An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

The overall goal of this procedure is to quantify the effect of alcohol on CL organs of different genotypes in a consistent manner. This is accomplished by first creating an agar plate containing alcohol with copper rings embedded in the surface to corral the animals. Next, the different genotypes are each corralled separately and exposed to alcohol During the exposure, video data is collected at set time points.

The final step is the measurement of the speed of each genotype using object recognition software. Ultimately, a comparison of speeds at early and late time points is used to uncover abnormal sensitivity or tolerance development to alcohol. The specific genotypes.

The main advantage of this technique over existing methods, which compare genotypes in different plates, is that the environmental variation is reduced. Generally, individuals new to this method will struggle with picking worms using a naked pick On the day before the assay. Pick L four stage worms to fresh NGM plates Seated with a lawn of OP 50 E coli culture, the plates at 20 degrees Celsius overnight, the assay requires standard unseated 60 by 15 millimeter.

NGM plates dry all of the plates to be used at 37 degrees Celsius for two hours with their lids off. Each trial requires four plates. The use of NGM plates is important here.

Differences in the composition of the plates. In particular, their osmolarity can strongly affect the dose responsive ethanol on behavior. This is due at least in part, to changes in the amount of ethanol accumulated by the animals Once dried, weigh each of the unseated NGM plates.

Use this weight to determine the volume of media on each plate, assuming that the media weighs as much as water. Next, plan to melt four copper rings with an inner diameter of 1.6 centimeters into each plate, so they can be viewed by the camera simultaneously. Using forceps.

Eat each ring in a flame for about three seconds and immediately put the ring on the plate without dropping it. Then press the ring gently in a few places to secure it along the periphery. Label the plates with the strain that will be in each ring.

Now, calculate the amount of 100%ethanol needed to get a 400 millimolar weight to volume concentration on the plate. Then load half the plates with ethanol by drizzling the ethanol from a pipette. Let all the prepared plates equilibrate for two hours before starting the experiment.

To begin, take two minutes or less to transfer 10 worms from each experimental group to the center of a copper ring on an acclimation plate. This is a plate with no ethanol. Avoid transferring food with the worms as it will reduce their movement.

Also, avoid damaging the agar surface because the worms will burrow. Record the time of loading onto the plate and be sure to stagger the transfer of worms to each plate by over two minutes, so each plate can be filmed individually at the appropriate time interval. Let each acclimation plate incubate at room temperature for 30 minutes.

Then transfer the worms to the assay plate following the order used to place the worms on the acclimation plate using a thin edged flattened worm. Pick without bacteria. Collect the worms on top of the flattened pick.

Using a scooping motion, then seal the plates with laboratory film to minimize loss of ethanol to vaporization. The speed at which the animals are transferred at this step is very important because the transfer to animals will be exposed to ethanol longer, so it is a good idea to rotate which strains are placed on the plate. First across experimental replicates.

A microscope with high resolution video camera is required to film the worms. Now, even illumination, such as from a three inch square backlight is vital for the analysis. Then to maintain contrast image the plate media side up, prepare the image analysis software to capture 120.

Frame 12 bit gray scale time-lapse movies in one frame per second. To reduce the size of the output file while still retaining sufficient image resolution, use a two by two bending mode. Now, record two minute movies from each plate.

10 minutes after the last worms were placed on that plate. Then make a second two minute recording. 30 minutes after worms were placed on the plate for this demonstration image.

Pro plus software is used, but a wide variety of other software can be adapted to the technique. Analyze the movies as two minute segments. First, apply a filter to the images that flattens the background and enhances the contrast of the worm objects.

Under the menu select process. Then filters, then enhancement, and then flatten. Set the parameter for a dark background and a 20 picks feature width.

Now analyze the locomotion of the animals in each ring separately with a circular region of interest that overlaps the copper ring. Identify and track the worms with the track objects command under the measure sub menu. In the tracking data table window, use the tracking options button to allow specific tracks to be excluded and to limit any experimental artifacts.

Under the auto tracking tab, set the track parameters to a velocity limit of 400 microns per frame. Set the acceleration limit to automatic, set the minimum total track length to 400 microns and set the predominant motion type to chaotic. Then under the track parameter, set the objects to allow partial tracks to have a minimum length of 21 frames, and to have a tracking prediction depth of one frame.

Now to initiate the tracking process first, click the find all tracks automatically function button. This brings up the count size options dialogue box and the tracking dialogue box. Select the manual option for the intensity range selection in the count size dialogue box.

This provides an important threshold step. Then adjust the intensity threshold sliders to create an inclusive range that highlights dark objects. A good starting point on a scale is between zero to 1, 500.

Now apply a size filter to exclude objects that are larger or smaller than a single worm. Go to the measure menu and then select measurement sub menu. This opens the count size options dialogue box there.

Set the area range to 28 to 120, 000 square microns and set the perimeter range to six to 2, 500 microns. Make adjustments to these parameters when using worms of abnormal size. Now, complete the tracking process by clicking.

Continue in the tracking dialogue box. Double check that the output tracks with the movie footage. Make sure everyone is represented unless there is a clear reason to filter it.

Then manually delete tracks produced by non worm objects. Next, calculate the velocity of each worm by averaging the distance traveled between each frame or each second and create an average velocity for the tracks in each population. Consider this final average to be one sample point for statistical analysis of the experiment.

Several genotypes with controls were analyzed using the described protocol. After 10 minutes of ethanol exposure, sensitive strains differed in locomotion from their control. This is marked by the degree of effect on the left axi of the graph.

The simple view is that strange, which showed a relative speed larger than that of the control were considered ethanol resistant, whereas strains with a lower relative speed to the controls were considered ethanol sensitive. Comparing data from 30 minutes of ethanol exposure to that from 10 minutes of ethanol exposure gave an index for acute functional tolerance to ethanol or a FT.An extended discussion of this and other results is provided in the text protocol. While attempting this procedure, it's important to remember to prevent the transfer of food to the assay plate.

Following this procedure, the impact of genotype on the behavioral effects of other drugs can be performed in order to answer additional questions like what are the shared mechanisms of actions between different drugs.