这项研究是由普通医学科学研究所（GM099883和GM074675）的赠款，以EAK的资助者在研究设计，数据收集和分析，发布决定，或准备的手稿没有作用的支持。我们感谢的人从我们在哈佛医学院加工车间的行为设备的设计（联系人<a href="http://mikesmachine.com">:http://mikesmachine.com）。</a> 作者的贡献:ST，BC设计的行为装置。 ST设计和优化实验方案。 ST和BC进行分析实验。 ST，EAK和BC写文章。

注:以下是实验性的协议，我们用它来 ​​允许可靠触发侵略对雄性果蝇之间（果蝇）的一步一步的说明。这个过程导致的"失败者"效应的形成。使用该协议，一个短期的"胜利者"效果也已被最近报道5,6-，图1示出了行为实验的时间线。 <img alt="图1" src="/files/ftp_upload/53395/53395fig1.jpg" /> 图1.时间线侵略的实验。第1天，后期雄蜂蛹（当翅膀变成黑色）被隔离在新配制的隔离小瓶。在新兴的成人后的5当日4日龄雄性果蝇麻醉与CO 2和涂上不同的颜色标识。第7天，食品杯和战斗室是准备设立的行为实验。两个6-7日龄成年男性被装入室，并能互动。 <a href="https://www.jove.com/files/ftp_upload/53395/53395fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 1.第一天:蛹的社会隔离<ol><li>提高，在25℃，50％相对湿度保持苍蝇股环境控制12小时光照/ 12小时黑暗周期的孵化器。 </li><li>准备隔离瓶:热鲜飞食品，直到融化。使用巴斯德吸管，转移大约的熔化飞食品1.5 ml的成单空玻璃试管（1.6×10厘米）。让食物凝固。 </li><li>隔离蛹:用细的画笔，轻轻地从股票瓶中取出后期雄蛹（当翅膀变成黑色），并把它放在一个新鲜烹制的食品含有试管的一侧。关闭隔离小瓶棉（ 图2）。 </li><lI&gt;含有蛹到12小时光照/ 12小时黑暗培养箱中在25℃和相对湿度50％放置隔离小瓶中。 </li></ol><img alt="图2" src="/files/ftp_upload/53395/53395fig2.jpg" /> 图2.社会隔离，一个后期男性蛹被放置在侧上新鲜制备的隔离管中。含蛹隔离瓶放置在12小时光照/ 12小时黑暗的孵化器在25℃，相对湿度50％。 <a href="https://www.jove.com/files/ftp_upload/53395/53395fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.第5天:绘画蝇的个人识别目的<ol><li>用4日龄雄性苍蝇和现有的行为实验2天执行此步骤在第5天，以减少的 CO 2麻醉的负面影响。 </li><li>麻醉蝇用CO 2。 </l我&gt; <li>用细牙签，放置一个小点上胸部丙烯酸涂料。避免将油漆的头，翅膀，腹部或腿部（ 图3）。 </li><li>让油漆干燥大约10秒钟，然后轻轻地传回苍蝇到原来孤立的小瓶。 </li><li>返回隔离小瓶于12小时光照/ 12小时黑暗培养箱中在25℃和相对湿度50％。 </li></ol><img alt="图3" src="/files/ftp_upload/53395/53395fig3.jpg" /> 图3.个体识别。麻醉带有CO 2，一小滴丙烯酸漆飞后涂抹在胸部。对于个体识别的目的，不同的颜色时（白色和蓝色在这里）。请参阅以下有关使用二氧化碳在这一步的讨论。 <a href="https://www.jove.com/files/ftp_upload/53395/53395fig3large.jpg" target="_blank">请点击此处查看本figu的放大版本。</a>回覆。 3，第7天:准备设备<ol><li>准备食物杯: <ol><li>热火新鲜飞食品，直到它融化。 </li><li>使用巴斯德吸管，转移大约0.8 ml的食物到螺旋盖小瓶（1.5厘米直径，1厘米高）的盖子，并允许冷却。避免气泡。 </li><li>通过混合干酵母几滴之水鲜酵母膏。当厚，应用一小滴酵母膏到食品杯表面的用牙签（图4）的中心。 </li></ol></li></ol><img alt="图4" src="/files/ftp_upload/53395/53395fig4.jpg" /> 图4.准备食物的杯子。一种食品杯新鲜飞食品准备。食品凝固后，酵母膏点放置在食用杯面的中心。 <a href="https://www.jove.com/files/ftp_upload/53395/53395fig4large.jpg" target="_blank">请速到v</a>IEW这个图的放大版本。 <ol start="2"><li>准备个人战斗钱伯斯: <ol><li>使用前用水每次彻底清洗装置和个人战斗腔室。干燥，使用前纸巾。 </li><li>将食物杯在每个战斗室的中心和地方解决了一滴腻子。 </li><li>覆盖战斗腔室与该装置的盖子（图5）。 </li><li>放置装置成高湿度环境（60％），在25℃。 </li><li>放置装置之上的光源以照亮战斗腔室，并设置一台摄像机用于从上方的记录。 </li></ol></li></ol><img alt="图5" src="/files/ftp_upload/53395/53395fig5.jpg" /> 图5.准备个别战斗室中。将新鲜制备的食品杯用酵母膏的表面上的点被固定到洗涤个人音响ghting室。钱伯斯被关闭的两片盖允许插入一个不透明的塑料分隔成单个的战斗室。 <a href="https://www.jove.com/files/ftp_upload/53395/53395fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 4，第7天:行为实验<ol><li>执行行为实验用6-7日龄雄蝇。 </li><li>将塑料分隔成各个作战室。 </li><li>从隔离管中取出棉塞和定位下方开放孔的管在装置的一侧。 </li><li>让飞进入战斗室由负趋地性，通常在几秒钟之内。 </li><li>关闭滑动壁飞已经离开所述管并进入该腔室后。 </li><li>重复此过程，介绍第二蝇进入战斗室的设备的另一端。 </li><li>放置装置背面被照明videotapiNG的位置。 </li><li>开始录像并取下塑料分隔，让苍蝇进行互动。 <ol><li>侵略实验: <ol><li>在这些实验中，记录打架至少20分钟，以确保强支配关系的形成。 </li><li>后所需的时间，停止录像。 </li><li>通过反转上述步骤卸下苍蝇战斗室。 </li><li>彻底用温和的清洁剂和水每次使用和清洗后清洗行为装置。 </li></ol></li><li>对于"失败者"的试验研究: 注意:在这些实验中，第一打架被认为是调节阶段和第二打斗作为测试阶段。休息之间的战斗的时间可能有所不同，但10分钟可形成强大的"失败者"的效果。 <ol><li>按照上述执行的第一次战斗的过程。 </li><li>关键的一步:为了诱导的"失败者"的心态，E的​​形成nsure首先打架至少有20分钟长，以建立强大的统治地位的关系。 20分钟后，重新插入塑料分隔成战斗室的苍蝇分离。 </li><li>经过10分钟休息时间，轻轻地撕下塑料分隔，让苍蝇进行交互的第二战。 </li><li>在第二个20分钟战斗结束，停止录像。 </li><li>删除苍蝇作为上述行为室。 </li><li>再次，彻底清洁行为装置。 </li></ol></li></ol></li></ol> 5.行为分析<ol><li>传输影片<ol><li>连接照相机到计算机。 </li><li>转换苍蝇.MTS为.mov格式的​​苍蝇，并加入电影剪辑在一起，使用的视频转换软件，并选择"加入所选的剪辑"。 </li><li>观察和分析每部电影与视频阅读器软件。 </li></ol></li><li>计分行为 注:这可以手动执行，我们荷兰国际集团的行为分析程序软件（如果可用）。下图描述了一个"失败者"的效果可能的人工分析。 <ol><li>得分苍蝇上的食物杯之间的第一次遭遇的时间（遭遇是对苍蝇至少持续2秒之间的相互作用）和外观第一刺的时间（一种行为模式，其中一个飞升至高它的后腿和捕捉下来，并试图抓住对手）。随后，比分延迟弓步（首先遇到的与第一弓步之间的间隔）。 </li><li>在战斗中遭遇数和箭步蹲的总数。 注意:如果需要，也可以得分等行为模式，如翼的威胁，拳击，击剑，撤退或遇到的第一个冲刺前的数字，例如。 </li><li>分数建立霸主地位（第一次遇到，当一只苍蝇务虚连续三次断食杯前后的时间之间的时间的时间呃接收弓步）。注意这飞是赢家，哪些战斗的失败者。 </li></ol></li><li>计算一个"失败者"的影响<ol><li>划分战斗在前面的失败者失去第二次战斗（失败者的损失:LL）的数量由打架的总数，由100所获得的比例代表"失败者"的效果倍增: [（＃苍蝇LL）/（＃总拼）]×100 </li><li>使用卡方检验来比较"失败者"效应是否是从50％的预期值统计学差异。 </li></ol></li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetising+Drosophila+for+behavioural+studies.">Anaesthetising Drosophila for behavioural studies.</a> J Insect Physiol. 46, 439-442 (2000).</li><li>Greenspan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fly Pushing: The theory and Practice of Drosophila Genetics. , (1997).</li><li>Cabral, L. G., Foley, B. R., Nuzhdin, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+sex+trade+with+violence+among+genotypes+in+Drosophila+melanogaster?.">Does sex trade with violence among genotypes in Drosophila melanogaster?.</a> PLoS One. 3 (4), e1986 (2008).</li></ol>

The authors have nothing to disclose.

利用果蝇以其强大的遗传方式作为模式生物的侵略研究的新时代开始了十几年前在可靠的战斗行为，可以单对苍蝇1,7所获得的引进新的实验舞台上， 8,17,19。这些领域包括所需的资源（食物杯，潜在配偶）和失败者的空间飞行，以退为进。当用这些领域获得的结果，加入到约果蝇拼行为以前的知识， 果蝇确立为用于检查像侵略一个复杂的行为非常理想的模型系统，用...

侵略在动物系统是紧密联系在一起的采集和如食物，领地和配偶的资源控股关联。鉴于这种起着个人健身的重要作用，这是不奇怪的侵略整个动物王国中发展而来的。作为一个自适应的特点是直接受益的个体，较强的学习和记忆组件与侵略有关 。在对社会地位的竞争，以往的战斗体验影响了后来的比赛结果。在一般情况下，以前输球阅历的增加，​​并赢得经验减少，失去以后的比赛的可能性（被称为"输家"和"赢家"的影响）。 "失败者"的影响已经在广泛的物种中观察到，一些报告表明，这些都可以持续数天，而"赢家"的影响通常是持续时间较短2,10,11的。 攻击行为的第一次报告在果蝇（D. ampelophila）是由斯特蒂文特于1915年在涉及性别认同和性别选择12纸。半个世纪后的今天，男性的水果更完整的检查飞行侵略所做，所描述的大多数在果蝇打架观察到的行为模式。这些实验主要是进行了小团体的男性和女性的苍蝇观察到小时，13-15。近来，通过加入强大的遗传工具，二进单一性别的战斗的设置和观测，D。更短的时间果蝇已成为侵略1,16的生物学研究的重要模式系统。同性对雄性和雌性果蝇的打架分析表明，侵略涉及刻板的行为模式，从一个到另一个在统计学上可靠的方式1,17过渡。一些观察到的行为模式是性别特异性，而其他人在战斗观察S在男女双方。男打架去更高强度级别比女性打架，结果有明确的"赢家"和"输家"的主导地位关系的形成。目前，许多实验室已经开始调查了生化18，神经7,19-21，和遗传22基础侵略。不幸的是，一元分析的研究深入了解拼动力学和的形成和维持支配关系是有问题的，由于使用的实验程序在不同实验室的多重的。基本上所有的文献中所描述的技术涉及处理苍蝇和操纵，导入到舞台19,23,24和行为实验3,4中转移出来的苍蝇的竞技场。轻柔抽吸是操纵苍蝇3,4,23-25 ​​最常见的方式，但冷或CO 2麻醉也使用9,26，即使previous研究已经报告说，这些程序都有飞的行为27,28有害影响。持续至少24小时的使用任何麻醉剂后，建议尽量减少其对行为的影响29,30。 苍蝇从以前的战斗经验和修改他们的行为模式的使用在新的情况下，这意味着学习和记忆陪伴，并激动遭遇的后果。在这个意义上，飞打架就像在其中飞得知一个战略工作，然后在随后的遭遇越来越多的时候使用它操作条件反射的学习情况。苍蝇改变自己的战斗策略后主导地位的关系都在打架成立至今，凭借赢家冲刺越来越多，失败者越来越少。经过一段时间的分离的，以往的失败者显示更顺从的行为和非常可能失去2与幼稚FL成对第二战斗IES或往届获奖者3,4。然而，由于缺乏一个实验的过程，不包括装卸苍蝇的取得了"失败者"的效果很难详尽的研究。在最近的研究中，我们比较常规用于实验室（吸入和冷麻醉）引入飞进行为室新方法，消除了处理两个实验程序。结果表明，冷麻醉有更大的负面影响侵略不是愿望，但即使抽吸苍蝇减少攻击性的水平。吸入没有，但是，造成伴随侵略的学习和记忆非常显著的影​​响。继相同的协议有两个实验程序（愿望，也没有处理），当苍蝇的处理是从实验过程5,6消除只观察到一个强大的"失败者"的效果。 理想情况下， 果蝇的侵略在Labo研究ratories应包括苍蝇通常在野外遇到（对资源的争夺，领土保卫和空间来逃脱）环境状况。此外，实验条件应进行优化，以可靠地诱导，观察和解释所检查的行为（尽量减少动物的处理，限制使用二氧化碳作为麻醉剂，规范的实验程序）。在试图解决大多数或所有这些问题，我们设计了消除处理苍蝇前，中，向他们介绍行为赛场后，新的行为装置。与这些设备中，蝇使用其固有负趋地性，以被移动，并转移进和移出的斗争​​腔室。通过消除处理苍蝇，该协议的目的是:（1）降低个体之间的变异性的行为; （二）减少所需的时间苍蝇相互作用并产生清晰显性关系（男性）; （三）可靠地诱发足够强大behavio拉尔发生变化，允许组建"失败者"的效果。 本协议描述一个新的实验设备和一步一步的过程来分析的侵略，并允许在D.强烈的"失败者"效应的形成果蝇。我们期望这一行为装置，可以很容易地适用于通过果蝇表现出其他社会行为的研究。

<table border="0" cellpadding="0" cellspacing="0" width="665">
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			<td height="42" style="height:42px;width:216px;">Drosophila melanogaster pupa</td>
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			<td height="84" style="height:84px;width:216px;">Computer</td>
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			<td style="width:167px;">With a minimum of 1.4 Ghz Processor, running Microsoft Windows or Machintosh HD</td>
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			<td height="42" style="height:42px;width:216px;">ClipWrap</td>
			<td style="width:163px;">Download online</td>
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			<td height="42" style="height:42px;width:216px;">QuickTime Player</td>
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</table>

a new approach that eliminates handling for studying aggression and the "loser" effect in drosophila melanogaster

果蝇攻击行为是由刻板行为模式的顺序表达（分析参见图1）。这种复杂的行为是由遗传，荷尔蒙和环境因素的影响。正如在许多生物，以往的战斗体验影响果蝇的战斗策略和以后比赛的结果:失去的斗争增加了失去以后的比赛的可能性，揭示了"失败者"效应可能涉及学习和记忆2-4。伴随像侵略复杂的社会行为表达的学习和记忆，是敏感的前期试验动物5,6的处理。许多实验程序被用在不同的实验室研究侵略7-9，然而，没有常规使用的协议，排除处理苍蝇当前可用。在这里，我们报告说，省去了处理果蝇的一个新的行为装置，使用insteað他们与生俱来的负面geotactic响应移动的动物进入或退出战斗室。在这个协议中，含有食品杯小圆形斗争领域被引入之前苍蝇分成相等的两半通过一个可移动的塑料滑块。苍蝇通过推拉房门及趋地进入他们的家庭隔离小瓶室。在去除塑料滑块，苍蝇可以自由交互。经过指定的时间段，苍蝇是通过滑块进行后续实验再次分离。所有这一切都没有办理个人苍蝇容易。该设备提供了一种新的方法来研究的侵略和相关的学习和记忆，其中包括在飞行战斗"失败者"效应的形成。此外，这种新的通用行为装置可以被用来研究苍蝇等社会行为和应，在一般情况下，是用于调查在基本行为过程经验相关的变化的兴趣。

本节介绍行为腔室的设计和一组典型的按照以上测定侵略和的"失败者"效应的形成描述的协议行为实验的分析。还示出的其他行为的样品，可以使用该装置来测量。 图6示出了新装置的示意图。该装置由三个圆筒形孔（尺寸:22.86毫米直径17毫米的高度），这些战斗的腔室。两片盖关闭顶部的战斗室和允许插入的薄不透明的塑料分频器，分割室分成相等的两部分。在...

Watch this Scientific Journal Video about A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster at JoVE.com

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

在果蝇打架的行为模式观察，争取动态，以及相关的学习和记忆都是通过实验条件的影响。这里介绍的协议描述了一种新的程序，完全消除了在实验操作苍蝇。这提高了战斗的动态，并允许形成强大的"失败者"的效果。

一种新的方法，省去了处理的研究侵略和"失败者"的影响<em&gt;果蝇</em

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This ...

a-new-approach-that-eliminates-handling-for-studying-aggression-loser

Research

JoVE Journal

Neuroscience

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

9.5K 次观看.  Harvard Medical School. 在果蝇打架的行为模式观察，争取动态，以及相关的学习和记忆都是通过实验条件的影响。这里介绍的协议描述了一种新的程序，完全消除了在实验操作苍蝇。这提高了战斗的动态，并允许形成强大的"失败者"的效果。 

视频: A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

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

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

粉条和Capellini处理测试：在大鼠和小鼠的灵巧的前爪功能简单的定量措施

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

电检测屏幕的配对Nanoinjection和生物活性化合物的使用果蝇巨人纤维系统

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

在自发活动的测定<em&gt;果蝇</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

简单的检测方法的社交回避的量化<em&gt;果蝇</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

在条件影响社会空间<em&gt;果蝇</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

在气味引导行为的高分辨率定量<em&gt;果蝇</em&gt;使用<em&gt; Flywalk</em&gt;范式

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

一个行为分析对机械感觉MARCM为基础的克隆<em&gt;果蝇</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

觅食路径长度协议<em&gt;果蝇</em&gt;幼虫

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...