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Endocrinology Vol. 138, No. 8 3476-3485
Copyright © 1997 by The Endocrine Society

ARTICLES

Hypothalamic-Pituitary-Adrenocortical Axis Changes in a Transgenic Mouse with Impaired Glucocorticoid Receptor Function1

Sharada Karanth, Astrid C. E. Linthorst, Günther K. Stalla, Nicholas Barden, Florian Holsboer and Johannes M. H. M. Reul

Max Planck Institute of Psychiatry (S.K., A.C.E.L., G.K.S., F.H., J.M.H.M.R.), Clinical Institute, Department of Neuroendocrinology, Section Neuroimmunoendocrinology, 80804 Munich, Germany, and Neuroscience Research Section (N.B.), CHUL Research Centre and Department of Physiology, Laval University, Ste-Foy, Quebec, Canada G1V 4G2

Address all correspondence and requests for reprints to: Johannes M. H. M. Reul, Ph.D., Section Neuroimmunoendocrinology, Department of Neuroendocrinology, Clinical Institute, Max Planck Institute of Psychiatry, Kraepelinstrasse 2, 80804 Munich, Germany. E-mail: reul{at}mpipsykl.mpg.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recently, a transgenic mouse with impaired glucocorticoid receptor (GR) function was created to serve as an animal model for the study of neuroendocrine changes occurring in stress-related disorders, such as major depression. Here, we investigated the hypothalamic-pituitary-adrenocortical (HPA) axis changes in these transgenic mice. There were no significant differences between basal early morning plasma ACTH and corticosterone levels in normal and transgenic mice. When animals were exposed to a mild stressor, an enhanced response in plasma ACTH was observed in the transgenic mice, whereas plasma corticosterone responses were not different. In view of these differences in plasma ACTH and corticosterone responses, we directed our studies toward the regulation of ACTH secretion on the hypothalamic-hypophyseal level in vitro. Therefore, an in vitro model, the pituitary-hypothalamic complex (PHc) was developed and its ACTH release profile was compared with that of the pituitary (PI) alone. The basal ACTH release by PHc and PI from normal and transgenic mice was similar. Regardless of the strain under study, the basal ACTH release by PI was significantly lower than the release by PHc. Stimulation of tissues with either high K+ (56 mM) or CRH (10 or 20 nM) produced an enhanced ACTH release from both PHc and PI, whereas the response in PI was larger than that in PHC. Moreover, the responses to these stimuli were markedly enhanced in tissues from transgenic mice. In tissues of normal mice, corticosterone inhibited both basal and CRH-stimulated ACTH release more potently in PHc than in PI. Furthermore, the feedback capacity of corticosterone to restrain both basal and CRH-stimulated ACTH release was highly impaired in tissues of transgenic mice, whereas the feedback in PHc appeared to be more affected than that in the PI of these animals.

In conclusion, the in vitro data on PHc and PI revealed intrahypothalamic mechanisms operating 1) to fine-tune stimulus-evoked ACTH responses; and 2) to facilitate the negative feedback action of glucocorticoids. Moreover, in the transgenic tissues, the impaired GR function was found to cause augmented stimulus-evoked ACTH responses and an impaired glucocorticoid feedback efficacy which appeared to be mainly defective at the hypothalamic level. Thus, in the transgenic mice with life-long central GR dysfunction we found impaired negative feedback combined with "normal" (i.e. noncompensated) in vivo plasma corticosterone responses. This is a condition with potentially grave pathophysiological consequences and, therefore, this transgenic animal may be regarded as a valuable model for the study of functional glucocorticoid insufficiency at the central nervous system level.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOID hormones are thought to exert negative feedback inhibition on hypothalamic-pituitary-adrenocortical (HPA) axis activity at the pituitary, hypothalamic, and extrahypothalamic level ultimately resulting in decreased synthesis and secretion of CRH and other corticotropic secretagogues, and in an inhibition of the production and secretion of ACTH (1, 2, 3, 4). These effects of glucocorticoids are mediated by intracellular soluble receptor molecules. Based on pharmacological, biochemical, and molecular studies, the glucocorticoid-binding receptors have been distinguished as mineralocorticoid (MR, type 1) and glucocorticoid receptors (GR, type 2; (5, 6, 7). In brain, highest MR levels are found in the pyramidal neurons of the hippocampus (8, 9, 10, 11) in which this receptor is thought, because of its substantial occupancy at all times (5), to exert a tonic control on neural circuits involved in the regulation of HPA axis activity and behavior (4, 5). Moderate levels of MR are present in the anterior pituitary where its function is still unknown (6, 12, 13, 14). GRs are rather ubiquitously distributed in the brain with higher concentrations in regions involved in the stress response such as the paraventricular nucleus of the hypothalamus and in the hippocampus, where these receptors appear to be colocalized with MR (9, 10, 11). High GR levels are also present in the anterior pituitary (6, 15, 16).

Neuroendocrine challenge tests, such as the dexamethasone suppression test (DST), have provided evidence for an aberrant GR function in major depressive illness and other diseases with known HPA axis disturbances (for review, see Refs. 17, 18). Clinical amelioration after antidepressant treatment has been observed to be associated with a normalization of HPA axis function. There is evidence that often this normalization develops before the clinical improvement (19, 20, 21). Recently, we and others have provided evidence suggesting that adjustments in brain and pituitary corticosteroid receptors may be involved in the attenuative effects of antidepressants on HPA axis activity in rats (22, 23, 24) and (transgenic) mice (25).

A transgenic mouse expressing antisense RNA directed against GR was created to study the disturbances in HPA axis regulation as evoked by the knockdown of brain GRs (26). It was envisaged that ultimately this transgenic mouse would serve as a neuroendocrine model for the HPA axis disturbances observed in depression. During the course of our studies, we observed that the transgenic mice exhibited diverse changes in the HPA axis including disparate hormonal responses after stressful stimuli (this study) and a decreased corticotropic sensitivity of the adrenal cortex (26a). These observations added an extra level of complexity to our in vivo experiments on HPA axis regulation in this animal model. Therefore, we used an in vitro model, the pituitary-hypothalamic-complex (PHc), to study the effects of GR knockdown on HPA regulation on the hypothalamic and pituitary level. The PHc retains both the anatomical orientation in situ and an intact neural linkage of the pituitary (27) and represents a valid system to study the interdependent hypothalamic-hypophyseal interactions in vitro (28). Thus, the PHc is a construct where the pituitary is still under hypothalamic control, whereas the sole pituitary (PI) is independent of any hypothalamic influence. Comparison of the ACTH release patterns of PHc and PI will provide information about the involvement of hypothalamic modulation of pituitary secretory activity. Thus, stimulus-evoked ACTH release and corticosterone-induced feedback efficacy on basal and stimulated ACTH release was investigated in tissues from normal and transgenic mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male mice of the B6C3F1 strain (the control mice) were purchased from Charles River Wiga (Sulzfeld, Germany). Male homozygous transgenic mice bearing a neurofilament promoter driven glucocorticoid receptor antisense construct (26; line1.3) were bred in the animal unit of our institute under controlled breeding conditions. The transgenic mice were created using animals of the B6C3F1 strain. The animals were maintained on a 12-h light, 12-h dark cycle (light on from 0600 to 1800 h) with controlled temperature (23 C) and humidity (60%). Food and tap water were available ad libitum. After arrival from the supplier the control mice were held at least 2 weeks before start of the experiment. All animals were 9–12 weeks old and had a body weight of 24–30 g at the time of the experiment.

Chemicals
Bacitracin and corticosterone were purchased from Sigma Chemical Co. (St. Louis, MO). Aprotinin was purchased from Bayer (Trasylol, Köln, Germany). Human/rat CRH was obtained from Bachem (Heidelberg, Germany). All reagents used were analytical grade.

Plasma ACTH and corticosterone
The experiments for assessment of basal and stress-induced HPA hormones were basically conducted as published previously (22, 29). Briefly, for estimation of basal early morning levels of ACTH and corticosterone, great care was taken to keep the mice undisturbed the night before the experiment. All neuroendocrine experiments were performed between 0700 and 0900 h. Control and transgenic mice were individually anesthetized (<15 sec) in a glass jar containing saturated halothane (Hoechst, Frankfurt am Main, Germany) vapor, after which the animals were decapitated immediately. This procedure was previously shown to yield valid basal HPA hormone values (22, 29). Trunk blood was collected in ice-chilled EDTA coated (1.5-ml) tubes containing 25 µg aprotinin. For determination of stress-induced plasma ACTH and corticosterone concentrations, control and transgenic mice were submitted to a novel environment stress procedure. Undisturbed mice were placed singly in new cages for 20 min, after which they were quickly anesthetized and decapitated, and trunk blood was collected as outlined above. Plasma samples for ACTH and corticosterone measurement by RIA (ICN Biomedicals, Costa Mesa, CA) were stored at -80 C and -20 C, respectively. The cross-reactivities of the polyclonal ACTH- and corticosterone-antisera with respective related substances was negligible. In addition, the ACTH-antiserum displayed similar specificity toward ACTH1–39 and ACTH1–24. The inter- and intraassay coefficient of variance for ACTH were 7% and 5%, respectively, with a detection limit of approximately 2 pg/ml. For corticosterone, the inter- and intraassay coefficient of variance were 7% and 4%, respectively, with a detection limit of 0.15 µg/100 ml.

Pituitary ACTH
The pituitary was immediately dissected from the above-mentioned animals killed under basal resting conditions and homogenized in 500 µl saline containing 28 µg/ml aprotinin. Next, the homogenates were centrifuged at low speed (3000 rpm) for 15 minutes at 4 C. Appropriate dilutions were made in Krebs Ringer Bicarbonate (KRB) buffer before measurement of ACTH by RIA (see above).

In vitro incubation: general procedures
After killing the animal by decapitation, the brain was removed and PHc was dissected as described previously (27). The hypothalamic island was demarcated by vertical cuts along the lateral hypothalamic sulci, the posterior edge of the optic chiasma, and the anterior edge of the mammillary bodies. A horizontal cut 2 mm from the base separated the island. The hypothalamic island attached to the pituitary via the hypophyseal stalk was then lifted from the skull. In some experiments, the hypothalamus (H) or the pituitary (PI) together, but disconnected from each other, were used. After dissection, tissues were incubated basically as previously described (30). Briefly, one PHc, one PI or one H plus one PI per tube was incubated in 400 µl of carbogenated KRB (pH 7.4) supplemented with the protease inhibitor Bacitracin (20 µM) in an atmosphere of 95% O2 and 5% CO2 with constant shaking at 50 cycles/min at 37 C. After a preincubation period of 1 h, medium was removed and discarded, and 400 µl of fresh KRB were added. Subsequently, depending on the type of experiment (see below), the incubation was continued for 1 or 2 h, after which the medium was collected. Next, 400 µl of KRB, KRB containing 56 mM KCl or CRH were added and incubated for 0.5–2 h after which the medium was collected. The high K+ medium was rendered isotonic by removal of an equivalent concentration of Na+. The collected medium was stored at -80 C until measurement of ACTH by RIA (see above).

In a separate set of experiments, after the 1-h preincubation period, PHc and PI were incubated with graded concentrations of corticosterone. The steroid was first dissolved in ethanol and then diluted in KRB. The final concentration of ethanol was less than 0.01%. After an incubation of 2 h, the medium was collected and either KRB or KRB containing 20 nM CRH was added for another incubation of 1 h. The collected media were frozen at -80 C and assayed for ACTH, as mentioned previously. At the end of the incubation procedure, the pituitaries were weighed. The pituitaries weighed between 1.2 and 2.0 mg and were not significantly different between the mouse strains. Data were expressed as pg ACTH/mg pituitary.

Experimental protocol of the in vitro incubations
The following in vitro experiments were conducted. Please note that all experiments were preceded by a preincubation period of 1 h.

  1. 1) Incubation of PHc, PI, and H + PI from normal and transgenic mice for 1 h or 2 h followed (PHc and PI only) by an incubation with medium containing high K+ (KRB + K+) or KRB only for 30 min.
  2. 2) Incubation of PHc and PI from normal and transgenic mice with CRH (10 nM) for 2 h followed by an incubation with medium containing either KRB only or KRB + K+ for 30 min.
  3. 3) Incubation of PHc and PI from normal mice with varying concentrations of corticosterone (10 nM to 1000 nM) for 2 h followed by an incubation with KRB containing only CRH (20 nM) for 1 h.
  4. 4) Incubation of PHc and PI from normal and transgenic mice with KRB or KRB + corticosterone (100 nM) for 2 h followed by an incubation with KRB or KRB containing only CRH (20 nM) for 1 h.

Statistics
Statistical analyses were conducted with Student’s t test or with ANOVA (one-way, two-way, or three-way where appropriate). In appropriate cases, ANOVA was followed by post-hoc comparisons of Duncan to test statistical differences between the experimental groups. As the level of significance, {alpha} < 0.05 was accepted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HPA axis hormone levels in control and transgenic mice
When mice were killed under basal early morning resting conditions, no differences in plasma ACTH and corticosterone levels were observed (Table 1Go). However, when mice were exposed to a novel environment situation, the resultant stress-induced plasma ACTH values in the transgenic mice were significantly higher (+39.2%) than those in the control animals. Stress-induced plasma corticosterone concentrations were not different between the two groups (Table 1Go). Despite the augmented stress-evoked plasma ACTH levels in the transgenic mice, no significant differences were found in pituitary ACTH content [65.6 ± 5.8 ng/pituitary in transgenic mice (n = 17) vs. 59.3 ± 3.6 ng/pituitary in normal animals (n = 24)].


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  		Table 1. Plasma ACTH and corticosterone in control and transgenic mice

 
ACTH release by PHc, H + PI and PI in normal mice after incubation with KRB for 1 or 2 h
In view of the differences between the in vivo stimulus-evoked responses in plasma ACTH and corticosterone, further studies in transgenic mice on HPA axis regulation at the hypothalamic-hypophyseal level were directed to the in vitro regulation of ACTH secretion from PHc and PI of these animals. Because no literature data are available on the release of ACTH by PHc (in this tissue neither in rats) or PI from mice, control experiments needed to be conducted. PHc, H + PI, and PI were incubated for 1 or 2 h with KRB. After an incubation of 1 or 2 h, PHc released significantly higher amounts of ACTH than both H + PI and PI (Table 2Go). However, after both 1 and 2 h, ACTH release by H + PI and PI were similar. Thus, an intact pituitary-hypothalamic link via the stalk has a significant stimulatory influence on basal ACTH release, whereas if the tissues are disconnected, there is no significant influence left of the hypothalamus on pituitary ACTH release. Subsequent experiments were performed with PHc and PI only.


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  		Table 2. In vitro ACTH release by PHc, H + PI and PI from control mice

 
Basal and depolarization-induced ACTH release by PHc and PI from normal and transgenic mice
Figure 1Go, A and C, shows that PHc from normal and transgenic mice release similar amounts of ACTH after incubation for either 1 or 2 h. The ACTH release by PI from normal and transgenic animals was not different but was significantly lower than the quantity of ACTH released by PHc (1 h, ANOVA; effect of tissue, F(1, 39) = 67.42, significance of F <= 0.0005, Fig. 1AGo; 2 h, ANOVA; effect of tissue, F(1, 48) = 121.41, significance of F <= 0.0005, Fig. 1CGo).



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  		Figure 1. Basal and K+-stimulated ACTH release from PHc and PI of normal and transgenic mice. Basal ACTH release was assessed by incubating tissues in KRB for 1 (A) or 2 h (C). After collection of medium, tissues were reincubated with KRB containing ("HIGH") 56 mM K+ or KRB only for 30 min. B and D, Data on K+-stimulation after previous incubations for 1 (A) or 2 h (C), respectively. ACTH was measured by RIA. For further experimental details, see text of Results and Materials and Methods. ir-ACTH, Immunoreactive ACTH. *, #, +, {alpha} < 0.05, Duncan multiple range test; A: n = 6–18; B: 5–9; C: n = 9–16; D: n = 6–12. *, {alpha} < 0.05 between PHc and PI within the same treatment and strain groups. #, {alpha} < 0.05 between high K+ and KRB within the same tissue and strain groups. +, {alpha} < 0.05 between transgenic and control within the same treatment and tissue groups.

 
To determine the effect of membrane depolarization on ACTH release by PHC and PI after the incubation for 1 and 2 h in KRB (for determination of basal release; see above), the medium was replaced by either KRB or KRB + K+ and incubated for 30 min. KRB + K+ produced a 2- to 3-fold increase in ACTH release by PHc and PI from normal mice as compared to their corresponding control groups incubated with KRB alone, irrespective of whether the tissues were previously incubated for 1 (ANOVA: effect of treatment: F(1, 39) = 191.37, significance of F <= 0.0005; Fig. 1BGo) or 2 h (ANOVA: effect of treatment: F(1, 46) = 117.93, significance of F <= 0.0005; Fig. 1DGo). The significant difference in ACTH release between PHc and PI observed under baseline conditions (Fig. 1Go, A and C) was totally abolished in the presence of high K+. Moreover, depolarization-induced ACTH release appeared to be higher when the pituitary was not under hypothalamic influence. However, statistical significance was only attained when tissues were preincubated for 1 h (ANOVA: interaction between tissue and treatment: F(1, 39) = 4.78, significance of F < 0.05; Fig. 1BGo), but not for 2 h (ANOVA: interaction between tissue and treatment: F(1, 46) = 0.23, significance of F > 0.05; Fig. 1DGo).

Furthermore, the data show a higher increase in K+-induced ACTH release in PHc and PI from transgenic mice than in that of control mice after previous incubations with KRB for either 1 h (ANOVA: interaction between strain and treatment: F(1, 39) = 60.63, significance of F <= 0.0005; Fig. 1CGo) or 2 h (ANOVA: interaction between strain and treatment: F(1, 46) = 12.45, significance of F = 0.001; Fig. 1DGo).

CRH-induced ACTH release by PHc and PI from normal and transgenic mice
To determine the responsiveness of PHc and PI of the mouse groups to an ACTH secretagogue, tissues were incubated with CRH (10 nM) for 2 h. CRH produced a 2- to 5-fold increase in ACTH release by PHc and PI from normal and transgenic mice (ANOVA: effect of treatment: F(1, 48) = 204.49, significance of F <= 0.0005; Fig. 2AGo). However, the CRH-induced ACTH release was significantly higher from PI than from PHc (ANOVA: interaction between treatment and tissue: F(1, 48) = 31.91, significance of F <= 0.0005). Moreover, the effect of CRH was much larger in tissues from transgenic mice than in those from the control animals (ANOVA: interaction between treatment and strain: F(1, 48) = 31.91, significance of F <= 0.0005; Fig. 2AGo).



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  		Figure 2. Effect of CRH on ACTH release from PHc and PI of normal and transgenic mice (A) and the effect of a subsequent K+-stimulation (B). Tissues were incubated for 2 h with 10 nM CRH or KRB, after which the medium was collected. The levels of ACTH in these media is depicted in A. Subsequently, the tissues were incubated with either KRB or KRB containing 56 mM K+ for 30 min (B). For further experimental details, see text of Results and Materials and Methods. ir-ACTH, Immunoreactive ACTH. #, +, {alpha} < 0.05, Duncan multiple range test; A: n = 5–9; B: n = 5–10, except for the KRB/KRB groups in B, n = 3. #, {alpha} < 0.05 between CRH and KRB (A) or high K+/KRB or high K+/CRH and KRB/KRB (B) within the same tissue and strain groups. +, {alpha} < 0.05 between transgenic and control within the same treatment and tissue groups.

 
Depolarization-induced ACTH release after CRH pretreatment
To study the effect of membrane depolarization on ACTH release by PHc and PI after incubation with CRH (to assess CRH-induced ACTH release, see above; Fig. 2AGo), the medium was replaced by KRB or KRB + K+ and incubated for another 30 min. After pretreatment with KRB, incubation of KRB + K+ produced a 2- to 3-fold increase in ACTH release by PHc and PI as compared to PHc and PI incubated with KRB only (ANOVA: effect of treatment: F(1, 24) = 64.48, significance of F <= 0.0005; Fig. 2BGo). However, K+ produced a larger effect in tissues of transgenic mice than in those of the normal animals (ANOVA: interaction between treatment and strain: F(1, 24) = 13.20, significance of F = 0.0001), which is consistent with the data shown in Fig. 1Go. A difference between the responsiveness of the tissues to K+ stimulation just escaped statistical significance (ANOVA: interaction between treatment and tissue: F(1, 24) = 3.88, significance of F = 0.06). Pretreatment with CRH had a significant stimulatory effect on the influence of K+ on ACTH release in both tissues (ANOVA: effect of CRH pretreatment: F(1, 44) = 9.0, significance of F = 0.01; Fig. 2BGo), which was similar in both mouse strains (no significant two-way interactions).

Effect of corticosterone on basal and CRH-induced ACTH release from PHc and PI of normal mice
To investigate the feedback efficacy of corticosterone in normal mice, medium was replaced with KRB containing various concentrations of corticosterone and incubated for 2 h. Figure 3AGo shows that corticosterone produced a significant dose-dependent suppression of basal ACTH release (ANOVA: effect of treatment: F(3, 55) = 108.18, significance of F <= 0.0005). It was observed that the inhibition by corticosterone was more effective on ACTH release from PHc than on that from PI (ANOVA: interaction between treatment and tissue: F(3, 55) = 6.13, significance of F = 0.0001). Clearly, a dose of 10 nM corticosterone produced a marked inhibition of ACTH release from PHc, whereas at this concentration no significant effect was observed on PI (Fig. 3AGo).



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  		Figure 3. Effect of corticosterone (CORT; A) and CRH (B) on ACTH release from PHc and PI of normal mice. Tissues were incubated for 2 h with various concentrations of corticosterone or KRB after which the medium was collected. The ACTH concentrations in these media are given in A. Next, fresh medium was added containing either KRB or KRB containing 20 nM CRH and tissues were incubated for another 1 h (B). For further experimental details, see text of Results and Materials and Methods. ir-ACTH, Immunoreactive ACTH. *, #, $, {alpha} < 0.05, Duncan multiple range test; A: n = 7–9; B: n = 6–8, except for the KRB/KRB group in B, n = 3. *, {alpha} < 0.05; A, between KRB-treated PHc and PI; B, between CRH/KRB and KRB/KRB, both within the same tissue groups. #, {alpha} < 0.05; A, between corticosterone groups and KRB within the same tissue groups; B, between CORT/CRH and KRB/CRH, both within the same tissue groups. $, {alpha} < 0.05; B, between CORT/CRH and KRB/KRB, within the same tissue groups.

 
After the incubation of PHc and PI with corticosterone for 2 h (to determine the effect of corticosterone on basal ACTH release, see above), the medium was replaced with KRB containing 20 nM CRH and the incubation proceeded for 1 h. In KRB-pretreated tissues, CRH evoked a 2- to 5-fold increase in ACTH release by PHc and PI (ANOVA: effect of treatment: F(1, 13) = 39.89, significance of F <= 0.0005; Fig. 3BGo). Pretreatment with corticosterone produced a dose-dependent decrease in CRH-induced ACTH release (ANOVA: effect of pretreatment: F(3, 50) = 36.13, significance of F <= 0.0005), albeit the steroid was more effective in PHc than in PI (ANOVA: interaction between pretreatment and tissue: F(3, 50) = 10.54, significance of F <= 0.0005; Fig. 3BGo). Hence, pretreatment with 10 nM corticosterone significantly inhibited CRH-induced ACTH release from PHc, whereas this concentration was ineffective in case of PI (Fig. 3BGo).

Effect of corticosterone on basal and CRH-induced ACTH release from PHc and PI of normal mice and transgenic mice
To evaluate the relative feedback efficacy of corticosterone in normal and transgenic mice, PHc and PI were incubated for 2 h with KRB containing 100 nM corticosterone. Figure 4AGo shows that incubation with 100 nM corticosterone significantly suppressed ACTH release from both PHc and PI of either experimental group (ANOVA: effect of treatment: F(1, 64) = 151.39, significance of F <= 0.0005). However, the effect of corticosterone was significantly less in tissues of transgenic mice than in those of control animals (ANOVA: interaction between treatment and strain: F(1, 64) = 5.61, significance of F = 0.021) and was overall more pronounced in PHc than in PI (ANOVA: interaction between treatment and tissue: F(1, 64) = 4.92, significance of F = 0.030), which is in line with the data presented in Fig. 3AGo. In addition, the statistical analyses also revealed that there was a significant difference in the degree of corticosterone-induced inhibition of ACTH release between PHc’s and PI’s of normal and transgenic animals (ANOVA: interaction between tissue and strain: F(1, 64) = 6.05, significance of F = 0.017; Fig. 4AGo), suggesting that the severity of the impairment in feedback efficacy was different between the tissues of the transgenic mice.



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  		Figure 4. Effect of corticosterone (CORT; A) and CRH (B) on ACTH release from PHc and PI of normal and transgenic mice. Tissues were incubated for 2 h with 100 nM corticosterone or KRB after which the medium was collected. The ACTH concentrations in these media are given in A. Next, fresh medium was added containing either KRB or KRB containing 20 nM CRH and tissues were incubated for another 1 h (B). For further experimental details, see text of Results and Materials and Methods. ir-ACTH, Immunoreactive ACTH. *, #, $, {alpha} < 0.05, Duncan multiple range test; A, n = 6–12; B: n = 7–9, except for the KRB/KRB groups in B, n = 3. *, {alpha} < 0.05; A, between KRB-treated PHc and PI; B, between CRH/KRB and KRB/KRB, both within the same tissue and strain groups. #, {alpha} < 0.05; A, between the corticosterone group and KRB; B, between CORT/CRH and KRB/CRH, both within the same tissue and strain groups. $, {alpha} < 0.05; B, between CORT/CRH and KRB/KRB, within the same tissue and strain groups. +, {alpha} < 0.05; B, between the KRB/CRH groups of transgenic and control tissues within the same tissue groups.

 
Figure 4BGo shows that incubation with 20 nM CRH after pretreatment with KRB produced a significant stimulation of ACTH release (ANOVA: effect of treatment: F(1, 31) = 141.18, significance of F <= 0.0005), which was more pronounced in PI than in PHc (ANOVA: interaction between treatment and tissue: F(1, 31) = 8.22, significance of F = 0.007). However, in contrast to the data shown in Fig. 2AGo, a strain difference in the effect of CRH just escaped statistical significance (ANOVA: interaction between treatment and strain: F(1, 31) = 3.10, significance of F = 0.088; Fig. 4BGo), which may be due to the higher concentration of CRH (20 nM vs. 10 nM in Fig. 2AGo) and/or the shorter incubation time (1 h vs. 2 h in Fig. 2AGo) used in this experiment.

As shown in Fig. 4BGo, pretreatment with 100 nM corticosterone significantly reduced the effect of CRH on ACTH release (ANOVA: effect of pretreatment: F(1, 64) = 184.58, significance of F <= 0.0005), but there were no significant differences between the mouse strains. However, Fig. 4BGo clearly shows that pretreatment with corticosterone suppressed the CRH-induced ACTH release from tissues of control animals toward a level not significantly different from that observed in the KRB/KRB control groups, whereas in case of the transgenic tissues a significant difference remained between the CORT/CRH and its respective KRB/KRB control groups (Fig. 4BGo; for details on the statistical analyses, see legend to Fig. 4BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that long-term impairment of glucocorticoid receptor function leads to multifarious changes in the HPA axis. Although baseline HPA hormone secretion was unaltered, a mild stress procedure provoked major differences between normal and transgenic animals in circulating ACTH levels, whereas no alterations were observed in the plasma corticosterone responses. These diverging hormonal responses dovetail with our previous observation that the adrenal cortex of the transgenic mice is hyporesponsive to adrenocorticotropic stimulation (26a). In view of these diverging in vivo neuroendocrine response and our aim to delineate the putatively aberrant central mechanisms responsible for the HPA axis dysfunction in the transgenic mice, we set out to investigate the in vitro ACTH release at the hypothalamic-pituitary level.

Our early experiments using PHc, H + PI, and PI indicated that the pituitary receives a stimulatory input from the hypothalamus, which is disrupted when the stalk is severed. It is well established that the secretion of ACTH is under the control of a variety of factors, the most important being CRH and arginine-vasopressin (AVP), but also other factors seem to be involved, such as oxytocin and angiotensin II (for review, see 2 . Thus, since in the PHc the in situ cytoarchitecture of the hypothalamus and the pituitary is maintained, an increased ACTH release by PHc may be due to concerted effects of several factors operating through the hypothalamic-hypophyseal axis. Because the ACTH releasing properties of H + PI and PI were similar, subsequent experiments were conducted with PHc and PI only. In addition, this observation indicates that the amount of ACTH possibly released from the hypothalamus may be negligible. The ACTH release by either tissue after 2 h of incubation was approximately two times higher than that after 1 h, suggesting that these in vitro explant systems are able to maintain a constant rate of ACTH release. Furthermore, no differences were found in the basal release of ACTH by PHc and PI from normal and transgenic mice, which corresponds with the similar plasma ACTH levels in normal and transgenic mice killed under basal early morning resting conditions.

The use of a high K+ concentration in the incubation medium in order to depolarize the tissue is a common method to study the release capacity of endocrine and nervous tissues for a variety of (neuro-)peptides and neurotransmitters (31). In the present study, depolarization produced a marked increase in ACTH release from both PHc and PI, thereby nullifying the differences in intrinsic release properties between the tissues observed under basal conditions. In addition, there was a profound difference between the K+-induced ACTH release from tissues of normal and transgenic animals, suggesting that the size of the releasable ACTH pool in the corticotrophs of transgenic mice was considerably larger than that in the control animals. However, the total pituitary content of ACTH was found not to be different between the animal groups, which may indicate that the releasable ACTH pool is not synonymous to but only part of the total ACTH pool. Other factors involved in the differential effect of depolarization on ACTH release by normal and transgenic tissues may include possible differences between the hypophyseal tissues in the density of the corticotrophic cells and their CA2+ gating properties. Changes in these various parameters may have been directly or indirectly induced by an altered GR function, because the antisense GR transgene has been shown not only to be expressed in brain but also in the pituitary gland of our transgenic mice (32).

We observed that exogenously administered CRH stimulated the ACTH release by both PHc and PI, although the response was much larger in PI than in PHc. Moreover, the absolute levels of ACTH attained after CRH were much lower in case of PHc than PI. Thus, it appears that in PHc CRH-stimulated ACTH release is inhibited by a factor from hypothalamic origin. The identity of this factor is unknown. Inhibitory [and, in some cases, stimulatory (33, 34)] effects of opioids, such as morphin and dynorphin, on basal and stimulated HPA axis activity have been reported in rats and humans (35, 36, 37, 38). However, in vivo and in vitro experiments conclusively showed that the opiate inhibition was not achieved by directly restraining pituitary ACTH release (36, 38, 39) but by suppressing hypothalamic CRH secretion (39, 40). These observations exclude endogenous opioids as putative attenuators of CRH-stimulated ACTH release in our PHc system. Besides glucocorticoid hormones, the peptide atrial natriuretic factor (ANF) has been found to directly inhibit pituitary ACTH release in rats (41) and in humans (42). Immunocytochemical studies have revealed the existence of ANF-positive neurons in the hypothalamus and ANF-positive fibers in the median eminence (43, 44, 45, 46). Moreover, ANF has been shown to be released in the hypophyseal portal blood (47). These data suggest that hypothalamic ANF appears to be involved in the regulation of pituitary secretion of POMC-derived peptides. Based on these literature data, we propose that the postulated hypothalamic ACTH release inhibiting factor is ANF. We found that treatment of tissues with CRH produced an augmented effect on a subsequent incubation with high K+, indicating 1) that the tissues were not depleted of ACTH; 2) that the tissues were able to respond to a second stimulus; and 3) that CRH may enlarge the releasable pool of ACTH.

In the present study, PHc and PI of the transgenic mice showed an augmented response in ACTH release upon stimulation by CRH as compared to that in tissues of normal animals. This result is in line with our in vivo observation of an amplified plasma ACTH response in the transgenic animals after a subcutaneous injection of CRH (26a). The degree to which the response was enhanced did not seem to differ between PHc and PI, as was also indicated by the absence of a significant three-way interaction (strain x tissue x treatment) in the ANOVA statistical analysis. To explain the strain differences in the ACTH release responses to CRH, the same reasons (i.e. releasable ACTH pool, corticotrophic cell density, Ca2+ gating properties) may apply as mentioned previously for the K+ stimulation (see above). In addition, an enhanced sensitivity of the pituitary corticotropes for CRH may exist in the transgenic mice. However, until now no information is available about any changes in the density and/or affinity of the pituitary CRH receptors in these animals. Furthermore, because the CRH-induced ACTH release was enhanced approximately to the same extent in the transgenic PHc and PI, it may be speculated that the activity of the postulated hypothalamus-derived inhibitory factor (ANF?, see above) is not changed in the transgenic animal.

We observed that corticosterone dose-dependently inhibited the basal ACTH release from both PHc and PI of normal mice. However, the ACTH release by PI was much less sensitive to the inhibitory action of the glucocorticoid than PHc. The observation that addition of 10 nM corticosterone lowered the ACTH release by PHc down to the level of release by PI (which was unaltered by 10 nM corticosterone), strongly suggests that the glucocorticoid effect on ACTH release was generated at the suprapituitary (hypothalamic) level, possibly by inhibition of corticotropic secretagogues. This observation corresponds with data reported by Vermes et al. (48), who elegantly showed in an in vitro hypothalamus-pituitary cell-adrenal cell perfusion system that pretreatment of the hypothalamic tissue blocks with dexamethasone resulted in a decline in corticosterone output from the hypothalamus-pituitary cell-adrenal cell system toward levels produced by a pituitary cell-adrenal cell set-up. Moreover, a lower sensitivity of corticotropic cells to the action of corticosterone has also been observed in rats (49) and seems to be due to the presence of corticosterone binding globuline (CBG) in these cells (50, 51, 52). However, regardless of the difference in tissue sensitivity and its underlying cause, it is striking that corticosterone was able to reduce basal ACTH release from PI, because in previous studies using incubated or perfused rat pituitaries, no glucocorticoid effects on basal secretion were observed at concentrations which were able to inhibit CRH-, vasopressin- or hypothalamic-stalk median eminence extract-stimulated ACTH secretion (for review, see 1 . The reason for this species difference is unknown.

In addition to the effects of corticosterone on basal ACTH release, we also found a marked influence of the glucocorticoid on the CRH-stimulated secretion, which is consistent with numerous in vitro studies on corticosteroid regulation of ACTH release from rat pituitaries (1, 2). However, similar to the findings on glucocorticoid suppression of basal ACTH release, a marked difference in tissue sensitivity to the inhibitory action of the glucocorticoid was evident. Whereas a profound suppression of ACTH release from PHc was observed at a concentration as low as 10 nM, this concentration was unable to restrain the CRH-evoked peptide secretion from the PI. Moreover, comparison of the glucocorticoid effect at this concentration on basal ACTH release with that on the succeeding CRH-evoked peptide secretion revealed that relatively the glucocorticoid had a much larger impact on the stimulated release than on the basal release (-50% vs. -25%, respectively). It has been suggested that exogenously added CRH increases endogenous CRH secretion via an ultrashort positive feedback mechanism (53). Such mechanism appears not to be extractable from our data and, thus, evidently the effect of corticosterone in the PHc was not precipitated by a suppression of this mechanism. Nevertheless, irrespective of the addition of CRH, the glucocorticoid effects found with the 10 nM concentration imply that corticosterone at this concentration acts within the hypothalamus to restrain pituitary ACTH secretion. In line with the previously mentioned, it may be suggested that corticosterone facilitates the action of an ACTH release inhibiting factor, such as ANF. This notion is supported by a report of Fink et al. (54), who showed that the inhibitory action of dexamethasone on plasma ACTH levels in long-term adrenalectomized rats was strongly reduced after iv infusion of anti-ANF antiserum. In addition, icv or iv infusion of an immunoneutralizing antiserum against ANF into rats produced an enhancement of the ether stress-induced plasma ACTH response (54, 55), suggesting that during stress ANF acts to attenuate the elevation in plasma ACTH. In view of these findings, it is tempting to speculate on whether CRH in conjunction with glucocorticoids would enhance hypothalamic ANF secretion, but as yet no information is available. Such mechanism would provide, first, an endogenous attenuator (ANF) controlling the amplitude of the stress response, and, second, a support for the negative feedback by glucocorticoids to rapidly restrain stress-induced ACTH release.

We found that the glucocorticoid negative feedback on basal and CRH-stimulated ACTH release from the transgenic tissues was greatly impaired, which is compatible with our recently published in vivo data (56). In the transgenic tissues, a reduced efficacy of corticosterone (100 nM) was observed both on baseline ACTH values and on the CRH-evoked release. Thus, CRH produced an escape from the glucocorticoid suppression in the transgenics but not in the controls. These observations are in line with the decline in GR capacity (~-50%) in these transgenic mice (26). Furthermore, statistical analysis confirmed that the feedback efficacy was more incapacitated in the PHc than in the PI of the transgenic mice, suggesting that the disturbance in glucocorticoid negative feedback is primarily at the hypothalamic level. A principal hypothalamic disturbance would be consistent with the main (primarily neuron-specific) expression pattern of the applied neurofilament promoter-driven antisense GR transgene (26). These data confirm the critical role of the GR as a mediator of glucocorticoid negative feedback on the hypothalamic-hypophyseal axis aimed to restrain the output of ACTH (1, 2, 3). Moreover, this study provides evidence for a supporting modulatory role of the hypothalamus in fine-tuning the effects of glucocorticoid hormone and CRH on hypophyseal ACTH secretion.

As described earlier, this transgenic mouse was created to serve as an animal model for the neuroendocrine changes in depression. Presently, it can be concluded that, in our transgenic mice, similarities as well as differences with the neuroendocrine features during this psychiatric illness are discernable. While the decreased feedback efficacy of corticosterone (this study) and dexamethasone (56), and the CRH-induced escape of ACTH from corticosterone suppression in the transgenics may be compatible with the often aberrant DST result (57) and the overshooting combined DST/CRH challenge test outcome in depressed patients (17, 58), other parameters were shown to be conspicuously different, such as basal HPA hormone levels, CRH-induced ACTH responses and adrenocortical sensitivity (17, 59, 60, 61, 62). These comparisons indicate that only regarding some aspects a concordance appears to exist between the HPA axis abnormalities in our transgenic mice and those in depressed patients. Thus, although it seems that a long-term GR dysfunction is involved in the HPA axis disturbances in depression, evidently additional factors must be implicated.

Given the nature of the defect in our transgenic mice, a resemblance with familial glucocorticoid resistance may be expected. Familial glucocorticoid resistance results from the partial inability of glucocorticoids to exert their effects on target tissues throughout the body (for review, see 63 . It appears to be caused by various molecular defects, such as point mutations or a microdeletion of the GR gene (64, 65). Of the multiple endocrine and clinical features of familial glucocorticoid resistance, it is of interest to note that the condition is associated with marked compensatory increases in circulating ACTH and cortisol, but not with Cushing-like bodily characteristics. These properties are in striking contrast with the absence of changes in basal circulating HPA hormone levels (this study) and the Cushing-like features of our transgenic mice (26). These disparities may originate from the different causes for the GR dysfunction in the transgenic mice as compared to that in glucocorticoid resistant humans and from the restricted (primarily nervous tissue in the transgenics) as opposed to the generalized localization (in glucocorticoid resistance) of the incapacitated GRs.

Thus, our transgenic mouse is a valuable model to study the consequences of life-long central GR dysfunction for HPA axis regulation and other glucocorticoid-controlled brain functions. The combination of "normal" in vivo plasma corticosterone responses and impaired negative feedback in the transgenic mice may be regarded as a condition with potentially grave pathophysiological consequences. From a neuroendocrine perspective, this study shows the major benefit of the use of PHc as compared to the sole PI, as the use of the PHc allows to include the investigation of integrative mechanisms operating within the hypothalamus and fine-tuning the release of ACTH and other pituitary hormones.


   Footnotes
 
1 This study was supported by grants of the Volkswagenstiftung (I/68 430 and I/70 543) and the EC (Grant No. CI1-CT93-0092). Back

Received October 21, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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