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.
