1 Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66,
CH-4058 Basel, Switzerland
2 Novartis Pharma AG, Lichtstrasse 35, CH-4056, Basel, Switzerland
3 Biomedizinische NMR Forschungs GmbH am Max-Planck-Institut für
biophysikalische Chemie, 37070 Göttingen, Germany
* Author for correspondence (e-mail: brian.hemmings{at}fmi.ch)
Accepted 14 April 2005
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SUMMARY |
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Key words: Pkb/Akt3 knockout, Brain development, Apoptosis
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Introduction |
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PKB is activated by numerous stimuli, including growth factors, hormones
and cytokines (Brazil and Hemmings,
2001; Chan et al.,
1999
; Datta et al.,
1999
). Activation of PKB occurs in response to signalling via
phosphoinositide 3 kinase (PI3K) and requires the membrane-bound second
messenger phosphatidylinositol-3,4,5-triphosphate
[PtdIns(3,4,5)P3 or PIP3]
(Burgering and Coffer, 1995
;
Cross et al., 1995
;
Franke et al., 1995
). The
current model for PKB regulation proposes that
PtdIns(3,4,5)P3, generated following PI3K activation,
interacts with the PH domain of PKB, recruiting the inactive kinase from the
cytoplasm to the plasma membrane and promoting a conformational change that
allows phosphorylation on two regulatory sites by upstream kinases at the
plasma membrane. One of these critical phosphorylation sites resides in the
activation loop of the kinase domain (Thr308 in PKB
) and the other is
located in the C-terminal regulatory domain (Ser-473 in PKB
), termed
the hydrophobic motif (Alessi et al.,
1996
; Brodbeck et al.,
1999
; Meier et al.,
1997
). The upstream kinase that phosphorylates Thr308 in the
activation loop of the kinase domain of PKB
in a PIP3-dependent-manner
has been identified and termed 3-phosphoinositide-dependent kinase 1 (PDK1)
(Alessi et al., 1997
;
Stokoe et al., 1997
). Thr308
phosphorylation is necessary and sufficient for PKB activation; however,
maximal activation requires additional phosphorylation at Ser473
(Alessi et al., 1996
;
Yang et al., 2002a
;
Yang et al., 2002b
). Several
different protein kinases and mechanisms have been proposed for the
phosphorylation of the hydrophobic motif
(Brazil and Hemmings, 2001
;
Yang et al., 2002b
).
Mice with targeted disruption of Pkb and/or
Pkbß have been obtained recently with Pkb
mutant
mice displaying an increased neonatal lethality and a reduction in body weight
of
30% (Chen et al.,
2001
; Cho et al.,
2001b
; Yang et al.,
2003
). Moreover, loss of Pkb
leads to placental
hypotrophy with impaired vascularisation
(Yang et al., 2003
). By
contrast, Pkbß-deficient mice are born with the expected
Mendelian ratio and exhibit a diabetes-like syndrome with elevated fasting
plasma glucose, hepatic glucose output, peripheral insulin resistance and an
compensatory increase of islet mass (Cho
et al., 2001a
). Compared with Pkb
mutant mice,
Pkbß-deficient mice are only mildly growth retarded
(Cho et al., 2001a
;
Garofalo et al., 2003
).
However, mice lacking both isoforms die after birth, probably owing to
respiratory failure (Peng et al.,
2003
). Pkb
ß double mutant newborns display a
severe reduction in body weight (
50%), prominent atrophy of the skin and
skeletal muscle, as well as impaired adipogenesis and delayed
ossification.
Here, we report the generation and characterisation of mice with targeted
disruption of the Pkb gene. Compared with
Pkb
-/- and
Pkbß-/- mice, Pkb
mutant
mice display a distinct phenotype without increased perinatal mortality,
growth retardation or altered glucose metabolism. However, loss of PKB
profoundly affects postnatal brain growth. Brains from adult
Pkb
mutant mice show a dramatic reduction in size and weight.
Taken together, our results reveal a novel and important physiological role
for PKB
in postnatal brain development.
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Materials and methods |
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Quantitative real time PCR
The levels of PKB isoforms in wild-type and mutant mice were determined by
quantitative Q-RT-PCR. The experiment was performed as described previously
(Yang et al., 2003). Briefly,
total RNA was purified using Trizol Reagent (Invitrogen). For the Q-PCR
reaction, 50 ng total RNA was mixed with 5' and 3' primers, Taqman
probe, MuLV reverse transcriptase, RNase inhibitor and the components of the
TaqMan PCR reagent kit (Eurogentec) in a total volume of 25 µl following
the TaqMan PCR reagent kit protocol.
Western blot analysis
For Western blot analysis, protein lysates were processed as previously
described (Yang et al., 2003).
PKB isoform-specific antibodies were obtained by immunizing rabbits with
isoform-specific peptides as previously described
(Yang et al., 2003
).
Antibodies against phospho-PKB (Ser473), phospho-GSK3
/ß (Ser21/9),
phospho-TSC2 (Thr1462) and phospho-p70S6K were purchased from Cell Signalling
Technologies. Antibodies against p27 and ERK were purchased from Santa Cruz
Biotechnology. The antibody against phospho-ERK (Thr202/Tyr204) was purchased
from Promega and the Pan-Actin antibody was obtained from NeoMarkers. Western
blots were scanned using a GS-800 BioRad densitometer with a resolution of
63.5 µm x 63.5 µm and bands were quantified using Proteomweaver
3.0.0.6 (Definiens).
Histological examination
For histological analysis, animals were perfused with phosphate-buffered
saline and 4% paraformaldehyde in phosphate-buffered saline. Organs were
dissected and kept in the same fixation solution overnight at 4°C. Samples
were embedded in paraffin following dehydration in ethanol. Tissues were cut
into 6 µm sections and stored for staining. For Haematoxylin-Eosin and
Cresyl-Violet (Sigma) staining, sections were freed of paraffin and
stained.
Cell number determination
To determine the number of cells in a whole brain, the DNA content was
determined as described by Labarca and Paigen
(Labarca and Paigen, 1980).
This method is based on the enhancement of fluorescence after binding of
bisbenzimid (Riedel-de Haen) to DNA. A linear standard curve (1-10 µg/ml)
was prepared to calculate the DNA concentration. The relative cell size in
posterior cortex was measured on plane-matched, parasagittal brain sections
stained with DAPI (Biotium). Image-Pro® Plus (Media Cybernetics) was used
to count the cell number and to calculate the mean area occupied by one cell.
The relative cell size is expressed as percent of wild type.
In vivo magnetic resonance imaging (MRI)
MRI studies of 4-month-old female Pkb wild-type
(n=5) and mutant mice (n=5) were performed at 2.35 T using a
MRBR 4.7/400 mm magnet (Magnex Scientific, Abingdon, England) equipped with
BGA20 gradients (100 mT m-1) and a DBX system (Bruker Biospin,
Ettlingen, Germany). For in vivo examinations, animals were anaesthetized
(1.0-1.5% halothane in 70:30 N2O:O2) and treated as
previously described (Natt et al.,
2002
). Briefly, radiofrequency (rf) excitation and signal
reception were accomplished with use of a Helmholtz coil (
100 mm) and
a surface coil (
20 x12 mm), respectively. Three-dimensional
T1-weighted (rf-spoiled 3D FLASH, repetition time TR=17 mseconds,
echo time TE=7.6 mseconds, flip angle 25°, measuring time 84 minutes) and
T2-weighted MRI data sets (3D fast spin-echo, TR/TE=3000/98
mseconds, measuring time 58 minutes) were acquired with an isotropic
resolution of 117 µm. Volumetric assessments were obtained by analysing
T1-weighted images using software provided by the manufacturer
(Paravision, Bruker Biospin, Ettlingen, Germany). After manually outlining the
whole brain and the ventricular spaces in individual sections, respective
areas were calculated (in mm2), summed and multiplied by the
section thickness.
Neuronal cell culture
E16.5 murine hippocampal neurons were isolated from timed matings. Cells
were kept in culture for 7 days on polylysine coverslips coated with
B27/Neurobasal (GIBCO Life Technologies). At day 7, cultures were treated with
glutamate (15 mM/24 hours), staurosporine (50 nM/12 hours) or were left
untreated. For the detection of apoptotic cells, five fixed cultures per
genotype were stained using a TUNEL-assay (Roche) according to the
manufacturer's instructions and counterstained with DAPI (Biotium). At least
200 cells per culture were counted and the percentage of apoptotic cells was
calculated.
Glucose and insulin tolerance test
Mice were housed according to the Swiss Animal Protection Laws in groups
with 12 hours dark/light cycles and with free access to food and water. All
procedures were conducted with the approval of the appropriate
authorities.
Random-fed and fasting blood glucose levels were determined in 5- to 6-month-old mice. Blood samples were collected from tail veins and glucose levels were determined using Glucometer Elite (Bayer). Mice (aged 5-6 months) were fasted overnight before the start of the glucose and insulin tolerance tests. Glucose (2 g/kg) was given orally to conscious mice. Insulin (1 U/kg) was administered by intraperitoneal injection to conscious mice. Blood samples were collected at indicated times from tail veins and glucose levels were determined using Glucometer Elite (Bayer). Blood glucose levels were expressed as percentage of initial value. Blood insulin levels were measured with a Mouse ultra-sensitive insulin ELISA (Immunodiagnostic Systems).
Microarray analysis
Microarray analysis was performed using murine MOE430A GeneChipsTM
(Affymetrix). Total RNA (10 µg) was reverse transcribed using the
SuperScript Choice system for cDNA synthesis (Life Technologies) and
biotin-labelled cRNA generated using the Enzo BioArray High Yield RNA
transcript labelling kit (Enzo Diagnostics) following the manufacturer's
protocol. cRNA fragmentation and hybridisation were performed as recommended
by Affymetrix. Expression data were calculated using the RMA algorithm from
BioConductor (Irizarry et al.,
2003). A gene was considered to be significantly altered in its
expression if it had an Affymetrix change P-value of less than 0.003
for either increase or decrease in at least two-thirds of replicate
comparisons and it had a minimum expression value of 100 in at least one
condition. A fold-change threshold of 1.5 was then applied and the resulting
genes were subjected to a one-way ANOVA with a P-value cut-off of
0.05. A Benjamini and Hochberg multiple testing correction and a Tukey
post-hoc test were applied.
Statistical analysis
To compare body weight, brain weight and volume, brain/body weight ratio,
DNA content, cell number and percentage of apoptotic cells between
Pkb+/+ and Pkb
-/-, an unpaired Students t-test was performed.
P values under 0.05 were considered as significant and values below
0.01 as highly significant.
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Results |
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Distribution of PKB in wild-type tissues
It has been reported that the PKB and ß isoform are widely
expressed in all organs, but with some isoform-specific features
(Altomare et al., 1998
;
Yang et al., 2003
). Less is
known about the tissue distribution of the PKB
isoform. Previous
reports suggest that PKB
has a more restricted distribution with high
levels in the adult brain and foetal heart and low levels in liver and
skeletal muscle (Brodbeck et al.,
1999
; Masure et al.,
1999
; Yang et al.,
2003
). We assessed the distribution of PKB
mRNA in 15 major
tissues of adult mice by quantitative RT-PCR and normalised to the level of
PKB
in the brain (Fig.
2A). PKB
mRNA was found at the highest level in brain and
testis, and at lower levels in lung, mammary gland, fat and spleen.
To investigate whether PKB ablation leads to compensatory increase
in PKB
and/or PKBß, total RNA isolated from brain, testis, lung,
mammary gland, fat and spleen of three Pkb
wild-type and three
mutant mice was subjected to quantitative RT-PCR. The levels of PKB
and
ß were normalised to the level of PKB
in wild-type brain and set
as 100%. Overall, no marked upregulation of PKB
and/or PKBß was
observed, including the brain (Fig.
2B,C). These results are consistent with Western blot analysis of
protein extracts of brains from Pkb
wild-type and mutant mice
(Fig. 1D). To investigate the
distribution and levels of individual PKB isoforms within the brain, protein
lysates were prepared from ten anatomically and functionally different
regions. In general, all three isoforms were expressed in all examined regions
but with certain isoform-specific features
(Fig. 2D). PKB
is
expressed in all regions at similar levels, whereas PKBß is expressed at
moderate levels in cortex, cerebellum, hippocampus and olfactory bulb, and at
lower levels in the hypothalamus, midbrain, brain stem and spinal cord.
PKB
is expressed in all examined regions but at higher levels in the
cortex and in the cerebellum.
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As PKB has been implicated in the regulation of cell and organ growth, body
weight was measured in male Pkb mutant mice and wild-type
littermate controls (n=5-8 per group) at different time points
(Chen et al., 2001
;
Cho et al., 2001b
;
Peng et al., 2003
;
Yang et al., 2003
). Body
weight did not differ significantly between male Pkb
mutant
mice and wild-type controls at any time point
(Fig. 4A). A similar result was
obtained with female Pkb
-/- mice and
Pkb
+/+ controls (n=5-8 per group, data not
shown), indicating that PKB
does not play a significant role in the
overall growth of mice.
To investigate a potential role of PKB in the regulation of glucose
metabolism, blood glucose levels were measured in adult (5-6 months)
Pkb
mutant mice under random-fed and fasting condition, and
compared with age- and gender-matched wild-type controls. Interestingly, both
random-fed and fasting blood glucose levels, were not significantly different
between wild-type and mutant mice (Fig.
4B). Additionally, blood insulin levels in random-fed condition
did not differ significantly between Pkb
+/+ and
Pkb
-/- mice (1.32±0.08 µg/l
versus 1.30±0.13 µg/l; n=6). To further investigate glucose
metabolism, overnight fasted mice were challenged with insulin (insulin
tolerance test) or glucose (glucose tolerance test). To test the insulin
responsiveness, insulin (1 U/kg) was applied by intraperitoneal injection und
blood glucose levels were measured at indicated time points using blood from
tail veins. No obvious differences of blood glucose levels in the insulin
tolerance test were found between the groups with mutant and control mice
(Fig. 4C). Additionally, mice
were challenged with orally applied glucose (2 g/kg) and blood glucose levels
were measured at indicated time. Compared with wild-type mice,
Pkb
-/- mice displayed a very similar response
to the glucose load (Fig. 4D).
Taken together, these results suggest that PKB
does not play a
significant role in the maintenance of glucose homeostasis.
Essential role of PKB in postnatal brain development
Next, adult Pkb+/+ and
Pkb
-/- mice were dissected and all major organs
were investigated macroscopically. Compared with
Pkb
+/+ littermate controls, the overall size of
brains from adult Pkb
-/- mice was strikingly
reduced. A representative example is shown in
Fig. 5B-D. Furthermore, the
weights of freshly dissected brains of Pkb
wild-type and
Pkb
mutant mice were measured at different ages
(Fig. 5A). Compared with age-
and gender-matched wild-type littermate controls, brains from adult
Pkb
-/- mice (3-12 months old) exhibited a highly
significant reduction in weight of about 25% (range 22%-29%), affecting both
males and females (data for females not shown, n=5-8). Interestingly,
at birth, brain weight did not differ significantly between
Pkb
+/+ and Pkb
-/- mice.
The reduction in brain size and weight was first observed at the age of 1
month, but was less pronounced compared with adult mice (
18%). In contrast
to Pkb
, Pkbß and IgfI-null mutant mice
(Beck et al., 1995
;
Cheng et al., 1998
;
Garofalo et al., 2003
;
Powell-Braxton et al., 1993
),
the brain/body weight ratio of Pkb
mutant mice was also
significantly reduced to a similar extent.
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Cell number in the brains of Pkb-/- mice
Next, we indirectly assessed the cell number by measuring the amount of DNA
in whole brains derived from Pkb+/+ and
Pkb
-/- mice. The amount of DNA is considered as an
indicator of cell number, whereas the amount of DNA per gram of tissue is an
indicator of cell density which is reciprocal to cell volume
(Zamenhof, 1976
). The DNA
contents of brains from newborns and 1-month-old mice were determined using
the method described by Labarca and Paigen
(Labarca and Paigen, 1980
).
Briefly, this method is based on the enhancement of fluorescence after binding
of bisbenzimid to DNA. Compared with wild-type control samples, whole brain
DNA content of Pkb
-/- newborns did not differ
significantly, indicating that cell number was not changed. Similarly, the DNA
content per gram of brain tissue was comparable between Pkb
wild-type and mutant mice, indicating a comparable cell size. In contrast to
newborns, the DNA content in brains of 1-month-old Pkb
mutant
mice was slightly, but significantly, reduced compared with the
Pkb
+/+ controls
(Table 1). However, the DNA
content per gram of tissue was, significantly increased in samples from
Pkb
-/- compared with wild-type littermate controls,
indicative of increased cell density (and reduced cell size).
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Susceptibility to glutamate and staurosporine induced cell death
It is established that the PI3K/PKB pathway plays a crucial role in cell
survival in the central nervous system
(Datta et al., 1999;
Dudek et al., 1997
;
Kim et al., 2002
). To
investigate the potential role of PKB
in apoptosis, primary cell
cultures were established from Pkb
+/+ and
Pkb
-/- hippocampal neurons. Immunocytochemistry at
day 28 using antibodies against tau (for axons) and Map2C (for dendrites) did
not reveal any obvious defects in the differentiation of
Pkb
-/- hippocampal neurons
(Fig. 7A-D). Consistent with
the analysis of the expression pattern of PKB isoforms in various brain
regions (Fig. 2D), we found
that PKB
, PKBß and PKB
, respectively, were expressed in
cultured wild-type primary hippocampal neurons. In accordance with the result
of Fig. 1D, we did not find a
compensatory upregulation of PKB
and PKBß in
Pkb
-deficient cells (Fig.
7E). To test the potential role of PKB
in the survival of
hippocampal neurons, cell cultures were challenged with glutamate (15 mM/24
hours) or staurosporine (50 nM/12 hours) after 7 days in culture. Apoptotic
cells were detected using the TUNEL assay. In untreated cells cultures, no
significant difference in the percentage of apoptotic cells between
Pkb
+/+ and Pkb
-/-
hippocampal neurons was observed (Fig.
7F). After treatment with glutamate or staurosporine, the
percentage of apoptotic cells was significantly increased (51% and 24%,
respectively, P<0.01) in cultures from
Pkb
-/- hippocampal neurons
(Fig. 7F). Additionally, we
analysed the number of apoptotic cells from adult brains (n=5 per
genotype) using TUNEL staining on parasagittal sections. Similar to the
results of untreated cultures, we did not find any difference between
Pkb
+/+ and Pkb
-/- mice
(data not shown).
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Discussion |
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The IGF1/PI3K/PKB pathway plays a crucial role in mammalian brain
development and function (D'Ercole et al.,
2002; Rodgers and Theibert,
2002
). Besides the severe growth retardation, adult mice with IGF1
deficiency exhibit a significant (38%) brain weight reduction
(Beck et al., 1995
). Similar to
the Pkb
mutant mice, all brain parts of the
Igf1-/- mice were affected but the general anatomical
organisation was normal. Furthermore, ablation of IGF1 resulted in a cell
type-dependent loss of neurons, as well as a reduced total number of
oligodendrocytes and hypomyelination (Beck
et al., 1995
; Ye et al.,
2002
). In addition, targeted deletion of IRS2 in mice also
produced a pronounced brain growth deficiency, but in contrast to
Pkb
mutants, the reduction was already apparent during
embryonic (E15.5) development (Schubert et
al., 2003
). By contrast, an increased brain mass was observed in
mice overexpressing IGF1 (Mathews et al.,
1988
; Ye et al.,
1995
). Moreover, mice with brain-specific deletion of PTEN, a
negative regulator of the PI3K/PKB pathway, exhibited an enlarged brain with
seizures and ataxia resembling Lhermitte-Duclos disease
(Backman et al., 2001
;
Kwon et al., 2001
). Less is
known about the consequences of Pkb
and Pkbß
inactivation for mouse brain development. Compared with
Pkb
-/- mice, adult Pkb
and
Pkbß mutant mice showed only a slight decrease in brain weight
(Garofalo et al., 2003
;
Yang et al., 2004
). In both
Pkb
and Pkbß mutant mice, no changes in the
gross brain morphology were reported.
However, inactivation of the Pkb gene resulted in a
significant reduction of brain weight and size. Interestingly,
Pkb
deficiency did not affect the general anatomical
organization of the brain. In vivo 3D MRI and histological analysis excluded
the absence of a specific brain region as the main cause of the weight
reduction, consistent with the result of the broad expression profile among
brain regions. More specifically, the proportionally reduced ventricular
system rules out major disturbances in production, circulation and absorption
of cerebrospinal fluid as a cause of reduced cell size/number in
Pkb
-/- mice. The biological relevance of the MRI
signal alterations in white matter such as the corpus callosum requires
further investigation. Nevertheless, it should be noted that the in vivo MRI
results are consistent with a pronounced, but not complete, deficit in myelin
deposition (Boretius, 2003
),
which is also strongly supported by the histological findings for myelin
staining. In agreement with our results, mice deficient in IGF1, a potent
activator of PKB
, the myelin-rich white matter regions, including
corpus callosum and anterior commissure, were overproportionally reduced by
about 70% (Beck et al., 1995
).
By contrast, mice overexpressing IGF1 display an increased brain weight and
the corpus callosum of the Igf1 transgenic mice was increased in
excess of proportionality (Carson et al.,
1993
).
The PI3-K/PKB signalling pathway plays a crucial role in the determination
of cell size (Scanga et al.,
2000; Shioi et al.,
2002
; Tuttle et al.,
2001
; Verdu et al.,
1999
). Results from transgenic mice overexpressing PKB show larger
cardiac myocytes and thymocytes, or hypertrophy and hyperplasia in the
pancreas (Kovacic et al.,
2003
; Mangi et al.,
2003
). An increase in neuronal soma size was observed in mice with
brain-specific deletion of PTEN (Backman et
al., 2001
; Kwon et al.,
2001
). By contrast, the size of skeletal muscle cells in
Pkb
ß double mutant mice was dramatically reduced
(Peng et al., 2003
). Our
results show that both cell number and cell size are affected, but that
reduced cell size contributes more than the reduced cell number. Additionally,
it has been shown that the mTOR signalling pathway is also involved in the
determination of cell size (Montagne et
al., 1999
; Oldham et al.,
2000
; Zhang et al.,
2000
). PKB modulates mTOR activity by phosphorylating TSC2, with a
subsequent disruption of the TSC1-TSC2 interaction
(Inoki et al., 2002
;
Potter et al., 2003
).
Recent publications have linked PI3-K/PKB with synaptic plasticity and
memory (Dash et al., 2004;
Kelly and Lynch, 2000
;
Lin et al., 2001
;
Robles et al., 2003
;
Sanna et al., 2002
). Wang and
colleagues demonstrated that the A-type
-aminobutyric acid receptors
(GABAAR), which mediate fast inhibitory synaptic transmission, is
phosphorylated by PKB (Wang et al.,
2003
). Phosphorylation of GABAAR leads to an increased
number of receptor on the cell membrane and an increased synaptic
transmission. Additionally, Lin et al. established a role of the PI3-K/PKB
pathway in fear conditioning in the amygdala
(Lin et al., 2001
). As we
found several genes involved in neuronal circuit activity in our microarray
experiment, future behavioural and electrophysiological studies of
Pkb
mutant brains will elucidate the specific role of
PKB
in synaptic transmission, learning and memory.
In summary, we have demonstrated that Pkb-deficient mice
display a phenotype distinct from Pkb
and Pkbß
mutant mice. Our results provide novel insights into the physiological
function of PKB
and suggest a crucial role in postnatal brain
development of mammals. Identification of PKB
specific substrates
involved in postnatal brain development is now of critical importance.
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ACKNOWLEDGMENTS |
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