Effect of the glucocorticoid receptor antagonist Org 34850 on basal and stress-induced corticosterone secretion.

The activity of the hypothalamic-pituitary-adrenal (HPA) axis is characterised both by an ultradian pulsatile pattern of glucocorticoid secretion and an endogenous diurnal rhythm. Glucocorticoid feedback plays a major role in regulating HPA axis activity and this mechanism occurs via two different receptors: mineralocorticoid (MR) and glucocorticoid receptors (GR). In the present study, the effects of both acute and subchronic treatment with the GR antagonist Org 34850 on basal and stress-induced HPA axis activity in male rats were evaluated. To investigate the effect of Org 34850 on basal diurnal corticosterone rhythm over the 24-h cycle, an automated blood sampling system collected samples every 10 min. Acute injection of Org 34850 (10 mg/kg, s.c.) did not affect basal or stress-induced corticosterone secretion, but was able to antagonise the inhibitory effect of the glucocorticoid agonist methylprednisolone on stress-induced corticosterone secretion. However, 5 days of treatment with Org 34850 (10 mg/kg, s.c., two times a day), compared to rats treated with vehicle (5% mulgofen in 0.9% saline, 1 ml/kg, s.c.), increased corticosterone secretion over the 24-h cycle and resulted in changes in the pulsatile pattern of hormone release, but had no significant effect on adrenocorticotrophic hormone secretion or on stress-induced corticosterone secretion. Subchronic treatment with Org 34850 did not alter GR mRNA expression in the hippocampus, paraventricular nucleus of the hypothalamus or anterior-pituitary, or MR mRNA expression in the hippocampus. Our data suggest that a prolonged blockade of GRs is required to increase basal HPA axis activity. The changes observed here with ORG 34850 are consistent with inhibition of GR-mediated negative feedback of the HPA axis. In light of the evidence showing an involvement of dysfunctional HPA axis in the pathophysiology of depression, Org 34850 could be a potential treatment for mood disorders.

Injections of urocortin 1 into the basolateral amygdala induce anxiety-like behavior and c-Fos expression in brainstem serotonergic neurons.

The amygdala plays a key role in emotional processing and anxiety-related physiological and behavioral responses. Previous studies have shown that injections of the anxiety-related neuropeptide corticotropin-releasing factor or the related neuropeptide urocortin 1 into the region of the basolateral amygdaloid nucleus induce anxiety-like behavior in several behavioral paradigms. Brainstem serotonergic systems in the dorsal raphe nucleus and median raphe nucleus may be part of a distributed neural system that, together with the basolateral amygdala, regulates acute and chronic anxiety states. We therefore investigated the effect of an acute bilateral injection of urocortin 1 into the basolateral amygdala on behavior in the social interaction test and on c-Fos expression within serotonergic neurons in the dorsal raphe nucleus and median raphe nucleus. Male rats were implanted with bilateral cannulae directed at the region of the basolateral amygdala; 72 h after surgery, rats were injected with urocortin 1 (50 fmol/100 nl) or vehicle (100 nl of 1% bovine serum albumin in distilled water). Thirty minutes after injection, a subgroup of rats from each experimental group was exposed to the social interaction test; remaining animals were left in the home cage. Two hours after injection rats were perfused with paraformaldehyde and brains were removed and processed for immunohistochemistry. Acute injection of urocortin 1 had anxiogenic effects in the social interaction test, reducing total interaction time without affecting locomotor activity or exploratory behavior. These behavioral effects were associated with increases in c-Fos expression within brainstem serotonergic neurons. In home cage rats and rats exposed to the social interaction test, urocortin 1 treatment increased the number of c-Fos-immunoreactive serotonergic neurons within subdivisions of both the dorsal raphe nucleus and median raphe nucleus. These results are consistent with the hypothesis that the basolateral amygdala and serotonergic neurons within the midbrain raphe complex are part of an integrated neural system modulating anxiety state.

Rapid intra-adrenal feedback regulation of glucocorticoid synthesis.

The hypothalamic-pituitary-adrenal axis is a vital neuroendocrine system that regulates the secretion of glucocorticoid hormones from the adrenal glands. This system is characterized by a dynamic ultradian hormonal oscillation, and in addition is highly responsive to stressful stimuli. We have recently shown that a primary mechanism generating this ultradian rhythm is a systems-level interaction where adrenocorticotrophin hormone (ACTH) released from the pituitary stimulates the secretion of adrenal glucocorticoids, which in turn feedback at the level of the pituitary to rapidly inhibit ACTH secretion. In this study, we combine experimental physiology and mathematical modelling to investigate intra-adrenal mechanisms regulating glucocorticoid synthesis. Our modelling results suggest that glucocorticoids can inhibit their own synthesis through a very rapid (within minutes), presumably non-genomic, intra-adrenal pathway. We present further evidence for the existence of a short time delay in this intra-adrenal inhibition, and also that at the initiation of each ACTH stimulus, this local feedback mechanism is rapidly antagonized, presumably via activation of the specific ACTH receptor (MC2R) signalling pathway. This mechanism of intra-adrenal inhibition enables the gland to rapidly release glucocorticoids while at the same time preventing uncontrolled release of glucocorticoids in response to large surges in ACTH associated with stress.

Clozapine, but not haloperidol, increases brain concentrations of neuroactive steroids in the rat.

The extrapyramidal side effects of typical antipsychotics, which are induced to a markedly reduced extent by clozapine, have been linked to a dysfunction of central gamma-aminobutyric acid (GABA)-mediated neurotransmission. The effects of clozapine on the brain concentrations of 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone, AP) and 3alpha,21-dihydroxy-5alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, THDOC), two potent and endogenous positive allosteric modulators of GABA-mediated chloride current intensities at GABA(A) receptors, were compared with those of the typical antipsychotic haloperidol. A single administration of clozapine (1.25-20 mg/kg, IP), but not of haloperidol (0.1 or 0.5 mg/kg, IP), induced dose- and time-dependent increases in the concentrations of progesterone, AP, and THDOC in the cerebral cortex and striatum of rats. Clozapine (at 10 mg/kg, but not at lower doses) also increased the concentrations of these steroids as well as that of corticosterone in plasma in intact rats, but failed to increase the cortical concentrations of AP and THDOC in adrenalectomized-orchidectomized rats. An acute challenge with clozapine (10 mg/kg), administered 48 h after the termination of daily treatment with the same dose for 19 days, still increased the cortical concentrations of progesterone, AP, and THDOC. These results suggest that the clozapine-induced increases in neuroactive steroid concentrations in the brain may contribute to the atypical pharmacological profile of this antipsychotic drug.

Opposite effects of short- versus long-term administration of fluoxetine on the concentrations of neuroactive steroids in rat plasma and brain.

RATIONALE: Recent preclinical and clinical studies have shown that selective serotonin re-uptake inhibitors modulate neurosteroid synthesis in an opposite manner. OBJECTIVES: The action of long-term administration of fluoxetine was investigated on the peripheral and central concentrations of 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-TH PROG) and 3alpha,5alpha-tetrahydrodeoxycorticosterone (of 3alpha,5alpha-TH DOC), progesterone, and pregnenolone in rats. We also investigated the effect of chronic treatment with fluoxetine on the foot-shock stress-induced increase in the plasma and brain concentrations of these steroids. METHODS: Fluoxetine was administered acutely (20 mg/kg) or chronically (10 mg/kg, once daily for 15 days). Steroids were extracted from plasma and brain, separated and purified by means of high-performance liquid chromatography, and quantified by means of radioimmunoassay. RESULTS: A single dose of fluoxetine (20 mg/kg, i.p.) induced in 20 min significant increases in the cerebral cortical and plasma concentrations of 3alpha,5alpha-TH PROG (+96% and +13%, respectively), 3alpha,5alpha-TH DOC (+129 and +31%, respectively), progesterone (+111 and +58%, respectively), and pregnenolone (+151 and +59%, respectively). In addition, the plasma concentration of corticosterone was also significantly increased (+24%) after acute administration of fluoxetine. In contrast, long-term administration of fluoxetine reduced the basal concentrations of these various steroids (ranging from -22 to -43%), measured 48 h after the last drug injection, in both brain and plasma. A challenge injection of fluoxetine (20 mg/kg, i.p.), however, was still able to increase the concentrations of steroids in both the brain and plasma of rats chronically treated with this drug. Acute foot-shock stress increased the cortical and plasma concentrations of steroids in rats chronically treated with fluoxetine to extents similar to those apparent in control rats. CONCLUSIONS: A repetitive increase in the brain concentrations of neuroactive steroids may contribute to the therapeutic action of fluoxetine.

Prevention of the stress-induced increase in frontal cortical dopamine efflux of freely moving rats by long-term treatment with antidepressant drugs.

Use of antidepressant drugs in the treatment of anxiety disorders has recently increased due to the anxiolytic effect of some of these agents. Because dopaminergic transmission in the prefrontal cortex is sensitive to anxiogenic or stressful stimuli, the effects of two antidepressant drugs with different mechanisms of action, imipramine and mirtazapine, on the response of rat cortical dopaminergic neurons to stress were investigated. A 2-week (but not single dose) administration of imipramine (10 mg/kg, i.p., twice daily) or mirtazapine (10 mg/kg, i.p., once daily) reduced and completely antagonized, respectively, the increase in dopamine release in the prefrontal cortex elicited by footshock stress. Long-term administration of imipramine or mirtazapine had no marked effect on the stress-induced increases in the brain or plasma concentrations of neuroactive steroids or corticosterone. An attenuation of the response of mesocortical dopaminergic neurons to stress induced by long-term treatment with antidepressants might contribute to the anxiolytic effects of such drugs.

Anthropometric variables, lifestyle and sports in school-age children: comparison between the cities of Bologna and Crotone.

Weight disorders are rapidly increasing in childhood. In Italy a strong geographic North-South gradient of overweight and obesity has been reported. The purpose of this study is to examine anthropometric variables, physical activity and lifestyles in school-age children in the cities of Bologna and Crotone. The sample consists of 963 children attending primary schools, aged 6-10 years. Height, weight, triceps, and subscapular skinfolds were measured for each child; body mass index (BMI) and percentage of fat mass were calculated. Physical activity was determined through an interview with each child. Another questionnaire was submitted to parents, in order to obtain information on sleep duration, means of transport and type of physical activity. In the city of Bologna, the mean values of anthropometric variables were: height (boys - 1,335 mm, girls - 1,332 mm), BMI (boys - 17.8 kg/m(2), girls - 17.3 kg/m(2)), and the proportion of overweight and obese individuals was: boys - 20.6%, girls - 12.7%. In Crotone the mean values were: height (boys - 1,275 mm, girls - 1,265 mm), BMI (boys - 19.4 kg/m(2), girls - 19.3 kg/m(2)), and the proportion of overweight and obese individuals was: boys - 46.7%, girls - 49.0%. The results have demonstrated significant correlations of lifestyle and physical activity with children's weight status. In Crotone there is a larger variation of weight disorders with higher percentages of overweight children than in Bologna. The children in Crotone are engaged in sedentary activities for a significantly longer time than children in Bologna, who are more physically active. The data show a strong North-South gradient concerning not only distribution of weight status, but also lifestyles especially including participation in sports and sedentary behavior. Our study also provides meaningful suggestions how to define lifestyles in order to achieve an optimal health status during growth.

Hippocampus remodeling by chronic stress accompanied by GR, proteasome and caspase-3 overexpression.

Chronic stress is a threat to homeostasis for many brain regions. While hippocampal formation is one of the most stress-sensitive areas of the cortex, molecular changes occurring as a result of increased glucocorticoid neurotoxicity in hippocampus are largely unknown. The aim of these studies was to investigate mRNA expression of mineralocorticoid and glucocorticoid receptors (MR, GR), proteasome subunits ß5 (constitutive subunit) and ß1i (inducible immunoproteasome subunit), mTOR (mammalian target of rapamycin), bcl-2; as well as caspase-3 immunoreactivity (confocal microscopy) in adult Wistar rat hippocampus following 10-day restraint stress (plastic restrainers, 6h daily). Chronic restraint led to a significant reduction in number of neuronal and astroglial cells in hippocampal regions CA1-3. This reaction was combined with substantial increase in GR and decrease in MR mRNA levels with the greatest response - 1.5-fold amplitude increase - observed in dentate gyrus and CA3 correspondingly. Stress did not change the expression of constitutive ß5 subunit but dramatically enhanced expression of inducible ß1i subunit and increased mTOR, and bcl-2 mRNA expression. Multiple scattered cells demonstrating caspase-3(+) profile were found in hippocampus of stressed animals. The study demonstrates that hippocampal remodeling induced by chronic restraint stress is associated with GR, immunoproteasome, mTOR, caspase-3 and bcl-2 overexpression in hippocampus.

Temporal effect of adrenocorticotrophic hormone on adrenal glucocorticoid steroidogenesis: involvement of the transducer of regulated cyclic AMP-response element-binding protein activity.

The availability of active steroidogenic acute regulatory protein (StAR) and side-chain cleavage cytochrome P450 (P450scc) are rate-limiting steps for steroidogenesis. Transcription of StAR and P450scc genes depends on cyclic AMP-response element-binding protein (CREB) phosphorylation and CREB co-activator, transducer of regulated CREB activity (TORC), which is regulated by salt-inducible kinase 1 (SIK1). In the present study, we investigated the relationship between TORC activation and adrenocorticotrophic hormone (ACTH)-induced steroidogenesis in vivo, by examining the time-course of the effect of ACTH injection (4 ng, i.v.) on the transcriptional activity of StAR and P450scc genes and the nuclear accumulation of transducer of regulated CREB activity 2 (TORC2) in rat adrenal cortex. ACTH produced rapid and transient increases in plasma corticosterone, with maximal responses between 5 and 15 min, and a decrease to almost basal values at 30 min. StAR and P450scc hnRNA levels increased 15 min following ACTH and decreased toward basal values at 30 min. Concomitant with an increase in nuclear phospho-CREB, ACTH injection induced nuclear accumulation of TORC2, with maximal levels at 5 min and a return to basal values by 30 min. The decline of nuclear TORC2 was paralleled by increases in SIK1 hnRNA and mRNA 15 and 30 min after injection, respectively. The early rises in plasma corticosterone preceding StAR and P450scc gene transcription suggest that post-transcriptional and post-translational changes in StAR protein mediate the early steroidogenic responses. Furthermore, the direct temporal relationship between nuclear accumulation of TORC2 and the increase in transcription of steroidogenic proteins, implicates TORC2 in the physiological regulation of steroidogenesis in the adrenal cortex. The delayed induction of SIK1 suggests a role for SIK1 in the declining phase of steroidogenesis.

Recovery from disrupted ultradian glucocorticoid rhythmicity reveals a dissociation between hormonal and behavioural stress responsiveness.

Ultradian release of glucocorticoids is thought to be essential for homeostasis and health. Furthermore, deviation from this pulsatile release pattern is considered to compromise resilience to stress-related disease, even after hormone levels have normalised. In the present study, we investigate how constant exposure to different concentrations of corticosterone affects diurnal and ultradian pulsatility. The rate of recovery in pulsatile hypothalamic-pituitary-adrenal (HPA) activity after withdrawal of exogenous corticosterone is also examined. Finally, the behavioural and neuroendocrine responsiveness to an audiogenic stressor is studied. Adrenally intact male rats were subcutaneously implanted with vehicle, 40% or 100% corticosterone pellets for 7 days. The continuous release of corticosterone from these implants abolished diurnal and ultradian corticosterone variation, as measured with high-frequency automated blood sampling. Pellet removal on post-surgery day 8 allowed rapid recovery of endogenous rhythms in animals previously exposed to daily average concentrations (40%) but not after exposure to high concentrations (100%) of corticosterone. Behavioural and neuroendocrine responsiveness to stress was distinctly different between the treatment groups. Audiogenic stimulation 1 day after pellet removal resulted in a similar corticosterone response in animals previously exposed to 40% corticosterone or vehicle. The 40% pellet group, however, showed less and shorter behavioural activity (i.e. locomotion, risk assessment) to noise stress compared to 100% corticosterone and vehicle-treated animals. In conclusion, unlike the animals impanted with 100% corticosterone, we find that basal HPA axis activity in the 40% group, which had mean daily levels of circulating corticosterone in the physiological range, rapidly reverts to the characteristic pulsatile pattern of corticosterone secretion. Upon reinstatement of the ultradian rhythm, and despite the fact that these animals did not differ from controls in their response to noise stress, they did show substantial changes in their behavioural response to stress.

A glucocorticoid sensitive biphasic rhythm of testosterone secretion.

Studies of the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-testicular (HPT) axis have revealed a reciprocal relationship between these two endocrine pathways. In rats, for example, disruption of the HPT axis alters the circadian secretion of corticosterone. Stress, on the other hand, can have varying effects on testosterone secretion in both rats and humans. Furthermore, in contrast to humans, where several pulses of testosterone secretion can be detected across the 24-h period with the largest in the morning, rats appear to exhibit a diurnal rhythm of testosterone secretion. In the present study, we used an automated blood sampling system to investigate the true circadian pattern of testosterone secretion under basal conditions and investigated how this responds to changes in levels of circulating corticosteroids. Analysis of plasma testosterone revealed the expected bimodal pattern of basal testosterone secretion. The two secretory episodes were 12.59 h +/- 41 min apart and 4.04 h +/- 16 min long, with one in the light phase and the other in the dark phase of the cycle. Interestingly, when both testosterone and corticosterone diurnal profile were compared, we found that the circadian rise in plasma corticosterone levels falls neatly between the two testosterone secretory episodes. Treatment of rats with the synthetic glucocorticoid methylprednisolone in their drinking water abolished the normal bimodal profile of testosterone secretion. These rats show transient pulses of testosterone throughout the 24 h, but no circadian pattern. By contrast, adrenalectomised rats maintain their bimodal circadian pattern, suggesting that an intact HPA axis is not necessary for generation of the endogenous HPT rhythm. Thus, although the circadian rhythm of testosterone does not depend on normal HPA function, increased levels of glucocorticoids can abolish normal HPT rhythmicity.

Inhibition of stress- or anxiogenic-drug-induced increases in dopamine release in the rat prefrontal cortex by long-term treatment with antidepressant drugs.

The effects of long-term treatment with imipramine or mirtazapine, two antidepressant drugs with different mechanisms of action, on the response of cortical dopaminergic neurons to foot-shock stress or to the anxiogenic drug FG7142 were evaluated in freely moving rats. As expected, foot shock induced a marked increase (+ 90%) in the extracellular concentration of dopamine in the prefrontal cortex of control rats. Chronic treatment with imipramine or mirtazapine inhibited or prevented, respectively, the effect of foot-shock stress on cortical dopamine output. Whereas acute administration of the anxiogenic drug FG7142 induced a significant increase (+ 60%) in cortical dopamine output in control rats, chronic treatment with imipramine or mirtazapine completely inhibited this effect. In contrast, the administration of a single dose of either antidepressant 40 min before foot shock, had no effect on the response of the cortical dopaminergic innervation to stress. These results show that long-term treatment with imipramine or mirtazapine inhibits the neurochemical changes elicited by stress or an anxiogenic drug with an efficacy similar to that of acute treatment with benzodiazepines. Given that episodes of anxiety or depression are often preceded by stressful events, modulation by antidepressants of the dopaminergic response to stress might be related to the anxiolytic and antidepressant effects of these drugs.