Name: A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila (Video) | JoVE Journal
Uploaded: 2014-02-19T15:30:00.000+00:00
Duration: 9 min 6 s
Description: Learn a low-cost method for analyzing seizure-like activity and locomotion in Drosophila melanogaster using a webcam and custom software. This technique enables researchers to study genetic and pharmacological effects on behavior.

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The authors also wish to express thanks to Kris Burner, Stephen McKinney, Laura Tobin, Jenny Gilbreath, Ashley Olley, Megan Hoffer, and Megan Hyde for their work in fine-tuning this assay.

1. Preparation of Individual Flies for Locomotion Assay
<ol>
	<li>Transfer flies by gently tapping into individual empty vials and cap the vials with a cotton plug. Allow flies to sit undisturbed 20-30 min before observation. It is important to not anesthetize the flies within the hours prior to behavioral observation as previous studies have found anesthesia exposure can alter behavior as compared to unanesthetized flies14.</li>
	<li>Gently tap the fly out of the vial and place individual flies under a 5 cm diameter Petri dish cover (5 mm high) with small slits to allow air passage. Illuminate from below and observe the flies using a webcam mounted above the Petri dish.</li>
</ol>
2. Preparation of Individual Flies for Seizure Assay
<ol>
	<li>Feed two-day old seizure sensitive flies either standard yeast/cornmeal/agar media or standard media mixed directly with drug. For the data presented here, flies were fed either 1 g of standard media or 1 g of standard media mixed with 25 mg of metformin.</li>
	<li>After the flies have fed for two days on the media or media plus drug, transfer them by gentle tapping into individual empty vials and cap the vials with a cotton plug. Allow flies to sit undisturbed for 20 min before observation. It is important to not anesthetize the flies within the hours prior to behavioral observation as previous studies have found anesthesia exposure can alter behavior as compared to unanesthetized flies14.</li>
	<li>Vortex individual vials containing a single fly on a lab vortexer using the highest setting for 10 sec. Place the immobilized fly on a blank white sheet of paper directly below a webcam mounted above the paper.</li>
</ol>
3. Video Recording
<ol>
	<li>Record the movement or SLA using the HandyAvi software program <a href="http://www.azcendant.com/download.htm">http://www.azcendant.com/download.htm</a>. This program uses the webcam to capture images based upon the user settings.</li>
	<li>Select the Time-Lapse images option under the Capture tab, When the window opens, select the webcam as the capture device. For most experiments the video frame size of 640 x 480 is adequate.</li>
	<li>Select Intel IYUV codec for the compression and choose 0.1 sec/frame for the seizure assay or choose 0.1-0.5 sec/frame for the movement assay. Under the advanced tab, select create a .bmp image file for each capture and select a folder for the program to store the images that it takes (the image stack).</li>
	<li>Place a dead fly on the sheet of paper. Click on the Video settings box and adjust the settings under the Device settings tab so that there is a bright white background and a clear contrast with the darker fly.</li>
	<li>Click the start button to begin the recording. Once the recording period has finished, click the stop button. The image stack should now be in the folder selected. Note: Before recording, ensure that the folder selected for the image stack is empty.</li>
</ol>
4. Data Analysis Using ImageJ
<ol>
	<li>Open NIH&#39;s free image processing software ImageJ, which can be downloaded at <a href="http://rsbweb.nih.gov/ij/download.html">http://rsbweb.nih.gov/ij/download.html</a>. Note: In addition to downloading the program, the Multitracker plug-in must be downloaded and installed as indicated at <a href="http://rsbweb.nih.gov/ij/plugins/index.html">http://rsbweb.nih.gov/ij/plugins/index.html</a>.</li>
	<li>Import the image stack into ImageJ by clicking on the File tab and then selecting Import &#8594; Image sequence.
	<ol>
		<li>Select the folder that contains the image stack, highlight one of the images and select open. A Sequence Options dialogue box will open. Click the Sort names numerically and then select OK and the image stack will be loaded into ImageJ.</li>
	</ol>
	</li>
	<li>Threshold all the images so that each consists of a white background with the fly being converted to a black dot. To begin this process, convert the image to an 8-bit format by selecting Type &#8594; 8 bit under the Image tab.
	<ol>
		<li>Click on the Image tab again and select Adjust &#8594; Threshold. This will open the Threshold dialogue box in which the threshold cut off point can be adjusted.</li>
		<li>Scan through the images to see if there are extra black dots or if a slice lacks a black dot. If this is the case, adjust the threshold to alleviate the problem. Usually the threshold does not need to be adjusted as the program defaults to an adequate value. 
		Note: Every slide in the stack should have just one black dot representing the position of the fly.</li>
		<li>Select apply in the Threshold dialogue box and a Convert to Mask dialogue box will open. Do not click any of the options but simply click OK to threshold the image stack.</li>
	</ol>
	</li>
	<li>After thresholding the image stack, select the Multitracker plug-in under the Plugins tab in order to measure the movement of the fly in pixels. The Object Tracker dialogue box will appear.
	<ol>
		<li>Click all of the four options given in the dialogue box and then select OK. The Multitracker plug-in will then attach coordinates to the black dot in each slice and list these in the Results window as well as the total path length in pixels. The Paths window gives a graphical display of the path the fly took.</li>
		<li>Copy the x-&#160;and y-coordinate list from the Results window and paste the data into an Excel spreadsheet. If any slide in the stack does not have a black dot in it, the Multitracker program will halt at that slide. If this occurs, it is necessary to go back to step 4.2 and re-threshold the stack to ensure that the fly registers as a black dot in the slide in question.</li>
	</ol>
	</li>
	<li>In order to convert the path lengths from pixels to cm, take a separate image of two dots that are spaced one cm apart on a blank white piece of paper.
	<ol>
		<li>Follow the above steps (3.1-4.4.2), to determine the x, y&#160;pixel coordinates for the two dots and use this information to determine the number of pixels corresponding to a cm. This conversion factor is used in step five to generate movement data in cm. Note: Once this is done, it is important to keep the camera fixed for all future recordings. Every time the camera is moved or adjusted, one needs to repeat this step to convert the pixels to cm.</li>
		<li>The path length that is generated by the Multitracker plug-in overestimates movement because slight fluctuations in body position and light intensity/reflection will affect the size and center of the black dot that is generated when thresholding the images. This noise must be removed in order to get an accurate estimation of fly movement.</li>
	</ol>
	</li>
</ol>
5. Data Analysis Using Excel
<ol>
	<li>Remove the noise from the recorded data by importing the series of x, y&#160;coordinates generated by the Multitracker program into Fly Analysis, an Excel Visual Basic program created by the authors. (The program is available by contacting the authors.)</li>
	<li>Click the Click Me button and select a threshold value cut-off. Select a threshold value of 0.5 cm/sec for SLA analysis or select a value between 0.1- 0.3 cm/sec for movement analysis. Click OK and the program will calculate the velocity, time and distance of movement. Note: The program uses a sliding window analysis to evaluate the data in sequential 0.5-1.0 sec bins and calculates the velocity during that window. Anything above the user-defined cutoff is treated as fly movement.</li>
</ol>

<ol><li>Stone, B., Evans, L., Coleman, J., Kuebler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+and+pharmacological+manipulations+that+alter+metabolism+suppress+seizure-like+activity+in%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Genetic and pharmacological manipulations that alter metabolism suppress seizure-like activity in.</a> 1496, 94-103 (2013).</li>
<li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+mutants+of+Drosophila+isolated+by+countercurrent+distribution%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Behavioral mutants of Drosophila isolated by countercurrent distribution.</a> Proc. Natl. Acad. Sci. U.S.A. 58, 1112-1119 (1967).</li>
<li>Slawson, J. B., Kim, E. Z., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-Resolution+Video+Tracking+of+Locomotion+in+Adult+Drosophila+melanogaster%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">High-Resolution Video Tracking of Locomotion in Adult Drosophila melanogaster.</a> J. Vis. Exp. (24), (2009).</li>
<li>Spink, A. J., Tegelenbosch, R. A., Buma, M. O., Noldus, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+EthoVision+video+tracking+systema+tool+for+behavioral+phenotyping+of+transgenic+mice%5BTitle%5D)+AND+%22Physiol.+Behav%22%5BJournal%5D)">The EthoVision video tracking systema tool for behavioral phenotyping of transgenic mice.</a> Physiol. Behav. 73, 731-744 (2001).</li>
<li>Kohlhoff, K. J., Jahn, T. R., Lomas, D. A., Dobson, C. M., Crowther, D. C., Vendruscolo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+iFly+tracking+system+for+an+automated+locomotor+and+behavioural+analysis+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22Integr.+Biol%22%5BJournal%5D)">The iFly tracking system for an automated locomotor and behavioural analysis of Drosophila melanogaster.</a> Integr. Biol. 3, 755-760 (2011).</li>
<li>Gomez-Marin, A., Partoune, N., Stephens, G. J., Louis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+tracking+of+animal+posture+and+movement+during+exploration+and+sensory+orientation+behaviors%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Automated tracking of animal posture and movement during exploration and sensory orientation behaviors.</a> PLoS ONE. 7, (2002).</li>
<li>Wolf, F. W., Rodan, A. R., Tsai, L. T., Heberlein, U. <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, 11035-11044 (2002).</li>
<li>Martin, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+portrait+of+locomotor+behaviour+in+Drosophila+determined+by+a+video-tracking+paradigm%5BTitle%5D)+AND+%22Behav.+Processes%22%5BJournal%5D)">A portrait of locomotor behaviour in Drosophila determined by a video-tracking paradigm.</a> Behav. Processes. 67, 207-219 (2004).</li>
<li>Meunier, N., Belgacem, Y. H., Martin, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+feeding+behaviour+and+locomotor+activity+by+takeout+in+Drosophila%5BTitle%5D)+AND+%22J.+Exp.+Biol%22%5BJournal%5D)">Regulation of feeding behaviour and locomotor activity by takeout in Drosophila.</a> J. Exp. Biol. 210, 1424-1434 (2007).</li>
<li>Koudounas, S., Green, E. W., Clancy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reliability+and+variability+of+sleep+and+activity+as+biomarkers+of+ageing+in+Drosophila%5BTitle%5D)+AND+%22Biogerontology%22%5BJournal%5D)">Reliability and variability of sleep and activity as biomarkers of ageing in Drosophila.</a> Biogerontology. 13, 489-499 (2012).</li>
<li>Catterson, J. H., Knowles-Barley, S., James, K., Heck, M. M., Harmar, A. J., Hartley, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dietary+modulation+of+Drosophila+sleep-wake+behaviour%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Dietary modulation of Drosophila sleep-wake behaviour.</a> PLoS One. 5, (2010).</li>
<li>Donelson, N., Kim, E. Z., Slawson, J. B., Vecsey, C. G., Huber, R., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-Resolution+positional+tracking+for+long-term+analysis+of+Drosophila+sleep+and+locomotion+using+the+%27%27Tracker%27%27+program%5BTitle%5D)+AND+%22PLos+ONE%22%5BJournal%5D)">High-Resolution positional tracking for long-term analysis of Drosophila sleep and locomotion using the ''Tracker'' program.</a> PLos ONE. 7, (2012).</li>
<li>Duboff, B., Fridovich-Keil, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mediators+of+a+long-term+movement+abnormality+in+a+Drosophila+melanogaster+model+of+classic+galactosemia%5BTitle%5D)+AND+%22Dis.+Model+Mech%22%5BJournal%5D)">Mediators of a long-term movement abnormality in a Drosophila melanogaster model of classic galactosemia.</a> Dis. Model Mech. 5, 796-803 (2012).</li>
<li>Seiger, M. B., Kink, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+anesthesia+on+the+photoresponses+of+four+sympatric+species+of+Drosophila%5BTitle%5D)+AND+%22Behav.+Genet%22%5BJournal%5D)">The effect of anesthesia on the photoresponses of four sympatric species of Drosophila.</a> Behav. Genet. 23, 99-104 (1993).</li>
<li>Pavlidis, P., Ramaswami, M., Tanouye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Drosophila+easily+shocked+gene%3A+a+mutation+in+a+phospholipid+synthetic+pathway+causes+seizure%2C+neuronal+failure%2C+and+paralysis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The Drosophila easily shocked gene: a mutation in a phospholipid synthetic pathway causes seizure, neuronal failure, and paralysis.</a> Cell. 79, 2333 (1994).</li>
<li>Royden, C. S., Pirrotta, V., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+tko+locus%2C+site+of+a+behavioral+mutation+in+D.+melanogaster%2C+codes+for+a+protein+homologous+to+prokaryotic+ribosomal+protein+S12%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The tko locus, site of a behavioral mutation in D. melanogaster, codes for a protein homologous to prokaryotic ribosomal protein S12.</a> Cell. 51, 165-173 (1987).</li>
<li>Parker, L., Padilla, M., Du, Y., Dong, K., Tanouye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+as+a+model+for+epilepsy%3A+bss+is+a+gain-of-function+mutation+in+the+para+sodium+channel+gene+that+leads+to+seizures%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Drosophila as a model for epilepsy: bss is a gain-of-function mutation in the para sodium channel gene that leads to seizures.</a> Genetics. , 187-534 (2011).</li>
<li>Hardie, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+AMP-activated+protein+kinase+in+the+metabolic+syndrome+and+in+heart+disease%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">Role of AMP-activated protein kinase in the metabolic syndrome and in heart disease.</a> FEBS Lett. 582, 81-89 (2008).</li>
<li>Fulgencio, J. P., Kohl, C., Girard, J., Pegorier, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+metformin+on+fatty+acid+and+glucose+metabolism+in+freshly+isolated+hepatocytes+and+on+specific+gene+expression+in+cultured+hepatocytes%5BTitle%5D)+AND+%22Biochem.+Pharma%22%5BJournal%5D)">Effect of metformin on fatty acid and glucose metabolism in freshly isolated hepatocytes and on specific gene expression in cultured hepatocytes.</a> Biochem. Pharma. 62, 439-446 (2001).</li>
</ol>

The authors declare that they have no competing financial interests.

When examining locomotor or movement patterns in Drosophila, it is useful to be able to extract information regarding the distance traveled, the velocity of movement and the pattern of movement. In order to extract this information, expensive equipment has traditionally been employed that is often prohibitive for smaller labs or labs that wish to use such assays sparingly4,8,9.
The assay described here can be used to determine the distance traveled (Figures 2</stron...

Given its short lifespan and the robust genetic tools available, Drosophila melanogaster is an excellent model system for investigating the etiology of various diseases and the underlying physiology of various biological processes. In many cases, it is advantageous to measure the effects that behavioral, genetic or pharmacological manipulations have on locomotion in these model organisms1,2.
There are a variety of methods that are commonly used for measuring fly movement in two-dimensions3-7. These systems can support the tracking of multiple flies simultaneously and can measure velocity, record path lengths and record the percentage of time a fly has spent moving. They have been used to study movement in a variety of contexts including the effects of drugs on locomotion and the sexual dimorphic nature of fly movement6-9. The major drawback of these systems is cost of the tracking system or the corresponding software and camera. In some cases, this can run in the thousands of dollars. Cost is a particular concern for a lab that would only need limited use of such a system, for example, quantifying locomotive patterns of a newly isolated mutant.
A simpler but less robust method is to use systems which record movement based on how many times a fly crosses an infrared light beam path that is placed in the middle of a closed tube10,11. While such systems can give valuable information on movement and sleep-wake cycles, they can over or under estimate movement because they fail to capture the actual path of the fly. For example, flies that exhibit considerable movement at the ends of the tube will register as low movement flies although additional high-resolution methods have been used to try and circumvent these limitations12.
Simpler and cheaper still are climbing devices that measure geotaxis, the upward movement of flies, through a single tube or a series of tubes2,13. While such systems are cheap and can easily identify geotaxis defects, they fail to capture many other aspects of movement that would be of interest to investigators.
For many labs, a low-cost robust analysis system that is simple to set-up and operate would be an advantageous tool for characterizing behavioral differences in D. melanogaster strains. Here we describe an assay that can be set-up for less than two hundred dollars and is able to give information about the path, velocity and duration of the fly&#39;s movement. To demonstrate the efficacy of the assay, we present data showing that it can be used to identify; 1) a locomotor defect in a Bang-sensitive (BS) mutant that is susceptible to seizures and 2) the ability of the drug metformin, which is commonly used to treat type II diabetes, to reduce the intensity of seizure-like activity (SLA) in two BS mutants.

<table><tbody><tr><td>WebCam</td><td>Logitech</td><td>Pro900</td><td>Any quality webcam will suffice.</td></tr><tr><td>HandiAVI time-lapse software</td><td>Azcendant software</td><td></td><td>Latest version can be found at http://www.azcendant.com/</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>Latest version can be found at http://rsbweb.nih.gov/ij/download.html</td></tr><tr><td>Multiracker Plug-In</td><td>NIH</td><td></td><td>Latest version can be found at http://rsbweb.nih.gov/ij/plugins/index.html</td></tr><tr><td>Vortexer</td><td>VWR</td><td>Vortex Genie 2</td><td>Most standard size vortexers such as the Vortex Genie 2 will suffice.</td></tr><tr><td>5 cm Petri dish cover</td><td>LabM Limited</td><td>D011</td><td>Smaller or larger Petri dish covers can be used for an arena in movement assay.</td></tr><tr><td>Light box</td><td>custom made</td><td></td><td>Built from scrap material. Illumination is used for the locomotion assay. Depending on the room lighting, it is possible to perform the assay without the light box.&amp;nbsp;</td></tr></tbody></table>

a low-cost method for analyzing seizure-like activity and movement in drosophila

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

The technique described here has been used previously to analyze differences in SLA in the Drosophila BS mutants1. The results presented here involve the BS strains easily shocked (eas), bang senseless (bss), and technical knockout (tko). The eas locus encodes an ethanolamine kinase involved in phosphatidyl ethanolamine synthesis15 and the tko locus encodes a mitochondrial riboprotein16. The bss muta...

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

The overall goal of this procedure is to identify alterations in locomotor or seizure behaviors in drosophila. This is accomplished by first separating the flies into individual vials with using anesthesia. Next, a simple webcam is used to record the Flies movement.The data are then imported into Image J to begin the analysis. Finally, custom made fly analysis software is used to determine the distance, velocity, and duration of movement during the recording period. Ultimately, this assay can show changes in locomotor or seizure behavior that are associated with genetic and pharmacological manipulations.Now, this low-cost recording technique that we're gonna describe can be used to both analyze seizure behavior and locomotion in fruit flies. In particular, we've used it to identify genetic and pharmacological manipulations that modify seizure behavior in those flies. But today, Chris Burner, an undergraduate research assistant in my lab, will be demonstrating this technique To begin set up the webcam that will record the flies during the locomotor and seizure assays.Once the camera is in place, open the handy a VI software program. To adjust the recording settings, select the time-lapse images option under the capture tab, and when the new window opens, select the webcam as the capture device. For most experiments, a video frame size of six 40 by four 80 is adequate Under the compression option, select the Intel IUV codec and set the seconds per frame according to the assay.Under the advanced tab, create a bitmap image file for each capture and select a folder for the program to store the image stack once recorded. Next place a dead fly on a sheet of paper. Click on the video settings box, and under the device settings tab, adjust the settings until the bright white background is in clear contrast with the darker fly.Click the start button to begin the recording. Once the recording period has finished, click the stop button. The image stack should now be in the folder selected to begin the locomotor activity assay.Transfer the flies into individual vials capped with a cotton plug. Allow the flies to sit undisturbed for 20 to 30 minutes before proceeding. After this time, gently tap the fly to be tested out of the vial and under a Petri dish cover.The cover should contain small slits to allow airflow position the fly under the webcam set up earlier. When ready, press start to begin recording locomotor activity. The first step of the seizure assay is to feed two day old seizure sensitive flies, either standard media or media mixed with the drug to be tested.After two days of feeding, transfer the flies by gentle tapping into individual empty vials and cap the vials with a cotton plug. Allow the flies to sit undisturbed for 20 minutes before observation. When ready, vortex a vial on a lab vortex or using the highest settings for 10 seconds.Immediately place the immobilized fly on a white sheet of paper directly below the webcam. Once in place, begin recording. This flies activity.The fly will undergo rapid uncoordinated movements interspersed with periods of paralysis. The recording is ended once the fly has righted itself. To begin analyzing the data, open the free image processing software image J once opened.Import the desired image stack by first clicking on the file tab and then selecting import. And finally, image sequence. Select the folder that contains the image stack and open an image.When the sequence options dialogue box opens, select sort names numerically and then select.Okay. The image stack will now be loaded into Image J.Next, select the image tab and set the type of value to eight bit. Click on the image tab again and select threshold under the adjust option.This sets the threshold in all of the images so that each consists of a white background with the fly being converted to a black dot. This also opens the threshold dialogue box where cutoff points can be adjusted. Scan through the images to see if any contain extra black dots or if the spot is missing.If necessary, adjust the threshold to alleviate any problems. Once adjustments have been made, select apply in the threshold dialogue box. When the convert to mask dialogue box opens, click okay to threshold the image stack.To measure the movement of the fly in pixels, select the multi tracker plugin under the plugins tab. When the object tracker dialogue box appears, click all four of the options given and select.Okay. The multi tracker plugin will then attach coordinates to the black.in each slice, and list these in the results window, as well as the total length in pixels. The paths window gives the graphical display of the path the fly took. Copy the X and Y coordinate list from the results window and paste the data into an Excel spreadsheet.If any slide in the stack does not have a black. in it, the multi tracker program will halt at that slide. If this occurs, re threshold the stack to ensure that the fly registers as a black.in the slide in question. To convert the path lengths from pixels to centimeters, take an image of two dots spaced a known distance apart on a blank white piece of paper. Determine the XY pixel coordinates for the two dots, and use this information to determine the number of pixels corresponding to one centimeter for more accurate results.Remove noise from the images by importing the XY coordinates into a custom made fly analysis program. Once opened, import data from each genotype or treatment into separate spreadsheets. Next, select the summary sheet tab and click on the click me button.Select a threshold value according to the type of analysis, click okay, and the program will calculate the velocity, time, and distance of movement. These representative paths show seizure-like activity in mutant flies with and without metformin treatment. Seizure activity was decreased with treatment in the two mutants tested.Metformin significantly reduced both the distance moved during the seizure assay and the total duration of seizure-like activity. In contrast, there was no change in the average velocity in these individual locomotor paths. The path of the wild type CS fly shows concentration around the periphery of the circular arena.While the path of A mutant BSS fly shows the same concentration, but with less movement overall. When tested for locomotion in a novel environment, the BSS mutants displayed a significant reduction in path length as compared to CS flies. The assay also detected changes in the velocity of movement while the CS fly explored during the locomotor assay.The BSS fly remained relatively inactive throughout the trial except for a few bursts of activity. One of the most important things to keep in mind when performing either of these behavioral assays with this technique is to ensure that you don't anesthetize the flies prior to doing the recordings. It turns out that anesthetization will affect both seizure behavior and locomotive behavior and can significantly alter your results.

a-low-cost-method-for-analyzing-seizure-like-activity-movement

Research

JoVE Journal

Neuroscience

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.

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus.  I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

This video describes how to track freely walking flies to assess their general locomotion ability. The featured protocol demonstrates the assay's setup and data analysis using video tracking software.

Tracking Freely Walking Flies: A Method to Assess Locomotion in Drosophila

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Measurement of Larval Activity in the Drosophila Activity Monitor

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.

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

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

A Simple Technique to Assay Locomotor Activity in Drosophila

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

This protocol details the use of Drosophila larvae to identify unique antiseizure compounds for the treatment of epilepsy.

Seizure Activity Induced by Electroshock in Drosophila Larvae

Learn a low-cost method for analyzing seizure-like activity and locomotion in Drosophila melanogaster using a webcam and custom software. This technique enables researchers to study genetic and pharmacological effects on behavior.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

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