The dividing population in aged cultures had a greater proportion of 2+ dividers, compared with the dividing population in young cultures (A, 56% vs. cells in aged cultures divide more frequently than ABT-046 mitotic cells in young cultures during a 48-hour period of live-cell time-lapse imaging. Double-thymidine-analog labeling also demonstrates that fewer aged cells are dividing at a given time, but those that do divide are significantly more likely to re-enter the cell cycle within a day, both in vitro and in vivo. Meanwhile, we observed that cellular survival is usually impaired in aged cultures. Using our live-cell imaging data, we developed a mathematical model describing cell cycle kinetics to predict the growth curves of cells over time in vitro and the labeling index over time in vivo. Together, these data surprisingly suggest that progenitor cells remaining in the aged SVZ are highly proliferative. Keywords:Neurogenesis, Aging, Mitotic rate, Mitotic activity, Survival, Progenitor == Introduction == Neural progenitor cells (NPCs) retaining the capacity to produce new neurons in the adult mammalian brain reside primarily in the subventricular zone (SVZ) and the subgranular zone of hippocampal dentate gyrus [1]. Neuron production declines in both areas during normal aging [2,3]; this phenomenon is usually correlated with cognitive decline [4,5]. However, the cellular mechanisms underlying age-related neurogenic decline are unclear. Neurogenesis is usually a complex, multistep process, and the documented age-related decline could KMT3B antibody be due to a decreased pool of neural stem cells, slower cell cycle progression, a lower survival rate, a deficit in migration capacity, or an inability to undergo neuronal differentiation and develop functional synapses. Although it can be challenging to examine these aspects of neurogenesis separately, previous investigations into the cellular mechanism of neurogenic decline have uncovered fewer neural stem cells existing in aged SVZ [6]. Of these remaining cells, a lower percentage are capable of differentiating into functional neurons; fewer aged NPCs could evoke action potentials after differentiation protocols, although those that did differentiate showed physiological characteristics indistinguishable by age [7]. In addition, researchers have observed a survival deficit in aging NPCs under both growth and differentiation conditions [7,8]. Much investigation of the proliferative capacity of aging NPCs in vitro has relied on gross analyses such as total cell or sphere counts that can be ABT-046 influenced by multiple confounding factors. Several in vivo studies, using single or sequential pulses of bromodeoxyuridine (BrdU), have concluded that aging leads to a loss of NPCs in the SVZ [6,7]. Others have suggested that neurogenic decline may be due to NPCs undergoing quiescence or a lengthening of the cell cycle, perhaps due to increased tumor suppressor expression [8,9] or decreased growth factor responsiveness [10,11]. However, cell cycle kinetics have not been empirically decided in aging NPCs. Thymidine-analog markers of S-phase can be used to determine the number of cells dividing at a given time (the mitotic index). The number of cells in S-phase at a given time gives no information about the rate of cell cycle transit or re-entry. However, several of these markers can be used in concert to calculate the time between successive S-phases. This measure is usually affected by the amount of time needed to transit through the cell cycle, as well as the latency and likelihood for NPCs to re-enter the cell cycle, all factors which could affect net proliferative activity. In this study, we quantified the time between successive S-phase labels and cytokinetic events in young adult and aged adult NPCs. Surprisingly, aged cultures appear to contain both a highly quiescent populace and a highly proliferative populace; in contrast, many young NPCs divide sporadically. This study demonstrates that, although a fewer number of aged cells are cycling at a given time, the actively cycling NPCs remaining in the aged mouse forebrain undergo more cell divisions in a given period of time than those in the young adult forebrain. == Materials and Methods == == NPC Isolation == All experiments were performed as approved by the University of Washington Institutional Animal Care and ABT-046 Use Committee. Female C57BL/6 mice were housed at 21C with access to food and water ad libitum. Adult NPCs were isolated as previously described [12]. Briefly, wild-type C57BL/6 mice, 3 months and 18 months of age, were overdosed with Beuthanasia and transcardially perfused with ice-cold saline. Brain tissue, not including olfactory bulbs or cerebellum, was mechanically and enzymatically dissociated.