We reported previously that the neuropeptide oxytocin attenuates stress-induced hypothalamo-pituitary-adrenal (HPA) activity and anxiety behavior. This study sought to identify forebrain target sites through which oxytocin may mediate its anti-stress effects. Ovariectomized, estradiol-treated rats received intracerebroventricular infusions of oxytocin (1 or 10 ng/hr) or vasopressin (10 ng/hr), and the patterns of neuronal activation after restraint stress were determined by semiquantitative mapping of c-fos mRNA expression. Oxytocin administration significantly attenuated the release of ACTH and corticosterone and the increase in corticotropin-releasing factor mRNA expression in the hypothalamic paraventricular nucleus (PVN) in response to 30 min restraint. Restraint also induced the expression of c-fos mRNA in selective regions of the forebrain, including the PVN, paraventricular thalamic nucleus, habenula, medial amygdala, ventrolateral septum (LSV), most subfields of the dorsal and ventral hippocampus, and piriform and endopiriform cortices. In most cases, this level of gene expression was unaffected by concomitant administration of oxytocin. However, in the PVN, LSV, and throughout all subfields of the dorsal hippocampus, restraint evoked no detectable increase in c-fos mRNA in animals treated with either dose of oxytocin. Vasopressin had no effects on either HPA axis responses or neuronal activation in response to restraint, indicating that the effects were highly peptide selective. These data show that central oxytocin attenuates both the stress-induced neuroendocrine and molecular responses of the HPA axis and that the dorsal hippocampus, LSV, and PVN constitute an oxytocin-sensitive forebrain stress circuit.

Characterization of a peripheral hormonal system identifies the origin and mechanisms of regulation of glucocorticoid hormone oscillations in rats.

Opioids are the most common drugs associated with unintentional drug overdose. Death results from respiratory depression. Prolonged use of opioids results in the development of tolerance but the degree of tolerance is thought to vary between different effects of the drugs. Many opioid addicts regularly consume alcohol (ethanol), and post-mortem analyses of opioid overdose deaths have revealed an inverse correlation between blood morphine and ethanol levels. In the present study, we determined whether ethanol reduced tolerance to the respiratory depressant effects of opioids. Mice were treated with opioids (morphine, methadone, or buprenorphine) for up to 6 days. Respiration was measured in freely moving animals breathing 5% CO2 in air in plethysmograph chambers. Antinociception (analgesia) was measured as the latency to remove the tail from a thermal stimulus. Opioid tolerance was assessed by measuring the response to a challenge dose of morphine (10 mg/kg i.p.). Tolerance developed to the respiratory depressant effect of morphine but at a slower rate than tolerance to its antinociceptive effect. A low dose of ethanol (0.3 mg/kg) alone did not depress respiration but in prolonged morphine-treated animals respiratory depression was observed when ethanol was co-administered with the morphine challenge. Ethanol did not alter the brain levels of morphine. In contrast, in methadone- or buprenorphine-treated animals no respiratory depression was observed when ethanol was co-administered along with the morphine challenge. As heroin is converted to morphine in man, selective reversal of morphine tolerance by ethanol may be a contributory factor in heroin overdose deaths.

In vivo glucocorticoid (GC) secretion exhibits a distinctive ultradian rhythmicity. The lipophilic hormone can rapidly diffuse into cells, although only the pulse peak is of sufficient amplitude to activate the low affinity glucocorticoid receptor (GR). Discrete pulses readily access brain regions such as the hippocampus where GR expression is enriched and known to regulate neuronal function, including memory and learning processes. In the present study, we have tested the hypothesis that GR brain targets are responsive to ultradian GC rhythmicity. We have used adrenalectomised rats replaced with pulses of corticosterone to determine the transcriptional effects of ultradian pulses in the hippocampus. Confocal microscopy confirmed that each GC pulse results in transient GR nuclear localisation in hippocampal CA1 neurones. Concomitant GR activation and DNA binding was demonstrated by synthetic glucocorticoid response element oligonucleotide binding, and verified for the Clock gene Period 1 promoter region by chromatin immunoprecipitation assays. Strikingly each GC pulse induced a 'burst' of transcription of Period 1 measured by heterogeneous nuclear RNA quantitative polymerase chain reaction. The net effect of pulsatile GC exposure on accumulation of the mature transcript was also assessed, revealing a plateau of mRNA levels throughout the time course of pulsatile exposure, indicating the pulse timing works optimally for steady state Per1 expression. The plateau dropped to baseline within 120 min of the final pulse, indicating a relatively short half-life for hippocampal Per1. The significance of this strict temporal control is that any perturbation to the pulse frequency or duration would have rapid quantitative effects on the levels of Per1. This in turn could affect hippocampal function, especially circadian related memory and learning processes.

Objectives-To characterize the dynamics of the pituitary-adrenal interaction during the course of coronary artery bypass grafting (CABG) both on and off pump. Since our data pointed to a major change in adrenal responsiveness to ACTH we used a reverse translation approach to investigate the molecular mechanisms underlying this change in a rat model of critical illness. Measurements and Results-Clinical studies: Blood samples were taken for 24 hours from placement of the first venous access. Cortisol and ACTH were measured every 10 and 60 minutes respectively, and corticosteroid binding globulin (CBG) was measured at the beginning and end of the 24 hour period and at the end of operation. There was an initial rise in both levels of ACTH and cortisol to supra-normal values at around the end of surgery. ACTH levels then returned towards pre-operative values. Ultradian pulsatility of both ACTH and cortisol was maintained throughout the peri-operative period in all individuals. The sensitivity of the adrenal gland to ACTH increased markedly at around 8 hours after surgery maintaining very high levels of cortisol in the face of 'basal' levels of ACTH. This sensitivity began to return towards pre-operative values at the end of the 24-hour sampling period.Animal studies: Adult, male Sprague-Dawley rats were either given lipopolysaccharide (LPS) or sterile saline via a jugular vein cannula. Hourly blood samples were subsequently collected for ACTH and corticosterone measurement. Rats were sacrificed 6 hours after the injection and the adrenal glands were collected for measurement of StAR, SF-1 and DAX1 mRNA and protein using RTqPCR and Western immunoblotting, respectively. Adrenal levels of the ACTH receptor (MC2R) mRNA and its accessory protein (MRAP) were also measured by RTqPCR. In response to LPS, rats showed a pattern of ACTH and corticosterone that was similar to patients undergoing CABG. We were also able to demonstrate increased intra-adrenal corticosterone levels and an increase in StAR, SF-1 and MRAP mRNAs and StAR protein, and a reduction in DAX1 and MC2R mRNAs, 6h after LPS injection.Conclusions-Severe inflammatory stimuli activate the HPA axis resulting in increased steroidogenic activity in the adrenal cortex and an elevation of cortisol levels in the blood. Following CABG there is a massive increase in both ACTH and cortisol secretion. Despite a subsequent fall of ACTH to basal levels, cortisol remains elevated and co-ordinated ACTHcortisol pulsatility is maintained. This suggested that there is an increase in adrenal sensitivity to ACTH, which we confirmed in our animal model of immune activation of the HPA axis. Using this model we were able to show that this increased adrenal sensitivity results from changes in the regulation of both stimulatory and inhibitory intra-adrenal signaling pathways. Increased understanding of the dynamics of normal HPA responses to major surgery will provide us with a more rational approach to glucocorticoid therapy in critically ill patients.Gibbison et al.