Name: A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products (Video) | JoVE Journal
Uploaded: 2020-05-30T14:00:00.000+00:00
Duration: 7 min 13 s
Description: Learn the Planarian Locomotor Velocity Test to assess stimulant and withdrawal effects of natural products on motility. This method generates reliable behavioral data for preclinical neuroscience research.

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The authors wish to acknowledge the office of Institutional Advancement, and the Morrisville College Foundation for a publication grant to support this work, as well as the SUNY Morrisville Collegiate Science and Technology Entry Program (CSTEP) for their ongoing assistance and support of undergraduate research at SUNY Morrisville. We also wish to thank Sophia Hutchens for helpful comments on the technique described.

1. Planarian husbandry
<ol>
	<li>Use planarians purchased from a biological supply company or wild-caught if needed. The planarians used in this protocol are Dugesia tigrina, as listed in the supply list. This species is also referred to as Girardia tigrina31. Other aquatic planarian species are also acceptable2,3. 
	NOTE: The protocol described is geared to animals purchased from a biological supply company. This assay system has not been tested with wild-caught planarians. However, if wild-caught planarians are used, it is recommended that they be habituated to the water used in the experiments, as well as the laboratory environment for at least 1 week before use.</li>
	<li>Upon arrival, transfer planarians to plastic food storage containers containing clean spring water and keep the lids ajar.</li>
	<li>Maintain planarians in a darkened environment.</li>
	<li>Feed newly delivered planarians after 24&#8211;36 h in their new environment.</li>
	<li>Allow planarians to acclimate to the laboratory at least 1 week prior to experimentation.</li>
	<li>Feed planarians on a regular twice-a-week schedule.
	<ol>
		<li>Allow planarians to feed ad libitum on chopped organic boiled eggs or blended organic beef liver for 1&#8211;2 h.</li>
		<li>Place fed planarians into a clean container after feeding.
		<ol>
			<li>Remove the soiled water off the planarians.</li>
			<li>Use a small flat watercolor paintbrush (number 3&#8211;6) to transfer food debris and slime adhering to the container from around planarians to a paper towel.</li>
			<li>With fresh spring water and gentle swirling or agitation, dislodge planarians and pour them into a clean container.</li>
			<li>Any planarians remaining adhered to the container can be transferred using a round watercolor paintbrush (number 3&#8211;6) or transfer pipette with a wide bore.</li>
			<li>Decant the transfer water.</li>
			<li>Cover the planarians with clean spring water.</li>
			<li>After 24 h, remove the planarians from any expelled food waste by transferring them to a clean container as described above (steps 1.6.2.1&#8211;1.6.2.6).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>To clean containers and utensils used for planarian husbandry, do not use soap or detergent. Clean these items by rinsing them well with clean water (tap water is acceptable) and drying them with a clean cloth or paper towel.</li>
</ol>
2. Preparation of planarians for experiments
<ol>
	<li>Allow newly delivered planarians to acclimate to their environment at least 1 week prior to experimentation.</li>
	<li>Starve planarians for 5&#8211;10 days prior to experimentation.</li>
	<li>Change the culture water at least 1x during the starvation period.</li>
</ol>
3. Planarian locomotor velocity (pLmV) test: Stimulant behaviors
<ol>
	<li>Prior to experimentation, ensure starved planarians are fully formed, with a complete and pigmented head and tail.</li>
	<li>Prepare a glass or plastic 10 cm Petri dish and a habituation container for the pLmV test prior to starting the experiment.
	<ol>
		<li>Place a clean 10 cm diameter Petri dish to be used for the pLmV test on prelaminated grid paper (with 0.5 cm squares).</li>
		<li>Add 20 mL of unadulterated spring water for controls, or spring water containing the appropriate concentration of the natural product being tested, to the 10 cm diameter Petri dish to be used for the pLmV test.</li>
		<li>Position a camera (e.g., cell phone or high-resolution camera) above the prepared 10 cm diameter Petri dish to record planarian motility over the grid paper during the experiment. A ring stand is a convenient way to position the camera at a distance that can record the entire view of the 10 cm Petri dish and grid lines.</li>
		<li>Prepare a habituation container with 5&#8211;10 mL of unadulterated spring water (controls), or spring water containing the appropriate concentration of the natural product being tested. A container similar to a scintillation vial or small 5 cm Petri dish is suitable.</li>
	</ol>
	</li>
	<li>Use a small, clean, flat, or round watercolor paintbrush to gently transfer a planarian from the stock container with spring water to the habituation container having 5&#8211;10 mL of unadulterated spring water or spring water containing the natural product being tested. 
	NOTE: When manipulating planarians for the pLmV assay, use a small, clean, flat, or round watercolor paintbrush (number 3&#8211;6). When moving planarians, the brush should be placed under the animal to lift it gently. To ensure planarians are not damaged when using the brush, the bristles of the brush should not be splayed out under the animal. Spreading out of the bristles could harm the planarian if it is caught between the fibers of the brush. 
	NOTE: A wide bore transfer pipette can also be used for transferring the planarian into a clean and dry habituation container.
	<ol>
		<li>If using the pipette, remove excess water moved with the planarian from the habituation container using the transfer pipette.</li>
		<li>Carefully add the habituation solution (i.e., spring water for controls or spring water containing the concentration of natural product being tested) to the habituation container containing the planarian.</li>
	</ol>
	</li>
	<li>Habituation periods will depend on the stimulation dynamics assessed for the natural product being tested. A habituation time of 2 min proved acceptable to detect stimulation in this work1.</li>
	<li>Following the 2 min habituation period use a watercolor paintbrush to gently transfer the planarian to the center of the prepared 10 cm Petri dish for the pLmV stimulation experiment.</li>
	<li>Start the camera to record the movement of the planarian. Record 10&#8211;11 min of video.</li>
	<li>Prepare the habituation container and the 10 cm Petri dish for the pLmV experiment with fresh solutions for each planarian.</li>
	<li>Use dedicated pipettes, dishes, containers, and paintbrushes for each experimental concentration of the natural product being tested to avoid inadvertently exposing planarians to the wrong solution during experimentation.</li>
	<li>Because planarians exhibit learned behaviors, each planarian (control or test) should only be used once21,22.</li>
</ol>
4. Planarian locomotor velocity (pLmV) test: Withdrawal behaviors
<ol>
	<li>Prior to experimentation, ensure starved planarians are fully formed, with a complete and pigmented head and tail.</li>
	<li>Prepare a 10 cm Petri dish (glass or plastic) for the pLmV experiment, a 5 cm Petri dish (glass or plastic) for rinsing the planarian following habituation, and a habituation container prior to starting the experiment.
	<ol>
		<li>Place a clean 10 cm diameter Petri dish to be used for the pLmV experiment on prelaminated grid paper with 0.5 cm squares).</li>
		<li>Add 20 mL of unadulterated spring water to the 10 cm diameter Petri dish to be used for the pLmV experiment.</li>
		<li>Position a camera above the prepared 10 cm diameter Petri dish as in step 3.2.3 to record planarian motility over the grid paper during the experiment.</li>
		<li>Prepare the planarian rinse container by adding 5 mL of spring water alone to the 5 cm Petri dish.</li>
		<li>Prepare a habituation container with 5&#8211;10 mL of unadulterated spring water (controls) or spring water containing the natural product being tested. A container similar to a scintillation vial or small 5 cm Petri dish (glass or plastic) is suitable.</li>
	</ol>
	</li>
	<li>Use a small, clean, flat, or round watercolor paintbrush to transfer a planarian from spring water to the prepared habituation container having 5&#8211;10 mL of unadulterated spring water (controls) or spring water containing the natural product being tested. Gently move the animal from the stock container to the habituation container. Ensure the planarian is not damaged by the brush. 
	NOTE: When manipulating planarians for the pLmV assay, use a small, clean, flat, or round watercolor paintbrush (number 3&#8211;6). When moving planarians, the brush should be placed under the animal to lift it gently. To ensure planarians are not damaged when using the brush, the bristles of the brush should not be splayed out under the animal. Spreading out of the bristles could harm the planarian if it is caught between the fibers of the brush. 
	NOTE: A wide bore transfer pipette can also be used to transfer the planarian into a clean and dry habituation container.
	<ol>
		<li>If using the pipette, excess water moved with the planarian should be removed from the habituation container using the transfer pipette.</li>
		<li>Carefully add the habituation solution (i.e., unadulterated spring water for controls or spring water containing the natural product being tested) to the habituation container containing the planarian.</li>
	</ol>
	</li>
	<li>Habituation periods for withdrawal will depend on the stimulation dynamics assessed for the natural product being tested; 2&#8211;5 min have proven sufficient.</li>
	<li>Following the habituation period use a watercolor paintbrush to gently transfer the planarian to the prepared 5 cm Petri dish containing spring water to rinse off any natural product from the habituation container. Ensure the planarian is not damaged by the brush.</li>
	<li>Immediately transfer the planarian to the center of the prepared 10 cm Petri dish containing spring water for the pLmV withdrawal experiment. Ensure the planarian is not damaged by the brush.</li>
	<li>Start the camera to record the movement of the planarian. Record 10&#8211;11 min of video.</li>
	<li>Prepare the habituation container, the rinse container, and the 10 cm Petri dish for the pLmV experiment with fresh solutions for each planarian.</li>
	<li>Use dedicated pipettes, dishes, containers, and paintbrushes for each experimental concentration of the natural product being tested to avoid inadvertently exposing planarians to the wrong solution during experimentation.</li>
	<li>Because planarians exhibit learned behaviors, use each planarian (control or test) only once26,27.</li>
</ol>
5. Data analysis
<ol>
	<li>Prepare a data collection table to document the behavior and the motility of planarians as the number of grid lines crossed for each minute during the pLmV run. The table should allow for the accumulated number of lines per minute crossed by the planarian to be documented as well. Include lines for notes and a table of definitions to tally the observation of behaviors during the experimental period, such as &#8216;wander&#8217; and &#8216;stop&#8217; (see Discussion).</li>
	<li>Using the video, count the number of full grid lines crossed by the planarian per minute for 10 min, and record that number on the data table. Typical planarian behavior consists of continuous velocity, forward-directed, horizontal movement, with periodic turns, and without stops.
	<ol>
		<li>Begin to time the experiment at the point that the planarian has moved off the paintbrush used to transfer it to the 10 cm Petri dish. Record this start time.</li>
		<li>To determine when the animal crosses a full grid square, focus on the head and score one line when the head fully crosses a square.</li>
		<li>To score a full grid when the worm moves around the edge of the dish, visualize a distance of 0.5 cm by referring to the lines as they extend out from the margins of the dish. If the planarian crosses the corner of a box, refer to the second line crossed to score one grid line. Again, focus on the head to make these determinations.</li>
		<li>Stop the video after each minute to record the data.</li>
		<li>When restarting the video to count the next minute, if the head of the worm was between grid lines when the video was stopped, record the first line crossed as a full box.</li>
		<li>Score the number of grid lines crossed for 10 min.</li>
	</ol>
	</li>
	<li>If the animal stops moving during a pLmV test and no longer crosses grid lines during the 10 min recording time, document the behavior of the planarian in the behavioral chart (e.g., &#8216;wander&#8217; or &#8216;stop&#8217;). Animals that cease their forward track during the pLmV assay should instead be taken note of and the data presented as a frequency of the total number of animals exposed to that reagent concentration. Coiling or convulsive behavior (known as a C behavior) preventing any forward movement during the habituation period indicates that the concentration of the natural product is not appropriate for use in a pLmV assay because the pLmV assay is motility-based. C-type behaviors can be analyzed using a different type of analysis (see Discussion).</li>
	<li>If possible, test multiple experimental concentrations of the natural product using at least 9&#8211;12 worms on different days and different times of the day if determining the overall effect of the reagent on planarian physiology. However, if researchers endeavor to reduce circadian rhythm-induced variability, experiments can be conducted with constant lighting at a set time of day using worms that are cultured with timed light/dark cycles and set feeding times. Have at least two experimenters involved in the project to allow for the option of having one individual record data, while the second individual counts the grid lines &#8216;blind&#8217; to the conditions used for data collection. Having different individuals involved in data collection, as well as statistical calculations and analysis, also reduces possible bias.</li>
	<li>Calculate grid line counts for each natural product concentration on each day as relative to control counts for each minute so that data from different days, times, and experimenters can be combined. These data can be averaged and then analyzed using Student&#8217;s T-tests. P-values for each test can be assessed per minute compared to the control and between reagent concentrations. ANOVA assessments using data sets derived from different experimental concentrations provide a further method of analysis.</li>
</ol>

<ol><li>Moustakas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guarana+provides+additional+stimulation+over+caffeine+alone+in+the+planarian+model%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Guarana provides additional stimulation over caffeine alone in the planarian model.</a> PloS One. 10 (4), 0123310(2015).</li>
<li>Raffa, R. B., Valdez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cocaine+withdrawal+in+Planaria%5BTitle%5D)+AND+%22European+Journal+of+Pharmacology%22%5BJournal%5D)">Cocaine withdrawal in Planaria.</a> European Journal of Pharmacology. 430 (1), 143-145 (2001).</li>
<li>Raffa, R. B., Holland, L. J., Schulingkamp, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+assessment+of+dopamine+D2+antagonist+activity+using+invertebrate+%28Planaria%29+locomotion+as+a+functional+endpoint%5BTitle%5D)+AND+%22Journal+of+Pharmacology+and+Toxicological+Methods%22%5BJournal%5D)">Quantitative assessment of dopamine D2 antagonist activity using invertebrate (Planaria) locomotion as a functional endpoint.</a> Journal of Pharmacology and Toxicological Methods. 45 (3), 223-226 (2001).</li>
<li>Thumé, I. S., Frizzo, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sertraline+induces+toxicity+and+behavioral+alternations+in+planarians%5BTitle%5D)+AND+%22Biomedical+Research+International%22%5BJournal%5D)">Sertraline induces toxicity and behavioral alternations in planarians.</a> Biomedical Research International. 2017, 5792621(2017).</li>
<li>Aggarwal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+a+novel+allosteric+modulator+of+the+human+dopamine+transporter%5BTitle%5D)+AND+%22ACS+Chemical+Neuroscience%22%5BJournal%5D)">Identification of a novel allosteric modulator of the human dopamine transporter.</a> ACS Chemical Neuroscience. 10 (8), 3718-3730 (2019).</li>
<li>Zhang, C., Tallarida, C. S., Raffa, R. B., Rawls, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sucrose+produces+withdrawal+and+dompamine-sensitive+reinforcing+effects+in+planarians%5BTitle%5D)+AND+%22Physiology+%26+Behavior%22%5BJournal%5D)">Sucrose produces withdrawal and dompamine-sensitive reinforcing effects in planarians.</a> Physiology & Behavior. 0, 8-13 (2013).</li>
<li>Zewde, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PLDT+%28planarian+light%2Fdark+test%29%3A+an+invertebrate+assay+to+quantify+defensive+responding+and+study+anxiety-like+effects%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">PLDT (planarian light/dark test): an invertebrate assay to quantify defensive responding and study anxiety-like effects.</a> Journal of Neuroscience Methods. 293, 284-288 (2018).</li>
<li>Risse, B., Otto, N., Berh, D., Jiang, X., Klämbt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FIM+Imaging+and+FIMtrack%3A+two+new+tools+allowing+high-throughput+and+cost+effective+locomotion+analysis%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">FIM Imaging and FIMtrack: two new tools allowing high-throughput and cost effective locomotion analysis.</a> Journal of Visualized Experiments. (94), e52207(2014).</li>
<li>Inoue, T., Hoshino, H., Yamashita, T., Shimoyama, S., Agata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Planarian+shows+decision-making+behavior+in+response+to+multiple+stimuli+by+integrative+brain+function%5BTitle%5D)+AND+%22Zoological+Letters%22%5BJournal%5D)">Planarian shows decision-making behavior in response to multiple stimuli by integrative brain function.</a> Zoological Letters. 1, 7(2015).</li>
<li>Hastrom, D., Cochet-Escartin, O., Zhang, S., Khuu, C., Collins, E. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Freshwater+planarians+as+an+alternative+animal+model+for+neurotoxicology%5BTitle%5D)+AND+%22Toxicological+Sciences%22%5BJournal%5D)">Freshwater planarians as an alternative animal model for neurotoxicology.</a> Toxicological Sciences. 147 (1), 270-285 (2015).</li>
<li>Risse, B., Berh, D., Otto, N., Klämbt, C., Jiang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FIMtrack%3A+an+open+source+tracking+and+locomotion+analysis+software+for+small+animals%5BTitle%5D)+AND+%22PLoS+One+Computational+Biology%22%5BJournal%5D)">FIMtrack: an open source tracking and locomotion analysis software for small animals.</a> PLoS One Computational Biology. 13 (5), 100553(2017).</li>
<li>Pagán, O. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Planaria%3A+an+animal+model+that+integrates+development%2C+regeneration+and+pharmacology%5BTitle%5D)+AND+%22International+Journal+of+Developmental+Biology%22%5BJournal%5D)">Planaria: an animal model that integrates development, regeneration and pharmacology.</a> International Journal of Developmental Biology. 61, 519-529 (2017).</li>
<li>Palladini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+pharmacological+study+of+cocaine+activity+in+planaria%5BTitle%5D)+AND+%22Comparative+Biochemistry+and+Physiology%22%5BJournal%5D)">A pharmacological study of cocaine activity in planaria.</a> Comparative Biochemistry and Physiology. 115 (1), 41-45 (1996).</li>
<li>Buttarelli, F. R., Pellicano, C., Pontieri, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuropharmacology+and+behavior+in+planarians%3A+translation+to+mammals%5BTitle%5D)+AND+%22Comparative+Biochemistry+and+Physiology+Part+C.+Toxicology+%26+Pharmacology%22%5BJournal%5D)">Neuropharmacology and behavior in planarians: translation to mammals.</a> Comparative Biochemistry and Physiology Part C. Toxicology & Pharmacology. 147 (4), 399-408 (2008).</li>
<li>Nishimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+glutamic+acid+decarboxylase+gene+and+distribution+of+GABAergeric+nervous+system+in+the+planarian+Dugesia+japonica%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Identification of glutamic acid decarboxylase gene and distribution of GABAergeric nervous system in the planarian Dugesia japonica.</a> Neuroscience. 153 (4), 1103-1114 (2008).</li>
<li>Raffa, R. B., Rawls, S. M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRaffa%2C+RB+%22A+model+for+drug+action+and+abuse%22">A model for drug action and abuse</a>. , Landes Bioscience. Austin, TX. (2008).</li>
<li>Hall, F., Morita, M., Best, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((neoplastic+transformation+in+the+planarian%3A+I+cocarcinogenesis+and+histopathology%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Zoology%22%5BJournal%5D)">neoplastic transformation in the planarian: I cocarcinogenesis and histopathology.</a> The Journal of Experimental Zoology. 240 (2), 211-227 (1986).</li>
<li>Voura, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Planarians+as+models+of+cadmium-induced+neoplasia+provide+measurable+benchmarks+for+mechanistic+studies%5BTitle%5D)+AND+%22Ecotoxicology+and+Environmental+Safety%22%5BJournal%5D)">Planarians as models of cadmium-induced neoplasia provide measurable benchmarks for mechanistic studies.</a> Ecotoxicology and Environmental Safety. 142, 544-554 (2017).</li>
<li>Van Roten, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+carcinogenic+trigger+to+study+the+function+of+tumor+suppressor+genes+in+Schmedtea+mediterranea%5BTitle%5D)+AND+%22Disease+Models+and+Mechanisms%22%5BJournal%5D)">A carcinogenic trigger to study the function of tumor suppressor genes in Schmedtea mediterranea.</a> Disease Models and Mechanisms. 11 (9), 032573(2018).</li>
<li>Mason, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemo-klino-kinesis+in+planarian+food+location%5BTitle%5D)+AND+%22Animal+Behaviour%22%5BJournal%5D)">Chemo-klino-kinesis in planarian food location.</a> Animal Behaviour. 23 (2), 460-469 (1975).</li>
<li>Van Huizen, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Weak+magnetic+fields+alter+stem+cell-mediated+growth%5BTitle%5D)+AND+%22Science+Advances%22%5BJournal%5D)">Weak magnetic fields alter stem cell-mediated growth.</a> Science Advances. 5 (1), 7201(2019).</li>
<li>Brown, H. M., Ogden, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+electrical+response+of+the+planarian+ocellus%5BTitle%5D)+AND+%22Journal+of+General+Physiology%22%5BJournal%5D)">The electrical response of the planarian ocellus.</a> Journal of General Physiology. 51 (2), 255-260 (1968).</li>
<li>Inoue, T., Yamashita, T., Agata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thermosensory+signaling+by+TRPM+is+processed+by+brain+serotonergic+neurons+to+produce+planarian+thermotaxis%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Thermosensory signaling by TRPM is processed by brain serotonergic neurons to produce planarian thermotaxis.</a> The Journal of Neuroscience. 34 (47), 15701-15714 (2014).</li>
<li>Byrne, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+ethanol+on+negative+phototaxis+and+motility+in+brown+planarians+%28Dugesia+tigrina%29%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">Effects of ethanol on negative phototaxis and motility in brown planarians (Dugesia tigrina).</a> Neuroscience Letters. 685, 102-108 (2018).</li>
<li>de Sousa, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptomic+analysis+of+planarians+under+simulated+microgravity+or+8g+demonstrates+that+alteration+of+gravity+induces+genomic+and+cellular+alterations+that+could+facilitate+tumoral+transformation%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Transcriptomic analysis of planarians under simulated microgravity or 8g demonstrates that alteration of gravity induces genomic and cellular alterations that could facilitate tumoral transformation.</a> International Journal of Molecular Sciences. 20 (3), 720(2019).</li>
<li>Best, J. B., Rubinstein, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maze+learning+and+associated+behavior+in+planaria%5BTitle%5D)+AND+%22Journal+of+Comparative+and+Physiological+Psychology%22%5BJournal%5D)">Maze learning and associated behavior in planaria.</a> Journal of Comparative and Physiological Psychology. 55, 560-566 (1962).</li>
<li>Shomrat, T., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+automated+training+paradigm+reveals+long-term+memory+in+planarians+and+its+persistence+through+head+regeneration%5BTitle%5D)+AND+%22The+Journal+Experimental+Biology%22%5BJournal%5D)">An automated training paradigm reveals long-term memory in planarians and its persistence through head regeneration.</a> The Journal Experimental Biology. 216, Pt 20 3799-3810 (2013).</li>
<li>Robarts-Galbraith, R. H., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+organ+trail%3A+insights+into+organ+regeneration+in+the+planarian%5BTitle%5D)+AND+%22Current+Opinion+in+Genetics+%26+Development%22%5BJournal%5D)">On the organ trail: insights into organ regeneration in the planarian.</a> Current Opinion in Genetics & Development. 32, 37-46 (2015).</li>
<li>Ivancovic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Model+systems+for+regeneration%3A+planarians%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Model systems for regeneration: planarians.</a> Development. 146 (17), 167684(2019).</li>
<li>Herath, S., Lobo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cross-inhibition+of+Turing+patterns+explains+the+self-organized+regulatory+mechanism+of+planarian+fission%5BTitle%5D)+AND+%22Journal+of+Theoretical+Biology%22%5BJournal%5D)">Cross-inhibition of Turing patterns explains the self-organized regulatory mechanism of planarian fission.</a> Journal of Theoretical Biology. 485, 110042(2019).</li>
<li>Animal Diversity. , Available from: 
 <a target="_blank" href="http://animaldiversity.ummz.umich.edu/accounts/Dugesia_tigrina/">http://animaldiversity.ummz.umich.edu/accounts/Dugesia_tigrina/</a> (2019).</li>
<li>Itoh, M. T., Shinozawa, T., Sumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circadian+rhythms+of+melatonin-synthesizing+enzyme+activities+and+melatonin+levels+in+planarians%5BTitle%5D)+AND+%22Brain+Research%22%5BJournal%5D)">Circadian rhythms of melatonin-synthesizing enzyme activities and melatonin levels in planarians.</a> Brain Research. 830 (1), 165-173 (1999).</li>
<li>Itoh, M. T., Igarashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circadian+rhythm+of+serotonin+levels+in+planarians%5BTitle%5D)+AND+%22Neuroreports%22%5BJournal%5D)">Circadian rhythm of serotonin levels in planarians.</a> Neuroreports. 11 (3), 473-476 (2000).</li>
<li>Hinrichsen, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photosensitivity+and+motility+in+planarian+Schmedtea+mediterranea+vary+diurnally%5BTitle%5D)+AND+%22Chronobiology+International%22%5BJournal%5D)">Photosensitivity and motility in planarian Schmedtea mediterranea vary diurnally.</a> Chronobiology International. 36 (12), 1789-1793 (2019).</li>
<li>Raffa, R. B., Desai, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Description+and+quantification+of+cocaine+withdrawal+signs+in+planaria%5BTitle%5D)+AND+%22Brain+Research%22%5BJournal%5D)">Description and quantification of cocaine withdrawal signs in planaria.</a> Brain Research. 1032 (1-2), 200-202 (2005).</li>
<li>Pagán, O. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cembranoid+from+tobacco+prevents+the+expression+of+induced+withdrawal+behavior+in+planarian+worms%5BTitle%5D)+AND+%22European+Journal+of+Pharmacology%22%5BJournal%5D)">A cembranoid from tobacco prevents the expression of induced withdrawal behavior in planarian worms.</a> European Journal of Pharmacology. 615 (1-3), 118-124 (2009).</li>
<li>Rawls, S. M., Patil, T., Yuvasheva, E., Raffa, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((First+evidence+that+drugs+of+abuse+produce+behavioral+sensitization+and+cross-sensitization+in+planarians%5BTitle%5D)+AND+%22Behavioural+Pharmacology%22%5BJournal%5D)">First evidence that drugs of abuse produce behavioral sensitization and cross-sensitization in planarians.</a> Behavioural Pharmacology. 21 (4), 301-313 (2010).</li>
<li>Venturini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+pharmacological+study+of+dopaminergic+receptors+in+planaria%5BTitle%5D)+AND+%22Neuropharmacology%22%5BJournal%5D)">A pharmacological study of dopaminergic receptors in planaria.</a> Neuropharmacology. 28 (12), 1377-1382 (1989).</li>
<li>Ouyang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+effects+of+Spenda%2C+Equal+and+sucrose%3A+Clues+from+planarians+on+sweeteners%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">Behavioral effects of Spenda, Equal and sucrose: Clues from planarians on sweeteners.</a> Neuroscience Letters. 636, 213-217 (2017).</li>
<li>Pagán, O. R., Montgomery, E., Deats, S., Bach, D., Baker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+of+nicotine-induced%2C+curare-sensitive%2C+behavior+in+planarians%5BTitle%5D)+AND+%22Neurochemical+Research%22%5BJournal%5D)">Evidence of nicotine-induced, curare-sensitive, behavior in planarians.</a> Neurochemical Research. 40 (10), 2087-2090 (2015).</li>
<li>Shibata, N., Agata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+interference+in+planarians%3A+feeding+and+injection+of+synthetic+dsRNA%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">RNA interference in planarians: feeding and injection of synthetic dsRNA.</a> Methods in Molecular Biology. 1774, 455-466 (2018).</li>
<li>Pagán, O. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reversal+of+cocaine-induced+planarian+behavior+by+parthenolide+and+related+sesquiterpene+lactones%5BTitle%5D)+AND+%22Pharmacology+Biochemistry+and+Behavior%22%5BJournal%5D)">Reversal of cocaine-induced planarian behavior by parthenolide and related sesquiterpene lactones.</a> Pharmacology Biochemistry and Behavior. 89 (2), 160-170 (2008).</li>
<li>Vouga, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stereochemistry+and+neuropharmacology+of+a+%27bath+salt%27+cathinone%3A+S-enantiomer+of+mephedrone+reduces+cocaine-induced+reward+and+withdrawal+in+invertebrates%5BTitle%5D)+AND+%22Neuropharmacology%22%5BJournal%5D)">Stereochemistry and neuropharmacology of a 'bath salt' cathinone: S-enantiomer of mephedrone reduces cocaine-induced reward and withdrawal in invertebrates.</a> Neuropharmacology. 91, 109-116 (2015).</li>
<li>Chan, J. D., Marchant, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacological+and+functional+genetic+assays+to+manipulate+regeneration+of+the+planarian+Dugesia+japonica%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Pharmacological and functional genetic assays to manipulate regeneration of the planarian Dugesia japonica.</a> Journal of Visualized Experiments. (54), e3058(2011).</li>
</ol>

The authors have nothing to disclose.

A straightforward and accessible planarian motility assay is described to determine the stimulant and withdrawal effects of natural products. As a behavioral model it is necessary to have stringent protocols for scoring movement and clear definitions of any behaviors to standardize observations between different experimenters. The ideas presented offer a demonstration of how this can be achieved. Each laboratory using this protocol should adapt the presented information to suit the effects of the particular product being...

The protocol described uses planarian motility to provide a means of assessing the biomodulating effects of natural substances. It was specifically adapted to determine if these substances function as stimulants, and if they then were associated with a measurable withdrawal behavior1. This assay, known as the planarian locomotor velocity (pLmV) test, was first used to test known pharmacological agents2,3. The application of this planarian motility-based assay has since grown in popularity and has been adopted by different laboratories interested in substances other than natural products4,5. For this assay, a planarian is placed in a Petri dish containing spring water or spring water containing a dissolved biomodulator. Because the dish itself is placed on graph paper, the number of grid lines crossed by the animal with time as it moves about the container can be used to determine&#160;the rate of movement in each condition. The light/dark test, otherwise referred to as the conditioned place preference test (CPP), is another variation on the theme of monitoring planarian motility, and assesses how quickly the animals respond and migrate to a darkened environment6,7. Video tracking of planarian movements can also be analyzed using computer programs and center of mass (COM) tracking8,9,10,11.
Using the planarian as an animal model for such studies offers several advantages over other animals in that the experimenter can easily control the assay environment. Specifically, starving the planarians before experimentation can prevent their exposure to other nutritional or pharmacological agents that might otherwise confound the results, and the specific biomodulator under investigation can be introduced to the planarians simply by adding it directly to the culture water, thereby standardizing exposure. Since planarians have a nervous system and neurotransmitters that are reminiscent of &#8216;higher-order&#8217; animals, the physiology and experimental responses of these animals to neuromuscular stimuli are considered biologically relevant to other organisms12,13,14,15,16. Also, because planarians are relatively inexpensive and straightforward to maintain in the laboratory, they offer an accessible biological model for many investigators.
As an experimental animal, planarians are suitable to a wide range of studies. For example, our group, as well as other investigators use planarians to study tumorigenesis17,18,19. Planarians also exhibit a host of response behaviors to chemical, thermal, gravitational, electrical, photo, and magnetic stimuli that have formed the basis of other assay systems. Some of these effects have been used to study learning and memory in these animals20,21,22,23,24,25,26,27. The primary use of the planarian model in the literature at present focuses on the activity of planarian pluripotent stem cells, called neoblasts, and their role in regeneration28,29,30. Thus, adopting the model described here allows for further study using other planarian-based assays to provide a broader understanding of how natural products and other biomodulators affect the organism.

<table><tbody><tr><td>Bottled Water - 1 Gal.</td><td>Poland Spring</td><td>N/A</td><td>Spring water for planarian culture and to prepare solutions</td></tr><tr><td>Brown Planaria (Dugesia tigrina)</td><td>Carolina Biological Supply Company</td><td>132954</td><td>Brown planaria living (other species are acceptable)</td></tr><tr><td>Flat Paintbrush</td><td>Royal Crafter&#039;s Choice</td><td>9159</td><td>Flat watercolor paintbrushes for cleaning planarian culture containers</td></tr><tr><td>Glass Petri Dish - 10 cm</td><td>Kimax</td><td>N/A</td><td>10 cm diameter (glass) Petri dishes for pLmV assay</td></tr><tr><td>Glass Petri Dish - 5 cm</td><td>Kimax</td><td>N/A</td><td>5 cm and Petri dishes for rinsing planarians during withdrawal experiments and for stimulant habituation</td></tr><tr><td>Grid Paper</td><td>Any</td><td>N/A</td><td>Standard 0.5 cm grid paper for pLmV assay</td></tr><tr><td>iPEVO Visualizer (software)</td><td>iPEVO</td><td>https://www.ipevo.com/software/visualizer</td><td>Document camera software for video capture and recording</td></tr><tr><td>Metalware Set with Support Stand and Retort Ring</td><td>Any</td><td>N/A</td><td>Standard chemistry lab ring stand to hold a cell phone camera if used</td></tr><tr><td>Organic Egg</td><td>Any</td><td>N/A</td><td>Organic egg or beef liver for feeding planarains</td></tr><tr><td>Polycarbonate Bottle w/ Screw-on Cap - 10 mL</td><td>Beckman</td><td>N/A</td><td>Plastic vials to hold 5 to 10 mL volumes for stimulant habituation</td></tr><tr><td>Round Storage Container - 10 cm</td><td>Ziploc</td><td>N/A</td><td>10 cm Round food storage containers for approximately 90 planarians or fewer</td></tr><tr><td>Round Water Paint Brush</td><td>LOEW-Cornell</td><td>N/A</td><td>Small round watercolor paint brushes (numbers 3 to 6) - soft</td></tr><tr><td>Transfer Pipette</td><td>Any</td><td>N/A</td><td>Wide bore (5 mL) plastic transfer pipettes to move planarians</td></tr><tr><td>USB Document Camera</td><td>iPEVO</td><td>CDVU-06IP</td><td>Document camera (or other camera or cell phone camera)</td></tr></tbody></table>

a planarian motility assay to gauge the biomodulating properties of natural products

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and withdrawal properties of natural products is described. Experimental assays benefitting from unique aspects of planarian physiology have been applied to studies on wound healing, regeneration, and tumorigenesis. In addition, because planarians exhibit sensitivity to a variety of environmental stimuli and are capable of learning and developing conditioned responses, they can be used in behavioral studies examining learning and memory. Planarians possess a basic bilateral symmetry and a central nervous system that uses neurotransmitter systems amenable to studies examining the effects of neuromuscular biomodulators. Consequently, experimental systems monitoring planarian movement and motility have been developed to examine substance addiction and withdrawal. Because planarian motility offers the potential for a sensitive, easily standardized motility assay system to monitor the effect of stimuli, the planarian locomotor velocity (pLmV) test was adapted to monitor both stimulation and withdrawal behaviors by planarians through the determination of the number of grid lines crossed by the animals with time. Here, the technique and its application are demonstrated and explained.

The laboratory set up and preparation of the workspace for running the pLmV assay should be completed before experimentation begins. This includes the preparation of the habituation container, rinse container if needed (for withdrawal experiments), Petri dish over laminated grid paper, and properly placed camera (Figure 1). Once all the videos are taken, it is advisable to use a common datasheet to standardize data collection and presentation between investigators (Figur...

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.

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

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 or

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Learn the Planarian Locomotor Velocity Test to assess stimulant and withdrawal effects of natural products on motility. This method generates reliable behavioral data for preclinical neuroscience research.

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

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