ARTICLE
Received 24 Nov 2015 | Accepted 26 Jan 2016 | Published 26 Feb 2016

DOI: 10.1038/ncomms10838

OPEN

Neurogenesis-mediated forgetting minimizes
proactive interference
Jonathan R. Epp1, Rudy Silva Mera1, Stefan Köhler2,3, Sheena A. Josselyn1,4,5,6 & Paul W. Frankland1,4,5,6

Established memories may interfere with the encoding of new memories, particularly when
existing and new memories overlap in content. By manipulating levels of hippocampal
neurogenesis, here we show that neurogenesis regulates this form of proactive interference.
Increasing hippocampal neurogenesis weakens existing memories and, in doing so, facilitates
the encoding of new, conﬂicting (but not non-conﬂicting) information in mice. Conversely,
decreasing neurogenesis stabilizes existing memories, and impedes the encoding of new,
conﬂicting information. These results suggest that reduced proactive interference is anadaptive beneﬁt of neurogenesis-induced forgetting.

1 Program in Neurosciences and Mental Health, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8. 2 Department of Psychology, Brain and
Mind Institute, University of Western Ontario, London, Ontario, Canada N6A 5B7. 3 Baycrest Centre, Rotman Research Institute, Toronto, Ontario, Canada
M6A 2E1. 4 Department of Psychology, University of Toronto, Toronto, Ontario, Canada M5S 3GM. 5 Department of Physiology, University of Toronto,
Toronto, Ontario, Canada M5S 1A8. 6 Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada M5S 1A8. Correspondence and requests
for materials should be addressed to P.W.F. (paul.frankland@sickkids.ca).

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ew neurons are continuously generated throughout
adulthood in the subgranular zone of the hippocampus.
These new neurons become synaptically integrated into
hippocampal circuits over the course of weeks, and, once
sufﬁciently mature, are thought to contribute to encoding of
new hippocampus-dependent memories1. Consistent with thisview, suppressing hippocampal neurogenesis typically impairs
memory encoding2, whereas increasing neurogenesis facilitates
this process3,4.
However, because neuronal integration necessarily remodels
hippocampal circuits, computational models predict that theaddition of new neurons may lead to the destabilization ofexisting memories, rendering them harder to access as neurogenesis unfolds5–8. Consistent with this prediction, we recently
showed that increasing neurogenesis after training promoted
forgetting of spatial and contextual memories in adult mice9.
The ﬁnding that increasing neurogenesis promotes forgetting
suggests a revised view of the role of adult neurogenesis in the
hippocampus, with ongoing neurogenesis impacting hippocampal
memory function in two interdependent ways. First, theintegration of new neurons may ‘clear out’ old memories and,second, facilitate encoding of new memories10. Since higher
levels of hippocampal neurogenesis are typically associated withimproved cognitive function11,12, in what way might neurogenesis-mediated clearance improve memory function?
Here, we manipulated levels of hippocampal neurogenesis in
adult mice and examined how it modulates interactions between
previously acquired memories (or old information) and encodingof new information in the same behavioural task. We hypothesized that neurogenesis-mediated weakening of old memories
might facilitate encoding of new, conﬂicting information, aprocess that could be characterized as a reduction of proactive
interference10.
Results
Post-training exercise weakens existing spatial memories. To
examine how hippocampal neurogenesis might regulate proactive
interference, we ﬁrst trained mice in the hidden platform versionof the water maze, a form of spatial learning that depends on the
hippocampus13 (Fig. 1a). Across the course of training, micefound the platform with decreasing latency (Supplementary
Fig. 1a) and, in a probe test conducted at training completion,
searched selectively in the region of the pool that formerly
contained the platform (Supplementary Fig. 1b). Mice were
subsequently allowed continuous access to a running wheel in
their home cage or housed conventionally, and then re-tested 1
month later. Consistent with previous reports14, running robustly
increased hippocampal neurogenesis (increased numbers ofproliferating cells (Supplementary Fig. 1c) and immatureneurons (Fig. 1d,e)). Furthermore, running-induced increases inneurogenesis were associated with a reduction in spatial
selectivity when mice were re-tested (Fig. 1b), suggesting that
post-training running induced forgetting of the spatial memory9.
Reversal learning is enhanced in mice that exercised. To theextent that post-training exercise weakened memory for theoriginal platform location, we predicted that mice should be able
to learn a reversal platform location more readily10. To test this,we shifted the platform to the opposite quadrant of the pool andcontinued to train the mice. Over the course of this reversal
training, mice in the running group found the reversal platform
more efﬁciently (Fig. 1c), suggesting that neurogenesis-mediatedweakening of memory for the original platform location allowed
more efﬁcient encoding of a new, conﬂicting memory. Supporting
this idea, the ease with which mice found the reversal location2

was inversely related to the strength of the memory for theoriginal platform location (r ¼  0.33, Po0.05). However, thisbeneﬁt was transient. With extended training, latencies to locate
the reversal platform converged between running and sedentarygroups, and, in a probe test at the completion of reversal training,both groups expressed an equivalent preference for the newplatform location (Supplementary Fig. 1d).
Reduced proactive interference depends on neurogenesis. In
addition to increasing hippocampal neurogenesis, runninginduces a number of physiological changes15. To examine theimportance of neurogenesis in our running effect, we next tested
whether preventing the running-induced increase in neurogenesis
would prevent both forgetting and subsequent facilitated reversal
learning. To do this, we used mice in which levels of neurogenesis
could be genetically decreased. Speciﬁcally, we trained nestin-tk(TK þ ) mice16 and their WT (wild-type; TK  ) littermate controls
in the water maze (Fig. 1f). Mice were subsequently allowed
continuous access to a running wheel in their home cage orhoused conventionally and then re-tested 4 weeks later.
During this 4-week retention delay, all mice were treated withvanganciclovir via their food. In TK þ mice, vanganciclovir
administration ablates only dividing cells expressing the tk gene.
Accordingly, vanganciclovir treatment in TK þ mice limited therunning-induced increase in neurogenesis to TK  sedentarylevels (Fig. 1i,j). Most importantly, preventing the runninginduced increase in neurogenesis prevented both the forgetting(Fig. 1g) and facilitated reversal learning (Fig. 1h) normallyinduced by running, indicating that the increase in neurogenesis
is necessary for these interrelated effects on memory.
Decreasing neurogenesis increases proactive interference.
To the extent that post-training increases in neurogenesis induceforgetting, we predicted that post-training reductions in neurogenesis would have the opposite effect and stabilize hippocampus-dependent memories10. In our initial experiments we did
not observe reliable forgetting in control mice when we used a
4-week retention delay. Therefore, to increase the probability ofobserving forgetting in control mice, we extended the retention
delay to 6 weeks in this experiment (Fig. 2a). Using this delay weobserved forgetting, with TK  mice searching less selectively inthe probe test conducted at the end of the retention delay(Fig. 2b,c). Critically, genetic suppression of hippocampal
neurogenesis (Fig. 2f,g) in the post-training window attenuatedthis forgetting, with vanganciclovir-treated TK þ mice searching
more selectively compared with vanganciclovir-treated TK  mice
(Fig. 2b,c). Consistent with this preserved spatial selectivity,
TK þ mice were slower at ﬁnding the platform when it was
subsequently moved to the opposite quadrant (Fig. 2d,e),
indicating that relative preservation of the original memory
interfered with subsequent learning of a conﬂicting platform
location.
Increasing neurogenesis decreases proactive interference. We
next evaluated whether this pattern of results would generalize toother forms of hippocampus-dependent learning. To do this wetrained TK  and TK þ mice in a hippocampus-dependent17,18
odour–context paired associates task. In this task, mice weretrained in two contexts, each containing two containers scented
with cinnamon or coffee. During training, digging in one of thesescented containers (for example, coffee) was reinforced in context
A, whereas digging in the other scented container (for example,
cinnamon) was reinforced in context B (Fig. 3a,b; Supplementary
Table 1). All mice reached stable, high levels of performance(that is, correct on 480% of trials) by the completion of training

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c

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SessionFigure 1 | Running-induced neurogenesis promotes forgetting of previous information and consequently facilitates reversal learning. (a) After water
maze training mice were housed conventionally (n ¼ 24) or given running wheel access (n ¼ 24) for 4 weeks before testing and reversal training.
(b) Running increased the number of doublecortin þ neurons in the dentate gyrus (F1,18 ¼ 142.03, Po0.001). (c) Examples of doublecortin þ immatureneurons in the dentate gyrus of sedentary versus runner mice. Scale bars, 50 mm. (d) Running reduced retention of the platform location (Group  Zone
interaction: F1,46 ¼ 9.56, Po0.005), and (e) facilitated reversal learning (Group  Day interaction: F1,46 ¼ 6.31, Po0.0001). (f) TK þ and TK  mice weretrained in the water maze and were then given oral vanganciclovir treatment for 4 weeks. During this period, roughly half of the mice received running
wheels and the other half remained sedentary (TK  sedentary n ¼ 14, TK þ sedentary n ¼ 17, TK  runner n ¼ 20, TK þ runner n ¼ 23). (g) The runninginduced increases in doublecortin þ cells were attenuated in TK þ mice (Genotype  Group interaction: F1,43 ¼ 7.08, Po0.05). (h) Examples ofdoublecortin þ immature neurons in the dentate gyrus of sedentary versus runner mice TK  and TK þ mice. Scale bars, 50 mm. (i) Post-training runninginduced forgetting in TK  (ZoneTarget4ZoneOther; Po0.05) but not in TK þ mice (P40.05; Group  Zone interaction: F3,74 ¼ 5.17, Po0.005). (j) Runningfacilitated reversal training in TK  but not TK þ mice (Group  Day interaction: F12,292 ¼ 1.86, Po0.05; T, target platform zone, O, average of threeequivalent platform zones in other quadrants). Data analysis used ANOVA (b,d,g,i) and repeated-measures ANOVA (e,j). *Po0.05 by Newman–Keuls post
hoc tests for multiple comparisons. Data shown are mean±s.e.m.

(Fig. 3c). Mice were subsequently given continuous access to arunning wheel in their home cage or housed conventionally, andthen re-tested 4 weeks later. During this period, all mice weretreated with vanganciclovir via their food. In the test, TK  micethat exercised after initial training showed inferior memory forthe paired associates compared with sedentary controls. In
contrast, exercise did not impair memory for the original
paired associates in TK þ mice (Fig. 3d). Consistent with thewater maze results, these data indicate that post-training exercise
weakens an established hippocampus-dependent memory.
Unaffected paired associate memory in the TK þ mice with
reduced hippocampal neurogenesis (Fig. 3e) suggests that theseforgetting effects are mediated via a neurogenic mechanism.
Mice were subsequently retrained with the original context/
odour contingencies reversed. Acquisition of these reversed
paired associates was facilitated only in the TK  mice that had
exercised following learning the original paired associates(Fig. 3f). Consistent with the water maze results, these data

indicate that post-training exercise weakens an establishedhippocampus-dependent memory and, in turn, this facilitatesacquisition of new paired associates that conﬂict with the original
learning. Supporting this idea, the ease with which mice learned
the reversed paired associates was inversely related to the strengthof the memory for the original paired associates (r ¼  0.51,
Po0.01). Importantly, attenuating running-induced increases inhippocampal neurogenesis prevented both the forgetting andfacilitated reversal learning in TK þ mice, indicating thathippocampal neurogenesis mediates these interrelated effects on
memory.
Reduced interference is speciﬁc to new conﬂicting memories.
In the above experiments, neurogenesis-induced forgetting might
facilitate reversal learning by reducing conﬂict between the original learning and the new learning. Alternately, improved
reversal learning might reﬂect a more general enhancement of

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vGAN

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Percent zone time

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Figure 2 | Post-training reduction of adult neurogenesis prevents forgetting of water maze platform location. (a) Nestin TK  (n ¼ 12) and TK þ (n ¼ 12)
mice were trained in the water maze and were then treated with vanganciclovir for 6 weeks. Spatial memory retention was probed, and then mice weretrained on a reversed platform location. (b) Probe test performance in TK  and TK þ mice before and after vanganciclovir treatment. Whereas TK  micesearched the target zone less post-vanganciclovir (indicating forgetting of the platform location), TK þ searched target zone equivalently pre- versus postvanganciclovir (indicating an absence of forgetting; Genotype  Delay interaction: F1,22 ¼ 4.07, P ¼ 0.056). (c) Discrimination of the platform zone(compared with the opposite zone) declined in vanganciclovir-treated TK  but not TK þ mice in the 6-week retention delay (Genotype  Delay interaction:
F1,22 ¼ 5.20, Po0.05). (d) Doublecortin þ neurons were reduced in TK þ mice (F1,22 ¼ 47.03, Po0.0001). (e) Examples of doublecortin þ neurons in thedentate gyrus of TK  and TK þ mice. Scale bars, 50 mm. (f) Suppression of neurogenesis impaired reversal learning in TK þ mice (Genotype main effect:
F1,25 ¼ 5.14, Po0.05). (g) TK þ mice spent less time in the target zone compared with TK  mice (Genotype  Zone interaction: F1,22 ¼ 4.42, Po0.05).
Data analysis used ANOVA (b,c,d,g) and repeated-measures ANOVA (f). *Po0.05 by Newman–Keuls post hoc tests for multiple comparisons. Data shown
are mean±s.e.m.

learning following elevation of hippocampal neurogenesis (independent of degree of conﬂict)3,4 (see also Supplementary Fig. 2).
To distinguish between these alternatives we modiﬁed the odour–context paired associates protocol so that the degree of conﬂict
between old and new paired associates could be systematicallymanipulated. In this case, mice were trained as previously andagain reached stable, high levels of performance (that is, correcton 480% of trials) by the completion of training (Supplementary
Fig. 3a). Mice were subsequently given continuous access to arunning wheel in their home cage or housed conventionally, andthen re-tested 4 weeks later. Running increased neurogenesis(Supplementary Fig. 3b), and weakened memory for the original
paired associates (tested using diluted odours; Supplementary
Fig. 3c). As expected, this indicates that running weakened thememory for the original paired associates, impeding patterncompletion when cue salience was diminished.
Mice were subsequently trained on new paired associates thatwere either in conﬂict (that is, reversal of original context/odour
contingencies) or not (that is, new context/odour pairs) with theoriginal learning (Fig. 4a). Running-induced increases inhippocampal neurogenesis facilitated the acquisition of newpaired associates, but only in the ‘high conﬂict’ condition(Fig. 4b). The absence of effects on learning the ‘low conﬂict’
paired associates (Fig. 4c) argues against the possibility thatelevated neurogenesis has a general facilitatory effect on learning,at least under these conditions. Rather, facilitation was only
observed under conditions where there was conﬂict between
previously stored memories and new learning. Consistent withthis interpretation, in separate experiments we conﬁrmedthat exercise does not facilitate acquisition of paired associates(or subsequent reversed paired associates) when it occurs before
training (Supplementary Figs 4,5).
4Discussion
Previously we showed that elevated levels of neurogenesis
promote forgetting of established memories9. Here wehypothesized that one beneﬁt of this neurogenesis-mediatedforgetting might be more efﬁcient encoding of new, conﬂicting
information. Using two distinct hippocampus-dependent tasks,
we provide support for this idea: Increasing hippocampal
neurogenesis after initial learning reduced this form of
proactive interference, whereas decreasing hippocampal
neurogenesis after learning increased this form of proactive
interference. Our results suggest that heightened levels of
hippocampal neurogenesis might allow animals to react more
rapidly to changing contingencies in otherwise stableenvironments.
Various roles have previously been assigned to the hippocampus with respect to mitigating interference (for example, seerefs 19–23). In particular, some earlier studies investigated therelationship between levels of hippocampal neurogenesis andmemory interference (as well as the closely related concept ofcognitive ﬂexibility)24–29. For example, in one study hippocampal
neurogenesis levels were experimentally suppressed in mice. Mice
were subsequently trained to ﬁnd a hidden platform in a ﬁxed
location in the water maze. In a second phase of training, theplatform was moved to a different location in the pool (that is,reversal learning). Reversal, but not initial, learning was impairedin mice in which hippocampal neurogenesis was reduced25.
However, in this and related studies24,26–29, manipulation ofhippocampal neurogenesis levels occurred before initial learning.
Therefore, it is unclear whether improved reversal learning isrelated to differences in initial learning or differences that emerge
at the reversal stage. By restricting our manipulations ofneurogenesis to the post-learning period, here we were able to

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a

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50

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A2, B1A1, B2A2, B1A1, B2A2, B1A1, B2A2, B1f

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odor 2(cinnamon)–

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1Figure 3 | Post-training induction of neurogenesis promotes forgetting of an odour–context paired-associates memory and consequently facilitates
reversal learning. (a) Mice were presented with two different cups of scented bedding in two different contexts and were trained to dig in the bedding toreceive a food reward. (b) TK  and TK þ mice were trained on odour–context pairings A1 and B2. Subsequently the mice either ran (TK  n ¼ 6, TK þ n ¼ 9)
for 4 weeks or remained sedentary (TK  n ¼ 5, TK þ n ¼ 8). During this period all mice received oral vanganciclovir treatment. After 4 weeks the mice
were tested on the original odour–context pairings and were then subjected to reversal learning (that is, A2 and B1). (c) At the end of training all groups
performed 480% correct (F1,24 ¼ 0.03, P40.86). (d) Post-training running induced forgetting in TK  but not in TK þ mice (P40.05; Group  Genotype
interaction: F1,24 ¼ 8.66, Po0.005). (e) The running-induced increase in doublecortin þ cells was attenuated in TK þ mice (Genotype  Group interaction:
F1,16 ¼ 5.29, Po 0.05). (f) Running facilitated reversal training in TK  but not TK þ mice (Group  Genotype interaction: F1,24 ¼ 8.66, Po0.005). Data
analysis used ANOVA (c,d,e) and repeated-measures ANOVA (f). *Po0.05 by Newman–Keuls post hoc tests for multiple comparisons. Data shown are
mean±s.e.m.

b

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Figure 4 | High- but not low-interference reversal learning is facilitated by running-induced neurogenesis. (a) Mice were trained on odour–context
pairings A1 and B2. Subsequently the mice either ran (n ¼ 16) for 4 weeks or remained sedentary (n ¼ 12). After 4 weeks the mice were tested on theoriginal odour–context pairings and were then subjected to either reversal learning (that is, A2 and B1 (high interference)) or were trained on two new
odour–context pairings (that is, C3 and D4 (low interference)). (b,c) Runners learned faster than sedentary mice in the high interference (Group main
effect: F1,72 ¼ 94.79, Po0.0001; Group  Session interaction: F6,72 ¼ 3.40, Po0.01) but not low-interference (Group main effect: F1,72 ¼ 0.00, P40.05)
condition. Data analysis used repeated-measures ANOVA (b,c). *Po0.05 by Newman–Keuls post hoc tests for multiple comparisons. Data shown
are mean±s.e.m.

show that decreased proactive interference is directly related toneurogenesis-mediated forgetting of the original learning. Thiseffect may ultimately contribute to increases in cognitive
ﬂexibility.
A perhaps unexpected observation was that increasing
hippocampal neurogenesis did not generally improve learning.
Rather, in the paired associate paradigm, facilitated acquisition
was only observed in the high interference condition
where paired associates were reversed. In contrast, in the lowinterference condition, as well as in related control experiments,

increasing hippocampal neurogenesis did not facilitate paired
associate learning. This contrasts with some previous studies
where increasing neurogenesis before training facilitated memory
formation (for example, see refs 3,4,30). Indeed, in the current
study we also observed improved acquisition in the water maze
when running occurred before training (Supplementary Fig. 2).
However, it is worth noting that these types of improvements are
not universally observed (for example, refs 9,31,32). There may be
a number of reasons why this is the case—ranging fromdifferences in amount and duration of training, task difﬁculty,

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as well as the timing of the increase in neurogenesis with respect
to the initiation of training.
Finally, what is forgotten? Experiences are thought to be
represented in parallel hippocampal–cortical networks. According to one prominent model33, hippocampal representations
contain high levels of contextual detail, whereas corticalrepresentations are more schematic in nature and contain fewer
contextual details. Therefore, continuous integration of newneurons into hippocampal circuits would be predicted to degrade
this contextually dense hippocampal representation. The
observed facilitation of reversal learning following elevation ofhippocampal neurogenesis in the water maze and pairedassociates task is consistent with such an account. In particular,
in the paired-associates task, uncoupling odour-reward
relationships from their contexts enables more efﬁcient reversal
learning.
Methods
Mice. Two strains of mice were used in these experiments. First, we used WT micethat were derived from a cross between 129Svev and C57Bl/6N mice (Taconic).
Second, to suppress adult neurogenesis we used transgenic mice expressing a
modiﬁed HSV-tk gene under control of the nestin promoter (TK þ mice)16,34.
These mice were maintained on a C57Bl/6N background (48 crosses), andexperimental mice were generated by crossing TK þ mice with 129Svev WT mice.
In all experiments, we used both male and female mice. Breeding was conducted in
the Hospital for Sick Children animal facility. Mice were housed in standard
laboratory conditions with 3–5 mice per cage and had free access to food andwater. Rooms were maintained on a 12 h light/dark cycle and behavioural testing
occurred during the light phase of the cycle. Experiments were conducted inaccordance with the Canadian Council on Animal Care guidelines and theprotocols were approved by the animal care committee at the Hospital for Sick
Children.
Manipulation of neurogenesis. To increase adult neurogenesis, mice were given
continuous access to a running wheel (Fast-Trac running wheels, Bioserv) in theirhome cages9. When the wheel was initially introduced to the cage each mouse wasgently placed on the wheel to facilitate running wheel use. In a subset of cages,
wheel revolutions were continuously recorded to track amount of running. Eachmouse ran an average of 4.7 km (±0.53 km) per day during the running period.
To decrease adult neurogenesis TK þ mice and non-transgenic littermate(TK  ) control mice were administered vanganciclovir orally. Vanganciclovir was
mixed into powdered chow (0.08%, B70 mg kg  1 daily)2, and mice had ad libitum
access to the chow in their home cage.
Water maze apparatus and general procedures. Before water maze training,mice were handled for 5 min per day over 5 consecutive days. Water maze training
was conducted in a pool 120 cm in diameter ﬁlled to within 10 cm of the rim.
The water was made opaque with non-toxic white paint. A hidden platform(10 cm diameter) was submerged in the centre of the northwest quadrant of thepool. Training consisted of ﬁve daily sessions each comprising six trials with amaximum trial length of 60 s. At the start of each trial, the mouse was lowered into
the water in one of four start positions (located in the north, south, east and west,
respectively), and the order of these start positions was varied pseudorandomly
across trials. At the end of each trial the mouse remained on the platform for an
additional 15 s. If the mouse failed to locate the platform within 60 s it was gently
guided to the platform by the experimenter at the completion of the trial.
To assess spatial preference, 24 h following the completion of training, mice
were given a 60 s probe trial. In this trial the platform was removed from the pool,and a southeast start position was used. The amount of time spent in a zone
centred on the former platform location (radius 15 cm) was compared with thetime spent in equivalent zones centred in the other three quadrants. The zone
represents 6.7% of total pool-surface area.
Reversal training used identical procedures except that the platform was
relocated to the middle of the southeast quadrant. Mice were trained for 5 days,
and each daily training session comprised three trials. Twenty-four hours following
reversal training, spatial preference was assessed in a probe test. In this reversal
probe test we additionally computed a discrimination score (time spent in thereversal platform zone/time spent in the original platform zone).
Water maze experiment 1. Does post-training exercise reduce retention andfacilitate reversal learning? WT mice were trained in the water maze for 5 days.
Twenty-four hours after the completion of training, mice were given a probe test,
and then either housed with a running wheel (runner group; n ¼ 24) or without arunning wheel (sedentary group; n ¼ 24). Four weeks later, retention was evaluatedin a probe test and, starting the following day, mice were trained to locate the6platform in the opposite quadrant of the pool for 5 days. Twenty-four hours after
the completion of this reversal training, a probe trial was conducted to assess
memory for the new platform location.
Water maze experiment 2. Are exercise-induced changes in retention andreversal learning dependent upon neurogenesis? TK þ and their WT littermate
control mice were trained in the water maze for 5 days, and spatial memory wasassessed 24 h following the completion of training. Starting the next day, all mice
were adminsitered vanganciclovir via chow in their home cage for a 4-weekretention period. During this period, some mice had access to running wheels(WT runner, n ¼ 20; TK þ runner, n ¼ 23), while others were housed conventionally (WT sedentary, n ¼ 14; TK þ sedentary, n ¼ 17). Spatial memory wastested in a probe test, and then, the following day, reversal learning was initiated.
Twenty-four hours following the completion of reversal training, mice were
presented with a second probe test.
Water maze experiment 3. Does post-training suppression of neurogenesis
increase retention and retard reversal learning? TK þ (n ¼ 12) and their WT
littermate control (n ¼ 12) mice were trained in the water maze for 5 days, andspatial memory was assessed 24 h following the completion of training. Starting thenext day, all mice were administered vanganciclovir via chow in their home cage
for a 6-week retention period. Spatial memory was then tested in a probe test.
One day later, reversal learning was initiated. Twenty-four hours following thecompletion of reversal training, spatial preference was assessed in a second
probe test.
Water maze experiment 4. Does pre-training exercise facilitate acquisition of
spatial learning? Starting at 8 weeks of age, WT mice were given access to running
wheels (n ¼ 15) or remained sedentary (n ¼ 16) for a period of 4 weeks. Mice weretrained in the water maze for 5 days, and spatial memory was assessed in a probe
test 24 h after the completion of training.
Paired-associates task. We trained TK þ and their WT littermate control miceon an odour/context paired-associates task17,18 in which the mice were required to
dig in a container of bedding scented with 1% spice (for example, coffee or
cinnamon) to receive a reward. Odour pairs were presented in two different
contexts. In context A odour 1 was rewarded while in context B odour 2 was
rewarded. The experimental procedures involved the following phases in sequence.
Habituation and shaping. Mice were ﬁrst habituated to the testing apparatus for 2days. Testing was conducted in empty mouse cages containing two 60  15 mm
petri dishes ﬁlled with corncob bedding. During the habituation sessions, thereward, chocolate-ﬂavoured sprinkles (10 mg, McCormick, Canada), was available
on top of the bedding. Next, the mice were shaped to dig in unscented bedding toreceive the reward by ﬁrst partially, and then completely, burying the sprinkles inthe bedding. Once the mice could consistently dig to retrieve a sprinkle, the reward
was no longer buried in the bedding. Instead, a single sprinkle was delivered on topof the bedding immediately after the mouse dug in the bedding. Shaping took ﬁvesessions.
Simple discrimination. Two different cups were scented with vegetable seasoningor tumeric, and mice were rewarded for digging only in the vegetable scented
bedding. Each trial ended after the mice made their ﬁrst choice. Simple
discrimination training typically took 5 sessions to reach a criterion of 80% correct.
Paired-associates training. We next introduced two new contexts and two new
odours. Context A was a white Plexiglass box and context B was a black and white
striped box (context B). The dimensions of both contexts were the same (32 
18  14 cm). The new odours were coffee (odour 1) and cinnamon (odour 2). In
context A, odour 1 was rewarded but in context B odour 2 was rewarded. Mice
were initially trained with 10 trials per day in a single context. Contexts alternatedacross days. Once a mouse reached 70% correct in both contexts we began presenting the mice with ﬁve trials of each context per session in a pseudorandom
pattern (A-B-A-AB-A-B-B-A-B). Mice were trained until they reached a criterionof at least 80% correct in 3 consecutive sessions. Once this criterion had beenreached, mice were housed conventionally (n ¼ 13) or given free home cage accessto a running wheels (n ¼ 15) for the next 4 weeks. All mice were fed a powderedfood diet containing vanganciclovir during the 4-week period.
Testing. Memory for the paired associates was tested following the 4-weekretention delay. This session comprised 10 trials, with 5 trials in each context.
Reversal training. Next, mice were trained on a reversal learning procedure. In
this task the original odour-context pairings were reversed. In context A, odour 2(for example, cinnamon) was now rewarded and in context B odour 1 (for example,

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10838

coffee) was rewarded. Mice were given 20 trials per session with 10 trials of eachcontext per session for 3 days.
Interference training. In a separate experiment we assessed the impact of varying
degrees of interference during the reversal learning period in WT mice. Memory
for the paired associates was tested in six daily sessions following the 4-weekretention delay. These sessions comprised 10 trials, with 5 trials in each context.
For sessions 1–3 we used the original (training) odour concentrations (100% odour
condition). For sessions 4–6 we used 50% (that is, 0.5% spices in corncob bedding)of the original odour concentration to evaluate retention with weaker retrieval cues.
Following testing WT mice were assigned to either high interference (sedentary,n ¼ 6; runner, n ¼ 8) or low interference (sedentary, n ¼ 6; runner, n ¼ 8) training.
High-interference training. In the high-interference condition, the originalodour–context pairings used during training were reversed. In context A, odour 2(for example, cinnamon) was now rewarded and in context B odour 1 (for example,
coffee) was rewarded. Mice were given 20 trials per session with 10 trials of eachcontext per session for 7 days.
Low-interference training. In the low-interference condition, two new odours andtwo new contexts were presented. Context C (polka dot wallpaper) and context D(silver metal box) were paired with odours 3 (ginger) and 4 (oregano). Mice were
given 20 intermixed trials per session for 7 days.
Pre-training running. In separate experiments mice were habituated, shaped andtrained in simple discrimination training. They were then housed conventionally(n ¼ 16) or given running wheel access (n ¼ 16) for 4 weeks before beginning thepaired associate training for 12 days. Two different odour–context pairings wereused for training. In the ﬁrst condition the mice were trained with the normal
odour–context pairings (contexts A and B and odours 1 and 2, n ¼ 8 per group). In
the second condition mice were trained with the odour–context pairings that wereotherwise used for the low-interference condition (contexts C and D and odours 3
and 4, n ¼ 8 per group). In a third condition mice were trained on the normal
odour–context pairings (A1, B2) for a period of 6 days and were then trained on
the reversal condition for an additional 6 days (A2, B1).
Histology. Mice were deeply anaesthetised with 4% chloral hydrate and were
perfused transcardially with 40 ml of 0.1 M PBS followed by 40 ml of 4% bufferedformaldehyde. Brains were extracted and post-ﬁxed in 4% formaldehyde for 24 h
and were then cryoprotected in 30% sucrose for 72 h. Sections of 50 mm were cut ona cryostat, and free-ﬂoating sections were stored in 0.02% sodium azide in 0.1 M
PBS. Sections were incubated for 48 h in goat anti-doublecortin primary antibody(Santa Cruz, SC-8066) diluted 1:200 in 0.3% Triton-X and 3% normal serum. The
sections were then incubated for 24 h in Alexa488 Donkey anti-goat secondary
antibody diluted 1:500 in PBS. The tissue was counterstained with DAPI andcoverslipped with PVA-DABCO. Doublecortin was quantiﬁed using an epiﬂuorescent microscope and  40 objective. Cells were counted through theanterior–posterior extent of the dentate gyrus using a 1/6 sampling fraction.
Statistical analysis. Data were analysed using ANOVA and repeated-measures
ANOVA with an a ¼ 0.05. Where applicable, Newman–Keuls post hoc comparisons were computed. All analyses were carried out using the software package
Statistica (Statsoft, version 10).

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Acknowledgements
This work was supported by grants from the Canadian Institutes of Health Research to
P.W.F. (MOP77561) and S.A.J. (MOP74650). J.R.E. received support from the Hospitalfor Sick Children and the Brain & Behavior Research Foundation.

Author contributions
J.R.E., S.K., S.A.J. and P.W.F conceived and designed the experiments. J.R.E. and R.S.M.
performed the experiments. J.R.E conducted the analyses. J.R.E., S.K., S.A.J. and P.W.F
wrote the paper.

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Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests.
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How to cite this article: Epp, J. R. et al. Neurogenesis-mediated forgetting minimizes
proactive interference. Nat. Commun. 7:10838 doi: 10.1038/ncomms10838 (2016).

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