This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.
This protocol uses two different semiautomated assays to quickly quantify C.elegans locomotor activity in liquid medium, and has the potential to be applied for drug screening in C.elegans disease models. The main advantages of these two techniques are their ease of use and ease of reproducibility. Both techniques have the potential to evaluate drug effects on worm behavior with variable throughput.
These motility assays are useful for evaluating the locomotor activity of C.elegans mitochondrial disease models in which the neuromuscular system of the animals is impaired. To analyze C.elegans locomotor activity in liquid medium on glass slides after synchronizing control in mutant worms strains on NGM plates with and without drug treatments of interest to the L4 stage, use a worm pick and a stereomicroscope to pick five worms per strain and condition into individual 20 microliter drops of S.basal solution on a glass slide. Add worms from the same experimental condition from different NGM plates to multiple droplets on the same slide to obtain multiple technical replicates.
When all of the worms have been collected, allow the animals to acclimate on the slide for one minute at room temperature before recording the worms swim activity in each drop for one minute at 15 frames per second per drop. To analyze the recorded C.elegans locomotor activity using ZebraLab software, use the ZEBRALAB AVI option to upload videos to the software. Click the Quantization with AVI Files option.
To create a new protocol, select Parameters, Protocol Parameters, and Time, and set the experiment duration to one minute. Select No Time Bin and unselect Integration Period according to the data output desired and select File and Open a Movie to upload each individual previously recorded video file. To build a single area of detection, use the circle tool to create an area around the entire liquid drop in which the worms are located.
The activity of all of the worms within the selected area will be detected. To indicate the distance to calibrate, draw a horizontal line from the left to the right of the video area and select Calibration, Draw, and Apply to Group. To allow detection of all the different C.elegans worm strains to be analyzed, unselect the Area icon and adjust the Detection Sensitivity in the Activity Threshold.
To visualize the tracks made by the animals while the activity analysis is underway, set the Display Scale to 70 and select Apply to Group. To analyze the videos, click Play, and select Experiment, and Execute to enter a file name for the video. After saving, a window will appear asking whether to analyze the video at the maximum computer speed.
Click Yes. The running experiment window will open. Click Start.
When the recording analysis is complete, click Experiment and stop to save the activity analyzed in the droplet in a spreadsheet. To analyze C.elegans locomotor activity in liquid medium in a 96-well plate format, when the worms reached the L4 stage, add 50 microliters of 2%weight per volume of E.coli OP50 and S.basal medium to each well of a clear 96-well flat bottom microplate. Use the stereomicroscope to manually pick 15 synchronized worms per well of the 96-well plate and allow the worms to acclimate for 20 minutes.
At the end of the acclimation period, use a standard flatbed scanner to scan each microplate two times with less than 10 seconds between scans To analyze the scans, use open-source software to align the two sequential scans and acquire the pixel change data between the two images and the relative worm scan score to determine the changes in locomotor response based on the light intensity produced by the scanner when set to a pixel threshold of five. In this analysis of C.elegans locomotor activity in a single drop of liquid medium, the mitochondrial complex I disease worms demonstrated a significant 38%decrease in their locomotor activity at the L4 stage compared to wild-type worms. Similarly, worms scan analysis of the same populations showed a significant reduction in locomotor activity for the L4 stage disease model worms compared to N2 Bristol wild-type worms.
This locomotive activity loss was also observed in adult day one stage mitochondrial complex I disease worms as assessed by worm scan analysis. It is essential to remember to use the correct number of worms when setting up each assay. Following this procedure, our lab frequently performs a drug screening.
These two techniques are used in our laboratory for evaluating the effects of drugs of interest on worm activity in preclinical animal models of mitochondrial disease.
