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.
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