Victor Viau

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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