感谢叶Zhang帮助示范痕迹，以及田基曾为图片提供帮助。这项工作得到了Ray Thomas Edwards Foundation早期职业奖和NIH授予（R01DC015519）的支持，其中包括C.-YS和NIH授权（R01DC009597和R01DK092640）给JWW

1.准备用于at4录制的硬件<ol><li>使用移液器拉拔仪器制备电极与铝硅酸盐玻璃毛细管（OD 1.0 mm，ID 0.64 mm）。稍微用一对细镊子钝化参比电极的尖端，以便于插入苍蝇的ly fly（ 即在头部前方的圆形板，在口部上方）。 注意:本研究中使用了7日龄的男性（柏林）。使用AHL盐溶液14作为两电极的电解质。 </li><li>通过混合900毫升蒸馏水与6.312克的NaCl，0.373克的KCl，0.337克的NaHCO 3制备1升AHL的，0.1120克的NaH 2 PO 4，1.892克海藻糖ּ2 H 2的O的，3.423克蔗糖， 1.192g HEPES和8.2mL 1M MgCl 2 。使用蒸馏水，总体积可达1 L。用1N NaOH将pH值调至7.4，并将溶液灭菌真空驱动过滤系统。对于长期储存，将AHL等分试样保持在4°C。 注意:一致地输送棕榈油酸取决于墨盒之间的均匀性。关键是每个墨盒以可重复的方式组装。 </li><li>使用剃刀刀片，从200μL移液器吸头尖端移除0.9厘米，创建第一个药筒部分，测量长度为4.1厘米;请参考图1A中详细描述的尺寸。使用另外200μL移液器吸头，分别从尖端和底部移除1.7厘米和1.5厘米，以产生测量1.8厘米的第二个墨盒部分（ 图1A ）。使用尺子来确保重复性。 </li><li>使用⅛"打孔机从滤纸切片。 </li><li>使用镊子将滤纸纸放在第二个墨盒部分的末端。目视确认空气可以通过的墨盒尖端有一个开口。 </li><li>将第一和第二盒部分连接在一起， 如图1A所示。将第二个墨盒部分向下倾斜，以方便对准准备的图形 （ 图1B ）。 </li><li>将墨盒连接到安装在显微操纵器上的气味输送管。 注意:此设计允许墨盒向外旋转以方便更换（ 图1C ）。 </li><li>在一个质量控制器中将恒定的加湿气流设置为2升/分钟，并在另一个质量控制器中将气味流量设置为500 mL / min。 </li><li>使用软件（参见材料表 ），编写施用500毫秒气味扑鼻的程序。 </li></ol> 2.制备用于交付的棕榈油酸性气味溶液注意:Or47b ORNs对顺式和反式 - 过饱和酸均有反应。由于棕榈油酸在R不稳定T，库存储存于-20°C，开箱后一个月内使用。乙醇是棕榈油酸的首选溶剂。 <ol><li>使用涡旋混合器将10μL 顺式或反式棕榈油酸储备液或稀释液与90μL100％乙醇混合，用于在1.7 mL微量管中进行十倍连续稀释。在一天内实验和使用之前，每天准备新鲜的棕榈油酸稀释液。 注意:对于不溶于乙醇的气味剂，推荐使用玻璃小瓶，用其他类型的有机溶剂制备气味稀释液。 </li><li>使用P10微量移液管，将5μL所需稀释度的顺式 - 葡萄糖酸溶液加到每个相应的药筒中的滤纸上。 注意:最高剂量（ 10-1 ）含有450μg化合物。对于反 - 葡萄糖酸溶液，应用4.5μL，以使最高剂量（10 -1 ）也含有450μgt他复合</li><li>为了完全蒸发溶剂，将棕榈油酸酯盒在真空干燥器中放置1小时，压力为7.59 mmHg。 注意:墨盒最多可在室内使用4小时。 </li></ol> 3.准备果蝇以准备进入at4感觉体内电生理记录注意:野生型（柏林）在标准的玉米面培养基中，在25°C的12:12光暗环境中饲养。苍蝇死亡后，苍蝇被性别划分为十组，由此分组饲养至七岁。雄性和雌性苍蝇中的Or47b ORN都响应于棕榈油酸。为了简单起见，目前的研究只检查了雄蝇。 <ol><li>组装飞行准备幻灯片:在玻璃滑块上，将玻璃盖玻片（18 x 18 mm 2 ）放在少量造型粘土上，与玻璃滑块形成〜3°的角度。在内部放置双面胶带e盖玻片的盖子和下面的幻灯片区域。用新鲜的磁带替换每一天的录音（ 图2A ）。 </li><li>使用吸气器15收集管道中的感兴趣的飞行物，然后在管道末端装入200μL移液器尖端。同时将管子向前吹，同时将空气吹入管中，将飞蝇推到移液器吸头末端。使用剃须刀切割正下方的飞行物体和2个头部长度在飞行之上。 </li><li>用造型粘土将吸头的底部捣实，向上推动飞行，直到天线和角质层都暴露（ 图2B ）。为了避免杀死飞行物，只添加足够的粘土来暴露天线和鸟类，因为这样可以防止飞行的腹部被压碎。此外，缓慢而轻轻地添加粘土以防止任何突然的收缩。通过检查天线确认飞行是否活着或长鼻运动。 </li><li>使用镊子来操纵容纳飞行的移液器吸头。使头部朝向，以使鹅掌戟面向观察者的右侧。使用细镊子沿着盖玻片调整准备，直到天线的侧面靠在胶带盖玻片表面上（ 图2B ）。 </li><li>在Arista上放置一个固定杆，将天线固定在双面胶带上以防止移动（ 图2B ）。 注意:用吸管拔出器从硼硅玻璃毛细管中拉出固定杆，并用模型粘土保持就位（ 图2A ）。 </li><li>将准备物放在钻机的平台上（ 图2C ）。使用显微镜，确认毛发沿着天线第三段的远侧边缘可见。 注意:理想情况下，感觉器应该在背景下清晰地显示出来，从而简化了它们<html>识别和便于记录（ 图3 ）。在这个准备中，大多数可触及的毛状感觉器都是at4型。 </li><li>保持准备下通过单独的空气输送管从来自该预备大约2cm（ 图4）的距离递送恒定加湿气流（2升/分钟），如先前2,15说明。 </li></ol> 4.响应于棕榈油酸的at4 Trichoids中Or47b ORNs的at4敏感活性的记录<ol><li>将参考电极插入鹅颈us（ 图3A ）。为了避免组织损伤，请确保电极正好插入表面下方，并可快速平滑地接触角质层下的血淋巴。 </li><li>使记录电极慢慢降低，进入与目标感光体相同的视野（="xfig"&gt;图3B）。记录在50X物镜下。 注意:坚韧的毛状角质层需要将记录电极插入感觉基底，其较宽的面积提供较大的靶，从而降低电极偏转的可能性（ 图3B ，插图）。 </li><li> 应用气味刺激感觉之前，请遵守以下选择标准;任何不符合这些标准的毛发都应该被拒绝，并选择另一种敏感剂。 <ol><li>观察到较高的信噪比（参见图3C ）。 </li><li>观察来自at4A和at4C神经元的可识别尖峰（ 图3C ）。 注意:值得注意的是，at4B尖峰振幅显示为非常类似于4A 10 ，无法轻松识别，无气味刺激。 </li><li>观察到at4A神经元的基础放电率为大约在或低于20Hz。 注意:该标准对于at4A是特异性的，因为神经元的基础放电速率高于基底神经元ORNs 2 。更高的基础射击表明神经元可能在电极插入期间被损坏。 </li></ol></li><li>将墨盒连接到加臭剂输送管。从溶剂控制开始，然后从低浓度到高浓度的气味剂。使用显微操纵器将墨盒朝向准备状态，同时将墨盒正好对准准备工作的头部。目视确认墨盒从几毫米处直接指向天线（ 图4 ）。 注意:目标是将墨盒的开口直接定位在天线处，并将其定位在靠近目标组织的位置。 </li><li>确保气味盒与记录电极在右侧分开1 - 2 mm，并从飞行前p在下面滑动约1毫米。 注意:在本文所述的设置中，气味盒与记录电极，参比电极和飞行准备滑块紧密接触（ 图4 ）。 注意:注意墨盒和记录/参比电极之间的距离。推荐距离约4毫米1 。无意的接触可以终止信号并破坏记录电极的尖端，损害当前的神经元并使进一步的记录变得复杂化。 注意:考虑分离墨盒和飞行准备幻灯片的距离。触摸盖玻片也可能会移动记录电极以破坏记录。 </li><li>按数据采集软件中的"记录"开始记录。 注意:对于每10秒的记录，如步骤1.9所述，将一个500 ms的气味脉冲直接传送到天线。 </li><li>加臭剂后，在更换下一个最高浓度的墨盒之前，请小心地取出墨盒。继续直到获得整个剂量范围。 注意:建议每次飞行中只记录一个Or47b ORN以避免任何可能的适应影响。 </li><li>完成录音后，用蒸馏水彻底冲洗记录电极。 </li><li>使用市售的离线分析软件分析和绘制数据。 </li></ol>

<ol>
	<li>Lin, H. -. H., 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=Hormonal+modulation+of+pheromone+detection+enhances+male+courtship+success.">Hormonal modulation of pheromone detection enhances male courtship success.</a> Neuron. 90 (6), 1272-1285 (2016).</li><li>Hallem, E. A., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coding+of+odors+by+a+receptor+repertoire.">Coding of odors by a receptor repertoire.</a> Cell. 125 (1), 143-160 (2006).</li><li>Silbering, A. F., 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=Complementary+function+and+integrated+wiring+of+the+evolutionarily+distinct+Drosophila+olfactory+subsystems.">Complementary function and integrated wiring of the evolutionarily distinct Drosophila olfactory subsystems.</a> J Neurosci. 31 (38), 13357-13375 (2011).</li><li>Min, S., Ai, M., Shin, S. A., Suh, G. S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedicated+olfactory+neurons+mediating+attraction+behavior+to+ammonia+and+amines+in+Drosophila.">Dedicated olfactory neurons mediating attraction behavior to ammonia and amines in Drosophila.</a> Proc Nat Acad Sci USA. 110, 1321-1329 (2013).</li><li>Kurtovic, A., Widmer, A., Dickson, B. J. <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+class+of+olfactory+neurons+mediates+behavioural+responses+to+a+Drosophila+sex+pheromone.">A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone.</a> Nature. 446 (7135), 542-546 (2007).</li><li>Van der Goes van Naters, W., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptors+and+neurons+for+fly+odors+in+Drosophila.">Receptors and neurons for fly odors in Drosophila.</a> Curr Biol. 17, 606-612 (2007).</li><li>Schlief, M. L., Wilson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+processing+and+behavior+downstream+from+highly+selective+receptor+neurons.">Olfactory processing and behavior downstream from highly selective receptor neurons.</a> Nat Neurosci. 10 (5), 623-630 (2007).</li><li>Laughlin, J. D., Ha, T. S., Jones, D. N. M., Smith, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Pheromone-sensitive+neurons+is+mediated+by+conformational+activation+of+pheromone-binding+protein.">Activation of Pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein.</a> Cell. 133 (7), 1255-1265 (2008).</li><li>Gomez-Diaz, C., Reina, J. H., Cambillau, C., Benton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligands+for+pheromone-sensing+neurons+are+not+conformationally+activated+odorant+binding+proteins.">Ligands for pheromone-sensing neurons are not conformationally activated odorant binding proteins.</a> PLoS Biol. 11 (4), e1001546 (2013).</li><li>Dweck, H. K. M., 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=Pheromones+mediating+copulation+and+attraction+in+Drosophila.">Pheromones mediating copulation and attraction in Drosophila.</a> Proc Nat Acad USA. 112, 2829-2835 (2015).</li><li>Cappa, C. D., Lovejoy, E. R., Ravishankara, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaporation+rates+and+vapor+pressures+of+the+even-numbered+C8-C18monocarboxylic+acids.">Evaporation rates and vapor pressures of the even-numbered C8-C18monocarboxylic acids.</a> J Phys Chem A. 112 (17), 3959-3964 (2008).</li><li>Kimura, K. -. I., Sato, C., Yamamoto, K., Yamamoto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+the+back+or+front:+the+courtship+position+is+a+matter+of+smell+and+sight+in+Drosophila+melanogaster+males.">From the back or front: the courtship position is a matter of smell and sight in Drosophila melanogaster males.</a> J Neurogenet. 29 (1), 18-22 (2015).</li><li>Grosjean, Y., 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=An+olfactory+receptor+for+food-derived+odours+promotes+male+courtship+in+Drosophila.">An olfactory receptor for food-derived odours promotes male courtship in Drosophila.</a> Nature. 478 (7368), 236-240 (2011).</li><li>Wang, J. W., Wong, A. M., Flores, J., Vosshall, L. B., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+calcium+imaging+reveals+an+odor-evoked+map+of+activity+in+the+fly+brain.">Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain.</a> Cell. 112 (2), 271-282 (2003).</li><li>Pellegrino, M., Nakagawa, T., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+sensillum+recordings+in+the+insects+Drosophila+melanogaster+and+Anopheles+gambiae.">Single sensillum recordings in the insects Drosophila melanogaster and Anopheles gambiae.</a> J Vis Exp. (36), e1-e5 (2010).</li></ol>

作者没有什么可以披露的。

在这里，我们描述了一种可以强烈诱导和记录Or47b ORN对棕榈油酸的反应的过程。我们修改了常规长途气味递送方法2，7，10解决信息素不足的加臭剂递送的问题。我们解决了低气味挥发性的问题，通过将化合物通过加味剂药筒递送，其开口位于制剂的毫米内。当考虑到每个气味盒的一致构造和放置时，该方案表现为以可再现的?...

<html> 果蝇 ORN响应广泛的气味，具有广泛的碳链长度和各种官能团，包括酯，醇，酮，内酯，醛，萜烯，有机酸，胺，硫化合物，杂环和芳族化合物2 ， 3 。气味物理化学特征不同，挥发性可能明显不同，化合物的蒸气压表示。值得注意的是， 黑腹果蝇的生物相关气味在其挥发性上有很大差异。例如，Ir92a ORN对20℃的蒸气压为6,432mmHg的氨4具有高挥发性。与此相反，Or67d ORNs到雄性信息素， 顺 -vaccenyl乙酸（C VA）5,6响应，则蒸气压的是在20℃43个毫米汞柱。 ove_content"&gt;研究对低挥发性气味剂的嗅觉反应对于常规的气味递送方法是特别有挑战性的，其中气味剂通过载体空气流在相对较长的距离（ 即几厘米）处输送），因此报道的嗅觉反应对于给定的低挥发性气味，可以根据气味递送系统的设计变化很大，例如，Or67d ORN对高剂量c VA的报告响应范围为〜40 7 - &gt; 200尖峰/秒6此外， c VA与常规递送方法的无效递送可能归因于假阴性结果，导致解释为c VA本身不足以激活Or67d ORN 8。这种解释后来受到另一项研究使用近距离的气味递送方法9因此是不正当的开发强有力的气味递送系统，以有效提供低挥发性的气味剂。 最近，我们确定了几种长链角质脂肪酸作为Or47b ORN的配体。它们位于4型Antennal Trichoid Sensillum（at4）。中长链脂肪酸增味剂，我们发现，棕榈油酸充当通过激活Or47b ORNs 1促进男性求偶春药信息素。然而，在使用常规气味递送方法的另一研究中，显示月桂酸甲酯引起Or47b ORNs的反应，而当从相同距离10出现时，棕榈油酸诱发无反应。与C VA，长链脂肪酸更不易挥发，具有蒸汽压低于0.001毫米汞柱，在25℃11。长链脂肪酸气味剂固有的低挥发性，这妨碍了天线通过的有效表现常规的气味递送系统，可能占假阴性结果10 。这种不一致性强调了传统气味递送系统在呈现低挥发性气味剂时的不足。之前已经表明，有效的蝇角状气味的传递需要气味源和目标组织6之间的紧密接近。因此，虽然从模仿它们通过果蝇在自然界中12，13可能遇到的距离，我们一致认为最小距离必须在我们的程序给予高度重视充分体现生物活性信息素的影响。 我们的方法具有进一步的优势，包括与标准电生理学钻机和技术的兼容性。预先存在的钻机设置需要最少的修改以适应此协议，大多数SSR步骤仅需要微小的调整。这个使我们的技术随着SSR经验丰富的研究人员可以轻松访问。此外，我们的技术允许呈现具有尖锐起始和抵消的低挥发性气味剂，将刺激递送与神经元反应相关联。最后，硬件布局便于加味剂盒之间的快速交换，从而加速在所需剂量范围内的数据收集。 我们首先审查参考和记录电极，成人血淋巴样（AHL）溶液，气味输送药筒和相应的嗅觉仪的准备。然后我们讨论制备棕榈油酸气溶液，然后制备用于记录的飞行物。在提供使用此方法获取的代表性数据之前，我们继续考虑选择毛状感器记录的标准，并更仔细地检查气味盒的位置。最后，我们通过探索该技术的有用应用得出结论ue，一些遇到的问题和他们的解决方案。

<table><tbody><tr><td>&lt;strong&gt;Prep Setup &amp;amp; Miscellaneous Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pipette Puller Instrument&amp;nbsp;</td><td>Sutter Instruments&lt;br/&gt; Novato CA USA</td><td>P97</td><td>Pipette Puller</td></tr><tr><td>Borosilicate Glass Capillaries</td><td>World Precision Instruments&lt;br/&gt; Sarasota FL USA</td><td>1B100F-4</td><td>to make holding rods</td></tr><tr><td>Aluminosilicate Glass Capillaries&amp;nbsp;</td><td>Sutter Instruments&lt;br/&gt; Novato CA USA</td><td>AF100-64-10</td><td>to make electrodes</td></tr><tr><td>Superfrost Microscope Slides</td><td>Fisher Scientific&lt;br/&gt; Pittsburgh PA USA</td><td>12-550-143</td><td>for fly-prep station</td></tr><tr><td>Permanent Double Sided Tape</td><td>Scotch&lt;br/&gt; St. Paul MN USA</td><td>NA</td><td>for fly-prep station</td></tr><tr><td>Upright microscope</td><td>Olympus&lt;br/&gt; Shinjuku Tokyo Japan</td><td>BX51</td><td>for recording rig</td></tr><tr><td>Plastalina modeling clay</td><td>Van Aken&lt;br/&gt; North Charleston SC USA</td><td>B0019QZMQQ</td><td>for prep station and to stablize the holding rod</td></tr><tr><td>Rapid-Flow Sterile Disposable Filter Unit with SFCA Membrane, 0.45 mm</td><td>Nalgene&lt;br/&gt; Rochester NY USA</td><td>#156-4045</td><td>to sterilize AHL solution</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Cartridge Materials&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td></tr><tr><td>200 &amp;micro;L pipette tip&amp;nbsp;</td><td>VWR&lt;br/&gt; Radnor PA USA</td><td>53508-810</td><td>to make odor cartridges and fly prep</td></tr><tr><td>Filter Paper</td><td>Whatman&lt;br/&gt; Maidstone Kent UK</td><td>740-E</td><td>to make odor cartridges&amp;nbsp;</td></tr><tr><td>Vacuum Desiccator&amp;nbsp;</td><td>Cole-Parmer&lt;br/&gt; Vernon Hills IL USA</td><td>VX-06514-30</td><td>to vaporize ethanol solvent</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Odorant Materials&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td></tr><tr><td>cis-palmitoleic acid</td><td>Cayman Chemical&lt;br/&gt; Ann Arbor MI USA</td><td>#10009871 (CAS # 373-49-9)</td><td>Or47b odorant</td></tr><tr><td>trans-palmitoleic acid</td><td>Cayman Chemical&lt;br/&gt; Ann Arbor MI USA</td><td>#9001798 (CAS # 10030-73-6)</td><td>Or47b odorant</td></tr><tr><td>Ethanol</td><td>Spectrum Chemical MFG.&amp;nbsp;&lt;br/&gt; New Brunswick NJ USA</td><td>E1028-500MLGL</td><td>to dilute palmitoleic acid&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Rig Setup Materials&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td></tr><tr><td>Odorant Cartridge Micromanipulator</td><td>Siskiyou&lt;br/&gt; Grants Pass OR USA</td><td>MX130R</td><td>to position the olfactometer</td></tr><tr><td>Flow Vision software&amp;nbsp;</td><td>Alicat&lt;br/&gt; Tuscon AZ USA</td><td>FLOWVISIONSC</td><td>software to control flow rate</td></tr><tr><td>Mass Controller</td><td>Alicat&lt;br/&gt; Tuscon AZ USA</td><td>MC-2SLPM-D</td><td>to control the flow rate for humidified air</td></tr><tr><td>Mass Controller</td><td>Alicat&lt;br/&gt; Tuscon AZ USA</td><td>MC-500SCCM-D</td><td>to control the flow rate for odor stimulation</td></tr><tr><td>Clampex</td><td>Molecular Devices&lt;br/&gt; Sunnyvale CA USA</td><td>Ver. 10.4</td><td>Data acquisition software</td></tr><tr><td>Air delivery tube</td><td>Ace Glass&lt;br/&gt; Vineland NJ USA</td><td>8802-936&amp;nbsp;</td><td>to deliver humidified air</td></tr><tr><td>50X objective lens&amp;nbsp;</td><td>Olympus&lt;br/&gt; Shinjuku Tokyo Japan</td><td>LMPLFL50X</td><td>recording rig</td></tr><tr><td>Clampfit 10</td><td>Molecular Devices&lt;br/&gt; Sunnyvale CA USA</td><td>Ver. 10.4</td><td>software for spike analysis&amp;nbsp;</td></tr><tr><td>Igor Pro 6</td><td>WaveMetrics&lt;br/&gt; Lake Oswego OR USA</td><td>Ver. 6.37</td><td>software for data analysis&amp;nbsp;</td></tr><tr><td>Audio Monitor</td><td>ALA Scientific Instruments&lt;br/&gt; Farmingdale NY USA</td><td>NPIEXB-AUDIS-08B</td><td>Aurally reports individual spikes</td></tr><tr><td>Extracellular Amplifier</td><td>ALA Scientific Instruments&lt;br/&gt; Farmingdale NY USA</td><td>NPIEXT-02F</td><td>to increase the amplitude of electrical signals</td></tr><tr><td>Valve Controller</td><td>Warner Instruments&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>VC-8</td><td>to control the opening of the valve for odor stimulation</td></tr><tr><td>Recording Electrode Micromanipulator</td><td>Sutter Instruments&lt;br/&gt; Novato CA USA</td><td>MP-285</td><td>to position recording electrode</td></tr><tr><td>Headstage Amplifier</td><td>ALA Scientific Instruments&lt;br/&gt; Farmingdale NY USA</td><td>EQ-16.0008</td><td>to increase the amplitude of electrical signals</td></tr><tr><td>Oscilloscope</td><td>Tektronix&lt;br/&gt; Beaverton OR USA</td><td>TDS2000C</td><td>Visual report of individual spikes</td></tr></tbody></table>

electrophysiological recording from drosophila trichoid sensilla in response to odorants of low volatility

昆虫依靠他们的嗅觉来引导对其生存至关重要的各种行为，例如寻找食物，捕食者避免，产卵和交配。不同挥发性的无数化学物质已被鉴定为激活昆虫嗅觉受体神经元（ORNs）的天然气味剂。然而，研究对低挥发性气味剂的嗅觉反应已经受到使用常规气味递送方法无法有效地呈现这种刺激的阻碍。在这里，我们描述了允许有效呈现低挥发性气味剂用于体内单敏感记录（SSR）的程序。通过最小化气味源和目标组织之间的距离，该方法允许应用生物显着但至今无法接近的气味剂，包括棕榈油酸，刺激性信息素，对参与求偶和交配行为的ORN有显着影响1 。因此，我们的方法为研究昆虫嗅觉和信息素通信提供了一种新的途径来测定大量低挥发性气味剂。

我们的技术成功应用于确定棕榈油酸的反式 （ 图5A ）与顺式 （ 图5B ）异构体的相对功效。我们的代表性数据表明，与顺式同种型相比， 反式 β-葡萄糖酸是Or47b ORNs的更有效配体（ 图5C ）。从每只苍蝇记录单个神经元，每个剂量曲线记录12只苍蝇，共24只苍蝇。集体数据从实验?...

Watch this Scientific Journal Video about Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility at JoVE.com

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

该协议的总体目标是演示如何呈现响应长链表皮信息素的果蝇嗅觉受体神经元的单敏感记录的低挥发性气味。

电生理记录<em&gt;果蝇</em&gt; Trichoid Sensilla响应低挥发性气味

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

electrophysiological-recording-from-drosophila-trichoid-sensilla

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

8.8K 次观看.  University of California San Diego. 该协议的总体目标是演示如何呈现响应长链表皮信息素的果蝇嗅觉受体神经元的单敏感记录的低挥发性气味。 

视频: Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

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

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

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

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

高分辨率测量的气味驱动在果蝇幼虫的行为

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

生物学

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

在气味诱发反应的钙成像果蝇触角叶

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

电生理记录从<em&gt;果蝇</em&gt; Labellar味感器

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

测量的生理反应<em&gt;果蝇</em&gt;感觉神经元脂质费洛蒙使用实时钙成像

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

免疫与感染

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 ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

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

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

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

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

通过计算机跟踪检测大鼠嗅觉行为<em&gt;果蝇</em&gt;在四象限嗅

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

Single Sensillum Recordings for Locust Palp Sensilla Basiconica

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially for the detection of non-volatile chemical cues through sensilla chaetica (previously named terminal sensilla or crested sensilla). There is now increasing evidence that these palps also have an olfactory function. An odorant receptor (LmigOR2) and an odorant-binding protein (LmigOBP1) have been localized in the neurons and accessory cells, respectively, in the sensilla basiconica of the palps. Single sensillum recording (SSR) is used for recording the responses of odorant receptor neurons, which is an effective method for screening active ligands on specific odorant receptors. SSR is used in functional studies of odorant receptors in palp sensilla. The structure of the sensilla basiconica located on the dome of the palps differs somewhat from the structure of those on the antennae. Therefore, when performing an SSR elicited by odorants, some specific advice may be helpful for obtaining optimum results. In this paper, a detailed and highly effective protocol for an SSR from insect palp sensilla basiconica is introduced.

This paper describes a detailed and highly effective protocol for single sensillum recordings from the sensilla basiconica on the palps of insect mouthparts.

蝗虫下颚触角 Basiconica 单触角记录

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially ...

一种增强昆虫味觉神经元活性分析的新方法

Base Recording: A Technique for Analyzing Responses of Taste Neurons in Drosophila

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential food source, compounds from the food source enter through the pore and activate neurons within. For over 50 years, these responses have been recorded using a technique called tip recording. However, this method has major limitations, including the inability to measure neural activity before or after stimulus contact and the requirement for tastants to be soluble in aqueous solutions. We describe here a technique that we call base recording, which overcomes these limitations. Base recording allows the measurement of taste neuron activity before, during, and after the stimulus. Thus, it allows extensive analysis of OFF responses that occur after a taste stimulus. It can be used to study hydrophobic compounds such as long-chain pheromones that have very low solubility in water. In summary, base recording offers the advantages of single-sensillum electrophysiology as a means of measuring neuronal activity - high spatial and temporal resolution, without the need for genetic tools - and overcomes key limitations of the traditional tip recording technique.

作者聚焦：化学接收的进步 – 从昆虫气味受体到非编码 RNA

A rarely used method of electrophysiological recording, base recording, allows analysis of features of taste coding that cannot be examined by conventional recording methods. Base recording also allows the analysis of taste responses to hydrophobic stimuli that cannot be studied using traditional electrophysiological methods.

基础记录：一种分析果蝇味觉神经元反应的技术

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential ...

电生理记录

摘要

探索更多视频

此视频中的章节

在昆虫单感器记录果蝇按蚊

高分辨率测量的气味驱动在果蝇幼虫的行为

在气味诱发反应的钙成像果蝇触角叶

电生理记录从

测量的生理反应

在气味引导行为的高分辨率定量

为记录细胞内电压响应电法

通过计算机跟踪检测大鼠嗅觉行为

蝗虫下颚触角 Basiconica 单触角记录

基础记录：一种分析果蝇味觉神经元反应的技术