(Application Abstract) This is a renewal proposal of DA07058-06. The primary goal of this research is to examine the actions of morphine on the brain-immune axis. The neuro-endocrine-immune axis is influenced by a network of soluble polypeptide cytokines, such as interleukin-l (IL-1). Both morphine and IL-1 have been shown to modulate the immune response through direct actions on immune cells and indirect actions on a regulatory cascade mediated via the hypothalamic-pituitary-adrenal (HPA) axis. During the current funding period, we have used FOS proto-oncogene protein as a neuronal marker to show that both morphine and IL-1 activate the hypothalamic paraventricular nucleus (PVN). The PVN is a brain area critical for the functioning of the HPA axis and endocrine system. Chronic exposure to morphine desensitizes the FOS response in the PVN to subsequent treatment with either morphine or IL-1, and also attenuates the expression of IL-1 in the brain. In vivo infusion of an antisense to c-fos attenuates IL- 1-induced FOS activation in the PVN. Taken together, these data indicate that morphine use attenuates both the action and expression of IL- 1 in the brain. It has been suggested that the HPA is involved in morphine's effect on immune responses. Leukocyte-endothelial adhesion (LEA) is the initial step of the immune response cascade. Our continuation studies will address whether blockage of IL-1-induced FOS activation in the PVN has biological significance related to the HPA axis and LEA in the course of morphine tolerance with the following specific aims: (1) to characterize the AP-1 binding activity in the rat brain following treatment with either morphine or lL-1 using gel-shift mobility assay and Western blot analysis; (2) to characterize the effects of blockage of FOS activation on IL-1's modulation of the HPA axis by examining the mRNA levels of corticotropin releasing factor (CRF), pro-opiomelanocortin (POMC) by reverse-transcriptase polymerase chain reaction (RTPCR), CRF peptide, adrenocorticotropic hormone (ACTH), and corticosterone by radioimmunoassay (RIA); (3) to characterize the modulatory role of the HPA products, such as glucocorticoid, on LEA both in animal and in vitro cell models using intravital microscopy; cell culture and light microscopy; and (4) to characterize the expression of IL- 1 in the central nervous system and periphery following chronic exposure to morphine using immunocytochemical staining, RT-PCR and Enzyme-linked immunosorbant assay (ELISA) techniques. These studies will elucidate the effects of morphine on the modulatory loop between the brain and the immune system. Specifically, these will establish the mechanisms by which morphine exposure affects IL-1-mediated pathways at the molecular, cellular and system levels.
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