This is a competing renewal application to study the mechanisms by which the brain coordinates the fever response to immune stimulation. In the first period of this grant, we found that low concentrations of circulating lipopolysaccharide (LPS) activates tissue macrophages in the meninges and microglial cells along blood vessels that penetrate the brain. These cells produce type 2 cyclooxygenase (COX2) and hence prostaglandins, which cross the blood-brain barrier and activate neurons in the ventromedial preoptic nucleus (VMPO). We hypothesize that the VMPO, and its direct and relayed projections to the paraventricular nucleus of the hypothalamus (PVH) are critical for producing fever. In this proposal, we test this hypothesis in the following experiments: 1.) We will use ibotenic acid to produce cell-specific lesions of the VMPO and PVH areas, and to determine the effects of these lesions on normal thermoregulation and fever. 2.) We will map the EP 1 -EP4 receptors for prostaglandin E2 by in situ hybridization to determine their cellular relationships with the VMPO and the PVH. We will also determine by double in situ hybridization whether the lesions in Aim 1 have removed cells in the VMPO or PVH region that are EP receptor positive and show Fos activation. 3.) We will examine the effect of knocking out the EP1-EP4 receptors on the fever responses and associated Fos expression in transgenic mice. 4.) We will then construct transgenic mice in which the relevant EP receptor genes have been modified to include loxP sites flanking the first coding exon. When we inject these mice with a novel adeno-associated viral vector, containing the gene for Cre recombinase, we will selectively delete the translation initiation site for the gene of interest, at a specific site in the hypothalamus. These experiments will determine the critical site at which expression of relevant EP receptors is necessary to allow fever responses. These studies should advance our understanding of the molecular and cellular mechanisms that underlie the autonomic, endocrine, and behaviora1 responses associated with fever
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