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The Journal of Neuroscience, November 15, 1999, 19(22):10098-10106
Expression of  1b Adrenoceptor mRNA in
Corticotropin-Releasing Hormone-Containing Cells of the Rat
Hypothalamus and Its Regulation by Corticosterone
Heidi E. W.
Day,
Serge
Campeau,
Stanley J.
Watson Jr, and
Huda
Akil
Mental Health Research Institute, University of Michigan, Ann
Arbor, Michigan 48109-0720
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ABSTRACT |
Considerable evidence supports a role for brainstem adrenergic and
noradrenergic inputs to corticotropin-releasing hormone (CRH) cells of
the hypothalamic paraventricular nucleus (PVN), in the control of
hypothalamic-pituitary-adrenocortical (HPA) axis function. However,
little is known about specific adrenoceptor (ADR) subtypes in
CRH-containing cells of the PVN. Here we demonstrate, using dual
in situ hybridization, that mRNA encoding
 1b ADR is colocalized with CRH in the rat PVN.
Furthermore, we confirm that these 1b ADR
mRNA-containing cells are stress-responsive, by colocalization with
c-fos mRNA after restraint, swim, or immune stress.
To determine whether expression of 1b ADR mRNA is
influenced by circulating glucocorticoids, male rats underwent
bilateral adrenalectomy (ADX) or sham surgery, and were killed after 1, 3, 7, or 14 d. In situ hybridization revealed
levels of 1b ADR mRNA were increased in the PVN 7 and
14 d after ADX, but were not altered in the hippocampus, amygdala,
or dorsal raphe. Additional rats underwent ADX or sham surgery and
received a corticosterone pellet (10 or 50 mg) or placebo for 7 d.
Corticosterone replacement (10 mg) reduced the ADX-induced increase in
PVN 1b ADR mRNA to control levels, whereas 50 mg of
corticosterone replacement resulted in a decrease in PVN
1b ADR mRNA as compared with all other groups. Furthermore, levels of plasma corticosterone were significantly correlated (inverse relationship) with 1b ADR mRNA in
the PVN.
We conclude that 1b ADR mRNA is expressed in
CRH-containing, stress-responsive cells of the PVN and is highly
sensitive to circulating levels of corticosterone. Because activation
of the 1B adrenoceptor is predominantly excitatory
within the brain, we predict that this receptor plays an important role
in facilitation of the HPA axis response.
Key words:
adrenergic; adrenoceptor; HPA axis; stress; CRH; hypothalamus; paraventricular nucleus; in situ
hybridization; adrenalectomy; glucocorticoid; corticosterone
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INTRODUCTION |
The
hypothalamic-pituitary-adrenocortical (HPA) axis is critical to an
animal's response to stress (for review, see Akil et al., 1999 ).
Parvocellular cells of the paraventricular nucleus of the hypothalamus
(PVN) project to the external zone of the median eminence and release
corticotropin-releasing hormone (CRH) and other corticotropin-releasing
factors into the portal circulation to stimulate corticotrophs of the
anterior pituitary. These cells subsequently release
adrenocorticotropin hormone (ACTH), which in turn stimulates release of
glucocorticoids (corticosterone in rat and cortisol in humans) from the
adrenal cortex, a defining feature of a stress response. Considerable
research has focused on elucidating the neural circuits that regulate
the HPA axis at the level of the PVN, and a number of direct and
indirect inputs have been demonstrated (Gaillet et al., 1991; Larsen
and Mikkelsen, 1995; Cullinan et al., 1996; Li et al., 1996; Herman and
Cullinan, 1997).
Direct adrenergic and noradrenergic inputs from the brainstem nucleus
tractus solitarius and ventrolateral medulla to the parvocellular PVN
have been described (Cunningham and Sawchenko, 1988; Cunningham et al.,
1990), and the importance of these pathways in regulation of the HPA
axis has been widely demonstrated (Gaillet et al., 1991; Itoi et al.,
1994; Parsadaniantz et al., 1995; Smith et al., 1995). Evidence
suggests that these inputs primarily have a facilitatory effect on HPA
axis regulation (Guillaume et al., 1987; Plotsky, 1987; Plotsky et al.,
1989). However, the adrenergic receptor (ADR) subtypes that may mediate
the effects of epinephrine or norepinephrine at the level of the PVN
have not been well characterized. Although considerable evidence
supports an involvement of 1 adrenoceptors at
the level of the PVN (Plotsky, 1987; Calogero et al., 1988; Kiss and
Aguilera, 1992; Whitnall et al., 1993), the subtype or subtypes of
1 adrenoceptors have not been determined, in
part because of the lack of selective ligands capable of discriminating between similar receptor subtypes. However, our laboratory and others
have demonstrated by in situ hybridization that mRNA
encoding the 1b ADR subtype is present at
moderate levels in parvocellular cells of the rat PVN (McCune et al.,
1993; Pieribone et al., 1994; Day et al., 1997). Stimulation of
1 adrenoceptors within the CNS is
thought to be predominantly excitatory, via decreased potassium conductance and slow depolarization (Bevan et al., 1977; McCormick and
Prince, 1988; Pan et al., 1994; Bergles et al., 1996), consistent with
the hypothesis that activation of the 1B ADR
subtype could be involved in stimulation of the HPA axis. To ascertain
more directly whether the 1B ADR subtype might
mediate excitation of CRH cells of the PVN, we determined, using dual
in situ hybridization histochemistry, whether
1b ADR mRNA is expressed within CRH cells of
the PVN. Furthermore, we investigated whether cells expressing this
receptor are active after stress, by the colocalization of c-fos with
1b ADR mRNA in cells of the PVN after
restraint, swim, or immune stress. In addition, because the gene for
this receptor has been shown to contain a glucocorticoid response
element (GRE) within its promoter region (Gao and Kunos, 1993), we went
on to examine the effect of circulating glucocorticoids on expression of 1b ADR mRNA in different brain regions
after bilateral adrenalectomy, with low or high dose corticosterone
replacement. Our data suggest that 1b ADR mRNA
is expressed in the majority of CRH-containing, stress-responsive cells
of the PVN and that the levels of expression of this receptor within
the PVN (but not other brain regions) are inversely related to
circulating corticosterone levels.
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MATERIALS AND METHODS |
Animals. All procedures described were approved by
the University of Michigan Committee on Use and Care of Animals. Adult male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA)
were used throughout. All animals were allowed to habituate to the
housing conditions for at least 10 d before any experimental manipulation. Rats were housed two or three per cage under conditions of constant temperature and humidity, on a 12 hr light/dark cycle (lights on at 7:00 A.M.), with ad libitum access to food and water.
Experiment 1: expression of 1b ADR mRNA in CRH and
stress-responsive cells of the PVN. Four animals, naive to any
experimental manipulation were used to determine the extent of
colocalization of 1b ADR mRNA in
CRH-containing cells of the PVN. These data, combined with previous
studies that have demonstrated that the majority of stress-induced
c-fos mRNA or Fos protein-containing cells of the medial parvocellular
PVN are CRH-positive (Ericsson et al., 1994; Rivest and Rivier, 1994;
Day et al., 1999), suggested that 1b ADR
mRNA-containing cells of the PVN are stress-responsive. To confirm
this, five animals were handled daily for 1 week, then subjected to
different stressors: 30 min restraint or swim stress (Cullinan et al.,
1995), n = 2 per group, or intraperitoneal
injection of human recombinant IL-1 , 5 µg/kg (Bachem), 30 min
before killing (n = 1). Previous data suggested
that the intraperitoneal injection procedure per se was not stressful,
as indicated by a lack of corticosterone secretion and c-fos mRNA
expression in the PVN after saline intraperitoneally (Day and Akil,
1996). Animals were killed by rapid decapitation 2-3 hr after lights
on (9:00-10:00 A.M.). The brains were removed and frozen in isopentane
cooled to  40 to 50°C, and stored at 80°C. Sections (10 µm)
were cut on a cryostat (Bright) through the hypothalamus. Tissue was
air-dried and stored at 80°C until processing for dual in
situ hybridization, as described previously (Day et al., 1999).
Briefly, cRNA probes complementary to CRH (courtesy of Dr. R. Thompson,
University of Michigan, Ann Arbor, MI; 770 mer) or c-fos (Dr. T. Curran, St. Jude Children's Research Hospital, Memphis, TN; 680 mer)
were generated and labeled with digoxigenin-UTP (dig-UTP; Boehringer Mannheim, Indianapolis, IN) using standard transcription methods. A
cRNA probe complementary to 1b ADR mRNA
(courtesy of Dr. R. Lefkowitz, Duke University Medical Center, Durham,
NC; 766 mer) was generated and labeled with
[35S]CTP and
[35S]-UTP. Brain sections were
hybridized overnight with both probes. The following day, sections were
treated with RNase A (200 µg/ml) for 1 hr at 37°C and washed to a
final stringency of 0.1× SSC at 65°C for 1 hr. Sections were then
processed for visualization of the digoxigenin-labeled probe. Briefly,
sections were incubated overnight with an antibody against digoxigenin,
conjugated to alkaline phosphatase (sheep anti-dig-AP; Fab fragments;
Boehringer Mannheim), diluted 1:20,000. After extensive washing,
sections underwent a color reaction by addition of 0.45% nitro blue
tetrazolium chloride (Boehringer Mannheim) and 0.35%
5-bromo-4-chloro-3-indoylphosphate, 4-toluidine salt (Boehringer
Mannheim). After completion of the color reaction (~18 hr) sections
were rinsed and stripped of antibody by incubation with 0.1 M glycine and 0.5% Triton-X 100, pH 2.2, for 10 min. Finally sections were fixed in 2.5% glutaraldehyde for 1 hr.
These last steps were found to help prevent the increase in background
after processing for radioactive signal. After exposure to x-ray film
(5 d), sections were dipped in liquid emulsion (Ilford KD-5;
Polysciences, Warrington, PA) and stored in light-tight boxes for 1 month. After this time sections were developed (Kodak D-19; Eastman
Kodak, Rochester, NY), dehydrated, and coverslipped in a xylene-based
mounting medium (Permount). The cellular distribution was determined
using a Leica (Nussloch, Germany; Leitz DMR) microscope. Nonradioactive
probes (CRH or c-fos) were visualized under bright field as a
blue-purple precipitate, whereas the radioactive probe (1b ADR) was visualized under dark field by
silver grain distribution. It should be noted that the nonradioactive
in situ hybridization technique is less sensitive than the
radioactive technique, hence the digoxigenin-labeled cell population
tends to be under-represented. Ideally, both combinations of
radioactive and nonradioactive probes would be analyzed. However, the
abundance of 1b ADR mRNA in the PVN was not
sufficient to obtain reliable labeling with the nonradioactive in
situ hybridization technique.
Sections at 50-80 µm intervals, three to five sections per animal,
in the region of the medial parvocellular PVN were analyzed. Cell
profile counts were determined at 40× magnification, with the aid of
an eyepiece grid. No attempt was made to establish absolute numbers of
cells within these structures. Rather, the numbers of cell profiles
counted for each animal were used to approximate the relative percent
colocalization of mRNA for that animal.
Experiment 2a: adrenalectomy time course. For the initial
time course, 35 rats were used (mean weight, 313 ± 4 gm). Of
these, 28 were anesthetized with pentobarbital and underwent bilateral adrenalectomy (ADX; four per group; n = 16) or sham
surgery (SHAM; three per group; n = 12). The
adrenalectomy was performed via a dorsolateral approach, and the
incision was closed with wound clips. Animals were housed two or three
per cage, and drinking water was replaced with a solution containing
0.9% saline and 5% dextrose. Animals were killed either 1 (22-24
hr), 3, 7, or 14 d after surgery. In addition, three unoperated
(UNOP) animals were included that did not undergo any surgical
manipulation, but were given the saline-dextrose solution to drink,
and four naive animals (NAIVE) that were not subjected to any
experimental manipulation. Animals were killed 2 hr before lights off
(5:00 P.M.) so that there were detectable levels of plasma
corticosterone in control animals against which to compare the
effectiveness of the ADX surgery. Trunk blood was collected in chilled
tubes containing EDTA, and plasma was separated and stored at
20°C for analysis of corticosterone. Brains were removed, frozen in isopentane cooled to 40 to 50°C, and stored at 80°C. Sections (10 µm) were cut on a cryostat through the hypothalamus, hippocampus, and brainstem, and stored at 80°C until processing for in
situ hybridization (as described by Day et al., 1997).
Experiment 2b: adrenalectomy and corticosterone replacement.
For the second part of the study, 30 animals were used, divided into
the following groups: NAIVE (n = 6), SHAM
(n = 6), ADX plus placebo (n = 6), ADX plus 10 mg of corticosterone (n = 6), and ADX
plus 50 mg of corticosterone (n = 6). The mean start
weight was 291 ± 3 gm. In addition to bilateral adrenalectomy,
animals were implanted with a subcutaneous pellet (Innovative Research of America) within the dorsal neck region. Rats received either placebo, 10 mg, or 50 mg of corticosterone in the form of 21 d release pellets. Surgery was performed over a 2 d period,
n = 3 per group per day. Drinking water for SHAM and
all ADX groups was replaced with an isotonic solution containing 0.45%
saline and 2.5% dextrose. Three days after surgery, a tail vein blood sample (75 µl) was taken from each animal 2 hr after lights on (9:00
A.M.). For this procedure, animals were restrained lightly, a lateral
tail vein was punctured with the corner of a razor blade, and blood was
collected in a heparinized capillary tube. Animals were killed 7 d
after surgery, 1-2 hr before lights off (5:00-6:00 P.M.). Trunk blood
and brains were collected as described in part 2a. Coronal brain
sections (10 µm) were cut through the hypothalamus and hippocampus
and stored at 80°C until processing for in situ hybridization (as described by Day et al., 1997).
Corticosterone analysis. Levels of plasma corticosterone
were analyzed as previously described (Day and Akil, 1996). Briefly, 10 µl duplicate samples of plasma were incubated overnight with an
antibody against corticosterone (raised in our laboratory) and
[3H]corticosterone (Amersham, Arlington
Heights, IL). Bound versus free corticosterone was separated by
charcoal extraction, and levels were calculated by comparison with a
standard curve.
Semiquantitative mRNA analysis. Levels of
1b ADR mRNA were analyzed by computer-assisted
optical densitometry. Brain section images from in situ
hybridization experiments were captured digitally (CCDcamera, model
XC-77; Sony, Tokyo, Japan), and the relative optical density of the
x-ray film was determined for each brain region using NIH Image 1.61 for Macintosh computer. A macro was written (Dr. Serge Campeau,
University of Michigan) that enabled signal above background to be
automatically determined. For each section, a background sample was
taken over an area of white matter, and the signal threshold was
calculated as mean gray value of background plus 3.5 × SD. The
section was automatically density-sliced at this value, so that only
pixels with gray values exceeding these criteria were included in the
analysis. Results are either expressed as mean signal above background,
which reflects mean gray values above background only, or as mean
integrated density, which reflects the number of pixels above
background (number of pixels above background × mean signal). In
cases in which the number of pixels was included in the measurement,
considerable care was taken to ensure equivalent areas were analyzed
between animals.
Photography and image processing. Figures
1, 2, and 4 were generated digitally.
Bright-field and dark-field images were captured with a Sony CCD video
camera (model DXC-970MD) attached to a Leica (Leitz DMR)
microscope, or from the x-ray film on a Northern Lights light-box
(Imaging Research), using MicroComputer Imaging Device (Ontario,
Canada) image analysis system. Composites were formed within Adobe
Photoshop and Adobe Illustrator. Brightness and contrast were altered
to generate photographic quality prints.
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Figure 1.
Dual in situ hybridization to show
coexpression of 1b ADR mRNA in CRH mRNA-containing cells
of the PVN. A, C, CRH mRNA in the PVN,
labeled with a nonradioactive (digoxigenin) probe, viewed under
bright-field illumination. B, D,
1b ADR mRNA in the PVN, labeled with a radioactive
(35S) probe, viewed under dark-field illumination. The
box in B represents the approximate area
over which cells were counted on each side of the PVN.
Arrows in C and D indicate
examples of double-labeled cells. Scale bars: A, B, 400 µm; C, D, 50 µm. Subdivisions of the PVN:
dp, dorsal parvocellular part; mpd,
medial parvocellular part, dorsal zone; mpv, medial
parvocellular part, ventral zone; pml, posterior
magnocellular part, lateral zone.
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Statistical analysis. Data were analyzed by one-way
ANOVA followed by Tukey-Kramer or Student-Newman-Keuls
post hoc multiple comparisons test, as indicated in
Results. Significance was set at p < 0.05.
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RESULTS |
Experiment 1: dual in situ hybridization
Expression of 1b ADR mRNA within the PVN
was highest in the parvocellular region. Dual in situ
hybridization revealed that virtually all CRH cells (97.7 ± 0.6%) expressed 1b ADR mRNA (Fig. 1; mean
number of double-labeled cells counted per animal = 554 ± 25; mean number of single-labeled CRH cells counted per animal = 13 ± 3; n = 4). In addition, the majority of
1b ADR mRNA cells (79.8 ± 0.5%), were
double-labeled for CRH (mean number of double labeled cells counted per
animal = 554 ± 25; mean number of single labeled
1b ADR cells counted per animal = 140 ± 5; n = 4). It should be noted that this is
likely an underestimation of the true degree of colocalization, because
of the relative insensitivity of the nonradioactive (CRH) compared with
radioactive (1b ADR) in situ
hybridization protocols.
Stress-induced c-fos mRNA expression in the PVN was found to be highly
colocalized with 1b ADR mRNA. For restraint
stress (n = 2), ~94 and 96% of c-fos-positive cells
were also labeled for 1b ADR mRNA, whereas 77 and 74% of 1b ADR-positive cells were also
labeled for c-fos mRNA (mean number of double labeled cells counted per
animal = 318; mean number of single labeled c-fos cells = 18;
mean number of single-labeled 1b ADR = 103). For swim stress (n = 2), ~83 and 94% of
c-fos-positive cells were also labeled for 1b
ADR mRNA, whereas 84 and 64% of 1b
ADR-positive cells were also labeled for c-fos mRNA (mean number of
double-labeled cells counted per animal = 149; mean number of
single-labeled c-fos cells = 26, mean number of single-labeled
1b ADR = 43). For intraperitoneal IL-1
(n = 1; Fig. 2), ~94%
of c-fos positive cells were also labeled for
1b ADR mRNA, whereas 77% of
1b ADR-positive cells were also labeled for
c-fos mRNA (number of double-labeled cells counted = 358; number
of single-labeled c-fos cells = 16; number of single-labeled
1b ADR = 123).
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Figure 2.
Dual in situ hybridization to show
coexpression of 1b ADR mRNA in c-fos mRNA-containing
cells of the PVN 30 min after IL-1 (5 µg/kg, i.p.). A,
C, c-fos mRNA in the PVN, labeled with a nonradioactive
(digoxigenin) probe, viewed under bright-field illumination. B,
D, 1b ADR mRNA in the PVN, labeled with a
radioactive (35S) probe, viewed under dark-field
illumination. The box in B represents the
approximate area over which cells were counted on each side of the PVN.
Arrows in C and D indicate
examples of double-labeled cells. Scale bars: A, B, 400 µm; C, D, 50 µm.
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Experiment 2a: ADX time course
Levels of corticosterone for all ADX animals were undetectable,
whereas naive, unoperated, and sham controls had levels between 4 and 6 µg/dl at the time of killing (Table 1).
Despite the addition of dextrose in the water, prolonged ADX resulted
in significantly lower weight gain as compared with sham controls. At
7 d the percentage of weight gain was not significantly different
between sham and ADX animals (sham, 14.0 ± 0.6%; ADX, 8.9 ± 1.1%). In contrast, after 14 d, the percentage of weight
gain of ADX animals was significantly lower (p < 0.01; Tukey post hoc comparison test) than sham controls (sham, 18.9 ± 4.2%; ADX, 0.2 ± 2.7%).
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Table 1.
Plasma levels of corticosterone at time of killing (2 hr
before lights off) in animals subjected to bilateral ADX or SHAM
surgery
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The effect of bilateral adrenalectomy on the expression of
1b ADR mRNA over time was studied. No
significant differences between naive, unoperated, and sham controls
were found. In contrast, levels of 1b ADR mRNA
were significantly increased in the PVN of ADX animals relative to sham
controls after 7 or 14 d (173 ± 19% and 193 ± 16%,
respectively; p < 0.05, Tukey post hoc
multiple comparison test; Fig. 3). This
increase appears to be specific to the PVN, because no significant
changes in expression of 1b ADR mRNA were
observed for the hippocampus, lateral amygdala, or dorsal raphe (Table
2).
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Figure 3.
Expression of 1b ADR mRNA in the
PVN 1, 3, 7, or 14 d after bilateral adrenalectomy
(ADX) or sham surgery. Data are expressed
as percentage of integrated density with respect to
(w.r.t.) sham. *p < 0.05; compared
with appropriate sham group, Tukey post hoc comparison
test.
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Table 2.
Levels of 1b ADR mRNA in the hippocampus,
dorsal raphe, and lateral amygdala after bilateral ADX or SHAM surgery
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Experiment 2b: ADX and corticosterone replacement
Levels of corticosterone (Table 3)
were undetectable in five of the six animals in the ADX plus placebo
group at either 3 d (A.M. sample) or 7 d (P.M. sample). The
sixth animal had low but detectable levels of corticosterone (0.02 µg/dl A.M.; 0.2 µg/dl P.M.). On this basis, the animal was excluded
from the study. The naive and sham groups had comparable levels of
corticosterone for both A.M. and P.M. samples. For the ADX plus 10 mg
of corticosterone group, levels of corticosterone were 2.42 ± 0.2 µg/dl after 3 d and 1.19 ± 0.29 µg/dl after 7 d.
This represented a significant decrease (p < 0.01; Student's t test), possibly caused by a decreased secretion of corticosterone from the pellet over time. For the ADX plus
50 mg of corticosterone group, levels were 7.47 ± 0.88 µg/dl
after 3 d and 6.83 ± 0.82 µg/dl after 7 d.
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Table 3.
Plasma levels of corticosterone after bilateral ADX with
low (10 mg) or high (50 mg) corticosterone replacement or SHAM surgery
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Weight gain for the animals varied across groups (Table 3). Naive and
sham groups exhibited a similar percentage of weight gain. ADX plus
placebo gained significantly less weight than sham or naive animals
(p < 0.01; Tukey post hoc multiple
comparisons test). In contrast, a low dose of corticosterone reversed
this effect, so that ADX plus 10 mg group exhibited a similar weight gain as the naive and sham animals. Animals treated with a high dose of
corticosterone also demonstrated significantly lower weight gain than
naive, sham, or ADX plus 10 mg groups (p < 0.01; Tukey post hoc multiple comparisons test).
The effect of ADX with corticosterone replacement on expression of
1b ADR mRNA in the PVN is shown in Figures
4 and 5.
Expression was not significantly different in the PVN of sham rats, as
compared to naive. As was observed in the initial time course
experiment, levels of 1b ADR mRNA were
increased in the PVN of ADX plus placebo animals (relative integrated
optical density = 832 ± 73 arbitrary units; 164% of sham
control), as compared with sham controls (relative integrated optical
density = 506 ± 48 arbitrary units; p < 0.05, Student-Newman-Keuls). This effect was reversed by a low dose of corticosterone, so that there were no significant differences between levels of 1b ADR mRNA in the ADX plus
10 group compared with sham controls. In ADX animals replaced with a
high dose of corticosterone (ADX plus 50), levels of
1b ADR mRNA were significantly decreased in
the PVN compared with all other groups (relative integrated optical
density = 215 ± 28 arbitrary units, 42% of sham control;
p < 0.01, for each group comparison,
Student-Newman-Keuls). It was determined that levels of
1b ADR mRNA in the PVN were significantly
correlated (inverse relationship) with plasma corticosterone levels at
the time of killing (Pearson analysis;
r2 = 0.638, p < 0.001 for all groups combined;
r2 = 0.717, p < 0.05 for ADX plus 10 mg of cortico-sterone group; Fig.
5b). As was observed in the initial time course experiment, these changes appeared specific to the PVN. No changes in
1b ADR mRNA levels were observed in any
hippocampal region in any of the treatment groups (Table
4).
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Figure 4.
In situ hybridization to show
expression of 1b ADR mRNA after bilateral ADX with low
(10 mg) or high (50 mg) corticosterone replacement. A,
Coronal brain section from a naive rat at the level of the PVN to show
general distribution of 1b ADR mRNA.
B-F, Expression of 1b ADR mRNA in the
PVN of naive (B), SHAM (C),
ADX (D), ADX plus 10 mg of corticosterone
(E), or ADX plus 50 mg of corticosterone
(F). Scale bar, 500 µm.
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Figure 5.
a, Expression of 1b
ADR mRNA in PVN after bilateral adrenalectomy
(ADX) with low (10 mg) or high (50 mg)
corticosterone replacement. Data are expressed as integrated optical
density (arbitrary units). *p < 0.05, **p < 0.01 compared with sham group;
 p < 0.05, p < 0.001 compared with ADX group; ##p < 0.01 compared with
ADX plus 10 group; Student-Newman-Keuls post hoc
multiple comparisons test. b, Correlation analysis
(Pearson; all groups, r2 = 0.638; p < 0.001; ADX plus 10 mg group,
r2 = 0.717;
p < 0.05) between levels of 1b ADR
mRNA in PVN and plasma corticosterone at time of killing, after
bilateral adrenalectomy with corticosterone replacement (0, 10, or 50 mg, s.c.) for 7 d.
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Table 4.
Levels of 1b ADR mRNA in hippocampal
subfields after bilateral ADX with low (10 mg) or high (50 mg)
corticosterone replacement or SHAM surgery
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DISCUSSION |
The data described in the present study demonstrate for
the first time that mRNA encoding one subtype of adrenergic receptor, 1b ADR, is localized in virtually every
CRH-containing parvocellular cell of the PVN. In addition, the majority
of PVN cells found to be responsive to either restraint, swim, or
immune stress, as indicated by expression of c-fos mRNA, were found
tocontain 1b ADR mRNA. Within the PVN,
1b ADR mRNA appears extremely
sensitive to levels of circulating glucocorticoids, because
removal of the adrenal glands led to an upregulation
of expression within 7 d, which was reversed by a low
replacement dose of corticosterone. Exposure of
adrenalectomized animals to a higher dose of corticosterone for 7 d led to a significant decrease in expression of
1b ADR mRNA in the PVN, as compared
with sham-operated controls. Furthermore, in
adrenalectomized rats with replaced corticosterone, levels of
1b ADR mRNA in the PVN were found to be
correlated significantly (inverse relationship) with plasma levels of corticosterone.
Potential role of the 1B ADR in PVN
activation or modulation
As outlined in the introductory remarks, many studies have
indicated the importance of the brainstem ascending noradrenergic and
adrenergic pathways in the control of the HPA axis. The data presented
in the current study indicate that 1B
adrenoceptors within CRH-containing cells of the PVN are potentially in
an excellent position to elicit or modify an HPA axis response.
Although 1b ADR mRNA appears highly
colocalized with c-fos mRNA after stress (restraint, swim, or
intraperitoneal IL-1), we certainly cannot determine from these data
whether this receptor subtype mediates excitation of CRH-containing PVN
neurons, and ultimately the HPA axis, in response to these stimuli.
Indeed, it seems most likely that a primarily systemic stress, such as
IL-1 administration, uses catecholaminergic medullary pathways to a
greater extent than a stressor with a significant neurogenic component,
such as footshock (Li et al., 1996), and probably restraint. Hence, the
function of PVN 1B adrenoceptors might be to
mediate the activity of CRH-containing neurons in the case of an immune
challenge, but may play a lessor role after other stressors. In keeping
with this, a previous study has suggested that the PVN Fos response and
corticosterone secretion after either restraint or hypertonic saline is
not dependent on the 1B adrenoceptor (Williams
and Morilak, 1997). However, it is of interest to note that in several brain regions excitation of 1 adrenoceptors
has been shown to result in slow depolarization, such that the membrane
potential is brought closer to the threshold for firing (Bevan et al.,
1977; McCormick and Prince, 1988; Pan et al., 1994; Bergles et al., 1996). Hence, 1B adrenoceptors appear to be in
an excellent position to modulate the activity of CRH-containing
PVN neurons, and may well be involved in the integration of
stress inputs at the level of the hypophysiotropic neurons of the PVN.
Glucocorticoid regulation of 1b ADR mRNA
Data from the present study demonstrate the regulation of
1b ADR mRNA in the PVN after removal of the
adrenal glands. The reversal of this effect by replacement of
corticosterone strongly suggests that expression of
1b ADR mRNA within the PVN is dependent on
circulating glucocorticoids, consistent with the observation that the
promoter region of the gene contains a GRE (Gao and Kunos, 1993).
However, the data are in contrast to that predicted from in
vitro studies, which demonstrated that dexamethasone and
aldosterone increased 1b ADR mRNA abundance in
DDT1 MF-2 smooth muscle cells because of increased transcriptional
activation (Sakaue and Hoffman, 1991). However, it is well known that
glucocorticoids regulate gene expression in a highly complex manner,
interacting in different ways depending on a vast number of different
factors, including cell type, glucocorticoid concentration, relative
abundance of mineralocorticoid receptors (MR) and glucocorticoid
receptors (GR) within a cell, and relative expression of other
transcription factors (Trapp and Holsboer, 1996; De Kloet et al., 1998;
Gottlicher et al., 1998; Spencer et al., 1998). Thus, the cellular
localization of a gene transcript will be important in determining the
effects of circulating levels of glucocorticoids in vivo,
above and beyond the presence or absence of a GRE. This may also
explain the specificity of 1b ADR regulation
to the PVN. In particular, despite the high expression of both MR and
GR in the hippocampus (Herman et al., 1989), no regulation of
1b ADR mRNA was observed in any hippocampal subfield. However caution should be exercised in interpretation of this
finding, because hippocampal expression of this receptor is extremely
low under basal conditions, and hence changes may be hard to detect.
Is regulation of 1b ADR mRNA mediated by MR
or GR?
In an effort to determine whether MR or GR are involved in the
regulation of 1b ADR mRNA, low and moderate
doses of corticosterone were used in the replacement study. MR has a 6- to 10-fold higher affinity for corticosterone than GR, and this
receptor is thought to be more important than GR in situations when
circulating levels of corticosterone are low, as occurs at the nadir of
the diurnal rhythm (light phase for rats). In contrast, when
circulating levels of corticosterone rise, either because of the normal
circadian rhythm or after HPA activation, although the occupation of MR remains high, the relative importance of GR is increased (Reul and De
Kloet, 1985; Dallman et al., 1991; De Kloet et al., 1998). The
data presented here indicated that a relatively low dose of corticosterone (10 mg pellet) was able to reverse the increase in
expression levels of 1b ADR mRNA obtained
after bilateral adrenalectomy, which may indicate an involvement of MR.
The demonstration that the higher dose of corticosterone (50 mg pellet)
further depressed expression of 1b ADR mRNA in
the PVN is consistent with the idea that this may be a GR-mediated
effect. The relative paucity of MR in the PVN, compared with the
abundance of GR (Reul and de Kloet, 1985), suggests that any role that
MR plays in the regulation of expression of this receptor mRNA is
probably indirect, whereas putative GR-mediated regulation may be
direct. Although expression levels of 1b ADR
mRNA in the PVN were significantly correlated (inverse relationship)
with plasma levels of corticosterone, regulation of this receptor mRNA
occurred over a relatively narrow range of corticosterone. It is
conceivable therefore, that regulation of 1b
ADR mRNA in the PVN is highly sensitive to the balance of occupancy of
MR and GR, and the use of compounds selective for these receptors will
be useful in investigating this further.
Limits of interpretation
At this point, it is not clear whether regulation of mRNA encoding
the 1b ADR is translated into functional
protein. Autoradiography of 1 ADR in the PVN,
using the nonspecific 1 ADR antagonist prazosin, has not demonstrated any changes in receptor level after ADX
(Jhanwar-Uniyal and Leibowitz, 1986; Cummings and Seybold, 1988).
However, we have shown previously that 1a ADR
mRNA is expressed at relatively high levels in the PVN, primarily in
magnocellular cells, but also at low levels in parvocellular cells (Day
et al., 1997), and data from our laboratory suggest that this receptor mRNA is not regulated by glucocorticoids in vivo (our
unpublished data). Hence, the effect of glucocorticoids on the
1B ADR may have been masked in
receptor-binding studies. As antibodies against this receptor become
available and as ligands with increased selectivity are generated that
are able to distinguish between the different 1 receptor subtypes, semiquantitative
immunoradiography and receptor autoradiography will be useful in
determining if levels of 1B ADR protein are
altered by glucocorticoids. In addition, the role of this receptor in
an animal's response to chronic stress, in which the HPA axis is
altered at many levels and often results in an increase in basal levels
of corticosterone, will be of particular interest. Further studies will
be needed to address these questions.
In conclusion, we have demonstrated the existence of a specific subtype
of adrenergic receptor mRNA, 1b ADR, in
CRH-containing, stress-responsive cells of the rat PVN. The levels of
mRNA encoding this receptor in the PVN, but not in other brain regions,
appear to be inversely related to circulating levels of corticosterone. Together these data indicate that the 1B
adrenergic receptor is in a strong position to play a significant role
in the complex and dynamic regulation of the HPA axis in response to stress.
| |
FOOTNOTES |
Received March 11, 1999; revised Aug. 4, 1999; accepted Aug. 25, 1999.
This work was supported by National Institute on Drug Abuse Grant 5RO1
DA02265-18, National Institute of Mental Health Grant 2PO1
MH42251-11, and the Pritzker Network for the study of depression.
Correspondence should be addressed to Heidi E. W. Day, Mental
Health Research Institute, University of Michigan, 205 Zina Pitcher
Place, Ann Arbor, MI 48109-0720. E-mail: heididay{at}umich.edu.
| |
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ABSTRACT
INTRODUCTION
