The authors want to thank Dr. Tadmiri Venkatesh for his encouragement to prepare this manuscript, Dova Brenman and Selwyn Chui for their constructive comments, Elyssa Burg and Wayne Rasband for designing the macro in ImageJ for all interfly distances and for nearest neighbor distances. The authors also want to thank the reviewers for their constructive comments.
A.A.A, M.C.C. and A.F.S were responsible for research design; S.N.J., A.A.A, M.N., Z.R., and A.J.M. performed the experiments. A.A.A, M.N., A.J.M. and M.C.C and A.F.S. analyzed the data; A.R.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.; by a Mathematics, Biology, Chemistry, and Geology majors scholarship for teachers-in-training and by a Louis Stokes Alliance for Minority Participation scholarship to A.A.A.

1.设备和试剂创造了内部（材料为他人见列表） <ol><li>制备果蝇冷麻醉装置如前所述8。 </li><li>准备一个飞吸气如前所述13。 </li><li>准备社会空间室和持有人。 <ol><li>命令或使玻璃板和丙烯酸垫片，以创造社会空间室。每个社会空间室由两个方形玻璃板（17.6厘米×17.6厘米，厚度为0.3厘米），两个直角三角形丙烯酸间隔物（16.5厘米，8.9厘米的碱基的高度，厚度为0.3厘米），并且两个矩形的空间（9厘米×1.5厘米，厚度为0.3厘米）（ 图1A）。 </li><li>装配玻璃和丙烯酸类社会空间室，使得其与 ​​图1B中的腔室。为此，通过将在玻璃上的一个方形窗格顶部的一个三角形垫片平面，使得启动直角三角形垫片的对准的平方窗格的一个角。下一步，放置在广场上窗格顶部的第二个三角形隔板持平，反映的第一个三角垫片。 注意：请一定要戴上手套，以免污染与你的手油和香料的作品。 </li><li>将两个小矩形隔板平放在方形面板的顶部，以及未涉及窗格的一面。广场上飞机前的丙烯酸垫片，现在应该形成一个三角形的舞台。 </li><li>放置玻璃的第二方形窗格上的丙烯酸系衬垫使得其上面低于玻璃原窗格对齐的顶部。 </li><li>使用四种长尾夹来固定窗格和隔板一起以形成社会空间室。将一个剪辑邻近的拐角处，在每个三角间隔物的长边，将它们固定在玻璃板内部。在这些相同侧，放置一个夹在相邻的拐角处，在每个矩形隔板，将它们固定在另一边的玻璃板。 </li><li>装配一个支座和一个烧瓶钳使得钳将保持在直立位置的社会空间室，作为社会空间室搁置在其上的工作表面的上边缘。 </li><li>对于实验中的每个重复，组建一个社会空间室，并支持立场。 </li></ol></li><li>准备攀登试验反应装置检查苍蝇测试的爬坡能力。 <ol><li>使用逆流装置，如前所述14,15。 </li></ol></li><li>通过执行实验上覆盖着白色的长椅盖在白色背景前一个作业面确保均匀的照明条件。 </li></ol> 2.准备蝇实验前<ol><li>维护苍蝇含标准果蝇的瓶子飞的食物。它们保存在一个孵育室在25℃在12小时光照/黑暗循环。 </li><li>一到两天之前，电子商务xperiment，收集并在冷麻醉性苍蝇，如前所述8。 <ol><li>寒意冷麻醉机在-4℃。将飞入50ml塑料管。完全淹没塑料管含在保温冰桶苍蝇成冰，等待至少5分钟，苍蝇变得动弹不得。 </li><li>将苍蝇在冰冷的麻醉设备的聚乙烯薄膜。在RT，使用立体显微镜来的男性从女性分开。保持苍蝇成群，含标准果蝇瓶飞的食物，不超过40苍蝇每小瓶。 </li></ol></li><li>在实验开始前两小时，确保在试验时将执行的房间的温度为24-25℃，湿度大约为50％。 </li><li>转移苍蝇到含有食品新的小瓶，并将它们放置2小时在哪里的实验将要执行的工作表面上。 注：对于这些experimenTS，所使用的蝇株果蝇品系：广-特殊或广-S（CS）， 瓦特1118铯10（或W - 瓦特1118异型杂交的10倍到广-S），俄勒冈州和萨马尔罕苍蝇都从我们的实验室库存16;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8。除了当指定，否则，给出的结果，得到与广东-S蝇（在红色色调）。 </li></ol> 3.执行实验<ol><li> （ - 以小时为单位的时间光发病后 - 时间授时ZT 4〜7）进行实验12点和下午3点之间。 </li><li>准备的社会空间，室内苍蝇的转移。 <ol><li>放置一个社会空间室平放在工作台上，用含有最接近你的身体矩形隔板一侧。 </li><li>删除ØNE夹最近你的身体，并向外滑动一个长方形的间隔，产生的矩形隔板之间大约为1厘米的差距。 </li><li>使用磁带和标记，标号社会空间室与性别，应变和重复次数的上角。注意确保磁带不包括在内三角舞台的一部分。 </li></ol></li><li>转移苍蝇进入社会空间室。 <ol><li>转让航班从含有食物进入一个新的空药瓶的瓶。吸苍蝇从空药瓶，并将其转移到社会空间室。 <ol><li>吸入吸引苍蝇入抽吸器的尖端。头放入的社会空间室的矩形隔板之间1厘米的差距，并在呼气一致的速度，迫使苍蝇进入内三角的舞台。 </li><li>立即矩形隔板滑回原位，关闭内三角竞技场的基部，然后将粘合剂剪辑重新打开。 </li></ol></l我&gt; </ol></li><li>上的冲击垫，它位于不同长凳比在该实验工作发生时，保持社会空间室直立，使得矩形隔板是在底侧。英镑三次，以确保所有的苍蝇已经下降到了舞台的底部。 注：由于亚克力间隔伸出最小室尺寸（ 图1D）中，砰的肘部在工作表面上，同时保持一个安全的扶住设备，以确保苍蝇已经跌到了谷底。 </li><li>启动计时器。 </li><li>放置在工作表面上的社会空间室和使用的支持立场，瓶夹，以保持直立。将尺子或已知长度的平贴在社会空间室的贴纸，但不涉及任何部位的内三角舞台。 <ol><li>对于水平放置的实验中，不要放在支撑支架上的社会空间室;而不是躺在工作台上的社会空间室持平。 </li></o升&gt; </li><li>当苍蝇纷纷落户，一般经过大约30分钟，以社会空间室的照片。确保帧包含整个内三角舞台上，统治者和标签。 </li><li>重复实验每个基因型和条件（最好3个内部重复和3个独立的重复）。 </li></ol> 4.分析的社会空间数据<ol><li>在导入ImageJ的17以下宏<ol><li> ImageJ的可以在这里找到：http://rsbweb.nih.gov/ij/。 </li><li>您可以通过保存在下面的宏在中的ImageJ / plugins文件夹命名为“Measure_Distances.txt”文件，在宏子文件夹中，并使用帮助&gt;更新菜单命令创建的插件菜单中“测量距离”命令。“ </li><li>在一个.txt复制文件中提供补充数据，并最初发表在3宏。 </li></ol></li><li>上传照片到电脑，并开立一个图像重新使用ImageJ。 <ol><li>在图片中的标尺上画线从0厘米〜1厘米的标记创建一个规模，并分析选项卡下，选择设置缩放功能可以设置一个比例为1厘米的距离（同样的方法进行的贴纸已知长度）。选择应用规模的图片前，在全局选项。 </li><li>使用图像选项卡，使得它包括所有的苍蝇下的裁剪功能裁剪图片，同时消除尽可能多的画面尽可能的休息。 </li><li>使画面的黑白从图片选项卡下的类型选项中选择8位。 </li><li>在图像选项卡的调节选项下将阈值特征取下图像中的所有的背景噪音。拖动滑块以增强或删除的对比度，使得每个蝇的本体可以清楚地看到，而不在图像中的任何其他标​​记。如果有斑纹是不是苍蝇，捕捉它们与矩形工具，并将其删除。 </li><li>摆好测量通过分析选项卡下的设置测量工具。选择面积，质量，质心的中心，并显示标签。接下来，选择分析颗粒分析选项卡下的功能来创建所有的黑点代表苍蝇编号列表。 <ol><li>当使用分析颗粒;集大小0.01-0.1（不选择像素单元）;圆0.00-1.00;显示轮廓;显示结果，并加入到经理。 </li><li>确保每个黑点（每个粒子）是在原始图片，通过编号的列表进行比较，以原始图象的苍蝇的精确表示。 注意：如果一些苍蝇非常接近一起被算作一次粒子，手动绘制白线分离两个蝇二进制图像上。 </li></ol></li><li>虽然列表中选择，使用最近邻 - 列出的距离宏插件标签第二个宏选项下创建一个包含距离一个新的列表，在厘米，从EACH飞到离他们最近的邻居。 </li></ol></li><li>从复制ImageJ的最近邻距离并粘贴到一个电子表格程序的一列。 <ol><li>编译所有从每个重复数据到同一个电子表格，并组织由基因型和条件。 </li></ol></li><li>使用统计软件进行数据分析。在这个实验中，图垫棱镜（6版本的MacOSX，格拉夫派得软件圣地亚哥加利福尼亚美国，www.graphpad.com）被用来进行单向ANOVA和秩和检验测试，Tukey的和邓尼特的事后检验。 </li><li>的距离的分布遵循非参数分布1，和数据被表示为框和Tukey的晶须（消除异常值）。 </li></ol> 5.登山分析（控制行为） <ol><li>使用登山法作为控制测试蝇攀爬能力14,15,18。 <ol><li>使用逆流装置，使得三个试验Ç一个可并行运行。 </li></ol></li><li>转移50到100天真果蝇（果蝇尚未前测试）在三个不同的测试小瓶，并且它们卡入第 1次，3次和最后时隙在逆流装置的底部，以在相反的位置新鲜空小瓶中。 </li><li>放置在空槽新鲜空小瓶在装置的底部。 </li><li>挖掘下来的装置三次，以使得所有的苍蝇都在每个管的底部，以启动试验。 </li><li>启动定时器，持续15秒（时间足以为3-7天老广-S〜100％，达到了顶级小瓶-见图3F）。 </li><li>滑动逆流装置的上部，由一个槽来置换顶小瓶中。 </li><li>通过点击设备下来，把所有的苍蝇在底部瓶收集苍蝇。 <ol><li> 1次和第2次管（或3次和第4次， 第 5次</sUp&gt;和6 次 ），分别对应于没有达到顶部苍蝇和爬到山顶小瓶苍蝇。 </li></ol></li><li>计算性能指数（PI）。 <ol><li>算蝇每个小瓶的数目。 </li><li>该PI是苍蝇能攀上上管的百分比。 </li><li>在PI的装置遵循正态分布1，和数据被表示为平均值加或减标准误差的平均值（SEM）的柱形图。 注意：可替换地，在PI可被计算为苍蝇能达到较低小瓶（老年人苍蝇的上部的百分比，5秒的足够的时间以量化的差在感染与非爬坡能力感染12个天龄广-S从静止图像量化：看到的结果）。而其他人使用的惊吓引起的负趋地表现不同的措施，同时也是适当的量化爬坡能力19-22。 </li></ol></li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+bacterial+infection+and+immune+defense+phenotypes+in+Drosophila+melanogaster.">Systemic bacterial infection and immune defense phenotypes in Drosophila melanogaster.</a> JoVE. , (2015).</li><li>Fernandez, R. W., Akinleye, A. A., Nurilov, M., Rouzyi, Z., Simon, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2013).</li><li>Suh, G. S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+population+of+olfactory+sensory+neurons+mediates+an+innate+avoidance+behaviour+in+Drosophila.">A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila.</a> Nature. 431, 854-859 (2004).</li><li>Helfrich-Fo&#776;rster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+control+of+morning+and+evening+components+in+the+activity+rhythm+of+Drosophila+melanogaster.--Sex-specific+differences+suggest+a+different+quality+of+activity.">Differential control of morning and evening components in the activity rhythm of Drosophila melanogaster.--Sex-specific differences suggest a different quality of activity.</a> J. Biol. Rhythms. 15, 135-154 (2000).</li><li>Fujii, S., Krishnan, P., Hardin, P. E., Amrein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nocturnal+male+sex+drive+in+Drosophila.">Nocturnal male sex drive in Drosophila.</a> Curr Biol. 17, 244-251 (2007).</li><li>Pellegrino, A. C., 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=Weather+forecasting+by+insects:+modified+sexual+behaviour+in+response+to+atmospheric+pressure+changes.">Weather forecasting by insects: modified sexual behaviour in response to atmospheric pressure changes.</a> PLoS One. 8, e75004 (2013).</li></ol>

The authors declare that they have no competing financial interests.

在这个协议中，对社会空间的量化的详细过程进行了说明。一些关键步骤，以确保实验成功的是：1）清理和设置设备，以保持自己的油脂和香料关闭设备的内室，2）确保苍蝇被收集之前至少有一天，当一直使用的手套实验，以减少冷麻醉的任何潜在影响，3）在实验前2小​​时，提供一种含有新鲜食物，以确保苍蝇不饿死，并已清洁自己新小瓶中，4）在这些2小时，让苍蝇使其恢复到其新的环境?...

社会互动是至关重要的一个组内的整体正常发展的个人和健康，并能在众多的品种可以观察到，从人类（ 智人 ），以简单的有机体，例如果蝇（ 果蝇 ）5,6。单个果蝇或人共享共同方式处理过程中这些互动的感觉信息，它是否是：听觉，视觉，嗅觉，触觉，味觉或。我们和其他人推测可能有是一种潜在的共享neurocircuitry底层行为反应社会互动和神经元细胞和参与可能是进化保守的基因7。一旦初始相互作用已经发生，相互作用的个人之间的社会空间要么增加（社交回避8）或减小（组形成/聚合5）。更复杂的相互作用，如侵略或求爱，就可以成行。 果蝇的稳定基团，如在以下的研究1-4,9使用。社会空间是指一种苍蝇和其最接近的邻居10之间的距离的量度。社会空间是D的一个特定人群一致当实验条件将被保留（平均约1-2个体的长度），并且改变相对于所述蝇社会经验，如果个体已经被保持在隔离1增加果蝇 。正确的目标是要维持正常的社会距离，而不是传统的添味剂或CVA感知1。因此，衡量社会空间可作为诊断工具来分析社会交往和量化D.社会行为黑1，我们在这里描述的细节如何执行此量化，并共同实验变量的影响程度如何这种行为。 我们表明，在该试验进行腔室，以及蝇的数目的取向 - 在一定程度上 - 做影响社会的空间。这是以前表明腔的几何形状影响的苍蝇11,12自发探索性运动，这种现象可能会最终影响他们决定定居。然而，只要在蝇密度（飞/厘米2）和腔室方向保持相同，苍蝇的社会空间也保持不变。该测定的鲁棒性的事实，使用不同的腔室的尺寸，形状独立实验室，和取向可以复制由WH的突变体所显示的结果示出ITE基因（影响眼部色素沉着），这是一个增加社会空间（1个垂直三角形或横向循环，用气流在3水平平方）。 我们的结果还表明，保持在该社会空间实验进行时间是至关重要的结果的一致性，因为我们表明，男性，但不是雌性，进一步分开，在晚上。然而，看到白天和晚上的时间之间的差异不是由于苍蝇的活动不同，和大家讨论的论点，表示不与社会空间相关的活动水平。 最后，有遗传基础的社会空间的测定，由白突变体所指示已经描述的1,3，和苍蝇，我们在这里介绍的各种近交系和捕获的野生株之间的差异。 因此，这个实验使一个很好的工具˚F或研究的遗传效应以及环境因素。

<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>Jazz-mix</td>
			<td>Fisher</td>
			<td>33545</td>
			<td>any other standard drosophila food would work</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>Medium size binder clips</td>
			<td>Staples</td>
			<td></td>
			<td>to hold the chambers together: 1-1/4&#34; (32mm) medium clips with 5/8&#34; capacity</td>
		</tr>
		<tr>
			<td>small SupportStands</td>
			<td>Carolina&#160;</td>
			<td>707161</td>
			<td rowspan="2">to hold the chambers in a vertical orientation</td>
		</tr>
		<tr>
			<td>Buret clamps</td>
			<td>Carolina</td>
			<td>707362</td>
		</tr>
		<tr>
			<td>Digital Camera</td>
			<td>Nikon</td>
			<td>&#160;Coolpix S8000&#160;</td>
			<td>to take the still pictures</td>
		</tr>
		<tr>
			<td>small ruler</td>
			<td></td>
			<td></td>
			<td>to be able to scale the picture</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>pounding pad</td>
			<td></td>
			<td></td>
			<td>any mouse pad works.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 6</td>
			<td>GraphPad Software Inc.</td>
			<td>Prism 6 for Mac OS X</td>
			<td>Any statistical analysis software with t-test, one-way and two ANOVA would work</td>
		</tr>
	</tbody>
</table>

conditions affecting social space in drosophila melanogaster

此处所描述的社会空间测定可用于定量的果蝇melanogasteř社会互动-或其它小昆虫-以直接的方式。如我们以前表明图1，在一个二维室，我们首先迫使苍蝇形成紧密基，随后使它们采取彼此其优选距离。之后苍蝇纷纷落户，我们测量到最近的邻居（或社会空间）的距离，处理静态图片，免费在线软件（ImageJ的）。到最近的邻居的距离的分析，使研究人员能够确定遗传和环境因素对社会互动的效果，同时控制了潜在的混杂因素。不同的因素，例如爬坡能力，天，性别的时间，和苍蝇的数目，可以修改蝇社会间距。因此，我们提出了一系列的实验控制，以减轻这些混杂影响。此法可用于至少两个目的。首先，研究人员能够确定自己喜欢的环境转变（如隔离，温度，压力或毒素）会影响社会的间距1,2。其次，研究人员可以剖析社会行为1,3这种基本形式的遗传和神经基础。具体而言，我们用它作为诊断工具，研究认为参与在其他生物，如候选基因孤独症在人中4社会行为直系同源基因的作用。

社会空间室可以作为一种工具，以量化果蝇的社会行为。丙烯酸垫片和玻璃板被夹在一起以形成内三角竞技场，提供了一个在其中苍蝇能形成稳定的基团没有许多潜在的混杂线索的存在二维区域。当苍蝇被转移到垂直领域，它们是由被窃听下来吓了一跳，他们通过逃逸行为作出回应：负趋地。他们爬到直立的三角形的顶点，强行纳入一个紧密的组，然后提供时间，以进一步将形成彼此，并?...

Watch this Scientific Journal Video about Conditions Affecting Social Space in Drosophila melanogaster at JoVE.com

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 ...

conditions-affecting-social-space-in-drosophila-melanogaster

Research

JoVE Journal

Neuroscience

Conditions Affecting Social Space in Drosophila melanogaster

12.0K 次观看.  University of Western Ontario. 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.

视频: Conditions Affecting Social Space 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

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 ...

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 ...

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 ...

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 ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

测量并改变配套驱动马累<em&gt;果蝇</em

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

适用于 果蝇 的微量呼吸测定法 - 深入了解 CASK 突变体代谢

Measuring O2 Consumption in Drosophila melanogaster Using Coulometric Microrespirometry

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable environment. A coulometric microrespirometer consists of an airtight chamber in which O2 is consumed and the CO2 produced by the organism is removed by an absorbent medium. The resulting pressure decrease triggers electrolytic O2 production, and the amount of O2 produced is measured by recording the amount of charge used to generate it. In the present study, the method has been adapted to Drosophila melanogaster tested in small groups, with the sensitivity of the apparatus and the environmental conditions optimized for high stability. The amount of O2 consumed by wildtype flies in this apparatus is consistent with that measured by previous studies. Mass-specific O2 consumption by CASK mutants, which are smaller and known to be less active, was not different from congenic controls. However, the small size of CASK mutants resulted in a significant reduction in O2 consumption on a per-fly basis. Therefore, the microrespirometer is capable of measuring O2 consumption in D. melanogaster, can distinguish modest differences between genotypes, and adds a versatile tool for measuring metabolic rates.

作者聚焦：温度对黑腹果蝇和沙漠适应性甲虫的影响 

Coulometric respirometry is ideal for measuring the metabolic rate of small organisms. When adapted for Drosophila melanogaster in the present study, measured O2 consumption was within the range reported for wildtype D. melanogaster by previous studies. Per-fly O2 consumption by CASK mutants, which are smaller and less active, was significantly lower than the wildtype.

使用库仑显微呼吸法测量黑腹果蝇的 O2 消耗量

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable ...