This work was supported by research grants to J.W.W. from the National Institute of Health (R01DK092640) and National Science Foundation (0920668).

1. Fly Collection and Starvation
<ol>
	<li>Rear the experimental flies under controlled temperature and humidity conditions (e.g. 21 &#176;C, 50-60% relative humidity) on a 12-hr light/dark cycle.</li>
	<li>Collect female flies on the day of eclosion and place them, along with 4-5 males, into new food vials (maximum 30 per vial). Age flies 2-5 days.</li>
	<li>Prepare the chambers for fly starvation.
	<ol>
		<li>Push a single tissue (4.8 x 8.4 inch) down to the bottom of an empty plastic vial. Completely soak the tissue with distilled water. Use an object to push down on the tissue and gently squeeze out excess water.</li>
		<li>Invert the vial to discard the extra water. There should be enough water to keep the flies hydrated and the starvation chamber moist, but not enough to drown the flies.</li>
	</ol>
	</li>
	<li>Transfer the flies from the food vial into a starvation chamber and plug the vial about 18-24 hr before the experiment begins. Store the vials under controlled temperature and humidity conditions overnight until the experiment begins the next day.</li>
</ol>
2. Preparation of the Food Odor
<ol>
	<li>Prepare a 1% agarose solution by adding 0.1 g of low melting temperature agarose to 10 ml of distilled water in a glass flask. Heat the agarose solution in a microwave just until it begins to boil but well before it boils over.
	<ol>
		<li>Stop the microwave and swirl the flask once. Repeat this step twice more until the agarose has completely dissolved. Keep the agarose solution in a liquid state by keeping the flask warm on a hotplate set at 50 &#176;C.</li>
	</ol>
	</li>
	<li>Add 990 &#956;l of the 1% agarose solution and 10 &#956;l of apple cider vinegar to a 1.5 ml Eppendorf tube to make a 1% apple cider vinegar solution. Vortex the solution until mixed and place in a dry bath incubator set to 50 &#176;C.</li>
</ol>
3. Testing Room and Behavior Chamber Setup
<ol>
	<li>Set the testing room to the desired conditions (e.g. temperature and humidity).</li>
	<li>Turn on the LED panel ( 660 nm).</li>
	<li>Rinse the sieves and testing plates with hot water and heat them in a drying oven until all moisture has evaporated. Cool the sieves and plates down to the testing room temperature before beginning experiments.</li>
	<li>Position a shallow dish on top of the diffuser plate and fill it with water to increase the local humidity and to mask the water in the agarose droplet.</li>
	<li>Position the sieves over the water dish.</li>
</ol>
4. Fly Loading into the Testing Plates
Diagrams with specifications for testing plates can be found in the Supplemental Files section. The testing plate is made of clear acrylic and consists of 6 testing arenas. A simple slider contains holding chambers that permit fly loading, temporary containment, and simultaneous release of 6 flies into their respective chambers at the start of the experiment. Cross-hairs etched at the center of each arena in the plate indicate where odorants should be pipetted.
<ol>
	<li>Insert the sliders into the acrylic testing plate.</li>
	<li>Gently slide the aspirator into the vial past the cotton plug and allow about 6 flies to walk into the aspirator.it is critical to be as gentle as possible in handling them. One may take advantage of phototactic fly behavior to induce flies to crawl towards the aspirator by pointing the vial opening towards a dim light source. If necessary, one may also apply gentle suction to aspirate approximately 6 female flies.</li>
	<li>Insert the tip of the aspirator into the first hole of the testing plate. Allow a single fly to pass into the holding cell and gently advance the slider forward to load a fly into the next hole. Continue until flies occupy all 6 holding cells of the plate.</li>
	<li>Pipette 5 &#956;l of 1% apple cider vinegar agarose solution directly onto the center of the cross-hairs on the inside face of the testing plate.</li>
</ol>
5. Positioning the Testing Plate
<ol>
	<li>To center the testing plate, open the file named &#34;Positioning Tool.vi.&#34; &#8220;LabVIEW VI for Positioning Tool. vi can be found in the Supplemental Files section. Run the file by clicking on the white arrow in the upper-left corner of the screen.</li>
	<li>Place the testing plate on top of the sieve such that the arena opening faces the sieve floor and the odor target is on the ceiling of the plate. Align the crosshairs etched into back of the testing plate with the cross hairs on the monitor screen.</li>
	<li>When alignment has been completed, abort execution by clicking on the red dot located near the upper-left corner of the monitor.</li>
</ol>
6. Record the Fly Position During the Experiment
<ol>
	<li>To track and record the coordinates of the fly during each food search trial, open the acquisition software file &#34;Fly Tracking--Six Zones.vi.&#34; LabVIEW VIs for &#8220;Fly Tracking--Six Zones.vi&#8221; can be found in the Supplemental Files section. Run the file by clicking on the white arrow in the upper-left corner of the monitor.</li>
	<li>Assign the file a name and click on &#34;OK.&#34;</li>
	<li>Advance the sliders in the testing chambers to release the flies into the testing arenas. Be careful not to move the testing chambers as this will lead to improper alignment with analysis software coordinates.</li>
	<li>Click on &#34;Start&#34; (recording begins) and ensure that the only source of light in the testing chamber is the 660 nm LED panel.</li>
	<li>When the trial is finished, remove the sieve and behavior chamber. Lift the testing plate from the sieve and remove the flies by submerging the plate in ice. Gently clean the plate with hot water and remove any agarose debris. Place the testing plates in a drying oven to remove moisture.</li>
	<li>Ventilate the testing area by turning on a small fan for approximately 2 min. Turn the fan off and load the next group of flies into the next testing plate.</li>
</ol>
7. Data Analysis Using Custom Software
&#8220;Data Analysis for Fly Tracking&#8212;Six Zones&#8221; can be found in the Supplemental Files section. During data acquisition, the acquisition software records individual fly position coordinates for each time point in a text file. A single digital camera positioned above the testing plates acquires images at a frame rate of 0.5 Hz. The analysis software program &#34;Data Analysis for Fly Tracking-Six Zones&#34; extracts information from that text file to a) calculate the average speed, b) determine the time point at which a fly successfully located the odor source, and c) build graphical windows that allow the user to view: fly location, distance of the fly from the odor source over time and average fly speed over time. It also formats the data for easy export into a spreadsheet program. In this macro, food search latency is defined as the time point at which flies spend at least 5 sec within a 5 mm radius of the center of the arena.
<ol>
	<li>Open the analysis software file &#34;Data Analysis for Fly Tracking--Six Zones&#34;. Under the &#34;Windows&#34; tab, click on &#34;Create New Table.&#34; Repeat this step until six tables have been created.</li>
	<li>Under the &#34;Macros&#34; tab, click on &#34;Foodfinding.&#34; A Main Panel should appear with the following options: Open Raw Data File for Layout; Open Raw Data File for Data File; Fly Location; Distance; Speed; Layout; FormatDataFile.</li>
	<li>To view raw data without appending values to a text file, click on &#34;Open Raw Data File for Layout.&#34; Locate and select the experimental data file in the browser window that appears. Click on &#34;Open.&#34;</li>
	<li>Click on &#34;Fly Location&#34; to view each fly&#39;s location in each of the six arenas ( six X-Y plots depicting each fly&#39;s position over time should appear on the screen).</li>
	<li>Click on &#34;Distance&#34; to view each fly&#39;s distance from the odor source ( six plots depicting the fly&#39;s distance from the odor source over time should appear on the screen). The horizontal line at y = 5 mm indicates the threshold at which the fly is considered to be located at the food source.</li>
	<li>Click on &#34;Speed&#34; to view each fly&#39;s average speed during the trial (six plots depicting the fly&#39;s speed over time should appear on the screen).</li>
	<li>Click on &#34;Layout&#34; to display a layout with all the fly location, distance, and speed graphs in addition to the average speed (during the first 50 sec) and latency of finding the odor source for each fly (Figure 1). To properly view the layout, it may be necessary to adjust the margins. To do this, first click on the layout window. Under the &#34;File&#34; tab, click on &#34;Page Setup for Layout.&#34; Reset the margins to 0.2 inches and click on &#34;OK.&#34; Immediately left of each location plot is a small table with the headings &#34;Speed&#34; and &#34;Latency.&#34; The values entered under each heading indicate the average speed in mm/sec and food search latency in seconds. Blank entries under Latency indicate the fly failed to locate the odor source. Food search latency is defined as the time point at which the flies spent at least 5 sec within a 5 mm radius from the center of the chamber.</li>
	<li>To print a layout, click on the layout area (updates layout to current file). Click on &#34;File&#34; and then click on &#34;Print Layout.&#34;</li>
	<li>To view the next file, simply click on &#34;Open Raw Data File for Layout.&#34; Click on the next raw data file you wish to view and click on &#34;OK.&#34; Click on the Layout window to update the window with the new data file.</li>
	<li>The settings may be saved in an experiment file for later use.</li>
</ol>
8. Export Data from the Data Analysis Software to a Spreadsheet Program
<ol>
	<li>To export speed and latency data for each file, click on &#34;Open Raw Data File for Data File.&#34; Select an experimental data file and click on &#34;Open.&#34; A new browser window will appear.</li>
	<li>In the new browser window, right-click on &#34;New&#34; and then click on &#34;Text Document.&#34; Name the new text document. Select the newly named text file and click on &#34;Open.&#34; This stores data from the raw data file into the text file.</li>
	<li>To export data from another file, click on &#34;Open Raw Data File for Data File.&#34; Click on another file and click on &#34;Open.&#34; Select the text file from step 8.2). Continue this process for the remaining data files you wish to export.</li>
	<li>Once all the desired data files have been added to the text file, click on &#34;Format Data File.&#34; Select the Text Document used to store data from the previous steps and click on &#34;Open&#34; (a new window will automatically open).</li>
	<li>Create a new Text Document in the window, assign the file a name, and click on &#34;Save.&#34; This creates a text file that contains the filename, average speed, and latency for each fly and can be imported into a spreadsheet program.</li>
	<li>Cumulative plots are constructed from data on the total number of flies reaching the food odor target as a function of time (Figure 3).</li>
	<li>Experimental data sets are analyzed for statistical significance using a z-test for proportions.</li>
</ol>

<ol>
	<li>Dethier, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The hungry fly : a physiological study of the behavior associated with feeding. , (1976).</li><li>Root, C. M., Ko, K. I., Jafari, A., Wang, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+facilitation+by+neuropeptide+signaling+mediates+odor-driven+food+search.">Presynaptic facilitation by neuropeptide signaling mediates odor-driven food search.</a> Cell. 145, 133-144 (2011).</li><li>Root, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+presynaptic+gain+control+mechanism+fine-tunes+olfactory+behavior.">A presynaptic gain control mechanism fine-tunes olfactory behavior.</a> Neuron. 59, 311-321 (2008).</li><li>Semmelhack, J. L., Wang, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Select+Drosophila+glomeruli+mediate+innate+olfactory+attraction+and+aversion.">Select Drosophila glomeruli mediate innate olfactory attraction and aversion.</a> Nature. 459, 218-223 (2009).</li><li>Faucher, C., Forstreuter, M., Hilker, M., de Bruyne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+responses+of+Drosophila+to+biogenic+levels+of+carbon+dioxide+depend+on+life-stage,+sex+and+olfactory+context.">Behavioral responses of Drosophila to biogenic levels of carbon dioxide depend on life-stage, sex and olfactory context.</a> J. Exp. Biol. 209, 2739-2748 (2006).</li><li>Quinn, W. G., Harris, W. A., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+behavior+in+Drosophila+melanogaster.">Conditioned behavior in Drosophila melanogaster.</a> Proc. Natl. Acad. Sci. U.S.A. 71, 708-712 (1974).</li><li>Fishilevich, E., Domingos, A. I., Asahina, K., Naef, F., Vosshall, L. B., Louis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+behavior+mediated+by+single+larval+olfactory+neurons+in+Drosophila.">Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila.</a> Curr. Biol. 15, 2086-2096 (2005).</li><li>Venken, K. J., Simpson, J. H., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+genes+and+cells+in+the+nervous+system+of+the+fruit+fly.">Genetic manipulation of genes and cells in the nervous system of the fruit fly.</a> Neuron. 72, 202-230 (2011).</li><li>Hamada, F. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+internal+thermal+sensor+controlling+temperature+preference+in+Drosophila.">An internal thermal sensor controlling temperature preference in Drosophila.</a> Nature. 454, 217-220 (2008).</li><li>Kitamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+modification+of+behavior+in+Drosophila+by+targeted+expression+of+a+temperature-sensitive+shibire+allele+in+defined+neurons.">Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons.</a> J. Neurobiol. 47, 81-92 (2001).</li><li>Sweeney, S. T., Broadie, K., Keane, J., Niemann, H., O'Kane, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+expression+of+tetanus+toxin+light+chain+in+Drosophila+specifically+eliminates+synaptic+transmission+and+causes+behavioral+defects.">Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects.</a> Neuron. 14, 341-351 (1995).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li></ol>

The authors declare no competing financial interest.

In this protocol, we describe a step-by-step procedure for the food search behavior assay. In addition to food related odors, it may also be adapted for the study of the fly&#39;s ability to locate other odor objects. For example, it may be applied toward the study of mate localization behavior in male flies3 .There are several additional considerations for this protocol that we will mention here regarding this procedure:
First, the rearing temperature determines how long experiment...

States of hunger promote two types of appetitive behaviors: food search and food consumption1. This simple behavioral assay is useful for the study of chemotactic behaviors associated with foraging2,3. Specifically, it tracks fly position, walking speed and latency to locating a food odor target. Latency of food finding serves as metric for measuring changes in the sensitivity of the fly&#39;s odor detection system downstream of changes in its internal appetitive state. A manual version of this assay was previously used to show GABA-B receptor signaling is important for odor localization behavior in adult flies3. The current automated version of the assay was instrumental in the study of how short neuropeptide-F (sNPF) signaling reshapes the olfactory map in Drosophila and influences appetitive behaviors2.
Testing is done in a dark, temperature and humidity controlled room. Digital video cameras set above the clear acrylic testing plates track flies backlit by 660-nm LED illumination. Information from the camera is processed in real time by a computer stationed next to the testing area. We use data acquisition software to record and save the coordinates of fly positions during the testing period.
In this paradigm, the subject is released into an arena that contains a food odor at the center; the odor object creates a food odor gradient within the arena that induces food search behavior in the fly. A similar odor search protocol has been applied toward the study of chemosensation in single Drosophila larvae7. While other behavioral assays such as the four-field olfactometer4,5 or the t-maze6 evaluate odor aversion or attraction behaviors, this paradigm is best suited to evaluate olfactory sensitivity and chemotaxis behaviors.
Several key advantages accompany this assay. First, it permits rapid acquisition of large data sets, because data collection and analysis are mostly automated. Second, this assay isolates and measures the behavior of single flies, thus eliminating social olfactory cues that may influence their behaviors. Third, the simplicity of the protocol and simple experimental design make the assay efficient and easy to teach others.
In addition, this assay may be used to further probe neural circuits underlying food search behavior by combining it with the extensive genetic toolkit available to Drosophila melanogaster8. Targeted expression of transgenes that silence or excite neurons can be achieved with tools such as the GAL4-UAS system as well as the UAS-shibirets1, UAS-tetanus-toxin, and UAS-TrpA1(B) transgenes9-12.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company </td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Apple Cider Vinegar</td>
			<td>Spectrum</td>
			<td></td>
			<td>commercially available</td>
		</tr>
		<tr>
			<td>Agarose, Type VII</td>
			<td>Sigma-Aldrich</td>
			<td>A0701</td>
			<td>low gelling temperature agarose</td>
		</tr>
		<tr>
			<td>Acrylic Testing Plate</td>
			<td></td>
			<td>custom</td>
			<td>Plate contains 6 arenas. Each arena is 60 mm in diameter 6 mm in height. See testing plate diagrams for specific measurements.</td>
		</tr>
		<tr>
			<td>LabVIEW V.8.5</td>
			<td>National Instruments</td>
			<td>776670-09</td>
			<td>platform for programs: PositioningTool.vi, FlyTracking--Six Zones.vi NOTE: &#34;elapsed time.vi&#34;, &#34;time into file.vi&#34;, and &#34;two object detect.vi&#34; are included subroutines that must be available in order for the main data acquisition program &#34;FlyTracking--Six zones.vi&#34; to run.</td>
		</tr>
		<tr>
			<td>LabVIEW Vision 8.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW Vision Acquisition Software 8.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW Vision Builder AI 3.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Igor Pro V.6</td>
			<td>Wavemetric, Inc.</td>
			<td></td>
			<td>platform for macro: Data Analysis for Fly Tracking--Six Zones</td>
		</tr>
		<tr>
			<td>Basler scA1390-17fm</td>
			<td>National Instruments</td>
			<td>779980-01</td>
			<td>Digital Camera NOTE: driver for camera available at Baslerweb.com</td>
		</tr>
		<tr>
			<td>8 mm lens</td>
			<td>National Instruments</td>
			<td>780024-01</td>
			<td>Lens for Basler Digital Camera</td>
		</tr>
		<tr>
			<td>Ground Glass Diffuser Plate</td>
			<td>Edmund Optics</td>
			<td>custom</td>
			<td>Diffuses light, 25 cm x 30 cm</td>
		</tr>
		<tr>
			<td>US Std. No. 100</td>
			<td>Fischer Scientific</td>
			<td>04-881X</td>
			<td>Sieve with nominal opening of 150 &#956;m</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Lighting Option 1</td>
		</tr>
		<tr>
			<td>LED backlight 660 nm (20 cm x 20 cm)</td>
			<td>Spectra West</td>
			<td>BL47192</td>
			<td>a simpler but more expensive lighting option.</td>
		</tr>
		<tr>
			<td>Power Supply for LED Backlight</td>
			<td>Spectra West</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Lighting Option 2</td>
		</tr>
		<tr>
			<td>660 nm LEDs</td>
			<td>Superbrightleds</td>
			<td>RL5R1330</td>
			<td>Wavelength 660 nm (approximately 7 x 7 LED array for a 14.7 inch x 9.75 inch panel)</td>
		</tr>
		<tr>
			<td>Linear DC Power Supply</td>
			<td>GW Instek</td>
			<td>GPS-1830D</td>
			<td>Power supply for LED Panel</td>
		</tr>
		<tr>
			<td>Solderless Breadboard</td>
			<td>Digikey</td>
			<td>922354-ND</td>
			<td>Breadboard for LEDs</td>
		</tr>
	</tbody>
</table>

a single-fly assay for foraging behavior in drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

The data analysis software and the layout, an example of which can be seen in Figure 1, are used to evaluate each fly&#39;s performance during its 10 min trial according to a set of analysis criteria. The following criteria are used to determine whether data from each fly will be used for data analysis and are designed to eliminate those flies that are unable to perform the food search task due to injury, illness, stress, or lack of motivation.
Flies that are inactive for more...

Watch this Scientific Journal Video about A Single-fly Assay for Foraging Behavior in Drosophila at JoVE.com

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

a-single-fly-assay-for-foraging-behavior-in-drosophila

Research

JoVE Journal

Neuroscience

13.1K Views.  University of California-San Diego. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. 

Video: A Single-fly Assay for Foraging Behavior in Drosophila

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.