Our protocol presents two assays, quantifying C.elegans movement. These methods evaluate motility phenotypes such as those exhibited by models of Amyotrophic Lateral Sclerosis, or ALS. The Radial Locomotion Assay is a cost-effective and easy method to detect crawling on a solid surface.
The Swimming Assay uses computer-based tracking for unbiased detection of thrashing movements in liquid. These methods are useful for quantifying movement differences in C.elegans. Although we study ALS, they can be used for any strain with altered motility.
Flip the NGM assay plates upside down. And label the bottom with an identifier for the C.elegans strains to be assayed. Make a small dot with the marker in the center of the upside down plate.
While working with a dissecting microscope, transfer worms to the center of the assay plate. And set a timer for 30 minutes. Put the lid back on the plate and set it aside.
Continue transferring worms until all strains are on the designated assay plates. After 30 minutes, begin scoring the first plate by removing the lid and placing the plate face down under the dissecting microscope. Adjust the microscope focus until all worms are visible through the Auger.
Using a different colored felt tip pen from the center point, put a small dot at the location of each worm. Check the edge of the plate as some worms may end up there. Also, count and record how many worms did not move from the center point.
Measure the distance from the center point to the final location markings for each worm, and record the distance using a ruler. The first worm point is marked with a dash. Record length data for each dot consecutively by rotating the plate in a clockwise fashion.
Open the associated software to set up and record the videos. Click the video capture icon. Press down on the dimmer knob and turn it clockwise to adjust the light.
In the video capture window, click on the settings tab and adjust the video mode to 2456x2052_Mono8, frame rate to 14, output as monochrome. Exposure of 0.00300 seconds. Gain to 1 dB.
Gamma to one. And rotate to 180. Move back to the capture tab, select the recording folder and assign a file name by entering it into the file prefix text box.
Set other capture settings like buffer, to 128 frames, and duration to one minute. Place the assay plate lit up on the device stage and center it in the video capture screen, then remove the lid. Use a micropipette to wash about 50 worms in 1 ml of M9 onto the assay plate.
Gently swirl the plate to bring the animals to the center, or use a micropipette to add a few drops of M9 to separate the animals. Set a timer for 60 seconds to allow worms to acclimate to swimming. Adjust the light knob such that the display is as bright as possible without overexposure.
Manually adjust the camera focus by turning the focusing ring on the lens body of the camera, while watching the display. After 60 seconds press the record button, select the first button on the workflow menu Import Image Sequence"and find and double click on a video. From the workflow menu select Set Sequence Info"In the new menu window check the naming scheme, add notes, and validate metadata.
Select Adjust Image"and a new pop-up menu called Image Adjustments"will open. Adjust image processing settings by setting the background smoothing to 10, Gaussian smoothing to five, Fill holes to two, Small object filter to zero, and skipping Integral Derivative Segmentation. Adjust the threshold level such that the animals are completely filled with green, but still distinct from the background.
Then click Apply"From the workflow menu, select Detect and Track"select three to seven worms and click the Detect Worms"button in the detection tab. Move to the tracking tab. In the tracking parameters check Use Backtracking"and uncheck Track worms at the edge of the image.
Set the max tracked hypothesis to five. Set the tracking mode to swimming. Move to the advanced settings section and set the frames worms can touch boundary to 50.
Frames worms can overlap to 500. Position tolerance to 0.50. And shape tolerance to 0.50.
Save these settings and deploy them for all videos in an experiment by saving them as a configuration. Navigate to the configuration manager in the upper left icon menu, and click the Save"icon. Give this configuration a name and description.
And click Okay"In the workflow menu, click Save Project"Track the videos by going to the workflow menu and clicking the Batch"icon. Click the Add"button under the file selection section of this batch-processing menu. navigate to and select all the project files to be processed.
Then click Open"Click the Start"button, and note the green progress indicator on the first file. Allow all the files to be processed. When all the files read has finished, close the software.
Select Analyze Data"Navigate to the track summary. And use the Export"button in the bottom right to export data in a spreadsheet readable format. Use the Turn Count"and Track Duration"to calculate turns per minute for each track in using spread sheeting functions.
Unstimulated dispersion of developmentally-staged L4 larva of five different strains, was measured using the Radial Locomotion Assay. And graphed as micro meter per minute traveled. The data displayed as bar graphs makes relative differences between strains clearer.
Whereas the final displacement of each worm plotted within the graph, allows the variation within the population to be better visualized. Rates of swimming in terms of thrashing or undulation frequency in the liquid, were measured using unbiased computer-assisted scoring and analysis. And graphed as thrashes per minute.
The bar graph data makes relative differences between strains easier to see. Whereas data from each individual worm scored are plotted within the graph, allowing the variation within the population to be better visualized. In the Radial Locomotion Assay, mild TDP-43 strain was not significantly different than the wild-type N2 strain.
However, in the Swimming Assay, both the mild and strong TDP-43 strains were not only significantly different from the wild-type N2, but also from one another. The ALS mutant TDP-43 strain have severe crawling impairment by Radial Locomotion, and they do not thrash in liquid. The tau-expressing strain have severe impairments in Radial Locomotion, but can thrash in the Swimming Assay.
The most important aspect to consider with these methods, is maintaining consistent controls between replicates. This ensures that variations in motility are caused by phenotypic differences and not environmental conditions. These methods provide a comprehensive assessment of two major C.elegans motility paradigms.
Follow-up methods could include additional behavioral characterization, biochemical analysis, or investigation of muscle or neuron function.
