Muddanna S. Rao and Bharathi Hattiangady contributed equally to this work.
The window and mechanisms of major age-related decline in the production of new neurons within the dentate gyrus of the hippocampus
Article first published online: 20 NOV 2006
Volume 5, Issue 6, pages 545–558, December 2006
How to Cite
Rao, M. S., Hattiangady, B. and Shetty, A. K. (2006), The window and mechanisms of major age-related decline in the production of new neurons within the dentate gyrus of the hippocampus. Aging Cell, 5: 545–558. doi: 10.1111/j.1474-9726.2006.00243.x
- Issue published online: 20 NOV 2006
- Article first published online: 20 NOV 2006
- Accepted for publication 18 September 2006
- dentate neurogenesis;
- granule cell layer;
- neural stem cells;
- stem cell proliferation;
- subgranular zone;
- stem cell differentiation
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 neurogenesis 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 progressively 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 differentiation (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.
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 neurogenesis 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 generally 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 and 24 months of age was insignificant. Moreover, it was found that decreased addition of new granule cells during aging was not linked to altered neuronal differentiation of newly born cells or to a decreased long-term survival of newly born cells because both of these parameters were comparable across the young adult, middle-aged and aged groups (Rao et al., 2005). Nevertheless, it was unclear whether the decrease in dentate neurogenesis between young adult age and middle age occurs progressively or abruptly at a certain age. Additionally, because of multiple injections of 5′-bromodeoxyuridine (BrdU) over a 12-day period, direct measurements of age-related alterations in the production of new cells per day, the extent of neuronal fate-choice decision by newly born cells shortly after birth, and the initial dendritic growth of newly differentiated neurons could not be determined. As elucidation of the above issues are important for developing apt intervention strategies that maintain greater levels of dentate neurogenesis during old age, we rigorously analyzed dentate neurogenesis at multiple time points during aging in this study.
We first quantified the status of dentate neurogenesis in F344 rats at multiple time points (4, 7.5, 9, 10.5, and 12 months of age) using doublecortin (DCX) immunostaining and optical fractionator counting of DCX-immunopositive (DCX+) neurons in the SGZ and GCL of the entire hippocampus. The choice of DCX immunostaining was based on earlier findings that DCX is an excellent marker of newly born neurons in the DG (Brown et al., 2003; Kempermann et al., 2003), and the vast majority of DCX+ neurons in the DG represent neurons that are born during the 12 days prior to euthanasia (Rao & Shetty, 2004). We next ascertained the overall production of new cells by NSCs, the neuronal fate-choice decision of newly born cells, initial survival of newly born cells and the overall dendritic growth of newly born neurons during the first 12 days after their birth. For this, we measured and characterized newly born cells in the SGZ and GCL of 4-, 12-, and 24-month-old rats at 6 h and 12 days after four intraperitoneal injections of BrdU (one injection every sixth hour for 18 h at a dose of 100 mg kg−1 body weight). We performed BrdU immunostaining and optical fractionator counting of BrdU+ cells, BrdU–DCX dual immunostaining/immunofluorescence, confocal microscopic analyses, and measurements of dendritic growth in 12-day-old neurons using Neurolucida (Microbrightfield Inc., Williston, VT, USA). Additionally, to determine age-related changes in the proliferation of putative NSCs, we quantified the total numbers of proliferating cells in the SGZ of 4-, 12- and 24-month-old rats using immunostaining for Ki67, an endogenous marker of proliferation expressed during late G1, S, M and G2 phases of the cell cycle (Cooper-Kuhn & Kuhn, 2002).
Distribution and number of newly born DCX+ neurons in different age groups of animals
We examined the hippocampi of 4-, 7.5-, 9-, 10.5-, and 12-month-old animals with DCX immunostaining and determined the age-related changes in dentate neurogenesis. DCX is a specific marker of newly born neurons in the adult DG and our recent study in adult animals has demonstrated that neurons identified with DCX immunostaining are fresh neurons that are predominantly born during the 12 days before euthanasia (Rao & Shetty, 2004). Hence, quantification of the absolute number of DCX+ neurons in the SGZ and GCL of the entire hippocampus reveals the recent status of dentate neurogenesis in the hippocampus. In all age groups, DCX immunostaining visualized newly formed neurons in the SGZ and the GCL throughout the antero-posterior extent of the DG (Fig. 1). Moreover, in all age groups, the cell bodies of DCX+ neurons were located in the SGZ or the inner third of the GCL (Fig. 1). In hippocampi of 4-, 7.5-, and 9-month-old animals (Fig. 1A–E), a significant fraction of DCX+ neurons had the phenotype of differentiated granule cells with vertically oriented dendrites extending into the outer two-thirds of the dentate molecular layer (Jones et al., 2003). In contrast, in the 10.5- and 12-month-old DG, DCX+ neurons were far fewer in number and only a few had vertically oriented dendrites reaching the outer two-thirds of the molecular layer (Fig. 1G,I). Furthermore, the dendritic branching of newly born neurons appeared less in the 10.5- and 12-month-old hippocampi, in comparison to younger age groups (Fig. 1H,J). A vast majority of DCX+ neurons in these groups appeared to have horizontally oriented dendrites, a feature of immature granule cells. Comparison of the absolute number of DCX+ neurons in the GCL and SGZ of the hippocampus between different age groups (n = 5 per age group) using one-way analysis of variance (anova) demonstrated that the extent of dentate neurogenesis in male F344 rats remains stable between 4 and 7.5 months of age, but decreases progressively thereafter until 12 months of age (Fig. 2). The decrease is 41% between 7.5 and 9.0 months of age (P < 0.001), 39% between 9.0 and 10.5 months of age (P < 0.001), and 34% between 10.5 and 12.0 months of age (P < 0.01; Fig. 2). Thus, the major decreases in dentate neurogenesis occur between 7.5 months and 12 months of age in male F344 rats.
Production of new cells per day from NSCs in the SGZ of young adult, middle-aged and aged rats
Examination of hippocampi at 6 h after four BrdU injections (over 18-h period) with BrdU immunostaining revealed newly born cells in the SGZ of all age groups of rats (Fig. 3A,C,E). The BrdU immunopositive cells were arranged in smaller clusters. However, individual clusters of BrdU-labeled cells were much smaller in the SGZ of middle-aged and aged rats because of fewer cells per cluster, in comparison to those in young adult rats. Additionally, the BrdU immunopositive cell clusters in the SGZ were less frequent in the middle-aged and aged hippocampi. Quantification of BrdU immunopositive cells at 6 h after BrdU injections in serial sections through the entire hippocampus revealed production of an average of 1723 new cells per day in the young adult SGZ (mean ± SEM = 1723 ± 90; n = 4; Fig. 3G). In contrast, the production of new cells was 501 ± 19 cells per day (n = 4) in the middle-aged SGZ and 370 ± 23 cells per day (n = 4) in the aged SGZ. The overall production of new cells in middle-aged (12-month-old) rats observed in this study is consistent with the production of new cells reported earlier for middle-aged Sprague-Dawley rats (McDonald & Wojtowicz, 2005). Overall, the decline in the production of new cells in the SGZ of 12- and 24-month-old rats was 71–78%, in comparison to the SGZ of 4-month-old rats (P < 0.001, one-way anova; Fig. 3G). However, the production of new cells per day was comparable between middle-aged and aged rats (Fig. 3G).
Early survival of newly born cells in the SGZ and GCL of young adult, middle-aged and aged rats
We ascertained the distribution and number of BrdU immunopositive cells at 12 days after four BrdU injections (over an 18-h period) in young adult, middle-aged, and aged rats. The BrdU immunopositive cells were more dispersed and isolated individual cells could be observed in the inner third of the GCL at this time point in all age groups (Fig. 3B,D,F). Quantification of the number of BrdU immunopositive cells for the entire SGZ and GCL revealed survival of 1244 newly born cells (1244 ± 90; n = 4) in 4-month-old rats, and 336 newly generated cells (336 ± 21; n = 4) in 12-month-old rats, and 275 new cells (275 ± 11; n = 4) in 24-month-old rats (Fig. 3G). Comparison of the numbers of newly born cells obtained at 12 days after BrdU injections with numbers of newly born cells measured at 6 h after BrdU injections demonstrated that the initial survival (i.e. for 12 days after birth) of newly born cells (67–74%) is similar across the three age groups of rats (Fig. 3G). However, two-way anova revealed that the decrease in the number of newly born cells from 6 h to 12 days is statistically significant for only 4-month-old rats (Fig. 3G).
Extent of proliferating (Ki67+) cells in the SGZ of young adult, middle-aged and aged rats
Investigation of hippocampi with Ki67 immunostaining demonstrated proliferating cells (putative NSCs) in the SGZ of all age groups of rats (Fig. 4). Similar to BrdU+ cells observed at 6 h after the last BrdU injection, Ki67+ cells were arranged in clusters. The individual clusters were relatively smaller and less frequently encountered in the SGZ of middle-aged and aged rats, in comparison to their counterparts in the young SGZ (Fig. 4A,C,E). Quantification of the total number of Ki67+ cells in the SGZ using serial sections through the entire hippocampus revealed that an average of 5074 cells are proliferative in the young adult SGZ (mean ± SEM = 5074 ± 285; n = 5; Fig. 4G). In contrast, the number of proliferating cells was 1440 ± 189 in the middle-aged SGZ (n = 5), and 731 ± 105 (n = 5) in the aged SGZ (Fig. 4G). When compared to the SGZ of 4-month-old rats, considerably fewer cells (putative NSCs) exhibited proliferation in the SGZ of 12- and 24-month-old rats (72–85% decline, P < 0.001, one-way anova; Fig. 4). Additionally, the number of proliferating cells in the SGZ exhibited further decline between middle age and old age (49% decline, P < 0.05; Fig. 4). Thus, decreased production of new cells per day observed in middle-aged and aged groups through BrdU labeling appears to be due to proliferation of a far fewer NSCs in the aging SGZ.
Neuronal differentiation of newly born cells in the young adult, middle-aged and aged SGZ
Characterization of neurons among newly born cells in animals killed at 6 h after BrdU injections was performed using DCX–BrdU dual immunofluorescence and Z-sectioning using confocal microscopy (Fig. 5A–F). Analyses of neurons among newly born cells in animals killed at 12 days after BrdU injections was accomplished using DCX–BrdU dual immunostaining using the two-chromogen method (Fig. 5G,I). With these, the DCX immunoreactivity was visualized in the soma and dendrites of newly born neurons and the BrdU immunoreactivity was detected in the nucleus of newly born cells. A great majority (75–81%) of newly born cells (BrdU positive cells) expressed the early neuronal marker DCX in all age groups at both 6 h and 12 days after BrdU injections (Fig. 6). Thus, most of the newly born cells differentiate into neurons in all age groups of rats. As similar percentages of newly born cells express DCX at 6 h and 12 days after BrdU injections, the results further suggest that DCX expression in newly born neurons persists for 12 days in all age groups of rats, which is consistent with our earlier results for young rats (Rao & Shetty, 2004). Extrapolation of the percentages of neurons with the absolute numbers of BrdU cells found at 6 h after four BrdU injections (over an 18-h period) revealed that the production of new neurons is 1395 per day in the young adult SGZ, 376 per day in the middle-aged SGZ, and 278 per day in the aged SGZ. Similarly, extrapolation of the percentages of neurons with numbers of BrdU cells found at 12 days after BrdU injections demonstrated that the addition of new neurons to the SGZ and GCL is 986 per day in the young adult, 263 in the middle-aged, and 205 in the aged.
Dendritic maturation of newly born neurons
Measurements of the soma and dendrites of 12-day-old neurons revealed that the numbers of dendritic nodes and endings per neuron were considerably greater in the young adult DG when compared to the middle-aged/aged DG (Table 1). In addition, the total dendritic length of new neurons in the middle-aged/aged DG is 42% less than the age-matched new neurons in the young adult DG (Table 1). The concentric circle analysis of Sholl revealed that the terminal branches of the apical dendrite in the young adult DG extend beyond the 200-µm distance from the soma (Fig. 7A). In contrast, in the middle-aged/aged DG, the terminal branches of the apical dendrite end between 150 and 200 µm distances from the soma (Fig. 7A). Greater numbers of dendritic segments intersect 50 and 100 µm distances from the soma in new neurons of young adult DG, in comparison to new neurons in the middle-aged/aged DG (Fig. 7A). Analyses of the dendritic nodes suggested that the majority of branching occurs at 50–100 µm distances from the soma in neurons of the young adult DG, in comparison to the middle-aged/aged DG where the majority of branching occurs between 0 and 50 µm from soma (Fig. 7B). Thus, the dendritic growth during the first 12 days after birth in the newly born neurons of the middle-aged/aged DG is retarded, in comparison to the newly born neurons of the young adult DG.
|Animal groups||Perimeter of soma (µm)||Cell body area (µm2)||Dendritic nodes (#)||Dendritic endings (#)||Total dendritic length (µm)|
|Young adult||27.5 ± 0.6||50.7 ± 2.2||7.9 ± 0.4||9.0 ± 0.4||505.3 ± 35.2|
|Middle-aged/aged||28.3 ± 1.0||49.0 ± 3.0||4.8 ± 0.4||6.3 ± 0.4||292.0 ± 25.2|
|P value||P > 0.05||P > 0.05||P < 0.0001||P < 0.0001||P < 0.0001|
The results of this quantitative study provide the following novel findings. (i) The window of major decrease in dentate neurogenesis during aging is between 7.5 months and 12 months of age in male F344 rats. This was evidenced by observations that the extent of dentate neurogenesis exhibits no change between 4 and 7.5 months of age, but declines considerably between 7.5 and 9 months (41% decline), 9 and 10.5 months (39% decline), and 10.5 and 12 months (34% decline) of age. Our earlier finding that the decrease in dentate neurogenesis between 12 and 24 months of age is insignificant (Rao et al., 2005) also supports this conclusion. (ii) Decreased production of new cells from NSCs underlies the decline in dentate neurogenesis during aging. This was established by the observation in BrdU labeling studies that dramatic decrease (71–78% decline) occurs in the production of new cells per day in the SGZ of 12- or 24-month-old rats in comparison to 4-month-old rats. (iii) Diminished production of new cells in the aging SGZ is linked to 72–85% reductions in the proliferation of putative NSCs. This was confirmed by the presence of considerably decreased number of Ki67+ cells in the SGZ of 12- or 24-month-old rats in comparison to 4-month-old rats. (iv) Aging does not impair the neuronal differentiation of newly born cells. This was substantiated by the observation that similar fractions (75–81%) of newly born cells differentiate into DCX+ neurons at 6–24 h after their birth. This finding is consistent with our earlier observation that similar fractions of newly born cells acquire DCX expression during 1–12 days after birth (Rao et al., 2005). (v) The extent of cell death of newly born cells is similar across the three age groups. This was attested by the indirect observation that comparable fractions (67–74%) of newly born cells exhibit 12 days survival in all age groups of rats. Our earlier finding that aging does not alter the long-term survival of newly born cells (Rao et al., 2005) also supports this conclusion. (vi) The early dendritic growth is retarded in newly born neurons of the middle-aged/aged DG. This was confirmed by the finding that newly born neurons in the middle-aged/aged DG exhibit considerably diminished dendritic growth at 12 days after their birth than their age-matched counterparts in the young adult DG. Thus, the window between young adult-to-adult ages is likely ideal for application of intervention strategies that are promising for both maintaining an enhanced proliferation of NSCs and promoting growth of newly born neurons in the SGZ. Such interventions may sustain greater levels of neurogenesis during aging.
New neuron production in the SGZ is decreased early during the course of aging
Characterization of newly born neurons positive for DCX reveals no significant change in dentate neurogenesis at 4–7.5 months of age, which is roughly equivalent to the phase of young adult-to-adult age. However, there is considerable progressive decrease in dentate neurogenesis between 7.5 and 12 months of age, which approximately corresponds to the period of adult-to-middle age. Analyses of newly born cells using BrdU labeling and BrdU and DCX immunofluorescence suggest that, between 4 months and 12 months of age, the production of new cells per day decreases dramatically, but neither the extent of neuronal fate-choice decision nor the initial survival of newly born cells is altered. This is consistent with the earlier report on dentate neurogenesis between 30-day-old and 12-month-old Sprague-Dawley rats (McDonald & Wojtowicz, 2005). Our results in addition show that the overall pattern of neurogenesis in 24-month-old rats is identical to that in 12-month-old rats, not only in terms of the production of new cells but also in terms of the extent of neuronal differentiation and initial 12-day survival. Considering these, it is clear that the major shift underlying decreased neurogenesis between adult-to-middle ages is decreased production of new cells. Analyses of Ki67+ cells further suggest that diminished production of new cells is linked to proliferation of far fewer putative NSCs in the aging SGZ. The overall decline in the number of proliferating NSCs in the SGZ was 72% in 12-month-old rats and 85% in 24-month-old rats. Thus, drastically diminished production of new neurons at middle age is a result of proliferation of fewer NSCs in the SGZ. Interestingly, the number of proliferating NSCs decreases further between 12 months and 24 months of age. However, the overall production of new neurons per day in 24-month-old rats remains statistically comparable to 12-month-old rats. This discrepancy might reflect other compensatory factors operating in the aged SGZ such as relatively decreased cell death of newly born cells immediately after their birth, in comparison to the middle-aged SGZ.
The above results raise an important question: why do fewer NSCs proliferate during adult-to-middle age period? This may be related to early changes in multiple neurogenesis regulatory factors. A decrease in the number of NSCs is however, unlikely, as analyses using putative markers of NSCs such as Sox-2, GFAP and vimentin suggest that the number of NSCs remains stable over the course of aging in the SGZ (Hattiangady & Shetty, 2006). Nevertheless, both increased quiescence and lengthening of cell cycle times observed in NSCs of the aging SGZ (Cameron & McKay, 1999; Hattiangady & Shetty, 2006) likely contribute to the diminished production of new cells during this period. Furthermore, as proliferation of NSCs is very sensitive to the concentration of multiple neurotrophic factors and signaling proteins in the milieu of the SGZ, it is possible that multiple factors that maintain proliferation of NSCs progressively decrease in the SGZ between adult-to-middle age period. These factors may include fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), phosphorylated cyclic adenosine monophosphate (AMP) response element binding protein (p-CREB), and neuropeptide Y (NPY). This is because each of these factors has been found to be mitogenic for stem/progenitor cell proliferation (Lichtenwalner et al., 2001; Jin et al., 2002a,b, 2003; Lee et al., 2002a,b; Nakagawa et al., 2002a,b; Howell et al., 2003; Fujioka et al., 2004; Sonntag et al., 2005). Moreover, the concentration of all of these factors has been shown to decline between young adult age and middle age (Shetty et al., 2004, 2005; Hattiangady et al., 2005). Besides, it is plausible that several other positive regulators of dentate neurogenesis, such as Wnt protein, serotonin, noggin, neurogenesin-1, and vascular niches (Gould, 1999; Palmer et al., 2000; Monje et al., 2002; Ueki et al., 2003; Fan et al., 2004; Lie et al., 2005) undergo progressive decline during this period. On the other hand, the negative regulators of dentate neurogenesis such as circulating corticosterone (Sapolsky, 1992; Gould et al., 1998) may exhibit progressive increase during this period. Indeed, studies show that basal corticosterone level doubles between 6 weeks and 12 months of age in rats (Heine et al., 2004) and removal of corticosterone from aged rats through adrenalectomy alleviates age-dependent decline in dentate neurogenesis (Cameron & McKay, 1999). Additionally, reduced cell death in the GCL observed during this period (Heine et al., 2004) likely contributes to the decreased neurogenesis, because it is believed that the presence of cell death in the GCL is a stimulatory factor for proliferation of NSCs (Gould & Tanapat, 1997). Thus, precise reasons for decreased proliferation of NSCs during adult-to-middle age period are not clear. Careful analyses of the above signaling proteins at multiple time points between adult-to-middle age period in future studies are required to address this issue.
Neuronal fate-choice decision and initial survival of newly born cells does not alter with aging
Analyses of newly born cells (BrdU labeled cells) for the expression of DCX at 6 h after four BrdU injections (over a period of 18 h) demonstrated that 75–81% of newly born cells differentiate into neurons, regardless of the age of the DG at the time of their birth. This is consistent with our earlier characterization of neuronal differentiation among newly born cells that are added to the SGZ and GCL over a period of 12 days (i.e. at 24 h after 12 daily injections of BrdU) in male F344 rats (Rao et al., 2005). Moreover, in the current study, the percentages of newly born cells expressing DCX were comparable at 6 h and 12 days after BrdU injections in all age groups, suggesting that DCX expression in newly born neurons persists for 12 days regardless of the age of the DG. The persistence of DCX for 12 days in virtually all newly born neurons is consistent with the suggestion in previous studies that DCX expression is present during neuronal progenitor, neuroblast, neuronal differentiation, and early dendritic and axon growth periods (Brown et al., 2003; Kempermann et al., 2003; Rao & Shetty, 2004). Thus, both the degree of neuronal differentiation from newly born cells and the period of DCX expression in newly generated neurons are not altered with aging, implying that the capability of the DG milieu to promote apt neuronal differentiation of newly born cells is not impaired with aging in F344 rats. Furthermore, the extent of cell death appears similar across the three age groups. This was confirmed by analyses of the initial survival of newly born cells and neurons, which revealed that similar percentages of newly born cells and neurons survive for 12 days in all age groups of rats. Along with our earlier observation that comparable percentages of newly born cells and neurons exhibit long-term survival (Rao et al., 2005), the results suggest that aging of the DG does not impair the survival of newly born cells and neurons at any stage of their development. From these results, it appears that the milieu of the aging DG contains adequate levels of factors that promote both neuronal differentiation and survival of newly born cells.
Early dendritic growth of newly born neurons is diminished in the aging DG
Analyses of dendritic growth at 12 days after their birth (based on BrdU–DCX dual immunostaining) revealed that newly born neurons in the middle-aged/aged DG have considerably diminished dendritic growth compared with their age-matched counterparts in the young adult DG. This is true for both dendritic branching and total dendritic length. The precise reasons for diminished dendritic growth in newly born neurons of the aging DG are unknown. It is possible that maturation of newly born neurons proceeds more slowly in the aging DG than in the young adult DG. This possibility is supported by our earlier observation that the acquisition of neuron-specific nuclear antigen (a marker of mature neurons) in newly born neurons added to the GCL over a period of 12 days is delayed in the middle-aged and aged DG, in comparison to young adult DG (Rao et al., 2005). In addition, a recent study on electrophysiological characteristics of newly born neurons in the young adult DG implies that the maturation of newly generated granule cells in the young adult DG is slower than new granule cells generated in the neonatal DG (Overstreet-Wadiche et al., 2006). The maturation process likely slows down further with increasing age resulting in significant differences in the dendritic growth of newly born neurons between young adult and aged groups. Alternatively, it is plausible that newly born neurons maintain a less complex dendritic tree and have altered function in the aging DG than their counterparts in the young adult DG. Comparison of morphological and electrophysiological characteristics of newly born neurons using retroviral markers at different time points after their birth between young adult and aging DG are needed in future to address these issues.
Diminished dendritic growth in newly born neurons of the aging DG could be linked to decreased γ-amino-butyric acid (GABA) signaling. This is because of the following. First, GABA signaling appears decreased in the aging hippocampus (Shetty & Turner, 1998b; Stanley & Shetty, 2004). Second, despite exhibiting functional glutamate receptors, newly born granule cells in the young adult DG initially receive only GABA-ergic synapses from local interneurons, and remain isolated from extrinsic excitatory input during the first 2 weeks of their existence (Wadiche et al., 2005; Overstreet-Wadiche & Westbrook, 2006). Third, both depolarizing GABA-ergic network activity and transcription factor activation were reduced in young adults relative to neonates (Overstreet-Wadiche et al., 2006). Fourth, maturation of newly born granule cells was altered in mice lacking the GABA synthesizing enzyme glutamic acid decarboxylase 65 (Overstreet-Wadiche et al., 2006). Fifth, previous studies suggest that early in development GABA can have trophic effects on cell proliferation, migration, and neurite outgrowth (Ben-Ari, 2002; Owens & Kriegstein, 2002). From these viewpoints, both decreased proliferation of NSCs and altered dendritic growth of newly born neurons in the aging DG may at least partially reflect decreased GABA signaling in the hippocampus with aging.
Implications of early decrease in dentate neurogenesis during aging
The results of this study suggest that the window of major decrease in dentate neurogenesis during aging occurs between 7.5 months and 12 months of age in male F344 rats. Additionally, earlier studies suggest that, by middle age, the hippocampi of these rats also exhibit reductions in several neurotrophic factors and other proteins that are important for dentate neurogenesis, learning, and memory. These include BDNF, NPY, p-CREB, FGF-2, IGF-1, and VEGF (Hattiangady et al., 2005; Shetty et al., 2005). However, interestingly, the time course of reductions observed in both neurogenesis and above proteins in the hippocampus is much before the period at which cognitive deficits become clearly apparent in rats (Bizon & Gallagher, 2003; Rosenzweig & Barnes, 2003). This discrepancy might reflect a different time course of other age-related changes in the hippocampus such as region-specific changes in neurotransmitters and their receptors, dendritic morphology, synaptic connectivity, Ca2+ dysregulation, gene expression, or factors that affect plasticity and alter the network dynamics of neural ensembles that support cognition (Burke & Barnes, 2006; Kelly et al., 2006). It is also possible that middle-aged rats are able to recruit some unknown compensatory mechanisms that prevent the potential cognitive dysfunctions associated with decreased neurogenesis and diminished concentration of neurotrophic factors. Furthermore, it is possible that neurogenesis-related cognitive deficits will not be apparent until the production/addition of new neurons to the GCL reaches drastically lower levels. Indeed, many studies have proposed that spatial learning in rodents can occur with a very small number of newly born neurons (Shors et al., 2002; Drapeau et al., 2003; Dupret et al., 2005; Montaron et al., 2006). For instance, in aged rats with preserved spatial memory, ∼50 new neurons are added to the GCL everyday whereas in aged rats with impaired spatial memory only 20 new neurons are added per day (Drapeau et al., 2003). Thus, although the overall neurogenesis dwindles dramatically at middle age, this may not be sufficient to induce significant deficits in cognitive function because > 200 new neurons are still added every day to the GCL.
Alternatively, it is also plausible that existing tests that ascertain cognitive deficits in rats are not efficient to detect milder cognitive deficits that may be present in middle-aged rats. Although traditionally age-related cognitive deficits such as memory losses have been considered as indicators of neuropathology in the aged and early stage Alzheimer's brains, recent studies imply that deterioration of memory begins much before old age. This is exemplified by the observation in human subjects that the magnitude of decline in memory is as great from 20 to 30 years as it is from 70 to 80 years (Park et al., 2002; Rex et al., 2006). Furthermore, in vitro brain slice studies demonstrate that the long-term potentiation, a presumed substrate of memory encoding, starts deteriorating during the transition from young adulthood to early middle age in the rat hippocampus (Rex et al., 2005, 2006). Thus, sensitive behavioral tests that identify milder cognitive deficits may be needed to understand the implications of decreased neurogenesis and many other changes observed at middle age in rats.
Conclusions and future directions
The results of this study suggest that the major age-related decrease in dentate neurogenesis occurs between adult-to-middle ages. Therefore, persistent application of intervention strategies that are efficacious for maintaining an enhanced proliferation of NSCs during this period may be useful for sustaining a greater level of neurogenesis during aging. The strategies that are likely useful include regular physical exercise, exposure to enriched environment and new learning (Churchill et al., 2002; Kempermann et al., 2002; Prickaerts et al., 2004; van Praag et al., 2005). Indeed, studies show that long-term environmental enrichment maintains adult hippocampal neurogenesis at a ‘younger’ level (Kempermann et al., 2002) and continued physical exercise from 3 to 9 months of age significantly reduces the age-dependent decline in cell proliferation in the SGZ (Kronenberg et al., 2006). However, it remains to be tested whether application of a combination of the above strategies, such as both physical exercise and new learning from a younger adult age, would maintain a greater level of hippocampal neurogenesis and cognitive function in the aged organism.
Animals, tissue processing, and DCX and Ki67 immunostaining
Male F344 rats were obtained from the National Institute on Aging colony at Harlan Sprague-Dawley. Five age groups of animals (4, 7.5, 9, 10.5, 12 months of age, n = 5 per age group) were used for DCX immunostaining in this study. Animals were perfused with 4% paraformaldehyde, and the brains were postfixed for 18 h in 4% paraformaldehyde and cryoprotected in 30% sucrose. Thirty-micrometer thick cryostat sections were cut coronally through the entire hippocampus and collected serially in phosphate buffer. In all age groups, serial sections (every 15th) through the entire hippocampus were chosen in each of the animals and processed for DCX immunostaining using a polyclonal antibody to DCX and avidin-biotin complex (ABC) method (Hattiangady et al., 2004; Rao & Shetty, 2004; Rao et al., 2005). As per the manufacturer's information, the DCX antibody used in this study is an affinity purified goat polyclonal antibody (1 : 200; Sc-8066, Santa Cruz Biotechnology, Santa Cruz, CA, USA), which was raised against a peptide mapping at the carboxy terminus of DCX of human origin. The peroxidase reaction was visualized using either vector grey or diaminobenzidine as the chromogen. Another series of serial sections (every 10th) through the entire hippocampus were chosen in each of the animals and processed for Ki67 immunostaining using a rabbit monoclonal antibody from Vector Laboratories (1 : 200; Burlingame, CA, USA) and ABC method. The peroxidase reaction was visualized using either vector grey or diaminobenzidine as the chromogen. Sections immunostained for DCX/Ki67 were mounted on clear slides, air-dried, counterstained with neutral red (for sections developed with vector grey) or hematoxylin (for sections developed with diaminobenzidine), dehydrated, cleared and cover slipped with permount. In each rat belonging to different groups, DCX+/Ki67+ cells in the SGZ and the GCL were counted from serial sections through the entire antero-posterior extent of the hippocampus using the optical fractionator method (Rao & Shetty, 2004).
BrdU injections and immunostaining
Young adult (4 months old), middle-aged (12 months old), and aged (24 months old) male F344 rats (n = 4 per age group per time point) received four intraperitoneal injections of BrdU (one injection every sixth hour for 18 h at a dose of 100 mg kg−1 body weight). Because of the possibility that single injection of BrdU might label only a very small number of cells in the aged groups and because the length of the S-phase of cell cycle in the SGZ of different age groups of rats is unknown, we chose multiple injection paradigm (i.e. four injections over a period of 18 h) to calculate the production of new cells per day in the SGZ. At 6 h and 12 days after the last BrdU injection, subgroups of rats were perfused with 4% paraformaldehyde solution and tissues processed for cryostat sectioning. Thirty-micrometer thick serial sections (every fifth) through the entire hippocampus were selected in each rat and processed for BrdU immunostaining (Rao & Shetty, 2004; Rao et al., 2005). In brief, sections were first treated with 0.1 M Tris-buffered saline (TBS) containing 3% hydrogen peroxide to remove the endogenous peroxidase, washed in TBS, incubated in formamide (50%) solution prepared in 2× saline sodium citrate buffer for 2 h at 65 °C, washed again in TBS, and incubated in 2 N HCl for 60 min at 37 °C. The sections were then neutralized with borate buffer (0.1 M, pH = 8.5), washed in TBS, blocked in 10% normal horse serum, incubated overnight at 4 °C in the mouse monoclonal BrdU antibody (1 : 50; Roche, Indianapolis, IN, USA) and washed in phosphate-buffered saline. The subsequent visualization procedure was performed by ABC method (Elite ABC kit; Vector) with diaminobenzidine as the chromogen. Sections were mounted on gelatin-coated slides, air-dried, counter-stained with hematoxylin, dehydrated, cleared and cover slipped. In each rat belonging to different age groups, BrdU-positive cells in the SGZ and the GCL were counted in every fifth section through the entire hippocampus using the optical fractionator method (Rao & Shetty, 2004; Rao et al., 2005).
Quantification of DCX+, BrdU+ and Ki67+cells using the optical fractionator method
In each of the chosen hippocampus (n = 5 for DCX+/Ki67+ cell counts and n = 4 for BrdU+ cell counts), DCX+/BrdU+/Ki67+ cells in the dentate GCL and the SGZ (two cell thick region from the inner margin of the dentate GCL) were counted in serial sections (every 15th for DCX+ cell counts, every 5th for BrdU+ cell counts, and every 10th Ki67+ cell counts) through the entire hippocampus using the StereoInvestigator system (Microbrightfield Inc., Williston, VT, USA). The StereoInvestigator system consisted of a color digital video camera (Optronics Inc., Muskogee, OK, USA) interfaced with a Nikon E600 microscope. In each animal, DCX+/BrdU+/Ki67+ cells were counted from 50 to 500 randomly and systematically selected frames (each measuring 40 × 40 µm, 0.0016 mm2 area) in every serial section using the ×100 lens. The numbers and densities of frames were determined by entering the parameter grid size (60 × 60 µm) in the optical fractionator component of the StereoInvestigator system. For these studies, we cut 30-µm thick sections through the DG using a cryostat that has been calibrated. Measurement of the thickness of sections immediately following sectioning using the StereoInvestigator system incorporating XYZ stage controller equipped with Z-axis position control (LEP Electronic Products Ltd, Hawthorne, NY, USA) has revealed that the variability between sections is minimal (i.e. ±1 µm). With BrdU–DCX immunostaining, sections showed significant shrinkage along the Z-axis (53–67%). The amount of shrinkage was calculated via measurement of the section thickness at the time of counting using the StereoInvestigator system incorporating XYZ stage controller equipped with Z-axis position control. In every animal, thickness of individual sections was measured in the area of GCL and the average section thickness for the animal was determined from section thickness values collected from multiple serial sections. For each animal, the actual thickness of sections at the time of counting (16–20 µm) was entered into the StereoInvestigator program to avoid errors in estimation of total cell counts.
For cell counting, in every section, the contour of GCL/SGZ area was delineated using the tracing tool, the optical fractionator component was activated, and the number and location of counting frames and the counting depth for that section was determined by entering parameters such as the grid size (60 × 60 µm), the thickness of top guard zone (4 µm) and the optical dissector height (i.e. 8 µm). A computer driven motorized stage then allowed the section to be analyzed at each of the counting frame locations. In every counting frame location, the top of the section was set, after which the plane of the focus was moved 4 µm deeper through the section (guard zone). This plane served as the first point of the counting process. All DCX+/BrdU+/Ki67+ cells that came into focus in the next 8-µm section thickness were counted if they were entirely within the counting frame or touching the upper or right side of the counting frame. For DCX+ cell counts, we relied on the cell body of DCX+ cells. As the cell body outlines of individual cells were clearly apparent in our sections counterstained with neutral red or hematoxylin, visualization of individual DCX+ cells projected on the computer screen from ×100 oil immersion lens was straightforward during counting. However, for BrdU+/Ki67+ cell counts, because of the presence of clusters of cells (particularly in younger groups), we did not rely solely on what was projected on the computer screen. We ascertained the number of cells in every cluster encountered in counting frame locations via direct observation through the microscope eyepieces and differential focusing. Thus, all DCX+/BrdU+/Ki67+ cells that were present within the 8-µm section depths were counted in every chosen section. Based on the above parameters and cell counts, the StereoInvestigator program calculated the total number of DCX+/BrdU+/Ki67+ cells per SGZ/GCL by utilizing the optical fractionator formula (Rao & Shetty, 2004; Rao et al., 2005).
Estimation of neurons among newly born cells in the SGZ and GCL at 6 h after BrdU injections
To ascertain the fraction of newly born cells that differentiate into neurons at 6 h after four BrdU injections (over 18-h period), we employed BrdU–DCX dual immunofluorescence and confocal microscopy. Sections were processed for BrdU–DCX dual immunofluorescence using established methods (Rao & Shetty, 2004; Rao et al., 2005) and antibodies from Santa Cruz (goat anti-DCX) and Roche (mouse anti-BrdU). In brief, sections were first incubated for 4 h at room temperature in the DCX antibody, washed in phosphate-buffered saline, incubated in the biotinylated horse anti-goat IgG (Vector) for 1 h, washed in PBS and the DCX+ cells visualized using streptavidin Texas red (red fluorescence). Following confirmation of the positive DCX immunofluorescence, sections were washed in TBS, incubated in formamide (50%) solution for 2 h at 65 °C, washed in TBS and incubated in 2N HCl for 60 min at 37 °C. Sections were then neutralized with borate buffer, washed in TBS, blocked in 3% normal serum, incubated overnight at 4 °C in the mouse monoclonal BrdU antibody and washed in TBS. To visualize BrdU as green fluorescence, sections were treated with goat anti-mouse IgG tagged with Alexa Fluor 488 for 1 h. Sections were then washed in TBS and cover slipped with slow fade/anti-fade mounting medium (Molecular Probes, Eugene, OR, USA). The newly born cells (i.e. BrdU-positive cells) that express DCX in the SGZ/GCL were quantified in hippocampi of animals belonging to young adult, middle aged and aged groups (n = 4 per group). This was accomplished via examination of individual BrdU+ cells using confocal laser scanning microscopy (LSM-410, Carl Zeiss, Jena, Germany). For this, Z-sectioning at 0.5- or 1-µm intervals was performed and optical stacks of 10–20 images were used for analysis of individual double-labeled cells.
Visualization and quantification of cells positive for both DCX and BrdU at 12 days after BrdU injections
Serial sections (every fifth) from the three age groups were first processed for DCX immunostaining using the ABC method described above and the immunoreaction was developed using Vector SG (Vector), which gave bluish gray reaction product in the cytoplasm of both soma and dendrites of DCX+ neurons. Sections were then treated with 0.1 M TBS containing 20% methanol and 3% hydrogen peroxide (to remove the peroxidase activity present after the first immunostaining) and processed for BrdU immunostaining using the procedure described earlier for single BrdU immunostaining above. To distinguish the blue-gray reaction product of DCX from the BrdU reaction product, BrdU immunoreaction was visualized using diaminobenzidine solution, which gave brown color to BrdU positive nuclei. Since DCX and BrdU immunostaining occurred in two different cell compartments, the reaction product resulting from DCX and BrdU could be distinguished unambiguously. The percentage of BrdU-positive cells expressing DCX was then quantified in different animals belonging to three age groups. This was accomplished by exhaustive counting of total BrdU-positive cells, and the double-labeled cells (positive for both BrdU and DCX) present in the dentate SGZ and the GCL. The counting was performed using a ×40 objective lens.
Measurement of the dendritic growth of new neurons at 12 days after birth
The neurons were chosen from sections of animals killed at 12 days after four BrdU injections and processed for DCX and BrdU sequential immunostaining using the two-chromogen method. As newly born neurons positive for both BrdU and DCX were infrequent and appeared to exhibit comparable dendritic growth in the middle-aged and aged groups, the neurons chosen for tracing from middle-aged and aged animals were pooled as one group. In each of the young adult and middle-aged/aged groups, we chose DCX+ neurons based on the following criteria. (i) Neurons were immunopositive for BrdU, exhibited vertically oriented dendrites and were located in or close to the middle third of the section thickness. (ii) Neurons did not have severed dendrites in close proximity to the soma. (iii) The dendrites of selected neurons had minimal overlap with the dendrites of adjacent cells so that all dendritic branches of chosen neurons could be traced unambiguously. The dendritic measurements of these DCX+ neurons were made using a semiautomatic neuron tracing system (Neurolucida, Microbrightfield Inc., Williston, VT, USA) linked to a Nikon microscope (Shetty & Turner, 1998a). In each group, 25 DCX+ neurons were traced in their entirety and data for various measurements were calculated, including the cell body size (area and perimeter), number of dendritic nodes and ends, and total dendritic length. To measure the extent of dendritic growth away from the soma and the branching of dendrites at different distances from the soma, the concentric circle analysis of Sholl (1953) was performed using the NeuroExplorer component of the Neurolucida program (Microbrightfield Inc., Williston, VT, USA).
This research was supported by grants from the National Institutes of Health (National Institute of Aging grant RO1 AG20924 to A.K.S.) and Department of Veterans Affairs (VA Merit Review Award to A.K.S.).
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