Name: a simple technique to assay locomotor activity in drosophila
Uploaded: 2023-02-24T14:00:00
Description: Watch this Scientific Journal Video about A Simple Technique to Assay Locomotor Activity in Drosophila at JoVE.com

这项工作得到了苏州大学和中国国家自然科学基金（82171414）的特别启动基金的支持。我们感谢刘春风教授的实验室成员的讨论和评论。

本研究使用W1118成年苍蝇和第三龄幼虫。
1. 实验准备
注意：用于 果蝇 运动跟踪的露天竞技场由无色无味硅胶制成。
<ol><li>根据制造商对硅胶试剂盒的说明，以 1：10 的比例混合试剂 A 和试剂 B（参见 材料表）。确保通过搅拌将碳酸氢钠添加到混合物中，直到颜色变为白色。将混合物转移到干净的培养皿中?...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+larval+activity+in+the+Drosophila+activity+monitor.">Measurement of larval activity in the Drosophila activity monitor.</a> Journal of Visualized Experiments. 98, e52684 (2015).</li><li>Walter, T., Couzin, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRex,+a+fast+multi-animal+tracking+system+with+markerless+identification,+and+2D+estimation+of+posture+and+visual+fields.">TRex, a fast multi-animal tracking system with markerless identification, and 2D estimation of posture and visual fields.</a> eLife. 10, (2021).</li><li>Maia Chagas, A., Prieto-Godino, L. L., Arrenberg, A. 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我们设计了一种基于与斐济图像处理程序兼容的开源材料 AnimalTracker API的方法，该方法可以使研究人员通过跟踪成年和个体幼蝇来系统地评估运动活动。 AnimalTracke 是用Java编写的工具，可以很容易地集成到现有的数据库或其他工具中，以促进对应用程序设计的动物跟踪行为的分析24。通过量化成虫和幼虫运动活动的软件计算公式逐帧分析，可以灵活分析几个参数，?...

黑腹果蝇 为研究由基因修饰1、药物治疗2 和衰老3 创建的神经元疾病模型中的细胞和分子功能提供了一种极好的模式生物。人类和果蝇之间生物途径、物理特性和疾病相关同系物基因的高度保守性使果 蝇 成为从分子到行为水平的理想模仿物4.在许多疾病模型中，行为缺陷是一个重要的指标，为各种人类神经病提供了有用的模型5，6。 果蝇 现在用于研究多种人类疾病、神经发育和神经退行性疾病，如帕金森病和肌萎缩侧索硬化症7，8。检测疾病模型的运动能力对于了解致病进展至关重要，并且可以提供与疾病过程背后的分子机制的表型相关性。
最近，已经为果蝇运动检测策略开发了商用软件工具和具有成本效益的程序，例如在分组果蝇中进行高通量测试9，10和实时测量运动11，12。一种这样的传统方法是快速交互式负地理分类（RING），也称为攀爬测定，它包括多个通道，允许包含具有相同性别和年龄的大量苍蝇种群，从而在收集数据的同时减少变异9，13。另一种用于分析运动行为的预测试方法是TriKinetics果蝇活动监测器（DAM），这是一种使用多个光束来检测细玻璃管内苍蝇活动运动的设备14。该设备连续记录位置，通过计算光束交叉来表示自动运动，以研究苍蝇在较长时间内的活动和昼夜节律15。尽管这些方法已广泛用于分析果蝇的行为缺陷以确定行为运动的变化，但它们总是需要特殊的测试设备或复杂的分析过程，并且限制了它们在某些模型中的应用，设备有限，简单。用于测试成年果蝇的基于动物追踪组的策略，例如 FlyGrAM11 和果蝇岛测定10，在预定义的区域实施社会招募和个体跟踪。然而，由于苍蝇的碰撞或重叠，在被蹂躏的区域，社会个体限制可能会对图像中的识别产生负面影响。尽管一些基于开源材料的方法，如TRex16、MARGO 12和FlyPi17，有紧急情况，但它们可以在行为测试中灵活使用，快速追踪苍蝇。这些测试方法与复杂的实验设备安装、特殊软件要求或专业计算机语言有关。对于幼虫，测量每单位时间穿过网格边界线数量的总距离18，或粗略计算个体的体壁收缩19，是评估其运动能力的主要方法。由于设备或装置和分析方法缺乏精确性，幼虫的一些行为运动可能逃脱检测，难以准确评估行为运动，尤其是精细运动15。
本开发的方法利用与斐济（ImageJ）图像处理程序兼容的 AnimalTracker 应用程序编程接口（API），通过分析成蝇和幼蝇的高清（HD）视频中的跟踪行为，系统地评估其运动活动。斐济是一个开源软件ImageJ发行版，可以将强大的软件库与多种脚本语言相结合，从而快速制作图像处理算法的原型，使其图像分析功能在生物学家中很受欢迎20。在当前的方法中，斐济与 AnimalTracker API 的集成被用来开发具有个性化算法插入的独特果 蝇 行为测定，并为详细文档和教程提供了一个有用的步骤，以支持运动行为的强大分析能力（图 1）。为了避免因苍蝇碰撞或重叠而导致图像中客观识别的复杂性，每个竞技场仅限于容纳一只苍蝇。在评估该方法的跟踪精度后，实施它来追踪和量化与有毒药物鱼藤酮一起给药的果 蝇 运动，该药物通常用于帕金森病的动物模型，最终发现药物治疗中的运动障碍21。这种方法采用开源和免费软件，不需要高成本的仪器，并且可以精确和可重复地分析 果蝇 的行为运动。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animal tracker</td>
			<td>Hungarian Brain Research Program</td>
			<td>version: 1.7</td>
			<td>pfficial website: http://animaltracker.elte.hu/main/downloads</td>
		</tr>
		<tr>
			<td>Camera software</td>
			<td>Microsoft</td>
			<td>version: 2021.105.10.0</td>
			<td>built-in windows 10 system</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>DELL</td>
			<td>Vostro-14-5480</td>
			<td>a comupter running win 10 system is available</td>
		</tr>
		<tr>
			<td>Drosophila carbon dioxide anesthesia workstation</td>
			<td>Wu han Yihong technology</td>
			<td>#YHDFPCO2-018</td>
			<td>official website: http://www.yhkjwh.com/</td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>Fiji team</td>
			<td>version: 1.53v</td>
			<td>official website: https://fiji.sc/</td>
		</tr>
		<tr>
			<td>Format factory software</td>
			<td>Pcfreetime</td>
			<td>version: X64 5.4.5</td>
			<td>official website: http://www.pcfreetime.com/formatfactory/CN/index.html</td>
		</tr>
		<tr>
			<td>Graph pad prism</td>
			<td>GraphPad Software</td>
			<td>version: 8.0.2</td>
			<td>official website: https://www.graphpad-prism.cn</td>
		</tr>
		<tr>
			<td>Hight definition camera</td>
			<td>TTQ</td>
			<td>Jingwang2 (HD1080P F1.6 6-60mm)</td>
			<td>official website: http://www.ttq100.com/product_show.php?id=35</td>
		</tr>
		<tr>
			<td>Office software</td>
			<td>Microsoft</td>
			<td>version: office 2019</td>
			<td>official website: https://www.microsoftstore.com.cn/software/office</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Bkman</td>
			<td>110301003</td>
			<td>size: 60 mm</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>DOW</td>
			<td>SYLGARD 184 Silicone Elastomer Kit</td>
			<td>Mix well according to the instructions</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Macklin</td>
			<td>#144-55-8</td>
			<td>Mix well with silica gel</td>
		</tr>
	</tbody>
</table>

a simple technique to assay locomotor activity in drosophila

黑腹果蝇 因其丰富的先进遗传操作技术和多样化的行为特征而成为研究各种疾病的理想模式生物。在动物模型中识别行为缺陷是衡量疾病严重程度的关键指标，例如，在神经退行性疾病中，患者经常经历运动功能障碍。然而，随着各种系统可用于跟踪和评估苍蝇模型中的运动缺陷，例如药物治疗或转基因个体，仍然缺乏从多个角度进行精确评估的经济且用户友好的系统。本文开发了一种基于 AnimalTracker 应用程序编程接口（API）的方法，该方法与斐济图像处理程序兼容，以系统地评估记录的视频中成年和幼虫个体的运动活动，从而可以分析其跟踪行为。该方法只需要一个高清摄像头和一个计算机外围硬件集成来记录和分析行为，使其成为筛选具有转基因或环境行为缺陷的苍蝇模型的一种经济有效的方法。给出了使用药理处理的苍蝇的行为测试示例，以展示该技术如何以高度可重复的方式检测成年苍蝇和幼虫的行为变化。

在本研究中，检查了用鱼藤酮处理的成蝇和第三龄幼虫的运动缺陷，并将其运动活性与用药物溶剂二甲基亚砜（DMSO）喂养的对照苍蝇的运动活性进行了比较。在果蝇中使用鱼藤酮治疗已被证明会导致大脑中的多巴胺能神经元丢失22并导致显着的运动缺陷23。如图11和图12所示，与喂食DMSO的对照苍蝇相比，用鱼藤...

Watch this Scientific Journal Video about A Simple Technique to Assay Locomotor Activity in Drosophila at JoVE.com

A Simple Technique to Assay Locomotor Activity in Drosophila

该协议通过使用开源软件斐济跟踪和分析手工竞技场中苍蝇的运动来评估 果蝇 的运动活动，该软件与插件兼容，以基于高清视频录制分割每帧的像素以计算速度、距离等参数。

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 ...

该方法基于开源软件，从录制的视频中系统评估成虫和幼虫苍蝇的跟踪行为的局部运动活动。这种方法需要低成本的设备集成来记录和分析行为，使其成为筛选苍蝇运动的一种经济有效的方法。该技术用于识别具有局部运动障碍的人类疾病的苍蝇模型中的行为改变，但不适用于治疗疾病，残疾或困难。在分析过程中必须清除相关的视频窗口，即原始视频窗口和处理视频窗口。首先，根据制造商的说明，以1比1的比例混合二氧化硅试剂盒的试剂A和试剂B，以准备用于果蝇运动跟踪的开放场地竞技场。接下来，将高清摄像机放在三脚架上，对其进行调整，使摄像机镜头垂直于二氧化硅竞技场的表面。然后打开斐济的视频。将进度条拖动到初始帧并默认批准。使用手绘选择工具选择苍蝇的整个身体。单击图像、调整以及亮度和对比度以调整白平衡，直到所选区域的灰度值接近宽背景。将麻醉的苍蝇转移到空旷的竞技场。一旦苍蝇从麻醉中恢复过来，将带有苍蝇的竞技场盘放在相机下方，并快速左右摇晃，以确保记录开始时苍蝇在运动。按相机应用程序上的录制按钮开始视频录制。录制完成后，按停止按钮终止视频录制。使用 M-J 异教徒编码将录制的视频转换为 AVI 格式，以便可以使用斐济打开和分析它们。此外，将视频的每秒帧数设置为成年苍蝇的 15 fps 和幼虫的 12 fps。要使用斐济打开视频，请在弹出窗口中选择两个选项，使用虚拟堆栈和转换为灰度。使用动物追踪器插件的设置活动图像工具获取处理窗口，并使用椭圆形工具在原始视频窗口中创建一个环绕竞技场的跟踪区域。为处理窗口中的第一个空白帧设置滤镜和两个滤镜的参数。然后选择原始视频窗口中的下一帧，并选择处理窗口的过滤表面。选择过滤后的处理窗口后，使用设置阈值工具将带有红色轮廓的 Tract Fly 转弯，该轮廓在处理窗口中覆盖。然后使用设置的斑点检测器让计算机识别具有红色配置文件的苍蝇，该配置文件覆盖在处理窗口中。为成蝇或幼虫设置最后一帧，如文本手稿中所述。使用显示斑点工具在原始视频窗口中显示跟踪矩形。启动跟踪，并在监视完成后导出跟踪文件。使用动物追踪器和追踪分析器插件加载轨迹和区域文件。使用区域设置选择所需的索引，并更改参数设置。使用帧速率计算帧间隔的时间。在跟踪分析仪中进行分析后，使用电子表格软件和图形板棱镜生成定量分析图表。打开曲目文件。将所有坐标复制到Microsoft Office Excel，并使用空格键拆分单元格。要计算每帧间隔的不动时间，请选择所有计算结果并插入柱形图，以按整个柱形图的边距直观地显示不动时间。为了说明方向角度的变化，请选择所有计算结果并插入散点图。用鱼藤酮处理的成蝇和第三龄幼虫与用溶剂DMSO喂养的对照苍蝇相比，具有显着的运动缺陷。对成蝇鱼藤酮处理的定量分析显示，行进距离和平均速度显着降低。并且不动时间显着增加。代表性图显示，与对照组相比，喂食鱼藤酮的苍蝇每帧间隔的运动速度峰值较少，表明运动活动缺陷的严重程度。与对照苍蝇相比，每帧移动像素的机构主义不动列也显示，鱼藤酮喂养的苍蝇在一分钟内的运动明显减少。代表性图表说明了鱼藤酮喂养的方向变化的移动角度，对照动物也揭示了苍蝇选择方向的变化。在第三龄幼虫中也观察到了类似的结果。结果表明，与对照组相比，喂食鱼藤酮的跟踪幼虫的行为运动明显受损。分析过程中最关键的一步是形成第一个空白帧，因为它证明了软件是否可以有效检查。这项技术可以让研究人员更深入地研究多种缺陷的潜在机制，如神经或肌肉损伤。

Research

JoVE Journal

Behavior

A Simple Technique to Assay Locomotor Activity in Drosophila

Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

鼠标短期和长期活动性分析视频跟踪软件

Locomotor  activity (LMA) is a simple and easily performed measurement of behavior in mice  and other rodents. Improvements in video tracking software ...

科研

行为学

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

行为和运动测量使用一个开放的领域活动监控系统的骨骼肌疾病

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

使用开放式现场迷宫来衡量小鼠的运动能力和焦虑样行为

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

跑步机疲劳试验：疲劳类似的行为，为鼠标操作简单，高通量检测

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

Using a Split-belt Treadmill to Evaluate Generalization of Human Locomotor Adaptation

用分体式跑步机评价人类运动适应的泛化

Understanding the mechanisms underlying locomotor learning helps researchers and clinicians optimize gait retraining as part of motor rehabilitation. ...

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

测试环境线索对交配行为的影响的方法<em&gt;黑腹果蝇</em

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

A Computational Method to Quantify Fly Circadian Activity

量化飞行昼夜活动的一种计算方法

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. ...

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

多色 FlpOut 技术在果蝇中的应用研究胶质细胞的高分辨率单细胞形态学

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between ...

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

一种简便、低成本的肌营养不良小鼠步行模型的测定方法

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...