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: 女性比率为 1:2. </li>
	<li> 分别为每个组和 #160;p er 实验收集新 eclosed 的雄性和雌性小瓶, 每只普通的苍蝇小瓶中放置10只苍蝇, 标准食物在和 #160; 25 和 #176; C. </li>
	<li> 根据实验, 将收集的苍蝇保持35天, 并将其用于5、15、25和35天的自动环分析. </li>
</ol> 2。自动环形检测 <ol> <li> 将每种基因型的10只收集的苍蝇 (中性) 转移到每个小瓶中, 然后用螺钉固定药瓶。分析控制苍蝇和苍蝇携带不同基因型一起为每组实验 (最多10瓶同时, 每瓶10只苍蝇). </li>
	<li> 打开放置在设备前面的数码相机, 一旦苍蝇都装好就开始录音. </li>
	<li> 在允许1分钟的苍蝇以小瓶结算后, 打开控制步骤驱动程序的步骤控制器; 这将驱动小型电动机控制连接的拉杆, 使其在2秒内连续上升并轻拍设备4次。请参见 图 1 。
	<ol> <li> 敲击后, 请注意, 苍蝇开始爬上墙。确保录制继续进行. </li>
	</ol> </li> <li> 在3到5连续的试验中重复步骤2.3 中描述的60秒间隔中的同步. </li>
	<li> 重复试验5、15、25和35天的老苍蝇。每组至少进行3项独立实验, 每次试验至少30只收集的苍蝇 (3 瓶). </li>
</ol> 3。数据分析 <ol> <li> 将录制的视频导入计算机. </li>
	<li> 在敲击后6秒拍摄视频的快照, 用于每个试用版. </li>
	<li> 将快照映像导入 RflyDetection 软件 (请参见 图 2 ), 使用 #39; 文件和 #39; 菜单. </li>
	<li> 通过使用工具栏上的基线图标, 然后使用光标标记图像上的上、下基线, 精确设置小瓶的上下基线. </li>
	<li> 输入每瓶的苍蝇数 (, 如 , 10 在这里) #39; 飞在矩形和 #39; 在和 #39 中的字段和瓶长 (, 如 , 14 厘米); 管高度和 #39; 设置栏中的字段。
	<ol> <li> 请注意, 每个小瓶都在屏幕上检测到各个飞行位置并标记有点. 
		注意: 图 2 指示所有菜单按钮单击的位置. </li>
	</ol> </li> <li> 注意, 软件自动确定每只苍蝇的爬升距离, 并在右侧面板的表格中显示每瓶10只苍蝇的平均值。(请参见 图 2 ) </li>
	<li> 使用统计软件 ( 如 、棱镜) 处理爬数, 以进行进一步的统计分析. </li>
	<li> 将数据显示为平均值和 #177; SEM. </li>
	<li> 计算 p 值的重要性 (用星号、* p 和 #60; 0.05、 p 和 #60; 0.01、*** p 和 #60; 0.001) 使用 one-way 方差分析 Bonferroni 多项比较试验. </li>
</ol>

<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>

作者没有什么可透露的。

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数的方法, 因此可以仔细地测量飞行成人攀爬过程的总体捕获。
接下来, 利用我们自行设计的 RflyDetection 软件, 可以确定准确的飞行位置。该软件考虑到整个2D 小瓶面积 (由矩形框架确定), 和顶部和底部基线, 在获得位置的所有苍蝇在指定区域内, 并提出他们作为点。此外, 对爬升距离和平均值的自动计算使得数据的处理变得更快、更容易。在一个合理的时间框架7,8,9中,可以振振有词地收集和分析一大群苍蝇的数据集。
尽管这里描述了与神经相关的攀岩活动的年龄依赖性下降, 但这种方法同样有效地分析了与发育缺陷相关的运动活动。为此, 自动环法是一种有效的方法来分析果蝇阴性 geotaxis 在任何成人阶段, 并在正常或病理状态的大脑功能。尽管如此, 尽管 RflyDetection 软件能够手动识别任何意外的 uncaptured, 但该检测方法在完全分析所有苍蝇方面的应用有限。

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重要的. 在 da 神经元中, 攀爬能力似乎不会受到auxGFP过度表达的影响, 而 da 神经元中的auxR16182 和auxGFP的表达则救出了 rna 干扰表型 (图 2和图 3)。除了验证自动环法作为高通量和有效的方法来评估与 DA 神经元变性相关的运动缺陷外, 这些结果还表明, 这种自动化的方法有助于识别潜在帕金森病的危险因素, 并提供了他们的神经退行性功能的线索。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56507/56507fig1.jpg"> 
图1。自动环形装置.(A和B) 设备的前后视图: 插座 (1), 立式钢杆 (2), 固定在车架 (3) 上的管子, 透明塑料瓶 (4), 泡沫棒 (5), 金属底座 (6), 微步驱动 (7), 电子控制器 (8),开关 (9), 小电机 (10), 和杠杆连接到小电机 (11)。长方形金属框架的大小是 46 cm x 26 cm x 16 cm, 藏品10透明塑料瓶 (2.1 cm 在直径, 14.5 cm 在高度) 巩固用螺丝。要打开仪器, 首先点击开关 (9), 打开 (8), (7), 和 (10) 因此。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56507/56507fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56507/56507fig2.jpg"> 
图2。RflyDetection 的典型快照图像分析.将快照图像导入到 RflyDetection 软件中, 并在该接口上的每个图标上都显示了右侧有文本的操作。在设置了顶部和底部基线后, 软件会自动检测并标记小瓶内的飞行位置。每只苍蝇的爬升距离 (cm) 为6秒, 每个小瓶的平均值都显示在表中。然后将这些数字输入统计软件, 以便进行进一步的统计分析。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56507/56507fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56507/56507fig3.jpg"> 
图3。在 DA 神经元中, 当辅助表达式为 downregulated 时, 统计条形图显示了随年龄的下降而上升的能力.采用自动环法分析了携带以下基因型的果蝇的负 geotaxis 行为: auxR16182/+ (无 Gal4), UAS-Dicer2;DDC-Gal4苍蝇越过w1118 (控制)、 UAS-mCD8GFP (控制)、 auxR16182、auxGFP和auxR16182; auxGFP (抢救)。请注意, 随着时间的推移, 当辅助表达式在 DA 神经元中减少时, 雄性和雌性果蝇的速度明显减慢. 在重新引入辅助表达式时, 会救出运动缺陷. 对5、15、25和35天的苍蝇进行了评估。雄性 (A) 和雌性 (B) 苍蝇分别显示。在右侧显示与不同条形颜色对应的基因型标签的图例。数据显示为平均± SEM. p-意义值 (用星号表示, * p和 #60; 0.05, ** p 和 #60; 0.01, ** p 和 #60; 0.001, ns: 没有意义, 在星号下面绘制了一个条形图来指示对象 比较) 计算比较mCD8GFP控件或辅助 rna 干扰, 使用 one-way 方差分析与 Bonferroni 多比较测试. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56507/56507fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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. ...

an-automated-rapid-iterative-negative-geotaxis-assay-for-analyzing

Nanyang Technological University

Research

JoVE Journal

Behavior

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

9.7K 次观看.  Tongji University School of Medicine. 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。

视频: 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

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 between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

背景和线索恐惧制约试验小鼠中使用视频分析系统

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 ...

科研

行为学

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III).
The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called &#34;stimulus mouse&#34;).
In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a stimulus mouse is placed under one of the inverted cups and the sociability of the test mouse is quantified by the amounts of time it spends near the cup containing the enclosed stimulus mouse vs. the empty inverted cup. In session III, the inverted cups are removed and both mice interact freely. The sociability of the test mouse in session III is quantified by the number of social approaches it makes toward the stimulus mouse and by the number of times it avoids a social approach by the stimulus mouse.
The automated evaluation of the movie detects the nose of the test mouse, which allows the determination of all described sociability measures in session I and II (in session III, approaches are identified automatically but classified manually). To find the nose, the image of an empty cage is digitally subtracted from each frame of the movie and the resulting image is binarized to identify the mouse pixels. The mouse tail is automatically removed and the two most distant points of the remaining mouse are determined; these are close to nose and base of tail. By analyzing the motion of the mouse and using continuity arguments, the nose is identified.
<img alt="Figure 1" src="/files/ftp_upload/52508/52508fig1.jpg" /> 
Figure 1. Assessment of Sociability During 3 sessions. Session I (top): Acclimation of test mouse to the cage. Session II (middle): Test mouse moving freely in the cage while the stimulus mouse is enclosed in an inverted cup. Session III (bottom): Both test mouse and stimulus mouse are allowed to move freely and interact with each other.

This protocol describes a method to quantify mouse sociability. Mice are videotaped as they move and interact in a special cage. Movie processing allows for the automated quantification of sociability with excellent accuracy and reliability.

使用自动图像处理方法鼠标社交性的实验评估

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression have been reported, the unpredictable chronic mild stress (UCMS) protocol is an established translationally-relevant model for inducing behavioral symptoms commonly associated with clinical depression, such as anhedonia, altered grooming behavior, and learned helplessness in rodents. The UCMS protocol also induces physiological (e.g., hypercortisolemia, hypertension) and neurological (e.g., anhedonia, learned helplessness) changes that are clinically associated with depression. Importantly, UCMS-induced depressive symptoms can be ameliorated through chronic, but not acute, treatment with common SSRIs. As such, the UCMS protocol offers many advantages over acute stress protocols or protocols that utilize more extreme stressors. Our protocol involves randomized, daily exposures to 7 distinct stressors: damp bedding, removal of bedding, cage tilt, alteration of light/dark cycles, social stresses, shallow water bath, and predator sounds/smells. By subjecting rodents 3-4 hr daily to these mild stressors for 8 weeks, we demonstrate both significant behavioral changes and poor health outcomes to the cardiovascular system. This approach allows for in-depth interrogation of the neurological, behavioral, and physiological alterations associated with chronic stress-induced depression, as well as for testing of new potential therapeutic agents or intervention strategies.

The unpredictable chronic mild stress (UCMS) protocol is a validated method for studying behavioral and physiological changes associated with chronic stress and depressive symptoms. Eight weeks of imposition of the UCMS protocol induces behavioral changes and poor health outcomes in rodents of either gender.

煽动抑郁症状，行为变化和不利健康结果鼠害一个不可预知的慢性轻度应激协议

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression ...

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

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

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

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly 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 ...

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

基于线虫人群的化学感应优选测定的自动分析

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

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 ...

An Automated T-maze Based Apparatus and Protocol for Analyzing Delay- and Effort-based Decision Making in  Free Moving Rodents

Many neurological and psychiatric patients demonstrate difficulties and/or deficits in decision making. Rodent models are helpful to produce a deeper understanding of the neurobiological causes underlying the decision-making problems. A cost-benefit based T-maze task is used for measuring decision making in which rodents choose between a high reward arm (HRA) and a low reward arm (LRA). There are two paradigms of the T-maze decision-making task, one in which the cost is a time delay and the other in which it is physical effort. Both paradigms require a tedious and labor-intensive management of experimental animals, multiple doors, pellet reward, and arm choice recordings. In the current work, we invented an apparatus based on traditional T-maze with full automation for pellet delivery, door management and choice recordings. This automated setup can be used for the evaluation of both delay- and effort-based decision making in rodents. With the protocol described here, our lab investigated the decision-making phenotypes of multiple genetically modified mice. In the representative data, we showed that the mice with ablated medial habenular showed aversions of both delay and effort and tended to choose the immediate and effortless reward. This protocol helps to decrease the variability caused by experimenter intervention and to enhance experiment efficiency. In addition, chronic silicon probe or microelectrode recording, fiber-optic imaging and/or manipulation of neural activity can be easily applied during the decision-making task using the setup described here.

This article introduces an automated T-maze apparatus that we invented, and a protocol based on this apparatus for analyzing delay-based decision making and effort-based decision making in free moving rodents.

基于自动 T 迷宫的自由移动啮齿动物延迟和努力决策分析装置及协议

Many neurological and psychiatric patients demonstrate difficulties and/or deficits in decision making. Rodent models are helpful to produce a deeper ...

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive decline, such as Alzheimer&#39;s disease (AD), have become a promising tool to elucidate the underlying mechanisms and promote the therapeutic advances. An important step is the validation and characterization of expected behavioral abnormality in the models, in the case of AD, cognitive decline. The long-term behavioral investigations of laboratory animals to study the effect of aging demand substantial efforts from researchers. The IntelliCage system is a high-throughput and cost-effective test battery for mice that eliminates the need for daily human handling. Here, we describe how the system is utilized in the long-term phenotyping of a genetic Alzheimer&#39;s disease model, specifically focusing on the cognitive functions. The experiment employs repeated battery of tests that assess spatial learning and executive functions. This cost-effective age-dependent phenotyping allows us to identify the transient and/or permanent effects of genes on various cognitive aspects.

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer&#39;s disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

基于 IntelliCage 的阿尔茨海默病多基因模型认知功能的自动、长期行为检测

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive ...

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

一种自动测定果蝇在空间和时间温度变化中的性能的方法

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

测量 果蝇 拉瓦的机械结节的改进的检测和工具

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