Alzheimer disease. Stem cell-based approaches have received considerable attention as a potential means of treatment, although it
remains to be determined whether stem cells can ameliorate memory dysfunction, a devastating component of these disorders. We
neural stem cells transplanted into the brain after neuronal ablation survive, migrate, differentiate and, most significantly, improve
Neuronal loss is a common feature of many neurological mala-
dies that affect the brain including traumatic brain injury (TBI),
and Dietrich, 2004). Transplantation of neural stem cells offers a
promising therapeutic strategy for minimizing the functional
damage that manifests in these disorders (Cao et al., 2002; Lind-
vall et al., 2004; Oliveira and Hodges, 2005; Lindvall and Kokaia,
2006; Vora et al., 2006). Transplanted cells may serve as a reser-
However, the ultimate measure of the therapeutic utility of stem
cells in the brain is recovery of function, particularly in regard to
vious studies report locomotor recovery resulting from stem cell
transplant after brain or spinal cord injury (Cummings et al.,
2005; Bernreuther et al., 2006; Yasuhara et al., 2006; Ziv et al.,
2006), it remains to be established whether stem cell therapy is a
viable approach for the treatment of neurological conditions re-
sulting in memory impairment.
to cognition, has been hampered in part by the complexity and
variability inherent in many of the commonly used animal mod-
surgical procedures that disrupt the blood–brain barrier by me-
chanical means, percussion or aspiration of brain matter (Jar-
ondary effects on other systems and also typically require
impaling the brain to achieve regional specificity, and ischemic
models deprive entire brain regions of nutrients and oxygen, of-
ten resulting in variable damage to multiple regions, and are also
complicated by reperfusion effects or a mixed population of cell
death, especially at the ischemic penumbra (Jarrard, 2002; Car-
michael, 2005). Moreover, these approaches for injuring the
brain typically cannot target precise cell populations, and wide-
spread regional damage limits the evaluation of complex cogni-
Here, we report the development of a unique transgenic
model of neuronal injury in which the tetracycline (tet)-
inducible promoter system is used to temporally and spatially
regulate the expression of diphtheria toxin A-chain (DTA), a po-
tent cytotoxin for eukaryotic cells (Kochi and Collier, 1993). Re-
gional specificity is conferred by utilization of the calcium-
calmodulin kinase II ? (CaMKII?)-regulatory region, thereby
show that CaMKII?-expressing neurons in the forebrain are se-
lectively vulnerable in a graded manner (i.e., CA1 region ? cor-
tex). Notably, the precision of this system renders it possible to
restrict the amount of neuronal loss and evaluate cognitive func-
tion both before and after a neuronally targeted and inducible
brain lesion. This system is ideal for exploring the therapeutic
potential of neural stem cells. Toward this end, we transplanted
stem cells in the brains of CaM/Tet-DTAmice with neuronal loss
cell transplantation also increases hippocampal synaptic density
and decreases neuronal death. Most significantly, we find that
Regenerative Medicine postdoctoral scholar award. We thank Drs. Henry Klassen (Children’s Hospital of Orange
County, Orange, CA) and Michael Young (Harvard Medical School, Boston, MA) for the GFP NSC cell line. We are
TheJournalofNeuroscience,October31,2007 • 27(44):11925–11933 • 11925
transplantation of neural stem cells produces a significant resto-
ration of memory. Our findings provide enticing evidence that
stem cell-based therapies may offer a viable approach for the
treatment of common neurological disorders and memory
Double transgenic mice were generated by crossing CaMKII?-tTA mice
(B6/CBA) to tetracycline-responsive element (TRE)-DTAmice (B6/
gosity, both lines were maintained separately and crossed together to
induction of DTA, mating pairs and offspring were maintained on 2000
parts per gram doxycycline chow (Research Diets, New Brunswick, NJ)
and switched at weaning to 2 mg/ml doxycycline (Sigma, St. Louis, MO)
supplemented with 5% sucrose in deionized, filtered drinking water.
Mice were genotyped by PCR, using primer pairs 5?-CGCATTA-
GAGCTGCTTAATG-3? and 5?-TCGCGATGACTTAGTAAAGC-3? for
and 5?-CCGCAGCGTCGTATTTATTG-3? for the TRE-DTAtransgene.
Induction of DTAexpression in adult bigenic mice was conducted by
administering water without supplemental doxycycline. All procedures
were performed in accordance with the regulations of the Institutional
Animal Care and Use Committee of the University of California, Irvine.
RNA was isolated from hemibrains that were homogenized in GuCNS
solution (4 M guanidine thiocyanate, 0.5% Sarkosyl, 0.025 M Na citrate,
100 mM ?-mercaptoethanol) and phenol/chloroform was extracted.
ison, WI) for 15 min in the presence of RNasin (Promega), followed by
reverse transcription with Superscript II RT using Oligo dT primers (In-
ers and PCR.
Mice used for histological analysis were either perfused or killed by car-
bon dioxide inhalation. For perfusions, mice were anesthetized with
perfused transcardially at a rate of 12 ml/min first with PBS followed by
4% paraformaldehyde in 0.01 M phosphate buffer, pH 7.4. Brains were
postfixed in 4% paraformaldehyde for 48 h, and sectioned at 40 ?m by
vibratome (Pelco, Redding, CA). Sections were subsequently stored in
PBS with 0.02% sodium azide.
For terminal deoxynucleotidyl transferase-mediated biotinylated UTP
nick end labeling (TUNEL), sections were incubated with 0.02 mg/ml
terminal transferase reaction mix (Roche, Indianapolis, IN) at 37°C for
biotin horseradish peroxidase system (Vector Laboratories, Burlingame,
CA). For anti-NeuN (1:10,000; Millipore, Temecula, CA) staining, tis-
sues were first quenched with 3%MeOH/hydrogen peroxide for 30 min,
oped on the second day with SG-1 (Vector Laboratories).
For immunofluorescence, sections were first blocked with 3% normal
serum with 2% BSA and 0.1% Triton-X in TBS, then incubated with
primary antibody in blocking solution overnight at 4°C. They were then
rinsed and incubated with secondary Alexa Fluor-conjugated antibodies
(1:200; Invitrogen) in block, rinsed, and mounted in Fluoromount G
(Southern Biotech, Birmingham, AL). Primary antibodies were as
follows: anti-GFAP (1:500; DAKO, Glostrup, Denmark), anti-NeuN
(1:10,000; Millipore), anti-NG2 chondroitin sulfate proteoglycan
(1:200; Millipore), and anti-green fluorescent protein (GFP; 1:2000;
Millipore). For anti-CNPase (1:20,000; Sigma), signal amplification
was performed with the Tyramide Signal Amplification System
(PerkinElmer, Waltham, MA). To determine percentage of differen-
tiation in stem cell injected animals, tissues double-stained for either
NeuN/GFP (neurons), GFAP/GFP (astrocytes), or NG2/GFP (oligo-
dendrocyte precursors) were analyzed at the 4.5 month post-
transplant time point. Because of the difficulty of consistently label-
with NG2, a marker of oligodendrocyte precursors (Polito and Reyn-
olds, 2005). Counts of GFP? cells at the dentate gyrus (including the
molecular layers) were first taken using a 10 ? 10 counting grid at
10? magnification, and then colocalization of GFP with each marker
(NeuN, GFAP, or NG2) was assessed at 20? magnification. The per-
centage differentiation was thus number of cells colocalized with the
given marker divided by total number of GFP? cells. Counts were
taken bilaterally on n ? 5 animals and then averaged. For colocaliza-
tion of labels in Figure 5, cells were double labeled with immunoflu-
orescent markers and analyzed by confocal microscopy (Olympus,
Tokyo, Japan) using the LaserSharp 2000 program. Z-scans were con-
ducted in 0.5 ?m steps with Kallman n ? 2. Lambda strobing and was
used in all scans to reduce nonspecific signal.
For each group of mice (n ? 5 per group), similar sections were stained
for NeuN in the same staining set. Three consecutive nonoverlapping
pictures were taken across the CA1 subfield at 40? magnification. All
pictures were taken in one sitting with no change in illumination. Pic-
tures were imported into ScionImage software program (National Insti-
tutes of Health, Bethesda, MD) and the threshold was set at 100 for all
pictures. Extraneous cells outside of the CA1 subfield were removed and
pixel intensity was determined.
All unbiased stereological assessments were performed using StereoIn-
vestigator software (MicroBrightField, Williston, VT). For each stereo-
logical evaluation, an optical fractionator probe was used to estimate
mean cell numbers and the Cavalieri principle was used to determine
volume of each brain region. Guard zones were set at 10% of measured
ing to delineate brain regions was done using a 4? objective and count-
ing was performed using a 100? oil objective. The counting was done in
every 12th section (40 ?m coronal sections) using n ? 5 animals per
group. A 25 ? 25 ?m counting frame was used in all stereological eval-
uations and all counts were performed in only one brain hemisphere.
induced, and noninduced animals was performed on five Nissl-stained
150 ?m sampling grid. Data are shown as mean estimated cell numbers
for this stereological assessment. Stereological evaluation of NeuN and
GFP in mice injected with stem cells was done on series of sections
throughout the region where GFP-labeled cells could be seen between
bregma ?1.28 mm and bregma ?2.92 mm. For GFP-labeled cell count-
ing, a 500 ? 500 ?m sampling grid was used to assess the entire hip-
pocampus and the entire cortex. Counting of NeuN-positive cells was
done in CA1 and dentate gyrus using 200 ? 200 ?m sampling grid.
parallel for the presynaptic vesicle protein synapsin-1 (1:500; Calbio-
chem, San Diego, CA) following standard protocols. Slides were coded
and images captured by a blinded observer using a Bio-Rad (Hercules,
CA) Radiance 2100 confocal microscope and identical scan settings.
Grayscale images were inverted and optical density was quantified using
radiatum of CA1 and the polymorph layer of the dentate gyrus were
defined in each image and average pixel intensity was measured from
each ROI. Measurements of white matter provided background levels
sections per animal were averaged and then animal pixel intensity was
11926 • J.Neurosci.,October31,2007 • 27(44):11925–11933Yamasakietal.•NeuralStemCellsImproveMemory
5-Bromo-2?-deoxyuridine (Sigma) was reconstituted in water with PBS
the experiment and volumes of injectate were calculated for 50 mg/kg
drug/animal weight. The labeling paradigm used was described by Jin et
al. (2004). Briefly, bromodeoxyuridine (BrdU) was injected twice a day
for the last four d of a 20 d induction period, with each injection sepa-
and then mice were returned to a dox water regimen to abrogate DTA
expression. Mice were perfused and killed 1 month after the last injec-
tion. To assess neurogenesis levels, the percentage of NeuN?BrdU?
colocalizing cells in the granule cell layer of the dentate gyrus was deter-
4–6 sections spaced 240 ?m apart (CaM/Tet-DTAmice) was imaged on
a confocal microscope at 60? or higher magnification. For determining
colocalization, BrdU? cells were scanned for colocalization of NeuN. If
NeuN did not colocalize definitively throughout the entire length of the
ing was ambiguous as to colocalization or no colocalization, the cell was
excluded. Settings for Z-scans were maintained throughout the entire
series as follows: green, iris, 1.5; gain, 39.0; offset, ?70.5; laser, 6.0; red,
iris, 1.7; gain, 21.2; offset, ?5.4; laser, 17.0). All scans were performed
with ? strobbing and Kallman n ? 2 stop to reduce nonspecific back-
ground and at a speed of 166 lines per second. Z-stacks were taken at 0.5
?m steps. The percentage of colocalization was determined by dividing
ous) colocalized and noncolocalized BrdU? cells.
Mouse enhanced GFP-expressing neural stem cells were grown in
DMEM/F12 with Glutamax and epidermal growth factor-2 (EGF-2) (20
ng/ml). On the day of the surgery, cells at postnatal day 14 (P14)–P17
were harvested, triturated, filtered and resuspended in 1? HBSS with
EGF-2 (20 ng/ml) at a concentration of 50,000 cells/?l. Aliquots of vehi-
cle and cell suspension were stored in sterile-O-ring-sealed tubes on ice
until they were removed for injection/transplantation, at which point
they were triturated and drawn up for injection in Flexifil tapertip sy-
ringes (World Precision Instruments, Sarasota, FL).
Adult CaM/Tet-DTAmice 4–6 months of age were induced by with-
drawal from doxycycline water. After 25 d on a regular food and water
diet, they were returned to doxycycline-supplemented water to abrogate
expression of diphtheria toxin. Eleven days after the start of abrogation,
each mouse was stereotactically injected bilaterally with 2 ul of either
vehicle or neural stem cells (NSCs) at a rate of 1 ?l/min. The syringe was
used for NSC injections and vehicle injections, and syringes were thor-
oughly rinsed with deionized water, and then buffer after each injection.
Mice were randomized to receive either vehicle or NSCs injections. Co-
mediolateral (M/L), ?1.75 mm; dorsoventral (D/V), ?1.75 mm. For
induced mice, because of hippocampal shrinkage with induction, coor-
dinates were altered slightly based on pilot studies so the injection site
would be comparable with the noninduced hippocampal site with coor-
dinates M/L ?2.00 mm and D/V ?1.85 mm. Surgery sites were sealed
with bone wax, sutured, closed with tissue-mend, and topical antibiotic
ness on heating pads, and then housed singly for a few weeks until the
surgery site had healed. At this point, females were group housed and
males were paired with nonbreeding females to prevent negative effects
of long-term isolation on future behavior.
Habituation. Each behavioral group consisted of n ? 8–13 animals.
Briefly, two Plexiglas rectangular chambers 18 ? 9.5 ? 10 inches were
used for the behavioral tests. Mice were handled for 3 consecutive days
(days 1–3), habituated for 15 min in groups of three or less (day 4), then
for 10 min with one mouse per cage (day 5), and 5 min with one mouse
per cage (day 6). We followed well documented behavioral procedures
only one behavioral test per day. On day 7, half of the mice were given
so that the half receiving the place test on day 7 received object tests on
were run simultaneously in adjacent cages separated by an opaque bar-
rier. Novel or displaced objects were counterbalanced in terms of loca-
tion in the cage. Objects were plastic or metal and ?1.5 inches in height.
of retained memory for objects. However, all pairs of objects (novel and
oughly with 70% EtOH before each trial. All exploratory segments and
tests were videotaped for scoring purposes. If an animal did not explore
both objects during the training phase, it was not scored during the test
fearful behavior on introduction to the chamber, scoring did not start
until the mice physically moved from their initial starting position: al-
ways in the corner closest to the familiar object. Exploration counted if
the mouse’s head was within one inch of the object with neck extended
and vibrissae moving. Simple proximity, chewing, or standing on the
object did not count as exploration.
Object recognition. Each mouse was placed in the chamber with two
identical objects spaced ?12 inches apart. The animals were allowed to
animal was returned to its home cage, the mouse was place back in the
chamber with the previously exposed object and a novel object for a 3
min probe test.
Place recognition. Each mouse was placed in the chamber with two
identical objects spaced by ?12 inches apart. After a 5 min exploration,
the chamber in which one of the two objects was displaced from its
otherwise indicated and considered significant at p ? 0.05.
To genetically lesion selective populations of neurons in the
brain, we generated a transgenic model with regulatable ex-
and Collier, 1993). The tet-inducible promoter system enables
temporal and spatial regulation, where regional specificity is
conferred by utilization of the CaMKII?-regulatory region.
Withdrawal of doxycycline from the drinking water of the
mice allows transactivator binding to the TRE of a second
transgene, which controls expression of DTA(Fig. 1A). Dou-
ing TRE-DTAmice (Lee et al., 1998) with CaMKII?-tTA mice
(Mayford et al., 1996).
Tight control of DTAexpression is crucial for limiting the
extent of neuronal loss and for avoiding leakiness during nonin-
duced periods. We evaluated DTAtransgene expression using
reverse transcriptase (RT)-PCR, a highly sensitive readout, and
Yamasakietal.•NeuralStemCellsImproveMemoryJ.Neurosci.,October31,2007 • 27(44):11925–11933 • 11927
find that DTAexpression is first apparent
at 8 d postinduction, but not in nonin-
duced mice (Fig. 1B) (data not shown).
Abrogating DTAexpression, which is im-
portant for limiting the magnitude of the
lesion, can be achieved within 7 d of re-
introducing dox (Fig. 1C). Collectively,
is very tightly regulated, and that DTAex-
pression can be induced and subsequently
abrogated with precision in the brains of
We next histopathologically evalu-
ated the consequences of progressive
DTAexpression in the brain. Nonin-
duced double-transgenic mice showed
no evidence of neuronal damage by he-
matoxylin and eosin (H&E) staining or
by NeuN, a marker for mature neuronal
nuclei (Fig. 2A–D). Depending on the
length of induction, we found that pyra-
midal neurons in the CA1 subfield are
the first neuronal cell type to die in re-
sponse to DTAinduction, with substantial degeneration from
this region apparent by 20 d postinduction (Fig. 2E–H).
Thirty days of induction leads to the marked destruction of
virtually all neurons in the CA1 subfield (Fig. 2I–L). Hence,
longer periods of DTAinduction lead to progressively greater
damage. Dying neurons appeared pyknotic, and were readily
apparent by their highly eosinophilic cytoplasm and con-
densed nuclei that were dark and basophilic after H&E stain-
ing (Fig. 2F,J).
We further quantified neuronal number by evaluating
brain sections stained with NeuN, using optical densitometry.
As expected, longer periods of transgene induction (20, 25,
and 30 d) led to significant reductions in NeuN optical density
at the CA1 subfield when compared with noninduced controls
(Fig. 2M). To quantitatively assess these changes, we per-
formed unbiased stereology at the CA1 subfield and dentate
gyrus in noninduced and 30 d induced mice. Consistent with
NeuN optical density measurements, stereology showed a sig-
nificant reduction in neuronal number at the CA1 subfield, as
well as at the dentate gyrus of the hippocampus (Fig. 2N).
TUNEL and Fluorojade B staining (supplemental Fig. 1, avail-
able at www.jneurosci.org as supplemental material) further
and NeuN. Thus, DTAexpression in an inducible transgenic
context provides an effective means for ablating select popu-
lations of neurons in the CNS.
In addition to the progressive neuronal loss within the hip-
pocampus, there is also a regional progression within the brain.
cortical areas, as assessed by Fluorojade B. At 15 d of induction,
there is defined loss from CA1 regions of the hippocampus, but
no apparent corresponding loss from cortical structures (Fig.
3A,B). At 20 d of induction, cell death is substantial in the CA1
region, but sparse at the cortical area (Fig. 3C,D). By 25 d of
induction, there is severe loss from the CA1 region, as well as
consistent loss from cortical subfields (Fig. 3E,F). Hence, longer
periods of DTAinduction lead to progressively greater damage,
and further, analysis indicates there is a spatial pattern of cell
ablation such that loss from the CA1 region initiates before cor-
tical loss occurs.
Neuronal loss has also been shown to affect neurogenesis in cer-
tain models, such as ischemia, traumatic brain injury, and epi-
lepsy (Gould and Tanapat, 1997; Parent et al., 1997; Jin et al.,
2001). To determine the impact of selective ablation of a sub-
enous neurogenesis, we induced CaM/Tet-DTAmice for 20 d, at
which time CA1 loss is apparent, and labeled newborn cells with
the thymidine analog, BrdU. To assess levels of neurogenesis,
DTAexpression was abrogated by the readdition of doxycycline
to the water regimen, and the percentage of the newly generated
cells expressing NeuN was determined at 1 month after BrdU
neurogenesis at the subgranular zone and granule cell layers of
the dentate gyrus after cell loss from the CA1 region in CaM/
Tet-DTAmice (supplemental Fig. 2, available at www.jneurosci.
org as supplemental material).
To define the effect of selective neuronal loss on cognitive func-
tion, we tested CaM/Tet-DTAmice for object recognition and
place memory following well described methods (Mumby et al.,
2002). Place memory is considered to be mostly hippocampal-
dependent in that it requires memory for the original spatial
location (Parkinson et al., 1988; Eichenbaum, 2000). In contrast,
object recognition relies mostly on cortical areas, including the
loss from the hippocampus is most significant from the CA1
region with minimal loss from cortical areas (Fig. 3C,D). These
mice have a significant deficit for place memory, the
hippocampal-dependent spatial task, and are not significantly
impaired relative to noninduced controls on object recognition
memory (Fig. 4). After 30 d of induction, a time point at which
lesioning in the cortex as well as hippocampus is significant, we
found that CaM/Tet-DTAmice were significantly impaired in
both object and place memory compared with noninduced con-
ing water or diet of CaM/Tet-DTAmice alleviates transcriptional repression, leading to activation and expression of the DTA
transgene exclusively in CaMKII?-expressing cells. B, RT-PCR shows that DTAexpression is first detectable by 8 d of induction
11928 • J.Neurosci.,October31,2007 • 27(44):11925–11933Yamasakietal.•NeuralStemCellsImproveMemory
object recognition task above chance levels, indicating that they
retain some memory for the object, but they were completely
impaired on the place memory test. As expected, there was no
significant difference in baseline performance of noninduced
CaM/Tet-DTAmice and nontransgenic mice on either the object
chance levels (Fig. 4). Thus, there is no detectable effect of the
CaM/Tet-DTAtransgene on performance of these tasks in non-
To determine whether NSC transplantation is a viable approach
expressing enhanced GFP or vehicle into the hippocampi of
CaM/Tet-DTAmice after 25 d of induction at which time point
?74% of their CA1 neurons were ablated
(Fig. 2M). As a control, we also implanted
NSCs into noninduced mice. Behavioral
testing was conducted at two time points
post-transplant and brains were then ana-
lyzed histologically (Fig. 5A).
The mouse NSCs were derived from
enhanced GFP-expressing mice at postna-
the chicken ?-actin promoter (Okabe et
al., 1997). Properties of these NSCs have
been well documented previously (Mizu-
moto et al., 2003) and we further con-
firmed that these they express nestin, as
well as markers of immature neurons
including TuJ1 (neuronal
?-tubulin) and DCX (doublecortin) in
vitro (data not shown).
In previous studies, local environmen-
tal factors have been shown to affect cell
migration and differentiation (Hoehn et
al., 2002; Kelly et al., 2004). To determine
the response of transplanted NSCs to the
microenvironment created by selective
neuronal ablation, we conducted a his-
topathological characterization of these
mice at 4.5 months post-transplantation.
We find that NSCs survive well out to this
time point. Interestingly, striking differ-
ences in the distribution and migration of
NSCs were readily apparent between the
brains of induced and noninduced mice.
In noninduced mice, NSCs migrate to-
ward cortical and white matter areas, in-
cluding the corpus callosum and travel as
far as the optic nerve (data not shown),
whereas in mice with induced neuronal
loss, NSCs remain within the hippocam-
pus but fail to migrate away from the le-
sion into the cortex and white matter (Fig.
5B). To quantify these distributions, we
used stereology to determine the number
of GFP? cells in the hippocampus and
cortex of induced and noninduced mice.
Overall, a significantly greater number of
NSCs are present in the cortex of nonin-
duced mice (Fig. 5C,D), whereas no significant difference was
evident within the hippocampus (Fig. 5D). Thus, the microenvi-
ronment created by the DTA-induced lesion may inhibit survival
into mature cell types, including neurons (Fig. 5E), astrocytes
ysis indicated percentages of differentiation as follows: 1.78 ?
0.515% neurons, 15.39 ? 6.76% astrocytes, and 17.07 ? 3.49%
The ultimate measure of the utility of stem cell transplantation
into the brain is functionality. To determine the effects of NSC
implantation on memory, mice were tested behaviorally at one
and 3 months after transplantation. These time points were se-
with 25 (25di) and 30 d of induction (30di) (ANOVA, F(4,21)? 32.77, p ? 0.0001, post hoc Bonferroni). N, Significant loss of
Yamasakietal.•NeuralStemCellsImproveMemory J.Neurosci.,October31,2007 • 27(44):11925–11933 • 11929
icantly different, we found that NSC-transplanted mice out-
perform their vehicle-injected counterparts in both object and
place memory ( p ? 0.1583, p ? 0.1311, respectively) (Fig. 6A).
However, by 3 months post-transplantation, mice that received
tion of place, a mostly hippocampal-dependent task, than
tion, which is more dependent on cortical regions, is not signifi-
cantly improved in NSC-transplanted mice ( p ? 0.2137). Thus,
in mice with a hippocampal lesion, transplanting NSCs into the
dependent task, whereas no effect is apparent for object recogni-
tion, a primarily cortical-dependent task.
Stem cells have been postulated to exert their effects in differ-
ent ways, one of which is through production of neurotrophins
(Llado et al., 2004; Yasuhara et al., 2006). Indeed, the GFP NSCs
used in this study produce brain-derived neurotrophic factor
mechanism. To determine whether stem cell transplant affects
synaptic plasticity in CaM/Tet-DTAmice, we compared optical
densities of the presynaptic vesicle protein synapsin in lesion-
before being killed. Two areas critically involved in hippocampal
circuitry were quantified including the stratum radiatum of CA1
and the polymorph layer of the dentate gyrus. Notably, a signifi-
cant increase in synaptic density was detected within the stratum
radiatum of CA1 in mice that received NSC-transplants versus
tate gyrus, although not significantly different, demonstrated a
similar trend (Fig. 6C,D). In addition to modulating synaptic
density, neurotrophins can also prevent the loss of vulnerable
neuronal populations after varying insults (for review, see
Tuszynski and Gage, 1995). Indeed, stereological assessment re-
vealed that significantly more neurons survive in both the CA1
region and the dentate gyrus after a lesion when NSCs are trans-
planted (Fig. 6E,F).
Here, we demonstrate the feasibility of using bigenic mice to
inducibly destroy a select population of neurons in the adult
mammalian CNS. By using the CaMKII? promoter to drive ex-
pression of the tet transactivator, inducible DTAexpression was
limited to forebrain neurons, and significantly, we showed that
the system is titratable such that CA1 pyramidal neurons are
In addition to precise spatial regulation, we further demonstrate
ing critical developmental periods. Likewise, our study indicates
that there was no DTAleakiness, as no cell loss was observed in
jneurosci.org as supplemental material). Furthermore, there is
regional progression to the lesion, such that the CA1 field of the
hippocampus is affected before cortical regions and the dentate
induction, but we further prove that expression can be readily
abrogated, thereby controlling the extent of the lesion. We con-
clude that inducible control of DTAoffers many advantages for
ablating distinct populations within the CNS, and by altering the
activator, this system can be readily adapted for knocking out
other neuronal subtypes.
The focal nature of the neuronal loss in our transgenic model
is demonstrated quite clearly on a histopathological level. We
days of induction results in loss localized to the CA1 region of the hippocampus (A), without
tial loss from the CA1 subfield (C) with sparse loss from cortical regions (D), whereas 30 d of
which the majority of loss occurs within the CA1 region of the hippocampus, hippocampal-
not significantly different from noninduced controls (NI). However, in mice induced for 30 d
11930 • J.Neurosci.,October31,2007 • 27(44):11925–11933 Yamasakietal.•NeuralStemCellsImproveMemory
on a hippocampal-dependent task, but notably did not cause
impairment on a cortically dependent task. By taking advantage
of the ability to progressively lesion regions of the brain with
increasing induction times, we are also able to induce to a time
point at which both hippocampal and cortically dependent be-
havioral tasks are impaired. Thus, cytotoxin expression targeted
to a specific neuronal subpopulation re-
sults in region-specific behavioral deficits
and underlines the utility of this model.
In this study, we used our model to de-
stem cells would lead to improved perfor-
mance on a short-term memory task. This
question is significant, as many neurolog-
ical disorders are marked by profound
cognitive impairments, and it is believed
ments would not be reversible. Notably,
most animal models of neurodegenerative
contrast, the novel model we present here
allows us to specifically address whether
memory can be improved after extensive
and selective neuronal loss.
Regarding the neural stem cell trans-
plantation, there are several significant
findings relevant to survival, migration,
and differentiation. First, we find that
transplanted NSCs survive for at least 4.5
months (the longest time point we exam-
ined). Second, we find that stem cells tend
to be distributed more widely in nonin-
duced mice, where they migrated from
their initial site of transplantation in the
hippocampus through white matter areas
fornix and into the deep layers of the cere-
bral cortex. It is tempting to speculate that
without neuronal loss may be attributable
lent cue to which neural stem cells re-
spond. Also, it is interesting to note that
there are, overall, a greater number of
duced mice, than in induced mice. It is
possible that the microenvironment cre-
ated by selective neuronal loss resulted in
firmed the multipotency of these GFP?
ferentiate into neurons, astrocytes, and
oligodendrocytes in vivo. Although we
find a small percentage of GFP? neurons,
it is also possible that GFP expression is
downregulated after differentiation. One
group has found that retrovirus- and
lentivirus-transfected neural progenitor
cells transplanted into the spinal cord lose
GFP expression after differentiation (Vro-
emen et al., 2005). If this is the case, then
the number of differentiated cells may
have been underestimated.
A common endpoint for studies examining functional recov-
al., 2006; Ziv et al., 2006). In contrast, few studies have examined
a cognitive endpoint, and of these, the results are conflicting. A
few have found improvement (Toda et al., 2001; Shear et al.,
on microenvironment, with significantly more GFP? cells migrating into cortical and white matter areas in noninduced mice
Yamasakietal.•NeuralStemCellsImproveMemory J.Neurosci.,October31,2007 • 27(44):11925–11933 • 11931
2004; Gao et al., 2006); however, other groups have found no
improvement (Veizovic et al., 2001; Hoane et al., 2004) or have
confusingly found improvement without concomitant survival
of cells (Jeltsch et al., 2003). Here, we show that neural stem cells
transplanted into the hippocampus of a transgenic model with
targeted neuronal loss are able to improve short-term memory
on a spatial task in a time-dependent manner. Furthermore, the
fact that the stem cells localize to the hippocampus in induced
mice is consistent with the selective improvement seen on the
hippocampal-dependent task, but not on the cortically depen-
studies examined transplants in models with lesions induced
types (neuronal, glial, endothelial, etc.). This is in direct contrast
to the focal and cell-specific nature of neuronal loss in our CaM/
Tet-DTAmice. Second, our behavioral paradigm was chosen for
its dual advantage of examining cognitive function on two tasks
primarily dependent on differing brain regions (Mumby et al.,
2002), hippocampus for place memory (Parkinson et al., 1988;
Eichenbaum, 2000) and perirhinal cortex for object recognition
(Buffalo et al., 1998; Brown and Aggleton, 2001; Winters et al.,
2004), and also for the fact that it makes use of the natural ten-
dency of the mouse to explore objects perceived as novel and is
thus less stressful than a water maze paradigm (Ennaceur and
Delacour, 1988; Mumby et al., 2002).
In our study, we find that it takes 3 months for a memory
effect to manifest. It has been shown that transplanted stem cells
take over a month in vivo to develop the electrophysiological
responsiveness of mature neurons (Auerbach et al., 2000; En-
glund et al., 2002), form long projecting axons in the brain, and
Thus, it is interesting that we find only a trend toward improve-
ment at 1 month and significant improvement of memory at 3
Trophic mechanisms are likely to contribute to the improve-
ment of memory in our model. Increases in the neurotrophic
factor BDNF occur after brain injury (Kokaia et al., 1998) and
BDNF has also been shown to upregulate synapsin (Causing et
al., 1997). The levels of synapsin we see in induced NSC trans-
injected controls at the CA1 region of the hippocampus, and are
consistent with neurotrophin-induced sprouting. Further, it is
likely that neurotrophins contribute to the neuronal sparing ef-
fect we see at both the dentate gyrus and CA1 region of NSC
transplanted mice resulting in memory improvement. Last, al-
though we do not see an increase in endogenous neurogenesis
with lesioning,another possible
neurotrophin-mediated augmentation of endogenous neuro-
genesis, is plausible and has, in fact, been described by other
groups (Zigova et al., 1998; Yoshimura et al., 2001).
Our study is the first to show that stem cells can help improve
to a neuronal subpopulation. Our approach is differentiated
neurons but also to other surrounding cells including glia and
endothelial cells. Because our genetic approach leads to specific
transplanted stem cells compensate for the lost neuronal func-
ral stem cell transplantation may offer a viable therapeutic ap-
proach to treat patients suffering from diseases and conditions
that involve neuronal loss and memory impairment.
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