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Keywords:

  • aging;
  • mice;
  • neural stem cells;
  • neurogenesis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

In the adult mouse brain, the subventricular zone (SVZ) is a neurogenic stem cell niche only 4–5 cell diameters thick. Within this narrow zone, a unique microenvironment supports stem cell self-renewal, gliogenesis or neurogenesis lineage decisions and tangential migration of newly generated neurons out of the SVZ and into the olfactory bulb. However, with aging, SVZ neurogenesis declines. Here, we examine the dynamic interplay between SVZ cytoarchitecture and neurogenesis through aging. Assembly of high-resolution electron microscopy images of corresponding coronal sections from 2-, 10- and 22-month-old mice into photomontages reveal a thinning of the SVZ with age. Following a 2-h BrdU pulse, we detect a significant decrease in cell proliferation from 2 to 22 months. Neuroblast numbers decrease with age, as do transitory amplifying progenitor cells, while both SVZ astrocytes and adjacent ependymal cells remain relatively constant. At 22 months, only residual pockets of neurogenesis remain and neuroblasts become restricted to the anterior dorsolateral horn of the SVZ. Within this dorsolateral zone many key components of the younger neurogenic niche are maintained; however, in the aged SVZ, increased numbers of SVZ astrocytes are found interposed within the ependyma. These astrocytes co-label with markers to ependymal cells and astrocytes, form intercellular adherens junctions with neighboring ependymal cells, and some possess multiple basal bodies of cilia within their cytoplasm. Together, these data reveal an age-related, progressive restriction of SVZ neurogenesis to the dorsolateral aspect of the lateral ventricle with increased numbers of SVZ astrocytes interpolated within the ependyma.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Neurogenesis continues in two major regions of the adult brain: the subgranular layers of the hippocampal dentate gyri and the subventricular zones (SVZ) along the lateral walls of the lateral ventricles (for reviews see Gage, 2000; Alvarez-Buylla et al., 2001; Peterson, 2002; Doetsch, 2003; Imura et al., 2003; Alvarez-Buylla & Lim, 2004; Kempermann et al., 2004; Wurmser et al., 2004). The largest of these, the SVZ, generates new neurons that migrate as chains along the lateral wall of the lateral ventricle culminating in the rostral migratory stream (RMS) where chains of neuroblasts transit the anterior forebrain along a restricted pathway to the olfactory bulb (OB) (Lois et al., 1996; Peretto et al., 1999). In the OB, SVZ neuroblasts differentiate into either granule cells or periglomerular interneurons.

The distinct microenvironment of the SVZ neural stem cell niche, with its scaffolding of intercellular matrix, support cells, and blood vessels, together with specific signaling molecules acting through precise cell–cell interactions, controls adult neurogenesis and gliogenesis. However, exactly how cell organization within the niche and, ultimately, intercellular communication regulates SVZ neurogenesis is not known. Electron microscopy (EM) imaging has revealed four major cell types that comprise the adult SVZ niche: ependymal cells, SVZ astrocytes, transitory amplifying progenitor cells and neuroblasts (Doetsch et al., 1997; Garcia-Verdugo et al., 1998). An ependyma, derived from radial glia during embryonic and postnatal development (Spassky et al., 2005), lines the ventricle. Adjacent to the ependyma are SVZ astrocytes that typically surround newly generated, highly migratory, chains of neuroblasts that course through the entire length of the SVZ (Doetsch & Alvarez-Buylla, 1996; Lois et al., 1996; Peretto et al., 1999). Interspersed among the chains of neuroblasts are immature, highly proliferative cells, best described as transitory amplifying progenitors (referred to here as type C cells). Blood vessels line the striatal boundary of the SVZ and astrocytic processes are found in contact with the basal lamina of endothelial cells lining blood vessels. Occasionally, astrocytic processes are also found interposed within the ependyma. SVZ astrocytes, or a subpopulation of SVZ astrocytes, have been identified as the neural stem cells (Chiasson et al., 1999; Doetsch et al., 1999a; Laywell et al., 2000) and both ventricle contact (Doetsch et al., 1999a; Conover et al., 2000) and association with blood vessels (Palmer et al., 2000; Capela & Temple, 2002; Shen et al., 2004) have been implicated in activating the neural stem cell phenotype.

With aging, SVZ neurogenesis has been shown to decrease by greater than 50% compared to that seen in a young adult (Tropepe et al., 1997; Jin et al., 2003; Maslov et al., 2004). This decline appears to be influenced in part by a reduction in epidermal growth factor receptor (EGFR) signaling and results in associated deficits in fine olfactory discrimination (Enwere et al., 2004). Levels of both EGFR and transforming growth factor-α (TGF-α) are reduced in aged mice (Tropepe et al., 1997; Enwere et al., 2004) and the TGF-α hypomorphic mutant mouse, waved-1, phenocopies aged mice in reduced levels of SVZ neurogenesis and loss of refined olfaction (Enwere et al., 2004). It is likely that other factors are also involved in the age-related decline of neurogenesis. For example, in the neighboring neural stem cell niche of the subgranular layers of the hippocampal dentate gyri, Shetty et al. (2005) found significant declines in the stem/progenitor cell factors FGF2, IGF-1 and VEGF in the aging hippocampus and suggested that these declines are responsible for corresponding reductions in neurogenesis. In apparent support of multiple growth factor involvement, intracerebroventricular infusion of fibroblast growth factor-2 (FGF2) or heparin-binding-EGF (HB-EGF) into the aged (23–25 months) mouse brain has been reported to restore SVZ neurogenesis to levels found in young adult (Jin et al., 2003). This ability to respond to exogenous growth factors suggests that, although the aging SVZ niche may be compromised, it still retains capacity for increased neurogenesis if properly stimulated. However, it is unclear which cell type is responding to the infused growth factors, how much of the neurogenic stem cell niche has been retained, or if increased neurogenesis can be sustained. Tropepe et al. (1997) provided some evidence that aging lengthens the cell cycle of proliferating progenitor cells, which typically have a cell cycle of 12.5 h in young adults (Morshead & van der Kooy, 1992). These cells, which are known to be EGF-responsive (Doetsch et al., 2002), may be stimulated by HB-EGF infusions, resulting in increased neurogenesis. Alternatively, the relatively quiescent neural stem cells, with a predicted cell cycle time of 28 days (Morshead et al., 1994), may be activated by FGF2 and HB-EGF infusions.

Here, we ask how cell composition and organization change in the aging SVZ niche. Using EM to provide high-resolution images of the cytoarchitecture of the SVZ, we assembled the electron micrographs into photomontages to reveal the entire SVZ of corresponding, anterior forebrain sections (coronal) for our aging series: young adult (2 months), middle-aged adult (10–13 months) and elderly (19–22 months) mice. These montages reveal progressive changes in the aging SVZ, including restriction of neurogenesis to the dorsolateral portion of the lateral ventricle and interpolation of multiple SVZ astrocytes within the ependyma.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

SVZ progenitor pool declines with aging

Previous studies have reported a 50% decline in BrdU-labeled cells in the SVZ of aged (20–25 months old) compared to 2-month-old mice (Tropepe et al., 1997; Jin et al., 2003; Maslov et al., 2004). In contrast, Kuhn et al. (1996) detected no difference in the level of BrdU incorporation by cells of the SVZ (6–21 months) in rat. To examine changes in SVZ cell proliferation and potential alterations to the progenitor pool in our aging series, we quantified the fraction of SVZ cells in S phase by counting the number of BrdU positive (BrdU+) cells relative to the number of cells positive for the general proliferation marker, Ki67, along the lateral wall of the lateral ventricle of young adult (2 months), mid-age (10–13 months) and elderly (19–22 months) CD-1 mice. Ki67 labels cells in G1, S, G2, and mitosis (Scholzen & Gerdes, 2000). Mice were injected with 50 mg BrdU kg−1 and perfused 2 h postinjection (2 h pulse). Anterior forebrain coronal sections (50 µm), from coordinates 0.5–1.42 mm anterior relative to bregma (Paxinos, 2001), were immunostained for BrdU (Fig. 1a–c) and Ki67, and positive cells were counted along the entire dorsoventral length of the lateral wall of the lateral ventricle. Similar to the work of others, we detected a dramatic 57% decrease in BrdU incorporation by mid-age and a 71% decrease in elderly mice (Fig. 1d). The ratio of BrdU+ cells/total Ki67+ cells (Fig. 1e) provided a labeling index for cell cycle length within the SVZ, allowing BrdU-incorporating cells to be evaluated based on total number of cycling cells within the anterior SVZ. In mammalian cells the length of S phase remains relatively constant, while the length of G1 regulates the rate of proliferation (DiSalvo et al., 1995; Chenn & Walsh, 2003). We detected no significant difference between young adult and mid-aged (10 months) mice in the ratio of BrdU+cells/total Ki67+ cells (Fig. 1e), indicating that older, mid-aged progenitor cells do not divide significantly slower than younger, 2-month-old progenitors. Instead, it appears that the total progenitor pool decreases in mid-aged mice. In elderly mice, we detected a decrease in the ratio of BrdU+cells/total Ki67+ cells (Fig. 1e). As has been previously suggested (Tropepe et al., 1997), this decrease may indicate a lengthening of the progenitor cell cycle, i.e. it takes longer for cells to enter S phase, or, alternatively, a loss of cycling progenitor cells may have occurred. To determine whether the decrease in progenitor pool results from a shift in the fraction of progenitors that remain as progenitors vs. those differentiating, we examined the fraction of cells that left the cell cycle 24 h following a single BrdU pulse labeling. Cells exiting the cell cycle were identified as BrdU+ and Ki67 and those remaining in the cell cycle as BrdU+ and Ki67+. By mid-age, we found a greater than 1.5-fold increase in progenitors exiting the cell cycle (Fig. 1f). These studies suggest that the progenitor pool is reduced with aging due, in part, to progenitors exiting the cell cycle.

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Figure 1. Changes in subventricular zone (SVZ) cell proliferation and the progenitor pool with aging. (a, b, c) BrdU+ cells, following a 2-h pulse, along the lateral wall of the lateral ventricle (red box in schematic) in young (a, 2 months), mid-age (b, 10 months) and elderly (c, 19-20 months) mice (V, ventricle). (d) Quantitation of BrdU+ cells along the SVZ shows a significant decrease by mid-age and a further decline in elderly mice compared to young adult mice (*, P < 0.05 versus young mice). (e) The percentage of progenitor cells (Ki67+) also labeled with BrdU after a 2-h pulse is not significantly different in young and mid-age SVZ, indicating similar cell cycle lengths. However, the ratio of BrdU+/Ki67+ cells decreases in elderly mice, possibly indicating a lengthening of the cell cycle in progenitor cells (*, P < 0.05 versus young mice). (f) The fraction of BrdU+ cells that were no longer dividing (BrdU+, Ki67) 24 h after a pulse label with BrdU, compared with BrdU+Ki67+ cells, increased in mid-age animals, indicating that more cells are leaving the cell cycle (*, P < 0.02 versus young mice).

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Programmed cell death has also been implicated in regulating the progenitor pool in proliferative ventricular zones during development (Thomaidou et al., 1997; Roth et al., 2000; Putz et al., 2005) and may be responsible for some of the decline in progenitor pool seen in aging. To examine cell death along the SVZ, active caspase-3 staining in young, mid-aged and elderly mice was evaluated. Caspase-3 is a key enzyme of the mammalian CNS apoptotic pathway (Blaschke et al., 1996, 1998; Thomaidou et al., 1997; Pompeiano et al., 2000). We detected similar levels of caspase-3+ cells along the SVZ of both young and mid-aged mice (Fig. 2a,b,d), suggesting that progenitor cell numbers are not regulated by cell death at mid-age. However, we do see an increase in cell death in the SVZ of elderly mice (Fig. 2c,d), which may account for decreased numbers of BrdU+ cells relative to cycling Ki67+ cells in elderly mice. Further examination reveals that a greater proportion of neuroblasts (DCX+) are entering apoptosis in old vs. young mice (Fig. 2e), but fewer GFAP+ astrocytes are apoptotic in old vs. young mice (Fig. 2f).

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Figure 2. Apoptotic cell death in the aging subventricular zone (SVZ). (a–c) Arrows indicate the location of caspase-3+ cells along the lateral wall of the lateral ventricle in young (a), mid-age (b) and elderly (c) mice (ventricle outlined in black). (d) Similar levels of caspase-3+ cells were detected in the SVZ of young and mid-age mice, whereas caspase-3+ staining increases in elderly mice. (e–h) The proportion of DCX+cas+/total cas+ cells increases in 16-month-old mice, as compared to 2-month-old mice (e), while the proportion of GFAP+cas+/total cas+ cells decreases in 16-month-old mice (f). Confocal images showing colocalization of DCX and caspase-3 (g) and GFAP and caspase-3 (h) immunostaining in the same cell population of the 16-month-old SVZ. Nuclei are labeled with TO-PRO-3 (blue).

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Changes in SVZ cytoarchitecture during aging

Detailed examination of the cytoarchitectural organization of the SVZ through aging was performed by transmission electron microscopy (EM). Ultra-thin, coronal sections were imaged and contiguous EM micrographs were assembled into a montage to show the lateral wall of the lateral ventricle. Montages were then colorized based on cell morphological criteria previously reported (Doetsch et al., 1997; Garcia-Verdugo et al., 1998; Conover et al., 2000) and presented here in Fig. 3. EM-based reconstructions of the aging SVZ in young, mid-aged and elderly mice revealed a thinning of the SVZ by mid-age, resulting in areas where striatal neurons and associated neuropil were in close apposition to the ependyma of the ventricle (Fig. 4). In elderly mice, the SVZ neurogenic region was restricted to primarily the dorsolateral aspect of the lateral ventricle, as stenosis of the ventral walls of the lateral ventricle occurred, sealing the lateral and medial ventricle walls (Fig. 5). This resulted in loss of both ependymal cells and, consequently, the associated SVZ in affected regions of the ventral aspect of the lateral ventricle. The dorsolateral region of the lateral ventricle retained pockets of active neurogenesis and key components typical of a younger SVZ neurogenic niche were maintained, including astrocytes surrounding tight clusters of neuroblasts, the presence of transitory amplifying progenitor (type C) cells and the close proximity of blood vessels to areas of neurogenesis. However, abnormalities were also found, including an increased number of astrocytes (arrows, green cells) positioned within the ependymal monolayer (yellow cells) and contacting the ventricular lumen (Fig. 4, young vs. elderly).

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Figure 3. Characteristics and coding of subventricular zone (SVZ) cell types, based on (Doetsch et al., 1997). (a) Ependymal cells (E): many mitochondria, lipid droplet (arrowhead), basal bodies of cilia (arrows), and microvilli; (b) astrocyte (As): irregular nuclei, frequently invaginated, intermediate filaments (not observable at magnification presented here), light cytoplasm; (c) neuroblasts (N): elongated cells, clustered with spaces between cells (*), microtubules (not observable at this magnification), dark cytoplasm; (d) transitory amplifying progenitor cell (type C): large, irregular nuclei with deep invaginations, many mitochondria.

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Figure 4. Transmission electron microscopy (EM) analysis of the anterior subventricular zone (SVZ) in young, mid-age and elderly mice reveals age-related changes. EM micrographs (of matched, coronal sections) were assembled into a montage and color-coded based on morphological and ultrastructural criteria. Shown here is the dorsolateral aspect of the lateral wall of the lateral ventricle for each age group (note that the young and mid-age SVZ has been truncated). SVZ reconstructions revealed a diminished SVZ with aging. However, pockets of neuroblasts (red cells) were detected in mid-age and elderly SVZs. In addition, increased numbers of astrocytes interpolated within the ependyma (arrows) in the elderly SVZ. Dorsal/ventral and medial/lateral orientations are shown (V, ventricle).

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Figure 5. Stenosis of ventral walls of the lateral ventricle resulted in loss of the ependymal monolayer and cells of the subventricular zone (SVZ). Photomontage of anterior lateral ventricle shows two intact ependymal monolayers apposed to each other in the ventral portion of the lateral ventricle in young mice (square bracket and insert). The same anterior region of the lateral ventricle in elderly mice shows stenosis of the ventral portion of the ventricle walls and the loss of the ependyma lining (boxed area and insert). Accompanying the loss of the ependyma is an associated loss of the cells of the lateral wall SVZ (V, ventricle; large arrows, nests of neuroblasts; bracket, region shown in insert at higher magnification).

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To assess changes in cell composition of the SVZ with aging, we counted SVZ cell types along the dorsolateral ventricle surface in matched, coronal sections (anterior sections were at 1.18 mm and posterior sections at 0.88 mm, relative to bregma) for all three age groups, using three mice per group (Fig. 6). Cell types were identified based on morphological criteria observed at the EM level, as previously described (Doetsch et al., 1997) and shown in Fig. 3. Both SVZ neuroblasts and type C cells decreased significantly by mid-age, but these levels were maintained in elderly mice (Fig. 6). Aging did not result in a significant reduction of either SVZ astrocytes or ependymal cells, although SVZ astrocytes showed a downward trend with age (Fig. 6). Our survey of both anterior and posterior SVZ regions revealed similar cell composition in these two different regions through aging (compare Fig. 6a and b).

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Figure 6. Counts of subventricular zone cell types along the lateral wall of the lateral ventricle at two different locations (A/P: 1.18mm and 0.88mm, relative to bregma) in young, mid-age and elderly mice (*, P < 0.05 when compared to same cell type in young mice).

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Chains of migrating neuroblasts are restricted to dorsal regions in aged mice

Newly generated neuroblasts migrate as chains along the lateral wall of the lateral ventricles and continue to use this form of chain migration during their transit through the rostral migratory stream (RMS) into the OB (Lois et al., 1996; Doetsch et al., 1997; Peretto et al., 1999). Once in the OB, the neuroblasts disperse and migrate as individual cells to their final site in either the granule cell or periglomerular layer. Whole mount preparations of the lateral wall of the lateral ventricle reveal that chain migration continues through aging, but becomes restricted to primarily the dorsal wall, as seen following doublecortin (DCX) labeling of SVZ neuroblasts of a 12-month-old (mid-aged) mouse (Fig. 7). This dorsolateral alignment of neuroblasts coincides with our EM observation of clusters of neuroblasts primarily in the dorsolateral aspect of the SVZ. In addition, we found that the diameter of the RMS diminishes significantly with age, suggesting that fewer neuroblasts are transported into the OB.

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Figure 7. Neuroblast chains in wholemount preparations of the lateral wall of the lateral ventricle wall (schematic indicates region of lateral wall shown). (a, b) Neuroblast chains (arrows), stained for doublecortin, are present at young and mid-age, but at mid-age the chains are found primarily in the dorsal portion of the lateral wall of the lateral ventricle. (c) The diameter of the RMS decreases with age (*P < 0.05; **P < 0.01).

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Interpolation of SVZ astrocytes into the aging ependyma

With increased age, EM analysis revealed that many SVZ astrocytes interpolated within the ependyma and established contact with the CSF of the ventricle. Astrocyte contact with the ventricle has been suggested to activate the neural stem cell phenotype (Doetsch et al., 1999a; Conover et al., 2000) and an occasional astrocytic process can be found interposed within the ependyma of younger mice. In elderly mice, the number of interposed astrocytes increased substantially (Fig. 4, young vs. elderly). In Fig. 8, we assembled a high-resolution EM photomontage showing one region of the lateral wall of the lateral ventricle where several astrocytes have penetrated the ependyma. These astrocytes appear to be firmly established within the ependyma based on the location of their nucleus, i.e. in alignment with nuclei of ependymal cells, and appearance of intercellular adherens junctions (zonulae adherens) with either neighboring ependymal cells or other interpolated astrocytes (Fig. 9). Previously, some interpolated astrocytes were found to have one cilium (Doetsch et al., 1999a,b; Conover et al., 2000), an apparent indicator of neuronal precursors and reminiscent of the cilium associated with neuronal precursors of the embryonic neuroepithelium and precursors of new neurons in the adult avian brain (Alvarez-Buylla et al., 1998). Surprisingly, as shown in Fig. 8 (b and c), an astrocyte interpolated within the ependyma of an elderly mouse contained three basal bodies of cilia in its cytoplasm. Double labeling of coronal anterior forebrain sections with the ependymal cell marker, S100β, and the astrocyte marker, GFAP, we detected several cells within the ependyma monolayer that co-labeled for both markers (Fig. 10). Together, these surprising findings suggest that SVZ astrocytes incorporated within the ependyma may assume characteristics of ependymal cells.

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Figure 8. Astrocytes interpolated within the ependyma of elderly mice. (a) EM photomontage of the lateral wall of the lateral ventricle of a 22-month-old mouse showing three astrocytes interposed within the ependyma (As, astrocytes; E, ependymal cells, N, neuroblasts; M, cell in mitosis; arrows, adherens junctions between astrocytes; arrowheads, adherens junctions between astrocytes and ependymal cells). (b) Higher magnification of interposed astrocyte, shown in boxed area in (a), revealed three basal bodies of cilia (boxed area) and adherens junctions between astrocyte and ependymal cells. (c) Basal bodies (arrows) found in interposed astrocyte. (d) Examples of basal bodies (arrows) typically found in ependymal cells.

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Figure 9. Astrocytes interpolated within the ependyma establish adherens junctions with neighboring ependymal cells or other interposed astrocytes. (a) High-resolution EM shows astrocyte (As) in contact with the ventricle. This astrocyte contacts an ependymal cell (E) on one side and another astrocyte on the other. In both cases, adherens junctions (brackets) are found between cells bounding this astrocyte within the ependymal wall. (b) EM micrograph reveals that neighboring astrocytes within the ependyma are linked by zonulae adherens. (c) Adherens junctions are typically found between ependymal cells (E) as shown here. (d) Astrocytes that do not have ventricle contact, such as those located within the subventricular zone, do not establish adherens junctions with neighboring ependymal cells (arrows point to cell membrane contact between ependymal cell and astrocyte). Insert shows lower magnification of contact between astrocyte and ependyma (As, astrocyte; E, ependymal cell; V, ventricle).

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Figure 10. Confocal image of a GFAP+/S100β+ cell within lateral ependymal monolayer (a) (dotted line indicates ventricle). (b) Colocalization of markers is demonstrated in YZ and XZ planes (nuclei stained blue with TO-PRO-3).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Neurogenesis declines with age in the two neurogenic regions of the adult brain, the dentate gyri of the hippocampus (Seki & Arai, 1995; Kuhn et al., 1996; Kempermann et al., 1998; Cameron & McKay, 1999) and the SVZ of the lateral ventricles (Tropepe et al., 1997; Jin et al., 2003). However, some capacity for neurogenesis is maintained in old age. The finding that the intraventricular infusion of growth factors in elderly mice can stimulate an increase in neurogenesis back to levels found in the young adult (Jin et al., 2003) suggests that the basic components for neurogenesis are retained. However, it is unclear which cells are targeted by the growth factors and it is unknown if this level of neurogenesis can be sustained past the 1-week observation period reported (Jin et al., 2003).

Cytoarchitecture of the aging SVZ

We present high-resolution EM micrographs of coronal sections through the SVZ in an aging series that includes young adult (2 months), mid-aged adult (10–13 months) and elderly (19–22 months) mice. Immediately apparent is the thinning of the SVZ with age. At mid-age, the SVZ has noticeably thinned from a diameter of 3–5 cells in young adults to a diameter of 1–3 cells thick (as viewed in coronal sections). In addition, instead of the relatively even distribution of clusters of neuroblasts along the entire dorsoventral aspect of the SVZ, as seen in the young adult, the mid-aged and elderly SVZ shows only residual pockets of neuroblasts. These pockets become restricted to the dorsolateral portion of the lateral ventricle in elderly mice, as stenosis of the lateral and medial walls occurs in the ventral portion. This closure of the ventral walls of the lateral ventricle results in loss of the ependyma and subsequent loss of cells composing the SVZ (Fig. 5). Interestingly, coronal sections of the anterior lateral ventricles from elderly brains revealed blood vessels cut in longitudinal section (Fig. 4, in all four elderly brains examined) vs. the typical cross-sectional orientation found in young and mid-aged mice (Fig. 4, young and mid-age). The change in the orientation of SVZ-associated blood vessels in elderly mice suggests some distortion of the SVZ occurs with age. It is possible that the 90° reorientation of SVZ blood vessels may be a consequence of ventral wall stenosis.

In elderly mice, the remaining neurogenic pockets, or nests, appear to possess cytoarchitectural elements thought to be important for a neurogenic stem cell niche, i.e. clusters of neuroblasts ensheathed by astrocytes, presence of blood vessels, and type C cells. However, it is not known how cell–cell interactions and signaling mechanisms may be altered with age. Additionally, the restriction of SVZ neurogenesis to the dorsolateral zone of the lateral ventricle brings into question the extent to which neurogenesis can be promoted in the aging brain. Our data suggest that only the dorsolateral zone of the lateral ventricle would be responsive to growth factors. Since astrocyte numbers are not significantly reduced in elderly mice, this suggests that neural stem cell numbers may be maintained. Therefore, it is tempting to suggest that the neural stem cells are the cells activated by infused growth factors. However, the absence of a specific neural stem cell marker prevents us from directly testing this possibility.

The increased number of SVZ astrocytes interposed within the aging ependyma is intriguing. Previous studies, including our own, suggested that astrocyte contact with the ventricle might be a requisite feature of SVZ neural stem cells (Doetsch et al., 1999a; Conover et al., 2000). However, focus on neural stem cell activation has since shifted to blood vessel/endothelial cell involvement (Palmer et al., 2000; Shen et al., 2004). It is possible that both CSF and endothelial cell contact are important for directing a population of SVZ astrocytes in their neural stem cell activity. Our finding of three basal bodies in the cytoplasm of an astrocyte that has penetrated the ependyma in an elderly mouse presents another possibility, that SVZ astrocytes, i.e. possibly neural stem cells, may be capable of repairing the aging ependyma. This hypothesis is not totally unprecedented, as radial glia have recently been shown to give rise to ependymal cells perinatally (Spassky et al., 2005). If SVZ astrocytes are required to maintain both ependyma repair and neurogenesis in aged animals, this may account for the lack of change in SVZ astrocyte numbers with age. The reduction in neuroblast numbers may be a result of the bi-potential role for SVZ astrocytes to repair the ependyma and contribute to neurogenesis in aged animals. Further studies are necessary to examine this possibility.

Age-related SVZ proliferation

Previously, others have reported a > 50% decrease in BrdU+ cells in the SVZ of elderly mice (22–25 months) (Tropepe et al., 1997; Jin et al., 2003; Maslov et al., 2004). We found a similar decrease, detected as early as 10 months, but which may occur even earlier. We initially examined whether this decrease in proliferating cells was due to a declining SVZ progenitor pool or to a lengthening of progenitor cell cycle, as suggested by Tropepe et al. (1997). At 10 months, we detected a significant decrease in BrdU+ cells, but the ratio of BrdU+ to the total number of cycling, Ki67+, cells did not change, suggesting that cell cycle dynamics were not changing in the mid-aged SVZ. At 22 months, we detected a further decrease in BrdU+ cells and in addition found a decrease in the ratio of cells in S phase (BrdU+) to the total number of cycling cells (Ki67+). This result could be explained in at least two ways: (i) cell cycle length for progenitors increased in elderly SVZ tissue, or (ii) increased cell death reduced the number of cells in S phase. Indeed, we detected increased cell death in the SVZ of elderly, but not mid-aged, mice, which may account for the reduction in BrdU+ cells.

In contrast to our work, and that of others, Kuhn et al. (1996) reported no decrease in BrdU+ cells in the SVZ with aging. Several differences between their analyses and ours may explain this discrepancy. First, their examination was in rats and second, they compared BrdU incorporation in 6- and 22-month-old animals, whereas, in our study we compared 2-, 10- and 22-month-old mice. We detected a significant decrease in BrdU incorporation from 2 to 10 months in mice and therefore it is possible that by 6 months the decline in proliferation may have already begun. Our studies showed that while a further decline in BrdU labeling occurs from 10 to 22 months in mice, this decrease in BrdU incorporation was not as dramatic as from 2 to 10 months. In addition, our BrdU+ cell counts were collected from the entire dorsoventral extent of the lateral wall of the lateral ventricle, to the level of the anterior commissure (from coronal sections, 50 µm, spanning coordinates 0.88 mm to 1.18 mm anterior, relative to bregma). Kuhn et al. (1996), however, counted cells within a 0.15 × 0.15-mm box placed along a dorsolateral portion of the lateral ventricle and counted every sixth section (40 µm). Our data suggested that the dorsolateral aspect of the SVZ remains the most prolific with age and therefore would show the least change. Together, these procedural differences, the use of rats vs. mice, 6-month vs. 2-month-old tissue and the limited area counted could all account for the discrepancies between the studies.

Age-related decreases in SVZ neurogenesis may also be linked to declines in telomerase activity associated with the aging SVZ. The young adult SVZ contains higher levels of telomerase activity than other brain regions (Caporaso et al., 2003) and this activity appears to mirror proliferation levels. Recent studies in mouse skin epithelium demonstrate that overexpression of TERT (telomerase reverse transcriptase), the protein component of telomerase involved in the de novo addition of telomere repeats to chromosome ends (Blackburn, 2001; Smogorzewska & de Lange, 2004; Sarin et al., 2005), promotes proliferation of quiescent, multipotent stem cells. This new function for TERT appears to operate independently of its traditionally accepted role in synthesizing telomere repeats (Sarin et al., 2005). Thus, it will be interesting to determine if increased TERT levels in the aged SVZ can act to stimulate neural stem cell proliferation or if additional growth factors would be required.

Neuroblast migration in aging SVZ

Newly generated neuroblasts migrate as chains along the lateral wall of the lateral ventricle and then travel anterior along the RMS into the OB. Using BrdU pulse-labeling, a 70% decrease in BrdU+ cells reaching the OB has been observed in elderly (24 months) compared to young adult (2 months) mice (Tropepe et al., 1997; Enwere et al., 2004). In mid-aged (12 months) mice, we detected robust chains of neuroblasts that are found primarily in the dorsal portion of the lateral wall of the lateral ventricle. This dorsolateral localization fits with our finding of stenosis of the ventral walls of the lateral ventricle and the restriction of neuroblasts to the dorsolateral zone as seen in EM micrographs of coronal forebrain sections.

Concluding remarks

Many factors appear to influence declines in adult neurogenesis, including reduced levels of specific growth factors, decreased telomerase activity, the increased presence of high levels of corticosteroids and inflammation (see Brazel & Rao, 2004, and references therein). Acting in concert, or alone, these biochemical changes may result in depletion of neural stem cells/progenitors or changes in their underlying properties. For example, the aging neurogenic regions appear somewhat receptive to increased neurogenesis, if properly stimulated. Adrenalectomy, reducing levels of corticosteroids, has improved levels of hippocampal neurogenesis in aged rats (Cameron & McKay, 1999; Montaron et al., 1999). Similarly, administration of growth (IGF-1, FGF2, EGF) and neurotrophic factors (BDNF) has also increased levels of progenitor cell proliferation in the dentate gyrus and SVZ. However, it is not clear by which mechanism this recovery occurs or the potential extent of the recovery. We present a detailed, cytoarchitectural analysis of the aging SVZ that defines a dorsolateral zone that remains neurogenic in the elderly mouse. This zone is marked by numerous astrocytes penetrating the ependyma and contacting the ventricle, but also possesses many of the components thought to be required of a neurogenic stem cell niche. An understanding of the cytoarchitectural requirements that make up a functional, neurogenic stem cell niche allows us to better assess the potential for neurogenesis in the aging brain.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Animals

Male CD-1 mice were purchased from Charles River and aged in our vivarium. Two-month-olds were designated young adult; 10- to 13-month–olds, middle-aged; and 19- to 22-month-olds, elderly. Animal procedures were performed under protocols approved by the Institutional Animal Care and Use Committee of the University of Connecticut and conform to National Institutes of Health guidelines.

Immunocytochemistry

Male mice were perfused transcardially with 0.9% saline followed by 3% paraformaldehyde in PBS. Brains were removed and further fixed overnight in 3% paraformaldehyde at 4 °C. Brains were washed in PBS three times for 40 min, prior to cutting 50 µm sections with a vibratome (VT-1000S, Leica, Wetzlar, Germany). Free-floating sections were washed in 0.1% Triton X-100 (Sigma, St. Louis, MO, USA) in PBS for 10 min, blocked in 10% goat serum (Sigma) in PBS/0.1% Triton X-100 for 1 h, and incubated with the following primary antibodies (concentrations are listed in µg mL−1, when suppliers provide this information, or otherwise by dilution factor): antibromodeoxyuridine (BrdU, 5 µg mL−1, cat. no. OBT0030, Accurate Chemical and Scientific Corporation, Westbury, NY, USA); anticaspase-3 (0.3 µg mL−1, cat. no. AF835, R&D Systems, Minneapolis, MN, USA); antidoublecortin (DCX, 1 µg mL−1, cat. no. SC-8066, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Ki67 (1 : 1000, cat. no. NCL-Ki67p, Novocastra, Newcastle upon Tyne, UK), anti-S100β (4.8 µg mL−1, cat. no. A5110, DAKO, Carpinteria, CA, USA), and anti-GFAP (1 : 400, cat. no. MAB360, Chemicon, Temecula, CA, USA). Sections were incubated with appropriate Alexa Fluor dye-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA) for 1 h, washed three times for 10 min in PBS and then incubated for 5 min in 2 µg mL−1 Hoechst 33342 (Sigma) or 15 min with TO-PRO-3 iodide (1 : 500, cat. no. T3605, Molecular Probes, Eugene, OR, USA). Secondary antibody alone was used as a control. Sections were washed for 5 min in PBS and coverslipped using aquapolymount (Polysciences, Inc., Warrington, PA, USA). Sections were imaged on either a Zeiss Axioskop 2 plus microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA), using a Retiga 1300 EX digital camera (Q-Imaging, Burnaby, BC, Canada) or on a Leica TCS SP2 confocal laserscan microscope (Bannockburn, IL, USA).

Bromodeoxyuridine (BrdU) immunocytochemisty

Mice (2-, 10-, and 19- to 20-month-old males) were injected with 50 mg BrdU kg−1 2 h prior to perfusion (2 h pulse). BrdU immunostaining of 50-µm sections (A/P coordinates 0.5–1.4 mm, relative to bregma) was conducted as described previously (Lie et al., 2002). Epifluorescence imaging of BrdU+ and Ki67+ cells along the lateral wall of the lateral ventricle was performed using a Zeiss Axioskop 2 plus microscope (Carl Zeiss MicroImaging, Inc.) and Retiga 1300 EX digital camera (Q-Imaging, Burnaby). BrdU+ and Ki67+ cells along the entire lateral wall of the lateral ventricle, from the dorsolateral aspect to the level of the anterior commisure, were counted using Openlab 3.1.5 imaging software (Improvision, Lexington, MA) in 18 anterior forebrain sections (50 µm), from coordinates 0.5–1.4 anterior, relative to bregma. At least three mice were used for each group and statistical analyses were performed using Student's t-test.

Cell death

Following anticaspase-3 immunostaining, caspase-3 positive cells within the SVZ were counted in 36 sections (25 µm) from coordinates 0.5–1.4 anterior/posterior, relative to bregma, as described above. At least three mice (aged 2, 13 and 19–20 months) were used for each group and statistical analyses were performed using Student's t-test.

Wholemounts

Wholemounts of the entire lateral wall of the lateral ventricle were prepared as previously described (Doetsch & Alvarez-Buylla, 1996) and immunostained for doublecortin (DCX, Santa Cruz Biotechnology). Wholemounts were placed onto glass slides, coverslipped with aquapolymount (Polysciences), and imaged by epifluorescence microscopy.

RMS diameters

To determine RMS diameters, 50-µm coronal sections (vibratome) of fixed brain were labeled for PSA-NCAM (1 : 400, AbCys) and GFAP (1 : 1000, cat. no. M0761, Dako, Carpinteria, CA, USA). Fluorescent images were captured (Axioskop 2+, Carl Zeiss and Retiga EX, Q-Imaging System) of the RMS. Measurements of the area of the RMS were performed using Openlab 3.1.5 (Improvision). Three RMS measurements were taken at 3.67, 3.72, 3.77 mm distal to the beginning of the olfactory bulbs and mean values ± SEM were calculated. At least three mice were used for each group and statistical analyses were performed using Student's t-test.

Electron microscopy

Male mice (2, 10 and 22 months old) were perfused transcardially with 0.9% saline followed by 2% paraformaldehyde/2.5% glutaraldehyde in 0.1 m PB. Heads were further fixed by immersion overnight in 2% paraformaldehyde/2.5% glutaraldehyde in 0.1 m PB; brains were removed and washed in PB three times for 40 min. Three 300-µm sections of the anterior forebrain were cut with a vibratome, pinned to plastic dishes to prevent buckling and processed as described previously (Conover et al., 2000). Briefly, sections were postfixed with 2% OsO4 in 0.1 m PB for 1.5 h, unpinned and dehydrated through a graded ethanol (EtOH) series. Sections were en bloc stained in 2% uranyl acetate at the 70% EtOH step for 1.5 h. Following dehydration, whole sections were twice washed in propylene oxide and embedded in a SPI-PON 812 (SPI Supplies, Westchester, PA, USA)/Araldite 506 (Ernest F. Fullam, Inc., Latham, NY, USA) mixture between aclar sheets. Following polymerization, the SVZ regions were cut from each section and re-embedded in the same epoxy mixture in capped inverted Beem capsules. Thin sections were cut with a diamond knife, placed onto Formvar-coated slot grids, and heavy-metal stained with uranyl acetate and lead citrate. Electron micrographs were digitized using an Epson 1680 scanner (1600 dpi) and montages were constructed of the SVZ using Adobe Photoshop. Cell types were identified based on previously described criteria (Doetsch et al., 1997) (see also Fig. 3). Cell counts were made along the lateral wall of the lateral ventricle, criteria included: (i) presence of nucleus, (ii) presence of an ependyma (in elderly mice, cells were not counted in regions where stenosis of the ventral walls of the lateral ventricle occurred), and (3) clearly defining characteristics of specific cell type. Image J (NIH) was used to perform measurements of SVZ regions evaluated.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

We gratefully acknowledge Dr Marie Cantino for her help with EM analysis and Kasey Baker for technical assistance throughout this project. We also thank Drs Salvatore Frasca, Jr, David Goldhamer and Joseph LoTurco for critically reading the manuscript.

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  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
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