ESTROGEN INFLUENCES ON NEUROENDOCRINE AGING

生殖衰老的特征是雌激素水平的变化；然而，大脑本身会受到与年龄相关的变化的影响，其中一些可能是雌激素水平改变的结果，而另一些可能与雌激素无关。因此，本研究将探讨雌激素与生殖衰老过程中大脑下丘脑神经元的相互作用。研究将重点关注雌激素对生殖轴关键细胞促性腺激素释放激素（GnRH）神经元的调节作用，以及谷氨酸能神经元通过NMDA受体介导雌激素对GnRH神经元的作用。为了更好地表征雌激素对下丘脑衰老的影响及其相互作用，我们将在卵巢切除术（OVX）的实验模型中，在不同的生殖功能阶段和内源性雌激素水平下，进行雌激素替代或不替代的研究，以及卵巢完整动物的自然模型。具体目的1和2将研究雌激素对年轻，中年（MA）和老年Sprague-Dawley OVX大鼠衰老过程中下丘脑功能的影响机制，给予长期或短期雌激素或载体替代（Aim 1）或在发情周期中卵巢完整的年轻，MA和老年大鼠（Aim 2）。在Aim 2中，动物在排卵前GnRH/LH激增，雌激素水平高的发情期或在发情期1，雌激素水平低的一天使用。无循环大鼠将被用于持续发情（高雌激素）或持续发情（高雌激素）或持续发情（低雌激素）。在这两个目的中，雌激素对GnRH和NMDA受体亚基mrna和蛋白质的影响将分别通过RNase保护实验和Western blots进行定量分析。NMDA受体亚单位在GnRH神经元上的定位及其受雌激素和衰老的调节将被确定。GnRH神经分泌系统对NMDA受体激动剂的反应性也将被评估。Specific Aims 3和4将以月经周期为28天的恒河猴为实验模型，研究雌激素对下丘脑神经元调控的生理变化。Aim 3将研究OVX幼猴和老年猴在长期或短期雌激素替代或不替代下下丘脑GnRH和谷氨酸释放的变化，以及NMDA受体激活对GnRH释放的调节。Aim 4将在高雌激素（卵泡晚期）或低雌激素（黄体晚期）的自然月经周期中，对卵巢完整的年轻和年老恒河猴进行类似的测量；将与闭经的老年猴子进行比较。实验预计将提供深入了解更年期雌激素缺乏的神经生物学效应。

Reproductive aging is characterized by changes in estrogen levels; however, the brain itself is subject to age-related changes, some of which may be consequences of altered estrogen levels, and others which may be estrogen-independent. Therefore, the present proposal will examine the interactions of estrogen with hypothalamic neurons in the brain during reproductive senescence. Studies will focus on estrogen regulation of gonadotropin-releasing hormone (GnRH) neurons, the key cells regulating the reproductive axis, as well as the mediation of estrogen's effects on GnRH neurons by glutamatergic neurons, acting via the NMDA receptor. In order to better characterize effects of estrogen and hypothalamic aging, as well as their interactions, studies will be performed in the experimental model of ovariectomy (OVX), with or without estrogen replacement, as well as the natural model of ovarian- intact animals, at different stages of reproductive function and endogenous estrogen levels. Specific Aims 1 and 2 will investigate mechanisms for estrogen's effects on hypothalamic function during aging in young, middle-aged (MA) and old Sprague-Dawley OVX rats, given long-term or short-term estrogen or vehicle replacement (Aim 1) or in ovarian-intact young, MA and old rats during the estrous cycle (Aim 2). For Aim 2, animals are utilized on pro-estrus the day of the pre-ovulatory GnRH/LH surge, with high estrogen levels, or on diestrus I, a day of low estrogen levels. Acyclic rats will be utilized on persistent estrus (high estrogen) or persistent estrus (high estrogen) or persistent diestrus (low estrogen). In both of these aims, effects of estrogen on GnRH and NMDA receptor subunit mRNAs and proteins will be quantitated by RNase protection assay and Western blots, respectively. The localization of NMDA receptor subunits on GnRH neurons, and their regulation by estrogen and aging, will be determined. The responsiveness of the GnRH neurosecretory system to administration of an NMDA receptor agonist will also be assessed. Specific Aims 3 and 4 will investigate the physiological changes in estrogen regulation of hypothalamic neurons, using the rhesus monkey, with its 28 day menstrual cycle, as an experimental model. Aim 3 will examine changes in hypothalamic GnRH and glutamate release and the regulation of GnRH release by NMDA receptor activation in OVX young and old monkeys, with or without long-term or short-term estrogen replacement. Aim 4 will make similar measurements in ovarian-intact young and old rhesus monkeys across the natural menstrual cycle during periods of high estrogen (late follicular phase) or low estrogen (late luteal phase); comparisons will be made with amenorrheic aged monkeys. Experiments are anticipated to provide insight into the neurobiological effects of an estrogen deficiency at menopause.