A Behavioral Assay to Study Effects of Retinoid Pharmacology on Nervous System Development in a Marine Annelid

  • M. Handberg-ThorsagerEmail author
  • V. Ulman
  • P. Tomançak
  • D. Arendt
  • M. Schubert
Part of the Methods in Molecular Biology book series (MIMB, volume 2019)


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 words

13-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).

Supplementary material

Movie 1

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)


  1. 1.
    Jékely G, Colombelli J, Hausen H et al (2008) Mechanism of phototaxis in marine zooplankton. Nature 456:395–399CrossRefGoogle Scholar
  2. 2.
    Fischer AH, Henrich T, Arendt D (2010) The normal development of Platynereis dumerilii (Nereididae, Annelida). Front Zool 7:31CrossRefGoogle Scholar
  3. 3.
    Randel N, Asadulina A, Bezares-Calderón LA (2014) Neuronal connectome of a sensory-motor circuit for visual navigation. elife 3:e02730CrossRefGoogle Scholar
  4. 4.
    Tosches MA, Bucher D, Vopalensky P et al (2014) Melatonin signaling controls circadian swimming behavior in marine zooplankton. Cell 159:46–57CrossRefGoogle Scholar
  5. 5.
    Conzelmann M, Offenburger S, Asadulina A et al (2011) Neuropeptides regulate swimming depth of Platynereis larvae. Proc Natl Acad Sci U S A 108:E1174–E1183CrossRefGoogle Scholar
  6. 6.
    Conzelmann M, Williams EA, Tunaru S et al (2013) Conserved MIP receptor-ligand pair regulates Platynereis larval settlement. Proc Natl Acad Sci U S A 110:8224–8229CrossRefGoogle Scholar
  7. 7.
    Niederreither K, Dollé P (2008) Retinoic acid in development: towards an integrated view. Nat Rev Genet 9:541–553CrossRefGoogle Scholar
  8. 8.
    Cunningham TJ, Duester G (2015) Mechanisms of retinoic acid signalling and its roles in organ and limb development. Nat Rev Mol Cell Biol 16:110–123CrossRefGoogle Scholar
  9. 9.
    Zieger E, Schubert M (2017) New insights into the roles of retinoic acid signaling in nervous system development and the establishment of neurotransmitter systems. Int Rev Cell Mol Biol 330:1–84CrossRefGoogle Scholar
  10. 10.
    Handberg-Thorsager M, Gutierrez-Mazariegos J, Arold ST et al (2018) The ancestral retinoic acid receptor was a low-affinity sensor triggering neuronal differentiation. Sci Adv 4:eaao1261CrossRefGoogle Scholar
  11. 11.
    Kane MA, Chen N, Sparks S et al (2005) Quantification of endogenous retinoic acid in limited biological samples by LC/MS/MS. Biochem J 388:363–369CrossRefGoogle Scholar
  12. 12.
    Escriva H, Bertrand S, Germain P et al (2006) Neofunctionalization in vertebrates: the example of retinoic acid receptors. PLoS Genet 2:e102CrossRefGoogle Scholar
  13. 13.
    Gutierrez-Mazariegos J, Nadendla EK, Studer RA et al (2016) Evolutionary diversification of retinoic acid receptor ligand-binding pocket structure by molecular tinkering. R Soc Open Sci 3:150484CrossRefGoogle Scholar
  14. 14.
    Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682CrossRefGoogle Scholar
  15. 15.
    Tinevez JY, Perry N, Schindelin J et al (2017) TrackMate: an open and extensible platform for single-particle tracking. Methods 115:80–90CrossRefGoogle Scholar
  16. 16.
    Meijering E, Dzyubachyk O, Smal I (2012) Methods for cell and particle tracking. Methods Enzymol 504:183–200CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • M. Handberg-Thorsager
    • 1
    Email author
  • V. Ulman
    • 1
  • P. Tomançak
    • 1
  • D. Arendt
    • 2
    • 3
  • M. Schubert
    • 4
  1. 1.Max Planck Institute of Molecular Cell Biology and GeneticsDresdenGermany
  2. 2.Developmental Biology UnitEuropean Molecular Biology LaboratoryHeidelbergGermany
  3. 3.Centre for Organismal StudiesUniversity of HeidelbergHeidelbergGermany
  4. 4.Sorbonne Université, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-Mer, Institut de la Mer de Villefranche-sur-MerVillefranche-sur-MerFrance

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