Victor Viau

Produced by the liver, plasma corticosteroid-binding globulin (CBG) binds glucocorticoids in the blood and regulates their access to target tissues. Glucocorticoids exert sexually dimorphic effects on development, metabolism, stress, and inflammation, but precisely how CBG modulates these actions remains unclear. Here, we examined how disrupting the SerpinA6 gene encoding CBG affects glucocorticoid actions in male and female rats. Initial observations in wild-type animals indicated that females display pubertal increases in adrenal weight in tandem with increases in plasma CBG, whereas no such changes were observed in males. Interestingly, adrenal weight gain was blunted in female rats lacking CBG, such that adrenal growth trajectories became similar between sexes. Loss of CBG also altered the expression of ~4,600 adrenal genes in adult females, but not males, including those related to steroidogenesis and growth. To understand the mechanisms underlying the deficit in adrenal growth associated with CBG deficiency, we then characterized the capacity of female CBG-knockout rats to show compensatory adrenal growth induced by unilateral adrenalectomy. The results of these experiments suggest that CBG normally enhances the proliferation and transdifferentiation of the glucocorticoid-producing region (zona fasciculata) of the adrenal gland, as these responses were blunted in rats without CBG. Because glucocorticoids control liver development and metabolic activities, hepatic transcriptomic profiling revealed a sex-specific effect of CBG ablation to alter the expression of genes related to cholesterol biosynthesis, inflammation, and metabolism in adult females. These studies suggest that the emergence of sex differences in CBG levels during puberty promotes the sexual dimorphism of both liver and adrenal function. Characterization of inflammatory and hypothalamic-pituitary-adrenal (HPA) axis responses to acute lipopolysaccharide and restraint stress continued to underscore how CBG calibrates the HPA axis and glucocorticoid actions in each sex. Interestingly, CBG-deficient males rather than females were particularly sensitive to immune challenge. These studies reveal that CBG shapes the sexual dimorphism of the adrenal and liver as well as responses to stress and inflammation, providing insight into how CBG deficiencies are linked to sex differences in glucocorticoid-dependent diseases.

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Few studies have explored the central mechanisms underlying sex differences in stress hypothalamic-pituitary-adrenal axis habituation, defined as a reduction in glucocorticoid steroid hormone responsiveness to non-life-threatening stimuli repeated in a predictable manner. As serotonin (5-hydroxytryptamine; 5-HT) is an important regulator of the HPA axis, the aim of this thesis was to determine how repeated stress-induced declines in HPA axis responsiveness is associated with changes in the 5-HT system. In the first study, males and females showed comparable declines in ACTH and corticosterone responses to repeated restraint, coupled to sex-specific changes in 5-HT 1A receptor mRNA levels in the hippocampus, hypothalamus and in the dorsal raphe nucleus, the latter representing a major source of serotonin in the central nervous system. These findings led me to hypothesize that stress habituation is met by sex differences in 5-HT 1A receptor availability and function. Subsequent studies using a challenge dose of the 5-HT 1A receptor agonist, 8-OH-DPAT, suggested that stress habituation is met by an increase in the sensitivity of postsynaptic 5-HT 1A receptors in both sexes, and by an increase in the sensitivity of presynaptic 5-HT 1A receptors only in males. Considering that stress is often accompanied by 5-HT dependent thermoregulatory responses, I then discovered that restraint-induced hyperthermia declines with repeated exposure, but only in males. Based on these findings to suggest that males and females use different serotonin substrates for adapting to stress, I subsequently characterized the nature by which males and females show differential transcriptional responses in the raphe nucleus, in addition to forebrain regions targeted by the raphe-serotonin system. The results revealed completely different transcriptional profiles between males and females in the raphe nucleus, hypothalamus, and lateral septum. The nature by which these sex-specific alterations contribute or respond to stress habituation remains to be seen. Nonetheless, the current findings continue to underscore how strikingly distinct factors controlling adaptive responses in males and females may be. As stress habituation minimizes the detrimental effects of excessive glucocorticoid exposure, the overall results provide a platform for distinguishing maladaptive from adaptive responses, in addition to understanding sex differences in stress resilience and vulnerability.

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The aim of this thesis was to investigate the brain pathways regulating the decline, or habituation of glucocorticoid responses to repeated exposure of psychological stress. In the first study, adult male rats were exposed to repeated bouts of restraint stress and tested for changes in endocrine responses and neuropeptide expression in the brain. Stress-induced elevations in glucocorticoids decreased as restraint was repeated, and this decline was associated with increased mRNA levels of the neuropeptide vasopressin in the medial amygdala and the bed nucleus of the stria terminalis. This suggested that central vasopressin signaling might be involved in coordinating glucocorticoid habituation. In a follow up experiment, adult male rats were again repeatedly restrained, however this time animals received continuous intracerebroventricular infusion of saline or an antagonist for the vasopressin V1a receptor. As expected, saline treated males showed stress-induced increases in vasopressin mRNA levels and habituation of glucocorticoid responses. Blocking activation of the vasopressin V1a receptor had no effect on basal or acute stress glucocorticoid levels. Antagonism did however attenuate habituation of glucocorticoid responses during subsequent restraint exposures. These findings indicated that habituation of endocrine stress responses depends on central vasopressin signaling. In the final set of experiments, saline and antagonist treated male rats were again repeatedly restrained, but this time brains were analyzed for changes in cellular activation using immunohistochemical detection of Fos protein. Antagonism had no effect on basal Fos expression, but blunted the habituation of repeated stress-induced Fos responses within the hypothalamus and select forebrain regions. Furthermore, a separate experiment revealed that repeated restraint induces region-specific changes in central V1a receptor binding levels which included decreases in the hippocampus, thalamus and central amygdala, but increases in the septum and bed nucleus of the stria terminalis. Overall, these studies show that habituation of glucocorticoid responses depends on central vasopressin V1a receptor signaling. The findings also suggest that stress-induced increases in V1a receptor binding within the bed nucleus and septum might be especially relevant in promoting habituation of glucocorticoid responses.

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Testosterone exposure during critical periods of development exerts major organizing effects on the hypothalamic-pituitary-adrenal (HPA) axis. The aim of this thesis was to determine how HPA axis activity is altered by neonatal testosterone, and where and how this might occur in the adult brain. In chapter two, I demonstrate that neonatal gonadectomy increased plasma corticosterone and Fos activation in the ACTH-regulating zone of the paraventricular nucleus of the hypothalamus under basal conditions and following restraint exposure. These responses were normalized with postnatal, but not adult testosterone replacement. Neonatal gonadectomized rats also had decreased numbers of AR and arginine vasopressin-positive cells in the bed nucleus of the stria terminalis and medial amygdala. This suggests that testosterone exposure during the neonatal period may prime adult HPA response to testosterone by altering AR levels and function within afferent mediators of HPA axis activity. Testosterone in the brain can be converted to estradiol by the aromatase enzyme, and estradiol usually impacts brain development directly. In chapter 3, I demonstrate that animals neonatally exposed to aromatase, or AR blockade fail to show a normal decline in corticosterone, or habituate, in response to repeat restraint exposure. By contrast, males castrated as adults show a significant reduction in corticosterone after repeated stress. These findings suggest an organizing influence of both ARs and estrogen conversion on HPA habituation, which occurs independently of activational effects of testosterone. The immediate early gene c-fos is rapidly induced in many brain regions following acute restraint stress and is an excellent tool for mapping functional differences in brain activation by stress. In chapter 4, we used c-fos mRNA as a tool to map changes in cellular activation in acutely stressed adult animals that received aromatase blockade neonatally. This treatment enhanced stress-induced c-fos expression in several limbic regions, including within the anterior cingulate and medial prefrontal cortex, lateral septum, anterior hypothalamic area, dorsal medial hypothalamus and medial amygdala, as well as at multiple levels of somatosensation. Based on these results, I propose estrogens exert effects during the neonatal period that result in systems-wide differences in adult neuroendocrine responses to homeostatic threat.

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The hypothalamic-pituitary-adrenal (HPA) axis is a critical mediator of the stress responsesystem. However, despite clear evidence for an inhibitory role of testosterone on stress-inducedactivation of the HPA axis, the routes and mechanisms have not been addressed. To firstdetermine where testosterone acts in the brain to regulate stress-related input to the HPA axis, Iused a combined retrograde transport and immunohistochemical procedure to characterize theanatomical nature by which androgen targets in the brain communicate with the paraventricularnucleus (PVN) of the hypothalamus, the initial point of the neuronally mediated stress response.The findings suggest that androgens could act throughout the brain, and on a large assortment ofbrain regions that innervate the PVN. Among the brain regions identified, neurons of the medialpreoptic nucleus (MPN), highly express androgen receptors and project abundantly to the PVN,suggesting that the MPN stands out as a potential site of integration between testosterone and theHPA axis. To test the functional role of these cells, I tested whether lesions of the MPN alter theinhibitory effects of testosterone on the HPA axis. By selectively removing cells in the MPN,testosterone regulation of the PVN and HPA axis was eliminated. Together, these findingsdemonstrated that the integrity of the MPN is essential in maintaining the regulatory effects oftestosterone on the brain's response to stress. Finally, to clarify whether the MPN is the seat of,or an obligatory relay for the central effects of testosterone, I tested the effects of implanting theandrogen receptor antagonist hydroxyflutamide into the MPN, on the stress-induced activation ofthe PVN and HPA output. The differential effects of androgen exposure in the MPN on thebiosynthetic capacity and activational responses of the PVN and its extended circuitriessuggested that the MPN is capable of bridging converging limbic influences to the HPA axiswith changes in gonadal status.

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