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...

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 border="0" cellpadding="0" cellspacing="0" width="643">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">6 oz square Drosophila bottle</td>
			<td style="width:101px;">Scimart</td>
			<td style="width:119px;">DR-103</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">agar</td>
			<td style="width:101px;">sigma</td>
			<td style="width:119px;">A1296</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">sucrose</td>
			<td style="width:101px;">sigma</td>
			<td style="width:119px;">S9378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">apple juice</td>
			<td></td>
			<td></td>
			<td>not from concentrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Tegosept</td>
			<td>Fisher</td>
			<td>T2300</td>
			<td>methyl-p-hydroxybenzoate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">35 x 10 mm round petri dish</td>
			<td style="width:101px;">Fisher</td>
			<td align="right">351008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">baker&#39;s yeast</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">PDMS casting mold</td>
			<td style="width:101px;">FlowJem</td>
			<td style="width:119px;"></td>
			<td>can be requested from authors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">isopropyl alcohol</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:119px;">A417-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">laboratory wipes</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:119px;">06-666-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">canned air</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:119px;">18-431</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">10 cm petri dish</td>
			<td style="width:101px;">BioPioneer</td>
			<td style="width:119px;">GS82-1473-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">agarose</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:119px;">50-444-176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">razor blade</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:119px;">12-640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">forceps</td>
			<td style="width:101px;">FST</td>
			<td style="width:119px;">11241-40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">22 x 40 cover glass, #1.5</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:119px;">50-365-605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Fiji (version 1.51d)</td>
			<td style="width:101px;">NIH</td>
			<td style="width:119px;"></td>
			<td>fiji.sc</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Excel 2016</td>
			<td style="width:101px;">Microsoft</td>
			<td></td>
			<td>www.microsoftstore.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">MATLAB R2016</td>
			<td style="width:101px;">Mathworks</td>
			<td style="width:119px;"></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?...

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

Research

JoVE Journal

Neuroscience

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

7.3K 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

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

在昆虫单感器记录果蝇按蚊</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

科研

神经科学

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3幼虫节段性神经的可视化 RD龄果蝇幼虫的筹备工作

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

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 ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

幼虫，蛹和成人的解剖和免疫组化<em&gt;果蝇</em&gt;视网膜

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

单苍蝇检测方法的觅食行为的<em&gt;果蝇</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their worlds when trying to understand their behavior and ecology. Sensitivity to the ultraviolet spectrum (UV; 300 - 400 nm) is found across multiple genera of birds, fish, reptiles, amphibians, and even mammals. This protocol describes a technique for examining organisms for the presence of UV-reflecting structures and a method for testing whether these cues are used as social signals in the context of mate choice. A spectrophotometer is used to detect the presence of UV reflectance and variation in reflective intensity between individuals and sexes. An example of this technique is presented in which a dichotomous mate choice test exposes sexually receptive individuals to opposite sex individuals whose visual appearance can be manipulated by filters that either transmit full spectrum or block UV wavelengths. This system allowed for the determination that female, but not male, sailfin mollies (Poecilia latipinna) were using UV markings as part of their mating decisions. These types of studies serve to expand our knowledge of the range of organisms that utilize UV and provide insight into how UV plays a role in their lives.

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection.

试验以检测紫外线反射结构，并使用茉莉花鳉确定其重要性伴侣偏好<em&gt;孔雀鱼latipinna</em

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...