虫从刚孵出的（0-4小时posthatch）通过第二龄期（24-48小时posthatch）所用的信道可以使用。除连续两天录音帽。 </li><li>丢弃后48小时全部大写。 </li><li>如果需要，重复步骤1.4.1-1.4.3长达5个附加Ð。后更少受精卵产生。 </li></ol></li></ol><ol><li>制备线性通道的PDMS（聚二甲基硅氧烷，硅氧烷弹性体）铸造模具（ 图1A）。 PDMS压铸模具是请直接从赫克歇尔实验室。 <ol><li>清洁用异丙醇铸模。空气干燥，DAB用干抹布实验室，或使用压缩空气。 </li><li>的铸模放入一个10cm培养皿盖子或其它容器。 </li></ol></li><li> 准备倒入琼脂糖溶液。 <ol><li>使水通过微波直至完全溶解3％的琼脂糖溶液（3克在100毫升）。 </li><li>冷却至55℃，使气泡上升到表面。 </li><li>倒入琼脂糖混合物到含有铸模使得模具刚好覆盖的培养皿。让琼脂糖设置，直至凝固。 </li></ol></li><li>移除铸模琼脂糖频道。用刀片修剪琼脂糖盘的边缘。 <ol><li>把通道S IN一个10cm培养皿浸入水中。它们可保持在4℃下7天。 </li></ol></li></ol> 3.装载幼虫进入通道到记录行为注:如果存放在4℃的渠道，允许使用行为记录之前渠道来RT。 <ol><li>使用干净的刀片切割的那些步骤2（ 图2A）制备一个单一信道。 </li><li>用一对镊子来放置通道上的玻璃盖玻片（ 图2B）开槽朝上。盖玻片的大小和厚度取决于用于成像的具体范围。 </li><li>用细镊子从步骤1制备琼脂/果汁瓶盖选择幼虫并放入切通道（ 图2C）。 <ol><li>不要挤幼虫。与钳齿尖轻轻把它捡起来。另外，也可以操纵幼虫与用细点画笔。 注意:这是重要的，因为幼虫很仙sitive触摸。如果不小心处理，伤害性反应可能会在实验的第一几秒钟内支配自己的行为。 </li><li>在水淹没简单地洗他们的幼虫。 </li><li>将幼虫到切通道后，使用镊子尖的尖端幼虫轻轻挑逗进入通道树林。 注:对于大多数应用幼虫的宽度和沟道的宽度应该匹配。幼虫在不同发展阶段的平均宽度如下:0小时posthatch - 140微米，24小时posthatch - 180微米，48小时posthatch - 320微米，72小时posthatch - 500微米，96小时posthatch - 750微米8。通道来在以下宽度:100，150，200，250，和300微米，所有通道150微米深。 </li><li>使用镊子齿或画笔的尖端来调整通道内的幼虫的位置。它可以在任何方向导向:腹侧起来，腹侧向下，腹面一侧，这取决于结构是将被成像。 </li></ol></li><li>使用镊子，轻轻翻转通道上，使得幼虫现在是在盖玻璃上。放置的水的一个或两滴在通道的端部，以便它用水（ 图2D）填充。 <ol><li>轻轻提起信道的两侧，以除去任何气泡。 </li><li>调整幼虫的方向。要做到这一点，轻轻轻推渠道，但不是玻璃盖滚幼虫。如果不改变方向，取下盖板玻璃和调整幼虫的位置。幼虫已准备好进行成像。 </li><li>图片频道/上倒置显微镜盖玻片。如果一个堂堂正正的显微镜成像，只需反转通道/盖玻片。 </li></ol></li></ol> 4.行为记录感兴趣的测量功能<ol><li>对于感兴趣的结构（ 例如 ，尾，中枢神经系统），测量的感兴趣特征（ 例如，荧光强度，位置）随时间。 注:结构如头部和尾部可以手动注释。例如，头部可以被识别为包含暗H形口钩，和尾部可以被识别为含有后气管spiricles。本文侧重于神经线。使用神经GAL4线ELAV-GAL4来驱动UAS-MYR-GFP转基因的（ELAV&gt; GFP）9,10荧光标记中枢神经系统（CNS）。中枢神经系统包含连接到神经索，其延伸至后（ 图3）二前脑叶。 </li><li>测量每个时间点（T）的神经线（F（NC））的像素强度。下面介绍一个人工标注的做法，但是这一步可以自动化。 <ol><li>在斐济（或类似软件）在"分析"菜单中的"设置测量..."对话框报告的平均灰度值和SLICË位置。 </li><li>在神经索的在电影（ 图3）的第一帧的中心手动绘制框。此步骤也可以与图像分割和配准算法自动化。 </li><li>在"分析"菜单中的"测量"命令在感兴趣的地区盒装报告的平均像素强度。这会产生一个"结果"窗口。 </li><li>在下一帧中，手动重新放置框而不使用箭头键调整。再次使用"测量"命令。更新后的结果将在"结果"窗口中。重复此步骤为每个感兴趣的帧。 </li><li>保存从文件菜单中的"结果"使用"另存为..."命令。结果将导出为.xls文件。 </li></ol></li></ol> 5.分析测量<ol><li> 规范化每个F（NC）值0和1之间（Z（T值）&lt;/ EM&gt;）。 <ol><li>在电子表格（或其他程序），打开Results.xls文件。该文件将有一个代表帧号和平均荧光强度两列。 </li><li>创建一个新列，并使用公式Z（T）= F（NC [T]）-min（F [NC]）/ MAX（F [NC]）-min（F [NC]）生成标准化的荧光值对应于每个时间点。 </li></ol></li><li> 确定在记录各个步幅的步幅时间。 注:一个箭步周期正向重复全身运动单元（和反向）爬行。按照惯例一大步向前开始（和结束）同尾，头和其他内脏器官的向前运动，如肠道或中枢神经系统1。 <ol><li>手动检查行为记录和记录每个步幅的开始时间（I）（I（步幅周期[N]））。 注意:在下面的例子中，前向运动CNS的作为步幅循环的开始，使用（ 图3）。 </li><li>对于每个步幅计算期间:ΔT（跨越周期）= I（步幅周期[N + 1]）-i（ 步幅周期[N]）。 </li></ol></li><li> 计算经过用于记录的每一个时间点的步幅周期的百分比。 <ol><li>使代表从一个箭步周期（C）开始经过时间的电子表格文件的附加列。设定的时间，因为在每一步幅的开始步幅开始到零（ 三（步周期[n]的）= 0）。因此开始后调整时间。 </li><li>使在电子表格文件一个附加列代表百分之步幅周期已过（％步幅周期 ）通过步幅期间除以调整时间和乘以100，以转换为步幅周期的百分比经过:％步幅周期 =（C（步周期[n]的））/（ΔT（跨越周期）* 100）。 </li></ol></li></ol> 6.生成极坐标图在抓取周期代表的利益结构的动力<ol><li>在MATLAB中，使用"导入数据"对话框中的选项卡导入更新Results.xls。 </li><li>创建一个新阵"西塔"包含％的步幅周期值（％步幅循环 ）。 （"西塔= VAR1"，其中VAR1代表％步幅循环 ）。 </li><li>创建一个新阵"卢"含有荧光强度值（Z（T））。 （"卢= VAR2"，其中VAR2代表Z（T））。 </li><li>使用&#39;polarplot&#39;命令来使极坐标曲线与百分之步幅周期作为角度，和荧光为半径（"polarplot（西塔，卢）"）。 </li></ol>

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The authors have nothing to disclose.

一种微流体装置建成，使线性琼脂糖渠道，可容纳果蝇幼虫（ 图1）。当果蝇幼虫被放置在这些线性琼脂糖渠道其行为库限于爬行，其允许在抓取周期幼虫结构的动力学的详细观察。 当幼虫进行一系列有节奏的步伐（ 图3）进行了一次成功的记录。如果这没有发生，检查喜欢在通道中的气泡障碍，并检查幼虫的身体健康。一个成功的记录的另一个重要因素是，幼虫被最佳地定向以可视化的利益的结构。如果幼虫不正确的方向，或者如果幼虫爬出来的通道，只需删除盖玻片并重新安装幼虫。根据我们的经验，幼虫〜20％最初安装的产量优良行为记录withouŤ调整。 在过去，测量是幼虫结构的位置的如口腔钩，肠，和爬行行为期间腹段。为了形象化这些结构在爬行步幅循环运动，产生了极坐标图。在本文中，神经索的荧光强度进行测定，并使用极坐标图上的爬行步幅周期（ 图4）以可视化的荧光的动态。有几个优点表示在极坐标图中的数据:它消除爬行速度作为一个变量，它可以总结从许多动物和许多进步数据，并允许两者总趋势和在数据11变化可视化。值得注意的是，它是可以测量的任何感兴趣的荧光标记的结构的动态。原则上，这种分析是适用于追踪发生在一个爬行周期的任何类型的动态变化。 </p&gt; 有一个宽的在本文中所描述的方法的应用程序阵列。在过去，线性琼脂糖通道已被用于在整个生物体，段和单个肌肉级1到记录幼虫的行为。这些数据表明，幼虫用于正向一个"内脏-腔内的窜动"机制和反向爬行，并且它们允许的神经肌肉机构进驱动两个和反向爬行待定1。今后，研究人员可以利用渠道来研究不同遗传背景爬行。另外，它应该是可能使用的通道来分析抓取期间使用钙成像幼虫的神经元的活性。这应导致该神经元激发与爬行周期的特定阶段运动的理解。最后，没有理由认为信道必须按照本文提出的线性设计;使用不同尺寸的渠道无疑将有助于回答一个变种有关的果蝇幼虫运动和电机控制作为一个整体问题iety。

这种方法的总体目标是详细研究果蝇幼虫爬行。在运动实验已经起到了开发和电机控制2测试理论具有重要作用。传统的运动进行了研究水生动物（ 如水蛭，七鳃鳗，蝌蚪）3。运动，在这些动物中的重复性质已经允许节律的研究中，生物物理事件驱动运动的分析，以及用于监测伴随运动神经放电模式。 对于运动的研究使用果蝇幼虫的呈现比其他模型系统的优点的独特组合:轻便遗传学，良好表征的发展，一个机构，是在第一和第二龄光学透明，并且整个的持续透射电镜重建神经系统4-6。然而， 的果蝇幼虫机车在平开运动的表面是有些复杂，包括暂停，打开和蜿蜒爬行7。本刊物使用线性琼脂糖渠道，引导的果蝇幼虫自发行为，幼虫进行持续的，直的，有节奏的爬行行为的方法。 研究果蝇幼虫行为在琼脂糖频道，而不是在平面开口面的行为，具有几个优点。首先，它可以让研究人员专门选择从众多运动是幼虫行为艺术的一部分爬行行为。第二，通过调节通道与幼虫体尺寸的宽度，爬行速度可以调整。三，渠道允许从背，腹，或侧面取决于幼虫如何加载和通道内面向查看的幼虫。这种多功能性幼虫方向允许任何利益结构要爬行过程中不断地可见。第四，信道是适合于与各种显微镜和目标的用途。例如，线性通道可用于在明场立体镜和/或用于旋转盘共焦显微镜1高分辨率成像低分辨率影像。第五，这种方法可以结合使用以任何遗传背景光遗传学/ thermogenetic神经元的操作。最后，因为无论是幼虫体（在第一和第二龄）和琼脂糖渠道光学透明，渠道可以学习动态运动，或由基因编码的荧光探针标记幼虫结构的荧光强度变化时使用。 所描述的方法适用于第一和第二龄果蝇幼虫行为详细运动学研究。该出版物分析向前爬行幼虫过程中CNS的荧光强度的动态变化来演示使用渠道，并为先导，以NEURonal公司钙成像。

<table><tbody><tr><td>6 oz square Drosophila bottle</td><td>Scimart</td><td>DR-103</td><td></td></tr><tr><td>agar</td><td>sigma</td><td>A1296</td><td></td></tr><tr><td>sucrose</td><td>sigma</td><td>S9378</td><td></td></tr><tr><td>apple juice</td><td></td><td></td><td>not from concentrate</td></tr><tr><td>Tegosept</td><td>Fisher</td><td>T2300</td><td>methyl-p-hydroxybenzoate</td></tr><tr><td>35 x 10 mm round petri dish</td><td>Fisher</td><td>351008</td><td></td></tr><tr><td>baker&#039;s yeast</td><td></td><td></td><td></td></tr><tr><td>PDMS casting mold</td><td>FlowJem</td><td></td><td>can be requested from authors</td></tr><tr><td>Isopropyl alcohol</td><td>Fisher</td><td>A417-1</td><td></td></tr><tr><td>laboratory wipes</td><td>Fisher</td><td>06-666-11</td><td></td></tr><tr><td>canned air</td><td>Fisher</td><td>18-431</td><td></td></tr><tr><td>10 cm petri dish</td><td>BioPioneer</td><td>GS82-1473-001</td><td></td></tr><tr><td>agarose</td><td>Fisher</td><td>50-444-176</td><td></td></tr><tr><td>razor blade</td><td>Fisher</td><td>12-640</td><td></td></tr><tr><td>forceps</td><td>FST</td><td>11241-40</td><td></td></tr><tr><td>22 x 40 cover glass, #1.5</td><td>Fisher</td><td>50-365-605</td><td></td></tr><tr><td>Fiji (version 1.51d)</td><td>NIH</td><td></td><td>fiji.sc</td></tr><tr><td>Excel 2016</td><td>Microsoft</td><td></td><td>www.microsoftstore.com</td></tr><tr><td>MATLAB R2016</td><td>Mathworks</td><td></td><td>www.mathworks.com</td></tr></tbody></table>

using linear agarose channels to study drosophila larval crawling behavior

的果蝇幼虫爬行正在成为一个强大的模型来研究感觉神经行为的控制。然而，在平开面幼虫爬行行为是复杂的，其中包括:暂停，车削和蜿蜒。这种复杂性在运动的剧目阻碍单个抓取步幅循环期间发生的事件的详细分析。为了克服这一障碍，线性琼脂糖渠道所做的限制幼虫的行为直，持续，有节奏地爬行。原则上，由于琼脂糖频道和果蝇幼虫体都是光学透明的，由遗传编码荧光探针标记的幼虫结构的运动可以在完整的，可自由移动的幼虫监测。在过去，幼虫放置在直线通道，并在整个生物体，段的水平爬行和肌肉进行分析1。在未来，幼虫在通道抓取可用于钙成像以监测神经最终活动。此外，这些方法可与任何基因型的幼虫，并与任何研究者设计的信道可以使用。因此，下面提出的协议是广泛适用于使用果蝇幼虫为模型，以了解电机控制的研究。

本文介绍了指导琼脂糖渠道的果蝇幼虫的行为和通过抓取周期测定幼虫结构动力学的方法。幼虫在直线渠道进行有节奏的爬行（ 图3）持续发作。因为这两个幼虫和渠道光学透明，渠道可以表达的幼虫在任何感兴趣的结构表达荧光探针可以使用。我们记录幼虫在所有神经元表达GFP（ELAV-Gal4的/ +; UAS-MYR-GFP / +）和监测在荧光强度中的神经索在爬行循环的动态变化。我们表明，在CNS向前移动在几乎相同的时间作为幼虫的头部和尾部（ 图4A-B）。作为肌肉收缩的波沿着主体轴线穿过，中枢神经系统移动进出焦点的平面引起的神经索的荧光改变（ 图4）。为了量化瓒<html>在神经索荧光强度GES在几个动物数进展我们表示上的极坐标图（ 图4C）中的数据。标绘在极坐标图中的数据表明，该神经索荧光超过步幅循环的动力学遵循可重复的图案。 <img alt="图1" src="/files/ftp_upload/54892/54892fig1.jpg" /> 图1:线性通道的设计指导果蝇幼虫爬行行为 （A）所示用来做线性琼脂糖渠道的微流体装置的设计。在该装置中的通道的宽度从100-300微米通过50微米的增量变化。的深度为150微米。 （B） 果蝇幼虫被装入琼脂糖通道。一个背视图显示与前（头）的权利。比例尺= 200微米。4892 / 54892fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54892/54892fig2.jpg" /> 图2:示出如何加载幼虫进入通道详见协议第3条<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54892/54892fig2large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54892/54892fig3.jpg" /> 图3:荧光标记的果蝇幼虫进行持续的，有节奏，直线爬行时放入琼脂糖通道左侧，在（ELAV&gt; GFP）显示所有神经元表达GFP幼虫的示意图。一个框显示了神经线的荧光强度（按区分该地区的细长形态）可以被测量。在右边是一个幼虫通过一个信道以一秒的间隔爬行的一个例子。一个背视图显示与前了。箭头指示了一大步的开始。比例尺= 200微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54892/54892fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54892/54892fig4.jpg" /> 图4:荧光变化的神经索在抓取周期的动态呈现极地坐标图 （A）一个步幅的示意图。百分比是指完成步幅周期的百分之一。尾巴，头部和内脏器官，如中枢神经系统的惯例向前运动标志着一个箭步（或步幅周期的0％）的开始。需要注意的是在CNS（白色）向前和向后，以及向上和向下。 A面争夺w被示出与前方的右侧。 （B）kymograph显示头部，尾部，以及中枢神经系统的运动。注意，步幅循环期间神经索的荧光强度是动态的。 （C）极坐标图显示在超过步幅周期的神经索的标准化荧光强度的动态变化。每个点代表在单个时间点（N = 3幼虫，每3步）单幼虫的归一化荧光强度。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54892/54892fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior at JoVE.com

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

果蝇幼虫是研究行为的神经控制功能强大的模型系统。该出版物描述了使用线性琼脂糖通道引发线性爬行和方法的持续发作期间重复爬行行为量化幼虫结构的动力学。

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

using-linear-agarose-channels-to-study-drosophila-larval-crawling

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

7.4K 次观看.  University of Chicago.  果蝇幼虫是研究行为的神经控制功能强大的模型系统。该出版物描述了使用线性琼脂糖通道引发线性爬行和方法的持续发作期间重复爬行行为量化幼虫结构的动力学。 

视频: Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

高分辨率测量的气味驱动在果蝇幼虫的行为

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

科研

生物学

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

生物工程

A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

Oxygen deprivation in animals can result from exposure to low atmospheric oxygen levels or from internal tissue damage that interferes with oxygen distribution. It is also possible that aberrant behavior of oxygen-sensing neurons could induce hypoxia-like behavior in the presence of normal oxygen levels. In D. melanogaster, development at low oxygen levels results in inhibition of growth and sluggish behavior during the larval phases. However, these established manifestations of oxygen deficit overlap considerably with the phenotypes of many mutations that regulate growth, stress responses or locomotion. As result, there is currently no assay available to identify i) cellular hypoxia induced by a mutation or ii) hypoxia-like behavior when induced by abnormal neuronal behavior.
We have recently identified two distinctive behaviors in D. melanogaster larvae that occur at normal oxygen levels in response to internal detection of hypoxia. First, at all stages, such larvae avoid burrowing into food, often straying far away from a food source. Second, tunneling into a soft substratum, which normally occurs during the wandering third instar stage is completely abolished if larvae are hypoxic. The assay described here is designed to detect and quantitate these behaviors and thus to provide a way to detect hypoxia induced by internal damage rather than low external oxygen. Assay plates with an agar substratum and a central plug of yeast paste are used to support animals through larval life. The positions and state of the larvae are tracked daily as they proceed from first to third instar. The extent of tunneling into the agar substratum during wandering phase is quantitated after pupation using NIH ImageJ. The assay will be of value in determining when hypoxia is a component of a mutant phenotype and thus provide insight into possible sites of action of the gene in question.

A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

The protocol describes a simple assay to identify Drosophila melanogaster larvae that are experiencing hypoxia under normal atmospheric oxygen levels. This protocol allows hypoxic larvae to be distinguished from other mutants that show overlapping phenotypes such as sluggishness or slow growth.

用于检测果蝇幼虫缺氧的挖洞/隧道法

Oxygen deprivation in animals can result from exposure to low atmospheric oxygen levels or from internal tissue damage that interferes with oxygen ...

行为学

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

对检测方法果蝇行为

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

神经科学

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

在幼虫馈电电路的功能分析果蝇</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

<meta charset="utf8"></head><body>一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

一个经济实惠且高效的“自制”果 蝇 行为研究平台，以及幼虫线粒体呼吸测定的配套方案

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

一种测定果蝇运动活性的简单技术

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

光斑分析分析果蝇拉瓦尔光轴

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

用线性琼脂糖渠道研究

摘要

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此视频中的章节

高分辨率测量的气味驱动在果蝇幼虫的行为

使用微流体芯片实时成像和损伤研究的响应

用于检测果蝇幼虫缺氧的挖洞/隧道法

对检测方法果蝇行为

在幼虫馈电电路的功能分析果蝇

一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

一种测定果蝇运动活性的简单技术

幼虫活动的测量

光斑分析分析果蝇拉瓦尔光轴

用线性琼脂糖渠道研究

摘要

探索更多视频

此视频中的章节

高分辨率测量的气味驱动在果蝇幼虫的行为

使用微流体芯片实时成像和损伤研究的响应

用于检测果蝇幼虫缺氧的挖洞/隧道法

对检测方法果蝇行为

在幼虫馈电电路的功能分析果蝇

一个经济实惠且高效的“自制”果 蝇 行为研究平台，以及幼虫线粒体呼吸测定的配套方案

一个经济实惠且高效的“自制”果 蝇 行为研究平台，以及幼虫线粒体呼吸测定的配套方案

一种测定果蝇运动活性的简单技术

幼虫活动的测量

光斑分析分析果蝇拉瓦尔光轴

一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案