Name: High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster (Video) | JoVE Journal
Uploaded: 2009-02-20T00:00:00.000+00:00
Duration: 9 min 8 s
Description: Learn high-resolution video tracking of locomotion in adult Drosophila melanogaster without anesthesia. This method enables detailed quantification of fly movement for behavioral neuroscience research.

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This work was supported by National Institutes of Health Grant R01 GM54408 awarded to L.C. Griffith. We would like to thank Fred Wolf for all his help in designing our square chamber and setting up our assay, Frank Mello at the Brandeis University machine shop for building our chamber, and Dan Valente and Tim Lebestky for helpful conversations regarding analysis issues.

Part 1: Feeding and Management of flies
<ol>
	<li>Flies should be grown in bottles containing yeast-free standard media. You will need large numbers of flies, so crosses need to be set up accordingly. Flies should be grown in a 12-hour light:dark cycle at 25ºC.</li>
	<li>Flies should be collected soon after eclosion (1-3 days). Flies can be worked with at this stage using a carbon dioxide diffuser, and should be sorted into test tubes containing autoclaved or yeast-free media. We use males only, stored 10 to a test tube.</li>
	<li>Allow at least a day or two after use of carbon dioxide before running the behavioral experiments. We run flies in our behavioral assay 3-5 days following ECLOSION.</li>
	<li>Assays should always be done within the same 2-3 hour time window each day to avoid circadian rhythm issues.</li>
</ol>
Part 2: Setting up the tracking system in a controlled environment
<ol>
	<li>All experiments are done in an environment room which maintains a constant temperature of 25ºC with 70% humidity.</li>
	<li>Suspend a digital video camera over the area to be recorded (camera facing downwards). Since our tracking software is contrast-based, we suspend the camera over a light box. The camera records videos directly onto a Dell computer via a firewire connection. We use a Sharp digital video camera, hooked up to a Dell computer with Windows Moviemaker (Vista version) for video acquisition.</li>
	<li>Air pulses will be administered in this protocol, so a source for air flow must be present in the room. Using rubber tubing, connect the air source to a carbon filter for air filtration.</li>
	<li>Using more rubber tubing, connect the other side of the carbon filter to an Erlenmeyer flask via a hollow rubber stopper, and fill the flask with about a half inch of water. This will humidify the air.</li>
	<li>The flask should also have a sidearm, which will connect to a Y-shaped valve via rubber tubing.</li>
	<li>One branch of the Y-valve should be connected to a flowmeter. The other branch should deliver air to the square locomotion chamber.</li>
</ol>
Part 3: Delivering flies to the locomotion chamber
<ol>
	<li>The square locomotion chamber is based on the design from Wolf et al. 20021, with the addition of small drop trays to prevent the flies from flying within the chamber. The chamber should be taken apart, and all components should be thoroughly wiped down with 70% ethanol and dried before use.</li>
	<li>Anesthesia immediately before conducting a behavioral assay can potentially compromise performance. To avoid this, flies are gently knocked into the chamber using a funnel with a small outlet. To make an outlet of the right size, attach a blue pipette tip (for P1000) to the end of a funnel. Use scissors to cut off the very end of the pipette tip to make the opening big enough for a fly to pass through.</li>
	<li>The top of our chamber is a small piece of plexiglass secured by screws around the edges. When the screws are removed, the holes can be used to deliver flies into the chamber. Position the plexiglass top so that the screw hole sits directly over the inner chamber instead of where a screw normally sits. Take a test tube containing the flies and place it upside down over the funnel, which should then be placed into the screw hole.</li>
	<li>Do NOT move the funnel, as this could hurt or damage the flies. Instead, gently bang the whole chamber, funnel and all, until all flies are inside the chamber. This knocking should be done on a mouse pad to absorb the shock of this banging. When flies are inside, remove the funnel, and re-position the plexiglass top over the screw beds. Then re-screw the plexiglass top into place.</li>
</ol>
Part 4: Running the Locomotor Assay
<ol>
	<li>Once the flies are properly loaded, let them acclimate in the chamber for 30 minutes. This acclimation period should take place in the controlled environment (25ºC, 70% humidity), and the chamber should be placed on top of the light box (which should be turned on). Turn other room lights off.</li>
	<li>Following the acclimation period, switch the Y-valve in the air flow system such that the air will only flow to the flowmeter. Turn on the air, and ramp the air up to the desired speed (4.0-6.0 L/min). We generally use 5.0-5.5 L/min, but any speed in that range will work.</li>
	<li>Once the desired airspeed is attained, switch the Y-valve so that the air flows to the locomotion chamber. Time this air pulse for 15 seconds, and then abruptly turn the air off. As you turn the air off, switch the camera to record mode. (This part takes practice to do all at once).</li>
	<li>Stop the recording after the desired time and SAVE. We record 30 second trials at 10 frames per second (at least 8 trials per genotype).</li>
</ol>
Part 5: Analysis of Videos
<ol>
	<li>We use the Dynamic Image Analysis System (DIAS) software 3.2 for motion tracking2. In order to use this software, we first convert our videos to an AVI file format with Quicktime 7.5.5.</li>
	<li>To track the flies based on contrast, we use the “Autotrace by Threshold” function. We then use the “Make Path from Trace” function to digitize these traces.</li>
	<li>To output the instantaneous speeds of each fly, we use the “Compute Parameters” function to produce a database file (DIAS-specific).  Using this function, we also smooth the data using a “5,15,60,15,5” Tukey Smoothing Window.</li>
	<li>Once this information is outputted as a database file, it can be opened in Microsoft Excel. We use a Matlab script to compile the instantaneous speeds within excel and calculate additional parameters necessary for understanding movement dynamics and bout structure.</li>
</ol>
Representative Results:
Figure 1 shows representative traces from wild type Canton S organisms (Figure 1, left panel) and flies null for the dCASK gene which were generated by crossing two large overlapping deletions (Df(3R)x307 and Df(3R)x313) (Figure 1, right panel). dCASK null flies have previously been shown to have locomotor problems using Buridan’s Paradigm3, and in our paradigm, compared to Canton S, they show greatly decreased locomotion. We always run wild type organisms first to make sure that conditions and behavior are within the normal range on a given day. We have observed deviations from standard responses in less than 5% of testing days. These differences can usually be attributed to problems with parameters of our controlled environment.
<img alt="Wild type vs mutant behavior paths; comparative tracking diagram, genetic influence on movement analysis." src="/files/ftp_upload/old/1096/1096fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1">
Figure 1. DIAS-generated traces of both Canton S wild type flies (above, left) and Locomotor-deficient dCASK Null flies generated from overlapping deletions (above,right). The traces represent what is seen in the recorded videos. Both pictures contrain traces from 8-10 flies run for 30 seconds in the video tracking assay following an air pulse.
<img alt="Bar chart comparing activity metrics under 'NO AIR' and 'AIR' conditions, including speed and acceleration." src="/files/ftp_upload/old/1096/1096fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2">
Figure 2. Wild type flies were run in the locomotor assay (above) following an air pulse (purple bars) and without an air pulse (blue). In all parameters calculated by the analysis program, there were no significant differences between the two conditions (determined with two-tailed student t-test). This demonstrates that following an air pulse, flies locomote normally in our setup.

<ol><li>Wolf, F. W., Rodan, A. R., Tsai, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-Resolution+Analysis+of+Ethanol-Induced+Locomotor+Stimulation+in+Drosophila%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">High-Resolution Analysis of Ethanol-Induced Locomotor Stimulation in Drosophila.</a> J Neurosci. 22 (24), 11035-11044 (2002).</li>
<li>Soll, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+computers+in+understanding+how+animal+cells+crawl%5BTitle%5D)+AND+%22International+review+of+cytology%22%5BJournal%5D)">The use of computers in understanding how animal cells crawl.</a> International review of cytology. 163, 43-104 (1995).</li>
<li>Martin, J. R., Ollo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+Drosophila+Ca2%2B%2Fcalmodulin-dependent+protein+kinase+%28Caki%29+is+localized+in+the+central+nervous+system+and+implicated+in+walking+speed%5BTitle%5D)+AND+%22The+EMBO+journal%22%5BJournal%5D)">A new Drosophila Ca2+/calmodulin-dependent protein kinase (Caki) is localized in the central nervous system and implicated in walking speed.</a> The EMBO journal. 15 (8), 1865-1876 (1996).</li>
<li>Levine, J. D., Funes, P., Dowse, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resetting+the+Circadian+Clock+by+Social+Experience+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Resetting the Circadian Clock by Social Experience in Drosophila melanogaster.</a> Science. 298, 2010-2012 (2002).</li>
</ol>

In our setup, a moderately strong air pulse transiently stops fly movement. When the air pulse ends, the flies are released from this stationary state and locomote normally, as shown in figure 2. Because this air pulse effectively synchronizes locomotion of the population, we use it to begin the trial so that we can also study the onset of movement following a momentum-breaking stimulus. The assay can, however, be done without an air pulse since locomotor parameters following initiation are unaffected by the air pulse...

<table><tbody><tr><td>Square Chamber</td><td>Tool</td><td>Machine Shop</td><td>N/A</td><td>Design from Wolf &lt;em&gt;et al.&lt;/em&gt; 2002&lt;sup&gt;1&lt;/sup&gt;</td></tr><tr><td>Digital Camera</td><td>Camera</td><td>Sharp</td><td>ViewcamZ VL-23</td><td></td></tr><tr><td>Flowmeter</td><td>Tool</td><td>Cole-Parmer</td><td>SY-32003-12</td><td></td></tr><tr><td>Light Box</td><td>Tool</td><td>DNASTAR</td><td>Seq-Easy</td><td></td></tr><tr><td>Charcoal Filter</td><td>Tool</td><td>Fisher Scientific</td><td>09-744-37</td><td></td></tr><tr><td>DIAS 3.2</td><td>Software</td><td>Soll technologies</td><td>N/A</td><td>www.solltechnologies.com</td></tr></tbody></table>

high-resolution video tracking of locomotion in adult drosophila melanogaster

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

In the video tracking assay, flies must be loaded into a square observation chamber without the use of anesthesia. To do this, a small funnel must be lined up with screw holes in the chamber, so as to knock the flies into the chamber. Using this funnel, screws then seal the holes of the chamber and an air pulse is administered through the air vents to initiate locomotion.A video camera suspended above the chamber, captures this locomotion and sends it to a computer where the paths of the flies are traced and analyzed. Hi, I'm Justin Slawson from the Griffith Lab here at the Biology Department at Brandeis University. Today we're going to show you a procedure for high resolution video tracking of locomotion.We're gonna use this procedure in our lab to study locomotive behavior in adult flies. So let's get started. To get started with the experiment, grow flies and bottles containing yeast-free standard media.This protocol requires a large number of flies, so crosses need to be set up accordingly. Flies should be grown in a 12 hour light dark cycle at 25 degrees Celsius. Collect the flies one to three days after Aus use a carbon dioxide diffuser to sort flies into test tubes containing yeast free media sort 10 males per test tube.Wait at least a day or two after use of carbon dioxide before running the behavioral experiments. Once the flies have been collected and sorted, they can be run in the video tracking system. The video tracking experiments are done in an environment room, which maintains a constant temperature of 25 degrees Celsius with 70%humidity.To videotape flies in motion, you'll need to suspend a digital video camera over the area to be recorded. With the camera facing downwards, we suspend the camera over a light box since our tracking software is contrast based. Next, using a fire wire cable, connect the camera to a computer.We use the software program, windows movie maker to record the video directly onto the computer. Since air pulses will be administered in this protocol, an airflow source must be present in the room. Rubber tubing connects the air source to a carbon filter for air filtration.Rubber tubing also connects the other side of the carbon filter to an erlenmeyer flask via a hollow rubber stopper. The flask should be filled with about a half inch of water, which will humidify the air. The flask should also have a small sidearm, which should be connected to a Ys shaped valve via more rubber tubing.One branch of the Y valve connects to a flow meter while the other branch connects to the square locomotion chamber. This square locomotion chamber is based on the design from Wolf Etal 2002 with the addition of small drop trays to prevent the flies from flying within the chamber before connecting the chamber to the tracking system, remember to use 70%ethanol to sterilize all the chamber components. Allow the components to drive before use.Now that the tracking system is ready, we can deliver the flies to the locomotion chamber to deliver the flies into the chamber. Remove the screws which normally keep the small plexiglass top of the chamber secure. Then position the plexiglass top so that the screw hole sits directly over the inner chamber.Anesthetizing the flies immediately before conducting. A behavioral assay can potentially compromise performance. To avoid anesthesia, use a funnel with a small outlet to gently knock flies into the chamber.To make a funnel with a small outlet, attach a P 1000 blue pipette tip to the end of the funnel. Use scissors to cut off the very end of the pipette tip to make the opening big enough for a fly to pass through. Next, take a test tube containing the flies and place it upside down over the funnel.Then place the end of the funnel into the screw hole. Do not move the funnel as this could hurt or damage the flies. Instead, gently bang the whole chamber funnel and all until all flies are inside the chamber.Use a mouse pad to absorb the shock of this banging. When flies are inside, remove the funnel and reposition the plexiglass top over the screw beds then scr the plexiglass top into place. Once the flies are properly loaded, place the chamber on top of a light box.Turn off the other lights in the environment room and let the flies acclimate in the chamber for 30 minutes. After the 30 minute acclimation period, proceed with the locomotor assay to begin the assay. Switch the Y valve in the airflow system such that the air will only flow to the flow meter, turn on the air and ramp the air up to the desired speed.Any speed within four to six liters per minute will work. We generally use five to five and a half liters per minute. Once the desired air speed is attained, switch the Y valve so that the air flows to the locomotion chamber.Time this air pulse for 15 seconds and then abruptly turn the air off as you turn the air off. Switch the camera to record mode. This step takes practice to do all at once.We record 32nd trials at 10 frames per second and at least eight trials per genotype. Stop the recording when the trial's complete and make sure you save the movie when the trials are complete. We can proceed with the video analysis to perform motion tracking of the locomotor assay videos.We use the dynamic image analysis system in order to use this software. Convert the videos to an A VI file format with QuickTime 7.5 0.5. Next, use the auto trace by threshold function to track the flies based on contrast, followed by the make path from trace function to digitize.These traces then apply the compute parameters function to output the instantaneous speeds of each fly into a database file. This function also allows for data smoothing. We use a 5 15 60 15 5 smoothing window.Once this information is outputted as a database file, we then open and save the files in Microsoft Excel. Excel files are processed using a MATLAB script to compile the instantaneous speeds and to calculate additional parameters necessary for understanding movement dynamics. And bout structure.Here is a representative result from this assay. The figure on the left shows traces from wild type Canton s organisms while the figure on the right shows flies null for the dask gene, which were generated by crossing two large overlapping deletions. As you can see in our paradigm, the Dask null flies show greatly decreased locomotion compared to Canton S, which has already been reported in the literature using a different type of locomotor assay.We've just shown you how to set up and run a locomotor video tracking assay. When doing this procedure, it's important to remember to perform the assays within the same two to three hour time window each day in order to avoid circadian rhythm issues and always run wild type organisms first to make sure that behavioral responses are within the normal range on a given day. So that's it.Thanks for watching and good luck with your experiments.

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