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>

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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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+and+functional+genetic+assays+to+manipulate+regeneration+of+the+planarian+Dugesia+japonica.">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 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...

Watch this Scientific Journal Video about A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products at JoVE.com

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 ...

a-planarian-motility-assay-to-gauge-biomodulating-properties-natural

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

5.6K Views.  State University of New York (SUNY) at Morrisville. 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.

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

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) controls several functions in C. elegans including learning and motor activity. Conditions that stimulate DA release (e.g., amphetamine (AMPH) treatments) or that prevent DA clearance (e.g., animals lacking the DA transporter (dat-1) which are incapable of reaccumulating DA into the neurons) generate an excess of extracellular DA ultimately resulting in inhibited locomotion. This behavior is particularly evident when animals swim in water. In fact, while wild-type animals continue to swim for an extended period, dat-1 null mutants and wild-type treated with AMPH or inhibitors of the DA transporter sink to the bottom of the well and do not move. This behavior is termed "Swimming Induced Paralysis" (SWIP). Although the SWIP assay is well established, a detailed description of the method is lacking. Here, we describe a step-by-step guide to perform SWIP. To perform the assay, late larval stage-4 animals are placed in a glass spot plate containing control sucrose solution with or without AMPH. Animals are scored for their swimming behavior either manually by visualization under a stereoscope or automatically by recording with a camera mounted on the stereoscope. Videos are then analyzed using a tracking software, which yields a visual representation of thrashing frequency and paralysis in the form of heat maps. Both the manual and automated systems guarantee an easily quantifiable readout of the animals' swimming ability and thus facilitate screening for animals bearing mutations within the dopaminergic system or for auxiliary genes. In addition, SWIP can be used to elucidate the mechanism of action of drugs of abuse such as AMPH.

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

Swimming induced paralysis (SWIP) is a well-established behavioral assay used to study the underlying mechanisms of dopamine signaling in Caenorhabditis elegans (C. elegans). However, a detailed method to perform the assay is lacking. Here, we describe a step-by-step protocol for SWIP.

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) ...

Behavior

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Biology

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Immunology and Infection

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor impairment that worsens with time. While considerable advances have been made in determining genetic drivers of ALS for a subset of patients, the majority of cases have an unknown etiology. Further, the mechanisms underlying motor neuron dysfunction and degeneration are not well understood; therefore, there is an ongoing need to develop and characterize representative models to study these processes. Caenorhabditis elegans can adapt their movement to the physical constraints of their surroundings, with two primary movement paradigms studied in a laboratory environment- crawling on a solid surface and swimming in liquid. These represent a complex interplay between sensation, motor neurons, and muscles. C. elegans models of ALS can exhibit impairment in one or both of these movement paradigms. This protocol describes two sensitive assays for evaluating motility in C. elegans: an optimized radial locomotion assay measuring crawling on a solid surface and an automated method for tracking and analyzing swimming in liquid (thrashing). In addition to the characterization of baseline motor impairment of ALS models, these assays can detect suppression or enhancement of the phenotypes from genetic or small molecule interventions. Thus, these methods have utility for studying ALS&#160;models and any C. elegans strain that exhibits altered motility.

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

This protocol describes two sensitive assays for discriminating among mild, moderate, and severe motor impairment in C. elegans models of amyotrophic lateral sclerosis, with general utility for C. elegans strains, with altered motility.

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor ...

Neuroscience

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes&#8212;many not associated with the oral apparatus (OA)&#8212;that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking

We present a protocol for the characterization of motility and behavior of a population of hundred micron- to millimeter-sized cells using brightfield microscopy and cell tracking. This assay reveals that Stentor coeruleus transitions through four behaviorally distinct phases when regenerating a lost oral apparatus.

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds ...

Culturing and Screening the Plant Parasitic Nematode Ditylenchus dipsaci

Plant-parasitic nematodes (PPNs) destroy over 12% of global food crops every year, which equates to roughly 157 billion dollars (USD) lost annually. With a growing global population and limited arable land, controlling PPN infestation is critical for food production. Compounding the challenge of maximizing crop yields are the mounting restrictions on effective pesticides because of a lack of nematode selectivity. Hence, developing new and safe chemical nematicides is vital to food security. In this protocol, the culture and collection of the PPN species Ditylenchus dipsaci are demonstrated. D. dipsaci is both economically damaging and relatively resistant to most modern nematicides. The current work also explains how to use these nematodes in screens for novel small molecule nematicides and reports on data collection and analysis methodologies. The demonstrated pipeline affords a throughput of thousands of compounds per week and can be easily adapted for use with other PPN species such as Pratylenchus penetrans. The techniques described herein can be used to discover new nematicides, which may, in turn, be further developed into highly selective commercial products that safely combat PPNs to help feed an increasingly hungry world.

Culturing and Screening the Plant Parasitic Nematode Ditylenchus dipsaci

The present protocol describes a reliable and straightforward method for culturing, collecting, and screening Ditylenchus dipsaci.

Plant-parasitic nematodes (PPNs) destroy over 12% of global food crops every year, which equates to roughly 157 billion dollars (USD) lost annually. With ...

A Toxicological and Ecotoxicological Assay Based on Mussel (Mytilus galloprovincialis) Hemocytes Motility

Hemocytes are the circulating immune-competent cells in bivalve mollusks and play a key role in several important functions of cell-mediated innate immunity. During the early stages of the immune response, hemocytes actively migrate to the site of infection. This inherent motility is a fundamental characteristic of these cells. It represents a key cellular function that integrates multiple processes, such as cell adhesion, cell signaling, cytoskeletal dynamics, and changes in cell volume. Therefore, alterations in cell motility following exposure to drugs or pollutants can serve as a useful toxicological endpoint. Despite the fundamental role of cell motility in cellular physiology, it has been poorly investigated from a toxicological perspective. This work proposes a novel in vitro method for the rapid and sensitive assessment of the toxicity and ecotoxicity of pollutants, based on evaluating the hemocyte motility of Mytilus galloprovincialis. We developed a cell motility assay on hemocytes adhering to the bottom of a 96-well polystyrene microplate. Following exposure to increasing concentrations of drugs, cell trajectories, and velocities were quantified by cell tracking under time-lapse microscopy, allowing us to measure the effects on hemocyte motility. Due to the ease of hemocyte collection from the animals in a relatively non-invasive manner, the proposed method offers an alternative test for screening the effects and mechanisms of action of pollutants and drugs. It aligns with the 3Rs (Replacement, Reduction, and Refinement) criteria, addressing ethical concerns and contributing to the reduction of vertebrate in vivo animal testing.

The article proposes a novel in vitro method for the rapid and sensitive assessment of the toxicity and ecotoxicity of pollutants, based on the motility of Mytilus galloprovincialis hemocytes. The method aims to contribute to the development of more ethical and sensitive toxicological and ecotoxicological exposure tests.

Hemocytes are the circulating immune-competent cells in bivalve mollusks and play a key role in several important functions of cell-mediated innate ...

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

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