Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The overall goal of this behavior recording setup and protocol is to quantify C Elegans'population-based chemosensory preference for soluble compounds using a multi-camera video acquisition system, followed by automated video analysis. This method can facilitate C Elegans'neurobiology research by enabling faster and more standardized behavioral analysis of genetic mutants. The main advantage of this technique is that multiple replicates can be assayed simultaneously under identical conditions while monitoring the temporal dynamics of the nematode population's response to soluble cues.

Demonstrating the behavior chamber assembly will be Christopher Cronin, a staff scientist from this laboratory. To begin, prepare the fabric covers, camera mount bars, corner brackets, extrusion bars and camera mount assembly as described in the accompanying text protocol. Beginning with the end bars attached to the center bar, attach the camera stand and holsters to the camera mount assembly.

Next, assemble the behavior chamber frame. Start by attaching four, one foot extrusion aluminum bars to a square polyester sheet. Slide one bar onto the two T-nuts of omni of the fabric sheet, centering the bar along the fabric width.

Tighten the fasteners to secure the fabric sheet to each bar. Spread the square polyester sheet on the work surface with the four attached aluminum bars on top. Then, slip eight corner brackets onto the top surfaces of the four aluminum bars, one at each end of each bar.

Position the corner brackets on the bars with the bracket's vertical legs flushed with the end of the bars and secure them into place by tightening the screws into the T-nuts. At each corner, stand a one foot aluminum extrusion upright, slipping the extrusion over the T-nuts on the two corner bracket legs. Then, secure the upright bar to the horizontal bars by tightening the fasteners, thus joining the two adjacent horizontal bars.

Slip the loose T-nuts of the H shaped camera mount assembly down into the uprights of the frame. Let the H slip all the way down to rest on top of the corner brackets at each corner of the frame. Secure the camera mount assembly in place, by tightening the four corner bracket screws.

Next, use that final four one foot extrusion aluminum bars to assemble the bottom layer of the chamber. Position the corner brackets with the brackets'legs flushed with the end of the bar and tighten the screws to secure it in place. Then, slip the bars down between the frame uprights with the loose T-nuts in the upright channels.

Position the bar so the top edges of their corner brackets are one inch below the open end of the uprights and secure in place by tightening the corner bracket screws. Next, slide the T-nuts of the four rectangular polyester sheets down into the channels of the upright extrusion bars to cover the sides of the chamber and tighten the fasteners to secure in place. Select one side to be a door for accessing the interior of the chamber and remove the two screws and T-nuts at the bottom layer of the chamber.

Place a plastic cap on the top end of each corner to serve as feet for the completed frame. Turn the frame right side up and insert two panel holders per slide inside the frame space layer, and twist to secure them in place. Then, place a 12 by 12 inch piece of clear acrylic on top of the panel holders to provide a stage for the assay plates.

Finally, position the camera and stage template as described in the accompanying text protocol. The day before the experiment, place 28 empty 35 millimeter plates in stacks of five or less on a flat surface. Fill each assay plate with five milliliters of nematode growth media agar and leave the assay plates to dry upright with their lids off, overnight at room temperature.

On the day of the experiment, turn on the backlight, boot the computer and launch the microscope camera's video capture software. Connect the microscope camera to the computer and click on the numbered camera thumbnail at the top of the software window to view the camera's live feed. To adjust the video recording settings, pull down the video format menu at the top left corner of the camera feed, and select MJPG 1280x1024.

Next, pull down the folder menu next to the video format menu, and select a folder in which to save the video files. Click on the auto-exposure icon at the top right corner of the camera feed and turn off the auto-exposure. Then, click on the LED icon immediately to the right of the auto-exposure icon to turn off the in-built microscope camera LEDs.

Click on the adjacent settings'icon and select, monochrome. Maximize contrast, and adjust the brightness. Align a solution placement template on top of the stage plate alignment template and click on the timelapse video recording icon on the left side of the camera feed.

Enter five seconds for duration and one second for the interval to record a five second calibration video at one frame per second. Use a five milliliter glass pipette to add 1.2 milliliters of nematode growth media buffer to a plate of synchronized worms and gently agitate the plate to displace the worms into the buffer. Pipette up buffer from the plate and dispense worms in buffer into a clean micro-centrifuge tube.

Let the tubes stand for one to two minutes to allow the worms to settle to the bottom. Then, use a plastic pipette tip to remove as much wash buffer from the tubes as possible without disturbing the pellet of aggregated worms at the bottom of the tubes. Refill the tube with one milliliter of clean nematode growth media buffer and repeat the wash procedure two more times.

Next, tape a solution placement template on top of the stage of a dissecting microscope. Then, place a strip of double-sided tape on either side of a plate alignment template. Align the perimeter of the assay plate with the circle on the plate alignment template and press down until the template adheres to the bottom of the plate.

Then, align the assay plate template member markers with the solution placement template member markers on the dissecting microscope. Next, use a P2 pipette to place one microliter of the M13 buffer, each in quadrants one and four. Use a new tip to place one microliter of 10 millimolar CuCl2 solution each into quadrants two and three.

Use a glass pasteur pipette to transfer the washed worms to the assay plates once the solution drops have been completely absorbed into the agar. Place a drop of worms on the areas above and below the circular treated regions. Fold the tissue four times and use the blunt edge to absorb excess wash buffer on the assay plates.

Immediately arrange the assay plates under the microscope cameras in the behavioral chamber by aligning the numbers of the template corners. Wait for two to three minutes to allow the worms to acclimate to the assay plates. Then, click on the timelapse video recording icon on the left side of the camera feed and enter 10 minutes for duration and one second for the interval to record a 10 minute video of one frame per second.

Finally, click on start, to start video recording. For each video frame, the preference index function counts the number of worm pixels in each region of interest and calculates the percentage of one pixel out of the total number of pixels in the region of interest. The preference index values obtained for different genotype and treatment pairings are shown here.

A positive preference index value indicates an attraction to the treatment while a negative preference index value indicates repulsion. A preference index of 0.02 is obtained when the M13 buffer solution was placed in both the control and experimental regions of interest, demonstrating that there is no spatial bias. This graph also shows that N2 worms strongly avoided the copper ions, resulting in a preference index of 0.67.

In addition, osm-3, which are defective in sensory cillia formation, and ocr-2 mutants which are defective in ASH mediated nociceptive responses shown decreased avoidance and even some mild attraction to copper ions. While attempting this protocol, it is important to work fast but also smoothly and consistently. The recording setup and assay protocol can be easily adapted to characterize population based behavioral responses that arise from the interactions between different C Elegans'sensory varieties.