Brian A. Kalman

Dexamethasone suppression of corticosteroid secretion: evaluation of the site of action by receptor measures and functional studies

A dose of dexamethasone was determined in rats (50 micrograms/kg s.c.) that suppressed the corticosterone response to restraint stress by 80%. Corticosteroid receptor occupancy estimates found that the 50 micrograms/kg s.c. dose of dexamethasone had no significant effect on available glucocorticoid receptor (GR) or mineralocorticoid receptor (MR) binding in brain regions (hypothalamus, hippocampus and cortex); on the other hand dexamethasone produced a selective and significant decrease in available GR in peripheral tissues (pituitary and spleen). Functional studies showed that the 50 micrograms/kg s.c. dose of dexamethasone completely blocked the effects of corticotropin-releasing hormone (CRH; 0.3-3.0 micrograms/kg i.p.) on corticosterone secretion, but did not inhibit the corticosterone response to an adrenocorticotropin hormone (ACTH; 2.5 I.U./kg i.p.) challenge. These studies indicate that this dose of dexamethasone exerts its inhibitory effects on the HPA axis primarily by acting at GR in the pituitary. The plasma dexamethasone levels produced by this dose of dexamethasone are similar to those present in humans the afternoon after an oral dexamethasone suppression test (DST), a time at which many depressed patients escape from dexamethasone suppression. These results support and extend other studies which suggest that the DST provides a direct test of the effects of increased GR activation in the pituitary on ACTH and cortisol secretion.

The role of the amygdala and dorsal raphe nucleus in mediating the behavioral consequences of inescapable shock

It has been argued that exposure to inescapable shock produces later behavioral changes such as poor shuttle box escape learning because it leads to the conditioning of intense fear, which later transfers to the shuttle box test situation and interferes with escape. Both fear, as assessed by freezing, and escape were measured in Sprague-Dawley rats 24 hr after exposure to inescapable shock. Lesions of the basolateral region and central nucleus of the amygdala eliminated the fear that transfers to the shuttle box after inescapable shock, as well as the fear conditioned in the shuttle box by the shuttle box shocks. However, the amygdala lesions did not reduce the escape learning deficit produced by inescapable shock. In contrast, dorsal raphe nucleus lesions did not reduce the fear that transfers to the shuttle box after inescapable shock, but eliminated the enhanced fear conditioning in the shuttle box as well as the escape deficit. The implications of these results for the role of fear and anxiety in mediating inescapable shock effects are discussed.

Selective Blockade of the Mineralocorticoid Receptor Impairs Hypothalamic‐Pituitary‐Adrenal Axis Expression of Habituation

The present study investigated the role of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) in the expression of habituation of the hypothalamic‐pituitary‐adrenal (HPA) axis response to stress. Male rats were restrained for 1 h per day for six consecutive days. On day 6, 1 h prior to restraint stress, both restraint‐naive and repeatedly restrained rats were injected s.c. with either vehicle (propylene glycol) or one of three corticosteroid receptor antagonist treatments: selective MR antagonist (RU28318 or spironolactone), selective GR antagonist (RU40555), or both MR and GR antagonists combined (RU28318 + RU40555). Blood samples were collected for corticosterone measurement at the beginning of stress, during stress, and 1 h after stress termination. Repeated restraint stress produced significant habituation of corticosterone responses. Acute treatment with the combined MR and GR antagonists prevented the expression of habituation. When tested alone, the MR antagonist also blocked the expression of corticosterone‐response habituation, whereas the GR antagonist had no effect. Neither the MR, nor the GR antagonists alone, significantly altered the corticosterone response to restraint in rats exposed to restraint for the first time. The final experiment examined the corticosterone response to a corticotropin releasing hormone (CRH, 3 µg/kg i.p.) challenge. Neither previous exposure to restraint or acute pretreatment with the combined MR and GR antagonists (RU28318 + RU40555) altered the corticosterone response to CRH challenge. This result indicates that the expression of habituation and its blockade by corticosteroid receptor antagonists is not a result of altered pituitary‐adrenal response to CRH. Overall, this study suggests that MR plays an important role in constraining the HPA axis response to restraint stress in restraint‐habituated rats. The dependence of the HPA axis on MR‐mediated corticosteroid negative feedback during acute stress may be an important mechanism that helps maximize the expression of stress habituation and thereby minimize exposure of target tissues to corticosteroids in the context of repeated stress.

Discrimination between changes in glucocorticoid receptor expression and activation in rat brain using western blot analysis.

These studies investigated autoregulation of glucocorticoid receptor (GR) protein expression and activation in rat brain using western blot methodology. By comparing GR immunoblot reactivity present in various tissue subcellular preparations we were able to discriminate between corticosterone-induced changes in GR activation or GR protein expression. Our cytosolic tissue preparation yielded a similar pattern of treatment effects on relative GR as measured by receptor binding assay or western blot. In both cases, short-term adrenalectomy (18 h) produced no change in cytosolic GR. On the other hand, long-term adrenalectomy (3-14 days) resulted in a large increase in cytosolic GR, whereas acute (1-2 h) treatment with high dose corticosterone produced a large decrease in cytosolic GR. Western blot measurement of GR levels in a nuclear extract or whole-cell extract from the same brains indicated that acute corticosterone treatment produced a large increase in nuclear GR and no change in whole-cell GR. Thus, all of the decrease in cytosolic GR observed after acute corticosterone treatment could be accounted for by receptor redistribution to the nuclear tissue fraction as opposed to rapid receptor protein downregulation. Long-term treatment of rats with adrenalectomy or high dose corticosterone produced a large increase and decrease, respectively, in whole-cell GR, indicating genuine changes in receptor protein expression. These studies indicate that in vivo regulation of GR protein expression in the rat brain can be studied using western blot analysis of a whole-cell tissue preparation. This procedure has an important advantage over receptor binding studies in that GR protein expression can be measured in adrenal-intact rats. These studies also support the validity of using cytosolic receptor binding assays to estimate relative changes in GR occupation/activation when appropriate comparison groups are included.

Chlordiazepoxide microinjected into the region of the dorsal raphe nucleus eliminates the interference with escape responding produced by inescapable shock whether administered before inescapable shock or escape testing.

Systemic administration of benzodiazepines before exposure to inescapable shock (IS) blocks the enhanced fear conditioning and escape learning deficits that follow exposure to IS, whereas administration before the subsequent behavioral testing eliminates the enhanced fear but not the interference with escape. The failure of benzodiazepines to reduce the IS-produced escape learning deficit when given before testing is inconsistent with a recent proposal that interference with escape is mediated by an IS-induced sensitization of dorsal raphe nucleus (DRN) activity. The present experiments demonstrate that chlordiazepoxide will block both the enhancement of fear and interference with escape responding when given before either IS or testing if microinjected in the region of the DRN. This suggests that systemic benzodiazepines fail to block escape deficits when given before testing because action at a site distant from the DRN counters the effect of the drug at the DRN.

Evaluation of RU28318 and RU40555 as Selective Mineralocorticoid Receptor and Glucocorticoid Receptor Antagonists, Respectively: Receptor Measures and Functional Studies

Corticosterone regulates a wide range of physiological parameters. Two receptors for corticosterone have been identified, the mineralocorticoid (type I) receptor (MR) and the glucocorticoid (type II) receptor (GR). To determine the relative role of these two receptors in mediating the effects of endogenous corticosterone, many studies have relied on the use of putative selective corticosteroid receptor antagonists. This study further examined the in vivo receptor selectivity of two compounds, RU28318 and RU40555 that are believed to be selective antagonists for MR and GR, respectively. Acute treatment of adrenalectomized rats with RU28318 (10-50 mg/kg) selectively decreased ex-vivo available MR binding in the hippocampus, whereas acute treatment with RU40555 (10-30 mg/kg) selectively decreased available GR binding in the hippocampus and pituitary. These receptor binding measures suggest that RU28318 in vivo selectively occupied MR, and that RU40555 in vivo selectively occupied GR. In functional studies, RU28318 (50 mg/kg) blocked the normalizing effect of aldosterone (120 microg/kg) on saline intake of adrenalectomized rats. RU40555 (30 mg/kg) blocked the suppressive effect of dexamethasone (50 microg/kg) on acute stress-induced corticosterone secretion. These studies further support the in vivo corticosteroid receptor selectivity of these two compounds and confirms their effective corticosteroid antagonistic properties.

Diazepam attenuation of restraint stress-induced corticosterone levels is enhanced by prior exposure to repeated restraint

Prior research has demonstrated that diazepam decreases hypothalamic-pituitary-adrenal cortex (HPA) axis activity in stressful contexts but, paradoxically, acts as a stimulator of basal axis activity. Also, several investigators have reported that low doses of diazepam are not effective in reducing stress-induced corticosterone (CORT) levels, yet similar doses typically produce anxiolytic effects on behavioral measures of fear and anxiety. We have examined the effects of diazepam on plasma CORT levels in male Sprague-Dawley rats utilizing a repeated restraint paradigm. Consistent with most literature, diazepam administered IP (1.5, 3.0, or 6.0 mg/kg) 1 h prior to restraint increased non-stress, baseline plasma CORT levels in a dose-dependent fashion. During the first exposure to the 1 h restraint-stress procedure, CORT levels of diazepam-injected rats did not differ from the stress levels of controls except at the 60-min stress time point in those subjects receiving 6.0 mg/kg. However, diazepam at all three doses was able to attenuate the stress-induced increase in CORT following 5 days of diazepam+restraint treatment. Using the 3.0 mg/kg dose as a probe, it was found that this effect was not dependent on the repeated administration of diazepam, but rather on repeated exposure to restraint. These results suggest that repeated restraint produces a change in neural sensitivity to benzodiazepines.

Acute exposure to a novel stressor further reduces the habituated corticosterone response to restraint in rats.

The present study sought to identify dishabituation of the hypothalamic-pituitary-adrenal (HPA) axis response to different psychological stressors. Young adult male Sprague Dawley rats were exposed to five, 1 h sessions of restraint stress on five consecutive days. On the sixth day, and 2 h before additional exposure to restraint, animals were subjected to 30 min of a small (27 cm square), elevated open field stressor (pedestal), which served as the dishabituating stimulus. We predicted HPA axis response dishabituation in chronically restrained rats exposed to the novel pedestal. Rats which underwent five days of restraint stress showed significantly blunted plasma corticosterone levels to restraint (habituation) as compared to restraint-naive rats. However, rats which underwent five sessions of restraint responded with an enhanced habituation response when confronted with restraint shortly after exposure to the novel pedestal. Instead of HPA axis response dishabituation, we observed enhanced habituation. Subsequent experiments determined that a 1.25mg/kg corticosterone injection could substitute for pedestal exposure to produce enhanced restraint habituation. Combined treatment with both the glucocorticoid receptor antagonist RU40555 (30 mg/kg) and the mineralocorticoid receptor antagonist RU28318 (50 mg/kg) blocked the expression of enhanced habituation after pedestal exposure. Thus, the delayed corticosterone negative feedback produced by novel stress led to enhanced expression of corticosterone response habituation.