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ÂDepartment 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

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 „ippocampus
— The YYY
 @elongs to
the lim@ic system and plays
important roles in long-term
memory and spatial navigation.

— It is located inside the medial

temporal lo@e @eneath the
cortical surface.

— Its curved shape reminded early

anatomists of the horns of a ram
(K
  [, or a seahorse.
The name was taken @y the 16th
century anatomist Julius Caesar
Aranzi from the Greek word
for seahorse (Greek:
·ÎÎ
, `   `
 ,
*
Î
, ë    
 

[.
 Importance of hippocampus
— Consolidation of new
memories
— Processing emotions,
especially emotional
memory
— Navigation
— Spatial orientation
 Department of Anatomy,
„istology JNMC„,AMU,
ALIGAR„ 2010

— Su@components are---
a[ Dentate gyrus
@[ Su@iculum
c[ Sectors referred to as
CA1,
CA2,
CA3,
CA4.

Department of
Anatomy
JNMC„,AMU,
ALIGAR„
2010
— It·s a tri-laminar
structure.

Superficial  

 consisting of nerve Molecular Granular Molecular
fi@ers and scattered small layer layer layer
neurons Polymorphic
Pyramidal
layer layer
The Y  
consisting of many large
pyramid-shaped neurons

The inner Y Y Polymorphic

 which is similar in layer
structure to the
polymorphic layer of the
cortex seen elsewhere.
Department of
Anatomy,
JNMC„,AMU,
ALIGAR„ 2010
 „YYY 3 Y
 4
Y

— The hippocampus has direct
connections to the entorhinal
cortex (via the su@iculum[ and
the amygdala.
— Entorhinal cortex projects to
the cingulate cortex, which has
a connections to the temporal
lo@e cortex, or@ital cortex,
and olfactory @ul@.Thus, all of
these areas can @e influenced
@y hippocampal output,
primarily from CA1.
— Neuro@iologists long @elieved that adult
@rains did not make new neurons, @ut
now we know otherwise. In the early
1960s, Joseph Altman at MIT reported
that new neurons were @eing produced
in the @rains of adult rats. Those findings
were somewhat forgotten for the next
30 years. Recently, this work has @een
revived and advanced.

— Eliza@eth Gould of Princeton University,

one of this article·s authors (Gage[ and
others have reported the @irth of new
neurons--neurogenesis--in the
hippocampus of adult rats, monkeys and
humans.
— Stem cells--primitive cells that are formed soon after fertilization and
that can divide indefinitely, which can divide a limited num@er of times
and give rise to cell types such as neurons and glia.

— Neurons continue to @e @orn throughout life in the olfactory @ul@,

dentate gyrus of the hippocampus. (Very recent evidence indicates
that some additional @rain areas might also produce new @rain cells.[

— These new neurons are derived from progenitor cells that reside in
the @rain·s su@ventricular zone(ventricles[ or in a layer of the
hippocampus called the su@granular zone.
— Investigators follow neurogenesis with tritiated-
thymidine or @romodeoxyuridine.

— The compound incorporated into cells can @e

visualized under the microscope with
autoradiographic or immunologic techniques
Department of
for tritiated-thymidine or @romodeoxyuridine, Anatomy,
JNMC„,AMU,
respectively. ALIGAR„ 2010

— These techniques show that progenitor cells in

the su@granular zone produce progeny that
migrate outward to the granule-cell layer and
differentiate into neurons. In this way, these new
granule cells join the population of existing
neurons.These newly @orn cells mature in the
granule-cell layer and send their dendrites
outward, whereas their cell processes go
inward and follow paths to other structures
within the hippocampus, such as the CA3 cell
fields.
 Major Depression
— Five (or more[ of the following symptoms have @een present during the
same 2-week period and represent a change from previous functioning; at
least one of the symptoms is either
(1[ Depressed mood or
(2[ Loss of interest or pleasure.

(1[ depressed mood most of the time, most every day

(2[ markedly diminished interest or pleasure in daily activities
(3[ significant weight loss when not dieting or weight gain, or appetite loss
(4[ insomnia or hypersomnia
(5[ psychomotor agitation or retardation (o@serva@le @y others[
(6[ fatigue or loss of energy
(7[ feelings of worthlessness or excessive or inappropriate guilt
(8[ diminished a@ility to think or concentrate
(9[ recurrent thoughts of death (not just fear of dying[, recurrent suicidal
ideation without a specific plan, or a suicide attempt or a specific plan for
committing suicide
— Various modes of stress
can @e used to induce
depression in the rat or an
individual.

— Studies in rats have shown

that the hippocampus of
stressed rats are on
average smaller and
microscopic examination
reveals neuronal loss.

— Repair can @e stimulated

@y many antidepressants.
 p21 gene
— p21WAF1/Cip1 @elongs to
the Cip/Kip family of cyclin
kinase inhi@itors (CKI[
(p21Waf1/Cip1, p27Kip1,
p57Kip1[

— p21Waf1/Cip1 was first

descri@ed as a potent and
universal inhi@itor of cyclin-
dependent kinases (Cdks[.

— p21 functions as a
checkpoint in the cell cycle
@y inhi@iting cdks at the
G1/S and G2/M interfaces.
In normal cells, p21
acts like a @rake to
@lock cell cycle
progression in the
event of DNA
damage, preventing
the cells from
dividing and
potentially @ecoming
cancerous.
 A@stract
— The su@granular zone (SGZ[ of the dentate gyrus of the
hippocampus is a @rain region where ro@ust neurogenesis
continues throughout adulthood.
— Cyclin-dependent kinases (CDKs[ have a primary role in
controlling cell division and cellular proliferation.
— p21cip1 (p21[ is a CDK inhi@itor that restrains cell cycle
progression.
— Chronic treatment with the tricyclic antidepressant
imipramine (10 mg/kg per day i.p. for 21 days[markedly
decreased hippocampal p21 mRNA and protein levels.
— These results suggest that p21 restrains neurogenesis in the
SGZ and imipramine-induced stimulation of neurogenesis
might @e a consequence of decreased p21 expression and the
su@sequent release of neuronal progenitor cells from the
@lockade of proliferation.
— Because many antidepressants stimulate neurogenesis, it is
possi@le that their shared common mechanism of action is
suppression of p21.
 Introduction
— In the central nervous system, developing neurons are
derived from quiescent multipotent or neural stem cells and
progenitors (1[. In the hippocampus, the neural progenitor
cells are located in the SGZof the DG, at the @order @etween
the hilus and the granular cell layer (GCL[ (2, 3[. New@orn
cells proliferate in SGZ, migrate into the GCL, develop the
morphological and functional properties of granule cell
neurons, and @ecome integrated into existing neuronal
circuitry (4[. This suggests an important role of intrinsic
stimulatory and inhi@itory factors in the regulation of
proliferation of neuronal precursor cells.

— In mammalian cells, the control of cellular proliferation

primarily is achieved in the G1 phase of the cell cycle. Cyclin
dependent kinases (CDKs[ tightly control the cell cycle
process. Cell cycle progression is negatively regulated @y two
families of CDK inhi@itors: Ink4/ARF type (p16, p15, p18, and
p19[ and Cip/Kip type (p21, p27, and p57[.
— p21Cip1 (p21[ acts in the G1phase of the cell cycle and
delays or @locks the progression of the cell into the S
phase (5[. p21 maintains cell quiescence, and chronic
activation of p21 can drive the cell into irreversi@le cell
growth arrest and senescence (5, 6[. Conversely,
inhi@ition of p21 increases cellular proliferation (7[.
— The induction of neurogenesis might @e part of the
molecular mechanisms underlying the therapeutic
effects of antidepressant treatment (8, 9[. All major
classes of antidepressants stimulate neurogenesis in
rodents (10²12[ and nonhuman primates (13[. The
present study evaluated the role of the CDK inhi@itor
p21 in neurogenesis. The results suggest that
antidepressants may stimulate SGZ neurogenesis @y
inhi@iting p21 expression.
 Materials and Methods
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.; Sigma²
Aldrich[. Twenty-four hours after the last injection,
the mice were su@jected to BrdU injections and
used for the @ehavioral experiment or killed for the
other studies. Cdk1atm1Tyj ( 




 

 ë

were o@tained from The Jackson
La@oratory. For real-time PCR, ELISA, and Western
@lot analyses, the mice were killed, the @rains were
removed and rapidly cooled in ice-cold saline, and
the hippocampi were dissected out (55[.
Immunohistochemistry.
Mice were anesthetized with isoflurane and perfused
with paraformaldehyde (4%[, and @rain paraffin sections
were processed as descri@ed previously (56[. The slides
were incu@ated with mouse anti-p21 monoclonal
anti@odies (BD Pharmingen[ and conjugated with Alexa
Fluor 488 fluorescent dye (Molecular Pro@es[. The
anti@ody for NeuN (Chemicon[ was conjugated with
Alexa Fluor 568 fluorescent dye (Molecular Pro@es[
and used to determine co-localization with p21
expression. DNA (nuclei[ was stained with ToPro3
(Molecular Pro@es[. Multi-parameter fluorescent
microscopy and Leica Confocal Software were used to
identify p21 intracellular localization and colocalization
with the neuronal marker NeuN. For compara@ility, all
images on confocal multi-image figure plates were
adjusted with identical contrast and @rightness settings.
˜

  
   
  



Intranuclear p21 expression in the SGZ of the
dentate gyrus. Dou@le-la@eling analysis with color-
coded fluorogram (58[ is shown. DNA (@lue[ p21
(green[
BrdU Immunochemistry.
The entire left half of the @rain
was cut into 5-m sagittal
sections and processed @y using
a BrdU La@eling and Detection
Kit (Roche Applied Biosystems[.
Sections were coded for @lind
o@servation. Un@iased 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 Representative images of
counted under a 100 o@jective, SGZ cells of 


and the sum was multiplied @y 3  




to estimate the total num@er of 
and DCX (red[.
BrdU-positive cells in the region.
Cells were counted if they were Cell nuclei were stained
in or touching the SGZ, and cells with the DNA-specific
were excluded if they were dye ToPro3 (@lue[.
more than two cell diameters
from the GCL (8[. Some
sections derived from p21-null
mice were dou@le-la@eled to
detect DCX (Santa Cruz
Biotechnology[, and at least 20
BrdU-positive cells were
examined @y confocal
microscopy to determine
colocalization with DCX.
FACS Analysis.
„ippocampi were dissociated with a Papain Dissociation
System (Worthington Biochemical[, and the cells were
processed according to a Flow Cytometry Staining Protocol
(Cell Signaling Technology[. Fixed cells were incu@ated
overnight with DCX anti@odies (Cell Signaling Technology[,
washed, and treated with secondary anti@odies (Alexa Fluor
586 fluorescent dye, red[, and samples were analyzed in a
FACS Cali@ur system (Becton Dickinson[. The cells were
gated on the @asis of forward/side scatter plot to eliminate
the de@ris. DCX-positive and DCX-negative cells were
sorted. For the dou@le-staining experiments, @oth DCX-
positive and DCX-negative cells were stained for p21 (BD
Pharmingen[ for 2 h at 4°C, then stained with a secondary
anti@ody (Alexa Fluor 488 fluorescent dye, green[, and the
num@er of DCX/ p21-positive cells was determined. Control
cells were stained with @oth secondary anti@odies.
Protein Isolation and Western Blot Analysis. Proteins
were isolated (Immunoprecipitation Kit; Roche
Diagnostics[, separated @y SDS/PAGE, electro@lotted
onto mem@ranes (Millipore[, incu@ated overnight with
primary anti@odies, and then incu@ated with
corresponding secondary anti@odies as descri@ed (56[.
Immunoreactive @ands were detected @y the ECL
immunodetection system. NeuN (Chemicon[ and
PCNA, DCX, and -actin (Santa Cruz Biotechnology[
anti@odies were used. For quantitative analysis @lots
were scanned @y using an Epson V750 PRO and
transferred to Ado@e Photoshop Elements 3. Intensity
analysis of the @ands (all multiple @ands simultaneously[
was performed with ImageJ Software (ImageJ; W. S.
Ros@and, National Institutes of „ealth;
http://rs@.info.nih.gov/ij[
Chronic treatment with imipramine suppresses p21 expression in the hippocampus. (
    
 


`

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with color-coded fluorograms (51[. Confocal images show dou@le immunofluorescence la@eling of hippocampal dentate
gyrus after chronic treatment with saline (




 
 

 





 



   





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a @lue overlay. Colocalized pixels in the fluorograms are
marked with @lue (


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su@granular low; GCL; granular
cell layer; ML, molecular layer. ( 
 

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(C[ or imipramine (IP[ (seven to eight mice per group[. ˜

   
     

 
  

 ˜   
   ! ˜ 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. ˜


 Results
— p21 isexpressed in neuro@lasts and newly developing
neurons in the SGZ of the hippocampus.

— P21 deletion increases proliferation of hippocampal neurons.

— Chronic treatment with imipramine decreases p21

expression in the SGZ of the hippocampus.

— Chronic treatment with imipramine increases neurogenesis

in the SGZ of the hippocampus and produces
antidepressant-like activity in the forced swim test.
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