Glucocorticoid Receptor Blockade Disinhibits Pituitary-Adrenal Activity during the Stress Hyporesponsive Period of the Mouse
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内分泌学杂志 2005年第3期
Leiden-Amsterdam Center for Drug Research/Leiden University Medical Center (M.S., M.S.O., M.v.d.M., E.R.d.K.), Leiden University, 2300RA Leiden, The Netherlands; Max Planck Institute for Psychiatry (M.S., M.B.M., F.H.), 80804 Munich, Germany; Department of Psychiatry (S.L.), University of California, Davis, California 95616
Address all correspondence and requests for reprints to: Dr. Mathias Schmidt, Max Planck Institute of Psychiatry, Molecular Stress Physiology, Kraepelinstrasse 2–10, 80804 München, Germany. E-mail: mschmidt@mpipsykl.mpg.de.
Abstract
During postnatal development, mice undergo a period of reduced responsiveness of the pituitary-adrenal axis, the stress hyporesponsive period (SHRP), which is largely under control of maternal signals. The present study was designed to test the hypothesis that this quiescence in hypothalamic-pituitary-adrenal (HPA) activity is mediated by glucocorticoid feedback. For this purpose, the role of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) in control of HPA activity was examined during the SHRP and in response to 24 h of maternal deprivation. Nondeprived or deprived (24 h) CD1 mice on postnatal d 8 were injected sc at 16 and 8 h before testing with the MR antagonist RU28318 or the GR antagonist RU38486. The results showed that, in nondeprived mice, blockade of GR rather than MR triggered a profound increase in anterior pituitary proopiomelanocortin mRNA, circulating ACTH, and corticosterone concentrations. In contrast, CRH mRNA in hypothalamus and GR mRNA in hippocampus and hypothalamus were decreased. Blockade of the GR during the deprivation period amplified the rise in corticosterone induced by maternal deprivation, whereas it reversed the deprivation effect on the other HPA markers, leading to profound increases in plasma ACTH, proopiomelanocortin mRNA expression in the anterior pituitary, CRH mRNA expression in the paraventricular nucleus, and MR mRNA expression in the hippocampus, but not in GR mRNA expression in the hippocampus and paraventricular nucleus. In conclusion, the data suggest that control of postnatal pituitary-adrenal activity during the SHRP involves GR-mediated feedback in the anterior pituitary, which is further potentiated in the absence of the mother.
Introduction
IN THE ADULT ANIMAL, hypothalamic-pituitary-adrenal (HPA) activity is under the tight control of glucocorticoid-negative feedback (1). This feedback action is mediated by two different corticosteroid receptor types, mineralocorticoid receptors (MR) and glucocorticoid receptors (GR), which differ distinctly in their distribution and pharmacological properties (2). In the rodent brain, the MRs have a very high affinity for corticosterone and are predominantly expressed in hippocampal pyramidal neurons and the dentate gyrus. In contrast, the GRs have an approximately 10-fold lower affinity for corticosterone and are found in fairly high concentrations throughout the brain and in the anterior pituitary (for review, see Refs.2 and 3).
The role of these two receptor types in the adult is relatively well defined (4, 5, 6, 7, 8, 9). However, the function of these receptors in the regulation of HPA axis activity during development has not yet been delineated. During a large part of the early postnatal development in rats and mice, the pituitary-adrenal axis of the pups is hyporesponsive to most stimuli (10), whereas the central component of the HPA axis is still responsive (11). This period has been termed the stress hyporesponsive period (SHRP). The main characteristics of this period are very low basal corticosterone levels and an inability of many stressors to elicit a corticosterone response (12). This hyporesponsiveness is time and stressor specific because some more severe stressors have been shown to induce a stress response (13, 14). Shortly after the first description of the SHRP, it was hypothesized that an enhanced GR-mediated negative feedback at the level of the anterior pituitary is the most proximal cause of the HPA hyporesponsiveness (15, 16, 17, 18). This hypothesis was supported by adult-like levels of GR in the anterior pituitary throughout postnatal ontogeny (19), low circulating levels of the plasma corticosterone-binding globulin during the SHRP (20), and an enhanced basal and stress-induced ACTH release after adrenalectomy or metyrapone treatment (21, 22). However, direct evidence on the role of pituitary glucocorticoid feedback in the maintenance of the SHRP is lacking.
A number of other studies were published that challenged this feedback hypothesis. Vazquez and Akil (23) showed that the expression of proopiomelanocortin (POMC) mRNA, the precursor of ACTH, increased steadily during postnatal development. Therefore, a high inhibitory tone via the GR on POMC expression seemed unlikely. Numerous studies have demonstrated that the activity of the HPA axis during development is under maternal regulation and that removal of the mother (i.e. 24 h of maternal deprivation) results in activation of HPA activity and strongly enhances stress responsiveness (24, 25, 26, 27, 28, 29). However, in a recent mouse study, maternal deprivation did not alter the expression of the GR in the pituitary (30). Furthermore, it was shown that suppression of a corticosterone increase during maternal deprivation by dexamethasone did not affect the central effects of maternal deprivation on gene expression of HPA markers (31). Maternal deprivation also ended the quiescence of the adrenal, suggesting that the stress hyporesponsiveness is actually a period of adrenal hyporesponsiveness (32, 33). In addition, direct blockade of GRs at the level of the paraventricular nucleus (PVN) resulted only in minimal effects on CRH expression and corticosterone secretion during the SHRP (34). These results challenged the role of GR in mediating the SHRP and the effects of maternal deprivation.
Thus far, little attention has been given to the possible role of the MR in regulating the stress hyporesponsiveness during development. In adult rats, it has been demonstrated that MR gene expression is under direct regulation of CRH, which also has functional consequences for MR-mediated control of the HPA axis (35). A functional connection between CRH and the MR has recently also been suggested during postnatal development of the mouse (36). Because MR expression is relatively high throughout the first weeks after birth in both mice (37) and rats (38), a regulatory role of the MR in maintaining the stress hyporesponsiveness seems possible.
In this study, two experiments were designed to investigate the role of MR or GR in maintaining the low postnatal pituitary-adrenal activity and in mediating the effects of maternal deprivation. For this purpose, control and maternally deprived animals were treated with the MR antagonist RU28318 or the GR antagonist RU38486. We find that GR-mediated feedback is an important mechanism for maintaining the low and stable pituitary-adrenal activity during the SHRP.
Materials and Methods
Animals
The offspring of CD1 mice (obtained from Charles River Laboratories, Wilmington, MA) were used in this study. After a habituation period of 1 wk, three or four females were mated with one male in polycarbonate boxes (820 cm3) containing sawdust bedding. Pregnant females were transferred to clean polycarbonate cages containing sawdust and two sheets of paper towels for nest material during the last week of gestation. Pregnant females were checked for litters daily at 0900 h. If litters were found, the day of birth was defined as d 0 for that litter. On the day after parturition (d 1), each litter was culled to eight healthy pups (four males and four females) and remained undisturbed until used in the experiment. All animals were housed under a 12-h light, 12-h dark cycle (lights on at 0700 h) and under constant temperature (23 ± 2 C) and humidity (55 ± 5%) conditions. Food and water were provided ad libitum.
The experiments were carried out in accordance with European Communities Council Directive 86/609/EEC. All efforts were made to minimize animal suffering during the experiments. The protocols were approved by the Animal Care Committee of the Faculty of Medicine, University of Leiden (Leiden, The Netherlands) as well as the Committee for the Care and Use of Laboratory Animals of the Government of Bavaria, Germany.
Experimental design
Two experiments were carried out. In experiment 1, the influence of MR or GR antagonist treatment on the HPA function of neonatal mouse pups during the SHRP was investigated. Five litters (40 pups) were used. All pups were randomly assigned to injections with vehicle (polyethylene glycol, two pups per litter), the MR antagonist RU28318 (three pups per litter, 50 μg/g body weight), or the GR antagonist RU38486 (three pups per litter, 100 μg/g body weight) and marked after the first injection with a marker pen. The injection volume was always adjusted to 30 μl per injection. All pups received two injections sc on postnatal d 8, always 16 and 8 h before testing at postnatal d 9. In addition to various reports in the literature (39, 40, 41), the dosage choice was based on a pilot study testing the effect of the MR antagonist in suppressing the activation of the MR-responsive gene SGK1 in the adrenal gland by aldosterone. The results of this study showed that, even 8 h after antagonist treatment, the induction of SGK1 by aldosterone was significantly reduced compared with vehicle-treated mouse pups (our unpublished data). The decision to start treating the deprived pups 8 h after the onset of deprivation was based on our recent study, which showed a significant elevation of corticosterone after 8 h of deprivation (42).
Experiment 2 studied the consequences of MR or GR antagonist treatment on the effects of maternal deprivation on HPA axis function. Eight litters (64 pups) were used. Litters were randomly assigned to either a maternally nondeprived or a maternally deprived condition. Maternal deprivation took place in a separate room in the animal facility under similar light and temperature conditions as mentioned earlier. If a nest was assigned to maternal deprivation, mothers were removed from their home cages 24 h before the day of experimentation (postnatal d 9). The home cage, which contained the litter, was then placed on a heating pad maintained at 30–33 C for 24 h. Neither food nor water was available during the deprivation period. During the deprivation period, pups were injected with either the vehicle polyethylene glycol (two pups per litter), the MR antagonist RU28318 (three pups per litter, 50 μg/g body weight), or the GR antagonist RU486 (three pups per litter, 100 μg/g body weight). The injection volume was always adjusted to 30 μl per injection. All deprived pups received two sc injections during the deprivation period, at 8 and 16 h after the onset of the deprivation (i.e. 16 and 8 h before testing). Nondeprived pups remained undisturbed with their mothers.
Testing procedure
Testing took place on postnatal d 9 between 0800 and 1100 h. At the time of testing, all pups were killed under basal conditions by decapitation. For nondeprived litters, the mother was removed from the home cage immediately before testing. Trunk blood from all pups was collected individually in labeled 1.5-ml EDTA-coated microcentrifuge tubes. All blood samples were kept on ice and later centrifuged for 15 min at 6000 rpm at 5 C. Plasma was transferred to clean, labeled, 1.5-ml microcentrifuge tubes. All plasma samples were stored frozen at –20 C until the determination of ACTH and corticosterone. ACTH and corticosterone were measured by RIA (MP Biomedicals Inc., Irvine, CA; sensitivity, 10 pg/ml and 0.125 μg/dl, respectively). Whole heads (without skin and jaw) were removed, frozen in isopentane at –40 C, and stored at –80 C for in situ hybridization.
In situ hybridization
Frozen brains were sectioned at –20 C in a cryostat microtome at 16 μm in the coronal plane through the level of the hypothalamic PVN, dorsal hippocampus, and pituitary. The sections were thaw-mounted on poly-L-lysine-coated slides, dried, and kept at –80 C. In situ hybridization using 35S-uridine triphosphate-labeled ribonucleotide probes (CRH, GR, and MR) or oligonucleotides (POMC) were performed as described previously (30). The cDNA probes for CRH, GR, and MR contained the full-length coding regions of CRH (rat) and GR and MR (mouse), respectively. The slides were apposed to Kodak Biomax MR film (Eastman Kodak Co., Rochester, NY) and developed. For POMC, slides were also dipped in Kodak NTB2 emulsion (Eastman Kodak Co.) and exposed at 4 C for 6 d. Slides were developed, counterstained with cresyl violet staining, and examined with a light microscope with both bright- and dark-field condensers.
Data analysis
Data were analyzed by ANOVA, with the level of significance set at P < 0.05. When appropriate, tests of simple main and interaction effects and subsequent post hoc comparisons were made by the Newman-Keuls procedures. The initial analysis included sex as a factor; once it was determined that sex was not a significant factor, the data were collapsed across this variable. Autoradiographs were digitized, and relative expression of CRH, MR, and GR mRNA was determined by computer-assisted optical densitometry [analySIS 3.1, Soft Imaging System GmbH (Münster, Germany); Scion Image, Scion Corporation, Frederick, MD]. The mean of four to eight measurements was calculated from each animal.
Results
Two separate experiments were carried out. In the first experiment, the effects of a MR or GR blockade were examined in 9-d-old mice during the SHRP under basal conditions. The second experiment examined the role of both receptors during the activation of the neonatal HPA axis by maternal deprivation. Because our results indicated a predominant role of the GR (but not of the MR) in the control of ACTH and corticosterone, gene expression analyses of the different HPA-related transcripts in the brain were only carried out in GR antagonist-treated and control animals.
Experiment 1
Hormonal responses.
For corticosterone and ACTH plasma concentrations (Fig. 1, A and B), ANOVA revealed a main effect of treatment (F(2,38) = 153.812, P < 0.0001; and F(2,38) = 403.582, P < 0.0001), respectively). Treatment with the MR antagonist RU28318 had no significant effect on ACTH secretion, but it slightly increased corticosterone levels. Treatment with the GR antagonist RU38486 increased ACTH and corticosterone levels by more than 10-fold compared with vehicle-injected controls.
FIG. 1. Plasma corticosterone (A) and ACTH (B) in 9-d-old mouse pups during the SHRP. All animals were injected with vehicle (polyethylene glycol, n = 10), the MR antagonist RU28318 (MR-A, n = 15), or the GR antagonist RU38486 (GR-A, n = 15). Data represent mean ± SEM. *, Significant from vehicle-treated animals; P < 0.05.
Effects of GR antagonist treatment in the brain and the anterior pituitary.
The results for CRH and POMC gene expression are presented in Fig. 2. For CRH and POMC transcripts, ANOVA revealed a significant effect of treatment (F(1,20) = 22.787, P < 0.001; and F(1,20) = 106.886, P < 0.0001, respectively). After GR antagonist treatment, CRH expression in the PVN decreased significantly, whereas POMC expression in the anterior part of the pituitary was largely increased.
FIG. 2. Expression levels of CRH in the PVN (A) and POMC in the anterior pituitary (B) in 9-d-old mouse pups treated with either vehicle or the GR antagonist RU38486 (GR-A). Data represent mean ± SEM. *, Significant from vehicle-treated pups; P < 0.05. C, Representative CRH expression autoradiograms in the PVN. D, Representative autoradiograms (a and b) of POMC expression in the pituitary. Measurements were taken in the anterior lobe, not in the intermediate lobe, which expresses POMC at a very high level. The area of the black squares is enlarged as dark-field pictures in c and d.
We also measured the expression of GR in the PVN and hippocampus (Fig. 3) as well as the expression of MR in the hippocampus (data not shown). For GR, ANOVA revealed a main effect of treatment (PVN: F(1,20) = 12.985, P < 0.01); CA1: F(1,20) = 7.768, P < 0.01). Mouse pups treated with the GR antagonist showed significantly reduced GR expression in the PVN and in the CA1 area of the hippocampus. In contrast, MR expression in the hippocampus did not change after GR antagonist treatment (CA1: F(1,17) = 0.247, not significant; CA2: F(1,17) = 0.151, not significant; CA3: F(1,17) = 0.206, not significant; DG: F(1,17) = 0.06, not significant).
FIG. 3. Expression levels of GR in the CA1 area of the hippocampus (A) and in the PVN (B). Data represent mean ± SEM. *, Significant from vehicle-treated pups; P < 0.05. C, Representative GR mRNA expression autoradiograms in the hippocampus. D, Representative autoradiograms of GR mRNA expression in the PVN.
Experiment 2
Hormonal responses.
With regard to the analysis of corticosterone and ACTH (Fig. 4), ANOVA revealed a main effect of treatment (F(3,59) = 354.22, P < 0.0001; F(3,61) = 143.741, P < 0.0001, respectively). Maternal deprivation increased circulating ACTH and corticosterone levels. When treated with the MR antagonist RU28318, the effect of maternal deprivation on corticosterone and ACTH increase was mildly but significantly enhanced. In contrast, maternally deprived pups treated with the GR antagonist RU38486 displayed highly elevated corticosterone (373% compared with vehicle-injected deprived pups) and ACTH levels (873% compared with vehicle-injected deprived pups).
FIG. 4. Plasma corticosterone (A) and ACTH (B) in nondeprived (NDEP, n = 24) or maternally deprived (DEP) 9-d-old mouse pups. Deprived animals were injected with vehicle (polyethylene glycol, n = 10), the MR antagonist RU28318 (MR-A, n = 15), or the GR antagonist RU38486 (GR-A, n = 15). Data represent mean ± SEM. *, Significant from control; #, significant from vehicle (veh)-injected deprived animals; P < 0.05.
Effects of GR antagonist treatment in the brain.
As in experiment 1, the gene expression of CRH and GR in the PVN, POMC in the anterior pituitary, and MR and GR in the hippocampus was examined.
For CRH, ANOVA revealed a main effect of treatment (F(2,24) = 41.579, P < 0.0001). Maternal deprivation resulted in a decrease of CRH expression in vehicle-treated animals (Fig. 5A). This decrease was prevented by treatment with the GR antagonist RU38486. The effects of GR antagonist treatment on POMC expression were also significant (F(2,24) = 78.059, P < 0.0001). After maternal deprivation, POMC expression was decreased in the anterior lobe of the pituitary (Fig. 5B). However, treatment of the pups with the GR antagonist RU38486 during the deprivation period reversed POMC expression to about twice the amount of deprived control pups, thereby also reaching significantly higher levels compared with nontreated control animals.
FIG. 5. Expression levels of CRH in the PVN (A) and POMC in the anterior pituitary (B) in maternally deprived (DEP) or nondeprived (NDEP) mouse pups. Deprived animals were injected with either polyethylene glycol as vehicle (veh) or the GR antagonist RU38486 (GR-A). Data represent mean ± SEM. *, Significant from control animals; P < 0.05. C, Representative CRH expression autoradiograms in the PVN. D, Representative POMC expression autoradiograms in the pituitary.
GR mRNA expression was measured in the CA1 area of the hippocampus (Fig. 6A) and in the PVN (Fig. 6B). In the CA3 area and the dentate gyrus of the hippocampus, GR expression was not measured because of the very low signal intensity in these areas. ANOVA revealed a main effect of treatment for the CA1 area and the PVN (F(2,27) = 8.336, P < 0.001; F(2,27) = 67.268, P < 0.0001, respectively). Maternal deprivation decreased GR expression in both areas. Treatment with the GR antagonist RU38486 further decreased GR expression in the PVN compared with the vehicle-injected deprived group. There was a trend (P < 0.09) for an increase in GR expression in the CA1 area compared with the deprived group.
FIG. 6. Expression levels of GR in the CA1 area of the hippocampus (A) and in the PVN (B) as well as MR expression levels in the hippocampus (E). Deprived animals were injected with either vehicle (DEP + veh) or the GR antagonist RU38486 (DEP + GR-A). Data represent mean ± SEM. *, Significant from control pups; #, significant from DEP + veh-injected pups; P < 0.05. C, Representative GR mRNA expression autoradiograms in the hippocampus. D, Representative GR mRNA expression autoradiograms in the PVN. F, Representative MR mRNA expression autoradiograms in the hippocampus. NDEP, Nondeprived; dg, dentate gyrus.
MR expression was measured in the CA1, CA2, CA3, and dentate gyrus subregions of the hippocampus (Fig. 6E). ANOVA revealed a main effect of treatment in all subregions [CA1: F(2,24) = 17.392, P < 0.0001; CA2: F(2,24) = 5.143, P < 0.05; CA3: F(2,24) = 6.24, P < 0.01; dentate gyrus: F(2,24) = 12.1, P < 0.001]. Maternal deprivation resulted in a decrease of MR expression. This decrease was significant for the CA1, CA2, and dentate gyrus regions. Treatment with RU38486 prevented the effects of maternal deprivation on MR expression in all subfields. In the CA1 area, treatment with the GR antagonist RU38486 after maternal deprivation even reversed MR expression to significantly higher levels compared with nondeprived animals.
Discussion
This study demonstrates that blockade of GR results in a profound increase of POMC gene expression in the anterior pituitary, ACTH release, and corticosterone secretion. This disinhibition observed after GR antagonist treatment provides evidence for the hypothesis that pituitary GRs mediate the suppression of ACTH production and release, providing strong support for the theory that negative feedback at the pituitary maintains the SRHP (17, 18). The remarkable efficacy of the GR antagonist is quite surprising because circulating corticosterone levels during development were thought to be too low to produce significant activation of the GR. However, corticosterone-binding globulin levels are also low during the SHRP (20), so that the effective (free) corticosterone concentration is likely to be much higher than the total corticosterone values would suggest. In contrast, treatment with the MR antagonist only slightly enhanced basal circulating corticosterone levels in the neonatal mouse. These results suggest that the control of HPA activity through MR already operates early in development (3), but in view of the small effect, a prominent role of MR in maintenance of the SHRP is unlikely.
There are several arguments that point to the pituitary as a main site of GR-mediated inhibitory control of HPA activity during the SHRP. First, in contrast to the brain, the GR is expressed in the pituitary and other peripheral organs at high levels throughout postnatal development (19, 43, 44, 45, 46). Second, the central CRH system has been shown to respond rapidly to mild stimuli during development, making it an unlikely target for GR-mediated control of the SHRP under basal conditions (11). Third, chronic blockade of PVN GR in the neonatal rat through a locally implanted cannula only slightly enhanced corticosterone secretion (34). In the same study, local GR blockade in the PVN failed to increase CRH expression during the first postnatal week and only slightly increased CRH expression during the second postnatal week. Finally, Grino et al. (47) and Walker et al. (48) demonstrated that adrenalectomy of neonatal rats greatly enhanced POMC gene expression in the anterior pituitary but did not alter CRH or vasopression synthesis in the PVN.
As previously reported, maternal deprivation resulted in an enhancement of circulating ACTH and corticosterone, whereas POMC, CRH, and GR expression decreased (30, 49, 50). A major difference between maternally deprived and GR antagonist-treated pups was found at the level of the pituitary, where POMC expression strongly increased after GR blockade but decreased in deprived animals. However, in a recent study, we demonstrated that the latter half of the 24-h deprivation period is characterized by enhanced glucocorticoid feedback, limiting or even reversing the initial effects of maternal deprivation, which results in suppressed POMC mRNA, ACTH, and CRH mRNA (42). Because this feedback signal is removed in the GR antagonist-treated pups, the enhanced ACTH release and POMC expression can be accounted to this fact.
Blockade of the GR during the deprivation period amplified the rise in corticosterone induced by maternal deprivation, whereas it reversed the deprivation effect on the other HPA markers. Consequently, a profound increase in plasma ACTH levels was found, whereas the deprivation-induced decreases in anterior pituitary POMC mRNA and CRH mRNA expression in the PVN were abolished. Thus, both maternal deprivation and GR antagonism decreased CRH mRNA expression in the PVN, whereas blockade of the GR in deprived animals reversed this effect. In this case, blockade of the GR appears to have opposite effects on CRH gene expression depending on the presence of the mother. To explain this paradox, it should be noted that, in adult animals, GR activation in the PVN has been shown to suppress CRH expression (51, 52). In addition, mice lacking a functional GR showed greatly enhanced CRH expression in the PVN (53) and immunoreactivity in the median eminence (54). Therefore, one interpretation of the present data is that the GR antagonist blocked an enhanced corticosterone feedback tone produced by elevated corticosterone levels induced by maternal deprivation, in particular in the latter half of the deprivation period. Alternatively, recent studies have indicated that, despite a close relationship between corticosterone levels and CRH expression during development, glucocorticoids suppress stress-induced changes of CRH expression rather than controlling basal expression levels (36). Another rather remote possibility is that RU486 might have both agonistic and antagonistic properties (55) or that it does not readily cross the blood-brain barrier, so that the observed effects on CRH mRNA expression would be a consequence of the sustained elevation of corticosterone.
In the previous paragraphs, GR-mediated feedback was considered to act in the core of the HPA axis. It is also conceivable that, beyond pituitary feedback, additional afferents to the HPA axis are disinhibited. One possibility is provided by metabolic signals that activate the central drive to the HPA axis. This hypothesis is supported by data from the maternally deprived rat, where elevated corticosterone levels as a result of deprivation can be abolished when food is provided to the pups (56). However, the replacement of only the licking behavior of the mother in the same study could also reverse some but not all maternal deprivation effects. Furthermore, in adult animals, metabolic signals were found to modulate HPA function in the absence of corticosterone (57, 58). The finding that maternal deprivation decreases CRH expression doesn’t necessarily contradict this hypothesis because we have previously shown that this decrease occurs only in the latter half of the deprivation period as a consequence of enhanced negative feedback (42). Collectively, the data suggest that the low pituitary-adrenal activity during postnatal development is due to an enhanced GR-mediated negative feedback on the pituitary involving possibly additional metabolic afferents to HPA activation.
Maternal deprivation has been shown to decrease GR expression in the PVN and the CA1 region of the hippocampus (30, 59). In the current study, GR expression was also decreased as a consequence of GR antagonist treatment as well as after maternal deprivation in combination with a GR blockade. A recent study in our laboratory indicated that this effect is not mediated by corticosterone or CRH because the decrease was still present in CRH receptor 1-deficient mice (36). The down-regulation of GR after antagonist treatment is puzzling because blockade of the GR was expected to result in an increase of GR expression, unless the excess antagonist itself exerts down-regulatory effects. A role for urocortins and CRH receptor 2 cannot be excluded in this respect because of the strong correlation between the ontogeny of urocortin 3 and GR (37). GR antagonist treatment prevented down-regulation of MR in the hippocampus after maternal deprivation, which is reminiscent of findings in the adult animal after the antagonist (40, 60) or after adrenalectomy (61). Because the GR was not active in the nondeprived animal, a positive feedback loop involving CRH cannot be excluded, as was observed by Gesing et al. (35) and recently in CRH receptor 1-deficient mice (37).
In conclusion, the present study shows that MR and GR play distinct roles in the control of HPA activity during development. We have provided direct evidence that GR-mediated feedback ensures the low pituitary-adrenal activity during the SHRP under basal conditions. After maternal deprivation, the GR antagonist revealed an enhanced inhibitory corticosterone tone.
Acknowledgments
The technical assistance of Steffanie Alam is gratefully acknowledged.
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Address all correspondence and requests for reprints to: Dr. Mathias Schmidt, Max Planck Institute of Psychiatry, Molecular Stress Physiology, Kraepelinstrasse 2–10, 80804 München, Germany. E-mail: mschmidt@mpipsykl.mpg.de.
Abstract
During postnatal development, mice undergo a period of reduced responsiveness of the pituitary-adrenal axis, the stress hyporesponsive period (SHRP), which is largely under control of maternal signals. The present study was designed to test the hypothesis that this quiescence in hypothalamic-pituitary-adrenal (HPA) activity is mediated by glucocorticoid feedback. For this purpose, the role of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) in control of HPA activity was examined during the SHRP and in response to 24 h of maternal deprivation. Nondeprived or deprived (24 h) CD1 mice on postnatal d 8 were injected sc at 16 and 8 h before testing with the MR antagonist RU28318 or the GR antagonist RU38486. The results showed that, in nondeprived mice, blockade of GR rather than MR triggered a profound increase in anterior pituitary proopiomelanocortin mRNA, circulating ACTH, and corticosterone concentrations. In contrast, CRH mRNA in hypothalamus and GR mRNA in hippocampus and hypothalamus were decreased. Blockade of the GR during the deprivation period amplified the rise in corticosterone induced by maternal deprivation, whereas it reversed the deprivation effect on the other HPA markers, leading to profound increases in plasma ACTH, proopiomelanocortin mRNA expression in the anterior pituitary, CRH mRNA expression in the paraventricular nucleus, and MR mRNA expression in the hippocampus, but not in GR mRNA expression in the hippocampus and paraventricular nucleus. In conclusion, the data suggest that control of postnatal pituitary-adrenal activity during the SHRP involves GR-mediated feedback in the anterior pituitary, which is further potentiated in the absence of the mother.
Introduction
IN THE ADULT ANIMAL, hypothalamic-pituitary-adrenal (HPA) activity is under the tight control of glucocorticoid-negative feedback (1). This feedback action is mediated by two different corticosteroid receptor types, mineralocorticoid receptors (MR) and glucocorticoid receptors (GR), which differ distinctly in their distribution and pharmacological properties (2). In the rodent brain, the MRs have a very high affinity for corticosterone and are predominantly expressed in hippocampal pyramidal neurons and the dentate gyrus. In contrast, the GRs have an approximately 10-fold lower affinity for corticosterone and are found in fairly high concentrations throughout the brain and in the anterior pituitary (for review, see Refs.2 and 3).
The role of these two receptor types in the adult is relatively well defined (4, 5, 6, 7, 8, 9). However, the function of these receptors in the regulation of HPA axis activity during development has not yet been delineated. During a large part of the early postnatal development in rats and mice, the pituitary-adrenal axis of the pups is hyporesponsive to most stimuli (10), whereas the central component of the HPA axis is still responsive (11). This period has been termed the stress hyporesponsive period (SHRP). The main characteristics of this period are very low basal corticosterone levels and an inability of many stressors to elicit a corticosterone response (12). This hyporesponsiveness is time and stressor specific because some more severe stressors have been shown to induce a stress response (13, 14). Shortly after the first description of the SHRP, it was hypothesized that an enhanced GR-mediated negative feedback at the level of the anterior pituitary is the most proximal cause of the HPA hyporesponsiveness (15, 16, 17, 18). This hypothesis was supported by adult-like levels of GR in the anterior pituitary throughout postnatal ontogeny (19), low circulating levels of the plasma corticosterone-binding globulin during the SHRP (20), and an enhanced basal and stress-induced ACTH release after adrenalectomy or metyrapone treatment (21, 22). However, direct evidence on the role of pituitary glucocorticoid feedback in the maintenance of the SHRP is lacking.
A number of other studies were published that challenged this feedback hypothesis. Vazquez and Akil (23) showed that the expression of proopiomelanocortin (POMC) mRNA, the precursor of ACTH, increased steadily during postnatal development. Therefore, a high inhibitory tone via the GR on POMC expression seemed unlikely. Numerous studies have demonstrated that the activity of the HPA axis during development is under maternal regulation and that removal of the mother (i.e. 24 h of maternal deprivation) results in activation of HPA activity and strongly enhances stress responsiveness (24, 25, 26, 27, 28, 29). However, in a recent mouse study, maternal deprivation did not alter the expression of the GR in the pituitary (30). Furthermore, it was shown that suppression of a corticosterone increase during maternal deprivation by dexamethasone did not affect the central effects of maternal deprivation on gene expression of HPA markers (31). Maternal deprivation also ended the quiescence of the adrenal, suggesting that the stress hyporesponsiveness is actually a period of adrenal hyporesponsiveness (32, 33). In addition, direct blockade of GRs at the level of the paraventricular nucleus (PVN) resulted only in minimal effects on CRH expression and corticosterone secretion during the SHRP (34). These results challenged the role of GR in mediating the SHRP and the effects of maternal deprivation.
Thus far, little attention has been given to the possible role of the MR in regulating the stress hyporesponsiveness during development. In adult rats, it has been demonstrated that MR gene expression is under direct regulation of CRH, which also has functional consequences for MR-mediated control of the HPA axis (35). A functional connection between CRH and the MR has recently also been suggested during postnatal development of the mouse (36). Because MR expression is relatively high throughout the first weeks after birth in both mice (37) and rats (38), a regulatory role of the MR in maintaining the stress hyporesponsiveness seems possible.
In this study, two experiments were designed to investigate the role of MR or GR in maintaining the low postnatal pituitary-adrenal activity and in mediating the effects of maternal deprivation. For this purpose, control and maternally deprived animals were treated with the MR antagonist RU28318 or the GR antagonist RU38486. We find that GR-mediated feedback is an important mechanism for maintaining the low and stable pituitary-adrenal activity during the SHRP.
Materials and Methods
Animals
The offspring of CD1 mice (obtained from Charles River Laboratories, Wilmington, MA) were used in this study. After a habituation period of 1 wk, three or four females were mated with one male in polycarbonate boxes (820 cm3) containing sawdust bedding. Pregnant females were transferred to clean polycarbonate cages containing sawdust and two sheets of paper towels for nest material during the last week of gestation. Pregnant females were checked for litters daily at 0900 h. If litters were found, the day of birth was defined as d 0 for that litter. On the day after parturition (d 1), each litter was culled to eight healthy pups (four males and four females) and remained undisturbed until used in the experiment. All animals were housed under a 12-h light, 12-h dark cycle (lights on at 0700 h) and under constant temperature (23 ± 2 C) and humidity (55 ± 5%) conditions. Food and water were provided ad libitum.
The experiments were carried out in accordance with European Communities Council Directive 86/609/EEC. All efforts were made to minimize animal suffering during the experiments. The protocols were approved by the Animal Care Committee of the Faculty of Medicine, University of Leiden (Leiden, The Netherlands) as well as the Committee for the Care and Use of Laboratory Animals of the Government of Bavaria, Germany.
Experimental design
Two experiments were carried out. In experiment 1, the influence of MR or GR antagonist treatment on the HPA function of neonatal mouse pups during the SHRP was investigated. Five litters (40 pups) were used. All pups were randomly assigned to injections with vehicle (polyethylene glycol, two pups per litter), the MR antagonist RU28318 (three pups per litter, 50 μg/g body weight), or the GR antagonist RU38486 (three pups per litter, 100 μg/g body weight) and marked after the first injection with a marker pen. The injection volume was always adjusted to 30 μl per injection. All pups received two injections sc on postnatal d 8, always 16 and 8 h before testing at postnatal d 9. In addition to various reports in the literature (39, 40, 41), the dosage choice was based on a pilot study testing the effect of the MR antagonist in suppressing the activation of the MR-responsive gene SGK1 in the adrenal gland by aldosterone. The results of this study showed that, even 8 h after antagonist treatment, the induction of SGK1 by aldosterone was significantly reduced compared with vehicle-treated mouse pups (our unpublished data). The decision to start treating the deprived pups 8 h after the onset of deprivation was based on our recent study, which showed a significant elevation of corticosterone after 8 h of deprivation (42).
Experiment 2 studied the consequences of MR or GR antagonist treatment on the effects of maternal deprivation on HPA axis function. Eight litters (64 pups) were used. Litters were randomly assigned to either a maternally nondeprived or a maternally deprived condition. Maternal deprivation took place in a separate room in the animal facility under similar light and temperature conditions as mentioned earlier. If a nest was assigned to maternal deprivation, mothers were removed from their home cages 24 h before the day of experimentation (postnatal d 9). The home cage, which contained the litter, was then placed on a heating pad maintained at 30–33 C for 24 h. Neither food nor water was available during the deprivation period. During the deprivation period, pups were injected with either the vehicle polyethylene glycol (two pups per litter), the MR antagonist RU28318 (three pups per litter, 50 μg/g body weight), or the GR antagonist RU486 (three pups per litter, 100 μg/g body weight). The injection volume was always adjusted to 30 μl per injection. All deprived pups received two sc injections during the deprivation period, at 8 and 16 h after the onset of the deprivation (i.e. 16 and 8 h before testing). Nondeprived pups remained undisturbed with their mothers.
Testing procedure
Testing took place on postnatal d 9 between 0800 and 1100 h. At the time of testing, all pups were killed under basal conditions by decapitation. For nondeprived litters, the mother was removed from the home cage immediately before testing. Trunk blood from all pups was collected individually in labeled 1.5-ml EDTA-coated microcentrifuge tubes. All blood samples were kept on ice and later centrifuged for 15 min at 6000 rpm at 5 C. Plasma was transferred to clean, labeled, 1.5-ml microcentrifuge tubes. All plasma samples were stored frozen at –20 C until the determination of ACTH and corticosterone. ACTH and corticosterone were measured by RIA (MP Biomedicals Inc., Irvine, CA; sensitivity, 10 pg/ml and 0.125 μg/dl, respectively). Whole heads (without skin and jaw) were removed, frozen in isopentane at –40 C, and stored at –80 C for in situ hybridization.
In situ hybridization
Frozen brains were sectioned at –20 C in a cryostat microtome at 16 μm in the coronal plane through the level of the hypothalamic PVN, dorsal hippocampus, and pituitary. The sections were thaw-mounted on poly-L-lysine-coated slides, dried, and kept at –80 C. In situ hybridization using 35S-uridine triphosphate-labeled ribonucleotide probes (CRH, GR, and MR) or oligonucleotides (POMC) were performed as described previously (30). The cDNA probes for CRH, GR, and MR contained the full-length coding regions of CRH (rat) and GR and MR (mouse), respectively. The slides were apposed to Kodak Biomax MR film (Eastman Kodak Co., Rochester, NY) and developed. For POMC, slides were also dipped in Kodak NTB2 emulsion (Eastman Kodak Co.) and exposed at 4 C for 6 d. Slides were developed, counterstained with cresyl violet staining, and examined with a light microscope with both bright- and dark-field condensers.
Data analysis
Data were analyzed by ANOVA, with the level of significance set at P < 0.05. When appropriate, tests of simple main and interaction effects and subsequent post hoc comparisons were made by the Newman-Keuls procedures. The initial analysis included sex as a factor; once it was determined that sex was not a significant factor, the data were collapsed across this variable. Autoradiographs were digitized, and relative expression of CRH, MR, and GR mRNA was determined by computer-assisted optical densitometry [analySIS 3.1, Soft Imaging System GmbH (Münster, Germany); Scion Image, Scion Corporation, Frederick, MD]. The mean of four to eight measurements was calculated from each animal.
Results
Two separate experiments were carried out. In the first experiment, the effects of a MR or GR blockade were examined in 9-d-old mice during the SHRP under basal conditions. The second experiment examined the role of both receptors during the activation of the neonatal HPA axis by maternal deprivation. Because our results indicated a predominant role of the GR (but not of the MR) in the control of ACTH and corticosterone, gene expression analyses of the different HPA-related transcripts in the brain were only carried out in GR antagonist-treated and control animals.
Experiment 1
Hormonal responses.
For corticosterone and ACTH plasma concentrations (Fig. 1, A and B), ANOVA revealed a main effect of treatment (F(2,38) = 153.812, P < 0.0001; and F(2,38) = 403.582, P < 0.0001), respectively). Treatment with the MR antagonist RU28318 had no significant effect on ACTH secretion, but it slightly increased corticosterone levels. Treatment with the GR antagonist RU38486 increased ACTH and corticosterone levels by more than 10-fold compared with vehicle-injected controls.
FIG. 1. Plasma corticosterone (A) and ACTH (B) in 9-d-old mouse pups during the SHRP. All animals were injected with vehicle (polyethylene glycol, n = 10), the MR antagonist RU28318 (MR-A, n = 15), or the GR antagonist RU38486 (GR-A, n = 15). Data represent mean ± SEM. *, Significant from vehicle-treated animals; P < 0.05.
Effects of GR antagonist treatment in the brain and the anterior pituitary.
The results for CRH and POMC gene expression are presented in Fig. 2. For CRH and POMC transcripts, ANOVA revealed a significant effect of treatment (F(1,20) = 22.787, P < 0.001; and F(1,20) = 106.886, P < 0.0001, respectively). After GR antagonist treatment, CRH expression in the PVN decreased significantly, whereas POMC expression in the anterior part of the pituitary was largely increased.
FIG. 2. Expression levels of CRH in the PVN (A) and POMC in the anterior pituitary (B) in 9-d-old mouse pups treated with either vehicle or the GR antagonist RU38486 (GR-A). Data represent mean ± SEM. *, Significant from vehicle-treated pups; P < 0.05. C, Representative CRH expression autoradiograms in the PVN. D, Representative autoradiograms (a and b) of POMC expression in the pituitary. Measurements were taken in the anterior lobe, not in the intermediate lobe, which expresses POMC at a very high level. The area of the black squares is enlarged as dark-field pictures in c and d.
We also measured the expression of GR in the PVN and hippocampus (Fig. 3) as well as the expression of MR in the hippocampus (data not shown). For GR, ANOVA revealed a main effect of treatment (PVN: F(1,20) = 12.985, P < 0.01); CA1: F(1,20) = 7.768, P < 0.01). Mouse pups treated with the GR antagonist showed significantly reduced GR expression in the PVN and in the CA1 area of the hippocampus. In contrast, MR expression in the hippocampus did not change after GR antagonist treatment (CA1: F(1,17) = 0.247, not significant; CA2: F(1,17) = 0.151, not significant; CA3: F(1,17) = 0.206, not significant; DG: F(1,17) = 0.06, not significant).
FIG. 3. Expression levels of GR in the CA1 area of the hippocampus (A) and in the PVN (B). Data represent mean ± SEM. *, Significant from vehicle-treated pups; P < 0.05. C, Representative GR mRNA expression autoradiograms in the hippocampus. D, Representative autoradiograms of GR mRNA expression in the PVN.
Experiment 2
Hormonal responses.
With regard to the analysis of corticosterone and ACTH (Fig. 4), ANOVA revealed a main effect of treatment (F(3,59) = 354.22, P < 0.0001; F(3,61) = 143.741, P < 0.0001, respectively). Maternal deprivation increased circulating ACTH and corticosterone levels. When treated with the MR antagonist RU28318, the effect of maternal deprivation on corticosterone and ACTH increase was mildly but significantly enhanced. In contrast, maternally deprived pups treated with the GR antagonist RU38486 displayed highly elevated corticosterone (373% compared with vehicle-injected deprived pups) and ACTH levels (873% compared with vehicle-injected deprived pups).
FIG. 4. Plasma corticosterone (A) and ACTH (B) in nondeprived (NDEP, n = 24) or maternally deprived (DEP) 9-d-old mouse pups. Deprived animals were injected with vehicle (polyethylene glycol, n = 10), the MR antagonist RU28318 (MR-A, n = 15), or the GR antagonist RU38486 (GR-A, n = 15). Data represent mean ± SEM. *, Significant from control; #, significant from vehicle (veh)-injected deprived animals; P < 0.05.
Effects of GR antagonist treatment in the brain.
As in experiment 1, the gene expression of CRH and GR in the PVN, POMC in the anterior pituitary, and MR and GR in the hippocampus was examined.
For CRH, ANOVA revealed a main effect of treatment (F(2,24) = 41.579, P < 0.0001). Maternal deprivation resulted in a decrease of CRH expression in vehicle-treated animals (Fig. 5A). This decrease was prevented by treatment with the GR antagonist RU38486. The effects of GR antagonist treatment on POMC expression were also significant (F(2,24) = 78.059, P < 0.0001). After maternal deprivation, POMC expression was decreased in the anterior lobe of the pituitary (Fig. 5B). However, treatment of the pups with the GR antagonist RU38486 during the deprivation period reversed POMC expression to about twice the amount of deprived control pups, thereby also reaching significantly higher levels compared with nontreated control animals.
FIG. 5. Expression levels of CRH in the PVN (A) and POMC in the anterior pituitary (B) in maternally deprived (DEP) or nondeprived (NDEP) mouse pups. Deprived animals were injected with either polyethylene glycol as vehicle (veh) or the GR antagonist RU38486 (GR-A). Data represent mean ± SEM. *, Significant from control animals; P < 0.05. C, Representative CRH expression autoradiograms in the PVN. D, Representative POMC expression autoradiograms in the pituitary.
GR mRNA expression was measured in the CA1 area of the hippocampus (Fig. 6A) and in the PVN (Fig. 6B). In the CA3 area and the dentate gyrus of the hippocampus, GR expression was not measured because of the very low signal intensity in these areas. ANOVA revealed a main effect of treatment for the CA1 area and the PVN (F(2,27) = 8.336, P < 0.001; F(2,27) = 67.268, P < 0.0001, respectively). Maternal deprivation decreased GR expression in both areas. Treatment with the GR antagonist RU38486 further decreased GR expression in the PVN compared with the vehicle-injected deprived group. There was a trend (P < 0.09) for an increase in GR expression in the CA1 area compared with the deprived group.
FIG. 6. Expression levels of GR in the CA1 area of the hippocampus (A) and in the PVN (B) as well as MR expression levels in the hippocampus (E). Deprived animals were injected with either vehicle (DEP + veh) or the GR antagonist RU38486 (DEP + GR-A). Data represent mean ± SEM. *, Significant from control pups; #, significant from DEP + veh-injected pups; P < 0.05. C, Representative GR mRNA expression autoradiograms in the hippocampus. D, Representative GR mRNA expression autoradiograms in the PVN. F, Representative MR mRNA expression autoradiograms in the hippocampus. NDEP, Nondeprived; dg, dentate gyrus.
MR expression was measured in the CA1, CA2, CA3, and dentate gyrus subregions of the hippocampus (Fig. 6E). ANOVA revealed a main effect of treatment in all subregions [CA1: F(2,24) = 17.392, P < 0.0001; CA2: F(2,24) = 5.143, P < 0.05; CA3: F(2,24) = 6.24, P < 0.01; dentate gyrus: F(2,24) = 12.1, P < 0.001]. Maternal deprivation resulted in a decrease of MR expression. This decrease was significant for the CA1, CA2, and dentate gyrus regions. Treatment with RU38486 prevented the effects of maternal deprivation on MR expression in all subfields. In the CA1 area, treatment with the GR antagonist RU38486 after maternal deprivation even reversed MR expression to significantly higher levels compared with nondeprived animals.
Discussion
This study demonstrates that blockade of GR results in a profound increase of POMC gene expression in the anterior pituitary, ACTH release, and corticosterone secretion. This disinhibition observed after GR antagonist treatment provides evidence for the hypothesis that pituitary GRs mediate the suppression of ACTH production and release, providing strong support for the theory that negative feedback at the pituitary maintains the SRHP (17, 18). The remarkable efficacy of the GR antagonist is quite surprising because circulating corticosterone levels during development were thought to be too low to produce significant activation of the GR. However, corticosterone-binding globulin levels are also low during the SHRP (20), so that the effective (free) corticosterone concentration is likely to be much higher than the total corticosterone values would suggest. In contrast, treatment with the MR antagonist only slightly enhanced basal circulating corticosterone levels in the neonatal mouse. These results suggest that the control of HPA activity through MR already operates early in development (3), but in view of the small effect, a prominent role of MR in maintenance of the SHRP is unlikely.
There are several arguments that point to the pituitary as a main site of GR-mediated inhibitory control of HPA activity during the SHRP. First, in contrast to the brain, the GR is expressed in the pituitary and other peripheral organs at high levels throughout postnatal development (19, 43, 44, 45, 46). Second, the central CRH system has been shown to respond rapidly to mild stimuli during development, making it an unlikely target for GR-mediated control of the SHRP under basal conditions (11). Third, chronic blockade of PVN GR in the neonatal rat through a locally implanted cannula only slightly enhanced corticosterone secretion (34). In the same study, local GR blockade in the PVN failed to increase CRH expression during the first postnatal week and only slightly increased CRH expression during the second postnatal week. Finally, Grino et al. (47) and Walker et al. (48) demonstrated that adrenalectomy of neonatal rats greatly enhanced POMC gene expression in the anterior pituitary but did not alter CRH or vasopression synthesis in the PVN.
As previously reported, maternal deprivation resulted in an enhancement of circulating ACTH and corticosterone, whereas POMC, CRH, and GR expression decreased (30, 49, 50). A major difference between maternally deprived and GR antagonist-treated pups was found at the level of the pituitary, where POMC expression strongly increased after GR blockade but decreased in deprived animals. However, in a recent study, we demonstrated that the latter half of the 24-h deprivation period is characterized by enhanced glucocorticoid feedback, limiting or even reversing the initial effects of maternal deprivation, which results in suppressed POMC mRNA, ACTH, and CRH mRNA (42). Because this feedback signal is removed in the GR antagonist-treated pups, the enhanced ACTH release and POMC expression can be accounted to this fact.
Blockade of the GR during the deprivation period amplified the rise in corticosterone induced by maternal deprivation, whereas it reversed the deprivation effect on the other HPA markers. Consequently, a profound increase in plasma ACTH levels was found, whereas the deprivation-induced decreases in anterior pituitary POMC mRNA and CRH mRNA expression in the PVN were abolished. Thus, both maternal deprivation and GR antagonism decreased CRH mRNA expression in the PVN, whereas blockade of the GR in deprived animals reversed this effect. In this case, blockade of the GR appears to have opposite effects on CRH gene expression depending on the presence of the mother. To explain this paradox, it should be noted that, in adult animals, GR activation in the PVN has been shown to suppress CRH expression (51, 52). In addition, mice lacking a functional GR showed greatly enhanced CRH expression in the PVN (53) and immunoreactivity in the median eminence (54). Therefore, one interpretation of the present data is that the GR antagonist blocked an enhanced corticosterone feedback tone produced by elevated corticosterone levels induced by maternal deprivation, in particular in the latter half of the deprivation period. Alternatively, recent studies have indicated that, despite a close relationship between corticosterone levels and CRH expression during development, glucocorticoids suppress stress-induced changes of CRH expression rather than controlling basal expression levels (36). Another rather remote possibility is that RU486 might have both agonistic and antagonistic properties (55) or that it does not readily cross the blood-brain barrier, so that the observed effects on CRH mRNA expression would be a consequence of the sustained elevation of corticosterone.
In the previous paragraphs, GR-mediated feedback was considered to act in the core of the HPA axis. It is also conceivable that, beyond pituitary feedback, additional afferents to the HPA axis are disinhibited. One possibility is provided by metabolic signals that activate the central drive to the HPA axis. This hypothesis is supported by data from the maternally deprived rat, where elevated corticosterone levels as a result of deprivation can be abolished when food is provided to the pups (56). However, the replacement of only the licking behavior of the mother in the same study could also reverse some but not all maternal deprivation effects. Furthermore, in adult animals, metabolic signals were found to modulate HPA function in the absence of corticosterone (57, 58). The finding that maternal deprivation decreases CRH expression doesn’t necessarily contradict this hypothesis because we have previously shown that this decrease occurs only in the latter half of the deprivation period as a consequence of enhanced negative feedback (42). Collectively, the data suggest that the low pituitary-adrenal activity during postnatal development is due to an enhanced GR-mediated negative feedback on the pituitary involving possibly additional metabolic afferents to HPA activation.
Maternal deprivation has been shown to decrease GR expression in the PVN and the CA1 region of the hippocampus (30, 59). In the current study, GR expression was also decreased as a consequence of GR antagonist treatment as well as after maternal deprivation in combination with a GR blockade. A recent study in our laboratory indicated that this effect is not mediated by corticosterone or CRH because the decrease was still present in CRH receptor 1-deficient mice (36). The down-regulation of GR after antagonist treatment is puzzling because blockade of the GR was expected to result in an increase of GR expression, unless the excess antagonist itself exerts down-regulatory effects. A role for urocortins and CRH receptor 2 cannot be excluded in this respect because of the strong correlation between the ontogeny of urocortin 3 and GR (37). GR antagonist treatment prevented down-regulation of MR in the hippocampus after maternal deprivation, which is reminiscent of findings in the adult animal after the antagonist (40, 60) or after adrenalectomy (61). Because the GR was not active in the nondeprived animal, a positive feedback loop involving CRH cannot be excluded, as was observed by Gesing et al. (35) and recently in CRH receptor 1-deficient mice (37).
In conclusion, the present study shows that MR and GR play distinct roles in the control of HPA activity during development. We have provided direct evidence that GR-mediated feedback ensures the low pituitary-adrenal activity during the SHRP under basal conditions. After maternal deprivation, the GR antagonist revealed an enhanced inhibitory corticosterone tone.
Acknowledgments
The technical assistance of Steffanie Alam is gratefully acknowledged.
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