Name: identification of olfactory volatiles using gas chromatography-multi-unit recordings (gcmr) in the insect antennal lobe
Uploaded: 2013-02-24T14:00:00
Description: Watch this Scientific Journal Video about Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe at JoVE.com

This work was supported by NSF grant IOS 1121692, and by the University of Washington's Research Foundation.

1. Volatile Follection
 <ol>
 <li>In this example, we use volatile samples from M. lewisii flowers - an alpine wildflower native to California. Volatiles are collected using dynamic sorption methods according to Riffell et al.14. Briefly, this method employs a closed loop trapping system where flowers are enclosed in a Teflon bag. Using an inert vacuum pump, the air around the flowers is sucked through a "trap" comprised of a Pasteur pipette filled with Porapak Q matrix. The return ...

<ol>
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Insect olfactory-mediated behaviors drive many different processes, including reproduction, host-site selection, and the identification of appropriate food resources. The study of these processes requires the ability to identify the volatiles emitted from the source, as well as the ability to identify those compounds that are mediating the behaviors. Complicating matters is that odors are comprised of tens to hundreds of individual compounds that together create a unique scent that is perceived differently than th...

<table border="1">
 <tbody>
 <tr>
 <td>Name of item</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Porapak Type Q 80-100 mesh</td>
 <td>Waters</td>
 <td>WAT027060</td>
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 <td>Reynolds Oven Bags</td>
 <td>Reynolds</td>
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 <td>GC</td>
 <td>Agilent</td>
 <td>7820A</td>
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 <td>GC column</td>
 <td>J&amp;W Scientific, Folsom, CA, USA</td>
 <td>DB-5 (30 m, 0.25 mm, 0.25 &mu;m)</td>
 <td></td>
 </tr>
 <tr>
 <td>Analytical helium carrier gas</td>
 <td>Praxair</td>
 <td>HE K</td>
 <td>1 cc/min</td>
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 <td>16-channel silicon electrode</td>
 <td>Neuronexus Technologies</td>
 <td>a4x4-3mm50-177</td>
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 <td>Fine wire NiCr, 0.012 mm diameter) </td>
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 <td>For making custom tetrodes and stereotrodes</td>
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 <td>Pre-amplifier</td>
 <td>Tucker-Davis System</td>
 <td>PZ-2</td>
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 <td>Amplifier </td>
 <td>Tucker-Davis System</td>
 <td>RZ-2</td>
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 <td>Data acquisition system - OpenEx suite</td>
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 <td>Online spike-sorting software - SpikePac</td>
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 <td>Offline spike-sorting software - Mclust Spike-sorting toolbox </td>
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identification of olfactory volatiles using gas chromatography-multi-unit recordings (gcmr) in the insect antennal lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

In the GCMR assay using the M. lewisii floral scent, we inject 3 &mu;l of the extract into the GC. The total number of volatiles eluting through the GC is typically 60-70 volatiles. The scent of M. lewisii is predominantly composed of monoterpenoids, including &beta;-myrcene (acyclic) and &alpha;-pinene, with the remainder of the scent composed of six-carbon volatiles, such as 2-hexanol, and sesquiterpenoids that comprise &lt; 1% of the headspace.
 GCM...

Watch this Scientific Journal Video about Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe at JoVE.com

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

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