我们要承认在马里实验室和Godenschwege的实验室，特别是艾琳米泽成员，征求意见，并帮助这个协议。这项工作是由国立神经疾病研究所和中风授予R21NS06637 FM和TAG; AB公司是由国家科学基金会奖号码082925，URM的资助：为未来的研究人员结合生物学。

1。电钻机设置<ol><li>详细描述了为电钻机设置所需的设备由奥古斯丁等 。在该杂志14。为所需的电生理仪器的详细解释，请参阅本文。 </li><li>加入了第六的微操作机器人，其持有的nanoinjector修改先前所描述的电钻机设置14。为方便应置于两者之间的刺激电极显微操作，在图2所示的动物的头。 </li><li>在实验开始前，确保你有一个旋转轴与所有显微操作的舒适范围，所有电极以及注射微量（见步骤2），可以达到动物。 </li></ol> 2。 nanoinjection集行动<ol><li>设置的nanoinjection需要1 Nanoliter2000（世界Precisi仪器，萨拉索塔，佛罗里达州，美国）或类似的喷油器的类型，允许在纳升数量控制注射。 </li><li>准备用玻璃微电极，电极拔拔80-100MΩ的电阻与喷油器提供的注射针头。 </li><li>为顺利注射，它需要的微电极锥在45度角的11-17微米开幕（ 图3）。 </li><li>慢慢回填注射微量合成油使用Hamilton注射器由Nanoliter2000手册的指示，确保无气泡存在。 </li><li>仔细确保上nanoinjector微量的和准备来载入排空多余的油由Nanoliter2000手册的指示，该化合物。 </li><li>放置在微操作机器人的喷油器和加载的复合在Nanoliter2000手册的指示。确保微量的一角，在此期间不中断程序。 </li><li>设置所需数量由Nanoliter2000手册的指示，在喷油器的控制盒注射纳升。请注意，总金额注入不应超过100 NL。我们发现，大批量的生理盐水对照解决方案可能会影响政府飞行服务队的电路功能。 </li><li>注射器拔出从控制箱与注射本身的异常，因为在拍摄过程中采集的nanoinjector电源干扰与录音，这是作为背景噪声（ 图4）可见，这是至关重要的。但是，不要断开电源控制箱，因为它会重新设置它。 </li></ol> 3。 果蝇准备<ol><li>麻醉与CO 2或冰2至6天的老苍蝇如前所述14,15。 </li><li>一旦动，使用镊子动物转移到中小板无线TH软牙科用蜡采摘它从它的腿。请注意，雄性果蝇的重量约1.0毫克，重约1.2毫克，一个女的果蝇，因此复合体的重量比是在男性和女性的苍蝇不同。因此，建议使用只有一个性别实验。 </li><li>正如先前所述14,15，精心安装飞背侧，确保胸部和头部被固定放置在身体周围的软牙科蜡。仔细传播的翅膀，让他们躺在胸部（ 图2，C），垂直。苍蝇应安装用尽可能少的损害。 </li></ol> 4。配对Nanoinjection /电<ol><li>将其头部对刺激电极的电钻机上安装飞。 </li><li>如前所述14,15（六钉与相应的刺激，地面和记录电极的动物igure 2）。除非需要，否则进入到TTM肌肉DLM和其他的地方之一记录电极。 DLM的肌肉位于胸部之间的前后背成中央毛发和苍蝇中线。过去一年肌肉靠近机翼附件，后部和前跨阿拉尔飞毛7之间。 </li><li>注射微量含有的化合物的三个单眼位于头后部内侧，但不注入尚未（ 图2，C），中心对齐。 </li><li>复方注射之前获得的GF基线记录 ​​过去一年和GF，DLM巨人光纤系统（政府飞行服务队， 图1）通过刺激大脑的途径。这样做，激活10列车10刺激（40-50毫伏）在100赫兹之间的列车14,15（ 图4）延迟1秒的持续时间为0.03毫秒每个巨纤维（GFS）。野生型飞SH乌尔德是能够遵循这种刺激率DLM的和TTM途径之一到一。丢弃飞DLM和GF GF的过去一年的途径，不遵循100 Hz刺激在1:1的比例。 </li><li>切换到连续刺激的GF在1 Hz的单脉冲（ 图4）。 </li><li>快速插入控制盒注射器。即使背景噪音会干扰1 Hz的刺激录音，不停止。 </li><li>小心地插入到略低于飞cuticule的头壳微量注射，注入飞淋巴化合物所需的金额，同时保持1 Hz的刺激（ 图4）。由于飞的开放式循环系统，整个神经系统会受到的化合物，在几秒钟内。虽然一个特定的注射部位是不能提供化合物进入淋巴的关键，我们发现单眼的地区，这是本地化ð在头壳最背侧，是一个方便的网站，允许一个简单的注射化合物的分布迅速，甚至导致内侧。 </li><li>立即从注射部位注射微量拔下喷油器从控制箱，1 Hz的快刺激，同时继续注射后1分钟（ 图4）。 </li><li>为了揭示政府飞行服务队的化合物更微妙的影响，强调在100赫兹的巨纤维（GFS）10 10刺激的列车与列车之间的延迟1秒。继续测试这个范例中，每5分钟至15分钟（ 图4）的GF通路的功能。然而，较短的时间间隔或更长的监测期间也有可能。 </li><li>为了检验该化合物是否具有在神经肌肉接头（NMJs）政府飞行服务队的作用，并可能缩小化合物的影响，继续激活运动神经元方向TLY由胸刺激。对于这一点，从眼睛的刺激电极，取代他们的胸部前两侧10 10刺激的列车，以刺激运动神经元，在100赫兹。 </li></ol> 注：电在视频显示的痕迹不相符的纯染料注射的效果。 5。代表结果拮抗剂对PSI DLM巨人光纤系统突触的影响 methyllycaconitine柠​​檬酸（MLA）是胆碱受体拮抗剂，是α7胆碱受体亚型的具体。 PSI的以DLMn GF-DLM的通路中的突触是正常功能Dα7胆碱受体亚型的依赖，同时去除Dα7胆碱受体亚型基因的GF过去一年通路5,6没有影响。为了证明我们检测的灵敏度和特异性，我们注入工作重点在不同浓度（0，0.02，0.04，0。08，注入0.12毫微克/毫克，46 NL）到动物的头（N = 10％复合处理; N = 15生理盐水治疗）。只雄蝇（野生型基因型野生10E），和化合物的效果监测，共注射后15分钟。 图5描述了注射前获得的基线的工作重点和生理盐水对照溶液注射后获得的录音和之间的差异。我们发现，注入的工作重点在GF-DLM的途径无法遵循通过刺激大脑中的政府飞行服务队一到100赫兹，而GF过去一年途径不受影响。 （ 图5，顶部和中间的痕迹，t-检验生理盐水对照组[1纳克/毫克和工作重点，在每个时间点不同浓度之间进行，除非数据非参数正态性和方差相等测试，否则，我们使用1 Mann-Whitney秩和检验。* P &lt;0.001）。然而，一到一个Response的DLM的观察时，直接刺激运动神经元（ 图5，底部跟踪），表明DLM的和TTM NMJ功能不影响工作重点。工作重点出现注射后达到其最大作用1分钟，0.04，0.08和0.12毫微克/毫克注射工作重点，因为没有进一步的显着变化，在接下来的15分钟的测试期间所指出的。此外，该化合物达到0.08毫微克/毫克的最大作用，因为更强的反应并没有观察到高剂量的0.12毫微克/毫克。 <img src="/files/ftp_upload/3597/3597fig1.jpg" alt="图1" /> 图1。系统（GFS）巨纤维纤维的巨系统图。（政府飞行服务队，以红色显示）巨人纤维突触电（峡路口）以及化学（胆碱）到外围形成突触中间神经（PSI，以绿色显示） Tergo Trochanteral肌肉神经元（TTMn，以黄色显示）5。在PS我要DLMn（背纵肌的神经元，以蓝色显示）连接是依赖于Dα7胆碱受体亚型6。最后到跳（TTM，紫色所示）和飞行肌的TTMn和DLMn器（DLM，紫色所示）神经肌肉接头（NMJ）是谷氨酸。 注：绿PSI的连接，电气和化学。然而，没有任何反应（缺乏差距路口）shakB突变，可以记录从DLM的政府飞行服务队的刺激后，在大脑中，这表明在没有电气连接的化学成分是不足以唤起动作电位PSI的5,16-18。 GF是因为PSI的连接缝隙连接依赖，这一数字仅显示为简单起见突触间隙连接。 <img src="/files/ftp_upload/3597/3597fig2.jpg" alt="图2" /> 图2。 显微操作小号ET-。 <ol class="ualpha"><li>一个修改先前公布的协议14设置使用适合配对队同时nanoinjections录音注射显微操作。安装的飞行准备是面向对实验者的苍蝇头。注射微操作机器人（＃1）被放置在前面的实验者之间的两个机器人钨刺激电极（2＃和3＃）。两个玻璃记录电极（＃4和＃5）显微操作，分别放在左边和右边。钨接地电极（＃6）的微操作机器人被放置在后面最远的左侧（如图所示），或在右侧。 </li><li>一位接近认为，从顶部的各种电极和注射微量的安排。 </li><li>一个正确安装D。黑电极和注射微量注射刺穿。注意动物的身体其胸部水平和它的翅膀上传播出去。蜡牢固地缠它的身体，防止动物移动。此外，接地电极（＃6，在腹部），记录电极的玻璃（＃4和＃5，在胸部，由黑暗的轮廓突出），（＃2和＃3，在每只眼睛刺激电极）被刺穿的地方，如前面所述14。微量注射液（＃1）正确对齐三个单眼（圆圈）的中心。在这方面，应放在插入注射微量。 </li></ol><img src="/files/ftp_upload/3597/3597fig3.jpg" alt="图3" /> 图3。斜面注射微量。适当斜面微量的图显示在这里。电极的开放应在45度角斜面，并有11至17微米之间的开放。一个适当的斜面注射微量是至关重要的顺利注入与最小ð豪悦国际来飞。 <img src="/files/ftp_upload/3597/3597fig4.jpg" alt="图4" /> 图4。总体方案的nanoinjection /电协议。一个代表图的整体方案，为nanoinjection /电协议。基线记录获得通过刺激巨纤维在100赫兹（GFS）10 10刺激（这里只显示了一列火车）列车启动。注射前，开始1 Hz的刺激一秒。在注射时间（喷油器插在控制箱），你会观察显着的背景噪音，但是，不要停止录音。注射后（从控制盒中拔出注射器），持续约1分钟为1 Hz的刺激。最后，继续强调政府飞行服务队10 10刺激列车在100赫兹，并继续测试这个范例中，每5分钟至15分钟的GF通路的功能。注：录音瓦特ERE操纵创建的总体方案，并不代表获得一个特定的结果。不向规模化，并非所有的痕迹。 <a href="http://www.jove.com/files/ftp_upload/3597/3597fig4large.jpg">点击图片放大</a> 。 <img src="/files/ftp_upload/3597/3597fig5.jpg" alt="图5" /> 图5。政府飞行服务队的工作重点。 <ol class="ualpha"><li>一个图形描述的影响α7胆碱受体拮抗剂Methyllycaconitine柠​​檬酸对果蝇GF-DLM的途径（MLA），在不同浓度（0，0.02，0.04，0.08，0.12毫微克/毫克N = 10％的复合治疗。 N = 15）生理盐水治疗。注射后只有一分钟，被视为一个重要和立竿见影的效果与0.04毫微克/毫克的工作重点。疗效显着，也看到与在0.8ng/mg和100Hz的刺激，政府飞行服务队的工作重点0.12ng/mg。盐水控制和0.02毫微克/毫克的工作重点之间无显着差异。此外，没有改变我1分钟后注射后，在测试的时间（15分钟），列印效果。生理盐水对照组（0毫微克/毫克）和工作重点，在每个时间点不同浓度之间进行t检验。报告为平均值+ / - 扫描电镜（SEM），* P &lt;0.001水平。 </li><li> DLM的反应的样品在100 Hz刺激的痕迹。上部曲线显示工作重点后，广发刺激大脑中注射前的肌肉反应。注意能够应对在100赫兹到每个刺激肌肉。中东跟踪显示DLM的答复后，工作重点注射液（0.12毫微克/毫克）。请注意，肌肉是无法应对每个刺激在100赫兹到一。 （星号）。底部跟踪显示相同的准备后，在胸部的运动神经元的直接刺激（0.12毫微克/毫克）的DLM的反应。 DLM的回应胸部的刺激在100赫兹到，大脑的刺激与反应的失败可以归因于胆碱能PSI - DLMn连接的。 </li><li>图形描述的工作重点不同浓度（0，0.02，0.04，0.08，0.12毫微克/毫克）的GF过去一年途径的影响。被视为在任何时间点盐水（1毫微克/毫克）和复方注射之间没有显着的影响。生理盐水对照组（1毫微克/毫克）和不同浓度的工作重点，在每个时间点之间进行t检验，* P &lt;0.001。 </li><li>在100 Hz刺激的TTM反应的样品的痕迹。上部曲线显示前工作重点注射后，刺激大脑中与广发激活肌肉的反应。请注意，是能够响应在100 Hz到所有刺激肌肉。中东跟踪显示后的工作重点注射液（0.12毫微克/毫克）的DLM反应。从TTM肌肉刺激大脑中的绿的反应保持一至一。底部的跟踪显示，过去一年的准备在胸部的运动神经元（0.12毫微克/毫克）100 Hz刺激的反应。 </li></ol>

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J., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Recordings+from+the+Giant+Fiber+Pathway+of+D.+melanogaster.">Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster.</a> J. Vis. Exp. (47), e2412 (2011).</li><li>Allen, M. J., Godenschwege, T., Zhang, B., Freeman, M. R., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila Neurobiology. , 215-224 (2010).</li><li>Blagburn, J. M., Alexopoulos, H., Davies, J. A., Bacon, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Null+mutation+in+shaking-B+eliminates+electrical,+but+not+chemical,+synapses+in+the+Drosophila+giant+fiber+system:+a+structural+study.">Null mutation in shaking-B eliminates electrical, but not chemical, synapses in the Drosophila giant fiber system: a structural study.</a> J. Comp. Neurol. 404, 449-458 (1999).</li><li>Thomas, J. B., Wyman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+altering+synaptic+connectivity+between+identified+neurons+in+Drosophila.">Mutations altering synaptic connectivity between identified neurons in Drosophila.</a> J. Neurosci. 4, 530-538 (1984).</li><li>Baird, D. H., Schalet, A. P., Wyman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Passover+locus+in+Drosophila+melanogaster:+complex+complementation+and+different+effects+on+the+giant+fiber+neural+pathway.">The Passover locus in Drosophila melanogaster: complex complementation and different effects on the giant fiber neural pathway.</a> Genetics. 126, 1045-1059 (1990).</li><li>Gorczyca, M., Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+cholinergic+synapse+in+the+giant+fiber+pathway+of+Drosophila+using+conditional+mutations+of+acetylcholine+synthesis.">Identification of a cholinergic synapse in the giant fiber pathway of Drosophila using conditional mutations of acetylcholine synthesis.</a> J. Neurogenet. 1, 289-313 (1984).</li><li>Allen, M. J., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+component+of+the+mixed+GF-TTMn+synapse+in+Drosophila+melanogaster+uses+acetylcholine+as+its+neurotransmitter.">The chemical component of the mixed GF-TTMn synapse in Drosophila melanogaster uses acetylcholine as its neurotransmitter.</a> The European journal of neuroscience. 26, 439-445 (2007).</li><li>Mejia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+for+in+vivo+screening+of+toxins+using+the+Drosophila+Giant+Fiber+circuit.">A novel approach for in vivo screening of toxins using the Drosophila Giant Fiber circuit.</a> Toxicon. 56, 1398-1407 (2010).</li><li>Stork, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+function+of+the+blood-brain+barrier+in+Drosophila.">Organization and function of the blood-brain barrier in Drosophila.</a> J. Neurosci. 28, 587-597 (2008).</li></ol>

没有利益冲突的声明。

这里介绍的nanoinjection /电生物活性，可快速筛选的化合物在果蝇的神经系统。这是一个小说在体内的技术，需要少量的化合物，在一个特点的神经电路引起的各种分子靶点的作用。这种方法可以用来测试不同的化合物的生物活性，从未知毒素市售药物制剂。 在这里，我们证明了我们的检测功能，使用工作重点，其中有巨人光纤系统（GFS）的果蝇（ 图5）效?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>录音玻璃电极：硼硅玻璃毛细管</td><td>世界精密仪器</td><td> 1B100F-4 </td><td> 1.0毫米外径，0.58毫米编号</td></tr><tr><td>刺激</td><td>草仪器</td><td>型号S48 </td><td></td></tr><tr><td>放大器</td><td>获得仪器公司</td><td>型号5A </td><td></td></tr><tr><td>数据采集​​软件：Digidata </td><td>分子器件</td><td>型号1440A </td><td></td></tr><tr><td>数据采集​​软件：pCLAMP </td><td>分子器件</td><td>第10版</td><td></td></tr><tr><td>体视显微镜瓦特第i个光纤显微镜环形灯</td><td> AmScope </td><td> SM-4T 型号HL250-AR </td><td></td></tr><tr><td>解剖的安装范围</td><td> AmScope </td><td> SM-2TZ </td><td></td></tr><tr><td>风筝手动微操作手和倾斜基地</td><td>世界精密仪器</td><td>型号：M3301 风筝：＃风筝M3-L的型号</td><td></td></tr><tr><td> 果蝇野生的10E基因型（野生型菌株） </td><td>布卢明顿联合中心</td><td>股票＃3892 </td><td></td></tr><tr><td>立式吸管拔</td><td>大卫Kopf仪器</td><td>型号700C </td><td></td></tr><tr><td>注射的玻璃micropipettes：硼硅玻璃毛细管</td><td>世界精密仪器</td><td>目录＃4878 </tD&gt; <td> 1.14毫米外径，内径0.5mm </td></tr><tr><td>硅油</td><td>费舍尔</td><td>目录＃S159-500 </td><td></td></tr><tr><td>倒角</td><td>萨特仪器有限公司</td><td>克拉的棕色类型 型号＃BV-10 </td><td></td></tr><tr><td> nanoliter2000 </td><td>世界精密仪器</td><td>目录＃B203XVY </td><td></td></tr><tr><td>蓝色食用色素</td><td>麦考密克</td><td> N / A </td><td>配料：水，丙烯乙二醇，FD＆C蓝色1和0.1％丙酯（防腐剂）。 </td></tr><tr><td> methyllycaconitine柠​​檬酸（工作重点） </td><td> tocris生命科学</td><td>目录＃1029 </td><td></td></tr><tr><td>塑料蜡棒</td><td> ？生公司 （阿克伦美国俄亥俄州） </td><td></td><td></td></tr></tboDY&gt; </table>

paired nanoinjection and electrophysiology assay to screen for bioactivity of compounds using the drosophila melanogaster giant fiber system

体内活性筛选化合物可以用来作为第一步，以确定可能到1,2药物制剂开发的候选人。我们开发了一种新型的nanoinjection /电检测，使检测的神经电路介导的在果蝇 3,4逃生响应功能的生物活性化合物的调节作用。我们在体内实验中，使用果蝇巨纤维系统（GFS， 图1）允许不同类型的化合物，如小分子肽的筛选，只需要最小数量引起的效果。此外，果蝇的飞行服务队提供了一个潜在的分子靶点神经或肌肉的种类繁多。 （GFS）巨纤维突触电（峡路口）以及化学（胆碱）到外围的中间神经突触（PSI）和“Tergo Trochanteral肌肉神经元（TTMn）5 </SUP&gt;。背侧纵肌神经元DLMn（）连接的PSI是依赖Dα7烟碱乙酰胆碱受体（nAChRs）的6。最后，神经肌肉接头（NMJ）的TTMn和DLMn跳（TTM）和飞行肌（DLM），是谷氨酸7-12。在这里，我们演示了如何注入化合物的纳升数量级，而获得巨人光纤系统，以及如何监察该电路的功能复合的影响的电生理细胞内记录。我们显示枸橼酸methyllycaconitine（MLA）的，胆碱受体拮抗剂，破坏PSI的DLMn连接，但不能连接或跳跃或飞行肌肉的功能NMJ的GF TTMn的的的特异性。 在这部影片开始之前，它是至关重要的，你仔细地观察和熟悉的朱庇特的视频名为“电生理记录，从四melan巨纤维通路“ogaster从奥古斯丁等 7，视频这里的目的是为这一现有技术的扩展。在这里，我们使用的细节，只对另外的的配对nanoinjections和监测技术的电生理记录方法和重点。

Watch this Scientific Journal Video about Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System at JoVE.com

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

迅速<em&gt;在体内</em&gt;分析测试neuromodulatory使用巨人光纤系统（GFS）的化合物<em&gt;果蝇</em&gt;描述。 nanoinjections动物的头，随着政府飞行服务队的电生理记录可以揭示生物活​​性化合物对神经或肌肉。

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 ...

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JoVE Journal

Neuroscience

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

13.0K 次观看.  Florida Atlantic University. 迅速在体内分析测试neuromodulatory使用巨人光纤系统（GFS）的化合物果蝇描述。 nanoinjections动物的头，随着政府飞行服务队的电生理记录可以揭示生物活​​性化合物对神经或肌肉。

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

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

从巨纤维通路的电生理记录 D。果蝇</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

科研

神经科学

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

成人整装准备果蝇巨纤维染料注射腹侧神经索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

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 ...

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 ...

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

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

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

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

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

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

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

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

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

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

一个行为分析对机械感觉MARCM为基础的克隆<em&gt;果蝇</em

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

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

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

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

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

高通量检测，以检查个体产蛋首<em&gt;果蝇</em

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

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 ...

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 ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...