Glucocorticoid-mediated responses of plasma ACTH and anterior pituitary pro-opiomelanocortin, growth hormone and prolactin mRNAs during adjuvant-induced arthritis in the rat.

Adjuvant arthritis (AA) in the rat leads to chronic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis and the loss of its diurnal rhythmicity. We have investigated the effects of adrenalectomy (ADX) and different levels of corticosterone replacement upon plasma ACTH levels and anterior pituitary pro-opiomelanocortin (POMC), GH and prolactin mRNAs during the development of AA. In control ADX animals, we observed the negative feedback effects of exogenous corticosterone on plasma ACTH and anterior pituitary POMC mRNA. In the ADX animal with AA, however, the increased POMC mRNA which was observed was not reduced by exogenous corticosterone on day 7 of AA, although the negative feedback effect of corticosterone on plasma ACTH was intact. On day 14, however, even high dose corticosterone replacement failed to have a significant feedback effect on the raised levels of plasma ACTH. In control ADX animals, corticosterone replacement resulted in increased anterior pituitary GH mRNA and reduced prolactin mRNA. In contrast, in ADX animals with AA, GH mRNA was reduced and there was a further decrease in prolactin mRNA. In these animals, corticosterone replacement did not affect GH or prolactin mRNA expression. These data demonstrate a disruption of the normal mechanisms underlying feedback inhibition of the HPA axis by glucocorticoids during AA. Similarly, the glucocorticoid-dependent regulation of GH and prolactin mRNA expression is altered in AA.     

Differential regulation of type II corticosteroid receptor messenger ribonucleic acid expression in the rat anterior pituitary and hippocampus

The present study was designed to characterize the regulation of the type II corticosteroid receptor (GR) mRNA in two tissues involved in the control of the hypothalamic-pituitary-adrenal axis. We have used a solution hybridization/S1 nuclease protection assay to quantitate GR mRNA levels in the rat hippocampus and anterior pituitary after CRF, dexamethasone (DEX), or corticosterone (CORT) treatment. In general, hippocampal GR mRNA levels increased after removal of endogenous corticosteroids by surgical adrenalectomy and decreased in response to glucocorticoid treatment. More specifically, in the hippocampus 1) GR mRNA expression was decreased when adrenalectomized (ADX) animals were replaced with a relatively low dose of CORT, but not with a low dose of DEX; 2) acutely, CRF was more effective than DEX in decreasing the levels of GR mRNA in intact animals; however, under the same paradigm in ADX animals, DEX decreased the level of GR mRNA, whereas CRF was ineffective; and 3) in contrast to the decrease in GR mRNA levels observed after acute and low doses of glucocorticoid treatment, chronic treatment with either DEX or CORT did not change the level of hippocampal GR mRNA. These results suggest that in the hippocampus the decrease in GR mRNA expression after CRF treatment is probably via the release of glucocorticoids, and that this tissue is more sensitive to endogenous glucocorticoids than DEX. Anterior pituitary GR mRNA was differentially regulated compared with that in the hippocampus. In marked contrast to Gr mRNA in the hippocampus, ADX did not alter anterior pituitary GR mRNA expression, and glucocorticoid treatment led to an increase in GR mRNA levels. In the anterior pituitary 1) glucocorticoid treatment led to an increase in GR mRNA expression, when replaced with a relatively low dose of DEX, but not when replaced with a low dose of CORT; 2) acutely, neither CRF nor DEX altered levels of GR mRNA in intact animals; however, under the same paradigm DEX increased levels in ADX animals; and 3) chronic DEX or CORT treatment of intact animals elevated levels of anterior pituitary GR mRNA. In summary, these data have demonstrated tissue-specific regulation of GR mRNA in the hippocampus and anterior pituitary, which is dependent on both the dose and length of treatment and, in addition, on the glucocorticoid itself.     

Glucocorticoid and mineralocorticoid receptor mRNA expression in rat brain

In the rat brain, the binding of corticosterone is mediated through two receptor types, the type I receptor and the type II receptor, which are presumed to be encoded by genes designated as MR and GR, respectively. We have studied the regulation of these receptors by glucocorticoids, utilizing a cytosol receptor binding assay. In addition, we have employed molecular probes for the GR and the MR to measure receptor mRNAs. The level of type II receptor binding is uniform across several brain regions, as is the expression of GR (type II) mRNA. In contrast, type I receptor binding is concentrated in the hippocampus, and the MR (type I) mRNA similarly shows a higher level of expression in hippocampus than in the other brain regions studied. Removal of endogenous glucocorticoids by adrenalectomy (ADX) induces an increase, and corticosterone administration results in a decrease, in the level of type I and type II binding in the hippocampus; however, no significant changes in the MR (type I) or GR (type II) mRNA levels are seen with these treatments. The diurnal variation of serum corticosterone in intact rats is correlated with a circadian regulation of type I receptor binding in the hippocampus, while MR (type I) mRNA expression is unaffected. Thus, the changes in type I and type II receptor binding capacity elicited by differing steroid conditions cannot be attributed to modulation of the steady state levels of MR (type I) or GR (type II) mRNA.     

Corticosterone infused intracerebroventricularly inhibits energy storage and stimulates the hypothalamo-pituitary axis in adrenalectomized rats drinking sucrose

When allowed to drink sucrose, bilaterally adrenalectomized (ADX) rats exhibit normal weight gain, food intake, sympathetic neural activity, and ACTH compared with sham-ADX rats. Furthermore, ADX rats drinking sucrose have normal corticotropin-releasing factor (CRF) mRNA throughout brain. In ADX rats without sucrose, all of these variables are abnormal. Systemic corticosterone (B) replacement also restores these variables in ADX rats to normal. To test whether B acts centrally, we infused B or saline intracerebroventricularly into ADX rats under basal conditions and after repeated restraint. Rats were exposed to no stress or 3 h/d restraint for 3 d. Body weights and food and fluid intakes were measured. Brains were analyzed using immunocytochemistry against glucocorticoid receptors (GR) and CRF. Intracerebroventricular B blocked the positive effects of sucrose on metabolism, increased basal ACTH concentrations, and augmented ACTH responses to restraint on d 3. B-infused rats exhibited nuclear GR staining in perirhinal cortex, hippocampus, and hypothalamic paraventricular nuclei, showing that infused B spreads effectively. CRF staining in the paraventricular nucleus of the hypothalamus was higher in B- than in saline-infused rats. We conclude that under basal conditions B acts systemically, but not in the brain, to restore metabolism and neuropeptides after adrenalectomy. By contrast, tonic GR occupancy in brain initiates metabolic and ACTH responses characteristic of stress.     

Modulatory role of locus coeruleus and estradiol on the stress response of female rats

The activity of the hypothalamic-pituitary-adrenal axis is modulated by the norepinephrinergic system and, in females, also by the ovarian hormones. We investigated the role of ovarian steroids and the locus coeruleus (LC) on stress-induced corticosterone secretion in female rats. Ovariectomized rats without hormonal replacement (OVX) or treated with estradiol (OVE) or estradiol plus progesterone (OVEP) were subjected to jugular cannulation. Immediately after that, each hormonal treatment group was subjected to LC lesion or sham surgery or no brain surgery. After 24 h, blood samples of all 9 groups were collected before and after ether inhalation. Other four groups (OVX control, sham and lesioned, and OVE) were perfused for glucocorticoid receptor (GR) immunocytochemistry in hippocampal CA1 neurons and paraventricular nucleus (PVN). Estradiol replacement decreased while LC lesions increased stress-induced corticosterone secretion. The effect of LC lesion was potentiated with the removal of ovarian steroids. Since GR expression of lesioned animals decreased in the hippocampus, but not in PVN, we suggest that the effect of LC lesion on corticosterone secretion could be due to a reduction in the efficiency of the negative feedback system in the CA1 neurons. However, this mechanism is not involved in the estradiol modulation on corticosteroid secretion, as no change in GR expression was observed in estradiol-treated animals.     

Effects of Prenatal Ethanol Exposure on Hypothalamic-Pituitary-Adrenal Regulation After Adrenalectomy and Corticosterone Replacement

BACKGROUND: Previous studies have demonstrated that rats prenatally exposed to ethanol (E) exhibit hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness, demonstrated by increased and/or prolonged elevations of adrenocorticotropin (ACTH) and/or corticosterone (CORT) after stress. This study investigated possible mechanisms of HPA hyperresponsiveness in E rats by manipulating CORT feedback regulation of HPA activity via adrenalectomy (ADX) with or without CORT replacement. METHODS: Male Sprague-Dawley rat offspring from prenatal E, pair-fed (PF) and ad libitum-fed control (C) groups were tested at 90 to 120 days of age. Rats were either sham-operated or underwent ADX, with or without CORT replacement. CORT (25 microg/ml) was replaced via the drinking water to achieve basal plasma CORT levels and maintain a phasic CORT signal. Seven days after surgery, animals were decapitated at the diurnal peak either under basal conditions or after a 15-min restraint stress, and trunk blood was collected. RESULTS: After ADX, loss of the CORT feedback signal resulted in increased plasma ACTH in all groups compared with those in sham animals. In addition, under basal conditions, ADX E rats had significantly greater plasma ACTH levels than both PF and C rats. However, no differences were seen in ADX rats after stress. CORT replacement after ADX was partially effective in normalizing ACTH levels under both basal and stress conditions, with no differences among E, PF, and C animals. CONCLUSIONS: These results suggest that E males may exhibit enhanced stimulatory inputs to the hypothalamus, increased pituitary sensitivity to secretagogues, or both, which may be revealed after ADX. In contrast, E animals seem similar to controls in their ability to use an exogenous CORT signal to regulate HPA activity.     

Inverse shift in circulating corticosterone and leptin levels elevates hypothalamic deiodinase type 2 in fasted rats

During food deprivation, plasma T(4) and T(3) levels are decreased. Under this metabolic condition, hypothalamic deiodinase type 2 (D2) activity and mRNA levels are elevated, whereas TRH mRNA levels are suppressed. Systemic T(4) administration does not reverse these hypothalamic changes. The mechanism(s) that underlies this paradoxical regulation of D2 during fasting is unknown. We hypothesize that leptin and/or glucocorticoids play a role in these mechanisms, and their interactions may be an important regulator of the hypothalamic-pituitary-thyroid axis. Thus, we assessed the effects of these hormones on D2 activity levels of food-deprived as well as fed animals using enzyme activity measurements. In food-deprived animals, corticosterone replacement reversed the inhibitory effect of adrenalectomy (ADX) on D2 induction, whereas ADX and ADX plus corticosterone replacement did not significantly affect D2 activity levels in rats fed ad libitum. Leptin administration to fed animals did not change D2 activity, whereas in fasted rats, leptin decreased D2 activity by reducing corticosterone plasma levels. When leptin was administered to fasted animals that were either ADX or ADX plus corticosterone treated at a high dose, D2 activity did not increase. Our results show that during fasting, diminishing leptin levels play a permissive role to enable glucocorticoid-induced up-regulation of D2. Thus, our observations suggest that appropriate induction of D2 activity during negative energy balance is dependent upon both leptin and glucocorticoid signaling.     

High neonatal leptin exposure enhances brain GR expression and feedback efficacy on the adrenocortical axis of developing rats

Leptin modifies the activity of the hypothalamic-pituitary-adrenal axis in adult rodents and inhibits the production of glucocorticoids from human and rat adrenals in vitro. During development, high levels of circulating leptin and low levels of corticosterone secretion are observed together with adrenal hyporesponsiveness to stress. As chronic neonatal leptin administration reduced stress-induced corticotropin-releasing factor mRNA expression and ACTH secretion in pups, we determined whether elevated leptin levels enhanced the feedback effect of glucocorticoids on the hypothalamic-pituitary-adrenal axis. In naive pups we found a highly significant inverse relationship between plasma levels of leptin and corticosterone (P < 0.01) during postnatal d 6-20. We tested the ability of dexamethasone (1 or 10 microg/kg BW, ip, -3 h before stress) to suppress ether-induced ACTH secretion in 10-d-old pups that were treated during the neonatal period (d 2-9) with either vehicle or leptin (1 or 3 mg/kg BW, ip, daily). The expressions of brain GR and MR in vehicle- or leptin-treated neonates were determined by in situ hybridization and Western blotting. Chronic leptin treatment enhanced the ability of dexamethasone to suppress ACTH secretion after stress, and the low dose of dexamethasone was discriminant. Leptin treatment increased GR mRNA levels in the hypothalamic paraventricular nucleus (P < 0.05) and in the dentate gyrus of the hippocampus in a dose-dependent fashion. Hippocampal GR protein concentrations were increased by leptin treatment (P < 0.05). Expression of MR mRNA was not modified. Thus, the ability of leptin to enhance glucocorticoid feedback in pups is mediated in part by changes in brain GR. The high circulating leptin concentrations found in developing pups might be critical to regulate glucocorticoid production, GR levels, and stress responses. As leptin levels in pups vary with maternal diet, leptin might represent an important mediator of the maternal environment on the infant.     

Ontogeny of Hypothalamic Corticotropin-Releasing Factor and Anterior Pituitary Pro-opiomelanocortin Expression in Male and Female Offspring of Alcohol-Exposed and Adrenalectomized Dams

This study was designed to determine whether the previously described sexually dimorphic changes in rat hypothalamic corticotrop-in-releasing factor (CRF) and anterior pituitary pro-opiomelanocortin (POMC) mRNA expression in response to fetal alcohol exposure (FAE) are present prepubertally and whether they are altered by maternal adrenalectomy. Hypothalamic CRF and anterior pituitary POMC mRNA levels were determined in male and female offspring of adrenalectomized (ADX) and sham-adrenalectomized (Sham) dams exposed to alcohol (FAE) or a pair-fed (PF) control diet during the last 2 weeks of gestation. CRF and POMC mRNA levels were measured by Northern blotting at 1, 7, 14, and 21 days of age. In offspring of control PF dams, CRF mRNA levels increased faster in females, increasing by day 7, followed by a decrease at days 14 and 21, whereas in males there was a gradual increase from days 1 to 21. FAE altered the ontogenic profile of CRF mRNA in female offspring by delaying and exaggerating the rise of CRF expression to day 14, but produced no effect in males. Maternal adrenalectomy, combined with FAE, resulted in an early rise of CRF mRNA on day 14 in male offspring. In females, the combined ADX/FAE treatment resulted in significantly increased CRF mRNA levels, compared with those of ADX/PF offspring, on days 7 and 14. By day 21, these differences in CRF mRNA levels between the ADX/FAE and ADX/PF offspring had disappeared. POMC mRNA levels generally increased by day 7, followed by a dramatic decrease by day 14 and another increase by day 21. FAE male offspring showed decreased levels of POMC mRNA, whereas females were not affected. Maternal adrenalectomy reversed this inhibition in male offspring, resulting in POMC mRNA levels similar to those measured in male offspring of PF control animals. In contrast, POMC mRNA levels of female offspring of ADX dams decreased in response to FAE. These data suggest that the previously observed switch from suppressed to enhanced POMC expression in FAE males is the result of developmental events beyond weaning. Because this sexually dimorphic regulation of CRF and POMC expression by prenatal alcohol exposure and maternal adrenalectomy occurs before the presence of adult levels of sex steroids, this suggests an organizational effect on the developing hypothalamic-pituitary-adrenal function.     

Differential response of type I and type II corticosteroid receptors to changes in plasma steroid level and circadian rhythmicity

Corticosterone (CORT) binds to two receptor systems in rat brain: the type I CORT-preferring receptor (CR) and the type II glucocorticoid receptor (GR). Discrimination between the two receptor types can be achieved with the 'pure' synthetic glucocorticoid RU 28362. In this study, we show that the binding capacity of GR in the rat hippocampus exhibits a strikingly different response from CR to adrenalectomy (ADX), chronic steroid replacement, hypophysectomy (HYPOX) and during circadian variation. Under those experimental conditions neither receptor site showed changes in binding affinity. After ADX, CR number remained relatively constant for a period of 13 days, while GR capacity increased by 133%, a level which was reached 5 days post-surgery. CR capacity showed circadian variation, since CR number was 65% higher in the evening than in the morning. GR capacities at those two time points were not significantly different. Replacement with subcutaneous CORT implants (100-mg pellets) for 7 days following ADX rats did not affect CR number, but caused a 38% decrease in GR number compared to control animals (cholesterol-treated, 7-day-ADX rats). On the other hand, dexamethasone (DEX) implants (5-, 15-, 25-mg pellets) elicited a dose-dependent increase in CR capacity (up to 99%) and a dose-dependent decrease in GR capacity (40-44%). Finally, 2 weeks after HYPOX, CR and GR numbers were increased by 60 and 38%, respectively. We conclude that the type II GR capacity responds in an autoregulatory manner to changes in circulating plasma glucocorticoid levels, while type I CR does not.     

Effects of prenatal ethanol exposure on basal limbic-hypothalamic-pituitary-adrenal regulation: role of corticosterone

BACKGROUND: Rats prenatally exposed to ethanol (E) exhibit hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness and changes in central HPA regulation following exposure to stressors. Whether ethanol-induced alterations in basal HPA regulation play a role in mediating HPA hyperresponsiveness remains unclear. We utilized adrenalectomy (ADX), with or without corticosterone (CORT) replacement, to investigate basal HPA function and the role of CORT in mediating ethanol-induced alterations. METHODS: Adult males and females from prenatal E, pair-fed (PF), and ad lib-fed control (C) groups were terminated at the circadian peak, 7 days following sham surgery or ADX, with or without CORT replacement. Plasma levels of CORT and adrenocorticotropin (ACTH), and mRNA levels of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) in the paraventricular nucleus, CRH Type 1 receptor (CRH-R1) and pro-opiomelanocortin (POMC) in the anterior pituitary, and mineralocorticoid (MR) and glucocorticoid (GR) receptors in the dorsal hippocampus were determined. RESULTS: Adrenalectomy resulted in significantly greater plasma ACTH elevations in E and PF males, and parallel CRH mRNA elevations in both E and PF males and females compared with their C counterparts. In contrast, pituitary CRH-R1 mRNA levels were lower in E compared with C males, with no differences in POMC. In addition, in response to ADX, E females showed a greater MR mRNA response, and E males showed a greater GR mRNA response compared with their C counterparts, and CORT replacement was ineffective in normalizing ADX-induced alterations in ACTH levels in E and PF females, hippocampal MR mRNA levels in E males, and AVP mRNA levels in PF males and females. CONCLUSIONS: Together, these data indicate that the prenatal ethanol exposure induces HPA dysregulation under basal conditions at multiple levels of the axis, resulting in alterations in both HPA drive and feedback regulation and/or in the balance between drive and feedback. While some effects may be nutritionally mediated, it appears that the mechanisms underlying basal HPA dysregulation may differ between E and PF animals rather than occurring along a continuum of effects on the same pathway. Altered basal HPA tone may play a role in mediating the HPA hyperresponsiveness to stressors observed in E offspring.     

Effects of leptin and corticosterone on the expression of corticotropin-releasing hormone, agouti-related protein, and proopiomelanocortin in the brain of ob/ob mouse

The present study was conducted to assess the effect of leptin and corticosterone on the expression of corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC) and agouti-related protein (AGRP) in the mouse brain. To this end, a 3 x 3 factorial experiment was designed in which adrenalectomized (ADX) ob/ob mice were treated with leptin and corticosterone. Leptin and corticosterone downregulated CRH expression in the paraventricular hypothalamic nucleus (PVH). Leptin prevented the stimulating effects of ADX on the expression of CRH and the combination of small doses of leptin and corticosterone was as potent as the high dose of corticosterone in suppressing CRH mRNA expression in the PVH. Leptin and corticosterone enhanced the expression of CRH in the central nucleus of amygdala and in the bed nucleus of the stria terminalis. In addition, the present results confirmed the downregulating effects of leptin on the expression of AGRP mRNA in the hypothalamic arcuate nucleus (ARC), and demonstrated that this effect was more apparent in ADX mice treated with corticosterone than in ADX mice not supplemented with corticosterone. Also, leptin and corticosterone had opposite effects on the expression of POMC in the ARC. The opposite effect of leptin and corticosterone on the expression of POMC and AGRP seems consistent with the reported effects that these hormones and peptides have on food intake and thermogenesis, suggesting that the modulation of POMC and AGRP expression can be a mechanism whereby leptin and corticosterone exert their effects in the regulation of energy balance. In contrast, the similarity in the action of leptin and corticosterone is not a priori consistent with a role of CRH in the effects of these hormones in the regulation of energy balance. The downregulating effect of leptin on the expression of CRH in the PVH strongly suggests that leptin can be a potent regulator of hypothalamic-pituitary-adrenal axis activity. Finally, the present results suggest that the effects of leptin on the expression of CRH, POMC and AGRP are not curbed by glucocorticoids.     

A revised role for P-glycoprotein in the brain distribution of dexamethasone, cortisol, and corticosterone in wild-type and ABCB1A/B-deficient mice

The ABCB1-type multidrug resistance efflux transporter P-glycoprotein (P-gp) has been hypothesized to regulate hypothalamic-pituitary-adrenal axis activity by limiting the access of glucocorticoids to the brain. In vivo systemic administration studies using P-gp-deficient mice have shown increased glucocorticoid entry to the brain compared with wild-type controls. However, these studies did not control for the presence of radiolabeled drug in the capillaries, verify an intact blood-brain barrier, or confirm stability of the glucocorticoids used. In the present study, an in situ brain perfusion method, coupled with capillary depletion and HPLC analyses, was used to quantify brain uptake of [3H]dexa-methasone, [3H]cortisol, and [3H]corticosterone in P-gp-deficient and control mice. A vascular marker was included in these experiments. The results show that brain uptake of [3H]dexamethasone was increased in the frontal cortex, hippocampus, hypothalamus, and cerebellum of P-gp-deficient mice compared with wild-type controls. Brain uptake of [3H]cortisol was increased in the hypothalamus of P-gp-deficient mice compared with wild-type controls, but no differences were detected in other regions. Brain uptake of [3H]corticosterone was not increased in P-gp-deficient mice compared with wild-type controls in any brain areas. After our systemic administration of the same radiolabeled glucocorticoids, HPLC analysis of plasma samples identified additional radiolabeled components, likely to be metabolites. This could explain previous findings from systemic administration studies, showing an effect of P-gp not only for dexamethasone and cortisol, but also for corticosterone. This in situ study highlights the different affinities of dexamethasone, cortisol, and corticosterone for P-gp, and suggests that the entry of the endogenous glucocorticoids into the mouse brain is not tightly regulated by P-gp. Therefore, our current understanding of the role of P-gp in hypothalamic-pituitary-adrenal regulation in mice requires revision.     

Corticotrope response to removal of releasing factors and corticosteroids in vivo

In the intact rat, adrenalectomy (ADX) is known to result in increased ACTH synthesis, content, and secretion from the anterior pituitary compared with those in the sham-adrenalectomized control. Treatment of adrenalectomized, rats with corticosterone prevents or reverses these changes in ACTH. Because corticosterone is known to act both at the corticotrope and at the level of CRF secretion, it is not clear to what extent the ACTH response to ADX is a result of removal of glucocorticoids from the pituitary per se. To test the role of brain input as well as the role of glucocorticoids on the corticotrope response to ADX, we performed the following experiment. Rats were prepared with anterolateral hypothalamic deafferentations (lesion) which severed CRF and arginine vasopressin cell bodies in the hypothalamus from their axonal endings in the median eminence and posterior pituitary. Control rats were subjected to sham lesions. Two days later, half of the rats in each group were subjected to either ADX or sham ADX; a subgroup of the lesioned rats was provided at the time of adrenal surgery with a constant infusion of rat CRF. Five days later, all rats were killed, and anterior pituitary levels of proopiomelanocortin (POMC) mRNA, ACTH, and protein; plasma ACTH and corticosterone, and adrenal and thymus weights were measured. In sham-lesioned rats, ADX resulted in increases in POMC mRNA, and plasma ACTH of 2.5- and 12-fold, respectively, compared to sham-adrenalectomized controls. In the absence of hypothalamic drive (lesion only), there were no responses of any of these variables to ADX. In lesioned rats driven with CRF, ADX resulted in increases in POMC mRNA and plasma ACTH of 2.2- and 2.6-fold, respectively, compared to sham ADX. After consideration of the three variables indicating ACTH synthesis, storage, and secretion and comparison of the results of ADX vs. sham ADX within and across the sets of animals, we conclude that 1) there is no autonomous response of the corticotrope to ADX; 2) the removal of corticosterone from the anterior pituitary may account for the majority of the effects of ADX on ACTH synthesis; and 3) the normal response to ADX requires secretion of CRF and increased secretion of another ACTH-releasing factor (possibly arginine vasopressin) that causes increased secretion but little synthesis of ACTH.     

Changes in the hypothalamo-corticotrope axis after bilateral adrenalectomy: evidence for a median eminence site of glucocorticoid action.

Bilateral adrenalectomy (ADX) leads to increased ACTH synthesis and secretion. It is thought that endogenous glucocorticoids exert a feedback mechanism at both pituitary and brain levels. The present study has been performed in order to determine the effect of ADX on the release of hypothalamic neuropeptides with corticotropin-releasing activity (CRA) and if there exists a median eminence site of glucocorticoid action to regulate hypothalamic-pituitary-adrenal (HPA) function. Adrenalectomized and sham-operated male rats were killed at different periods after surgery (2, 5, 7 and 14 days) and trunk blood was collected for ACTH and corticosterone (B) concentrations measurement. Brain (median eminence, ME; and medial basal hypothalamus, MBH) and pituitary (anterior lobe, AP; and neurointermediate lobe, NIL) tissues were dissected in order to evaluate either peptide content or in vitro hormone release. The results indicate that ADX blunted plasma B levels and increased AP ACTH content and secretion in a time-related fashion up to the 14th day. ADX significantly decreased both CRF and CRA contents in the ME at all periods studied; ME arginine-vasopressin (AVP) increased 7 and 14 days after ADX. MBH CRF decreased after ADX, but returned to sham value 2 weeks later; similarly, MBH AVP decreased at all periods after ADX. Removal of endogenous glucocorticoids did not vary neither oxytocin (OXY) content in the ME and MBH nor AVP and OXY contents in the NIL. In our superfusion experiments, we found that ADX increased basal AVP release and did not change spontaneous CRF secretion from ME terminals. Dexamethasone (Dxm, 10 nM) diminished AVP but not CRF output by ME tissues from adrenalectomized rats. A direct relationship was found between ME CRF and 28 mM KCl (hK+)-induced CRF release by MEs from adrenalectomized rats. ME fragments from adrenalectomized rats were hyperresponsive to kH+ stimulation of AVP release. Dxm (10 nM) decreased the hK(+)-evoked CRF and AVP release by MEs from adrenalectomized rats. ADX and dexamethasone treatment did not influence basal and hK(+)-elicited ME OXY release. Additionally, a rapid glucocorticoid inhibitory effect on ACTH secretion by isolated AP cells from both sham and adrenalectomized rats was found, and an in vitro corticotrope hyporesponse to 0.63 nM CRF and 9.25 nM AVP stimulation during several days after ADX.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of pro-opiomelanocortin gene expression by glucocorticoids in the denervated rat intermediate pituitary gland

The effect of frontal deafferentation of the medial basal hypothalamus on pro-opiomelanocortin (POMC) gene expression was studied in the intermediate lobe (IL) and anterior lobe (AL) of the pituitary gland in the absence and presence of corticosterone (CORT). The lesion in the basal hypothalamus removed neural inputs to the IL and induced the glucocorticoid receptor (GR) in this tissue. The GR was visualized in the denervated IL by immunocytochemistry. Induction of the GR had a slow onset and was detectable at 3 weeks after lesion, but not at one week after the lesion. In order to study the effect of IL denervation on pro-opiomelanocortin (POMC) gene expression, the level of messenger RNA specifically encoding POMC was measured 1 and 3 weeks after lesion, in the IL and AL. In adrenalectomized (ADX) animals, the changes in POMC mRNA levels were not significant 1 week after lesion in the IL. Three weeks after denervation there was a 3-fold decrease in POMC mRNA in the IL in ADX rats which was blocked by chronic CORT replacement via subcutaneously implanted pellets. In the AL, CORT reduced the level of POMC gene expression in both the lesioned and control animals. It is concluded that (1) removal of neural input induces GR in the denervated IL cells; (2) with the appearance of the GR, POMC gene expression in the IL becomes sensitive to circulating glucocorticoids; (3) under these conditions, CORT may stimulate POMC gene expression in the IL as opposed to its inhibitory effect in the AL.     

Analysis of the stress response in rats trained in the water-maze: differential expression of corticotropin-releasing hormone, CRH-R1, glucocorticoid receptors and brain-derived neurotrophic factor in limbic regions

Glucocorticoids and corticotropin-releasing hormone (CRH) are key regulators of stress responses. Different types of stress activate the CRH system; in hypothalamus, CRH expression and release are increased by physical or psychological stressors while in amygdala, preferentially by psychological stress. Learning and memory processes are modulated by glucocorticoids and stress at different levels. To characterize the kind of stress provoked by a hippocampal-dependent task such as spatial learning, we compared the expression profile of glucocorticoid receptor (GR), pro-CRH and CRH-R1 mRNAs (analyzed by RT-PCR), in amygdala, hippocampus and hypothalamus and quantified serum corticosterone levels by radioimmunoassay at different stages of training. mRNA levels of brain-derived neurotrophic factor (BDNF) were also quantified due to its prominent role in learning and memory processes. Male Wistar rats trained for 1, 3 or 5 days in the Morris water-maze (10 trials/day) were sacrificed 5-60 min the after last trial. A strong stress response occurred at day one in both yoked and trained animals (increased corticosterone and hypothalamic pro-CRH and CRH-R1 mRNA levels); changes gradually diminished as the test progressed. In amygdala, pro-CRH mRNA levels decreased while those of BDNF augmented when stress was highest, in yoked and trained animals. Hippocampi, of both yoked and trained groups, had decreased levels of GR mRNA on days 1 and 3, normalizing by day 5, while those of pro-CRH and CRH-R1 increased after the 3rd day. Increased gene expression, specifically due to spatial learning, occurred only for hippocampal BDNF since day 3. These results show that the Morris water-maze paradigm induces a strong stress response that is gradually attenuated. Inhibition of CRH expression in amygdala suggests that the stress inflicted is of physical but not of psychological nature and could lead to reduced fear or anxiety.     

Dexamethasone-induced suppression of vasopressin gene expression in the bed nucleus of the stria terminalis and medial amygdala is mediated by changes in testosterone

Vasopressin (VP) neurons in the bed nucleus of the stria terminalis (BNST) and medial amygdala (AME) are sensitive to changes in circulating levels of testosterone (T). To determine whether these cells are responsive to changes in glucocorticoid levels, in situ hybridization and quantitative autoradiography were used to measure VP mRNA in cells of the BNST and AME in rats that were adrenalectomized (ADX; 14 days) or ADX with dexamethasone (DEX) replacement. These treatments produced the predicted changes in VP gene expression in the medial parvocellular group of the paraventricular nucleus. The VP mRNA content within cells of the BNST or AME was unaffected by adrenalectomy. Treatment with DEX significantly decreased both the number and labeling intensity of VP cells in the BNST and AME. Measurement of plasma T in these animals showed that DEX treatment significantly lowered mean T levels compared with those in either sham-operated or ADX animals. Adrenalectomy alone did not significantly alter T levels. To determine whether DEX influenced VP gene expression via a glucocorticoid action or secondarily by a suppression of T, the above experiment was repeated with groups that were castrated and implanted with Silastic capsules containing T to maintain physiological levels of T. Administration of DEX again decreased both VP cell number and labeling intensity of cells in the BNST and AME in sham-implanted animals. However, VP gene expression was unaffected in those animals that received T capsules. Administration of corticosterone did not alter T levels or the number of cells in the BNST or AME. These results suggest that, in contrast to paraventricular nucleus neurons, adrenalectomy (14 days) is not a potent stimulus in altering VP activity in the BNST or AME. The DEX-induced decrease in VP gene expression is mediated by a secondary suppression of T levels. These results support the finding that gonadal steroids are essential in maintaining the biosynthetic integrity of VP neurons in the BNST and AME.     

Effects of adrenalectomy and glucocorticoids on rat brain dopamine receptors.

The effects of adrenalectomy (ADX) and dexamethasone (DEX) treatment on brain dopamine (DA) receptors of ovariectomized (OVX) rats were investigated by autoradiography using binding of the D1 and D2 antagonists ligands [3H]SCH 23390 and [3H]spiperone, respectively. Fourteen days after ADX, D1 receptors decreased in the middle striatum (M.CPu) and in the dorsal area of the posterior striatum (P.CPu). A more pronounced decrease was observed in the substantia nigra (SN) and no significant changes occurred in the anterior striatum (A.CPu), globus pallidus (GP) and accumbens (Acb). D2 receptors decreased in the M.CPu and dorsal area of the P.CPu and remained unchanged in the A.CPu as compared to OVX rats. Twenty-eight days after ADX, D1 and D2 receptors decreased in the A.CPu (D2), in the M.CPu (D2) and substantially in the SN (D1). DEX treatment (14 days, 0.5 mg/kg, b.i.d., IM, starting 14 days after ADX) reversed these effects in the A.CPu (D2), M.CPu (D2) and SN (D1) when compared to ADX+OVX rats. DEX also increased the density of D1 receptors in Acb, A.CPu and M.CPu when compared to OVX rats. Striatal homogenates of rats treated chronically with ACTH and corticosterone had an increased density of D1 receptors while these treatments alone left these receptors unchanged, thus suggesting either a minor role or no role of the changes in ACTH levels following glucocorticoids manipulations. Our results suggest that the adrenals play a role in the modulation of DA receptors activity in the rat brain.