Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

This protocol allows researchers to clarify planarian behavior and the sec mechanisms underlying this behavior by combining quantitative behavioral analysis with molecular and chemical perturbations. This technique is easily accessible to all skill levels as it does not require advanced instruments or specialized software to get quantitative behavioral readouts of free moving planarians. Scrunching can be used to study exception in planarians, and serves as sensitive endpoint to assay, disrupting nervous system function caused by Xenobiotics and disease.

Begin by placing a dimmable LED Panel upon a flat surface, which will provide a uniform white background and can be used as an adjustable light source to obtain appropriate contrast. Place a 100 millimeter Petri dish arena on the LED Panel. Mount a camera on a ring stand above the arena and adjust its position, height and focus as necessary so that the entire arena is centered within the field of view and is in focus.

Fill the arena with approximately 25 milliliters of the appropriate exposure media to have maximum volume, turn on the LED Panel and turn off any other light sources that may negatively affect recording quality. Drop a planarian at the center of the arena using a transfer Pipette. When testing a chemical solution, transfer the planarian with as little of planarian water as possible, so that the concentration of the chemical is not significantly changed.

Begin recording data as image sequences in a native Fiji format. For gliding experiments, record one to two minutes of gliding behavior, for scrunching or peristalsis experiments, record long enough to capture at least three consecutive oscillations occurring in a straight line. Once the experiment is completed, terminate the recording.

If the planarian reaches the boundary of the arena without satisfying the termination criterion, pipette the planarian back to the center of the arena. Open the raw image sequence for an experiment in Fiji, convert it to eight bit and use the arrow tool or slider at the bottom of the image stack to pan through the image sequence. To extract a time period and region of interest, draw a region of interest encompassing the full path of a planarian using the rectangle tool.

Right click on the image stack and select duplicate, check the box where duplicate stack. Enter the first and last frames of the sequence of interest and click okay. For gliding experiments, extract a period of gliding where the planarian moves at least twice its body length for scrunching or peristalsis experiments.

Extract an instance when the planarian undergoes a minimum of three consecutive and complete body oscillations in a straight line. Apply a threshold to the duplicated image stack to binarize the image and extract the planarian from the background, adjust the sliding bars so that the entire planarian is highlighted in red. The exact values are dependent on imaging quality.

Leave the boxes for dark background, stack histogram and don't reset range unchecked. Scroll through the image stack to ensure a good threshold range and then click apply. In the convert stack to binary window, set the method to default and the background to light.

Uncheck all boxes in this window and then click okay. A binarized image showing a black planarian on a white background will appear. Make sure that the entire planarian is visible in all frames of the image sequence.

Set measurements by clicking analyze and set measurements. Check the boxes for area, center of mass, stack position and fit ellipse, then click okay. Select the open image stack and select analyze, and analyze particles.

In the analyze particles window, select show and masks to open a new stack showing all the objects that were detected with the chosen parameters. Set a size filter to remove unwanted noise by entering the approximate area of the planarian. Then check the boxes for display results and clear results and click okay.

Pan through the mask image stack using the slider at the bottom of the Panel to make sure that there is no noise or frames without a planarian. On the results window, save the data using file and save as, add the CSV extension to the file name, to save the data as comma separated values. Once the data for the image stack is saved, close the respective image stack results and mask windows.

To induce scrunching via noxious temperature, heat planarian water in a glass beaker to 65 degrees Celsius on a hot plate. Place a planarian in the center of the arena, and wait until it orients itself upright and begins gliding, then begin recording. Use a P 200 Pipette to slowly and consistently pipette 100 microliters of the preheated planarian water, post pharyngeal onto the tail end of the planarian to induce scrunching.

Stop the recording, once scrunching has ceased. Place the planarian in a recovery container and exchange the media in the Petri dish with fresh room temperature planarian water, if running more experiments. To induce scrunching via amputation, transfer a planarian to the center of the arena and wait until the planarian orients itself upright and begins gliding.

Then begin recording, amputate the planarian with a clean razor blade. Making sure that the cut location is consistent across experiments. Stop the recording once the anterior piece has ceased scrunching.

Remove both pieces, place them in a separate container and allow them to regenerate for seven days. Amputated planarians can be reincorporated into the home container once they have regenerated. This protocol was used to test whether near UV light exposure, induces scrunching in S.mediterranea and D.japonica planarians, while D.japonica planarians scrunch, when exposed to near UV light, S.mediterranea planarians either exhibit tail thinning or no response.

A quantification of the scrunching parameters for the D.japonica planarians that exhibited at least three consecutive straight line scrunches, reveals characteristics scrunching parameters for this species In contrast exposure to 250 micromolar cinnamaldehyde unknown TRPA one agonist in mice, causes scrunching and S.mediterranea. Whereas D.japonica planarians display a mixture of snake-like and oscillatory motion interrupted by gliding or vigorous head turns. A quantification of the samples with at least three consecutive oscillations, yielded significantly lower values for three out of four parameters then expected for scrunching in D.japonica, indicating that the observed oscillatory motion is not scrunching.

RNAi confirms the specificity of scrunching and response to cinnamaldehyde exposure in S.mediterranea. Within 180 seconds of exposure in planarian water, all control RNAi planarian scrunched. Compared to none of the SMTRPA one RNAi planarians demonstrating that S.mediterranea scrunching in cinnamaldehyde requires SMTRPA one.

It is essential to be consistent and how the animals are manipulated to reduce noise and ensure reproducibility in the behavioral measurements. This projects can be expanded to include body shape analysis, which would allow for identification and quantification of other planarian behaviors that are not captured here.