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 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.
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 – independent of cilia function – 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.
1. Quantitative planarian behavior assays
2. Scrunching induction
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) when exposed to near-UV light, S. mediterranea planarians either exhibit tail thinning (7/10) as previously described17 or no response (3/10) (Figure 4A,4B). A quantification of the scrunching parameters, as outlined in Section 1.4, for the D. japonica planarians that exhibited at least 3 consecutive straight-line scrunches reveals characteristic scrunching parameters for this species7,28 (νm = 0.84 ± 0.14, |Δε|max = 0.56 ± 0.06, v*m = 0.47 ± 0.07, and felong = 0.56 ± 0.03, values reported as mean ± standard deviation for N=7).
In contrast, exposure to 250 µM cinnamaldehyde, a known TRPA1 agonist in mice34, causes scrunching in S. mediterranea7,28 (νm = 0.46 ± 0.08, |Δε|max = 0.36 ± 0.08, v*m = 0.16 ± 0.04, and felong = 0.58 ± 0.04, values reported as mean n ± standard deviation for N=8) (Figure 5A), whereas D. japonica planarians at the same (and 1.6x the concentration) display a mixture of snake-like and oscillatory motion, interrupted by gliding and/or vigorous head turns (Figure 5A). A quantification of the (8/24) samples with at least three consecutive oscillations yields significantly lower values for 3 out of 4 parameters than expected for scrunching in this species (νm = 0.43 ± 0.08, |Δε|max = 0.39 ± 0.03, v*m = 0.17 ± 0.02, and felong = 0.54 ± 0.06, values reported as mean ± standard deviation for N=8). Thus, while D. japonica appear to scrunch upon cinnamaldehyde exposure, a comparison of the calculated parameters with the literature values for this species7,28 shows that the observed oscillatory motion is not scrunching. This example highlights the importance of quantitative measurements in conjunction with careful inspection of the raw behavioral data to properly interpret observed behaviors.
RNAi confirms the specificity of scrunching in response to cinnamaldehyde exposure in S. mediterranea. Within 180 seconds of exposure to 250 µM cinnamaldehyde in planarian water 15/15 unc22 (control) RNAi S. mediterranea planarians scrunched, whereas 0/16 SmTRPA1 RNAi planarians scrunched (Figure 5B), demonstrating that S. mediterranea scrunching in cinnamaldehyde requires SmTRPA1. Knockdown of SmTRPA1 was confirmed through a 60 second exposure to a 100 μM AITC bath28.
Figure 1: Planarian behavior experimental setup.
(A) Sample experimental setup for studying planarian behavior. (B) 100 mm Petri dish arena centered in the field of view of the camera. Please click here to view a larger version of this figure.
Figure 2: Representative examples of the Fiji image analysis of planarians in arena.
(A) Selected region of interest, encompassing the full planarian path, indicated by the yellow rectangle. (B) Sample frames from the region of interest after duplication. (C) Subtracting the planarian from background and noise via thresholding (i) 8-bit image of planarian with noise, denoted by the asterisk. (ii) Binarized image of planarian after thresholding. (iii) Mask of planarian after setting filtering by size to remove noise. Please click here to view a larger version of this figure.
Figure 3: Plotting planarian length with respect to time.
(A) Raw plot of planarian length versus time for a scrunching S. mediterranea planarian. The asterisk denotes a moment when the planarian turned while scrunching. (B) Possible ways to trim scrunching data. (i) A correctly trimmed plot that removes the turning event data. (ii) An incorrectly trimmed plot that does not remove the turning event data. Please click here to view a larger version of this figure.
Figure 4: Species specific responses to near-UV light.
(A) Sample frames of D. japonica scrunching and S. mediterranea tail thinning in response to near-UV light. (B) Representative oscillation plots of S. mediterranea and D. japonica in response to near-UV light. Please click here to view a larger version of this figure.
Figure 5: Species specific response to 250 μM cinnamaldehyde, a TRPA1 agonist.
(A) Representative oscillation plots for D. japonica and S. mediterranea planarians in a 250 µM cinnamaldehyde bath. (B) Representative oscillation plots showing loss of scrunching in 250 µM cinnamaldehyde in SmTRPA1 RNAi S. mediterranea planarians. Please click here to view a larger version of this figure.
Supplemental Materials. Please click here to download these materials.
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 planarians can be screened at once by recording multiple planarians simultaneously. When screening more than one planarian in a single arena, regions of interest can be drawn in Fiji to isolate individual planarians as described here or more advanced multi-object tracking can be employed. One issue with having multiple planarians in the same arena is that they can cross paths. This problem can be solved through the use of multi-well plates to isolate planarians from each other while still enabling simultaneous recording of many individuals to quantify behavior23,24. However, planarians will spend relatively more time at the wall in smaller arenas, requiring adjustments to the image analysis and limiting the resolution for scrunching/peristalsis quantification.
When stimuli are administered locally (e.g., pipetting7, amputation7,28, laser pointer17), it is crucial that the planarians are consistently stimulated in the same region because stimulating other body regions can potentially induce different behaviors. Different methods of delivery (such as pipetting or bath of a chemical) can also affect the consistency of the behavioral phenotype. Additionally, planarians can desensitize quickly28, which needs to be taken into consideration when planning experiments as the same planarians should not be immediately reused for multiple experiments, either using the same or different stimuli. Finally, as shown here for near-UV exposure and cinnamaldehyde, it is important to be aware that the same stimulus can induce distinct behaviors in different planarian species. D. japonica scrunched when stimulated with near-UV light near the tail tip, while S. mediterranea planarians displayed tail thinning. In contrast, cinnamaldehyde exposure induced scrunching in S. mediterranea but not in D. japonica planarians. Thus, while scrunching is a conserved response of various planarian species to noxious stimuli7, it has species specific parameters7,28, sensitivities28, and inducers28. Therefore, for a new species for which scrunching has not yet been parameterized, it is best to start with a well-conserved inducer, such as amputation7, to determine the species-specific parameters before testing the response to other stimuli.
One limitation of the analysis described here is that it does not account for turns and/or mixed behaviors, such as intermittent scrunching with head wiggling, gliding, or other body shape changes. However, close inspection of the raw data can help mitigate these issues if these instances are manually excluded from the analysis, as demonstrated in Figure 3. In addition, it is possible to add body shape analysis to the center of mass and length tracking described here and expand the protocol to quantify these other planarian behaviors. Given that the analysis does not make any assumptions about the studied organism, the protocol could in principle also be applied to other organisms that show similar types of behaviors.
The method of quantifying the different planarian gaits and distinguishing scrunching from peristalsis, as described here, assumes no prior training in computational image analysis or behavioral studies and does not require specialized equipment or software. To facilitate protocol adaptation, example data is provided in the Supplemental Material. The ease of obtaining and culturing planarians, as well as the ability to record behaviors without specialized equipment, makes planarian behavioral studies broadly accessible to research across all levels, from primary school classrooms to academic labs. A modified version of this protocol has been successfully used in a teaching laboratory setting that was primarily composed of freshmen and sophomore students and included both prospective STEM and non-STEM majors.
The combination of molecular (RNAi) and chemical tools with quantitative behavioral analysis, as described in this protocol, allow researchers to gain mechanistic insights into the molecular control of behavior. Such work has uncovered some of the key mediators and neuronal circuits involved in planarian gliding19,20, phototaxis17,35,36, thermotaxis9,37, and scrunching9,28,29. Although planarian behaviors may not have direct corollary behaviors in higher organisms, such as humans, these behaviors represent fundamental neuronal functions important to all organisms – the ability to sense and process specific stimuli and react appropriately. Because of the conservation of key neuronal functions across different organisms, mechanistic studies in planarians can teach us more broadly about neuronal control of behavior. Additionally, analyzing planarian behavior in response to chemical exposure can be used to study the chemical’s effects on the planarian nervous system23,24,25, which may inform on potential risks to the human brain. In particular, scrunching induced by noxious heat was found to be a sensitive and specific endpoint for assaying neurotoxicity, because it becomes disrupted by exposure to certain classes of chemicals22,24,25,30. Finally, the planarian’s unique regenerative capabilities allow researchers to dissect the dynamics of how different behaviors are restored during neuroregeneration.
The authors have nothing to disclose.
The authors thank Mr. Tapan Goel for comments on the manuscript. This work was funded by NSF CAREER Grant 1555109.
Allyl isothiocyanate, 95% (AITC) | Sigma-Aldrich | 377430-5G | CAUTION: Flammable and acutely toxic; handle in a fume hood with appropriate PPE. |
Camera lens, 2/3 25mm F/1.4 | Tamron | 23FM25SP | |
Cell culture plates, 6 well, tissue culture treated | Genesee Scientific | 25-105 | |
Centrifuge tubes, 50 mL polypropylene, sterile | MedSupply Partners | 62-1019-2 | |
Cinnamaldehyde, >95% | Sigma-Aldrich | W228613-100G-K | |
Dimmable A4 LED Tracer Light Box | Amazon | B07HD631RP | |
Flea3 USB3 camera | FLIR | FL3-U3-13E4M | |
Heat resistant gloves | Fisher Scientific | 11-394-298 | |
Hot plate | Fisher Scientific | HP88854200 | |
Instant Ocean Sea Salt, prepared in deionized water | Instant Ocean | SS15-10 | Prepare in deionized water at 0.5 g/L. |
Montjüic salts, prepared in Milli-Q water | Sigma-Aldrich | various | 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. |
Petri dishes, 100 mm x 20 mm, sterile polystyrene | Simport | D210-7 | |
Pipette, 20-200 μL range | Rainin | 17008652 | |
PYREX 150 mL beaker | Sigma-Aldrich | CLS1000150 | |
Razor blade, 0.22 mm | VWR | 55411-050 | |
Roscolux color filter: Golden Amber | Rosco | R21 | Alternatively purchase the Roscolux Designer Color Selector (Musson Theatrical product #SBLUX0306) which includes all 3 color filters together. |
Roscolux color filter: Medium Red | Rosco | R27 | |
Roscolux color filter: Storaro Red | Rosco | R2001 | |
Samco transfer pipette, 62 µL large aperture | Thermo Fisher | 691TS | |
Support stand | Fisher Scientific | 12-947-976 | |
Thermometer | VWR | 89095-600 | |
UV laser pointer | Amazon | B082DGS86R | This is a Class II laser (405nm ±10nm) with output power <5 mW. |