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

目标。在这里，我们建议测试新生儿性类固醇对下丘脑-垂体-肾上腺（HPA）神经内分泌轴编程的需求和参与部位。我们目前的研究结果表明，在新生儿时期雄激素受体的激活和睾酮向雌激素的转化都是HPA轴正常发育所必需的。在这一成功的基础上，目前建议的所有实验都将使用成年雄性大鼠（75至85天），在出生后12小时内皮下植入抗雄激素（氟他胺）或芳香化酶抑制剂（ATD），然后在断奶后第21天取出。我们假设睾酮永久改变或组织大脑、行为和生理的能力与雄激素受体的变化有关，雄激素受体位于下丘脑室旁核（PVN）主要驱动HPA轴上游的大脑区域。在AIM-1中，将采用一系列解剖学技术来识别投射到PVN区域的细胞，对抑制应激作出反应，并显示雄激素受体。我们还假设睾酮对组织压力相关PVN通路的持久影响涉及神经肽精氨酸抗利尿激素（AVP）及其主要受体V1A受体中枢活性的关键变化。重要的是，这一假设与睾酮对大脑中AVP合成的刺激作用，以及AVP中枢含有通路和睾酮在HPA轴上运作的共同抑制特征是一致的。在AIM-2中，我们将比较在侧脑室注射载体或V1A受体拮抗剂的动物中HPA轴激素对抑制应激的反应。新生儿V1A受体的依赖性变化不可能局限于调节神经内分泌反应的途径，但也可能发生在产生积极应对反应的脑回路中。因此，我们建议使用相同的药理学方法来测试防御性掩埋行为的改变。在AIM-3中，研究旨在直接检测AVP V1A受体表达和功能的变化。我们将采用原位杂交，受体放射自显影和受体偶联检测方法。先前的实验旨在揭示与驱动PVN神经内分泌反应的刺激途径的男性化相关的过程。这也可能包括下丘脑轴糖皮质激素负反馈调节的潜在变化。在AIM-4中，我们将测试新生儿操作在多大程度上改变皮质酮抑制应激诱导的HPA轴激活的能力，以及糖皮质激素受体的表达和激活。利用细胞、电路和系统水平的方法，目前的建议有望提供新的发现，并增加我们对新生儿睾酮如何促进HPA轴和行为反应的发展的理解。目前出现的情况是，各种因素可以影响围产期睾酮增加的开始和持续时间（例如环境压力、产妇行为、营养、药物）。基于睾酮的普遍组织效应，我们预测任何影响睾酮释放的早期生活事件都有能力改变任何数量的体内平衡过程。因此，提出的工作肯定会影响生理学和神经生物学的几个学科和领域。

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.