The long-term goal of this project is to resolve the structural and functional organization of the neural networks responsible for controlling neuroendocrine CRH neurons in the paraventricular nucleus of the hypothalamus (PVH). The PVH is the key convergence point for the neural regulation of the hypothalamo-pituitary-adrenal axis. Dysfunction in this control network leads to the aberrant glucocorticoid secretion seen a wide range of diseases. The proposed studies focus on the functional organization of the pre-motor network in and around the PVH that acts as a major control element for CRH neurons in the PVH. In particular, we will focus on interactions between catecholaminergic inputs to the PVH region and this control network. Catecholaminergic neurons provide one of the densest and most widely studied inputs to the PVH. They play a critical role in generating ACTH and glucocorticoid responses to virtually all stressors, but particularly those generated within the body such as cardiovascular and metabolic stimuli. During psychological stress, they also help the PVH to coordinate neuroendocrine and autonomic responses with responses generated by the telencephalon. The pre-motor network is proposed to actively and dynamically controls the level of CRH synthesis and release, and critically, it can impose differential control over each process. The proposed experiments use a hierarchically ordered model of CRH neuronal afferents, with three testable hypotheses: 1) Excitatory drive to neuroendocrine CRH neurons is exerted by pre-motor glutamate neurons, which are tonically inhibited by GABAergic neurotransmission; 2) The pre-motor Glu/GABA network is necessary for catecholaminergic activation of CRH neurons; 3) Corticosterone targets the pre-motor network to shift the balance away from alpha1 adrenoreceptor actions in CRH neurons towards alpha1 actions in GABA neurons, so inhibiting Crh expression. The experiments use two approaches: first, an acutely-prepared ex vivo hypothalamic/PVH slice to investigate catecholaminergic interactions with pre-motor GABAergic and glutamatergic control of CRH synthesis and release. Second, in vivo experiments will use intra-venous insulin injections as a stimulus to activated the catacholaminergic pathways that project into the hypothalamus. We will then examine the requirement for catechoaminergic interactions with pre-motor glutamate mechanisms to drive synthesis and release programs in CRH neurons. Endpoints will target CRH synthesis and release, including changes in neuronal spike frequencies, CRH hnRNA, phospho-ERK1/2, phospho-CREB, and circulating ACTH. Analytical methods include in situ hybridization, electrophysiology, fluorescence immunocytochemistry, confocal and conventional microscopy, qRT-PCR, and radioimmunoassay. These experiments are designed to generate a new and innovative view of the functional organization of the neural networks responsible for controlling CRH neuroendocrine function.
Dysfunctional ACTH/glucocorticoid secretion is a feature of many clinical problems. Much of this dysfunction involves important but poorly understood neural control networks in the hypothalamus that integrate stress-related information to drive ACTH secretion and peptide gene expression in a manner appropriate for the ongoing situation. The proposed studies will clarify how these local control networks are functionally organized using a range of whole animal and ex vivo experimental designs.
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