Evaluating Motor Impairment in C. elegans Models Using the Radial Locomotion Assay

Protocol

1. Radial locomotion assay

1. Prepare 100 mm or 150 mm diameter Petri dishes about half full of nematode growth media (NGM) and seeded with a uniform lawn of OP50 bacteria to cover the entire surface of the agar.
2. Flip the NGM assay plates upside-down and label the bottom with an identifier for the C. elegans strains to be assayed, 2 plates per strain (Figure 1A).
3. Make a small dot with the marker in the center of the upside-down plate (Figure 1A).
4. While working with a dissecting microscope, transfer 15-20 staged matched worms to the center of the assay plate directly over the center dot (visible through the NGM) using a platinum-wire pick (Figure 1B).
   NOTE: Try to transfer all the worms at the same time or as quickly as possible.
   1. Set a timer for 30 min after worms are placed in the center of the plate. Put the lid back on the plate and set it aside (Figure 1C).
5. Continue transferring worms until all strains are on the designated assay plates, making a note of approximately how long it takes to transfer between plates (aim for ~ 1 min between plates). Keep all plates in the same order that they were set up.
6. After 30 min, begin scoring the first plate.
   1. Remove the lid and place the plate face down under the dissecting microscope, such that the labeled back of the plate is facing up, and the NGM agar is between the line of sight and the worms. The plate will be upside-down from normal usage.
   2. Adjust the microscope focus until worms are visible through the agar.
   3. Using a different colored felt tip pen from the center point, put a small dot at the location of each worm - follow the worm tracks through the bacterial lawn to find the most distal animals.
   4. Check the edge of the plate as some worms may end up there. Also, count and record the number of worms in the center point dot (Figure 1D).
7. Continue marking all assay plates in order, such that all plates have 30 min of activity time, being careful to account for the time it took to set up the plate. Then, perform manual or digital measurements as described in steps 1.8 or 1.9.
8. Perform manual measurements using a ruler.
   1. Use a ruler to measure in mm, the distance from the center point to the final location markings for each worm and record the distance. To help keep track of progress during scoring, label the first scored point with a small marking line.
   2. Record length data for each dot consecutively by rotating the plate in a clockwise fashion (Figure 1E).
9. Perform digital measurement using a scanner and ImageJ.
   1. Using a flatbed scanner, scan the back of the assay plate(s) next to a ruler (numbers facing the scanning surface) (Figure 2A).
   2. Open the scanned plate image in ImageJ or a similar program.
      ​NOTE: ImageJ is a free Java-based image processing software hosted by the NIH.
   3. Click on the Straight-Line Tool and then draw a line connecting the 1 cm and 2 cm points on the scanned image of the ruler (Figure 2B).
      NOTE: Holding the Shift computer key will force the line to the nearest 45° angle.
   4. Navigate to the Analyze menu and use it to set the scale (Analyze | Set Scale) (Figure 2C).
   5. Enter the Known Distance (10) and Units of Length (mm) in the appropriate boxes, do not adjust the distance in pixels number. Check Global to apply the same scale to all images being analyzed in each ImageJ session; otherwise, set the scale for each image upon opening (Figure 2D). Click Ok.
      NOTE: Steps 1.9.2-1.9.5 are performed to set the measurement scale. Measurements for this image will now automatically relate the length of lines to the defined pixel: length relationship. Images scanned at 300 dpi will have an approximate relationship of 11.8 pixels per mm.
   6. Use the Paintbrush Tool to mark just above the first point to be scored.
      NOTE: This is a visual indicator for the first worm scored.
   7. Use the Straight-Line Tool to draw a line from the center point to the first worm data point.
   8. Click the M key on the keyboard to measure the distance. Alternatively, navigate to the Analyze menu and choose Measure (Analyze | Measure). In both cases, the measurement will appear in a new window; this window will collect all measurements per image.
   9. Keeping the center point constant, move the end of the line touching the first worm point to the next worm point, moving clockwise, and click the M key to measure.
   10. Continue measuring each point in a clockwise fashion until all points are scored.
   11. Copy and Paste length measurement data into a spreadsheet to save it. Then, repeat the process for each scanned plate image.
       ​NOTE: Steps 1.9.6 - 1.9.11 measure the worm displacement values (Figure 2E-F).
10. Perform statistical analysis.
    1. Combine replicates within a single experiment so that a total number scored is between 30-40 worms.
    2. Perform independent experimental replicates on different days and with independent populations of worms to end up with 3 independent experimental replicates.
    3. Analyze data using appropriate statistical analyses such as Student's t-test for 2 strains or 1-way ANOVA for three or more strains.

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Results

Figure 1: Radial locomotion assay workflow. Panels A-E show the generalized steps of the radial locomotion assay. The steps are as follows: (A) NGM Plates, with OP50 seeded to the edges, are prepared by labeling and marking central dot, (B) worms are placed in the center of the agar as marked by the central dot, (C) worms are allowed to move freely for a set amount of time, (D) plate is flipped bottom up and the final location of each worm is marked in a different color than the central dot, (E) The distance from the center dot to each final worm location in measured either by hand or digitally.

Figure 2: Digital measurement of final location using ImageJ. Distance from center measurements can be measured by hand or digitally, to measure digitally using ImageJ (A) scan backside of the plate with a ruler in the frame. (B) Draw a known length using the line tool. (C) Use the known length drawn in (B) to set scale [Analyze | Set Scale…]. (D) Use the line tool to draw a line from the center point to a final location mark, repeat for each mark. (E) A paintbrush line marking the first mark measured aids in scoring (squiggly black line near the 1 mark). Final location marks are numbered in yellow to illustrate the directionality of scoring. (F) Measurements are recorded in the Results window. Save results elsewhere for statistical analysis.

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Materials

Name	Company	Catalog Number	Comments
C. elegans	Caenorhabditis Genetics Center (CGC)	-	Aquire your strains as desired, N2 is a useful control strain
Disposable pasteur pipets, borosilicate glass	VWR	14673-010	Glass pipet used to create worm pick - hold glass pipette in one hand and ~1" of platinum wire (held by pliers) in the other over a flame to join.
Disposable petri dishes, 35x10mm	VWR	10799-192	Assay plates for WormLab Imaging System
Disposable petri dishes, 60x15mm	VWR	25384-090	Stock plates for worms
Disposable petri dishes, 100x15mm	VWR	25384-302	Standard radial locomotion assay plate
Disposable petri dishes, 150x15mm	VWR	25384-326	Longer time frame radial locomotion assay plate
Dissecting microscope	Leica	M80	Scope for maintaining worms and setting up radial locomotion assays
Fine-tipped markers	VWR	52877-810	Need at least 2 colors for radial locomotion assays. Fine tips required for accuracy.
Flatbed Scanner	Amazon	Epson Perfection V850	Optional for radial locomotion assay. Protocol assumes a resolution of 300dpi, most scanners would work fine
NGM (Nematode Growth Medium)	VWR	76347-412	Medium used to cultivate C. elegans. Can be made from scratch, see WormBook: Maintenance of C. elegans
OP50 bacteria	Caenorhabditis Genetics Center (CGC)	OP50	Primary food source for C. elegans
p1000 pipettor	VWR	76207-552	Pipettor, used in swimming assay
p1000 tips	VWR	83007-384	Tips for pipettor, used in swimming assay
Platinum wire, 0.2032mm diameter	VWR	BT136585-5M	Fine gauge platinum wire used to create worm pick - hold glass pipette in one hand and ~1" of platinum wire (held by pliers) in the other over a flame to join.
Ruler	VWR	56510-001	Need to score radial locomotion assays
ImageJ	NIH	-	Optional free software provided by the NIH - https://imagej.nih.gov/ij/
WormLab Imaging System	MBF Bioscience	WormLab	The Imaging System includes WormLab hardware (bright field stage, camera, and housing) and WormLab software. https:// www.mbfbioscience.com/wormlabimaging- system