Critical influence of neonatal testosterone on stress pathways in the adult brain.

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OBJECTIVES. Here we propose to test the requirements for and sites of involvement of neonatal sex steroids in programming the hypothalamic-pituitary-adrenal (HPA) neuroendocrine axis. Our current findings indicate that the activation of androgen receptors and the conversion of testosterone to estrogens during the neonatal period are both required for the normal development of the HPA axis to occur. Building on this success, all of the experiments of the current proposal will employ adult male rats (75 to 85 days) bearing subcutaneous implants of anti-androgen (flutamide) or an inhibitor of aromatase (ATD) within 12 h of birth, that are then removed on day 21 of weaning. We hypothesize that the capacity of testosterone to permanently alter or organize brain, behavior and physiology is linked to changes in androgen receptors, localized to brain regions located upstream from the principle driver of the HPA axis, the paraventricular nucleus (PVN) of the hypothalamus. In AIM-1, a battery of anatomical techniques will be employed to identify cells that project to the PVN region, respond to restraint-stress, and show androgen receptors. We also hypothesize that the enduring influence of testosterone to organize stress-related pathways to the PVN involves critical changes in the central activity of the neuropeptide arginine vasopressin (AVP) and its principle receptor, the V1A receptor. Importantly, this hypothesis is consistent with the stimulatory effect of testosterone on AVP synthesis in the brain, and the shared inhibitory characteristics by which central AVP-containing pathways and testosterone operate on the HPA axis. In AIM-2 we will compare HPA axis hormone responses to restraint-stress in animals receiving vehicle or V1A receptor antagonist injections into the lateral brain ventricles. Neonatal dependent changes in V1A receptors are not likely restricted to pathways regulating neuroendocrine responses, but might also occur within brain circuits generating active coping responses. Hence, we propose to use the same pharmacological approach to test for alterations in defensive burying behavior. In AIM-3, studies are designed to examine changes in AVP V1A receptor expression and function directly. We will employ in situ hybridization, receptor autoradiography and receptor coupling detection methods. Preceding experiments are geared towards unmasking processes related to the masculinization of stimulatory pathways driving PVN neuroendocrine responses. This may also include underlying changes in glucocorticoid negative-feedback regulation of the HPA axis. In AIM-4 we will test the extent to which neonatal manipulations alter the capacity of corticosterone to suppress the stress-induced activation of the HPA axis, in addition to glucocorticoid receptor expression and activation. SIGNIFICANCE. Using cellular, circuit, and system level approaches, the current proposal promises to provide novel findings and to increase our understanding of how neonatal testosterone contributes to the development of HPA axis and behavioral responses. What is emerging is that a variety of factors can influence the onset and duration of perinatal increases in testosterone (e.g. environmental stress, maternal behavior, nutrition, drugs). Based on the pervasive organizational effects of testosterone, we predict that any early life event that impacts testosterone release has the capacity to redirect any number of homeostatic processes. Thus, the work proposed is certain to impact several disciplines and fields of interest in physiology and neurobiology.