Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Summary

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.

Abstract

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.

Introduction

In addition to their popularity for stem cell and regeneration research^1,2,3, freshwater planarians have long been used in behavioral studies^4,5, 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 studies^6,7,8,

Protocol

1. Quantitative planarian behavior assays

1. Experimental setup
   1. 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 arena^23,24, but larger arenas facilitate automated image analysis.
   2. Mount a camera on a ring stand above the arena (Figure 1A). Adjust the camera position, hei....

Results

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).......

Discussion

Using this protocol, one can quantitatively study the effects of physical and chemical stimuli^7,28,29 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.......

Materials

Name	Company	Catalog Number	Comments
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.