The absence of specific NSC markers has traditionally limited direct investigation of the effects of age on endogenous NSCs, forcing investigators to rely on indirect functional readouts such as the neurosphere assay. As such, only three studies have attempted to address this question to date, with agreement that aging results in a decline in proliferating (i.e., bromodeoxyuridine‐positive, BrdU +ve ) cells in the rostral periventricular region (PVR, a 3‐ to 5‐cell‐thick region lining the lateral ventricles enriched in stem and progenitor cells) [ 8 , 9 ], but disagreement that a decline in neurosphere‐forming cells (SFCs) also occurs [ 10 ]. Therefore, we undertook a systematic analysis of the effects of age on stem and progenitor cells in the mouse brain by examining the number of BrdU +ve cells (assaying all dividing cells) and SFCs (assaying NSCs and more restricted progenitor cells) within the PVR along the lateral ventricles. Given our recent demonstration that only ∼5% of neurospheres are NSC derived, we also used the neural colony‐forming cell assay (N‐CFCA), which (based on its semisolid culture system) prohibits the recently reported fusion of neurospheres [ 11 ], and more importantly for the first time, enables NSCs to be discriminated from progenitors based on colony size [ 12 , 13 ]. Rather than examining only young (∼2 months) and aged (∼24 months) mice, we examined mice at 6‐8 weeks and at 6, 12, 18, and 24 months to provide a more comprehensive analysis of the age‐related changes within the PVR.

In addition to its beneficial effects in humans such as enhanced memory and learning and improved executive function, physical exercise is known to increase brain volume in regions prone to age‐related cognitive decline, suggesting a biological basis for its ability to reduce the risk of developing neurodegenerative disorders including Alzheimer's disease [ 1 , 2 ]. Rodent studies have corroborated human trials by demonstrating that voluntary running in both young and aged mice increases hippocampal neurogenesis, resulting in improved performance in water and radial arm mazes, and in object recognition [ 3 - 6 ]. Moreover, running can also prevent the age‐related decline in hippocampal progenitors and slow the decline in hippocampal neurogenesis that accompanies old age [ 7 ]. Because neural stem cells (NSCs) are considered to underpin the regenerative capacity of the brain, we hypothesized that running would increase NSC frequency in aged mice, resulting in an augmented regenerative response.

A one‐way analysis of variance (ANOVA) or Student's two‐tailed t tests were used to analyze data as appropriate (Prism 4; GraphPad Software, San Diego, http://www.graphpad.com ). Significant ANOVA values were followed by post hoc Tukey's or Bonferroni multiple comparisons test where appropriate. All values are expressed as mean ± standard error of the mean unless otherwise indicated. The level of significance for all comparisons was p < .05.

Mice were restrained within a plastic chamber and placed in a lead‐shielded container (reducing exposure by 95%) leaving only the head exposed for irradiation. Irradiation was induced by exposure to a Co 60 source in a Gamma Cell 200 (Atomic Energy, Ottawa, Canada, http://www.aecl.ca ) irradiator until a 3.5‐Gy dose had been given. Two cohorts of GH‐ or vehicle‐infused animals were harvested at survival periods of 1, 2, or 3 weeks after irradiation to determine the repopulation kinetics of the PVR. In one cohort, PVR tissue was harvested from the dorsal‐lateral corner of the lateral ventricles and ultimately cultured in the neurosphere assay. A second cohort received a single 150‐μl i.p. injection of BrdU (18 mg/ml, 45 mg/kg BW, dissolved in 0.07 N NaOH in 0.9% NaCl; Sigma‐Aldrich) 2 hours prior to sacrifice.

Mice were housed in cohorts of three, in cages (51 × 22 × 13 cm) fitted with a single hanging running wheel attached to a high top lid for each cage (Able Scientific, Welshpool, WA, Australia, http://www.ablescientific.com.au ). Distance traveled per 24‐hour period was measured by an Enduro 8 cyclometer (Cat Eye Co., Osaka, Japan, http://www.cateye.com ). Control mice were similarly housed three per cage without the presence of the running wheel. No significant difference in total running distance per 24 hours (assayed over a 7‐day period) was detected when mice were housed singly, or in cohorts up to four mice.

Following the generation of a single‐cell suspension, PVR cells were cultured in 35‐mm cell culture dishes in a collagen semisolid matrix with a 2‐mm grid (Nunc) at a density recommended by the manufacturer of mouse NeuroCult Neural Colony‐forming Cell Assay kit (Stem Cell Technologies). The semisolid matrix contained serum‐free media supplemented with EGF (20 ng/ml), bFGF (10 ng/ml), and heparin (20 ng/ml), and cells were incubated for 21 days in vitro (DIV) in 5% CO 2 as previously described [ 12 , 13 ]. After 21 DIV, the total number of colonies and the diameter of each colony were determined using an eyepiece graticule on an inverted Leica light microscope with phase contrast.

Adult mice were sacrificed by cervical dislocation, and the brains removed immediately to prewarmed Petri dishes containing HEPES‐buffered minimum essential medium (HEM), which consisted of minimum essential medium (Gibco/Invitrogen, Grand Island, NY, http://www.invitrogen.com ) supplemented with 16 mM HEPES (Sigma‐Aldrich) and 100 units/ml penicillin/streptomycin (Gibco/Invitrogen). In the case of traditional PVR dissections, adult brains were sectioned coronally at the level of the optic chiasm, and PVR tissue was harvested, pooled, and diced with a scalpel for 1 minute before enzymatic digestion. In the case of vibratome sectioning, whole brains were harvested and cut into 400‐μm sections using a Leica VT 1000s vibratome. Individual coronal vibratome sections were collected serially starting at the level of 4.28 mm rostral to Bregma and continuing to Bregma −2.66 mm. The entire PVR (encompassing a 3‐ to 5‐cell‐layer‐thick region immediately adjacent to the ependymal lining of the ventricles) was microdissected from each section under ×2 magnification using an ultrafine scalpel. Harvested tissue was diced with a scalpel for 1 minute, and then enzymatically brought to a single‐cell suspension. Regardless of tissue harvest technique, neural tissue was digested with 0.1% trypsin‐EDTA (Gibco/Invitrogen) for 7 minutes at 37°C, followed by a wash with 0.014% wt/vol trypsin inhibitor (type I‐S from soybean; Sigma‐Aldrich) dissolved in HEM. Following centrifugation at 100 g relative centrifugal force (rcf) for 7 minutes, the resulting pellet was resuspended in 1,000 μl 0.1 M Dulbecco's phosphate‐buffered saline (PBS, calcium and magnesium free; Gibco/Invitrogen) and mechanically triturated until smooth. Cells were incubated at 5% CO 2 in complete medium (CM)—consisting of mouse NeuroCult NSC Basal Medium plus Proliferation Supplements (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com )—supplemented with 20 ng/ml of purified mouse receptor‐grade epidermal growth factor (EGF; BD Biosciences, North Ryde, NSW, Australia, http://www.bd.com/anz ), 10 ng/ml basic fibroblast growth factor (bFGF; Roche, Dee Why, Australia, http://www.roche‐australia.com ), and 20 ng/ml heparin (Sigma‐Aldrich) at a density of 2,500 cells/cm 2 . For serial expansion experiments, neurospheres were collected via centrifugation (5 minutes at 100 g rcf), then resuspended and incubated for 2 minutes in 0.1% trypsin‐EDTA, followed by washing in trypsin inhibitor in HEM. Spheres were brought to a single‐cell suspension by mechanical dissociation in PBS. Viability counts were performed on the resulting cell suspensions and cells were plated in CM+EGF/bFGF at a density of 2.5 × 10 5 cells/cm 2 in T‐25 tissue culture flasks (Nunc, Rochester, NY, http://www.nuncbrand.com ).

Twelve hours prior to surgery, osmotic mini pumps (1007D; 7‐day infusion at 0.5 μl/hour; Alzet Osmotic Pumps, Cupertino, CA, http://www.alzet.com ) were loaded with growth hormone (GH, 10 μg/ml, recombinant rat, Novozymes GroPep, Thebarton SA, Australia http://www.gropep.com.au ) or vehicle solution (0.9% sterile physiological saline; Sigma‐Aldrich), attached to the infusion cannula and the entire apparatus was incubated at 37°C. Mice were anesthetized via intramuscular injection as per [ 12 ], mounted onto a stereotaxic frame and a 1.5‐cm incision was made to expose the skull. A single hole was drilled in the skull (1.25 mm width) directly above the lateral ventricle (anterior/posterior: −0.5 lateral: +1.0 depth: −3.7), and the 30‐gauge cannula was lowered and fixed into place on the skull by the addition of cyanoacrylate adhesive, so as to enable unilateral infusion directly into the lumen of the lateral ventricle. Mice implanted with pumps containing 10 μg/ml of GH received 120 ng of GH per day or 840 ng over a 7‐day period. At the completion of the infusion period, mice were anesthetized via the inhalation of halothane, and the pumps surgically removed, while the cannulas remained in place.

Age‐dependent decline in the number and location of dividing cells in the periventricular (PVR) of the lateral ventricles. (A): Histogram showing the estimated total number of BrdU +ve cells detected along the entire length of the lateral ventricles (between Bregma points +1.42 mm and −2.8 mm) of young and aging C57Bl/6 mice. Compared with juveniles, the number of BrdU +ve cells was significantly reduced in 6‐month‐old mice ( p < .001). Although reduced numbers of BrdU +ve cells were also detected in 12‐month‐old mice (compared with 6 months), only 24‐month‐old mice differed significantly ( p < .05). Three mice used at each time point (one‐way analysis of variance, Bonferroni post hoc). (B): Representative images showing the distribution of BrdU +ve cells (at similar coordinates) in the periventricular of juvenile, 6‐, 12‐, and 24‐month‐old mice. Abbreviations: BrdU +ve , bromodeoxyuridine‐positive; D, dorsal; Juv, juvenile; L, lateral.

Individual cells counted were identified by a bright green positive nuclear staining consistent with BrdU‐Alexa 488 labeling (Fig. 1 B), and counted if they were within the counting frame but not touching exclusion lines. The area of the contour was recorded and monitored throughout the counting, and was found to be fairly consistent in size throughout each of the brains. Every second subsequent tissue section was counted until 10 sections were enumerated in total. In all cases, this corresponded to a completion at the most caudal portion of the ventricle. Images were captured on a Canon EOS digital camera (Canon, Lake Success, NY, http://www.usa.canon.com ) using an Olympus Axiophot upright fluorescence microscope (Tokyo, http://www.olympus‐global.com ).

Having located the most rostral aspect of the PVR in the series, counts were initiated at a random start using StereoInvestigator software (StereoInvestigator, v8; MicroBrightField, Williston, VT, http://www.mbfbioscience.com ) and a Leica DMBL microscope (Leica). A ×63 oil objective was used to count the cells using a counting frame size of x: 60 μm × y: 40 μm to obtain between 1‐5 cells per frame. A fractionator size of x: 120 μm × y: 90 μm was used for an average of 20 sites per section using a systematic random sampling protocol. These parameters were selected to minimize the coefficient of error of the estimate, while maximizing the efficiency of sampling.

The total number of BrdU +ve cells detected within the entire length of the PVR (a 3‐ to 5‐cell‐thick layer surrounding the entire ventricle) of one hemisphere was then enumerated by stereological methods. Identical methods were used to quantify all sections. Briefly, a ×10 objective (HC PL Fluotar ×10, Leica, Heerbrugg, Switzerland, http://www.leica.com , NA: 0.30) was used to delineate the contour of the area to be counted. The PVR was traced, bounded by a visible border 2‐3 cells thick (visible by 4′,6′‐diamidino‐2‐phenylindole [DAPI]). This visible anatomical boundary was followed to its limits at each corner of the ventricle and was used to demarcate the counting area. The ventricle was not included in the contour area.

Animals were housed and handled in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. The University of Queensland Animal Ethics Committee approved all experimentation prior to onset. Growth hormone receptor‐null (GHR −/− ) mice [ 14 ] were kindly provided by J.J. Kopchick (Edison Labs, OH). A single cohort of juvenile (6‐8 weeks old) female C57BL/6J mice were obtained from the Central Animal House (University of Queensland) and housed together for the duration of the study. Two hours prior to death adult mice were administered a single, 150‐μL (18 mg/ml, 45 mg/kg body weight [BW]) injection (intraperitoneal, i.p.) of BrdU (Sigma‐Aldrich, St. Louis, http://www.sigmaaldrich.com ; dissolved in 0.07 N NaOH in 0.9% NaCl). At sacrifice, mice were deeply anesthetized with sodium pentobarbitone (260 mg/kg BW, Lethabarb; Virbac, Sydney, Australia, http://www.virbac.com ), then transcardially perfused with ice‐cold 0.9% saline followed by ice‐cold 4% paraformaldehyde (Sigma‐Aldrich) in 0.1 M phosphate buffer (pH 7.4). Brains were then harvested, postfixed, and cryoprotected as previously described [ 12 ]. Serial frontal sections (14 μm) were cut with a MICROM cryostat, and mounted on three series of SuperFrost Plus slides (Thermo Fisher Scientific Inc., Waltham, MA, http://www.thermofisher.com ) so as to produce three essentially identical progressions of sections along the entire length of the lateral ventricle (from Bregma point +1.42 mm to −2.80 mm). Tissue sections were processed for BrdU immunocytochemistry as previously described [ 12 ].

RESULTS

An Age‐Related Decline in Stem and Progenitor Cells Occurs in the Periventricular Region of the Lateral Ventricles To provide support as to whether aging results in the preservation [10] or decline [8, 9] of neural precursor cells (i.e., stem and progenitor cells), we first replicated earlier studies by examining the number of dividing (i.e., BrdU+ve) cells within the PVR of the entire lateral ventricle by administering a single injection of BrdU in wild‐type (WT) mice aged 6‐8 weeks (juvenile) to 24 months, 2 hours prior to sacrifice. Consistent with prior reports [8, 9, 15], the frequency of BrdU+ve cells detected in aged (24 months) mice was significantly reduced compared with juvenile and 6‐month‐old mice (Fig. 1A). We also observed an age‐related change in the distribution of dividing cells, with BrdU+ve cells encircling the ventricles in juvenile mice, but increasingly absent from the dorsal and medial walls in animals 6 months of age and older (Fig. 1B). More importantly, however, by examining mice at multiple ages we unexpectedly detected a precipitous decline (i.e., ∼40% of juvenile) in BrdU+ve cells in 6‐month‐old mice, a finding not previously reported. To directly test whether aging results in a decline in proliferative precursor cells, we next microdissected the PVR from serial vibratome sections (8 × 400 μm) across the entire rostral‐caudal aspect of the lateral ventricles, and enumerated the number of SFCs and colony‐forming cells (CFCs) cultured from each section [12, 13]. Data across all eight sections were collated (for a given age) to provide a representative measure across the entire lateral ventricle. Consistent with previous reports [9, 16] we observed an age‐related decline in SFCs (which was mirrored by CFCs, attesting to the detection of similar cell populations), resulting in the loss of ∼75% of these populations by 24 months (Fig. 2A). Importantly, SFC and CFC frequency was also significantly reduced in 6‐month‐old versus juvenile mice. Figure 2 Open in figure viewer PowerPoint Age‐dependent decline in stem and progenitor cells in the periventricular of the lateral ventricles. (A): A significant decline in the total number of SFCs (black bars) generated across eight vibratome sections (Bregma points +2.34 mm to −0.70 mm) was observed between juvenile (1828 ± 109, n = 6) and 6‐month‐old (1054 ± 101, p < .001) mice and 18‐ versus 24‐month‐old mice (472 ± 23, p < .05). A significant decline in CFC frequency (white bars) was also apparent between juvenile (2010 ± 67) and 6‐month‐old (1175 ± 42, p < .001) mice, and 6‐ versus 12‐month‐old mice (794 ± 21, p < .01), but not between 18‐month‐old (627 ± 101, n = 5) versus 24‐month‐old (457 ± 19, p > .05) mice. (B) The frequency of large, neural stem cell‐derived colonies declined significantly at each time point except between 6 and 12 months, from a maximum of 70 ± 4 (Juv), to 40 ± 3 at 6 months, 34 ± 0.3 at 12 months (p = .089), 19 ± 3 at 18 months (n = 5), and 6 ± 2 at 24 months. n = 3 mice used unless otherwise stated (*, p ≤ .05, **, p ≤ .01, one‐way analysis of variance, Tukey's post hoc). Abbreviations: CFCs, colony‐forming cells; Juv, juvenile; SFCs, neurosphere‐forming cells. Using the N‐CFCA and counting large (i.e., NSC‐derived) colonies, we now report an age‐related decline in endogenous NSCs, starting with an ∼37% reduction in number at 6 months of age, and ultimately resulting in ∼90% loss by 24 months (Fig. 2B). No significant difference was observed in the diameter of the large colonies generated from 12‐, 18‐, or 24‐month‐old mice after 21 days in culture, suggesting a slowing of the cell cycle cannot account for fewer large colonies being detected. We also excised individual large colonies generated from 24‐month‐old mice from their collagen matrix and cultured them in the neurosphere assay to confirm that they were NSC derived. All (7/7) of the harvested large colonies demonstrated the cardinal NSC properties of proliferation, self‐renewal (i.e., passaged > 5 times), and multipotency in vitro. These results demonstrate, for the first time, that aging results in a significant decline in NSCs, and that the onset of both stem and progenitor loss occurs unexpectedly early in life.

Physical Exercise Stimulates the Proliferation of Endogenous NSCs in Extrahippocampal Regions It is now clear that neurogenesis declines significantly with age in both laboratory‐housed and wild mice, and that exposure to an enriched environment or voluntary exercise augments hippocampal neurogenesis in both young and aged mice [3-6, 17]. Based on these studies, we hypothesized that physical exercise would activate NSCs outside the hippocampal formation, and tested whether this was the case by culturing PVR cells harvested from runners in the N‐CFCA and neurosphere assay. Given the paucity of information regarding the activation of SFCs, and more importantly, NSCs, we first determined the duration of physical exercise necessary to activate endogenous SFCs and NSCs. Middle‐aged (12 months) female mice were housed three per cage [18] and provided ad libitum access to a running wheel for 10 or 21 days, then sacrificed and PVR cells harvested from vibratome sections and cultured either in the neurosphere assay or the N‐CFCA. As illustrated in Figure 3, 10 days of running increased SFC (Fig. 3A) and CFC (Fig. 3B) frequency by 30% and 43%, respectively, and more importantly also resulted in a significant increase in NSC frequency (∼44%, Fig. 3C). Of interest, although an additional 11 days did not alter SFC or CFC frequency, it significantly increased NSC frequency to 137% of nonrunners. Figure 3 Open in figure viewer PowerPoint Effect of physical exercise on periventricular stem and progenitor cells in the lateral ventricles of 12‐month‐old mice. Twelve‐month‐old mice were provided unlimited access to a running wheel for 10 or 21 days. (A): Compared with nonrunners (naïve, 906 ± 66), the frequency of neurosphere‐forming cells (SFCs) was significantly increased following 10 days (10d) of voluntary exercise (1175 ± 53, p < .05). Access for an additional 11 days (21d) did not alter SFC frequency (1182 ± 52). (B): The frequency of total colonies (794 ± 21) also increased significantly following 10 days (1136 ± 62, p < .01) of physical exercise, remaining unchanged at 21 days (1126 ± 33). (C): Compared with naïve mice (34 ± 0.3) the frequency of large colonies was significantly increased following 10 (49 ± 4.5, p < .05) and 21 (80 ± 5.2, p < .001) days of physical exercise. n = 3 mice harvested at each time point (*, p ≤ .05, **, p ≤ .01, one‐way analysis of variance with Tukey's post hoc). (D): Total number of colonies generated from PVT tissue (800 μm sections) harvested from naïve, 10 day and 21 day runners. (E): The frequency of BrdU+ve cells detected in the PVR of 21 day runners (21d) was significantly increased as compared to age‐matched non‐runners (naïve). Abbreviation: d, day. To date, the exercise‐induced activation of neural precursors was thought restricted to the hippocampus, as Brown et al. [19] reported the lack of an observed effect either on the dividing (i.e., BrdU+ve) cells in the lateral ventricles, or on the addition of new neurons to the olfactory bulb. Although seemingly contradictory, a closer examination of our data reveals agreement between these two studies. Indeed, by presenting the total number of colonies generated per 800‐μm section (Fig. 3D) rather than collating eight 400‐μm sections into a single value (Fig. 3B), regional differences become apparent. Looking at sections 15‐16 (Fig. 3D) the anatomical region examined by Brown et al. (i.e., +0.38 mm to −0.34 mm from Bregma), we see no effect; however, immediately rostral to this region (i.e., sections 11‐14) a robust exercise‐induced increase in CFCs is evident. To demonstrate that exercise also increases the frequency of periventricular stem/progenitor cells in sections 11‐14, 12‐month‐old mice were housed with a running wheel, and given a single i.p. injection of BrdU on day 10, and then sacrificed 11 days later. Consistent with our in vitro evidence, the frequency of BrdU+ve cells detected in the PVR of runners was significantly increased compared with age‐matched nonrunners (Fig. 3E). Taken together, these data demonstrate the ability of physical exercise to stimulate endogenous stem/progenitor cells. To determine whether increasing age altered the effects of exercise on endogenous stem and progenitor cells, PVR cells were harvested (as above) from young and aging 21‐day runners and cultured in the N‐CFCA to assay. Compared with age‐matched nonrunners, runners aged 6, 12, and 18 months were characterized by 58% ± 2%, 137% ± 16%, and 67% ± 9% increases in NSC frequency, respectively (Fig. 4A). However, although 18‐ and 24‐month‐old mice ran equal distances per day (Fig. 5), 24‐month‐old runners exhibited an unexpected halving in NSC frequency. This was also the case for progenitor populations, as evidenced by a significant increase in SFC (Fig. 4C) and CFC (Fig. 4B) frequency in mice ≤ 18 months of age, followed by a significant (∼50%) decline in these populations in 24‐month runners. Taken together, these results demonstrate, for the first time, that physical exercise induces endogenous NSCs (and progenitors) within the lateral ventricles to divide, and that in mice of advanced age, physical exercise is potentially detrimental. Figure 4 Open in figure viewer PowerPoint Age‐related decline in the exercise‐induced activation of endogenous forebrain stem and progenitor cells. (A): The frequency of neural stem cell‐derived colonies increased significantly following exercise in 6‐month‐old (40 ± 3 to 64 ± 1, p < .01), 12‐month‐old (34 ± 0.3 to 80 ± 5, p < .001), and 18‐month‐old (19 ± 3 to 32 ± 2, p = .035) mice. Although a ∼50% decline occurred in 24‐month‐old mice (6.3 ± 2 to 3.0 ± 2, p = .245). (B): Colony‐forming cell frequency also increased significantly in 6‐month‐old (1175 ± 42 vs. 1,739 ± 31, p = .001) and 12‐month‐old (794 ± 21 to 1,126 ± 33, p = .001) mice, but not 18‐month‐old runners (627 ± 101, n = 5 to 936 ± 38, p = .068), declining once again in 24‐month‐old runners from 457 ± 19 to 222 ± 97 (n = 6) (p = .026). (C) Compared with nonrunners (naïve), neurosphere‐forming cell frequency significantly increases in 6‐month‐old (1054 ± 101 vs. 1,689 ± 66, p = .010), 12‐month‐old (906 ± 66 vs. 1182 ± 53, p = .026), and 18‐month‐old (683 ± 52, n = 4 vs. 927 ± 22, p = .013) runners, declining significantly in 24‐month‐old runners (472 ± 23 vs. 221 ± 25, p = .0018). n = 3 runners harvested at each time point unless otherwise stated (*, p ≤ .05, **, p ≤ .01, Student's t test). Naïve data taken directly from Figure 2. Abbreviation: m, month. Figure 5 Open in figure viewer PowerPoint Running distance not predictive of neural stem cell‐stimulatory effects. The daily running distances of individual cohorts of juvenile, 12‐, 18‐, and 24‐month‐old mice were calculated by dividing the total distance (km) traveled per 24 hours by the number of mice per cage (i.e., 3). Three cages were measured for each time point. Over a period of 21 days, running distances fell into two distinct groups, with juvenile (3.77 ± 0.2) and 12‐month‐old (3.88 ± 0.5) mice averaging significantly greater distances per day than either 18‐month‐old (1.14 ± 0.4) or 24‐month‐old (0.81 ± 0.2, p < .01) mice, which did not differ significantly (p > .05). (repeated measures analysis of variance with least square deconvolution post hoc test). Abbreviations: Juv, juvenile; km, kilometer; m, month.

Physical Exercise Augments the Regenerative Capacity of the Brain To determine whether the exercise‐induced increase in NSCs results in a physiological change (i.e., augmented regeneration) we adopted a previously used in vivo ablation/regeneration approach where dividing cells are ablated (thereby largely sparing relatively quiescent NSCs) by exposing the head to a sublethal dose of ionizing irradiation [9, 20-22]. Control experiments determined the regenerative response of nonrunners of increasing age by exposing cohorts to a single dose of ionizing radiation (3.5 Gray) and then sacrificing mice at 24 hours, 1 week, and 3 weeks after irradiation. The repopulation of all dividing cells in the lateral ventricles (+2.54 to +0.94 in relation to Bregma) was assayed by administering a single BrdU injection (45 mg/kg body weight) 2 hours prior to sacrifice and then counting the number of BrdU+ve cells at each survival point. For all ages, after ablation of the majority of dividing cells, proliferating cells rebounded to preirradiation levels within 3 weeks after irradiation, suggesting an absence of an age‐related effect. However, a different picture emerged when we specifically assessed the repopulation of neural precursors (rather than all dividing cells) by tracking the reappearance of SFCs, which include both progenitor cells and NSCs. Consistent with previous reports [20], SFC frequency was restored to control levels by 3 weeks after irradiation in both juvenile and 6‐month‐old mice (Fig. 6B). However, SFC frequency failed to reach preirradiation (time [T] = 0) levels in 12‐month‐old (p = .003), 18‐month‐old (p = .005), and 24‐month‐old (p = .002) animals, reflecting a reduced regenerative response. Interestingly, the first age at which repopulation was incomplete (i.e., 12 months) corresponds to the time at which ∼50% of endogenous NSCs are lost (Fig. 2B), suggesting an age limit beyond which the brain can no longer effectively maintain homeostasis. Figure 6 Open in figure viewer PowerPoint Periventricular repopulation is significantly improved in 12‐ and 18‐month‐old runners. (A): Following the irradiation‐induced depletion of dividing cells (24 hours), the frequency of BrdU+ve cells returned to naïve levels (T = 0) by 3 weeks in all ages assayed (p > .05). (B): At 3 weeks after irradiation, neurosphere‐forming cell (SFC) frequency significantly exceeded preirradiation levels (T = 0) in juvenile mice (p < .05), was equal to control (T = 0) levels in 6‐month‐old mice (p = .125), and was significantly reduced in mice ≥ 12 months of age (p < .01). (C): Compared with 12‐month‐old nonrunners (naïve), significantly greater SFCs were detected at 1 (571 ± 10 vs. 1,454 ± 35, p < .001) and 3 (462 ± 38 vs. 859 ± 143, p < .01) weeks after ablation. (D): Compared with 18‐month‐old nonrunners (naïve), significantly greater SFCs were detected at 1 (528 ± 34 vs. 870 ± 28, n = 6, p < .001) and 3 (377 ± 13 vs. 667 ± 64, n = 4, p < .001) weeks after ablation. (One‐way analysis of variance with Tukey's post hoc, n = 3 per group.) Abbreviations: BrdU+ve, bromodeoxyuridine‐positive; Juv, juvenile; h, hour; m, month; T, time; wk, week. To determine whether the exercise‐induced increase in NSCs (Fig. 4A) could augment PVR regeneration we repeated the ablation/repopulation experiment using mice that exhibited a reduced regenerative capacity, yet remained amenable to exercise‐induced activation of NSCs (i.e., 12‐ and 18‐month‐old mice). As opposed to the repopulation of age‐matched nonrunners, which failed to restore the PVR to nonirradiated control levels up to 3 weeks after ablation, 12‐month‐old mice that ran for 21 days prior to irradiation exhibited a heightened regenerative response (Fig. 6C). Indeed, by 1 week after ablation the number of SFCs in the PVR of 12‐month‐old runners significantly exceeded control levels, reaching a frequency typically observed in a naïve 6‐month‐old mice (Δ, T = 0, Fig. 6B). Moreover, although SFC number significantly declined by 3 weeks after irradiation, it nonetheless remained significantly higher than nonrunners, similar to that of nonirradiated 12‐month‐old mice (•, T = 0, Fig. 6C). Eighteen‐month‐old runners likewise exhibited a heightened regeneration of the PVR, with SFC frequency at 1 week after irradiation once again significantly exceeding control levels (Fig. 6D), equaling the frequency typical of a 12‐month‐old naïve animal (•, T = 0, Fig. 6C). By 3 weeks SFC frequency declined, mirroring that of age‐matched naïve mice. Taken together, these results demonstrate the augmented regeneration of the PVR in both 12‐ and 18‐month‐old runners and demonstrate the ability of exercise to reset the frequency of endogenous NSCs.