EFFECTS OF PRENATAL DEXAMETHASONE ON HIPOPCAMPAL SEROTONIN 1A RECEPTORS IN ADULT MALE RATS

The main activation route for the stress response is the hypothalamo-pituitaryadrenal axis (HPA) and the sympatho-adrenomedullary system. The HPA axis is a neuroendocrine feedback loop mediated by an array of tissue specific hormones, receptors and neurotransmitters that regulate glucocorticoid (GC) release. GCs are steroidal hormones produced by the adrenal glands and are key players in a negativefeedback loop controlling HPA activity. They influence the HPA axis through glucocorticoid receptors in the hypothalamus and pituitary and through both glucocorticoid (GR) and mineralcorticoid receptors (MR) that are co-localized in the hippocampus. Repeated or chronic stress exerts a negative influence on these HPA axis regulatory sites and contributes to potentially pathological conditions, especially during early development. For example, chronic stress promotes increased maternal adrenal gland secretion of glucocortiocoid, leading to abnormally high concentrations of GC inthe fetal environment. The timing and maturation of the HPA axis relative to birth is highly species specific and is closely linked to landmarks in fetal development. In rats this development of the HPA axis takes place in utero and continues even shortly after birth. It is likely that the maternal endocrine environment will affect fetal development during this critical time point and may alter the overall set point for the expression ofgenes and their protein products that mediate fetal HPA axis function. Dexamethasone (DEX) is a synthetic glucocorticoid (sGC) and is a consensus treatment in preterm pregnancies used to expedite fetal lung development. However it has been shown that DEX causes long term physiological and behavioral disorders in prenatally-exposed laboratory animals. Previous studies have also shown that it alters the MR: GR receptor ratio in the hippocampus. Taking into consideration corticosteroid regulation of serotonin receptors, especially 5HT1A receptors and their putative interaction with glucocorticoid receptors in the hippocampus, we hypothesized that prenatal DEX exposure would lead to changes in the expression and function of 5HT1A receptors in the hippocampus. We administered DEX to rat dams during the last trimester of gestation and investigated the changes in these receptors in the adult rat offspring. Radioligand receptor binding assays were used to study hippocampal 5HT1A receptor binding affinity and number. Our results demonstrate that hippocampal 5HT1A receptors are increased in the DEX animalscompared with controls by 36%, with no change in binding affinity. The efficiency of ligand-induced receptor signal transduction via G-protein activation was also studied using [35S]GTPγS incorporation assay. Using this technique, we showed that there was no significant difference in the maximum ligand mediated stimulation (Emax) of 5HT1Areceptors between control and dex exposed animals. However, the intracellular signalling efficiency of hippocampal 5HT1A receptors was diminished, since a significant increase in EC50 values was obtained with the dex exposed group showing a value 51% higherEC50 than controls. Taken together these data illustrate a considerable change in the 5HT1A component of the serotonergic system following prenatal DEX exposure.

Occupational role stress is associated with higher cortisol reactivity to acute stress

We investigated whether occupational role stress is associated with differential levels of the stress hormone cortisol in response to acute psychosocial stress. Forty-three medication-free nonsmoking men aged between 22 and 65 years (mean ± SEM: 44.5 ± 2) underwent an acute standardized psychosocial stress task combining public speaking and mental arithmetic in front of an audience. We assessed occupational role stress in terms of role conflict and role ambiguity (combined into a measure of role uncertainty) as well as further work characteristics and psychological control variables including time pressure, overcommitment, perfectionism, and stress appraisal. Moreover, we repeatedly measured salivary cortisol and blood pressure levels before and after stress exposure, and several times up to 60 min thereafter. Higher role uncertainty was associated with a more pronounced cortisol stress reactivity (p = .016), even when controlling for the full set of potential confounders (p < .001). Blood pressure stress reactivity was not associated with role uncertainty. Our findings suggest that occupational role stress in terms of role uncertainty acts as a background stressor that is associated with increased HPA-axis reactivity to acute stress. This finding may represent a potential mechanism regarding how occupational role stress may precipitate adverse health outcomes.

Cortisol levels and history of depression in acute coronary syndrome patients

BACKGROUND: Depressed mood following an acute coronary syndrome (ACS) is a risk factor for future cardiac morbidity. Hypothalamic-pituitary-adrenal (HPA) axis dysregulation is associated with depression, and may be a process through which depressive symptoms influence later cardiac health. Additionally, a history of depression predicts depressive symptoms in the weeks following ACS. The purpose of this study was to determine whether a history of depression and/or current depression are associated with the HPA axis dysregulation following ACS. METHOD: A total of 152 cardiac patients completed a structured diagnostic interview, a standardized depression questionnaire and a cortisol profile over the day, 3 weeks after an ACS. Cortisol was analysed using: the cortisol awakening response (CAR), total cortisol output estimated using the area under the curve method, and the slope of cortisol decline over the day. RESULTS: Total cortisol output was positively associated with history of depression, after adjustment for age, gender, marital status, ethnicity, smoking status, body mass index (BMI), Global Registry of Acute Cardiac Events (GRACE) risk score, days in hospital, medication with statins and antiplatelet compounds, and current depression score. Men with clinically diagnosed depression after ACS showed a blunted CAR, but the CAR was not related to a history of depression. CONCLUSIONS: Patients with a history of depression showed increased total cortisol output, but this is unlikely to be responsible for associations between depression after ACS and later cardiac morbidity. However, the blunted CAR in patients with severe depression following ACS indicates that HPA dysregulation is present.

The Role of Cortisol in the Cycle of Violence

Child abuse and neglect are universal risk factors for delinquency, violence and aggression; this phenomenon is known as the cycle of violence. Despite a wide body of research demonstrating this phenomenon, the processes which mediate this relationship remain largely unknown. One potentially relevant result of abuse and neglect may be disruptions in the development of the body’s stress response, specifically the function of the Hypothalamic-Pituitary-Adrenal (HPA) axis. The HPA-axis, and its end-product, cortisol, may play a role in regulating aggressive behavior, but this function may be disrupted following abuse and neglect. Another risk factor for aggression, psychopathy, may mediate the cycle of violence or independently contribute to aggressive behavior. This study examined the relationship between child abuse and neglect, HPA-axis function, psychopathy and aggression. History of abuse was measured using a self-report questionnaire, the Childhood Trauma Questionnaire. Using a within-subject, placebo-controlled, counter-balanced dosing design, 67 adults were given an acute dose of 20mg cortisol as a challenge to the HPA-axis. Following dosing, measures of cortisol response were obtained through saliva samples, and state-aggressive behavior was measured by a laboratory task, the Point-Subtraction Aggression Paradigm (PSAP). Basal measures of cortisol were obtained prior to dosing. Psychopathy and a trait-measure of aggression were assessed through self-report questionnaires. PSAP data and trait-aggression scores were normalized and summed for an overall aggression score. Linear regression analyses indicated that a history of abuse and neglect robustly predicted aggression, supporting the cycle of violence hypothesis. Further, abuse and neglect predicted a diminished HPA-axis response to the cortisol challenge. Although a diminished HPA-axis response significantly predicted increased aggression, mediation analysis revealed that HPA-axis reactivity did not mediate a significant portion of the effect of abuse and neglect on aggression. However, HPA-axis reactivity did mediate part of the effect, indicating that HPA-axis function may be a factor in the cycle of violence. Psychopathy robustly predicted increased aggression. Although the results indicate that cortisol, psychopathy and HPA-axis function are involved in the cycle of violence, further research is required to better understand the complex interaction of these factors.

Influence of transrectal and transabdominal ultrasound examination on salivary cortisol, heart rate, and heart rate variability in mares

Pregnancy diagnostics in equine reproduction are routinely performed using transrectal ultrasonography, although it is also possible to visualize the fetus by transabdominal ultrasound examinations from the 90th day of gestation onward. We hypothesized that ultrasound examinations may stress the mare and that the gestational stage status and lactation may influence the mare's stress reaction. To investigate the stress reaction, 25 thoroughbred mares of different age, pregnancy and lactational status underwent a transrectal examination. In pregnant mares, an additional transabdominal examination was performed. Salivary cortisol concentration, mean heart rate, and heart rate variability of mares were assessed to evaluate the reactions of hypothalamic–pituitary–adrenal (HPA) axis and of the autonomic nervous system. Significant differences were observed between lactating and nonlactating mares; with a lower responsiveness to stress in lactating mares. The transrectal ultrasound examination in nonlactating mares induced a significant increase in salivary cortisol (P < 0.05), and in the heart rate variability parameter, ratio of low to high frequencies (P < 0.05). This reflects an activation of the HPA axis and a shift to more sympathetic dominance. In contrast, a transabdominally performed pregnancy check did not induce an activation of the HPA axis over basal level but increased the mean heart rate and low to high frequency ratio. The results of this study indicate that checks of advanced pregnancies can be easily performed by transabdominal ultrasonography. With regard to animal welfare, this technique should be preferred during midgestation in nonlactating mares.

The implicit affiliation motive moderates cortisol responses to acute psychosocial stress in high school students

It has been previously shown that the implicit affiliation motive – the need to establish and maintain friendly relationships with others – leads to chronic health benefits. The underlying assumption for the present research was that the implicit affiliation motive also moderates the salivary cortisol response to acute psychological stress when some aspects of social evaluation and uncontrollability are involved. By contrast we did not expect similar effects in response to exercise as a physical stressor. Fifty-nine high school students aged M = 14.8 years were randomly assigned to a psychosocial stress (publishing the results of an intelligence test performed), a physical stress (exercise intensity of 65–75% of HRmax), and a control condition (normal school lesson) each lasting 15 min. Participants’ affiliation motives were assessed using the Operant Motive Test and salivary cortisol samples were taken pre and post stressor. We found that the strength of the affiliation motive negatively predicted cortisol reactions to acute psychosocial but not to physical stress when compared to a control group. The results suggest that the affiliation motive buffers the effect of acute psychosocial stress on the HPA axis.

Methylation of NR3C1 is related to maternal PTSD, parenting stress and maternal medial prefrontal cortical activity in response to child separation among mothers with histories of violence exposure.

Prior research has shown that mothers with Interpersonal violence-related posttraumatic stress disorder (IPV-PTSD) report greater difficulty in parenting their toddlers. Relative to their frequent early exposure to violence and maltreatment, these mothers display dysregulation of their hypothalamic pituitary adrenal axis (HPA-axis), characterized by hypocortisolism. Considering methylation of the promoter region of the glucocorticoid receptor gene NR3C1 as a marker for HPA-axis functioning, with less methylation likely being associated with less circulating cortisol, the present study tested the hypothesis that the degree of methylation of this gene would be negatively correlated with maternal IPV-PTSD severity and parenting stress, and positively correlated with medial prefrontal cortical (mPFC) activity in response to video-stimuli of stressful versus non-stressful mother-child interactions. Following a mental health assessment, 45 mothers and their children (ages 12-42 months) participated in a behavioral protocol involving free-play and laboratory stressors such as mother-child separation. Maternal DNA was extracted from saliva. Interactive behavior was rated on the CARE-Index. During subsequent fMRI scanning, mothers were shown films of free-play and separation drawn from this protocol. Maternal PTSD severity and parenting stress were negatively correlated with the mean percentage of methylation of NR3C1. Maternal mPFC activity in response to video-stimuli of mother-child separation versus play correlated positively to NR3C1 methylation, and negatively to maternal IPV-PTSD and parenting stress. Among interactive behavior variables, child cooperativeness in play was positively correlated with NR3C1 methylation. Thus, the present study is the first published report to our knowledge, suggesting convergence of behavioral, epigenetic, and neuroimaging data that form a psychobiological signature of parenting-risk in the context of early life stress and PTSD.

Prospective analysis of adrenal function in patients with acute exacerbations of COPD: the Reduction in the Use of Corticosteroids in Exacerbated COPD (REDUCE) trial

OBJECTIVE To analyze prospectively the hypothalamic-pituitary-adrenal (HPA) axis and clinical outcome in patients treated with prednisone for exacerbated chronic obstructive pulmonary disease (COPD). DESIGN Prospective observational study. SUBJECTS AND METHODS Patients presenting to the emergency department were randomized to receive 40 mg prednisone daily for 5 or 14 days in a placebo-controlled manner. The HPA axis was longitudinally assessed with the 1 μg corticotropin test and a clinical hypocortisolism score at baseline, on day 6 before blinded treatment, at hospital discharge, and for up to 180 days of follow-up. Prednisone was stopped abruptly, irrespective of the test results. Patients discharged with pathological test results received instructions about emergency hydrocortisone treatment. RESULTS A total of 311 patients were included in the analysis. Mean basal and stimulated serum total cortisol levels were highest on admission (496±398 and 816±413 nmol/l respectively) and lowest on day 6 (235±174 and 453±178 nmol/l respectively). Pathological stimulation tests were found in 63, 38, 9, 3, and 2% of patients on day 6, at discharge, and on days 30, 90, and 180 respectively, without significant difference between treatment groups. Clinical indicators of hypocortisolism did not correlate with stimulation test results, but cortisol levels were inversely associated with re-exacerbation risk. There were no hospitalizations or deaths as a result of adrenal crisis. CONCLUSION Dynamic changes in the HPA axis occur during and after the treatment of acute exacerbations of COPD. In hypocortisolemic patients who were provided with instructions about stress prophylaxis, the abrupt termination of prednisone appeared safe.

Predictors of benzodiazepine self -administration behavior in anxious patients

The purpose of this study was to examine factors that may be associated with benzodiazepine (BZ) self-administration and risks of dependence in anxious patients. Preliminary work included examination of psychosocial characteristics and subjective drug response as potential predictors of medication use. Fifty-five M, F patients with generalized anxiety or panic disorder participated in a 3-week outpatient Choice Procedure in which they self-medicated “as needed” with alprazolam (Alz) and placebo. Findings showed that a large amount of variance in alprazolam preference, frequency, and quantity of use could be predicted by measures of anxiety, drug liking, and certain personality characteristics. The primary study extended this work by examining whether individual differences in Alz sensitivity also predict patterns of use. Twenty anxious patients participated in the study, which required 11 weekly clinic visits. Ten of these also participated in a baseline assessment of HPA-axis function that involved 24-hour monitoring of cortisol and ACTH levels and a CRH Stimulation Test. This assessment was conducted on the basis of prior evidence that steroid metabolites exert neuromodulatory effects on the GABA A receptor and that HPA-axis function may be related to BZ sensitivity and long-term disability in anxious patients. Patients were classified as either HIGH or LOW users based on their p.r.n. patterns of Alz use during the first 3 weeks of the study. They then participated in a 4-week dose response trial in which they received prescribed doses of medication (placebo, 0.25, 0.5, and 1.0mg Alz), each taken TID for 1 week. The dose response trial was followed by a second 3-week Choice Procedure. Findings were not indicative of biological differences in Alz sensitivity between the HIGH and LOW users. However, the HIGH users had higher baseline anxiety and greater anxiolytic response to Alz than the LOW users. Anxiolytic benefits of p.r.n. and prescribed dosing were shown to be comparable, and patients' conservative patterns of p.r.n. medication use were not affected by the period of prescribed dosing. Although there was not strong evidence to suggest relationships between HPA-axis function and Alz use or sensitivity, interesting findings emerged about the relationship between HPA-axis function and anxiety. ^

(Table 1) Cortisol concentration in hair of East Greenland polar bears (Ursus maritimus)

To demonstrate the ability to assess long-term hypothalamic-pituitary-adrenocortical (HPA) axis activity in polar bears (Ursus maritimus), a pilot study was conducted in which cortisol concentration was analyzed in hair from 7 female (3-19 years) and 10 male (6-19 years) East Greenland polar bears sampled in 1994-2006. The hair was chosen as matrix as it is non-invasive, seasonally harmonized, and has been validated as an index of long-term changes in cortisol levels. The samples were categorized according to contamination: eight were clean (2 females, 6 males), 5 had been contaminated with bear blood (2 F, 3 M), and 4 with bear fat (3 F, 1 M). There was no significant difference in cortisol concentration between the three categories after external contamination was removed. However, contaminated hair samples should be cleaned before cortisol determination. Average hair cortisol concentration was 8.90 pg/mg (range: 5.5 to 16.4 pg/mg). There was no significant correlation between cortisol concentration and age (p = 0.81) or sampling year (p = 0.11). However, females had higher mean cortisol concentration than males (females mean: 11.0 pg/mg, males: 7.3 pg/mg; p = 0.01). The study showed that polar bear hair contains measurable amounts of cortisol and that cortisol in hair may be used in studies of long-term stress in polar bears.

Early-life exposure to endotoxin alters hypothalamic–pituitary–adrenal function and predisposition to inflammation

We have investigated whether exposure to Gram-negative bacterial endotoxin in early neonatal life can alter neuroendocrine and immune regulation in adult animals. Exposure of neonatal rats to a low dose of endotoxin resulted in long-term changes in hypothalamic–pituitary–adrenal (HPA) axis activity, with elevated mean plasma corticosterone concentrations that resulted from increased corticosterone pulse frequency and pulse amplitude. In addition to this marked effect on the development of the HPA axis, neonatal endotoxin exposure had long-lasting effects on immune regulation, including increased sensitivity of lymphocytes to stress-induced suppression of proliferation and a remarkable protection from adjuvant-induced arthritis. These findings demonstrate a potent and long-term effect of neonatal exposure to inflammatory stimuli that can program major changes in the development of both neuroendocrine and immunological regulatory mechanisms.

Psicobiología del estrés prenatal:implicaciones en el eje hipotálamo-hipofisiario-adrenal de los bebés

The womb is the first developmental environment. After developmental psychobiologists started to investigate intrauterine evolution of infant and its long-term impact, they found that prenatal and postnatal development is influenced by mother’s psychological health. Specifically, scientific research evidence indicates that prenatal stress is a possible cause of subsequent psychopathological vulnerability. This vulnerability comes from stress sensitivity and is the basis of many childhood disorders. In the last decade, there are evidences for a fetal origin of stress sensitivity in the context of the fetal programming theory (Entringer et al., 2009, Grant et al., 2009, Gutteling et al., 2004, Huizink et al., 2004, O’Connor et al., 2005). According to fetal programming hypothesis, babies that have been exposed to high levels of prenatal stress would develop elevated HPA axis reactivity and thus increased stress sensitivity in the postnatal period. In the field of animal psychobiology, several studies have shown that prenatal stress could play some role on fetal programming of neurodevelopment and HPA axis (Glover, 2010, Weinstock, 2005, 2008). In human psychobiology, evidences are less clear (Glover, 2010). Although research in this regard has been growing during the last few years, more studies are warranted to investigate the relationship between maternal stress and fetal programming of neurodevelopment and the HPA axis in humans, to confirm the findings which are evident from animal psychobiology...

Evidence that the Bed Nucleus of the Stria Terminalis Contributes to the Modulation of Hypophysiotropic Corticotropin-Releasing Factor Cell Responses to Systemic Interleukin-1β

Systemic infection activates the hypothalamic-pituitary-adrenal (HPA) axis, and brainstem catecholamine cells have been shown to contribute to this response. However, recent work also suggests an important role for the central amygdala (CeA). Because direct connections between the CeA and the hypothalamic apex of the HPA axis are minimal, the present study investigated whether the bed nucleus of the stria terminalis (BNST) might act as a relay between them. This was done by using an animal model of acute systemic infection involving intravascular delivery of the proinflammatory cytokine interleukin-1 (IL-1, 1 g/kg). Unilateral ibotenic acid lesions encompassing the ventral BNST significantly reduced both IL-1-induced increases in Fos immunoreactivity in corticotropin-releasing factor (CRF) cells of the hypothalamic paraventricular nucleus (PVN) and corresponding increases in adrenocorticotropic hormone (ACTH) secretion. Similar lesions had no effect on CRF cell responses to physical restraint, suggesting that the effects of BNST lesions were not due to a nonspecific effect on stress responses. In further studies, we examined the functional connections between PVN, BNST, and CeA by combining retrograde tracing with mapping of IL-1-induced increases in Fos in BNST and CeA cells. In the case of the BNST, these studies showed that systemic IL-1 administration recruits ventral BNST cells that project directly to the PVN. In the case of the CeA, the results obtained were consistent with an arrangement whereby lateral CeA cells recruited by systemic IL-1 could regulate the activity of medial CeA cells projecting directly to the BNST. In conclusion, the present findings are consistent with the hypothesis that the BNST acts as a relay between the CeA and PVN, thereby contributing to CeA modulation of hypophysiotropic CRF cell responses to systemic administration of IL-1.

Performance and endocrine responses of group housed weaner pigs exposed to the air quality of a commercial environment

The growth performance and endocrine responses of male weaner pigs (3 to 8 weeks of age) was evaluated in two different environments (clean and dirty) and housing (single or groups of 10 pigs/pen) conditions. The dirty environment contained significantly elevated ammonia, carbon dioxide and dust levels compared with the clean environment. Pigs grew faster and consumed more feed in the clean environment and this was associated with reduced plasma cortisol concentrations compared with pigs in the dirty environment. Pigs housed in groups in the dirty environment had increased β-endorphin and decreased IGF-I concentrations compared to group housed pigs in the clean environment. Feed conversion efficiency did not differ due to environment or group housing. Plasma concentration of cortisol, p-endorphin, IGF-I and IGF-II did not differ between single and group housed pigs. Activity of the hypothalamic-pituitary-adrenal (HPA) axis was greater in response to environmental conditions than group housing, and this was associated with reduced growth in weaner pigs. © 2004 Elsevier B.V. All rights reserved.

Down-regulation of central glucocorticoid receptor expression using antisense oligonucleotide technology

Endogenous glucocorticoids and serotonin have been implicated in the pathophysiology of depression, anxiety and schizophrenia. This thesis investigates the potential of downregulating expression of central Type II glucocorticoid receptors (GR) both in vitro and in vivo, with empirically-designed antisense oligodeoxynucleotides (ODN), to characterise GR modulation of 5-HT2A receptor expression using quantitative RT-PCR, Western blot analysis and radioligand binding. The functional consequence of GR downregulation is also determined by measuring 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane hydrochloride (DOI) mediated 5-HT2A receptor specific headshakes. Using a library of random antisense ODN probes, RNAse H accessibility mapping of T7-primed, in vitro transcribed GR mRNA revealed several potential cleavage sites and identified an optimally effect GR antisense ODN sequence of 21-mer length (GRAS5). In vitro efficacy studies using rat C6 glioma cells showed a 56% downregulation in GR mRNA levels and 80% downregulation in GR protein levels. In the same cells a 29% upregulation in 5-HT2A mRNA levels and 32% upregulation in 5-HT2A protein levels was revealed. This confirmed the optimal nature of the GRAS5 sequence to produce marked inhibition of GR gene expression, and also revealed GR modulation of the 50-HT2A receptor subtype in C6 glioma cells to be a tonic repression of receptor expression. The distribution of a fluorescently-labelled GRAS5 ODN was detected in diverse areas of the rat brain after single ICV administration, although this fluorescence signal was not sustained over a period of 5 days. However, fluorescently-labelled GRAS5 ODN, when formulated in polymer microspheres, showed diverse distribution in the brain which was maintained for 5 days following a single ICV administration. This produced no apparent neurotoxic effects on rat behaviour and hypothalamic-pituitary-adrenal (HPA) axis homeostasis. Furthermore, a single polymer microsphere injection ICV proved to be an effective means of delivering antisense ODNs and this was adopted for the in vivo efficacy studies. In vivo characterisation of GRAS5 revealed marked downregulation of GR mRNA in rat brain regions such as the frontal cortex (26%), hippocampus (35%), and hypothalamus (39%). Downregulation of GR protein was also revealed in frontal cortex (67%), hippocampus (76%), and hypothalamus (80%). In the same animals upregulation of 5-HT2A mRNA levels was shown in frontal cortex (13%), hippocampus (7%), and hypothalamus (5%) while upregulation in 5-HT2A protein levels was shown in frontal cortex (21 %). This upregulation in 5-HT2A receptor density as a result of antisense-mediated inhibition of GR was further confirmed by a 55% increase in DOl-mediated 5-HT2A receptor specific headshakes. These results demonstrate that GR is involved in tonic inhibitory regulation of 5-HT2A receptor expression and function in vivo, thus providing the potential to control 5-HT2A-linked disorders through corticosteroid manipulation. These experiments have therefore established an antisense approach which can be used to investigate pharmacological characteristics of receptors.