Summary

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

Published: July 27, 2017
doi:

Summary

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。

Abstract

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。

Introduction

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, -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“>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( 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 – > 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。

Protocol

1.准备用于at4录制的硬件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。 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…

Representative Results

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?…

Discussion

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?…

Declarações

The authors have nothing to disclose.

Acknowledgements

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

Materials

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

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Ng, R., Lin, H., Wang, J. W., Su, C. Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility. J. Vis. Exp. (125), e56147, doi:10.3791/56147 (2017).

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