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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Prep Setup &#38; Miscellaneous Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Puller Instrument&#160;</td>
			<td>Sutter Instruments 
			Novato CA USA</td>
			<td>P97</td>
			<td>Pipette Puller</td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments 
			Sarasota FL USA</td>
			<td>1B100F-4</td>
			<td>to make holding rods</td>
		</tr>
		<tr>
			<td>Aluminosilicate Glass Capillaries&#160;</td>
			<td>Sutter Instruments 
			Novato CA USA</td>
			<td>AF100-64-10</td>
			<td>to make electrodes</td>
		</tr>
		<tr>
			<td>Superfrost Microscope Slides</td>
			<td>Fisher Scientific 
			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 
			St. Paul MN USA</td>
			<td>NA</td>
			<td>for fly-prep station</td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus 
			Shinjuku Tokyo Japan</td>
			<td>BX51</td>
			<td>for recording rig</td>
		</tr>
		<tr>
			<td>Plastalina modeling clay</td>
			<td>Van Aken 
			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 
			Rochester NY USA</td>
			<td>#156-4045</td>
			<td>to sterilize AHL solution</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;&#160;&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cartridge Materials</td>
			<td>&#160;&#160;&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L pipette tip&#160;</td>
			<td>VWR 
			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 
			Maidstone Kent UK</td>
			<td>740-E</td>
			<td>to make odor cartridges&#160;</td>
		</tr>
		<tr>
			<td>Vacuum Desiccator&#160;</td>
			<td>Cole-Parmer 
			Vernon Hills IL USA</td>
			<td>VX-06514-30</td>
			<td>to vaporize ethanol solvent</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;&#160;&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Odorant Materials</td>
			<td>&#160;&#160;&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cis-palmitoleic acid</td>
			<td>Cayman Chemical 
			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 
			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.&#160; 
			New Brunswick NJ USA</td>
			<td>E1028-500MLGL</td>
			<td>to dilute palmitoleic acid&#160;</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;&#160;&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Rig Setup Materials</td>
			<td>&#160;&#160;&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Odorant Cartridge Micromanipulator</td>
			<td>Siskiyou 
			Grants Pass OR USA</td>
			<td>MX130R</td>
			<td>to position the olfactometer</td>
		</tr>
		<tr>
			<td>Flow Vision software&#160;</td>
			<td>Alicat 
			Tuscon AZ USA</td>
			<td>FLOWVISIONSC</td>
			<td>software to control flow rate</td>
		</tr>
		<tr>
			<td>Mass Controller</td>
			<td>Alicat 
			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 
			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 
			Sunnyvale CA USA</td>
			<td>Ver. 10.4</td>
			<td>Data acquisition software</td>
		</tr>
		<tr>
			<td>Air delivery tube</td>
			<td>Ace Glass 
			Vineland NJ USA</td>
			<td>8802-936&#160;</td>
			<td>to deliver humidified air</td>
		</tr>
		<tr>
			<td>50x objective lens&#160;</td>
			<td>Olympus 
			Shinjuku Tokyo Japan</td>
			<td>LMPLFL50X</td>
			<td>recording rig</td>
		</tr>
		<tr>
			<td>Clampfit 10</td>
			<td>Molecular Devices 
			Sunnyvale CA USA</td>
			<td>Ver. 10.4</td>
			<td>software for spike analysis&#160;</td>
		</tr>
		<tr>
			<td>Igor Pro 6</td>
			<td>WaveMetrics 
			Lake Oswego OR USA</td>
			<td>Ver. 6.37</td>
			<td>software for data analysis&#160;</td>
		</tr>
		<tr>
			<td>Audio Monitor</td>
			<td>ALA Scientific Instruments 
			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 
			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&#160;&#160;&#160;</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 
			Novato CA USA</td>
			<td>MP-285</td>
			<td>to position recording electrode</td>
		</tr>
		<tr>
			<td>Headstage Amplifier</td>
			<td>ALA Scientific Instruments 
			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 
			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

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

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

含量自发活动，研究昼夜节律和睡眠参数果蝇</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

科研

神经科学

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

斑马鱼幼虫的前脑的电生理记录

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Vertical T-maze Choice Assay for Arthropod Response to Odorants

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, various technologies are being developed as control tools. A subset of these tools focuses on modifying the behavior of arthropods by attraction or repulsion. Therefore, arthropods are often the focus of behavioral investigations. Various tools have been developed to measure arthropod behavior, including wind tunnels, flight mills, servospheres, and various types of olfactometers. The purpose of these tools is to measure insect or arachnid response to visual or more often olfactory cues. The vertical T-maze oflactometer described here measures choices performed by insects in response to attractants or repellents. It is a high throughput assay device that takes advantage of the positive phototaxis (attraction to light) and negative geotaxis (tendency to walk or fly upward) exhibited by many arthropods. The olfactometer consists of a 30 cm glass tube that is divided in half with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the test subjects to make a choice between two potential odor fields in assays involving attractants. In assays involving repellents, lack of normal response to known attractants can also be measured as a third variable.

A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device.

的垂直T-迷宫的选择含量，节肢动物响应加味剂

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

同时前和突触后电生理记录<em&gt;爪蟾</em神经肌肉文化

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

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 ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同时电生理记录和抑制剂在啮齿动物大脑微注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

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 ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.