The Journal of Neuroscience, October 19, 2011 • 31(42):15113–15127 • 15113

Behavioral/Systems/Cognitive

Posttraining Ablation of Adult-Generated Neurons Degrades
Previously Acquired Memories
Maithe Arruda-Carvalho,1,3* Masanori Sakaguchi,1,2,3* Katherine G. Akers,1,2,3 Sheena A. Josselyn,1,2,3
and Paul W. Frankland1,2,3
1

Program in Neurosciences and Mental Health, The Hospital for Sick Children, Toronto, Canada M5G 1X8, 2Department of Physiology, University ofToronto, Toronto, Canada M5S 1A8, and 3Institute of Medical Science, University of Toronto, Toronto, Canada M5S 1A8

New neurons are continuously generated in the subgranular zone of the adult hippocampus and, once sufficiently mature, are thought tointegrate into hippocampal memory circuits. However, whether they play an essential role in subsequent memory expression is notknown. Previous studies have shown that suppression of adult neurogenesis often (but not always) impairs subsequent hippocampusdependent learning (i.e., produces anterograde effects). A major challenge for these studies is that these new neurons represent only asmall subpopulation of all dentate granule cells, and so there is large potential for either partial or complete compensation by granule cells
generated earlier on during development. A potentially more powerful approach to investigate this question would be to ablate adultgenerated neurons after they have already become part of a memory trace (i.e., retrograde effects). Here we developed a diphtheria
toxin-based strategy in mice that allowed us to selectively ablate a population of predominantly mature, adult-generated neurons either
before or after learning, without affecting ongoing neurogenesis. Removal of these neurons before learning did not prevent the formationof new contextual fear or water maze memories. In contrast, removal of an equivalent population after learning degraded existing
contextual fear and water maze memories, without affecting nonhippocampal memory. Ablation of these adult-generated neurons even1 month after learning produced equivalent memory degradation in the water maze. These retrograde effects suggest that adultgenerated neurons form a critical and enduring component of hippocampal memory traces.

Introduction
Division of progenitor cells in the subgranular zone leads to thecontinuous addition of new neurons to the adult hippocampus, abrain region that plays a central role in memory formation (Denget al., 2010). These newly generated neurons initially migrate into
the granule cell layer of the dentate gyrus (DG) and, over thecourse of several weeks, gradually establish functional afferent
and efferent connections (Zhao et al., 2006; Toni et al., 2007,2008). Previous studies have shown that, once sufficiently mature, adult-generated neurons are activated during memory formation and/or expression (Kee et al., 2007; Tashiro et al., 2007;
Trouche et al., 2009; Stone et al., 2011), suggesting that they
become integrated into hippocampal memory traces. However,these correlative studies do not establish whether these neurons
Received July 5, 2011; revised Aug. 16, 2011; accepted Aug. 31, 2011.
Author contributions: M.A.-C., M.S., S.A.J., and P.W.F. designed research; M.A.-C., M.S., and K.G.A. performed
research; M.A.-C., M.S., and K.G.A. analyzed data; and S.A.J. and P.W.F. wrote the paper.
This work was supported by grants from the Canadian Institutes of Health Research to P.W.F. (MOP86762) and
S.A.J. (MOP74650). M.A.-C., M.S., and K.G.A. were supported by fellowships from the Ontario Graduate Scholarshipprogram (M.A.-C.), The Hospital for Sick Children (M.A.-C.), Japanese Society for the Promotion of Science (M.S.), andthe Canadian Institutes of Health Research (K.G.A.). We thank R. Kageyama and I. Imayoshi for the nestin-Cre ERT2
mice, A. Waisman for the iDTR mice, D. Lagace for technical advice, R. Aleixo for artwork, and C. Teixeira, A. Corvelo,
and members of the Frankland/Josselyn laboratories for discussions and technical assistance.
M.A.-C. and M.S. contributed equally to this work.
The authors declare no competing financial interests.
Correspondence should be addressed to Paul W. Frankland at the above address. E-mail: paul.
frankland@sickkids.ca.
DOI:10.1523/JNEUROSCI.3432-11.2011
Copyright © 2011 the authors 0270-6474/11/3115113-15$15.00/0

represent an essential component of a hippocampal memory. A
direct way to test this would be to examine the impact of removing only this population of neurons after memory formation on
subsequent expression of that memory. Although transgenic,
pharmacological, and irradiation-based approaches have previously been used to manipulate adult neurogenesis before memory formation (Shors et al., 2001; Saxe et al., 2006; Dupret et al.,2008; Imayoshi et al., 2008; Zhang et al., 2008; Clelland et al.,2009; Deng et al., 2009; Garthe et al., 2009; Kitamura et al., 2009),
it has been technically challenging to manipulate adult-generated
neurons after learning.
To address this question, we developed a “tag and ablate”
transgenic strategy that allowed us to tag adult-generated neurons, allow them to mature, and ablate them either before or after
training (see Fig. 1a). To ablate neurons, we used a diphtheria
toxin (DT)-based system (Buch et al., 2005; Han et al., 2009).
Apoptotic cell death is reliably induced after DT binds to the DT
receptor (DTR). Since mice do not express functional DTRs, andare therefore normally insensitive to DT (Middlebrook and Dorland, 1977), we used transgenic mice that express simian DTRs in
a Cre recombinase-inducible manner (iDTR mice) (Buch et al.,2005). To restrict DT-induced apoptosis to adult-generated neurons, we crossed iDTR mice with nestin-Cre ERT2 mice, in which atamoxifen (TAM)-inducible Cre recombinase is expressed under
the control of a nestin promoter (Imayoshi et al., 2008). In adultoffspring from this cross, TAM administration induces permanent expression of DTRs in neural progenitor cells and theirprogeny, and subsequent administration of DT ablates only thisArruda-Carvalho et al. • Adult Neurogenesis and Retrograde Amnesia

15114 • J. Neurosci., October 19, 2011 • 31(42):15113–15127

tagged population of adult-generated neurons. We found that
selective ablation of these tagged, adult-generated neurons immediately (or up to 1 month) after training impaired memory
expression using three distinct hippocampus-dependent tasks.
Therefore, our data indicate that adult-generated neurons, if
available at the time of learning, come to form an essential andenduring component of hippocampal memory traces.

Materials and Methods
Cell culture
To verify that murine-derived cells are insensitive to DT, we conducted
cell viability assays using two cell lines, one from mice (3T3 cells) and one
from monkey (2-2 cells). Cells were seeded in six-well plates (2 ⫻ 10 5
cells per plate). Twenty-four hours later, DT (0, 0.01, 0.1, or 1 ng/ml) wasapplied to fresh medium. Supernatant and trypsinized cells were collected 72 h later. Cell viability was assessed by exclusion of trypan blue
(Sigma). Internal duplicates were run for each condition.

Mice

Nestin-CreERT2 mice. Nestin-Cre ERT2⫹ mice express TAM-inducible Cre
recombinase under the control of a nestin promoter and have been described previously (Imayoshi et al., 2008). The line of nestin-Cre ERT2
mice we used in our experiments corresponds to line 4 in the study by
Imayoshi et al. (2008) and has the highest recombination efficiency in thesubgranular zone of the hippocampus.
iDTR mice. iDTR mice have been described previously (Buch et al.,2005; Gropp et al., 2005). In iDTR ⫹ mice, the gene encoding 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 recombinasemediated removal of a transcriptional STOP cassette. It is important tonote that neither high doses of DT in wild-type mice (Saito et al., 2001)
nor expression of DTR alone (without DT) (Buch et al., 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, which 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 ⫹, and nestin-Cre ERT2⫺/iDTR ⫺ offspring. An equivalent breeding strategy was used for the LacZ reporterline. Except in the case of wild-type mice, all of the transgenes were kept
as heterozygote in the chromosome of each transgenic mouse to avoid
possible complications of overexpressing Cre recombinase (Forni et al.,2006) or loss of the Rosa allele (Zambrowicz et al., 1997). In all ablation
experiments, we compared double-transgenic mice (2xTg; nestin-CreERT2⫹/iDTR ⫹) with control, single-transgenic littermate mice (CTR;
nestin-Cre ERT2⫹/iDTR ⫺ or nestin-Cre ERT2⫺/iDTR ⫹). Both CTR and
2xTg mice were treated with TAM and, subsequently, DT (see below).
Importantly, this design ensures that group effects cannot be attributed to nonspecific effects of TAM or DT. To characterize TAM-induced
recombination, we compared nestin-Cre ERT2⫹/Rosa-LacZ ⫹ versus NestinCre ERT2⫺/Rosa-LacZ ⫹ mice.
Mice were bred in our colony at The Hospital for Sick Children andmaintained on a 12 h light/dark cycle with ad libitum access to food andwater. Before all behavioral experiments, mice were handled for 2 min
twice per day for 5 d. Male and female offspring were used in all experi-ments. All experiments were performed in the light cycle and conductedin accordance with The Hospital for Sick Children Animal Care and Use
Committee.

Drugs
TAM treatment. TAM (Sigma) was dissolved in minimal ethanol andsuspended in sunflower seed oil (Lagace et al., 2007). Mice received threerounds of TAM treatment. In each round, mice received daily injections(180 mg/kg, i.p.) for 5 d, and each round was 4 weeks apart.
DT treatment. Preparation and delivery of DT was described previously (Han et al., 2009). DT (Sigma) was dissolved in PBS and readilycrosses the blood– brain barrier (Wrobel et al., 1990). In the majority ofexperiments, mice received daily injections of DT (16 ␮g/kg, i.p) for 7 d.
In two experiments (the visual discrimination and remote water mazeexperiments), mice received injections for 2 d. We chose 2 d of DT
treatment in these experiments because we observed significant forgetting after a week-long retention delay in preliminary visual discrimination experiments.

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 avibratome (VT1200S; Leica). The following primary antibodies wereused: rabbit monoclonal anti-calbindin (D28K; 1:600; Cell Signaling
Technology), mouse monoclonal anti-calretinin (1:1500; Swant), goatpolyclonal anti-doublecortin (DCX; 1:4500; Santa Cruz Biotechnology),
rabbit polyclonal anti-Egr1 (Zif268; 1:10000; Santa Cruz Biotechnology),
rabbit polyclonal anti-ER␣ (1:1000; Santa Cruz Biotechnology), mousemonoclonal anti-glial fibrillary acidic protein (GFAP; 1:120,000; Cell
Signaling Technology), rabbit polyclonal anti-Iba1 (1:1200; Wako
Chemicals), mouse monoclonal anti-NeuN (1:1000; Millipore), rabbit
polyclonal anti-LacZ (1:6000; Invitrogen), mouse monoclonal antinestin (1:150; BD PharMingen), rabbit polyclonal anti-Ki67 (1:10,000;
Abcam), and goat anti-human heparin binding-EGF (DTR; 1:150; R&D
Systems). All sections were treated with 1% hydrogen peroxidase. Sections were then incubated overnight with the primary antibody and thenfor 60 min at 20°C with HRP-conjugated or biotinylated secondary antibodies (1:750; Jackson Immunoresearch Laboratories). Signals were
amplified and visualized using the Vectastain Elite ABC kit (Vector
Laboratories), tyramide signal amplification, Alexa-Fluor conjugatedstreptavidin (Invitrogen), or DAB. Sections were mounted on slides
with Permafluor antifade medium. For Ki67 staining, slides werecounterstained with methyl green and mounted with Cytoseal 280
mounting medium (Thermo Fisher Scientific).

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 one-fifth systematic section sampling fractions covering the entire anteroposterior extent of the DG. 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 theanteroposterior extent of the DG using a 10⫻ objective on the Eclipse 80i
epifluorescence microscope (Nikon). We estimated the total number of
LacZ ⫹ cells after TAM treatment using the optical fractionator methodon the BX61 epifluorescence microscope (Olympus) using a 60⫻, 1.45
NA objective and a motorized XYZ stage attached to a computer with
StereoInvestigator 9.1 (MBF Bioscience) (Chen et al., 2004). A random
systematic sampling was used for these stereological analyses (section
interval, one-fifth; grid size, 250 ⫻ 250 ␮m; 2D counting frame, 90 ⫻ 90
␮m; fractionators, 30 ␮m in thickness). Tissue thickness measured ineach counting frame was used to estimate the total number of LacZ ⫹ cells
in the entire DG. Conditions were optimized to obtain a Gundersen
coefficient of error below 0.05 (Gundersen et al., 1999). We quantified
GFAP ⫹ cells and Iba1 ⫹ cells in the DG and CA1 throughout the entire
anteroposterior extent of the hippocampus. Using StereoInvestigator
software and the BX61 epifluorescence microscope (Olympus), we cre-

Arruda-Carvalho et al. • Adult Neurogenesis and Retrograde Amnesia

ated separate contours for the DG and the CA1. All GFAP ⫹ and Iba1 ⫹cells within the contours were counted using a 40⫻ objective. Cell density was calculated by the total number of cells divided by the total area ofthe contours.

General behavioral apparatus and procedures
Context fear conditioning. In the fear conditioning experiments, threecontexts were used. Context A (the training context) consisted of a stainless steel conditioning chamber (31 ⫻ 24 ⫻ 21 cm; MED Associates),
containing a stainless steel shock grid floor. Shock grid bars (diameter,
3.2 mm) were spaced 7.9 mm apart. The grid floor was positioned over astainless-steel drop pan, which was lightly cleaned with 70% ethyl alcohol
to provide a background odor. The front, top, and back of the chamber
were made of clear acrylic, and the two sides were made of modular
aluminum. For context B, a white plastic floor covered the shock gridbars, and a plastic, triangular insert was placed inside the same conditioning chamber used for context A. One of the walls of this insert had ablack/white-striped pattern. The other two walls were white. Context B
was cleaned with water. As contexts A and B were located in the same
windowless room and used common apparatus, they shared some overlapping features. In contrast, context C (37 ⫻ 16 ⫻ 27 cm) was located ina different room and contained features that were essentially distinct
from context A or B. It was made of opaque acrylic walls, with bedding
covering the floor. In contexts A and B, mouse freezing behavior wasmonitored via overhead cameras. Freezing was assessed using an automated scoring system (Actimetrics), which digitized the video signal at 4
Hz and compared movement frame by frame to determine the amount offreezing. Freezing in context C was scored manually.
During training, mice were placed in context A. After 2 min, mice were
presented with a 30 s tone (2800 Hz, 85 dB) that coterminated with a 2 s
footshock (0.5 mA). Mice remained in the context for an additional 30 s
before being returned to their home cage. Responsivity to the shock
during training was estimated by comparing mouse velocity immediately
preceding versus during shock presentation using the following formula:(velocityshock ⫺ velocitypre-shock)/(velocityshock ⫹ velocitypre-shock).
Eight days after training, freezing was assessed in 5 min tests in contexts A and B (⬃4 h intertest interval). In context B, the tone was presented after a 2 min delay. Three hours later, freezing was assessed in a 2
min test in context C. Discrimination of contexts A and B was computedby comparing freezing in contexts A and B during the first 2 min of
testing (i.e., before the tone was presented). As in previous studies, weused the following discrimination index (Corvelo and Eyras, 2008; Wanget al., 2009): (freezingcxt A ⫺ freezingcxt B)/max(freezingcxt A, freezingcxt B).
Discrimination scores ranged from ⫺1 to ⫹1, with positive scores reflecting greater levels of freezing in context A compared with context B.
This measure changes at the same rate regardless of the relative differencein freezing levels in contexts A and B, and therefore it is more sensitive in
detecting small differences in relative freezing levels than other commonly used discrimination indices [e.g., (freezingcxt A ⫺ freezingcxt B)/(freezingcxt A ⫹ freezingcxt B)] (McHugh et al., 2007) that change slowly
when differences are small and more rapidly when differences are large(Corvelo and Eyras, 2008). Importantly, we found no difference between
statistical analyses based on our discrimination index compared withother commonly used discrimination indices.
Water maze (hidden platform version). The apparatus and behavioral
procedures have been described previously (Teixeira et al., 2006; Kee etal., 2007). Behavioral testing was conducted in a circular water maze tank(120 cm in diameter, 50 cm deep), located in a dimly lit room. The pool
was filled to a depth of 40 cm with water made opaque by adding white,
nontoxic paint. Water temperature was maintained at 28 ⫾ 1°C by aheating pad located beneath the pool. A circular escape platform (10 cm
diameter) was submerged 0.5 cm below the water surface, in a fixed
position in one of the quadrants. The pool was surrounded by curtains atleast 1 m from the perimeter of the pool. The curtains were white and had
distinct cues painted on them.
Water maze training took place over 5 d. On each day, mice received
three training trials (intertrial interval, ⬃15 s). For each trial, mice werereleased into the pool, facing the wall, at one of four pseudorandomly
varied start locations. The trial was complete once the mouse found theJ. Neurosci., October 19, 2011 • 31(42):15113–15127 • 15115

platform or 60 s had elapsed. If the mouse failed to find the platform ona given trial, the experimenter guided the mouse onto the platform. Afterthe completion of training, spatial memory was assessed in a 60 s probe
test with the platform removed from the pool. Behavioral data fromtraining and the probe tests were acquired and analyzed using an automated tracking system (Actimetrics). Using this software, we recorded anumber of parameters during training, including escape latency andswim speed. In probe tests, we measured the amount of time micesearched the target zone (23.6 cm in radius, centered on the location of
the platform during training) versus the average of three other equivalentzones in other areas of the pool (Moser et al., 1993). Each zone represents
15% of the total pool surface.
Water maze (visual discrimination version). For the visual discrimination task, we modified the water maze apparatus described above. The
pool contained two visual cues. These cues were cylindrical (4 cm indiameter, 4 cm in height), with either a vertical or horizontal black/white-striped pattern. One of these cues (counterbalanced across mice)
was always positioned above the submerged escape platform (10 cm indiameter, 0.5 cm below the surface of the water). A transparent plastic
cylindrical bar (1 cm in diameter, 13 cm in height) connected the cue tothe platform. The other nonreinforced cue was also positioned 13 cm
above the surface of the water. For the nonreinforced cue, an identicaltransparent plastic cylindrical bar connected the cue to base of the pool.
Nonpatterned white curtains replaced the curtains containing distal cues
to minimize reliance on spatial strategies.
Visual discrimination training took place over 5 d. Across training
trials, the locations of the reinforced and nonreinforced cues were varied
pseudorandomly. On each day mice, received six training trials (intertrial
interval, ⬃10 min). On each trial, mice were released into the pool, facing
the wall, at one of four pseudorandomly varied start locations. The trial
was complete once the mouse found the escape platform or 60 s had
elapsed. If the mouse failed to find the platform on a given trial, theexperimenter guided the mouse onto the platform. Three days after thecompletion of training, discrimination memory was assessed in a 30 sprobe test with both cues present, but with the platform removed fromthe pool. As before, behavioral data from training and the probe tests
were acquired and analyzed using an automated tracking system. During
the probe test, searching was highly focused around the two cues. Accordingly, we compared time spent in two zones (15 cm in radius) centered on the cue locations. Additionally, heat maps representing averaged
group data were generated using Matlab (MathWorks). In the heat map,the average time(s) mice spent in a 6 ⫻ 6 cm area of the pool was normalized for n ⫽ 10 mice per group. Behavioral procedures were adaptedfrom previous studies which established that posttraining lesions of the
hippocampus disrupt visual discrimination memory (Sutherland et al.,
2001; Epp et al., 2008).

Specific experimental protocols

Characterization of TAM-induced recombination. Nestin-Cre ERT2⫹/
Rosa-LacZ ⫹ or nestin-Cre ERT2⫺/Rosa-LacZ ⫹ mice were treated with
TAM. Seven weeks after the completion of TAM treatment, the number,
distribution, and cellular phenotype of recombined LacZ ⫹ cells wasquantified using immunohistochemical methods.
Characterization of TAM-induced recombination and ablation in 2xTg
mice. 2xTg mice were treated with TAM. Seven weeks after the completion of TAM treatment, mice received daily injections of PBS or DT for
7 d. Twenty-four hours after the final DT injection, mice were perfused,and DTR, nestin, calretinin, and DCX expression were quantified
immunohistochemically.
Characterization of basal proliferation rates in 2xTg mice. To evaluate
whether our tagging impacted ongoing proliferation in the adult hippocampus, we examined expression of Ki67 in CTR and 2xTg mice 7
weeks after the completion of TAM treatment. Ki67 is a cell-cycle-related
nuclear protein, expressed by proliferating cells in all phases of the active
cell cycle (Kee et al., 2002).
Contextual fear: posttraining ablation. CTR and 2xTg mice were treatedwith TAM. Seven weeks after the completion of TAM treatment, mice
were trained in context A. Beginning 1 d later, mice received daily injec-

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Arruda-Carvalho et al. • Adult Neurogenesis and Retrograde Amnesia

tions of DT for 7 d. One week after training,
freezing was assessed in contexts A and B andthen in context C in a subset of mice.
Contextual fear: pretraining ablation. CTR
and 2xTg mice were treated with TAM. Sevenweeks after the completion of TAM treatment,
mice were trained in context A. One week after
training, freezing was assessed in contexts A
and B and later in context C. In this experiment, mice received daily injections of DT for7 d during the week preceding training.
Conditioned taste aversion: posttraining
ablation. To evaluate whether posttraining DTinduced ablation impairs subsequent expression of a conditioned taste aversion memory,
we trained CTR and 2xTg mice 7 weeks after
the completion of TAM treatment, using previously described methods (Ding et al., 2008).
During training, mice had access to a single
bottle containing saccharin (0.2%) for 30 min.
Forty minutes later, mice were treated with
LiCl (0.15 M, i.p., 2% body weight). Beginning
the next day, mice received daily injections ofDT-based ablation. a, Schematic of the tag and ablate strategy for ablating mature, adult-generated neurons. In adult
DT for 7 d. One day later, conditioned taste Figure 1. ERT2⫹
nestin-Cre/iDTR ⫹ mice (2xTg), TAM administration leads to permanent expression of DTRs in neural progenitor cells andaversion was assessed in a choice test in which
theirprogeny.
Subsequentadministration of DT ablates this tagged population of adult-generated neurons only. b, c, In vitro assay
mice had access to bottles containing either
demonstrating
insensitivityof wild-type mouse cell lines to DT. Whereas mouse-derived 3T3 cells were insensitive to increasingwater or saccharin. An aversion index was calculated as saccharin consumed/total fluid con- concentrations of DT (n ⫽ 2) (b), DT dose-dependently reduced the number and viability of monkey-derived 2-2 cells (n ⫽ 3) (c).
sumed. An additional group of mice weretions of DT for 2 d. One day later, discrimination was assessed in a probe
treated identically except that they received an injection of saline (instead
test.
of LiCl) during training.
General behavioral characterization after DT-induced ablation. CTR
Water maze, hidden platform version: posttraining ablation, recent
and 2xTg mice were treated with TAM. Seven weeks after the completiongroup. CTR and 2xTg mice were treated with TAM. Seven weeks after theof TAM treatment, mice received daily DT injections for 7 d, and behavcompletion of TAM treatment, mice were trained in the hidden platform
ior was evaluated in a battery of tests. All mice were given the following
version of water maze for 5 d. Beginning 1 d after the completion of water
tests in the same order (open field, visual discrimination, beam walking,maze training, mice received daily injections of DT for 7 d. One day later,
hanging grip, sticky dot/adhesive removal, forced swim).
spatial memory was assessed in a probe test. Additional groups of CTR
For open-field testing, mice were placed in the center of a squareand 2xTg mice were treated identically, except that they received PBS
shaped arena (45 ⫻ 45 ⫻ 20 cm height) and allowed to explore for 20
rather than DT during the 7 d before the probe test.
min. The open-field apparatus was constructed of Plexiglas and was
Water maze, hidden platform version: pretraining ablation. CTR anddimly lit from above. Mouse location was tracked by a camera located
2xTg mice were treated with TAM. Seven weeks after the completion ofabove the open field, and total distance traveled and time spent in three
TAM treatment, mice were trained in the hidden platform version of thedifferent zones (outer, middle, inner) was measured (Limelight2; Acwater maze for 5 d, and spatial memory was tested in a probe test 1 week
timetrics). Total distance traveled was used as a measure of spontaneous
later. In this experiment, mice received daily injections of DT for 7 d
motor activity, and distribution of activity in different regions of theduring the week preceding training.
arena was used as a measure of anxiety-related behavior (Archer, 1973).
Water maze, hidden platform version: posttraining ablation, remote
To evaluate vision, we trained mice in the visual discrimination watergroup. CTR and 2xTg mice were treated with TAM. Seven weeks after themaze task across 3 consecutive days (six trials per day). The latency tocompletion of TAM treatment, mice were trained in the hidden platform
reach the platform and swim speed were recorded.
version of water maze for 5 d. Mice were treated identically to above
To assess sensorimotor control and locomotor activity, we used theexcept that 35 d after the completion of water maze training, mice rebeam walking task (Chen et al., 2004). Mice were placed on a beam (70ceived daily injections of DT for 2 d. One day later, spatial memory was
cm long, 2 cm wide, elevated 30 cm above floor) for 60 s, and paw slips
assessed in a probe test.
(forelimb and hindlimb) were recorded. Each mouse received three conWater maze visual discrimination version: posttraining lesion. One day
secutive trials, and paw slips were calculated per distance traveled.
after training in the visual discrimination task, wild-type mice received
To evaluate muscle strength, the hanging grip test was used (Chen etsham lesions or NMDA lesions of the entire hippocampus as describedal., 2004). In this test, mice were allowed to hang vertically from a metal
previously (Wang et al., 2009). Two days later, discrimination was aswire using their forepaws. The latency to fall was measured. Importantly,
sessed in a probe test. Mice were perfused after the probe test. Brains weremouse weights did not differ between genotypes.
removed, fixed overnight in PFA, and transferred to 30% sucrose. CorTo assess somatosensory function, we used the sticky dot or adheonal sections (50 ␮m) were cut using a cryostat. Sections were mounted
sive removal task (Schallert et al., 2000). Briefly, a small piece of tapeon gelatin-coated slides, stained with neutral red, and coverslipped with
was placed on the plantar surface of the mouse’s forepaw. Time taken
Cytoseal. Using StereoInvestigator software, the entire hippocampus andfor mice to sense (i.e., shaking its paw or bringing its paw to its
the area of the hippocampus sustaining damage were outlined separately
mouth) and remove the adhesive tape were recorded. Each mouse wasfor every fourth section. The proportion of total hippocampal tissuetested three times, and data were averaged. Each test lasted a maxidamaged was 66.2 ⫾ 7.3%.
mum of 3 min.
Water maze visual discrimination version: posttraining ablation. CTR
To assess depressive-like behaviors, we used the forced swim test (Porand 2xTg mice were treated with TAM. Seven weeks after the completion
solt, 1979). Mice were placed in a cylinder (radius, 6.5 cm; height, 20 cm)
of TAM treatment, mice were trained in the visual discrimination task.
for 6 min. The cylinder was filled to a depth of 12 cm with 25 ⫾ 1°C water.
The time spent motionless was recorded for each mouse.
Beginning 1 d after the completion of training, mice received daily injec-

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J. Neurosci., October 19, 2011 • 31(42):15113–15127 • 15117

from monkey (2-2 cells). As expected,
DT dose-dependently reduced the number and viability of 2-2, but not 3T3,
cells (Fig. 1b,c) (Middlebrook and Dorland, 1977).
Characterization of tag and ablate mice
Tagging new neurons
We first characterized TAM-induced recombination (tagging) in nestin-Cre ERT2
mice by crossing them with rosa-LacZ reporter mice. In these reporter mice, Cremediated excision of a STOP cassette
induces LacZ expression under the control of the Rosa26 promoter (the samepromoter as in the iDTR mice). Consistent with previous results, at 4 weeks ofage, Cre ERT2 protein expression in nestinCre ERT2⫹ mice was robust, restricted toprogenitor cells (Fig. 2a, Table 1), andlimited to adult neurogenic regions including the subgranular zone (Fig. 2b– g)(Imayoshi et al., 2008). As expected, TAM
induced recombination (LacZ ⫹ cell) in
nestin-Cre ERT2⫹/Rosa-LacZ ⫹ reporter
mice, and no recombination was observedin nestin-Cre ERT2⫺/Rosa-LacZ ⫹ littermate
controls (Fig. 3a). We next examinedtagged cells 7 weeks after TAM administration was complete. As observed previously (Imayoshi et al., 2008), TAMinduced recombination in the dividing
progenitor population was highly efficient, with 86.2 ⫾ 2.7% (mean ⫾ SEM) of
Ki67 ⫹ cells and 77.7 ⫾ 7.8% of nestin ⫹
ERT2
ERT2⫹
Figure 2. Cre expression is restricted to progenitor cells and limited to adult neurogenic regions. a, In nestin-Cre
cells in the DG expressing LacZ. Furthermice, Cre ERT2 protein expression (green) was found in nestin ⫹ and DCX ⫹ cells (red) but not mature neurons (NeuN; red) in the DG more, LacZ ⫹ cells were uniformly distrib(scale bar, 10 ␮m). b– g, In these mice, Cre ERT2 protein expression was limited to the subgranular zone of the DG and the uted throughout the anteroposterior
subventricular zone of the lateral ventricle (LV) (millimeters relative to bregma; scale bars, 250 ␮m). Mo, Molecular layer; GCL, extent of the DG (Fig. 3b), suggesting no
granule cell layer; CPu, caudate–putamen; Pir, piriform cortex; Thl, thalamus; OB, olfactory bulb; Gr, granular layer; Gl, glomerular
intraregional differences in TAMlayer; CA, cornu ammonis.
induced recombination efficiency. Using
stereological methods, we estimated there
Table 1. Cell maturity markers
were 31,709 ⫾ 2984 LacZ ⫹ cells and 496,266 ⫾ 46,556 NeuN ⫹NestinDCX
NeuN
cells in the DG. Therefore, our tagged population of cells repreERT2⫹
sents ⬃6% of the entire population of DG neurons (i.e., 31,709 ofPercentage of Cre
97 ⫾ 0.94
85 ⫾ 2.3
0.72 ⫾ 0.26
cells
expressing marker (⫾SEM)
496,266).
Cells analyzed
490
460
474
To evaluate the phenotype of tagged cells, we next stainedfor proteins expressed at different stages of cell differentiation(Kempermann et al., 2004) (Fig. 3c). Ninety-four percent oftagged (LacZ ⫹) cells were also positive for markers of mature
Data analysis
neurons (NeuN, calbindin) with far fewer (⬍5%) that were
Data were analyzed using ANOVAs followed by t tests. Because both
positive for progenitor cell or immature neuronal markers
male and female mice were used, sex was initially included as a factor(nestin, doublecortin, calretinin) (Fig. 3d, Table 2). Therefore,
in the ANOVAs. Consistent with previous studies (Jonasson, 2005),
we found that males performed better than females in one of the water
this predominantly mature population of tagged neurons cormaze experiments (hidden platform version, posttraining ablation,
responds to the population of adult-generated neurons thatrecent group). However, we found no further effects of sex and noare activated during the formation and/or expression ofsignificant interactions between sex and genotype; therefore, this fachippocampus-dependent memories (Kee et al., 2007; Stone et
tor was dropped from analysis.
al., 2011). Consistent with this, at this time point LacZ ⫹ cells
expressed activity-dependent genes such as zif268 after trainResultsing (Fig. 3d). Importantly, basal levels of proliferation in the
Murine cells are insensitive to DT
hippocampus were unaltered by tagging (unpaired t test, p ⬎We verified that murine cells are insensitive to DT using two0.05) (Fig. 3e).
cell lines, one derived from mice (3T3 cells) and the otherArruda-Carvalho et al. • Adult Neurogenesis and Retrograde Amnesia

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Figure 3. Tagging new neurons. a, TAM-induced recombination occurs only in nestin-Cre ERT2⫹ mice (scale bar, 50 ␮m). b, Recombination occurred throughout the anteroposterior extent of theDG (millimeter relative to bregma; scale bars, 150 ␮m). c, Schematic showing markers associated with different developmental stages of adult hippocampal neurogenesis. d, Seven weeks after thecompletion of TAM treatment, most LacZ ⫹ cells (green) costained for mature neuronal markers (NeuN, calbindin; red), with far less staining for progenitor cell or immature neuronal markers (nestin,
DCX, calretinin; red). LacZ ⫹ cells additionally expressed activity-dependent gene zif268 (red) after behavioral testing. e, Seven weeks after the completion of TAM treatment, Ki67 expression levels
were similar in control (CTR; n ⫽ 11) and 2xTg (n ⫽ 7) mice, indicating that DTR expression has no effect on ongoing proliferative activity in the adult hippocampus (scale bar, 250 ␮m). Mo,
Molecular layer; GCL, granule cell layer.
Table 2. Cell types
Cell maturity marker
Percentage of LacZ ⫹ cells expressing marker (⫾SEM)
Cells analyzed

Type 1, 2a, 2b

Type 2b, 3, immature neuron

Immature neuron

Immature to mature neuron

Postmitotic mature neuron

Nestin3.1 ⫾ 1.1455

DCX
5.1 ⫾ 1.8
513

Calretinin
0.72 ⫾ 0.46
457

NeuN
94 ⫾ 1.4
374

Calbindin
94 ⫾ 2.1
461

Ablating tagged neurons
To ablate these tagged neurons, we crossed nestin-Cre ERT2 micewith iDTR mice, in which Cre-mediated excision of a STOP cassette renders cells sensitive to DT (Buch et al., 2005). In TAMtreated nestin-Cre ERT2⫹/iDTR ⫹ (2xTg) mice, DTR-expressing
cells were localized to the subgranular zone and innermost layerof the DG (consistent with the pattern of LacZ expression in thereporter mice above) (Fig. 4a). Subsequent systemic injection ofDT (but not PBS) virtually abolished these DTR-expressing cells(100% reduction; unpaired t test: t(8) ⫽ 1.93, p ⬍ 0.05) (Fig. 4a),indicating that DT treatment efficiently ablated the tagged neurons (Buch et al., 2005; Han et al., 2009). In the same mice, we
additionally quantified numbers of progenitor cells and immature neurons in the DG after DT versus PBS treatment. We foundthat DT treatment greatly reduced overall numbers of doublecortin ⫹ (Fig. 4b), nestin ⫹ (⬃94% reduction; unpaired t test: t(7) ⫽4.16, p ⬍ 0.01) (Fig. 4c), and calretinin ⫹ (⬃86% reduction; unpaired t test: t(6) ⫽ 2.03, p ⬍ 0.05) (Fig. 4d) cells in the DG of 2xTg
mice. Whereas this subpopulation represents only a small proportion of all tagged cells (⬃94% are NeuN ⫹ or calbindin ⫹),nonetheless these data are consistent with the reduction in DTRexpressing cells and suggests that DT-induced ablation is highly
efficient.
After DT treatment, there were similar numbers of astrocytes(unpaired t tests, p ⬎ 0.05) but increased microglia in the hippocampus (Fig. 5). The increase in microglia was limited to theDG (unpaired t test: t(7) ⫽ 3.46, p ⬍ 0.01) and not observed in
CA1 (unpaired t test, p ⬎ 0.05); therefore, it likely reflects localized phagocytosis after DT-induced apoptotic cell death. Furthermore, TAM and DT treatment produced no changes inmouse weight (unpaired t tests, p ⬎ 0.05) (Fig. 6a) or in a range oftests assessing emotion, vision, motor, or somatosensory func-

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tantly, this design ensures that groupeffects cannot be attributed to nonspecificeffects of TAM or DT.
Posttraining ablation of tagged neurons
degrades a contextual fear memory
We used our tag and ablate strategy totest whether deletion of adult-generated
neurons after training would impair
subsequent memory expression. To test
memory, we first used a contextual fear
conditioning task in which mice learn anassociation between a context and anaversive event (i.e., the delivery of a mildfootshock). When returned to the samecontext, contextual fear memory is inferredfrom an increase in freezing behavior (Kimand Fanselow, 1992). The specificity of thememory may then be evaluated by comparing freezing levels in the trained versus
alternate contexts (Wang et al., 2009).
This task is hippocampus dependent (Kimand Fanselow, 1992) and engages dentate
granule cells (including those generated
Figure 4. Ablating tagged neurons. a, DT (n ⫽ 4) but not PBS (n ⫽ 6) efficiently ablated DTR-expressing cells in the DG (scale during adulthood) (Stone et al., 2011).
bar, 10 ␮m). b– d, Consistent with this, DT treatment reduced overall numbers of doublecortin ⫹ (DCX; scale bar, 150 ␮m) (b), We trained 2xTg and control mice with anestin ⫹ (scale bar, 10 ␮m) (c), and calretinin ⫹ (scale bar, 10 ␮m) (d) cells in the DG of 2xTg mice. Mo, Molecular layer; GCL, tone–shock pairing 7 weeks after TAM
granule cell layer. *p ⬍ 0.05.
treatment (Fig. 7a), a time point when thevast majority of tagged, adult-generated
neurons have matured (i.e., ⬃94% express the mature neuronal marker calbindin, indicating that they are ⬃4 weeks ofage or older (Zhao et al., 2008). During
training, both 2xTg and control mice responded similarly to the shock (unpaired t
test, p ⬎ 0.05) (Fig. 7b) and exhibited
equivalent levels of freezing immediately
before and after the footshock (Genotype ⫻ Training phase ANOVA, effect ofTraining phase only, F(1,20) ⫽ 31.85, p ⬍0.001; planned comparison for aftershock freezing indicated CTR vs 2xTg,
p ⬎ 0.05) (Fig. 7c), indicating that tagging
neurons (DTR expression) alone does notalter responsivity to shock or general activity levels. After DT treatment, control
mice exhibited robust freezing in the
trained context (context A) and less freezing in an alternate context (context B) thatshared a number of overlapping featureswith the training context. In contrast,
Figure 5. DT-induced ablation produces minimal inflammation. a, GFAP (red) and ionized calcium binding adaptor molecule 1 2xTg exhibited robust but equivalent(Iba1; green) expression in CTR (n ⫽ 5) and 2xTg (n ⫽ 4) mice 24 h after administration of DT. b, GFAP levels were similar for CTR levels of freezing in both (Genotype ⫻and 2xTg mice in both the DG and CA1 regions. c, Iba1 expression was increased in 2xTg mice only in the DG and not in the CA1 Context ANOVA, Genotype ⫻ Context
region. Note that Iba1 expression was mainly limited to the subgranular zone and innermost layer of the DG, a pattern that matches interaction, F(1,21) ⫽ 17.66, p ⬍ 0.005;
the distribution of tagged (i.e., DTR ⫹ or LacZ ⫹) cells after TAM treatment. *p ⬍ 0.05.
Newman–Keuls post hoc tests indicated
ACTR ⬎ BCTR only, p ⬍ 0.05) (Fig. 7d),
indicating that this posttraining ablation
tion (ANOVAs and unpaired t tests; Genotype effects, p ⬎ 0.05)
abolished the ability to discriminate two similar contexts (un(Fig. 6b–j). In subsequent behavioral experiments, 2xTg and litpaired t test, t(21) ⫽ 4.27, p ⬍ 0.001) (Fig. 7e). To test whether thisposttraining ablation abolished the ability to discriminate distermate control mice (either nestin-Cre ERT2⫹ or iDTR ⫹ but notboth) were used. All mice were treated with TAM and subsesimilar contexts, we next placed mice in a third context (context
quently treated with DT either before or after training. ImporC), which had no overlapping features with the original training

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Figure 6. General health and behavior are not altered by DT-induced ablation. Seven weeks after the completion of TAM treatment, CTR (n ⫽ 6) and 2xTg (n ⫽ 7) mice were treatedwith DT. a, Body weights were not different after the completion of TAM or DT treatments. b–j, The behavior of TAM- and DT-treated CTR and 2xTg mice was characterized in a batteryof tests. We observed no effect on time spent immobile in the forced swim test (b); total exploration in the open field (c); time spent in the outer, middle, and innermost regions of theopen field (d); latency to find platform in the visual discrimination water maze (WM) (e); swim speed during training in the visual discrimination water maze (f ); paw slips in the beam
walk test (g); latency to fall in the bar hanging test (h); latency to detect adhesive tape in the sticky dot test (i); or latency to remove adhesive tape in the sticky dot test (j).

context. In context C, both 2xTg and control mice exhibited
similarly low levels of freezing (unpaired t test, p ⬎ 0.05) (Fig. 7f ).
Therefore, these results indicate that posttraining ablation of
adult-generated neurons led to the degradation (but not the erasure) of a contextual fear memory: whereas recognition of the
training context was unaffected, the ability to discriminate between similar (but not dissimilar) contexts was impaired.
The present experimental design rules out several alternativeinterpretations of the data. First, the absence of freezing in context C indicates that the discrimination deficit in 2xTg mice was
not attributable to an overall increase in propensity to freeze.
Second, because DT was administered in the home cage, memory degradation effects cannot be caused by blockade of areactivation-induced phenomenon, such as reconsolidation (Nader et al., 2000). Third, memory for the tone–shock association
was unaltered after the posttraining ablation (unpaired t test, p ⬎0.05) (Fig. 7g). Because this type of memory is supported by the
amygdala (Han et al., 2009), this suggests that deleting adultgenerated neurons affects hippocampus-dependent memory
only. Consistent with this, in a separate group of mice, similar
posttraining ablation did not affect expression of a previously
acquired conditioned taste aversion memory (Group ANOVA,Group effect, F(4,23) ⫽ 5.53, p ⬍ 0.01; Newman–Keuls post hoc
tests indicated stronger saccharin preference in the CTR/SAL
group compared with the CTR/LiCL and 2xTg/LiCl groups, p ⬍0.05) (Fig. 7h).
Pretraining ablation of tagged neurons does not prevent
formation of new contextual fear memory
We next tested whether ablation of a similar population of adultgenerated neurons immediately before training would impair ac-

quisition of a new contextual fear memory. As in the previous
experiment, 2xTg and control mice were treated with TAM,
trained 7 weeks later, and tested after a 1 week delay. In this case,however, DT was administered during the week before training
rather than during the week after training (Fig. 8a). This experimental design ensures that DT targets an equivalent populationof neurons (in terms of number and maturity) and that the retention delay is identical to the first experiment. During training,both 2xTg and control mice responded similarly to the shock(unpaired t test, p ⬎ 0.05) (Fig. 8b) and exhibited equivalentlevels of freezing immediately before and after shock delivery(Genotype ⫻Training phase ANOVA, effect of Training phase
only, F(1,25) ⫽ 40.95, p ⬍ 0.0001; planned comparison for aftershock freezing indicated CTR vs 2xTg, p ⬎ 0.05) (Fig. 8c), indicating that the pretraining DT-induced ablation did not affectshock reactivity or general activity levels. One week after training,both 2xTg and control mice froze more in context A comparedwith context B (Genotype ⫻ Context ANOVA, effect of Context
only, F(1,25) ⫽ 9.41, p ⬍ 0.005; planned comparisons indicated
ACTR ⬎ BCTR and A2xTg ⬎ B2xTg, p ⬍ 0.05) (Fig. 8d), and, furthermore, the degree of discrimination did not differ between groups(unpaired t test, p ⬎ 0.05) (Fig. 8e). As expected, both 2xTg andcontrol mice exhibited similarly low levels of freezing in context
C (unpaired t test, p ⬎ 0.05) (Fig. 8f ), and tone freezing was also
unaffected by pretraining ablation (unpaired t test, p ⬎ 0.05)(Fig. 8g).
In the first experiment, ablation of a population of predominantly mature, adult-generated neurons immediately
after training impaired subsequent memory expression, presumably because these neurons had become an integral component of the memory trace. In contrast, deletion of anArruda-Carvalho et al. • Adult Neurogenesis and Retrograde Amnesia

J. Neurosci., October 19, 2011 • 31(42):15113–15127 • 15121

address this, we used the hidden platformversion of the water maze task, in which
both acquisition and expression dependon the hippocampus (Riedel et al., 1999;
Teixeira et al., 2006). As before, we trained
2xTg and control mice in the water maze 7
weeks after TAM treatment (Fig. 9a).
During training, latency to find the platform declined similarly in both groups(Genotype ⫻ Training day ANOVA, effect of Training day only, F(4,22) ⫽ 19.21,
p ⬍ 0.0001), indicating that tagging (DTR
expression) does not interfere with swimming, motivation, and vision required for
acquisition of a spatial memory (Fig. 9b).
After the completion of training, 2xTg
and control mice were administered DT,
and their spatial memory was assessed in aprobe test 7 d later. In this test, 2xTg micesearched less selectively than control
mice, spending less time in the target zone
(Genotype ⫻ Zone ANOVA, Genotype ⫻Zone interaction, F(1,22) ⫽ 5.67, p ⬍ 0.05;
planned comparisons indicated TCTR ⬎T2xTg, TCTR ⬎ OCTR, and T2xTg ⬎ O2xTg,
p ⬍ 0.05) (Fig. 9c). We additionally conducted a similar experiment in which
TAM-treated 2xTg and control mice weretrained in the water maze but were administered PBS rather than DT before theprobe test (Fig. 9d). During training, latency to find the platform declined similarly in both groups (Training day ⫻Genotype ANOVA, effect of Training day
only, F(4,23) ⫽ 22.93, p ⬍ 0.0001) (Fig. 9e),and in the probe test, both 2xTg and control mice exhibited robust spatial memory
Figure 7. Posttraining ablation of adult-generated neurons degrades contextual fear memory. a, Mice were treated with (Genotype ⫻ Zone ANOVA, effect of
TAM and trained in context A. After DT-induced ablation of adult-generated neurons, contextual memory was assessed in Zone only, F(1,22) ⫽ 38.50, p ⬍ 0.0001;
contexts A–C. b, c, During training, CTR (n ⫽ 12) and 2xTg (n ⫽ 11) mice exhibited similar response to shock (b) and planned comparisons indicated TCTR ⬎freezing (c) levels before and after shock delivery. d, After DT treatment, CTR mice froze more in the trained context (A) O
CTR and T2xTg ⬎ O2xTg, p ⬍ 0.05, TCTR vsversus a similar context (B). In contrast, 2xTg mice froze equally in both. e, DT-induced ablation abolished context T
2xTg, p ⬎ 0.05) (Fig. 9f ). Together, these
discrimination. f, g, Freezing in a dissimilar context (C) (f) and tone fear (g) were similar in CTR and 2xTg mice. h, Mice were
findings indicate that neither DTR tagtreated with TAM and trained in a conditioned taste aversion task. During training, saccharin was paired with 0.15 M LiCl.
ging (in the absence of DT) nor DT adAfter DT-induced ablation of adult-generated neurons, preference for saccharin versus water was evaluated. CTR (n ⫽ 12)
and 2xTg (n ⫽ 11) mice exhibited equivalent preference for saccharin. Importantly, this preference was lower compared ministration alone (in the absence of DTR
tagging) impairs memory. Instead a comwith mice (n ⫽ 11) for which saccharin was paired with saline (rather than LiCl) during training. *p ⬍ 0.05.
bination of DTR tagging and DT is necessary to induce memory loss.
equivalent population of neurons immediately before training
Consistent with our fear-conditioning data, these results indid not prevent the formation of a new contextual fear memdicate that selective removal of adult-generated neurons after
ory, indicating that memory formation may be supported bytraining impairs subsequent memory expression. In contrast,
existing dentate granule cells when this population of adultDT-induced ablation of an equivalent population of adultgenerated neurons is absent at the time of training. Moreover,generated neurons immediately before training had no effect on
the preserved ability to discriminate between both similar
the formation of a new spatial memory (Fig. 9g). After DT treatand dissimilar contexts suggests that pretraining removal ofment, both 2xTg and CTR mice learned to find the platform withadult-generated neurons did not significantly impact memory
progressively shorter latencies (Genotype ⫻ Training day
quality.
ANOVA, effect of Training day only, F(4,23) ⫽ 9.48, p ⬍ 0.01)(Fig. 9 h). Likewise, in the probe test 1 week later, both 2xTg and
CTR mice searched selectively, spending equivalent amounts ofPosttraining ablation of tagged neurons degradestime in the target zone (Genotype ⫻ Zone ANOVA, effect of
spatial memory
Zone only, F(1,23) ⫽ 8.14, p ⬍ 0.01; planned comparisons indiThe hippocampus is engaged by multiple forms of learning. To
cated TCTR ⬎ OCTR, T2xTg ⬎ O2xTg, p ⬍ 0.05, TCTR vs T2xTg, p ⬎evaluate the generality of our findings, we next asked whether
0.05) (Fig. 9i). This suggests that new spatial learning may besimilar posttraining ablations would impact spatial memory. To

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Arruda-Carvalho et al. • Adult Neurogenesis and Retrograde Amnesia

supported by existing dentate granulecells when adult-generated neurons are
not present at the time of training. Importantly, this pretraining ablation did not affect swimming, navigation, vision, or
motivation required for acquisition andexpression of spatial memory. Therefore,
as pretraining and posttraining ablations
targeted equivalent populations of adultgenerated neurons, these results exclude
the possibility that such performance factors could account for spatial memory
loss after posttraining ablations.
Posttraining ablation of tagged neurons
degrades remote spatial memory
Expression of water maze memory depends on the hippocampus for at least 1
month after training (Clark et al., 2005;
Teixeira et al., 2006). To address whether
adult-generated neurons play a persistentrole in memory expression, we next
treated additional groups of control and
2xTg mice with DT 1 month (rather than
1 d) after training (Fig. 9j). As before, bothgroups learned to locate the platform during training (Genotype ⫻ Training day
ANOVA, effect of Training day only,
F(4,24) ⫽ 26.83, p ⬍ 0.0001) (Fig. 9k) and Figure 8. Pretraining ablation of adult-generated neurons does not prevent the formation of a new contextual fear memory. a,control mice exhibited robust spatial Mice were trained in context A after DT-induced ablation of adult-generated neurons. Contextual memory was assessed in contexts
memory even when tested more than 1 A–C 1 week later. b, c, During training, CTR (n ⫽ 13) and 2xTg (n ⫽ 14) mice exhibited similar response to shock (b) and freezing(c) levels before and after shock delivery. d, e, After DT treatment, both CTR and 2xTg mice discriminated between contexts A andmonth later. In contrast, at this remote B (d), and the degree of discrimination did not differ (e). f, g, Freezing in a dissimilar context (f ) and tone fear (g) were equivalent
time point, 2xTg mice searched less selec- in CTR and 2xTg mice. *p ⬍ 0.05.
tively compared to control mice, spending
less time in the target zone (Genotype ⫻(planned paired t test, t(8) ⫽ 4.27, p ⬍ 0.05). In contrast, mice
Zone ANOVA, Genotype ⫻ Zone interaction, F(1,24) ⫽ 3.53, p ⫽with cytotoxic hippocampal lesions spent similarly little time
0.07; planned comparisons indicated that TCTR ⬎ T2xTg, TCTR ⬎close to either cue (planned paired t test, p ⬎ 0.05) (Fig. 10c). Our
OCTR, and T2xTg ⬎ O2xTg, p ⬍ 0.05) (Fig. 9l ). These findings
lesions affected ⬃66 ⫾ 7% of hippocampal tissue (Fig. 10d).
indicate that adult-generated neurons play an integral and endurSimilar hippocampal lesions in rats produce equivalent deficits
ing role in the expression of a spatial memory.
(Clark et al., 2007) and indicate that an intact hippocampus is
Posttraining ablation degrades visual discrimination memory
Posttraining ablation of adult-generated neurons impaired theability to discriminate between two similar contexts in the fearconditioning experiment. To further explore the nature of thisdeficit, we next developed a water maze visual discrimination
task. In this task, two similar cues (one vertically striped, one
horizontally striped) were positioned above the surface of thewater. Across training trials, the locations of these two cues varied
pseudorandomly. However, one of the cues was always locatedabove a hidden platform (e.g., horizontal stripes). To evaluate
whether this form of visual discrimination memory depends on
the hippocampus, we first examined the impact of posttraining
cytotoxic hippocampal lesions (Fig. 10a). During training, bothgroups of mice learned to discriminate between the reinforced
and nonreinforced cues (Lesion ⫻ Training day ANOVA, effectof Training day only, F(4,68) ⫽ 52.69, p ⬍ 0.0001) (Fig. 10b). Micereceived lesion or sham surgery 1 d after training, and discrimination memory was subsequently assessed in a probe test inwhich both cues were present but neither reinforced. In thisprobe test, control mice spent significantly more time searchingclose to the previously reinforced cue than the nonreinforced cue

necessary for the expression of this form of visual discrimination
memory.
We next evaluated whether posttraining ablation of adultgenerated neurons would similarly impair the expression of avisual discrimination memory (Fig. 10e). During training, 2xTg
and control mice learned to discriminate between the reinforced
and nonreinforced cues (Genotype ⫻ Training day ANOVA, effect of Training day only, F(4,21) ⫽ 24.44, p ⬍ 0.0001) (Fig. 10f ).
After training, both groups of mice were administered DT, anddiscrimination memory was assessed in a probe test. As before,
control mice spent more time searching close to the previously
reinforced cue compared with the nonreinforced cue in the probe
test (planned paired t test, t(11) ⫽ 3.02, p ⬍ 0.01). Interestingly,
DT-induced ablation produced a more subtle deficit than cytotoxic hippocampal lesions. Although discrimination was abolished in the 2xTg mice (planned paired t test, p ⬎ 0.05), theirpropensity to search close to either of the cues remained intact(Fig. 10g,h). Therefore, these results indicate that posttraining
ablation of a population of predominantly mature, adultgenerated neurons led to the degradation (rather than complete
erasure) of a visual discrimination memory: some general features of the memory were retained (e.g., cue–platform associa-

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Figure 9. Posttraining (but not pretraining) ablation of adult-generated neurons impairs spatial memory expression. a, Mice were treated with TAM and trained in the water maze.
After DT-induced ablation of adult-generated neurons, spatial memory was assessed in a probe test. b, During training, latency to find platform declined equivalently in CTR (n ⫽ 12) and2xTg (n ⫽ 12) mice. c, After DT-induced ablation, 2xTg mice searched less selectively compared with CTR mice, spending less time in the target zone (T). d, Additional groups of
TAM-treated mice were trained in the water maze. However, mice were treated with PBS (rather than DT) during the week preceding memory testing. e, During training, latency to findplatform declined equivalently in CTR (n ⫽ 11) and 2xTg (n ⫽ 14) mice. f, In the probe test, both CTR and 2xTg mice searched selectively, spending more time in the target zone comparedwith other (O) nontarget zones in the pool. g, Mice were trained in the hidden version of the water maze after the completion of TAM treatment. During the week before training, mice
were treated with DT. h, During training, latency to find the platform declined equivalently in CTR (n ⫽ 14) and 2xTg (n ⫽ 11) mice. i, In the probe test, both CTR and 2xTg mice searchedselectively at the target zone. j, Mice were treated with TAM and trained in the water maze. One month later, mice were treated with DT, and spatial memory was assessed in a probe test.
k, During training, latency to find platform declined equivalently in CTR (n ⫽ 14) and 2xTg (n ⫽ 12) mice. l, After DT-induced ablation at the remote time point, 2xTg searched lessselectively compared with CTR mice, spending less time in the target zone. *p ⬍ 0.05.

tion), but the ability to discriminate between similar cues was
impaired.

Discussion
In these experiments, we used a tag and ablate transgenic strategy
to examine the role of adult-generated neurons in hippocampal
memory. This approach offered two key advantages. First, unlike
previous approaches that targeted neural stem or progenitor cells
to produce a global disruption of neurogenesis (Clelland et al.,2009; Deng et al., 2009; Dupret et al., 2008; Garthe et al., 2009;
Imayoshi et al., 2008; Kitamura et al., 2009; Saxe et al., 2006;
Shors et al., 2001; Zhang et al., 2008), our tag and ablate systemallowed us to specifically target a population of adult-generated
neurons that were predominantly born several weeks before
training, without impacting ongoing proliferative activity in theadult hippocampus. Second, whereas previous approaches primarily manipulated neurogenesis before learning, our strategy
allowed us control over the timing of the ablation. Therefore,

using this system, we were able to ablate adult-generated neurons
before or after memory formation. We found that selective ablation of adult-generated neurons immediately (or up to 1 month)after training impaired subsequent memory expression in three
distinct hippocampus-dependent tasks. Previous studies provided correlative evidence that adult-generated neurons are activated during the formation and expression of hippocampal
memories. The present findings, that their posttraining ablation
disrupts expression of a previously acquired memory, provides
direct experimental evidence that these neurons, if available atthe time of learning, come to form an essential and enduringcomponent of hippocampal memory traces.
To ablate populations of adult-generated granule cells in a
temporally specific manner, we took advantage of the well characterized DT system (Buch et al., 2005). DT reliably induces apoptosis following receptor-mediated endocytosis (Dorland et al.,1979), but wild-type mouse cells are 10,000-fold less sensitive to15124 • J. Neurosci., October 19, 2011 • 31(42):15113–15127

Arruda-Carvalho et al. • Adult Neurogenesis and Retrograde Amnesia

Figure 10. Posttraining ablation of adult-generated neurons impairs visual discrimination memory. a, During training, a submerged platform was located beneath one of two visual cues (e.g.,
horizontal stripes). After training, the hippocampus was lesioned, and discrimination between the reinforced and nonreinforced cues was evaluated in a probe test. b, Before surgery, latency tolocate the platform declined at similar rates in lesion (n ⫽ 10) and sham (n ⫽ 9) mice. c, After surgery, whereas sham mice searched selectively at the reinforced cue, lesion mice did not search ateither cue. d, Representative images of brains from sham and lesion mice. e, CTR (n ⫽ 12) and 2xTg (n ⫽ 11) mice were treated with TAM and trained in the visual discrimination task. After
DT-induced ablation of adult-generated neurons, visual discrimination memory was assessed in a probe test. f, Across training days, latency to locate the platform declined at similar rates in CTR and
2xTg mice. g, In the probe test, whereas CTR mice searched selectively at the reinforced cue, 2xTg mice spent equivalent amounts of time close to the reinforced and nonreinforced cues. h, Heat maps
reflect preferential searching close to previously reinforced (left peak) versus nonreinforced (right peak) cue in CTR but not 2xTg mice. Note that the peak at top of pool corresponds to the release pointat the start of the probe test. *p ⬍ 0.05.

DT than human or monkey cells (Eidels et al., 1983; Middlebrookand Dorland, 1977; Stenmark et al., 1988). We exploited the insensitivity of mouse cells to DT by using a transgenic line of micein which a functional simian DTR receptor is expressed in a Crerecombinase-inducible fashion (iDTR mice). Crossing iDTR
mice with mice that express an inducible Cre-recombinase innestin ⫹ cells allowed us to permanently express DTRs in neuralprogenitor cells and their progeny. Importantly, we found thatneither DT administration nor DTR expression alone affected
memory, consistent with previous findings (Han et al., 2009).
Rather, only the combination of DTR tagging and DT administration induced retrograde memory loss, showing the specificityof the system.
The cells targeted for ablation included progenitor cells, immature neurons, and mature neurons. Therefore, our posttraining ablation effects on memory might be caused by loss of any (orall) of these different cell populations. Indeed, genetic deletion of
predominantly immature, adult-generated neurons is associatedwith long-term retention deficits in a water maze task (Deng etal., 2009), suggesting that this population of cells contributes tomemory robustness. However, cellular imaging approaches suggest that adult-generated neurons are not maximally activatedduring memory formation and/or expression until they reach amore mature stage [⬎4 weeks of age (Kee et al., 2007; Stone et al.,2011)]. In our experiments, the majority of tagged cells weremature (e.g., 94% were calbindin ⫹ and therefore ⬎4 weeks of age(Zhao et al., 2008)), and therefore it is very likely that loss of thispopulation of mature, adult-generated neurons contributed significantly to memory loss.
In contrast to the impairment produced by posttraining ablation of adult-generated neurons, similar pre training ablation did
not prevent the formation of new memories. These effects parallel those of partial hippocampal lesions in water maze [both hidden (Moser and Moser, 1998) and visual discrimination (Epp etal., 2008) versions] and contextual fear tasks (Frankland et al.,
1998; Maren et al., 1997; Wiltgen et al., 2006). For example,
whereas chemical lesions ablating ⬃30% of hippocampal tissue
disrupt the expression of a previously acquired water maze memory, similar-sized lesions do not prevent the acquisition of a newwater maze memory (Moser and Moser, 1998). This suggests
that, whereas spatial memories may normally be distributed
throughout the hippocampus, new learning may be supported byresidual tissue when the lesion precedes training. Analogously,
here we show that in the absence of a large population of adultgenerated neurons, memory formation may be supported by existing dentate neurons without an obvious impact on memory
quality. This potential for compensation by existing, developmentally generated granule cells may account for variable impact
of pretraining disruptions of adult neurogenesis on memory formation. Although memory formation may be impaired by pretraining suppression of adult neurogenesis (Clelland et al., 2009;
Deng et al., 2009; Drew et al., 2010; Dupret et al., 2008; Garthe etArruda-Carvalho et al. • Adult Neurogenesis and Retrograde Amnesiaal., 2009; Imayoshi et al., 2008; Saxe et al., 2006; Shors et al., 2001;
Tronel et al., 2010; Zhang et al., 2008), sometimes the effects ofthese types of manipulations are mild or even nonexistent (Denget al., 2010; Hernandez-Rabaza et al., 2009; Jaholkowski et al.,2009). Many factors differ across studies and likely contribute to
the discrepant results. The advantage of the current approach isthat we were able to directly contrast pretraining and posttraining
ablations under identical conditions (i.e., method of ablation,
number and type of cells targeted, training paradigm, apparatus,etc.). Our finding that posttraining lesions had greater impact
than pretraining lesions is consistent with the idea that targeting
neurons after memory formation is likely more disruptive because these neurons have already become committed to thememory trace.
Because cell death is induced by apoptosis, rather than necrosis, using our DT system, impact on surrounding cells is
minimized. Indeed, after DT-induced ablation, there was no
hippocampus-wide increase in astrocyte number, and the increase in microglia number was restricted to the DG. Nonetheless, our ablation affected a large number of cells (⬎30,000)
and therefore might nonspecifically impact hippocampal
function. However, the dissociable effects of pretraining andposttraining ablation on memory suggest that such off-target
effects cannot account for the results. For example, the absence of pretraining effects in our water maze experiment indicates that DT-induced ablation did not simply affectswimming, navigation, vision, or motivation necessary for the
expression of a spatial memory. Likewise, in our contextual
fear experiment, the absence of pretraining effects indicatesthat DT-induced ablation did not simply affect the ability toperceive differences between similar contexts. Finally, it isworth noting that our DT-induced ablation should, in addition, lead to loss of subventricular zone-generated cells in theolfactory bulb. However, it is unlikely that the loss of theseneurons can account for our retrograde memory effects since
we observed consistent memory loss in three different tasks,
each with different stimulus properties and performance demands. In particular, the two water maze-based tasks do notdepend on olfactory information (Morris et al., 1982).
Neurogenesis in the DG can be classified into three broad
periods: embryonic, postnatal, and adult. Embryonic and postnatal neurogenesis occurs as a result of proliferation in the primary, secondary, and tertiary dentate matrices. However, these
germinal regions decline by postnatal day 10. By postnatal days
20 –30, and continuing throughout adulthood, neurogenesis becomes restricted to the subgranular zone (Altman and Bayer,1990). Therefore, in our experiments we initiated TAM treatment in mice at 4 weeks of age [for similar experimental strategies, see Ables et al. (2010) and Sierra et al. (2010)]. Because
proliferation rates are higher in this postjuvenile period, this approach enabled us to tag large numbers of granule cells. Even so
this tagged population (including both cells generated during a
post-juvenile period as well as during adulthood) represents nomore than 6% of the entire population of granule cells at the timeof ablation. That deletion of this relatively small proportion ofdentate neurons was sufficient to produce robust retrograde
memory deficits across three different tasks is perhaps surprisingand suggests that this population of subgranular zone-generated
neurons plays an especially important role within a broader network of dentate neurons supporting contextual fear, water mazeand visual discrimination memories. Indeed, it is necessary tolesion ⬎15% of the entire hippocampus to produce similar ret-

J. Neurosci., October 19, 2011 • 31(42):15113–15127 • 15125

rograde memory deficits in the water maze (Moser and Moser,
1998). Since cells generated at different stages of development
and adulthood seem to be integrated into hippocampal memory
circuits at similar rates (Stone et al., 2011), this suggests that thispopulation of subgranular zone-derived neurons may disproportionately influence hippocampal memory function and raises thepossibility that granule cells derived from the subgranular zone
may make distinct contributions to hippocampal memory relative to those generated in the primary, secondary, and tertiary
matrices earlier on during development. What is the nature ofthis role? In the fear conditioning and visual discrimination tasks,
posttraining ablation led to memory degradation rather than erasure. In both tasks, although some general features of the memory
were retained (e.g., context–shock, cue–platform associations),
the ability to discriminate between similar contexts and patterns
was impaired. Consistent with recent reports (Clelland et al.,2009; Sahay et al., 2011), these deficits may suggest a specializedrole for adult-generated neurons in disambiguating similar (butnonetheless discrete) representations (or pattern separation).

References
Ables JL, Decarolis NA, Johnson MA, Rivera PD, Gao Z, Cooper DC,
Radtke F, Hsieh J, Eisch AJ (2010) Notch1 is required for maintenance of the reservoir of adult hippocampal stem cells. J Neurosci
30:10484 –10492.
Altman J, Bayer SA (1990) Migration and distribution of two populations ofhippocampal granule cell precursors during the perinatal and postnatal
periods. J Comp Neurol 301:365–381.
Archer J (1973) Tests for emotionality in rats and mice: a review. Anim
Behav 21:205–235.
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.
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.
Clark RE, Broadbent NJ, Squire LR (2005) Hippocampus and remote spatial memory in rats. Hippocampus 15:260 –272.
Clark RE, Broadbent NJ, Squire LR (2007) The hippocampus and spatial
memory: findings with a novel modification of the water maze. J Neurosci
27:6647– 6654.
Clelland CD, Choi M, Romberg C, Clemenson GD Jr, Fragniere A, Tyers P,
Jessberger S, Saksida LM, Barker RA, Gage FH, Bussey TJ (2009) A functional role for adult hippocampal neurogenesis in spatial pattern separation. Science 325:210 –213.
Corvelo A, Eyras E (2008) Exon creation and establishment in human genes.
Genome Biol 9:R141.
Deng W, Saxe MD, Gallina IS, Gage FH (2009) Adult-born hippocampal
dentate granule cells undergoing maturation modulate learning andmemory in the brain. J Neurosci 29:13532–13542.
Deng W, Aimone JB, Gage FH (2010) New neurons and new memories:
how does adult hippocampal neurogenesis affect learning and memory?
Nat Rev Neurosci 11:339 –350.
Ding HK, Teixeira CM, Frankland PW (2008) Inactivation of the anterior
cingulate cortex blocks expression of remote, but not recent, conditioned
taste aversion memory. Learn Mem 15:290 –293.
Dorland RB, Middlebrook JL, Leppla SH (1979) Receptor-mediated internalization and degradation of diphtheria toxin by monkey kidney cells.
J Biol Chem 254:11337–11342.
Drew MR, Denny CA, Hen R (2010) Arrest of adult hippocampal neurogenesis in mice impairs single- but not multiple-trial contextual fear conditioning. Behav Neurosci 124:446 – 454.
Dupret D, Revest JM, Koehl M, Ichas F, De Giorgi F, Costet P, Abrous DN,
Piazza PV (2008) Spatial relational memory requires hippocampal adultneurogenesis. PLoS One 3:e1959.

15126 • J. Neurosci., October 19, 2011 • 31(42):15113–15127
Eidels L, Proia RL, Hart DA (1983) Membrane receptors for bacterial toxins.
Microbiol Rev 47:596 – 620.
Epp J, Keith JR, Spanswick SC, Stone JC, Prusky GT, Sutherland RJ (2008)
Retrograde amnesia for visual memories after hippocampal damage inrats. Learn Mem 15:214 –221.
Forni PE, Scuoppo C, Imayoshi I, Taulli R, Dastru 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.
Frankland PW, Cestari V, Filipkowski RK, McDonald RJ, Silva AJ (1998)
The dorsal hippocampus is essential for context discrimination but notfor contextual conditioning. Behav Neurosci 112:863– 874.
Garthe A, Behr J, Kempermann G (2009) Adult-generated hippocampal
neurons allow the flexible use of spatially precise learning strategies. PLoS
One 4:e5464.
Gropp E, Shanabrough M, Borok E, Xu AW, Janoschek R, Buch T, Plum L,
Balthasar N, Hampel B, Waisman A, Barsh GS, Horvath TL, Bruning JC(2005) Agouti-related peptide-expressing neurons are mandatory forfeeding. Nat Neurosci 8:1289 –1291.
Gundersen HJ, Jensen EB, Kieu K, Nielsen J (1999) The efficiency of systematic sampling in stereology—reconsidered. J Microsc 193:199 –211.
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.
Hernandez-Rabaza V, Llorens-Martin M, Velazquez-Sanchez C, Ferragud A,
Arcusa A, Gumus HG, Gomez-Pinedo U, Perez-Villalba A, Rosello J,
Trejo JL, Hernandez-Rabaza V, Llorens-Martin M (2009) Inhibition of
adult hippocampal neurogenesis disrupts contextual learning but spares
spatial working memory, long-term conditional rule retention and spatial
reversal. Neuroscience 159:59 – 68.
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.
Jaholkowski P, Kiryk A, Jedynak P, Ben Abdallah NM, Knapska E, Kowalczyk
A, Piechal A, Blecharz-Klin K, Figiel I, Lioudyno V, Widy-Tyszkiewicz E,
Wilczynski GM, Lipp HP, Kaczmarek L, Filipkowski RK (2009) New
hippocampal neurons are not obligatory for memory formation; cyclin
D2 knockout mice with no adult brain neurogenesis show learning. Learn
Mem 16:439 – 451.
Jonasson Z (2005) Meta-analysis of sex differences in rodent models oflearning and memory: a review of behavioral and biological data. Neurosci Biobehav Rev 28:811– 825.
Kee N, Sivalingam S, Boonstra R, Wojtowicz JM (2002) The utility of Ki-67
and BrdU as proliferative markers of adult neurogenesis. J Neurosci
Methods 115:97–105.
Kee N, Teixeira CM, Wang AH, Frankland PW (2007) Preferential incorporation of adult-generated granule cells into spatial memory networks inthe dentate gyrus. Nat Neurosci 10:355–362.
Kempermann G, Jessberger S, Steiner B, Kronenberg G (2004) Milestonesof neuronal development in the adult hippocampus. Trends Neurosci
27:447– 452.
Kim JJ, Fanselow MS (1992) Modality-specific retrograde amnesia of fear.
Science 256:675– 677.
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.
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.
Maren S, Aharonov G, Fanselow MS (1997) Neurotoxic lesions of the dorsal
hippocampus and Pavlovian fear conditioning in rats. Behav Brain Res
88:261–274.
McHugh TJ, Jones MW, Quinn JJ, Balthasar N, Coppari R, Elmquist JK,
Lowell BB, Fanselow MS, Wilson MA, Tonegawa S (2007) Dentate gyrus
NMDA receptors mediate rapid pattern separation in the hippocampal
network. Science 317:94 –99.
Middlebrook JL, Dorland RB (1977) Response of cultured mammalian cells

Arruda-Carvalho et al. • Adult Neurogenesis and Retrograde Amnesia
to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity. Can J Microbiol 23:183–189.
Morris RG, Garrud P, Rawlins JN, O’Keefe J (1982) Place navigation impaired in rats with hippocampal lesions. Nature 297:681– 683.
Moser E, Moser MB, Andersen P (1993) Spatial learning impairment parallels the magnitude of dorsal hippocampal lesions, but is hardly present
following ventral lesions. J Neurosci 13:3916 –3925.
Moser MB, Moser EI (1998) Distributed encoding and retrieval of spatial
memory in the hippocampus. J Neurosci 18:7535–7542.
Nader K, Schafe GE, Le Doux JE (2000) Fear memories require protein
synthesis in the amygdala for reconsolidation after retrieval. Nature
406:722–726.
Porsolt RD (1979) Animal model of depression. Biomedicine 30:139 –140.
Riedel G, Micheau J, Lam AG, Roloff EL, Martin SJ, Bridge H, de Hoz L,
Poeschel B, McCulloch J, Morris RG (1999) Reversible neural inactivation reveals hippocampal participation in several memory processes. Nat
Neurosci 2:898 –905.
Sahay A, Scobie KN, Hill AS, O’Carroll CM, Kheirbek MA, Burghardt NS,
Fenton AA, Dranovsky A, Hen R (2011) Increasing adult hippocampal
neurogenesis is sufficient to improve pattern separation. Nature
472:466 – 470.
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.
Saxe MD, Battaglia F, Wang JW, Malleret G, David DJ, Monckton JE, Garcia
AD, Sofroniew MV, Kandel ER, Santarelli L, Hen R, Drew MR (2006)
Ablation of hippocampal neurogenesis impairs contextual fear conditioning and synaptic plasticity in the dentate gyrus. Proc Natl Acad Sci
U S A 103:17501–17506.
Schallert T, Fleming SM, Leasure JL, Tillerson JL, Bland ST (2000) CNS
plasticity and assessment of forelimb sensorimotor outcome in unilateral
rat models of stroke, cortical ablation, parkinsonism and spinal cord injury. Neuropharmacology 39:777–787.
Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E (2001) Neurogenesis in the adult is involved in the formation of trace memories. Nature
410:372–376.
Sierra A, Encinas JM, Deudero JJ, Chancey JH, Enikolopov G, OverstreetWadiche LS, Tsirka SE, Maletic-Savatic M (2010) Microglia shape adulthippocampal neurogenesis through apoptosis-coupled phagocytosis. Cell
Stem Cell 7:483– 495.
Stenmark H, Olsnes S, Sandvig K (1988) Requirement of specific receptors
for efficient translocation of diphtheria toxin A fragment across theplasma membrane. J Biol Chem 263:13449 –13455.
Stone SSD, Teixeira CM, Zaslavsky K, Wheeler AL, Martinez-Canabal A,
Wang AH, Sakaguchi M, Lozano AM, and Frankland PW (2011) Functional convergence of developmentally- and adult-generated granule cells
in dentate gyrus circuits supporting hippocampus-dependent memory.
Hippocampus Advance online publication. Retrieved September 7, 2010.
doi:10.1002/hipo.20845.
Sutherland RJ, Weisend MP, Mumby D, Astur RS, Hanlon FM, Koerner A,
Thomas MJ, Wu Y, Moses SN, Cole C, Hamilton DA, Hoesing JM (2001)
Retrograde amnesia after hippocampal damage: recent vs. remote memories in two tasks. Hippocampus 11:27– 42.
Tashiro A, Makino H, Gage FH (2007) Experience-specific functional modification of the dentate gyrus through adult neurogenesis: a critical period
during an immature stage. J Neurosci 27:3252–3259.
Teixeira CM, Pomedli SR, Maei HR, Kee N, Frankland PW (2006) Involvement of the anterior cingulate cortex in the expression of remote spatial
memory. J Neurosci 26:7555–7564.
Toni N, Teng EM, Bushong EA, Aimone JB, Zhao C, Consiglio A, van Praag
H, Martone ME, Ellisman MH, Gage FH (2007) Synapse formation onneurons born in the adult hippocampus. Nat Neurosci 10:727–734.
Toni N, Laplagne DA, Zhao C, Lombardi G, Ribak CE, Gage FH, Schinder AF(2008) Neurons born in the adult dentate gyrus form functional synapses
with target cells. Nat Neurosci 11:901–907.
Tronel S, Belnoue L, Grosjean N, Revest JM, Piazza PV, Koehl M, Abrous DN(2010) Adult-born neurons are necessary for extended contextual discrimination. Hippocampus. Advance online publication. Retrieved
November 3, 2010. doi:10.1002/hipo.20895.
Trouche S, Bontempi B, Roullet P, Rampon C (2009) Recruitment of adultgenerated neurons into functional hippocampal networks contributes toArruda-Carvalho et al. • Adult Neurogenesis and Retrograde Amnesia
updating and strengthening of spatial memory. Proc Natl Acad Sci U S A
106:5919 –5924.
Wang SH, Teixeira CM, Wheeler AL, Frankland PW (2009) The precision of
remote context memories does not require the hippocampus. Nat Neurosci 12:253–255.
Wiltgen BJ, Sanders MJ, Anagnostaras SG, Sage JR, Fanselow MS (2006)
Context fear learning in the absence of the hippocampus. J Neurosci
26:5484 –5491.
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.
Zambrowicz BP, Imamoto A, Fiering S, Herzenberg LA, Kerr WG, Soriano P

J. Neurosci., October 19, 2011 • 31(42):15113–15127 • 15127(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.
Zhang CL, Zou Y, He W, Gage FH, Evans RM (2008) A role for adult TLXpositive neural stem cells in learning and behaviour. Nature 451:1004 –1007.
Zhao C, Teng EM, Summers RG Jr, Ming GL, Gage FH (2006) Distinct
morphological stages of dentate granule neuron maturation in the adultmouse hippocampus. J Neurosci 26:3–11.
Zhao C, Deng W, Gage FH (2008) Mechanisms and functional implicationsof adult neurogenesis. Cell 132:645– 660.