Biochemistry
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A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products
Chapters
Summary May 30th, 2020
Planarian motility is used to gauge the stimulant and withdrawal properties of natural products when compared to the movement of the animals in spring water alone.
Transcript
The Planarian Locomotor Velocity Test is a means for quickly assessing stimulant and possible withdrawal effects for a variety of biological modifying agents that might affect motility. This essay is easily controlled and provides a straightforward and accessible method for generating reliable behavioral data in even the most basic research settings. Since the planarian nervous system has a centralized rudimentary brain, a bilateral symmetry and analogous neurotransmitter systems, behavioral studies using these organisms offer preclinical insights into possible vertebrate responses.
Experiments can be further affected by physiological pathway modifiers, drugs or even genetically modified planarians to provide insight into the associated biological mechanisms. It is important to have the planarian so that the transfers between containers do not stress them and to standardize the grid line curves between lab members. The best way to obtain consistent results from this behavior essay experiment, is by seeing how experienced users performed them.
After setting up a planarian culture, feed the worms ad libitum on a regular twice a week schedule of chopped organic boiled eggs or blended organic beef liver for one to two hours. After feeding, remove any soiled water from the container and use a flat watercolor paint brush to transfer any food debris and slime adhering to the container to a paper towel. Use fresh spring water and gentle swirling or agitation to dislodge the planarians and decamp the worms into a clean container.
Slide a Wide Bore Pipette Tip under any planarians remaining adhered to the old container to facilitate their transfer to the new dish and to carefully decant the transfer water. Then, cover the planarians in the new container with fresh spring water. To perform a Planarian Locomotor Velocity Test, first ensure that the five to 10 days starved planarians are fully formed with complete and pigmented heads and tails.
If the planarians are ready place one clean 10 centimeter diameter Petri dish onto one piece of pre-laminated grid paper with 0.5 centimeter squares per experimental group and add 20 milliliters of unadulterated spring water for the control group or 20 milliliters of spring water containing the appropriate concentration of the natural product being tested to each dish. Position a camera above the prepared locomotor velocity stimulation dish and add five to 10 milliliters of unadulterated spring water or spring water containing the appropriate concentration of the experimental compound to an appropriately sized habituation container. Next, use a small clean watercolor paint brush to gently transfer one planarian from the culture container to the prepared habituation container and allow the planarian to habituate to the solution in the container for two minutes.
Research will allow the planarians to move onto the brush and to carefully lift the animals without forcing them. At the end of the habituation period, use the brush to carefully transfer the planarian to the center of the prepared locomotor velocity stimulation dish containing the same solution as the habituation container and start the camera to record 10 to 11 minutes of movement of the planarian. To test the effects of withdrawal at the end of the habituation period, rinse the planarian in a five centimeter dish containing spring water only.
Before transferring the worm to the center of the prepared locomotor velocity dish containing fresh spring water and start the camera to record 10 to 11 minutes of movement. The camera setup allows a clear view of the planarian and the grid paper and permits an accurate assessment of the progress of the animal for the duration of the experiment. The data collection should include the total number of grid lines crossed as well as the cumulative total number of lines crossed per minute of the experiment.
To standardize the results from each trial, the planarian locomotor velocity runs can be calculated and plotted as the number of boxes crossed relative to the progress of the matching control worm. As a benchmark, planarians in spring water typically cover approximately 25 boxes in three minutes. A range of test reagent concentrations can be examined to determine planarian motility in response to the test compounds of interest.
Withdrawal data can be assessed by quantifying the number of grid lines crossed in spring water relative to the spring water control after the animals were habituated in the bio modulating product being examined. Spring water control planarians move over grid lines for the duration of the experiment. At times however, the test planarians cease crossing grid lines during the essay with behaviors that are characteristic for the product being tested.
This suggests that a different method of analysis might be required. Such instances can be tracked and the data can be illustrated as the percentage of the total number of animals exposed to a particular reagent concentration. It is essential to handle the planarian so that they are not damaged during the transfer between dishes.
After determining the effects of different concentrations of a natural product, biological pathway modifiers can be added to observe any dynamic changes to the initial results. The Planarian Locomotor Velocity Test was used to test for stimulation and withdrawal using known pharmacological agents. We adapted the system to study these same concepts with natural products.
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