236

Brain Research, 549 (1991) 236-246 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391166122

BRES 16612

Corticosterone regulation of Type I and Type II adrenal steroid receptors in brain, pituitary, and immune tissue Robert L. Spencer 1, Andrew H. Miller 2, Marvin Stein 2 and Bruce S. McEwen 1 1Laboratory of Neuroendocrinology, Rockefeller University, New York, NY 10021 (U.S.A.) and 2Department of Psychiatry, Mt. Sinai School of Medicine, New York, NY 10029 (U.S.A.)

(Accepted 11 December 1990) Key words: Glucocorticoidreceptor; Receptor down-regulation; Stress; Hippocampus; Spleen; Thymus; Rat

Type I and Type II adrenal steroid receptor levels were compared in the brain, pituitary and immune system of adrenalectomized rats in the presence or absence of several replacement doses of corticosterone. Six days of adrenalectomyproduced an up-regulation of Type II adrenal steroid receptors in the brain and spleen. The lowest replacement dose of corticosterone (equivalent to resting levels of this hormone) blocked this Type II receptor up-regulation, while higher replacement doses of corticosterone were associated with widespread Type I and Type II adrenal steroid receptor down-regulation. However, the dose of corticosterone required for receptor down-regulation varied between tissues. Specifically, hippocampal receptors were most sensitive to corticosterone, whereas pituitary receptors were the least sensitive. All tissues examined, except the pituitary, exhibited a down-regulation of Type II receptors with a high corticosterone replacement dose which approximated acute stress levels of this hormone. In summary, physiologicallyrelevant concentrations of corticosterone were capable of down-regulating Type I and Type II adrenal steroid receptors in multiple brain areas and peripheral immune tissues, including peripheral blood mononuclear cells. In contrast, adrenal steroid receptor levels in the pituitary were relatively insensitive to regulation by corticosterone. INTRODUCTION Adrenal steroids, especially glucocorticoids, play an important role in the interaction between nervous, endocrine and immune systems 31. Two different cytosolic receptors for adrenal steroids have been described by receptor binding studies 33'52 and, recently, cDNA clones corresponding to the 2 receptor types have been identified 2'19. The 2 adrenal steroid receptor types differ in their affinities for corticosterone, the primary glucocorticoid in the rat, and differ in their distribution throughout the body. The Type I adrenal steroid receptor, also referred to as the mineralocorticoid receptor, has a high affinity for corticosterone and is most concentrated in the hippocampus. Much lower levels of the Type I adrenal steroid receptor are found in peripheral tissues including the spleen, while no Type I adrenal steroid receptor binding has been detected in the thymus 3°. The Type II adrenal steroid receptor, also referred to as the glucocorticoid receptor, has a 3-10 fold lower affinity for corticosterone than does the Type I receptor. The Type II receptor is fairly evenly distributed throughout central and peripheral tissues and is found in high concentrations in the spleen and thymus 23'3°. Several in vivo studies have reported that glucocorti-

coid treatment or chronic stress were able to downregulate adrenal steroid receptor level 34'37-39'45-48, whereas, removal of endogenous glucocorticoids by adrenalectomy resulted in adrenal steroid receptor upregulation 26"49. However, not all chronic stress paradigms have produced a down-regulation of adrenal steroid receptors 42"47'53 and the parameters for adrenal steroid receptor down-regulation by glucocorticoids in vivo have not been well characterized. The threshold dose of corticosterone necessary for adrenal steroid receptor down-regulation has not been determined and whether or not this dose is within the physiological range for endogenous corticosterone is unclear. In addition, most in vivo studies of adrenal steroid receptor regulation have focused only on adrenal steroid receptor levels in the hippocampus and have suggested that the hippocampus is uniquely sensitive to glucocorticoid and stress effects on receptor level. However, the susceptibility of other brain regions or peripheral tissues to adrenal steroid receptor down-regulation has not been extensively evaluated. Finally, potential differences in the regulation of Type I vs. Type II adrenal steroid receptors by corticosterone have not been examined in detail. We report here studies examining the ability of systemic corticosterone to down-regulate both Type I and

Correspondence: R.L. Spencer, Rockefeller University, Box 290, 1230 ~ork Ave., New York, NY 10021, U.S.A.

237

T y p e II a d r e n a l s t e r o i d r e c e p t o r s in a v a r i e t y o f b r a i n a r e a s , p i t u i t a r y , a n d s e v e r a l i m m u n e tissues, i n c l u d i n g peripheral blood mononuclear

cell ( P B M s ) .

For these

studies we examined a physiologically relevant range of corticosterone

doses

so

that

the

threshold

dose

of

c o r t i c o s t e r o n e n e c e s s a r y t o d o w n - r e g u l a t e a d r e n a l ster o i d r e c e p t o r s in e a c h t i s s u e c o u l d b e a p p r o x i m a t e d . MATERIALS AND METHODS

Subjects. Male Sprague-Dawley rats (300-350 g) were obtained from Charles River (Kingston) and for the duration of treatment were housed (2-3 per chamber) in standard clear plastic housing chambers. The animal room was maintained on a 14:10 lights on:off schedule with lights on at 5 a.m. The rats were given ad lib rat chow and tap water. Following adrenalectomy, 0.9% saline was substituted as the sole drinking solution. Steroids. [1,2,6,7-3H(N)]corticosterone (87.1 Ci/mmol) was obtained from New England Nuclear (Boston, MA). [1,2,6, 7-3H(N)]aldosterone (75 Ci/mmol) and [1,2,4-3H(N)]dexametha sone (40 Ci/mmol) were obtained from Amersham (Arlington Heights, IL). Unlabeled corticosterone was obtained from Steraloids (Wilton, NH). The selective Type II receptor agonist, RU28362, was a gift from Roussel-Uclaf (RomainviUe, France). Adrenalectomy and corticosterone replacement. Bilateral adrenalectomy was performed on rats that were fully anesthetized with the inhalant, methoxyflurane (Metofane, Pitman-Moore, Washington Crossing, NJ). Corticosterone pellets were surgically implanted subcutaneously, and for replacement studies were implanted at the same time that the animals were adrenalectomized. Corticosterone pellets were made according to the method of Meyer 2s and weighed approximately 100 rag. For the low dose corticosterone treatment the pellets were made of a 50% mixture of corticosterone and cholesterol. For all other corticosterone treatment doses the pellets were made of 100% corticosterone. Pellets were placed under the skin of the back region and, in the case where rats received 4 pellets, also under the skin of the posterior lateral flank. No more than 2 pellets were placed at any one location. Experimental procedures Experiment 1. There were 6 treatment groups with 4 animals per group. Four groups of rats were adrenalectomized 6 days before sacrifice and 2 groups were adrenalectomized 24 h before sacrifice. One group served as a 6 day ADX group. The 3 other 6 day ADX groups were given a replacement dose of corticosterone, via subcutaneously placed corticosterone pellets. The 3 replacement doses were one 50% corticosterone pellet (low dose), one 100% corticosterone pellet (medium dose) or 4 100% corticosterone pellets (high dose). The corticosterone pellets were removed 24 h before sacrifice, a time period that our pilot studies indicated was sufficient for total clearance of corticosterone from the blood plasma, even for the high dose corticosterone treatment. A 5th group was also implanted with 4 100% corticosterone pellets for 5 days, but the adrenals were left in place until 24 h before sacrifice at which time both the pellets and the adrenals were removed. The 6th group was a 24 h A D X group. Adrenal steroid receptor levels measured in animals that were ADX for 24 h were used to approximate the basal level of adrenal steroid receptors in an intact animal. This is a time period of ADX that is routinely used to assess adrenal steroid receptor levels in rats since it provides sufficient time for clearance of endogenous steroids which interfere with the binding measures but is believed to be less than the time necessary for significant up-regulation of adrenal steroid receptors 35"47. The inclusion of the 24 h ADX animals allowed for assessment of the degree of up-regulation that occurred with 6 days of adrenalectomy. Furthermore, we were able to distinguish between replacement doses of corticosterone that

blocked the up-regulation of adrenal steroid receptors vs. doses of corticosterone that produced a down-regulation of adrenal steroid receptors relative to the 24 h A D X level of adrenal steroid receptors. Both type I and type II adrenal steroid receptor binding was examined in the hippocampus and spleen of each of the 6 treatment groups. Adrenal steroid receptor binding was also examined in pituitaries from the 6 day A D X group, 6 day A D X with high dose corticosterone replacement group, and in the 24 h ADX group. Experiment 2. The basic design of the first experiment was repeated except only 4 treatment groups were examined with 5-6 rats per group. The treatment groups were a 6 day A D X group, 6 day A D X groups with 5 days of intermediate (2 pellets) or high (4 pellets) dose corticosterone replacement, and a 24 h A D X group. For this experiment type I and type II adrenal steroid receptor binding of the 4 treatment groups was measured in the immune tissues, spleen and thymus, in a variety of brain areas (hippocampus, cortex overlying the hippocampus, septum + diagonal band of Broca, cerebellum, and hypothalamus) and in the pituitary. Type II adrenal steroid receptor binding was also examined in isolated peripheral blood mononuclear cells from rats in all but the intermediate corticosterone replacement group. Isolation of peripheral blood mononuclear cells. Trunk blood was collected into heparinized tubes and diluted 1:3 with Hanks balanced salt solution (HBSS; GIBCO, Grand Island, NY). The resultant suspension was then layered on a Ficoli-Hypaque gradient and centrifuged for 45 min at 500 g. The white blood cell layer was removed and washed twice with HBSS. The cells were then counted, pelleted and frozen for subsequent adrenal steroid receptor measures. When necessary, cells were pooled from 2 or 3 animals within a treatment group to provide a sufficient number of cells for the binding assay. This procedure resulted in a final n of 3 independent samples per treatment group. The mean cell counts and protein concentration for the samples from the 24 h A D X group (4.1 x 1 0 6 cells, 1.1 mg/ml) and high dose corticosterone replacement group (4.3 x 1 0 6 cells, 1.1 mg/ml) did not differ, whereas, the cell count and protein concentration for the 6 day A D X group (11.8 × 106 cells, 1.7 mg/ml) was higher than for the other 2 groups. The receptor binding level measured for each sample was normalized by expressing adrenal steroid receptor binding as the concentration of specific binding relative to protein content. Type I and Type H adrenal steroid receptor binding. An in vitro cytosolic receptor binding assay was used to measure adrenal steroid receptor levels. The binding assay procedure, as has been described in detail elsewhere 43, consisted of tissue homogenization, centrifugation at 105,000 g for 60 min at 4 °C, and then placement of the resulting supernatant (cytosol) in incubation solutions containing radiolabeled steroids with or without unlabeled competitors. After overnight incubation (18-22 h, 4 °C), cytosol/incubation solutions were filtered through columns containing 1.25 ml of LH-20 Sephadex (Pharmacia, Piscataway, N J). The eluate, containing bound steroid, was collected directly into scintillation vials. Scintillation fluor (Ready Safe, Beckman, Fullerton, CA) was added to the vials and tritium radioactivity counted on a scintillation counter (Packard Series 1500 Tri-Carb; 38% efficiency). The homogenization and incubation buffer contained 10 mM Tris, 1 mM EDTA, 20 mM molybdic acid, 5 mM dithiothreitol and 10% glycerin in double distilled water (pH = 7.4). For saturation binding assays, 5 different concentrations of [3H]dexamethasone (0.3-10 nM) or [3H]aldosterone (0.3-6 nM) were used. Type II binding was derived by subtracting [3H]dexamethasone binding in the presence of a selective type II competitor, RU28362 (0.5 #M) ~°, from total binding. This concentration of RU28362 was used based on competition binding studies that indicated that 0.5 # M RU28362 would displace [3H]dexamethasone binding (10 nM) to Type II, but not Type I, hippocampal binding sites (data not shown). Type I binding was derived by subtracting non-specific binding (binding in the presence of 2.5 g M corticosterone) from [3H]aldosterone binding in the presence of RU28362 (0.5 pM). For single point determinations of Type II adrenal steroid receptor binding a single

238 saturating concentration of [3H]dexamethasone (10 nM) with or without the presence of RU28362 (0.5/~M) was used. For single point determinations of Type I adrenal steroid receptor binding a single saturating concentration of [3H]aldosterone (6 nM) in the presence of RU28362 (0.5/~M) or corticosterone (2.5 pM) was used. [3H]Dexamethasone (10 nM) was substituted for [3H]aldosterone when measuring Type I binding in the spleen due to the low Type I signal in that tissue. [3H]Dexamethasone is an effective ligand for measuring Type I adrenal steroid receptors in vivo24"43. Furthermore, LH-20 is much more efficient at trapping free [-~H]dexamethasone than [3H]aldosterone25 and consequently, we have found that when measuring the low level of Type I receptors that are present in the spleen that [3H]dexamethasone provides a cleaner signal than does [3H]aldosterone. Specific binding was expressed as fmol per mg of cytosol protein. Protein content was determined by the method of Bradford 3, with use of bovine serum albumin as a standard. Corticosterone plasma measurement. Plasma corticosterone was measured by radioimmunmoassay using a commercially available rabbit antiserum raised against corticosterone-3-oxime BSA (B3163, Endocrine Sciences, Tarzana, CA). The antiserum has very low cross reactivity with other major steroids. Assay sensitivity was 10 pg of corticosterone, and coefficients of variation within and between assays were 5% (n = 4) and 10% (n = 4), respectively. Data analysis and statistics. For saturation binding studies the binding parameters, dissociation constant (Kd) and binding maximum (Bmax), were derived from Scatchard analysis 22. For single point receptor binding measures and organ weight measures analysis of variance was used to test for overall differences between treatment groups. Both the Student's t-test and Tukey test were used for post hoc tests of significant differences between means in order to include both a conservative (Tukey test) and powerful (Student's t-test) assessment of statistical significance. Means that differed significantly according to the Student's t-test were only indicated in the cases where they did not also differ significantly according to the Tukey test. Data are expressed as mean _+ S.E.M.

80

I

.t

High CORT (Ex~ 1) High CORT (Ex~ 2)

*

Intmnedi~ CORT(Exl~ 2) m , ~ C O R T ~ E ~ t) I

20.

b

~

1

3

5

Day After CORT Replacement Fig. 1. Plasma corticosterone levels of adrenalectomized rats with corticosterone (CORT) pellet replacement. The CORT pellets (approximately 100 mg) were surgically placed under the skin (see Materials and Methods) and the replacement doses used in the first and second experiment (Expt. 1 and Expt. 2) were as follows: high CORT, 4 100% CORT pellets; intermediate CORT, 2 100% CORT pellets; medium CORT, one 100% CORT pellet; low CORT, one 50% CORT pellet.

T h e a d r e n a l s t e r o i d r e c e p t o r b i n d i n g in t h e 24 h A D X g r o u p was u s e d as a r e f e r e n c e a n d was c o n s i d e r e d to reflect the steady-state level of adrenal steroid receptors t h a t a r e p r e s e n t in a n o r m a l i n t a c t r a t (see M a t e r i a l s a n d Methods).

Plasma corticosterone levels and organ weights. So t h a t

RESULTS

the replacement doses of corticosterone could be related to p h y s i o l o g i c a l

Experiment 1

levels o f e n d o g e n o u s

corticosterone,

low,

c o r t i c o s t e r o n e levels o f a s e p a r a t e g r o u p o f i n t a c t rats was

m e d i u m a n d h i g h r e p l a c e m e n t d o s e o f c o r t i c o s t e r o n e to

evaluated during the nadir and the peak of their diurnal

produce a down-regulation of adrenal steroid receptors.

cycle a n d a f t e r 1 h o f r e s t r a i n t stress. T h e a . m . a n d p . m .

This

experiment

evaluated

the

ability

of

a

TABLE I

Body, thymus and spleen weights of animals in experiment 1 Rats were adrenalectomized (ADX) either 6 days or 24 h before sacrifice. Three groups of 6 day ADX rats were given corticosterone replacement with subcutaneous corticosterone (CORT) pellets (approximately 100 mg), placed in the rat for 5 days and then surgically removed 24 h before sacrifice. One group of rats was left adrenal intact during 5 days of high dose CORT treatment and then 24 h before sacrifice had CORT pellets and adrenals removed. Treatment doses were: low dose, one 50% CORT pellet; medium dose, one 100% CORT pellet; high dose, 4 100% CORT pellets.

Treatment

Body weight (b. wt.) (g)

Thymus weight (mg)

% Thymus weight (mg/lOOgb. wt.)

Spleen weight (rag)

% Spleen weight (mg/lOOgb. wt.)

24 h A D X 6 Day A D X 6 Day ADX + low CORT 6 Day A D X + medium CORT 6 Day A D X + high CORT 24 h ADX + high CORT

348 + 320 + 322 + 331 + 292 ± 304 ±

717 + 806 + 568 + 663 + 185 ± 207 +

206 + 253 + 176 + 200 + 64 + 68 +

717 + 690 + 718 + 675 + 485 + 447 +

206 + 13 216 ± 16 222 + 5 204 +_ 6 166 + 4 ~ 147 + 10"*

3 3 7 8 15"* 5*

39 38 62 25 9** 32**

11 14a 20 7 2** 12"*

* P < 0.05, **P < 0.01, for significantly different from the 24 h ADX group, Tukey test (n = 3-4). a p < 0.05, for significantly different from the 24 h ADX group, Student's t-test (n = 3-4).

43 47 25 28 29** 36**

239 100 250,

l~ 2A h ADX 6 day ADX

i

I! 200 ¸

Low CORT Medium CORT

24 h ADX 6 day ADX CORT HighCORT

150

100,.n m

50-

[-

o

s

Pituitary

Spleen

Hippocampus

Cortex

Hippocs~np~

Cortex

Selmim

Hypothalam~ Ce,~.ll~

700 600.

Z"

500, v 300.

300 200. v

100. 0

100

0 Hippocampus

Pituitary

Spleen

Fig. 2. Type I and Type II adrenal steroid receptor binding in hippocampus, pituitary and spleen after adrenalectomy (ADX) and corticosterone (CORT) replacement (Experiment 1). Replacement doses of CORT were removed 24 h before animals were sacrificed for receptor binding measurements. Single point binding determinations were made on tissue from individual animals (n = 3-4). Statistically significant differences from the 24 h ADX group are indicated: *P < 0•05, **P < 0.01, Tukey test.

basal corticosterone levels were 1.2 + 0.2/~g% and 25.2 + 2.9/~g%, respectively• The corticosterone levels after 1 h of restraint stress were 41.7 + 3.0 ~g%. The plasma corticosterone levels of the corticosterone replaced rats was measured on the 1st, 3rd and 5th day of replacement (Fig. 1). The low corticosterone dose (one 50% corticosterone pellet) produced plasma corticosterone levels (9.2 + 0.5/~g%) throughout the 5 days of treatment that were intermediate between the a.m. and p.m. basal levels of intact rats. The medium corticosterone dose (one 100% corticosterone pellet) produced plasma corticosterone levels (13.5 + 1.0/tg%) that were only slightly higher than the low dose levels. The high corticosterone dose (4 100% corticosterone pellets) produced plasma corticosterone levels in the high

Selmim

HypothLhtmus C~ehelltan

Fig• 3. Type I and Type II adrenal steroid receptor binding in brain tissue after adrenalectomy (ADX) and corticosterone (CORT) replacement (Experiment 2). Replacement doses of CORT were removed 24 h before animals were sacrificed for receptor binding measurements. Single point binding determinations were made on tissue from individual animals (n = 5-6). Statistically significant differences from the 24 h ADX group are indicated: *P < 0.05, **P < 0.01, ***P < 0.001, Tukey test; ap < 0.05, Student's t-test. physiological stress range. The high dose plasma corticosterone levels progressively decreased across the 5 treatment days from 54.8 _+ 1.8/~g% after 1 day to 28.2 _ 2 . 7 / a g % after 5 days of treatment. The decline in circulating corticosterone levels over the 5 days of treatment observed in rats receiving the high corticosterone dose may have been a consequence of induced metabolism of corticosterone rather than a decline in pellet secretion rate. In a separate group of animals (n = 3), the 4 corticosterone pellets were replaced after 5 days of treatment with 4 new pellets. There was no increase in circulating corticosterone levels after replacement with new pellets (data not shown). There was a significant increase in thymus weight with 6 days of A D X which was blocked by both low and medium corticosterone replacement. Neither the low or medium corticosterone doses had a negative effect on body weight, thymus weight or spleen weight (Table I). The high dose corticosterone treatment, on the other hand, produced a significant decrease in body weight,

240 TABLE II

Body, thymus and spleen weightsof animals in experiment2 Rats were adrenalectomized (ADX) either 6 days or 24 h before sacrifice. Two groups of 6 day ADX rats were given corticosterone replacement with subcutaneous corticosterone (CORT) pellets (approximately 100 mg), placed in the rat for 5 days and then surgically removed 24 h before sacrifice. Treatment doses were: intermediate dose, 2 100% CORT pellets; high dose, 4 100% CORT pellets.

Treatment

Body weight (b. wt.) (g)

Thymus weight (mg)

% Thymus weight (mg/lOOgb. wt.)

Spleenweight (mg)

% Spleen weight (mg/lOOgb. wt.)

24 h ADX 6DayADX 6 Day ADX + intermediate CORT 6 Day ADX + high CORT

332 + 14 292 + 11 310 + 10 287 _+ 11a

604 + 17 666 + 22" 259 + 33*** 138 + 21"**

183 + 230 + 84 + 47 +

756 + 66 709 + 41 668 + 54 509 _+54*

229 + 22 245 _+ 18 215 __.13 176 _+ 13

8 15" 12"** 5***

* P < 0.05, ***P < 0.001, for significantly different from the 24 h ADX group, Tukey test (n = 5-6). " P < 0.05, for significantly different from the 24 h ADX group, Student's t-test (n = 5-6). and a significant decrease in the percent of thymus and spleen weight relative to body weight. Rats that were given high dose corticosterone treatment without prior A D X had high plasma corticosterone levels very similar to the high dose corticosterone r e p l a c e m e n t group (data not shown) and c o m p a r a b l e decreases in body, thymus and spleen weights (Table I). A l t h o u g h all rats were A D X at time of euthanasia, one of the rats from the 6 day A D X group and one of the rats from the high dose corticosterone r e p l a c e m e n t group had low, but detectable, corticosterone levels in their trunk blood collected at the time of euthanasia and their data were excluded from the analyses. All of the other rats had undetectable corticosterone levels. Adrenal steroid receptor levels. Type I adrenal steroid receptors showed no up-regulation in any of the tissues e x a m i n e d after 6 days of adrenalectomy. There was a significant down-regulation of Type I adrenal steroid receptors in the hippocampus with the medium corticosterone r e p l a c e m e n t dose and in the hippocampus and spleen with the high corticosterone replacement dose (Fig. 2). Type II adrenal steroid receptors in the hippocampus showed a significant up-regulation after 6 days of adrenalectomy (Fig. 2). The low and medium corticosterone r e p l a c e m e n t doses were sufficient to block the upregulation of h i p p o c a m p a l Type II adrenal steroid receptors but did not p r o d u c e a down-regulation of Type I1 adrenal steroid receptors. However, the high replacement dose of corticosterone p r o d u c e d a significant down-regulation of Type II adrenal steroid receptors in the h i p p o c a m p u s and spleen. The degree of Type II adrenal steroid r e c e p t o r down-regulation with the high dose corticosterone t r e a t m e n t was the same for rats that were either A D X or intact during the 5 days of corticosterone treatment. The pituitary stood out as uniquely insensitive to the effects of corticosterone treatment on adrenal steroid receptor regulation. There was no down-regulation of

Type I or Type II adrenal steroid receptors with high dose corticosterone t r e a t m e n t in the pituitary. F u r t h e r m o r e , there was no up-regulation of adrenal steroid receptors after 6 days of A D X (Fig. 2).

Experiment 2 In the second e x p e r i m e n t the basic design of the first experiment was r e p e a t e d in o r d e r to replicate the effect of high corticosterone t r e a t m e n t on adrenal steroid receptor down-regulation. The second e x p e r i m e n t included an i n t e r m e d i a t e dose of corticosterone replacement (2 100% corticosterone pellets) that was higher than the m e d i u m dose used in the first e x p e r i m e n t in an attempt to m o r e closely d e t e r m i n e the threshold dose of corticosterone that would down-regulate adrenal steroid receptors. The second e x p e r i m e n t also included measures of adrenal steroid r e c e p t o r level in a variety of brain regions, in the pituitary, thymus and in isolated peripheral blood m o n o n u c l e a r cells (PBMs). Peripheral blood m o n o n u c l e a r cells are an easily o b t a i n a b l e source of cells from human patients. If there is a parallel regulation of adrenal steroid r e c e p t o r levels in PBMs and in other tissues then PBMs may serve as useful m a r k e r cells, reflecting altered adrenal steroid receptor levels in other tissues.

Plasma corticosterone levels and organ weights. The intermediate r e p l a c e m e n t dose of corticosterone produced plasma corticosterone levels that after one day of t r e a t m e n t (24.1 + 2.2 ktg%) were similar to the diurnal peak level of e n d o g e n o u s corticosterone in intact rats (Fig. 1). By the 5th day of t r e a t m e n t the plasma corticosterone levels of the i n t e r m e d i a t e replacement group had tailed off to 10.5 + 2.0 /~g%. The high corticosterone dose p r o d u c e d plasma corticosterone levels very similar to those o b s e r v e d with high dose treatment in the first e x p e r i m e n t (Fig. 1). None of the treatment groups had detectable corticosterone levels in their trunk blood. As in the first e x p e r i m e n t , there was a significant

241 70

TABLE III

l

i 2,t h AD 6 day AI

60. 50-

Type I and Type H adrenal steroid receptor binding in hippocampal cytosol: Scatchard analysis

Intml~ HighOC

Treatment conditions are described in Materials and Methods and Table II. Scatchard analysis was performed on Type I and Type II adrenal steroid receptor saturation binding curves. Cytosol was pooled from one half hippocampus of each rat within a treatment group (5-6 rats per group). Single point binding measures on the remaining half hippocampus for the individual animals are presented in Fig. 3.

3020-

°

Type I

10-

0 Thymus 1000

ND Spleen Pituitary

PBM

I

I~0

*a,

0

Thymus

Spleen Pituitary PBM

Fig. 4. Type I and Type II adrenal steroid receptor binding in peripheral tissue after adrenalectomy (ADX) and corticosterone (CORT) replacement (Experiment 2). Replacement doses of CORT were removed 24 h before animals were sacrificed for receptor binding measurements. Single point binding determinations for thymus, spleen and pituitary were made on tissue from individual animals (n = 5-6). Peripheral blood mononuclearcells (PBM) were sometimes pooled between 2 or 3 animals within a treatment group in order to have sufficient number of cells for receptor binding determinations (n = 3). No Type I binding was detected in thymus tissue and Type I binding in PBM was not determined (ND). Statistically significant differences from the 24 h ADX group are indicated: *P < 0.05, **P < 0.01; ***P < 0.001, Tukey test; ap < 0.05, Student's t-test.

increase in thymus weight relative to total body weight after 6 days of A D X (Table II). Both the intermediate and the high dose corticosterone treatments produced a significant decrease in absolute thymus weight and in thymus weight relative to body weight when compared to thymus weights of the 24 h A D X group. The high dose corticosterone treatment also produced a decrease in body weight and in absolute spleen weight (Table II). Adrenal steroid receptor level. As was the case in the first experiment, there was not an up-regulation of the Type I adrenal steroid receptor in any of the tissues

24 h ADX 6 Day ADX 6 Day ADX + intermediate CORT 6 Day ADX + high CORT

Type H

Ka

B,,~

g d

Bma x

1.2 1.4

192.4 190.9

0.6 0.4

363.4 471.3

1.3

93.5

0.5

265.4

1.2

114.0

0.6

260.7

examined after 6 days of ADX, although there was a trend in that direction for the hippocampus. There was an especially high variability in hippocampal Type I binding level of the 6 day A D X group, suggesting a variable degree of Type I receptor up-regulation between individual animals. On the other hand, there was a significant and consistent down-regulation of the Type I receptor in the hippocampus with the intermediate corticosterone replacement dose and in the hippocampus, septum, cerebellum and spleen with the high corticosterone replacement dose (Figs. 3, 4). As we have reported before, we were not able to detect any Type I signal in the thymus 3°. The Type II adrenal steroid receptor was up-regulated after 6 days of A D X in all of the brain areas examined and in the spleen (Figs. 3, 4). There was a downregulation of the Type II adrenal steroid receptor in the hippocampus, septum, spleen and the thymus with the intermediate corticosterone replacement dose and in all of the tissues examined, except the pituitary, with the high corticosterone replacement dose. The magnitude of the Type II receptor down-regulation was similar across brain areas and in the spleen (16-31%), whereas in the thymus and the peripheral blood mononuclear cells there was a much larger percent down-regulation (65%). Scatchard analysis was performed on pooled hippocampi from each treatment group (Table III) and the pattern of relative Bmax'S for each treatment group corresponded to the single point binding results; there was a higher Type II adrenal steroid receptor Bmax for the 6 day A D X group relative to the 24 h A D X group and both the Type I and Type II adrenal steroid receptor Bmax'S were lower in the intermediate and high corticosterone replacement groups. There was no difference

242 in Ko's between treatment groups for either the Type I or Type II adrenal steroid receptor. This indicates that there was no residual corticosterone in the tissue of any of the treatment groups, confirming that total clearance of corticosterone had occurred by 24 h after corticosterone pellet removal and ADX. There was an overall greater level of Type I and Type II adrenal steroid receptor binding in the second experiment compared to the first experiment. Although the different tissue regions within each experiment were assayed separately, all of the tissues examined (and therefore all of the assays) from the second experiment yielded higher adrenal steroid receptor levels than were obtained from the first experiment. We have noted previously considerable variability in absolute adrenal steroid receptor level between experiments. However, when re-assaying tissue from the same animals within an experiment we obtain very similar inter-assay binding levels (Spencer and Miller, unpublished observations). There may be genuine differences in absolute adrenal steroid receptor binding level between different batches of rats, nevertheless, the relative differences in binding level between receptor subtypes, tissue regions and treatment groups was very similar in our 2 experiments (compare Figs. 2, 3 and 4). DISCUSSION

Tissue differences in adrenal steroid receptor regulation by corticosterone. Sustained elevation of corticosterone for 5 days resulted in Type I and Type II adrenal steroid receptor down-regulation in a number of tissue regions, both in the brain and in peripheral immune tissues. All tissues examined, except the pituitary, exhibited Type II adrenal steroid receptor down-regulation with a high dose of corticosterone treatment that produced circulating corticosterone levels in the acute stress range. The ubiquity of the down-regulation of adrenal steroid receptors observed in this study has not been described before. Although 2 other studies have suggested that only hippocampal adrenal steroid receptors down-regulate after corticosterone treatment, neither study examined a wide range of tissues nor did they utilize more than one treatment dose of corticosterone 39"4s. Although adrenal steroid receptor down-regulation was evident throughout the brain and in immune tissue, there were differences, between tissues in the threshold dose of corticosterone necessary to produce adrenal steroid receptor down-regulation. The hippocampus was the most sensitive site, with hippocampal Type I adrenal steroid receptors down-regulating at a lower dose than in any other tissue. The sensitivity of the hippocampus for Type II adrenal steroid receptor down-regulation, how-

ever, was matched by the septum, spleen and thymus. The pituitary stood out as uniquely insensitive to corticosterone, as evidenced by a lack of adrenal steroid receptor down-regulation with the range of corticosterone doses used in these studies. A number of investigators have proposed that PBMs, which are an easily obtainable source of cells from human patients, may serve as peripheral markers, reflecting adrenal steroid receptor changes in brain tissue. Our study confirms that a down-regulation of adrenal steroid receptors can be measured in PBMs and may parallel receptor levels changes in the brain, at least for the Type II adrenal steroid receptor. However, it must be emphasized that with traditional adrenal steroid receptor binding studies an accurate measure of adrenal steroid receptors can only be made when endogenous glucocorticoids are absent 8"43'46. Consequently, when measuring adrenal steroid receptors in PBMs from human patients, an increased occupation and activation of adrenal steroid receptors by elevated endogenous glucocorticoids may not be differentiated from a down-regulation of adrenal steroid receptors. Even in the present study, in which endogenous glucocorticoids were absent, there is the potential that the differences in adrenal steroid receptor binding of the different corticosterone replaced groups was partially a consequence of the different clearance times of corticosterone after pellet removal. The recent use of antibodies raised against adrenal steroid receptors to measure adrenal steroid receptor level may provide a solution to this problem in future studies 1. The relationship between adrenal steroid receptor activation by acute stress and adrenal steroid receptor downregulation. In the brain, the tissue differences in the threshold dose of corticosterone necessary to downregulate adrenal steroid receptors may be a result of tissue differences in the extent of adrenal steroid receptor occupation and activation with varying doses of chronic corticosterone treatment. In a previous study in which we estimated the degree of adrenal steroid receptor occupation and activation by basal and stress levels of endogenous glucocorticoids we found differences in the sensitivity to glucocorticoids between brain regions and the pituitary that parallels the differences reported here for sensitivity to adrenal steroid receptor downregulation 43. It appears that there may be factors within the various tissues that can regulate the availability of glucocorticoids to the adrenal steroid receptors within those tissues 4. A factor that may contribute to a diminished regulation of adrenal steroid receptors in the pituitary by corticosterone is corticosteroid binding globulin. Corticosterone binding globulin is found in high concentration within pituitary tissue, possibly even intracellularly, and may act to buffer the pituitary from

243 circulating glucocorticoids 12'13'2°'21. The intrinsic regulation of adrenal steroid receptors in the pituitary may also differ from other tissues. In a recent study41, Type II adrenal steroid receptor mRNA levels in the pituitary, but not the hippocampus increased with glucocorticoid treatment. Interestingly, adrenal steroid receptors of the spleen and thymus were as sensitive to the down-regulatory effects of chronic corticosterone treatment as were the adrenal steroid receptors in brain tissue. In contrast, we have noted in a previous study that Type II adrenal steroid receptors in both spleen and thymus did not exhibit significant occupation/activation by endogenous glucocorticoids after acute stress; only Type I adrenal steroid receptors in the spleen were occupied and activated 3°. Thus, with sustained elevation of glucocorticoids there is eventual access of adrenal steroids to the adrenal steroid receptors in these immune tissues. Perhaps factors that are able to buffer adrenal steroid receptors of the spleen and thymus from glucocorticoids under acute stress conditions become saturated and ineffective under conditions of sustained glucocorticoid elevation.

Type I vs. Type H adrenal steroid receptor regulation by corticosterone. These studies indicate that the concentrations of both Type I and Type II adrenal steroid receptors in vivo are subject to autoregulation by their endogenous ligand, corticosterone. The ability of the Type I adrenal steroid receptor to be down-regulated by corticosterone treatment is not consistent with a previous report 34. However, in the previous report the only dose of corticosterone tested (one 100 mg corticosterone pellet implanted s.c.) was equivalent to the medium corticosterone dose of our first experiment, a dose that was near the borderline for producing Type I adrenal steroid receptor down-regulation. Removal of endogenous glucocorticoids with adrenalectomy resulted in an up-regulation of Type II adrenal steroid receptors, suggesting that Type II adrenal steroid receptor levels are subject to some tonic inhibition by normal circulating concentrations of endogenous adrenal steroids. Our lowest replacement dose of corticosterone was sufficient to prevent the up-regulation of Type II adrenal steroid receptors in the hippocampus, whereas, a considerably higher dose of corticosterone was necessary to produce a down-regulation of Type II adrenal steroid receptors. It is possible that in some tissues the Type I adrenal steroid receptor mediates tonic depression of Type II adrenal steroid receptor levels. Recent studies provide support for co-localization of both Type 1 and Type II adrenal steroid receptors in some cells within the hippocampus 5°. Luttge et al. 27 report that aldosterone, a mineralocorticoid with predominantly Type I adrenal

steroid receptor specificity, was able to down-regulate Type II adrenal steroid receptors in the hippocampus and cortex of the mouse brain. Furthermore, the selective Type I adrenal steroid receptor antagonist, RU26752, was able to block the down-regulatory effect of aldosterone. Since the study by Luttge et al. 27 used adrenal steroid receptor level from 4 day adrenalectomized mice as their reference point, it is possible that much of the aldosterone effect on Type II adrenal steroid receptor level was to block the up-regulation of Type II adrenal steroid receptors that occurs after ADX rather than an actual down-regulation of receptors. In the present study a down-regulation of Type I and Type II adrenal steroid receptors was only observed with intermediate to high doses of corticosterone. The low corticosterone replacement dose of the first experiment produced plasma corticosterone levels well above a.m. basal levels and therefore presumably saturated Type I receptors 33, but did not result in a down-regulation of Type I or Type II receptors. Consequently, our data suggest that corticosterone must act at the Type II adrenal steroid receptor before Type I and Type II adrenal steroid receptor down-regulation by corticosterone can occur. Certainly the requirement of the high corticosterone replacement dose to down-regulate adrenal steroid receptors in some of the tissue areas examined points to a Type II adrenal steroid receptor mediation of down-regulation in those regions. It is possible that some co-operation between Type I and Type II adrenal steroid receptors contributes to adrenal steroid receptor down-regulation. Adrenal steroid receptors are intracellular proteins that can bind directly to DNA and apparently act as ligand-dependent transcription factors 5. Consequently, there is the potential for a direct negative feedback effect of activated adrenal steroid receptors on subsequent adrenal steroid receptor transcription. The autoregulation of adrenal steroid receptors by their endogenous ligand has been reported in a variety of in vitro cell lines 44. In the in vitro case, the down-regulation of adrenal steroid receptors has been correlated with decreases in adrenal steroid receptor mRNA levels 5,32,36,51. On the other hand, the effect of high dose glucocorticoid treatment on adrenal steroid receptor mRNA level in vivo has not been consistently demonstrated 7,~s,41. This raises the possibility that elevated glucocorticoids can produce a down-regulation of adrenal steroid receptors by a post-transcriptional mechanism. Several in vitro studies have also concluded that there may be some post-transcriptional effects of glucocorticoids that contribute to adrenal steroid receptor down-regulation 14,51.

Physiological implications of adrenal steroid receptor down-regulation. A down-regulation of adrenal steroid receptors in the brain may lead to impaired negative

244 feedback function of the HPA axis, resulting in a net increase in endogenous glucocorticoid levels. Abnormalities in HPA axis function have been noted in a number of clinically depressed and Alzheimer's patients 6'11'16'17. These abnormalities include hypercortisolism and impaired ability of dexamethasone to suppress cortisol levels. A selective down-regulation of hippocampal adrenal steroid receptors has been proposed to account for the impaired H P A axis function 4°. However, due to the widespread down-regulation of adrenal steroid receptors observed in this study, a decrease of adrenal steroid receptors in brain areas other than the hippocampus in response to chronic elevation of glucocorticoids should not be ruled out. On the other hand, the lack of adrenal steroid receptor down-regulation in the pituitary with chronic corticosterone treatment suggests that the pituitary may be a less likely site of adrenal steroid receptor down-regulation with hypercortisolism. The impact of elevated glucocorticoid levels on immune function, may be counteracted, at least partially, by a down-regulation of adrenal steroid receptors in immune tissues. In the spleen the magnitude of the down-regulation of Type II adrenal steroid receptors was about 30% with high dose corticosterone treatment. The functional impact of a 30% decrease in adrenal steroid receptor level has yet to be determined. We have found that there is almost a 1:1 relationship between amount of splenic Type II adrenal steroid receptor activation by dexamethasone treatment and amount of inhibition of splenocyte proliferation, with evidence for very few, if any, spare adrenal steroid receptors 29. Thus, even a small degree of adrenal steroid receptor down-regulation in the spleen would be expected to lead to a diminished effect of glucocorticoids on lymphocyte proliferation. Aside from a direct down-regulation of receptors by corticosterone, the large decrease (65%) of adrenal steroid receptors in PBMs and thymus may also reflect a change in the cellular composition of these immune compartments in response to chronic corticosterone treatment. Glucocorticoids have been shown to lyse immune cells and lead to redistribution of immune cells between the different immune compartments 9. ThereREFERENCES 1 Antakly, T., Raquidan, D., O'Donnell, D. and Katnick, L., Regulation of glucocorticoid receptor expression: I. Use of a specific radioimmunoassay and antiserum to a synthetic peptide of the N-terminal domain, Endocrinology, 126 (1990) 18211828. 2 Arriza, J.L., Weinberger, C., Cerelli, G., Glaser, T.M., Handelin, B.L., Housman, D.E. and Evans, R.M., Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoidreceptor, Science, 237 (1987) 268-275. 3 Bradford, M.M., A rapid and sensitive method for the quanti-

fore, it is possible that corticosterone treatment resulted in a selective retention of cells within the thymus and blood stream having a lower adrenal steroid receptor concentration than the total population of cells present in the untreated conditions. Adrenal steroid receptor down-regulation: effect o f exogenous corticosterone vs. chronic stress. The studies described in this paper indicate that adrenal steroid receptors are capable of being down-regulated by sustained exposure to exogenous corticosterone levels in the diurnal peak to stress level range. A chronic stress paradigm capable of maintaining circulating corticosterone levels in a similar range would be expected to result in some down-regulation of adrenal steroid receptors both in the brain and immune tissues. The ability to down-regulate adrenal steroid receptors of the brain with chronic stress has been demonstrated in some studies TM 37,38, but has not been seen with all chronic stress paradigms 42'47'53. Due to the negative feedback action of glucocorticoids on their own secretion and the ability of the HPA axis to adapt to chronic stimulation 41, many chronic stress paradigms may not be able to maintain glucocorticoid levels of sufficient magnitude and duration to produce a direct down-regulation of adrenal steroid receptors. However, there may be neural and hormonal factors other than the direct action of glucocorticoids that are able to modify adrenal steroid receptor levels during sustained periods of stress. Our data indicates that adrenal steroid receptor levels throughout the brain and in immune tissue are responsive to systemic treatment with high physiologic doses of corticosterone. Whether or not chronic stress can produce such an extensive effect on adrenal steroid receptor levels has yet to be determined. The time course and other parameters contributing to the down-regulation of adrenal steroid receptors by exogenous glucocorticoids or by chronic stress needs to be examined in more detail.

Acknowledgements. This work was supported in part by an NIMH Grant (MH-41256) to B.S. McEwen and by a Research Scientist Development Award (MH-00680) and Small Grant Award (MH46504) to A.H. Miller.

tation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 4 Brinton, R.E. and McEwen, B.S., Regional distinctions in the regulation of type I and type II adrenal steroid receptors in the central nervous system, Neurosci. Res. Commun., 2 (1988) 37-45. 5 Burnstein, K.L., Jewell, C.M. and Cidlowski, J.A., Human glucocorticoid receptor cDNA contains sequences sufficient for receptor down-regulation, J. Biol. Chem., 265 (1990) 72847291. 6 Carroll, B.J., The dexamethasone suppression test for melancholia, Br. J. Psychiatry, 140 (1982) 292-304. 7 Chao, H.M., Choo, P.H., McEwen, B.S., Glucocorticoid and

245 mineralocorticoid receptor mRNA expression in rat brain, Neuroendocrinology, 50 (1989) 365-371. 8 Chou, Y.-C. and Luttge, W.G., Activated type II receptors in brain cannot rebind glueocorticoids: relationship to progesterone's antiglucocorticoid actions, Brain Research, 440 (1988) 67-78. 9 Cohen, J.J. and Crnic, L.S., Glucocorticoids, stress, and the immune response. In D.R. Webb (Ed.), lmmunopharmacology and the Regulation of Leukocyte Function, Immunology Series Vol. 19, Marcel Dekker, Inc., New York, 1982, pp. 61-91. 10 Coirini, H., Magarinos, A.M., de Nicola, A.F., Rainbow, T.C. and McEwen, B.S., Further studies of brain aldosterone binding sites employing new mineralocorticoid and glucocorticoid receptor markers in vitro, Brain Research, 361 (1985) 212-216. 11 Davis, K.L., Davis, B.M., Greenwald, B.S., Mohs, R.C., Math6, A.A., Johns, C.A. and Horvath, T.B., Cortisol and Alzheimer's disease, I: basal studies, Am. J. Psychiatry, 143 (1986) 300-305. 12 de Kloet, E.R., Burbach, P. and Mulder, G.H., Localization and role of transcortin-like molecules in the anterior pituitary, Mol. Cell. Endocrinol., 7 (1977) 261-273. 13 de Kloet, E.R., Voorhuis, Th.A.M., Leunissen, J.L.M. and Koch, B., Intracellular CBG-like molecules in the rat pituitary, J. Steroid Biochem., 20 (1984) 367-371. 14 Dong, Y., Poellinger, L., Gustafsson, J.-A. and Okret, S., Regulation of glucocorticoid receptor expression: evidence for transcriptional and posttranslational mechanisms, Mol. Endocrinol., 2 (1988) 1256-1264. 15 Eldridge, J.C., Brodish, A., Kute, T.E. and Landfield, P.W., Apparent age-related resistance of type II hippocampal corticosteroid receptors to down-regulation during chronic escape training, J. Neurosci., 9 (1989) 3237-3242. 16 Evans, D.L., Burnett, G.B. and Nemeroff, C.B., The dexamethasone suppression test in the clinical setting, Am. J. Psychiatry, 140 (1983) 586-589. 17 Greenwald, B.S., Math~, A.A., Mohs, R.C., Levy, M.I., Johns, C.A. and Davis, K.L., Cortisol and Alzheimer's disease, II: dexamethasone s~uppression, dementia severity, and affective symptoms, Am. J. Psychiatry, 143 (1986) 442-446. 18 Herman, J.P., Patel, P.D., Akil, H. and Watson, S.J., Localization and regulation of glucocorticoid and mineralocorticoid receptor messenger RNAs in the hippocampal formation of the rat, Mol. Endocrinol., 3 (1989) 1886-1894. 19 Hollenberg, S.M., Weinberger, C., Ong, E.S., Cerelli, G., Oro, A., Lebo, R., Thompson, E.B., Rosenfeld, M.G. and Evans, R.M., Primary structure and expression of a functional human glucocorticoid receptor cDNA, Nature, 318 (1985) 635-641. 20 Koch, B., Sakly, M., Lutz-Bucher, B. and Briaud, B., Glucocorticoid binding and control of ACTH secretion, J. Physiol. (Paris), 77 (1981) 923-933. 21 Kuhn, R.W., Green, A.L., Raymoure, W.J. and Siiteri, P.K., Immunocytochemical localization of corticosteroid-binding globulin in rat tissues, J. Endocrinol., 108 (1986) 31-36. 22 Limbird, L.E., Cell Surface Receptors: a Short Course on Theory and Methods, Martinus Nijhoff Publishing, Boston, 1986, pp. 69-79. 23 Lowy, M.T., Quantification of type I and II adrenal steroid receptors in neuronal, lymphoid and pituitary tissues, Brain Research, 503 (1989) 191-197. 24 Luttge, W.G., Davda, M.M., Rupp, M.E. and Kang, C.G., High affinity binding and regulatory actions of dexamethasonetype I receptor complexes in mouse brain, Endocrinology, 125 (1989) 1194-1203. 25 Luttge, W.G., Emadian, S.M., Densmore, C.L. and Slatick, R.S., Efficacy of five methods for the bound-free separation of gluco- and mineralocorticoids from type I, II and III receptors found in HEPES- and TRIS-buffered mouse brain cytosol, Steroids, 43 (1984) 429-443. 26 Luttge, W.G. and Rupp, M.E., Differential up- and downregulation of type I and type II receptors for adrenocorticoste-

roid hormones in mouse brain, Steroids, 53 (1989) 59-76. 27 Luttge, W.G., Rupp, M.E. and Davda, M.M., Aldosteronestimulated down-regulation of both type I and type II adrenocorticosteroid receptors in mouse brain is mediated via type I receptors, Endocrinology, 125 (1989) 817-824. 28 Meyer, J.S., Mioco, D.J., Stephenson, B.S., Krey, L.C. and McEwen, B.S., Subcutaneous implantation method for chronic glucocorticoid replacement therapy, Physiol. Behav., 22 (1979) 867-870. 29 Miller, A.H., Spencer, R.L., Trestman, R.L., Kim, C., McEwen, B.S. and Stein, M., Adrenal steroid receptor activation and immune function, Am. J. Physiol., in press. 30 Miller, A.H., Spencer, R.L., Stein, M. and McEwen, B.S., Adrenal steroid receptor binding in spleen and thymus after stress or dexamethasone, Am. J. Physiol.: Endocr. Metab., 259 (1990) E405-E412. 31 Munck A., Guyre, P.M. and Holbrook, N.J., Physiological functions of glucocorticoids in stress and their relation to pharmacological actions, Endocr. Rev., 5 (1984) 25-44. 32 Okret, S., Poellinger, L., Dong, Y. and Gustafsson, J.-A., Down-regulation of glucocorticoid receptor mRNA by glucocorticoid hormones and recognition by the receptor of a specific binding sequence within a receptor cDNA clone, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 5899-5903. 33 Reul, J.M.H.M. and de Kloet, E.R., Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation, Endocrinology, 117 (1985) 2505-2511. 34 Reul, J.M.H.M., van den Bosch, F.R. and de Kloet, E.R., Differential response of type I and type II corticosteroid receptors to changes in plasma steroid level and circadian rhythmicity, Neuroendocrinology, 45 (1987) 407-412. 35 Reul, J.M.H.M., van den Bosch, ER. and de Kloet, E.R., Relative occupation of type-I and type-II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications, J. Endocr., 115 (1987) 459-467. 36 Rosewicz, S., McDonald, A.R., Maddux, B.A., Goldfine, I.D., Miesfeld, R.L. and Logsdon, C.D., Mechanism of glucocorticoid receptor down-regulation by glucocorticoids, J. Biol. Chem., 263 (1988) 2581-2584. 37 Sapolsky, R.M., Krey, L.C. and McEwen, B.S., Glucocorticoidsensitive hippocampal neurons are involved in terminating the adrenocortical stress response, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 6174-6177. 38 Sapolsky, R.M., Krey, L.C. and McEwen, B.S., Stress downregulates corticosterone receptors in a site-specific manner in the brain, Endocrinology, 114 (1984) 287-292. 39 Sapolsky, R.M. and McEwen, B.S., Down-regulation of neural corticosterone receptors by corticosterone and dexamethasone, Brain Research, 339 (1985) 161-165. 40 Sapolsky, R.M. and Plotsky, P.M. Hypercortisolism and its possible neural bases, Biol. Psychiatry, 27 (1990) 937-952. 41 Sheppard, K.E., Roberts, J.L. and Blum, M., Differential regulation of type II corticosteroid receptor messenger ribonucleic acid expression in the rat anterior pituitary and hippocampus, Endocrinology, 127 (1990) 431-439. 42 Spencer, R.L. and McEwen, B.S., Adaptation of the hypothalamic-pituitary-adrenal axis to chronic ethanol stress, Neuroendocrinology, 52 (1990) 481-489. 43 Spencer, R.L., Young, E.A., Choo, P. and McEwen, B.S., Adrenal steroid type I and type II receptor binding: estimates of in vivo receptor number, occupancy, and activation with varying level of steroid, Brain Research, 514 (1990) 37-48. 44 Svec, E, Glucocorticoid receptor regulation, Life Sci., 36 (1985) 2359-2366. 45 Svec, E, The biopotency of dexamethasone at causing hepatic glucocorticoid receptor down-regulation in the intact mouse, Biochim. Biophys. Acta, 970 (1988) 90-95. 46 Svec, E, Corticosterone regulates the level of hepatic glucocorticoid receptors in mice, Proc. Soc. Exp. Biol. Med., 188 (1988) 474-479.

246 47 Svec, E, Gordon, S. and Tate, D., Glucocorticoid receptor regulation: the effects of adrenalectomy, exogenous glucocorticold, and stress on hepatic receptor number in male and female mice, Biochem. Med. Metab. Biol., 41 (1989) 224-233. 48 Tornello, S., Orti, E., de Nicola, A.E, Rainbow, T.C. and McEwen, B.S., Regulation of glucocorticoid receptors in brain by corticosterone treatment of adrenalectomized rats, Neuroendocrinology, 35 (1982) 411-417. 49 Turner, B.B., Tissue differences in the up-regulation of glucocorticoid-binding proteins in the rat, Endocrinology, 118 (1986) 1211-1216. 50 Van Eekelon, J.A.M., Jiang, W., de Kloet, E.R. and Bohn, M.C., Distribution of the mineralocorticoid and the glucocorti-

coid receptor mRNAs in the rat hippocampus, J. Neurosci. Res., 21 (1988) 88-94. 51 Vedeckis, W.V., Ali, M. and Allen, H.R., Regulation of glucocorticoid receptor protein and mRNA levels, Cancer Res.. 49 (1989) 2295s-2302s. 52 Veldhuis, H.D., van Koppen, C., van Ittersum, M. and de Kloet, E.R., Specificity of the adrenal steroid receptor system in rat hippocampus, Endocrinology, 110 (1982) 2044-2051. 53 Young, E.A., Spencer, R.L. and McEwen, B.S., Changes at multiple levels of the hypothalamic-pituitary-adrenal axis following repeated electroconvulsive shock, Psychoneuroendocrinology, 15 (1990) 165-172.