The brain is thought to have decreased neuronal turnover with aging, leading to a progressive decline in neuron number, as well as to increased susceptibility to disease. The hippocampal dentate gyrus and the olfactory bulb continue to generate new neurons postnatally, but this process becomes less prominent during adulthood and especially during aging. Other regions, like the cerebral cortex, can generate new neurons in infant and juvenile animals, but not in adulthood. However, the changes in neuronal turnover rates with age have never been assessed. In this project we will use the GFAP- CreERT2 (GCE) transgenic mice, in which GFAP+ cells express a constitutively inactive Cre recombinase that can be transiently activated by a tamoxifen injection in vivo, allowing to genetically "mark" a time-specific cohort of astroglial cells with EGFP to follow their fate over subsequent epochs of development. We will tag GFAP+ cells with permanent EGFP expression in juvenile, adult and aged mice, and test the specificity and efficiency of reporter gene expression in astroglial cells at these different ages. We will then use this genetic fate mapping system to quantitatively assess neural stem cell fates and neuronal turnover in juvenile and adult mice, with the aim of eventually extending these studies to the aged brain. The total number of cells arising from GFAP precursors, visualized via EGFP reporter gene expression, will be estimated by stereological unbiased counting techniques and characterized by immunocytochemical markers and electron microscopy (EM). Two lines of studies will be pursued;(1) a quantitative assessment of the number and proportions of neurons, oligodendrocytes and astrocytes arising from GFAP+ cells in juvenile as compared to adult brains, and (2) an estimation of the rate of neuronal replacement in three key brain regions, comparing juvenile versus adult brains. In Aim 1, we will characterize the fate attained by GFAP+ cells in the cerebral cortex, hippocampus and olfactory bulb of adult animals reared in standard or enriched environment. The hypothesis is that the ability of GFAP+ cells to give rise to neurons and oligodendrocytes decreases as a function of aging, and that this effect will be less marked in mice reared under environmental stimulation. In Aim 2, we will compare the rate of neuronal turnover in the cerebral cortex, hippocampal dentate gyrus and olfactory bulb of juvenile and adult animals. At different times after tagging (up to 6-7 months), the total number of new and pre-existing neurons and the proportion of new versus total NeuN+ neurons will be ascertained. The experiment will be done in standard and enriched rearing conditions to assess the ability of the brain to modify turnover rates as a result of increased sensorimotor stimulation and cognitive activity. PUBLIC HEALTH RELEVANCE: Decreased production of neurons and increased cell death are common in the aged central nervous system and contribute to increased susceptibility to brain injury and degenerative disorders. The goal of this project is to develop rigorous methods for assessing neuronal turnover and how this turnover is affected by environmental enrichment, with the intent of applying these methods to study the aging CNS. Understanding how neuronal turnover differs in the immature and aged brain tissue will provide new ideas to improve plasticity in the aged brain.