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Endocrinology Vol. 146, No. 12 5587-5595
Copyright © 2005 by The Endocrine Society

Low Doses of Dexamethasone Can Produce a Hypocorticosteroid State in the Brain

A. M. Karssen, O. C. Meijer, A. Berry, R. Sanjuan Piñol and E. R. de Kloet

Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research and Leiden University Medical Center, 2300 RA Leiden, The Netherlands

Address all correspondence and requests for reprints to: E. R. de Kloet, Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research, P.O. Box 9503, 2300 RA Leiden, The Netherlands. E-mail: e.kloet{at}lacdr.leidenuniv.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The synthetic glucocorticoid dexamethasone (dex) blocks stress-induced hypothalamic-pituitary-adrenal (HPA) activation primarily at the level of the anterior pituitary because multidrug resistance P-glycoprotein hampers its penetration in the brain. Here, we tested the hypothesis that central components of the HPA axis would escape dex suppression under conditions of potent peripheral glucocorticoid action. We subchronically treated rats with low or high doses of dex. The animals were subjected on the last day of treatment for 30 min to a restraint stressor after which central and peripheral markers of HPA axis activity were measured. Basal and stress-induced corticosterone secretion, body weight gain, adrenal and thymus weight, as well as proopiomelanocortin mRNA in the anterior pituitary were reduced in a dose-dependent manner by dex administered either 5 d sc or 3 wk orally. In the brain, the highest dose dex suppressed CRH mRNA and CRH heteronuclear RNA in the paraventricular nucleus (PVN). However, in the peripherally active low-dose range of dex CRH mRNA and heteronuclear RNA showed resistance to suppression, and CRH mRNA expression in the PVN was in fact enhanced under the long-term treatment condition. In the PVN, c-fos mRNA was suppressed by the highest dose of dex, but this effect showed a degree of resistance after long-term oral treatment. c-fos mRNA responses in the anterior pituitary followed those in PVN and reflect central drive of the HPA axis even if corticosterone responses are strongly reduced. The results support the concept that low doses of dex can create a hypocorticoid state in the brain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SYNTHETIC GLUCOCORTICOID dexamethasone (dex) is a potent and rather selective glucocorticoid receptor (GR) ligand in vivo (1). It has profound effects on energy metabolism, the immune system, and hypothalamic-pituitary-adrenal (HPA) axis activity. In the clinic, it is commonly used, often in combination with a CRH challenge, for diagnostic and research purposes to test HPA axis function in, e.g. Cushing’s disease and affective disorders. Several studies have demonstrated a pituitary rather than a brain site of action in the suppression of HPA axis activity if moderate amounts of dex are administered (2, 3, 4, 5, 6). In support of these findings, it was demonstrated that the efflux transporter P-glycoprotein, expressed at the luminal side of the blood-brain barrier (7), hampers the penetration of dex into the brain (8, 9).

In the present study, the hypothesis was tested that dex exclusion from the rat brain by P-glycoprotein provides a subtle way to create a brain-selective alternative for adrenalectomy, dissociated from the abundant exposure of peripheral corticosteroid targets to glucocorticoids, with adrenal medullary functions and aldosterone secretion intact. This state would arise because treatment with small amounts of dex results in suppression of endogenous corticosterone secretion, and, at the same time, dex would poorly substitute for corticosterone in the brain. As a consequence, a reduced feedback of glucocorticoids to its central targets should ensue and create in the brain a tissue-specific hypocorticoid condition.

All doses of dex are expected to inhibit adrenal output of corticosterone and, thus, lead to decreased occupancy of the high-affinity mineralocorticoid receptor (MR) in the brain. Although high doses of dex lead to the aphysiological situation in which GR gets occupied, but MR becomes depleted from endogenous corticosterone (10), the low poorly penetrating dose would lead to lower activation of both MR and GR. This could yield a model to study the effects of a hypocorticoid brain state, dissociated from peripheral glucocorticoid exposure.

To test this hypothesis, male rats were treated with different doses of dex for either 5 d sc or 3 wk orally. The rats treated with dex were compared with two different control groups. Adrenalectomized (ADX) rats served as control for the central (and peripheral) hypocorticoid state, and a high-dose dex group represented chronic GR activation as present in hypercorticism. To reveal glucocorticoid feedback, the animals were exposed the last day to a restraint stressor. As markers for peripheral glucocorticoid effects, body, adrenal and thymus weight, and plasma corticosterone levels were measured as well as the expression of anterior pituitary proopiomelanocortin (POMC) mRNA. As central markers, the CRH mRNA and heteronuclear RNA (hnRNA) were measured in the parvocellular part of the hypothalamic paraventricular nucleus (PVN). cFos mRNA was used as marker for the excitatory input to the PVN and the anterior pituitary (11).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Young adult male Wistar rats (Charles River, Sulzfeld, Germany), weighing around 185–265 g at the time of arrival, were used. Animals were group-housed except for the drinking water experiment during which they were solitary housed, under a 12-h light, 12-h dark cycle with lights off at 20 h in a temperature (21 C) and humidity-controlled room. They had free access to food and drinking water. The experiments took place 2 wk after arrival at the lab. During this period, rats were handled daily. All experiments were carried out in accordance with the European Communities Council Directive 86/609/EEC and with approval from the animal care committee of the Faculty of Medicine, Leiden University (Leiden, The Netherlands).

Experiment 1: 5-d treatment with dex sc
At the start of experiment, the rats weighed 303 ± 15 g (mean ± SD). They were divided into four groups each consisting of six to 10 animals. One group (ADX) was ADX under gas anesthesia (isoflurane) by dorsal approach at the start of the experiment. After ADX, animals had free access to 0.9% saline and normal drinking water. All other animals were sham-operated. For 5 d, one group (DEXlow) was sc injected with a low dose of 50 µg/kg dex-21-phosphate (Sigma-Aldrich, St. Louis, MO; dissolved in 0.9% saline) twice daily at 0900 and 2100 h. A second group (DEXhigh) was treated with a high dose of dex (500 µg/kg twice daily). Sham-operated (VEH) and ADX groups were treated with vehicle. During the whole experimental period, body weight was monitored. At d 4, blood was sampled 1 h after lights off to determine corticosterone plasma levels at the circadian peak. At d 6, the animals received one last injection in the morning. Six hours later at 1500 h, half of each group (three to five rats per group) was exposed to restraint stress in a wire mesh/plastic cylinder for 30 min. At the start and after 15 min, blood samples were taken using the tail incision method (12). After 30 min, animals were removed from the restrainers and immediately decapitated. Nonstressed animals (the second half of each group) were decapitated directly after removal from their home cage. Trunk blood was collected in EDTA-coated tubes and centrifuged. Plasma was kept at –20 C until determination of corticosterone plasma levels. Brains were rapidly removed from the skull and quickly frozen in isopentane precooled on dry ice/ethanol. Thymus, pituitary, and adrenal glands were dissected and frozen on dry ice. All tissues were stored at –80 C until further use. Thymus and adrenals were cleaned and weighed.

Experiment 2: 3-wk treatment with dex in drinking water
At the start of the drinking water experiment, rats weighed 225 ± 9 g (mean ± SD). They were divided into six treatment groups consisting of five to eight animals. Three groups were treated with different concentrations (0.5, 1.0, and 10 µg/ml) of dex 21-phosphate in their drinking water for 3 wk (DEX0.5, DEX1.0, and DEX10 groups). One group was ADX at the start of the experiment. After adrenalectomy, animals had free access to 0.9% saline and normal drinking water. All other animals were SHAM operated including control animals (VEH) that received normal drinking water during the whole experiment. Each day, animals and bottles were weighed to determine the body weight gain and volume of drinking solution that each animal had drunk over 24 h. One animal of the highest concentration group died before the end of the experiment.

After 3 wk, rats were stressed by restraint in a wire mesh/plastic cylinder and decapitated after 30 min as described for experiment 1. A control nonstress, nontreated group (VEH nonstress) was decapitated immediately after removal from the home cage.

In situ hybridization
Coronal sections of 14 µm through the PVN of the hypothalamus and hippocampus were cut in a cryostat. Pituitaries were sectioned at 12 µm. Sections were thaw-mounted on poly-L-lysine-coated microscopic slides. These slides were stored at –80 C until hybridization. The sections were postfixed in a freshly prepared 4% paraformaldehyde solution (pH 7.2) for 60 min at room temperature and rinsed twice in PBS for 5 min at room temperature. In case of in situ mRNA hybridizations, sections were permeabilized with proteinase K [1 µg/ml in 0.1 M Tris (pH 8.0)] at 37 C for 10 min. After a brief rinse in diethyl pyrocarbonate-treated water, they were treated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min at room temperature and finally rinsed in 2x standard sodium citrate (SSC) (SSC = 0.15 M NaCl and 0.015 M sodium citrate) for 10 min at room temperature. Subsequently, the sections were dehydrated through a graded series of ethanol and air dried.

Preparation of probes
To visualize mRNAs, in situ mRNA and oligonucleotide hybridizations were performed. Different 33P- and 35S-labeled cRNA antisense probes were employed to hybridize with complementary brain tissue c-fos and CRH mRNA and CRH hnRNA. The c-fos mRNA probe was transcribed from a pBluescript (pBS) KS plasmid containing a 2.1-kb full-length rat c-fos cDNA sequence (courtesy of Dr. T. Curran, St. Jude Children’s Research Hospital, Lauderdale, Memphis, TN) in the presence of 33P-UTP (ICN Biomedicals, Costa Mesa, CA; Isoblue stabilized, specific activity. 3000 Ci/mmol). This probe was hydrolyzed by incubation in 90 mM 0.2 M Na2CO3 and 60 mM 0.2 M NaHCO3 at 60 C for 10 min to facilitate the tissue penetration. The CRH hnRNA 33P-UTP-labeled probe was transcribed from a 687-bp fragment (courtesy of P. Sawchenko, The Salk Institute, San Diego, CA) covering the single intron of the rat CRH gene subcloned into a pBS vector. A full-length probe for CRH mRNA (1.2 kb subcloned into pBS; courtesy of Dr. K. Mayo, Northwestern University, Evanston, IL) was synthesized in presence of 35S-UTP. Incorporation of labeled UTP was at least 75%.

A 42-nucleotide mouse POMC oligonucleotide (GGT-TTT-CAG-TCA-GGG-GCT-GTT-CAT-CTC-CGT-TGC-CAG-GAA-ACA; 90% homology with rat POMC; Eurogentec, Seraing, Belgium) was end-labeled with 33P-dATP (NEN Life Science Products, Hoofddorp, The Netherlands; 2000 Ci/mmol, 10 mCi/ml) using terminal transferase with the manufacturer’s protocol (Roche Molecular Biochemicals, Almere, The Netherlands). A 0.33-pmol oligonucleotide was labeled at molar ratio of 1:20 (oligo:label). Incorporation was typically between 50 and 75%, resulting in a tail of 5- to 7.5-A residues per oligonucleotide.

Hybridization procedures
For riboprobes, each slide, containing four sections, was loaded with a 100-µl mix containing 70% deionized formamide, 10% dextran sulfate, 3x SSC, 50 mM dithiothreitol, 1x Denhardt’s solution, 0.1 mg/ml yeast tRNA, 0.1 mg/ml sheared herring sperm DNA, and 1–3 x 106 dpm of the probe, and covered with microscopic coverslips. Overnight hybridization was performed in a moist chamber at 55 C. As a control, a few slides were hybridized with sense probe. The next day, coverslips were removed, and the slides were washed in 2x SSC at room temperature for 10 min, treated with Rnase A [2 mg/100 ml in 0.5 M NaCl (pH 7.5)] at 37 C for 10 min, and washed three times in 2x SSC/50% formamide at 60 C for 15 min. After a short wash with 2x SSC, sections were dehydrated in an ethanol series and air dried. Finally, the slides were put in an x-ray exposure holder and apposed to Biomax MR film (Kodak, Rochester, NY) for 3–12 d (13).

In situ hybridization using oligonucleotides was performed essentially as described (14). Labeled oligonucleotide (0.5–0.8 x 106 dpm) per 100 µl hybridization mix was applied to each slide. Hybridization mix consisted of 50% formamide, 10% dextran sulfate, 4x SSC, 25 mM sodium phosphate (pH 7.0), 1 mM sodium pyrophosphate, 20 mM DTT, 5x Denhardt’s, 100 µg/ml poly-A, and 100 µg/ml sheared herring sperm DNA. Sections were coverslipped and hybridized overnight in a moist chamber at 42 C. The next morning, coverslips were removed, rinsed in 1x SSC at room temperature, washed twice for 30 min in 1x SSC at 50 C, washed for 5 min in 1x SSC at room temperature, dehydrated in an ethanol series, and air dried. Then, sections were apposed to Kodak Biomax MR film that were developed after 17–20 h.

Densitometric quantification
Optical density was quantified with analysis performed on a Macintosh computer using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image). Nissl staining and light microscopy were used to confirm the presence of the PVN in the section. Optical densities were determined by outlining the parvocellular part of the PVN. The OD of the area dorsolateral from the PVN was used to correct for tissue background. Measurements of three to five sections were averaged per animal with the mean value from each animal used in subsequent statistical analysis.

Corticosterone plasma levels
The plasma corticosterone concentration was determined using a standard in-house RIA procedure (15). Antiserum raised in sheep against corticosterone-21-hemisuccinate BSA was a gift from Dr. F. Sweep (University of Nijmegen, The Netherlands). The detection limit was 0.2 µg/dl. Animals were considered ADX when basal trough plasma corticosterone levels were lower than 1.0 µg/dl.

Determination of apoptosis
The presence of apoptotic granule cells in the dentate gyrus was evaluated by qualitatively scoring cell nuclei with fragmented DNA (i.e. pyknotic cells) in Nissl-stained sections of the hippocampus at a magnification of 400x (Fig. 1DGo).



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  		FIG. 1. Dissociation of nonstress-reactive markers for glucocorticoid exposure in periphery and brain after 5 d of sc injections with low (black bars) or high (dark gray bars) dex, compared with vehicle-treated control (light-gray bars) and ADX (open bars) rats. Thymus weight (A) and anterior pituitary POMC mRNA expression (B) are decreased after 5-d treatment with low and high doses of dex, whereas CRH mRNA (C) is lowered only after the high dose. *, Significantly different from vehicle control; #, significantly different from DEXlow (Tukey HSD, P < 0.05). D, Apoptotic cells in the dentate gyrus as identified by fragmented DNA in cresyl-violet staining (arrows) were observed in 70% of the ADX animals but never in dex-treated rats.

 
Statistics
Completeness of ADX was determined based on corticosterone plasma levels and on relative saline intake (Richter, 1941); one animal was excluded from the analysis because of incomplete ADX. Plasma corticosterone data were analyzed using one-way ANOVA or repeated measurements ANOVA, where appropriate. All other data were analyzed using two-way ANOVA with treatment and stress as factors (experiment 1) or one-way ANOVA with treatment as factor and stress as covariate (experiment 2; only one nonstress control group was included in this case). When a significant stress effect or treatment x stress interaction was absent, data of stress and nonstress groups were combined for further analysis on treatment effects. In case stress or interaction effects were significant, stress and nonstress groups were separately analyzed applying one-way ANOVA. When appropriate, post hoc comparisons were performed using Tukey’s honestly significant difference (HSD) test. Statistical difference was accepted at P < 0.05. All data are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In both experiments, we evaluated basal as well as restraint stress-induced parameters of the HPA axis. Because POMC mRNA in the pituitary and CRH mRNA in the PVN did not react to the 30-min stressor, we have included these in the basal, nonstress-reactive parameters.

Experiment 1: 5-d treatment with dex sc
Basal activity: peripheral.
Two daily sc injections of dex for 5 d suppressed basal plasma corticosterone levels both in the morning immediately before the stress response and at the circadian peak (P < 0.05, Table 1Go). Further, adrenal and thymus weights were significantly reduced in both DEXlow and DEXhigh groups (P < 0.05; Table 1Go, Fig. 1AGo). Body weight decreased from the start of the treatment in contrast to vehicle-treated animals that gained weight at a normal rate resulting in a significant difference between both groups at the end of the treatment (Table 1Go). Two-way ANOVA did not reveal an effect of 30-min restraint stress on POMC mRNA expression in the anterior pituitary [F(1,21) = 0.319; P > 0.05], which is consistent with previous studies (16), but there was a significant treatment effect; dex treatment significantly reduced POMC mRNA expression in the anterior pituitary in a dose-dependent manner (P < 0.05, Fig. 1BGo). ADX animals had undetectable corticosterone plasma levels, increased POMC mRNA expression, and increased thymus weight (P < 0.05, Table 1Go and Fig. 1Go, A and B). Pituitary intermediate lobe POMC mRNA expression was not affected by treatment nor by stress (data not shown).


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  		TABLE 1. Peripheral glucocorticoid markers affected by 5-d dex treatment or ADX (experiment 1)

 
Basal activity: central.
CRH mRNA expression was not affected by 30-min restraint stress. Basal CRH mRNA levels were strongly reduced in the DEXhigh group but in DEXlow group were not different from vehicle-treated animals (Fig. 1CGo). No significant up-regulation was observed after ADX. In 70% of the ADX animals, pyknotic cells, which are indicative of apoptosis, were abundantly present in the granule cell layer of the dentate gyrus, mainly located in the tip of the inner blade (Fig. 1DGo). Apoptosis of dentate gyrus cells was not found in the brains of any of the DEXlow animals and in DEXhigh animals (0%).

Restraint stress-induced activity: peripheral.
Repeated measurements ANOVA on the stress-induced corticosterone response revealed a significant interaction effect of stress and treatment [F(6,24) = 46.7; P < 0.05; Fig. 2AGo]. The levels at 30 min after the initiation of stress were considerably lower in the DEXlow group (P < 0.05) compared with those in VEH group (Fig. 2AGo), although the stress-induced rise of corticosterone was not completely blocked to the extent observed in the DEXhigh group. In agreement with a previous report (17), restraint stress-induced a response of c-fos mRNA expression in the anterior pituitary [F(1,21) = 17.5; P < 0.05], which was significant in low-dose dex and control rats but reduced in the DEXhigh group (Fig. 2BGo).



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  		FIG. 2. Responses to 30-min restraint stress are different after 5 d of injections with low or high doses of dex. A, Plasma corticosterone levels after stress are completely suppressed in the DEXhigh rats (open diamonds) and significantly decreased in the DEXlow rats (closed triangles) in comparison with vehicle treated (black circles). ADX rats are shown as black squares. Basal levels at the start of the stress are also given in Table 1Go but repeated here for the sake of clarity. B, c-fos mRNA responses to stress in the anterior pituitary are reduced only after the high dose of dex (dark-gray bars). Vehicle-treated controls (light gray), DEXlow (black bars), and ADX (open bars) rats have similar responses to stress. C, c-fos mRNA responses in the PVN and the CRH hnRNA response (D) are specifically reduced in the DEXhigh group. $, Significantly different from corresponding nonstressed group; *, significantly different from stressed vehicle control group; #, significantly different from corresponding DEXlow group (Tukey HSD; P < 0.05).

 
Restraint stress-induced activity: central.
In the brain, the stress-induced responses of c-fos mRNA and CRH hnRNA expression in the parvocellular part of the PVN were strongly reduced in the DEXhigh group (Fig. 2Go, C and D). However, in the DEXlow animals, the rapid increases of c-fos mRNA and CRH hnRNA after stress were not different from the VEH animals or the 5-d ADX animals. With respect to the control measurements for stress induction, dex treatment had no effect on the basal level of c-fos mRNA in the PVN or on basal CRH hnRNA levels, although the latter showed a tendency toward differences, possibly reflecting lower expression in the DEXhigh group [F(3,13) = 3.236, P = 0.06; Fig. 2DGo].

Experiment 2: 3-wk treatment with dex in drinking water
As described above, the 5-d treatment with a low dose of dex led to a clear dissociation between peripheral and central effects of corticosterone. However, we hoped to create by dex administration a model in which we would induce an ADX-like central state in terms of glucocorticoid exposure, rather than a low-corticosterone one. Because we could not distinguish between the two states and because the 5-d low-dose dex treatment was insufficient to completely block stress-induced increase in corticosterone levels, we treated rats for 3 wk with low and high doses of dex via the drinking water and subjected the animals to restraint stress after this period.

Basal activity: peripheral effects.
dex treatment did not affect the volume of drinking solution drunk by the animals (Table 2Go). The amounts of dex ingested by the animals were calculated based on the volume drunk and the concentration of dex. Animals treated with 0.5, 1.0, or 10 µg dex/ml in their drinking water ingested per day 42 ± 1, 91 ± 4, and 1447 ± 145 µg dex/kg body weight, respectively. Control animals gained 27–33% body weight during the experimental period. DEX0.5 and DEX1.0 lost weight during the first 5 d and stabilized later on, such that they had not gained substantial weight at the end of the experimental period. In the DEX10 animals, the treatment resulted in a severe loss of body weight (Table 2Go).


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  		TABLE 2. Peripheral glucocorticoid markers after 3-wk treatment with dexamethasone through drinking water (experiment 2)

 
ADX and treatment with dex in the drinking water had the expected effects on peripheral glucocorticoid markers. In all dex treatment groups, basal trough levels of plasma corticosterone were below detection level of the assay. Also, circadian peak corticosterone levels in the DEX10 and DEX1.0 groups were completely suppressed, whereas hormones levels in the DEX0.5 group were significantly lowered compared with control rats, but not suppressed completely. Adrenal and thymus weights were dose dependently decreased by dex treatment, whereas thymus weight was increased in ADX animals (P < 0.05, Table 2Go and Fig. 3AGo).



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  		FIG. 3. Dissociation of basal indicators of glucocorticoid exposure in periphery and brain after 3 wk of dex treatment via the drinking water with 0.5 (black bars), 1.0 (hatched bars), or 10 (dark-gray bars) µg/ml, compared with untreated control (light-gray bars) and ADX rats (open bars). A, Thymus weight is dose-dependently decreased by dex. B, Anterior pituitary POMC mRNA is decreased after the highest dose of dex, tends to decrease after 1.0 µg/ml (P = 0.07), but is not lower in DEX0.5 animals compared with untreated controls. C, CRH mRNA in the PVN is strongly reduced after the highest dose of dex but increased in ADX rats and after treatment with the lowest dose of dex, indicative of hypocorticism in the brain. In DEX1.0 animals, CRH mRNA levels are not different from untreated controls. *, Significantly different from control; #, significantly different from DEX0.5 (Tukey HSD, P < 0.05).

 
Anterior pituitary POMC mRNA levels were significantly and strongly reduced in the DEX10 group and strongly increased in ADX animals. Although the average anterior pituitary POMC mRNA expression was reduced by 35% in the Dex1.0 groups, this difference did not reach statistical significance (P = 0.06; Fig. 3BGo).

Basal parameters: central.
Similar to the injection study, central markers were differentially affected by treatment with small amounts of dex compared with treatment with large amounts of dex. Both DEX0.5 and ADX animals showed increased levels of CRH mRNA in the PVN compared with the control group (P < 0.05), whereas the DEX10 animals showed a clear reduction in CRH mRNA levels (Fig. 3CGo). The CRH mRNA expression in the DEX1.0 animals was not changed compared with untreated control animals. No signs of apoptosis within the granule cell layer of the dentate gyrus could be found in any of the non-ADX treatment groups.

Restraint stress-induced: peripheral.
The stress-induced increase of corticosterone was completely blocked in the DEX10 and the DEX1.0 groups. In the DEX0.5 group, dex treatment reduced corticosterone plasma levels at 30 min after onset of stress to 35% of control levels (Fig. 4AGo).



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  		FIG. 4. Responses to 30-min restraint stress of peripheral (A and B) and central (C and D) parameters differ after 3 wk of treatment with various doses of dex. A, Plasma corticosterone levels after stress are completely suppressed in the DEX10 (open diamonds) and DEX1.0 (closed diamonds) rats and strongly decreased in the DEX0.5 rats (closed triangles) in comparison with untreated control rats (open circles). Basal levels at the start of the stress are also given in Table 2Go but repeated here for the sake of clarity. B, c-fos mRNA in anterior pituitary is higher in stressed (forward hatch) than in control rats (light gray). Three weeks of dex treatment did not lead to diminished responses but even tended to increase the response in the DEX0.5 (black bars) and DEX1.0 (backward hatch). Stress responses of DEX10 rats (dark-gray bars) were not different from untreated controls. C, PVN c-fos mRNA responses to stress were significantly lower in DEX10 animals compared with untreated controls. Stress induced c-fos in DEX0.5, DEX1.0, and ADX rats did not differ from stressed vehicle-treated controls. D, PVN CRH hnRNA responded to stress significantly in DEX0.5 rats only, in line with a hypocorticoid state. The DEX10 group was the only stress group that had significantly lower values than the DEX0.5 and the vehicle-treated stress groups, indicating a complete lack of responsiveness to the stressor. $, Significantly different from nonstressed group; *, significantly different from stressed vehicle control group; #, from corresponding DEX0.5 group; %, from the ADX group (Tukey HSD; P < 0.05).

 
The c-fos mRNA expression in the anterior pituitary clearly responded to stress in all groups (Fig. 4BGo). Treatment also significantly affected expression levels between different groups of stressed animals [F(4,27) = 3.676, P < 0.05]. Low doses of dex treatment did not suppress the stress-induced c-fos expression in anterior pituitary but if anything seemed to induce an increased c-fos mRNA (although this effect was only found to be statistically significant when compared with the ADX group).

Restraint stress-induced: central.
c-fos mRNA showed a strong response to stress in all groups. This response showed a significant attenuation in the DEX10 animals (Fig. 4CGo), whereas it was not different from control animals in the two other dex-treated groups and in ADX animals. Stress induced a modest increase in CRH hnRNA in the PVN of untreated control animals, which in fact did not reach statistical significance by Tukey HSD post hoc test (P = 0.12; Fig. 4DGo). Only the DEX0.5 group showed a highly significant increase in CRH hnRNA levels after stress. There was a very clear dissociation between the lowest and highest dose of dex because CRH hnRNA levels clearly did not react to stress in the DEX 10 group.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that hypothalamic CRH mRNA as well as restraint stress-induced CRH hnRNA and c-fos mRNA expression are resistant to dex under conditions in which peripheral glucocorticoid targets like anterior pituitary POMC expression, adrenal corticosteroid secretion, and thymus weight are suppressed. Although we were not able achieve a true brain-selective adrenalectomy, this finding supports the concept that, using the right conditions, the partial exclusion of dex from the brain combined with the suppression of peripheral pituitary-adrenal activity can create the state of a central hypocorticoid condition.

Although in both experiments the effects of low dex clearly dissociated between brain and periphery, the case for an actual hypocorticoid central state, based on centrally measured parameters, can only be made for the long-term oral treatment, when CRH mRNA was increased relative to the stressed control group to a similar extent in the DEX0.5 and ADX groups. Interpretation of the first 5-d experiment is hampered by the fact that ADX group did not function as the positive control we expected. Apart from the unexpected lack of significantly increased thymus weight, we also did not observe any increase in brain markers, such as CRH mRNA. These atypical results remain unexplained. They do not reflect incomplete ADX, because other markers did react: plasma hormone levels, amount of ingested saline, POMC mRNA levels, and apoptosis in the dentate gyrus of the hippocampus. Importantly, the other tested paradigm of 3-wk oral low-dex administration most likely does lead to a hypocorticoid state, as is evident from the increased CRH mRNA levels and the CRH hnRNA response to restraint stress.

The underpinning of the concept of exclusion of dex from brain originates from studies demonstrating that in vivo 3H-corticosterone administered in the circulation can easily enter the brain (4, 18, 19), whereas the entrance of 3H-dex is hampered. The brain does express high amounts of GR (18, 20), and in vitro brain GRs retain both 3H-dex and 3H-corticosterone (4, 21). Accordingly, the existence of a blood-brain barrier limiting the in vivo uptake of dex was postulated (2, 4, 22). Conclusive evidence for this barrier was obtained more recently, when it was demonstrated that the efflux transporter P-glycoprotein at the blood-brain barrier hampered the penetration of dex, prednisone, and cortisol, which are exogenous steroids for mice and rats (8, 9, 19, 23, 24). If the P-glycoprotein gene was disrupted as in the mdr1a knockout mice, tracer amounts of 3H-dex and 3H-cortisol were taken up and retained in the brain of these mutants, accumulating in hippocampal neurons as 3H-corticosterone did (9, 19). However, in intact animals, as a result of its poor central access to the brain, moderate doses of dex primarily act on the anterior pituitary to suppress pituitary-adrenal activity (3). Our data corroborate therefore a pituitary site of dex action on stress-induced ACTH release. However, our data also suggest that for the presently used stressor in rats a complete chronic suppression of the HPA axis requires doses of dex that can also act at (and may involve) central binding sites.

Our findings are consistent with reports studying receptor occupancy in pituitary and brain tissue after either short-term or acute dex treatment (5, 6, 10, 11, 25). The latter studies have demonstrated that low doses of dex substantially occupy GR in the pituitary and other peripheral target tissues, whereas GR activity in the brain was relatively little affected. With regards to the MR, data have demonstrated that it will become to some extent depleted after a single dose of dex that suppresses endogenous corticosterone secretion (10). Using chronic high doses of dex, the responses to restraint stress were clearly reduced, and CRH mRNA expression was diminished after administration of large amounts of dex. This indicates that the barrier formed by P-glycoprotein is of course relative (8) and can be overcome by high amounts of dex to activate the GR (10, 25, 26). Indeed, studies reporting effects of dex on glucocorticoid targets in the brain used very high systemic doses or local brain implants of dex (27, 28, 29, 30, 31).

Qualitative analysis revealed that pyknotic cells in the dentate gyrus were clearly visible in the ADX animals, reflecting apoptosis as a consequence of lack of MR activation (32, 33). However, no dose of dex treatment caused apoptosis, even though high doses of dex failed to protect against ADX-induced apoptosis (34), and in fact have been reported to induce apoptosis in the hippocampus in rats (6 months but not 1 month old) (35). In the low-dex groups, the PM levels of corticosterone were still elevated significantly above the detection limit, which likely protects against apoptosis via MR occupation. The lack of apoptosis even in the high-dex groups may indicate that corticosterone levels were not suppressed to the level that no MR activation was present, or that aldosterone levels were high enough to activate MR and protect against apoptosis.

Although not all changes as observed after ADX were found in the brains of dex-treated rats, the amount of circulating corticosterone in blood and brain was strongly reduced after dex, also after low-dose treatment. Accordingly, the animals will be characterized by a profound change in the activation of the MR and GR. Such changes in MR/GR balance are considered a risk for coping with stress and are thought to enhance vulnerability to stress-related disease (36, 37). The low-dose dex-treated animal in particular may therefore present an interesting animal model because it ensures a relatively stable hypocorticoid climate in the brain that is not disturbed by (acute) stress-induced rises in corticosterone. Such a model may be useful in further defining the input of metabolic factors and cognitive processes in glucocorticoid-dependent brain mechanisms (38, 39, 40, 41).

c-fos mRNA and CRH hnRNA on the PVN were resistant to dex suppression at low doses. In these groups as well as in the ADX groups, the responses were not augmented compared with those of the control groups in the present study. The latter is at variance with the previously reported amplified central stress response 5 d to 1 wk after ADX (29, 42) or after acute glucocorticoid withdrawal (43). However, in agreement with our finding are several other studies reporting no effect of 5-d ADX on induction of c-fos mRNA in hypothalamus after stress (44, 45, 46). As was pointed out in a recent report (11), an acute injection of the glucocorticoid RU28362 was capable to suppress stress-induced CRH expression rather than c-fos mRNA. Thus, acute suppression by RU28362 may target CRH and does not affect the excitatory input activating c-fos. It may be relevant that although CRH may be a direct target gene for glucocorticoids, at least in vitro (47), this has to our knowledge not been shown for c-fos. Our present data show that PVN c-fos expression does not escape blockade with high doses of dex, suggesting that under those conditions, these excitatory inputs are also suppressed.

Interestingly, c-fos mRNA induction in the anterior pituitary in the 5-d experiment reacted similar to central markers and was only suppressed by the high dose of dex. Possibly (parallel to the PVN discussed above) the pituitary c-fos response is governed primarily by hypothalamic secretagogs, rather than subject to direct negative feedback. Support for the dissociation of pituitary POMC and c-fos regulation comes from a study that used AtT-20 cells to show that induction of c-fos was less strongly suppressed by dex than POMC mRNA (48). After 3 wk of administration through the drinking water, no suppression of this c-fos response was observed, not even in the DEX10 group, whereas at lower doses, the c-fos response was even enhanced. This is striking because the measures for PVN activity, c-fos, and CRH hnRNA were suppressed by the highest dose of dex. Possibly, the chronically disturbed MR/GR balance led to adaptations in parameters that were not evaluated here, such as vasopressin or other secretagogs. Alternatively, the assumption that the anterior pituitary c-fos response to restraint stress reflects solely corticotropic cells can be questioned. Anterior pituitary POMC mRNA expression is reduced after treatment with dex for 5 d, but after treatment for 3 wk, the effects of low doses of dex were surprisingly less dramatic. This to us is also suggestive of adaptive responses to the imbalanced activation of the two corticosteroid receptor types, with consequences for pituitary function.

The impaired penetration of dex into the brain has important implications for the interpretation of the dex suppression test. This test is used in the clinic, often in combination with a CRH challenge, to evaluate the dysregulation of the HPA axis in, e.g. depressive patients (49, 50). A characteristic feature is the hyperactive HPA axis, which is not suppressed by a low dose of dex administered to these patients the night before. This escape of dex suppression observed in hypercortisolemic depressive patients is exaggerated by exogenous CRH. The present study offers an explanation for this phenomenon. The low-dex concentration will create a hypocorticoid condition in the brain and therefore trigger a response from the CRH-producing neurons of the PVN, which may be reflected by the enhanced response in the anterior pituitary. dex suppression in healthy subjects can indeed be overcome by concurrent infusion of CRH and vasopressin (51).

In contrast to dex, the naturally occurring glucocorticoid corticosterone can easily enter the brain. In the mdr1a knockout and wild-type mice, 3H-corticosterone was taken up in brain and retained by brain MR to a similar extent (9, 19). Mdr1b-coded P-glycoprotein has been suggested to hamper corticosterone entrance to rodent brain, based on studies using mdr1a/1b knockout animal (24). It should be pointed out however that the mdr1b gene is expressed at the blood-brain barrier at much lower levels than mdr1a (52, 53) but is highly expressed in the adrenal. We would argue that the small effect in the mutants reported previously by others (24) probably is caused by factors beyond the brain affecting the distribution and metabolism of corticosterone. Interestingly, evidence based on measurement of steroid concentrations in postmortem brain points to preferential brain uptake of corticosterone also in humans (19). Thus, the much better penetration of corticosterone than dex in brain makes this steroid a much better candidate to MR and GR function (37, 54, 55, 56, 57), perhaps also in humans.

In conclusion, our findings suggest that treatment with small amounts of dex can produce a hypocorticoid state selectively in the brain concomitant with feature of modestly increased glucocorticoid action in the periphery. This condition will specifically affect through imbalance of MR and GR central glucocorticoid target areas modulating HPA axis, behavioral adaptation, and synaptic plasticity, particularly after stress. Thus, divergence of peripheral and central glucocorticoid effects due to hampered access of small amounts of dex to the brain may serve as a model for states with disturbed glucocorticoid signaling in the brain.


   Acknowledgments
 
The skillful assistance of Peter Steenbergen and Marleen Visser is highly appreciated. We also are grateful to Sergiu Dalm, Heidi Lesscher, Maaike van der Mark, and Servane Lachize for technical assistance. We thank Liesbeth de Lange for critical reading of the manuscript.


   Footnotes
 
This work was supported by The Netherlands Organisation for Scientific Research (NWO Vidi Grant to O.C.M.), by the European Union ERASMUS (European Community Action Scheme for the Mobility of University Students) exchange program (to R.S.P.), and by the Royal Netherlands Academy of Arts and Sciences (to E.R.d.K.).

Present address for A.M.K.: Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Palo Alto, California 94304-5485.

First Published Online September 8, 2005

Abbreviations: ADX, Adrenalectomized; dex, dexamethasone; GR, glucocorticoid receptor; hnRNA, heteronuclear RNA; HPA, hypothalamic-pituitary-adrenal; HSD, honestly significant difference; MR, mineralocorticoid receptor; pBS, pBluescript; POMC, proopiomelanocortin; PVN, paraventricular nucleus; SSC, standard sodium citrate.

Received April 27, 2005.

Accepted for publication August 31, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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