p21Cip1restricts neuronal proliferation in the
subgranular zone of the dentate gyrus
of the hippocampus
Robert N. Pechnick*†, Svetlana Zonis‡, Kolja Wawrowsky‡, Jonathan Pourmorady‡, and Vera Chesnokova‡§
‡Department of Medicine, Division of Endocrinology, and *Department of Psychiatry and Behavioral Neurosciences, Cedars-Sinai Medical Center, 8700
Beverly Boulevard, Los Angeles, CA 90048; and†Brain Research Institute, University of California, Los Angeles, CA 90024
Communicated by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, November 23, 2007 (received for review July 2, 2007)
The subgranular zone (SGZ) of the dentate gyrus of the hippocam-
pus is a brain region where robust neurogenesis continues
throughout adulthood. Cyclin-dependent kinases (CDKs) have a
primary role in controlling cell division and cellular proliferation.
p21Cip1(p21) is a CDK inhibitor that restrains cell cycle progression.
Confocal microscopy revealed that p21 is abundantly expressed in
the nuclei of cells in the SGZ and is colocalized with NeuN, a marker
for neurons. Doublecortin (DCX) is a cytoskeletal protein that is
primarily expressed by neuroblasts. By using FACS analysis it was
found that, among DCX-positive cells, 42.8% stained for p21,
indicating that p21 is expressed in neuroblasts and in newly
developing neurons. p21-null (p21?/?) mice were examined, and
the rate of cellular proliferation, as measured by BrdU incorpora-
tion, was increased in the SGZ of p21?/?compared with WT mice.
In addition, the levels of both DCX and NeuN protein were
increased in p21?/?mice, further demonstrating increased hip-
antidepressant imipramine (10 mg/kg per day i.p. for 21 days)
markedly decreased hippocampal p21 mRNA and protein levels,
produced antidepressant-like behavioral changes in the forced
swim test, and stimulated neurogenesis in the hippocampus. These
results suggest that p21 restrains neurogenesis in the SGZ and
imipramine-induced stimulation of neurogenesis might be a con-
sequence of decreased p21 expression and the subsequent release
of neuronal progenitor cells from the blockade of proliferation.
Because many antidepressants stimulate neurogenesis, it is possi-
ble that their shared common mechanism of action is suppression
antidepressant ? neurogenesis ? depression ? cyclin-dependent kinase
itors (1). In the hippocampus, the neural progenitor cells are
located in the subgranular zone (SGZ) of the dentate gyrus, at
the border between the hilus and the granular cell layer (GCL)
(2, 3). Newborn cells proliferate in SGZ, migrate into the GCL,
develop the morphological and functional properties of granule
cell neurons, and become integrated into existing neuronal
circuitry (4). This suggests an important role of intrinsic stimu-
latory and inhibitory factors in the regulation of proliferation of
neuronal precursor cells.
In mammalian cells, the control of cellular proliferation
primarily is achieved in the G1phase of the cell cycle. Cyclin-
dependent kinases (CDKs) tightly control the cell cycle process.
Cell cycle progression is negatively regulated by two families of
CDK inhibitors: Ink4/ARF type (p16, p15, p18, and p19) and
Cip/Kip type (p21, p27, and p57). p21Cip1(p21) acts in the G1
phase of the cell cycle and delays or blocks the progression of the
cell into the S phase (5). p21 maintains cell quiescence, and
chronic activation of p21 can drive the cell into irreversible cell
increases cellular proliferation (7).
n the central nervous system, developing neurons are derived
from quiescent multipotent or neural stem cells and progen-
The induction of neurogenesis might be part of the molecular
mechanisms underlying the therapeutic effects of antidepressant
treatment (8, 9). All major classes of antidepressants stimulate
neurogenesis in rodents (10–12) and nonhuman primates (13).
Irradiation of the brain blocks hippocampal neurogenesis and
abolishes the behavioral effects of antidepressants (14). Fur-
of the therapeutic effects of these medications (15). The present
study evaluated the role of the CDK inhibitor p21 in neurogen-
esis. The results suggest that antidepressants may stimulate SGZ
neurogenesis by inhibiting p21 expression.
p21 Is Expressed in Neuroblasts and Newly Developing Neurons in the
SGZ of the Hippocampus. Confocal microscopy images revealed
that p21 is abundantly expressed in the nuclei of cells in the SGZ
(Fig. 1A) and that it is colocalized with NeuN, a marker for
neurons. (Fig. 1B). p21 is not colocalized with NeuN in the GCL,
a region where postmitotic neurons reside. Some p21-positive
cells were negative for NeuN. Because p21 was not found in glial
cells (data not shown), this indicates that p21 also is likely
expressed in premature, NeuN-negative neuroblasts. Thus, p21
expression might be confined to neuronal precursors or/and
newly developing neurons.
Doublecortin (DCX) is a cytoskeletal protein that is primarily
expressed by neuroblasts, and a subpopulation of NeuN/DCX-
positive cells is found in the SGZ (16, 17). FACS analysis was
used to determine whether p21 is expressed in DCX-positive
cells. Whole hippocampi from 10 mice were pooled and disso-
ciated, and DCX-positive cells were selected by sorting. Approx-
imately 5 ? 104DCX-positive cells were obtained. They were
small in size (Fig. 1C Upper), in agreement with data showing
that DCX is expressed in small, compact cells corresponding to
neuroblasts (17). After sorting, both DCX-positive and DCX-
negative cells were stained for p21. Among the DCX-positive
cells, 42.8% stained for p21 (Fig. 1C Lower). In a separate
experiment, among the DCX-negative cells, ?1% stained for
p21 (data not shown). These results demonstrate that p21 is
expressed in neuroblasts and in newly developing neurons.
p21 Deletion Increases Proliferation of Hippocampal Neurons. To
further evaluate the role of p21 in hippocampal neurogenesis,
p21-null (p21?/?) mice were examined. Heterozygous (p21?/?)
male and female mice were bred to obtain WT and p21?/?mice
from the same breeding. BrdU, which incorporates into nuclear
Author contributions: R.N.P. and S.Z. contributed equally to this work; R.N.P. and V.C.
designed research; S.Z., K.W., and J.P. performed research; R.N.P., S.Z., K.W., and V.C.
analyzed data; and R.N.P. and V.C. wrote the paper.
The authors declare no conflict of interest.
§To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2008 by The National Academy of Sciences of the USA
January 29, 2008 ?
vol. 105 ?
DNA during the S phase of the cell cycle, was used to assess
cellular proliferation. WT and p21?/?mice were injected with
BrdU every 2 h for a total of three injections and then killed 24 h
after the first injection. In the SGZ, BrdU incorporation was
P ? 0.05) (Fig. 2 A and B). The average number of BrdU-positive
cells per section also was higher in p21?/?mice than in WT mice
(9.6 ? 1.04 vs. 5.03 ? 0.48, P ? 0.05) (Fig. 2C). These results
indicate that the rate of cellular proliferation was increased in
the SGZ of p21?/?mice. Separate groups of mice were killed,
and protein levels of DCX and NeuN were measured in whole
hippocampi by Western blot. The levels of both DCX and NeuN
were increased in p21?/?mice, further demonstrating increased
neuronal proliferation in these animals (Fig. 2 D and E). To
confirm that the neuroblasts were proliferating, slides were
double-stained for BrdU and DCX. BrdU was colocalized with
DCX in the SGZ of p21?/?mice (Fig. 3), showing that the in-
creased cellular proliferation was due to enhanced neurogenesis.
The number of DCX-positive cells in the hippocampi of
p21?/?mice also was assessed. Ten whole hippocampi from WT
and p21?/?mice were dissociated, and DCX-positive cells were
identified by FACS analysis. In the WT mice 2.6% of hippocam-
pal cells were positive for DCX, whereas in the p21?/?mice 4.1%
of cells were DCX-positive (data not shown). The increased
number of DCX-positive cells in the SGZ of p21?/?mice
confirms that the enhanced neurogenesis was the result of the
increased proliferation of neuroblasts.
Chronic Treatment with Imipramine Decreases p21 Expression in the
SGZ of the Hippocampus. To investigate whether p21 is involved in
the antidepressant-dependent increase of neuronal prolifera-
tion, p21 expression was measured in mice chronically treated
with the tricyclic antidepressant imipramine. Mice were treated
daily for 21 days with saline (0.9%) or imipramine hydrochloride
(10 mg/kg per day i.p.) and killed 24 h after the last injection.
Confocal microscopy indicated that treatment with imipramine
markedly decreased the number of cells positive for p21 in the
SGZ compared with saline-treated controls (Fig. 4 A–H). Other
from whole hippocampi. p21 gene expression was detected by
quantitative real-time PCR, and levels of p21 protein were
analyzed by ELISA. p21 mRNA levels were lower in the
hippocampi after imipramine treatment (Fig. 4I), as were levels
of p21 protein (Fig. 4J). There were no differences in p27Kipand
p18INK4CmRNA or protein levels (data not shown). Thus,
chronic treatment with imipramine specifically suppresses p21
expression in the SGZ of the hippocampus.
of the Hippocampus and Produces Antidepressant-Like Activity in the
Forced Swim Test (FST). To confirm that the imipramine-induced
decrease in p21 is associated with increased neurogenesis, sep-
arate groups of mice were chronically treated with imipramine
(or saline) as described above. Twenty-four hours after the last
injection of imipramine (or saline), the mice were injected every
2 h with BrdU for a total of three injections and then killed 24 h
after the first BrdU injection. The number of BrdU-positive cells
in the SGZ was significantly higher in mice treated with imip-
ramine, indicating increased BrdU incorporation and increased
cellular proliferation in this region (Fig. 5A).
Other mice were similarly treated with imipramine (or saline),
u l i
ANDn o i t a z i l ac o l oC 12 p /AND12p
of DNA and p21 staining (DNA, blue; p21, green). Colocalized pixels in the fluorogram are marked with a yellow gate. Colocalization of DNA and p21 is marked
zone; GCL, granular cell layer; ML, molecular layer. The box delineates the region imaged by confocal microscopy. The confocal image illustrates p21-positive
Scatter plot obtained from dissociated hippocampi with the cell size on the x axis (forward scatter, FSC) and cell granularity on the y axis (side scatter, SSC).
DCX-positive cells are gated. (Lower) Gated DCX-positive cells were collected and analyzed for p21 fluorescence. Dual-parameter fluorescence intensity of DCX
versus p21 is shown. Cells appearing in the upper right quadrant are strongly positive for both antigens (42.8%), whereas the upper left quadrant represents
DCX-positive cells negative for p21 (48.8%). The experiment was performed two times with similar results, and a representative graph is shown.
p21 is expressed in newly developing neurons in the SGZ of hippocampus. (A) Intranuclear p21 expression in the SGZ of the dentate gyrus.
Pechnick et al. PNAS ?
January 29, 2008 ?
vol. 105 ?
no. 4 ?
and protein was isolated from whole hippocampi and analyzed
by Western blot. The levels of PCNA, a marker of cellular
proliferation, were higher in imipramine-treated mice, confirm-
ing increased cellular proliferation. Levels of NeuN also were
elevated in the hippocampi of the imipramine-treated mice (Fig.
5B), demonstrating that there was an increase in the number of
neurons in the hippocampus.
To show that the imipramine treatment regimen produced a
behavioral response, additional mice were treated for 21 days
with imipramine (or saline), and 24 h after the last injection they
were subjected to the FST, a widely accepted animal model used
to screen drugs for antidepressant activity (18–20). In the FST,
antidepressant activity is reflected by a reduction in time spent
immobile and an increase in the time spent in active, escape-
oriented behaviors. Immobility was significantly decreased after
the imipramine treatment regimen produced antidepressant-like
In the adult, neurogenesis in the hippocampus is restricted to the
SGZ of the dentate gyrus (21, 22). The results of the present
study show that p21 immunoreactivity is prominent within the
dentate gyrus and restricted to the SGZ. The small, compact
morphology of cells expressing p21 is consistent with migrating
neuroblasts that express DCX (17). The results of the flow
cytometry studies show that 42% of newly developing neurons
(DCX-positive cells) in the hippocampus are also positive for
p21. Double-staining for p21 and NeuN indicates that p21 is
localized in neurons in the SGZ. The absence of p21 in the GCL,
postmitotic neurons do not express DCX, indicate that p21 is
expressed in developing neurons. Although NeuN is considered
to be a marker of mature neurons, a population of immature, still
dividing neuronal cells (D1 cells) localized in the SGZ express
both DCX and NeuN (17, 23). Others have also found type 2
neuroblasts (NB2) that express both DCX and NeuN and remain
- / - 12 p TW
n i t ca
BrdU Positive Cells
4 . 0
8 . 0
2 . 1
6 . 1
- / - 12p
- / - 12pTW
BrdU Positive Cells
gyrus of WT and p21?/?WT mice. (B) Quantification of BrdU-positive cells in the SGZ of the hippocampus in p21?/?and WT mice. Three mice per group were
analyzed. (C) Average numbers of BrdU-positive cells per section in the SGZ of p21?/?and WT mice. (D) Western blot analysis of NeuN and DCX in whole
hippocampus of WT and p21?/?mice. Mouse DCX monoclonal antibodies recognize two bands in the 41- to 44-kDa range. Mouse NeuN monoclonal antibodies
recognize two to three bands in the 46- to 48-kDa range and a band at ?66 kDa. For each sample, hippocampi from three mice per group were pooled. The
experiment was repeated three times, and a representative blot is shown. (E) Quantitative analysis of three independent experiments. The intensity of the DCX
and NeuN bands in each experiment was quantified and corrected with respect to the loading control (?-actin), and the ratios were corrected to WT mice to
calculate relative changes.*, P ? 0.05.
p21 deletion increases neuronal proliferation in the dentate gyrus. (A) Immunoperoxidase-labeled BrdU in representative sections from the dentate
e g r eM) i e l cu n ( 3 o rPoTXCDU d rB
and DCX (red). Cell nuclei were stained with the DNA-specific dye ToPro3 (blue). (B) The orthogonal slices are shown to confirm double labeling throughout the
extent of two positive cells. The area imaged in the confocal micrographs is 100 ? 100 ?m.
www.pnas.org?cgi?doi?10.1073?pnas.0711030105 Pechnick et al.
confined to the SGZ (16). Taken together, these results suggest
that p21 expression in the hippocampus is confined to dividing,
immature neurons. However, p21 also might be expressed at
earlier stage of neuronal differentiation, including stem cells.
CDKs play a critical role in regulating the cell division cycle
(6, 24, 25). Inhibitors of these kinases, such as p21, block the G1-
example, p21 has been shown to be an important regulator of
fore, decreased levels of p21 would be expected to be associated
with increased cellular proliferation. The data from the present
of p21?/?mice. BrdU incorporation was greater in the SGZ of
the p21?/?mice, and there was up-regulation of DCX and NeuN
proteins. In addition, there was a higher number of DCX–
positive cells in the hippocampi of p21?/?mice compared with
WT animals. In support of our finding, p21?/?mice have been
found to exhibit increased forebrain neural stem cell prolifera-
tion and more cell divisions (27), as well as greater neurogenesis
in response to experimental ischemia (28). In addition, p21 is a
transcriptional target for p53. When p53 is absent, p21 is
down-regulated, and there is increased proliferation and survival
of neural stem cells (29).
Others have found that cell cycle inhibitors are expressed in
the adult brain. For example, p15INK4Bhas been found in the
forebrain, p21 has been found in the cerebellum (30, 31), and
p27Kiphas been found in the cerebellum and postmitotic cortical
neurons (31, 32). It is likely that other CDK inhibitors also affect
neurogenesis. Double knockout animals obtained from crossing
p19-null and p27-null mice exhibit increased neuronal prolifer-
ation in all parts of the brain (33). Given the capacity of p21 to
restrain the cell cycle, its localization in the SGZ, and its
confinement to DCX-positive cells, p21 might play an important
and unique role in regulating neuronal proliferation in the
hippocampus of adult mice.
antidepressants increases the total number of neurons in the
hippocampus (8) and that SGZ neuronal progenitor cells are the
target of fluoxetine treatment (16). Increased neurogenesis in the
adult hippocampus occurs after chronic administration of different
classes of antidepressants, including selective serotonin and nor-
and after electroconvulsive shock therapy, a procedure that is
efficacious in the treatment of depression (4, 12, 34). Consistent
a tricyclic antidepressant stimulates neuronal proliferation in the
hippocampus, as shown by increased BrdU incorporation and
elevated levels of NeuN and PCNA. Imipramine treatment also
decreased immobility in the FST. The increased neurogenesis and
the behavioral effects were associated with decreased p21 mRNA
and protein levels in the SGZ. Thus, suppression of p21 might
mediate these effects of imipramine.
The mechanism of action underlying imipramine-induced
suppression of p21 is not known. It is possible that imipramine
suppresses p21 promoter activity secondary to its effects on
neurotransmitters such as serotonin and/or norepinephrine.
Imipramine inhibits the reuptake of both neurotransmitters, and
its effects on p21 expression could involve either or both
neurotransmitters. Local hippocampal factors also could be
involved in the regulation of p21 expression. For example, the
interplay between TGF?2 and brain-derived neurotrophic factor
in the cerebellum has been suggested to account for antiprolif-
erative and prodifferentiating activities observed in postmitotic
cerebellar neurons (30).
If a mechanism of action of antidepressants is to reduce
p21-induced suppression of neurogenesis, then the etiology of
with color-coded fluorograms (51). Confocal images show double immunofluorescence labeling of hippocampal dentate gyrus after chronic treatment with
saline (A–D) or imipramine (E–H). p21-positive cells are red, and neurons (NeuN-labeled) are green. Cells where p21 and NeuN are colocalized are marked with
a blue overlay. Colocalized pixels in the fluorograms are marked with blue (D and H). The area imaged in the confocal micrographs is 250 ? 250 ?m. SGZ,
(C) or imipramine (IP) (seven to eight mice per group).*, P ? 0.05. (J) p21 protein levels after chronic treatment with imipramine (IP) as measured by ELISA. p21
protein expression in normal saline-treated mice (C) was taken as 100%. Shown are results summarized from three independent experiments. For each
experiment whole hippocampi from three mice per group were pooled.**, P ? 0.01.
Chronic treatment with imipramine suppresses p21 expression in the hippocampus. (A–H) Double-labeling analysis of the hippocampal dentate gyrus
Pechnick et al. PNAS ?
January 29, 2008 ?
vol. 105 ?
no. 4 ?
some forms of depression might involve stimulation or up-
regulation of p21. Stress is thought to play an important role in
the pathogenesis of depression (35, 36), and glucocorticoids
released in response to stressors can stimulate p21 promoter
activity (37). Inflammatory processes also have been implicated
in the pathogenesis of major depression (38), and proinflam-
matory cytokines, including IL-6 (39) and INF?, can arrest
neuronal proliferation (40). These proinflammatory cytokines
activate the Jak–STAT signaling pathway (41), and several
binding sites for STAT1 and STAT3 are present on the p21
promoter (37). Antidepressants might indirectly reduce p21
levels by decreasing hypothalamo-pituitary-adrenal axis activity
(42, 43) or by restraining the expression of inflammatory cyto-
The role of neurogenesis in the hippocampus is not completely
clear, but it has been suggested to be involved in memory
formation and mood regulation (14, 45, 46). In depressed
patients, hippocampal volume is decreased, and the magnitude
of the volume reduction is directly related to the length and
severity of the illness (15, 47–49). In rodents, chronic stress-
induced ‘‘depression’’ also leads to hippocampal atrophy (50–
52). Because neurogenesis is required for some of the behavioral
effects of chronic antidepressant treatment in rodents (14), the
final target for different classes of drugs and treatments that
produce antidepressant effects.
In summary, the present data demonstrate that p21 is ex-
pressed in the SGZ of the dentate gyrus of the hippocampus,
where it might act to restrain neurogenesis. Chronic treatment
releasing neuronal progenitor cells from the blockade of pro-
liferation. Because many antidepressants have been found to
stimulate neurogenesis (8), it is possible that a shared common
mechanism of action of antidepressants is suppression of p21. It
must be acknowledged that the causal linkages between de-
creases in hippocampal neurogenesis and depression, and be-
tween therapeutic effects of antidepressants and increases in
neurogenesis, have not been proven (53, 54). However, if these
relationships are confirmed, then the results of the present study
suggest that new therapies for major depression could be de-
signed to directly target the expression of CDK inhibitors such
Experimental Animals. Two-month-old, male C57BL/6j mice were treated daily
for 21 days with saline (0.9%) or imipramine hydrochloride (10 mg/kg per day
i.p.; Sigma–Aldrich). Twenty-four hours after the last injection, the mice were
subjected to BrdU injections and used for the behavioral experiment or killed
for the other studies. Cdk1atm1Tyj(p21?/?) mice on 129S2 genetic background
were obtained from The Jackson Laboratory. For real-time PCR, ELISA, and
Western blot analyses, the mice were killed, the brains were removed and
rapidly cooled in ice-cold saline, and the hippocampi were dissected out (55).
The Institutional Animal Care and Use Committee approved all experimental
Immunohistochemistry. Mice were anesthetized with isoflurane and perfused
with paraformaldehyde (4%), and brain paraffin sections were processed as
described previously (56). The slides were incubated with mouse anti-p21
fluorescent dye (Molecular Probes). The antibody for NeuN (Chemicon) was
conjugated with Alexa Fluor 568 fluorescent dye (Molecular Probes) and used
to determine colocalization with p21 expression. DNA (nuclei) was stained
with ToPro3 (Molecular Probes). Multiparameter fluorescent microscopy and
Leica Confocal Software were used to identify p21 intracellular localization
and colocalization with the neuronal marker NeuN. For comparability, all
images on confocal multiimage figure plates were adjusted with identical
contrast and brightness settings.
BrdU Immunochemistry. The entire left half of the brain was cut into 5-?m
sagittal sections and processed by using a BrdU Labeling and Detection Kit
(Roche Applied Biosystems). Sections were coded for blind observation. Un-
biased random sampling from 0.36 to 0.6 mm lateral to the midline (55) was
carried out. Every third section (of a total of 30 sections) was counted under
a ?100 objective, and the sum was multiplied by 3 to estimate the total
or touching the SGZ, and cells were excluded if they were more than two cell
diameters from the GCL (8). Some sections derived from p21-null mice were
double-labeled to detect DCX (Santa Cruz Biotechnology), and at least 20
BrdU-positive cells were examined by confocal microscopy to determine co-
localization with DCX.
(Worthington Biochemical), and the cells were processed according to a Flow
Cytometry Staining Protocol (Cell Signaling Technology). Fixed cells were
incubated overnight with DCX antibodies (Cell Signaling Technology),
washed, and treated with secondary antibodies (Alexa Fluor 586 fluorescent
dye, red), and samples were analyzed in a FACSCalibur system (Becton Dick-
inson). The cells were gated on the basis of forward/side scatter plot to
double-staining experiments, both DCX-positive and DCX-negative cells were
stained for p21 (BD Pharmingen) for 2 h at 4°C, then stained with a secondary
antibody (Alexa Fluor 488 fluorescent dye, green), and the number of DCX/
p21-positive cells was determined. Control cells were stained with both sec-
Protein Isolation and Western Blot Analysis. Proteins were isolated (Immuno-
precipitation Kit; Roche Diagnostics), separated by SDS/PAGE, electroblotted
onto membranes (Millipore), incubated overnight with primary antibodies,
and then incubated with corresponding secondary antibodies as described
(56). Immunoreactive bands were detected by the ECL immunodetection
system. NeuN (Chemicon) and PCNA, DCX, and ?-actin (Santa Cruz Biotech-
using an Epson V750 PRO and transferred to Adobe Photoshop Elements 3.
Intensity analysis of the bands (all multiple bands simultaneously) was per-
Mean Immobility Score
n i t ca
BrdU Positive Cells
of protein levels of NeuN and PCNA in whole hippocampus after chronic treatment with saline or imipramine. Mouse NeuN monoclonal antibodies recognize
two to three bands in the 46- to 48-kDa range and a band at ?66 kDa. For each sample hippocampi from three mice per group were pooled. The experiment
*, P ? 0.05 (n ? 15–17 mice per group).
Chronic treatment with imipramine increases neuronal proliferation in the hippocampus and decreases immobility. (A) Quantification of BrdU-positive
www.pnas.org?cgi?doi?10.1073?pnas.0711030105Pechnick et al.
formed with ImageJ Software (ImageJ; W. S. Rosband, National Institutes of Download full-text
ELISA. p21 protein levels in the hippocampus were determined by using a
Proteins were isolated by using an Immunoprecipitation Kit (Roche Diagnos-
tics). The hippocampi from three mice per group were pooled, and lysates
from three independent experiments were assessed in triplicate.
Quantitative Real-Time PCR. Total RNA was isolated with TRIzol reagent
according to the manufacturer’s instructions (Invitrogen). Quantitative real-
time PCR (56) was performed to detect p21 mRNA expression. Relative quan-
tification of p21 mRNA in experimental samples was determined from the
expressed as arbitrary units.
was performed as described previously (57). Briefly, mice are individually
water maintained at 22–24°C. Every 30 sec for a total of 6 min the mice are
rated for immobility, defined as the absence of active, escape-oriented be-
haviors such as swimming, jumping, rearing, sniffing, or diving. At each time
present or absent. The raters were blind to the experimental treatment. The
number of time points where immobility was scored during the last 4 min of
the experimental session was summed for each subject to yield a total immo-
bility score (18).
Statistical Analysis. The total immobility scores for the FST were analyzed by
ELISA, and BrdU injection were analyzed by using unpaired t tests.
ACKNOWLEDGMENTS. We are grateful to Dr. Shlomo Melmed for his con-
tinuing support and for the generous gift of the p21?/?mice. We thank Drs.
a NARSAD Young Investigator Award and National Institutes of Health Grant
MH 079988 (to V.C.) and by the Levine Family Fund Research Endowment
1. Gage FH (2000) Mammalian neural stem cells. Science 287:1433–1438.
2. Cameron HA, Woolley CS, McEwen BS, Gould E (1993) Differentiation of newly born
neurons and glia in the dentate gyrus of the adult rat. Neuroscience 56:337–344.
adult rat: Age-related decrease of neuronal progenitor proliferation. J Neurosci
4. Warner-Schmidt JL, Duman RS (2006) Hippocampal neurogenesis: Opposing effects of
stress and antidepressant treatment. Hippocampus 16:239–249.
5. Sherr CJ, Roberts JM (1999) CDK inhibitors: Positive and negative regulators of G1-
phase progression. Genes Dev 13:1501–1512.
6. Sharpless NE, DePinho RA (2004) Telomeres, stem cells, senescence, and cancer. J Clin
7. Gartel AL, Radhakrishnan SK (2005) Lost in transcription: p21 repression, mechanisms,
and consequences. Cancer Res 65:3980–3985.
8. Malberg JE, Eisch AJ, Nestler EJ, Duman RS (2000) Chronic antidepressant treatment
increases neurogenesis in adult rat hippocampus. J Neurosci 20:9104–9110.
9. Perera TD, et al. (2007) Antidepressant-induced neurogenesis in the hippocampus of
adult nonhuman primates. J Neurosci 27:4894–4901.
10. Malberg JE (2004) Implications of adult hippocampal neurogenesis in antidepressant
action. J Psychiatry Neurosci 29:196–205.
11. van Praag H, et al. (2002) Functional neurogenesis in the adult hippocampus. Nature
12. Duman RS (2004) Depression: A case of neuronal life and death? Biol Psychiatry
effects of antidepressants. Science 301:805–809.
15. Wong EY, Herbert J (2006) Raised circulating corticosterone inhibits neuronal differ-
entiation of progenitor cells in the adult hippocampus. Neuroscience 137:83–92.
in the adult brain. Proc Natl Acad Sci USA 103:8233–8238.
J Comp Neurol 467:1–10.
18. Porsolt RD, Le Pichon M, Jalfre M (1977) Depression: A new animal model sensitive to
antidepressant treatments. Nature 266:730–732.
19. Borsini F, Meli A (1988) Is the forced swimming test a suitable model for revealing
antidepressant activity? Psychopharmacology (Berlin) 94:147–160.
20. Willner P, Wilkes M, Orwin A (1990) Attributional style and perceived stress in endog-
enous and reactive depression. J Affect Disord 18:281–287.
neural stem cells. Mol Cell Neurosci 8:389–404.
22. Gage FH, Kempermann G, Palmer TD, Peterson DA, Ray J (1998) Multipotent progen-
itor cells in the adult dentate gyrus. J Neurobiol 36:249–266.
types, lineage, and architecture of the germinal zone in the adult dentate gyrus.
J Comp Neurol 478:359–378.
24. Pardee AB (1989) G1 events and regulation of cell proliferation. Science 246:603–608.
25. Morgan DO (1995) Principles of CDK regulation. Nature 374:131–134.
27. Kippin TE, Martens DJ, van der Kooy D (2005) p21 loss compromises the relative
quiescence of forebrain stem cell proliferation leading to exhaustion of their prolif-
eration capacity. Genes Dev 19:756–767.
28. Qiu J, et al. (2004) Regenerative response in ischemic brain restricted by p21cip1/waf1.
J Exp Med 199:937–945.
29. Meletis K, et al. (2006) p53 suppresses the self-renewal of adult neural stem cells.
30. Lu J, Wu Y, Sousa N, Almeida OF (2005) SMAD pathway mediation of BDNF and TGF
beta 2 regulation of proliferation and differentiation of hippocampal granule neu-
rons. Development 132:3231–3242.
31. Legrier ME, Ducray A, Propper A, Kastner A (2001) Region-specific expression of cell
cycle inhibitors in the adult brain. NeuroReport 12:3127–3131.
32. Yoshikawa K (2000) Cell cycle regulators in neural stem cells and postmitotic neurons.
Neurosci Res 37:1–14.
33. Zindy F, et al. (1999) Postnatal neuronal proliferation in mice lacking Ink4d and Kip1
inhibitors of cyclin-dependent kinases. Proc Natl Acad Sci USA 96:13462–13467.
Pharmacol Sci 26:631–638.
35. Holsboer F, Barden N (1996) Antidepressants and hypothalamic-pituitary-adrenocor-
tical regulation. Endocr Rev 17:187–205.
36. Gold PW, Chrousos GP (2002) Organization of the stress system and its dysregulation
in melancholic and atypical depression: High vs low CRH/NE states. Mol Psychiatry
37. Gartel AL, Tyner AL (1999) Transcriptional regulation of the p21((WAF1/CIP1)) gene.
Exp Cell Res 246:280–289.
38. Maes M (1999) Major depression and activation of the inflammatory response system.
Adv Exp Med Biol 461:25–46.
pal neurogenesis. Science 302:1760–1765.
40. Tanabe T, Kominsky SL, Subramaniam PS, Johnson HM, Torres BA (2000) Inhibition of
the glioblastoma cell cycle by type I IFNs occurs at both the G1 and S phases and
correlates with the upregulation of p21(WAF1/CIP1). J Neurooncol 48:225–232.
41. Ihle JN (1996) STATs: Signal transducers and activators of transcription. Cell 84:331–
42. McEwen BS, Olie JP (2005) Neurobiology of mood, anxiety, and emotions as revealed
by studies of a unique antidepressant: Tianeptine. Mol Psychiatry 10:525–537.
43. Sapolsky RM (2004) Is impaired neurogenesis relevant to the affective symptoms of
depression? Biol Psychiatry 56:137–139.
44. Abdel-Salam OM, Baiuomy AR, Arbid MS (2004) Studies on the anti-inflammatory
effect of fluoxetine in the rat. Pharmacol Res 49:119–131.
45. Shors TJ (2001) Neurogenesis in the adult is involved in the formation of trace
memories. Nature 410:372–376.
for neurogenesis in adult brain. Proc Natl Acad Sci USA 100:13632–13637.
47. Sheline YI, Wang PW, Gado MH, Csernansky JG, Vannier MW (1996) Hippocampal
atrophy in recurrent major depression. Proc Natl Acad Sci USA 93:3908–3913.
in mood disorders. Proc Natl Acad Sci USA 95:13290–13295.
49. Rajkowska G (1999) Morphometric evidence for neuronal and glial prefrontal cell
pathology in major depression. Biol Psychiatry 45:1085–1098.
50. McEwen BS, Tanapat P, Weiland NG (1999) Inhibition of dendritic spine induction on
hippocampal CA1 pyramidal neurons by a nonsteroidal estrogen antagonist in female
rats. Endocrinology 140:1044–1047.
51. Duman RS, Heninger GR, Nestler EJ (1997) A molecular and cellular theory of depres-
sion. Arch Gen Psychiatry 54:597–606.
53. Feldmann RE, Jr, Sawa A, Seidler GH (2007) Causality of stem cell based neurogenesis
and depression—to be or not to be, is that the question? J Psychiatr Res 41:713–723.
55. Paxinos G, Franklin KBJ (1997) The Mouse Brain in Stereotaxic Coordinates (Academic,
New York), 2nd Ed.
56. Chesnokova V, Kovacs K, Castro AV, Zonis S, Melmed S (2005) Pituitary hypoplasia in
Pttg?/? mice is protective for Rb?/? pituitary tumorigenesis. Mol Endocrinol
57. Pechnick RN, et al. (2004) Reduced immobility in the forced swim test in mice with a
targeted deletion of the leukemia inhibitory factor (LIF) gene. Neuropsychopharma-
58. Demandolx D, Davoust J (1997) J Microscopy 185:21–36.
Pechnick et al. PNAS ?
January 29, 2008 ?
vol. 105 ?
no. 4 ?