Name: an automated rapid iterative negative geotaxis assay for analyzing adult climbing behavior in a drosophila model of neurodegeneration
Uploaded: 2017-09-12T15:00:00
Description: Watch this Scientific Journal Video about An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration at JoVE.com

我们感谢布卢明顿股票中心和维也纳的果蝇的飞行股票 rna 干扰中心。该设备的专利属于中国科学院上海市先进研究院。对 RflyDetection 软件的要求应由富煌 (见作者名单)。这项工作得到了中国国家基础研究项目 (973 项目 2013CB945602) 和中国国家自然科学基金 (31270825 和 31171043) 的资助。我们感谢何议员的讨论和意见。

1. 飞集 <ol> <li> 在标准飞行食物上保持苍蝇在25和 #176; C、70% 湿度和 12 h/12 h 光/暗循环. </li>
	<li> 收集 UAS-Dicer2;DDC-GAL4 飞行 virginsunder 二氧化碳 (CO 2 ) 麻醉. </li>
	<li> 将这些处女带到2天大的成年雄蝇携带以下基因型: UAS-mCD8GFP (控制), auxR 16182 ( 辅助 干扰) , auxGFP ( 辅助 干扰), 和 auxR 16182 ;auxGFP (抢救), 与男性: 女性比?...

<ol>
	<li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> J Vis Exp. (49), (2011).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> J Vis Exp. (61), (2012).</li><li>Madabattula, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+of+Climbing+Defects+in+a+Drosophila+Model+of+Neurodegenerative+Disorders.">Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders.</a> J Vis Exp. (100), e52741 (2015).</li><li>Podratz, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+climbing+apparatus+to+measure+chemotherapy-induced+neurotoxicity+in+Drosophila+melanogaster.">An automated climbing apparatus to measure chemotherapy-induced neurotoxicity in Drosophila melanogaster.</a> Fly (Austin). 7 (3), 187-192 (2013).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp Gerontol. 40 (5), 386-395 (2005).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+rapid+iterative+negative+geotaxis+assay+and+its+use+in+a+genetic+screen+for+modifiers+of+Abeta(42)-induced+locomotor+decline+in+Drosophila.">Automated rapid iterative negative geotaxis assay and its use in a genetic screen for modifiers of Abeta(42)-induced locomotor decline in Drosophila.</a> Neurosci Bull. 31 (5), 541-549 (2015).</li><li>Shen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SH2B1+is+Involved+in+the+Accumulation+of+Amyloid-beta42+in+Alzheimer's+Disease.">SH2B1 is Involved in the Accumulation of Amyloid-beta42 in Alzheimer's Disease.</a> J Alzheimers Dis. 55 (2), 835-847 (2017).</li><li>Song, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxilin+Underlies+Progressive+Locomotor+Deficits+and+Dopaminergic+Neuron+Loss+in+a+Drosophila+Model+of+Parkinson's+Disease.">Auxilin Underlies Progressive Locomotor Deficits and Dopaminergic Neuron Loss in a Drosophila Model of Parkinson's Disease.</a> Cell Rep. 18 (5), 1132-1143 (2017).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+RBO-PI4KIII&#945;+Facilitates+A&#946;42+Secretion+and+Ameliorates+Neural+Deficits+in+A&#946;42-Expressing+Drosophila.">Downregulation of RBO-PI4KIII&#945; Facilitates A&#946;42 Secretion and Ameliorates Neural Deficits in A&#946;42-Expressing Drosophila.</a> J Neurosci. 37 (19), 4928-4941 (2017).</li><li>Riemensperger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+dopamine+pathway+underlies+progressive+locomotor+deficits+in+a+Drosophila+model+of+Parkinson+disease.">A single dopamine pathway underlies progressive locomotor deficits in a Drosophila model of Parkinson disease.</a> Cell Rep. 5 (4), 952-960 (2013).</li></ol>

在这里描述的自动环分析可以同时对数以百计的苍蝇的飞行负 geotaxis 行为进行高通量研究。以前的分析成人攀登的策略涉及单个小瓶中单飞的观察, 而苍蝇的位置则由眼睛手动检测。这个相当繁琐的过程有时可能会导致对数据的误读或误解, 以及 labor-intensive 的工作。我们的自动环分析从一个简单的点击开始, 该仪器自动同步和评估高达100苍蝇的攀登能力。电同步提供了更精确的控制时间和其他参?...

为分析果蝇的成人攀爬行为制定了各种协议和方法。比较费力的是, 传统的分析主要涉及将一只苍蝇放入单个小瓶中, 并使用手动的力量来为同步1,2,3,4。这是单调乏味和费时, 不适合大规模高通量的研究, 并有潜在的变化, 手动力量, 用来打击苍蝇以及其他限制。为了改进这种检测, 我们开发了一种快速的迭代负 Geotaxis (环) 试验, 它允许高通量分析在同一时间的大量苍蝇5。然而, 该检测仍需要人工施加力来同步飞行动作。我们的版本的环化验, 修订后, 先前的化验, 包括一个金属基地承载多个装有苍蝇的小瓶自动控制由电机驱动飞行同步6。录音后, 同步后立即爬升, 然后使用自行设计的软件进行分析。我们的自动环试验已经消除了繁琐和 labor-intensive 的过程, 收集数据从一个单一的苍蝇, 一次一个, 并使数据采集过程更有效。此外, 在一些研究中使用了自动环法来阐明阿尔茨海默氏症和帕金森病的发病机制, 并以高效率的方法验证了7,8,9.
在本文中, 我们演示了使用DDC-Gal4驱动的 rna 干扰飞行的自动环检测。DDC-Gal4是在多巴胺能 (DA) 和素神经元中特别表达的 Gal4 线, 因此它代表了分析与神经10相关的运动缺陷的靶基因效应的一个很好的工具。此外, 我们还合并了UAS-Dicer2, 这是一条提高 rna 干扰效率的飞行线, 用于生成UAS-Dicer2;DDC-Gal4工具行。我们选择使用的 rna 干扰的苍蝇是auxilin (辅助) 的 rna 干扰 v16182 (auxR16182), 一个我们先前确定的基因, 以展示对飞行运动活动的影响8。auxGFP苍蝇也准备用于分析在辅助上的效果. 我们将展示如何使用自动环法测量苍蝇负 geotaxis, 提出结果, 并讨论从结果获得的任何影响。

<table>
	<tbody>
		<tr>
			<td>Forma Environmental Chamber</td>
			<td>Thermo&#160;</td>
			<td>3949</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide cylinders</td>
			<td>FuLian GAS Technology</td>
			<td>GB/T6052</td>
			<td></td>
		</tr>
		<tr>
			<td>HDR-Camcorder</td>
			<td>SONY</td>
			<td>HDR-CX220E</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular stereomicroscope</td>
			<td>Xin Zhen</td>
			<td>SMZ-168BL</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic scales</td>
			<td>MinQiao</td>
			<td>SL1002N</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>Haier</td>
			<td>SC-350</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar-agar powder</td>
			<td>Sinopharm</td>
			<td>10000561</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sinopharm</td>
			<td>10010518</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn meal</td>
			<td>Sinopharm</td>
			<td>5464654</td>
			<td></td>
		</tr>
		<tr>
			<td>Brown sugar</td>
			<td>LiuCaiYuan</td>
			<td>45467936</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant dry yeast</td>
			<td>AB MAURI</td>
			<td>20886</td>
			<td></td>
		</tr>
		<tr>
			<td>AuxR16182</td>
			<td>VDRC</td>
			<td>7187</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-Dicer2</td>
			<td>Bloomington</td>
			<td>24650</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-mCD8GFP</td>
			<td>Bloomington</td>
			<td>32185</td>
			<td></td>
		</tr>
		<tr>
			<td>DDC-Gal4</td>
			<td></td>
			<td></td>
			<td>A gift from Fude Huang</td>
		</tr>
		<tr>
			<td>AuxGFP</td>
			<td></td>
			<td></td>
			<td>A gift from Henry Chang</td>
		</tr>
	</tbody>
</table>

an automated rapid iterative negative geotaxis assay for analyzing adult climbing behavior in a drosophila model of neurodegeneration

神经退行性疾病往往与运动能力的逐渐丧失, 缩短寿命, 和年龄相关的神经。为了了解这些细胞事件的机制, 以及它们之间的因果关系,果蝇, 用其复杂的遗传工具和多样的行为特征, 作为疾病模型来评估神经退行性表型。在这里, 我们描述了一个高通量的方法来分析果蝇成人负 geotaxis 行为, 作为可能的运动缺陷的迹象, 与神经。一个自动化的机器是设计和开发, 以驱动飞行同步使用最初的电脉冲, 后来允许记录的负 geotaxis 行为的过程中秒到分钟。然后用自行设计的 RflyDetection 软件对数字记录的视频图像进行统计处理。不同于基于单飞的人工控制的负 geotaxis 检测, 这种精确、快速和高通量的协议允许同时获取超过数百个苍蝇的数据, 提供了一种有效的方法来推进我们与神经相关的运动缺陷的基本机制的理解。

本文说明了在评估苍蝇负 geotaxis 行为的使用自动环法。与以前的环分析不同的是, 我们的检测包括一个自动装置, 它提供了一种同步飞行动作的电动力量, 并同时对数以百计的苍蝇进行解析 (图 1)。分析了Dicer2;DDC 和 #62; auxR16182 苍蝇在6秒的时间内显示出在攀爬距离上的年龄依赖性下降, 这表明在 DA 神经元中, 辅助表达对飞行活动是至关?...

Watch this Scientific Journal Video about An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration at JoVE.com

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

此 step-by 步骤协议使用自动缸系统来分析果蝇的负 geotaxis 行为, 它承载数以百计的苍蝇并使其动作与电动机同步。在同步时, 采用自行设计的 RflyDetection 软件对飞行负 geotaxis 行为进行检测、数字记录和分析。

一种自动快速迭代阴性 Geotaxis 分析方法在神经的果蝇模型中的成人攀爬行为

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...

Research

JoVE Journal

Behavior

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

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The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

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Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

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