We thank the past and present members of the Scholz lab for discussion and Helga D&#246;ring for excellent technical support. We especially thank the members of the Biocenter workshop of the University of Cologne for their support and creativity. The work is supported by SFB 1340, SysMedAlc, and DAAD-Siemens.

1. CAFE分析注:该试剂盒由三个部分组成:一个实验小瓶中，一个特定的盖子和微毛细血管。带盖的塑料盒用于传输准备好的小瓶，并更有效地控制湿度。 <ol><li>使用果蝇培养塑料瓶（可选的8厘米高，3.3厘米直径的），为用于测定的管。 </li><li>密封用含有一个O形圈（ 图1A，1B）生产的有机玻璃盖小瓶中。负荷通过敲击或与通过盖的中心开口（直径0.9厘米），这也允许空气流通和供水吹管苍蝇，和用海绵塞关闭孔。六小锥形孔（0.4厘米上部直径，0.3厘米内径）围绕中心孔，适合2的枪头 - 20微升量保持毛细血管到位。 （见盖的技术细节补充的数字。） 注:使用与用于毛细管代替在我们的手稿中使用的定制的盖的开口的海绵塞是可能的。我们定制的盖子允许准备小瓶减少毛细血管跌倒风险的安全处理。 </li><li>为了呈现液态食品，用1μL标记5μL微毛细管。 20μL的枪头和插入尖端插入孔（ 图1B中，标有红色边） -通过切断2的顶部在盖的锥形开口的毛细血管的位置。为了防止苍蝇逃跑，插入一个完整无缺的2 - 20微升的枪头到同一开幕。 </li><li>为了安全地处理多个小瓶的准备，将它们放入一个塑料盒与网格镶嵌（ 图2A）。 </li></ol> 2.苍蝇的制备<ol><li>保持在标准的食品苍蝇在25℃，60％相对湿度和12小时/ 12小时明 - 暗循环。 </li><li>为了控制繁殖条件，引进35处女的女性D 15-男性每个实验组进一个塑料瓶的文化（9.8厘米高，4.8厘米口径）含50毫升飞的食物。允许苍蝇产卵的第3天，然后转移成蝇到新鲜食品的小瓶，让他们产卵两天。在此之后再次重复传输。经过2天多弃成蝇。 </li><li>如食物摄取取决于飞的大小，由麻醉2到3天的成人确定一组100苍蝇的重量苍蝇使用CO 2蝇垫并将它们收集到1.5毫升的塑料管中并用一个标准的测量实验室规模。确定由性别（ 表1）排序的至少四个独立的飞组的湿重;用重量来计算每毫克飞微升食品消费。使用该值，以确定食品的单个飞每个实验饲料的量，并相应调整食品填充的毛细管的数目通过将避免毛细管排空。 <ol><li>对于3小时检测，使用20苍蝇和两个填充的毛细管。对于长期的试验（&gt; 3小时，长达9天），使用一组八个苍蝇与四个填充的毛细管的供给（可靠的结果，不能少于8苍蝇所描述的条件下得到）。 </li></ol></li><li>独立飞进下CO 2的曝光测量体重后组（8或20苍蝇）。该集团转移到一个新的食品小瓶（含15毫升标准的食品）允许从CO 2的镇静48小时之前实验中复苏。使用4〜6日龄苍蝇的CAFE法。 </li><li>作为非饥饿的野生型果蝇饲料仅略微19， 第21，前饥饿蝇3小时喂养实验。当食品消费数天监测空腹无要求。对于空腹，转让轻轻拍打他们入含约0.5毫升的DDH只蘸45毫米直径的折叠滤纸小瓶飞行测试前16〜20小时2 O（双蒸水），并关闭了插入CAFE法盖子。 </li></ol> 3.液体食品的研制<ol><li>通过填充102.6克蔗糖（C 12 H 22 O 11），以100毫升的DDH 2 O移液器3微升，33微升，333微升，3.3毫升和6.6毫升制备3 M（10％，重量/体积）蔗糖原液的原液成15毫升的塑料管;加2食色液（红色:胭脂虫[E124];蓝色:靛蓝胭脂红[E132]），并填写至10毫升的DDH 2 O.由此产生的浓度是0.001，0.01，0.1，1，和2蔗糖。 注:食用色素允许更容易地可视化半月板。然而，染料可能对食物摄入的影响。为了避免偏差，由于该染料分配所述食品染料或实验和组中随机染料的食品样品的使用。 </li><li>测试对于酒精偏好吸管在一个15毫升的塑料管3蔗糖原液的333微升。添加100％的EtOH（乙醇）的1.5毫升（2.3毫升）和添加DDH 2 O的高达10毫升以产生15％（0.25毫摩尔）和23％（0.39毫摩尔）的工作的解决方案。 </li><li>保持在4℃-20℃股票方案和工作方案;在1周内使用。 </li><li>填充到10的毛细管同时用有色食品溶液中，通过毛细力。插入毛细管的端部到蔗糖溶液（保持毛细管以45°角）的溶液。停止，如果该液体到达顶端毛细管（5微升）标记，并在外部和内部用纸巾除去过量溶液。 </li></ol> 4.大会和执行毛细管馈线分析<ol><li>如果不需要禁食，通过敲击或通过吹塑管转移实验苍蝇测定。确保包括三个控制小瓶无苍蝇量化蒸发。 </li><li>小心地取出枪头 - 即关闭外openin之一（2 20μL体积）GS，并且插入填充玻璃毛细管，底端。通过将枪头回旁边的毛细管固定毛细管。如果一些食品的解决方案正在测试，因此重复此过程。 </li><li>放置毛细管在同一级别的所有样品瓶内结束，以避免如果食物源分别位于不同的高度可能发生偏差（3 - 从盖4厘米）;保持距离，以滤纸来防止毛细管由无意中触摸滤纸或食物来源不同粘度泄漏。 </li><li>标记用标记笔（马克开始 ）的着色液体的上端。为了确保不同的毛细管可以被识别，标记它们单独使用一种颜色或条纹码。 </li><li>放置多个制备CAFE测定用网格嵌的塑料盒内，并在实验室条件下，或在一个与温度，光，湿度控制CLIM传送盒（ 图2A）到安全位置吃室（参数:25℃，相对湿度60％，12小时/ 12小时明-暗循环）为实验期间（ 例如 3小时或天）。 </li><li>仿佛在数天进行测定底部滤纸变干，应用清水通过海绵塞（100微升），每24小时，以保持湿度恒定试验中。使用装有30毫升DDH 2 O的湿度设备四个独立小瓶（8厘米高，3.3厘米口径）旁边放置在塑料盒的CAFE检测。使用盖的塑料盒来创建实验（ 图2A）中的湿度控制的环境。 注:更广泛的变化在实验室条件下进行;但是，它是可行的执行在室温CAFE测定（ 例如 ，在一教室）。使用加湿装置（滤纸，具有或不具有湿海绵塞，充满水的小瓶和盖的塑料盒）强烈鼓励降低蒸发（ <st荣&gt;图2B）。 </li><li>长期实验，每24小时更换新鲜那些充满毛细血管。记死苍蝇每24小时间隔之前，用活苍蝇的数量来计算每飞消费放置一段时间。测量液面下降（见5.1）后，丢弃旧的毛细血管。 注:在一个3小时实验中，我们几乎看不到任何死苍蝇。在4天的研究，我们通常会发现，第1 - 3死苍蝇。 </li><li>在测定或更换毛细管之前结束，标志着毛细管（标记结束 ）用记号笔下弯月面，而CAFE法仍处于直立位置。丢弃的数据。如果标志到底是不是最初的标记（ 开始标记）。不要取下盖子，因为这可能会改变的半月板。 </li><li>小心地从试验中移除毛细管并将它们存储为数据收集。检查，如果毛细管内的液体到达下端如果不抛弃它D中的数据，因为食物是不是苍蝇访问。收集每瓶所有毛细管为一组。将未切割枪头到所有的开口，防止苍蝇逃跑。拆的安装并在室温下供进一步使用洗小瓶，盖子和海绵塞子在肥皂浴和干燥过夜。 注:蝇可以在测定后进行进一步分析。用肉眼或解剖显微镜下确认的食物摄取。 </li><li>上的至少三个不同的日子的相同基因型的重复实验。 </li></ol> 5.数据收集和分析<ol><li>测量用卡尺或尺子毛细管标记开始和结束标记之间的距离。将数据直接传送到电子表格，使用连接数显卡尺（ 图1E）USB（通用串行总线）。丢弃测量后的毛细血管。 </li><li>账户毛细管大小来计算食物的摄取或蒸发。例如，考虑帽illary的73毫米长，并且包含食品液5微升。一个14.6毫米减少弯月反映了1μL解决方案的摄取。用下面的公式计算食物的摄取: 食物摄取（μL）=测得的距离（mm）/14.6毫米</li><li>以排除对食物摄入的蒸发的效果，计算平均的三（至少）控制小瓶蒸发而不苍蝇。减去从苍蝇的食品消费所得到的值这个平均值。 </li><li>使用下面的公式来确定每飞总消费量: 食物消耗（μL）=（食物的摄取[μL] - 蒸发损耗[μL]）/在小瓶苍蝇总数。对于长期实验使用蝇的24小时间隔的开始之前存活的数目。 </li><li>为了说明在车身尺寸上的差异，比如男性和女性之间的苍蝇，规范食品消费体重（μL食品/毫克飞）。 </li><li>用于数据分析统计软件。对于规范同盟分布式数据，使用学生t -tests确定两个飞组之间的差异，并使用ANOVA（方差分析）与事后杜克克拉默测试两个以上的组。在选择的情况下，分析使用非参数单样本符号检验由随机选择的差异。 </li></ol>

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H., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+regulation+in+Drosophila.">Feeding regulation in Drosophila.</a> Curr. Opin. Neurobiol. 29, 57-63 (2014).</li><li>Luo, J. N., Lushchak, O. V., Goergen, P., Williams, M. J., N&#228;ssel, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+insulin-producing+cells+are+differentially+modulated+by+serotonin+and+octopamine+receptors+and+affect+social+behavior.">Drosophila insulin-producing cells are differentially modulated by serotonin and octopamine receptors and affect social behavior.</a> Plos One. 9 (6), e99732 (2014).</li></ol>

作者什么都没有透露。

该报告描述了一步一步的时尚CAFE法，着眼于技术设置并在实验室的成功表现。由于其简单，该测定也可以教育上用作校实验。该实施例表明该测定允许食物感测，在短和更长的时间周期（数小时至数天）的偏好和消费在果蝇的调查。网吧法已在该领域广泛使用的调查对象包括食品和药品消费，吸毒，能量平衡和喂养16神经元控制<sup cl...

饮食是必不可少的;然而，导致进食障碍如食欲过盛，厌食或暴饮暴食强加的一般倾向的食物摄取的失调对个人和社会1，2，3，收费。目前研究的目的是揭开食物摄入量的监管机制，并提供规避障碍形成的战略。使用哺乳动物模型生物体许多研究在进食障碍4，5，6中提供的电路和信号系统的作用的新的见解。然而，我们的这些疾病背后的神经细胞和分子基础知识没有完成仍远。在最近几年，果蝇果蝇已成为解开基本机械分析上市metabolis的调节中有价值的模型系统M 7，8，9。对果蝇毛细管进纸器（CAFE）法成立于西摩·本泽在2007年的绿头苍蝇10,11灵感来自早期的工作由Dethier实验室。网吧分析使我们能够直接测量果蝇食物的摄入量。在这种行为测试系统，苍蝇喂上放在瓶内分级玻璃毛细管提供流质食物。毛细管半月板的衰落表明通过蒸发和食品消费的食品解决方案的损失。确定由小瓶蒸发率没有苍蝇允许摄入食物的准确定量。 网吧测定是用来测量果蝇喂养几个行为范式之一，研究人员选择最合适他们的具体题。使用特定试验应考虑以下几点决定:提供食物的性质;饲养条件;摄取或营养物的摄取和调查食物消耗或响应于食物的测量。 本报告中所描述的CAFE法是理想的直立饲养条件下以下的液体食物来源的食物的摄入量。可替代地，食物摄入量可以为苍蝇组来测量在小瓶或在平板上的着色的食物来源。蝇通常杀死或麻醉后喂养和摄取的染料的量由法或染色腹部的目测来确定。蝇开始排泄摄取食物摄入后仅30分钟，因此这种方法很难用于连续长馈送的分析行为12,13。 相比之下蝇保持完整时吸收染料s的放射性示踪剂的使用和它们的放射性同位素的消费在闪烁计数器14，15被评分。由飞消化系统的放射性标记的吸收，使长期摄取食物测量可能的，但可能会导致因非吸收而排出体外示踪分子的消费被低估。另一种方法来测量响应果蝇的食物是黑的长鼻扩展响应（PER），它通常出现在食物中摄取16。这间优雅的方法措施，食物刺激的初步反应，但不记录的摄入量。食物摄入量是使用用于馈送17，18的规临界几个后消化反馈信号馈送期间动态调整。多次尝试在最近几年已经进行了半自动化数据集合中的每个试验<s补课="外部参照"&gt; 19，20。该PER由电垫或电极的组合检测到的，并通过计算机计算。结合PER检测放射性同位素的摄取透露，这个实验是由低灵敏度限于检测量喂养差异18。手动进测定（MAFE）21，在其中一个苍蝇用玻璃毛细管手动馈送，最近开发用于测量在单一固定的飞食品摄取。该MAFE法消除觅食和进食引发的干扰，有几秒钟的时间分辨率，PER的启动和食品消费可以独立在试验进行监控。然而，其中一路飞的固定化的影响摄食行为的某些方面（ 如运动，动机）仍然需要进行调查。对于不同的检测方法用于测量果蝇我食品消费出色的横向评测lanogaster并帮助研究人员找到最合适的，看到德什潘德和马克思报告 13，22。 网吧法避免了一些上述其它测定法的缺点，并结合简单易用的食物的摄入量能够可靠地计量。这里，提供了CAFE测定的详细说明，我们示出了简单的设置的修改，以减少蒸发。代表性的成果包括两个食物选择实验（短期和长期）和苍蝇的蔗糖的吸收是证明。在讨论中，我们比较我们用其他的方法来执行的CAFE法，并突出显示潜在的局限性所描述的方法。

<table border="0" cellpadding="0" cellspacing="0" width="582">
	<tbody>
		<tr height="31">
			<td height="31" style="height:31px;width:161px;">Vials (breeding)</td>
			<td style="width:117px;">Greiner Bio-One</td>
			<td style="width:123px;">960177</td>
			<td style="width:183px;">www.greinerbioone.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Vials (CAFE assay)</td>
			<td>Greiner Bio-One</td>
			<td>217101</td>
			<td>www.greinerbioone.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Lid-CAFE assay</td>
			<td>Workshop</td>
			<td>&#8211;</td>
			<td>&#8211;</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Plastic box, low wall</td>
			<td>Plastime</td>
			<td>353</td>
			<td>www.plastime.it</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Cover for the plastic box</td>
			<td>Workshop</td>
			<td>&#8211;</td>
			<td>&#8211;</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Capillaries</td>
			<td>BLAUBRAND&#160;</td>
			<td>REF 7087 07</td>
			<td>www.brand.de</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Pipette tips</td>
			<td>Greiner Bio-One</td>
			<td>771290</td>
			<td>www.greinerbioone.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Filter paper circles</td>
			<td>Whatman</td>
			<td>10 311 804</td>
			<td>www.sigmaaldrich.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">D(+)-Sucrose</td>
			<td>AppliChem</td>
			<td>57-50-1</td>
			<td>www.applichem.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Ethanol absolute</td>
			<td>VWR Chemicals</td>
			<td>20,821,330</td>
			<td>www.vwr.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Food color (red, E124)</td>
			<td>Backfun</td>
			<td>10027</td>
			<td>www.backfun.de</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Food color (blue, E133)</td>
			<td>Backfun</td>
			<td>10030</td>
			<td>www.backfun.de</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Soap solution (CVK 8)</td>
			<td>CVH</td>
			<td>103220</td>
			<td>www.cvh.de</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Digital caliper</td>
			<td>GARANT</td>
			<td>412,616</td>
			<td>www.hoffmann-group.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Vials (breeding)</td>
			<td></td>
			<td></td>
			<td>Height 9.8 cm, diameter 4.8 cm&#160;</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Vials (CAFE assay)</td>
			<td></td>
			<td></td>
			<td>Height 8 cm, diameter 3.3 cm</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Lid-CAFE assay</td>
			<td></td>
			<td></td>
			<td>Produced in university workshop, technical drawing supplied</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Plastic box, low wall</td>
			<td></td>
			<td></td>
			<td>A plastic grid inlay was custom-made for 8 x 10 vial positions&#160;</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Cover for the plastic box</td>
			<td></td>
			<td></td>
			<td>Dimensions (37 x29 x18 cm)</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Capillaries</td>
			<td></td>
			<td></td>
			<td>DIN ISO 7550 norm,&#160; IVD-guideline 98/79 EG, ends polished</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Pipette tips</td>
			<td></td>
			<td></td>
			<td>Pipettes for the outer circle are cut according to the lid</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Filter paper circles</td>
			<td></td>
			<td></td>
			<td>45 mm diameter works nicely if folded for the vials used</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">D(+)-Sucrose</td>
			<td></td>
			<td></td>
			<td>Not harmful</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Ethanol absolute</td>
			<td></td>
			<td></td>
			<td>Highly flammable liquid and vapor</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Food color (red, E124)</td>
			<td></td>
			<td></td>
			<td>Not stated</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Food color (blue, E133)</td>
			<td></td>
			<td></td>
			<td>Not stated</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Soap solution (CVK 8)</td>
			<td></td>
			<td></td>
			<td>Odor neutral soap</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Digital caliper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Standard fly food</td>
			<td></td>
			<td></td>
			<td>(for 20 L)</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Agar</td>
			<td></td>
			<td></td>
			<td>160 g</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Brewer`s Yeast</td>
			<td></td>
			<td></td>
			<td>299.33 g</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Cornmeal</td>
			<td></td>
			<td></td>
			<td>1200g</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Molasses</td>
			<td></td>
			<td></td>
			<td>1.6 L</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Propionic acid</td>
			<td></td>
			<td></td>
			<td>57.3 mL</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Nipagin 30%</td>
			<td></td>
			<td></td>
			<td>160 mL</td>
		</tr>
	</tbody>
</table>

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.

将W 1118基因型的苍蝇用于演示如何执行该测定。将W 1118的突变体通常用于产生转基因株系，并控制转基因的遗传背景标有白基因。通常情况下，对行为实验，所有的转基因品系进行回交五代到同一个W 1118的库存，这是用来作为实验对照。我们显示不同的实验:蒸发损失为我们的改性设置的比较，一个短期的食物选择的实验...

Watch this Scientific Journal Video about The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster at JoVE.com

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 ...

the-capillary-feeder-assay-measures-food-intake-drosophila

Research

JoVE Journal

Neuroscience

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

18.5K 次观看.  Albertus-Magnus University of Cologne. 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.

视频: The CApillary FEeder Assay Measures Food Intake 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

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 ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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 ...

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 ...

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. ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...