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Mood disorders are associated with dysregulation of prefrontal cortex (PFC) function, circadian rhythms, and diurnal glucocorticoid (corticosterone [CORT]) circulation. Entrainment of clock gene expression in some peripheral tissues depends on CORT. In this study, we characterized over the course of the day the mRNA expression pattern of the core clock genes Per1, Per2, and Bmal1 in the male rat PFC and suprachiasmatic nucleus (SCN) under different diurnal CORT conditions. In experiment 1, rats were left adrenal-intact (sham) or were adrenalectomized (ADX) followed by 10 daily antiphasic (opposite time of day of the endogenous CORT peak) ip injections of either vehicle or 2.5 mg/kg CORT. In experiment 2, all rats received ADX surgery followed by 13 daily injections of vehicle or CORT either antiphasic or in-phase with the endogenous CORT peak. In sham rats clock gene mRNA levels displayed a diurnal pattern of expression in the PFC and the SCN, but the phase differed between the 2 structures. ADX substantially altered clock gene expression patterns in the PFC. This alteration was normalized by in-phase CORT treatment, whereas antiphasic CORT treatment appears to have eliminated a diurnal pattern (Per1 and Bmal1) or dampened/inverted its phase (Per2). There was very little effect of CORT condition on clock gene expression in the SCN. These experiments suggest that an important component of glucocorticoid circadian physiology entails CORT regulation of the molecular clock in the PFC. Consequently, they also point to a possible mechanism that contributes to PFC disrupted function in disorders associated with abnormal CORT circulation.

The hypothalamus-pituitary-adrenal (HPA) axis diurnal cycle of activity is manifest in circadian rhythms of ACTH and corticosterone secretion, which in the rat peak around the onset of the dark period. This cycle is thought to be driven by daily fluctuations in activity of CRH neurons within the paraventricular nucleus of the hypothalamus (PVN), controlled by suprachiasmatic nucleus inputs. In this study we examined whether the circadian drive that regulates ACTH and corticosterone basal secretion in the rat is reflected in PVN immediate early gene expression and, if so, whether different genes respond uniformly or uniquely to circadian stimulatory input. In addition, we examined how circadian drive and acute stress, two categories of stimuli that induce HPA axis activation, comparatively affect gene expression within different components of the HPA axis (c-fos mRNA, CRH heteronuclear RNA, and zif268 mRNA in PVN; c-fos mRNA, proopiomelanocortin heteronuclear RNA, and zinc finger 268 mRNA in anterior pituitary; c-fos mRNA and nerve growth factor I-B mRNA in adrenal cortex). Finally, we examined whether circadian differences in gene expression depend on endogenous glucocorticoids and, if so, whether the dependence is on an acute or permissive influence of the hormone. We found that a circadian drive that regulates HPA axis basal hormone secretion is also manifest on basal c-fos gene expression in the PVN. Moreover, we show that different immediate early genes within the HPA axis anatomical components display different diurnal patterns of gene expression. These differential patterns result, in part, from gene-specific responses to circadian signals and acute and/or permissive glucocorticoid actions.

Hippocampal glucocorticoid receptors (GRs) are believed to play a major role in age-related cognitive decline and cellular vulnerability. It has been proposed that these receptors mediate damaging effects of elevated glucocorticoid secretion on cellular function. In the present report we present evidence that intracellular trafficking of the GR is impaired with hippocampal aging, manifest as decreased nuclear translocation and deficient DNA binding. We also present evidence that chaperone proteins responsible for GR nuclear translocation are decreased with hippocampal aging. Age-related nuclear GR decreases are not observed in hypothalamus, indicating regional specificity of trafficking deficits. Aging did not affect nuclear or cytosolic MR levels. These data suggest that GR signaling is diminished, rather than enhanced, during hippocampal aging. Diminished GR signaling capacity may attenuate the beneficial effects of glucocorticoids on hippocampal regulation of memory and stress integration.

Neuronal mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) proteins are glucocorticoid-activated transcription factors that bind identical DNA response elements yet transduce distinct physiological/transcriptional actions. The present study assessed regulation of adrenocorticosteroid receptor RNA and protein following intermittent stress exposure, using Sprague Dawley (S-D) and stress-hyperresponsive Fischer 344 (F344) rat strains. The F344 (but not S-D) strain showed enhanced acute stress responsivity and enhanced corticosterone secretion following prolonged stress. F344 rats also showed reduced responsiveness to a novel stressor after prolonged stress exposure, suggestive of enhanced glucocorticoid negative feed-back. Upon prolonged stress, F344 rats down-regulated MR hnRNA in CA1, CA3, and dentate gyrus. Transcriptional changes were accompanied by decreased expression of the alpha 5' messenger RNA (mRNA) form, consistent with altered promoter utilization. In contrast, alpha 5' splice variant, full-length mRNA, and MR protein expression were not affected by stress in either strain, implying that transcriptional changes do not affect overall mRNA or protein expression. GR protein was increased in pyramidal and granule cell somata/nuclei of F344 rats despite lack of a change in mRNA expression. These data suggest that prolonged stress elicits restricted changes in MR and GR expression in the F344 strain only. Overall, stable expression of adrenocorticosteroid receptors is rigorously defended in hippocampal neurons, apparently through transcriptional and posttranscriptional mechanisms.

The nonpeptide CRH antagonist antalarmin has been shown to block both behavioral and endocrine responses to CRH. However, it's potential activity in blunting behavioral and endocrine sequelae of stressor exposure has not been assessed. Because antagonism of central CRH by alpha-helical CRH attenuates conditioned fear responses, we sought to test antalarmin in this regard. In addition, it remains unclear as to whether this is a result of receptor blockade during conditioning or during testing. Thus, we explored whether CRH mediates the induction or expression of conditioned fear (freezing in a context previously associated with 2 footshocks; 1.0 mA, 5 sec each). Furthermore, because rats previously exposed to inescapable shock (IS; 100 shocks, 1.6 mA, 5 sec each), demonstrate enhanced fear conditioning, we investigated whether this effect would be blocked by antalarmin. Antalarmin (20 mg/kg x 2 ml i.p.) impaired both the induction and expression of conditioned fear. In addition, antalarmin blocked the enhancement of fear conditioning produced by prior exposure to IS. Despite the marked behavioral effects observed in antalarmin-treated rats, antalarmin had no effect on IS-induced rises in ACTH or corticosterone. However, antalarmin did block the ACTH response produced by exposure to 2 footshocks.

Adrenal glucocorticoids play an important role in mediating many of the behavioral and physiological effects of exposure to stressors. Focus has been primarily on the acute stress-induced rise in glucocorticoids [corticosterone (CORT) in the rat]. There are reports, however, that exposure to chronic stressors can produce an increase in basal CORT and a decrease in corticotropin-binding globulin (CBG). These changes occur subsequent to the stress-induced rise in CORT. The following experiments examined whether exposure to an acute stressor (100 5-set inescapable tail shocks; IS) could also produce long term changes in basal CORT and CBG. We report that a single session of IS results in an increase in basal total serum CORT that persists 48-96 h after IS termination. The increase is present only at the diurnal trough (morning). CBG levels are also decreased for 24-48 h. The decrease is present at both the diurnal peak (evening) as well as the trough (morning). These changes result in an increase in the percent and amount of biologically active CORT (unbound or free). Thus, glucocorticoid-sensitive targets are exposed to high levels of free CORT for several days after IS termination. The long term increase in free CORT reported here may play an important role in mediating some of the effects produced by IS as well as those produced by other acute stressors. (Endocrinology 136: 5336-5342, 1995)

CRH is the principal hypothalamic neurohormone responsible for hypothalamic-pituitary adrenal (HPA) axis activity (1). CRH also serves as an important transsynaptic signal in various neurocircuits in the brain that regulate behavioral and emotional responses to stress (2). Thus, CRH appears to be a key molecule in the coordination of behavioral and neuroendocrine responses to stressful events. Dysregulation of CRH neuronal function is associated with stress-related psychiatric disorders, and abnormal CRH neuropeptide production levels appear to underlie some of this neuronal dysregulation (3). Consequently, there is much interest in determining the regulation of CRH gene expression, with a particular eye on identifying new regulatory proteins that may point to candidate genes for disease susceptibility (4). Studies find that there is high basal CRH gene expression within the medial parvocellular portion of the hypothalamic paraventricular nucleus. However, CRH gene expression is also rapidly increased by various excitatory stimuli that lead to HPA axis activation (5, 6). The coupling of neuronal excitation with CRH gene induction appears to be largely driven by protein kinase A (PKA)-mediated activation of the transcription factor cAMP response element binding protein (CREB) (7, 8). But some recent findings have thrown a wrench in this straightforward understanding of CRH gene expression. Liu et al. (9) noted that, in a hypothalamic-derived neuronal cell line (4B cells) and hypothalamic primary neuronal cultures, treatment with either the phorbol ester phorbol-12-myristate-13-acetate (PMA; protein kinase C activator) or forskolin (adenylate cyclase activator) led to CREB activation (phosphorylation of CREB ser133). Paradoxically, however, only forskolin treatment was sufficient to stimulate CRH gene promoter activity and CRH gene expression, even though this stimulatory activity required CREB activation (9). A related phenomenon has been observed in vivo: microinfusion of forskolin but not phorbol ester in the rat paraventricular nucleus led to increased CRH gene expression, even though both drugs led to increased HPA axis hormone secretion (10). These findings prompted Liu et al. (9) to consider the possible role of a CREB coregulatory factor in the regulation of CRH gene expression. Such a factor would have to meet three conditions. It would have to be rapidly activated by stimulatory intercellular signals, activated by forskolin but not PMA, and dependent on CREB for CRH gene transactivation capability. In the current issue of Endocrinology, Liu et al. (11) explored the possibility that the recently discovered transducer of regulated CREB activity (TORC), which is also known as CREB-regulated transcription coactivator, could be such a factor. Two groups independently discovered this evolutionarily conserved family of CREB coactivators (TORC1, TORC2, and TORC3) by use of high-throughput screening strategies for proteins that potentiated CREB-dependent gene expression (12, 13). Subsequent studies in various peripheral tissue cell lines have found that TORC has a number of interesting biochemical features. These features not only account for TORC’s ability to interact with CREB protein and thereby permit CREB-dependent gene expression, but they also provide for diverse ways in which TORC can be activated or deactivated. TORC binds to the dimerization and DNA binding domain of CREB. Although this interaction does not appear to alter CREB’s DNA binding capability, it enhances CREB’s association with TAFII130, a component of the general transcription

Corticosteroid hormones regulate many aspects of neural function via mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Although GR expression is negatively regulated by endogenous corticosteroids, the autologous regulation of MR expression has been less well studied, partly due to limitations of receptor binding assays that cannot measure the ligand-activated form of MR. Using MR-reactive antibodies and Western blot, we examined relative MR protein expression in rat brain and its potential autoregulation by corticosteroids. We found that MR protein expression is autoregulated in a negative fashion by adrenal steroids. Compared with GR, we see a more rapid regulation of MR, such that there is a substantial increase in MR protein within 12 h after adrenalectomy, whereas GR levels show very little increase until more than 24 h after adrenalectomy. Also, in contrast to GR, which has been found to be regulated by both MR and GR, adrenalectomy-induced increase in MR was prevented by treatment with the MR selective agonist, aldosterone, but not the GR selective agonist, RU28362. Interestingly, acute treatment of adrenalectomized rats with corticosterone produced a significant decrease in whole-cell MR protein within 45 min, suggesting ligand-induced rapid degradation of MR. Chronic high levels of corticosterone also produced a significant decrease in MR protein levels below adrenal-intact rat levels. These results have important implications for previous studies that estimated the proportion of MR that are occupied in vivo by various circulating levels of corticosterone. Those studies compared available MR binding levels in adrenal-intact rats with 24-h adrenalectomized rats, with the assumption that there were no differences between the various conditions in total receptor expression. Those studies concluded that MR is nearly fully occupied by even the lowest circulating corticosterone levels. Given the 2- to 3-fold increase in MR protein that we have observed within 24 h after adrenalectomy, it is likely that those studies significantly overestimated the proportion of MR that were occupied by low basal corticosterone levels. These results support the prospect that MR as well as GR can participate in the transduction of phasic corticosteroid signals.

These studies further evaluated the relative role of mineralocorticoid (type I) and glucocorticoid (type II) receptors in mediating corticosteroid feedback regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Acute treatment of rats with the selective mineralocorticoid receptor antagonist, RU28318 (50 mg/kg sc), produced elevated basal corticosterone levels in the morning, but had no effect on basal corticosterone levels in the evening or on restraint stress corticosterone levels at either time of day. Acute treatment with the selective glucocorticoid receptor antagonist, RU40555 (30 mg/kg sc) had no effect on basal or restraint stress corticosterone levels at either time of day. However, combined treatment with RU28318 and RU40555 produced an elevation of evening basal corticosterone levels (and morning basal on one occasion) and produced an increase in corticosterone levels during and after stress at both times of day. In a separate experiment conducted in the morning, the combined RU28318 and RU40555 treatment also produced elevated ACTH responses during restraint stress. Based on available corticosteroid receptor measures, the RU28318 treatment was estimated to selectively occupy approximately 85% of mineralocorticoid receptors in rat brain, whereas the RU40555 treatment was estimated to selectively occupy approximately 50% of glucocorticoid receptors in rat brain. We conclude that mineralocorticoid receptor activation is necessary and sufficient to maintain low basal corticosterone levels during the circadian trough, whereas glucocorticoid receptor activation is necessary to constrain corticosterone secretion during the circadian peak or during acute stress. However, even during the circadian peak or acute stress, mineralocorticoid receptor activation plays an important role in facilitating the glucocorticoid receptor dependent regulation of HPA axis activity by corticosterone.

Adrenal steroids exert their effects through two distinct adrenal steroid receptor subtypes; the high affinity type I, or mineralocorticoid, receptor and the lower affinity type II, or glucocorticoid, receptor. Adrenal steroids have well known effects on immune cell distribution, and although both type I and II receptors are expressed in immune cells and tissues, few data exist on the relative effects mediated through these two receptor subtypes. Accordingly, we administered selective type I and II adrenal steroid receptor agonists to young adult male Sprague-Dawley rats for 7 days and then measured immune cell distribution in the peripheral blood and spleen. Results were compared with those of similar studies using the naturally occurring glucocorticoid of the rat, corticosterone, which binds both type I and II receptors. The majority of the well characterized effects of adrenal steroids on peripheral blood immune cells (increased neutrophils and decreased lymphocytes and monocytes) were reproduced by the type II receptor agonist, RU28362. RU28362 decreased the numbers of all lymphocyte subsets [T-cells, B-cells, and natural killer (NK) cells] to very low absolute levels. The largest relative decrease (i.e. in percentage) was seen in B-cells, whereas NK cells exhibited the least relative decrease and actually showed a 2-fold increase in relative percentage during RU28362 treatment. Similar to RU28362, the type I receptor agonist, aldosterone, significantly reduced the number of lymphocytes and monocytes. In contrast to RU28362, however, aldosterone significantly decreased the number of neutrophils. Moreover, aldosterone decreased the number of T-helper cells and NK cells, while having no effect on the number of B-cells or T-suppressor/cytotoxic cells. Corticosterone at physiologically relevant concentrations had potent effects on immune cell distribution, which were indistinguishable from those of the type II receptor agonist, RU28362. Taken together, these results indicate that effects of adrenal steroids on immune cell distribution are dependent on the receptor subtype involved as well as the specific cell type targeted. These factors allow for varied and complex effects of adrenal steroids on the immune system under physiological conditions.

To examine diurnal differences in the proportions of receptors that were occupied and activated by basal and stress levels of corticosterone, we measured available type I (mineralocorticoid) and type II (glucocorticoid) adrenal steroid receptor levels in brain, pituitary, and immune tissues of unstressed and acutely stressed rats at the times of day when basal corticosterone secretion was at its trough [morning (AM)] and peak [evening (PM)]. In general, the estimated adrenal steroid receptor activation was greater in brain than in pituitary or immune tissue, and within a particular tissue, there was a greater degree of estimated activation of the adrenal steroid high affinity type I receptor than of the type II receptor. There was a greater activation of brain type II receptors by basal corticosterone in the PM (30-35%) than the AM (5-15%). As acute stress produced similar levels of receptor activation at both times of day (45-50%), the net change in type II receptor activation in the brain after acute stress was much smaller in the PM than in the AM. This suggests that there may be diurnal differences in the role of type II receptors in corticosterone negative feedback on the hypothalamic-pituitary-adrenal axis. In immune tissues, type II receptor activation by acute stress was especially heterogeneous, depending on both the immune compartment and the time of day, suggesting that these are important factors contributing to a differential impact of corticosterone on immune responses during acute stress. Taken together our results suggest that the tonic and phasic influences of corticosterone on target tissue responses very not only with the diurnal and stress secretion patterns of corticosterone, but also with target tissue factors, such as type I and type II receptor expression and hormone bioavailability. All of these factors contribute to considerable selectivity of action for the systemic hormone corticosterone.

We examined the influence of the type I adrenal steroid receptor agonist, aldosterone, on type II adrenal steroid receptor binding in the rat spleen and thymus after adrenalectomy. In the spleen, adrenalectomy was associated with a significant up-regulation of type II receptors, which was blocked by the concurrent administration of aldosterone (1 microgram/h) via sc osmotic minipumps. Neither adrenalectomy nor aldosterone treatment altered type II receptor binding in the thymus. Despite high doses of aldosterone (10 micrograms/h), which resulted in supra-physiological blood concentrations of this hormone, there was no evidence of type II receptor decreases in spleen or thymus below receptor levels found in sham-adrenalectomized rats. The effect of aldosterone on type II receptor binding appeared to be mediated by the type I receptor, since there was no aldosterone effect on the thymus, which did not exhibit detectable levels of type I receptor binding. Moreover, there was no evidence that aldosterone competed for the type II receptor in vivo or in vitro as determined by measurements of the type II receptor dissociation constant in the spleen of adrenalectomized, aldosterone-treated animals. Since selective activation of the type I receptor occurs in the spleen under physiological conditions, these results indicate that type I receptors may play a tonic inhibitory role in type II receptor expression in immune cells which express both receptor subtypes and reside in this tissue. Furthermore, the findings suggest that there may be different mechanisms involved in the up-regulation vs. the down-regulation of type II adrenal steroid receptors, and effects mediated solely via the type I adrenal steroid receptor appear only to influence the former process.