Fever is an adaptive reaction, mediated by the CNS, in response to immune stimuli. Recent studies, including ones from previous cycles of this grant, have indicated that lipopolysaccharide (LPS) and pyrogenic cytokines activate production of prostaglandin E2 (PGE2) by endothelial cells lining small venules at the margins of the brain. PGE2 acts upon specific EP receptors in the preoptic area to cause fever responses. There is evidence that both EP1 and EP3 receptors cause hyperthermia, and EP4 receptors cause hypothermia, but the mechanisms for the interplay of these receptors in activating hyperthermic and hypothermic pathways in response to immune stimuli remain largely unknown. In addition, the melanocortin 4 (MC4) receptor is known to cause antipyresis, but its mechanism of action is also not understood. We have developed a new model for the interactions of these receptor types in the preoptic area in producing fever responses and new genetically based tools for analyzing these pathways. We will use mice with conditional knock-in or knock-out gene constructs for the EP1, EP3, EP4, and MC4 receptors in combination with an adeno-associated viral vector (AAV) we have developed containing the gene for Cre recombinase, to selectively turn these genes on or off in specific populations of cells in the preoptic area, to study their roles in fever responses. Both the EP3 and EP4 receptors have been engineered with loxP sites in strategic locations, so that in cells expressing Cre, the genes are deactivated. We have placed a transcriptional blocker surrounded by loxP sites into the EP1 and MC4 genes, so that they are not transcribed (the animals are null mutants) but gene expression can be reactivated in cells that express Cre. We will study the fever response to LPS in animals with these gene constructs and with injections of AAV-Cre into specific preoptic cell groups that express these receptors, and which we hypothesize to play critical roles in fever responses. We will then correlate the physiological response with the changes in gene expression and the neurotransmitters and other receptor types expressed by these cells. This information should allow us to understand the specific neural pathways involved in producing fever responses and to design pharmacological interventions to limit hypothermia and vasodilation in septic shock, as well as controlling disabling fever responses in non-infectious inflammatory disorders.
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