Validation of a new method to assess the long-term Hypothalamic-Pituitary-Adrenal Activity using hair glucocorticoids as biomarkers. - Studies on pigs and laboratory Sprague-Dawley Rats

The evaluation of chronic activity of the hypothalamic-pituitary-adrenal (HPA) axis is critical for determining the impact of chronic stressful situations. The potential use of hair glucocorticoids as a non-invasive, retrospective, biomarker of long term HPA activity is of great interest, and it is gaining acceptance in humans and animals. However, there are still no studies in literature examining hair cortisol concentration in pigs and corticosterone concentration in laboratory rodents. Therefore, we developed and validated, for the first time, a method for measuring hair glucocorticoids concentration in commercial sows and in Sprague-Dawley rats. Our preliminary data demonstrated: 1) a validated and specific washing protocol and extraction assay method with a good sensitivity in both species; 2) the effect of the reproductive phase, housing conditions and seasonality on hair cortisol concentration in sows; 3) similar hair corticosterone concentration in male and female rats; 4) elevated hair corticosterone concentration in response to chronic stress manipulations and chronic ACTH administration, demonstrating that hair provides a good direct index of HPA activity over long periods than other indirect parameters, such adrenal or thymus weight. From these results we believe that this new non-invasive tool needs to be applied to better characterize the overall impact in livestock animals and in laboratory rodents of chronic stressful situations that negatively affect animals welfare. Nevertheless, further studies are needed to improve this methodology and maybe to develop animal models for chronic stress of high interest and translational value in human medicine.

A feedback-controlled ensemble model of the stress-responsive hypothalamo-pituitary-adrenal axis

The present work develops and implements a biomathematical statement of how reciprocal connectivity drives stress-adaptive homeostasis in the corticotropic (hypothalamo-pituitary-adrenal) axis. In initial analyses with this interactive construct, we test six specific a priori hypotheses of mechanisms linking circadian (24-h) rhythmicity to pulsatile secretory output. This formulation offers a dynamic framework for later statistical estimation of unobserved in vivo neurohormone secretion and within-axis, dose-responsive interfaces in health and disease. Explication of the core dynamics of the stress-responsive corticotropic axis based on secure physiological precepts should help to unveil new biomedical hypotheses of stressor-specific system failure.

Effects of aqueous extract from Asparagus officinalis L. roots on hypothalamic-pituitary-gonadal axis hormone levels and the number of ovarian follicles in adult rats

Background: Asparagus is a plant with high nutritional, pharmaceutical, and industrial values. Objective: The present study aimed to evaluate the effect of aqueous extract of asparagus roots on the hypothalamic-pituitary-gonadal axis hormones and oogenesis in female rats. Materials and Methods: In this experimental study, 40 adult female Wistar rats were divided into five groups, which consist 8 rats. Groups included control, sham and three experimental groups receiving different doses (100, 200, 400 mg/kg/bw) of aqueous extract of asparagus roots. All dosages were administered orally for 28 days. Blood samples were taken from rats to evaluate serum levels of Gonadotropin releasing hormone (GnRH), follicular stimulating hormone (FSH), Luteinal hormone (LH), estrogen, and progesterone hormones. The ovaries were removed, weighted, sectioned, and studied by light microscope. Results: Dose-dependent aqueous extract of asparagus roots significantly increased serum levels of GnRH, FSH, LH, estrogen, and progestin hormones compared to control and sham groups. Increase in number of ovarian follicles and corpus luteum in groups treated with asparagus root extract was also observed (p<0.05). Conclusion: Asparagus roots extract stimulates secretion of hypothalamic- pituitary- gonadal axis hormones. This also positively affects oogenesis in female rats.

Infralimbic cortex controls the activity of the hypothalamus-pituitary adrenal axis and the formation of aversive memory: effects of environmental enrichment

The aim of the present study was to investigate the effects of the stimulation and inhibition of the ventral part of the medial prefrontal cortex (infralimbic cortex) on basal and stress-induced plasma levels of corticosterone and on the acquisition of aversive memory in animals maintained in control and environmental enrichment (EE) conditions. Intracortical microinjections of the GABAA antagonist picrotoxin and agonist muscimol were performed in male Wistar rats to stimulate and inhibit, respectively, the activity of the infralimbic cortex. Injections were performed 60 min before foot shock stress and training in the inhibitory avoidance task. Picrotoxin injections into the infralimbic cortex increased basal plasma levels of corticosterone. These increases were higher in EE rats which suggest that EE enhances the control exerted by infralimbic cortex over the hypothalamus-pituitary-adrenal (HPA) axis and corticosterone release. Muscimol injections into the infralimbic cortex reduced the stress-induced plasma levels of corticosterone and the retention latency 24 h after training in the inhibitory avoidance performance in control and EE animals, respectively. These results further suggest that the infralimbic cortex is required for the activation of the HPA axis during stress and for the acquisition of contextual aversive memories.

Mice behaviorally selected for high Pavlovian fear memory exhibit fear generalization, HPA-axis alterations and increased basal activity in cortico-limbic circuits

Biological factors underlying individual variability in fearfulness and anxiety have important implications for stress-related psychiatric illness including PTSD and major depression. Using an advanced intercross line (AIL) derived from C57BL/6 and DBA/2J mouse strains and behavioral selection over 3 generations, we established two lines exhibiting High or Low fear behavior after fear conditioning. Across the selection generations, the two lines showed clear differences in training and tests for contextual and conditioned fear. Before fear conditioning training, there were no differences between lines in baseline freezing to a novel context. However, after fear conditioning High line mice demonstrated pronounced freezing in a new context suggestive of poor context discrimination. Fear generalization was not restricted to contextual fear. High fear mice froze to a novel acoustic stimulus while freezing in the Low line did not increase over baseline. Enhanced fear learning and generalization are consistent with transgenic and pharmacological disruption of the hypothalamic-pituitary-adrenal axis (HPA-axis) (Brinks, 2009, Thompson, 2004, Kaouane, 2012). To determine whether there were differences in HPA-axis regulation between the lines, morning urine samples were collected to measure basal corticosterone. Levels of secreted corticosterone in the circadian trough were analyzed by corticosterone ELISA. High fear mice were found to have higher basal corticosterone levels than low line animals. Examination of hormonal stress response components by qPCR revealed increased expression of CRH mRNA and decreased mRNA for MR and CRHR1 in hypothalamus of high fear mice. These alterations may contribute to both the behavioral phenotype and higher basal corticosterone in High fear mice. To determine basal brain activity in vivo in High and Low fear mice we used manganese-enhanced magnetic resonance imaging (MEMRI). Analysis revealed a pattern of basal brain activity made up of amygdala, cortical and hippocampal circuits that was elevated in the High line. Ongoing studies also seek to determine the relative balance of excitatory and inhibitory tone in the amygdala and hippocampus and the neuronal structure of its neurons. While these heterogeneous lines are selected on fear memory expression, HPA-axis alterations and differences in hippocampal activity segregate with the behavioral phenotypes. These differences are detectable in a basal state strongly suggesting these are biological traits underlying the behavioral phenotype (Johnson et al, 2011).

The relationship between stress, HPA axis functioning and brain structure in first episode psychosis over the first 12 weeks of treatment

Stress and abnormal hypothalamic-pituitary-adrenal axis functioning have been implicated in the early phase of psychosis and may partly explain reported changes in brain structure. This study used magnetic resonance imaging to investigate whether biological measures of stress were related to brain structure at baseline and to structural changes over the first 12 weeks of treatment in first episode patients (n=22) compared with matched healthy controls (n=22). At baseline, no significant group differences in biological measures of stress, cortical thickness or hippocampal volume were observed, but a significantly stronger relationship between baseline levels of cortisol and smaller white matter volumes of the cuneus and anterior cingulate was found in patients compared with controls. Over the first 12 weeks of treatment, patients showed a significant reduction in thickness of the posterior cingulate compared with controls. Patients also showed a significant positive relationship between baseline cortisol and increases in hippocampal volume over time, suggestive of brain swelling in association with psychotic exacerbation, while no such relationship was observed in controls. The current findings provide some support for the involvement of stress mechanisms in the pathophysiology of early psychosis, but the changes are subtle and warrant further investigation.

Pituitary volume in teenagers with first-presentation borderline personality disorder

This study used magnetic resonance imaging to examine pituitary gland volume (PGV) in teenage patients with a first presentation of borderline personality disorder (BPD). No difference in PGV was observed between healthy controls (n=20) and the total BPD cohort (n=20). However, within the BPD cohort, those exposed to childhood trauma (n=9) tended to have smaller pituitaries (-18%) than those with no history of childhood trauma (n=10). These preliminary findings suggest that exposure to childhood trauma, rather than BPD, per se, might be associated with reduced PGV, possibly reflecting hypothalamic-pituitary-adrenal axis dysfunction.

Pituitary volume predicts future transition to psychosis in individuals at ultra-high risk of developing psychosis

Background We examined pituitary volume before the onset of psychosis in subjects who were at ultra-high risk (UHR) for developing psychosis. Methods Pituitary volume was measured on 1.5-mm, coronal, 1.5-T magnetic resonance images in 94 UHR subjects recruited from admissions to the Personal Assessment and Crisis Evaluation Clinic in Melbourne, Australia and in 49 healthy control subjects. The UHR subjects were scanned at baseline and were followed clinically for a minimum of 1 year to detect transition to psychosis. Results Within the UHR group, a larger baseline pituitary volume was a significant predictor of future transition to psychosis. The UHR subjects who later went on to develop psychosis (UHR-P, n = 31) had a significantly larger (+12%; p = .001) baseline pituitary volume compared with UHR subjects who did not go on to develop psychosis (UHR-NP, n = 63). The survival analysis conducted by Cox regression showed that the risk of developing psychosis during the follow-up increased by 20% for every 10% increase in baseline pituitary volume (p = .002). Baseline pituitary volume of the UHR-NP subjects was smaller not only compared with UHR-P (as described above) but also compared with control subjects (−6%; p = .032). Conclusions The phase before the onset of psychosis is associated with a larger pituitary volume, suggesting activation of the HPA axis.

Waterborne exposure to PFOS causes disruption of the hypothalamus-pituitary-thyroid axis in zebrafish larvae

Thyroid hormones (THs) play an important role in the normal development and physiological functions in fish. Environmental chemicals may adversely affect thyroid function by disturbing gene transcription. Perfluorooctane sulfonate (PFOS), a persistent compound, is widely distributed in the aquatic environment and wildlife. In the present study, we investigated whether PFOS could disrupt the hypothalamic-pituitary-thyroid (HPT) axis. Zebrafish embryos were exposed to various concentrations of PFOS (0, 100, 200 and 400 mu g L-1) and gene expression patterns were examined 15 d post-fertilization. The expression of several genes in the HIPT system, i.e., corticotropin-releasing factor (CRF), thyroid-stimulating hormone (TSH), sodium/iodide symporter (NIS), thyroglobulin (TG), thyroid peroxidase (TPO), transthyretin (TTR), ioclothyronine deiodinases (Dio1 and Dio2) and thyroid receptor (TR alpha and TR beta), was quantitatively measured using real-time PCR. The gene expression levels of CRF and TSH were significantly up-regulated and down-regulated, respectively, upon exposure to 200 and 400 mu g L-1 PFOS. A significant increase in NIS and Diol gene expression was observed at 200 mu g L-1 PFOS exposure, while TG gene expression was down-regulated at 200 and 400 mu g L-1 PFOS exposure. TTR gene expression was down-regulated in a concentration-dependent manner. Up-regulation and down-regulation of TR alpha and TR beta gene expression, respectively, was observed upon exposure to PFOS. The whole body thyroxine (T-4) content remained unchanged, whereas triiodothyronine (T-3) levels were significantly increased, which could directly reflect disrupted thyroid hormone status after PFOS exposure. The overall results indicated that PFOS exposure could alter gene expression in the HPT axis and that mechanisms of disruption of thyroid status by PFOS could occur at several steps in the synthesis, regulation, and action of thyroid hormones. (C) 2009 Elsevier Ltd. All rights reserved.

Waterborne exposure to PFOS causes disruption of the hypothalamus-pituitary-thyroid axis in zebrafish larvae

Thyroid hormones (THs) play an important role in the normal development and physiological functions in fish. Environmental chemicals may adversely affect thyroid function by disturbing gene transcription. Perfluorooctane sulfonate (PFOS), a persistent compound, is widely distributed in the aquatic environment and wildlife. In the present study, we investigated whether PFOS could disrupt the hypothalamic– pituitary–thyroid (HPT) axis. Zebrafish embryos were exposed to various concentrations of PFOS (0, 100, 200 and 400 lg L 1) and gene expression patterns were examined 15 d post-fertilization. The expression of several genes in the HPT system, i.e., corticotropin-releasing factor (CRF), thyroid-stimulating hormone (TSH), sodium/iodide symporter (NIS), thyroglobulin (TG), thyroid peroxidase (TPO), transthyretin (TTR), iodothyronine deiodinases (Dio1 and Dio2) and thyroid receptor (TRa and TRb), was quantitatively measured using real-time PCR. The gene expression levels of CRF and TSH were significantly up-regulated and down-regulated, respectively, upon exposure to 200 and 400 lg L 1 PFOS. A significant increase in NIS and Dio1 gene expression was observed at 200 lg L 1 PFOS exposure, while TG gene expression was down-regulated at 200 and 400 lg L 1 PFOS exposure. TTR gene expression was down-regulated in a concentration-dependent manner. Up-regulation and down-regulation of TRa and TRb gene expression, respectively, was observed upon exposure to PFOS. The whole body thyroxine (T4) content remained unchanged, whereas triiodothyronine (T3) levels were significantly increased, which could directly reflect disrupted thyroid hormone status after PFOS exposure. The overall results indicated that PFOS exposure could alter gene expression in the HPT axis and that mechanisms of disruption of thyroid status by PFOS could occur at several steps in the synthesis, regulation, and action of thyroid hormones.

Systemic apomorphine alters HPA axis responses to interleukin-1 beta adminstration but not sound stress

Apomorphine is a dopamine receptor agonist that was recently licensed for the treatment of erectile dysfunction. However, although sexual activity can be stressful, there has been little investigation into whether treatments for erectile dysfunction affect stress responses. We have examined whether a single dose of apomorphine, sufficient to produce penile erections (50 mug/kg, i.a.), can alter basal or stress-induced plasma ACTH levels, or activity of central pathways thought to control the hypothalamic-pituitary-adrenal axis in rats. An immune challenge (interleukin-1beta, 1 mug/kg, i.a.) was used as a physical stressor while sound stress (100 dB white noise, 30 min) was used as a psychological stressor. Intravascular administration of apomorphine had no effect on basal ACTH levels but did substantially increase the number of Fos-positive amygdala and nucleus tractus solitarius catecholamine cells. Administration of apomorphine prior to immune challenge augmented the normal ACTH response to this stressor at 90 min and there was a corresponding increase in the number of Fos-positive paraventricular nucleus corticotropin-releasing factor cells, paraventricular nucleus oxytocin cells and nucleus tractus solitarius catecholamine cells. However, apomorphine treatment did not alter ACTH or Fos responses to sound stress. These data suggest that erection-inducing levels of apomorphine interfere with hypothalamic-pituitary-adrenal axis inhibitory feedback mechanisms in response to a physical stressor, but have no effect on the response to a psychological stressor. Consequently, it is likely that apomorphine acts on a hypothalamic-pituitary-adrenal axis control pathway that is unique to physical stressors. A candidate for this site of action is the nucleus tractus solitarius catecholamine cell population and, in particular, A2 noradrenergic neurons. (C) 2003 Elsevier Science Ltd. All rights reserved.

Medial prefrontal cortex suppression of the hypothalamic–pituitary–adrenal axis response to a physical stressor, systemic delivery of interleukin-1β

Previous studies have shown that the medial prefrontal cortex can suppress the hypothalamic-pituitary-adrenal axis response to stress. However, this effect appears to vary with the type of stressor. Furthermore, the absence of direct projections between the medial prefrontal cortex and corticotropin-releasing factor cells at the apex of the hypothalamic-pituitary-adrenal axis suggest that other brain regions must act as a relay when this inhibitory mechanism is activated. In the present study, we first established that electrolytic lesions involving the prelimbic and infralimbic medial prefrontal cortex increased plasma adrenocorticotropic hormone levels seen in response to a physical stressor, the systemic delivery of interleukin-1beta. However, medial prefrontal cortex lesions did not alter plasma adrenocorticotropic hormone levels seen in response to a psychological stressor, noise. To identify brain regions that might mediate the effect of medial prefrontal cortex lesions on hypothalamic-pituitary-adrenal axis responses to systemic interleukin-1beta, we next mapped the effects of similar lesions on interleukin-1beta-induced Fos expression in regions previously shown to regulate the hypothalamic-pituitary-adrenal axis response to this stressor. It was found that medial prefrontal cortex lesions reduced the number of Fos-positive cells in the ventral aspect of the bed nucleus of the stria terminalis. However, the final experiment, which involved combining retrograde tracing with Fos immunolabelling, revealed that bed nucleus of the stria terminalis-projecting medial prefrontal cortex neurons were largely separate from medial prefrontal cortex neurons recruited by systemic interleukin-1beta, an outcome that is difficult to reconcile with a simple medial prefrontal cortex-bed nucleus of the stria terminalis-corticotropin-releasing factor cell control circuit.