Stress-Induced Transcriptional Regulation in the Developing Rat Brain Involves Increased Cyclic Adenosine 3',5'-Monophosphate-Regulatory Element Binding Activity
Carolyn G. Hatalski and
Tallie Z. Baram
Departments of Anatomy and Neurobiology and
Pediatrics, University of California, Irvine, California
92697-4475
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ABSTRACT
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The cAMP-regulatory element (CRE) binding protein
(CREB) functions as a trans-acting regulator of genes
containing the CRE sequence in their promoter. These include a number
of critical genes, such as CRF, involved in the hypothalamic response
to stressful stimuli in the adult. The ability of the developing rat
(during the first 2 postnatal weeks) to mount the full complement of
this stress response has been questioned. We have previously
demonstrated the stress-induced up-regulation of the transcription of
hypothalamic CRF during the second postnatal week in the rat. The focus
of the current study was to explore the mechanism of transcriptional
regulation in response to stress through the physiological induction of
transcriptional trans-activators that bind to the CRE in
the developing rat brain. CRE-binding activity was detected via gel
shift analysis in extracts from both the hypothalamus and the cerebral
cortex of the developing rat. CREB was identified in these extracts by
Western blot analysis and was shown to be the major contributor to the
CRE-binding activity by gel shift analysis with two specific antibodies
directed against CREB. After acute hypothermic stress, the abundance of
CRE-binding activity (but not of total immunoreactive CREB), increased
in hypothalamic extracts. This enhanced CRE-binding activity was
blocked by an antiserum directed against CREB and was accompanied by an
apparent increase in CREB phosphorylation. These results indicate that
posttranslational enhancement of CRE-binding activity is likely to
constitute an important mechanism for up-regulation of genes possessing
the CRE sequence in the developing rat hypothalamus by adverse external
signals.
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INTRODUCTION
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The complement of cellular gene expression is determined by
complex interactions of trans-acting transcriptional
regulatory factors with specific DNA sequences. The ability to rapidly
and precisely alter the expression of individual genes in response to
environmental stimuli is believed to result from modulating the
interactions of these transcription factors with their corresponding
DNA-regulatory elements.
Modulation of gene expression by extracellular signals can occur via
activation of G protein-coupled membrane receptors leading to increased
levels of intracellular messengers such as calcium and cAMP (reviewed
in Refs. 14). Genes responsive to cAMP have been found to have a
common DNA promoter sequence, the cAMP-regulatory element (CRE) (5).
Elevation of intracellular cAMP concentration results in
phosphorylation of specific proteins by protein kinase A (2). An
important protein target of protein kinase A-mediated phosphorylation
is the transcriptional trans-acting factor CRE-binding
protein (CREB) (1). CREB and other proteins that bind to the CRE are
members of the bZip superfamily, containing a basic leucine zipper.
These regulatory proteins interact to form homo- or heterodimers to
bind DNA and regulate transcription of responsive genes (2).
Transcriptional regulation leading to altered expression of critical
genes is a fundamental aspect of the response to adverse external or
internal signals, i.e. stress. A key mediator activating
both the hormonal and behavioral responses to stress is the
neuropeptide CRF. Like a number of genes that participate in the stress
response, the CRF promoter contains a CRE consensus sequence, and its
transcription is regulated by alterations in cAMP concentration and CRE
binding (6, 7).
It has been documented that changes in CRE-binding activity and CREB
phosphorylation can occur rapidly in the adult rat brain in response to
diverse environmental stimuli, resulting in activation (or in some
cases inhibition) of transcription (2). However, it is unclear whether
the same transcriptional regulatory factors are activated in the
central nervous system (CNS) of the developing rat. Differences between
the mature and developing hormonal responses to stress signals have
been established. The first 2 postnatal weeks are considered a stress
hyporesponsive period with respect to hypothalamic-pituitary-adrenal
activation (8, 9, 10, 11, 12). This developmental period has been characterized by
attenuated hormonal responses and altered gene regulation in response
to stress as compared with the adult. For example, expression of
c-fos, an immediate early gene that is rapidly up-regulated
by a variety of stress and activation signals in the mature brain, is
not up-regulated in the immature rat brain even by the profound
neuronal activation associated with kainic acid-induced seizures (13).
Similarly, stress-induced up-regulation of CRF mRNA levels occurs only
after the first postnatal week (14). Transcription of both the CRF and
c-fos gene is believed to be regulated (at least in part) by
binding of trans-acting regulatory proteins to the CRE
sequence in the promoters of these genes (6, 7, 15, 16).
This study addressed two principal questions: First, do hypothalamic
and cortical cells express CRE-binding activity during the first 2
postnatal weeks and is this CRE-binding capacity related to CREB?
Second, is the developing organism capable of rapid, adaptive
regulation of its gene repertoire by increasing the activity of
transcriptional regulatory factors to alter expression of a group of
genes such as those containing the CRE sequence?
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RESULTS
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CRE-Binding Activity and CREB in the Anterior Hypothalamus of the
Developing Rat
CRE-binding activity in protein extracts of 12- to 13-day-old rat
brain was demonstrated using gel shift analysis. Protein extracts from
both the anterior hypothalamus (Fig. 1
)
and the cerebral cortex (data not shown) from stress-free, control rats
resulted in band retardation consistent with the presence of binding
activity for the CRE consensus sequence in these extracts. Increasing
concentrations of protein extract resulted in a corresponding increase
in signal intensity of the retarded band (Fig. 1
).

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Figure 1. The CRE-Binding Activity Is Present in Extracts
from the Anterior Hypothalamus of the Developing Rat
Gel shift analysis of a radioactively labeled oligonucleotide
containing the CRE consensus sequence that was preincubated with
increasing concentrations of protein extract (1.25, 2.5, 5, 10, and 20
µg) from the anterior hypothalamus of 12- to 13-day-old rats. No
shift of the DNA fragment was detectable in the absence of brain
extract (lane 1).
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The specificity of the DNA-binding activity to the CRE consensus
sequence was confirmed via competition experiments. CRE-binding
activity was essentially eliminated by preincubation of the tissue
extracts with 100-fold excess of unlabeled oligonucleotide containing
the CRE consensus sequence (Fig. 2
, compare lane 2 with lane 3). This DNA binding was not eliminated by
100-fold excess of unlabeled oligonucleotides containing other DNA
consensus sequences including AP1, glucocorticoid response element
(GRE), octomer binding protein 1 (Oct1), nuclear factor (NF)
B, and
promoterspecific transcription factor (SP1) (Fig. 2
).

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Figure 2. The CRE-Binding Activity in Hypothalamic Extracts
Is Specific
Gel shift analysis of a radioactively labeled oligonucleotide
containing the CRE consensus sequence incubated with 10 µg protein
extract from the anterior hypothalamus of 12- to 13-day-old rats (lanes
28). DNA-binding activity (lane 2) is eliminated by 100-fold excess
unlabeled DNA fragment containing the CRE consensus sequence (lane 3)
but not by 100-fold excess DNA fragments containing consensus sequences
for AP1 (lane 4), GRE (lane 5), Oct1 (lane 6), NF B (lane 7), or SP1
(lane 8).
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The presence of the protein CREB was determined in extracts from the
anterior hypothalamus and the cerebral cortex using Western immunoblot
analysis (Fig. 3
). Two different
antibodies were used to identify CREB: the first was a polyclonal
antiserum directed against CREB-1 (epitope corresponding to amino acids
295321), and the other was a polyclonal antiserum specific for the
phosphorylated form of CREB (epitope corresponding to amino acids
128141) (17). Use of each antibody in Western immunoblot analysis of
size-fractionated, membrane-bound extracts from both the anterior
hypothalamus and the cerebral cortex resulted in a single predominant
band with a molecular mass of approximately 43 kDa, consistent with the
molecular mass of CREB (18).

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Figure 3. CREB and Phosphorylated CREB Are Present in Both
Anterior Hypothalamic and Cortical Extracts of the Developing Rat
Western blot analysis of 10 µg protein extract from the anterior
hypothalamus (lanes 1 and 3) and cerebral cortex (lanes 2 and 4) of 12-
to 13-day-old rats using antisera directed against CREB-1 (lanes 1 and
2) and phosphorylated CREB (lanes 3 and 4). A predominant, 43-kDa
immunolabeled band is evident in all lanes, consistent with CREB. CREB
and the phosphorylated form of CREB are present in the anterior
hypothalamus (lanes 1 and 3) and in the cerebral cortex (lanes 2 and
4).
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Further confirmation that CREB was at least partially responsible for
the CRE-binding activity of anterior hypothalamic extracts was obtained
using gel shift analysis of this DNA-binding activity after incubation
of the protein extract with the same anti-CREB antibodies used for the
Western immunoblot analysis. The antiserum directed against
phosphorylated CREB resulted in a supershifted band (Fig. 4
, lane 3). The antiserum directed
against CREB-1 blocked the CRE-binding activity of the extract, likely
via steric hindrance (Fig. 4
, lane 4).

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Figure 4. Functional and Antigenic Specificity of the
CRE-Binding Activity of Rat Hypothalamic Extract
Gel shift analysis of a radioactively labeled oligonucleotide
containing the CRE consensus sequence with 10 µg protein extract from
the anterior hypothalamus of 12- to 13-day-old rats (lanes 25).
Before incubation with the labeled oligonucleotide, samples were
treated as follows. A, Incubation with 100-fold excess of unlabeled
oligonucleotide containing the CRE consensus sequence resulted in
elimination of the retarded band representing CRE-binding activity
(lane 2); B, incubation with an antiserum directed against
phosphorylated CREB ( pCREB) caused additional retardation with a
supershifted band (lane 3, indicated by arrow in the
margin); C, incubation with antiserum directed against CREB-1
( CREB-1) resulted in nearly complete inhibition of DNA-binding
activity (lane 4); D, incubation with buffer alone (lane
5).
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Hypothermic Stress Results in an Increase of CRE-Binding Activity
in the Anterior Hypothalamus but Not the Cerebral Cortex
Experiments were performed to determine whether there was an
alteration in CRE-binding activity in the developing rat brain in
response to stress. Hypothermic stress, which is a potent age-specific
stressor for the immature rat (14), was employed. Animals were killed
at several time points after the hypothermia, and plasma was analyzed
for corticosterone levels as a measure of stress. Consistent with
previous results (11, 14), hypothermic stress resulted in a significant
increase in plasma corticosterone levels (Fig. 5
).

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Figure 5. Plasma Corticosterone Levels after Acute
Hypothermic Stress
Plasma corticosterone (CORT) was measured in stress-free rats and at
several time points after the termination of hypothermic stress. A
robust stress-induced elevation of plasma CORT is evident by the end of
the hypothermia period and peaks 60 min later. Each column represents
the mean of at least four 12- to 13-day-old rats with bars indicating
the SEM. Asterisks represent a significant
difference from the stress-free group (P < 0.0001)
determined by unpaired Students t test.
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Extracts from the anterior hypothalamus and the cerebral cortex were
analyzed for CRE-binding activity by gel shift analysis. Acute
hypothermic stress resulted in an increased CRE-binding activity in
hypothalamic extracts as indicated by enhanced signal intensity of the
retarded band (Fig. 6
). Maximal amounts
of CRE-binding activity were detected 30 min after the termination of
the hypothermic stress. Levels of CRE-binding activity at 180 min
remained elevated relative to tissue derived from stress-free controls.
The CRE-binding activity of these extracts was substantially reduced by
competition with 100-fold excess unlabeled oligonucleotide containing
the CRE consensus sequence (Fig. 6
), suggesting that this DNA-binding
activity is specific for the CRE. Unlike the stress-induced
increase in CRE-binding activity in the anterior hypothalamus, there
was no consistent change in the CRE-binding capacity of cortical
extracts after hypothermic stress (Fig. 7
). The CRE-binding activity in samples
from both the anterior hypothalamus and the cerebral cortex of rats
subjected to hypothermic stress was blocked by exposure of the extracts
to the antiserum directed against CREB-1 (Fig. 8
). In addition, Southwestern blot
analysis of hypothalamic protein extracts indicated that a protein with
molecular mass consistent with that of CREB was capable of binding an
oligonucleotide containing the CRE consensus sequence (Fig. 9A
). This CRE-binding capacity was
increased in extracts obtained from animals subjected to hypothermic
stress, although overall CREB immunoreactivity did not increase in
response to stress (Fig. 9B
).

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Figure 6. Increased CRE-Binding Activity in the Anterior
Hypothalamus in Response to Hypothermic Stress
Gel shift analysis of the DNA containing the CRE consensus sequence
using rat anterior hypothalamic extracts at several time points after
hypothermic stress. Each lane represents 10 µg of protein extract
from 12- to 13-day-old rats. Samples in lanes 1 and 7 were derived from
rats under stress-free conditions. For the other samples, rats were
killed at 0 min (lanes 2 and 8), 15 min (lanes 3 and 9), 30 min (lanes
4 and 10), 60 min (lanes 5 and 11) or 180 min (lanes 6 and 12) after
termination of hypothermic stress. A progressive increase in the
CRE-binding capacity of the hypothalamic extracts is evident after
stress, peaking at the 30-min time point (lane 4). Specificity of the
CRE-binding activity was determined by competing with 100-fold excess
of an unlabeled oligonucleotide containing the CRE consensus sequence.
All of the stress-induced CRE-binding activity is eliminated by excess
unlabeled CRE (lanes 712)
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Figure 7. Stress Increases the CRE-Binding Activity in the
Anterior Hypothalamus but Not the Cerebral Cortex
Gel shift analysis of DNA containing the CRE consensus sequence
using 10 µg protein extracts from the anterior hypothalamus (lanes
15) and the cerebral cortex (lanes 610). Lanes represent protein
extracts from 12- to 13-day-old rats under stress-free conditions
(lanes 1 and 6) or 0 min (lanes 2 and 7), 15 min (lanes 3 and 8), 30
min (lanes 4 and 9), and 60 min (lanes 5 and 10) after maximal
hypothermic stress. Increased intensity of the CRE-binding activity
signal is evident in the hypothalamic extracts after stress (lanes
35) as compared with the stress-free condition (lane 1). Signal
intensity of the CRE- binding activity of cortical extracts is not
consistently influenced by exposure to stress (lanes 710 compared
with lane 6).
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Figure 8. Stress-Induced CRE-Binding Activity Is Blocked with
an Antiserum Directed against CREB-1
Gel shift analysis of DNA containing the CRE consensus sequence using
10 µg protein extracts from the anterior hypothalamus (lanes 14 and
9 and 10) and the cerebral cortex (lanes 58 and 11 and 12) of 12- to
13-day-old rats. Samples were incubated with antiserum directed against
CREB-1 ( CREB-1), which blocks the DNA-binding activity of CREB
(lanes 18), or with buffer (lanes 912) before addition of the
labeled oligonucleotide containing the CRE consensus sequence. Protein
extracts were obtained from rats under stress-free conditions (lanes 1,
5, 9, and 11), or 0 min (lanes 2 and 6), 30 min (lanes 3, 7, 10, and
12), and 60 min (lanes 4 and 8) after maximal hypothermic stress. The
stress-free levels of CRE-binding activity in the hypothalamus (lane 9)
and cortex (lanes 11 and 12) and the stress-induced increase in
CRE-binding activity in the hypothalamus (lane 10) are eliminated by
preincubation of the extracts with the CREB-1 antibody (lanes
18).
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Figure 9. Stress-Induced CRE-Binding Activity in the
Hypothalamus Is Specific
A, Southwestern blot analysis of protein extracts from the anterior
hypothalamus. Protein-bound membranes were incubated with
32P-labeled double-stranded oligonucleotide containing the
CRE consensus sequence. Protein extracts obtained from 12- to
13-day-old rats exposed to stress shows reveal increased signal
intensity compared with stress-free control. Size of resultant bands
are consistent with CREB (43 kDa). B, Western blot analysis of same
membrane using an antiserum directed against CREB-1. No increase in
CREB immunoreactivity in extracts from animals exposed to stress is
apparent.
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The augmented CRE-binding activity in the anterior hypothalamus after
hypothermic stress was not associated with an overall increase of CREB
immunoreactivity in these extracts (Fig. 10
, top). However,
immunoreactive phosphorylated CREB was increased with stress (Fig. 10
, bottom) as indicated by Western blot analysis. This increase
in phosphorylated CREB abundance was also specific to the anterior
hypothalamus since it was not observed in cortical extracts.

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Figure 10. Stress Increases Phosphorylated CREB, but not CREB
Immunoreactivity, in the Hypothalamus
Western blot analysis of 20 µg protein extract from the anterior
hypothalamus (left) or the cerebral cortex
(right) of the 12- to 13-day-old rat under stress-free
conditions, or 0 min, 15 min, and 30 min after the termination of
hypothermic stress. Blots were bound with an antiserum directed against
CREB ( CREB) and phosphorylated CREB ( pCREB). There is no apparent
stress-induced change in the abundance of CREB immunoreactivity in
either the hypothalamic or cortical extracts. However, there is an
apparent increase in phosphorylated CREB immunoreactivity in
hypothalamic (left bottom) but not cortical
(right bottom) extracts after hypothermic stress as
compared with extracts from the stress-free rats.
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These results, shown here for 12- to 13-day-old rats, were replicated
using 4- to 6-day-old rats; i.e. CRE-binding activity and
the stress-induced enhancement of both CRE-binding and phosphorylated
CREB in the anterior hypothalamus were demonstrated throughout the
first 2 postnatal weeks.
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DISCUSSION
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The current study demonstrates increases in CRE-binding activity
and phosphorylation of CREB that provide a mechanism for stress-induced
up-regulation of the transcription of CRE-containing genes (such as
CRF) in the developing hypothalamus. First, these results provide
evidence that CRE-binding activity is present in the developing rat
hypothalamus and cerebral cortex. Furthermore, CREB is likely to be the
principal contributor to this CRE-binding activity since DNA binding is
essentially blocked by an antiserum directed against CREB-1 (Fig. 4
).
Previous investigations have demonstrated DNA binding activity of CREB
(19) and constitutive expression of CREB-mRNA (20) in the developing
rat brain. This study documents that phosphorylated CREB is present and
is involved in DNA binding in hypothalamic and cortical extracts of the
developing rat, as suggested by the supershift (Fig. 4
) and Western
blot (Fig. 3
) analyses using an antiserum specific for the
phosphorylated form of CREB as well as a Southwestern blot (Fig. 9A
)
showing that a protein with molecular mass consistent with CREB binds
to DNA containing the CRE consensus sequence.
The observed stress-induced increase in CRE binding was specific, as
indicated by the ability of excess unlabeled oligonucleotide containing
the CRE consensus sequence to compete for this binding (Fig. 6
). CREB
is likely to be a major constituent not only of the basal CRE-binding
activity as shown above, but also of the stress-induced increase in
CRE-binding activity. The evidence for this fact is provided by the
elimination of the majority of CRE-binding capacity of the hypothalamic
and cortical extracts by exposure to antiserum directed against CREB-1,
which blocks the interaction of CREB with the CRE-DNA fragment (Fig. 8
), and by the observed increase of CRE binding by a protein of a size
consistent with CREB (43 kDa) by Southwestern blot analysis (Fig. 9A
).
In addition to enhanced CRE-binding capacity, hypothermic stress
resulted in increased phosphorylated CREB in extracts from the anterior
hypothalamus, but not the cerebral cortex (Fig. 10
). These findings
indicate that the developing hypothalamus exhibits plasticity in its
complement of activated genes in response to environmental
alterations.
The mechanism for the stress-induced enhancement of CREB-mediated DNA
binding may be deduced from analysis of the data presented above.
Stress did not lead to increased levels of total CREB in the
hypothalamus or cortex (Fig. 10
). In contrast, phosphyrylated CREB and
CRE-binding activity were both enhanced in the hypothalami of
cold-stressed rat pups. These findings are consistent with a
posttranslational mechanism by which stress acts to activate existing
CREB and increase CRE-binding capacity. CREB phosphorylation may be
accompanied by activation of other components of the physiological CRE-
binding protein complex.
CRE-binding activity and CREB play an important role in gene
regulation. The promoters of numerous genes possess the CRE consensus
sequence in a position suggesting a role for this element in
transcriptional modulation. For example, the immediate early gene
c-fos (15, 16), as well as the genes for arginine
vasopressin (AVP) (21), proenkephalin (22, 23), and CRF (6, 7) are
likely regulated by transcriptional factors that bind to the CRE
sequence in their promoters. The regulation of these genes is of
particular interest because their expression is enhanced in the adult
rat hypothalamus after stress (15, 24, 25, 26, 27, 28, 29). A recent comparative,
quantitative time course analysis of the levels of mRNA of these genes
in the adult rat hypothalamus has shown concurrent expression of
phosphorylated CREB and CRF heteronuclear RNA, but delayed expression
of c-fos and AVP heteronuclear RNA (29). This observation
suggests that, in addition to phosphorylated CREB, other regulatory
factors (such as AP1) may contribute to expression of certain genes in
response to stress. In addition, the presence, phosphorylation, and
dimerization of CREB is required but not always sufficient to activate
a CRE-possessing promoter (1, 2).
In contrast to the significant information available regarding the
regulation of stress-responsive, CRE-possessing genes in the mature
CNS, relatively little is known about the mechanisms of transcriptional
regulation by adverse external signals during development. Early
studies suggested that the immature rat does not possess a robust
hormonal response to stress. However, previous studies from our
laboratory have revealed that age-appropriate, hypothermic stress
increased steady-state CRF mRNA in the hypothalamus of rats starting in
the second postnatal week (14). The current study demonstrates that
phosphorylated CREB- and CRE-binding activity are present and inducible
in the rat anterior hypothalamus during the first 2 postnatal weeks.
Enhanced CREB phosphorylation and binding activity may thus provide the
mechanism for augmentation of the transcription of stress-responsive
genes in the developing hypothalamus. Specifically, CREB-mediated
increased transcription of CRE-possessing genes, such as CRF, in
response to hypothermic stress may provide a critical adaptive capacity
to the neonatal rat: the stress neurohormone CRF activates both
hormonal and behavioral responses that mediate survival in adverse
situations.
As determined in the current study, hypothermic stress increased
CRE-binding activity in the hypothalamus, but not in the cerebral
cortex, of developing rats (Fig. 7
). This finding is consistent with
several hypotheses: First, the lack of alteration in CRE binding in the
cerebral cortex may derive from the functional immaturity of this brain
region in the rat during the first 2 weeks of life. Alternatively, the
regulatory mechanisms of CREB-responsive genes in the hypothalamus may
differ from those in the cerebral cortex. Finally, the stimulus of
hypothermic stress, which activates relevant genes in the hypothalamus,
may not be transmitted to the cortex in a context leading to gene
activation.
The ability to adapt rapidly to stimuli is likely to be critical for
the survival of the organism in a changing environment. This issue has
been discussed in detail in a body of work addressing the stress
response in the developing rat (8, 9, 10, 11, 12). Early work suggested that
during the first 2 weeks of life there is little hormonal stress
response with only small changes in plasma levels of the stress
hormones ACTH and corticosterone. More recent studies have demonstrated
that the developing hypothalamus does respond to external stimuli via
secretion of CRF (14) and vasopressin (30, 31) which, in
turn, leads to elevated plasma ACTH and corticosterone (11). The
current study shows that transcriptional regulation of gene expression
in the developing hypothalamus can be modulated by envirinmental
changes (i.e. stress). Thus, these environmental stimuli are
capable of inducing both immediate (hormonal) and long-term alterations
in hypothalamic function by qualitatively and quantitatively modulating
the repertoire of gene expression in this critical CNS region.
In conclusion, this study demonstrates that transcription of
hypothalamic genes that are regulated by CRE binding is likely to be
modulated by environmental stimuli during the first 2 postnatal weeks.
This is consistent with physiological observations and confirms the
critically important adaptability of the functions subserved by this
brain region.
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MATERIALS AND METHODS
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Animals and Stress Paradigm
Timed pregnancy Sprague Dawley-derived rats (Zivic-Miller,
Zelienople, PA) were housed under a 12-h light, 12-h dark cycle and
given unlimited access to food and water. Delivery was verified at 12-h
intervals and the day of birth was considered day 0; litters were
culled within 24 h of delivery to 12 pups of both sexes. Each
experiment included all of the experimental groups and was performed at
least twice. Samples from each timepoint consisted of a pool of tissue
from two to three pups without attention to sex. To avoid potential
effects of litter size on brain maturation, members of each litter were
used at a single age so that litter size was stable from birth to the
time of death. Before each experiment, cages were undisturbed for
24 h, and all experiments were performed at 08001100 h. All
experiments were conducted in accordance with the NIH Guide for the
Care and Use of Laboratory Animals and approved by the Institutional
Animal Care Committee.
Twelve or 13-day-old rats (four litters) were individually subjected to
maximally tolerated hypothermic stress, defined by development of
rigor, little response to tactile stimulus, and average core
temperature of 9.8 C, as previously described in detail (14). This
required cold exposure for 40100 min for individual rats. These
experiments were repeated on 4- to 6-day-old rats (three litters, data
not shown). Maximal cold stress at this age required 3060 min. After
cold-separation stress, pups were placed on a euthermic pad where they
regained normal core temperature of 3334 C in 1015 min. Rats were
rapidly decapitated at 0, 15, 30, 60, or 180 min following the
hypothermic stress. Stress-free controls were killed within 2 min of
disturbance. Trunk blood was collected at time of death for analysis of
plasma corticosterone levels by RIA (ICN, Irvine, CA) as previously
described (32). Assay sensitivity was 0.5 µg/dl and interassay
variability was less than 5%.
Preparations of Brain Extracts
Brains were dissected on ice for isolation of the anterior
hypothalamus and the cerebral cortex. The anterior hypothalamus
consisted of a tissue block including the paraventricular,
suprachiasmatic, and supraoptic nuclei, as well as the anterior
hypothalamic and medial preoptic areas. The cerebral cortex tissue
block consisted of the occipital cortical area. At each timepoint,
tissue from two pups was pooled, immersed into 1 ml cold PBS (pH 7.4),
and homogenized (micro tissue grinder, Kontes, Vineland, NJ) on ice.
The homogenate was centrifuged at 4,000 x g for 10 min at 4
C and the resulting pellet was frozen at -80 C. The pellet was
resuspended in 5 volumes of extraction buffer [10 mM
HEPES, pH 7.9, 400 mM NaCl, 100 µM EGTA, 500
µM dithiothreitol (DTT), 500 µM
phenylmethylsulfonylfluoride, and 5% (vol/vol) glycerol] by
trituration, vortex, and homogenization. Membranes and debris were
separated by centrifugation at 10,000 x g for 15 min
at 4 C. The resulting supernatant was aliquoted and frozen at -80 C.
Extracts were analyzed for protein concentration by protein assay
(Bio-Rad, Hercules, CA) using BSA as a standard.
Oligonucleotide Preparation
For gel shift analysis, 50 ng (3.5 pmol) of double-stranded DNA
oligonucleotide (22 nucleotides in length) containing the CRE consensus
sequence (Stratagene, La Jolla, CA) were labeled with
[32P]ATP (New England Nuclear, Boston, MA) using 30 U of
T4 polynucleotide kinase (Promega Corp., Madison, WI) according to the
manufacturers protocol. The oligonucleotide was extracted first with
phenol-chloroform-isoamyl alcohol (48:48:1) and then with
chloroform-isoamyl alcohol (48:1) followed by column chromotography
(NucTrap, Stratagene). Incorporation of 32P was determined
as 3575% with specific activity of 5 x 105-2
x 106 cpm/µg. For Southwestern blot analysis, 2 pmol CRE
oligonucleotide were labeled with 20 pmol
[32P]-
-deoxy-ATP (New England Nuclear) using terminal
deoxynucleotidyl transferase (Promega Corp.) according to the
manufacturers instructions. The labeled oligonucleotide was purified
by column chromotography (NucTrap, Stratagene).
Gel Shift Analysis
Gel shift analysis was modified from published protocols (33, 34). Briefly, 10 µg protein extract were incubated for 1 h at
room temperature with 100 pg labeled oligonucleotide containing the CRE
consensus sequence. The final incubation volume was 20 µl containing
10 mM Tris pH, 7.5, 50 mM NaCl, 5
mM MgCl2, 1 mM EDTA, 1
mM DTT, 1 µg poly (dI-dC) (Sigma, St. Louis, MO), and
0.1% (wt/vol) Triton X-100. After incubation, 2 µl of loading buffer
(50% glycerol, 0.5% bromphenol blue, 0.5% xylenecyanole) was added,
and the samples were electrophoresed on a 5% polyacrylamide gel
(acrylamide-bisacrylamide, 37.5:1) in 0.5 x TBE buffer (44.5
mM Tris-borate, 44.5 mM boric acid, and 1
mM EDTA) at 160 V. Gels were dried and exposed to film
(Reflection NEF, Dupont, Boston, MA) using a Reflection intensifying
screen. Each assay was repeated at least once to verify results.
Competition experiments were performed by including 10 ng unlabeled
double-stranded DNA in the reaction mixture. The oligonucleotides used
for competition were each 22 nucleotides long and included the
consensus sequence for CRE, AP1, GRE, Oct1, NF
B, or SP1
(Stratagene).
Supershift and antibody-blocking experiments were performed by
incubating the protein extract with antisera (1:100) for 2 h at 4
C before the addition of radiolabeled oligonucleotide.
Western Immunoblot and Southwestern Blot Analysis
Samples were analyzed by Western immunoblot and Southwestern
blot by size fractionation of 20 µg protein extract through
denaturing 10% SDS-PAGE (35) under reducing conditions, followed by
transfer to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH)
(36). Prestained molecular mass markers (Bio-Rad) were included on each
gel. All incubations were performed at room temperature unless
otherwise noted.
For Western immunoblots, membranes were stained with Ponceau S (Sigma)
(37) to verify transfer. Membranes were then blocked for 1 h with
buffer consisting of 0.5% nonfat dry milk (NFDM, Carnation) and 0.05%
Tween-20 (Fisher Scientific, Pittsburgh, PA) in Tris-balanced saline
(TBS, 50 mM Tris-HCl, pH 7.5, and 150 mM NaCl)
followed by overnight incubation at 4 C with 1:1000 dilution of
antisera in the same buffer. The membranes were washed three times in
TBS, incubated for 2 h with 1:1000 dilution of horseradish
peroxidase-conjugated secondary antibody (goat anti-rabbit IgG, Sigma),
washed five times in TBS, and visualized using a chemiluminescent
substrate (ECL, Amersham, Arlington Heights, IL).
Southwestern blots were performed based on published protocols (38, 39). First, membranes were rehydrated in HEPES buffer (25
mM HEPES, pH 7.9, 25 mM NaCl, 5 mM
MgCl2, 0.5 mM DTT) for 30 min. Then, membranes
were blocked for 1 h in 5% NFDM in HEPES buffer, followed by a
rinse in 0.25% NFDM in HEPES buffer. Probe was bound by incubating
membranes overnight with 2 pmol of 32P-labeled CRE
oligonucleotide in 5 ml of HEPES buffer containing 0.25% NFDM and 5
µg poly(dI-dC) (Sigma). Membranes were rinsed three to five times in
HEPES buffer containing 0.25% NFDM, air dried, and exposed to
film.
Antibodies
Rabbit polyclonal antiserum 5322, specific for the
phosphorylated form of CREB (17) (epitope corresponding to amino acids
128141, with antibodies to the unphosphorylated polypeptide removed
by adsorbtion), was a generous gift from Dr. M. Montminy. Rabbit
polyclonal antiserum specific for CREB-1 (epitope corresponding to
amino acids 295321) and non-cross-reactive with other ATF/CREB
transcription factors was purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. M. Montminy for kindly providing the antiserum
specific for phosphorylated CREB. The technical support of L. Schultz
is greatly appreciated. We also thank Dr. M. Villalba, B.
Tantayanubutr, and M. Eghbal-Ahmadi for comments on this
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Tallie Z. Baram, M.D, Ph.D., Med Sci I, 4475; University of California at Irvine, Irvine, California 92697-4475.
This work was supported by NIH Grant NS-28912 (to T.Z.B.).
Received for publication May 5, 1997.
Revision received October 1, 1997.
Accepted for publication October 1, 1997.
 |
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