These experiments are designed to investigate how catecholaminergic afferents control expression of the crh gene in neuroendocrine neurons of the hypothalamic paraventricular nucleus (PVH). CRH neuroendocrine neurons are motor neurons that release CRH into the hypophysial vasculature to stimulate ACTH release, and initiate the stress response. The mechanisms they use to integrate a broad array of neural and humoral inputs is the foundation of their overall function, both in the basal and the stressed state. Dysfunction of these processes occurs in depression, post-traumatic stress disorder, and a number of metabolic diseases. Catecholaminergic inputs from the hindbrain are a major input to the PVH, and convey important viscerosensory information that impacts the secretion of ACTH. These experiments will investigate the mechanisms that catecholaminergic neurons use to control crh gene expression. The hypotheses, specific aims, and associated experiments are designed to address four facets of this control process. They will determine: 1) if catecholaminergic afferents interact with glutamatergic mechanisms; 2) the nature of signal transduction mechanisms engaged within CRH neurons, particularly with regard to mitogen-activated protein kinase pathways; 3) their role in encoding corticosterone-related information; 4) how catecholaminergic afferents integrate with other concurrently activated afferents. Experiments will each employ one of two ways to drive the system. The first will use intravenous 2-deoxy-D-glucose (2DG) to rapidly stimulate crh gene expression in the PVH. This increase is absolutely dependent on catecholaminergic afferents, which can be specifically lesioned by injections of an anti-dopamine-a-hydroxylase saporin-conjugate into the PVH. The second will use local PVH injections of norepinephrine. Depending on the specific experiment, these models will be combined with local PVH injections of adrenergic or glutamatergic receptor antagonists, or specific inhibitors of signaling kinases to explore the mechanisms underlying catecholaminergic control of crh gene expression. The measured dependent variables will be CRH heteronuclear RNA, which is the primary transcript of the crh gene, and the phosphorylation state of various signal transduction intermediaries. In situ hybridization or immunocytochemistry will be used for this purpose. Collectively, these experiments are designed to provide the basis for future investigations into how the stress response is organized in terms of circuitry and neuronal function, with the overall goal of explaining how both interosensory and cognitive stressors are elaborated by the neuroendocrine hypothalamus in health and disease.
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