ARTICLES© 2007 Nature Publishing Group http://www.nature.com/natureneuroscience

Preferential incorporation of adult-generated granulecells into spatial memory networks in the dentate gyrus
Nohjin Kee1–3,5, Cátia M Teixeira1–5, Afra H Wang1,3 & Paul W Frankland1–3
Throughout adulthood, new neurons are continuously added to the dentate gyrus, a hippocampal subregion that is important inspatial learning. Whether these adult-generated granule cells become functionally integrated into memory networks is not known.
We used immunohistochemical approaches to visualize the recruitment of new neurons into circuits supporting water mazememory in intact mice. We show that as new granule cells mature, they are increasingly likely to be incorporated into circuits
supporting spatial memory. By the time the cells are 4 or more weeks of age, they are more likely than existing granule cells to be
recruited into circuits supporting spatial memory. This preferential recruitment supports the idea that new neurons make a uniquecontribution to memory processing in the dentate gyrus.

New neurons are continuously generated in the subgranular zone of the
hippocampus throughout adulthood1–7. The majority of these newborn neurons become integrated into existing dentate gyrus circuitry
within 3 weeks, extending axons along the mossy fiber tract to CA3 andreceiving excitatory synaptic input from perforant path afferents8–13.
Whether these new neurons are subsequently integrated into memory
networks is unknown. Previous studies have used irradiation or
antimitotic agents such as methylazoxymethanol acetate (MAM) to
suppress adult neurogenesis in rodents. Such manipulations impair
learning in trace eye-blink14 and fear15 protocols, but not in several
other hippocampus-dependent tasks15–17. Because suppression of adultneurogenesis after these manipulations is typically incomplete18,19,
learning may be supported by either residual adult-generated neurons
or even existing granule cells1.
To circumvent this issue, we used immunohistochemical approaches
to visualize the recruitment of new neurons into circuits supportingwater maze memory in intact mice. We labeled adult-generated granulecells with the proliferation marker 5-bromo-2¢-deoxyuridine(BrdU)20,21. To identify dentate gyrus neurons processing spatial
information, we quantified expression of the immediate-early genes
c-fos (also known as Fos) and Arc (encoding the activity-regulated
cytoskeletal-associated protein), after behavioral testing22. A majoradvantage of the water maze is that training in the hidden version ofthis task produces stable, long-lasting spatial memories in mice23. In
addition, unlike with some forms of hippocampal learning such as
contextual fear24 or trace eye-blink25 conditioning, the expression ofthis memory is continuously dependent on the hippocampus, including the dentate gyrus23,26–28. Our experiments reveal that adultgenerated neurons are recruited into dentate gyrus circuits supporting
spatial memory in an age-dependent manner. Furthermore, we showthat by the time new neurons are 4–8 weeks of age, they are

preferentially recruited into circuits supporting spatial memory compared with existing granule cells.
RESULTS
Recruitment of new neurons into spatial memory networks
Changes in the expression of immediate-early genes such as c-fos arecorrelated with neuronal firing, and therefore they can be used asneuronal activity markers in the brain22. In preliminary studies, we firstestablished that Fos expression is limited to neurons (Supplementary
Results and Supplementary Fig. 1 online) and is selectively upregulated in the dentate gyrus after both water maze training and testing(Supplementary Results and Supplementary Fig. 2 online).
We next examined whether adult-generated granule cells are incorporated into spatial memory circuits in the dentate gyrus. Mice were
initially treated with BrdU and different groups of mice were thentrained in the water maze either 1, 2, 4, 6 or 8 weeks later. Spatial
memory was tested in all groups of mice 10 weeks after BrdU treatment(Fig. 1a). Mice were killed 90 min after this test, and Fos and BrdU
expression were quantified using immunohistochemical approaches.
We predicted that if BrdU-labeled neurons are incorporated into
dentate gyrus circuits supporting spatial memory at the time oftraining, then there should be Fos expression in BrdU+ cells following
spatial memory testing.
Over the course of water maze training, mice required progressively
less time to escape to the platform, and escape latencies did not differ
between groups (data not shown). This training produced stable andlong-lasting spatial memory in all groups: mice showed a strong spatial
bias for the area of the pool where the platform was located during
training in each of the three probe tests (Fig. 1b; Supplementary
Results and Supplementary Fig. 3 online). After the probe test, we
identified many Fos+ and BrdU+ cells in the dentate gyrus (Fig. 1c,d).

1Program in Neurosciences and Mental Health, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. 2Department of Physiology and
3Institute of Medical Science, University of Toronto, Toronto, Ontario M5S 1A8, Canada. 4Graduate Program in Areas of Basic and Applied Biology, Universidade do Porto,

4050-465 Porto, Portugal. 5These authors contributed equally to this work. Correspondence should be addressed to P.W.F. (paul.frankland@sickkids.ca).
Received 20 December 2006; accepted 12 January 2007; published online 4 February 2007; doi:10.1038/nn1847

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delay (weeks)(c,d) Representative confocal images of Fos+ (red), BrdU+ (green) and Fos+BrdU+(arrow) cells in the dentate gyrus following probe testing in the 1-week (c) and 6-week (d)groups (scale bar, 20 mm). (e) After probe testing, similar numbers of Fos+ cells were identified in the dentate gyrus in all groups (P 4 0.05; ANOVA).
(f) Numbers of BrdU+ cells in the dentate gyrus were higher in the 1-week and 2-week groups (P o 0.05; ANOVA). (g) The number of Fos+BrdU+ cells
increased as the treatment-training interval lengthened (P o 0.01; ANOVA). (h) Fos expression was elevated in BrdU+ (orange) cells relative to NeuN+ (blue)
cells in groups of mice with longer delays between BrdU treatment and training (P o 0.01; ANOVA, significant delay  cell type interaction). The probabilityof Fos expression was higher in BrdU+ cells than in NeuN+ cells only in the 6- and 8-week groups (all P o 0.05; Fishers LSD test). (i) Ten weeks after BrdU
treatment, Fos expression in BrdU+ cells was much greater in PTZ-treated mice compared with home cage (HC) control mice (P o 0.001; ANOVA). Error bars
represent ± s.e.m.

Fos expression was similar in all groups (Fig. 1e), consistent with theidea that activity in these neurons is always required for the expressionof water maze memory23,26,27. We also identified BrdU+ cells in thedentate gyrus in all groups of mice. These numbers of BrdU+ cells werehigher in the mice trained 1 or 2 weeks after BrdU treatment (Fig. 1f),
indicating that exercise, learning or enrichment associated with water
maze training promotes survival of 1–2-week-old neurons, as previously reported29–31. Most notably, the overlap between the Fos+ and
BrdU+ populations depended on the time of training. As the delay
between BrdU treatment and water maze training lengthened, thenumber of Fos+BrdU+ cells increased, suggesting that as adult-generated granule cells mature, they are increasingly likely to be incorporated
into dentate gyrus circuits supporting spatial memory (Fig. 1g).
Because Fos is only expressed in neurons after behavioral testing(Supplementary Results and Supplementary Fig. 1), all doublelabeled cells identified in the dentate gyrus are neuronal.
The design used here rules out several alternative interpretations ofthese data. First, mice were the same age at the time of BrdU treatment,
and so age-dependent differences in the proliferation and differentiation of adult-generated granule cells cannot account for differences in
Fos expression in these new neurons32. Second, the fixed time between
BrdU treatment and mouse death ensured that all BrdU+ cells were thesame age at the time of death, and so developmental stage cannot
account for differences in Fos expression in these new neurons. Third,
mice in each group underwent exactly the same experience before
death, and therefore group differences in numbers of Fos+BrdU+ cells
are specifically related to spatial memory processing and not related tononspecific aspects of the probe tests (for example, swimming or
physical exercise, stress or arousal).

2

New neurons are preferentially recruited
To determine the age at which these adult-generated granule cells areincorporated into spatial memory networks, we next estimated thetotal number of neurons in the granule cell layer by counting thenumber of cells expressing the mature neuronal marker neuronalspecific nuclear protein, NeuN. This analysis indicated that the probability of existing granule cells expressing Fos after a probe test ranged
between 2.1% and 2.4% across groups (Fig. 1h). In mice in the 1- and2-week groups, the probability of Fos expression in BrdU+ cells was
lower than in the mature neuron population, suggesting suboptimal
recruitment of these new neurons at this age. In the 4-week group, theamount of Fos expression in the BrdU+ population was similar to thatof mature neurons, indicating that by 4 weeks of age, new neurons arerecruited into spatial memory networks in the dentate gyrus. Perhaps
most notably, by the time new neurons are 6–8 weeks of age, they are as
much as twice as likely as mature neurons to be recruited into spatial
memory networks. This preferential recruitment was most pronounced
in the innermost portion of the dentate gyrus (Supplementary Results
and Supplementary Fig. 4 online), where the vast majority of BrdU+
cells were located33. This raises the possibility that there are regional
differences in the propensity of new granule cells to be recruited into
spatial memory circuits. Throughout the granule cell layer, Fos expression in BrdU+ cells after probe tests was higher than in home-cage
control mice and much lower than in mice treated with the chemical
convulsant pentylenetetrazol (PTZ) (Fig. 1i).
These data show that as new neurons mature, they become preferentially recruited into spatial memory networks. In all groups, Fos
expression was quantified 10 weeks after BrdU treatment. At this time,
approximately 90% of BrdU+ cells in the granule cell layer are neuronal

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between Arc+ and BrdU+ populations depended on the time of training.
Representative confocal images of Arc+ (red), BrdU+ (green) and Arc+BrdU+(arrow) cells in the dentate gyrus following probe testing in either the 1-weekor 6-week groups are shown (scale bar, 20 mm). (b) Comparison of Arc
expression in mature (NeuN+, blue) versus adult-generated (BrdU+, orange)
granule cells. Arc expression was elevated in BrdU+ cells relative to NeuN+
cells in groups of mice with longer delays between BrdU treatment andtraining (P o 0.05; ANOVA, significant delay  cell type interaction). The
probability of Arc expression was higher in adult-generated (BrdU+) cells
compared to mature (NeuN+) cells only in the 4-week, 6-week and 8-weekgroups (all P o 0.05; Fishers LSD test). Error bars represent ± s.e.m.

(Supplementary Results and Supplementary Fig. 5 online). As Fos
expression is limited to neurons, our analyses likely underestimate therecruitment of new neurons into spatial memory networks in thedentate gyrus. Nevertheless, this preferential recruitment suggests thatthese neurons have a competitive advantage compared with existing
granule cells, and, consistent with this, immature neurons have a lower
threshold for long-term potentiation and are more excitable thanmature granule cells5,34,35.
Arc induction in new neurons
To evaluate whether this temporally specific pattern would apply toanother marker of neuronal activation, we examined Arc expression in
the same mice. Activity that is associated with both memory formationand memory retrieval induces robust Arc expression in neurons36–38(Supplementary Results and Supplementary Fig. 1). Like Fos
expression, Arc expression likely mediates the stabilization of
new and reactivated memory circuits22,36–38. Similar to our resultswith Fos, we found that the overlap between the Arc+ and BrdU+
populations depended on the time of training (Fig. 2a). The percentage
of BrdU+ cells expressing Arc increased as the delay between
BrdU treatment and training increased (Fig. 2b). In the 2-weekgroup, Arc expression was similar in BrdU+ and existing cells,suggesting that recruitment of new neurons into spatial memory
networks in the dentate gyrus may occur as early as 2 weeks. However,in mice in the 4–8-week groups, there was a threefold increase in
Arc expression in BrdU+ cells relative to the existing granule cell

population, indicating that maximal recruitment does not occur
until later on.
We confirmed this preferential recruitment in an additional experiment where the delay between BrdU treatment and training was varied,
but the delay between training and testing was kept constant (Supplementary Results and Supplementary Fig. 6 online). These data
support the conclusion that as adult-generated granule cells mature,they are increasingly likely to be incorporated into dentate gyrus
circuits supporting spatial memory. The data confirm that as newneurons mature, they are preferentially recruited. Finally, these analyses
indicate that our effects apply to another immediate-early gene and arethus not specific to c-fos.
Recruitment is specific to the dentate gyrus
The subgranular zone is one of two major neurogenic regions in thebrain. Olfactory precursor cells divide in the subventricular zone andnew neurons migrate via the rostral migratory stream to the granuleolfactory bulb4,39. Because neurons in the olfactory bulb would not be
expected to participate in spatial memory, we next examined the patternof Fos and BrdU expression in the olfactory bulb in the same mice.
After the probe test, we found many Fos+, BrdU+ and Fos+BrdU+
cells in the granule cell layer of olfactory bulb (Fig. 3a–c). However, theprobability of Fos expression in BrdU+ cells did not differ between
groups (Fig. 3d), indicating that the age-dependent recruitment of
new neurons into spatial memory networks was specific to thedentate gyrus.
One-week-old neurons are not transiently recruited
The low number of double-labeled neurons in the 1-week groupsuggests that new neurons are not recruited into spatial memory
networks at this young age. An alternative possibility, however, is

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Figure 3 Age-dependent integration of new neurons into circuits supporting
spatial memory does not occur in the olfactory bulb. (a) Low-magnification
examples of Fos immunofluorescence, BrdU immunofluorescence andmerged images of the olfactory bulb (scale bar, 1 mm). (b,c) Highmagnification confocal images of the olfactory bulb showing Fos+, BrdU+
and Fos+BrdU+ cells following probe tests in either the 1-week (b) or
6-week (c) groups (scale bar, 20 mm). (d) Quantification revealed similar
numbers of Fos+ and BrdU+ cells in both groups (all P 4 0.05, ANOVA).
Notably, the probability of BrdU+ cells expressing Fos did not vary as afunction of treatment-training interval (P 4 0.05, ANOVA), indicating thatage-dependent recruitment of new neurons into spatial memory networks is
specific to the dentate gyrus. Cells counts are per 100-mm  100-mm area.
Error bars represent ± s.e.m.

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Figure 4 Are 1-week-old neurons transiently incorporated into dentate gyrus
circuits supporting spatial memory? (a) Mice were trained either 1 week or
6 weeks following BrdU treatment, and Fos and BrdU expression wasquantified after the third day of training. (b) The probability of Fos expression in the mature neuronal population (NeuN+) was similar in bothgroups. However, Fos expression in BrdU+ cells depended on the delay
between BrdU treatment and training (P o 0.01; ANOVA, significant delay cell type interaction). In the 6-week group, Fos expression in the BrdU+ cells
was significantly greater than in the NeuN+ cells (P o 0.05, Fishers LSDtest), indicating that these cells are preferentially activated following water
maze training. Error bars represent ± s.e.m.

that these new neurons are transiently incorporated into spatial
networks, but do not subsequently survive and therefore cannot be
detected at later time points2,29. To evaluate this possibility, we trained
mice either 1 week or 6 weeks after BrdU treatment and examined Fos
expression induced by training alone (Fig. 4a). Following training,many Fos+BrdU+ cells were identified in the 6-week group. In contrast,
no Fos+BrdU+ cells were identified in the 1-week group (Fig. 4b),
suggesting that 1-week-old neurons are not transiently recruited into
spatial memory networks in the dentate gyrus. Notably, Fos expression
in the mature neuron population in the dentate gyrus was similar inboth groups of mice. Furthermore, in the 6-week group, Fos expression
in the BrdU+ cell population exceeded that in the matureneuron population, indicating that 6-week-old neurons are preferentially activated during water maze training, consistent with our
first experiment.
The lack of recruitment of 1-week-old neurons was confirmed in an
additional experiment where spatial memory was tested 1 d after thecompletion of training. Under these conditions, we found a complete
absence of Fos+BrdU+ cells following the probe test (Supplementary
Results and Supplementary Fig. 7 online). Together, these results
indicate that 1-week-old neurons do not become functionallyintegrated into spatial memory circuits in the dentate gyrus, noteven transiently.
Recruitment depends on the BrdU treatment-training delay
Our results suggest that as adult-generated neurons mature, they
become increasingly likely to be incorporated into dentate gyrus
networks supporting spatial memory. In the first series of experiments,

however, a fixed 10-week survival period was used, and therefore
retention delays varied across groups. To control for this potential
confound, we trained three additional groups of mice and then tested
retention 4 weeks later. These mice were treated with BrdU either
6 weeks before, 1 week before or 1 week after the start of water mazetraining (Fig. 5a). During the retention test, all mice searched selectively in the region of the pool that formerly contained the escapeplatform (Fig. 5b). Although Fos+ and BrdU+ cells were identified in
the dentate gyrus in all groups, significant overlap between these two
populations was only observed in the mice trained 6 weeks after BrdU
treatment. In contrast, there were significantly fewer Fos+BrdU+ cells ingroups of mice that were treated with BrdU either 1 week before or
1 week after training (Fig. 5c). These results confirm that the degree ofoverlap between the Fos+ and BrdU+ populations is dependent on the
delay between BrdU treatment and training, and not on the delay
between training and testing. They further indicate that once recruited,
adult-generated neurons are stably maintained in spatial memory
networks for at least 4 weeks.
In addition, the mice which were treated with BrdU post-training
provide an ideal control for Fos expression in BrdU+ cells associatedwith non-mnemonic aspects of the probe test (such as swimming or
physical exercise, stress or arousal). In these mice there were very few
Fos+BrdU+ cells, indicating that these nonspecific aspects of the testingprocedure do not significantly contribute to gene expression in these
new neurons in the dentate gyrus, and furthermore, that these newneurons are not simply more likely to be activated during memory
retrieval. There were similarly few Fos+BrdU+ cells in mice trained
1 week after BrdU treatment. The fact that the numbers of Fos+BrdU+
cells in groups of mice treated with BrdU either 1 week before or 1 week
after training were similar suggests that there must be very little
recruitment of 1-week-old neurons into spatial memory networks inthe dentate gyrus.
Fos expression correlates with memory strength
To more closely examine the relationship between memory strength andFos expression, we next trained mice that have deficits in spatial
learning. To do this, we used mice carrying a point mutation of theaCaMKII gene (T286A), which abolishes this enzyme’s ability toautophosphorylate40. In this experiment, CaMKIIT286A homozygous
mice and their wild-type littermate controls were treated with BrdU andtrained 6 weeks later (Fig. 6a). CaMKIIT286A homozygous mice wereimpaired in a probe test 1 d after training (Fig. 6b), which is consistent
with the idea that autophosphorylation of CaMKII at the Thr286
residue is important for spatial learning40. The learning impairments
in CaMKIIT286A homozygous mice are likely to be due to deficits insynaptic plasticity throughout the hippocampus (and possibly otherforebrain regions). In contrast, similar numbers of BrdU+ cells wereidentified in wild-type and CaMKIIT286A homozygous mice (Fig. 6c),Figure 5 Overlap between Fos+ and BrdU+
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Figure 6 Fos expression in the dentate gyrus is correlated with memory strength. (a) To explore the relationship between Fos expression and memory strength,a-CaMKIIT286A mice and their wild-type (wt) littermate controls were trained in the water maze 6 weeks after BrdU treatment. Spatial memory was tested
1 d later. (b) In the probe test, wild-type mice searched more selectively than a-CaMKIIT286A mice (density plot, left). Although both groups of mice spent
more time searching the target zone (T, dark blue) compared with other (O, light blue) zones, wild-type mice spent significantly more time in the target zone
compared to a-CaMKIIT286A mice (P o 0.05; ANOVA, significant genotype  zone interaction). (c) The number of BrdU+ cells in the dentate gyrus was similar
in wild type and a-CaMKIIT286A mutant (mut) mice (P 4 0.05; ANOVA). (d) Following the probe test, the number of Fos+ cells was reduced in
a-CaMKIIT286 mutants (mut) relative to wild-type controls (P o 0.05; ANOVA). (e) Similarly, the percentage of BrdU+ cells expressing Fos was reduced
in a-CaMKIIT286 mutants (mut) relative to wild-type controls (P o 0.05; ANOVA). (f) Fos expression in the dentate gyrus was similar in wild type anda-CaMKIIT286A mutant (mut) mice following restraint stress (P 4 0.05; ANOVA). Error bars represent ± s.e.m.

suggesting that adult neurogenesis was normal in the CaMKIIT286A
homozygous mice (although it is still possible that maturation of newneurons is affected). Consistent with the learning deficits, the numbersof both Fos+ (Fig. 6d) and Fos+BrdU+ (Fig. 6e) cells in the dentate
gyrus were reduced by approximately two-thirds in the CaMKIIT286A
homozygous mice. This reduction in numbers of Fos+ and Fos+BrdU+
cells was not due to an overall disruption of Fos regulation in
CaMKIIT286A homozygous mice: induction of Fos after restraint stress
was normal in these mutants (Fig. 6f).
In this experiment, wild-type and CaMKIIT286A homozygous mice
were subjected to the same behavioral procedures and showed similarlevels of adult neurogenesis and similar regulation of Fos expression
following restraint stress. Therefore, the finding that the probe test fails
to induce Fos in BrdU+ cells in CaMKIIT286A homozygous micestrengthens the support for a relationship between activation of newneurons and spatial memory processing. In addition, it provides further
evidence that nonspecific aspects of the test procedure (such as
swimming or physical exercise, stress or arousal) do not contribute to
Fos expression in both new and existing neurons in the dentate gyrus.
DISCUSSION
The continuous generation and integration of new neurons in thedentate gyrus has led to the idea that these adult-generated neurons
have a function in memory. Using immunohistochemical approaches tovisualize new neurons in intact animals, we showed that these newneurons are incorporated into dentate gyrus circuits supporting spatial
memory in an age-dependent manner. Significant recruitment of newneurons did not occur until they were at least 2 weeks old, and beyond
4 weeks new neurons were more likely than mature neurons to be
recruited into dentate gyrus circuits supporting spatial memory. Thispreferential recruitment is consistent with a decreased threshold forplasticity in new neurons5,34,35 and supports the idea that new neurons
make a unique contribution to memory processing in the dentate gyrus1.
Previous studies showed that spatial learning enhances the survival of 1–
2-week-old neurons, perhaps because it promotes integration of these
adult-generated neurons into dentate gyrus circuits29. However, ourresults indicate that maximal integration of new neurons into spatial
memory–related circuits does not occur until much later, suggesting that
integration into existing dentate gyrus circuitry and subsequent integration into memory-related circuits are distinct processes.
To identify BrdU-labeled cells processing spatial memory, we quantified the expression of two immediate-early genes, c-fos and Arc. The
expression profiles for Fos and Arc were remarkably similar following
water maze testing. First, the expression of both Fos and Arc was

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limited to neurons. Second, consistent with the idea that spatial
information is sparsely encoded in the dentate gyrus41, we found thatboth Fos and Arc were expressed in a relatively small proportion of
mature neurons following water maze testing36. Previous studies usedsimilar immunohistochemical approaches to show that adult-generated granule cells can be activated by a variety of stimulation, including
high-frequency electrical perforant path stimulation42, kainate-induced
seizure activity43, water maze training43 and spatial exploration44.
Although these latter two behavioral studies establish that adultgenerated granule cells respond to environmental stimulation, theycannot distinguish between gene expression related to memory encoding and gene expression associated with nonspecific aspects of the task(for example, swimming or physical exercise, stress or arousal).
Notably, these studies do not address whether new neurons are stably
incorporated into memory circuits.
We addressed this question by examining the activation of BrdUlabeled cells following recall of a spatial memory. Six key observations
establish a role for these new neurons in spatial memory. First,
activation of new neurons depended on behavioral expression of thespatial memory. Second, this activation was reduced in mutant micethat were unable to form a spatial memory. Third, activation of newneurons was not associated with nonspecific aspects of the probe test,
such as swimming or physical exercise, stress or arousal. Fourth,activation depended on the age of new neurons at the time of training(that is, the delay between BrdU treatment and training, rather than the
delay between training and testing) and did not simply reflect a lower
threshold for activation at the time of retrieval. Fifth, this pattern ofresults was observed only in the dentate gyrus and did not extend toother brain regions that do not have a role in spatial memory. Finally,
we found that new neurons were stably recruited into spatial memory
circuits in the dentate gyrus, consistent with an enduring role for the
hippocampus in the expression of water maze memories23,26,27,45.
These experiments reveal that as adult-generated granule cells
mature, they are increasingly likely to be recruited into spatial memory
circuits in the dentate gyrus. This age-dependent recruitment tracks themorphological and physiological maturation of new neurons3–6,11,12,33.
After migrating into the granule cell layer, adult-generated neurons
initially extend axons into CA3 and dendritic processes into themolecular layer8–10. At this stage, immature neurons may receive
GABA-mediated excitatory synaptic inputs11,12,46. However, major
glutamatergic synaptic activation from perforant path afferents does
not occur until new neurons are 2 (ref. 12) or more11 weeks old, whenspines begin to form10. Consistent with this, our data indicate thatsignificant recruitment does not occur until new neurons reach 2 weeks5© 2007 Nature Publishing Group http://www.nature.com/natureneuroscience

ARTICLES
of age, and by the time new neurons are 4–8 weeks of age they are more
likely than existing granule cells to be recruited into spatial memory
circuits. This preferential recruitment was evident during acquisition,
as well as during recall, and in experiments using either c-fos or Arc as
markers of neuronal activation. Consistent with the idea that these newneurons are competitively advantaged compared to existing granulecells, by the time new neurons are 3 weeks or older, they have a lower
threshold of activation and a lower threshold for inducing long-term
potentiation relative to existing granule cells34,35. These distinct physiological characteristics likely promote integration into spatial memory circuits, and a recent study suggests that new neurons may
maintain this competitive advantage for several months44.
Previous studies have used irradiation or antimitotic agents such as
MAM to suppress adult neurogenesis in rodents. Such manipulations
have impaired learning in trace eye-blink14 and fear15 paradigms. Similar
treatments, however, do not prevent learning in other hippocampusdependent tasks16. For example, suppression of adult neurogenesis does
not prevent acquisition in the Morris water maze15,17 (although longterm retention may be affected17). Several issues have complicated theinterpretation of these ablation-based approaches. First, these treatments have typically targeted relatively immature neurons (for example,
less than 2 weeks of age14,15), an age at which new neurons are not yet
integrated into dentate gyrus circuitry10. The current experimentssuggest maximal recruitment does not occur until at least 4 weeks ofage. Second, suppression of adult neurogenesis following irradiation or
MAM treatment is typically incomplete18,19, and therefore it is possible
for learning to be supported by residual adult-generated neurons. Third,
even complete suppression of adult-generated granule cells may notprevent learning, because learning may be supported by existing granulecells1. Rather, memories formed without the contribution of newneurons may contain degraded temporal information1, be more prone
to interference47,48 or be less durable17. Here we circumvented theseissues by using immunohistochemical approaches to visualize therecruitment of new neurons into spatial memory networks in intact
mice. We identified a window in which new neurons are preferentially
recruited into spatial memory circuits. The preferential recruitment of
adult-generated granule cells suggests that they make a unique, but as yet
unspecified, contribution to memory processing in the dentate gyrus1.
METHODS
Mice. Male offspring from a cross between C57Bl/6NTacfBr (C57B6) and
129Svev (129) mice (Taconic) were used in the majority of these experiments.
In one experiment, we used a-CaMKIIT286A knock-in mice, generated previously40. The a-CaMKIIT286A mutation has been maintained in a C57B6
background (ten or more generations). To generate experimental mice,a-CaMKIIT286A heterozygous mice in the C57B6 background were first crossedwith 129 mice to obtain F1 hybrid mice. F1 heterozygous mice were thencrossed again to obtain F2 hybrid mice. Genotypes were determined by PCR
analysis of tail DNA samples, and only male wild-type and a-CaMKIIT286A
homozygous mice were used in the experiments. All mice were bred in ourcolony at The Hospital for Sick Children, and mice were maintained on a 12 h
light/12 h dark cycle with free access to food and water. Mice were at least
8 weeks of age at the start of experiments, and behavioral procedures wereconducted during the light phase of the cycle. Experiments were conducted
blind to the treatment condition of the mouse and according to protocols
approved by the Animal Care Committee at The Hospital for Sick Children.
BrdU administration. BrdU was dissolved in 0.1 M phosphate-buffered saline,
pH 7.4 (PBS), and heated to 50–60 1C. BrdU was injected intraperitoneally for
5 d according to the specific experimental protocols described below.
Water maze and general training procedures. The apparatus and behavioral
procedures have been previously described23. Behavioral testing was conducted6

in a circular water maze tank (120 cm in diameter, 50 cm deep), located in adimly lit room. The pool was filled to a depth of 40 cm with water madeopaque by adding white, nontoxic paint. The water temperature was maintained at 28 ± 1 1C by a heating pad located beneath the pool. A circular escapeplatform (10 cm in diameter) was submerged 0.5 cm below the water surface,in a fixed position in one of the quadrants. The pool was surrounded bycurtains; hung at least 1 m from the perimeter of the pool. The curtains werewhite and had distinct cues painted on them.
Before the commencement of training, mice were individually handled for
2 min per d over 7 consecutive days. In the majority of experiments, mice weretrained over 5 d. On each training day, mice received six training trials(presented in two blocks of three trials; interblock interval was B1 h, intertrial
interval was B15 s). On each trial they were placed into the pool, facing thewall, in one of four start locations. The order of these start locations was
pseudorandomly varied throughout training. The trial was complete once themouse found the platform or 60 s had elapsed. If the mouse failed to find theplatform on a given trial, the experimenter guided the mouse onto theplatform. Following the completion of training, spatial memory was assessedin a series of probe tests with an intertest interval of approximately 3 min. In
this test, the platform was removed from the pool, and the mouse was allowed
60 s to search for it. In Experiment 2, Fos and BrdU expression were examinedfollowing training. Procedures were as described above, except that mice weretrained for 3 d. On the third day of training, mice received only the first blockof three trials.
Behavioral data from the training and the probe tests were acquired andanalyzed using an automated tracking system (Actimetrics). Using this software, we were able to record a number of parameters during training, including
escape latency and swim speed. In probe tests, we quantified performance in
two ways. First, we measured the amount of time mice searched the target zone
(20 cm in radius, centered on the location of the platform during training)
versus the average of the time spent searching three other equivalent zones inother areas of the pool23. These zones each represent approximately 11% of thetotal pool surface. Second, we represented probe test performance as a colormap (or density plot), with hot colors corresponding to areas of the pool thatwere more frequently visited23.
Experimental procedures. Experiment 1. In the first experiment, we determined the age at which adult-generated granule cells are incorporated into
spatial memory networks. To label the dividing cell population, mice were
initially injected with BrdU for 5 consecutive days (50 mg per kg of bodyweight; two injections per d). Separate groups of mice were then trained in thewater maze for 5 d, either 1 week (n ¼ 9), 2 weeks (n ¼ 7), 4 weeks (n ¼ 7),
6 weeks (n ¼ 8) or 8 weeks (n ¼ 9) later. Spatial memory was assessed in all
mice 10 weeks after BrdU treatment. In this test, mice were given a series ofthree probe tests. In addition, Fos expression in BrdU+ cells was examined inhome cage control mice (n ¼ 5) and mice treated with the chemical convulsant,PTZ (50 mg per kg, n ¼ 2), 10 weeks after BrdU treatment (50 mg per kg, two
injections per d).
Experiment 2. In this experiment we evaluated whether new granule cells are
only transiently incorporated into spatial networks in the dentate gyrus.
Two groups of mice were trained in the water maze 1 week (n ¼ 8) or
6 weeks (n ¼ 8) after BrdU treatment (100 mg per kg; 5 d, two injections perd). These mice were trained for 3 d. On the third day of training, mice received
only the first block of three trials. We chose this abbreviated training schedule
because, in our previous experiments, we found that escape latencies reachedasymptote by the fourth day of training23. Therefore, at this time point
significant learning is still occurring and we avoid the high levels of stresstypically associated with the first day of training.
Experiment 3. In this experiment, we varied the delay between BrdU
treatment and training, although the retention delay between training andtesting was fixed at 4 weeks. Mice were treated with BrdU (100 mg per kg; 5 d,
two injections per d) either 6 weeks before (n ¼ 7), 1 week before (n ¼ 6) or
1 week after (n ¼ 6) training. On the test day, all mice were presented with aseries of three probe tests.
Experiment 4. In this experiment, we examined the relationship between
memory strength and Fos expression. Six weeks after BrdU treatment (100 mg
per kg; 5 d, two injections per d), a-CaMKIIT286A homozygous mice (n ¼ 8)

ADVANCE ONLINE PUBLICATION NATURE NEUROSCIENCEARTICLES© 2007 Nature Publishing Group http://www.nature.com/natureneuroscienceand their wild-type littermates (n ¼ 8) were trained in the water maze. One day
following the completion of training, spatial memory was tested in a series ofthree probe tests. Additionally, Fos expression was examined following restraint
stress in wild-type (n ¼ 3) and a-CaMKIIT286A homozygous (n ¼ 3) mice.
Mice were placed in a well-ventilated Plexiglas cylinder for 60 min (volume,
60 cm3) and perfused 60 min later.
Tissue preparation. Ninety minutes following the completion of behavioral
testing, mice were deeply anesthetized and perfused transcardially with PBS andthen 4% paraformaldehyde (PFA). Brains were removed, fixed overnight in
PFA and then transferred to 30% sucrose solution and stored at 4 1C. Coronal
sections of 50 mm were cut and divided into four sets. Each of these sets
included sections at 200-mm intervals, and covered the full anterior-posterior
extent on the dentate gyrus. Free-floating sections were prepared for immunohistochemistry, and additional sets were stored in a PBS solution containing
0.02% sodium azide for later processing.

BrdU+ and Fos+BrdU+ cells using the optical dissector, stereological method50.
Cells were counted within a single z-stack (area, 165  225 mm). Cells in theuppermost 5-mm focal plane were not included in counts.
Imaging. Representative images were acquired using either a Nikon Eclipse 80i
epifluorescence microscope or an Olympus IX81 DSU confocal microscope.
The signal for Alexa-488 labeling was detected using a Nikon HQ FITC filter,
and the Alexa-568 labeling was detected using a Nikon HQ R filter. Images weredigitized with a Nikon DXM 1200F CCD camera or with a Photometrics
CoolSnap CCD camera, respectively.
Statistical analyses. To evaluate whether mice searched selectively in probe tests,
paired t-tests were performed on the zone (target versus other) data. The effects
of experimental manipulations on numbers of Fos+, BrdU+ and Fos+BrdU+ cells
were evaluated with ANOVAs. Significant main effects or interactions were
followed up with post hocs (Fishers LSD test), where appropriate.

BrdU, Fos and NeuN immunohistochemistry. For BrdU immunohistochemistry, the BrdU antigen was exposed by incubating the sections in 1 N HCl at
45 1C for 30 min. Immunohistochemistry was performed using primary
antibodies to Fos (rabbit anti-Fos polyclonal antibody; 1:1,000, Calbiochem),
BrdU (rat anti-BrdU monoclonal antibody; 1:500, Accurate Chemicals), Arc(rabbit anti-arc polyclonal antibody;1:500, generously donated by P. Worley)
and NeuN (mouse anti-NeuN Alexa Fluor 488 conjugated; 1:1,000, Chemicon).
We used Alexa-488 goat anti-rat, Alexa-568 goat anti-mouse and Alexa-568
goat anti-rabbit (1:500, Molecular Probes) as secondary antibodies. Sections
were incubated in primary antibodies overnight at room temperature(20–25 1C) for Fos and BrdU labeling or at 4 1C for Arc and BrdU labeling.
Sections were incubated with secondary antibodies and anti-NeuN primary
antibody for 2 h at room temperature. Antibodies were diluted in blocking
solution containing 2% goat serum, 1% bovine serum albumin and 0.2%
Triton X-100 dissolved in PBS. Sections were mounted on slides with Permafluor anti-fade medium (Lipshaw Immunon).

Note: Supplementary information is available on the Nature Neuroscience website.

Quantification. We quantified Fos+, BrdU+ and Fos+BrdU+ cells throughout
the anterior-posterior extent of the granule cell layer. The average number of
Fos+, BrdU+ and Fos+BrdU+ cells per section was then normalized for theentire dentate gyrus by multiplying this average by the number of 50-mm
sections corresponding to the entire dentate gyrus2,19,29,30,43.
Quantification of Fos+cells. The density of Fos+ cells was relatively low(around 2.1–2.4% of mature granule cells expressed Fos following behavioral
testing) and therefore it was possible to quantify the number of Fos+ cells fromtwo-dimensional images of the entire dentate gyrus. These were acquired using
a 10 objective on a Nikon Eclipse 80i epifluorescence microscope (Nikon
Instruments) and Fos+ cells were counted using ImageJ software (National
Institute of Health). In a subset of animals we performed equivalent stereological counts of Fos+ cells using a confocal microscope (Olympus IX81 DSU).
These yielded similar estimates, verifying the accuracy of our method.
Quantification of BrdU+and Fos+BrdU+cells. Numbers of BrdU+ and
Fos+BrdU+ cells were first determined using the epifluorescence microscopewith a 40 objective. A subset of BrdU+ and Fos+BrdU+ cells were confirmedwith the confocal microscope. To verify that Fos+BrdU+ cells were doublelabeled, we collected 20 focal planes, and the resulting three-dimensional image
was examined to ensure that the fluorochromes originated from the same cell.
Partially labeled or sectioned cells were not counted.
Quantification of NeuN+cells in the dentate gyrus. To estimate the total
number of mature neurons in the dentate gyrus, sections were stained for themature neuronal marker, NeuN. Using a method adapted from a previous
report36, two z-stacks per dentate gyrus (one each from the upper and lower
blades) were collected from four mice at 40 magnification using the confocal
microscope. The total number of NeuN+ cells in each 50-mm stack was counted
manually and the total area of the middle plane was measured. From this we
calculated the density of NeuN+ cells in the dentate gyrus. From our analysis of
Fos+ cells, we then determined the proportion of mature neurons expressing
Fos following behavioral testing.
Quantification of Fos+, BrdU+and Fos+BrdU+cells in the olfactory bulb. Three
50-mm sections containing the granule cell layer of the olfactory bulb wereselected from each mouse49. A confocal microscope was used to quantify Fos+,

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Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions

NATURE NEUROSCIENCE ADVANCE ONLINE PUBLICATIONACKNOWLEDGMENTS
We thank S. Josselyn for comments on this manuscript. This work was
supported by grants from the Canadian Institutes of Health Research and the
EJLB Foundation (P.W.F.). N.K. and A.H.W. were supported by Hospital for Sick
Children Restracomp awards. C.M.T. received support from the Graduate
Program in Areas of Basic and Applied Biology and the Portuguese
Foundation for Science and Technology.
AUTHOR CONTRIBUTIONS
P.W.F., N.K. and C.M.T. conceived the experiments. C.M.T., P.W.F. and
A.H.W. conducted the water maze studies, N.K. and C.M.T. conducted theimmunohistochemistry and quantification and C.M.T. conducted the statisticalanalyses. PW.F. supervised the project and wrote the paper.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.

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