Methods in Brain Development of Molluscs
- 952 Downloads
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.
KeywordsImmunocytochemistry Fluorescence Antibody staining Neurogenesis Vibratome section Gene expression Complexity Cephalopod Brain Lophotrochozoa In situ hybridization
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.
- 2.Bullock TH, Horridge GA (1965) Structure and function in the nervous systems of invertebrates. Freeman and Company, London, p 1611Google Scholar
- 5.Chase R (2002) Behavior & its neural control in gastropod molluscs. Oxford University Press, Oxford, p 314Google Scholar
- 6.Nixon M, Young JZ (2003) The brains and lives of cephalopods. Oxford University Press, Oxford, p 392Google Scholar
- 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
- 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.Jékely G, Arendt D (2007) Confocal detection of NBT/BCIP in situ hybridization samples by reflection microscopy. Biochemica 4:12–14Google Scholar