Helen C. Atkinson

Corticosterone Levels in the Brain Show a Distinct Ultradian Rhythm but a Delayed Response to Forced Swim Stress

Circulating corticosterone levels show an ultradian rhythm resulting from the pulsatile release of glucocorticoid hormone by the adrenal cortex. Because the pattern of hormone availability to corticosteroid receptors is of functional significance, it is important to determine whether there is also a pulsatile pattern of corticosterone concentration within target tissues such as the brain. Furthermore, it is unclear whether measurements of plasma corticosterone levels accurately reflect corticosterone levels in the brain. Given that the hippocampus is a principal site of glucocorticoid action, we investigated in male rats hippocampal extracellular corticosterone concentrations under baseline and stress conditions using rapid-sampling in vivo microdialysis. We found that hippocampal extracellular corticosterone concentrations show a distinct circadian and ultradian rhythm. The PULSAR algorithm revealed that the pulse frequency of hippocampal corticosterone is 1.03 +/- 0.07 pulses/h between 0900 and 1500 h and is significantly higher between 1500 and 2100 h (1.31 +/- 0.05). The hippocampal corticosterone response to stress is stressor dependent but resumes a normal ultradian pattern rapidly after the termination of the stress response. Similar observations were made in the caudate putamen. Importantly, simultaneous measurements of plasma and hippocampal glucocorticoid levels showed that under stress conditions corticosterone in the brain peaks 20 min later than in plasma but clears concurrently, resulting in a smaller exposure of the brain to stress-induced hormone than would be predicted by plasma hormone concentrations. These data are the first to demonstrate that the ultradian rhythm of corticosterone is maintained over the blood-brain barrier and that tissue responses cannot be reliably predicted from the measurement of plasma corticosterone levels.

The significance of glucocorticoid pulsatility.

Glucocorticoids are secreted in discrete pulses resulting in an ultradian rhythm in all species that have been studied. In the rat there is an approximately hourly rhythm of corticosterone secretion, which appears to be regulated by alternating activation and inhibition of the HPA axis. At the level of signal transduction, the response to these pulses of corticosterone is determined by its dynamic interaction with the two transcription factors--the glucocorticoid and mineralocorticoid receptors. While the mineralocorticoid receptor remains activated throughout the ultradian cycle, the glucocorticoid receptor shows a phasic response to each individual pulse of corticosterone. This phasic response is regulated by an intranuclear proteasome-dependent rapid downregulation of the activated glucocorticoid receptor.

Gonadectomy Reverses The Sexually Diergic Patterns Of Circadian and Stress-Induced Hypothalamic-Pituitary-Adrenal Axis Activity In Male and Female Rats

Enhanced corticosterone release by female compared to male rats under basal and stress conditions is well documented. The demonstration that gonadectomy enhances stress‐induced corticosterone secretion in male rats, but reduces such levels in female rats, suggests a causal association between gonadal steroids and corticosterone release. The present study examined the corticosterone profile of sham gonadectomized and gonadectomized female and male rats under basal and stress conditions. An automated sampling system collected blood from each freely moving, unanaesthetized rat every 10 min (i) over a 24‐h period; (ii) following noise stress; and (iii) following an immune‐mediated stress (lipopolysaccharide, LPS). Plasma was analysed for corticosterone content using radioimmunoassay. Castration resulted in a significant increase in basal corticosterone release compared to the sham‐castrated male rats. Pulsar analysis revealed a significant two‐fold increase in the number of corticosterone pulses over 24 h. Corticosterone increases in response to noise stress and to LPS injection were enhanced following castration. Conversely, ovariectomy resulted in a two‐fold reduction in the number of corticosterone pulses as well as the stress response compared to sham‐ovariectomized female rats. Arginine vasopressin (AVP), corticotrophin‐releasing hormone (CRH) and glucocorticoid receptor mRNAs in the paraventricular nucleus and pro‐opiomelanocortin (POMC) mRNA in the anterior pituitary were analysed post‐LPS administration by in situ hybridization. Significantly higher values were found for AVP, CRH and POMC mRNAs examined for sham females and castrated males compared to sham males and ovariectomized females. This study confirms previous reports concerning the influence of gonadal factors in regulating HPA axis activity and stress responsiveness. The present results extend these observations to the regulation of the dynamic pattern of corticosterone release under basal conditions and suggests that this alteration in pulsatility is important for the differences in stress responsiveness when comparing males and females.

Gonadal Steroid Replacement Reverses Gonadectomy-Induced Changes in the Corticosterone Pulse Profile and Stress-Induced Hypothalamic-Pituitary-Adrenal Axis Activity of Male and Female Rats

We investigated the effects of gonadal hormone replacement on the pulsatile parameters underlying basal circadian corticosterone secretion in castrated male and ovariectomized female rats using an automated sampling system. Blood was collected from freely moving, unanaesthetized rats every 10 min over a 24‐h period and sampling was continued during a noise stress and after lipopolysaccharide (LPS) administration. Castrated male rats had markedly higher corticosterone levels than intact controls. This was reflected by increased number and frequency of pulses in addition to an increase in the pulse height and amplitude under both basal circadian and stress conditions. Hormone replacement with either testosterone or dihydrotestosterone returned these corticosterone levels and circadian profile to those found in intact males, confirming an androgen‐mediated effect. Ovariectomized females had significantly lower basal and stress‐induced corticosterone levels with lower frequency and amplitude of corticosterone pulses than intact females. 17β‐oestradiol replacement returned basal levels, pulsatile measurements and stress‐induced corticosterone levels to those found in intact females. Three hours post‐LPS administration, castrated males demonstrated significantly higher values of parvocellular paraventricular nucleus (PVN) arginine vasopressin and corticotrophin‐releasing factor and anterior pituitary pro‐opiomelanocortin mRNA while ovariectomized females showed significantly lower levels of all three transcripts compared to intact controls. PVN glucocorticoid receptor mRNA levels 3 h post‐LPS administration were significantly decreased in castrated males and significantly increased in ovariectomized female rats. Replacement of gonadal steroids resulted in a return to the levels found in intact controls after LPS. Gonadal steroid replacement is sufficient to reverse changes in the pulsatile characteristics of corticosterone release after gonadectomy. In addition, gonadal steroid replacement reverses stress‐induced alterations in hypothalamic‐pituitary‐adrenal (HPA) activity. These data demonstrate a major contribution of gonadal steroids to the regulation of HPA axis activity and to the pulsatile characteristics of corticosterone release.

The Relationship between Maternal and Umbilical Cord Androgen Levels and Polycystic Ovary Syndrome in Adolescence: A Prospective Cohort Study

CONTEXT The prenatal antecedents of polycystic ovary syndrome (PCOS) are not known, but prenatal androgen exposure is thought to contribute. This has not previously been investigated in large prospective studies of normal human pregnancy. OBJECTIVE The aim of the study was to establish the prospective relationship between early life androgen exposure and PCOS in adolescence. DESIGN AND SETTING A prospective cohort study was conducted in the general community. PATIENTS OR OTHER PARTICIPANTS A total of 2900 pregnant women were recruited at 18 wk gestation. Prenatal androgen exposure was measured from maternal blood samples (at 18 and 34-36 wk) and umbilical cord blood. Timed (d 2-5 menstrual cycle) blood samples were collected, clinical hyperandrogenism was assessed, and transabdominal ultrasound examination of ovarian morphology was performed in 244 unselected girls from the Raine cohort aged 14-17 yr. MAIN OUTCOME MEASURE(S) We examined the relationship between early life androgen exposure and PCOS in adolescence. RESULTS We did not observe a statistically significant relationship between early life androgen exposure and PCOS in adolescence. CONCLUSIONS This is the first prospective study to evaluate the relationship between prenatal androgen exposure and PCOS in adolescence in normal pregnancy. Our findings do not support the hypothesis that maternal androgens, within the normal range for pregnancy, directly program PCOS in the offspring.

Clinical, ultrasound and biochemical features of polycystic ovary syndrome in adolescents: implications for diagnosis

BACKGROUND Diagnosing polycystic ovary syndrome (PCOS) in adolescence is clinically challenging. The prevalence of clinical, ultrasound and biochemical features of PCOS in a community-based adolescent population using current diagnostic criteria has not previously been described. METHODS This was a prospective cohort study with 244 unselected post-menarchal girls, mean age 15.2 years, of whom 91% were Caucasian. Subjects were recruited from a large population-based birth cohort (the Raine cohort). Clinical hyperandrogenism (HA) was quantified using Ferriman-Gallwey scores. In the early follicular phase (Day 2-6), we measured circulating androgens and sex hormone-binding globulin by immunoassay, and ovarian morphology was assessed by transabdominal ultrasound examination. BMI and waist-hip ratio were measured. RESULTS Normal ranges for early follicular phase androgens in adolescence were derived for this population. The top 5 and 10% of circulating free testosterone levels were 45.6 and 34.5 pmol/l, respectively. Fifty-one percent of girls reported menstrual irregularity. Clinical HA was uncommon, being observed in only 3.5% of girls. Mean ovarian volume was greater than that reported by others in adult women and 35% of girls had polycystic ovary morphology on transabdominal ultrasound. Taking the upper 5% of free testosterone as HA, 42 girls (18.5%) would have met the Rotterdam criteria for PCOS, 11 girls (5%) the Androgen Excess Society criteria and 7 girls (3.1%) the National Institutes of Health criteria. CONCLUSIONS Menstrual irregularity is common in adolescence and does not relate to clinical or biochemical HA. Diagnostic criteria for PCOS which include ovarian volume and morphology may be of limited use in adolescence.

Diurnal variation in the responsiveness of the hypothalamic-pituitary-adrenal axis of the male rat to noise stress

Basal activity of the rat hypothalamic‐pituitary‐adrenal (HPA) axis is highly dynamic and displays both circadian and ultradian rhythmicity in corticosterone secretion. This study investigated the relationship between basal corticosterone pulsatility and the corticosterone response to noise during the early light phase when there are no endogenous corticosterone pulses and during the early dark phase when there are hourly pulses of corticosterone. An automated blood sampling system was used to collect blood in conscious male rats at 5‐min intervals before, during and after exposure to 10‐min periods of white noise (104 dB). Behavioural responses to noise were also monitored during these periods. During the early light phase (morning), there was a consistent corticosteroid response to noise with corticosterone concentrations rising rapidly and reaching peak values 10–15 min after the noise had ceased, following which circulating concentrations declined at a rate comparable to the hormones half‐life. A second noise stress, 80 min later, resulted in adaptation of the corticosterone response. During the early dark phase (evening), the corticosterone response to the noise was similar to that seen in the morning, although there was no adaptation to a second stimulus. During the evening, the inhibition of endogenous HPA activity after the sound was limited to 40 min following stress.

Postnatal masculinization alters the HPA axis phenotype in the adult female rat

The ability of postnatal testosterone propionate (TP) to masculinize both behaviour and gonadal cyclicity in the female rat is well documented. We have investigated whether postnatal androgen also has an organizational effect on another sexually dimorphic neuroendocrine system – the hypothalamo‐pituitary‐adrenal (HPA) axis. Female rats were exposed to a single injection of testosterone propionate (TP) or oil within 24 h of birth. As adults, rats were either ovariectomized and given 17β‐oestradiol replacement (OVXE2) or sham ovariectomized with cholesterol implants (SHOVX). An automated sampling system collected blood from unanaesthetized adult female rats every 10 min over a 24‐h period, during a mild psychological stress (noise) and following an immunological lipopolysaccharide stress (LPS). Neonatal TP‐treated SHOVX rats had a significant reduction in the number, height, frequency and amplitude of corticosterone pulses over the basal 24‐h period, compared to both the neonatal oil‐treated and TP‐treated OVXE2 animals. The corticosterone response to both noise and LPS was also significantly decreased for the TP‐treated SHOVX females. Three hours post‐LPS administration, TP females had significantly lower values of paraventricular nucleus (PVN) corticotrophin releasing hormone (CRH), arginine vasopressin (AVP) and anterior pituitary proopiomelanocortin (POMC) mRNAs and greater PVN glucocorticoid receptor (GR) mRNA expression compared to the oil‐treated controls. E2 replacement in adult TP rats normalized all the mRNA levels, except for PVN GR mRNA which did fall towards the levels of the oil‐control animals. A single injection of TP within 24 h of birth disrupts the development of the characteristic female pattern of corticosterone secretion and the normal female HPA response to stress, resulting in a pattern similar to that seen in males. These effects can be reversed by E2 treatment in the adult TP female rat.

Corticosteroids mediate fast feedback of the rat hypothalamic-pituitary-adrenal axis via the mineralocorticoid receptor.

The aim of this study was to investigate fast corticosteroid feedback of the hypothalamic-pituitary-adrenal (HPA) axis under basal conditions, in particular the role of the mineralocorticoid receptor. Blood samples were collected every 5 min from conscious rats at the diurnal peak, using an automated blood sampling system, and assayed for corticosterone. Feedback inhibition by rapidly increasing concentrations of ligand was achieved with an intravenous bolus of exogenous corticosteroid. This resulted in a significant reduction in plasma corticosterone concentrations within 23 min of the aldosterone bolus and 28 min of methylprednisolone. Evaluation of the pulsatile secretion of corticosterone revealed that the secretory event in progress at the time of administration of exogenous steroid was unaffected, whereas the next secretory event was inhibited by both aldosterone and methylprednisolone. The inhibitory effect of aldosterone was limited in duration (1 secretory event only), whereas that of methylprednisolone persisted for 4-5 h. Intravenous administration of canrenoate (a mineralocorticoid receptor antagonist) also had rapid effects on the HPA axis, with an elevation of ACTH within 10 min and corticosterone within 20 min. The inhibitory effect of aldosterone was unaffected by pretreatment with the glucocorticoid receptor antagonist RU-38486 but blocked by the canrenoate. These data imply an important role for the mineralocorticoid receptor in fast feedback of basal HPA activity and suggest that mineralocorticoids can dynamically regulate basal corticosterone concentrations during the diurnal peak, a time of day when there is already a high level of occupancy of the cytoplasmic mineralocorticoid receptor.

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.