Aging Cell (2006) 5, pp545–558 Doi: 10.1111/j.1474-9726.2006.00243.x © 2006 The Authors 545 Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2006 Blackwell Publishing Ltd The window and mechanisms of major age-related decline in the production of new neurons within the dentate gyrus of the hippocampus Muddanna S. Rao, 1,2 Bharathi Hattiangady 1,2 and Ashok K. Shetty 1,2 1 Department of Surgery (Division of Neurosurgery), Duke University Medical Center, Durham, NC 27710, USA 2 Medical Research Service, Veterans Affairs Medical Center, Durham, NC 27705, USA Summary While it is well known that production of new neurons from neural stem/progenitor cells (NSC) in the dentate gyrus (DG) diminishes greatly by middle age, the phases and mechanisms of major age-related decline in DG neu- rogenesis are largely unknown. To address these issues, we first assessed DG neurogenesis in multiple age groups of Fischer 344 rats via quantification of doublecortin- immunopositive (DCX + ) neurons and then measured the production, neuronal differentiation and initial survival of new cells in the subgranular zone (SGZ) of 4-, 12- and 24-month-old rats using four injections (one every sixth hour) of 5′-bromodeoxyuridine (BrdU), and BrdU–DCX dual immunostaining. Furthermore, we quantified the numbers of proliferating cells in the SGZ of these rats using Ki67 immunostaining. Numbers of DCX + neurons were stable at 4 –7.5 months of age but decreased pro- gressively at 7.5–9 months (41% decline), 9–10.5 months (39% decline), and 10.5–12 months (34% decline) of age. Analyses of BrdU + cells at 6 h after the last BrdU injection revealed a 71–78% decline in the production of new cells per day between 4-month-old rats and 12- or 24-month-old rats. Numbers of proliferating Ki67 + cells (putative NSCs) in the SGZ also exhibited similar (72–85%) decline during this period. However, the extent of both neuronal differ- entiation (75–81%) and initial 12-day survival (67–74%) of newly born cells was similar in all age groups. Additional analyses of dendritic growth of 12-day-old neurons revealed that newly born neurons in the aging DG exhibit diminished dendritic growth compared with their age- matched counterparts in the young DG. Thus, major decreases in DG neurogenesis occur at 7.5–12 months of age in Fischer 344 rats. Decreased production of new cells due to proliferation of far fewer NSCs in the SGZ mainly underlies this decline. Key words: 5′-bromodeoxyuridine; aging; dentate neuro- genesis; doublecortin; granule cell layer; neural stem cells; rat; stem cell proliferation; subgranular zone; stem cell differentiation. Introduction Neural stem/progenitor cells (NSC) in the subgranular zone (SGZ) of the dentate gyrus (DG) produce new cells all through life via proliferation (Altman & Das, 1965; Kaplan & Hinds, 1977; Cameron et al., 1993; Eriksson et al., 1998; Gage, 2002; Gould & Gross, 2002; Abrous et al., 2005). The majority of these newly born cells differentiate into granule cells, which incorporate first into the dentate granule cell layer (GCL) and then into the hippocampal circuitry (Cameron & McKay, 2001; van Praag et al., 2002; Dayer et al., 2003; Christie & Cameron, 2006). Although the precise functions of dentate neurogenesis during adulthood are still unclear, multiple studies imply that the ongoing dentate neurogenesis is important for a broad range of hippocampal functions, which include learning, long-term spatial memory, and mood (Barnea & Nottebohm, 1994; Gould et al., 1999; Gross, 2000; Kempermann, 2002; Madsen et al., 2003; Santarelli et al., 2003; Malberg, 2004; Abrous et al., 2005; Snyder et al., 2005; Leuner et al., 2006; Winocur et al., 2006). However, addition of new neurons to the dentate GCL declines drastically by middle age (Kuhn et al., 1996; Nacher et al., 2003; Rao et al., 2005). Additionally, it has been shown that aged rats with preserved spatial memory exhibit relatively higher levels of dentate neuro- genesis than aged rats with spatial memory impairments (Drapeau et al., 2003). While a direct link between decreased neurogenesis and impaired memory during old age is controversial (Bizon & Gallagher, 2003; Merrill et al., 2003; Bizon et al., 2004), it is gen- erally believed that decreased dentate neurogenesis contributes to cognitive impairments during old age. Hence, there is great interest in comprehending the mechanisms and implications of an age-related decrease in dentate neurogenesis. Recently, using Fischer 344 (F344) rats, we quantified age- related changes in the quantity of new cells/neurons added to the GCL over a period of 12 days, and neuronal differentiation, maturation and long-term survival of newly born granule cells (Rao et al., 2005). This study suggested that most of the decrease in dentate neurogenesis occurs by middle age (12 months), as the decrease between 4 months and 12 months of age was very dramatic, but the decrease between 12 months Correspondence Professor Ashok K. Shetty, PhD, Division of Neurosurgery, DUMC Box 3807, Duke University Medical Center, Durham, NC 27710, USA. Tel.: 919-286- 0411 (Ext. 7096); fax: 919-286-4662; e-mail: ashok.shetty@duke.edu Muddanna S. Rao and Bharathi Hattiangady contributed equally to this work. Accepted for publication 18 September 2006