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Received 3 Sep 2012 | Accepted 6 Nov 2012 | Published 4 Dec 2012

DOI: 10.1038/ncomms2261

Suppression of adult neurogenesis impairs
population coding of similar contexts
in hippocampal CA3 region
Yosuke Niibori1, Tzong-Shiue Yu1, Jonathan R. Epp1, Katherine G. Akers1, Sheena A. Josselyn1,2& Paul W. Frankland1,2Different places may share common features, but are coded by distinct populations of CA3
neurons in the hippocampus. Here we show that chemical or genetic suppression of adultneurogenesis in the hippocampus impairs this population-based coding of similar (but notdissimilar) contexts. These data provide a neural basis for impaired spatial discrimination
following ablation of adult neurogenesis, and support the proposal that adult neurogenesis
regulates the efﬁciency of a pattern separation process in the hippocampus.

1 Program in Neurosciences and Mental Health, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8. 2 Department of Physiology and Instituteof Medical Science, University of Toronto, Toronto, Ontario, Canada M5S 1A8. Correspondence and requests for materials should be addressed to P.W.F.
(email: paul.frankland@sickkids.ca).

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T

he ability to distinguish between different places is an
essential feature of the hippocampal episodic memory
system1. Computational models initially proposed that thisis mediated by a pattern separation process within the
hippocampal dentate gyrus (DG)-CA3 circuit2, and recent
electrophysiological recording studies in rodents provide direct
support for this view3–5. The DG and CA3 appear to code context
changes differently. In the DG, context shifts produce changes inﬁring rate of the same population of active neurons (that is, a ratecode), whereas the same context shifts alter the populations ofcells ﬁring in CA3 (that is, a population code)3,4. This CA3
population-based code of distinct environments may also be
revealed using activity-dependent gene mapping of neuronal
activation following exposure to the same versus different
contexts6.
As new neurons are added to the DG throughout adulthood7,
this putative process of pattern separation occurs in acontinuously evolving DG-CA3 circuit. Therefore, here we ask
whether adult neurogenesis in the DG modulates the efﬁciency ofcontext coding in the downstream CA3 region.
To visualize the population code in CA3 we used a cellularimaging approach that takes advantage of the transcriptional timecourse of the activity-regulated cytoskeleton-associated protein(arc) (also known as Arg3.1)8. Following neuronal activation, arc
mRNA is rapidly induced in the nucleus (within 5 min), but
subsequently shuttled to the cytoplasm (within 25 min).
Therefore, the localization of arc RNA (either in the nucleus,
cytoplasm or both) identiﬁes neuronal ensembles activated byseparate context exposures. Accordingly, we used this techniqueto examine the effects of suppressing adult neurogenesis on
activation of populations of CA3 neurons following exposure to
the same, similar or dissimilar contexts. We found thatsuppression of adult neurogenesis impairs CA3 population
coding of similar contexts.
Results
Chemical reduction of neurogenesis impairs population
coding. We ﬁrst used a chemical approach to suppress adultneurogenesis (see Supplementary Methods). Wild-type (WT)
mice were treated with the DNA-alkylating agent Temozolomide(TMZ) or vehicle (VEH) for 4 weeks. Consistent with previous
studies9, we observed that TMZ treatment suppressed adultneurogenesis, reﬂected by both a reduction in the number of DG
cells expressing the proliferation marker, Ki67, and cells
expressing the immature neuronal marker, NeuroD (Fig. 1a,b).
Importantly, TMZ did not appear to compromise immune
function (leukocyte recovery), alter general behaviour or lead toweight loss (Supplementary Fig. S1). Next, separate cohorts of
VEH- and TMZ-treated mice were habituated to two similar
contexts (A, triangular-shaped; B, square-shaped), located in thesame room. Twenty-four hours later, mice were either exposed tocontext A twice (AA condition), context A then B (AB condition)or context A then a novel, dissimilar context (C, rectangularshaped and located in a different room; see Supplementary Fig.
S2) (AC condition). Context exposures were separated by 20 min
and brains were removed immediately after the second exposure
and processed for arc ﬂuorescent in situ hybridization to identify
populations of CA3 neurons activated by the ﬁrst (cytoplasmicarc) and/or second (intranuclear arc) context exposure (Fig. 1cand Supplementary Fig. S3).
Relative to home cage, context exposures robustly induced arc
in CA3 neurons (range 38.1–58.0%; Fig. 1d and Supplementary
Fig. S4), consistent with previous experiments showing thatspatial exploration reliably increases arc mRNA8. Overall levels ofcytoplasmic and nuclear arc were similar in VEH- and TMZ2

treated mice (Supplementary Table S1), indicating that TMZ
treatment did not alter the likelihood of arc induction. However,the degree of overlap between populations of CA3 neurons
activated by the ﬁrst versus second context exposure varied acrossgroups and across conditions (Fig. 1d,e). In VEH-treated mice,re-exposure to the same context (AA) was more likely to activate
the same population of CA3 neurons than exposure to similar(AB) or dissimilar (AC) contexts. Consistent with previous
electrophysiological recording and cellular imaging experiments
in rats3–6, these data indicate that different contexts are
represented, in part, by distinct populations of CA3 neurons(or a population code) (although the magnitude of separation isless than in rats6). In contrast, TMZ treatment impaired thispopulation-based coding of different contexts. Activation of thesame population of CA3 neurons was equally likely in the AA andAB conditions (but reduced in the AC condition) (Fig. 1d,e).
Therefore, these data indicate that reducing DG neurogenesis
impairs population coding of similar, but not dissimilar (ornovel), contexts by downstream CA3 neurons. In the TMZtreated mice, the population of CA3 neurons activated by thesecond context exposure was larger than that activated by the ﬁrst
context exposure (Fig. 1f, Supplementary Table S2). In an
additional experiment, where we reversed the order of context
presentation (BA rather than AB; Supplementary Fig. S5), we
found a similarly expanded population of CA3 neurons active on
the second exposure in TMZ-treated mice (Fig. 1f, Supplementary
Table S1). Therefore, this suggests that increased overlap of
population codes in TMZ-treated mice is primarily driven by an
expansion of the population of cells activated by the second
exposure.
Genetic reduction of neurogenesis impairs population coding.
We next used a genetic approach to reduce adult neurogenesis.
To do this, we used transgenic mice in which a modiﬁed HSV-tk
gene is expressed from the nestin promoter/enhancer (TK þmice)10. In these mice, the administration of ganciclovir (GAN), a
nucleoside analogue, ablates only the dividing cells expressing tk.
Accordingly, adult TK þ mice and their WT littermate controls
were treated with GAN for 4 weeks, and then population codingof similar contexts by CA3 neurons was evaluated as above.
Neurogenesis was reduced in TK þ mice treated with GAN(reduced number of cells expressing Ki67 and NeuroD; Fig. 2a,b),
without any obvious change in general health or behaviour(Supplementary Fig. S6). As before, in WT mice re-exposure to
the same context (AA) was more likely to activate the same
population of CA3 neurons than exposure to similar (AB)contexts. In contrast, activation of the same population of CA3
neurons was equivalently likely in the AA and AB conditions in
the GAN-treated TK þ mice (Fig. 2c–e and Supplementary Fig.
S7). Therefore, although chemical and genetic reduction of adultneurogenesis differed in magnitude, they produced equivalentdisruption of population coding. This suggests that CA3
population coding of similar context is highly sensitive toﬂuctuations in neurogenesis, with even modest reductions being
sufﬁcient to abolish population-based discrimination of similar
contexts.
Neurogenesis regulates behavioural context discrimination.
Using the same context manipulations as above, we conﬁrmedthat suppression of adult neurogenesis produces parallel behavioural effects11,12. To do this, we used a fear-learning task toevaluate discrimination between similar or dissimilar contexts. In
the ﬁrst experiment, VEH- and TMZ-treated WT mice were preexposed to two similar contexts, located in the same room. The
next day they were placed in one of the contexts and shocked

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Figure 1 | Chemical Suppression of adult neurogenesis impairs coding of similar contexts. (a) Ki67 þ and (b) NeuroD þ staining was reduced in TMZtreated (n ¼ 4) compared with VEH-treated (n ¼ 4) mice, indicating that TMZ treatment reduced proliferation (t6 ¼ 5.36, Po0.01) and the number of
immature neurons (t6 ¼ 5.09, Po0.01) in the DG, respectively. Scale, 200 mm. Mo, molecular layer, GCL, granule cell layer. (c) Experimental design. TMZtreated and VEH-treated mice were habituated to contexts A (triangle) and B (square), and then tested in context A consecutively (AA; VEH, n ¼ 8, TMZ,
n ¼ 8), context A then context B (AB; VEH, n ¼ 9, TMZ, n ¼ 10) or context A then context C (AC; VEH, n ¼ 10, TMZ, n ¼ 8) or home cage (H; VEH, n ¼ 4,
TMZ, n ¼ 5). (d) Venn diagrams indicating populations of CA3 cells activated either by ﬁrst (t1), second (t2) or both (overlap, shaded) context exposures,as reﬂected by arc expression (context exposures4home cage, t-tests, Po0.05). (e) Percent of CA3 cells activated by both context exposures in VEHversus TMZ-treated mice (Treatment  Context interaction, F3,54 ¼ 4.43, Po0.01; Tukey post-hoc tests, *Po0.05). (f) Percent of CA3 cells activated on
second (t2) versus ﬁrst (t1) context exposure in VEH-treated (n ¼ 7) versus TMZ-treated (n ¼ 7) mice (t24t1 in AB [t9 ¼ 3.11, Po0.05] and BA [t6 ¼ 2.41,
P ¼ 0.05] only in TMZ-treated mice). In these graphs, error bars represent s.e.m., and signiﬁcantly different contrasts are marked by * (where Po0.05).

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*

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Figure 2 | Genetic suppression of adult neurogenesis impairs hippocampal coding of similar contexts. (a) Ki67 þ and (b) NeuroD þ staining was
reduced in GAN-treated TK þ compared with WT mice ((Ki67: t15 ¼ 3.20, Po0.01; WT, n ¼ 9, TK þ , n ¼ 8); (NeuroD: t9 ¼ 10.30, Po0.001; WT, n ¼ 6,
TK þ , n ¼ 5)). Scale, 200 mm. Mo, molecular layer, GCL, granule cell layer. (c) Experimental design. Mice were habituated to contexts A and B, and theneither tested in context A consecutively (AA; WT, n ¼ 4, TK þ , n ¼ 4) or context A then context B (AB; WT, n ¼ 5, TK þ , n ¼ 4). (d) Venn diagrams
indicating populations of CA3 cells activated either by ﬁrst (t1), second (t2) or both (overlap, shaded) context exposures, as reﬂected by arc expression.
(e) Percent of CA3 cells activated by both context exposures in GAN-treated WT versus TK þ mice (Genotype  Context interaction, F1,13 ¼ 8.28, Po0.05;
WTAA versus WTAB, t7 ¼ 4.91, Po0.001). In these graphs, error bars represent s.e.m., and signiﬁcantly different contrasts are marked by * (where
Po0.05).

immediately, and then conditioned freezing was assessed inboth the shock-paired versus shock-unpaired context (Fig. 3a).
During testing, both VEH- and TMZ-treated mice froze morein the context paired with shock versus the no shock context(Fig. 3b). However, the degree of discrimination differed, with
TMZ-treated mice exhibiting reduced levels of discriminationof these similar contexts compared with VEH-treated controls(Fig. 3c). Importantly, discrimination depended upon context
pre-exposure, as both VEH- and TMZ-treated mice that were
not pre-exposed exhibited low and equivalent levels of freezingin either context when tested (Supplementary Fig. S8). In
contrast, discrimination of dissimilar contexts was not affectedby chemical reduction of adult neurogenesis (Fig. 3d). During
testing, both VEH- and TMZ-treated mice froze more in thecontext paired with shock versus the no shock context (Fig. 3e),and the degree of discrimination of these dissimilar contexts
did not differ (Fig. 3f). We found similar impairments inbehavioural discrimination following genetic reduction of
adult neurogenesis in TK þ mice (Supplementary Fig. S9).
Therefore, consistent with our ﬁnding that suppression of adultneurogenesis in DG impaired population coding of contexts
A and B, these results indicate that suppression of adultneurogenesis impaired behavioural discrimination of these same
4

contexts. We note that freezing levels in the context pairedwith shock were generally reduced following suppression of
adult neurogenesis. Using standard contextual fear-conditioning
paradigms, previous studies have sometimes found deﬁcits
following suppression of adult neurogenesis (for example,13,14),
whereas others have not (for example,14,15). The present results
therefore additionally indicate that prior experience in thecontexts may modulate the impact of neurogenesis suppression
on contextual learning.
Discussion
Pattern separation is a neurocomputational construct, referring to
the process of transforming similar (overlapping) representations
into distinct, orthogonalized codes2,16. Using a cellular imaging
approach, we found that different contexts were coded by distinct
populations of neurons in the hippocampal CA3 region in intact
WT mice, consistent with the idea that DG-CA3 circuits
disambiguate representations of similar contexts3–6. In contrast,
both chemical and genetic suppression of adult neurogenesis in
DG impaired this CA3 population coding of contexts. In
particular, we found that adult neurogenesis suppression
impaired coding of similar (but not dissimilar) contexts,

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Pre exposure

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VEH TMZ

TMZ

Figure 3 | Chemical suppression of adult neurogenesis impairs behavioural discrimination of similar contexts. (a) Experimental design (similar
contexts). Mice were preexposed to contexts A (triangle) and B (square). The following day they received an immediate shock in context B, and then 24 hlater freezing was assessed in contexts A and B. (b) During the test, both VEH-treated (n ¼ 11) and TMZ-treated (n ¼ 12) mice froze more in context B
versus A (Context main effect, F1,21 ¼ 56.83, Po0.001). (c) However, discrimination (freezingA  freezingB) was reduced in TMZ-treated mice (unpaired ttest, t21 ¼ 3.54, Po0.01). (d) Experimental design (dissimilar contexts). Mice were preexposed to a rectangular context (located in a different room) andthe square context. The following day they received an immediate shock in the square context, and then 24 h later freezing was assessed in the squareversus rectangular contexts. (e) During the test, both VEH-treated (n ¼ 12) and TMZ-treated (n ¼ 12) mice froze more in the square versus rectangular
context (Context main effect, F1,44 ¼ 165.06, Po0.001). (f) Discrimination (freezingA  freezingB) was similar in TMZ-treated mice (unpaired t-test,
t22 ¼ 0.82, P40.05). In these graphs, error bars represent s.e.m., and signiﬁcantly different contrasts are marked by * (where Po0.05).

consistent with the idea that reducing neurogenesis would hinder
resolution of small (but not larger) differences in mnemonic
input. This impaired population coding was reﬂected by anincrease in the number of cells that were co-activated by bothcontexts. However, it appeared that this increased overlap was
associated with an expansion of the population of CA3 neurons
activated by the second context exposure rather than a decrease insegregation of populations of CA3 neurons activated by eachcontext. The latter is the process of orthogonalization that has
been historically associated with pattern separation in theliterature, and therefore our results indicate that experimental
reduction of neurogenesis leads to less distinct population codesof similar contexts, but not necessarily via a reduction inorthogonalization. In parallel, we found chemical or genetic
reduction of adult neurogenesis impaired behavioural
discrimination of the same contexts. These results are
consistent with recent ﬁndings that reducing neurogenesis
impedes spatial discrimination11,12,17, whereas enhancing
neurogenesis promotes spatial discrimination11,18, and provide
an essential link between the effects of manipulating levels ofadult neurogenesis on behaviour and changes in information
processing in hippocampal networks. New neurons have been
proposed to facilitate population coding of contexts either bydirectly promoting remapping in place cells in CA3 or indirectly
by activating dentate inhibitory neurons that lead to increased
sparsiﬁcation of DG codes that then feed forward into CA3(ref. 19,20). Our data indicate that new neurons facilitate
disambiguation of contexts by regulating activity of CA3
neurons, and thereby reducing overlap in population codes.

Methods
Mice. In these experiments we used two lines of mice. First, in the chemical
suppression of adult neurogenesis experiments we used male WT offspring froma cross between C57Bl/6NTacfBr (C57B6) and 129Svev (129) mice (Taconic).
Second, in genetic suppression of adult neurogenesis experiments we usedtransgenic mice expressing a modiﬁed HSV-tk under control of the nestin
promoter (TK þ mice)10. These mice were maintained on a C57B6 background(48 crosses), and experimental mice were generated by crossing TK þ mice with
C57B6 WT mice (for arc ﬂuorescent in situ hybridization experiments)
or with 129 WT mice (for fear conditioning experiments). Male and female TK þ
and WT littermates were used in experiments. All mice were bred in our colony
at The Hospital for Sick Children, maintained on a 12 h light/dark cycle with freeaccess to food and water. Behavioural procedures were conducted during thelight phase of the cycle, blind to genotype of the mouse and according toprotocols approved by the Animal Care Committee at The Hospital for Sick
Children.
Chemical suppression of adult neurogenesis. Starting at 6 weeks of age,
WT mice were treated with the DNA-alkylating agent TMZ (Sigma) for 4 weeks.
We used an established injection protocol that has previously been shown toreliably suppress neurogenesis in adult mice9. During each of the 4 weeks oftreatment, mice received daily injections of TMZ (25 mg kg  1 intraperitoneally)for 3 consecutive days, followed by 4 days of no injections. TMZ was dissolved in
10% dimethyl sulfoxide. The VEH group received injections of 10% dimethyl
sulfoxide.
Genetic suppression of adult neurogenesis. For arc in situ hybridization
experiments, TK þ mice and their WT littermate controls were treated with GAN(200 mg kg  1 per day, Cytovene-IV, Roche Pharmaceuticals) delivered via osmotic
minipumps (model 2002, Alzet). Pumps were implanted subcutaneously at 8 weeks
of age as previously described10. For behavioural experiments, GAN was mixed
into powdered chow (0.08%, B70 mg kg  1 per day) (ref. 21), and TK þ mice andtheir WT littermate controls had ad lib access to the chow in their home cage for 28
days, starting at 8 weeks of age.

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Immunohistochemistry. Following testing, mice were decapitated and brains werefrozen on dry ice. Coronal sections (20 mm) were cut using a cryostat. All sections
were treated with 1% hydrogen peroxidase. Sections were then incubated overnightwith the primary antibodies (anti-Ki67 (1/100; Chemicon) or anti-NeuroD (1/150;
Santa Cruz)) at 4 1C and then for 60 min at 20 1C with horseradish peroxidase(HRP)-conjugated secondary antibodies (1:300; Jackson Immuno-research, PA),and developed with an Alexa 488-conjugated tyramide ampliﬁcation system (PerkinElmer) (See refs 8,22). Sections were mounted on slides with Permaﬂuor antifade medium, and counter-stained with Hoechst 33258 to mark nuclei. Ki67 þ and
NeuroD þ cells were quantiﬁed using an epiﬂuorescent microscope (Nikon Eclipse80i) with  40 objective. To generate cell counts we used 1/6 systematic section
sampling fractions covering the entire anterior–posterior extent of the DG.
arc ﬂuorescent in situ hybridization. Coronal sections (20 mm) were cut andthaw-mounted on slides, such that each slide contained sections from different
treatment conditions (to maximize relevant between-group comparisons withineach slide). Digoxigenin (DIG)-labelled antisense and sense riboprobes for arc
mRNA were generated using a commercial transcription kit (MaxiScript, Ambion)
and RNA labelling mix (Roche Diagnostics) from a plasmid encoding arc open
reading frame. arc antisense riboprobe was detected with an anti-DIG antibody
conjugated with HRP FAB fragments (1/300; JacksonImmunoResearch) anddeveloped with an Alexa 488-conjugated tyramide ampliﬁcation system(PerkinElmer) (See refs 8,22).
Brieﬂy, sections were ﬁxed with 4% paraformaldehyde ﬁxation buffer, washed in
2  0.3 M sodium chloride and 0.03 M SSC, treated with 0.5% acetic anhydride/
1.5% triethanolamine, and 50% methanol/50% acetone before being incubated in
hybridization buffer (with 50% formamide, 5  SSC, 0.1 mg ml  1 yeast transfer
RNA, 0.1% Tween-20 and 50 mg ml  1 Heparin) for 30 min at 20 1C. Sections wereincubated with arc riboprobe (1 ng ml  1 in hybridization buffer) at 56 1C
overnight. Twenty-four hours later, sections were treated sequentially with RNaseA(10 mg ml  1 in 2  SSC at 37 1C for 30 min), followed by two washes in 0.5  SSC
at 56 1C. Sections were then treated with hydrogen peroxide (1% for 30 min),blocking reagent (Roche Diagnostics) with goat serum (4%) (30 min) andincubated with an anti-DIG HRP-conjugated antibody (described above) inblocking solution for 2 h at 20 1C. Tyramide ampliﬁcation was performed for
30 min at 20 1C. Nuclei were visualized with Hoechst 33258 (Sigma) counterstain.
Images were captured using a confocal microscope (Zeiss LSM 710 equipped
argon diode laser). Images were acquired using a X40 oil-immersion lens andz-sectioned in 1 mm thick optical sections. Images were collected from CA3 (AP:B  2.0 mm from bregma)23, and every third section was analysed. Microscopesettings (pinhole, gain and contrast) were kept constant for all images on a given
slide. Localization of arc signal was analysed using Image-Pro PLUS software(MediaCybernetics). Small, bright uniformly DAPI stained nuclei (roughly 5 mm indiameter) from putative glial cells were not counted. All other whole nuclei cells(putative neurons) in the ﬁeld of view within the CA3 region were counted.
Nuclear arc was deﬁned as focal arc puncta in the nucleus whereas cytoplasmic arc
was deﬁned as arc-positive dots surrounding the nucleus. Nuclei were only
considered if they could be discerned in their entirety through the z-sectioned
images. Manual cell counts were performed by an observer unaware of thetreatment condition. Two to four sections were analysed per mouse, and thenumber of CA3 cells having intranuclear foci and/or cytoplasmic signals of arc
mRNA were counted.
Contexts. The three contexts (A (triangular), B (square) and C (rectangular);
Supplementary Fig. S2) used in these experiments have been previously described24. Contexts A and B were located in the same room. Context C was located in adifferent room.
Arc in situ ﬂuorescent hybridization experiments. Before behavioural testing,mice were handled daily for 2 min each across 4 days. Behavioural testing was
conducted on 2 consecutive days, starting immediately following completion of the
4-week TMZ/GAN treatment. On the ﬁrst day (habituation), mouse home cages
were transported from the vivarium to a quiet, holding area in the behavioural lab.
After 1 hour acclimation, mice were placed in context A for 5 min, and thenreturned to their home cage. Approximately 1 h later, mice were placed in context
B for 5 min and then returned to their home cage and taken back to the vivarium.
The following day (testing), mice were acclimated for 1 h, and then placed incontext A for 5 min. They were immediately returned to their home cage, and then
20 min later placed in context A (AA condition), context B (AB condition) orcontext C (AC condition) for 5 min. Additional groups of VEH- and TMZ-treatedmice were treated identically, but tested in B then A (BA condition). Immediately
following this, mice were killed by decapitation and brains were removed andfrozen on dry ice. Mice in the home cage group were decapitated in the vivarium.
Fear conditioning experiments. To evaluate whether suppression of adult neurogenesis impairs context discrimination we adapted a fear-conditioning paradigm25,26. During the last 4 days of TMZ/VEH (chemical suppression) or GAN(genetic suppression) treatments, mice were handled daily for 2 min each.
Behavioural testing was conducted over 3 consecutive days, starting following
6

completion of TMZ treatment/handling. On the ﬁrst day (preexposure), mousehome cages were transported from the vivarium to a quiet, holding area in the
behavioural lab. After 1 hour acclimation, mice were placed in context A for 5 min,and then returned to their home cage. Approximately 1 h later, mice were placed incontext B for 5 min and then returned to their home cage and taken back to thevivarium. The following day, mice were placed in context B, where they received an
immediate shock (1.0 mA, 2 s duration). They remained in the context for an
additional minute and were then taken back to the vivarium. The following day,mice were tested in both context A and context B, in a counter-balanced order.
Tests were 5 min in duration, and spaced 1 h apart. To test whether context
discrimination depended on context preexposure, additional groups of mice weretreated as above, but not preexposed to the contexts. To test whether chemical
reduction of neurogenesis impaired discrimination of dissimilar contexts, we
conducted an additional experiment. All procedures were identical, except that thesquare and rectangular (located in different room) contexts were used. During thepreexposure, training and testing phases, mouse behaviour was monitored via four
overhead cameras. Freezing behaviour was assessed using an automated scoring
system (Actimetrics, Wilmette, IL), which digitized the video signal at 4 Hz andcompared movement frame by frame to determine the amount of freezing.
Statistical analyses. Effects of manipulating adult neurogenesis on arc induction
and behaviour were evaluated by analysis of variance followed by t-tests or Tukey
post-hoc tests where appropriate. In graphs, data are presented as mean±s.e.m.

References
1. Yassa, M. A. & Stark, C. E. Pattern separation in the hippocampus. Trends
Neurosci. 34, 515–525 (2011).
2. Treves, A. & Rolls, E. T. Computational analysis of the role of the hippocampus
in memory. Hippocampus 4, 374–391 (1994).
3. Leutgeb, J. K., Leutgeb, S., Moser, M. B. & Moser, E. I. Pattern separation in thedentate gyrus and CA3 of the hippocampus. Science 315, 961–966 (2007).
4. Leutgeb, S., Leutgeb, J. K., Treves, A., Moser, M. B. & Moser, E. I. Distinct
ensemble codes in hippocampal areas CA3 and CA1. Science 305, 1295–1298(2004).
5. McHugh, T. J. et al. Dentate gyrus NMDA receptors mediate rapid patternseparation in the hippocampal network. Science 317, 94–99 (2007).
6. Vazdarjanova, A. & Guzowski, J. F. Differences in hippocampal neuronal
population responses to modiﬁcations of an environmental context: evidence
for distinct, yet complementary, functions of CA3 and CA1 ensembles.
J. Neurosci. 24, 6489–6496 (2004).
7. Zhao, C., Deng, W. & Gage, F. H. Mechanisms and functional implications ofadult neurogenesis. Cell 132, 645–660 (2008).
8. Guzowski, J. F. et al. Mapping behaviorally relevant neural circuits withimmediate-early gene expression. Curr. Opin. Neurobiol. 15, 599–606 (2005).
9. Garthe, A., Behr, J. & Kempermann, G. Adult-generated hippocampal neurons
allow the ﬂexible use of spatially precise learning strategies. PLoS One 4, e5464(2009).
10. Yu, T. S., Zhang, G., Liebl, D. J. & Kernie, S. G. Traumatic brain injury-induced
hippocampal neurogenesis requires activation of early nestin-expressing
progenitors. J. Neurosci. 28, 12901–12912 (2008).
11. Sahay, A. et al. Increasing adult hippocampal neurogenesis is sufﬁcient toimprove pattern separation. Nature 472, 466–470 (2011).
12. Tronel, S. et al. Adult-born neurons are necessary for extended contextual
discrimination. Hippocampus 22, 292–298 (2012).
13. Saxe, M. D. et al. Ablation of hippocampal neurogenesis impairs contextual fear
conditioning and synaptic plasticity in the dentate gyrus. Proc. Natl Acad. Sci.
USA 103, 17501–17506 (2006).
14. Drew, M. R., Denny, C. A. & Hen, R. Arrest of adult hippocampal neurogenesis
in mice impairs single- but not multiple-trial contextual fear conditioning.
Behav. Neurosci. 124, 446–454 (2010).
15. Deng, W., Saxe, M. D., Gallina, I. S. & Gage, F. H. Adult-born hippocampal
dentate granule cells undergoing maturation modulate learning and memory inthe brain. J. Neurosci. 29, 13532–13542 (2009).
16. Marr, D. Simple memory: a theory for archicortex. Philos. Trans. R Soc. Lond. B
Biol. Sci. 262, 23–81 (1971).
17. Clelland, C. D. et al. A functional role for adult hippocampal neurogenesis inspatial pattern separation. Science 325, 210–213 (2009).
18. Nakashiba, T. et al. Young dentate granule cells mediate pattern separation,
whereas old granule cells facilitate pattern completion. Cell 149, 188–201(2012).
19. Aimone, J. B., Deng, W. & Gage, F. H. Resolving new memories: a critical lookat the dentate gyrus, adult neurogenesis, and pattern separation. Neuron 70,
589–596 (2011).
20. Sahay, A., Wilson, D. A. & Hen, R. Pattern separation: a common function for
new neurons in hippocampus and olfactory bulb. Neuron 70, 582–588 (2011).
21. Snyder, J. S., Soumier, A., Brewer, M., Pickel, J. & Cameron, H. A. Adult
hippocampal neurogenesis buffers stress responses and depressive behaviour.
Nature 476, 458–461 (2011).

NATURE COMMUNICATIONS | 3:1253 | DOI: 10.1038/ncomms2261 | www.nature.com/naturecommunications& 2012 Macmillan Publishers Limited. All rights reserved.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2261

22. Han, J. H. et al. Selective erasure of a fear memory. Science 323, 1492–1496(2009).
23. Franklin, K. B. J. & Paxinos, G. The Mouse Brain in Stereotaxic Coordinates(Academic Press, 2007).
24. Wang, S. H., Teixeira, C. M., Wheeler, A. L. & Frankland, P. W. The precisionof remote context memories does not require the hippocampus. Nat. Neurosci.
12, 253–255 (2009).
25. Fanselow, M. S. Conditioned and unconditional components of post-shock
freezing. Pavlov. J. Biol. Sci. 15, 177–182 (1980).
26. Frankland, P. W. et al. Consolidation of CS and US representations inassociative fear conditioning. Hippocampus 14, 557–569 (2004).

Author contributions
P.W.F., S.A.J. and Y.N. conceived and designed the experiments. Y.N. performed the arc
in situ ﬂuorescent hybridization analyses and statistical analyses. Y.N. and J.R.E.
performed the fear-conditioning experiment and associated analyses. Y.N., T.-S.Y. and
K.G.A. characterized adult neurogenesis and general behaviour in the TK þ mice. S.A.J.
and P.W.F. supervised the project and wrote the paper.

Additional information
Supplementary Information accompanies this paper on http://www.nature.com/
naturecommunications
Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests.

Acknowledgements
This work was supported by grants from the Canadian Institutes of Health Research to
P.W.F. (MOP86762) and S.A.J. (MOP74650). We thank Blake Richards for discussion ofthis work. J.R.E. and K.G.A. received support from the Ontario Ministry of Research andInnovation and Canadian Institutes of Health Research, respectively.

Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Niibori, Y. et al. Suppression of adult neurogenesis impairs
population coding of similar contexts in hippocampal CA3 region. Nat. Commun. 3:1253
doi: 10.1038/ncomms2261 (2012).

NATURE COMMUNICATIONS | 3:1253 | DOI: 10.1038/ncomms2261 | www.nature.com/naturecommunications& 2012 Macmillan Publishers Limited. All rights reserved.

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