用气相色谱 - 多单元的录音（GCMR）昆虫触角叶嗅觉挥发物的鉴定
Summary
嗅觉介导许多不同的昆虫的行为，并经常由数十至数百种挥发性化合物组成的复杂混合物。气相色谱法与多通道记录在昆虫触角叶中，我们描述的生物活性化合物的鉴定方法。
Abstract
所有的生物体生活在一个世界充满感官刺激，确定其所处的环境行为和生理反应。嗅觉是特别重要的昆虫，它用自己的嗅觉系统响应和歧视之中，复杂的刺激气味。这些气味引起的行为，调解过程，如繁殖和栖息地选择1-3。此外，化学传感由昆虫介导的行为，农业和人类健康是非常重要的，其中包括4-6，草食动物的粮食作物7，授粉和传播疾病8,9。因此，在昆虫行为的嗅觉信号，其作用是识别重要的，了解生态过程和人类的食物资源和福祉。
至目前为止，已经难以识别驱动昆虫行为的挥发性物质，往往是乏味。目前的技术包括：气相色谱耦合电生理记录（GC-EAG）和气相色谱耦合的单感器录音（GC-SSR）10-12。这些技术被证明是重要的生物活性化合物的鉴定。我们已经开发出一种方法，使用气相色谱多通道电生理的录音（称为“GCMR”）的神经元在触角叶（AL;昆虫的嗅觉中枢）13,14。这个国家的最先进的技术使我们能够探测气味信息是如何表示的昆虫的大脑。此外，因为在这个级别的嗅觉处理神经元对气味的反应是非常敏感的，因为天线AL神经元的受体神经元到收敛的程度，AL录音可以让活性成分自然的气味，高效和高灵敏度的检测。在这里，我们描述GCMR举一个例子，它的使用。
一些一般的步骤是国际富豪的ved检测生物活性挥发物和昆虫响应。挥发物，首先需要收集的利益来源（在这个例子中，我们使用花沟酸浆属 （属Phyrmaceae）），其特征为需要使用标准的GC-MS技术14-16。昆虫准备用最小的解剖研究，之后记录电极插入触角叶，多渠道的神经开始录制的。处理后的神经数据，然后显示特定的气味分子的昆虫的神经系统造成显着的神经反应。
虽然我们在这里给出的例子中特定授粉研究，GCMR可以扩大到范围广泛的研究生物和挥发性来源。例如，此方法可用于在吸引或排斥矢量昆虫和作物害虫的加臭剂的识别。此外，GCMR也可以被用来识别有益昆虫的引诱剂，如婆llinators。该技术可以扩展到非昆虫科目以及。
Protocol
1。挥发性Follection 在这个例子中，我们使用挥发性样品M. lewisii花-高山野花原产于美国加州。收集挥发物，采用动态吸附方法，根据到Riffell 等。14。简单地说，该方法采用一个闭环特氟隆包括在花捕集系统。使用的惰性的真空泵，花的周围空气被吸入，通过一个“陷阱”巴斯德吸管包括充满PORAPAK Q矩阵。从泵的返回空气通过活性炭过滤。一规定时间周期之后，在我们…
Representative Results
在使用M. GCMR分析的lewisii花香的味道，我们3微升提取物注入到GC。挥发物的纯化，通过GC的总数通常为60-70挥发物。香味M. lewisii是主要组成的单萜类化合物，包括β-月桂烯（非周期性）和α-蒎烯，用剩余六碳挥发物，如2 -己醇，倍半萜类化合物，包括<1％的顶空组成的气味。 GCMR利用触角叶神经元的敏感性，以及神经元的处理用的重要的生物…
Discussion
昆虫的嗅觉介导的行为驱动许多不同的流程，包括复制，主机选址，并确定适当的食物资源。这些过程的研究需要从源头抓起，以及识别能力，调解行为的化合物，这些化合物的挥发物排放的识别能力。复杂的问题是，气味是由几十到几百，共同创造一个独特的气味，被认为比单个的成分6,7,13,19,20不同的单个化合物。 ，最初研究中的性信息素系统12进行的，最近更是在食品和产卵…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由国家科学基金会资助IOS 1121692，和大学的华盛顿研究基金会的支持。
Materials
| Name of item | Company | Catalog Number | Comments |
| Porapak Type Q 80-100 mesh | Waters | WAT027060 | |
| Reynolds Oven Bags | Reynolds | ||
| GC | Agilent | 7820A | |
| GC column | J&W Scientific, Folsom, CA, USA | DB-5 (30 m, 0.25 mm, 0.25 μm) | |
| Analytical helium carrier gas | Praxair | HE K | 1 cc/min |
| 16-channel silicon electrode | Neuronexus Technologies | a4x4-3mm50-177 | |
| Fine wire NiCr, 0.012 mm diameter) | Sandvik Kanthal HP Reid | PX000004 | For making custom tetrodes and stereotrodes |
| Pre-amplifier | Tucker-Davis System | PZ-2 | |
| Amplifier | Tucker-Davis System | RZ-2 | |
| Data acquisition system – OpenEx suite | Tucker-Davis System | ||
| Online spike-sorting software – SpikePac | Tucker-Davis System | ||
| Offline spike-sorting software – Mclust Spike-sorting toolbox | David Redish, Department of Neuroscience, University of Minnesota | Free download at http://redishlab.neuroscience.umn.edu/MClust/MClust.html | MATLAB toolbox |
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Tags
IdentificationOlfactory VolatilesGas Chromatography-multi-unit RecordingsGCMRInsect Antennal LobeSensory StimuliBehavioral ResponsePhysiological ResponseOlfactionInsectsOdor StimuliReproductionHabitat SelectionChemical SensingAgricultureHuman HealthPollinationHerbivoryTransmission Of DiseaseEcological ProcessesHuman Food ResourcesVolatile Identification TechniquesGas Chromatography-coupled Electroantennogram Recording (GC-EAG)Gas Chromatography-coupled Single Sensillum Recordings (GC-SSR)Bioactive CompoundsMulti-channel Electrophysiological Recordings
Cite This Article
Byers, K. J. R. P., Sanders, E., Riffell, J. A. Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe. J. Vis. Exp. (72), e4381, doi:10.3791/4381 (2013).