Confocal fluorescence imaging of embryonic mouse brain

Abstract

How do neurons develop and form a precise and stereotyped arrangement of connections? What sort of neural networks are found in biological systems? These are fundamental problems in neurobiology the answers to which have profound implications in our understanding of cellular differentiation, information processing and complex systems formation. The vertebrate brain, perhaps the most complex and the most developed of neural structures, is known to develop through a well-regulated interplay of inductive signals in the early embryo, to give rise to a dorsal anterior tube-like structure of neuroepithelial tissue. Within the neural tube, cells divide, some differentiate into neurons, migrate and extend axons to various target tissues in the body. The complexity of the resulting mature nervous system renders tracing and imaging of all the neural connections in the adult brain technically difficult. Thus, transparent mouse embryos whose initial axon tracts and neural connections are simpler and are still being formed make ideal subjects to study the growing complexity of neural network formation during the early stages of neurogenesis in the mammalian brain. Here, a method is presented to image the whole mouse brain using three different neuronspecific antibodies probed with fluorophore-conjugated secondary antibodies. The method exploits the optical sectioning capability of the confocal laser microscope to render a 3-dimensional reconstruction of the embryonic mouse brain.