Name: Testing Drosophila Olfaction with a Y-maze Assay (Video) | JoVE Journal
Uploaded: 2014-06-12T13:00:00.000+00:00
Duration: 6 min 45 s
Description: Learn a simple Y-maze assay to test olfactory behavior in Drosophila melanogaster using starvation and odorant exposure. This method helps researchers study sensory processing and preference responses.

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5 minutes

We thank 4 anonymous reviewers for their work to improve the manuscript. We thank the Centre National de la Recherche Scientifique for its financial support to MBG and YG, and the Universit&#233; de Bourgogne and the French Ministry of Research to MMS. Research in YG laboratory is funded by the European Research Council (ERC Starting Grant, GliSFCo-311403), the Agence Nationale de la Recherche (ANR-JCJC, GGCB-2010), the Conseil R&#233;gional de Bourgogne (Faber), and the CNRS.

1. Before Starting
<ol>
	<li>Use an isogenized reference stock bearing stable and robust behavioral phenotypes. There is no general rule for choosing this stock, since all potential controls may carry heterogeneous background alleles.</li>
	<li>Use this control strain to backcross every other stock necessary for later steps. This backcrossing step is typically represented by at least 5 successive crosses of a single virgin female (to allow possible crossing-over between homologous chromosomes) to 2&#8211;3 isogenic reference males5. This step is important to homogenize the genetic background between the different fly stocks.</li>
	<li>Maintain Drosophila stocks on a standard corn flour (9%), yeast (10%), and agar medium (1.5%) complemented with antibiotic (0.4% methyl para-hydroxy-benzoate) in a 12-hr light/dark cycle at 25 &#176;C.</li>
	<li>Achieve chemosensory experiments in a temperature-controlled room (25 &#176;C) under far red light (to eliminate the contribution of visual cues, and to focus on chemosensory signals). Regularly renew the air of the room to ventilate the area between each experiment.</li>
</ol>
2. Olfactory Response using a Y-maze Assay
<ol>
	<li>Starve the flies for 16&#8211;18 hr at 25 &#176;C in glass tubes containing wet paper towel before testing.</li>
	<li>Join a Y-shape connector to two glass vials and to a smaller plastic vial (loading vial). Use 1 ml pipette tips that pass through the foam stoppers to link the connector to the three vials, and to obtain a tightly sealed Y-maze. Cut the narrow ends of two pipette tips (~2 mm diameter, to avoid any return of the fly once it has made its decision) to form two &#8220;trap&#8221; vials, and a large end of one pipette tip to form the &#8220;loading&#8221; tube (Figure 1A).</li>
	<li>Just before connecting the &#8220;trap&#8221; vials (Figure 1B), place one ~6 mm diameter filter paper in each vial. Add 40 &#956;l of odorant solution on one filter paper, and 40 &#956;l of the corresponding solvent on the second filter paper.</li>
	<li>Introduce ten 4 to 9 day-old flies into the &#8220;loading&#8221; vial. Do not use CO2 anesthesia during this transfer, since it has a strong effect on behavior10. Rather use brief cooling on&#160;ice. Proper manipulation of anaesthetized flies is important to limit stress on the subjects as much as possible.
	<ol>
		<li>Perform a series of Y-maze tests at 25 &#176;C under far-red light (using LED bulbs to limit possible heating source) to avoid visual stimuli as much as possible. Be careful to alternate the orientations of the Y-mazes (odorant containing tube on the left, or on the right, and loading tube in front or in the back; Figure 1C).</li>
		<li>Allow several hours for the flies to enter in the trap vial containing the odorant or the solvent. Count flies after 24 hr to increase the participation up to more than 80% and provide the maximum olfactory index value (Simonnet, personal communication).</li>
		<li>Calculate the resulting olfactory index using the following formula: (number in the odor tube - number in the solvent tube)/total number of loaded flies.</li>
	</ol>
	</li>
	<li>Wash Y-maze set-up as follows: soak the dismantled set-up in RBS 35 MD overnight. Thoroughly rinse out with tap water. Finally rinse with deionized water and let dry out.</li>
</ol>
3. Statistical Analysis of the Data
<ol>
	<li>Perform a t-test, a one-way ANOVA or a two-way ANOVA depending on data and variables.</li>
</ol>

<ol><li>Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genes+and+ligands+for+odorant%2C+vomeronasal+and+taste+receptors%5BTitle%5D)+AND+%22Nat.+Rev.+Neurosci%22%5BJournal%5D)">Genes and ligands for odorant, vomeronasal and taste receptors.</a> Nat. Rev. Neurosci. 5, 263-278 (2004).</li>
<li>Silbering, A. F., Benton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ionotropic+and+metabotropic+mechanisms+in+chemoreception%3A+%27chance+or+design%5BTitle%5D)+AND+%22EMBO+Rep%22%5BJournal%5D)">Ionotropic and metabotropic mechanisms in chemoreception: 'chance or design.</a> EMBO Rep. 11, 173-179 (2010).</li>
<li>Ziegler, A. B., Berthelot-Grosjean, M., Grosjean, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+smell+of+love+in+Drosophila%5BTitle%5D)+AND+%22Front.+Physiol%22%5BJournal%5D)">The smell of love in Drosophila.</a> Front. Physiol. 4, (2013).</li>
<li>Silbering, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complementary+function+and+integrated+wiring+of+the+evolutionarily+distinct+Drosophila+olfactory+subsystems%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Complementary function and integrated wiring of the evolutionarily distinct Drosophila olfactory subsystems.</a> J. Neurosci. 31, 13357-13375 (2011).</li>
<li>Grosjean, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+olfactory+receptor+for+food-derived+odours+promotes+male+courtship+in+Drosophila%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">An olfactory receptor for food-derived odours promotes male courtship in Drosophila.</a> Nature. 478, 236-240 (2011).</li>
<li>Woodard, C., Huang, T., Sun, H., Helfand, S. L., Carlson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+analysis+of+olfactory+behavior+in+Drosophila%3A+a+new+screen+yields+the+ota+mutants%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Genetic analysis of olfactory behavior in Drosophila: a new screen yields the ota mutants.</a> Genetics. 123, 315-326 (1989).</li>
<li>Semmelhack, J. L., Wang, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Select+Drosophila+glomeruli+mediate+innate+olfactory+attraction+and+aversion%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Select Drosophila glomeruli mediate innate olfactory attraction and aversion.</a> Nature. 459, 218-223 (2009).</li>
<li>Budick, S. A., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Free-flight+responses+of+Drosophila+melanogaster+to+attractive+odors%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Biology%22%5BJournal%5D)">Free-flight responses of Drosophila melanogaster to attractive odors.</a> The Journal of Experimental Biology. 209, 3001-3017 (2006).</li>
<li>Martin, F., Charro, M. J., Alcorta, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutations+affecting+the+cAMP+transduction+pathway+modify+olfaction+in+Drosophila%5BTitle%5D)+AND+%22J.+Comp.+Physiol.+A%22%5BJournal%5D)">Mutations affecting the cAMP transduction pathway modify olfaction in Drosophila.</a> J. Comp. Physiol. A. 187, 359-370 (2001).</li>
<li>Barron, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anaesthetising+Drosophila+for+behavioural+studies%5BTitle%5D)+AND+%22Journal+of+Insect+Physiology%22%5BJournal%5D)">Anaesthetising Drosophila for behavioural studies.</a> Journal of Insect Physiology. 46, 439-442 (2000).</li>
<li>Ai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acid+sensing+by+the+Drosophila+olfactory+system%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Acid sensing by the Drosophila olfactory system.</a> Nature. 468, 691-695 (2010).</li>
<li>Becher, P. G., Bengtsson, M., Hansson, B. S., Witzgall, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flying+the+fly%3A+long-range+flight+behavior+of+Drosophila+melanogaster+to+attractive+odors%5BTitle%5D)+AND+%22J.+Chem.+Ecol%22%5BJournal%5D)">Flying the fly: long-range flight behavior of Drosophila melanogaster to attractive odors.</a> J. Chem. Ecol. 36, 599-607 (2010).</li>
</ol>

The authors have nothing to disclose.

Our Y-maze protocol is based on a previously described protocol9. However, we introduce two major differences. First, we use narrow pipette tips to prevent the flies from returning once they decide to enter in the vial containing the solvent or the solvent plus the odorant. These narrow tips are also useful to limit the odorant diffusion in the Y-maze. Second, we use a smaller loading vial to force the flies to enter in the Y-maze. It is important to have a high participation of these flies (80% to 100% after ...

Chemoreception through taste or olfaction is a key sensory modality for animal survival. It gives vital cues necessary for the detection of a danger or food sources, as well as for social interactions. It also helps animals to find a sex partner necessary for their reproduction. For more than 20 years, intensive research, including Nobel prize winning work by Richard Axel and Linda Buck in 2004 &#34;for their discoveries of odorant receptors and the organization of the olfactory system&#34;, has been carried out to reveal the molecular and cellular bases of olfaction1,2.
One of the favorite animal models for scientists to dissect olfactory perception is D. melanogaster. This insect shares a similar cellular and molecular odor-coding strategy with mammals. The scientific community uses diverse behavioral paradigms to study the role of odorants in this fruit fly. These tests include multimodal assays such as courtship tests where various sensory modalities, including olfaction, are important to elicit male courtship3. Other assays have also been developed to tackle the role of odorants more specifically; these include T-mazes, Y-mazes, trap assays, four-field arenas and wind-tunnels4,5,6,7,8.
In this article we present a simple modified Y-maze assay, which provides robust olfactory responses using D. melanogaster. Our set-up uses end-tips in contrary to a previously described method9. Thus, our Y-maze has two advantages. First, it avoids any return in the system once the fly has made her choice. Second, it limits the exchange of odorants in all areas of the Y-maze. This last advantage is important since Drosophila are very sensitive to air flow which is often used to avoid odorant saturation. To adjust the experimental set-up with an air flow would be time and cost consuming. Therefore, our Y-maze assay represents an efficient and fast way to test olfactory performance of Drosophila.

<table><tbody><tr><td>Drosophila Polystyrene tube</td><td>VWR europe</td><td>734-2255</td><td>30 x 25 mm&amp;nbsp;&lt;br/&gt; Y-maze</td></tr><tr><td>Drosophila Borosilicate tube</td><td>Dijon verre</td><td></td><td>95 X 25 mm&lt;br/&gt; Y-maze</td></tr><tr><td>Foam stopper</td><td>Dutscher</td><td>999038</td><td>&lt;br/&gt; Y-maze</td></tr><tr><td>Y-shaped connector</td><td>Europrix</td><td>11020605</td><td>&lt;br/&gt; Y-maze</td></tr><tr><td>100-1,000 &amp;micro;l pipette tips</td><td>Corning</td><td>4868</td><td>Join the following pipette tips to the Y-shaped connector. Cut 2 pipette tips at 65 mm from the wide end, and connect the narrow end (with a &amp;sim;2 mm opening) to 2 test vials. These openings will limit the U-turns once the flies enter the tubes containing the odors. Cut 1 pipette tip at 35 mm from the wide end, and connect it to the loading vial.&lt;br/&gt; Y-maze</td></tr><tr><td>Far-Red LED Bulb</td><td>Rubin-Lacaque</td><td>0RB180238</td><td>625-630 nm</td></tr><tr><td>Acetic Acid</td><td>Sigma-Aldrich</td><td>45725</td><td></td></tr><tr><td>Phenylacetic Acid</td><td>Sigma-Aldrich</td><td>P16621</td><td></td></tr><tr><td>Yeast</td><td>Sensient Flavors Strasbourg</td><td>1018880464</td><td></td></tr><tr><td>Cornmeal</td><td>eurogerm</td><td>Farine de ma&amp;iuml;s</td><td></td></tr><tr><td>Agar</td><td>Kalys</td><td>HP-697-25</td><td></td></tr><tr><td>Methyl hydroxy 4 benzoate</td><td>VWR international</td><td>25605293</td><td></td></tr></tbody></table>

testing drosophila olfaction with a y-maze assay

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

Figure 1D shows two representative responses using this Y-maze assay. Canton-S males strongly avoid 10% acetic acid diluted in distilled water, whereas they do not significantly avoid 10% phenylacetic acid. These assays are based on 10 males per replicate placed together in the loading vials. This protocol can sometimes lead to large standard error of the mean. If needed, it is possible to reduce this drawback by using 20 males per replicates instead of only 10. Mathematically, the choice of one individu...

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze assay.

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

The overall goal of this procedure is to provide a simple Y maze assay to test olfaction in Drosophila melon Gaster. This is accomplished by first starving flies for 16 to 18 hours, so they're motivated to explore new environments with volatile stimuli. The second step of the procedure is to assemble the Y maze setup containing the odor sources on filter paper, such as acetic acid in solvent versus solvent alone.The third step is to quickly and gently introduce the flies into the loading vial. After several hours, the final step is to score the flies location in the maze. Ultimately, the results can show a response toward odorant from the data obtained with the Y maze.We first as the idea for this method when we decided to have a simple and inexpensive method to test the olfactory response of phyla. An advantage of this technique over other option is that it allows the flies to make a decision whether too much stress Demonstrating the procedure will be Megan Simon, a PhD student from my laboratory For this protocol have ready an ISO inized fly line with stable, robust behavioral phenotypes. Any such stock will serve as a reference control for the olfactory test, for each genetic strain to be used for the experiment back, cross the strain to the reference strain for five generations.Pair single experimental strain females to a few isogenic males for each back cross. For olfactory experiments, maintain the drosophila on standard corn flour medium with an antibiotic under 1212 light cycle. And at 25 degrees Celsius.The experiments should always be executed at 25 degrees Celsius and under light in the far red spectra, not visible to drosophila. It must also be possible to fully ventilate the room between experiments before conducting the assay the following morning. At the end of the day, transfer cohorts of 10 to 20 experimental lines to glass vials with moisture provided from a wet towel.The flies must be tested in 16 to 18 hours, or they could die due to de or starvation the next day. Assemble the Y maze. Start by joining the Y shaped connector to two glass vials using foam connectors with trimmed pipette tips, which make the conduits their tapering shape makes these one-way passages.The third branch of the Y is attached to a loading tube made from the large portion of a pipette tip and allows passage from the loading vial to the choice point of the maze. Beyond each tapering tunnel is a glass vial, one of which must be loaded with an odorant load. These as 40 microliter boluses soaked into 28 millimeters squares of tissue paper.Which of the two contains the odorant should alternate randomly to conduct the test first.Cool. The flies on ice briefly, so the flies are slowed down. This should be under far red light from LED bulbs, and at 25 degrees Celsius, gently drop the cohort of flies into the loading vial and load the Y maze with the test flies.Do not use gas anesthesia. It is really critical to transfer the flies to the loading vial both quickly and gently. It is actually the most difficult step of this experiment because flies have to wake up rapidly before the diffusion of others in the Y mass.The flies should be allowed to explore the maze for 24 hours, so a maximum number of flies move to one or the other glass vial. The number of flies present in the different tubes serves to calculate the olfactory index. Standard statistical methods should be applied to this value gathered from the behavior of up to 10 flies.If 10 fly cohorts are not providing consistent results, the trials can be run with 20 fly cohorts. To clean up the maze components, soak them dismantled in RBS 35 MD overnight. Follow this with rinses in tap water and a final rinse in DI water before air drying.Standard responses to an odorant. Using the Y maze include attraction, indifference, or avoidance. For example, cantons flies strongly avoid 10%acetic acid, whereas they do not avoid 10%pheno acetic acid.While attempting this procedure, it's important to remember that this wave asay represent an artificial environment for the flies. Following this procedure as a method like teammates fulfilled arena or internal assays can be performed in order to confirm the olfactory responses of flies and the different conditions. After watching this video, you should have a good understanding of how to simply test olfactory responses of filano gaste using the SW asay.

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Learn a simple Y-maze assay to test olfactory behavior in Drosophila melanogaster using starvation and odorant exposure. This method helps researchers study sensory processing and preference responses.

Testing Drosophila Olfaction with a Y-maze Assay

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