Methods in Brain Development of Molluscs

Representatives of the phylum Mollusca have long been important models in neurobiological research. Recently, the routine application of immunocytochemistry in combination with confocal laser scanning 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 protocols for both whole-mount preparations of developmental stages—usually 70–300 μm in size—as well as for vibratome sections of complex brains. Although our procedures have been optimized for marine molluscs, they may easily be adapted for other (marine) organisms by the creative neurobiologist.