We thank Terry Shelley for manufacturing the fly arena and the light-tight enclosure, Liz Marr for help with fly stock maintenance, and Xiaojing Gao and Junjie Luo for help with the Matlab code used for data analysis. We thank Johan Lundstr&#246;m at the Monell Chemical Senses Center for demonstrating his odor delivery setup. This work was supported by grants from the Whitehall Foundation (CJP) and NIH NIDCD (R01DC013070, CJP).

1.安装装配<ol><li>根据提供的图纸（补充材料，SupplementalSketch_StarShapedArena.pdf）制造星形舞台（19.5厘米19.5厘米0.7厘米）出聚四氟乙烯（PTFE）的。竞技场可以由商业或定制设施来制造。 </li><li>获得两个玻璃板（20.25厘米20.25厘米2毫米厚），和钻一个孔（〜直径0.7厘米）恰恰在使用金刚石涂层钻头玻璃板之一的中心。 </li><li>制造的行为舞台上不透光的行为框。还根据所提供的附图（补充材料，SupplementalSketch_LightTightBox.pdf）制造的不透光照相机箱的红外线CCD视频摄像机。盒子可以由商业或定制设施来制造。 </li><li>安装在后壁和行为箱的侧壁上的LED阵列的空调单元。放置温度探头在通过即时温度反馈和调整的侧孔的行为框（参见图1和2的详细信息）。 </li><li>红外过滤器和圆偏振器安装到相机，并安装组件插入相机框。行为箱和照相机箱由一个玻璃窗口的行为框的更好的温度控制（ 见图1和2的详细信息）分隔。 </li><li>红外CCD相机连接到摄像机适配器。摄像机适配器连接到USB转换器。将USB转换器连接到USB端口的计算机进行数据采集上。 </li><li>根据制造商的说明在计算机上安装的视频转换器的驱动程序。任选地，安装由USB视频转换器的制造商提供的访问范围更广的照相机设置和采集参数的图像处理软件。 </li><li>空调机（器通过"输出"温度控制器背面）和温度探头（通过"关于温控器的背面）的温度控制器热电偶"。将探头插入行为框。 注意:在我们的布局的温度控制系统能够保持18℃和30℃之间的框的温度。较高或较低的环境温度可能是thermogenetic（dTrpA1，TRPM8或shibire TS）是有用的实验来操纵神经元的活动或抑制突触传递。对于大多数实验中，温度保持在25℃。 </li><li>组装气味输送系统在下面的步骤（请参阅图1B的详细示意图和连接件）: <ol><li>使用空气压力调节器，以控制从中央空气系统中的空气输入端。碳的空气过滤器（填充有木炭）连接到压力调节器，以从中央一净化空气IR系统。 </li><li>组装包含由高分辨率的流量计管调节多个通道的流动控制系统。 </li><li>经由歧管从碳空气过滤器到流量计管的输出连接， 如图1B和2F。直接通过电子控制的三路电磁阀，流量计管的输出，如果清洁空气离开流量计排出进入房间或进入定做气味室24来调节。 </li><li>根据制造商的手册安装电磁阀控制器。 </li></ol></li><li>通过将其连接到一个数据采集设备（DAQ），并根据制造商的手册的电源安装电子空气流量计。安装数据采集接口软件，以验证每一个实验前的舞台上的每个象限等于流量。 </li></ol> 2.嗅觉刺激准备<ol><li>准备5气味即由一个塑料外容器的蚂蚁腔室24中 ，玻璃内部容器，一个特制的聚四氟乙烯盖插入，原来的容器盖与中央部分除去，两个单向阀。 注意:围绕聚四氟乙烯盖O形环可用于防止气味灌注过程中从气味室空气泄漏。参见图1原理图和图2的分庭的照片。 </li><li>使用溶剂能够控制四个气味室，并为测试气味一个腔室。装满1000微升溶剂或气味稀释的玻璃容器（测试增味剂+适当的溶剂中，实验前调匀），将对应的塑料室内的玻璃容器（不要溅出液体到塑料室），并拧紧盖子。确保始终用干净室测试气味和溶剂的控制。 注:嗅觉的吸引力可以通过1/16稀释使用w苹果醋（5％酸度）的触发亚特。与此相反，嗅觉斥力行为可以通过在矿物油中使用丙酸乙酯的10％的稀释度进行研究。在这些情况下控制刺激气味都与商会纯矿物油。 </li></ol> 3.粉煤灰制备<ol><li>后苍蝇标准玉米面介质上。将30名男性和一个标准瓶30母本苍蝇，并让他们在25°C或室温产卵5天。 </li><li>对于每个实验，收集新eclosed（&lt;1日龄），男25例，女25例短暂的二氧化碳麻醉下飞行。 </li><li>防止苍蝇与2-4天标飞介质的小瓶。 </li><li>实验前40-42小时，转移苍蝇不二氧化碳麻醉&#160;用约10毫升1-％琼脂糖凝胶的小瓶。这将让没有食物，这有助于增加他们的自发活动加湿苍蝇。 注:90％以上的苍蝇应该生存的饥饿。一些基因型愈合少你不得通过40小时饥饿做到这一点。在这些情况下，更短的时间，如24-28小时是可接受的，但应保持所有实验条件并重复相同的。 </li></ol> 4.行为反应吸引力和驱避剂加味剂<ol><li>切换温度控制器上，并设置为25℃。 </li><li>通过油管插入到加臭剂室的出口和连接气味室（对照和测试气味物质）推到连接上的行为框接头。 </li><li>通过使用气流计，以确保控制和气味气流是等于100毫升/分钟检查每个象限的流速。 </li><li>清洁聚四氟乙烯飞舞台和用70％乙醇2-3次玻璃板并且允许它们完全空气干燥（〜3-4分钟）。 </li><li>加盖玻璃板用夹子竞技场。 </li><li>转印蝇无CO 2麻醉成通过在玻璃板之一的孔竞技场。前前后后呃转移，放置一个圆形网状的孔，防止苍蝇逃跑。 注:CO 2麻醉已被证明影响果蝇行为25和不应该被一个行为实验中的24小时内使用。 </li><li>将苍蝇放入不透光室的舞台上，通过连接连接到管路连接四个控制气流推到连接上的行为框竞技场角件，关闭房间的门，等待10 15分钟让果蝇适应新的环境。如果可能的话，开关灯灭在执行试验室内，以避免可能的最小的漏光那可就偏见实验结果。 </li><li>运行5-10分钟的对照实验，其中苍蝇暴露于4个控制空气流。 </li><li>立即分析数据（见下面的数据分析一节），以确保苍蝇在赛场上均匀分布，并且吸引指数接近为0，这一步是必不可少的，因为它验证有在舞台上的偏好或规避无失控来源（ 例如来自外部的光，温度分布不均匀，凹凸不平的舞台，气味污染，泄漏等 ）。如果苍蝇分配不公或的自发活动低，丢弃苍蝇，再次清洁竞技场（步骤4.4），并使用新一批果蝇重复实验。 </li><li>通过在3路阀或重新插入所述连接器管切换连接测试加臭剂室向设置。 </li><li> 5-10分钟跑的测试实验，如下面的第5节讨论的分析数据（见参考文献14和图3）。记录超过20分钟延长可导致数据文件可能难以计算过程。如果再实验录音需要，迅速停止和重新启动跟踪程序。这导致实验记录之间一〜10秒的差距。 </li><li>抛弃它ð苍蝇。 </li><li>用70％乙醇清洁舞台和玻璃板（步骤4.4），并更换透光外壳内的连接器的管。为了加快实验中，新的清洁竞技场可以使用，并且在执行实验运行脏竞技场清洗。 </li><li>用新一批的苍蝇运行另一个实验中，如果需要的话。如果几个实验在同一天运行，需要格外小心，确保没有加臭剂在系统中留下从以前的测试运行。这通常是不与低浓度的增味剂的 CO 2或一个问题，但可能需要高度浓缩的刺激高达实验运行之间的24小时间隙。此外，流管毕竟管道可以如果加臭剂污染在对照实验怀疑被替换。要始终保持干燥的空气对实验之间不断冲洗系统</li></ol> 5.数据分析注:建议跟踪飞收购softwarE（在材料的详细），采集过程中实时跟踪苍蝇，节省了时间标记，并在* .dat文件格式，所有检测到的苍蝇的坐标。我们已经开发出一种定制的Matlab的例程来将数据转换成一个Matlab格式，并分析数据。在补充的材料被提供的代码的例子，但实现的细节将取决于用于数据采集的软件。 <ol><li>加载的原始数据。创建跟随舞台的轮廓空间mask和掩码应用到的原始数据删除落在领域之外，因为它们代表噪声（ 图4A， 补码 MaskSpatialFiltering.m，Score.m，DrawCircularMask所有数据点。米）。 </li><li>删除移动的速度低于0.163厘米/ s的超过3秒，因为这样的数据很可能是噪声或由非移动蝇（ 图4B，补充代码TemporalFiltering.m）生成的所有数据点。 </li><li>剩下的可视化数据通过一次全部绘制它们，或单点的轨迹（ 图3， 补码 SingleTrajectoryViewer.m）。 注意:气味边界在四场的位置可能取决于许多因素，如每个加臭剂的特性和气流速率被使用。例如，极易挥发气味物质可能会更充分地填补了气味象限比不易挥发气味物质。因此它很可能是每个加臭剂可以表现出略微不同的气味边界。因为它使用真空以从特定点采样空气，因此破坏在该点的加臭剂浓度使用光离子化检测的测量气味的边界可能会产生问题。尽管如此，气味边界可以迅速根据实时行为数据估算。例如，根据累计飞行轨迹响应于不同的气味的气味边界可以清楚地在图3C和3D观察。 </li><li>卡尔culate吸引力索引来确定对照实验是否产生没有偏好响应，并且还获得了加臭剂（或光遗传学9）刺激的反应。要计算的吸引力指数（AI），使用最后5分钟的控制或测试记录。为了获得吸引力的措施，+1（绝对的吸引力）和-1（绝对排斥）介于下列公式用于计算AI: <img alt="式（1）" src="/files/ftp_upload/54346/54346eq1.jpg" /> 其中N 测试数据点的在测试象限的数目，N 控制为数据点中的三个控制象限的平均数目。这项措施是直观没有偏好会接近零值来表示。然而，它不能正确地表示，它们位于所述的加臭剂象限蝇的总数的比例。为了得到该措施，一个百分比指数（PI）可用于: <img alt="公式（2）" src="/files/ftp_upload/54346/54346eq2.jpg"/&gt; 其中N 测试数据点的在测试象限的数目，并且N 总是数据点的所有四个象限的总数。此公式提供了0和1之间落下，与对应于没有行为偏好（ 图3E和图4C， 补充代码 AttractionIndex.m）0.25的量度。 </li><li>运行的每个实验条件下重复5-10，使用苍蝇每个重复的新组。通过使用洛夫-斯米尔诺夫非参数检验（ 图3F，在Matlab kstest2功能）比较条件控制之间或针对吸引力索引。 </li></ol>

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C., Boesveldt, S., Albrecht, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+building+an+inexpensive+computer-controlled+olfactometer+for+temporally-precise+experiments.">Methods for building an inexpensive computer-controlled olfactometer for temporally-precise experiments.</a> Int J Psychophysiol. 78 (2), 179-189 (2010).</li><li>Colinet, H., Renault, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+effects+of+CO2+anaesthesia+in+Drosophila+melanogaster.">Metabolic effects of CO2 anaesthesia in Drosophila melanogaster.</a> Biology Letters. 8 (6), 1050-1054 (2012).</li><li>Ramdya, P., 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=Mechanosensory+interactions+drive+collective+behaviour+in+Drosophila.">Mechanosensory interactions drive collective behaviour in Drosophila.</a> Nature. 519 (7542), 233-236 (2015).</li><li>Ofstad, T. A., Zuker, C. S., Reiser, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+place+learning+in+Drosophila+melanogaster.">Visual place learning in Drosophila melanogaster.</a> Nature. 474 (7350), 204-207 (2011).</li><li>Beshel, J., Zhong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graded+encoding+of+food+odor+value+in+the+Drosophila+brain.">Graded encoding of food odor value in the Drosophila brain.</a> J Neurosci. 33 (40), 15693-15704 (2013).</li><li>Steck, K., 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+high-throughput+behavioral+paradigm+for+Drosophila+olfaction+-+The+Flywalk.">A high-throughput behavioral paradigm for Drosophila olfaction - The Flywalk.</a> Sci Rep. 2, 361 (2012).</li><li>Thoma, M., Hansson, B. S., Knaden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+Quantification+of+Odor-guided+Behavior+in+Drosophila+melanogaster+Using+the+Flywalk+Paradigm.">High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm.</a> J. Vis. Exp. (106), (2015).</li><li>Claridge-Chang, A., 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=Writing+memories+with+light-addressable+reinforcement+circuitry.">Writing memories with light-addressable reinforcement circuitry.</a> Cell. 139 (2), 405-415 (2009).</li><li>Parnas, M., Lin, A. C., Huetteroth, W., Miesenbock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+discrimination+in+Drosophila:+from+neural+population+codes+to+behavior.">Odor discrimination in Drosophila: from neural population codes to behavior.</a> Neuron. 79 (5), 932-944 (2013).</li></ol>

The authors declare that they have no competing financial interests.

这里所描述的四场嗅觉是研究大量人口的野生型和突变体果 ​​蝇苍蝇的嗅觉反应一个通用行为系统。每个实验以〜1小时（包括设置，实验运行和清洁），并且4-6实验每天可常规进行。用40-50苍蝇5分钟的典型试验产生大约45万跟踪的数据点进行分析。所描述的结构也可使用，以小的修改，并监控其他昆虫或昆虫幼虫的运动响应于嗅觉或其它感觉刺激在一段时间内，从最小到几天。四象限检...

适应并响应外部环境的能力，对于所有动物的生存至关重要。动物需要避免危险，寻找食物和寻找配偶，并从以前的经验中学习。感觉系统功能，以接收各种刺激，如视觉，化学和mechanosensory的，和发送这些信号到中枢神经系统来解释和解码。大脑然后指示基于感知环境适宜的运动行为，如觅食或捕食逃逸。了解感觉系统如何检测外部世界，大脑如何解码并指导决策，是神经生物学的一个重大挑战。 果蝇是研究如何神经回路引导行为的强大模型系统。除了 ​​是简单和廉价的维护， 果蝇表现出许多不同的和复杂的刻板行为，但用COMPAC这样做ŧ约10万神经元的神经系统。强大遗传技术用于操纵果蝇基因组的存在，以及数以千计的转基因品系已生成的选择性和可重复性地标记的神经元10-13的相同的子集。这些转基因品系可以用来选择性地操纵标记神经元（激活或抑制）的活性，并且这些操作可以被用来研究如何神经功能导向的行为。 多种行为检测已制定了各种研究果蝇的行为。 果蝇 ，像许多动物，用他们的指导许多行为选择，比如寻找食物，寻找配偶，避免危险的嗅觉。因此嗅觉是研究外部刺激是如何检测和动物的神经系统解释，引导适当的选择一个良好的感觉系统。这样，一些试验中已经开发了用于investiga婷幼虫和成虫嗅觉行为。传统上，在果蝇嗅觉行为是由两选择T-迷宫范例，它可用于测定先天和了解到嗅觉行为3测定。在该试验中，约50苍蝇给出了两种管之间:一个管包含有问题的气味和其它含有一个控制气味（通常是气味溶剂）。苍蝇给出的设定时间做出选择，然后是在不同的腔室蝇的数目进行计数。尽管T-迷宫是许多实验的简单测定中，也有一些限制。例如，嗅觉行为仅在一个时间点测量的，并且该时间点之前进行不同的选择将被丢弃。同样，人口内苍蝇的个体行为被忽略。另外，T型迷宫要求苍蝇人工计数，这可能会引入错误。最后，因为只有两种测量的选择，这减少经常需要察觉的细微的行为变化的统计力量。到两选择T-迷宫一个替代方案是一个四象限（四场）嗅觉14-18。在该试验中，动物探究在其中每个竞技场的四个角的填充有加臭空气的潜在来源的舞台。竞技场有皱褶星形最大化四个实验确定的气味象限的形成。如果气味在随后的一个角供给它包含只在那一个象限。动物的行为可以相比，其在三个控制象限行为，因为他们进入和离开气味象限被跟踪，和容易。因此四象限嗅觉试验记录到刺激气味在大的试验舞台空间和时间的行为反应。 四象限嗅觉首先由彼得森等人的 15和兽医等人开发的。17，调查醇个别寄生蜂的工厂行为反应。福彻等 18 Semmelhack和王16改编设置监视单个果蝇的嗅觉反应。四象限嗅觉是吸引和排斥反应同样敏感，允许广泛的测试增味剂和条件。自定义编写飞跟踪软件，由Alex Katsov 19开发，目前由儒略布朗（在材料详述）保持，引入额外的优点，以更近的四象限嗅觉14,20-23的实现。现在有可能以测定到在高空间同时100蝇（27.5像素/厘米）和分辨率，它允许提取的各种参数，如在任何时间点的位置，速度和苍蝇的加速度随时间（每秒30帧）。这使得调查果蝇的行为反应的动力学气味20 </s了&gt;。然而，应该指出的是，在人口中的各个蝇在整个跟踪期间的身份不能保持。相反，每个飞行轨道被记录为只要两个飞轨道不相交。在这一点，苍蝇发散后新的轨道进行分配。通过将其它视频俘获软件（在材料表详述），相同的配置允许灵活的跟踪时段，并且可以使用通过以较低的帧速率拍摄图像来跟踪苍蝇长达24小时。此选项被用来研究果蝇产卵行为和喜好产卵14比较他们的身体姿势。四字段嗅觉也可用于研究的反应在多峰（ 例如嗅觉和视觉）刺激，或以光遗传学9或thermogenetic 21刺激感官刺激的演示相结合。此外，高时间分辨率允许轨迹的FO提取R IN乐团数据集的每一个人飞。因此，该方法允许调查嗅觉引导人口行为和也从个人的社交互动。通过该测定所产生的数据是健壮和高度可再现的，允许使用的四场嗅觉用于行为屏幕。 在这里，我们描述了一个四象限嗅觉安装组件。我们进一步展示其在应对苹果醋和排斥响应高度集中丙酸乙酯测定嗅觉吸引力的使用。最后，我们描述和用于记录飞跟踪数据的分析提供了示例代码。

<table><tbody><tr><td>&lt;strong&gt;Air delivery system&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;(Quantity needed)&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Tubing and connectors&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Thermoplastic NPT(F) Manifolds</td><td>Cole-Parmer, IL, USA</td><td>R-31522-31</td><td>1</td></tr><tr><td>Hex reducing&amp;nbsp; nipple (1/4MNPT-&gt;1/8MNPT)</td><td>McMaster-Carr, IL, USA</td><td>5232T314</td><td>1</td></tr><tr><td>Tubing (ID:1/8)</td><td>McMaster-Carr, IL, USA</td><td>5108K43</td><td>50 Ft</td></tr><tr><td>Tubing (ID:1/16)</td><td>McMaster-Carr, IL, USA</td><td>52355K41</td><td>100 Ft</td></tr><tr><td>Barbed tube fittings</td><td>McMaster-Carr, IL, USA</td><td>5117K71</td><td>1 pack</td></tr><tr><td>Push-to-connect tube fittings</td><td>McMaster-Carr, IL, USA</td><td>5779K102</td><td>4</td></tr><tr><td>Barbed Tube Fittings (1/4MNPT-&gt;1/8BF)</td><td>McMaster-Carr, IL, USA</td><td>5463K439</td><td>1 pack (10)</td></tr><tr><td>Barbed Tube Fittings (1/8MNPT-&gt;1/8BF)</td><td>McMaster-Carr, IL, USA</td><td>5463K438</td><td>2 pack (10)&amp;nbsp;</td></tr><tr><td>Barbed Tube Fittings (1/8MNPT-&gt;1/16BF)</td><td>McMaster-Carr, IL, USA</td><td>5463K4</td><td>2 pack (10)&amp;nbsp;</td></tr><tr><td>Barbed Tube Fittings (1/4MNPT-&gt;1/4BF)</td><td>McMaster-Carr, IL, USA</td><td>&amp;nbsp; 5670K84</td><td>1</td></tr><tr><td>Hex head plug</td><td>McMaster-Carr, IL, USA</td><td>48335K152</td><td>1</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Air pressure regulator, air filter and flowmeters&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td>&lt;strong&gt;(Quantity needed)&lt;/strong&gt;</td></tr><tr><td>Labatory gas drying unit</td><td>W A HAMMOND DRIERITE CO LTD, OH, USA</td><td>Model: L68-NP-303; stock #26840</td><td>1</td></tr><tr><td>Multitube frames for 150 mm flowtubes</td><td>Cole-Parmer, IL, USA</td><td>R03215-30</td><td>1</td></tr><tr><td>Multitube frames for 150 mm flowtubes</td><td>Cole-Parmer, IL, USA</td><td>R03215-76</td><td>1</td></tr><tr><td>150 mm flowtubes</td><td>Cole-Parmer, IL, USA</td><td>R-03217-15</td><td>9</td></tr><tr><td>Valve Cartridge</td><td>Cole-Parmer, IL, USA</td><td>R-03218-72</td><td>9</td></tr><tr><td>Precision Air regulator</td><td>McMaster-Carr, IL, USA</td><td>6162K13</td><td>1</td></tr><tr><td>Soleniod valves</td><td>Automate Scientific, Berkeley, CA</td><td>02-10i</td><td>4</td></tr><tr><td>Solenoid valve controller</td><td>ValveLink 8.2, Automate Scientific, Berkeley, CA</td><td>01-18</td><td>1</td></tr><tr><td>Electronic flow meter</td><td>Honeywell</td><td>AWM3100V</td><td>1</td></tr><tr><td>DAQ (NI USB-6009, National Instruments) and a&amp;nbsp;</td><td>National Instruments</td><td>NI USB-6009</td><td>1</td></tr><tr><td>Power supply</td><td>Extech Instruments</td><td>382200</td><td>1</td></tr><tr><td>Odor chambers</td><td></td><td></td><td></td></tr><tr><td>Polypropylene Wide Mouth jar 2 oz; 60 ml</td><td>Nalgene</td><td>562118-0002</td><td>At least 5 are required per experiment, but a separate chamber is required for each dillution of each odorant. Available at Container Store, part #635114)</td></tr><tr><td>Glass odor chamber, 0.25 oz</td><td>Sunburst Bottle</td><td>LB4B</td><td>At least 5 are required per experiment&amp;nbsp;</td></tr><tr><td>&quot;In&quot; valve for odor chamber</td><td>Smart Products, Inc., CA, USA</td><td>214224PB-0011S000-4074</td><td>1 of these parts is used per odor chamber but they need to be replaced frequently</td></tr><tr><td>&quot;Out&quot; valve for odor chamber</td><td>Smart Products, Inc., CA, USA</td><td>224214PB-0011S000-4074</td><td>1 of these parts is used per odor chamber but they need to be replaced frequently</td></tr><tr><td>O ring</td><td>RT Dygert International, MN, USA</td><td>AS568-029 Buna-N O-R</td><td>1 pack (100)</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Fly arena, camera and behavior boxes&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td>&lt;strong&gt;(Quantity needed)&lt;/strong&gt;</td></tr><tr><td>Behavior and camera box material</td><td>Interstate plastics, CA, USA</td><td>ABS black extruded (https://www.interstateplastics.com/Abs-Black-Extruded-Sheet-ABSBE~~ST.php)</td><td>1803 sq inch</td></tr><tr><td>Teflon for fly arena and odor chamber inserts, 3/8&quot; thick, 12&quot; x 12&quot;</td><td>McMaster-Carr, IL, USA</td><td>8545K27&amp;nbsp;</td><td>1</td></tr><tr><td>Glass plates, 1/8&quot; Thick, 9&quot; x 9&quot;</td><td>McMaster-Carr, IL, USA</td><td>8476K191&amp;nbsp;</td><td>2</td></tr><tr><td>Dual action thermoelectric controller</td><td>WAtronix Inc, CA, USA</td><td>DA12V-K-0</td><td>1</td></tr><tr><td>IR LED array</td><td>Advanced Illumination, Rochester, VT, USA</td><td>AL4554-88024, PS24-TL</td><td>2 LED arrays and one power supply</td></tr><tr><td>Air conditioner Unit</td><td>Melcor Store</td><td>&amp;nbsp;MAA280T-12</td><td>1</td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Imaging system&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td>&lt;strong&gt;(Quantity needed)&lt;/strong&gt;</td></tr><tr><td>Cosmicar/Pentax C21211TH (12.5 mm F/1.4) C-mount Lens</td><td>B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td><td>PEC21211 KP</td><td>1</td></tr><tr><td>CCXC-12P05N Interconnect Cable</td><td>B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td><td>SOCCXC12P05N</td><td>1</td></tr><tr><td>DC-700 Camera Adapter</td><td>B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td><td>SODC700</td><td>1</td></tr><tr><td>B+W 40,5 093 IR filter</td><td>B AND H PHOTO AND ELECTRONICS CORP, NY, USA</td><td>65-072442</td><td>1</td></tr><tr><td>TiFFEN 40.5 mm Circular polarizer</td><td>Amazon</td><td></td><td>1</td></tr><tr><td>IR Videocamera</td><td>Industrial Vision Source, FL, USA</td><td>Sony XC-EI50 (SY-XC-E150)</td><td>1</td></tr><tr><td>USB video converter</td><td>The Imagingsource, NC, USA</td><td>DFG/USB2-It</td><td>1</td></tr><tr><td>iFlySpy2 (fly tracking software)</td><td>Julian Brown, Stanford, Calfornia: julianrbrown@gmail.com</td><td>iFlySpy2</td><td>1</td></tr><tr><td>IC Capture 2.2 software</td><td>The Imagingsource, NC, USA (http://www.theimagingsource.com/en_US/products/software/iccapture/)</td><td></td><td></td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Miscellaneous&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td>&lt;strong&gt;(Quantity needed)&lt;/strong&gt;</td></tr><tr><td>Dremel rotary tool</td><td>Dremel, Racine, WI, USA</td><td>Dremel 8000-03&amp;nbsp;</td><td>1</td></tr><tr><td>Diamond-coated drill bits for glass cutting</td><td>Available from various suppliers; MSC industrial Supply Co, Melville, NY</td><td>90606328</td><td>1</td></tr></tbody></table>

olfactory behaviors assayed by computer tracking of drosophila in a four-quadrant olfactometer

神经生物学的一个主要挑战是要了解如何电路的神经，由于其复杂的行为，强大的基因技术，紧凑的神经系统功能，引导适当的动物行为， 果蝇是这种调查的一个极好的模型系统。实验室行为分析已经长期用于与果蝇模拟自然环境性质和研究下面的相应的行为的神经机制（ 例如趋光性，趋化性，感官学习和记忆）1-3。随着最近的那个标签特定的神经元亚群的转基因果蝇线大集合的可用性，行为检测已采取了突出的作用与行为4-11链接神经元。多功能和可重复的模式，以数据分析的基本计算程序一起，是各种genotyp候选飞线的快速检测不可缺少的上课。特别有用的是设置了在试验动物，实验持续时间和呈现刺激性质的数目灵活。选择的测定法也应生成可再生的数据，很容易获取和分析。在这里，我们提出了测定果蝇的行为反应的系统和协议的详细说明，在一个大的四场舞台上飞起来。设置在这里用来测定苍蝇单个嗅觉刺激的反应;然而，相同的设置可以被修改以测试多个嗅觉，视觉或光遗传学刺激，或这些的组合。嗅觉设置记录蝇种群响应气味的活性，并计算分析方法被应用到量化苍蝇的行为。所收集的数据进行分析，以得到一个快速读出一个实验运行，这是有效的数据收集和实验条件的优化必不可少的。

四象限嗅觉检测记录并分析在一个大的行为空间许多苍蝇的行走活动。添味剂可以被引入到该输入一个，两个，三个或全部四个象限的空气流。在没有气味，苍蝇就会随意四个象限之间移动。此行为是观察关键，因为它表明未故意偏差没有被引入化验。这些偏差可以包括光，温度波动，在空气流的差异，或气味的污染物。 图3B示出在25个男性的四象限嗅觉的行...

Watch this Scientific Journal Video about Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer at JoVE.com

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

通过计算机跟踪检测大鼠嗅觉行为<em&gt;果蝇</em&gt;在四象限嗅

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

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Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

15.6K 次观看.  Johns Hopkins University School of Medicine. We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

视频: Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they yield data relevant to the cognitive processing that occurs under ecologically valid conditions. These two goals often are at odds, since well controlled stimuli often are too repetitive to sustain subjects' motivation. Studies employing electroencephalography (EEG) are often especially sensitive to this dilemma between ecological validity and experimental control: attaining sufficient signal-to-noise in physiological averages demands large numbers of repeated trials within lengthy recording sessions, limiting the subject pool to individuals with the ability and patience to perform a set task over and over again. This constraint severely limits researchers' ability to investigate younger populations as well as clinical populations associated with heightened anxiety or attentional abnormalities. Even adult, non-clinical subjects may not be able to achieve their typical levels of performance or cognitive engagement: an unmotivated subject for whom an experimental task is little more than a chore is not the same, behaviourally, cognitively, or neurally, as a subject who is intrinsically motivated and engaged with the task. A growing body of literature demonstrates that embedding experiments within video games may provide a way between the horns of this dilemma between experimental control and ecological validity. The narrative of a game provides a more realistic context in which tasks occur, enhancing their ecological validity (Chaytor &amp; Schmitter-Edgecombe, 2003). Moreover, this context provides motivation to complete tasks. In our game, subjects perform various missions to collect resources, fend off pirates, intercept communications or facilitate diplomatic relations. In so doing, they also perform an array of cognitive tasks, including a Posner attention-shifting paradigm (Posner, 1980), a go/no-go test of motor inhibition, a psychophysical motion coherence threshold task, the Embedded Figures Test (Witkin, 1950, 1954) and a theory-of-mind (Wimmer &amp; Perner, 1983) task. The game software automatically registers game stimuli and subjects' actions and responses in a log file, and sends event codes to synchronise with physiological data recorders. Thus the game can be combined with physiological measures such as EEG or fMRI, and with moment-to-moment tracking of gaze. Gaze tracking can verify subjects' compliance with behavioural tasks (e.g. fixation) and overt attention to experimental stimuli, and also physiological arousal as reflected in pupil dilation (Bradley et al., 2008). At great enough sampling frequencies, gaze tracking may also help assess covert attention as reflected in microsaccades - eye movements that are too small to foveate a new object, but are as rapid in onset and have the same relationship between angular distance and peak velocity as do saccades that traverse greater distances. The distribution of directions of microsaccades correlates with the (otherwise) covert direction of attention (Hafed &amp; Clark, 2002).

Procedures for recording high-density EEG and gaze data during computer game-based cognitive tasks are described. Using a video game to present cognitive tasks enhances ecological validity without sacrificing experimental control.

结合电玩与高密度的脑电图和红外视线跟踪基于行为的实验

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they ...

科研

神经科学

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

高分辨率测量的气味驱动在果蝇幼虫的行为

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

生物学

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

在果蝇的飞行过程中可视介导的气味追踪

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

单苍蝇检测方法的觅食行为的<em&gt;果蝇</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

Testing Drosophila Olfaction with a Y-maze Assay

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze assay.

测试<em&gt;果蝇</em&gt;用Y型迷宫测定嗅觉

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

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 ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

跟踪果蝇幼虫行为对嗅神经元 Optogenetic 刺激的反应

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

果蝇 自闭症谱系障碍的遗传和行为特征模型

Paradigms for Behavioral Assessment in Drosophila Model of Autism Spectrum Disorder

Autism Spectrum Disorder (ASD) encompasses a heterogeneous group of neurodevelopmental disorders with common behavioral symptoms including deficits in social interaction and ability for communication, enhanced restricted or repetitive behaviors, and also, in some cases, learning disability and motor deficit. Drosophila has served as an unparalleled model organism for modeling a great number of human diseases. As many genes have been implicated in ASD, fruit flies have emerged as a powerful and efficient way to test the genes putatively involved with the disorder. As hundreds of genes, with varied functional roles, are implicated in ASD, a single genetic fly model of ASD is unfeasible; instead, individual genetic mutants, gene knockdowns, or overexpression-based studies of the fly homologs of ASD-associated genes are the common means for gaining insight regarding molecular pathways underlying these gene products. A host of behavioral techniques are available in Drosophila which provide easy readout of deficits in specific behavioral components. Social space assay and aggression and courtship assays in flies have been shown to be useful in assessing defects in social interaction or communication. Grooming behavior in flies is an excellent readout of repetitive behavior. Habituation assay is used in flies to estimate the ability for habituation learning, which is found to be affected in some ASD patients. A combination of these behavioral paradigms can be utilized to make a thorough assessment of the human ASD-like disease state in flies. Using Fmr1 mutant flies, recapitulating Fragile-X syndrome in humans, and POGZ-homolog row knockdown in fly neurons, we have shown quantifiable deficits in social spacing, aggression, courtship behavior, grooming behavior, and habituation. These behavioral paradigms are demonstrated here in their simplest and straightforward forms with an assumption that it would facilitate their widespread use for research on ASD and other neurodevelopmental disorders in fly models.

作者聚焦：探索果蝇的自闭症谱系障碍症状 — 遗传模型和行为测试

Autism Spectrum Disorder (ASD) is associated with impaired social and communicative behavior and the emergence of repetitive behavior. For studying the interrelation between ASD genes and behavioral deficits in the Drosophila model, five behavioral paradigms are described in this paper for assaying social spacing, aggression, courtship, grooming, and habituation behavior.

自闭症谱系障碍果 蝇 模型的行为评估范式

Autism Spectrum Disorder (ASD) encompasses a heterogeneous group of neurodevelopmental disorders with common behavioral symptoms including deficits in ...

通过计算机跟踪检测大鼠嗅觉行为

摘要

探索更多视频

结合电玩与高密度的脑电图和红外视线跟踪基于行为的实验

高分辨率测量的气味驱动在果蝇幼虫的行为

在果蝇的飞行过程中可视介导的气味追踪

单苍蝇检测方法的觅食行为的

测试

在气味引导行为的高分辨率定量

跟踪果蝇幼虫行为对嗅神经元 Optogenetic 刺激的反应

自闭症谱系障碍果蝇模型的行为评估范式

自闭症谱系障碍果蝇模型的行为评估范式

自闭症谱系障碍果蝇模型的行为评估范式

通过计算机跟踪检测大鼠嗅觉行为

摘要

探索更多视频

结合电玩与高密度的脑电图和红外视线跟踪基于行为的实验

高分辨率测量的气味驱动在果蝇幼虫的行为

在果蝇的飞行过程中可视介导的气味追踪

单苍蝇检测方法的觅食行为的

测试

在气味引导行为的高分辨率定量

跟踪果蝇幼虫行为对嗅神经元 Optogenetic 刺激的反应

自闭症谱系障碍果 蝇 模型的行为评估范式

自闭症谱系障碍果 蝇 模型的行为评估范式

自闭症谱系障碍果 蝇 模型的行为评估范式

自闭症谱系障碍果蝇模型的行为评估范式

自闭症谱系障碍果蝇模型的行为评估范式

自闭症谱系障碍果蝇模型的行为评估范式