The Psychiatric Institute, Department of Psychiatry, University of
Illinois at Chicago, Chicago, Illinois; and Veterans Affairs Chicago
Health Care System (West-Side Division), Chicago, Illinois
To define the molecular mechanisms of abnormal hypothalamic pituitary
adrenal (HPA) axis during ethanol dependence, we investigated the
effect of chronic ethanol treatment (15 days) and its withdrawal (24 h)
on the expression of glucocorticoid receptors (GRs) and glucocorticoid
response element (GRE)-DNA binding in the rat brain. The effects of
chronic mianserin [serotonin (5-HT)2A/2C antagonist] treatment on these parameters in various brain structures of control diet-fed and ethanol-fed rats were also investigated. It was found that
ethanol treatment and withdrawal significantly decreased the GR protein
levels in cortical (cingulate gyrus, frontal, parietal, and piriform
cortex) and amygdaloid (central, medial, and basolateral) structures
and paraventricular nucleus (PVN) of hypothalamus of rats. It was also
observed that ethanol treatment produced significant reductions in GR
protein levels in various hippocampal structures (CA1, CA2, CA3, and
dentate gyrus), but these changes were normalized during ethanol
withdrawal. Ethanol treatment also significantly decreased GRE-DNA
binding in rat cortex and hippocampus, which remained decreased in the
cortex but reverted to normal in hippocampus during ethanol withdrawal.
Chronic mianserin (alone) treatment had no effect on cortical GRE-DNA
binding and GR protein levels in cortical, amygdaloid, or PVN
structures but significantly decreased the GR protein expression in
various hippocampal structures and GRE-DNA binding in whole
hippocampus. However, when administered concurrently with ethanol
treatment, mianserin significantly antagonized the reductions in
cortical GRE-DNA binding and in GR protein expression in cortical, PVN,
and central, but not medial and basolateral, amygdaloid structures
during ethanol withdrawal. On the other hand, mianserin treatment along
with ethanol administration significantly decreased the hippocampal
GRE-DNA binding and GR protein expression in various hippocampal
structures during ethanol withdrawal. Furthermore, ethanol treatment
and its withdrawal or mianserin treatment had no effect on the
neuron-specific nuclear protein levels in the various brain structures.
Taken together, these results indicate that interaction of
5-HT2A/2C receptors with GRs in cortical, central
amygdaloid, and PVN structures may play a role in neural mechanisms of
alcohol dependence. It is possible that decreased GR expression in PVN
may be responsible for the abnormal HPA axis during ethanol exposure
and withdrawal.
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Introduction |
The
hypothalamic pituitary adrenal (HPA) axis is one of the primary
physiological systems activated during stress situations (Herman and
Cullinan, 1997
; Plotsky et al., 1998). The release of
corticotropin-releasing factor (CRF) from the paraventricular nucleus
(PVN) of the hypothalamus activates the release of adrenocorticotropin from pituitary, which stimulates the production of glucocorticoids from
the adrenals. The glucocorticoids regulate the HPA axis through a
negative feedback mechanism via binding to soluble glucocorticoid receptors at the pituitary, hypothalamic, and extra hypothalamic levels
and, thus, inhibit the release of CRF and adrenocorticotropin (Plotsky,
1991; Jacobson and Sapolsky, 1991; Feldman and Weidenfeld, 1995). The
glucocorticoid binding receptor has been divided into type
1-mineralocorticoid receptor (MR) and type 2-glucocorticoid receptor
(GR) (Reul and De Kloet, 1986; Funder, 1992). The GR act as a
ligand-inducible gene transcription factor, and after activation, the
GR complex translocates into nucleus where it binds to glucocorticoid
response element (GRE) of gene promoters and, thus, regulates the
expression of selected genes (Simons et al., 1992; Gower, 1993).
It has been shown that acute and chronic ethanol consumption leads to a
hyperactive HPA axis both in animals and humans, and this may be
related to increased hypothalamic CRF secretion (Rivier et al., 1984;
Wand, 1993; Rivier, 1996). The hyperactive HPA axis has also been shown
during ethanol withdrawal after long-term exposure in animals and
humans (Tabakoff et al., 1978; Wand, 1993). Furthermore, it has been
shown that adrenalectomy prevents the development of ethanol preference
in rats (Fahlke and Eriksson, 2000). These results indicate that the
HPA axis seems to be one of the physiological systems implicated in the
action of acute and chronic ethanol exposure. Ethanol exposure and
withdrawal possibly disrupts the negative feedback mechanisms of HPA
axis regulation, however, the molecular mechanism by which this process takes place is not well understood. It is also unknown how ethanol exposure and withdrawal modify the GRE-DNA binding in rat brain structures. Therefore, one goal of the present study was to examine the
effect of chronic ethanol treatment and its withdrawal on the
expression of GR in various brain structures including PVN and also on
GRE-DNA binding in rat cortex and hippocampus.
There are several lines of evidence, which indicate that the HPA
axis is regulated by several neurotransmitters including serotonin
(5-HT) (Feldman and Weidenfeld, 1995; Herman and Cullinan, 1997). It
has been shown that drugs that have ability to enhance 5-HT function
can stimulate the HPA axis (Van de Kar, 1991; Bagdy et al., 1989).
Among multiple 5-HT receptors that exist in the brain, it was found
that at least 5-HT1A,
5-HT2A, and 5-HT2C
receptors in the PVN have stimulatory effects on HPA axis functions
(Bagdy and Makara, 1994; Bagdy, 1996; Van de Kar et al., 2001). The
neuroendocrine studies suggest that GRs in PVN are the major site for
the feedback action of glucocorticoids (Plotsky,1991). It is possible
that 5-HT2A/2C receptors in the neural circuitry
of PVN may interact with GR to maintain the normal HPA axis, and this
interaction may be disturbed by ethanol exposure. We, therefore, also
examined the effect of mianserin (5-HT2A/2C
antagonist) on GR protein expression in various brain structures
including PVN during chronic ethanol exposure and its withdrawal.
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Materials and Methods |
Animals and Treatment.
Male Sprague-Dawley rats weighing 250 to 260 g (at the beginning of the experiment) were used in all
experiments. All animal procedures were in accordance with the National
Institute of Health "Guide for Care and Use of Laboratory Animals"
and were approved by the Animal Care Committee of the University of
Illinois at Chicago and VA Chicago Health Care System (West Side
Division), Chicago. Ethanol administration to rats was performed by
oral ethanol feeding as described previously (Pandey, 1996; Pandey et
al., 1999, 2001). After a brief acclimation period, rats were housed in
individual cages and offered 100 ml of Lieber-DeCarli control diet
(Lieber-DeCarli Diet 82; Bioserve Inc., Frenchtown, NJ) as their sole
source of food and fluid. Both control and ethanol liquid diets are
nutritionally complete diets (Bioserve Inc.; Lieber and DeCarli, 1982).
Fresh diet was provided between 5 and 6 PM every day. For chronic
ethanol study, one group of rats was also introduced gradually to
ethanol and maintained on the liquid diet containing ethanol (9%, v/v)
for 15 days and another group of rats received the control diet for 15 days (pair-fed control group). Ethanol-fed rats were withdrawn for 0, 12, 24, and 72 h and sacrificed, and brains were then used to
study the time course for changes in GRE-DNA binding.
To study the expression of GR, another batch of rats were treated with
ethanol or control diet for 15 days as described above. For chronic
mianserin treatment studies, the control liquid diet-fed and the
ethanol diet-fed groups also received mianserin (10 mg/kg; i.p.) or
vehicle once daily at 8 to 9 AM for 15 days. It has been shown that the
drug mianserin disappeared from the rat brain with a half-life of 1 to
3 h (Sanders-Bush et al., 1987). Mianserin-treated rats were used
for neurochemical studies 24 h after the mianserin injection on
the 15th day. For ethanol withdrawal studies, groups of ethanol-fed
rats with and without mianserin treatment were withdrawn for 24 h
after 15 days of ethanol treatment. Thus, there were five groups of
rats in the chronic ethanol and chronic mianserin study: (1) control
liquid diet-fed plus vehicle, (2) ethanol-fed (0-h withdrawal) plus
vehicle, (3) ethanol-withdrawn (24 h after 15 days of ethanol
treatment) plus vehicle, (4) control liquid diet-fed plus chronic
mianserin-treated (15 days of i.p. 10 mg/kg/day), and (5)
ethanol-withdrawn (24 h after 15 days of ethanol treatment) plus
chronic mianserin-treated rats. In all experiments, we measured the
daily ethanol-diet intake of the various ethanol-fed rats and
control-diet intake in control rats. All these rats were anesthetized with pentobarbital (50 mg/kg, i.p.) and perfused with 4%
paraformaldehyde, and the brains were collected for
immunohistochemistry as described below. Another batch of rats was
prepared as described above, the rats were decapitated, and their
brains were removed. The cerebral cortices and hippocampi were
dissected out and frozen at 
80°C until used for measurement of
GRE-DNA binding activities as described below. There were no
differences in mean body weight among the different groups of rats.
Measurement of GRE-DNA Binding Activity by Gel Mobility Shift
Assay: Preparation of Nuclear Extracts.
Nuclear extracts from the
cortices, and hippocampi areas were prepared according to the method of
Pandey et al. (1999). Tissues were homogenized in buffer I [10 mM
4-(2-hydroxy ethyl-)1-piperazineethane sulfonic acid (HEPES), pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 µg/ml pepstatin]. The homogenates were centrifuged at 100,000g for 30 min. The resulting pellet was
resuspended in buffer II (20 mM HEPES, pH 7.9, 0.84 M NaCl, 1.5 mM
MgCl2, 0.4 mM EDTA, 0.5 mM DTT, 50% glycerol,
and protease inhibitors as in buffer I). After 15 min of incubation on
ice with frequent agitation, nuclear extracts were collected by
centrifugation at 20,000g for 15 min, and protein content of
the nuclear extracts was determined.
Preparation of DNA Probes.
Commercially available
(Stratagene, La Jolla, CA) oligonucleotides carrying regulatory
elements of GRE sequence (5'-GATCA GAACA CAGT GTTCTCTA-3') were used.
The probes were end-labeled with [
-32P]ATP
using T4 polynucleotide kinase according to the manufacturer's method
(US Biochemicals, Cleveland, OH).
Gel Mobility DNA Binding Assay.
GRE-DNA binding reactions
were carried out by incubating 15 µg of the nuclear protein with 1 µg of poly(dI-dC) and 6 µg of bovine serum albumin in a reaction
mixtures [20 mM HEPES, pH 7.9, 1 mM DTT, 0.3 mM EDTA, 0.2 mM ethylene
glycol bis (
aminoethyl ether)
N,N,N',N'-tetraacetic acid,
80 mM NaCl, 10% glycerol, and 0.2 mM phenylmethylsulfonyl fluoride]
for 15 min at room temperature. Approximately 40,000 dpm of
32P-labeled GRE oligonucleotide was added, and
the incubation was continued for an additional 30 min. DNA-protein
complexes were resolved on a 4% nondenaturing polyacrylamide gel in a
buffer containing 25 mM Tris borate (pH 8.2) and 0.5 mM EDTA. The gel was dried and autoradiographed with intensifying Kodak film (Eastman Kodak, Rochester, NY) at 80°C. The optical densities of the bands of the DNA-protein complexes on the autoradiogram were measured using
Loats Image Analysis System (Loats Associates, Inc., Westminster, MD),
and values were expressed as percentage of control. For the competitive
experiment, the nuclear extract protein first incubated with unlabeled
GRE oligonucleotides (50 or 100 ng) and then with 32P-labeled GRE oligonucleotide probes as
described above.
Gold-Immunolabeling of Glucocorticoid Receptor and
Neuron-Specific Nuclear (NeuN) Proteins.
Rats were anesthetized
and intracardially perfused with normal saline (100 ml) followed by 400 ml of 4% ice-cold paraformaldehyde fixative. Brains were removed and
placed in the fixative for 20 h at 4°C. After postfixation,
brains were soaked in 10% sucrose, followed by 20% sucrose, and then
30% sucrose (prepared in 0.01 M phosphate buffer, pH 7.4). Brains were
then frozen, and 20-µm coronal sections were cut using a cryostat.
The sections were placed in 0.1 M phosphate-buffered saline (PBS) at
4°C.
The gold immunolabeling procedure was performed according to the
procedure described by Pandey et al. (2001). Brain sections were washed
with PBS (twice for 10 min) and then blocked with RPMI 1640 with
L-glutamine (Invitrogen, Carlsbad, CA) for 30 min followed
by 10% normal goat serum (diluted in PBS containing 0.25% Triton
X-100) for 30 min at room temperature. Sections were then incubated
with 1% BSA (prepared in PBS containing 0.25% Triton X-100) for 30 min at room temperature. Sections were further incubated with anti-GR
or anti-NeuN (Chemicon International, Temecula, CA) antibody (1:500
dilution for GR and 1:200 for NeuN in PBS containing 0.25% Triton
X-100) for 18 h at room temperature. After two 10-min washes with
PBS and two 10-min washes in 1% BSA in PBS, sections were incubated
with gold particles (1 nm) conjugated with the secondary antibody (Ted
Pella Inc., Redding, CA) for 1 h at room temperature. Sections
were further rinsed several times with 1% BSA in PBS followed by
water. Gold-immunolabeling was then silver enhanced for approximately
15 min and washed several times with water. Sections were then mounted
on slides, dehydrated, and examined under a light microscope. For
negative brain sections, an identical protocol was used except that 1%
BSA in PBS was substituted for primary antibody. The quantification of
gold-immunolabeled GR and NeuN proteins were performed using the Loats
Image Analysis System (Loats Associates, Inc.) connected to the light
microscope, which calculated the number of gold particles in a defined
brain region at high magnification (100×). The threshold of each image was set up in such a way that areas without staining should give zero
counts. Under this condition, gold particles in the defined areas of
three adjacent brain sections of each rat were counted and then values
were averaged for each rat. Results were expressed as number of
gold particles/100 µm2 area in a specific brain
region. GR rabbit polyclonal antibody was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). We also characterized the GR antibody
by Western blot using cytosolic fraction of cortex according to the
procedure as described by Pandey et al. (2001).
Statistical Analyses.
Differences among the groups were
evaluated by using nonparametric Kruskal-Wallis test. Specific subgroup
comparisons (between two groups) were performed using the Mann-Whitney
U test. A value of p < 0.05 was considered significant.
| |
Results |
Pattern of Alcohol Consumption and Blood Ethanol Levels.
All
rats gained weight during the 15 days of ethanol treatment or mianserin
treatment, and there were no significant differences in mean ± S.E.M. (n = 12) body weight (at the end of 15 days of treatment) among the ethanol-fed plus vehicle (285 ± 5.1 g),
ethanol-withdrawn plus vehicle (283 ± 5.4 g),
ethanol-withdrawn plus mianserin (282 ± 4.7 g), control plus
vehicle (284 ± 5.1 g), and control plus mianserin (292 ± 2.1 g)-treated rats. We also measured the daily intakes of
control and ethanol diets during mianserin treatment. The patterns of
ethanol consumption (milliliters per day or grams per kilogram per day)
are shown in Fig. 1. As can be seen,
mianserin treatment has no effect on daily ethanol intake in rats.
Because animals were pair fed, the control groups also consumed similar amounts (ml/day) of liquid control diet. It was found that control groups consumed about 53 ± 0.6 ml/day (n = 11)
control liquid diet. We also measured the morning (8-10 AM) blood
ethanol levels (BELs) using an Analox alcohol analyzer (Lunenburg, MA)
just before the initiation of withdrawal of ethanol-fed rats (15 days)
concurrently treated with or without mianserin. There were no
significant differences in mean BELs among the ethanol-fed rats plus
vehicle (198 ± 32 mg %), and ethanol-fed rats plus mianserin
(182 ± 19 mg %). The BELs in both groups after 24 h
withdrawal was 0 mg %. The BELs in ethanol-fed rats plus vehicle (0 h
withdrawal) was 210 ± 26 mg %. These blood ethanol values for
chronic studies (15 days) are similar to the values reported by us in
several previous publications (Pandey, 1996; Pandey et al., 1999).
These results indicate that ethanol-fed rats treated with or without
mianserin consumed similar amount of ethanol diet.
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Fig. 1.
Effects of mianserin treatment on daily alcohol
consumption (ml/day or g/kg/day) in rats under forced ethanol treatment
paradigm. Rats were fed with ethanol diet (containing 9% ethanol) with
concurrent treatment with mianserin or vehicle for 15 days and then
withdrawn for 0 and 24 h. Values are mean ± S.E.M. of 12 rats at each day.
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Time Course for Changes in GRE-DNA Binding in Rat Cortex and
Hippocampus during Ethanol Withdrawal.
We first characterized
GRE-DNA binding in the nuclear extract of cortex using competitive
experiment with excess of unlabeled GRE oligonucleotides (50 or 100 ng). It was found that unlabeled oligonucleotide dose dependently
attenuated the GRE-DNA binding in the nuclear extract of rat cortex
(data not shown). We then measured the GRE-DNA binding in the nuclear
extracts of cortices and hippocampi of pair-fed control, ethanol-fed,
and ethanol-withdrawn (12, 24, and 72 h) rats. The patterns of the
GRE-DNA protein complexes in cortex and hippocampus are shown in Fig.
2A. It was found that chronic ethanol
treatment significantly decreased GRE-DNA binding activity in the rat
cortex and hippocampus, whereas ethanol withdrawal produced opposite
effect in the cortex and hippocampus. GRE-DNA binding activity remained
decreased at 12 and 24 h of ethanol withdrawal and returned to
normal levels after 72 h of ethanol withdrawal in the rat cortex
(Fig. 2B). In the hippocampus, GRE-DNA binding was not significantly
altered at any time point (12, 24, and 72 h.) of ethanol
withdrawal after 15 days of ethanol treatment (Fig. 2B). These results
indicate that GRE-DNA binding is decreased in the nuclear extract of
cortex during ethanol treatment and early phases of withdrawal. On the
other hand, GRE-DNA binding is decreased in the nuclear extract of
hippocampus during ethanol treatment and quickly reverted to normal
levels during ethanol withdrawal.
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Fig. 2.
A, representative autoradiogram of the gel-mobility
shift assay showing the time course of changes in nuclear GRE-DNA
binding activity in the rat cortex and hippocampus during ethanol
withdrawal after 15 days of ethanol treatment. Rats were treated with
ethanol (9% v/v) or control-liquid diet. Ethanol-treated rats were
withdrawn from ethanol for 0, 12, 24, and 72 h, and cortices and
hippocampi from these rats were used for the measurement of GRE-DNA
binding activity. Fifteen micrograms of nuclear extract protein were
incubated with 32P-labeled GRE oligonucleotides and GRE-DNA
protein complexes were separated out by gel mobility shift assay as
described under Materials and Methods. B, the effect of
various time points of ethanol withdrawal (0, 12, 24, and 72 h)
after 15 days of ethanol exposure on GRE-DNA binding activity in the
rat cortex and hippocampus. Values are the mean ± S.E.M. of 6 to
12 experiments and are represented as percents of the normal controls.
*, significantly different from the pair-fed control group
(p < 0.05).
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Effects of Chronic Mianserin Treatment on GRE-DNA Binding in Rat
Cortex and Hippocampus during Ethanol Withdrawal.
We studied the
effects of chronic mianserin treatment on GRE-DNA binding in the
nuclear extracts of cortices and hippocampi of control liquid diet-fed
and ethanol diet-fed rats. It was found that mianserin treatment alone
has no effect on GRE-DNA binding in nuclear extract of the cortex but
produced significant reduction in GRE-DNA binding in the nuclear
extract of hippocampus (Fig. 3).
Interestingly, when administered concurrently with ethanol treatment,
mianserin significantly antagonized the down-regulation of cortical
GRE-DNA binding during ethanol withdrawal (Fig. 3). As mentioned above,
GRE-DNA binding in hippocampus returned to normal levels at 24 h
of ethanol withdrawal, but mianserin treatment produced significant
decrease in GRE-DNA binding in hippocampus of ethanol-withdrawn rats.
These results indicate that mianserin treatment produced opposite
effects in the cortex and hippocampus during ethanol withdrawal.
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Fig. 3.
Effect of ethanol withdrawal (0 and 24 h)
after 15 days of ethanol treatment and the effect of chronic mianserin
(5-HT 2A/2C receptor antagonist) with or without ethanol
administration on the GRE-DNA binding activity in the rat cortex and
hippocampus. Values are the mean ± S.E.M. of 6 to 12 experiments
and are represented as percents of the normal controls. *,
significantly different from the pair-fed control group
(p < 0.05).
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Effects of Ethanol Treatment and Withdrawal on the Expression of GR
in Rat Cortex and Hippocampus.
To determine whether decreased
GRE-DNA binding in rat cortex and hippocampus is related to decreased
protein levels of GR, we determined the protein expression of GR in rat
cortex and hippocampus during ethanol treatment and withdrawal. We
first characterized GR antibody using Western blot technique. It was
found that this antibody recognized a single band of GR proteins (~95
kDa) in the cytosolic fraction of cortex (Fig.
4). We used this antibody to determine
the subcellular distribution of GR in various structures of cortex and
hippocampus during ethanol treatment and withdrawal. The
gold-immunolabeling of GR protein is specific because negative brain
sections do not show any labeling (data not shown). The GR-positive
cell bodies can be seen in Fig. 5A for
cortical and Fig. 6A for hippocampal
structures. It was found that chronic ethanol treatment significantly
decreased the protein expression of GR in layers II/III and IV/IV of
cingulate gyrus, frontal, and parietal and in layer II of piriform
cortex and further decreased in these structures during ethanol
withdrawal (24 h) (Fig. 5, A and B). On the other hand, the protein
expression of GR was significantly decreased in CA1, CA2, CA3, and
dentate gyrus (DG) of hippocampus during ethanol treatment but reverted
to normal levels during ethanol withdrawal (Fig. 5, A and B). These
results indicate that decreases in GRE-DNA binding in rat cortex during ethanol treatment and withdrawal may be related to decreases in GR
protein expression.
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Fig. 4.
Representative Western blot of GR protein in the
cytosolic fraction of rat cortex. Different concentrations of protein
were resolved on SDS-polyacrylamide gel, transferred to nitrocellulose
membrane, and incubated with GR primary antibody and then secondary
antibody.
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Fig. 5.
A, low magnification views of GR gold-immunolabeling
in various cortical structures of control diet-fed plus vehicle,
ethanol-fed plus vehicle, ethanol-withdrawn plus vehicle, control
diet-fed plus mianserin, and ethanol-withdrawn plus mianserin-treated
rats. A through D, GR-positive cell bodies in layer IVV of cingulate
gyrus (CG), the frontal cortex (Fr), the parietal cortex (Par), and
layer II of piriform (Piri) cortex, respectively, of control diet-fed
rats. E through H, GR-positive cell bodies in CG, Fr, Par, and Piri
structures, respectively, of ethanol-fed rats. I through L, GR-positive
cell bodies in CG, Fr, Par, and Piri structures, respectively, of
ethanol-withdrawn rats. M through P, GR-positive cell bodies in CG, Fr,
Par, and Piri structures, respectively, of ethanol-withdrawn plus
mianserin-treated rats. Q through T, GR-positive cell bodies in CG, Fr,
Par, and Piri structures, respectively, of mianserin (alone)-treated
rats. Arrows indicate some of the GR-positive cell bodies. Scale bar,
40 µm in A through T. B, effect of ethanol withdrawal (0 and 24 h) after 15 days of ethanol treatment and the effect of chronic
mianserin treatment with or without ethanol administration on GR
protein levels in various layers of the cortical structures. Values are
mean ± S.E.M. of five to six rats in each group. *,
significantly different from the pair-fed control group
(p < 0.05).
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Fig. 6.
A, low magnification views of GR gold-immunolabeling
in various hippocampal structures (CA1 and CA2) of control diet-fed
plus vehicle, ethanol-fed plus vehicle, ethanol-withdrawn plus vehicle,
control diet-fed plus mianserin, and ethanol-withdrawn plus
mianserin-treated rats. A through E, GR-positive cell bodies in CA1 of
control diet-fed, ethanol-fed, ethanol-withdrawn, ethanol-withdrawn
plus mianserin, mianserin (alone)-treated rats, respectively. F through
J, GR-positive cell bodies in CA2 of control diet-fed, ethanol-fed,
ethanol-withdrawn, ethanol-withdrawn plus mianserin, mianserin
(alone)-treated rats, respectively. Arrows indicate some of the
GR-positive cell bodies. Scale bar, 40 µm in A through J. B, effect
of ethanol withdrawal (0 and 24 h) after 15 days of ethanol
treatment and the effect of chronic mianserin treatment with or without
ethanol administration on GR protein levels in various hippocampal
structures. Values are mean ± S.E.M. of five rats in each group.
*, significantly different from the pair-fed control group
(p < 0.05).
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Effects of Ethanol Treatment and Withdrawal on the Expression of GR
in Hypothalamus and Amygdala.
We also investigated the subcellular
distribution of GR in the PVN and central, medial, and basolateral
amygdaloid structures during ethanol treatment and withdrawal. The
GR-positive cell bodies can be seen in central and medial amygdaloid
(Fig. 7A) and PVN structures (Fig.
8A). It was found that chronic
ethanol treatment significantly decreased the protein expression of GR in the PVN (Fig. 8B) and in the central, medial, and basolateral amygdala (Fig. 7B) and further decreased in these structures during ethanol withdrawal (Figs. 7B and 8B). These results indicate that GR
protein expression is decreased in the PVN and in various amygdaloid structures during chronic ethanol treatment and its withdrawal.
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Fig. 7.
A, low magnification views of GR gold-immunolabeling
in various amygdaloid structures (central and medial amygdala) of
control diet-fed plus vehicle, ethanol-fed plus vehicle,
ethanol-withdrawn plus vehicle, control diet-fed plus mianserin, and
ethanol-withdrawn plus mianserin-treated rats. A through E, GR-positive
cell bodies in medial amygdala of control diet-fed, ethanol-fed,
ethanol-withdrawn, ethanol-withdrawn plus mianserin, mianserin
(alone)-treated rats, respectively. F through J, GR-positive cell
bodies in central amygdala of control diet-fed, ethanol-fed,
ethanol-withdrawn, ethanol-withdrawn plus mianserin, mianserin
(alone)-treated rats, respectively. Arrows indicate some of the
GR-positive cell bodies. Scale bar, 40 µm in A through J. B, effect
of ethanol withdrawal (0 and 24 h) after 15 days of ethanol
treatment and the effect of chronic mianserin treatment with or without
ethanol administration on GR protein levels in various amygdaloid
structures. Values are mean ± S.E.M. of five rats in each group.
*, significantly different from the pair-fed control group
(p < 0.05).
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Fig. 8.
A, low magnification views of GR gold-immunolabeling
in PVN structures of control diet-fed plus vehicle, ethanol-fed plus
vehicle, ethanol-withdrawn plus vehicle, control diet-fed plus
mianserin, and ethanol-withdrawn plus mianserin-treated rats. A through
E, GR-positive cell bodies in PVN structures of control diet-fed,
ethanol-fed, ethanol-withdrawn, ethanol-withdrawn plus mianserin,
mianserin (alone)-treated rats, respectively. Arrows indicate some of
the GR-positive cell bodies. Scale bar, 40 µm in A through F. B,
effect of ethanol withdrawal (0 and 24 h) after 15 days of ethanol
treatment and the effect of chronic mianserin treatment with or without
ethanol administration on GR protein levels in PVN structures. Values
are mean ± S.E.M. of five rats in each group. *, significantly
different from the pair-fed control group (p < 0.05).
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Effects of Chronic Mianserin Treatment on the Expression of GR in
the Rat Brain during Ethanol Withdrawal.
We studied the
effects of mianserin treatment on GR protein levels in the cortical,
amygdaloid, hippocampal, and PVN structures of control liquid diet-fed
and ethanol diet-fed rats. It was found that mianserin treatment alone
had no effect on GR protein levels in cortical (Fig. 5, A and B),
amygdaloid (Fig. 7, A and B), or PVN (Fig. 8, A and B) structures but
produced significant reductions in the GR protein levels in hippocampal
structures (CA1, CA2, CA3, and DG) (Fig. 6, A and B). It was also
observed that mianserin treatment, when administered concurrently with
ethanol treatment, significantly antagonized the decreased expression
of GR in the PVN (Fig. 8, A and B), cortical (Fig. 5, A and B), and
central but not in medial and basolateral amygdaloid (Fig. 7, A and B) structures during ethanol withdrawal. We observed that decreased expression of GR protein in various structures of hippocampus during
ethanol treatment returned to normal levels during withdrawal. Interestingly, mianserin treatment produced significant decrease in GR
protein levels in hippocampal (CA1, CA2, CA3, and DG) structures of
ethanol-withdrawn rats (Fig. 6, A and B). These results indicate that
blockade of 5-HT2A/2C receptors during ethanol
exposure antagonizes the ethanol-induced decrease in GR expression in
PVN, cortical, and central amygdaloid structures but not in
hippocampal, medial, and basolateral amygdaloid structures.
Effects of Chronic Ethanol Treatment and Its Withdrawal on Number
of Neurons in Rat Brain Structures.
To examine whether decreased
expression of GR in various brain structures during ethanol exposure
and its withdrawal is related to loss of neurons, we investigated the
protein levels of NeuN in the nuclei. The NeuN-positive nuclei can be
seen in Fig. 9A. It was found that
chronic ethanol exposure and its withdrawal has no effects on the NeuN
protein levels in the various structures of hippocampus, amygdala,
cortex, and PVN (Fig. 9B). We also examined the effects of mianserin
treatment on NeuN protein levels in brain structures of control and
ethanol-withdrawn rats. It was found that mianserin treatment has also
no effects on NeuN protein levels in various brain structures of
control diet- and ethanol diet-fed rats. These results suggest that
neither ethanol treatment nor mianserin treatment is associated with
loss of neurons in various brain structures.
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Fig. 9.
A, low magnification views of NeuN
gold-immunolabeling in frontal cortex (layer IV/V), central amygdala,
and PVN of control diet-fed plus vehicle, ethanol-fed plus vehicle,
ethanol-withdrawn plus vehicle, control diet-fed plus mianserin, and
ethanol-withdrawn plus mianserin-treated rats. A through E,
NeuN-positive nuclei in frontal cortex of control diet-fed,
ethanol-fed, ethanol-withdrawn, ethanol-withdrawn plus mianserin,
mianserin (alone)-treated rats, respectively. F through J,
NeuN-positive nuclei in central amygdala of control diet-fed,
ethanol-fed, ethanol-withdrawn, ethanol-withdrawn plus mianserin,
mianserin (alone)-treated rats, respectively. K through O,
NeuN-positive nuclei in PVN of control diet-fed, ethanol-fed,
ethanol-withdrawn, ethanol-withdrawn plus mianserin, mianserin
(alone)-treated rats, respectively. Scale bar, 40 µm in A through O. B, effect of ethanol withdrawal (0 and 24 h) after 15 days of
ethanol treatment and the effect of chronic mianserin treatment with or
without ethanol administration on NeuN levels in various brain
structures. Values are mean ± S.E.M. of five rats in each
group.
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Discussion |
Neuroadaptation in the HPA Axis during Chronic Ethanol Exposure and
Its Withdrawal.
The first key finding of the present investigation
is that expression of GR is decreased in PVN, cortical, hippocampal,
and amygdaloid structures during chronic ethanol exposure. Furthermore, the reduction in GR protein levels is more in PVN, cortical, and amygdaloid structures but reverted to normal levels in hippocampus during ethanol withdrawal. It was also found that reduction in GR
protein levels in cortical and hippocampal structures are associated with GRE-DNA binding in these structures during ethanol treatment.
The mechanisms by which ethanol treatment and withdrawal produced
down-regulation of GR in various brain structures are not clear, but
may be related to high levels of circulating glucocorticoids as
reported by several investigators in rodent models and human alcoholics
during ethanol exposure and its withdrawal (Tabakoff et al., 1978;
Wand, 1993; Rivier, 1996). It has been shown that high levels of
glucocorticoids leads to down-regulation of GR in several cell systems
(Okret et al., 1986; Rosewicz et al., 1988). Hyperactive HPA axis has
been shown in human alcoholics (Risher-Flowers et al., 1988; Wand,
1993). However, there are inconsistent reports about hyperactive HPA
axis in animal models, and this may be due to variations in blood
ethanol levels, methods, and duration of ethanol administration (Lee
and Rivier, 1994; Rivier, 1996; Ogilvie et al., 1997; Rasmussen et al.,
2000). In the present study, rats were treated with 9% ethanol for 15 days, which produced blood ethanol levels in the range of 210 to 198 mg
%. Under this ethanol treatment paradigm, there are significant reductions in the protein levels of GR in the PVN and other
nonhypothalamic regions (cortex, hippocampus, and amygdala).
The GRs in the PVN and hippocampus have been shown to be an important
regulator of HPA axis via feedback mechanism (Jacobson and Sapolsky,
1991; Plotsky, 1991). In the present study, GR expression is decreased
both in PVN and hippocampus during ethanol exposure. In PVN, GR
expression further decreased whereas in hippocampus, GR expression
reverted to normal levels during ethanol withdrawal. This suggests that
GRs are regulated differentially in PVN and hippocampus during ethanol
withdrawal. Furthermore, the hyperactive HPA axis during ethanol
treatment and withdrawal may be related to decreased expression of GR
in PVN, but may not be related to GR in hippocampus. The dichotomy
between regulation of HPA axis by GR in PVN and hippocampus was shown
earlier using local infusion of GR antagonist and agonist in stressed
rats. It was found that GR antagonist and agonist infusion in
hippocampus has no effect on corticosterone levels whereas GR
antagonist and agonist infusion in PVN is able to increase or suppress
the corticosterone levels, respectively (De Kloet et al., 1988; Kovacs
and Makara, 1988). The results suggest that GR in PVN but not in
hippocampus is crucial in feedback inhibition of HPA axis.
It has been established that effects of glucocorticoids in the brain
are mediated by MR and GR and both of these receptors are important in
mediating the negative feedback action of glucocorticoids(De Kloet,
1991). MR is rich in hippocampus and GR is located through out the
brain (Reul and De Kloet, 1986; Feldman and Weidenfeld, 1995). The
chronic ethanol exposure has no effects on GR and MR levels as
determined by binding techniques in the cytosolic fraction of
hippocampus (Spencer and McEwen, 1990). One previous study investigated
the effects of chronic ethanol exposure (10 days) on mRNA levels of GR
in the hippocampal structures and found that mRNA levels of GR are
significantly decreased in the CA1, CA3, and DG of hippocampus (Eskay
et al., 1995). Here we found that chronic ethanol (15 days) exposure
causes significant reductions in the GR protein levels in hippocampal
structures and in other brain structures. Because GR protein levels are
decreased in cortical, amygdaloid, hippocampal, and PVN structures
during ethanol exposure, this suggests that GR in these brain
structures may play a role in the neuromechanisms of alcohol tolerance.
Furthermore, GR protein levels are normalized in the hippocampus
whereas in PVN, cortical and amygdaloid structures remained decreased
during ethanol withdrawal; this suggests that GR in nonhippocampal area
such as PVN and central amygdala may play a role in the process of
ethanol dependence. The one caveat of the present study is that we have
not investigated the neuroadaptational changes in MR protein levels in
various brain structures during ethanol exposure or its withdrawal. It is possible that the imbalance between MRs and GRs in hippocampus or
PVN may be crucial in the abnormal HPA axis during ethanol dependence.
Future studies will investigate this possibility.
Hyperactive HPA Axis and Neuronal Loss in the Rat Brain during
Chronic Ethanol Exposure.
It has been hypothesized that
hippocampal structure is more vulnerable to endangering or weakening
effects of elevated circulating levels of glucocorticoids (Walker et
al., 1981; Eskay et al., 1995; Lukoyanov et al., 1999). Thus, other
possible explanation for decreased GR protein expression in hippocampus
or other brain structures may be related to neurotoxicity in part to
chronically elevated glucocorticoids during ethanol treatment. We
tested this possibility by measuring neuronal marker (NeuN protein) in
the neurons and found that neither chronic ethanol treatment nor
withdrawal had any effects on the NeuN protein levels. These results
indicate that decreased expression of GR in various brain structures is not due to loss of neurons during 15 days ethanol treatment or its
withdrawal. It has been shown that long-term treatment with ethanol
(more than 1 month) leads to loss of neurons in the hippocampus (Walker
et al., 1981; Lukoyanov et al., 1999). Taken together, this suggests
that long but not short-term ethanol exposure causes loss of neurons in
the hippocampus.
5-HT2A/2C Receptor Interactions with HPA Axis during
Chronic Ethanol Exposure.
The second key observation of the
present study is that blockade of 5-HT2A/2C
receptors during alcohol drinking antagonized the ethanol-induced
decreased expression of GR in PVN, cortical, and central but not medial
and basolateral amygdaloid structures. On the other hand, mianserin
treatment significantly decreased the GR expression in the hippocampal
structures of control and ethanol-withdrawn rats. Thus GR expression in
the neurocircuitry of hippocampus and other brain structures (cortex,
PVN and central amygdala) behaves differentially during serotonergic
manipulations. Because mianserin treatment has no effects on daily
ethanol intake under forced treatment paradigm, this suggests that
prevention of ethanol-induced decreases in GR protein levels in various
brain structures by mianserin treatment may be related to direct action of drug on 5-HT2A/2C receptors.
It has been shown that 5-HT2A/2C receptor
agonist stimulates HPA axis and this action, primarily due to
stimulation of 5-HT2A receptor, mediated CRF
release in the PVN (Van de Kar, 1991; Feldman and Weindenfeld, 1995;
Van de Kar et al., 2001). The notion that 5-HT2A/2C receptors in the PVN may regulate the
HPA axis is supported by the fact that lesioning of PVN prevents this
receptor-mediated release of corticosterone in rats (Bagdy and Makara,
1994). Regardless of mechanisms, these results indicate that
interaction between 5-HT2A/2C receptors and GRs
in the PVN, cortical, or central amygdaloid structures may play an
important role in the pathophysiology of HPA axis during ethanol
dependence. The decreased GRs in the limbic structures such as central
amygdala and frontal cortex may be involved in anxiety developing
during ethanol withdrawal. We have shown earlier that ethanol
withdrawal (24 h) after 15 days treatment produced anxiety-like
behavior in rats (Pandey et al., 1999). It has been also shown that
local infusion of corticosterone in to central amygdala increase CRF
mRNA levels and also anxiety-like behaviors in rats (Shepard et al.,
2000). Increased CRF levels in the central amygdala have been shown to
be involved in anxiety-like behaviors in rats during ethanol withdrawal
(Koob et al., 1998). Furthermore mianserin treatment is also able to
prevent anxiety during ethanol withdrawal (Lal et al., 1993). It is
possible that 5-HT2A/2C receptor interaction with
GRs and CRF in the central amygdala may be one of the mechanisms
responsible for the ethanol withdrawal-related anxiety. Future
experiments are needed to explore such relationship during ethanol withdrawal.
| |
Conclusions |
The data presented here provide the first evidence that decreased
GR protein levels in PVN, cortical, hippocampal, and amygdaloid structures may be associated with neuroadaptational mechanisms to
chronic ethanol exposure. The GR expression normalized in hippocampal but further decreased in cortical, amygdaloid, and PVN structures during ethanol withdrawal, this suggests that GR in these
nonhippocampal structures may be associated with the process of alcohol
dependence. Because GRs in the neural circuitry of PVN are the
important regulator of HPA axis, it is possible that decreased GRs in
this brain structures may be responsible for the compromised HPA axis
during ethanol exposure and withdrawal. Moreover the decreased
expression of GR in various brain structures during ethanol exposure
and withdrawal is not related to neuronal loss.
This work was supported by National Institute on Alcohol Abuse
and Alcoholism Grant AA10005 and by a Department of Veterans Affairs
Merit Review Grant (to S.C.P.).
GR, glucocorticoid receptor;
GRE, glucocorticoid response element;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin;
PVN, paraventricular nucleus;
HPA, hypothalamic-pituitary adrenal;
5-HT, serotonin;
HEPES, 4-(2-hydroxy
ethyl-)1-piperazine ethane sulfonic acid;
DTT, dithiothreitol;
NeuN, neuron-specific nuclear protein;
CRF, corticotropin-releasing factor;
MR, mineralocorticoid receptor;
BEL, blood ethanol levels.
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Abstract
Introduction
subunit protein and phospholipase C isozymes in the rat brain.
J Neurochem
67:
2355-2361[Medline].
