Both cortisol and corticosterone circulate in human plasma however corticosterone has been relatively neglected in human research to date. There is evidence of distinct regulation within different tissues with the transmembrane transporter ABCB1, highly expressed in the brain, exporting cortisol but not corticosterone. This may account for the relative accumulation of corticosterone in the CNS. In contrast, ABCC1, highly expressed in adipose tissue and skeletal muscle, exports corticosterone but not cortisol, suggesting cortisol is the principal glucocorticoid acting in these tissues. We tested the hypotheses that: (i) corticosterone physiology in humans is different to that of cortisol; (ii) inhibition of ABCC1 increases binding of corticosterone to corticosteroid receptors in adipose tissue and skeletal muscle but has no central CNS effect; and (iii) corticosterone is superior to cortisol as a basis for glucocorticoid replacement therapy with fewer metabolic side effects. We compared paired salivary and plasma samples from 10 healthy individuals. Plasma corticosterone showed a similar diurnal variation to cortisol but salivary corticosterone was low and did not correlate with plasma concentrations. A placebo-controlled randomised crossover study was carried out in 14 healthy individuals comparing receptor occupancy of glucocorticoids centrally and peripherally with and without ABCC1 inhibition. Receptor occupancy was assessed through displacement with MR and GR antagonists potassium canrenoate and mifepristone. Centrally, ABCC1 inhibition caused increased activation of the HPA axis after MR and GR antagonism. Peripherally, we were unable to show displacement from adipose tissue or skeletal muscle. A further placebo-controlled randomised crossover study is still ongoing in 16 patients with congenital adrenal hyperplasia, comparing metabolic effects of placebo, cortisol and corticosterone infusions over 6 hours. We present interim data for n=8. ACTH and 17-OHP were suppressed with corticosterone. Metabolic parameters were similar between placebo, cortisol and corticosterone phases. These data suggest corticosterone physiology is distinct compared with cortisol in humans. We have shown ABCC1 inhibition alters the HPA axis after receptor antagonism which suggests ABCC1 may play more of a key role centrally than previously thought. Corticosterone suppresses ACTH and 17-OHP in the short term in congenital adrenal hyperplasia, highlighting the possibility of its use as an alternative glucocorticoid replacement therapy in the future.

acase@tulane.edu / Stress is a major determinant of quality of life and chronic stress plays an important contributing role in the occurrence of psychiatric and physiological pathologies. Ascending brainstem noradrenergic afferents provide a critical excitatory drive to the hypothalamic-pituitary-adrenal (HPA) axis in response to stress by activating corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN). The stimulatory role of noradrenergic synapses on CRH neurons in the regulation of the HPA axis has long been known, but the cellular mechanisms have been perplexing. We demonstrate a retrograde inter-neuronal communication stimulated by norepinephrine that utilizes a trans-astroglial signaling mechanism to activate upstream neurons and recurrent synaptic inputs. We found that NE activates postsynaptic 1-adrenoceptors in PVN CRH neurons, which triggers a calcium response in astrocytes via the dendritic release of vasopressin. Activated astrocytes stimulate upstream glutamate neurons via ATP release and P2X receptor activation, resulting in the recurrent excitation of the CRH neurons. The NE excitation of CRH neurons is strengthened by simultaneous presynaptic 2-adrenoceptor-mediated suppression of GABA release but is restrained at higher NE concentrations by activation of upstream GABAergic neurons via the same postsynaptic α1-adrenoceptor-mediated retrograde neuronal-glial signaling mechanism. Thus, the NE stimulation of CRH neurons in the PVN is mediated by a novel retrograde signaling mechanism that enlists a trans-neuronal-astroglial circuit to activate upstream glutamate and GABA neurons. This mechanism is stress-sensitive. Acute stress desensitizes this α1-receptor mediated circuit via glucocorticoid receptor-dependent signaling. The desensitization of the excitatory circuit, through which NE exerts its major stimulatory drive to CRH neurons, indicates a cellular mechanism or a target site of negative feedback. Chronic stress desensitized the CRH neurons to both α1 and α2-adrenoceptor activation. Thus, the noradrenergic regulation of CRH neurons was lost entirely following chronic stress, rendering the CRH neurons completely insensitive to this major regulatory input. Our research revealed the cellular mechanisms of the NE regulation of CRH neurons under control, acute stress and chronic stress conditions. It fills an important gap in knowledge concerning the noradrenergic excitatory drive to the CRH neurons and the stress-induced glucocorticoid feedback control of the HPA axis. / 1 / Chun Chen

No description available.

No description available.

This study creates and analyzes a model of the Hypothalamus-Pituitary-Adrenal axis to better understand cortisol rhythmicity perpetuated by circadian inputs, system dynamics and feedback inherent within the system. Differential equations are created to model human physiology with cortisol and precursor hormone outputs fit to physiologic data. The model is created with an input of circadian cues from the hypothalamus which are designed to create a more realistic stimulation of the cortisol cascade over predecessors. The study also incorporates additional signaling pathways unique to this model. The project explores the properties of the model under mathematical analysis; then, the simulation of known medical pathologies is used to analyze the model's predictive ability. It is found that incorporating the additional signaling pathway of Arginine Vasopressin increases the model's predictive capability in certain pathological conditions over predecessor models. Additionally, the origination of ultradian rhythm is explored through simulation and two possible explanations are found. First, pulsatile release of Adrenocorticotropic Hormone combined with negative feedback into the system from glucocorticoid receptors elicits the observed ultradian oscillations in humans. Additionally, simulations of increased hypothalamic monitoring and control of cortisol concentrations create a natural oscillation within the desired period. Results from numerical perturbation simulations and dynamic sensitivity analysis are employed to offer justification for known pathological conditions developing from circadian dysregulation. / Master of Science / This study aims to better understand the body's natural cortisol rhythm by creating a mathematical model of the Hypothalamus-Pituitary-Adrenal axis. The model uses differential equations to simulate human physiology and includes circadian cues from the suprachiasmatic nucleus to create a more accurate representation of how cortisol is released in the body. The study also incorporates additional signaling pathways and interactions unique to this model. By analyzing the model and simulating known medical conditions, it was found found that incorporating these additional signaling pathways improved the model's predictive ability in certain situations. Then, numerical simulations were used to investigate how circadian dysregulation can lead to pathological conditions.The study also explored the origin of ultradian rhythm, or short-term fluctuations in cortisol levels, and found two possible explanations. One explanation is the pulsatile release of Adrenocorticotropic Hormone combined with negative feedback from glucocorticoid receptors. Another explanation is increased hypothalamic control of cortisol concentrations. Overall, this study provides insights into the complex dynamics of the Hypothalamus-Pituitary-Adrenal axis and the origination of pathology in the system.

Callous-unemotional (CU) traits have been shown to designate a particular subgroup of antisocial youth that are particularly violent, recidivistic, and more likely to continue offending in adulthood. Disordered neuroendocrine function may be a mechanism for the development of CU traits. We examined whether altered stress responsivity served as a mechanism linking stress exposure and the expression of CU traits. Participants were 15 incarcerated adolescent girls with CU traits. Measures of CU traits, stress exposure, and salivary cortisol were collected. Results revealed girls with CU traits had higher morning levels of cortisol, an intact cortisol awakening response (CAR), and flatter diurnal rhythms. Results indicated the type of stressor being measured and time since stressor onset are crucial to the interpretation of neuroendocrine function. We also found support for a neurobiological model for the development of CU traits drawing on the Adaptive Calibration Model. Implications of the study and directions for further research are discussed.

Os Transtornos por Uso de Substâncias (TUS) são propostos pela Associação Norte-Americana de Psiquiatria e englobam diversas dependências químicas, incluindo o tabagismo e a dependência de crack. O uso de crack tem aumentado consideravelmente nos últimos anos, segundo dados de levantamentos brasileiros e mundiais. Com mecanismo de ação semelhante ao da cocaína, porém com efeito mais rápido, o crack é uma droga que causa grave prejuízo a seus usuários e conduz à dependência em pouco tempo. O tabagismo também é uma condição bastante prevalente no mundo inteiro, sendo que os prejuízos relacionados a esse transtorno envolvem principalmente o desenvolvimento de doenças respiratórias e câncer. Diversos trabalhos têm buscado identificar fatores de suscetibilidade a esses transtornos, sendo que estudos de associação envolvendo genes do sistema de resposta a estresse vêm ganhando importância. O eixo Hipotálamo- Pituitária-Adrenal (HPA) é o sistema de resposta a estresse mais conhecido em humanos e alvo do presente trabalho, uma vez que o funcionamento dessa cascata de sinalização da produção de cortisol pode desempenhar um papel relevante no desenvolvimento de psicopatologias. Além disso, o eixo HPA pode ser considerado um mediador da relação entre situações adversas na infância e o surgimento de TUS. O objetivo desse trabalho, portanto, é investigar o papel de polimorfismos no gene CRHR1 (que codifica o receptor tipo 1 do hormônio liberador de corticotrofina – CRH, hormônio inicial do eixo HPA) sobre o desenvolvimento e características clínicas da dependência química. Para isso, foram realizados dois estudos, envolvendo amostras de mulheres. No primeiro artigo, a partir de três amostras – (1) 136 usuárias de crack, (2) 55 tabagistas e (3) 262 voluntárias saudáveis – foram identificadas diferenças de freqüências alélicas e genotípicas do SNP rs12944712; além disso, a análise de haplótipos envolvendo tal SNP e o rs110402 revelou maior frequência do haplótipo contendo o alelo G nos dois polimorfismos nos grupos de usuárias de crack e tabagistas. No segundo artigo, foi avaliado o efeito da interação geneambiente envolvendo os mesmos polimorfismos e situações adversas na infância – estimada pela escala Childhood Trauma Questionnaire (CTQ) –sobre características clínicas da amostra de usuárias de crack, como craving e gravidade da dependência – estimados, respectivamente, pelas escalas Cocaine Selective Severity Assessment (CSSA) e Addiction Severity Index (ASI6). A análise não revelou uma interação significativa. A presente dissertação, portanto, revela um efeito do gene CRHR1 sobre o desenvolvimento de TUS, embora esse efeito pareça não se manifestar sobre a gravidade. Mais estudos são necessários visando esclarecer o real efeito do gene e do eixo HPA como um todo sobre os fenótipos e comportamentos aditivos, bem como os mecanismos subjacentes a essa relação. / The Substance Use Disorders (SUD) are proposed by the American Psychiatric Association and comprise several addictions, including tobacco and crack cocaine smoking. The prevalence of crack cocaine dependence has grown considerably, according to survey data from Brazil and worldwide. With mechanism of action similar to cocaine but with faster effect, crack is a drug that causes severe impairments to its users and leads to dependence in a short time. Tobacco smoking is also a highly prevalent condition worldwide, and the impact related to this disorder includes cancer and respiratory diseases. Several studies have attempted the identification of susceptibility factors to these disorders, and association studies involving stress response system genes have been performed. The Hypothalamic-Pituitary-Adrenal (HPA) axis is the best known stress response system, and the signaling cascade production of cortisol may play a role in the development of psychopathology. In addition, HPA axis can be considered a mediator of the relationship between early life adversity and SUD. The aim of this study is to investigate the role of polymorphisms in CRHR1 gene (encoding the corticotrophin releasing factor – CRH – receptor type 1, an initial factor in HPA axis response) on the development and clinical traits of drug dependence. Two studies were conducted on women samples. In the first article, three groups were compared - (1) 136 crack smokers, (2) 55 tobacco smokers and (3) 262 healthy volunteers. Differences in allele and genotype frequencies of the SNP rs12944712 were identified; in addition, the haplotype analysis involving this SNP and rs110402 revealed a higher frequency of the haplotype containing G allele in both polymorphisms in the groups of nicotine dependents and crack smokers. In the second article, we evaluated the effect of a gene-environment interaction involving the same polymorphisms and childhood adversity - estimated by Childhood Trauma Questionnaire (CTQ) scale – on clinical characteristics of the sample of crack users, such as craving and severity of dependence - estimated respectively by the Cocaine Selective Severity Assessment (CSSA) and Addiction Severity Index (ASI6) scales. The analysis did not reveal a significant interaction. This dissertation thus shows an effect of CRHR1 genepolymorphism on the development of SUD, although this effect does not seem to impact disorder severity. More studies are needed in order to clarify the actual effect of the gene and of the HPA axis as a whole on addictive behaviors.

Em função da categoria dos estressores, vias neurais específicas são envolvidas e respostas distintas podem ser induzidas. A literatura tem reportado que o estrógeno (E 2 ) através de seus receptores de tipos (ER) e (ER) influencia a atividade do eixo hipotálamo hipófise adrenal (HPA). Além disso, há evidências de que o E2 exerça efeitos protetores em situação de choque hemorrágico. O objetivo deste trabalho foi verificar a participação dos receptores ER e ER na atividade do eixo HPA durante estresse hemorrágico. Foram utilizadas ratas Wistar ovariectomizadas que receberam injeções s.c. de DMSO (veículo), PPT (agonista ER) ou DPN (agonista ER), durante 3 dias. No segundo dia, as ratas foram canuladas para coleta seriada de sangue na manhã seguinte. Os animais receberam (controle) ou não (hemorrágicos) reposição imediata com salina. Os hormônios corticosterona (CORT), ocitocina (OT) e vasopressina (AVP) foram dosados por radioimunoensaio. Ao final do experimento, os ratos foram perfundidos e os cérebros processados para imuno-histoquímica de FOS, tirosina hidroxilase (TH) e hormônio liberador de corticotrofina (CRH). Nos animais tratados com veículo, a hemorragia gradual moderada aumentou a secreção de CORT, OT e AVP, a expressão de neurônios TH ativados na região A1C1 e de FOS no mpPVN. O PPT reduziu a secreção de CORT, na situação controle atuando no LC e mpPVN; e também após hemorragia atuando no LC, NTS, A1C1 e mpPVN. O DPN reduziu a secreção de CORT apenas após estresse hemorrágico atuando no LC, A1C1 e mpPVN. O PPT bloqueou o aumento da secreção de OT e aumentou a secreção de AVP, após hemorragia. O DPN, por sua vez, reduziu a concentração plasmática de OT e aumentou a concentração plasmática de AVP, independentemente da hemorragia. Em conclusão: o estrógeno pode exercer uma ação inibitória sobre a secreção basal de CORT somente através da ação do ER sobre o LC e mpPVN; a secreção de CORT aumenta em resposta à hemorragia gradual moderada e o estrógeno pode exercer um controle inibitório nessa resposta através de ER atuando sobre LC, NTS, A1C1 e mpPVN, bem como através de ER atuando sobre LC, A1C1 e mpPVN. / Depending on the stressors category, specific neural pathways are involved and different responses can be selected. It has been reported in the literature that estrogen (E2 ) can affect hypothalamus pituitary adrenal (HPA) axis activity through its receptors type (ER) and (ER). Moreover, there is evidence that E 2 has protecting properties after hemorrhagic shock. The aim of this work was to assess the participation of ER and ER on HPA axis activity during hemorrhagic stress. It was used ovariectomized Wistar rats that received s.c. injections of: DMSO (vehicle), PPT (ER agonist) or DPN (ER agonist), during 3 days. In the second day the rats were catheterized for serial blood collect in the next morning. Animals received (control) or not (hemorrhagic) immediate reposition with same volume of isotonic saline. The hormones corticosterone (CORT), oxytocin (OT) and vasopressin (AVP) were measured by radioimmunoassay. At the end of the experiment, animals were perfused and their brains were processed for immuno-histochemistry for FOS, tyrosine hydroxylase (TH) and corticotropin releasing hormone (CRH). In vehicle treated animals, the gradual hemorrhage enhanced CORT, OT and AVP secretion, TH activated neurons expression in A1C1 and FOS expression in mpPVN. PPT decreased plasma CORT in control situation acting on LC and mpPVN, and also after hemorrhage acting on LC, NTS, A1C1 and mpPVN. DPN reduced plasma CORT only after hemorrhagic stress acting on LC, A1C1 and mpPVN. PPT blocked the increase of OT secretion and increased AVP secretion, after hemorrhage. The agonist DPN reduced OT and increased AVP levels, despite hemorrhage. In conclusion: E2 can exert an inhibitory effect on CORT basal secretion only through ER action on LC and mpPVN; CORT secretion increases after gradual moderate hemorrhage and E2 inhibit this secretion through ER action on LC, NTS, A1C1 and mpPVN, as well through ER action on LC, A1C1 and mpPVN.

Corticotroph cells from the anterior pituitary are an integral component of the hypothalamic-pituitary-adrenal (HPA) axis, which controls the neuroendocrine response to stress. Following stressful stimuli, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) from the hypothalamus act synergistically to stimulate adrenocortiotrophin hormone (ACTH) secretion from corticotroph cells. ACTH is released into the circulation where it stimulates the secretion of glucocorticoids from the adrenal cortex. The HPA axis is kept in fine balance through an elegant negative feedback system where elevation of plasma glucocorticoids results in inhibition at the level of both the pituitary and the hypothalamus. During acute stress, glucocorticoids can be beneficial however chronic elevation of glucocorticoids can have many adverse effects on health. Corticotroph cells are electrically excitable and have been shown to fire single-spike action potentials as well as complex bursting patterns. Stimulation of corticotrophs with physiological concentrations of CRH/AVP results in a robust increase in firing frequency and a transition from spiking to bursting. Intracellular Ca2+ increases to a greater extent during bursting which has been proposed to drive hormone secretion. There is evidence to suggest that large conductance calcium- and voltage-gated potassium (BK) channels promote bursting behaviour in anterior pituitary cells. Glucocorticoids have been shown to regulate ACTH secretion and also modulate BK channel activity. However, the effects of glucocorticoids on native corticotroph excitability are currently unknown. The aim of this study was to first characterise the electrical properties of corticotrophs under basal conditions and following exposure to CRH/AVP. Secondly, to investigate the regulation of corticotroph excitability by glucocorticoids. Finally, establish the role of the BK channel in regulating bursting behaviour and CORT regulation in corticotroph cells. Corticotroph cells were acutely isolated by trypsin digestion from mice aged 2-5 months constitutively expressing GFP under control of the POMC promoter (POMC-GFP). Mice used for pituitary cell culture were male unless otherwise stated. Cells were maintained in a serum free media and electrophysiological recordings obtained 24-96 hours post-isolation. Current clamp recordings were obtained from corticotrophs using the perforated patch technique. Although spontaneous activity of corticotroph cells was variable, they displayed predominantly single-spike action potentials under basal conditions. Stimulation with physiological concentrations of CRH and AVP (0.2 nM and 2 nM respectively) resulted in a membrane depolarisation accompanied by an increase in firing frequency and a transition to bursting. Individually, CRH and AVP were able to increase corticotroph excitability. However, only CRH was able to drive an increase in bursting suggesting that bursting is primarily regulated through the cAMP/PKA pathway. Experiments were performed to investigate the modulation of corticotroph activity by glucocorticoid negative feedback. Acute exposure (< 10 min) to corticosterone resulted in a decrease in spontaneous activity as well as shortening the response to CRH/AVP. Pretreatment of corticotrophs with 100 nM corticosterone (90 min) resulted in a membrane hyperpolarisation and a decrease in spontaneous firing frequency. Following corticosterone pretreatment, CRH/AVP failed to induce a significant transition from spiking to bursting. Increasing the pretreatment time to 150 minutes resulted in a further suppression of both spontaneous and CRH/AVPevoked activity. Fast activation of BK channels during the upstroke of an action potential has been proposed to promote bursting behaviour in other pituitary cells. Corticotrophs treated with a BK channel blocker (1 μM paxilline) or isolated from BK-/- mice showed no significant difference in basal activity but displayed a reduction in CRH/AVPevoked bursting activity. In both cases, bursting was significantly reduced but not completely abolished. Corticosterone treatment of BK-/- cells resulted in a further decrease in both firing frequency and bursting behaviour. Taken together, these results suggest that although BK channels play an important role in bursting, they are not the only component. Comparisons of male and female corticotrophs revealed subtle differences in their properties. Following CRH/AVP stimulation, male cells displayed a high degree of bursting activity whereas female cells exhibited predominantly an increase in singlespike action potential frequency. Treatment of female corticotrophs with corticosterone (150 min) resulted in a significant reduction in firing frequency but no measurable change in bursting behaviour. BK-/- cells from female mice showed no difference in bursting activity following CRH/AVP compared to wild types. This data suggests that modulation of firing frequency is the more important component in female corticotroph cells. In conclusion, CRH/AVP is proposed to drive ACTH secretion in male corticotroph cells through an increase in bursting activity. Corticosterone pretreatment suppresses both spontaneous and CRH/AVP-evoked activity. It is possible that corticosterone regulates corticotroph excitability through two mechanisms. Corticosterone suppresses bursting activity following CRH/AVP stimulation through multiple targets which might include the BK channel. Additionally, corticosterone reduces firing frequency through a mechanism independent of BK channels. It is important to further characterise the physiology of corticotroph cells and how ACTH secretion is regulated through their electrical excitability. This would lead to a greater understanding of the role of corticotrophs in the HPA axis. Further study of corticotrophs could potentially lead to pharmacological manipulation of the stress response and novel treatments for stress-related disorders.

Epidemiological studies in humans have revealed a relationship between altered development in utero and an increased incidence of pathophysiology during postnatal life. One of the mechanisms underlying this relationship is thought to be exposure to excess glucocorticoids during critical phases of brain development. The aim of the current set of studies was to determine the effects of prenatal stress during discrete developmental windows on behaviour and endocrine function in male and female guinea pig offspring. Guinea pigs were used as the model for these studies as they are a long-gestation species that give birth to neuroanatomically mature young and fetal brain development is well characterized. Pregnant guinea pigs were exposed to a high frequency strobe light during the period of rapid fetal brain growth or the period of rapid brain myelination. Pregnant guinea pigs were allowed to deliver normally and guinea pig offspring were tested for ambulatory activity, anxiety and hypothalamic-pituitary-adrenal (HPA) axis function. Male offspring whose mothers were exposed to stress during the period of rapid brain growth exhibited increased anxiety behaviour, increased basal plasma cortisol levels and decreased plasma testosterone levels. We found that replacing testosterone in these animals reversed the behavioural and endocrine differences. Male offspring whose mothers were exposed to stress during the period of rapid myelination exhibited an increased plasma cortisol response to activation of the HPA axis. Female offspring whose mothers were exposed to stress during the period of rapid brain myelination exhibited decreased ambulatory activity and a blunted salivary cortisol response to the stress of the strobe light, but only during the estrous phase of the reproductive cycle. Therefore, the current set of studies has demonstrated the effects of prenatal stress on behaviour and HPA axis activity are dependent on; 1) the timing of the prenatal stress and 2) the hypothalamic-pituitary-gonadal axis in both male and female offspring. These studies have begun to uncover the mechanisms underlying programming and provide the basis for continuing research in humans.