Summary

在昆虫单感器记录果蝇按蚊</em

Published: February 17, 2010
doi:

Summary

在使用单一感器录音昆虫气味的嗅觉感官神经元的电生理反应,可测。在这个视频文章,我们将演示如何执行单一飞醋的触角感器录音(。<em>果蝇</em>)和疟疾的蚊子的上颌触须(<em>按蚊</em>)。

Abstract

昆虫的嗅觉寻找食物,配偶,天敌和产卵场所3,。昆虫的嗅觉感觉神经元(OSNs)被称为感受器的感觉毛,其中包括嗅觉器官表面封闭。每个感器的表面覆盖着微小的孔隙,通过气味传递和化解感器淋巴液的液体,沐浴安置在一个给定的感器OSNs的感官树突。通过OSN树突表达气味受体(OR)的蛋白质,它在昆虫气味门控离子通道4,5的功能。互动的ORs要么增加气味或减少通过OSN基础射速。这在动作电位形成的神经元活动体现的质量,强度和时间特征的气味6,7的第一个代表性。

鉴于方便地访问这些感觉毛,它可以执行从单一OSNs引入感器淋巴结记录电极,参比电极放置在昆虫的眼睛或身体的淋巴结外的录音。在果蝇 ,一至四个OSNs感器的房子,但每个OSN通常会显示一个特征的尖峰幅度。穗排序技术使人们有可能分配扣球个别OSNs的反应。这种单一感器记录(SSR)技术监控潜在OSNs 1,2,8受体活性产生的尖峰电脉冲感器淋巴液和参比电极之间的区别。在尖峰数量变化的气味代表的气味编码在昆虫细胞的基础。这里,我们描述的制备方法,目前在我们的实验室用来执行对果蝇和按蚊的SSR,并显示在感器特定方式的气味引起的代表痕迹。

Protocol

1。气味稀释大多数气味溶于石蜡油。然而,DMSO或乙醇也可以被用来作为对特定气味的替代溶剂。准备适当稀释(例如1:10体积:体积,V:V)从纯粹的玻璃小瓶气味。大多数的气味稀释,在室温下稳定,但极易挥发的化合物,它是更好的每周工作稀释。每个感器的响应,以不同浓度范围内不同的气味。对于果蝇 ,一个有用的查表的,适当的浓度,使用一个给定的感器,可以发现…

Discussion

用于生物识别的食物来源,潜在的队友,和食肉动物的嗅觉线索。嗅觉感觉神经元(OSNs)之间的外部刺激,并进一步处理信息的大脑中心的第一个接力中心。在果蝇按蚊 ,OSNs方便,可以监视他们的电活动而刺激的气味喷。

单感器记录(SSR)技术的解释,该视频已被广泛应用于从OSNs记录和研究它们的电反应,大量的气味6, 7。嗅觉受体(ORS)6,11…

Materials

Material Name Type Company Catalogue Number Comment
Paraffin oil Odors Fluka 76235  
High purity odors (>98%) Odors Sigma-Aldrich   Methyl acetate
#296996
1-octen-3-ol
#74950
Filter paper strips Odors Fisherbrand 05-714-1 Chromatography paper
Connectors Odors Cole-Parmer EW-06365-40 1/16×1/8″
Glass vials Odors Agilent Technologies 5182-0556  
Air line plastic tubing Odor Delivery Python Products 500PAL  
1 serological pipette Odor Delivery Corning 4101 10 mL
Plastic tubing Odor Delivery Cole-Parmer EW-06418-0 0.050″x0.090″OD
Disposable borosilicate glass Pasteur pipettes Odor Delivery FisherBrand 13-678-20A 5-3/4 inches
Programmable stimulus controller Odor Delivery Syntech CS-55  
Anti-vibration table Electrophysiology Equipment TMC 63533 36”Wx30”Dx29”H
Faraday cage Electrophysiology Equipment TMC MI8133303  
Inverted microscope Electrophysiology Equipment Nikon E600FN ECLIPSE Recording microscope
10x and 100x objectives Electrophysiology Equipment Nikon 10x Plan Fluor 100x L Plan  
Dissecting microscope Electrophysiology Equipment Nikon EZ645 electrode sharpening/insect prep microscope
Magnetic stands Electrophysiology Equipment Newport MODEL 150  
IDAC Electrophysiology Equipment Syntech IDAC-4  
Acquisition software Electrophysiology Equipment Syntech Autospike  
1 macromanipulator Electrophysiology Equipment NARISHIGE MN-151 Joystick manipulator
Used for positioning reference electrode
1 micromanipulator Electrophysiology Equipment EXFO PCS-6000 Used for positioning recording electrode
Crocodile clip Electrophysiology Equipment Pomona AL-B-12-0  
Electric cable Electrophysiology Equipment Pomona B-36-0 Test Cable Assembly
2 electrode holders Electrophysiology Equipment Syntech N/A Electrode holders (set of 2) for tungsten wire electrode
AC probe Electrophysiology Equipment Syntech N/A Universal single ended probe (10xAC)
Tungsten electrodes Electrophysiology Equipment Microprobes M210 straight tungsten rods, 0.005“x3“
Potassium hydroxide Electrophysiology Equipment Sigma-Aldrich 221473  
Syringe Electrophysiology Equipment BD 301625 20 mL
Power supply Electrophysiology Equipment WILD HEERBRUGG 6V 40W e.g MTR32  
Vertical puller Insect prep Narishige PB-7  
Razor blade Insect prep VWR 55411-050  
Dental wax Insect prep Patterson 091-1503  
Microscope slide Insect prep FisherBrand 12-550A  
Cover glass Insect prep FisherBrand 12-541A 18X18 #1.5
Polypropylene mesh Insect prep Small Parts inc. CMP-0500-B  
Glass electrode Insect prep Frederick Haer & Co. 27-32-0-075 Capillary tubing borosilicate 1.5mm OD x 1.12mm ID x 75 mm
Double-sided tape (3M) Insect prep 3M MMM6652P3436 Double-sided tape (3M)
Forceps Insect prep Fine Science Tools 021×0053 Dumont #5 Mirror Finish Forceps
Small plastic cup Insect prep VWR 89009-662 7 x 5.7 (23/4 x 21/4)
Electric aspirator Insect prep Gempler’s RHM200  

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Cite This Article
Pellegrino, M., Nakagawa, T., Vosshall, L. B. Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae. J. Vis. Exp. (36), e1725, doi:10.3791/1725 (2010).

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