ИСТИНА

Complex patterns of cell division, migration, elimination, and differentiation define brain structure and function. Ability to image, quantify, and analyze dividing and migrating cells in the whole brain (3D) is critical for revealing the features that are not recognized on conventional flat sections. Moreover, a time series of 3D images (pseudo-4D) may uncover dynamics and hidden patterns of the processes that underlie brain development, aging, disease, and therapy. This holds especially true for the studies of neurogenesis both in the developing and the adult nervous system where neural stem and progenitor cells divide in restricted regions and migrate along intricate trajectories to reach distant areas of the brain. We developed a new histological technique for 3D imaging of proliferating cells in the whole brain of developing and adult mice, based on labeling the dividing cells with 5-ethynyl-2'-deoxyuridine (EdU) and detecting them with fluorescent azide using whole-mount click-reaction (WM-CLICK). We also developed novel methods for automatic volume registration, cell counting, and morphing of 3D images for pseudo-4D data representation. We have now applied these techniques for visualizing patterns of cell division and migration in the early postnatal and adult mouse brain. We describe 3D patterns of division and migration of cells, most of them neural progenitors, and arrange a 3D time series into a pseudo-4D representation of cell division and migration in the perinatal brain. We also discovered three distinct proliferation/migration streams in the subventricular zone of the adult mouse brain – dorsolateral, dorsomedial and ventral, which merge together into a common rostral migration stream (RMS) and traced the 4D dynamics of their formation. Furthermore, we developed new computational algorithms to reveal the changes in the 3D patterns of cell division induced by pro- or anti-neurogenic factors, such as memantine and gamma-radiation, finding several brain areas affected by memantine treatment, including CA regions and dentate gyrus of the hippocampus, subcallosal zone, postpiriform transition area, and caudal piriform cortex. We also used WM-CLICK and computational algorithms to reveal the differences in early brain development of the wild type vs. autism model mice. Together, these examples demonstrate the utility of our approach for the quantitative and descriptive analysis of neurogenesis.