Nuclear factor-κB is a critical mediator of stress-
impaired neurogenesis and depressive behavior
Ja Wook Kooa,b, Scott J. Russob, Deveroux Fergusonb, Eric J. Nestlerb, and Ronald S. Dumana,1
aLaboratory of Molecular Psychiatry, Center for Genes and Behavior, Departments of Psychiatry and Pharmacology, Yale University School of Medicine, New
Haven, CT 06519; andbFishberg Department of Neuroscience, Mount Sinai School of Medicine, New York, NY 10029
Edited* by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved December 28, 2009 (received for review September 16, 2009)
Proinflammatory cytokines, such as IL-1β, have been implicated in
the cellular and behavioral effects of stress and in mood disorders,
although the downstream signaling pathways underlying these
effects have not been determined. In the present study, we demon-
strate a critical role for NF-κB signaling in the actions of IL-1β and
stress. Stress inhibition of neurogenesis in the adult hippocampus,
which has been implicated in the prodepressive effects of stress, is
blocked by administration of an inhibitor of NF-κB. Further analysis
reveals that stress activates NF-κB signaling and decreases prolifer-
ation ofneuralstem-likecellsbut notearly neural progenitorcellsin
the adult hippocampus. We also find that depressive-like behaviors
caused by exposure to chronic stress are mediated by NF-κB signal-
ing. Together, these data identify NF-κB signaling as a critical medi-
ator of the antineurogenic and behavioral actions of stress and
cytokine|IL-1|inflammation|neural progenitor cell|depression
ing in enormous personal and economic costs and, in many cases,
suicide (1, 2). Despite significant efforts, the neurobiological
mechanisms underlying depression have not been characterized.
Both genetic and environmental factors contribute to depression,
and traumatic or repeated stress is known to precipitate or exac-
erbate mood disorders (3–5). In addition, proinflammatory cyto-
and infection as well as by psychological stress have been impli-
cated in depressive behavior in rodent models and depressed
brain regionsthatcontrol emotion andmood, including inhibition
of neurogenesis in the adult hippocampus (5, 9, 10). Inhibition of
(NSCs) or intermediate transient amplifying neural progenitor
cells (ANPs), that are influenced have not been characterized (9,
11).Arole forproinflammatorycytokines is supportedby a recent
report that IL-1β signaling is necessary and sufficient for the
antineurogenic and behavioral effects of stress (6). One possible
which is activated by IL-1β and other cytokines both in peripheral
immune cells and in the brain (8, 12). Chronic stress enhances the
activation of NF-κB in response to inflammatory stimuli (13, 14),
andsocial stress increases NF-κB signaling in healthy subjects and
produces an exaggerated response in depressed patients (15, 16).
In the present study, we investigate the role of NF-κB in the
cellular and behavioral responses to acute and chronic stress. The
results demonstrate that the inhibition of neurogenesis by stress
occurs via activation of NF-κB in NSCs and that stress-induced
ood disorders represent a major health concern, affecting
over 15% of the population in developed countries, result-
Neurogenesis Caused by Acute and Chronic Stress. To examine the
role of NF-κB in the effects of acute stress, rats were adminis-
SignalingMediates the Suppressionof Hippocampal
tered an inhibitor of NF-κB before acute immobilization stress
(45 min) (Fig. 1A). Two selective structurally different NF-κB
inhibitors were tested: JSH-23 (JSH) and SC-514 (SC) (17, 18).
Immediately after immobilization, rats were administered BrdU,
a thymidine analogue that is incorporated into dividing cells (2 h
post-BrdU injection). Post hoc analysis revealed that immobili-
zation stress significantly decreases the number of BrdU+cells in
the subgranular zone (SGZ) of the dentate gyrus (DG), and this
effect is completely blocked by preadministration of either JSH
or SC (Fig. 1 B–D).
We also examined the role of NF-κB in the antiproliferative
effects of chronic unpredictable stress (CUS), a model of
depression with face, predictive, and construct validity (19, 20).
Exposure to CUS significantly decreased the number of BrdU+
cells, and this effect was completely blocked by infusion of JSH
(i.c.v., minipump) (Fig. 1F). At the time point examined (14 days
after BrdU) (Fig. 1E), the majority of the BrdU+cells express
doublecortin (DCX), a marker of immature neurons (Fig. 1H).
Analysis of the BrdU+DCX+double-labeled cells demonstrates
effect was blocked by JSH (Fig. 1G).
Acute Stress Decreases the Proliferation of NSCs: Blockade by NF-κB
Inhibition. We next examined the influence of acute immobiliza-
tion stress (45 min) on the two major classes of progenitors. NSCs
undergoasymmetric division, areself-renewing, have aradial glial
morphology, and express GFAP and the transcription factor sex-
2A). ANPs undergo symmetric division and express SOX2 but not
GFAP (22, 23). In the current study, we found that nearly all
BrdU+cells were SOX2+(97.1 ± 1.5%), and exposure to acute
immobilization stress significantly decreased the number of
BrdU+SOX2+double-labeled cells. Importantly, this effect of
stress was completely blocked by pretreatment with the NF-κB
inhibitor JSH (Fig. 2B). We also found that acute stress sig-
nificantly decreases the number of triple-labeled NSCs (BrdU+
SOX2+GFAP+) (Fig. 2C) but not the number of ANPs (BrdU+
SOX2+GFAP−) (Fig. 2D), and this effect is blocked by JSH
Previously, we have shown that the antineurogenic effects of
stress occur via IL-1β/IL-1receptor type I (RI) signaling, both in
vivo and in vitro (6). We found that IL-1RI is expressed in both
NSCs and ANPs, albeit at different levels (27.6 ± 2.2% and 49.2
± 6.3%, respectively) (Fig. S1). Because the majority of the
SOX2+cells are GFAP+NSCs (81.3 ± 1.4%), there are more
NSCs than ANPs that express IL-1RI.
Author contributions: J.W.K., E.J.N., and R.S.D. designed research; J.W.K., S.J.R., and D.F.
performed research; J.W.K. and R.S.D. analyzed data; and J.W.K., S.J.R., E.J.N., and R.S.D.
wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
| February 9, 2010
| vol. 107
| no. 6
Acute Stress Activates NF-κB Signaling in NSCs. We examined the
cellular localization of NF-κB activation using transgenic NF-κB/
LacZ reporter mice. Immunohistochemical analysis demon-
strates that β-gal is expressed in the granule cell layer of the DG
(Fig. 3 A and B), as previously reported (24). Triple-labeling
studies demonstrate that β-gal is expressed in both NSCs
(GFAP+SOX2+) (Fig. 3 E–I) and ANPs (GFAP−SOX2+) (Fig.
3J) in the SGZ as well as in cells in the SGZ/DG that express
markers of immature (DCX) (Fig. 3K) or mature (NeuN) (Fig.
3L) neurons. Notably, proliferating newborn cells labeled with
BrdU (2 h after injection) did not express β-gal (Fig. 3 A–D),
suggesting that NF-κB activation blocks proliferation.
We also found that acute stress significantly increased the
number of NF-κB/β-gal+NSCs but not ANPs in the SGZ, and
this effect was blocked by pretreatment with the IL-1 receptor
antagonist (Ra) (Fig. 3 M and N). This suggests that acute stress
preferentially activates NF-κB in NSCs. Stress did not change the
ratio of NSCs to total progenitors in the SGZ [F2,9= 0.537, P =
not significant (n.s.)]. Together, these data demonstrate that
acute stress stimulates NF-κB signaling in NSCs and that this
effect is mediated by IL-1β.
NF-κB Signaling Underlies Depressive-Like Behavioral Effects of CUS.
To determine if NF-κB signaling underlies the effect of CUS on
depressive-like behaviors, SC was administered during 4 weeks of
exposure to CUS (Fig. 4A). The novelty-induced hypophagia
(NIH) test is a model of anxiety that is responsive to chronic
antidepressant administration (25). CUS exposure significantly
increased the latency to drink relative to nonstressed control mice
is blocked by continuous infusion of SC (i.c.v., minipump). There
was no difference in latency to drink in the home cage, a control
for this paradigm (F2,38= 0.315, P = n.s.; n = 13–15 per group)
and no difference between nonstressed SC-infused (66.2 ± 14.4 s)
and control (75.0 ± 19.6 s) groups in the novel cage (t9= 0.345,
P = n.s.; n = 5–6 per group). We next tested the animals on
sucrose consumption, a measure of anhedonia, which is a core
symptom of depression (19, 20). CUS significantly decreased
sucrose consumption, and this effect was also blocked by SC
infusion (Fig. 4C). There was no difference in the consumption
of tap water, a control for this test (F2,21= 0.578, P = n.s.; n = 5–
13 per group) and no difference in the sucrose consump-
tion between nonstressed SC-infused (75.9 ± 2.8 mg/kg) and
control (73.3 ± 3.8 mg/kg) groups (t12= 0.517, P = n.s.; n = 6–8
Inhibition of NF-κB Blocks the Antiproliferative Effects of IL-1β in
Neural Progenitor Cells in Vitro. To investigate further the mech-
anisms by which stress inhibits neurogenesis, studies were con-
neurogenesis in rats. (A) Schematic depicting the experimental
procedures for acute stress (aSTR). After 8–10 days of recovery
and handling, rats were infused (i.c.v.) with the NF-κB inhibitors
JSH or SC or with vehicle (DMSO) before immobilization and
were then killed (sac) 2 h after BrdU administration. injt.,
injection. (B) aSTR decreased the number of BrdU+cells in the
DG of the hippocampus. The effect of aSTR was blocked by
either JSH or SC (one-way ANOVA: F3,26= 3.527, P < 0.05; n = 7–8
per group). Dividing BrdU+cells (black arrowheads) in the DG of
acute-stressed (C) and JSH-infused stressed (D) rats. GCL, gran-
ular cell layer. (Scale bar: 100 μm.) (E) Schematic for CUS. Rats
were implanted with a cannula (i.c.v.) and minipump (s.c.), were
exposed totwo stressorsper dayfor 21days, received BrdU daily
the effects of CUS on BrdU+cells (F3,19= 5.013, P < 0.05; n = 5–6
per group) (F), and immature neurons were measured as the
number of BrdU+(red) DCX+(green) double-labeled cells per
hippocampal DG (F3,12= 6.250, P < 0.01; n = 4 per group) (G and
H). (Scale bar: 25 μm.) By one-way ANOVA followed by the
Fisher’s PLSD test, *P < 0.05 and **P < 0.01 compared with
control (DMSO) and#, P < 0.05 and##, P < 0.01 compared with
Effects of stress and NF-κB inhibitors on hippocampal
liferation in acute stress. (A) Representative photograph of proliferating
NSCs [GFAP+(red) SOX2+(blue) BrdU+(green) cells] in the SGZ. (B) Acute
stress decreased the proliferation of total progenitors (SOX2+BrdU+cells),
and there was no significant effect in CUS animals receiving JSH (F2,11=
4.215, P < 0.05; n = 4–5 per group). aSTR, acute stress; CTRL, control. Stress-
induced impairment of NSC (F2,11= 5.814, P < 0.05) (C) but not ANP (F2,11=
0.852, P = n.s.) (D) proliferation was blocked by JSH. (Scale bar: 25 μm.) By the
Fisher’s PLSD test, *, P < 0.05 compared with CTRL group and#, P < 0.05
compared with aSTR group.
Role of NF-κB signaling in the regulation of NSC and ANP cell pro-
| www.pnas.org/cgi/doi/10.1073/pnas.0910658107Koo et al.
ducted on cultured adult hippocampal progenitors (AHPs).
Under the culture conditions used (20 ng/mL FGF-2), ∼90% of
DAPI+cells expressed nestin, a marker of AHPs (Fig. S2B).
Most of the nestin+cells (∼90%) also incorporate BrdU, indi-
cating that the majority of the AHPs are actively proliferating
(6). Incubation with IL-1β (2 h) significantly decreases AHP
proliferation compared with vehicle, and this effect is blocked by
coincubation with JSH or IL-1Ra (Fig. 5 A–C). The effect of IL-
1β was not influenced by inhibitors of corticosterone (CORT;
RU486) or p38 MAPK (SB203580) (Fig. 5C). IL-1β and CORT
decreased the ratio of BrdU+to nestin+cells but did not
influence the total number of DAPI+cells or the ratio of nestin+
to DAPI+cells (Fig. S2 A-C). In contrast, the antiproliferative
effect of CORT was not influenced by JSH but was blocked by
RU486 as expected and by a p38 MAPK inhibitor (SP600125;
Fig. S3A). These data suggest that CORT-mediated suppression
of AHP proliferation is mediated by p38 MAPK signaling and
that different mechanisms underlie the antiproliferative effects of
CORT and IL-1β. Incubation with IL-1β or CORT did not alter
the ratio of TUNEL+to DAPI+cells (Fig. S3 B and C), indi-
cating that the decrease in cell number is not a result of cell death.
The results of the current study demonstrate that inhibition of
neurogenesis by acute or chronic stress is blocked by inhibition of
NF-κB signaling. A requirement for NF-κB is shown using two
different selective inhibitors, one that blocks IkB kinase (IKK)
(SC) and the dissociation of IκB and NF-κB and one that directly
antineurogenic effects of acute stress result from inhibition of
NSC proliferation (Fig. 6). Triple-labeling studies show that the
number of GFAP+SOX2+BrdU+NSCs but not ANPs was sig-
nificantly decreased by acute stress. These effects were confirmed
in cultured hippocampal progenitor cells. Because NSCs undergo
asymmetric division, giving rise to another NSC and an inter-
mediate progenitor (i.e., ANP), the long-term consequences of
inhibiting NSC proliferation (i.e., in response to CUS) would be a
reduction in the pool ofNSCs as well as intermediate progenitors.
The ANPs then give rise to additional neurons, and decreased
numbers of these cells could account for the reduction in imma-
ture neurons observed after exposure to long-term stress in the
The results also demonstrate that acute stress activates NF-κB
signaling in GFAP+NSCs in adult hippocampus (Fig. 6), con-
sistent with the hypothesis that NF-κB signaling in NSCs but not
reporter expression in the hippocampus
in NF-κB/LacZ reporter mice. (A–D) Most
hippocampal progenitors [∼94%; BrdU+
express the NF-κB
(green), yellow arrowheads]. (Scale bar:
reporter in NSCs [β-gal+(F, green) SOX2+
(G, blue) GFAP+(H, red)]. (Scale bar: 50
μm.) (I) Enlarged view of NSCs from E
(Inset). (J) NF-κB/β-gal was observed in
ANPs (β-gal+SOX2+GFAP−) as well as
NSCs in the SGZ. Immature [β-gal+DCX+
(I) neurons also expressed the NF-κB
reporter. (M–P) Acute immobilization
stress (aSTR) resulted in more NF-κB acti-
vation (β-gal+) in NSCs but not in ANPs or
in total progenitors compared with con-
trol (CTRL) [NSCs, F2,9= 11.381, P < 0.01
(M); ANPs, F2,9= 0.134, P = n.s. (N); total
progenitors, F2,9= 2.994, P = n.s. (O); n =
4 per group]. (Scale bar: 25 μm.) By the
Fisher’s PLSD test, **, P < 0.01 compared
withCTRL and##,P <0.01 compared with
Acute stress effects on NF-κB
were exposed to two or three stressors per day for 28 days and received BrdU
daily for the last 4 days of CUS. On days 21 and 28, NIH and the SCT, respec-
tively, were conducted. Mice were killed (sac) 24 h after the last BrdU
administration. (B)CUS significantly increased latency todrink, and this effect
15 per group). CTRL, control. (C) SC administration also blocked the effect of
CUS on sucrose consumption (F2,21= 3.497, P < 0.05; n = 5–13 per group). By
the Fisher’s PLSD test, *, P < 0.05 compared with CTRL and#, P < 0.05 com-
pared with CUS.
Effects of CUS on depressive-like behaviors in NIH and sucrose con-
Koo et al.PNAS
| February 9, 2010
| vol. 107
| no. 6
in ANPs underlies the reduction in cell proliferation. However,
the relatively small percentage of NSCs that exhibit NF-κB/β-gal
staining (20–25%) raises a question as to whether inhibition of
this population could account for a 50% reduction in NSC
proliferation? There are several potential explanations for this
discrepancy. One is that NF-κB-induced gene expression is not
an accurate reflection of the total level of NF-κB activity in
NSCs. This could result from low sensitivity of β-gal gene
expression (i.e., NF-κB is activated but without an increase in
β-gal expression) and/or other gene transcription-independent
signaling mechanisms that could influence NSC proliferation.
Stress could also activate NF-κB in surrounding cells (e.g., ANPs,
neurons, glia) that indirectly influence NSC proliferation. For
example, stress-induced NF-κB activation results in generation
of nitric oxide (NO) (26–28), and NO has been shown to inhibit
neurogenesis in the adult hippocampus and to contribute to the
inhibition of neurogenesis by stress (29, 30).
Our current results also demonstrate that activation of NF-κB
in response to acute stress is blocked by an inhibitor of IL-1RI.
This is consistent with previous reports that IL-1β/IL-1RI sig-
naling is rapidly activated (31) and is necessary and sufficient for
suppression of neurogenesis by acute stress (6). Localization
studies demonstrate that IL-1RI is expressed in both NSCs and
ANPs in adult hippocampus. In addition, although the percent-
age of NSCs that express IL-1RI is lower than that of ANPs
(∼25% and 50%, respectively), the ratio of IL-1RI+NSCs to IL-
1RI+ANPs is ∼3:1, because the total number of NSCs is much
greater than the total number of ANPs. However, given the
expression of IL-1RI in intermediate progenitors, it is surprising
that stress selectively activates NF-κB and inhibits proliferation
of NSCs. This could result from differential localization/avail-
ability of IL-1β in proximity to NSCs vs. ANPs or from expression
of different signaling in these two populations of progenitors.
Previous studies report that NF-κB differentially affects pro-
liferation, maturation, and survival depending on cell type,
localization, and conditions. Much of this work has focused on
immune and cancer/tumor cells, in which NF-κB is associated
with cell proliferation and antiapoptotic pathways but also with
proapoptotic pathways (32, 33). There is also evidence that NF-
κB activation has either prosurvival or cell death effects in the
brain depending on the conditions (34–36). These effects could
be explained by differential expression and activation of the
multiple subtypes and components of the NF-κB family. For
example, the proapoptotic actions of NF-κB that occur pos-
tischemia involve p50/p65, whereas c-Rel-containing dimers
increase resistance to ischemia by activating antiapoptotic
pathways (36). There is one report that RelA is expressed in
NSCs in the SVZ, whereas p50 is expressed in migrating neural
precursors (37). In p50 deletion mice, there is no effect on cell
proliferation in adult hippocampus under nonstress conditions
(38), consistent with the negative results of the current study.
Additional studies of stress in p50 mutant mice, or in conditional
deletion mutants, would further characterize the role of NF-κB
Activation of the hypothalamic-pituitary-adrenal axis and
elevation of glucocorticoids are typically associated with antiin-
flammatory responses, including inhibition of NF-κB signaling,
via inhibition of IKK and inhibition of NF-κB transcription (17,
18). However, there are also reports that stress can increase the
effects of NF-κB, including enhancement of inflammatory
responses (13, 14, 27). The current study provides another
example wherein stress results in selective activation of NF-κB
signaling in a discrete population of cells, the NSCs.
We also examined the role of NF-κB in anxiety- and depres-
sion-related behaviors after exposure to CUS. Inhibition of NF-
κB blocked the increase in latency to feed in response to CUS in
the novelty suppressed feeding test (NSF) paradigm, consistent
with a previous report that anxiety-related behaviors are
decreased in NF-κB/p50 null mice (39). We also found that in-
hibition of NF-κB blocks the reduction in sucrose consumption
resulting from CUS, an antidepressant-like effect. These findings
are consistent with human studies, which demonstrate that social
stress/anxiety increases NF-κB signaling and that this effect is
enhanced in depressed patients (16). Although we have focused
on models that are responsive to chronic antidepressants, which
are more relevant to the therapeutical response times, it would
be interesting to examine other models predictive of anti-
depressant response (e.g., forced swim test, learned helplessness
paradigm). It would also be interesting to determine if blockade
of NF-κB after exposure to stress rather than before stress, as
was done in the current study, would be sufficient to reverse NF-
κB signaling and produce antidepressant behavioral effects.
In summary, the results demonstrate that IL-1β/NF-κB sig-
naling is activated by stress and indicate that this signaling
pathway is required for the antineurogenic and anhedonic effects
of repeated stress (Fig. 6). Blockade of NF-κB could inhibit the
actions of other proinflammatory cytokines implicated in stress
and depression, including IL-6 and TNF-α. Also, blockade of
NF-κB in both peripheral immune cells and the brain could
provide beneficial antiinflammatory and antistress actions. The
diverse and cell type-dependent actions of NF-κB make it a
complex drug target, although there may be specific cases of
elevated inflammatory processes in which NF-κB inhibitors
would be useful and efficacious for the treatment of depression.
IL-1β + JSH
Nestin+ BrdU+ DAPI+
JSHRa SB RU
images of proliferating AHPs [BrdU+(red) nestin+(green)] in the presence of
JSH blocked the effect of IL-1β on the ratio of BrdU+to nestin+compared with
control (CTRL) (F5,16= 11.174, P < 0.001; n = 3–4 per group). By the Fisher’s
PLSD test, **, P < 0.01 and ***, P < 0.001 compared with CTRL and##, P < 0.01
and###, P < 0.001 compared with IL-1β. (Scale bar: 50 μm.)
Analysis of IL-1β signaling in cultured AHPs. (A and B) Representative
hippocampus. Stress activates IL-1β/NF-κB signaling, resulting in impairment
of hippocampal neurogenesis and thereby contributing to depressive-like
behaviors. Notably, the results demonstrate that acute stress decreases NSC
proliferation and that this effect is mediated by IL-1β/NF-κB signaling. In
contrast, antidepressant treatment increases neurogenesis by inducing NSC
[electroconvulsive seizure (ECS)] or ANP (chronic fluoxetine) proliferation.
Model of stress-induced impairment of NSC proliferation in the
| www.pnas.org/cgi/doi/10.1073/pnas.0910658107Koo et al.
Animals. Adult male Sprague–Dawley (SD) rats (Charles River) with initial
weights of 230–250 g and adult male NF-κB/LacZ transgenic reporter mice
(24, 40) and their WT littermates with initial weights of 20–30 g were used
for experiments. Animals were housed, two to five per cage, under standard
illumination parameters (12-h light/dark cycle) and with ad libitum access to
food and water. All experiments were conducted in accordance with
guidelines of the Society for Neuroscience and National Institutes of Health
as well as those of the institutional animal care and use committees at Yale
University and Mount Sinai Schools of Medicine.
Cannula Implantation and Microinjections. A 26-gauge guide cannula (Plastics
One) was implanted for i.c.v. infusion of the NF-κB inhibitors JSH and SC (15 μg
per 2.5 μL per rat; Calbiochem) or 100% (vol/vol) DMSO (vehicle) into either
the left or right (randomly assigned) lateral ventricle of the rats and for i.c.v.
infusion of IL-1Ra (0.25 μg per 1 μL per mouse; R&D Systems) or 0.1% BSA/PBS
(vehicle) into the mouse lateral ventricle. For details, see SI Methods.
Acute Stress Exposure. For acute immobilization stress, SD rats were
restrained in a Plexiglas immobilization tube (3.5-in diameter × 7-in length)
for 45 min. For acute stress experiments with mice, animals were stressed for
50 min with a combination of restraint stress and rotation stress on an
orbital shaker. All experiments for acute stress exposure were performed
between 1100 and 1400 hours.
BrdU Labeling and Quantitative Analysis. BrdU immunohistochemistry and
quantitative analysis using a modified unbiased stereology protocol was
conducted as described in SI Methods.
Surgical Procedure and Treatments for CUS. Ani.c.v.cannula(BrainInfusionKit2
20 μg/μL) during CUS. A miniosmotic pump (Alzet 2004 for rats, Alzet 1004 for
mice), with a flow rate of 60 μg/day for rats and 48 μg/day for mice, was
implanted s.c. and connected to the cannula. For details, see SI Methods.
CUS Procedure. For our CUS procedure, we used various stressors of which the
sequence was intentionally designed to maximize unpredictability. All CUS
rats were exposed to two stressors a day for 21 days (for details, see Table S1).
All rats received BrdU injection (i.p., 75 mg/kg) for 4 days of the last CUS
period. Two weeks after BrdU injection, rats were perfused, BrdU immu-
nohistochemistry was performed, and BrdU+cells were quantified in the
hippocampus using a modified unbiased stereology protocol. BrdU+cells
colabeled with DCX were also quantified in DG (three to four sections per
animal) using confocal laser microscopy (Zeiss). In the case of mice, two or
three stressors a day were used for 28 days (for details, see Table S2).
NIH and Sucrose Consumption Test. All mice were singly housed 2 days before
NIH and the sucrose consumption test were performed. For details of the
tests, see SI Methods.
Neural Progenitor Cell Isolation and Culture. Neural progenitor cells were
isolated from the hippocampi of adult female SD rats (body weight of 160–
200 g; Charles River) and cultured as previously described (6, 41). For details,
see SI Methods.
Statistical Analysis. All data are expressed as the mean ± SEM. Experiments
with two groups were analyzed statistically using an unpaired t test.
Experiments with three or more groups were subjected to one-way ANOVA,
followed by the Fisher’s probable least-squares difference (PLSD) post hoc
test. The level of statistical significance for all analyses was set at P < 0.05.
Additional Details. The reader is referred to SI Methods for additional routine
procedures, including immunofluorescence and TUNEL assays.
ACKNOWLEDGMENTS. We thank A. Bhakar for NF-κB/LacZ reporter mice and
E. Mouzon and X. Li for technical help. This work was supported by US Public
Health Service Grants MH45481, 2 PO1 MH25642, and R01 MH51399; a Veter-
ans Administration National Center Grant for Post-Traumatic Stress Disorder;
and the Connecticut Mental Health Center.
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