Name: a planarian motility assay to gauge the biomodulating properties of natural products
Uploaded: 2020-05-30T14:00:00
Description: Watch this Scientific Journal Video about A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products at JoVE.com

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 ...

<ol>
	<li>Moustakas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guarana+provides+additional+stimulation+over+caffeine+alone+in+the+planarian+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocaine+withdrawal+in+Planaria.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+dopamine+D2+antagonist+activity+using+invertebrate+(Planaria)+locomotion+as+a+functional+endpoint.">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>Thum&#233;, I. S., Frizzo, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sertraline+induces+toxicity+and+behavioral+alternations+in+planarians.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+allosteric+modulator+of+the+human+dopamine+transporter.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sucrose+produces+withdrawal+and+dompamine-sensitive+reinforcing+effects+in+planarians.">Sucrose produces withdrawal and dompamine-sensitive reinforcing effects in planarians.</a> Physiology &amp; Behavior. , 8-13 (2013).</li><li>Zewde, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLDT+(planarian+light/dark+test):+an+invertebrate+assay+to+quantify+defensive+responding+and+study+anxiety-like+effects.">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., Kl&#228;mbt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FIM+Imaging+and+FIMtrack:+two+new+tools+allowing+high-throughput+and+cost+effective+locomotion+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planarian+shows+decision-making+behavior+in+response+to+multiple+stimuli+by+integrative+brain+function.">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. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planarians+as+models+of+cadmium-induced+neoplasia+provide+measurable+benchmarks+for+mechanistic+studies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+carcinogenic+trigger+to+study+the+function+of+tumor+suppressor+genes+in+Schmedtea+mediterranea.">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. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrical+response+of+the+planarian+ocellus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermosensory+signaling+by+TRPM+is+processed+by+brain+serotonergic+neurons+to+produce+planarian+thermotaxis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+ethanol+on+negative+phototaxis+and+motility+in+brown+planarians+(Dugesia+tigrina).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maze+learning+and+associated+behavior+in+planaria.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+training+paradigm+reveals+long-term+memory+in+planarians+and+its+persistence+through+head+regeneration.">An automated training paradigm reveals long-term memory in planarians and its persistence through head regeneration.</a> The Journal Experimental Biology. 216, 3799-3810 (2013).</li><li>Robarts-Galbraith, R. H., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+organ+trail:+insights+into+organ+regeneration+in+the+planarian.">On the organ trail: insights into organ regeneration in the planarian.</a> Current Opinion in Genetics &amp; Development. 32, 37-46 (2015).</li><li>Ivancovic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+systems+for+regeneration:+planarians.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-inhibition+of+Turing+patterns+explains+the+self-organized+regulatory+mechanism+of+planarian+fission.">Cross-inhibition of Turing patterns explains the self-organized regulatory mechanism of planarian fission.</a> Journal of Theoretical Biology. 485, 110042 (2019).</li><li>Itoh, M. T., Shinozawa, T., Sumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythms+of+melatonin-synthesizing+enzyme+activities+and+melatonin+levels+in+planarians.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+of+serotonin+levels+in+planarians.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosensitivity+and+motility+in+planarian+Schmedtea+mediterranea+vary+diurnally.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+and+quantification+of+cocaine+withdrawal+signs+in+planaria.">Description and quantification of cocaine withdrawal signs in planaria.</a> Brain Research. 1032 (1-2), 200-202 (2005).</li><li>Pag&#225;n, O. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cembranoid+from+tobacco+prevents+the+expression+of+induced+withdrawal+behavior+in+planarian+worms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+evidence+that+drugs+of+abuse+produce+behavioral+sensitization+and+cross-sensitization+in+planarians.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pharmacological+study+of+dopaminergic+receptors+in+planaria.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+effects+of+Spenda,+Equal+and+sucrose:+Clues+from+planarians+on+sweeteners.">Behavioral effects of Spenda, Equal and sucrose: Clues from planarians on sweeteners.</a> Neuroscience Letters. 636, 213-217 (2017).</li><li>Pag&#225;n, O. R., Montgomery, E., Deats, S., Bach, D., Baker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+nicotine-induced,+curare-sensitive,+behavior+in+planarians.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+in+planarians:+feeding+and+injection+of+synthetic+dsRNA.">RNA interference in planarians: feeding and injection of synthetic dsRNA.</a> Methods in Molecular Biology. 1774, 455-466 (2018).</li><li>Pag&#225;n, O. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+cocaine-induced+planarian+behavior+by+parthenolide+and+related+sesquiterpene+lactones.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereochemistry+and+neuropharmacology+of+a+'bath+salt'+cathinone:+S-enantiomer+of+mephedrone+reduces+cocaine-induced+reward+and+withdrawal+in+invertebrates.">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. 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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&#39;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 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...

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

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 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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