Newly born cells in the ageing dentate gyrus display normalmigration, survival and neuronal fate choice but endureretarded early maturation

Muddanna S. Rao,1,2 Bharathi Hattiangady,1,2 Ali Abdel-Rahman,2 Dirk P. Stanley1,2 and Ashok K. Shetty1,21Division of Neurosurgery, DUMC Box 3807, Duke University Medical Center, Durham, NC 27710, USA2Medical Research Service, Veterans Affairs Medical Center, Durham, NC 27705, USA

Keywords: 5¢-bromodeoxyuridine, adult neurogenesis, ageing, dendritic growth, doublecortin, hippocampus, neural progenitors,neural stem cells, rat

Abstract

Addition of new granule cells to the dentate gyrus (DG) from stem or progenitor cells declines considerably during ageing. However,potential age-related alterations in migration, enduring survival and neuronal fate choice of newly born cells, and rate of maturationand dendritic growth of newly differentiated neurons are mostly unknown. We addressed these issues by analysing cells that arepositive for 5¢-bromodeoxyuridine (BrdU), doublecortin (DCX), BrdU and DCX, and BrdU and neuron-specific nuclear antigen (NeuN)in the DG of young adult, middle-aged and aged F344 rats treated with daily injections of BrdU for 12 consecutive days. Analysesperformed at 24 h, 10 days and 5 months after BrdU injections reveal that the extent of new cell production decreases dramaticallyby middle age but exhibits no change thereafter. Interestingly, fractions of newly formed cells that exhibit appropriate migration andprolonged survival, and fractions of newly born cells that differentiate into neurons, remain stable during ageing. However, in newlyformed neurons of the middle-aged and aged DG, the expression of mature neuronal marker NeuN is delayed and early dendriticgrowth is retarded. Thus, the presence of far fewer new granule cells in the aged DG is not due to alterations in the long term survivaland phenotypic differentiation of newly generated cells but solely owing to diminished production of new cells. The results alsounderscore that the capability of the DG milieu to support neuronal fate choice, migration and enduring survival of newly born cellsremains stable even during senescence but its ability to promote rapid neuronal maturation and dendritic growth is diminished asearly as middle age.

Introduction

Endogenous stem or progenitor cells continually insert new neuronsinto the dentate gyrus (DG) of the hippocampus throughout life(Kaplan & Hinds, 1977; Cameron et al., 1993; Eriksson et al., 1998;Gage, 2002; Gould & Gross, 2002; Steindler & Pincus, 2002; Jinet al., 2003, 2004; Alvarez-Buylla & Lim, 2004). A considerablenumber of newly generated neurons in the adult DG differentiate intofull-fledged functional granule cells with appropriate dendritic growthand axon projections and survive for prolonged periods (van Praaget al., 2002; Cameron & McKay, 2001; Dayer et al., 2003; Rao &Shetty, 2004). Further, the extent of dentate neurogenesis correlateswith the hippocampal functions of learning and memory (Gross, 2000;Feng et al., 2001; Shors et al., 2001; Dobrossy et al., 2003; Monjeet al., 2003; Kempermann et al., 2004a). Additionally, the helpfulbehavioural effects of chronic antidepressants are mediated by thestimulation of dentate neurogenesis (Santarelli et al., 2003).Nevertheless, addition of new neurons to the DG from stem or

progenitor cells declines drastically during ageing (Seki & Arai, 1995;Kuhn et al., 1996; Cameron&McKay, 1999; Lichtenwalner et al., 2001;Kempermann et al., 2002; Jin et al., 2003;Nacheret al., 2003).Althougha direct relationship between decreased neurogenesis and impairments

in learning and memory during old age is contentious (Bizon &Gallagher, 2003; Merrill et al., 2003), aged rats with preservedspatial memory indeed exhibit greater levels of new neuronproduction in the DG than do rats with spatial memory impairments(Drapeau et al., 2003), implying that substantially decreased DGneurogenesis in old age can lead to deficits in memory. Moreover, itis envisaged that a reduced fraction of new granule cells in old age isone of the causes of age-related deleterious alterations in thehippocampus (Cameron & McKay, 1999). This is because fullymature granule cells are more easily depolarized and fire largeraction potentials than young granule cells (Liu et al., 1996) andhence it is hypothesized that a greater ratio of mature granule cells inold age (due to decreased neurogenesis) would induce a surge ofhyperactivity and damage to the hippocampus (Cameron & McKay,1999). Thus, it is imperative to analyse the proportional contributionof distinct regulatory phases of neurogenesis to the diminishednumber of new granule cells in the ageing DG. Elucidation of theseissues is essential for the development of appropriate therapeuticstrategies that restore the number of new granule cells in the ageingDG to the level observed in the young adult DG.While it is well established that addition of new granule cells to the

DG from stem or progenitor cells declines considerably during ageing,the potential age-related alterations in migration, enduring survival,neuronal fate choice of newly born cells and rate of maturation anddendritic growth of newly differentiated neurons are mostly unknown.

Correspondence: Dr Ashok K. Shetty, as above.E-mail: ashok.shetty@duke.edu

Received 28 April 2004, revised 28 October 2004, accepted 4 November 2004

European Journal of Neuroscience, Vol. 21, pp. 464–476, 2005 ª Federation of European Neuroscience Societies

doi:10.1111/j.1460-9568.2005.03853.x

Therefore, we investigated whether the microenvironment of theageing DG remains efficacious for supporting migration, long-termsurvival and adequate neuronal differentiation of newly born cells, andmaturation and dendritic growth of newly differentiated granule cells.We analysed cells that are positive for 5¢-bromodeoxyuridine (BrdU),doublecortin (DCX), BrdU and DCX, and BrdU and neuron-specificnuclear antigen (NeuN) in the DG of young adult, middle-aged andaged Fischer 344 (F344) rats treated with daily injections of BrdU for12 consecutive days. The production, early migration and neuronalfate choice of newly born cells, and maturation and dendritic growthof newly born neurons, were analysed at 24 h and 10 days after thelast BrdU injection, and the long-term survival and final location ofneurons generated during the BrdU injection period was analysed5 months after their birth.

Materials and methods

Animals and BrdU injections

Male F344 rats were obtained from the National Institute of Ageingcolony at Harlan Sprague-Dawley (Indianapolis, IN, USA). Three agegroups of animals, young adult (4 months old, n ¼ 19), middle-aged(12 months old, n ¼ 19), and aged (24 months old, n ¼ 19) wereused in this study. The animals were individually housed in anenvironmentally controlled room (� 23 �C) with a 12-h light : 12-hdark cycle, and were given food and water ad libitum. We used F344rats in this study as this rat strain has several advantages for studiesfocused on ageing. The genetic background is known and the normallife span and development are quite well defined. Generally, 3–6-month-old rats are considered young adults, 12–21-month-old rats aremiddle-aged and 22-month-old or older rats are considered aged. Theirlife expectancy is � 29 months, with a maximal survival time of� 36 months (Coleman et al., 1977). Animals received intraperitonealinjections of BrdU (Sigma, St Louis, MO, USA) once daily (at a doseof 100 mg ⁄ kg body weight) for 12 consecutive days. All experimentswere performed as per the animal protocol approved by theInstitutional Animal Care and Use Committee of the Duke UniversityMedical Center and the animal studies subcommittee of the DurhamVeterans Affairs Medical Center.

Tissue processing, and BrdU and DCX immunohistochemistry

Five rats from every age group were killed at each of the three time-points: 24 h, 10 days and 5 months after the last BrdU injection.Animals were deeply anaesthetized with halothane and perfusedtranscardially with 4% paraformaldehyde. The brains were postfixedin 4% paraformaldehyde overnight at 4 �C and cryoprotected in 30%sucrose solution. Thirty-micrometer-thick cryostat sections were cutcoronally through the entire hippocampus and collected serially inphosphate buffer. At each time-point, every 15th section through thehippocampus was selected in each of the animals and processed forsingle BrdU immunostaining using a monoclonal antibody to BrdU.Another series (every 15th) of sections from animals killed at 24 hafter the last BrdU injection were processed for DCX immunostainingusing a polyclonal antibody to DCX. Additional series of sections (6–10 per animal) were processed for the following dual immunostainingmethods: BrdU and DCX (at 24-h and 10-day time-points), DCX andNeuN (at the 24-h time-point) and BrdU and NeuN (at 24-h, 10-dayand 5-month time-points). Additional groups of rats (n ¼ 4 ⁄ agegroup) were killed at the 24-h time-point. From these, 60-lm-thickcoronal sections were cut through the entire hippocampus and

processed for the analyses of the dendritic growth of newly generatedneurons using DCX immunostaining.The BrdU immunostaining was performed using the avidin–biotin

complex (ABC) method (Elite ABC kit; Vector, Burlingame, CA,USA) with diaminobenzidine (DAB) as the chromogen (Shetty &Turner, 2000). The detailed methods for visualization of BrdU in newlyborn cells are described in our recent report (Rao & Shetty, 2004). Theimmunostained sections were mounted on gelatin-coated slides, air-dried, counterstained with haematoxylin, dehydrated, cleared andcover-slipped. The DCX immunostaining was also done using theABC method, as described in our recent report (Rao & Shetty, 2004).As per the manufacturer’s information, the DCX antibody used in thisstudy is an affinity-purified goat polyclonal antibody (1 : 200; Sc-8066, Santa Cruz Biotechnology, Santa Cruz, CA, USA), which wasraised against a peptide mapping at the carboxy terminus of DCX ofhuman origin. The peroxidase reaction was visualized using eitherDAB or vector grey as the chromogen. For visualizing the entiredendritic morphology in DCX-positive cells, 60-lm-thick sectionscollected from additional animals in each age group were incubated inthe DCX antibody for 2 days, treated with biotinylated horse antigoatIgG for 2 h, washed in phosphate-buffered saline, incubated in theABC reagent for 1 h and developed with vector grey.

Quantification of BrdU-positive cells and DCX-positive neuronsusing the optical fractionator method

In each animal killed at 24 h, 10 days and 5 months after the lastBrdU injection (n ¼ 5 per age group and time point), BrdU-positivecells in the dentate granule cell layer (GCL) and the dentatesubgranular zone (SGZ; two-cell-thick region from the inner marginof the dentate GCL) were counted in every 15th section through theentire antero-posterior extent of the hippocampus (450 lm apart)using the StereoInvestigator system (Microbrightfield Inc., Williston,VT, USA). In addition, in animals killed at 24 h after the last BrdUinjection (n ¼ 5 per age group), DCX-positive neurons in the GCLand SGZ were similarly counted. The StereoInvestigator systemconsisted of a colour digital video camera (Optronics Inc., Muskogee,OK, USA) interfaced with a Nikon E600 microscope.

Size and number of counting frames, section thickness, and shrinkageof tissue along the Z-axis after immunostaining

In each animal, BrdU- and DCX-positive cells were counted from 50–400 randomly and systematically selected frames (each measuring40 · 40 lm, 0.0016 mm2 area) in every 15th section using the 100·oil-immersion objective lens. The numbers and densities of frameswere determined by entering the parameter grid size (60 · 60 lm) inthe optical fractionator component of the StereoInvestigator system(Rao & Shetty, 2004). Counting of cells from 50–400 randomly andsystematically chosen frames in every 15th section through the dentateGCL–SGZ guaranteed that effectively every BrdU- and DCX-positivecell within the DG had equal odds of being counted. This is imperativebecause the scattering of BrdU- and DCX cells within the GCL–SGZwas visibly heterogeneous in all age groups. For these studies, we cut30-lm-thick sections through the DG using a cryostat that has beencalibrated. Measurement of the thickness of sections immediatelyfollowing sectioning using the StereoInvestigator system incorpor-ating an XYZ stage controller equipped with Z-axis position control(LEP Electronic Products Ltd, Hawthorne, NY, USA) has revealedthat the variability between sections is minimal (i.e. ±1 lm). WithBrdU or DCX immunostaining, sections showed significant shrinkagealong the Z-axis. Measurement of the thickness of sections in different

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regions of the DG using the StereoInvestigator system describedabove revealed that, in sections processed for BrdU immunostaining,the average thickness was reduced to 67% of the initial thickness inyoung adults and 53% of the initial thickness in middle-aged and agedanimals. Hence, at the time of BrdU data collection the thickness ofsections was 20 lm in young adults and 16 lm in the middle-agedand aged animals. Thus, the shrinkage of brain sections along theZ-axis following BrdU immunostaining varied depending upon theage of the animal. However, within each age group the degree ofdeformation of the sections in regions of the DG was uniform (i.e.homogenous uniform deformation). In sections processed for DCXimmunostaining, the average thickness was reduced to 67% of theinitial thickness in all age groups. Thus, the degree of shrinkage ofbrain sections along the Z-axis following DCX immunostaining wasuniform across the three age groups.

Cell counting method

In every section, the contour of GCL–SGZ area was first delineated forcounting using tracing function of the StereoInvestigator. Followingthis, the optical fractionator component was activated and the numberand location of counting frames and the counting depth for that sectionwas determined by entering parameters such as the grid size(60 · 60 lm), the thickness of the top guard zone (4 lm) and theoptical dissector height (i.e. 8 lm). A computer driven motorizedstage then allowed the section to be analysed at each of the countingframe locations. In every counting frame location the top of the sectionwas set, after which the plane of the focus was moved 4 lm deeperthrough the section (guard zone) to get rid of the problem of unevensection surface. This plane served as the first point of the countingprocess. All BrdU- and DCX-positive cells that came into focus in thenext 8 lm section thickness were counted if they were entirely withinthe counting frame or touching the upper or right side of the countingframe. Based on the above parameters and cell counts, the StereoIn-vestigator program calculated the total number of BrdU- andDCX-positive cells per DG by utilizing the optical fractionatorformula, N ¼ (1 ⁄ ssf).(1 ⁄ asf).(1 ⁄ hsf).EQ– (Dorph-Petersen et al.,2001). The abbreviation ‘ssf’ represents the section sampling fraction,which was 15 in this study; ‘asf’ symbolizes the area samplingfraction, which is calculated by dividing the area sampled with thetotal area of the GCL–SGZ (i.e. the sum of GCL–SGZ areas in every15th section); ‘hsf’ stands for the height sampling fraction, which iscalculated by dividing the height sampled (i.e. 8 lm in this study) withthe section thickness at the time of analysis (i.e. 20 lm in young adultsand 16 lm in middle-aged and aged animals for BrdU cell counts, and16 lm in all age groups for DCX cell counts); EQ– denotes the totalcount of particles sampled for the entire DG.

Demonstration and measurement of cells positivefor both DCX and BrdU

To determine the fractions of DCX-positive neurons that expressBrdU, sections from animals belonging to different age groups killedat 24 h and 10 days after BrdU injections were processed for DCXand BrdU dual immunostaining using the two-chromogen methoddescribed in our recent report (Rao & Shetty, 2004). The percentage ofDCX-positive neurons expressing BrdU and the percentage of BrdU-positive cells expressing DCX were then quantified in differentanimals. This was accomplished by exhaustive counting of total DCX-positive cells, total BrdU-positive cells and the double-labelled cells(positive for both DCX and BrdU) present in the dentate GCL-SGZ.Six to ten sections were used from each animal and the counting was

performed at a magnification of 400· using a 40· objective lens. Thetotal number of BrdU-positive cells analysed in different groups was550 in young adults, 239 in the middle-aged and 201 in the aged.Although the two-chromogen double immunohistochemical methodemployed for quantification of BrdU labelling in DCX-positive cellsin this study was very reliable in our hands, this method is not awidely used technique for labelling two antigens within the same cells.Therefore, we crosschecked the efficiency of the above method byemploying a sequential dual immunofluorescence method on addi-tional tissue sections from animals belonging to the three age groups,as described in our earlier report (Rao & Shetty, 2004).

Analyses of NeuN expression in DCX-positive cellsand BrdU-positive cells

To determine the fractions of DCX-positive cells that express themature neuronal marker NeuN, sections from animals killed at the24-h time point were processed for DCX and NeuN dual immuno-fluorescence, as detailed in Rao & Shetty (2004). The fraction ofDCX-positive cells that expressed NeuN was quantified by examina-tion of individual DCX-positive cells at 400· in sections fromdifferent animals (n ¼ 4 per age group, 50–100 cells ⁄ animal). Cellsthat exhibited DCX and NeuN coexpression were also verified usingconfocal laser scanning microscopy (LSM-410, Carl Zeiss). For this,Z-sectioning at 1-lm intervals was performed and optical stacks of8–10 images were used for analysis of individual double-labelledcells. To visualize the fraction of BrdU-positive cells that differentiateinto mature neurons, double immunofluorescence methods for dem-onstrating NeuN and BrdU were employed. Sections from animalskilled at 24 h, 10 days and 5 months after the last BrdU injection wereused for this analysis. In brief, sections were processed first for variousBrdU preincubation treatments as described elsewhere (Rao & Shetty,2004), blocked in normal serum, incubated in a cocktail solutioncontaining rat BrdU antibody (1 : 50; Accurate Chemicals, Westbury,NY, USA) and mouse NeuN antibody (1 : 1000; Chemicon, Teme-cula, CA, USA). Sections were then treated with a mixture of goatantimouse IgG tagged with Alexa Fluor 488 and biotinylated rabbitantirat IgG (Vector), and incubated in streptavidin Texas Red. Sectionswere coverslipped with slow fade–antifade mounting medium andvisualized with a Nikon E600 fluorescence microscope. The fractionof BrdU-positive cells that express NeuN was quantified byexamination of individual BrdU-positive cells at 400· in sectionsfrom different animals (n ¼ 4 per age group and time point,50–100 cells ⁄ animal). Cells that exhibited BrdU and NeuN coexpres-sion were also carefully verified using confocal laser scanningmicroscopy (LSM-410, Carl Zeiss). For this, Z-sectioning at 1-lmintervals was performed and optical stacks of 8–10 images were usedfor analysis of individual double-labelled cells.

Measurement of the dendritic maturation of newly generatedneurons in the three age groups

We first compared the overall dendritic maturation of newly generatedneurons in the three age groups during the DCX expression phase,based on the orientation of dendrites in relation to the GCL. This wasaccomplished by measuring the fractions of DCX neurons withvertical dendrites emanating from the soma and extending into thedentate molecular layer through the GCL (presumably more maturenew neurons) and DCX-positive neurons with horizontally orientatedsoma and dendrites in the SGZ (presumably immature new neurons).Further, because some of the DCX-immunopositive neurons with

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vertical dendrites also exhibited basal dendrites and the presence ofbasal dendrites is one of the immature features of granule cells, wealso quantified the fractions of DCX-positive neurons with or withoutbasal dendrites as another measure of dendritic maturation of newlyformed neurons in the three age groups. For these measurements, weanalysed 492 neurons in the young adult group (� 123 ⁄ animal,n ¼ 4), 480 neurons in the middle-aged group (� 120 ⁄ animal, n ¼ 4)and 284 in the aged group (� 71 ⁄ animal, n ¼ 4). In addition, wequantified the overall dendritic growth in neurons that are relativelymore mature in all three age groups. For this, in each age group wechose the well-differentiated (or best looking) DCX-positive neuronsin 60-lm-thick sections, based on the following criteria: (i) neuronsexhibited vertically orientated dendrites that extended into the dentatemolecular layer and were mostly located in or close to the middle thirdof the section thickness; (ii) neurons did not have severed dendrites inclose proximity to the soma; and (iii) dendrites of selected neurons hadminimal overlap with the dendrites of adjacent cells so that alldendritic branches of chosen neurons could be traced unambiguously.The dendritic and other morphological measurements of these well-differentiated DCX-positive neurons were made at 1150· magnifica-tion, using a semiautomatic neuron-tracing system (Neurolucida;Microbrightfield, Colchester, VT) linked to a Nikon microscope(Shetty & Turner, 1998). In each age group, 24 DCX-positive neurons(n ¼ 6 ⁄ animal) from 60-lm sections processed for DCX immuno-staining were traced in their entirety and data for various measure-ments were calculated, including the cell body size (area andperimeter), number of dendritic nodes and ends and total dendriticlength. To measure the extent of dendritic growth away from the somaand the branching of dendrites at different distances from the soma,the concentric circle analysis of Sholl (1953) was performed using theNeuroExplorer component of the Neurolucida program.

Measurement of the migration of newly generated cellsinto the granule cell layer

We quantified the migration of newly generated (i.e. BrdU-immuno-positive) cells into the GCL in the three age groups by measuring therelative position of these cells in the GCL at 10 days and 5 monthsafter the last BrdU injection. Following short-term survival (i.e. at10 days), the number of BrdU-immunopositive cells in two regions(SGZ and GCL) was measured and the relative fractions of cells inthese regions were calculated. This measurement comprised analysesof the location of 799 cells in the young adult group (n ¼ 4), 460 cellsin the middle-aged group (n ¼ 4) and 268 cells in the aged group(n ¼ 4). Following prolonged survival (i.e. at 5 months), the numberof BrdU-immunopositive cells was measured in three separate regions(inner, middle and outer thirds of the GCL) and the fractions of cells inthese regions were then calculated. This involved characterization ofthe location of 510 cells in the young adult group (n ¼ 4), 247 cells inthe middle-aged group (n ¼ 4) and 222 cells in the aged group(n ¼ 4).

Statistical analyses

For every parameter, the value was first calculated separately for eachanimal before the means and SEM were determined for the totalnumber of animals included per group. The values from different agegroups of animals for different parameters were compared using eitherone-way anova with the Student–Newman–Keuls multiple compar-ison post hoc test or two-way anova.

Results

Production of new cells in the young adult, middle-aged andaged DG

In all age groups, BrdU immunostaining revealed newly generatedcells in the GCL and the SGZ. However, both middle-aged and agedDG exhibited ostensibly fewer BrdU-positive cells than the young DG(Fig. 1, 24-hour A1–C2; top panel). In all age groups, BrdU-positivecells in the GCL were mostly dispersed whereas those in the SGZwere in 2–4 cell clusters, which is consistent with previous studies(Gray & Sundstrom, 1998; Gould et al., 1999; Parent et al., 1999;Palmer et al., 2000). The total number of new cells (i.e. BrdU-positivecells) generated over a period of 12 days in the GCL and SGZ wasmuch greater (P < 0.001) in the young adult DG (Mean ±SEM ¼ 20 673 ± 1,696, n ¼ 5; Fig. 1D) than in either middle-agedDG (5402 ± 597, n ¼ 5, Fig. 1D) or the aged DG (4644 ± 112;n ¼ 5, Fig. 1D). Thus, the addition of new cells to the SGZ–GCL overa period of 12 days decreased substantially by middle age (74–78%reduction) and was maintained at the same level between middle ageand old age, which is consistent with several earlier studies in rats andmice (Kuhn et al., 1996; Lichtenwalner et al., 2001; Nacher et al.,2003 Bondolfi et al., 2004).

Migration of newly born cells in the young adult, middle-agedand aged DG

Newly born cells persist at 10 days after BrdU injections in all threeage groups (Fig. 1, 10 days; lower left panel). The cells appearedmostly isolated at this time point. In the young adult DG, the majorityof cells (82%) were already in the GCL and some had reached themiddle and outer thirds of the GCL whereas in the middle-aged andaged DG 64–65% of cells were in the GCL, suggesting that the earlymigration of newly born cells is slower in the ageing DG. Examinationat 5 months after BrdU injections revealed the persistence of newlyborn cells for prolonged periods in all three age groups (Fig. 1,5 months; lower middle panel). The surviving BrdU cells were clearlyisolated and virtually all of them were in the GCL; some of them werein the outer third of the GCL. Measurement of the location of BrdUcells revealed that the distribution of cells that were added 5 monthspreviously to the GCL was highly similar in the three age groups. In allgroups, 67–69% of cells were in the inner third of the GCL, 14–20% ofcells were in the middle third of the GCL and 12–15% of cells were inthe outer third of the GCL. Thus, despite an early delay the migration ofnewly born cells was not impaired in the middle-aged and aged DG.

Long-term survival of newly born cells in the young adult,middle-aged and aged DG

To determine the long-term survival of newly born cells, we quantifiedand compared the total number of BrdU-positive cells at 24 h, 10 daysand 5 months after 12 daily injections of BrdU between the three agegroups, using two-way anova (Fig. 1D). The loss of newly generatedcells was significant between the 24-h and 10-day time-points in theyoung adult DG (P < 0.01) but not in the middle-aged and aged DG.Further, the loss of newly generated cells between the 10-day and5-month time-points was significant in the young adult andmiddle-agedgroups (P < 0.01–0.001) but not in the aged group. Nevertheless, thetwo-way anova revealed that the overall loss of newly generated cellsbetween the 24-h and 5-month survival time-points was significant in allthree age groups and the percentage decrease was similar across the

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Fig. 1.

Fig. 2.

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three age groups (young adults, 56% reduction, P < 0.001; middle-aged, 47% reduction, P < 0.05; aged, 51% reduction, P < 0.05). Theinteraction between age and the number of cells surviving was notsignificant at the 10-day time-point but was significant at the 5-monthtime-point. Overall, it is clear that the percentage of newly born cells thatexhibit long-term (5-month) survival remains steady (47–56%),regardless of the age of the DG at the time of their birth.

Production of new neurons in the young adult, middle-agedand aged DG

DCX is a specific marker of newly born neurons in the adult DGand our recent study in adult animals has demonstrated that neuronsidentified with DCX immunostaining are new neurons that arepredominantly born during the 12 days before euthanasia (Rao &Shetty, 2004). In all age groups, DCX immunostaining revealednewly formed neurons in the SGZ and the GCL (Fig. 2A–C). Thecell bodies of DCX-positive neurons were located in the SGZ andthe inner third of the GCL in all groups (Fig. 2A–C). However, inthe young adult DG the vast majority of DCX-positive neurons hadthe phenotype of differentiated granule cells with verticallyorientated dendrites extending into the outer two-thirds of thedentate molecular layer (Jones et al., 2003). In contrast, theproportion of DCX-positive neurons with such vertically orientateddendrites was lower in the middle-aged and aged DG. Quantifica-tion of the total number of DCX-positive cells (Fig. 2D) demon-strated an average of 21 000 new neurons in young adult DG(Mean ± SEM, 20 944 ± 1028, n ¼ 5), 5000 new neurons in themiddle-aged DG (5380 ± 333, n ¼ 5) and 4000 new neurons in theaged DG (3763 ± 190, n ¼ 5). Thus, the production of newneurons in the DG decreased substantially (74% reduction) bymiddle-age and was maintained at the same level thereafter (82%reduction in old age), which is consistent with the results of BrdUcell counts described earlier.

Neuronal fate choice of newly generated cells in the youngadult, middle-aged and aged DG

We characterized neurons (DCX-positive cells) among BrdU-positivecells at 24 h after BrdU injections, using sections stained for DCX andBrdU dual immunostaining (Fig. 3, A1–C2). This revealed that thevast majority of newly born cells (BrdU-positive cells) expressed theearly neuronal marker DCX in all age groups. Both DCX-positiveneurons having the phenotype of differentiated granule cells (i.e. cellswith vertically orientated dendrites; Fig. 3, A1, B1 and C1) and DCX-

positive neurons having a relatively immature morphology (i.e. cellswith horizontally orientated dendrites in the SGZ; Fig. 3, A2, B2 andC2) were positive for BrdU. Analyses of DCX-immunopositiveneurons in sections processed sequentially for DCX and BrdU dualimmunofluorescence confirmed the above findings (Fig. 3, D1–E3).Quantification demonstrated that 73–76% of newly born cellsexpressed the early neuronal marker DCX, regardless of the age ofthe DG (young DG, 76 ± 7%; middle-aged DG, 74 ± 2%; aged DG,73 ± 2%; Fig. 3F), suggesting that most of the newly born cellsdifferentiate into neurons in all age groups. Further, DCX expressionin similar fractions of newly born cells in the three age groupssuggests that neuronal fate choice of newly born cells (i.e. the fractionof newly born cells that exhibit phenotypic differentiation intoneurons) is not altered with ageing. Further analyses with BrdU andDCX dual immunostaining at 10 days after BrdU injections revealedthat only 30–31% of BrdU cells expressed DCX at this stage in all agegroups (Fig. 3F), which is consistent with the clearance of DCXobserved over time in newly born neurons of young animals (Brownet al., 2003; Kempermann et al., 2003). Thus, the clearance of DCX innewly born cells over time is similar across the three age groups. TheBrdU cells that express DCX at this time point are probably the cellsthat are born during the last third (i.e. the last 4 days) of the BrdUinjection period.Evaluation of BrdU immunoreactivity among DCX-positive neu-

rons at 24 h after the last BrdU injection revealed that the vastmajority of DCX-positive neurons were densely BrdU-positive in allthree age groups (Fig. 3, A1–C2). Quantification demonstrated that> 87% of DCX-positive neurons in the DG were newly born in allthree age groups (Fig. 3G). Thus, in all three age groups the vastmajority of DCX-immunopositive neurons were new neurons thatwere generated during the preceding 12 days. Analyses at 10 daysafter BrdU injections demonstrated that only 16–20% of DCX-positivecells express BrdU (Fig. 3G), suggesting that the great majority ofDCX-positive neurons visualized at 10 days after BrdU injectionswere born after the BrdU injection period. Thus, the overall durationof DCX expression in newly formed neurons in the DG was similaracross the three age groups. In addition, the data demonstrate that thedose (100 mg ⁄ kg body weight) and frequency (once daily) of BrdUadministered in this study was suitable for labelling the vast majorityof new granule cells that were produced over a period of 12 days. Themost likely explanation for the efficiency of BrdU labelling in thisstudy is that, at the time of BrdU injection every day, the greatmajority of proliferating stem or progenitor cells happened to be in theS-phase, which usually lasts for 8–12 h. Thus, our data are suggestiveof synchronized cycling of progenitor cells in the DG, which contrastswith the results of Cameron & McKay (2001). Differences in the strain

Fig. 2. Arrangement of newly generated neurons in the dentate gyrus (DG) of (A) young adult, (B) middle-aged and (C) aged rats visualized with doublecortin(DCX) immunostaining. Note that the density of DCX-positive neurons is much greater in the young adult DG. DCX-positive neurons in the young adult are presentthroughout the DG along the subgranular zone (SGZ) and the granule cell layer (GCL) whereas DCX-positive neurons in the middle-aged and aged DG are inclusters of 2–4 cells separated by much larger gaps along the SGZ and GCL. (D) Comparison of the total number of DCX-positive neurons between the youngadult, middle aged and aged DG. Note that the total number is reduced dramatically as early as middle age but exhibits no significant change between the middle ageand the old age. DH, dentate hilus; IML, inner molecular layer; MML, middle molecular layer; OML, outer molecular layer. Scale bar, 50 lm (A–C).

Fig. 1. Distribution of newly generated (BrdU-positive) cells in the dentate gyrus (DG) of young adult, middle-aged and aged rats at 24 h (upper panel), 10 days(lower left panel) and 5 months (lower middle panel) after daily injections of BrdU for 12 consecutive days. Sections were counterstained with haematoxylin afterBrdU immunostaining. A1, B1 and C1 illustrate newly generated cells (brown reaction product) in the granule cell layer (GCL) and the dentate hilus (DH). Note thatthe density of newly born cells is much greater in the young adult DG than in either the middle-aged or aged DG at all time points. A2, B2 and C2 show magnifiedviews of boxed regions from A1, B1 and C1. (D) Comparison between the three age groups of the survival of newly born cells at 24 h, 10 days and 5 months afterBrdU injections. Note that the pattern of loss of newly born cells between 24 h and 10 days and between 10 days and 5 months varies depending on the age of theDG. However, the overall reductions in newly born cells between 24 h and 5 months are significant in all three age groups. Moreover, the percentage reductionsbetween 24 h and 5 months are similar in all three age groups. Scale bars, 200 lm (A1, B1, C1, all panels); 25 lm (A2, B2, C2, upper panel), 50 lm (A2, B2, C2,lower left and lower middle panels).

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and age-groups of rats used probably explain the discrepancy infindings between the two studies.

Acquisition of NeuN by newly born neurons during the DCXexpression phase

We examined the maturation of newly born neurons during the DCXexpression phase by analysing the expression of mature neuronalmarker NeuN in DCX-immunopositive neurons. Figure 4 illustratesexamples of cells that express DCX and NeuN in the DG of youngadult (Fig. 4, A1–A3) and aged (Fig. 4, B1–B3) rats using confocalmicroscopy. Analysis of cells that express DCX and NeuN demon-strated that only 11–13% of DCX-positive cells express the matureneuronal marker NeuN in the middle-aged and aged groups, incomparison to 40% of DCX cells expressing NeuN in the young adultgroup (Fig. 4C). Thus, the expression of NeuN is delayed in newlyborn neurons of the middle-aged and aged DG.

Early dendritic growth of newly born neurons in the young adult,middle-aged and aged DG

This was analysed in thicker sections immunostained for DCX. In theyoung adult DG, 69% of newly generated neurons exhibited dendritesthat are orientated vertically and traversed both GCL and the inner andmiddle regions of the molecular layer (Fig. 5, A1 and 2). The remaining31% of newly formed neurons had both soma and dendrites orientatedhorizontally in the SGZ (Fig. 3, A2). In contrast, in the middle-agedand the aged DG, only a smaller fraction (28–30%) of newly bornneurons exhibited vertically orientated dendrites. Examples of suchneurons are illustrated in Fig. 5, B1 and 2, and C1 and 2. As the DCX-immunopositive neurons with vertically orientated dendrites arerelatively more mature among the DCX-expressing newly bornneurons (Rao & Shetty, 2004), a smaller fraction of such neurons inthe middle-aged and aged DG suggests that the early dendritic growthof newly born neurons is retarded in the ageing DG. Indeed, appraisalof newly generated neurons for basal dendrites revealed that a greaternumber of newly generated neurons (56–60%) in the middle-aged andaged DG exhibit basal dendrites, an immature feature of granule cells inrodents (Seress & Pokorny, 1981; Marti-Subirana et al., 1986; Lubbers& Frotscher, 1988; Jones et al., 2003). This is in contrast to the youngadult DG where only 39% of DCX-positive neurons exhibited basaldendrites. The fraction of DCX-positive neurons having basal dendritesobserved in young adults in this study was less than in the observationsof Ribak et al. (2004). The discrepancy could be related to the differentstrains of rats used in the two studies. The morphology and the patternof dendritic growth among newly generated neurons with verticallyorientated dendrites appeared similar across the three age groups.

These neurons exhibited one thick apical dendrite, which branchedwithin the dentate GCL and displayed a number of short terminalbranches mostly in the middle third of the molecular layer (Fig. 5, A2,B2 and C2). However, measurements of the soma and dendritesrevealed that neurons in the young adult DG exhibited a significantlygreater number of dendritic nodes and endings than those in the middle-aged and aged DG (Table 1). In addition, the total dendritic length wasgreater in neurons of the young adult DG (Table 1). The concentriccircle analysis of Sholl (1953) revealed that, in all age groups, most ofthe dendritic branches of the apical dendrite of newly generatedneurons extended beyond the 100-lm distances from the soma andended between 100 and 150 lm distant from the soma (Fig. 5D).However, neurons in the young adult DG had a greater number ofnodes at 50–100- and 100–150-lm distances from the soma thanneurons in the middle-aged and aged DG (Fig. 5D). Neurons from theyounger DG also exhibited a greater number of dendritic endingsbetween 100 and 150 lm distant from the soma than neurons in themiddle-aged and aged DG. Thus, during the DCX expression phase,newly born neurons in the ageing DG had retarded dendritic growth incomparison to neurons in the young adult DG.

Fraction of mature neurons among newly born cells at differenttime points after BrdU injections

Examples of newly born cells that express the mature neuronal markerNeuN at 24 h and 5 months after BrdU injections are illustrated inFig. 6 (upper panel). Quantification of NeuN expression at 24 h afterBrdU injections revealed that only 14% of BrdU cells expressed NeuNin the middle-aged and aged groups, in comparison to 38% of BrdUcells expressing NeuN in the young adult group (Fig. 6C). However, at10 days after BrdU injections the number of newly born cells thatacquired NeuN expression was similar across the three age groups.Thus, newly born neurons in the middle-aged and aged rats exhibited adelay in the expression of the mature neuronal marker NeuN but theycaught up with NeuN expression during the additional 10-day survivalperiod. Quantification of NeuN expression among the surviving BrdU-positive cells at 5 months post-BrdU injections revealed that 80–84%of BrdU-positive cells expressed NeuN in all age groups (Fig. 6C).Thus, among the newly generated cells that survived 5 months thevast majority in all age groups were neurons. Extrapolation of thesepercentages with the absolute number of BrdU cells revealed thenumber of newly added neurons that exhibited 5-month survival indifferent age groups. The average number of newly added neurons(over a 12-day period) that exhibit long-term survival was 7703 in theyoung adult DG, 2268 in the middle-aged DG and 1814 in the agedDG. Comparison of these numbers with numbers of neurons measuredusing BrdU–DCX double immunostaining at 24 h after the last BrdU

Fig. 3. Newly generated neurons in the dentate gyrus (DG) of (A1 and A2) young adult, (B1 and B2) middle aged and (C1 and C2) aged rats receiving dailyinjections of BrdU for 12 consecutive days and visualized with 5¢-bromodeoxyuridine (BrdU) and doublecortin (DCX) double immunostaining. (A1, B1 and C1)Examples of newly born neurons with vertically orientated soma and dendrites in the three age groups. (A2, B2 and C2) Examples of newly born neurons withhorizontally orientated soma and dendrites. Note that the majority of newly born neurons are clearly positive for both BrdU (brown reaction product in the nucleus)and DCX (blue-grey reaction product in the cytoplasm of the soma and dendrites) in all age groups suggesting their birth during the 12-day BrdU injection period.Examples of DCX cells that are not positive for BrdU are also seen in A1 and C1 (indicated by arrows). D1–E3 demonstrate newly generated neurons visualized withDCX (red) and BrdU (green) dual immunofluorescence from young adult (D1–D3) and middle-aged (E1–E3) animals. Note that the red DCX-positive cells have aclear green BrdU immunofluorescence in their nucleus. (F) Comparison of the percentages of BrdU cells that are positive for DCX at 24 h and 10 days after BrdUinjections. Note that 73–76% of BrdU cells are positive for DCX in all three age groups at 24 h after the last BrdU injection, implying that most of the newly borncells differentiate into neurons in all age groups. At 10 days after BrdU injections, only 30–31% of BrdU cells express DCX in all age groups, suggesting that theclearance of DCX over time is similar across the three age groups. (G) Comparison of the percentages of DCX cells that are positive for BrdU in the three agegroups at 24 h and 10 days after BrdU injections. Note that the vast majority of DCX-positive neurons are immunopositive for BrdU at 24 h after the last BrdUinjection, suggesting that DCX-positive cells in all three age groups represent neurons that are mostly born during the 12-day BrdU injection period. However, only16–20% of DCX-positive cells express BrdU at 10 days after BrdU injections in all age groups, suggesting that most of the DCX-positive cells seen at 10 days afterBrdU injections were born after the BrdU injection period. Scale bar, 20 lm (A1–C2), 25 lm (D1–E3).

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Fig. 4. The left panel illustrates examples of newly born cells that express doublecortin (DCX) and the mature neuronal marker NeuN in (A1–A3) the young adultDG and (B1–B3) the aged DG. The expression of NeuN is depicted in green and the expression of DCX is in red. Arrowheads denote cells that are dual-labelled forNeuN and DCX. (C) Comparison of the percentage of newly born neurons that are positive for DCX and NeuN at 24 h after the last BrdU injection. Note that onlysmaller fractions (11–13%) of DCX neurons expressed NeuN in the middle-aged and aged groups in comparison to 40% of DCX cells expressing NeuN in the youngadult group (P < 0.001), suggesting that the expression of NeuN is delayed in the newly born neurons of middle-aged and aged rats. Scale bar, 25 lm (A1–B3).

Fig. 3.

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Fig. 5.

Fig. 6.

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injection suggests that 49–57% of newly generated neurons exhibitedprolonged survival regardless of the age of the DG at the time of theirbirth.

Discussion

This quantitative study provides five novel findings pertaining to DGneurogenesis and ageing. (i) Newly generated cells in the middle-aged and aged DG exhibit a delay in migration but this early delaydoes not hamper the eventual migration of newly born cells into theGCL, as fractions of new cells that occupy different zones of theGCL at 5 months after their birth are similar in the three age groups.(ii) Ageing does not impair the microenvironment of the DG thatsupports the long-term survival of newly differentiated granule cells,as 49–57% of newly differentiated granule cells exhibit enduring(5-month) survival in all age groups. (iii) The extent of neuronal fatechoice of newly generated cells in the middle-aged and aged DGremains akin to that observed in the young adult DG. This wassubstantiated by differentiation of 73–76% of BrdU-labelled newlygenerated cells into DCX-positive neurons in all three age groups.(iv) Newly generated granule cells mature slowly in the middle-agedand aged DG. This was demonstrated by a delay in the expression ofthe mature neuronal marker NeuN among newly born neurons. (v)Newly born granule cells in the middle-aged and aged DG haveretarded dendritic growth during the DCX expression phase.Additionally, our data confirm that the extent of new neuronproduction in the DG decreases dramatically as early as middle age

but exhibits no change thereafter. These results collectively under-score the fact that the far fewer new granule cells in the middle-agedand aged DG is not due to impairments in the enduring survivaland ⁄ or neuronal fate choice of newly generated cells but is ratherdue to diminution in the production of new cells. Because thecompetence of the DG milieu to support migration, enduring survivaland neuronal fate choice of newly born cells remains stable evenduring senescence, strategies that augment the production of newcells in the ageing DG (Lichtenwalner et al., 2001; Jin et al., 2003)may ameliorate age-related deficits in hippocampal-dependent learn-ing and memory.

Migration and long-term survival of newly generated cellsin the middle-aged and the senescent DG

The migration of newly born neurons at 10 days after the last BrdUinjection showed that the movement of cells across the GCL is slowerin the middle-aged and aged DG than in the young adult DG.However, the position of new cells in the GCL at 5 months after theirbirth was similar across the three age groups, suggesting that newlyborn cells in the ageing DG eventually acquire positions in the GCLthat are similar to their counterparts in the young adult DG. Thus, theoverall migration of newly born cells is not altered in the middle-agedand aged DG. Quantification of the survival of newly generated cellsat 5 months after their birth demonstrated that 47–56% of newly borncells (equivalent to 49–57% of newly born neurons observed at 24 hafter the last BrdU injection) exhibit prolonged survival, regardless of

Table 1. Morphometric measurements of DCX-immunopositive new neurons with vertically orientated dendrites in the young adult, middle-aged and aged dentategyrus

Animalgroups

Perimeter(lm)

Cell bodyarea (lm2)

Dendriticnodes (n)

Dendriticendings (n)

Total dendriticlength (n)

Young adult 32.6 ± 1.0 65.9 ± 3.5 10.5 ± 0.9 11.7 ± 0.9 518.3 ± 43.9Middle-aged 31.6 ± 0.9 65.3 ± 3.5 5.3 ± 0.5 6.5 ± 0.4 382.8 ± 31.6Aged 33.2 ± 1.1 68.4 ± 5.0 5.8 ± 0.5 6.9 ± 0.5 344.2 ± 21.7P-value > 0.05 > 0.05 < 0.001 < 0.001 < 0.01

Values represent mean ± SEM of 24 DCX-positive neurons. The neurons were traced at 1150· magnification using Neurolucida. Neurons were traced in theirentirety and data for the above parameters were collected individually for each neuron using NeuroExplorer before calculating the mean ± SEM for the totalpopulation in every age group.

Fig. 5. Visualization of newly generated [doublecortin (DCX)-positive] neurons in thicker (60 lm) sections of the dentate gyrus from young adult, middle-agedand aged rats. (A1, B1 and C1) Examples of newly born neurons from the three age groups; the neurons have well-developed apical dendrites emanating from thesoma. Note that the vertically orientated dendrites of these neurons cross the inner and middle molecular layers (IML and MML) and extend into the outer molecularlayer (OML) in all three age groups. However, the number of such neurons is considerably greater in the young adult DG than in either middle-aged or aged DG.(A2, B2 and C2) Examples of DCX-positive neurons with vertically orientated dendrites from A1, B1 and C1 (indicated by arrows) traced with Neurolucida. GCL,granule cell layer. (D) Sholl’s concentric circle analyses (Sholl, 1953) of the number of intersections, nodes and endings in the apical dendrites of well-differentiateddoublecortin (DCX)-positive newly born neurons from young adult, middle-aged and aged dentate gyrus. Note that most of the dendritic branches in these neuronsextend beyond the 100-lm distances from the soma and end between 100 and 150 lm distances from the soma in all three age groups (see top bar chart). A fewdendritic branches also cross 200 lm distances from the soma. However, the extent of branching of apical dendrites (measured by the number of nodes) between 50-and 100-lm and between 100- and 150-lm distances from the soma is considerably diminished in the middle-aged and aged groups in comparison to the young adultgroup (see middle bar chart). Further, the number of dendritic endings is significantly less in the 100–150-lm distances from the soma in the middle-aged and agedgroups. Thus, comparison of even the most well developed newly born neurons during the DCX expression period from the three age groups suggests delayedmaturation of dendrites in newly born neurons of middle-aged and aged DG. Scale bar, 50 lm (A1–C2).

Fig. 6. Expression of NeuN by newly generated cells at different time points after their birth in distinct age groups of rats. The dual-labelling was verified usingZ-sectioning at 1-lm intervals in a confocal laser scanning microscope. The upper left panel illustrates newly generated cells (i.e. BrdU-positive cells, shown by redfluorescence) in the granule cell layer that are positive for NeuN (shown by green fluorescence) at 24 h after the last BrdU injection. A1–A3 are from a young adultDG and B1–B3 are from an aged DG. The upper right panel shows examples of newly generated cells that are positive for NeuN at 5 months after BrdU injections.A1–A3 are from a young adult DG and B1–B3 are from an aged DG. (D) Comparison between the three age groups of rats of the percentages of NeuN-positiveneurons among BrdU-positive cells at 24 h, 10 days and 5 months after BrdU injections. Note that the fractions of BrdU cells that express NeuN at the 24-h timepoint are greater in the young adult DG than in the middle-aged and aged DG. However, the fractions of BrdU cells that express NeuN are similar between the threeage groups of rats at 10-day and 5-month time points, suggesting that NeuN expression in newly born cells is delayed in the middle-aged and aged DG during theinitial survival period. Scale bar, 25 lm.

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the age of the DG at the time of their birth. These results underscorethe fact that both middle-aged and aged DG are capable ofsupporting the survival of newly differentiated granule cells forprolonged periods, possibly the rest of lifespan in the aged DG. Theresults obtained in the young adult DG are consistent with a previousstudy (Dayer et al., 2003). As the long-term survival of newlygenerated granule cells in the DG reflects their incorporation into thehippocampal circuitry (Stanfield & Trice, 1998; Markakis & Gage,1999), it appears that the fraction of newborn granule cells that areincorporated into the hippocampal circuitry remains stable over thecourse of ageing.

Neuronal fate choice of newly generated cellsin the middle-aged and the aged DG

A previous neurogenesis study using mice showed that the extent ofneuronal fate choice of newly born cells declines with ageing(Kempermann et al., 1998). Further, a recent study in rats using dualimmunofluorescence for rat collapsin response-mediated protein-4(CRMP-4) and BrdU, reports that the degree of neuronal differen-tiation from newly born cells is 22% in the middle-aged DG and 2%in the aged DG, in sharp contrast to neuronal differentiation of 84%of newly born cells in the young adult DG (Nacher et al., 2003).However, DCX and BrdU dual immunostaining analyses at 24 hafter the last BrdU injection in this study reveals that 73–76% ofnewly born cells differentiate into neurons in all three age groups.The discrepancy between the current study and the study byKempermann et al. (1998) probably reflects differences in age-related changes in neurogenesis between mice and rats. On the otherhand, the discrepancy between the present study and the study byNacher et al. (2003) is probably linked to several differencesbetween the two studies, including the choice of neuronal marker(CRMP-4 vs. DCX) employed, the sex and housing conditions ofrats used, the duration of BrdU injections and the analysis time-pointafter BrdU injections. However, differences in the neuronal markeremployed probably played a major role for this discrepancy. This isbecause the neuronal marker (i.e. CRMP-4) used by Nacher et al.(2003) is a protein thought to be involved in axonal guidance duringdevelopment (Quinn et al., 1999) and hence not expressed untilsome time after the cells have become postmitotic. Indeed, a recentstudy by Seki (2002) demonstrates that most of the newly born cellsin young rats express CRMP-4 at 2 weeks after BrdU injections,suggesting that the expression of CRMP-4 in newly born cellsdepends on the activity of cells. In contrast, the expression of DCXis not only present in newly formed granule cells for � 2 weeks aftertheir birth (Brown et al., 2003; Rao & Shetty, 2004) but is alsorobust during the initial stages of the neuronal development, whichinclude the dividing neuronal progenitors and neuroblasts (Kemper-mann et al., 2003, 2004b). Thus, our finding that the extent ofneuronal fate choice of newly born cells is not impaired with ageingis reliable. The results also imply that the ability of the DG milieu topromote neuronal fate choice of a considerable fraction of newlyborn cells remains steady even during senescence. Additionalanalyses revealed that the duration of DCX expression in the newlygenerated neurons is not altered with ageing. These results probablyreflect the persistence of adequate amounts of factors that promoteneuronal fate choice of newly born cells during ageing in the DG.Further, a recent study suggests that two independent populations ofprogenitors (neuronal and glial progenitors) exist in the SGZ (Steineret al., 2004). From this perspective, it is plausible that age-relatedreductions in the number and ⁄ or proliferation of these progenitorsare similar.

Expression of NeuN and dendritic growth in newly generatedneurons of the ageing DG

Our results suggest that the maturation of newly born neurons isdelayed in the middle-aged and aged DG. This was shown by thedelayed expression of NeuN in newly born neurons of the middle-agedand aged DG. At 24 h after the last BrdU injection, only 11–13% ofnewly born (i.e. DCX-positive) neurons expressed NeuN in themiddle-aged and aged DG in comparison to 40% of newly bornneurons expressing NeuN in the young adult group. However, at10 days after BrdU injections the fraction of BrdU-positive cells thatexpress NeuN is similar across the three age groups (56–60%),implying that the early delay in the acquisition of NeuN by newly bornneurons of the middle-aged and aged DG is resolved during theadditional 10-day survival period. Delayed maturation of newly bornneurons during the early period after their birth in the ageing DGprobably involves age-related decreases in the levels of neurotrophicfactors that support rapid neuronal maturation. A greater fraction ofnewly generated (DCX-immunopositive) neurons in the middle-agedand aged DG exhibited horizontal and ⁄ or basal dendrites, immaturefeatures of granule cells in rodents (Seress & Pokorny, 1981; Marti-Subirana et al., 1986; Lubbers & Frotscher, 1988; Jones et al., 2003;Ribak et al., 2004). This is in sharp contrast to the young adult DGwhere a predominant fraction of newly generated neurons exhibitedvertically orientated dendrites, which is a relatively more maturefeature of granule cells and is consistent with the granule cellsobserved during the postnatal period (Rihn & Claiborne, 1990; Rao &Shetty, 2004). Additionally, the overall dendritic growth of newly borngranule cells having extensive vertical dendrites was greater in theyoung adult DG than in the middle-aged and aged DG. Theseobservations suggest that the dendritic growth of newly born neuronsis retarded during the DCX expression period in the middle-aged andaged DG. However, it is not clear from the results whether this is just adelay or newly born neurons in aged groups will exhibit smallerdendritic trees forever. Dendritic reconstruction studies at multipletime points following injections of retroviral markers encoding greenfluorescent protein (van Praag et al., 2002) are needed in future tofully analyse the differences in the sequential dendritic growth ofnewly born neurons in the three age groups.

Potential reasons for diminished production of new cellsin the middle-aged and the senescent DG

Addition of new cells to the DG over a period of 12 days exhibited a74% decrease at middle age and a 77% decrease at old age in maleF344 rats in this study. Measurement of the total number of newneurons added during the same period revealed the same trend. Theseresults are consistent with multiple earlier reports using both femaleF344 rats and other strains of rats (Seki & Arai, 1995; Kuhn et al.,1996; Lichtenwalner et al., 2001; Nacher et al., 2003). The extent ofproduction of new neurons in the young adult DG depends uponmultiple factors. These include the number and rate of proliferation ofstem or progenitor cells in the SGZ, the presence of significant celldeath in the GCL as a stimulatory factor (Gould & Tanapat, 1997), theconcentration of hormones such as glucocorticoids (Gould et al., 1998)and stem or progenitor cell proliferation factors such as epidermalgrowth factor, fibroblast growth factor-2 (FGF-2), insulin growthfactor-1 (IGF-1), vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) (Lichtenwalner et al., 2001; Jinet al., 2002a,b, 2003; Lee et al., 2002a,b), neurotransmitters such asserotonin (Gould, 1999), and vascular niche (Palmer et al., 2000;Monje et al., 2002). As the intensities or amounts of many of the above

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factors alter considerably during the course of ageing (Limke & Rao,2003), it is plausible that a dramatic decline in the production of newneurons in the middle-aged and aged DG is a consequence of age-related changes in multiple factors. This is supported by the followingobservations in previous studies. First, cells that exhibit stem orprogenitor cell properties such as radial glia (Seri et al., 2001) decreaseconsiderably with ageing in the SGZ (Nacher et al., 2003), suggestingthat the actual number of stem or progenitor cells declines with ageing.Second, the rate of stem or progenitor cell proliferation in the SGZdiminishes with ageing suggesting a lengthening of the cell cycleduration with ageing (Cameron &McKay, 1999). Third, the rate of celldeath in dentate granule cell layer decreases over the course of ageing(Heine et al., 2004) implying reduced cell death-derived stimulatoryfactors for stem cell proliferation in the ageing SGZ. Fourth, the levelsof circulating corticosteroids are elevated during ageing (Sapolsky,1992) and higher levels of corticosteroids are associated with decreasedneurogenesis (Gould et al., 1998). Fifth, the neurotransmitter serotonindecreases with ageing (Stemmelin et al., 2000) and greater levels ofserotonin are associated with enhanced neurogenesis (Gould, 1999).Finally, the multiple stem cell proliferation factors such as FGF-2,IGF-1, VEGF, and BDNF decrease considerably by middle age in thehippocampus (Shetty et al., 2004a,b). Additionally, other secretoryfactors such as neurogenesin-1 from granule cells and astrocytes (Uekiet al., 2003) may also decline with ageing. Thus, a dramatic declineobserved in the production of new neurons in the middle-aged and agedDG is probably a consequence of multiple age-related alterations in themilieu of the SGZ.

Acknowledgements

This research was supported by grants from the National Institutes of Health(National Institute for Ageing grant RO1AG20924 to A.K.S.) and Departmentof Veterans Affairs (VA Merit Review Award to A.K.S.)

Abbreviations

BrdU, 5¢-bromodeoxyuridine; CRMP-4, collapsin response-mediated protein-4;DAB, diaminobenzidine; DCX, doublecortin; DG, dentate gyrus; F344, Fischer344 (rats); GCL, granule cell layer; NeuN, neuron-specific nuclear antigen;SGZ, subgranular zone.

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