The Journal of Neuroscience, November 19, 2014 • 34(47):15793–15803 • 15793

Systems/Circuits

Posttraining Ablation of Adult-Generated Olfactory Granule
Cells Degrades Odor–Reward Memories
X Maithe Arruda-Carvalho,1,2 X Katherine G. Akers,1 Axel Guskjolen,1,3 Masanori Sakaguchi,1 Sheena A. Josselyn,1,2,3,4
and Paul W. Frankland1,2,3,4
1Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada, M5G 1X8, and 2Institute of Medical Science andDepartments of 3Physiology and 4Psychology, University of Toronto, Toronto, Ontario, Canada, M5S 1A8Proliferation of neural progenitor cells in the subventricular zone leads to the continuous generation of new olfactory granule cells (OGCs)
throughout life. These cells synaptically integrate into olfactory bulb circuits after ⬃2 weeks and transiently exhibit heightened plasticity
and responses to novel odors. Although these observations suggest that adult-generated OGCs play important roles in olfactory-related
memories, global suppression of olfactory neurogenesis does not typically prevent the formation of odor–reward memories, perhaps
because residual OGCs can compensate. Here, we used a transgenic strategy to selectively ablate large numbers of adult-generated OGCs
either before or after learning in mice. Consistent with previous studies, pretraining ablation of adult-generated OGCs did not prevent theformation of an odor–reward memory, presumably because existing OGCs can support memory formation in their absence. However,ablation of a similar cohort of adult-generated OGCs after training impaired subsequent memory expression, indicating that if these cells
are available at the time of training, they play an essential role in subsequent expression of odor–reward memories. Memory impairment
was associated with the loss of adult-generated OGCs that were ⬎10 d in age and did not depend on the developmental stage in which they
were generated, suggesting that, once sufficiently mature, OGCs generated during juvenility and adulthood play similar roles in theexpression of odor–reward memories. Finally, ablation of adult-generated OGCs 1 month after training did not produce amnesia,
indicating that adult-generated OGCs play a time-limited role in the expression of odor–reward memories.
Key words: adult neurogenesis; memory; olfactory

Introduction
Neural progenitor cells in the subventricular zone (SVZ) continuously generate cells that migrate via the rostral migratory stream
to populate the olfactory bulb (OB) throughout life (Ming andSong, 2005; Belvindrah et al., 2009; Lepousez et al., 2013). The
vast majority of these cells (⬎90%; Winner et al., 2002; Lazariniand Lledo, 2011) differentiate into olfactory granule cells (OGCs)and extend dendrites into the external plexiform layer. After ⬃2weeks, they form reciprocal dendrodendritic contacts with mitralcells, the principal neurons in the OB. Around the same time,
input synapses from higher-order regions form, and, soon after,
OGCs exhibit heightened plasticity at these input synapses (Carleton et al., 2003; Gao and Strowbridge, 2009; Nissant et al.,2009), are more responsive to novel odors (Magavi et al., 2005;
Received June 1, 2013; revised Oct. 7, 2014; accepted Oct. 12, 2014.
Author contributions: M.A.-C., K.G.A., M.S., S.A.J., and P.W.F. designed research; M.A.-C., K.G.A., and A.G. performed research; M.A.-C. and K.G.A. analyzed data; M.A.-C., K.G.A., S.A.J., and P.W.F. wrote the paper.
This work was supported by grants from Brain Canada (P.W.F.). and Canadian Institutes of Health Research
Grants MOP86762 (P.W.F.) and MOP74650 (S.A.J.). M.A.-C. and K.G.A. were supported by fellowships from theOntario Graduate Scholarship program (M.A.-C.), the Hospital for Sick Children (M.A.-C.), and the Canadian Institutesof Health Research (K.G.A.). We thank members of the Frankland/Josselyn laboratories for discussions and technicalassistance.
The authors declare no conflicts of interest.
Correspondence should be addressed to Paul W. Frankland, SickKids, Hospital for Sick Children, 686 Bay Street,Toronto, ON, Canada, M5G 0A4. E-mail: paul.frankland@sickkids.ca.
DOI:10.1523/JNEUROSCI.2336-13.2014
Copyright © 2014 the authors 0270-6474/14/3415793-11$15.00/0

Belnoue et al., 2011), and are preferentially activated by recall of
an odor–reward memory (Sultan et al., 2010). Furthermore, subsequent optical stimulation of this population of cells facilitates
the formation of an odor–reward memory (Alonso et al., 2012).
These studies suggest that adult-generated OGCs play an important role in the formation of odor memories, but whether they
represent an essential component of the odor-related memory
trace or, more simply, modulate the efficiency of OB processing(e.g., by synchronizing mitral cell activity; Mouret et al., 2009;
Urban and Arevian, 2009; Cleland, 2010), is less clear.
Examinations of the effect of suppressing olfactory neurogenesis on subsequent odor learning are potentially useful in exploring the role of adult-generated OGCs in odor memory. However,these types of interventions have produced mixed results, withexperimental reduction in olfactory neurogenesis frequently notpreventing the acquisition of odor memory (Imayoshi et al.,2008; Breton-Provencher et al., 2009; Lazarini et al., 2009; Valley
et al., 2009; Sultan et al., 2010), likely because of the potential forexisting OGCs to compensate and mask potential deficits. A
potentially more powerful approach would be to target adultgenerated OGCs after training, once they have had an opportunity to integrate into an odor-related memory trace (Frankland,2013). To address this question, we used a transgenic strategy toconditionally express diphtheria toxin receptors (DTRs) in a Cre
recombinase-dependent manner (Buch et al., 2005; ArrudaCarvalho et al., 2011). To restrict DT-induced apoptosis to adult-

Arruda-Carvalho et al. • Adult-Generated Olfactory Granule Cells and Odor Memory

15794 • J. Neurosci., November 19, 2014 • 34(47):15793–15803

Figure 1. DT-based ablation. Schematic of the tag-and-ablate strategy for ablating mature
adult-generated neurons. In adult nestin–Cre ERT2⫹/iDTR ⫹ mice (2⫻Tg), TAM administration
leads to permanent expression of DTRs in neural progenitor cells and their progeny. Subsequentadministration of DT ablates this tagged population of adult-generated neurons only.

generated neurons, we crossed inducible DTR (iDTR) mice withnestin–Cre ERT2 mice in which a tamoxifen (TAM)-inducible Cre
recombinase is expressed under the control of a nestin promoter(Imayoshi et al., 2008). In adult offspring from this cross, TAM
administration induces permanent expression of DTRs in neuralprogenitor cells and their progeny, and subsequent administration of DT ablates only this tagged population of cells (Fig. 1).
By using a line of nestin–Cre ERT2 mice with highest expression
in the hippocampus, we previously used this “tag-and-ablate”
approach to examine whether adult-generated dentate granulecells play an essential role in hippocampal memory (ArrudaCarvalho et al., 2011). Here, we use a line of nestin–Cre ERT2 micewith highest expression in the SVZ to ask whether adultgenerated OGCs play an essential role in odor–reward memory.
Our main finding is that posttraining ablation of this populationof adult-generated OGCs impairs subsequent expression of anodor–reward memory. Memory loss is associated with loss of
OGCs that are ⬎10 d in age and does not depend on the developmental stage (juvenility vs adulthood) in which OGCs weregenerated. Finally, ablation of the same population of cells at
remote time points after training is ineffective, suggesting that
adult-generated OGCs play a time-limited role the expression ofthese types of memories.

Materials and Methods
Micenestin–CreERT2 mice. nestin–Cre ERT2⫹ mice express a TAM-inducible
form of Cre recombinase under the control of a nestin promoter, as
described previously (Imayoshi et al., 2008). We compared recombination in two lines of nestin–Cre ERT2 mice corresponding to lines 4 and 5-1
in the study by Imayoshi et al. (2008), and we chose to use line 5-1
because the recombination rate was significantly higher in the SVZ.
iDTR mice. iDTR mice have been described previously (Buch et al.,2005; Gropp et al., 2005). In iDTR-expressing (iDTR ⫹) mice, the geneencoding DTR [simian Hbegf (heparin-binding epidermal growth
factor-like growth factor)] is under the control of the ubiquitous Rosa26
locus promoter, but expression of the DTR transgene is dependent on the
Cre recombinase-mediated removal of a transcriptional STOP cassette. It
is important to note that neither high doses of DT in wild-type mice
(Saito et al., 2001) nor expression of DTR alone (without DT) (Buch etal., 2005) produces behavioral abnormalities or cell death.
Rosa–LacZ mice. The Rosa–LacZ reporter mice have been described
previously (Zambrowicz et al., 1997). Similar to iDTR mice, the geneencoding LacZ is under the control of the ubiquitous Rosa26 locus promoter, and expression of the LacZ transgene is dependent on the Cre
recombinase-mediated removal of a transcriptional STOP cassette. We
crossed this reporter line with nestin–Cre ERT2 mice to initially characterize TAM-induced recombination because, unlike LacZ that is expressed in the nucleus, DTRs are expressed as membrane proteins andare therefore difficult to quantify precisely. Importantly, we observed

similar patterns of LacZ and DTR expression after TAM treatment whencrossed with nestin–Cre ERT2 mice.
All lines were maintained on a C57BL/6 background (Taconic Farms).
Genotypes were determined by PCR analysis of tail DNA samples as
described previously (Zambrowicz et al., 1997; Buch et al., 2005; Imayoshi et al., 2008). nestin–Cre ERT2⫹ mice were bred with iDTR ⫹/⫺ or
iDTR ⫹/⫹ mice, resulting in nestin–Cre ERT2⫹/iDTR ⫹, nestin–Cre ERT2⫹/
iDTR ⫺, nestin–Cre ERT2⫺/iDTR ⫹, or nestin–Cre ERT2⫺/iDTR ⫺ offspring.
An equivalent breeding strategy was used for the LacZ reporter line. Except
in the case of wild-type mice, all of transgenes were kept as heterozygous
in the chromosome of each transgenic mouse to avoid possible complications by overexpressing Cre recombinase (Forni et al., 2006) or loss of
the Rosa allele (Zambrowicz et al., 1997). In most ablation experiments,
we compared double transgenic mice (2⫻Tg, nestin–Cre ERT2⫹/iDTR ⫹)to control, single transgenic littermate mice (CTR, nestin–Cre ERT2⫹/
iDTR ⫺ or nestin–Cre ERT2⫺/iDTR ⫹). In one experiment, nestin–
Cre ERT2⫹ mice from a Rosa–LacZ cross were also used as controls. BothCTR and 2⫻Tg mice were treated with TAM and subsequently DT (seebelow).
All mice were bred in our colony at the Hospital for Sick Children andmaintained on a 12 h light/dark cycle with access to food and water ad
libitum. Before all behavioral experiments, mice were handled for 2
min/d for 5 d. Male and female offspring were used in all experiments. All
experiments were performed during the light cycle and approved by the
Hospital for Sick Children Animal Care and Use Committee.

Drugs
TAM treatment. TAM (Sigma) was dissolved in minimal ethanol (10%)
and suspended in sunflower seed oil (30 mg/ml; Lagace et al., 2007). Micereceived daily injections (180 mg/kg, i.p.) for 5 d. TAM injections started
when mice were 8 (adult) or 4 –5 (juvenile) weeks old.
DT treatment. Preparation and delivery of DT has been described previously (Han et al., 2009). DT (Sigma) was dissolved in PBS (500 ng/␮l)
and readily crosses the blood– brain barrier (Wrobel et al., 1990). Micereceived daily injections of DT (16 ␮g/kg, i.p) for 5 d.

Immunohistochemistry
Mice were perfused transcardially with PBS (0.1 M) and 4% paraformaldehyde (PFA). Brains were removed, fixed overnight in PFA, and transferred to 0.1 M PBS. Coronal sections (40 ␮m) were cut using a vibratome(Leica VT1200S). The following primary antibodies were used: goatpolyclonal anti-doublecortin (DCX; 1:4500; Santa Cruz Biotechnology),
rabbit polyclonal anti-ER␣ (1:1000; Santa Cruz Biotechnology), mousemonoclonal anti-NeuN (1:1000; Millipore), rabbit polyclonal anti-LacZ(1:6000; Invitrogen), mouse monoclonal anti-nestin (1:150; BD
Pharmingen), rabbit polyclonal anti-Ki67 (1:200; Abcam), rabbit polyclonal anti-cleaved caspase-3 (Asp175; 1:1000; Cell Signaling Technology), and rabbit polyclonal anti-Egr-1 (588; zif268; Santa Cruz
Biotechnology). All sections were treated with 1% hydrogen peroxidase.
Sections were then incubated overnight with the primary antibody andthen for 60 min at 20°C with HRP-conjugated or biotinylated secondary
antibodies (1:750; Jackson ImmunoResearch). Signals were amplified
and visualized using Vectastain Elite ABC kit (Vector Laboratories), tyramide signal amplification, and/or Alexa Fluor-conjugated streptavidin(Invitrogen). Sections were mounted on slides with Permafluor anti-fademedium.

Imaging and quantification
All images were acquired using epifluorescent (either a Nikon Eclipse 80i
or an Olympus BX61) or confocal (LSM 710 Zeiss) microscopes. To
calculate cell number, cell density, or proportion of double-positive cells,
we used 1⁄5 systematic section sampling fractions covering the entire anterior–posterior extent of the SVZ or OB. To calculate the proportion of
double-labeled cells, confocal 1 ␮m Z-stack images were obtained using
ZEN software (Zeiss) with a minimal interval of 15 ␮m to prevent duplicate counts of the same cell. We quantified Ki67 ⫹ cells throughout theanterior–posterior extent of the SVZ using a 10⫻ objective on the Nikon
epifluorescence microscope. We estimated the total number of LacZ ⫹cells after TAM treatment using the optical fractionator method on the
Olympus epifluorescence microscope using a 60⫻, 1.45 numerical aper-Arruda-Carvalho et al. • Adult-Generated Olfactory Granule Cells and Odor Memory

ture objective and a motorized x–y–z stage attached to a computer with
Stereoinvestigator 9.1 software (MBF Bioscience; Chen et al., 2004). A
random systematic sampling was used for these stereological analyses(section interval of 1⁄5, grid size of 300 ⫻ 300 ␮m, two-dimensionalcounting frame of 60 ⫻ 60 ␮m using fractionators of 30 ␮m thickness).
Tissue thickness measured in each counting frame was used to estimate
the total number of LacZ ⫹ cells in the entire OB. Conditions were optimized to obtain a Gundersen coefficient of error below 0.05 (Gundersen
et al., 1999).We quantified cleaved caspase-3 ⫹ cells throughout the entire anterior–posterior extent of the OB using the Nikon microscope.

General behavioral apparatus and procedures
Odor–reward memory task. The apparatus and training schedule havebeen described previously (Imayoshi et al., 2008; Akers et al., 2011). Mice
were food restricted to maintain ⬃90% of normal body weight andtrained for 4 d in the odor–reward task. On each day of training, mice
were given one reinforced and one non-reinforced trial. In all trials, theodorant solution [the enantiomers (⫹) or (⫺) carvone, diluted in mineral oil at a concentration of 10 ⫺2] was pipetted onto filter paper. The
filter paper was placed inside a Petri dish (60 ⫻ 15 mm) with a perforated
cover to allow for odor flow. The scented Petri dish was covered in
bedding and placed at one end of a mouse cage (32 ⫻ 18 ⫻ 14 cm).
During the reinforced trial, six sugar pellets (10 mg each) were placed
beneath the bedding on top of the scented Petri dish. During the nonreinforced trial, no sugar was buried below the bedding. The reinforced
odor and order of odor exposure were counterbalanced across mice.
Training trials were 5 min in duration.
For the probe tests, mice were placed in a cage with both odors present
but in the absence of sugar for 10 min. Odor–reward memory was measured as time spent digging over the correct odor. All probe tests werevideo recorded and manually coded. A preference index was calculated as[(diggingreinforced odor ⫺ diggingnon-reinforced odor)/(diggingreinforced odor
⫹ diggingnon-reinforced odor)]. Performance in this task has been shown tobe hippocampus independent (Akers et al., 2011).

Specific experimental protocols
Characterization of TAM-induced recombination. Four- to five- andeight-week-old nestin–Cre ERT2⫹/Rosa–LacZ ⫹ mice (n ⫽ 5–7) weretreated with TAM. Three weeks after the completion of TAM treatment,
the number, distribution, and cellular phenotype of recombined LacZ ⫹cells were quantified using immunohistochemical methods.
Characterization of TAM-induced recombination and ablation in 2⫻Tg
mice. Four- to 5-week-old 2⫻Tg mice were treated with TAM. Three
weeks after the completion of TAM treatment, mice received daily injections of PBS (n ⫽ 4) or DT (n ⫽ 5) for 5 d. Twenty-four h after the final
DT injection, mice were perfused, and cleaved caspase-3 expression wasquantified using immunohistochemical methods.
Open field. CTR (4 –5 weeks old, n ⫽ 15; 8 weeks old, n ⫽ 10) and2⫻Tg (4 –5 weeks old, n ⫽ 10; 8 weeks old, n ⫽ 7) mice were treated with
TAM. Three weeks later, mice received daily injections of DT for 5 d.
Twenty-four hours after the final DT injection, mice were placed in thecenter of a square-shaped open-field arena (45 ⫻ 45 ⫻ 20 cm) andallowed to explore for 10 min. The open-field apparatus was constructedof Plexiglas and was dimly lit from above. Mouse location was tracked bya camera located above the open field, and total distance traveled as wellas time spent in three different zones (outer, middle, inner) was recorded(Limelight2; Actimetrics). Total distance traveled was used as a measureof spontaneous motor activity, and distribution of activity in different
regions of the arena was used as a measure of anxiety-related behavior(Archer, 1973).
Odor–reward memory task: posttraining ablation (adult mice). Eightweek-old CTR (n ⫽ 16) and 2⫻Tg (n ⫽ 26) mice were treated with TAM.
Three weeks later, mice were trained in the odor–reward memory task for
4 d. Twenty-four hours after the completion of training, odor preference
was assessed (pre-DT probe). Starting the following day, mice received
daily DT injections for 5 d. Twenty-four hours after the final injection,
odor preference was reassessed (post-DT probe). In a second experiment, CTR (n ⫽ 8) and 2⫻Tg (n ⫽ 10) mice were treated identically but
were trained 10 d (not 3 weeks) after TAM treatment.

J. Neurosci., November 19, 2014 • 34(47):15793–15803 • 15795

Odor–reward memory task: posttraining ablation (juvenile mice). Fourto five-week-old CTR (n ⫽ 18) and 2⫻Tg (n ⫽ 19) mice were treatedwith TAM. Three weeks later, mice were trained in the odor–reward
memory task for 4 d. Twenty-four hours after the completion of training,odor preference was assessed (pre-DT probe). Starting the following day,mice received daily DT injections for 5 d. Twenty-four hours after the
final injection, odor preference was reassessed (post-DT probe).
Odor–reward memory task: pretraining ablation (adult mice). Eightweek-old CTR (n ⫽ 10) and 2⫻Tg (n ⫽ 11) mice were treated with TAM.
Three weeks later, mice were trained in the odor–reward memory task for
4 d. Twenty-four hours after the completion of training, odor preference
was assessed in a probe test. Mice received daily injections of DT for 5 d
immediately preceding training.
Odor–reward memory task: pretraining ablation (juvenile mice). Fourto five-week-old CTR (n ⫽ 15) and 2⫻Tg (n ⫽ 15) mice were treatedwith TAM. Three weeks later, mice were trained in the odor–reward
memory task for 4 d. Twenty-four hours or 28 d after the completion oftraining, odor preference was assessed in a probe test. Mice received daily
injections of DT for 5 d immediately preceding training.
Odor–reward memory task: remote posttraining ablation. Eight-weekold CTR (n ⫽ 16) and 2⫻Tg (n ⫽ 8) mice were treated with TAM. Three
weeks later, mice were trained in the odor–reward memory task for 4 d.
Twenty-four hours after the completion of training, odor preference wasassessed (pre-DT probe). Starting 23 d later, mice received daily injections of DT for 5 d. Twenty-four hours later, odor preference was reassessed (post-DT probe).

Data analysis
Cleaved caspase-3 data were analyzed using a planned, one-tailed independent samples t test, and open-field data were analyzed using ANOVA.
Odor–reward task data (i.e., dig durations and preference indices) wereanalyzed using ANOVAs, followed by Bonferroni’s post hoc tests orplanned, two-tailed paired and independent samples t tests. We initially
included sex as a factor in our analyses but found no significant sex
effects; therefore, this factor was dropped from analysis.

Results
Characterization of tag-and-ablate mice
We first characterized TAM-induced recombination (“tagging”)
in nestin–Cre ERT2 mice by crossing them with Rosa–LacZ reporter mice. In these reporter mice, Cre-mediated excision of a
STOP cassette induces LacZ expression under the control of the
Rosa26 promoter (the same promoter as in the iDTR mice). Consistent with previous results (Arruda-Carvalho et al., 2011),
Cre ERT2 protein expression was limited to adult neurogenic regions (including the SVZ) in both 4- and 8-week-old mice (Fig.
2a,b). Within the SVZ, Cre ERT2 protein was exclusively colocalized with the neural progenitor marker nestin (Fig. 2c,d; Table 1)
but not the neuroblast (Type A cell) marker DCX (Fig. 2e,f; Table
1; Imayoshi et al., 2008).
As expected, in 8-week-old nestin–Cre ERT2⫹/Rosa–LacZ ⫹ reporter mice, TAM-induced recombination was efficient, with
58.5 ⫾ 4.8% (mean ⫾ SEM) of Ki67 ⫹ cells coexpressing LacZ
(Fig. 2g). Three weeks after TAM treatment, LacZ ⫹ cells were
found throughout the granule cell layer of the OB (Fig. 2h), suggesting that large numbers of recombined cells differentiated into
OGCs. Using stereological methods, we estimated that there were141,337 ⫾ 4106 LacZ ⫹ cells, which corresponds to ⬃10% of theentire population of OGCs (Imayoshi et al., 2008). There werefewer LacZ ⫹ cells located in the glomerular layer, indicating thata much smaller fraction of cells differentiated into periglomerularcells, as expected. TAM-induced recombination was similarly efficient in 4-week-old mice, with 146,452 ⫾ 16,033 LacZ ⫹ cells in
the granule cell layer of the OB after a 3 week delay (Fig. 2i). These
numbers of LacZ ⫹ cells in the OB using the current Cre-driverline (corresponding to line 5-1 in the study by Imayoshi et al.,

15796 • J. Neurosci., November 19, 2014 • 34(47):15793–15803

Arruda-Carvalho et al. • Adult-Generated Olfactory Granule Cells and Odor Memory

Table 1. CRE ERT2 expression pattern
4 weeks
Cell maturity marker
Percentage ⫾ SEM of CRE
cells expressing marker
Cells analyzed

ERT2⫹

8 weeks

NestinDCX

NestinDCX

95.9 ⫾ 1.2

2.6 ⫾ 0.3

98.1 ⫾ 0.7

1.8 ⫾ 0.4

537

536

649

706

2008) were much higher than those observed in our previous
study using an alternate nestin–Cre ERT2⫹ line (corresponding toline 4 in the study by Imayoshi et al., 2008; Fig. 2j).
To evaluate the phenotype of these cells, we next stained forproteins expressed at different stages of cell differentiation. In
8-week-old mice, 50% of tagged (LacZ ⫹) cells were DCX ⫹,whereas 67% were NeuN ⫹ (Fig. 3a,b; Table 2). In 4-week-old
mice, 33% of tagged (LacZ ⫹) cells were DCX ⫹, whereas 63%
were NeuN ⫹ (Fig. 3c,d;Table 2). Because DCX is expressed inneuroblasts and immature neurons and NeuN is expressed onlyin neurons, this suggests that the population of tagged neurons includes large numbers of late-stage DCX ⫹ cells, corresponding to a time point when OGCs display enhanced input
plasticity (Nissant et al., 2009) and responses to novel odors(Magavi et al., 2005). Accordingly, at this time point, LacZ ⫹cells expressed activity-dependent genes such as zif268 after
training (Fig. 3e).
To ablate these tagged neurons, we crossed nestin–Cre ERT2
mice with iDTR mice in which Cre-mediated excision of a STOP
cassette renders cells sensitive to DT (Buch et al., 2005). In previous studies, we and others showed that administration of DT
leads to apoptosis of DTR-tagged cells (Buch et al., 2005; Han etal., 2009; Arruda-Carvalho et al., 2011; Vukovic et al., 2013;
Whiddon and Palmiter, 2013). Three weeks after TAM treatment, we confirmed that systemic injection of DT (but not PBS)
led to an increase in cleaved caspase-3 ⫹ cells in 2⫻Tg mice (t(7) ⫽2.24, p ⬍ 0.05; Fig. 3f,g). This increase was limited to the granulecell layer of the OB, matching the location of DTR-expressing
cells. As we have observed previously, there were no overtchanges in behavior after DT-induced ablation. In the open field,
2⫻Tg and littermate CTR mice (either nestin–Cre ERT2⫹ or
iDTR ⫹ but not both) traveled an equivalent distance (4 weeks
old: 2⫻Tg, 5.0 ⫾ 0.7 m; CTR, 4.1 ⫾ 0.1 m, t(22) ⫽ 1.53, p ⫽ 0.14;
8 weeks old: 2⫻Tg, 4.9 ⫾ 1.0 m; CTR, 6.8 ⫾ 0.3 m, t(15) ⫽ 1.97,
p ⫽ 0.068) and spent equivalent time in different regions of thearena (4 weeks old: genotype ⫻ zone ANOVA, zone main effect,
F(2,46) ⫽ 374.3, p ⬍ 0.0001; genotype ⫻ zone interaction, F(2,46) ⫽1.54, p ⫽ 0.23; 8 weeks old: genotype ⫻ zone ANOVA, zone main
effect, F(2,30) ⫽ 1248.9, p ⬍ 0.0001; genotype ⫻ zone interaction,
F(2,30) ⫽ 0.01, p ⫽ 0.99; Fig. 3h,i). In all subsequent behavioral
experiments, CTR and 2⫻Tg mice were treated with TAM andsubsequently treated with DT either before or after training. Importantly, this design ensures that group effects cannot be attributed to nonspecific effects of TAM or DT.
Figure 2. Cre ERT2 expression is restricted to progenitor cells and limited to adult neurogenic
regions. In nestin–Cre ERT2⫹ mice, Cre ERT2 protein expression was limited to adult neurogenic
regions, including the SVZ of the lateral ventricle (LV) in 4-week-old (a) and 8-week-old (b)mice. Scale bar, 100 ␮m. In these mice, Cre ERT2 protein expression (green) was found in nestin ⫹ (c,d)butnotDCX ⫹ (e,f)cells(red)in4-week-oldand8-week-oldmice,respectively.Scalebar,10 ␮m.
g, TAM-induced recombination was efficient, with ⬃59% of LacZ ⫹ cells in the SVZ also expressing
the proliferation marker Ki67. Scale bar, 10 ␮m. Three weeks after TAM treatment, LacZ ⫹ cells were
found throughout the granule cell layer (Gr) of the OB in 4-week-old (h) and 8-week-old (i) mice.
FewerLacZ ⫹ cellswerefoundintheglomerularcelllayer(Gl),likelycorrespondingtoperiglomerularcells. j, TAM-induced recombination (LacZ ⫹ cells) in the OB was greatly reduced in an alternate
Cre-driver line (line 4 from the study by Imayoshi et al., 2008). Scale bar, 100 ␮m.

Posttraining ablation of adult-generated OGCs impairs
expression of an odor–reward memory
Adult-generated OGCs synaptically integrate ⬃2 weeks after
birth (Carleton et al., 2003). From 2– 4 weeks of age, adultgenerated OGCs exhibit enhanced responsivity to novel odors(Magavi et al., 2005) and enhanced plasticity at input synapses(Nissant et al., 2009) compared with preexisting OGCs. Therefore, to test whether this population of adult-generated OGCs
form an essential component of an odor-related memory,
8-week-old (adult) CTR and 2⫻Tg mice were treated with TAM

Arruda-Carvalho et al. • Adult-Generated Olfactory Granule Cells and Odor Memory

J. Neurosci., November 19, 2014 • 34(47):15793–15803 • 15797

Figure 3. Tagging new neurons. Three weeks after the completion of TAM treatment in 4-week-old (a, b) and 8-week-old (c, d) adult mice, most LacZ ⫹ cells (green) costained for neuronalmarkers (NeuN, DCX; red). e, LacZ ⫹ cells additionally expressed activity-dependent gene zif268 (red) after behavioral testing. Scale bar, 10 ␮m. f, g, In 2⫻Tg mice, DT treatment increased thenumber of cleaved caspase-3 ⫹ (Cl-caspase 3) cells in the granule cell layer. Scale bar, 100 ␮m. Insets show higher magnification of selected areas. Scale bar, 50 ␮m. After DT-induced ablation, timespent in the outer, middle, and inner regions of the open field were unaltered in 4-week-old (h) and 8-week-old (i) mice. *p ⬍ 0.05.
Table 2. Phenotype of tagged cells
4 weeks
Cell maturity marker
Percentage ⫾ SEM of LacZ
cells expressing marker
Cells analyzed⫹

8 weeks

DCX

NeuN

DCX

NeuN

50.2 ⫾ 3.2

67.2 ⫾ 1.6

32.6 ⫾ 2.7

63.2 ⫾ 2.3

675

694

836

658

and trained in an associative odor–reward task 3 weeks later (Fig.
4a). This post-TAM delay corresponds to a time point when large
numbers of adult-generated cells have reached the OB (Fig. 2h)and express mature neuronal markers, such as NeuN (Fig. 3b;
Table 1). In the odor–reward task (Schellinck et al., 2001), foodrestricted mice learn to discriminate between two similar odors[the enantiomers (⫹) and (⫺) carvone], one of which is reinforced with sugar pellets. Twenty-four hours after the completionof training, memory for the odor–reward association was assessed in a probe trial in which both odors were present in theabsence of sugar. This form of odor–reward memory does notdepend on the hippocampus (Akers et al., 2011). In the (pre-DT)probe test, both CTR and 2⫻Tg mice spent more time digging
near the previously reinforced odor (CTR, t(15) ⫽ 8.02, p ⬍ 0.001;
2⫻Tg, t(25) ⫽ 7.45, p ⬍ 0.0001; Fig. 4b,c), indicating that expression of DTRs in OGCs did not affect motivation or motor skills
necessary to acquire the odor–reward memory.
CTR and 2⫻Tg mice were subsequently treated with DT, andodor memory was reassessed 6 d later. In this (post-DT) probe
test, CTR mice continued to spend more time digging near thepreviously reinforced odor (t(15) ⫽ 3.79, p ⬍ 0.005), indicatingthat the odor–reward memory was stable and did not undergo
extinction through retesting (Fig. 4b). In contrast, 2⫻Tg micespent equivalent time digging over both odors (t(25) ⫽ 0.97, p ⫽0.34; Fig. 4c), indicating that posttraining ablation of adultgenerated OGCs led to memory loss. Comparisons of pre-DT
versus post-DT preference indices support this conclusion. An
ANOVA with genotype (CTR, 2⫻Tg) as a between-subjects variable and test time (pre-DT, post-DT) as a within-subjects vari-able revealed main effects of genotype (F(1,40) ⫽ 7.05, p ⬍ 0.005),
test time (F(1,40) ⫽ 10.09, p ⬍ 0.005), and a genotype ⫻ test timeinteraction (F(1,40) ⫽ 4.55, p ⬍ 0.05). Whereas preference for thepreviously reinforced odor was similar before and after DT treatment in CTR mice ( p ⬎ 0.05), it was significantly reduced afterDT treatment in 2⫻Tg mice ( p ⬍ 0.05). Furthermore, preference
for the reinforced odor during the post-DT probe test wassignificantly reduced in 2⫻Tg compared with CTR mice ( p ⬍0.05; Fig. 4d).
Although these results suggest that adult-generated cells form
an essential component of the odor–reward memory trace, thetagged population of cells is heterogeneous in terms of locationand maturity. Therefore, memory loss might be related to a lossof relatively mature adult-generated OGCs in the OB or, alternatively, a loss of less mature cell types located in the OB or the
rostral migratory stream. To address this limitation, we next
treated adult CTR and 2⫻Tg mice with TAM and trained them inthe odor–reward task 10 d, rather than 3 weeks, later (Fig. 4e). In
this case, posttraining ablation did not affect the expression of theodor–reward association. Both CTR and 2⫻Tg mice expressed apreference for the reinforced odor in probe tests before and afterDT treatment (pre-DTCTR, t(7) ⫽ 3.09, p ⬍ 0.05; post-DTCTR,t(7) ⫽ 4.99, p ⬍ 0.005; pre-DT2⫻Tg, t(9) ⫽ 4.31, p ⬍ 0.005; postDT2⫻Tg, t(9) ⫽ 3.32, p ⬍ 0.01; Fig. 4f,g), and the magnitude of thispreference was similar at both time points for both CTR and2⫻Tg mice (genotype ⫻ test time ANOVA, no main effects orinteractions, all F values ⬍1; Fig. 4h). These findings suggest thatthe loss of adult-generated cells ⬎10 d in age is responsible formemory impairment after posttraining DT treatment. This population primarily corresponds to OGCs that have reached the OB
and formed input/output connections with mitral cells within the
OB and higher association areas, including the piriform cortex,
anterior olfactory nucleus, and cortical amygdaloid nucleus(Lepousez et al., 2013). The results also suggest that secondary
effects of the death of large numbers of cells do not generally
disrupt OB function.

15798 • J. Neurosci., November 19, 2014 • 34(47):15793–15803

Arruda-Carvalho et al. • Adult-Generated Olfactory Granule Cells and Odor Memory

Figure 4. Posttraining ablation of adult-generated OGCs impairs expression of odor–reward memory. a, Adult (8-week-old) mice were treated with TAM and trained in the odor–reward task 3
weeks later. Odor–reward memory was evaluated 1 d after training (pre-DT probe) and again after DT treatment (post-DT probe). b, CTR mice spent more time digging over the previously reinforced
odor in the probe tests before and after DT treatment. c, Although 2⫻Tg mice spent more time digging over the previously reinforced odor in the pre-DT probe test, they spent equivalent time
digging over the reinforced versus non-reinforced odors in the post-DT test. d, Preference indices for CTR and 2⫻Tg mice in the pre-DT and post-DT tests. e, Adult mice were treated with TAM andtrained in the odor–reward task 10 d later. Odor–reward memory was evaluated 1 d after training (pre-DT probe) and again after DT treatment (post-DT probe). Both CTR (f ) and 2⫻Tg (g) micespent more time digging over the previously reinforced odor in the probe tests before and after DT treatment. h, Preference indices for CTR and 2⫻Tg mice in the pre-DT and post-DT tests. *p ⬍ 0.05.

Pretraining ablation of adult-generated OGCs does notprevent formation of an odor–reward memory
We next tested whether ablation of a similar population of adultgenerated OGCs immediately before training would impair acquisition of a new odor–reward memory. As in the firstexperiment, adult CTR and 2⫻Tg mice were treated with TAM,
trained 3 weeks later, and then tested. In this case, however, DT
was administered during the week before training rather than theweek after training (Fig. 5a). This experimental design ensuresthat DT targets an equivalent population of cells (in terms ofnumber and maturity).
In a probe test 1 d after the completion of training, both CTR(t(9) ⫽ 3.99, p ⬍ 0.005) and 2⫻Tg (t(10) ⫽ 3.66, p ⬍ 0.005) miceexpressed a preference for the reinforced odor, indicating that
pretraining ablation of adult-generated OGCs does not preventthe formation of a new odor–reward memory (Fig. 5b,c). Using

different strategies to suppress olfactory neurogenesis before
training in adult rodents, previous studies have similarly foundthat the ability to form an olfactory associative memory is unaffected (Imayoshi et al., 2008; Breton-Provencher et al., 2009;
Lazarini et al., 2009; Sultan et al., 2010). In some instances, however, deficits in the persistence of odor–reward memories havebeen detected in rodents with suppressed olfactory neurogenesis(Breton-Provencher et al., 2009; Lazarini et al., 2009; Sultan et al.,2010). Therefore, CTR and 2⫻Tg mice were retested 28 d after
the completion of training. In this remote test, both CTR (t(9) ⫽6.61, p ⬍ 0.0001) and 2⫻Tg (t(10) ⫽ 3.28, p ⬍ 0.01) mice continued to express a preference for the reinforced odor (Fig. 5b,c),
indicating that pretraining ablation of OGCs did not alter thepersistence of an odor–reward memory. The lack of effect ofpretraining ablation on the formation and persistence of odor–
reward memories was supported by comparisons of preferenceArruda-Carvalho et al. • Adult-Generated Olfactory Granule Cells and Odor Memory

J. Neurosci., November 19, 2014 • 34(47):15793–15803 • 15799

Figure 5. Pretraining ablation of adult-generated OGCs does not prevent acquisition of an odor–reward memory. a, Adult mice were treated with TAM and trained in the odor–reward task 3
weeks later. Mice were treated daily with DT for 5 d immediately preceding training. Odor–reward memory was evaluated 1 and 28 d after the completion of training. Both CTR (b) and 2⫻Tg (c)mice spent more time digging over the previously reinforced odor in probe tests 1 and 28 d after training. d, Preference indices for CTR and 2⫻Tg mice in the 1 and 28 d tests. *p ⬍ 0.05.

Figure 6. Posttraining ablation of juvenile-generated OGCs impairs odor–reward memory. a, Juvenile (4 –5 weeks old) mice were treated with TAM and trained in the odor–reward task 3 weeks
later. Odor–reward memory was evaluated 1 d after training (pre-DT probe) and again after DT treatment (post-DT probe). b, CTR mice spent more time digging over the previously reinforced odor
in the probe tests before and after DT treatment. c, Although 2⫻Tg mice spent more time digging over the previously reinforced odor in the pre-DT probe test, they spent equivalent time digging
over the reinforced versus non-reinforced odors in the post-DT test. d, Preference indices for CTR and 2⫻Tg mice in the pre-DT and post-DT tests. *p ⬍ 0.05.

indices. An ANOVA with genotype and test time as factors revealed no main effects or genotype ⫻ test time interactions (Fig.
5d). Therefore, whereas ablating adult-generated OGCs after
training leads to memory loss, ablating an equivalent populationof cells before training does not prevent the formation of anodor–reward memory. This suggests that existing OGCs may
support the formation of odor–reward memory.
Posttraining ablation of juvenile-generated OGCs impairs
expression of an odor–reward memory
We next tested whether a similar dissociation between the effects
of pretraining versus posttraining ablation of OGCs would beobserved in younger, 4- to 5-week-old mice (an age that corresponds to juvenility; Wei et al., 2011). Using an protocol identicalto that used for adult mice, we first assessed the effect of posttraining ablation of OGCs generated during juvenility (Fig. 6a).
Similar to adult mice, we found that posttraining ablation led tomemory loss. Whereas CTR mice expressed a preference for thereinforced odor both before (t(17) ⫽ 6.80, p ⬍ 0.0001) and after(t(17) ⫽ 5.80, p ⬍ 0.0001) DT treatment (Fig. 6b), 2⫻Tg mice only
expressed a preference for the reinforced odor before DT treatment (t(18) ⫽ 4.77, p ⬍ 0.0005). After DT treatment, 2⫻Tg miceinvestigated the reinforced and non-reinforced odors equivalently (t(18) ⫽ 1.70, p ⫽ 0.11; Fig. 6c).
This pattern of results was confirmed by comparisons of preference indices. An ANOVA with genotype (CTR, 2⫻Tg) as a
between-subjects variable and test time (pre-DT, post-DT) asa within-subjects variable revealed main effects of genotype

Arruda-Carvalho et al. • Adult-Generated Olfactory Granule Cells and Odor Memory

15800 • J. Neurosci., November 19, 2014 • 34(47):15793–15803(F(1,35) ⫽ 6.44, p ⬍ 0.05) and test time(F(1,35) ⫽ 12.11, p ⬍ 0.005). However, the
ANOVA indicated that the genotype ⫻test time interaction only approached significance (F(1,35) ⫽ 3.02, p ⫽ 0.09), and sothese results should be interpreted withmore caution. Nonetheless, planned comparisons indicated that, whereas preference for the reinforced odor was similar
before and after DT treatment for CTR
mice (t(17) ⫽ 1.13, p ⫽ 0.28), it was significantly reduced after DT treatment in2⫻Tg mice (t(18) ⫽ 4.19, p ⬍ 0.001; Fig.
6d). Furthermore, during the post-DT
test, preference for the reinforced odor
was significantly reduced in 2⫻Tg compared with CTR mice (t(35) ⫽ 2.70, p ⫽0.011). Importantly, these contrasts remain significant even after correcting for
multiple comparisons.
We next assessed the effect of pretraining ablation of OGCs generated during juvenility. Again, using a protocol identicalto that used for adult mice (Fig. 7a), we
found that pretraining ablation did notprevent the formation of a persistentodor–reward memory. Both CTR and2⫻Tg mice expressed preference for thereinforced odor when tested 1 d (CTR,
t(14) ⫽ 5.33, p ⬍ 0.0001; 2⫻Tg, t(14) ⫽5.14, p ⬍ 0.0001) or 28 d (CTR, t(14) ⫽3.76, p ⬍ 0.005; 2⫻Tg, t(14) ⫽ 2.71, p ⬍0.05) after training (Fig. 7b,c). Moreover,the degree of preference did not changewith time or genotype (genotype ⫻ test
time ANOVA, no main effects or interactions; Fig. 7d). Similar to adult mice, theseresults suggest that existing OGCs maycompensate for the pretraining loss ofjuvenile-generated OGCs.

Figure 7. Pretraining ablation of juvenile-generated OGCs does not prevent acquisition of an odor–reward memory. a, Juvenile
mice were treated with TAM and trained in the odor–reward task 3 weeks later. Mice were treated daily with DT for 5 d immediately
preceding training. Odor–reward memory was evaluated 1 and 28 d after the completion of training. Both CTR (b) and 2⫻Tg (c)mice spent more time digging over the previously reinforced odor in probe tests 1 and 28 d after training. d, Preference indices for
CTR and 2⫻Tg mice in the pre-DT and post-DT tests. *p ⬍ 0.05.

Figure 8. Posttraining ablation of adult-generated OGCs does not impair remote odor–reward memories. a, Adult mice weretreated with TAM and trained in the odor–reward task 3 weeks later. Odor–reward memory was evaluated 1 d after training(pre-DT probe). Mice were treated with DT 4 weeks later, and then odor–reward memory was assessed in a second probe test
(post-DT probe). Both CTR (b) and 2⫻Tg (c) mice spent more time digging over the previously reinforced odor in the probe tests
before and after DT treatment. d, Preference indices for CTR and 2⫻Tg mice in the pre-DT and post-DT tests. *p ⬍ 0.05.

Adult-generated OGCs are only transiently necessary forexpression of an odor–reward memory
Odor–reward memories may persist for many weeks. For example, using training conditions similar to those used here, mice
were able to express an odor preference up to 28 d after training(Schellinck et al., 2001). Therefore, we next asked whether adultgenerated OGCs are always necessary for expression of an odor–
reward memory once learned or whether memory expression
becomes independent of this population of cells over time. To dothis, adult CTR and 2⫻Tg mice were treated with TAM andtrained 3 weeks later, and memory was tested 1 d after training(pre-DT probe). Mice were then treated with DT 4 weeks later(rather than immediately) and memory was reassessed (post-DTprobe; Fig. 8a). We found that ablating adult-generated OGCs 1
month after learning did not affect subsequent expression of thatmemory. Both CTR and 2⫻Tg mice expressed a preference forthe reinforced odor in pre-DT (CTR, t(15) ⫽ 6.22, p ⬍ 0.0001;
2⫻Tg, t(7) ⫽ 6.36, p ⬍ 0.0005) and post-DT (CTR, t(15) ⫽ 3.52,
p ⬍ 0.005; 2⫻Tg, t(7) ⫽ 3.28, p ⬍ 0.05) tests, and the magnitudeof this preference was similar at both time points for both CTR
and 2⫻Tg mice (genotype ⫻ test time ANOVA, no main effects
or interactions, all F values ⬍ 1; Fig. 8b– d). These results contrast

with the effects of similar ablations conducted immediately after
training and therefore suggest that, although adult-generated
OGCs may initially play an essential role in the expression ofodor–reward memory, with time these memories may be expressed independently of this population of cells.

Discussion
We used a DTR-based ablation strategy to study the role of adultgenerated OGCs in odor–reward memories. There are five main
findings. First, posttraining ablation of adult-generated OGCs
impaired subsequent odor preference, establishing that thesecells play an essential role in the expression of odor–reward memories. Second, this role depends on cell age at the time of training,with memory loss associated with cells ⬎10 d in age. Third, pretraining ablation of the same population does not prevent theformation of a new odor–reward memory, highlighting the potential for compensation by existing OGCs. Fourth, ablation of
OGCs generated during juvenility produced a similar pattern ofresults, suggesting that OGCs play similar roles in odor–reward
memories whether generated during juvenility versus adulthood.
Fifth, ablation of OGCs at remote time points after training did
not induce memory loss, indicating that adult-generated OGCs
may play a time-limited role in the expression of odor–reward
memories.

Arruda-Carvalho et al. • Adult-Generated Olfactory Granule Cells and Odor Memory

In previous ablation studies, neural stem and/or progenitor
cells were targeted and therefore behavior was studied after a
global reduction in adult neurogenesis (Imayoshi et al., 2008;
Breton-Provencher et al., 2009; Lazarini et al., 2009; Moreno etal., 2009; Valley et al., 2009; Feierstein et al., 2010; Sultan et al.,2010; Sakamoto et al., 2011; Cushman et al., 2012; Feierstein,2012). The present DTR-based ablation approach differs in so far
as we were able to study the effect of depletion of large numbers ofadult-generated OGCs that were already mature and, presumably, synaptically integrated into OB circuits at the time of training. In particular, this tag-and-ablate approach allowed us
control over the timing of the target and ablation of cells after (aswell as before) training. This latter feature is important because it
allowed us to ablate adult-generated OGCs that have had theopportunity to become integrated into odor memory circuits.
Consistent with the idea that targeting “memory-committed”
cells would be more powerful (Frankland, 2013), we found that
posttraining (but not pretraining) ablations impaired memory.
Our finding that posttraining ablations impaired memory
suggests that, if available at the time of training, adult-generated
OGCs are essential for expression of an odor–reward memory.
These loss-of-function data complement a recent gain-offunction study that showed that optical stimulation of adultgenerated OGCs facilitated the formation of odor–reward
memories (Alonso et al., 2012). In our study, memory loss wasmost likely associated with ablation of cells that were ⬎10 d ofage, because similar posttraining ablations of cells that were ⱕ10d of age at the time of training did not lead to memory loss. This
corresponds to a time point when OGCs have synaptically integrated into OB circuits and display heightened plasticity at input
synapses (Nissant et al., 2009) and responsivity to novel odors
and olfactory learning (Magavi et al., 2005; Sultan et al., 2010;
Belnoue et al., 2011; Lepousez et al., 2014). Furthermore, thedevelopmental stage at which OGCs were generated did not appear to matter. Both posttraining ablation of OGCs generated
during juvenility or adulthood led to memory loss. Previous
studies suggest that OGCs generated during earlier developmental stages/juvenility may make distinct contributions to olfactorymediated social affiliative behaviors (Wei et al., 2011). The
current results suggest that these findings do not generalize toodor–reward memories. Finally, although we used a Cre-driverline with highest recombination efficiency in the SVZ (line 5-1
from the study of Imayoshi et al., 2008), DT-induced ablation
should also effect significant numbers of adult-generated dentate
granule cells in the hippocampus. However, it is unlikely that the
memory deficits we observed were attributable to a loss of dentate
granule cells, because lesions of the entire hippocampus do notaffect this type of odor–reward memory (Akers et al., 2011).
DT injections efficiently ablate DTR-tagged cells. Based on
our fate-mapping analyses, we estimate that our DT injections led
to the loss of large numbers of adult-generated OGCs (⬎140,000
cells). Although DT-induced ablations are associated with minimal
inflammation (Buch et al., 2005; Han et al., 2009; Arruda-Carvalho
et al., 2011), it is possible that widespread apoptotic cell death might
generally disrupt OB function. However, neither posttraining ablation of predominantly immature cells (⬍10-d-old cells) nor ablationof a similar population of OGCs before training disrupted odor–
reward memory. This suggests that the DT-induced ablation did not
generally impair odor perception or alter motivation and motor
skills required for learning the odor–reward association.
Control over the timing of the ablation made it possible toadditionally explore the role of adult-generated OGCs in the continued expression of odor–reward memories, long after initial

J. Neurosci., November 19, 2014 • 34(47):15793–15803 • 15801

training. Whereas ablation of adult-generated OGCs soon after
training led to a loss of odor–reward memory, similar ablations 4
weeks after training were ineffective. Odor–reward memories arelikely supported by a distributed circuit including populations ofcells that are both intrinsic and extrinsic to the OB. Our results
suggest that these circuits are dynamic and change over time, with
OGCs playing only a transient role in the expression of odor–
reward memories. Consistent with this evidence for timedependent reorganization of circuits supporting odor–reward
memories, a recent immediate early gene mapping study suggeststhat ⬃5-week-old adult-generated OGCs are engaged duringodor learning but are no longer activated when that memory isrecalled 4 weeks later (Belnoue et al., 2011). This time-dependentrole for adult-generated OGCs in odor–reward memories mirrors the role of the hippocampus in episodic-like memories(Frankland and Bontempi, 2005). For example, expression ofcontextual fear memories are initially dependent on the hippocampus, but after ⬃1 month, these types of memories may be
expressed independently of the hippocampus (Kim and Fanselow, 1992). An interesting parallel between the OB and the
hippocampus is that, in both systems, there is continuous integration of new neurons into existing circuits. In the hippocampus, this continuous neurogenesis-associated remodeling may
degrade information already stored in these circuits (Feng et al.,
2001; Meltzer et al., 2005; Kitamura et al., 2009; Josselyn andFrankland, 2012; Frankland et al., 2013; Akers et al., 2014). Thisdegradation of the hippocampal trace, coupled with the strengthening of corticocortical connections, is thought to underlie thetransformation of the memory from a hippocampus-dependent
to hippocampus-independent form (Frankland and Bontempi,
2005). Similarly, it is possible that continuous integration of
adult-generated OGCs degrades information stored within the
OB such that, with time, expression of olfactory memories nolonger critically depends on OGCs but may be supported by cortical networks alone. One caveat is worth noting here. An alternative possibility is that conditionally expressed DTRs aredegraded during this period, rendering “tagged” cells no longer
sensitive to DT at more remote time points. Although this ispossible, our previous studies using this system in the hippocampus suggest that this is unlikely (Arruda-Carvalho et al., 2011). In
these studies, DT-induced ablations induced amnesia, even when
initiated several weeks after TAM-induced DTR expression.
What role might adult-generated OGCs play in the expressionof odor–reward memories? There are at least two possibilities.
First, adult-generated OGCs might represent informationstoring units themselves in a broader circuit, likely including
higher-order cortical regions such as the piriform cortex, anterior
olfactory nucleus, and cortical amygdaloid nucleus, and loss of
this population of cells soon after training degrades the representation of the odor–reward association sufficiently that mice are
no longer able to discriminate between similar reinforced andnon-reinforced odors. An alternate possibility is that, rather thanstoring information, adult-generated OGCs might modulate theefficiency of retrieval of information stored elsewhere (e.g., inother OB cells). For example, adult-generated OGCs regulate
synchronous firing of mitral cells via dendrodendritic synapses(Urban and Arevian, 2009), and disrupted synchrony may impair
retrieval and/or discrimination between similar odors. These two
possibilities are difficult to disentangle. However, our findingthat ablation of adult-generated OGCs only produced memory
loss under a limited set of conditions favors an informationstorage account. In particular, the absence of memory loss after15802 • J. Neurosci., November 19, 2014 • 34(47):15793–15803

pretraining ablation of OGCs suggests that this type of ablation
does not lead to a generalized dysregulation of retrieval.

References
Akers KG, Kushner SA, Leslie AT, Clarke L, van der Kooy D, Lerch JP, Frankland PW (2011) Fetal alcohol exposure leads to abnormal olfactory bulb
development and impaired odor discrimination in adult mice. Mol Brain
4:29. CrossRef Medline
Akers KG, Martinez-Canabal A, Restivo L, Yiu AP, De Cristofaro A, Hsiang
HL, Wheeler AL, Guskjolen A, Niibori Y, Shoji H, Ohira K, Richards BA,
Miyakawa T, Josselyn SA, Frankland PW (2014) Hippocampal neurogenesis regulates forgetting during adulthood and infancy. Science 344:
598 – 602. CrossRef Medline
Alonso M, Lepousez G, Sebastien W, Bardy C, Gabellec MM, Torquet N,
Lledo PM (2012) Activation of adult-born neurons facilitates learningand memory. Nat Neurosci 15:897–904. CrossRef Medline
Archer J (1973) Tests for emotionality in rats and mice: a review. Anim
Behav 21:205–235. CrossRef Medline
Arruda-Carvalho M, Sakaguchi M, Akers KG, Josselyn SA, Frankland PW(2011) Posttraining ablation of adult-generated neurons degrades
previously acquired memories. J Neurosci 31:15113–15127. CrossRef
MedlineBelnoue L, Grosjean N, Abrous DN, Koehl M (2011) A critical time window
for the recruitment of bulbar newborn neurons by olfactory discrimination learning. J Neurosci 31:1010 –1016. CrossRef Medline
Belvindrah R, Lazarini F, Lledo PM (2009) Postnatal neurogenesis: fromneuroblast migration to neuronal integration. Rev Neurosci 20:331–346.
MedlineBreton-Provencher V, Lemasson M, Peralta MR 3rd, Saghatelyan A (2009)
Interneurons produced in adulthood are required for the normal functioning of the olfactory bulb network and for the execution of selected
olfactory behaviors. J Neurosci 29:15245–15257. CrossRef Medline
Buch T, Heppner FL, Tertilt C, Heinen TJ, Kremer M, Wunderlich FT, Jung S,
Waisman A (2005) A Cre-inducible diphtheria toxin receptor mediates
cell lineage ablation after toxin administration. Nat Methods 2:419 – 426.
CrossRef Medline
Carleton A, Petreanu LT, Lansford R, Alvarez-Buylla A, Lledo PM (2003)
Becoming a new neuron in the adult olfactory bulb. Nat Neurosci 6:507–
518. CrossRef Medline
Chen GH, Wang YJ, Zhang LQ, Zhou JN (2004) Age- and sex-related disturbance in a battery of sensorimotor and cognitive tasks in Kunming
mice. Physiol Behav 83:531–541. CrossRef Medline
Cleland TA (2010) Early transformations in odor representation. Trends
Neurosci 33:130 –139. CrossRef Medline
Cushman JD, Maldonado J, Kwon EE, Garcia AD, Fan G, Imura T, Sofroniew
MV, Fanselow MS (2012) Juvenile neurogenesis makes essential contributions to adult brain structure and plays a sex-dependent role in fear
memories. Front Behav Neurosci 6:3. CrossRef Medline
Feierstein CE (2012) Linking adult olfactory neurogenesis to social behavior. Front Neurosci 6:173. CrossRef Medline
Feierstein CE, Lazarini F, Wagner S, Gabellec MM, de Chaumont F, OlivoMarin JC, Boussin FD, Lledo PM, Gheusi G (2010) Disruption of adultneurogenesis in the olfactory bulb affects social interaction but not maternal behavior. Front Behav Neurosci 4:176. CrossRef Medline
Feng R, Rampon C, Tang YP, Shrom D, Jin J, Kyin M, Sopher B, Miller MW,
Ware CB, Martin GM, Kim SH, Langdon RB, Sisodia SS, Tsien JZ (2001)
Deficient neurogenesis in forebrain-specific presenilin-1 knockout miceis associated with reduced clearance of hippocampal memory traces. Neuron 32:911–926. CrossRef Medline
Forni PE, Scuoppo C, Imayoshi I, Taulli R, Dastrù W, Sala V, Betz UA, Muzzi
P, Martinuzzi D, Vercelli AE, Kageyama R, Ponzetto C (2006) High levels of Cre expression in neuronal progenitors cause defects in brain development leading to microencephaly and hydrocephaly. J Neurosci 26:
9593–9602. CrossRef Medline
Frankland PW (2013) Neurogenic evangelism: comment on Urbach et al.
(2013). Behav Neurosci 127:126 –129. CrossRef Medline
Frankland PW, Bontempi B (2005) The organization of recent and remotememories. Nat Rev Neurosci 6:119 –130. CrossRef Medline
Frankland PW, Köhler S, Josselyn SA (2013) Hippocampal neurogenesis
and forgetting. Trends Neurosci 36:497–503. CrossRef Medline
Gao Y, Strowbridge BW (2009) Long-term plasticity of excitatory inputs to

Arruda-Carvalho et al. • Adult-Generated Olfactory Granule Cells and Odor Memory
granule cells in the rat olfactory bulb. Nat Neurosci 12:731–733. CrossRef
MedlineGropp E, Shanabrough M, Borok E, Xu AW, Janoschek R, Buch T, Plum L,
Balthasar N, Hampel B, Waisman A, Barsh GS, Horvath TL, Brüning JC(2005) Agouti-related peptide-expressing neurons are mandatory forfeeding. Nat Neurosci 8:1289 –1291. CrossRef Medline
Gundersen HJ, Jensen EB, Kiêu K, Nielsen J (1999) The efficiency of systematic sampling in stereology—reconsidered. J Microsc 193:199 –211.
CrossRef Medline
Han JH, Kushner SA, Yiu AP, Hsiang HL, Buch T, Waisman A, Bontempi B,
Neve RL, Frankland PW, Josselyn SA (2009) Selective erasure of a fear
memory. Science 323:1492–1496. CrossRef Medline
Imayoshi I, Sakamoto M, Ohtsuka T, Takao K, Miyakawa T, Yamaguchi M,
Mori K, Ikeda T, Itohara S, Kageyama R (2008) Roles of continuous
neurogenesis in the structural and functional integrity of the adult forebrain. Nat Neurosci 11:1153–1161. CrossRef Medline
Josselyn SA, Frankland PW (2012) Infantile amnesia: a neurogenic hypothesis. Learn Mem 19:423– 433. CrossRef Medline
Kim JJ, Fanselow MS (1992) Modality-specific retrograde amnesia of fear.
Science 256:675– 677. CrossRef Medline
Kitamura T, Saitoh Y, Takashima N, Murayama A, Niibori Y, Ageta H, Sekiguchi M, Sugiyama H, Inokuchi K (2009) Adult neurogenesis modulates the hippocampus-dependent period of associative fear memory. Cell
139:814 – 827. CrossRef Medline
Lagace DC, Whitman MC, Noonan MA, Ables JL, DeCarolis NA, ArguelloAA, Donovan MH, Fischer SJ, Farnbauch LA, Beech RD, DiLeone RJ,
Greer CA, Mandyam CD, Eisch AJ (2007) Dynamic contribution of
nestin-expressing stem cells to adult neurogenesis. J Neurosci 27:12623–
12629. CrossRef Medline
Lazarini F, Lledo PM (2011) Is adult neurogenesis essential for olfaction?
Trends Neurosci 34:20 –30. CrossRef Medline
Lazarini F, Mouthon MA, Gheusi G, de Chaumont F, Olivo-Marin JC, Lamarque S, Abrous DN, Boussin FD, Lledo PM (2009) Cellular and behavioral effects of cranial irradiation of the subventricular zone in adultmice. PLoS One 4:e7017. CrossRef Medline
Lepousez G, Valley MT, Lledo PM (2013) The impact of adult neurogenesis
on olfactory bulb circuits and computations. Annu Rev Physiol 75:339 –
363. CrossRef Medline
Lepousez G, Nissant A, Bryant AK, Gheusi G, Greer CA, Lledo PM (2014) Olfactory learning promotes input-specific synaptic plasticity in adult-bornneurons. Proc Natl Acad Sci U S A 111:13984 –13989. CrossRef Medline
Magavi SS, Mitchell BD, Szentirmai O, Carter BS, Macklis JD (2005) Adultborn and preexisting olfactory granule neurons undergo distinct
experience-dependent modifications of their olfactory responses in vivo.
J Neurosci 25:10729 –10739. CrossRef Medline
Meltzer LA, Yabaluri R, Deisseroth K (2005) A role for circuit homeostasis
in adult neurogenesis. Trends Neurosci 28:653– 660. CrossRef Medline
Ming GL, Song H (2005) Adult neurogenesis in the mammalian central
nervous system. Annu Rev Neurosci 28:223–250. CrossRef Medline
Moreno MM, Linster C, Escanilla O, Sacquet J, Didier A, Mandairon N(2009) Olfactory perceptual learning requires adult neurogenesis. Proc
Natl Acad Sci U S A 106:17980 –17985. CrossRef Medline
Mouret A, Murray K, Lledo PM (2009) Centrifugal drive onto local inhibitory interneurons of the olfactory bulb. Ann N Y Acad Sci 1170:239 –254.
CrossRef Medline
Nissant A, Bardy C, Katagiri H, Murray K, Lledo PM (2009) Adult neurogenesis promotes synaptic plasticity in the olfactory bulb. Nat Neurosci
12:728 –730. CrossRef Medline
Saito M, Iwawaki T, Taya C, Yonekawa H, Noda M, Inui Y, Mekada E, Kimata
Y, Tsuru A, Kohno K (2001) Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice. Nat Biotechnol
19:746 –750. CrossRef Medline
Sakamoto M, Imayoshi I, Ohtsuka T, Yamaguchi M, Mori K, Kageyama R(2011) Continuous neurogenesis in the adult forebrain is required for
innate olfactory responses. Proc Natl Acad Sci U S A 108:8479 – 8484.
CrossRef Medline
Schellinck HM, Forestell CA, LoLordo VM (2001) A simple and reliable testof olfactory learning and memory in mice. Chem Senses 26:663– 672.
CrossRef Medline
Sultan S, Mandairon N, Kermen F, Garcia S, Sacquet J, Didier A (2010)
Learning-dependent neurogenesis in the olfactory bulb determines longterm olfactory memory. FASEB J 24:2355–2363. CrossRef Medline

Arruda-Carvalho et al. • Adult-Generated Olfactory Granule Cells and Odor Memory
Urban NN, Arevian AC (2009) Computing with dendrodendritic synapses in the olfactory bulb. Ann N Y Acad Sci 1170:264 –269. CrossRef
MedlineValley MT, Mullen TR, Schultz LC, Sagdullaev BT, Firestein S (2009) Ablation of mouse adult neurogenesis alters olfactory bulb structure and olfactory fear conditioning. Front Neurosci 3:51. CrossRef Medline
Vukovic J, Borlikova GG, Ruitenberg MJ, Robinson GJ, Sullivan RK, Walker
TL, Bartlett PF (2013) Immature doublecortin-positive hippocampal
neurons are important for learning but not for remembering. J Neurosci
33:6603– 6613. CrossRef Medline
Wei L, Meaney MJ, Duman RS, Kaffman A (2011) Affiliative behavior requires juvenile, but not adult neurogenesis. J Neurosci 31:14335–14345.
CrossRef Medline
Whiddon BB, Palmiter RD (2013) Ablation of neurons expressing melanin-

J. Neurosci., November 19, 2014 • 34(47):15793–15803 • 15803
concentrating hormone (MCH) in adult mice improves glucose tolerance
independent of MCH signaling. J Neurosci 33:2009 –2016. CrossRef
MedlineWinner B, Cooper-Kuhn CM, Aigner R, Winkler J, Kuhn HG (2002) Longterm survival and cell death of newly generated neurons in the adult rat
olfactory bulb. Eur J Neurosci 16:1681–1689. CrossRef Medline
Wrobel CJ, Wright DC, Dedrick RL, Youle RJ (1990) Diphtheria toxin effects on brain-tumor xenografts. Implications for protein-based braintumor chemotherapy. J Neurosurg 72:946 –950. CrossRef Medline
Zambrowicz BP, Imamoto A, Fiering S, Herzenberg LA, Kerr WG, Soriano P(1997) Disruption of overlapping transcripts in the ROSA beta geo 26
gene trap strain leads to widespread expression of beta-galactosidase inmouse embryos and hematopoietic cells. Proc Natl Acad Sci U S A 94:
3789 –3794. CrossRef Medline