A Behavioral Assay to Study Effects of Retinoid Pharmacology on Nervous System Development in a Marine Annelid
Autonomous animal locomotion, such as swimming, is modulated by neuronal networks acting on cilia or muscles. Understanding how these networks are formed and coordinated is a complex scientific problem, which requires various technical approaches. Among others, behavioral studies of developing animals treated with exogenous substances have proven to be a successful approach for studying the functions of neuronal networks. One such substance crucial for the proper development of the nervous system is the vitamin A-derived morphogen retinoic acid (RA). In the larva of the marine annelid Platynereis dumerilii, for example, RA is involved in the specification and differentiation of individual neurons and responsible for orchestrating the swimming behavior of the developing larva. Here, we report a workflow to analyze the effects of RA on the locomotion of the P. dumerilii larva. We provide a protocol for both the treatment with RA and the recording of larval swimming behavior. Additionally, we present a pipeline for the analysis of the obtained data in terms of swimming speed and movement trajectory. This chapter thus summarizes the methodology for analyzing the effects of a specific drug treatment on larval swimming behavior. We expect this approach to be readily adaptable to a wide variety of pharmacological compounds and aquatic species.
Key words13-cis and all-trans retinoic acid Behavioral analysis Live imaging Marine invertebrate larvae Movement trajectory Pharmacological treatments Platynereis dumerilii Swimming speed
The authors are indebted to Miquel Vila Farré and Jochen Rink for help with microscopy recordings. Mette Handberg-Thorsager is supported by the Deutsche Forschungsgemeinschaft (DFG, grant number TO563/7-1), Vladimir Ulman by the German Federal Ministry of Research and Education (BMBF) under the code 031L0102 (de.NBI), and Detlev Arendt by the European Molecular Biology Laboratory and the European Research Council (BrainEvoDevo No. 294810).
Curated swimming tracks of wild-type Platynereis dumerilii larvae or P. dumerilii larvae treated with dimethyl sulfoxide (DMSO, control larvae), 0.5 μM all-trans retinoic acid (ATRA All-trans), 1 μM all-trans retinoic acid 13-cis and all-trans (ATRA), 0.5 μM 13-cis retinoic acid (13cRA), or 1 μM 13-cis retinoic acid (13cRA). The trajectories are overlaid on the live imaging recordings. The first 254 time points are shown. The color of the track indicates the starting time point in the movie, following the color code in Fig. 6a. Scale bar: 500 μm (mp4 22846 kb)