Name: electrophysiological recording from drosophila trichoid sensilla in response to odorants of low volatility
Uploaded: 2017-07-27T15:00:00
Description: Watch this Scientific Journal Video about Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility at JoVE.com

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N...

<ol>
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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>

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, ...

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JoVE Journal

Neuroscience

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

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

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

Forebrain Electrophysiological Recording in Larval Zebrafish

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

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

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

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, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

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

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

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

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

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

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

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

全细胞电压钳记录的电生理方法<em&gt;果蝇</em&gt;光感受器

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...