Identifying and quantitating neural stem and progenitor cells in the adult brain

Abstract

Adult brain contains neural stem and progenitor cells that are capable of generating new neurons. Active continuous neurogenesis is limited to the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Newborn neurons gradually become fully functional and integrated into the existing circuitry of the olfactory bulb and the hippocampus. Transition from stem cells to fully differentiation neurons, the neuronal differentiation cascade, occurs through defined steps, and different classes of neuronal precursors can be distinguished by their morphology, expressed markers, and mitotic activity. Cells in these classes can be identified by immunophenotyping, labeling with thymidine analogues, and infection with retro- and lentiviral vectors. We here describe a transgenic approach that allows identification, in vivo visualization, isolation, and accurate enumeration of various classes of stem and progenitor cells in the adult brain. We generated a series of reporter mouse lines in which neural stem and progenitor cells express various fluorescent proteins (GFP, CFPnuc, H2B-GFP, DsRedTimer, and mCherry) under the control of the regulatory elements of the nestin gene. Using these lines, we were able to dissect the neuronal differentiation cascade into several discrete steps and to evaluate the changes induced by various neurogenic and antineurogenic stimuli. In particular, nuclear localization of the fluorescent signal in nestin-CFPnuc mice greatly simplifies the distribution pattern of neural stem and progenitor cells and allows accurate quantitation of changes induced by neurogenic agents in distinct classes of neuronal precursors. We present protocols for applying confocal microscopy, stereology, and electron microscopy to evaluate changes in the neurogenic compartments of the adult brain.