We thank Rachelle Kanippayoor for her help in identifying the new wild-type strain as being of the melanogaster species. R.W.F, O.F. and A.F.S were responsible for research design; R.W.F, M.N. and O.F. performed the experiments. R.W.F, M.N., O.F. and A.F.S. analyzed the data; R.W.F., I.S.M. and A.F.S. wrote the manuscript. 
This work was supported by PSC-CUNY research awards, jointly funded by The Professional Staff Congress and The City University of New York to A.F.S.; by internal funding from Western University to A.F.S. and I.S.M.; by a training support from the National Alliance for Hispanic Health&#8217;s Alliance/Merck Ciencia (Science) Hispanic Scholars Program and a University Fellowship from the Yale Graduate School of Arts and Sciences to R.W.F.

1.设备和试剂创造了内部（见材料清单等） <ol><li>准备果蝇冷麻醉机进行飞行工作。 <ol><li>切的多孔聚乙烯片以覆盖一个小塑料盒（12.7厘米长，10.2厘米宽），一个移液管尖端盒子通常顶盖（12.7厘米长，10.2厘米宽，3.8厘米深）。 </li><li>装满碎冰的箱子，盖上多孔聚乙烯片材。 </li></ol></li><li>准备一个T-迷宫器具;适应先前在细节28-31所描述的装置。 </li><li>准备描述32苍蝇吸引。 </li><li>准备趋光反应装置取代苍蝇进出测试小瓶无压力： <ol><li>使用逆流装置中，所描述的29,33。 </li></ol></li></ol>注：T迷宫也可用于其它目的，如所描述的28。 <ol start="5"><li>确保坎ogeneous照明条件：执行上覆盖有白色长椅罩长凳的实验中，并在一个白色板的前面。 注：对于这些实验，所用的飞株果蝇野生类型：广-S，俄勒冈州和撒马尔罕航班从我们的实验室库存34;埃尔伍德苍蝇被收集在2011年秋季在亨廷顿的埃尔伍德附近，长岛，纽约，美国。 </li></ol> 2.准备蝇实验前在进行实验前1-2天： <ol><li>保持苍蝇标准玉米面琼脂-糖蜜酵母培养基中于25℃在12小时光照/黑暗周期。 </li><li>收集响应苍蝇在冷麻醉前1-2天进行实验<ol><li>从它们已经提出入50ml Falcon管，用漏斗将瓶子转移苍蝇。 </li><li>将猎鹰管冰下一级苍蝇，成保温冰桶。 </li><li>将冷麻醉设备用在-4℃，寒意碎冰。 </li><li>等待5分钟，苍蝇进入寒意昏迷。 </li><li>转移苍蝇到冷麻醉装置的多孔聚乙烯片材。 </li><li>收集响应苍蝇在冰冷的麻醉设备。在室温下，体视显微镜，分离3-7日龄的男性从女性的苍蝇，在40组维持这些苍蝇。 </li></ol></li><li>收集发射苍蝇1-2天前进行的实验。 <ol><li>轻轻地收集广-S混合性苍蝇从他们的瓶子，用口吸气，控制了他们的号码。 </li><li>在组的60-100维持这些苍蝇，因为它们将是发射器。 </li><li>根据需要，数据点准备发射的许多样本。 </li></ol></li><li>允许恢复至少一晚集合反应和发射器后，以减少冷麻醉任何混杂效应和对行为的嘴吸集合。 </li><li>开放测试瓶的袋子，以允许陈旧的塑料气味的空气袋被新鲜空气取代，实验至少1天前。 </li></ol> 2小时进行实验前： <ol start="6"><li>确保在将实验进行房间内的温度是大约23-25​​℃，甚至光，湿度30％以上。 </li><li>转移反应和发射果蝇到新鲜食品小瓶，以确保这些苍蝇不饿死。 </li><li>让果蝇适应环境2小时，在该实验将进行在板凳上。 </li></ol> 3.执行的社交回避实验<ol><li>在Zeitgeber时间ZT5和ZT9之间下午进行每个实验在一天的同一时间，在范围3-4小时的。 </li><li>广场上的冲击垫的T-迷宫，并拧紧螺丝夹因此T他的电梯是稳定的。 </li><li>传送应答苍蝇到一个新的测试瓶。管理单元该小瓶含有响应苍蝇到T迷宫的顶部。 </li><li>倾斜的T-迷宫设备和挖掘它的冲击垫使果蝇倒在了电梯。 </li><li>移动电梯的顶部向下所以苍蝇是T型迷宫的顶部和底部部分之间的，但要注意，以保持上述的选择点的电梯 - 防止苍蝇逸出。 </li><li>获得小瓶DSO（见第5生成以前由苍蝇占领DSO-免费瓶）。 <ol><li>而响应苍蝇正在适应这个新的环境中，将发射器苍蝇在测试小瓶。 </li><li>将一块棉花的收小瓶使果蝇不要逃避。 </li><li>机械搅动此小瓶上的小型涡旋如下：涡流15秒，从涡流取出小瓶，持续5秒。重复3次，总共55秒。 </li><li>雷姆奥雅纳发射苍蝇出新鲜小瓶 - 通过在食品小瓶转移 - 和快速地将本DSO填充小瓶插入T迷宫装置的两个侧面中的一个。 </li><li>交替放置一边在每个新运行的DSO填补小瓶。 </li></ol></li><li>放置一个新鲜的测试小瓶在T迷宫的另一侧。 </li><li>把电梯完全放下，让果蝇可以新鲜的测试瓶和小瓶测试与DSO之间选择，并开始计时。 </li><li>让我们，新鲜瓶和小瓶DSO之间的苍蝇选择，1分钟，除非另有说明。 </li><li> 1分钟后，将电梯向上分开苍蝇在新鲜小瓶中，小瓶DSO，和那些被困在电梯。 </li><li>算在每个小瓶蝇的数目，并在电梯。 </li><li>重复此为每个基因型/条件。 </li></ol> 4.分析了社交回避数据<ol><li>算在每个小瓶的每个热诺果蝇数YPE，并且将数据传输到电子表格。 </li><li>使用电子表格程序，通过减去苍蝇在DSO小瓶的数量从果蝇中DSO无小瓶的数量，再由蝇的总数除以计算出的性能指数（PI）为每个基因型。 </li><li>通过比较统计分析软件对结果的分析督察“的意思。 <ol><li>理想情况下，预备2-3内部重复每个实验，和3个独立的重复序列，在不同的日子进行的，用不同的瓶子或跨越30。 </li><li>使用高斯分布的统计测试，作为数据遵循正态分布。 </li><li>当测试一个效果（ 即 ，密度- 图2），使用单向方差分析，具有检测后对多重比较（逐列），或简单t检验每个结果比较的对照。 </li><li>当组实验中进行，同时测试条件和性别（空药瓶，强调了ð未强调苍蝇;和性别的影响- 图1），使用两因素ANOVA。 </li></ol></li></ol> 5.生成瓶由非重读苍蝇此前占用 - 执行取而代之3.5〜3.6 <ol><li>使用的趋光性，制备由苍蝇，但没有强调占用小瓶，作为对照。 </li><li>转让苍蝇在测试瓶中，并卡入逆流装置，在相反的位置，新鲜的空药瓶。 </li><li>与远端瓶直接在一个15瓦荧光灯冷白光前水平奠定了设备，并盖上黑布。 </li><li>让苍蝇在新鲜小瓶轻轻移动。这可能需要几分钟的时间。一旦大多数苍蝇的行走朝向远端小瓶的光，启动计时器，持续1分钟。 </li><li>轻轻取出黑色布，开关的光的位置，恢复与黑色布，并且允许苍蝇离开测试小瓶中。 </li><li>在那个时候，我nitiate选择实验在3.2至3.5。跳过3.6和3.7。 </li><li>移动装置的滑动件，该腾空从另一侧的测试小瓶苍蝇分离。 </li><li>使用在T型迷宫的测试腾空小瓶马上供选择的实验，如3.7。 </li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+decline+in+behavioral+performance+of+Drosophila+melanogaster+with+age.">Differential decline in behavioral performance of Drosophila melanogaster with age.</a> Mechanisms of Ageing and Development. , 127-647 (2006).</li></ol>

The authors declare that they have no competing financial interests.

本协议描述为社会避免检测的详细过程。广-S只会避免在飞行以前一直强调机械小瓶和应答者的性别和数量不影响社交回避的性能。然而，反应的遗传背景有重要的影响。 以下是几个关键的步骤成功完成这个实验：1）实验前总是转移苍蝇2小时，并确保不影响他们的环境; 2）在一天的同一时间进行的实验总是理想地之间Zeitgeber时间光的发病后ZT5（小时）和ZT9减少的变化表现挂?...

该方法的目标是很容易量化的果蝇简单的社会行为，独立于求偶和侵略一个新的方面。 社会互动是至关重要的内一个社会的正常发育和健康个体，以及一个社会群体作为一个整体的功能。这些相互作用的高复杂性就必须大的样本大小和一个系统，它允许为行为简化为社会行为的遗传和神经基础仍然知之甚少。 果蝇是可用于识别基因的有力的遗传模型，并社会互动的神经基础。事实上，D.果蝇有复杂的社会行为剧目和社会化的一些直接测量已经完成1-7。不过，这些努力都集中在相对复杂的社会行为，如aggres西伯相互作用3,6，求爱3,8-12的各个方面，以及如何社会经验影响其他行为，如学习，或昼夜节律13-17。此外，许多这些测定依赖于分析蝇基的复杂的交互模式，利用视频跟踪和计算机软件来分析得到的数据的丰度。这样的分析是非常宝贵的，并导致重要的新见解，如在组7苍蝇飞相互作用的动态。一个限制，但是，这些检测到社会大众的交通不便，和基本认同他人的机制的了解有限。换句话说，信号由一个单独的和其识别由另一个发射的基础仍然知之甚少18。 相比之下，苍蝇也表现出一个简单的行为，社交回避，使个人从强调苍蝇发出的信号搬开：该D.黑素ogaster应力加臭剂或DSO 19。在高通量测定法中，此行为可以量化为避免由其他蝇，或社交回避19射出的应力的信号。苍蝇被放置在一个T型迷宫装置和给予的选择，以避免含有DSO的小瓶。使用此测定，CO 2被证明是在DSO的组分必要响应为CO 2解剖19所述的神经电路，和一部分。 这里介绍的社交回避法类似概念在西摩·本泽的实验室开发的简单行为检测的启用几代科研人员解剖复杂的行为20。社交回避的分析，可以进行低成本地使用T型迷宫测定中，允许社会行为的更广泛的研究。例如，使用这种测定法，我们最近证明了孤独症不同的遗传风险在社会B对比效果ehavior检测。突变的候选基因自闭症- neurobeachin 21,22 -目前存在的缺陷无论是在社会空间（23别处描述）和社交回避24。异常的多巴胺信号也提出了自闭症在人类25,26的病因中发挥作用。与此相反，以与neurobeachin所获得的结果，我们发现，社交回避表现是不受增加或多巴胺能细胞减少果蝇囊泡单胺转运（VMAT）的水平，尽管社会空间直接相关，这些水平VMAT 27。与neurobeachin和VMAT得到的对比结果强调了识别各种形式的反社会行为，并且因此不同的底层神经电路系统调节响应于他人的可能性。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Stereo Zoom Microscope&#160;</td>
			<td>Nikon&#160;&#160;</td>
			<td>SMZ-645</td>
			<td>Any other standard scope for fly handling would work</td>
		</tr>
		<tr>
			<td>Small paint brushes&#160;</td>
			<td></td>
			<td></td>
			<td>for pushing flies</td>
		</tr>
		<tr>
			<td>Porous Polyethylene, 12&#34; x 12&#34; Sheet</td>
			<td>Flystuff - Genesee</td>
			<td>46-100</td>
			<td>http://www.flystuff.com/ProductInfo.php?productID=46-100 
			Porous Plastic sheet for the cold anesthesia box</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Alarm Timer/Stopwatch</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Sharpie pens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini vortex</td>
			<td>Fisher</td>
			<td>14-955-151&#160;</td>
			<td>http://www.fishersci.com/ecomm/servlet/itemdetail 
			For mechanical agitation of the flies - any vortex would work.</td>
		</tr>
		<tr>
			<td>Corning Life Sciences DL No.:352017, Falcon test tube; round bottom; disposable; no closure, 14mL;&#160; 17 x 100mm</td>
			<td>Fisher</td>
			<td>14-959-8 &#160;&#160;</td>
			<td>http://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalogId=29104&#38;productId=2771811&#38;distype=0&#38;highlightProductsItemsFlag=Y&#38;fromSearch=1&#38;searchType=PROD&#38;hasPromo=0 
			These snap in place in the in-house made T-maze and counter-current apparatus (see text)</td>
		</tr>
		<tr>
			<td>cotton balls</td>
			<td></td>
			<td></td>
			<td>to close the vials after the experiment.</td>
		</tr>
		<tr>
			<td>trifold board and white bench cover</td>
			<td></td>
			<td></td>
			<td>to provide a white background, and a homogeneous light.</td>
		</tr>
		<tr>
			<td>white bench cover</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pounding pad</td>
			<td></td>
			<td></td>
			<td>any mouse pad works.</td>
		</tr>
		<tr>
			<td>large black cloth</td>
			<td></td>
			<td></td>
			<td>to cover the counter-current apparatus in phototaxis response.</td>
		</tr>
		<tr>
			<td>cool-white light&#160;</td>
			<td>Home Depot</td>
			<td>1000516563</td>
			<td>http://www.homedepot.ca/product/illume-26-fluorescent-plug-in-linear/911423 
			any similar linear light with fluorescent light bulb cool-white at 13-15W would work</td>
		</tr>
	</tbody>
</table>

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.

社交回避试验是一个强大的测试定量果蝇识别一个应力信号（DSO）被其他蝇发射的能力，并因此评估社会互动的一个方面。使用已知作为T迷宫，其具有两个不同的选项之间进行选择呈现苍蝇各种行为测定常用的装置进行测定-左或右19,28-31。在这种情况下，效率在该苍蝇避免DSO是通过计算和比较它们的性能指数（PI）的定量。零P​​I表示50-50分布或没有回避，而100 PI表明，所有的苍?...

Watch this Scientific Journal Video about Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster at JoVE.com

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 ...

straightforward-assay-for-quantification-social-avoidance-drosophila

Research

JoVE Journal

Neuroscience

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

11.1K 次观看.  Yale University. 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.

视频: Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

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 ...

科研

神经科学

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenetic刺激的逃避行为<em&gt;果蝇</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

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 ...

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 ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

高通量检测，以检查个体产蛋首<em&gt;果蝇</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

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 ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

毛细管馈线分析测量的摄食<em&gt;果蝇</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...