Notch regulates cell fate and dendrite morphology
of newborn neurons in the postnatal dentate gyrus
Joshua J. Breunig*†, John Silbereis‡, Flora M. Vaccarino*‡, Nenad Sˇestan*†, and Pasko Rakic*†§
*Department of Neurobiology,†Kavli Institute for Neuroscience, and‡Child Study Center, Yale University School of Medicine, New Haven, CT 06520
Contributed by Pasko Rakic, October 25, 2007 (sent for review September 15, 2007)
The lifelong addition of neurons to the hippocampus is a remark-
able form of structural plasticity, yet the molecular controls over
proliferation, neuronal fate determination, survival, and matura-
tion are poorly understood. Expression of Notch1 was found to
change dynamically depending on the differentiation state of
neural precursor cells. Through the use of inducible gain- and
loss-of-function of Notch1 mice we show that this membrane
receptor is essential to these distinct processes. We found in vivo
that activated Notch1 overexpression induces proliferation,
whereas ?-secretase inhibition or genetic ablation of Notch1 pro-
motes cell cycle exit, indicating that the level of activated Notch1
regulates the magnitude of neurogenesis from postnatal progen-
itor cells. Abrogation of Notch signaling in vivo or in vitro leads to
a transition from neural stem or precursor cells to transit-amplifying
cells or neurons. Further, genetic Notch1 manipulation modulates
survival and dendritic morphology of newborn granule cells. These
results provide evidence for the expansive prevalence of Notch
signaling in hippocampal morphogenesis and plasticity, suggest-
ing that Notch1 could be a target of diverse traumatic and envi-
ronmental modulators of adult neurogenesis.
adult neurogenesis ? Mash1 ? proneural ? stem cell
precursor cells in almost all regions of the adult brain, neurons
are actively generated in large numbers only in the dentate gyrus
(DG) of the hippocampus and subependymal zone (SEZ) of the
lateral ventricles. Although much progress has been made in
identifying factors regulating these progenitors (4–6), our
knowledge of the molecular mediators that permit neurogenesis
in these regions remains limited (3, 7).
Notch is a transmembrane receptor (Notch1–4 in mammals)
that upon ligand binding (Jagged1/2 or Delta 1–4 in mammals)
is cleaved, releasing the intracellular portion (NICD) that trans-
locates to the nucleus (8, 9). There, it binds Rbpsuh (also known
as RBPjk) and the coactivator Mastermind to initiate transcrip-
tion of target genes (10). The role of Notch in neural progenitors
during postnatal hippocampal neurogenesis has not been inves-
tigated in vivo.
Here, we used an inducible form of Cre recombinase spatially
restricted to astroglia. Here, we report that ablation or overex-
pression of Notch1 in glial fibrillary acidic protein (Gfap)-
expressing astroglial cells dramatically affects the proliferation,
cell fate, and survival of progenitors as well as the maturation of
newly generated neurons, displaying the central role of Notch1
in postnatal hippocampal plasticity.
he great majority of neurons in the mammalian brain are
generated prenatally (1–3). Despite the existence of neural
Notch1 and Notch-Signaling Components Are Expressed in the Post-
natal Hippocampal Neurogenic Niche. We first examined the ex-
pression pattern of Notch1 in the postnatal murine forebrain by
using in situ hybridization. Notch1 signal was enriched in the
germinal areas, which include the SEZ and the subgranular zone
(SGZ) of the DG (Fig. 1A). Multiple components of the Notch
pathway, including Hes1, Rbpsuh, Dll-1, and Jag1, were also
expressed in this region (supporting information (SI) Fig. 6). An
antibody raised against the intracellular portion of Notch
(NICD) was used to precisely examine the cell types expressing
Notch and determine the subcellular localization of NICD. This
antibody should recognize all forms of Notch1 containing the
intracellular aspect of the protein, including full-length Notch1
and NICD. NICD was present in the cytoplasm of many Gfap?
cells including the radial glia-like neural progenitors that reside
in the SGZ of the DG (94 ? 2% of Gfap?cells were NICD?, n ?
231 cells, three animals; Fig. 1 B and C). This pattern did not
significantly change in all ages examined from P5 to 6 months
(data not shown). Among the Doublecortin?(Dcx?) young
neurons, NICD was typically absent from the nucleus of the most
immature cells—as evidenced by their lack of a prominent
process or dendritic arborization in comparison with neighbor-
ing, ramified Dcx?cells (Fig. 1C, SI Fig. 7, data not shown).
These more immature Dcx?cells (also known as Type-3 cells)
are thought to be a subpopulation of the transit-amplifying cells
(TACs) in the postnatal brain (11, 12). However, mature Dcx?
cells exhibited higher levels of nuclear NICD, which was posi-
tively correlated with the age/maturation state of the cell as
determined by dendritic complexity, size, and nuclear localiza-
tion of the cdk inhibitor p27Kip1, an early indicator of postmi-
totic status (SI Fig. 7).
Embryonically, as the radial glial cells differentiate, proneural
basic helix–loop–helix genes are activated to initiate cell cycle
withdrawal and migration in cells with low Notch activity (13,
14). We found a similar pattern as the proneural genes Ascl1 and
granule cell layer, or molecular layer (Fig. 1D, SI Figs. 6 and 8).
Ngn2 was also found to frequently colocalize with Dcx (SI Fig.
6). Ascl1-expressing cells were present in substantially greater
numbers compared with Ngn2 (data not shown), which is
consistent with previous reports (15), and they were largely
proliferative in nature as determined by colocalization with the
cell cycle marker Ki67 (SI Fig. 8). There was frequently an
inverse correlation between the intensity of cytoplasmic NICD
and Ascl1 immunostaining (Fig. 1D). However, clear nuclear
exclusively in Ascl1? radial glia (SI Fig. 9). Taken together, the
expression of Notch1 in hippocampal radial glia-like precursors
and its down-regulation in TACs suggests that Notch1 is involved
in the regulation of SGZ radial glia differentiation into com-
mitted neural progenitor cells during postnatal neurogenesis in
the DG. Furthermore, the positive correlation between more
mature NeuN?/Dcx?cells and nuclear NICD intensity suggests
that Notch signaling may also be important for newborn neuron
performed research; J.J.B., J.S., F.M.V., and N.Sˇ. contributed new reagents/analytic tools;
J.J.B., F.M.V., N.Sˇ., and P.R. analyzed data; and J.J.B., F.M.V., N.Sˇ., and P.R. wrote the paper.
The authors declare no conflict of interest.
§To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
December 18, 2007 ?
vol. 104 ?
Genetic Manipulation of Notch1 Regulates Proliferation of Gfap?
Progenitor Cells in Vivo. Because of the high toxicity of Notch-
related side effects in the gut following the administration of
?-secretase inhibitors (GSIs) and because GSIs can potentially
interfere with a host of signaling pathways, injection of GSIs
allows for only an acute observation of the effects of chemical
inhibition of Notch signaling (16, 17). Thus, we investigated the
Notch signaling pathway further in a more precise, genetic
manner. Mice that express a tamoxifen-inducible form of Cre
recombinase under the human Gfap promoter (GCE) (18) were
crossed with either loxP-flanked Notch1 mice (19) or, con-
versely, with conditional NICD transgenic mice (20) to condi-
tionally ablate (GCE; Notch1fl/fl hereafter referred to as
‘‘Notch1 cKO’’ mice) or overexpress activated Notch1 (GCE;
NICD hereafter referred to as ‘‘NICD Tg’’ mice) in Gfap?glial
cells and all of their progeny on induction of Cre recombination
(Fig. 2 A and B). A dose of 1 mg of tamoxifen was given at
postnatal day (P) 10, P12, and P14 to induce recombination. This
dosing paradigm was chosen because it limited outward signs of
toxicity, permitted mice to gain weight normally, and allowed
100% survival of animals while permitting recombination as
determined by reporter expression in significant numbers of
Gfap?cells (Fig. 2C). The control group in all experiments
consisted of littermates that were either tamoxifen-treated
GCE?; Notch1?/?, GCE; NICD?, GCE?; NICD/?, or GCE?;
Notch1fl/flmice, or vehicle-treated Notch1 cKO or NICD Tg
mice (Fig. 2 A and B). No significant differences in proliferation
or neuronal differentiation were noted between these groups at
the time points examined (data not shown), indicating that Cre
toxicity was properly controlled for (21). The efficacy of recom-
bination in Notch1 cKO and NICD Tg mice was assessed by
RT-PCR for Hes5. Levels of hippocampal Hes5 mRNA showed
equal and opposite changes when compared with control mice
(SI Fig. 10). This is consistent with the role of Hes5 as one of the
primary effectors of Notch signaling and displays the effective-
ness of these inducible mice in recombination of nonreporter,
floxed alleles. Similarly, immunostaining for NICD generally
showed the expected cell autonomous change in Notch protein
levels when used in combination with GFP reporter staining in
the Notch1 cKO; GFP and NICD Tg; GFP mice (SI Fig. 10).
Compared with controls, Notch1 cKO mice killed 1 week after
the final tamoxifen treatment displayed a modest but significant
drop in proliferation in the hilus (Fig. 3 B, C, and E). This was
similar to in vivo results obtained with gamma secretase inhib-
itors (SI Fig. 11). Rather remarkably, there was a dramatic and
widespread 3- to 4-fold increase in Ki67?cells in NICD Tg mice,
which included an overall change in the pattern of proliferation
as marker positive cells were widely present in the hilus and
molecular layer, regions not noted for such high levels of
proliferation at this age (Fig. 3 D and E). We then performed cell
cycle analysis by using a Ki67/iodeoxyuridine (IdU) double-
labeling method, which allows for cell cycle exit analysis due to
the 24-h survival period after IdU injection, allowing some cells
to enter G0after incorporating the thymidine analogue into their
DNA—while others reenter (22). Cells exiting the cycle should
be IdU?/Ki67?, whereas reentering cells would be IdU?/Ki67?.
The percentage of cells exiting the cell cycle in the SGZ (number
of cells IdU?/Ki67?divided by number of cells IdU?) was 42 ?
2% in controls vs. 73 ? 6% in Notch1 cKO or 4 ? 2% in NICD
Tg mice (Fig. 3F). Also, compared with controls and NICD Tg
mice, fewer Gfap?cells proliferated in Notch1 cKO mice a week
postrecombination when the IdU was given (9 ? 1% in Notch1
cKO mice vs. 20 ? 1% in controls; P ? 0.001; Fig. 3G).
Next, double immunolabeling was performed for IdU with
chlorodeoxyuridine (CldU), another marker of DNA synthesis,
and two days after the final tamoxifen injection was performed at
immunohistochemical localization of Notch1 in P24 hippocampal coronal
sections. Immunoreactivity is present in virtually all mature neurons and
astrocytes. (C) Confocal image of a section stained for NICD (red), NeuN
(green), Dcx (blue), and Gfap (magenta) taken through the dentate gyrus.
as significant localization of NICD in the cytoplasm of Gfap?astrocytes (filled
arrowhead) and an absence of NICD in the nearby Dcx?cell (empty arrow-
head). (D) Immunostaining for NICD (red), Ascl1 (green), Dcx (blue), and Gfap
(magenta). Ascl1?doublet with one Ascl1?nucleus colocalizing with Gfap
(filled arrowhead) and the other Gfap-(empty arrowhead). NICD did not
colocalize with the mostly nuclear Ascl1 protein in either case. GCL, granule
cell layer; SGZ, subgranular zone. (Scale bars: B, 100 ?m; C and D, 10 ?m.)
breedings and ligand-induced Cre recombination. (A) GCE mice are crossed
with loxP-flanked (‘‘floxed’’) Notch1 mice. GCE; Notch1fl/flmice are given
heat shock protein 90 in the cytoplasm and thus is inactive—to translocate to
the nucleus where it recombines paired loxP sites, ablating the Notch1 pro-
tein. (B) GCE mice are crossed with NICD transgenic mice. GCE; NICD mice are
given tamoxifen, causing nuclear translocation of the CreER protein which
recombines the loxP sites, excising the ‘‘STOP’’ codon and inducing NICD
protein transcription and translation. (C) Three doses of Tamoxifen induce
recombination in a significant population of SGZ cells as determined by
reporter expression (GFP, green). (Scale bar: C, 50 ?m.)
Breunig et al.
December 18, 2007 ?
vol. 104 ?
no. 51 ?
P14. Monoclonal antibodies specific for these two thymidine ana-
logues permit specific recognition of either compound in tissue,
allowing for the identification of two cohorts of S-phase cells (23).
Again, a more than twofold increase in singly labeled CldU?or
IdU?cells was observed in NICD Tg animals compared with
controls and Notch1 cKO mice. When counting CldU and IdU
doubly positive cells, which would be indicative of a cell prolifer-
ating at both P16 and P23, there was little difference between
control animals and Notch1 cKO mice, but there were 3-fold more
CldU?/IdU?cells in NICD Tg mice compared with both other
groups (Fig. 3K). Also, the pattern of labeled cells in Notch1 cKO
mice was altered in that most CldU?cells were located in the GCL
compared with controls where the proportion of cells in the SGZ
vs. GCL was more balanced, indicating increased migration of
CldU?cells in Notch1 cKO mice (Fig. 3 I and M). Conversely, very
few cells appeared to migrate into the GCL in the NICD Tg
animals, which would be consistent with the lack of cell cycle exit
observed (Fig. 3 J and M). These results show that ablating Notch1
cell cycle while overexpressing NICD decreases cell cycle exit.
Alteration of Newborn Neuron Number After Genetic Manipulation of
Notch1. As these groups are genetic mosaics, we bred the Rosa26
(53) and CAG-CAT-GFP (24, 25) reporter strains into each
group. By using the same TM delivery protocol and perfusion
time as pictured in Fig. 3A, animals in each group were examined
for the percentage of SGZ/GCL cells expressing GFP alone
(miscellaneous cell types), or in combination with Gfap/Sox2
(astroglia/progenitors), or Dcx (new neurons). In agreement
with the cell cycle exit data, Notch1 loss caused a significant
increase in new neurons at the expense of progenitors and
miscellaneous cell types (Fig. 4B). Conversely, GFP cells in
NICD Tg animals overwhelmingly colocalized with Gfap and
Sox2, indicating the NICD functions to maintain glial progenitor
cells in the SGZ/GCL (Fig. 4B). There was no apparent differ-
ence in the average recombination rate or density of recombined
cells across the groups or between reporter strains (data not
shown). Overall cell death was increased in Cre? controls,
Notch1 cKO and NICD Tg animals but examination of the
phenotype of dying cells did not yield a pattern indicating that
apoptosis was a significant cause of the shift in cell fates (SI Fig.
12). The increase in death seen in Cre? controls when compared
with Cre? animals indicates that Cre toxicity is a significant
factor in this assay and thus warrants caution in interpreting
survival data from such Cre lines (21). This reciprocal change in
cell fate was also seen in comparable young adult mice but the
These results display that cell autonomous manipulation of
Notch1 causes a dynamic shift in the ratio of Gfap-positive stem
cells versus newborn neurons.
Notch is a Critical Modulator of Dendritic Arborization in Newly
Generated Neurons. Because of its known effects on neurite
outgrowth in vitro (26–28), we examined the morphology of
newly generated, migrating/differentiating neurons at P37 by
using Dcx immunostaining, which labels the maturing den-
dritic arbor of these young cells. Dcx marks newborn neurons
that have been born after tamoxifen treatment at P10, P12, and
P14 (11, 18). Despite a lack of significant alteration in the
totals number of Dcx?cell bodies in NICD Tg and Notch1 cKO
groups (Fig. 5G), there was a remarkable and disproportionate
cells are preferentially found in the granule cell layer. The average number of
CldU-positive cells per mm3in control animals was used for normalization.
Note n ? 6 for each experimental group. Asterisks indicate a statistical
difference between experimental groups (*, P ? 0.05;**, P ? 0.001; Student’s
cell cycle exit. (A) Schematic of the injection paradigm for tamoxifen, CldU,
and IdU. (B–D) Ki67 (red)/IdU (green) immunostaining of the dentate gyrus
of control (Ctrl), NICD overexpressing (NICD Tg), and Notch1 ablated
(Notch1 cKO) animals. (B?–D?) Ki67 signal from B–D shown with enhanced
contrast. (E) NICD overexpression drastically increases the number of pro-
liferating cells in the subgranular zone (SGZ), hilus, and molecular layer
when compared with controls and Notch1 cKO groups. The average num-
ber of Ki67 positive cells per mm3in control animals was used for normal-
ization. (F) Opposite effects on cell cycle exit are seen when comparing
NICD Tg and Notch1 cKO animals with controls. Significantly more cells
leave the cell cycle in Notch1 cKO animals than in controls or NICD Tg
animals where only 7% of cells leave the cell cycle. (G) The phenotype of
proliferate at a reduced level. (Immunostaining for Dcx/Gfap is not shown
for the sake of clarity.) (H–J) CldU (red)/IdU (green) immunohistochemistry
on DG tissue sections. (H?-J?) CldU signal from H–J is shown with enhanced
contrast and the upper limit of the SGZ is labeled with a dotted red line. (K)
The number of cells labeled by CldU and IdU increase in the NICD Tg group.
was used for normalization. (L) CldU/IdU double-positive cell number
increases almost threefold in NICD Tg mice over control and Notch1 cKO
animals. The average number of CldU/IdU double-positive cells per mm3in
control animals was used for normalization. (M) CldU?cells, which synthe-
sized DNA one week before perfusion, remain preferentially in the sub-
granular zone in NICD Tg animals whereas in Notch1 cKO animals CldU?
Genetic manipulation of Notch influences cell proliferation, and
www.pnas.org?cgi?doi?10.1073?pnas.0710156104 Breunig et al.
change in dendritic arborization and branching (Fig. 5 A–F).
Notch1 cKO animals had significantly less complex arboriza-
tion than controls (Fig. 5 A, B, D, E, and H). NICD Tg animals
showed the greatest amount of dendritic complexity, stubby
arbors, and more numerous varicosities (Fig. 5 C, F, H, and I).
The varicosities are thought to be indicative of more immature
granule neurons and typically have no relationship with spine
development (29). Thus, in addition to regulating prolifera-
tion, and differentiation of neural progenitors, Notch signaling
regulates dendritic morphology in the newborn, maturing
Notch Mediates a Binary Switch Between Neural Stem or Precursor
Cells and Committed Progenitor Cell Types. By using inducible loss-
and gain-of-function mice, our data demonstrate that Notch1
plays a role in the proliferation, cell fate determination, and
maturation of cells in a context-dependent manner that can be
correlated with subcellular distribution of the protein (SI Fig.
13). Previous studies have indicated that Notch biases cells
toward an astrocytic fate at the expense of neurons and glia
(30–32). These results have been found in vivo and in vitro. In
vitro studies were performed by using retrovirally transduced,
growth factor-dependent neural stem cells expressing NICD
(31). In vivo studies used retrovirus or electroporation to express
NICD in proliferating cells (30, 32–34). Indeed, we have con-
firmed that these methods do largely block neurogenesis (SI Fig.
14, data not shown). NICD overexpression leads to the main-
tenance of Gfap-expressing neural stem cells in vivo and pro-
motes astrogliogenesis in vitro under differentiating conditions.
We have seen that the reciprocal phenotype is observed on cell
autonomous loss of Notch1 or on forced expression of a dom-
inant-negative form of Mastermind (DN-MAML) (35, 36)—
which functions to block the nuclear signaling of all four notch
receptors (SI Fig. 14). Neural stem cells expressing DN-MAML
continued to proliferate in the presence of EGF/FGF2 (data not
shown) but largely lost the ability to generate glia and proliferate
under differentiation conditions, suggesting that Notch1 func-
(green) reporter expression in the SGZ in tissue immunostained for Dcx (red),
Sox2 (blue), and Gfap (magenta). Example of a GFP? glial cell (A?) showing
expression of Sox2 and Gfap. (A?) GFP?/Dcx? neurons in the GCL. (B) Notch1
cKO animals show a preferential generation of neurons , but NICD Tg mice
same reciprocal change in cell fates is seen in 4- to 6-month-old mice given
tamoxifen 1 week before killing. Note n ? 4 for each experimental group.
0.05;**, P ? 0.001; Student’s t test; B and E). Error bars represent SEM. (Scale
bars: A-A?, C-C?, 10 ?m; D, 50 ?m.)
Cell autonomous changes in cell fate in the DG. (A) Induced GFP
the dentate gyrus at P37. Representative examples of Dcx?/GFP? neurons in
cells. Representative examples of the more ramified cells in each group are
show. (G) Dcx?cell numbers are not significantly different in the SGZ of NICD
Tg and Notch1 cKO animals when compared with controls. The average
number of Dcx?cells per mm3in control animals was used for normalization.
(H) NICD Tg animals display more dendrites per Dcx?cell body. Notch1 cKO
animals show a significant drop in dendritic complexity. (I) NICD Tg animals
have more varicosities per Dcx?cell whereas Notch1 cKO mice show a signif-
icant decrease. Note n ? 6 for each experimental group. Asterisks indicate a
statistical difference between experimental groups (*, P ? 0.05;**, P ? 0.001;
Student’s t test; G–I). Error bars represent SEM. (Scale bars: A–C, 30 ?m.)
Notch-signaling modulates the dendritic arborization of maturing
Breunig et al.
December 18, 2007 ?
vol. 104 ?
no. 51 ?
tions to maintain neural stem cells whereas transit-amplifying
cells use other signaling pathways or noncanonical Notch sig-
naling (SI Fig. 13). The inability of DN-MAML expression to
promote neurogenesis—as is seen after Notch1 ablation in the
dentate gyrus—indicates that active neurogenic signals from the
microenvironment are needed to promote neurogenesis even in
the absence of antineuronal Notch signaling.
Reactive Neurogenesis in the Postnatal Brain. It is noteworthy that
the pattern of hyperproliferation and morphological plasticity
shares similarities with the alterations in neurogenesis seen after
trauma, indicating that Notch1 could be an in vivo modulator of
posttrauma neurogenic responses (37–40). Consistent with this
SGZ after status epilepticus (41) and ischemia (42). Seizures are
known to be one of the most profound stimulators of dentate
gyrus neurogenesis. Different experimental models show vary-
ing degrees of neurogenesis. Strikingly, increased neurogenesis
has been seen concurrently with dramatic dendritic changes in
the newborn population (40). Furthermore, because of the
transient increase in neurogenesis we observed, it is conceivable
that Notch signaling is activated in a graded manner by other
potent stimulators of adult neurogenesis such as running (43),
ischemia (44), and focal lesion (45). In particular, it has recently
been observed that Notch signaling mediates profound postisch-
emia responses (46), and dynamic changes in the expression
pattern of Notch-related molecules suggests the involvement of
Notch in the postlesion (47) and postischemia neurogenic re-
sponse (42, 44).
Dendritic Alterations in Newborn Neurons. We also show that
genetic manipulation of Notch in newborn neurons in the
hippocampus alters the dendritic morphology. In vitro, Notch
was found to have profound effects on the arborization of
cortical neurons (26–28). Our results are consistent with this,
showing that the dendritic arborization of newly generated
neurons is modulated in vivo in a dosage-dependent manner
based on the cleavage of the Notch receptor. Nevertheless, as
Notch signaling levels vary with seizure (41), ischemia (46), and
potentially many other stimuli, such changes in dendrite ar-
borization may not be entirely artificial and can reflect physio-
voluntary exercise can significantly alter dendritic morphology
and complexity in the dentate gyrus (48, 49), in addition to its
known function in neuronogenesis (43).
Our data illuminate the complexity of Notch signaling as a
whole in the transition from progenitor to mature neuron,
displaying its central role during postnatal neurogenesis and
raising the possibility that it plays a part in the plasticity resulting
from trauma or environmental stimulus. Notch acts in one
context to stimulate proliferation of endogenous progenitors, yet
in maturing neurons modulates structural plasticity and survival
pathways, suggesting that differential clinical manipulation of
Notch could aid in multiple aspects of functional recovery after
disease, trauma, or pathological aging.
Note. After the completion of these experiments, Mizutani et al.
(50) reported that neural stem cells and committed progenitors
display differential levels of Notch signaling in the embryonic
brain, consistent with our findings in the postnatal and adult
Materials and Methods
Mice. GCE; R26R/R26R, GCE; Z/EG/?, GCE;?/?,
GCE;Notch1fl/fl(Notch1 cKO), or GCE;NICD (NICD Tg) at the ages noted were
gavage at the times noted. [No differences were noted between delivery
methods, as has been described (51).] Littermates were used as controls. CldU
or IdU (Sigma) were given at the times noted as a single i.p. injection equimo-
lar to 100 mg/kg of bromodeoxyuridine. (Most experiments were performed
of DAPT (Sigma) 200 mg/kg, DBZ (Calbiochem) 4 mg/kg, or DMSO (Sigma)
every 12 h for 3 days based on studies characterizing in vivo activity (16, 52).
Statistical Analysis. A two-tailed unpaired Student’s t test was used for anal-
yses of all experiments presented in Figs. 3–5. A P value ? 0.05 was considered
Details on other methods are available in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank C. Anderson, J. Bao, and M. Pappy for
excellent experimental assistance; D. Anderson, J. Aster, M. Colbert, A. Israel,
J. Johnson, W. Pear, F. Radtke, and J. Shen for mice and reagents; and J.
Arellano, K. Burns, R. Duman, M. Givogri, K. Hashimoto-torii, K. Herrup, C. Y.
Kuan, J. Loturco, R. Rasin, M. Sarkisian, T. Town, and members of the P.R. and
N.Sˇ. laboratories for helpful discussions, technical support, and comments.
This work was supported by National Institutes of Health Grants R01
MH067715, HD045481, AG019394, and NS047200; the March of Dimes Birth
Defects Foundation Grant FY05-73; and the Kavli Institute.
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