Quantitative characterization of planarian wild-type behavior as a platform for screening locomotion phenotypes

Abstract

SUMMARYChanges in animal behavior resulting from genetic or chemical intervention are frequently used for phenotype characterizations. The majority of these studies are qualitative in nature, especially in systems that go beyond the classical model organisms. Here, we introduce a quantitative method to characterize behavior in the freshwater planarian Schmidtea mediterranea. Wild-type locomotion in confinement was quantified using a wide set of parameters, and the influences of intrinsic intra-worm versus interworm variability on our measurements was studied. We also examined the effect of substrate, confinement geometry and the interactions with the boundary on planarian behavior. The method is based on a simple experimental setup, using automated center-of-mass tracking and image analysis, making it an easily implemented alternative to current methods for screening planarian locomotion phenotypes. As a proof of principle, two drug-induced behavioral phenotypes were generated to show the capacity of this method. Supplementary material available online at

On-chip immobilization of planarians for in vivo imaging

“…We compared the performance of the PIC to standard methods used in planarian research for immobilization, such as anesthetizing worms with chloretone or ethanol or immobilization in agarose1617. In all cases, we performed brightfield and fluorescence imaging because in vivo studies of planarian regeneration require observation both of anatomical changes in the whole organism and of cellular-level changes in specific structures.…”

“…Three typical protocols for application of anesthetics were compared to mounting on the PIC: long (2.5 hours) exposure to dilute (0.08% by volume) chloretone16, short (5 minutes) exposure to concentrated (0.2%) chloretone, and 1 hour exposure to 3% ethanol17. Planarians were kept in the low-concentration chloretone during imaging, but were removed from the high concentration chloretone and the ethanol prior to imaging to minimize toxicity, in keeping with previously published procedures1617.…”

“…Planarians were kept in the low-concentration chloretone during imaging, but were removed from the high concentration chloretone and the ethanol prior to imaging to minimize toxicity, in keeping with previously published procedures1617. Under brightfield illumination, all conventional chemical methods led to insufficient immobilization for proper imaging (Fig.…”

“…Worms were kept in 1× planarian water16 in the dark at 20°C except during feeding, cleaning, and experiments. For fluorescent in vivo labeling, we used Hoechst 33342, the Chromeo live cell mitochondrial staining kit (Active Motif, Carlsbad, CA), dextran-fluorescein (MW 2,000,000; Life Technologies, Grand Island, NY), and dextran-tetramethylrhodamine (MW 2,000,000; Life Technologies).…”

“…Planarians are negatively phototactic in the entire visible spectrum15, making immobilization of an animal for in vivo imaging at (sub-)cellular resolution a major challenge. Current methods for planarian immobilization include anesthetizing with chloretone (1,1,1-trichloro-2-methyl-2-propanol)16 or ethanol17, or embedding in low melting point agarose. There are major drawbacks to all existing immobilization techniques.…”