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

Summary

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.

Abstract

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.

Introduction

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 agents^2,3. 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 products....

Protocol

1. Planarian husbandry

1. 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 acceptable^2,3.
   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 t....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bottled Water - 1 Gal.	Poland Spring	N/A	Spring water for planarian culture and to prepare solutions
Brown Planaria (Dugesia tigrina)	Carolina Biological Supply Company	132954	Brown planaria living (other species are acceptable)
Flat Paintbrush	Royal Crafter's Choice	9159	Flat watercolor paintbrushes for cleaning planarian culture containers
Glass Petri Dish - 10 cm	Kimax	N/A	10 cm diameter (glass) Petri dishes for pLmV assay
Glass Petri Dish - 5 cm	Kimax	N/A	5 cm and Petri dishes for rinsing planarians during withdrawal experiments and for stimulant habituation
Grid Paper	Any	N/A	Standard 0.5 cm grid paper for pLmV assay
iPEVO Visualizer (software)	iPEVO	https://www.ipevo.com/software/visualizer	Document camera software for video capture and recording
Metalware Set with Support Stand and Retort Ring	Any	N/A	Standard chemistry lab ring stand to hold a cell phone camera if used
Organic Egg	Any	N/A	Organic egg or beef liver for feeding planarains
Polycarbonate Bottle w/ Screw-on Cap - 10 mL	Beckman	N/A	Plastic vials to hold 5 to 10 mL volumes for stimulant habituation
Round Storage Container - 10 cm	Ziploc	N/A	10 cm Round food storage containers for approximately 90 planarians or fewer
Round Water Paint Brush	LOEW-Cornell	N/A	Small round watercolor paint brushes (numbers 3 to 6) - soft
Transfer Pipette	Any	N/A	Wide bore (5 mL) plastic transfer pipettes to move planarians
USB Document Camera	iPEVO	CDVU-06IP	Document camera (or other camera or cell phone camera)