Differential effects of learning on neurogenesis: learning increases or decreases the number of newly born cells depending on their birth date

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Abstract

The hippocampal formation, to which new neurons are added on a daily basis throughout life, is important in spatial learning. Surviving de novo produced cells integrate into the functional circuitry, where they can influence both normal and pathological behaviors. In this study, we examined the effect of the water-maze (a hippocampal-dependent spatial task) on neurogenesis. Learning in this task can be divided into two phases, an early phase during which performance improves rapidly, and a late phase during which asymptotic levels of performance are reached. Here we demonstrate that the late phase of learning has a multifaceted effect on neurogenesis depending on the birth date of new neurons. The number of newly born cells increased contingently with the late phase and a large proportion of these cells survived for at least 4 weeks and differentiated into neurons. In contrast, late-phase learning decreased the number of newly born cells produced during the early phase. This decline in neurogenesis was positively correlated with performance in the water-maze. Thus, rats with the highest de novo cell number were less able to acquire and use spatial information than those with low numbers of new cells. These results show that learning has a complex effect on hippocampal neurogenesis, and reveals a novel mechanism through which neurogenesis may influence normal and pathological behaviors.

Introduction

The hippocampal formation has long been associated with the execution of higher-order cognitive functions, and impairment of this structure following severe stress and aging has been linked to cognitive disturbances.1 More recently, the hippocampal formation has also been involved in the regulation of motivation of rewards, and considered a new target for the understanding of disorders such as depression2,3,4,5,6,7 and addiction.8,9,10,11 In order to understand the involvement of the hippocampal formation in the mediation of normal and pathological behaviors, much attention has recently been devoted to hippocampal neurogenesis.

Within the hippocampal formation, the dentate gyrus (DG) has the unique ability to generate new neurons throughout the entire life of an individual.12,13,14 The newly born cells develop into granule neurons and are capable of extending axonal projections to the CA3 area and integrating into functional circuits.15,16,17 Neurogenesis has been proposed to play a role in hippocampal-mediated learning and has been implicated in the appearance of behavioral pathologies associated with the hippocampal formation.18,19,20,21

In this report, we studied the influence of spatial learning on neurogenesis in order to develop our understanding of the relationship between the two. Specifically, we analyzed the effects of different phases of learning in the water-maze. In this task, two phases of learning can be distinguished: an early phase during which performance improves rapidly, and a late phase during which asymptotic levels of performance are reached. These two phases seem to involve different brain processes28,29,30,31 and, consequently, may differentially influence neurogenesis.

Neurogenesis was studied by injecting 5-bromo-2′-deoxyuridine (BrdU), a thymidine analogue that is incorporated by dividing cells. The injection protocols and dates of killing were adapted in order to distinguish the effects of the early and late phases of learning on neurogenesis. The neuron-specific marker NeuN was used to identify the phenotype of the newly born neurons.

Methods

Housing conditions

In all, 185 male Sprague–Dawley rats (Iffa Credo, France), weighing 200 g and aged 2 months at the start of the experiment, were used. The animals were housed individually in plastic cages, had ad libitum access to food and water, and were maintained on a regular 12 h light : dark cycle with constant temperature and humidity.

Water-maze training

Animals were tested 2 weeks following their arrival. The apparatus consisted of a circular swimming pool built of gray plastic (180 cm diameter × 60 cm height), filled with water (20°C) and made opaque by the addition of milk powder. Before the start of the training, the animals were habituated to the pool for 3 days for 1 min per day. During training, the animals were required to locate a submerged platform, hidden 1.5 cm under the water, in a fixed location using the spatial cues available within the testing room. Animals were divided into three experimental groups: Learning, Yoked and Unmanipulated. The Learning group was trained for four trials per day (90 s, with an intertrial interval of 20 s) for 4–8 days (see Table 1). The Yoked animals, a control for the stress and motor activity associated with the water-maze training, were placed into the pool without the platform and were paired for the duration of the trial with the Learning animals. The Unmanipulated group was left in the animal house and was not exposed to the water-maze. Results are expressed as mean±SEM.

Table 1 Summary of water-maze training and BrdU injection regime

BrdU injections

Animals of the Learning and Yoked groups received one daily injection of BrdU (50 mg/kg i.p. in phosphate buffer) 30 min before the first daily trial in the water-maze. Following the injection and prior to the testing, they were kept in their home cage. The Unmanipulated rats were injected within the same daytime period as the other two groups.

Immunohistochemistry

Rats were deeply anesthetized with chloral hydrate (400 mg/kg i.p.), and were perfused transcardially with 150 ml of phosphate-buffered saline (PBS; pH=7.3) containing heparin (5 × 104 IU/ml), followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH=7.3). Following a 48 h postfixation period in paraformaldehyde, 50 μm frontal sections were cut on a vibratome and collected in PBS (0.1 M, pH 7.4). For BrdU labeling, free-floating sections were treated with 2 N HCl (30 min at 37°C), and then rinsed in borate buffer for 5 min (0.1 M, pH 8.4). They were extensively washed with PBS and then preincubated for 45 min with PBS containing 0.3% Triton X-100 and 3% of horse normal serum (blocking solution). After blocking, the sections were incubated under agitation for 72 h at 4°C in mouse monoclonal anti-BrdU antibody (1/200; Dako, Trappes, France) diluted in PBS containing 0.3 Triton X-100 and 1% of horse normal serum. The sections were then incubated under agitation for 2 h with a biotin-labeled horse anti-mouse IgG antibody (1/200, Vector, Valbiotec, Paris, France). Sections from all animals were processed in parallel and immunoreactivities (IRs) were visualized by the biotin–streptavidin technique (ABC kit, Dako) using 3,3′-diaminobenzidine (DAB) as chromogen.

The phenotypes of the BrdU-labeled cells of the Learning, Yoked and Unmanipulated groups were examined 27 days (4 weeks) following the last BrdU injection (batch 3, n=8 per group) by immunofluorescent double labeling. Bound anti-BrdU molecules (1/1000; Acurate) were revealed using a Cy3-labeled anti-rat IgG antibody and bound anti-NeuN antibodies (1/1000, Chemicon) were visualized with an Alexa 488 goat anti-rabbit IgG antibody.22 The percentage of BrdU-labeled cells that expressed NeuN was determined using a Molecular Dynamics Sarastro 2000™ CLSM confocal microscope. Five animals per group, on average 50 cells per animal, were examined for colabeling by BrdU and NeuN. To verify that the cells were double labeled, approximately 25 sequential layers of 1 μm each were obtained from the selected area allowing for the in-depth exploration of the section in the z-axis.

Quantitative evaluation of peroxidase staining

The number of BrdU-IR cells in the left and right dentate gyrus was estimated by counts made systemetically on every 10 consecutive 50 μm thick sections along the rostrocaudal axis of the hippocampal formation. On each section, BrdU-IR cells were counted in the granule and subgranule layers, excluding those in the outermost focal plane. The resulting numbers were tallied and multiplied by the inverse of the section sampling fraction (1/ssf=10). The volume of reference was estimated according to the Cavalieri method: Vref=T × ∑A × 1/ssf, where ‘T’ is the mean thickness of the vibratome section (50 μm) and ‘A’ is the area of the granular and subgranular cell layers. The results are expressed as mean±SEM. In order to control for a nonspecific effect of learning, BrdU-IR cells were also counted (batch 2) within the dorsolateral corner of the subventricular zone22,23,24,25,26,27,28,29,30,31,32 (0.7 anterior to the bregma according to the atlas of Paxinos and Watson;33 its surface was measured and results were expressed as the mean number of BrdU-IR cells per mm2. Results are expressed as mean±SEM.

Procedures

Six batches of animals were used and each batch incorporated the three experimental groups. The BrdU injection schedule, the days of training in the water-maze and the days of killing for each batch of animals are outlined in Table 1.

In all the experiments, the Yoked and Unmanipulated animals did not differ (Table 2, Group effect: F1,90=2.27, P=0.14; Group × Experiment interaction: F5,90=0.57, P=0.72) and, thus, the combined data are shown in all figures as Controls.

Table 2 Influence of training in the water-maze on neurogenesis

Statistical analysis

Data were analyzed by a Student's t-test or by an ANOVA followed by post hoc comparisons when appropriate, using the Newman–Keuls test. Relationships between learning performances and the number of BrdU-IR cells were evaluated using the Pearson' correlation test.

Results

In the water-maze task, animals are required to locate a hidden platform, submerged in a circular swimming pool, using the spatial cues available in the testing room. Learning of this task is reflected by a progressive decrease in the latency to find the escape platform (Figure 1a,c,e,g). The evolution of performance is characterized by two consecutive phases. During the first one, which lasted for 4 days in our paradigm, there is a fast and large (more than 80%) improvement in performance. During the second phase, the following 4 days in this study, there is a smaller and slower increase in performance (less than 20%) and a stable baseline is reached. These two phases have been defined here as ‘early’ and ‘late’.

Figure 1
figure1

Influence of the early (first 4 days) and late (last 4 days) phases of learning on neurogenesis. Latency to find the escape platform in a water-maze task (a,c,e,g). Number of BrdU-IR cells in rats injected with BrdU during the early (b), late (d,f) and early+late phase of learning (h). The late phase of learning induced an increase in the number of BrdU-IR cells (d) that survived for 4 weeks (f). The black bar represents the period of BrdU injections. Arrows indicate the day of killing.

Learning increases the number of cells produced during the late phase of learning

In order to distinguish the effect of different phases of learning on neurogenesis (Figure 2), animals were injected with BrdU either during the first 4 days of training, corresponding to the early phase (batch 1), or during the last 4 days of training, corresponding to the late phase (batch 2). The results of these experiments indicated that the early phase of learning does not modify cell proliferation, while the late phase of learning increases it.

Figure 2
figure2

BrdU-labeled cells in the dentate gyrus. (a) Microphotography of BrdU staining (in red) in a Fluoro Nissl Green counterstained section of a control rat. (b) Optical section (of 0.7 μm) obtained by confocal microscopy, showing that BrdU-labeled cells (red nuclear stain, Cy3) are double stained with the neuronal marker NeuN (green stain, Alexa 488). (a) 50 μm, (b) 10 μm.

When animals were injected with BrdU during the early phase (Figure 1a), the Control and Learning groups did not differ in the number of BrdU-IR cells (Figure 1b, t28=0.51, P=0.61). In contrast, when animals were injected with BrdU during the late phase of learning (Figure 1c), the Learning group showed a higher number of BrdU-stained cells than Controls (Figure 1d, t40=3.04, P=0.004). These differences were not due to differences in the reference volume (Control group: 1.157±0.032 mm3; Learning group: 1.182±0.038 mm3; t40=0.49, P=0.63). To control for a nonspecific effect of learning on BrdU staining, another neurogenic brain area, the subventricular zone, was examined.22,32 Learning did not modify cell proliferation in this region (Unmanipulated group: 1505.3±194.4 BrdU-IR cells; Yoked group: 1230.0±120.4 BrdU-IR cells; Learning group: 1220.0±102.4 BrdU-IR cells; Group effect: F2,13=1.25, P=0.32). These results indicate that learning-induced upregulation of cell proliferation occurs specifically in the DG and is not mediated by nonspecific changes in BrdU bioavailability.

In an additional experiment (batch 3), we analyzed the survival of cells produced during the late phase of training. For this experiment, animals were injected with BrdU during the last 4 days of training (Figure 1e), but were killed 27 days after the last BrdU injection. It was also found in this case that Learning animals had a higher number of BrdU-IR cells than Controls (Figure 1f, t28=2.91, P=0.007). These differences were not due to differences in the reference volume (Control group: 1.037±0.028 mm3, Learning group: 1.119±0.046 mm3; t28=1.61, P=0.12). The number of BrdU-IR cells found in this experiment was approximately 20% lower than that measured in animals killed 24 h after the end of training (delay effect: F1,68=19.21, P<0.0001). Furthermore, this decrease was proportionally similar in all the experimental groups (delay × group interaction: F1,68=0.003, P=0.99). These results probably reflect the fact that not all newly produced cells survive after birth.15,34 We also examined whether learning influenced the phenotype of the newly born cells (Figure 2a,b). At 27 days after the last BrdU injection, confocal analysis revealed that in the Learning, Yoked and Unmanipulated groups, 74, 75 and 69%, respectively, of the cells marked for BrdU were double labeled for the neuronal marker NeuN (Group effect: F2,12=1.95, P=0.18).

Previously, it has been shown that when animals are injected with BrdU during the entire period of training, learning does not modify neurogenesis.21 Consequently, we wanted to replicate the effects of this protocol in our conditions (batch 4; Figure 1g). We found that when the animals are injected with BrdU for the entire duration of training (here 8 days), the Control and the Learning groups did not differ for the number of BrdU-IR cells (Figure 1h, t22=0.99, P=0.33).

Late phase of learning decreases the number of cells born during the first phase of learning

The results of the previous experiments indicate that the late phase of learning increases neurogenesis, while the early phase has no effect. However, it is difficult to understand why there is no visible effect on neurogenesis when animals are injected during the entire period of learning. This could be the outcome if the late phase of learning decreases the survival of the cells that have been produced during the early phase.

In order to address this question, animals were injected with BrdU during the first 4 days of training only (batch 5). However, this time, the Learning group was allowed to complete the 8 days of training. Strikingly, in this case, a significant decrease in the number of BrdU-IR cells was observed (Figure 3b; t39=3.18, P=0.002). These differences were not due to differences in the reference volume (Control group: 1.210±0.030 mm3; Learning group: 1.136±0.032 mm3; t39=−1.65, P=0.11). In the last experiment (batch 5), animals were killed 5 days after the last BrdU injection, while in the first experiment (batch 1, Figure 1a), they were killed 24 h later. Consequently, it could be hypothesized that differences between the two experiments in the number of BrdU-IR cells could be due to a decrease in cell survival over time. In order to address this question, the learning group was injected with BrdU during the first 4 days of training and left undisturbed in the animal house for 5 days, before being killed (batch 6). In this case, the Learning and Control groups did not differ in the number of BrdU-IR cells (Figure, t24=0.67 3c, t24=0.67, P=0.51). These results indicate that the decrease in BrdU-IR cells observed when animals are injected during the early phase of learning is specifically induced by the late phase of learning.

Figure 3
figure3

Influence of the late phase of learning on the survival of cells produced during the early phase. (a) Latency to find the hidden platform in a water-maze task in rats submitted to a complete (black symbols) or a partial training (gray symbols). The number of BrdU-IR cells in rats injected with BrdU during the first 4 days of training and killed when the training is complete (b), or is partial (c). When animals experienced a complete training (8 days (b)), a significant decrease in the number of BrdU-IR cells was found in the learning group (L) compared to controls (C). In contrast, when training was restrained to the first 4 days, no significant difference was found between the two groups (c). The black bar represents the period of BrdU injections. The arrow indicates the day of killing.

In conclusion, taken together, these results indicate that the late phase of learning has contradictory effects. It apparently decreases the survival of newborn cells produced during the early phase of the training, but also increases the number of cells produced during the late phase. These opposing effects could explain why no effects were found when animals were injected with BrdU during both phases.

Learning-induced decrease in cell proliferation is related to performance in the water-maze

In order to further characterize the relationship between changes in neurogenesis and learning, we correlated the performance in the water-maze with the number of BrdU-IR cells. First, we analyzed the relationship between performance and the increase in cell proliferation induced by the late phase of learning (batch 2). In this case, no significant correlation was found between performance in the water-maze and the number of BrdU-IR cells (n=16, r=−0.12, P=0.66). We then studied the relationship between learning and the decrease in the number of BrdU-IR cells induced by the late phase (batch 5). In this case, a positive correlation was found between the mean latency to reach the platform during the last 4 days of training (from day 5 to day 8) and the number of BrdU-IR cells (Figure 4, n=21, r=0.55, P=0.01). Thus, the best performance (lower latency to reach the platform) was found in animals with the lowest number of BrdU-IR cells, that is, in animals in which learning induced the highest decrease in neurogenesis. This was due to a deficit to retain and use what was learned on the precedent day, as there was a positive correlation between the mean of the latency to reach the platform on the first trials of each day of training and the number of BrdU-IR cells (n=21, r=0.60, P=0.004). No correlation was found with the subsequent trials (second trial: r=0.39, P=0.074, third trial: r=0.15, P=0.521, fourth trial: r=0.34, P=0.124).

Figure 4
figure4

Correlation between behavioral performances in the water-maze and the number of BrdU-IR cells generated during the early phase of learning. The mean of the latencies to find the platform during the last 4 days of training (from day 5 to 8) was positively correlated with the number of BrdU-IR cells. Consequently, rats with the lowest number of BrdU-IR cells were the ones with the best performances in the spatial memory task.

To better illustrate the relationship between behavioral performance and learning-induced decrease in BrdU-IR cell number, animals of the learning group (batch 5) were ranked on the basis of their number of BrdU-IR cells. The subjects with the five highest and lowest BrdU-IR cell counts35,36 were compared for their performance in the water-maze. As shown in Figure 5, animals with the lowest number of BrdU-IR cells had a better performance in the water-maze than animals with the highest number of BrdU-IR cells (Group effect, F1,8=13.07, P=0.007). Rats with the lowest number of BrdU-IR cells rapidly learnt the platform location and performance quickly reached a stable plateau (from day 5 to day 8: Day effect, F3,12=0.69, P=0.57, Trial effect, F3,12=0.36, P=0.78). This was not the case for the group with a high number of BrdU-IR cells (from day 5 to day 8: Day effect, F3,12=3.52, P=0.049; Trial effect, F3,12=6.81, P=0.006). Animals with the poor learning-induced decrease in the number of BrdU-IR cells showed a specific deficit in the between day performance. In other words, they had a deficit in reference memory.

Figure 5
figure5

Latency to find the escape platform during the four trials of each training day in the water-maze in animals injected with BrdU during the first 4 days of training, which showed the highest and the lowest number of BrdU-IR cells. The low BrdU cell group contained the five animals with the lowest number of BrdU-IR cells. The high BrdU cell group contained the five animals with the highest number of BrdU-IR cells.

Discussion

This report shows that the different phases of learning in the water-maze have distinct effects on neurogenesis in the dentate gyrus of the hippocampal formation.

The early phase of learning, corresponding to acquisition of the task, did not modify cell proliferation in line with previous observations that this phase does not effect neurogenesis in the dentate gyrus.34 This absence of change in cell proliferation could be due to the stress generated by the early trials in the water-maze. Stressful events and the concomitant release of corticosterone are known to inhibit cell proliferation.35,36,37,38 However, our experiments were specifically designed to control for the potential effects of stress. Animals were habituated to the pool before training and two control groups were used: Unmanipulated animals that were not exposed to the pool and Yoked animals that were exposed to the pool without the platform. Although in batch 1 the number of BrdU-IR cell in the Yoked rats tended to be lower than that measured in Unmanipulated rats, this difference was not statistically significant and no difference was found for the other batches of animals. Thus, in the present experiment, stress cannot explain the absence of modification of neurogenesis observed in the early phase of training.

In contrast, the late phase of learning increased the number of newborn cells. This increase in cell proliferation probably results in the addition of new neurons to the dentate gyrus network. Indeed, the newly generated cells survived for at least 4 weeks and differentiated into neurons. However, this increase in cell proliferation does not seem to directly sustain learning, since no correlation was found with performance in the water-maze. Furthermore, since physical activity increases the production of trophic factors within the hippocampus and could also change BrdU availability, it could be argued that the increase in cell proliferation observed here simply results from swimming in the water-maze. This hypothesis seems unlikely on the basis of three observations. First, the effect of swimming on growth factors is most prominent for massed training that involves higher levels of physical activity than the spaced training used in our experiments.29,30,31 Second, the number of BrdU-labeled cells in Yoked animals, which were submitted to exactly the same level of physical activity as that of the Learning group, did not statistically differ from that of Unmanipulated controls. Higher levels of activity such as the ones observed in a running wheel are necessary for modifying hippocampal neurogenesis.21 Finally, learning had no significant effects on cell proliferation within the subventricular zone, which indicates that the increase in cell proliferation is specific to a learning-related structure such as the dentate gyrus and does not result from nonspecific changes in BrdU bioavailability.

The late phase of learning also decreased the number of cells produced during the first phase of training. Interestingly, the decrease in the number of BrdU-IR cells was associated with learning performance. Thus, rats with the highest levels of learning-induced decrease in BrdU-labelled cells learned the task the most rapidly. In contrast, the absence of such a decrease was associated with a delayed acquisition of new spatial memories. In particular, a deficit in using the information acquired the previous day was observed. Such a deficit suggests a specific impairment of reference memory that is considered one of the major functions of the hippocampal formation.39 A possible explanation for the decrease in the number of BrdU-IR cells is a dilution of the BrdU-labeling, resulting from the further divisions of the labeled cells. Such a dilution, which would be higher in the Learning group because of the increase in cell proliferation, may render the newborn neurons undetectable. An alternative hypothesis is that the learning-induced decrease in BrdU-IR cells results from an active phenomenon, namely the death of newborn cells. This hypothesis is supported by the observation that learning in the water-maze increases the expression of proteins involved in apoptosis40 and that intrahippocampal administration of anticaspases, which blocks cell death, impaired long-term spatial memory.41

The mechanisms of the learning-dependent changes in hippocampal neurogenesis that we have observed here are so far unknown. However, it is interesting to note that the different phases of learning in the water-maze also have distinct effects on the expression of trophic factors in the hippocampal formation. These trophic factors, such as basic fibroblast growth factor (bFGF) and brain-derived neurotrophic factor (BDNF), are able to modify neurogenesis. In particular, the levels of hippocampal messenger RNA (mRNA) for bFGF increase during the early phase of water-maze learning, while they decrease below the baseline during the late phase of learning.29 In contrast, the levels of mRNA for BDNF, TrkB (a tyrosine kinase receptor mediating the effects of BDNF) and synapsin I (a downstream effector for the BDNF tyrosine kinase pathway) are increased in the hippocampal formation during the late phase of learning.30,31 Thus, variations in bFGF and BDNF could be involved in determining the modifications in cell survival and proliferation observed here during the learning of a hippocampal-dependant task.

Our findings, and those of previous studies, allow us to speculate that complex changes in hippocampal neurogenesis, including an increase in cell survival, proliferation and death, accompany hippocampal-dependant learning. An increase in cell survival occurs during the early phase of learning and involves cells that have been produced in a previous stage. Indeed, it has been shown that 4 days of training in the water-maze, corresponding to the early phase of our experiment, increases the survival of cells whose birth predate the beginning of learning.34 This increase in cell survival could facilitate the establishment of synaptic connections from the dentate gyrus to the CA3 region, the major projection area of the newborn cells, promoting in this way the establishment of the memory trace. This idea is consistent with findings showing that conditions that increase the basal rate of neurogenesis also increase learning,18,19,20,21 while conditions that decrease neurogenesis impair learning.19,20 An increase in cell proliferation also occurs during the late phase of learning and seems to involve cells generated during this phase. This increase in cell proliferation, occurring once asymptotic level of performances are reached, could constitute a resetting process that renders the dentate gyrus available to integrate new information and store new memories. This hypothesis is consistent with observations made in presenilin-1 knockout mice, in which environment-induced increase in neurogenesis is deficient. Thus, in these mutants, a longer storage of contextual fear memories, interpreted as a reduced clearance of hippocampal memory traces,42 has been observed. Although further studies are needed to confirm this phenomenon, it can be hypothesized that cell death, which seems to occur during the consolidation of the memory trace, could constitute a trimming mechanism that suppresses neurons that have not established learning-related synaptic connections.

In conclusion, our results show that learning has complex effects on neurogenesis in the adult hippocampus and that an alteration of the chain of events leading to neurogenesis could determine the behavioral performances of an individual.

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Acknowledgements

We are grateful to Dr P Ciofi (INSERM U378, Bordeaux, France), Mr Dakhli (Institut François Magendie, Bordeaux, France), Dr J Ralphs (University of Cardiff, UK) and Mr Hommolle V (Perkin-Elmer, France) for their help and suggestions. This work is supported by the INSERM, University of Bordeaux II, ‘Région Aquitaine’ and ‘Fondation pour la Recherche Médicale’. MD was supported by the INSERM ‘Poste Vert’ and ‘Fondation pour la Recherche Médicale’ fellowships.

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Correspondence to D N Abrous.

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Döbrössy, M., Drapeau, E., Aurousseau, C. et al. Differential effects of learning on neurogenesis: learning increases or decreases the number of newly born cells depending on their birth date. Mol Psychiatry 8, 974–982 (2003) doi:10.1038/sj.mp.4001419

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Keywords

  • neurogenesis
  • hippocampus
  • learning
  • memory
  • water-maze
  • BrdU

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