NEURAL REGULATION OF THE FETAL PARAVENTRICULAR NUCLEUS

我们已经在绵羊身上发现，胎儿的下丘脑室旁核 核（PVN）是必要的：1）分泌ACTH的 下丘脑-垂体-肾上腺轴（HHAA）对低氧血症的反应， 2)导致分娩的HHAA活性的延长增加。 在 成年动物脑干和海马的神经元输入有很强的 对HHAA活性的修饰作用。 此外，脑干和 海马体是大脑的一个区域， 糖皮质激素反馈受体（GR）。 GR的实验操作 对HHAA产量有着深远的影响。 我们建议继续我们的 胎羊室旁核在低氧血症中作用的研究 和分娩时 具体目标1将确定6-羟基多巴胺（6-OHDA）的作用 在PVN的激素反应中， 胎儿HHAA与低氧血症和分娩时机的关系。 这将是 a）立体定位和尼氏染色技术， 制作妊娠120天胎羊脑立体定位图谱; B）荧光金逆行追踪和免疫细胞化学（ICC）染色 多巴胺β-羟化酶和c-fos蛋白的表达 早期基因，以确定哪些脑干儿茶酚胺神经元项目， PVN，这些神经元在低氧血症期间被激活， 胎儿分娩; c）立体定向技术分娩 微量注射6-OHDA，一种广泛用于研究 儿茶酚胺能神经元，进入PVN本身，以去除敏感的 神经元输入 具体目标2将确定神经元输入的作用， 海马体通过穹窿在大脑的激素反应中移动， HHAA与低氧血症和分娩时机有关。 这将是 完成使用我们的立体定位手术技术， 穹窿 具体目标3将确定糖皮质激素反馈在 脑干和海马对胎儿HIM的激素反应 通过立体定向植入皮质醇晶体到 脑干和海马体的预选区域。 特定位点 选择将基于通过ICC和mRNA测量的GR浓度 分析.在所有三种特定目的中，外周血浆 CRH的ACTH、AVP、皮质醇和PVN含量， 将测量AVP mRNA作为终点。 低氧血症和早产是发病和死亡的重要原因 在人类怀孕期间。 此外，低氧血症在本提案中被用作 可重复的，精确确定的急性应激刺激使我们能够 微调我们的方法，并与长期的研究进行比较， 更复杂和更少了解的原因开始分娩。 这些研究从逻辑上讲是从以前的项目I和II中产生的。 在这段融资期间，我们证明，除其他成果外， 室旁核的损伤消除了胎儿ACTH对低氧血症的反应， 低血压和妊娠期延长。 详细控制实验 胎儿对低氧血症的反应和体内启动机制的数据 只有在动物胎儿中才能获得。 这个项目 与项目I和项目III在跨物种和 机械水平。我们的实验对 胎儿损伤和浪费的问题。 此外，这些研究将改善 我们对控制神经和内分泌相互作用的理解 HHAA功能。

We have shown in sheep that the fetal hypothalamic paraventricular nucleus (PVN) is necessary for: 1) secretion of ACTH by the hypothalamo-hypophyseal-adrenal axis (HHAA) in response to hypoxemia and 2) the prolonged increase in HHAA activity that leads to parturition. In adult animals neuronal inputs from brainstem and hippocampus have strong modifying effects on HHAA activity. In addition, the brainstem and hippocampus are areas of the brain with high concentrations of glucocorticoid feedback receptors (GR). Experimental manipulation of GR has profound influence on HHAA output. We propose to continue our investigation in fetal sheep of the role of the fetal PVN in hypoxemia and in parturition. Specific Aim 1 will determine the role of 6-hydroxydopamine (6-OHDA) susceptible neuronal inputs to the PVN in the hormonal response of the fetal HHAA to hypoxemia and in timing of parturition. This will be accomplished by using: a) stereotaxic and nissl staining techniques to produce a stereotaxic atlas of the 120 days gestation fetal sheep brain; b) retrograde tracing of fluorogold and immunocytochemical (ICC) staining for dopamine beta-hydroxylase and protein expression of c-fos immediate early gene to determine which brainstem catecholamine neurons project to the PVN and that these neurons are activated during hypoxemia and parturition in the fetus; c) stereotaxic technique to deliver micro-injections of 6-OHDA, a neurotoxin used extensively to study catecholaminergic neurons, into the PVN itself to remove susceptible neuronal input. Specific Aim 2 will determine the role of neuronal inputs from the hippocampus that travel via the fornix in the hormonal response of the HHAA to hypoxemia and in timing of parturition. This will be accomplished using our stereotaxic surgical technique to transect the fornix. Specific Aim 3 will determine the role of glucocorticoid feedback in brainstem and hippocampus on hormonal response of the fetal HIM to hypoxemia by stereotaxically implanting cortisol crystals into preselected areas of the brainstem and hippocampus. Specific site selection will be based on GR concentration as measured by ICC and mRNA analysis. In all three Specific Aims, peripheral plasma radioimmunoassayable ACTH, AVP, and cortisol and PVN content of CRH and AVP mRNA's will be measured as end points. Hypoxemia and prematurity are important causes of morbidity and mortality in human pregnancy. In addition, hypoxemia is used in this proposal as a repeatable, precisely determined acute stress stimulus allowing us to fine tune our methods and make comparison to the long term studies of the more complex and less understood causes of initiation of parturition. These studies arise logically out of Project I and II of the previous funding period in which we demonstrated, among other results, that lesions of the PVN abolished the fetal ACTH response to hypoxemia and hypotension and prolonged gestation. Detailed controlled experimental data on fetal responses to hypoxemia and in vivo mechanisms of initiation of parturition can only be obtained in fetal animals. This project closely interacts with Projects I and III at both cross-species and mechanistic levels. Our experiments have important relevance to the problem of fetal damage and wastage. Also, these studies will improve our understanding of nervous and endocrine interactions in control of HHAA function.