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Methods in Brain Development of Molluscs

  • Andreas WanningerEmail author
  • Tim Wollesen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2047)

Abstract

Representatives of the phylum Mollusca have long been important models in neurobiological research. Recently, the routine application of immunocytochemistry and gene expression analyses in combination with confocal laserscanning microscopy has allowed fast generation of highly detailed reconstructions of neural structures of even the smallest multicellular animals, including early developmental stages. As a consequence, large-scale comparative analyses of neurogenesis—an important prerequisite for inferences concerning the evolution of animal nervous systems—are now possible in a reasonable amount of time. Herein, we describe immunocytochemical staining and in situ hybridization protocols for both, whole-mount preparations of developmental stages—usually 70–300 μm in size—as well as for vibratome and cryostat sections of complex brains. Although our procedures have been optimized for marine molluscs, they may easily be adapted to other (marine) organisms by the creative neurobiologist.

Keywords

Immunocytochemistry Fluorescence Antibody staining Neurogenesis Vibratome section Gene expression Complexity Cephalopod Brain Lophotrochozoa In situ hybridization 

Notes

Acknowledgments

This work was supported by the FWF (Austrian Science Fund) grant P24276-B22 to A.W. T.W. acknowledges support by the FWF (J-4198—Schrödinger fellowship) and the European Molecular Biology Laboratory Heidelberg. We thank Emanuel Redl (University of Vienna) for critical comments on an earlier version of this chapter.

References

  1. 1.
    Haszprunar G, Wanninger A (2012) Molluscs. Curr Biol 22(13):R510–R514CrossRefGoogle Scholar
  2. 2.
    Bullock TH, Horridge GA (1965) Structure and function in the nervous systems of invertebrates. Freeman and Company, London, p 1611Google Scholar
  3. 3.
    Lin MF, Leise EM (1996) Gangliogenesis in the prosobranch gastropod Ilyanassa obsoleta. J Comp Neurol 374:180–193CrossRefGoogle Scholar
  4. 4.
    Shigeno S, Tsuchiya K, Segawa S (2001) Embryonic and paralarval development of the central nervous system of the loliginid squid Sepioteuthis lessoniana. J Comp Neurol 437:449–475CrossRefGoogle Scholar
  5. 5.
    Chase R (2002) Behavior & its neural control in gastropod molluscs. Oxford University Press, Oxford, p 314Google Scholar
  6. 6.
    Nixon M, Young JZ (2003) The brains and lives of cephalopods. Oxford University Press, Oxford, p 392Google Scholar
  7. 7.
    Marois R, Carew TJ (1997) Projection patterns and target tissues of the serotonergic cells in larval Aplysia californica. J Comp Neurol 386:491–506CrossRefGoogle Scholar
  8. 8.
    Friedrich S, Wanninger A, Brückner M, Haszprunar G (2002) Neurogenesis in the mossy chiton, Mopalia muscosa (Gould) (Polyplacophora): evidence against molluscan metamerism. J Morph 253:109–117CrossRefGoogle Scholar
  9. 9.
    Wanninger A, Haszprunar G (2003) The development of the serotonergic and FMRF-amidergic nervous system in Antalis entalis (Mollusca, Scaphopoda). Zoomorphology 122:77–85Google Scholar
  10. 10.
    Braubach OR, Dickinson AJG, Evans CCE, Croll RP (2006) Neural control of the velum in larvae of the gastropod, Ilyanassa obsoleta. J Exp Biol 209:4676–4689CrossRefGoogle Scholar
  11. 11.
    Voronezhskaya EE, Nezlin LP, Odintsova NA, Plummer JT, Croll RP (2008) Neuronal development in larval mussel Mytilus trossulus (Mollusca: Bivalvia). Zoomorphology 127:97–110CrossRefGoogle Scholar
  12. 12.
    Todt C, Wanninger A (2010) Of tests, trochs, shells, and spicules: development of the basal mollusk Wirenia argentea (Solenogastres) and its bearing on the evolution of trochozoan larval key features. Front Zool 7:6CrossRefGoogle Scholar
  13. 13.
    Wollesen T, Loesel R, Wanninger A (2009) Pygmy squids and giant brains: mapping the complex cephalopod CNS by phalloidin staining of vibratome sections and whole-mount preparations. J Neurosci Methods 179:63–67CrossRefGoogle Scholar
  14. 14.
    Wollesen T, Cummins SF, Degnan BM, Wanninger A (2010) FMRFamide gene and peptide expression during central nervous system development of the cephalopod mollusk, Idiosepius notoides. Evol Dev 12:113–130CrossRefGoogle Scholar
  15. 15.
    Wollesen T, Nishiguchi MK, Seixas P, Degnan BM, Wanninger A (2012) The VD1/RPD2 α1-neuropeptide is highly expressed in the brain of cephalopod mollusks. Cell Tiss Res 348:439–452CrossRefGoogle Scholar
  16. 16.
    Rodriguez J, Deinhardt F (1960) Preparation of a semipermanent mounting medium for fluorescent antibody studies. Virology 12:316–317CrossRefGoogle Scholar
  17. 17.
    Moltschaniwskyj N, Hall K, Lipinski M, Marian J, Nishiguchi M, Sakai M, Shulman D, Sinclair B, Sinn D, Staudinger M, Gelderen R, Villanueva R, Warnke K (2007) Ethical and welfare considerations when using cephalopods as experimental animals. Rev Fish Biol Fish 17:455–476CrossRefGoogle Scholar
  18. 18.
    Andrews PLR, Darmaillacq AS, Dennison N, Gleadall IG, Hawkins P, Messenger JB, Osorio D, Smith VJ, Smith JA (2013) The identification and management of pain, suffering and distress in cephalopods, including anaesthesia, analgesia and humane killing. J Exp Mar Biol Ecol 447:46–64CrossRefGoogle Scholar
  19. 19.
    Jékely G, Arendt D (2007) Confocal detection of NBT/BCIP in situ hybridization samples by reflection microscopy. Biochemica 4:12–14Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Integrative Zoology, Faculty of Life SciencesUniversity of ViennaViennaAustria
  2. 2.European Molecular Biology LaboratoryHeidelbergGermany

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