Name: planarian scrunching as a quantitative behavioral readout for noxious stimuli sensing
Uploaded: 2020-07-30T14:30:00
Description: Watch this Scientific Journal Video about Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing at JoVE.com

The authors thank Mr. Tapan Goel for comments on the manuscript. This work was funded by NSF CAREER Grant 1555109.

1. Quantitative planarian behavior assays
<ol>
	<li>Experimental setup
	<ol>
		<li>Place a dimmable LED panel upon a flat surface. The LED panel serves two purposes: (1) to provide a uniform white background and (2) to be used as an adjustable light source to obtain appropriate contrast. Place a 100 mm Petri dish arena upon the LED panel. 
		NOTE: To increase throughput, a multi-well plate may be used as an arena23,24, but larger arenas facilitate automate...

<ol>
	<li>Rink, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+systems+and+regeneration+in+planaria.">Stem cell systems and regeneration in planaria.</a> Development Genes and Evolution. 223, 67-84 (2013).</li><li>Reddien, P. W., Alvarado, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+Planarian+Regeneration.">Fundamentals of Planarian Regeneration.</a> Annual Review of Cell and Developmental Biology. 20, 725-757 (2004).</li><li>Cebri&#224;, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+the+central+nervous+system:+how+easy+for+planarians.">Regenerating the central nervous system: how easy for planarians.</a> Development Genes and Evolution. 217, 733-748 (2007).</li><li>Pearl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memoirs:+The+Movements+and+Reactions+of+Fresh-Water+Planarians:+A+Study+in+Animal+Behaviour.">Memoirs: The Movements and Reactions of Fresh-Water Planarians: A Study in Animal Behaviour.</a> Journal of Cell Science. , 2-46 (1903).</li><li>Mc Connell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Manual of Psychological Experimentation on Planarians. , (1967).</li><li>Talbot, J., Sch&#246;tz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+characterization+of+planarian+wild-type+behavior+as+a+platform+for+screening+locomotion+phenotypes.">Quantitative characterization of planarian wild-type behavior as a platform for screening locomotion phenotypes.</a> Journal of Experimental Biology. 214, 1063-1067 (2011).</li><li>Cochet-Escartin, O., Mickolajczk, K. J., Collins, E. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrunching:+a+novel+escape+gait+in+planarians.">Scrunching: a novel escape gait in planarians.</a> Physical Biology. 12, 055001 (2015).</li><li>Inoue, T., 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=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, 1-15 (2015).</li><li>Arenas, O. 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=Activation+of+planarian+TRPA1+by+reactive+oxygen+species+reveals+a+conserved+mechanism+for+animal+nociception.">Activation of planarian TRPA1 by reactive oxygen species reveals a conserved mechanism for animal nociception.</a> Nature Neuroscience. 20, 1686-1693 (2017).</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> Journal of Experimental Biology. 216, 3799-3810 (2013).</li><li>Blackiston, D., Shomrat, T., Nicolas, C. L., Granata, C., 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=A+Second-Generation+device+for+automated+training+and+quantitative+behavior+analyses+of+Molecularly-Tractable+model+organisms.">A Second-Generation device for automated training and quantitative behavior analyses of Molecularly-Tractable model organisms.</a> PLoS One. 5, 1-20 (2010).</li><li>Ross, K. G., Currie, K. W., Pearson, B. J., Zayas, R. 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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=SoxB1+Activity+Regulates+Sensory+Neuron+Regeneration,+Maintenance,+and+Function+in+Planarians.">SoxB1 Activity Regulates Sensory Neuron Regeneration, Maintenance, and Function in Planarians.</a> Developmental Cell. 47, 331-347 (2018).</li><li>Nishimura, 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=Reconstruction+of+Dopaminergic+Neural+Network+and+Locomotion+Function+in+Planarian+Regenerates.">Reconstruction of Dopaminergic Neural Network and Locomotion Function in Planarian Regenerates.</a> Developmental Neurobiology. 67, 1059-1078 (2007).</li><li>Birkholz, T. R., Beane, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+planarian+TRPA1+homolog+mediates+extraocular+behavioral+responses+to+near-ultraviolet+light.">The planarian TRPA1 homolog mediates extraocular behavioral responses to near-ultraviolet light.</a> Journal of Experimental Biology. 220, 2616-2625 (2017).</li><li>Currie, K. W., Molinaro, A. M., Pearson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+sources+of+hedgehog+modulate+neurogenesis+in+the+adult+planarian+brain.">Neuronal sources of hedgehog modulate neurogenesis in the adult planarian brain.</a> Elife. 5, (2016).</li><li>Talbot, J. A., Currie, K. W., Pearson, B. J., Collins, E. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smed-dynA-1+is+a+planarian+nervous+system+specific+dynamin+1+homolog+required+for+normal+locomotion.">Smed-dynA-1 is a planarian nervous system specific dynamin 1 homolog required for normal locomotion.</a> Biology Open. , 1-8 (2014).</li><li>Currie, K. W., Pearson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors+lhx1/5-1+and+pitx+are+required+for+the+maintenance+and+regeneration+of+serotonergic+neurons+in+planarians.">Transcription factors lhx1/5-1 and pitx are required for the maintenance and regeneration of serotonergic neurons in planarians.</a> Development. 140, 3577-3588 (2013).</li><li>Hagstrom, 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=Planarian+cholinesterase:+molecular+and+functional+characterization+of+an+evolutionarily+ancient+enzyme+to+study+organophosphorus+pesticide+toxicity.">Planarian cholinesterase: molecular and functional characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity.</a> Archives of Toxicology. 92, 1161-1176 (2018).</li><li>Hagstrom, D., Cochet-Escartin, O., Collins, E. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planarian+brain+regeneration+as+a+model+system+for+developmental+neurotoxicology.">Planarian brain regeneration as a model system for developmental neurotoxicology.</a> Regeneration. 3, 65-77 (2016).</li><li>Hagstrom, D., Cochet-Escartin, O., Zhang, S., Khuu, C., Collins, E. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freshwater+planarians+as+an+alternative+animal+model+for+neurotoxicology.">Freshwater planarians as an alternative animal model for neurotoxicology.</a> Toxicological Sciences. 147, 270-285 (2015).</li><li>Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-behavioral+endpoint+testing+of+an+87-chemical+compound+library+in+freshwater+planarians.">Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater planarians.</a> Toxicological Sciences. , 26-44 (2019).</li><li>Zhang, S., Hagstrom, D., Siper, N., Behl, M., Collins, E. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+neurotoxic+potential+of+15+flame+retardants+using+freshwater+planarians.">Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.</a> Neurotoxicology and Teratology. 73, 54-66 (2019).</li><li>Wu, J. P., Li, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+freshwater+planarians+in+environmental+toxicology+studies:+Advantages+and+potential.">The use of freshwater planarians in environmental toxicology studies: Advantages and potential.</a> Ecotoxicology and Environmental Safety. 161, 45-56 (2018).</li><li>Rompolas, P., Azimzadeh, J., Marshall, W. F., King, 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=Analysis+of+ciliary+assembly+and+function+in+planaria.">Analysis of ciliary assembly and function in planaria.</a> Methods in Enzymology. 525, 245-264 (2013).</li><li>Sabry, Z., 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=Pharmacological+or+genetic+targeting+of+Transient+Receptor+Potential+(TRP)+channels+can+disrupt+the+planarian+escape+response.">Pharmacological or genetic targeting of Transient Receptor Potential (TRP) channels can disrupt the planarian escape response.</a> PLoS One. , 753244 (2019).</li><li>Cochet-Escartin, O., Carter, J. A., Chakraverti-Wuerthwein, M., Sinha, J., Collins, E. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slo1+regulates+ethanol-induced+scrunching+in+freshwater+planarians.">Slo1 regulates ethanol-induced scrunching in freshwater planarians.</a> Physical Biology. 13, 1-12 (2016).</li><li>Hagstrom, D., Truong, L., Zhang, S., Tanguay, R. L., Collins, E. M. 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=Comparative+analysis+of+zebrafish+and+planarian+model+systems+for+developmental+neurotoxicity+screens+using+an+87-compound+library.">Comparative analysis of zebrafish and planarian model systems for developmental neurotoxicity screens using an 87-compound library.</a> Toxicological Sciences. , (2019).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9, 676-682 (2012).</li><li>Akiyama, Y., Agata, K., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+Behaviors+and+Wall-Curvature+Lead+to+Apparent+Wall+Preference+in+Planarian.">Spontaneous Behaviors and Wall-Curvature Lead to Apparent Wall Preference in Planarian.</a> PLoS One. 10, 0142214 (2015).</li><li>Paskin, T. R., Jellies, J., Bacher, J., Beane, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planarian+Phototactic+Assay+Reveals+Differential+Behavioral+Responses+Based+on+Wavelength.">Planarian Phototactic Assay Reveals Differential Behavioral Responses Based on Wavelength.</a> PLoS One. 9, 114708 (2014).</li><li>Petrus, 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=A+role+of+TRPA1+in+mechanical+hyperalgesia+is+revealed+by+pharmacological+inhibition.">A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition.</a> Molecular Pain. 3, 40 (2007).</li><li>Takano, T., 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=Regeneration-dependent+conditional+gene+knockdown+(Readyknock)+in+planarian:+Demonstration+of+requirement+for+Djsnap-25+expression+in+the+brain+for+negative+phototactic+behavior.">Regeneration-dependent conditional gene knockdown (Readyknock) in planarian: Demonstration of requirement for Djsnap-25 expression in the brain for negative phototactic behavior.</a> Development, Growth &amp; Differentiation. 49, 383-394 (2007).</li><li>Nishimura, 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=Identification+of+glutamic+acid+decarboxylase+gene+and+distribution+of+GABAergic+nervous+system+in+the+planarian+Dugesia+japonica.">Identification of glutamic acid decarboxylase gene and distribution of GABAergic nervous system in the planarian Dugesia japonica.</a> Neuroscience. 153, 1103-1114 (2008).</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> Journal of Neuroscience. 34, 15701-15714 (2014).</li></ol>

Using this protocol, one can quantitatively study the effects of physical and chemical stimuli7,28,29 or genetic manipulation (RNAi)28,29 on planarian locomotion. To maximize spatial resolution, it is best to move the camera as close as possible to the arena while ensuring the entire arena is in the field of view. To increase throughput, the behavior of multiple planaria...

In addition to their popularity for stem cell and regeneration research1,2,3, freshwater planarians have long been used in behavioral studies4,5, taking advantage of their comparatively large size (a few millimeters in length), ease and low cost of laboratory maintenance, and broad spectrum of observable behaviors. The introduction of computer vision and automated tracking to planarian behavior studies6,7,8,9,10,11 have enabled quantitative differentiation of behavioral phenotypes. Animal behavior is a direct readout of neuronal function. Because the planarian nervous system is of medium size and complexity, but shares conserved key elements with the vertebrate brain12,13,14, studying planarian behavior can provide insight into conserved mechanisms of neuronal action which may be hard to directly probe in more complex organisms. Thus, planarians are a valuable model for comparative neurobiology studies8,12,15,16,17,18,19,20,21. In addition, the aquatic environment allows for rapid and facile exposure to chemicals to study their effect on brain function in regenerating and adult planarians, making them a popular system for neurotoxicology22,23,24,25,26.
Planarians possess three distinct gaits, referred to as gliding, peristalsis, and scrunching. Each gait is exhibited under specific circumstances: gliding is the default gait, peristalsis occurs when ciliary function is compromised7,27, and scrunching is an escape gait &#8211; independent of cilia function &#8211; in response to certain noxious stimuli7. We have shown that scrunching is a specific response, elicited by the sensation of certain chemical or physical cues, including extreme temperatures or pH, mechanical injury, or specific chemical inducers, and thus is not a general stress response7,28,29.
Because of its specificity and stereotypical parameters, which can easily be quantified using this protocol, scrunching is a powerful behavioral phenotype that enables researchers to perform mechanistic studies dissecting sensory pathways and neuronal control of behavior25,28. Additionally, scrunching has been shown to be a sensitive endpoint to assay adverse chemical effects on nervous system development and function in neurotoxicology studies22,24,25,30. As several different sensory pathways seem to converge to induce scrunching through various mechanisms28, scrunching differs from other planarian behaviors because various, but specific, stimuli can be used to dissect distinct neuronal circuits and study how different signals are integrated to produce the scrunching phenotype.
Importantly, species differences exist, wherein one chemical may elicit scrunching in one planarian species, but a different behavioral response in another. For example, we have found that anandamide induces scrunching in the planarian species Dugesia japonica but induces peristalsis in Schmidtea mediterranea28. This example highlights the importance of being able to reliably distinguish between the different gaits, because they are the phenotypic manifestations of distinct molecular mechanisms. However, distinction of scrunching from peristalsis is difficult using qualitative observational data, because both gaits are musculature-driven and share qualitative similarities7,28. Thus, to distinguish the gaits it is necessary to perform cilia imaging or a quantitative behavioral study, which allows distinction based on characteristic parameters7,28. Because cilia imaging is experimentally challenging and requires specialized equipment such as a high-magnification compound microscope and a high-speed camera7,28, it is not as broadly accessible to researchers as quantitative behavioral analysis.
Here, we present a protocol for (1) the induction of scrunching using various physical (noxious temperature, amputation, near-UV light) and chemical (allyl isothiocyanate (AITC), cinnamaldehyde) stimuli and (2) the quantitative analysis of planarian behavior using freely available software. By quantifying four parameters (frequency of body length oscillations, relative speed, maximum amplitude, and asymmetry of body elongation and contraction)7, scrunching can be differentiated from gliding, peristalsis, and other behavioral states reported in the literature, such as snake-like locomotion15 or epilepsies15. Furthermore, while scrunching is conserved among different planarian species7, each species has its own characteristic frequency and speed; therefore, once the gliding and scrunching speeds of a species have been determined, speed alone can be used as a means to distinguish scrunching from gliding and peristalsis29. The protocol assumes no prior training in computational image analysis or behavioral studies and thus can also be applied for planarian behavioral experiments in a teaching laboratory context at the undergraduate level. Example data to facilitate protocol adaptation is provided in the Supplemental Material.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Allyl isothiocyanate, 95% (AITC)</td>
			<td>Sigma-Aldrich</td>
			<td>377430-5G</td>
			<td>CAUTION:&#160; Flammable and acutely toxic; handle in a fume hood with appropriate PPE.</td>
		</tr>
		<tr>
			<td>Camera lens, 2/3 25mm F/1.4&#160;</td>
			<td>Tamron</td>
			<td>23FM25SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates, 6 well, tissue culture treated</td>
			<td>Genesee Scientific&#160;</td>
			<td>25-105</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 50 mL polypropylene, sterile</td>
			<td>MedSupply Partners</td>
			<td>62-1019-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cinnamaldehyde, &#62;95%</td>
			<td>Sigma-Aldrich</td>
			<td>W228613-100G-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimmable A4 LED Tracer Light Box</td>
			<td>Amazon</td>
			<td>B07HD631RP</td>
			<td></td>
		</tr>
		<tr>
			<td>Flea3 USB3 camera</td>
			<td>FLIR</td>
			<td>FL3-U3-13E4M</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat resistant gloves</td>
			<td>Fisher Scientific</td>
			<td>11-394-298</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher Scientific</td>
			<td>HP88854200</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant Ocean Sea Salt, prepared in deionized water</td>
			<td>Instant Ocean</td>
			<td>SS15-10</td>
			<td>Prepare in deionized water at 0.5 g/L.</td>
		</tr>
		<tr>
			<td>Montj&#252;ic salts, prepared in Milli-Q water</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td>Prepare in milli-Q water at 1.6 mM NaCl, 1.0 mM CaCl2, 1.0 mM MgSO4, 0.1 mM MgCl2, 0.1 mM KCl, 1.2 mM NaHCO3; adjust pH to 7.0 with HCl.</td>
		</tr>
		<tr>
			<td>Petri dishes, 100 mm x 20 mm, sterile polystyrene</td>
			<td>Simport</td>
			<td>D210-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette, 20-200 &#956;L range</td>
			<td>Rainin</td>
			<td>17008652</td>
			<td></td>
		</tr>
		<tr>
			<td>PYREX 150 mL beaker</td>
			<td>Sigma-Aldrich</td>
			<td>CLS1000150</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade, 0.22 mm</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Roscolux color filter:&#160; Golden Amber</td>
			<td>Rosco</td>
			<td>R21</td>
			<td>Alternatively purchase the Roscolux Designer Color Selector (Musson Theatrical product #SBLUX0306) which includes all 3 color filters together.</td>
		</tr>
		<tr>
			<td>Roscolux color filter:&#160; Medium Red</td>
			<td>Rosco</td>
			<td>R27</td>
			<td></td>
		</tr>
		<tr>
			<td>Roscolux color filter:&#160; Storaro Red</td>
			<td>Rosco</td>
			<td>R2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Samco transfer pipette, 62 &#181;L large aperture</td>
			<td>Thermo Fisher</td>
			<td>691TS</td>
			<td></td>
		</tr>
		<tr>
			<td>Support stand&#160;</td>
			<td>Fisher Scientific</td>
			<td>12-947-976</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>VWR</td>
			<td>89095-600</td>
			<td></td>
		</tr>
		<tr>
			<td>UV laser pointer</td>
			<td>Amazon</td>
			<td>B082DGS86R</td>
			<td>This is a Class II laser (405nm &#177;10nm) with output power &#60;5 mW.</td>
		</tr>
	</tbody>
</table>

planarian scrunching as a quantitative behavioral readout for noxious stimuli sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Extraocular near-UV perception in S. mediterranea planarians is TRPA1-dependent and has been proposed to be linked to H2O2 release17. Because H2O2 exposure induces TRPA1-dependent scrunching in S. mediterranea and D. japonica planarians28, the steps in Section 2.1.4 can be used to test whether near-UV light exposure induces scrunching in both species. While D. japonica planarians scrunch (10/10)...

Watch this Scientific Journal Video about Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing at JoVE.com

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...

This protocol allows researchers to clarify planarian behavior and the sec mechanisms underlying this behavior by combining quantitative behavioral analysis with molecular and chemical perturbations. This technique is easily accessible to all skill levels as it does not require advanced instruments or specialized software to get quantitative behavioral readouts of free moving planarians. Scrunching can be used to study exception in planarians, and serves as sensitive endpoint to assay, disrupting nervous system function caused by Xenobiotics and disease.Begin by placing a dimmable LED Panel upon a flat surface, which will provide a uniform white background and can be used as an adjustable light source to obtain appropriate contrast. Place a 100 millimeter Petri dish arena on the LED Panel. Mount a camera on a ring stand above the arena and adjust its position, height and focus as necessary so that the entire arena is centered within the field of view and is in focus.Fill the arena with approximately 25 milliliters of the appropriate exposure media to have maximum volume, turn on the LED Panel and turn off any other light sources that may negatively affect recording quality. Drop a planarian at the center of the arena using a transfer Pipette. When testing a chemical solution, transfer the planarian with as little of planarian water as possible, so that the concentration of the chemical is not significantly changed.Begin recording data as image sequences in a native Fiji format. For gliding experiments, record one to two minutes of gliding behavior, for scrunching or peristalsis experiments, record long enough to capture at least three consecutive oscillations occurring in a straight line. Once the experiment is completed, terminate the recording.If the planarian reaches the boundary of the arena without satisfying the termination criterion, pipette the planarian back to the center of the arena. Open the raw image sequence for an experiment in Fiji, convert it to eight bit and use the arrow tool or slider at the bottom of the image stack to pan through the image sequence. To extract a time period and region of interest, draw a region of interest encompassing the full path of a planarian using the rectangle tool.Right click on the image stack and select duplicate, check the box where duplicate stack. Enter the first and last frames of the sequence of interest and click okay. For gliding experiments, extract a period of gliding where the planarian moves at least twice its body length for scrunching or peristalsis experiments.Extract an instance when the planarian undergoes a minimum of three consecutive and complete body oscillations in a straight line. Apply a threshold to the duplicated image stack to binarize the image and extract the planarian from the background, adjust the sliding bars so that the entire planarian is highlighted in red. The exact values are dependent on imaging quality.Leave the boxes for dark background, stack histogram and don't reset range unchecked. Scroll through the image stack to ensure a good threshold range and then click apply. In the convert stack to binary window, set the method to default and the background to light.Uncheck all boxes in this window and then click okay. A binarized image showing a black planarian on a white background will appear. Make sure that the entire planarian is visible in all frames of the image sequence.Set measurements by clicking analyze and set measurements. Check the boxes for area, center of mass, stack position and fit ellipse, then click okay. Select the open image stack and select analyze, and analyze particles.In the analyze particles window, select show and masks to open a new stack showing all the objects that were detected with the chosen parameters. Set a size filter to remove unwanted noise by entering the approximate area of the planarian. Then check the boxes for display results and clear results and click okay.Pan through the mask image stack using the slider at the bottom of the Panel to make sure that there is no noise or frames without a planarian. On the results window, save the data using file and save as, add the CSV extension to the file name, to save the data as comma separated values. Once the data for the image stack is saved, close the respective image stack results and mask windows.To induce scrunching via noxious temperature, heat planarian water in a glass beaker to 65 degrees Celsius on a hot plate. Place a planarian in the center of the arena, and wait until it orients itself upright and begins gliding, then begin recording. Use a P 200 Pipette to slowly and consistently pipette 100 microliters of the preheated planarian water, post pharyngeal onto the tail end of the planarian to induce scrunching.Stop the recording, once scrunching has ceased. Place the planarian in a recovery container and exchange the media in the Petri dish with fresh room temperature planarian water, if running more experiments. To induce scrunching via amputation, transfer a planarian to the center of the arena and wait until the planarian orients itself upright and begins gliding.Then begin recording, amputate the planarian with a clean razor blade. Making sure that the cut location is consistent across experiments. Stop the recording once the anterior piece has ceased scrunching.Remove both pieces, place them in a separate container and allow them to regenerate for seven days. Amputated planarians can be reincorporated into the home container once they have regenerated. This protocol was used to test whether near UV light exposure, induces scrunching in S.mediterranea and D.japonica planarians, while D.japonica planarians scrunch, when exposed to near UV light, S.mediterranea planarians either exhibit tail thinning or no response.A quantification of the scrunching parameters for the D.japonica planarians that exhibited at least three consecutive straight line scrunches, reveals characteristics scrunching parameters for this species In contrast exposure to 250 micromolar cinnamaldehyde unknown TRPA one agonist in mice, causes scrunching and S.mediterranea. Whereas D.japonica planarians display a mixture of snake-like and oscillatory motion interrupted by gliding or vigorous head turns. A quantification of the samples with at least three consecutive oscillations, yielded significantly lower values for three out of four parameters then expected for scrunching in D.japonica, indicating that the observed oscillatory motion is not scrunching.RNAi confirms the specificity of scrunching and response to cinnamaldehyde exposure in S.mediterranea. Within 180 seconds of exposure in planarian water, all control RNAi planarian scrunched. Compared to none of the SMTRPA one RNAi planarians demonstrating that S.mediterranea scrunching in cinnamaldehyde requires SMTRPA one.It is essential to be consistent and how the animals are manipulated to reduce noise and ensure reproducibility in the behavioral measurements. This projects can be expanded to include body shape analysis, which would allow for identification and quantification of other planarian behaviors that are not captured here.

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