 |
 Previous Article | Next Article 
The Journal of Neuroscience, May 1, 2002, 22(9):3673-3682
Regulation of Neurogenesis in Adult Mouse Hippocampus by cAMP and
the cAMP Response Element-Binding Protein
Shin
Nakagawa,
Ji-Eun
Kim,
Rena
Lee,
Jessica E.
Malberg,
Jingshan
Chen,
Cathy
Steffen,
Ya-Jun
Zhang,
Eric J.
Nestler, and
Ronald S.
Duman
Division of Molecular Psychiatry, Abraham Ribicoff Research
Facilities, Connecticut Mental Health Center, Yale University School of
Medicine, New Haven, Connecticut 06508
 |
ABSTRACT |
The cAMP cascade, including the cAMP response element-binding
protein (CREB), is known to play an important role in neuronal survival
and plasticity. Here the influence of this cascade on neurogenesis in
adult hippocampus was determined. Activation of the cAMP cascade by
administration of rolipram, an inhibitor of cAMP breakdown, increased
the proliferation of newborn cells in adult mouse hippocampus. In
addition, rolipram induction of cell proliferation resulted in mature
granule cells that express neuronal-specific markers. Increased cell
proliferation is accompanied by activation of CREB phosphorylation in
dentate gyrus granule cells, suggesting a role for this transcription
factor. This possibility is supported by studies demonstrating that
cell proliferation is decreased in conditional transgenic mice that
express a dominant negative mutant of CREB in hippocampus. The results
suggest that the cAMP-CREB cascade could contribute to the actions of
neurotransmitters and neurotrophic factors on adult neurogenesis.
Key words:
rolipram; phosphodiesterase; transgenic mice; phosphorylation; proliferation; granule cell; transcription factor
| |
INTRODUCTION |
Neurogenesis occurs in adult brain
of several different species, including human (Eriksson et al., 1998 ;
Pincus et al., 1998; Kukekov et al., 1999), and is a dynamic process
that is regulated in both a positive and negative manner by
environmental, endocrine, and pharmacological stimuli. For example,
enriched environment, exercise, or hippocampal-dependent models of
learning increase neurogenesis in adult (Kempermann et al., 1997; Gould
et al., 1999; van Praag et al., 2000). In contrast, aging,
environmental, or psychosocial stress, as well as administration of
adrenal glucocorticoids, decrease neurogenesis in the hippocampus (Kuhn
et al., 1996; Gould et al., 1997; Cameron and McKay, 1999).
Downregulation of adult neurogenesis in response to stress and high
levels of adrenal glucocorticoids could contribute to the decreased
volume of hippocampus that has been observed in the brains of patients
with mood disorders (Sheline et al., 1996; Steffens et al., 2000). In
contrast, hippocampal neurogenesis is upregulated by antidepressant
treatment (Malberg et al., 2000a), and this effect could reverse or
block the damaging effects of stress on hippocampus. These findings
indicate that neurogenesis is a form of neural plasticity that
contributes to the ability of adult hippocampus to adapt and respond to
a variety of stimuli.
Although progress has been made in characterizing the neurotransmitters
and trophic factors that regulate neurogenesis (Cameron et al., 1998;
Gage, 2000), much less is known about the intracellular signal
transduction cascades that influence this process in adult hippocampus.
The cAMP signal transduction cascade, including the cAMP response
element-binding protein (CREB), is one potential candidate. Activation
of this pathway is known to increase performance in cellular and
behavioral models of learning and memory and to mediate neurotrophic
factor-induced cell survival (Silva et al., 1998; Finkbeiner, 2000).
Studies in cultured progenitor cells demonstrate that activation of the
cAMP pathway increases neuronal differentiation and neurite outgrowth
(Palmer et al., 1997; Takahashi et al., 1998). In addition,
antidepressant treatment upregulates the cAMP signal transduction
cascade in hippocampus (Duman et al., 1997b, 2000), and this could
mediate the action of antidepressants on neurogenesis (Malberg et al.,
2000a).
The current study examines the influence of the cAMP system on
neurogenesis in adult rodent hippocampus. The cAMP cascade can be
pharmacologically regulated in vivo by administration of rolipram, an inhibitor of phosphodiesterase type IV (PDE4). PDE4 is a
subfamily of high-affinity, cAMP-specific enzymes that degrade cAMP
(Conti et al., 2000). In addition, the role of CREB in neurogenesis is
examined. CREB is a transcription factor that is activated by its
phosphorylation on Ser133 via
cAMP-dependent protein kinase, as well as by
Ca2+- and neurotrophic factor-dependent
signaling pathways (Duman et al., 2000). We generated an inducible
transgenic mouse that overexpresses a dominant negative phosphorylation
mutant of CREB (Ser133 to Ala) in the
granule cell layer (GCL) of hippocampus for these studies. The results
demonstrate that activation of the cAMP pathway increases the
proliferation of hippocampal granule cells and that inhibition of CREB
decreases this process.
| |
MATERIALS AND METHODS |
Drug treatment. Male C57BL/6 mice, 8-10 weeks old
(Charles River Laboratories, Wilmington, MA), were used for the study
with rolipram. For the chronic paradigm, mice were given saline
containing 2% DMSO as control or rolipram (1.25 mg/kg, i.p; Sigma, St.
Louis, MO) in saline containing 2% DMSO daily for 14 d. To
evaluate the effect of rolipram on cell proliferation,
bromodeoxyuridine (BrdU) (75 mg/kg, i.p; Sigma) was administered
to label dividing cells 2 hr after the last injection of rolipram or
vehicle. Mice were killed 2 hr (control, n = 7;
rolipram, n = 8) or 24 hr (control, n = 6; rolipram, n = 6) after BrdU injection. For the acute
paradigm, saline (n = 5) or rolipram (n = 8) was administered once. Two hours after the injection, mice were
given a single injection of BrdU once and killed 24 hr later. To
evaluate the effect of chronic rolipram on survival of newly born
cells, mice were given BrdU every 2 hr three times after chronic saline
(n = 7) or rolipram (n = 7)
administration and killed 4 weeks after BrdU injection. All mice were
given BrdU at postnatal week 10. All animal procedures were in strict
accordance with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals and were approved by the
Yale Animal Care and Use Committee.
Transgenic mice. To assess the effect of dominant negative
mutants of CREB on the cell proliferation in the adult hippocampus, we
generated transgenic mice expressing CREB mutant (mCREB) under the tetracycline responsive promoter (Furth et al., 1994; Chen et al.,
1998). The CREB mutant contains a conservative serine to alanine
substitution at position 133, which destroys the protein kinase A
phosphorylation site but maintains charge balance (Gonzalez and
Montminy, 1989). Although not phoshorylated, mCREB can still bind to
the CRE. Thus, mCREB inhibits CREB action by occupying the CRE and
preventing access by wild-type CREB and other CRE-binding factors
(Shaywitz and Greenberg, 1999). The mCREB construct, a gift from
Michael E. Greenberg (Harvard University, Boston, MA) was engineered
with a FLAG tag peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) at the N
terminus so that mCREB could be distinguished from endogenous CREB. A
1.1 kb fragment of the vector containing mCREB was released by
digestion with HindIII and XbaI and subcloned
into the pTet-splice (Shockett et al., 1995) in place of the TetOP
minimal cytomegalovirus promoter. The new plasmid was designated as
pTetOP-mCREB. DNA fragment [containing the promoter, open reading
frame, SV40 intron, and poly(A+) signal]
from pTetOP-mCREB was purified by electroelution and microinjected into
the pronuclei of oocytes from SJL × C57BL/6 mice. The mice
expressing the tTA gene under the control of
Ca2+-calmodulin-dependent kinase II  (CaMKII) promoter, CaMKII-tTA, were provided by Eric R. Kandel
(Columbia University, New York, NY) (Mayford et al., 1996). Tail DNA
from both mice was isolated using Tissue Amp DNA kit (Quiagen,
Chatsworth, CA) and analyzed for the transgene by PCR. Of these
techniques, PCR was used for routine genotyping of the transgenic mice.
The CaMKII-tTA transgene was detected by PCR with the following
primers: TTAB1, 24-mer, 5' GCG ACT TGA TGC TCT TGA TCT TCC 3'; and
TTAF1, 24-mer, 5' GAG CTG CTT AAT GAG GTC GGA ATC 3'. The TetOP-mCREB
transgene was detected by PCR with the following primers: CREB-B1,
24-mer, 5' GCT GCA TTG GTC ATG GTT AAT GTC 3'; and CREB-F1, 25-mer, 5'
CAG CCA TCA GTT ATT CAG TCT CCA C 3'. The founder mice were crossbred with the ICR outbred mouse line to generate F1 mice. F2 homozygous transgenic mice were obtained by crossbreeding F1 siblings; homozygous genotype was confirmed by crossbreeding them with wild-type mice. We
used only one line for each single transgenic mouse for the present
study. During the prenatal period to postnatal week 6, the transgenic
mice were given doxycycline (100 µg/ml) (Sigma), an analog of
tetracycline, in drinking water to turn off the expression of mCREB in
mice carrying the CaMKII-tTA gene plus TetOP-mCREB (Chen et al., 1998).
From postnatal weeks 6-10, mice were fed without doxycycline to wash
out doxycycline and express the target gene. At postnatal week 10, mice
(CaMKII-tTA single transgenic mice, n = 4; TetOP-mCREB
single transgenic mice, n = 7; CaMKII-tTA × TetOP-mCREB bitransgenic mice, n = 5) were given BrdU
once and killed 2 hr later to evaluate the cell proliferation in the
adult hippocampus. All of the transgenic mice used in this study were maintained in strict accordance with National Institutes of Health and
institutional animal care guidelines.
Immunohistochemistry. All mice were killed via intracardial
perfusion with 4% paraformaldehyde under anesthetization with sodium
pentobarbital (100 mg/kg, i.p.). A freezing microtome was used to
collect serial coronal 30 µm sections through the entire hippocampus.
Every sixth or ninth section was slide mounted for peroxidase BrdU
immunolabeling. The sections were incubated in 0.01 M citric acid at 90°C, digested in trypsin
(0.1%) in Tris buffer containing 0.1% CaCl2 for 10 min, denatured in
2N HCl for 30 min, blocked in 3.0% normal horse serum for 20 min, and
incubated overnight at 4°C in mouse monoclonal antibody against BrdU
(1:100; Becton Dickinson, San Jose, CA) in PBS containing 3% normal
horse serum and 0.1% Tween 20. On the next day, the sections were
incubated in biotinylated mouse secondary antisera (1:200; Vector
Laboratories, Burlingame, CA) for 60 min, incubated in
avidin-biotin-horseradish peroxidase (1:50; Vector Laboratories) for
60 min, and reacted in the solution of 3,3'-diaminobenzidine
containing nickel ammonium sulfate (Vector Laboratories). The sections
were counterstained with cresyl violet. For peroxidase FLAG
immunolabeling, free-floating 30 µm sections from transgenic mice
were used. Sections were incubated in 0.5% Triton X-100 in TBS for 45 min at 4°C, blocked in 5.0% normal horse serum-0.1% Triton X-100
in TBS for 45 at 4°C, and incubated overnight at 4°C in mouse
monoclonal antibody against FLAG (1:1000; Sigma). The avidin-biotin
blocking kit (Vector Laboratories) was used at these steps to reduce
the nonspecific labeling. The following step was done as described
above except for the counterstaining. For immunofluorescence staining,
free-floating 30 µm sections were used. The following DNA
denaturation steps preceded the incubation with anti-BrdU antibody: 2 hr incubation in 50% formamide-2× SSC (0.3 M
NaCl and 0.03 M sodium citrate) at 65°C, 5 min
rinse in 2× SSC, 30 min incubation in 2N HCl at 37°C, and 10 min
rinse in 0.1 M boric acid, pH 8.5. Sections were
incubated in TBS-0.1% Triton X-100-3% normal goat serum (TBS-Tds)
for 30 min and with primary antibodies in TBS-Tds for 1-3 d at 4°C.
The primary antibody used for immunofluorescence staining were as
follows: rat anti-BrdU, 1:100 (Herlan Sera Lab, Loughborough, UK);
mouse anti- neuronal-specific nuclear protein (NeuN), 1:100
(Chemicon, Temecula, CA); rabbit anti S100 , 1:2500 (Swant,
Bellinoza, Switzerland); rabbit anti-pCREB, 1:400 (New England Biolabs,
Beverly, MA); and mouse anti-FLAG (1:500; Sigma). The fluorescent
secondary antibodies used were anti-rat FITC, anti-rabbit Cy3,
anti-mouse Cy3 or Cy5, 1:200 (Jackson ImmunoResearch, West Grove, PA).
Stereology. The number of BrdU-positive cells in the
bilateral entire hippocampus region were counted with a coded
one-in-nine series section for the rolipram study and a one-in-six
series section the for transgenic mouse study. Using this approach, the number of sections analyzed to cover the entire hippocampus was 10 or
11 for the rolipram study and 15 or 16 for the transgenic mouse study.
We used a modified version of the optical fractionator method for
unbiased stereological analysis of the total number of BrdU-positive
cells in the hippocampal subdivisions as reported previously (West et
al., 1991) and as used and cited in a recent report (Gould et al.,
1999). A cell was counted as being in the subgranular zone (SGZ)
of the dentate gyrus if it was touching or in the SGZ. Cells that were
located more than two cells away from the SGZ were classified as hilar.
All BrdU-positive cells, regardless of size or shape, were counted
through a 100× objective [Olympus BX-60 (Olympus Optical, Toyko,
Japan) or Zeiss (Oberkochen, Germany)] throughout the
rostrocaudal extent of the granule cell layer. The total numbers of
BrdU-positive cells in both sides of granule cell layer or hilus were
multiplied by 6 or 9, respectively and reported as total number of
cells per region.
Analysis of phenotypes. A one-in-nine series of sections
from control (n = 6) and chronic rolipram-treated
(n = 6) animals surviving 4 weeks after the injection
of BrdU was triple labeled for BrdU, NeuN, and S100 as described
above and analyzed by confocal laser microscopy (LSM 510; Zeiss). Fifty
BrdU-positive cells per animal were analyzed for coexpression of BrdU
and NeuN for neuronal phenotype and S100 for glial phenotype. Six
chronic rolipram-treated and six control mice were analyzed. Ratios of
BrdU-positive cells colabeling with NeuN, with S100, or with neither
NeuN nor S100 were determined.
| |
RESULTS |
Chronic rolipram administration increases the proliferation of
granule cells in adult hippocampus
The number of newly born cells in the adult dentate gyrus was
determined by immunohistochemical detection of BrdU within the nuclei
of dividing cells. Chronic or acute rolipram-treated and vehicle-treated control mice were killed 24 hr after BrdU incorporation to detect proliferating cells (Fig.
1a,b). In chronic
rolipram-treated animals, the number of BrdU-positive cells in the GCL
was 6538 ± 363 (mean ± SEM) cells per bilateral entire
dentate gyri (BDG) compared with 4768 ± 439 cells per BDG in the
controls, which corresponds to a 37% increase
(p < 0.05) (Fig. 1a). In the hilus, the number of BrdU-positive cells was 601 ± 32 cells per BDG in rolipram-treated animals compared with 544 ± 85 cells per BDG in
controls. In acute rolipram-treated animals, the number of BrdU-positive cells in GCL was 6442 ± 394 cells per BDG compared with 6210 ± 209 cells per BDG in controls (Fig. 1b).
In the hilus, the number of BrdU-positive cells in acute
rolipram-treated animals was 871 ± 102 cells per BDG compared
with 1018 ± 141 cells per BDG in controls. These data indicate
that chronic, but not acute, rolipram treatment results in more newborn
cells in the dentate gyrus granule cell layer relative to control
mice.
View larger version (46K):
[in this window]
[in a new window]
|
Figure 1.
Effect of rolipram administration on cell
proliferation in the adult hippocampus (a-c). The
number of BrdU-positive cells in the entire bilateral dentate gyri 24 hr (a, b), 2 hr
(c), or 4 weeks (d) after BrdU
administration. Repeated (14 d) rolipram administration increased the
number of proliferating cells in the granule cell layer compared with
vehicle-treated control mice (a, c).
Acute (1 d) rolipram treatment had no effect on the number of newborn
cells (b). Chronic rolipram increased the number
of surviving cells in the granule cell layer relative to control mice 4 weeks after BrdU administration (d). The results
are expressed as the mean ± SEM number of BrdU-positive cells in
bilateral dentate gyri (n = 6 animals).
*p < 0.05 compared with the corresponding control
(Student's t test).
|
|
The length of S phase of the cell cycle at which BrdU is incorporated
was estimated to be ~8.0 hr in the proliferating zone of adult
hippocampus (Nowakowski et al., 1989). To confirm the results of
chronic rolipram treatment on cell proliferation, a 2 hr time point
after a single BrdU injection, which detects just a single round of
dividing cells, was examined (Fig. 1c). In chronic rolipram-treated animals, the number of BrdU-positive cells in GCL was
4902 ± 244 cells per BDG compared with 3994 ± 160 cells per BDG in controls, which corresponds to a 22% increase
(p < 0.05). In the hilus, the number of
BrdU-positive cells was 761 ± 75 per BDG in rolipram-treated
animals compared with 753 ± 104 cells per BDG in controls.
BrdU-positive nuclei of proliferating cells were localized
predominantly in the subgranular layer (the border between granule cell
layer and hilus) or within GCL in both chronic rolipram-treated and
control mice (Fig.
2a,b). The nuclei
were darkly stained and exhibited variable shapes (Fig.
2c,d). In addition, the cells appeared frequently
in clusters of two or more (Fig. 2e), and mitotic-like
figures were observed in most sections (data not shown). This indicates
that incorporation of BrdU occurs during proliferation of granule cells
and cannot be explained by incorporation of the nucleotide during the
repair of damaged DNA. The morphology of nuclei of newly generated
cells was not different between rolipram-treated and control
animals.
View larger version (89K):
[in this window]
[in a new window]
|
Figure 2.
Effect of chronic rolipram administration on the
morphology and distribution of proliferating cells in the adult
hippocampus. As quantified in Figure 1, chronic rolipram treatment
(b) increased the number of proliferating cells
relative to control mice (a). BrdU-positive cells
were mostly in the subgranular layer between GCL and hilus 24 hr
after BrdU injection. c-e, Examples of proliferating cells
24 hr after BrdU administration. Nuclei of BrdU-positive cells were
dark and irregular in shape. Many proliferating cells occurred in
clusters (arrow in e). No apparent
difference in morphology and distribution of proliferating cells was
seen between rolipram-treated and control mice. Scale bars:
a, b, 200 µm; c-e, 10 µm.
|
|
Survival and phenotype of BrdU-labeled cells
The number of [3H]thymidine-labeled
newly born cells decreases during differentiation (Cameron et al.,
1993). To determine whether the newborn cells observed after chronic
rolipram treatment can survive, cells labeled with BrdU were allowed to
mature for 4 weeks (Figs. 1d,
3). In chronic rolipram-treated animals,
the number of BrdU-positive cells in GCL was 2012 ± 74 (mean ± SEM) cells per BDG compared with 1491 ± 145 cells per BDG in
controls, which corresponds to a 35% increase
(p < 0.05) (Fig. 1d). In the hilus,
the number of BrdU-positive cells was 412 ± 40 cells per BDG in
rolipram-treated animals compared with 324 ± 24 cells per BDG in
controls. Bright-field microscopic analysis shows that all
BrdU-positive cells 4 weeks after BrdU administration are round and
large with granular or dark-stained nuclei and are located within the
GCL (Fig. 3a,b). BrdU-positive cells in
rolipram-treated and control animals did not apparently differ in terms
of their morphology and location.
View larger version (78K):
[in this window]
[in a new window]
|
Figure 3.
Effect of chronic rolipram administration on the
morphology, distribution, and phenotype of surviving cells in the adult
hippocampus. BrdU-positive cells 4 weeks after BrdU administration
showed mature cell morphology within the GCL: sparse, multipunctate
(a) or dark, uniform (b)
BrdU-labeled nuclei with pale cytoplasm. Confocal images of
triple-labeled cells with the mitotic marker BrdU
(green; c, g), the
glial marker S100 (red; d,
h), the neuronal marker NeuN (blue;
e, i), and merged images of the three
labels (f, j) demonstrate cells
with a neuronal (c-f) or glial
(g-j) phenotype. Scale bars: a,
b, 10 µm; c-j, 20 µm.
Rolipram-treated and control mice did not differ in depth and
morphology of BrdU-positive cells within the granule cell layer. The
number of BrdU-labeled cells expressing either NeuN or S100 was
determined for each group. The results are expressed as percentage and
are the mean ± SEM of a total of 50 cells counted for each group.
There was no significant difference between the control and chronic
rolipram-treated mice.
|
|
To examine the phenotype of mature BrdU-positive cells in the GCL,
triple labeling for BrdU, the mature neuronal marker NeuN (Mullen et
al., 1992), and the glial marker S100 (Boyes et al., 1986) was
performed (Figs. 3c-j, 4).
Confocal microscopic analysis showed that the majority of BrdU-positive
cells colocalized with NeuN [79.3 ± 3.2 and 76.3 ± 3.2%
for control and chronic rolipram-treated animals, respectively
(mean ± SEM)], and a much smaller percentage colocalized with
S100 [5.6 ± 1.0 and 8.3 ± 0.9% for control and chronic
rolipram-treated animals, respectively (mean ± SEM)]. The
percentage of cells that mature into neurons or glia was not different
in vehicle- and rolipram-treated groups (Fig. 3k).
View larger version (84K):
[in this window]
[in a new window]
|
Figure 4.
a-d, Immunostaining of pCREB in the adult
dentate gyrus of acute or chronic rolipram-treated mice. Confocal
images with low magnification showed that pCREB-immunopositive cells
were aligned in the deepest part of GCL in the control animals
(a, c). Although the same immunostaining
pattern was seen in the acute rolipram-treated mice
(b), the entire granule cell layer was strongly
stained with pCREB antibody in the chronic rolipram-treated mice
(d). e-j, Double immunolabeling of
BrdU (green) and pCREB (red) in
the adult dentate gyrus of chronic rolipram-treated mice 24 hr after
BrdU administration. Confocal images with high magnification showed
that BrdU-labeled cells (arrows) did not show
immunoreactivity of pCREB in either acute (e-g) or
chronic (h-j) rolipram-treated animals. Scale bars:
a-d, 200 µm; e-j, 20 µm.
|
|
Rolipram increases the phosphorylation of CREB in the granule cell
layer of hippocampus
Rolipram is a specific inhibitor of the high-affinity cAMP
phosphodiesterase IV, which can activate the cAMP system by inhibiting cAMP breakdown, and could result in increased phosphorylation of CREB
(Conti and Jin, 2000). To examine the phosphorylation of CREB in adult
dentate GCL with acute or chronic rolipram treatment, immunofluorescence staining with antibodies against phosphorylated CREB
(pCREB) was performed (Fig. 4a-d). In animals with acute or
chronic saline treatment, the pCREB immunohistochemistry was observed
in nuclei of cells aligned in the lowest part of GCL, adjacent to or
including the SGZ, of adult dentate gyrus (Fig. 4a,c). Although a similar staining pattern was
seen in the acute rolipram-treated animals (Fig. 4b),
chronic rolipram treatment increased pCREB immunostaining in whole GCL,
including the subgranular zone, as well as CA4 and hilus region (Fig.
4d). This magnitude of pCREB labeling was observed in
~70% of the animals that received chronic rolipram treatment. In the
remaining 30% of the animals tested, pCREB was still increased
relative to vehicle or acute rolipram treatment but in a smaller
portion of the GCL. There was no difference in the rostrocaudal extent
of the staining pattern.
To determine the relationship between newly born cells and
pCREB-expressing cells in the subgranular zone of the GCL, brain sections from acute or chronic rolipram-treated animals killed 24 hr
after BrdU injection were double stained with antibodies against BrdU
and pCREB (Fig. 4e-j). Similar results were found 2 hr
after injection of BrdU. BrdU-positive newborn cells were localized in
the subgranular layer but did not colocalize with pCREB-expressing
cells in either the acute (Fig. 4e-g) or chronic (Fig.
4h-j) rolipram-treated animals. Together, these findings demonstrate that pCREB immunohistochemistry is induced by chronic rolipram administration in granule cells throughout the GCL but not in
newborn BrdU-labeled cells.
Overexpression of a dominant negative mutant of CREB (mCREB)
decreases granule cell proliferation
To assess the role of CREB phosphorylation in adult hippocampal
neurogenesis, we used transgenic mice with inducible, region-specific expression of a FLAG-tagged dominant negative CREB (mCREB) by means of
crossbreeding CaMKII-tTA mice (Mayford et al., 1996) with mice
containing the TetOP-mCREB gene (Fig. 5).
CaMKII-tTA and TetOP-mCREB single transgenic mice and CaMKII-tTA × TetOP-mCREB bitransgenic mice were bred with doxycycline in the
drinking water from conception to postnatal week 6, followed by a 4 week period without doxycycline. All three genotypes displayed normal
voluntary movement and activity during development and approximately
the same body weight at postnatal week 10 (CaMKII-tTA mice, 24.7 ± 0.3 gm, n = 4; TetOP mCREB mice, 25.1 ± 0.7 gm, n = 7; CaMKII-tTA × TetOP-mCREB mice,
24.1 ± 1.1 gm, n = 5).
View larger version (27K):
[in this window]
[in a new window]
|
Figure 5.
Schematic diagram of the tetracycline-regulated
gene expression system. Gene 1 encodes the
tetracycline-regulated transactivator protein tTA under the control of
CaMKII promoter. Gene 2 encodes the FLAG-tagged CREB
mutant (mCREB) gene with modified phosphorylation site
(Ser133 to Ala) under the control of the
tetracycline-responsive promoter TetOP. In the absence of doxycycline,
a tetracycline analog, tTA binds to and activates TetOP and increases
the expression of the downstream target gene mCREB. When doxycycline is
added, it binds to tTA, causes a conformational change of tTA, and
prevents it from activating TetOP, thereby turning off the expression
of target genes. We used transgenic mice that received doxycycline in
the drinking water during development (i.e., pregnant mothers received
doxycylcine) to postnatal week 6, followed by 4 weeks without
doxycycline.
|
|
The expression of the FLAG-tagged mCREB was visualized by
immunohistochemistry using an anti-FLAG antibody. At postnatal week 6 with doxycycline in the drinking water, both single transgenic and
bitransgenic mice showed no FLAG immunoreactivity (data not shown).
This finding indicates that the dosage of doxycycline (100 µg/ml in
drinking water) used was sufficient to turn off the expression of
mCREB. At postnatal week 10, after 4 weeks off doxycycline, strong
expression of FLAG was seen in several forebrain regions of
CaMKII-tTA × TetOP-mCREB bitransgenic mice, including olfactory
bulb, superficial and deep layers of cortex, caudate putamen, nucleus
accumbens, amygdala, hippocampus, and Purkinje cells of the cerebellar
lobule X (Fig. 6a-d).
FLAG-mCREB staining was not observed in the thalamus, midbrain, or
medulla oblongata (Fig. 6a,b). Bright-field
microscopic images with higher magnification showed that most of, but
not all, granule cells in hippocampal dentate gyrus expressed mCREB
(Fig. 6d,e). This expression pattern was very
similar to that seen for the first line of CaMKII promoter-tTA transgenic mice described by Kandel and his colleagues (Mayford et al.,
1996). In contrast, no immunoreactivity was observed in CaMKII-tTA
(data not shown) or TetOP-mCREB (Fig. 6c) single transgenic mice. These findings demonstrate that the expression of mCREB was
regulated by doxycycline in a time-limited and region-specific manner.
View larger version (80K):
[in this window]
[in a new window]
|
Figure 6.
Inducible expression of mCREB decreases cell
proliferation in hippocampus. mCREB expression was detected in
olfactory bulb (OB), cortex (Cx),
piriform cortex (Pir), caudate putamen
(CP), nucleus accumbens (Acb), amygdala
(Amy), hippocampus (Hi), and cerebellum
(Cb) but not in the thalamus (Th) and
midbrain and medulla oblongata (a, b) in
CaMKII-tTA × TetOP-mCREB bitransgenic mice. No immunoreactivity
was shown in TetOP-mCREB single transgenic mice
(c). In the dentate gyrus, most of, but not all,
granule cells (d) and a few cells in (Figure legend continued.) the CA3 region
expressed mCREB (d). Double immunolabeling for
BrdU and mCREB in the adult hippocampus of bitransgenic mice 2 hr after
BrdU administration is shown in f. There was no mCREB
immunoreactivity in the BrdU-positive cells. Scale bars:
a, 1 mm; b-d, 200 µm;
e, f, 20 µm. Mice expressing mCREB in the
hippocampus or genotype controls were treated with BrdU and 2 hr later
harvested for immunohistochemical analysis. The number of proliferating
cells in the granule cell layer was significantly decreased in the
bitransgenic mice relative to single transgenic mice
(g). CaMKII-tTA, CaMKII-tTA single
transgenic mice with no mCREB expression; TetOP-mCREB,
TetOP-mCREB single transgenic mice with no mCREB expression;
CaMKII-tTA × TetOP-mCREB,
CaMKII-tTA × TetOP-mCREB bitransgenic mice that express mCREB in
hippocampus. *p < 0.05 compared with the single
transgenic control (ANOVA, followed by Scheffe's post
hoc comparison).
|
|
Both single transgenic and bitransgenic mice at postnatal week 10 were
killed 2 hr after a single injection of BrdU, and the proliferation of
newborn cells was determined by immunodetection of BrdU. In the GCL of
the CaMKII-tTA and TetOP-mCREB single transgenic lines, the numbers of
BrdU-positive cells were 3904 ± 581 and 3729 ± 97 cells per
BDG, respectively (Fig. 6g). However, there was a
significant decrease in the number of BrdU-positive cells in the GCL of
the CaMKII-tTA × TetOP-mCREB bitransgenic mice (2462 ± 280 cells per BDG; p < 0.05). There was no difference in
the number of BrdU-positive cells in the hilus region among the three groups. The morphology and distribution of BrdU-positive cells in the
dentate gyrus of the three transgenic lines tested did not differ from
that in the wild-type mice (data not shown). This demonstrates that
there are no gross abnormalities that preclude the use of these
conditional lines of mCREB mice for these studies. Finally, newborn
BrdU-labeled cells were not colocalized with mCREB-immunopositive cells
in the bitransgenic mice, although the BrdU-labeled cells were
surrounded by mCREB-stained cells (Fig. 6f).
| |
DISCUSSION |
The results of both pharmacological and conditional transgenic
approaches used for this study demonstrate that activation of the
cAMP-CREB cascade increases the number of BrdU-labeled cells in the
hippocampus. Chronic, but not acute, administration of rolipram
increases the number of BrdU-labeled cells in the granule cell layer.
The requirement for repeated treatment is somewhat unexpected because
rolipram should increase cAMP within a relatively short period of time
(i.e., minutes to hours after reaching the brain). However, the
increase in cAMP levels is dependent not only on the rate of
degradation but also on the rate of synthesis. It is possible that
synthesis is not sufficiently high to support a significant increase of
cAMP levels, even in the presence of the PDE4 inhibitor. This is
supported by studies in brain slices that demonstrate a relatively
small effect of rolipram on basal cAMP levels but a much greater
increase in the presence of forskolin, an activator of cAMP synthesis
(Barad et al., 1998). However, it is important to point out that the
exact status of cAMP accumulation in hippocampal cells is not known and
that there may be an alternate explanation for the requirement for
chronic rolipram administration. One possibility is that the induction
of proliferation is dependent on long-term adaptations that occur in
response to repeated rolipram treatment. A similar scenario is proposed
for antidepressants, which acutely increase monoamine levels but
require repeated administration to induce hippocampal proliferation
(Malberg et al., 2000a). For example, chronic antidepressant
administration upregulates the cAMP cascade, including increased
expression of CREB (Nibuya et al., 1996; Duman et al., 1997b, 2000).
Yet another possibility is that rolipram is acting indirectly via
regulation of factors or endocrine systems in other parts of the brain
or even in other tissues that then lead to regulation of neurogenesis
in hippocampus. For example, peripheral regulation of insulin-like
growth factor-1 (IGF-1) is reported to increase neurogenesis in adult
hippocampus (Aberg et al., 2000).
The influence of rolipram treatment on the fate of newborn cells was
examined 4 weeks after BrdU administration. Because many newborn cells
do not survive, the total number of BrdU-labeled cells in control and
rolipram-treated hippocampus decreases proportionally at this time
point, as reported previously (Gould et al., 1999). However, there is
still a significant increase in the number of BrdU-labeled cells in
rolipram-treated animals relative to controls. At this later time,
~80% of the cells express a neuronal marker (NeuN), and 5-10%
express a glial marker (S100), as reported previously (van Praag et
al., 1999). The cells expressing NeuN have migrated into the granule
cell layer and have characteristics of mature granule cells. The
percentage of cells expressing these markers was not significantly
influenced by rolipram treatment. These results demonstrate that the
newborn cells resulting from the rolipram treatment have a cellular
phenotype that is the similar to that of vehicle-treated controls but
that the net number of BrdU-labeled neurons is
significantly increased by chronic rolipram administration. It should
also be pointed out that the survival of newborn cells at even longer
time points could be different from that observed in the present study
because a recent report demonstrates that there is a further loss of
BrdU-labeled cells 5-9 weeks after BrdU administration in monkeys
(Gould et al., 2001).
CREB regulates gene expression in response to activation of the cAMP
system (Duman et al., 2000) and could contribute to the observed
upregulation of cell proliferation. One striking finding of this study
is that there is marked localization of pCREB immunoreactivity in the
subgranular zone, suggesting a possible relationship with cell
proliferation. Chronic, but not acute, administration of rolipram
increases the number of pCREB-immunopositive cells throughout the
granule cell layer, including cells adjacent to the subgranular zone.
This is consistent with the time course for the induction of cell
proliferation by repeated, but not acute, rolipram administration. Double-labeling studies confirm that pCREB staining is localized to the
subgranular zone but also demonstrate that pCREB is not colocalized
with newborn BrdU-labeled cells. There are two possible interpretations
of these results. First, rolipram may not directly influence the
cAMP-CREB cascade in the progenitor cells but influences surrounding
cells that then increase proliferation via release of a factor that
increases proliferation. The second possibility is that rolipram does
influence the progenitor cells but that this effect is transient and
occurs during a brief time interval preceding and/or shortly after cell division.
The role of CREB in regulation of cell proliferation was directly
examined using conditional transgenic mice that over express a dominant
negative CREB mutant. The CaMKII promoter was used to drive expression
of tTA and subsequent regulation of TetOp-mCREB and was shown to be
sensitive to inhibition by doxycycline. The pattern of mCREB expression
was similar to that reported previously in mice using this same line of
CaMKII-tTA mice crossed with another TetOp-regulated transgene (Mayford
et al., 1996). This distribution includes robust expression in the
granule cell layer of hippocampus. When mCREB was overexpressed, there
was a significant reduction in the number of BrdU-labeled cells. This
is consistent with the hypothesis that the cAMP-CREB cascade exerts a
positive influence on proliferation in adult hippocampus.
Because the CaMKII promoter is turned on during late embryonic or early
postnatal development, the expression of mCREB is not expected to occur
in progenitor cells or newborn cells. This was confirmed by
double-labeling studies. Expression of mCREB is found in most granule
cells in the dentate gyrus but not in newborn cells that are
immunopositive for BrdU. The localization of mCREB in cells that
surround, but not in, the BrdU-labeled cells is consistent with the
hypothesis that the cAMP-CREB cascade regulates cell
proliferation indirectly via expression of a factor(s) that is released
from granule cells that surround progenitor cells. Some of the factors
that are reported to increase cell proliferation during development or
in adult brain and that could mediate the effect of the cAMP system
include IGF-1, epidermal growth factor, transforming growth factor-,
and fibroblast growth factor-2 (Cameron et al., 1998).
Another aspect of neurogenesis that could be influenced by the
cAMP-CREB cascade is the maturation and survival of newborn cells. The
experimental design to directly examine survival is to label newborn
cells with BrdU and then administer rolipram for 4 weeks during the
time when cells are maturing and when there is natural cell death.
Preliminary studies using this paradigm demonstrate that rolipram
treatment also influences the survival of cells (Nakagawa et al.,
2000). In addition, we found that pCREB immunoreactivity is colocalized
with, and may regulate the expression of, markers of immature neurons
in the BrdU-labeled cells (Nakagawa et al., 2000). Further
characterization of the actions of the cAMP-CREB cascade on cell
survival and the factors that mediate these effects warrant an
independent and in depth series of experiments and is the focus of
current investigations.
The cAMP-CREB cascade could underlie the action of different
neurotransmitters, neurotrophic factors, or conditions known to
influence neurogenesis. One possibility is learning and memory, which
is known to be influenced by activation of cAMP-CREB cascade and
associated with increased CREB phosphorylation in the hippocampus (Barad et al., 1998; Silva et al., 1998). Hippocampal-dependent learning is also reported to increase neurogenesis (Gould et al., 1999). Although the time course for the regulation of learning and
memory in both cellular and behavioral models occurs in a much shorter
time frame, it is possible that upregulation of cell proliferation
enhances the maintenance or processing of memories.
The results of this study may also have relevance to our understanding
of the action of antidepressants. Previous studies demonstrate that
repeated antidepressant treatment upregulates the cAMP-CREB pathway
(Nibuya et al., 1996; Thome et al., 2000), suggesting that this
second-messenger cascade may be involved in the actions of
antidepressant treatment (Duman et al., 1997b, 2000). In addition,
repeated antidepressant treatment increases hippocampal granule cell
proliferation (Malberg et al., 2000a). Rolipram is also reported to
have antidepressant effects in animal models of depression and has been
shown to have efficacy in depressed patients (Duman et al., 1997b,
2000). Together, the results are consistent with the hypothesis that
upregulation of the cAMP pathway underlies the induction of granule
cell proliferation in response to antidepressant treatment. Although it
is difficult to extrapolate from these findings to studies in humans,
it is interesting to speculate that the induction of hippocampal
proliferation could block or reverse the atrophy of hippocampus
reported in clinical studies (Duman et al., 1997b, 2000). Additional
brain imaging and postmortem studies will be needed to determine
whether there is a decrease in the number of granule cells in the
hippocampus of unmedicated depressed patients and whether hippocampal
atrophy and cell loss is reversed by antidepressant treatment.
| |
FOOTNOTES |
Received Oct. 5, 2001; revised Dec. 6, 2001; accepted Jan. 15, 2002.
This work is supported by United States Public Health Service Grants
MH45481 and 2 PO1 MH25642 and a Veterans Administration National Center
Grant for post-traumatic stress disorder.
Correspondence should be addressed to Ronald S. Duman, 34 Park Street,
New Haven, CT 06508. E-mail: ronald.duman{at}yale.edu.
C. Steffen's and E. J. Nestler's present address: Department of
Psychiatry, University of Texas Southwestern Medical Center, 5323 Harry
Hines Boulevard, Dallas, TX 75390-9070.
| |
REFERENCES |
-
Aberg M,
Aberg ND,
Hedbacker H,
Oscarsson J,
Eriksson PS
(2000)
Peripheral infusion of IGF-1 selectively induces neurogenesis in the adult rat hippocampus.
J Neurosci
20:2896-2903[Abstract/Free Full Text].
-
Barad M,
Bourtchouladze R,
Winder DG,
Golan H,
Kandel E
(1998)
Rolipram, a type IV-specific phosphodiesterase inhibitor, facilitates the establishment of long-lasting long-term potentiation and improves memory.
Proc Natl Acad Sci USA
95:15020-15025[Abstract/Free Full Text].
-
Boyes B,
Kim SU,
Lee V,
Sung SC
(1986)
Immunohistochemical colocalization of S-100b and the glial fibrillary acidic protein in rat brain.
Neuroscience
17:857-865[Web of Science][Medline].
-
Cameron H,
McKay RDG
(1999)
Restoring production of hippocampal neurons in old age.
Nat Neurosci
2:894-897[Web of Science][Medline].
-
Cameron H,
Wooley CS,
McEwen BS,
Gould E
(1993)
Differentiation of newly boron neurons and glia in the dentate gyrus of the adult rat.
J Neurosci
56:337-344.
-
Cameron H,
Hazel TG,
McKay RD
(1998)
Regulation of neurogenesis by growth factors and neurotransmitters.
J Neurobiol
36:287-306[Web of Science][Medline].
-
Chen J,
Kelz MB,
Zeng G,
Sakai N,
Steffen C,
Shockett PE,
Picciotto MR,
Duman RS,
Nestler EJ
(1998)
Transgenic animal models with inducible, targeted gene expression in brain.
Mol Pharmacol
54:495-503[Abstract/Free Full Text].
-
Conti M,
Jin S-L
(2000)
The molecular biology of cyclic nucleotide phosphodiesterases.
Prog Nucleic Acid Res Mol Biol
63:1-38.
-
Duman R,
Heninger GR,
Nestler EJ
(1997)
A molecular and cellular theory of depression.
Arch Gen Psychiatry
54:597-606[Abstract/Free Full Text].
-
Duman R,
Malberg J,
Nakagawa S,
D'Sa C
(2000)
Neuronal plasticity and survival in mood disorders.
Biol Psychiatry
48:732-739[Web of Science][Medline].
-
Eriksson P,
Perfileva E,
Bjork-Eriksson T,
Alborn A,
Nordborg C,
Peterson D,
Gage F
(1998)
Neurogenesis in the adult human hippocampus.
Nat Med
4:1313-1317[Web of Science][Medline].
-
Finkbeiner S
(2000)
CREB couples neurotrophin signals to survival messages.
Neuron
25:11-14[Web of Science][Medline].
-
Furth P,
Onge LS,
Böger H,
Gruss P,
Gossen M,
Kistner A,
Bujard H,
Hennighausen L
(1994)
Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter.
Proc Natl Acad Sci USA
91:9302-9306[Abstract/Free Full Text].
-
Gage F
(2000)
Mammalian neural stem cells.
Science
287:1433-1438[Abstract/Free Full Text].
-
Gonzalez G,
Montminy MR
(1989)
Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133.
Cell
59:675-680[Web of Science][Medline].
-
Gould E,
McEwen BS,
Tanapat P,
Galea LAM,
Fuchs E
(1997)
Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and NMDA receptor activation.
J Neurosci
17:2492-2498[Abstract/Free Full Text].
-
Gould E,
Beylin A,
Tanapat P,
Reeves A,
Shors TJ
(1999)
Learning enhances adult neurogenesis in the hippocampal formation.
Nat Neurosci
2:260-265[Web of Science][Medline].
-
Gould E,
Vail N,
Wagers M,
Gross CG
(2001)
Adult-generated hippocampal and neocortical neurons in macaques have a transient existence.
Proc Natl Acad Sci USA
98:10910-10917[Abstract/Free Full Text].
-
Kempermann G,
Kuhn HG,
Gage F
(1997)
More hippocampal neurons in adult mice living in an enriched environment.
Nature
386:493-495[Medline].
-
Kuhn H,
Dickinson-Anson H,
Gage FH
(1996)
Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation.
J Neurosci
16:2027-2033[Abstract/Free Full Text].
-
Kukekov V,
Laywell ED,
Suslov O,
Davies K,
Scheffler B,
Thomas LB,
O'Brien TF,
Kusakabe M,
Steindler DA
(1999)
Multipotent stem/progenitor cells with similar properties arise from two neurogenic regions of adult human brain.
Exp Neurol
156:333-344[Web of Science][Medline].
-
Malberg J,
Eisch AJ,
Nestler EJ,
Duman RS
(2000a)
Chronic antidepressant treatment increases neurogenesis in adult hippocampus.
J Neurosci
20:9104-9110[Abstract/Free Full Text].
-
Mayford M,
Bach ME,
Huang YY,
Wang L,
Hawkins RD,
Kandel ER
(1996)
Control of memory formation through regulated expression of a CaMKII transgene.
Science
274:1678-1683[Abstract/Free Full Text].
-
Mullen R,
Buck CR,
Smith AM
(1992)
NeuN, a neuronal specific nuclear protein in vertebrates.
Development
116:201-211[Abstract].
-
Nakagawa S,
Kim J-E,
Lee R,
Chen J,
Duman RS
(2000)
CREB plays a critical role in the survival of newborn cells in the adult hippocampus.
Soc Neurosci Abstr
26:2317.
-
Nibuya M,
Nestler EJ,
Duman RS
(1996)
Chronic antidepressant administration increases the expression of cAMP response element binding protein (CREB) in rat hippocampus.
J Neurosci
16:2365-2372[Abstract/Free Full Text].
-
Nowakowski R,
Lewin SB,
Miller MW
(1989)
Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and the DNA-synthetic phase for an anatomically defined population.
J Neurocytol
18:311-318[Web of Science][Medline].
-
Palmer T,
Takahashi J,
Gage FH
(1997)
The adult rat hippocampus contains primordial neural stem cells.
Mol Cell Neurosci
8:389-404[Web of Science][Medline].
-
Pincus D,
Keyoung HM,
Harrison-Restelli C,
Goodman RR,
Fraser RA,
Edgar M,
Sakakibara S-I,
Okano H,
Nedergaard M,
Goldman SA
(1998)
Fibroblast growth factor-2/brain-derived neurotrophic factor-associated maturation of new neurons generated from adult human subependymal cells.
Ann Neurol
43:576-585[Web of Science][Medline].
-
Shaywitz A,
Greenberg ME
(1999)
CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals.
Annu Rev Biochem
68:821-861[Web of Science][Medline].
-
Sheline Y,
Wany P,
Gado MH,
Csernansky JG,
Vannier MW
(1996)
Hippocampal atrophy in recurrent major depression.
Proc Natl Acad Sci USA
93:3908-3913[Abstract/Free Full Text].
-
Shockett P, Difilippantonio M, Hellman N, Schatz DG (1995) A
modified tetracycline-regulated system provides autoregulatory,
inducible gene expression in cultured cells and transgenic mice. Proc
Natl Acad Sci USA 6522-6526.
-
Silva A,
Kogan JH,
Frankland PW,
Kida S
(1998)
CREB and memory.
Annu Rev Neurosci
21:127-148[Web of Science][Medline].
-
Steffens D,
Byrum CE,
McQuoid DR,
Greenberg DL,
Payne ME,
Blitchington TF,
MacFall JR,
Krishnan KRR
(2000)
Hippocampal volume in geriatric depression.
Biol Psychiatry
48:301-309[Web of Science][Medline].
-
Takahashi J,
Palmer TD,
Gage FH
(1998)
Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult-derived neural cell cultures.
J Neurobiol
38:65-81.
-
Thome J,
Sakai N,
Shin K,
Steffen C,
Zhang YJ,
Impey S,
Storm D,
Duman RS
(2000)
cAMP response element-mediated gene transcription is upregulated by chronic antidepressant treatment.
J Neurosci
20:4030-4036[Abstract/Free Full Text].
-
van Praag H,
Kempermann G,
Gage FH
(1999)
Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus.
Nat Neurosci
2:266-270[Web of Science][Medline].
-
van Praag H,
Kempermann G,
Gage FH
(2000)
Neural consequences of environmental enrichment.
Nat Rev Neurosci
1:191-198[Web of Science][Medline].
-
West M,
Slomianka L,
Gundersen H
(1991)
Unbiased stereological estimation of the total number of neurons in the subdivision of the rat hippocampus using the optical fractionator.
Anat Rec
231:482-497[Medline].
Copyright © 2002 Society for Neuroscience 0270-6474/02/2293673-10$05.00/0
This article has been cited by other articles:
|
|
|
|
 
J.-S. Lee, D.-J. Jang, N. Lee, H.-G. Ko, H. Kim, Y.-S. Kim, B. Kim, J. Son, S. H. Kim, H. Chung, et al.
Induction of Neuronal Vascular Endothelial Growth Factor Expression by cAMP in the Dentate Gyrus of the Hippocampus Is Required for Antidepressant-Like Behaviors
J. Neurosci.,
July 1, 2009;
29(26):
8493 - 8505.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Jagasia, K. Steib, E. Englberger, S. Herold, T. Faus-Kessler, M. Saxe, F. H. Gage, H. Song, and D. C. Lie
GABA-cAMP Response Element-Binding Protein Signaling Regulates Maturation and Survival of Newly Generated Neurons in the Adult Hippocampus
J. Neurosci.,
June 24, 2009;
29(25):
7966 - 7977.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Segi-Nishida, J. L. Warner-Schmidt, and R. S. Duman
Electroconvulsive seizure and VEGF increase the proliferation of neural stem-like cells in rat hippocampus
PNAS,
August 12, 2008;
105(32):
11352 - 11357.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C. Garza, M. Guo, W. Zhang, and X.-Y. Lu
Leptin Increases Adult Hippocampal Neurogenesis in Vivo and in Vitro
J. Biol. Chem.,
June 27, 2008;
283(26):
18238 - 18247.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Grumolato, H. Ghzili, M. Montero-Hadjadje, S. Gasman, J. Lesage, Y. Tanguy, L. Galas, D. Ait-Ali, J. Leprince, N. C. Guerineau, et al.
Selenoprotein T is a PACAP-regulated gene involved in intracellular Ca2+ mobilization and neuroendocrine secretion
FASEB J,
June 1, 2008;
22(6):
1756 - 1768.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kvajo, H. McKellar, P. A. Arguello, L. J. Drew, H. Moore, A. B. MacDermott, M. Karayiorgou, and J. A. Gogos
A mutation in mouse Disc1 that models a schizophrenia risk allele leads to specific alterations in neuronal architecture and cognition
PNAS,
May 13, 2008;
105(19):
7076 - 7081.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-W. Wang, D. J. David, J. E. Monckton, F. Battaglia, and R. Hen
Chronic Fluoxetine Stimulates Maturation and Synaptic Plasticity of Adult-Born Hippocampal Granule Cells
J. Neurosci.,
February 6, 2008;
28(6):
1374 - 1384.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Thakker-Varia, J. J. Krol, J. Nettleton, P. M. Bilimoria, D. A. Bangasser, T. J. Shors, I. B. Black, and J. Alder
The Neuropeptide VGF Produces Antidepressant-Like Behavioral Effects and Enhances Proliferation in the Hippocampus
J. Neurosci.,
November 7, 2007;
27(45):
12156 - 12167.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Tchantchou, Y. Xu, Y. Wu, Y. Christen, and Y. Luo
EGb 761 enhances adult hippocampal neurogenesis and phosphorylation of CREB in transgenic mouse model of Alzheimer's disease
FASEB J,
August 1, 2007;
21(10):
2400 - 2408.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. L. Gur, A. C. Conti, J. Holden, A. J. Bechtholt, T. E. Hill, I. Lucki, J. E. Malberg, and J. A. Blendy
cAMP Response Element-Binding Protein Deficiency Allows for Increased Neurogenesis and a Rapid Onset of Antidepressant Response
J. Neurosci.,
July 18, 2007;
27(29):
7860 - 7868.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Sasaki, K. Kitagawa, E. Omura-Matsuoka, K. Todo, Y. Terasaki, S. Sugiura, J. Hatazawa, Y. Yagita, and M. Hori
The Phosphodiesterase Inhibitor Rolipram Promotes Survival of Newborn Hippocampal Neurons After Ischemia
Stroke,
May 1, 2007;
38(5):
1597 - 1605.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Bruel-Jungerman, S. Davis, C. Rampon, and S. Laroche
Long-term potentiation enhances neurogenesis in the adult dentate gyrus.
J. Neurosci.,
May 31, 2006;
26(22):
5888 - 5893.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Giachino, S. De Marchis, C. Giampietro, R. Parlato, I. Perroteau, G. Schutz, A. Fasolo, and P. Peretto
cAMP Response Element-Binding Protein Regulates Differentiation and Survival of Newborn Neurons in the Olfactory Bulb
J. Neurosci.,
November 2, 2005;
25(44):
10105 - 10118.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. P. Wasilewska-Sampaio, M. S. Silveira, O. Holub, R. Goecking, F. C. A. Gomes, V. M. Neto, R. Linden, S. T. Ferreira, and F. G. De Felice
Neuritogenesis and neuronal differentiation promoted by 2,4-dinitrophenol, a novel anti-amyloidogenic compound
FASEB J,
October 1, 2005;
19(12):
1627 - 1636.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Contestabile, T. Fila, R. Bartesaghi, and E. Ciani
Cyclic AMP-mediated Regulation of Transcription Factor Lot1 Expression in Cerebellar Granule Cells
J. Biol. Chem.,
September 30, 2005;
280(39):
33541 - 33551.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. S. Naylor, A. I. Persson, P. S. Eriksson, I. H. Jonsdottir, and T. Thorlin
Extended Voluntary Running Inhibits Exercise-Induced Adult Hippocampal Progenitor Proliferation in the Spontaneously Hypertensive Rat
J Neurophysiol,
May 1, 2005;
93(5):
2406 - 2414.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Nixon and F. T. Crews
Temporally Specific Burst in Cell Proliferation Increases Hippocampal Neurogenesis in Protracted Abstinence from Alcohol
J. Neurosci.,
October 27, 2004;
24(43):
9714 - 9722.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Cancedda, E. Putignano, A. Sale, A. Viegi, N. Berardi, and L. Maffei
Acceleration of Visual System Development by Environmental Enrichment
J. Neurosci.,
May 19, 2004;
24(20):
4840 - 4848.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Fujioka, A. Fujioka, and R. S. Duman
Activation of cAMP Signaling Facilitates the Morphological Maturation of Newborn Neurons in Adult Hippocampus
J. Neurosci.,
January 14, 2004;
24(2):
319 - 328.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. S. Newton, J. Thome, T. L. Wallace, Y. Shirayama, L. Schlesinger, N. Sakai, J. Chen, R. Neve, E. J. Nestler, and R. S. Duman
Inhibition of cAMP Response Element-Binding Protein or Dynorphin in the Nucleus Accumbens Produces an Antidepressant-Like Effect
J. Neurosci.,
December 15, 2002;
22(24):
10883 - 10890.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
