Our goal in this project is to understand the ontogeny and regulation of endogenous opioid systems and their contribution to developmental and analgesic processes. We will integrate morphological, genetic, and behavioral approaches to elucidate several aspects of opioid peptide expression, maturation and function. We will continue to delineate the prenatal expression patterns for gene families involved in processing propeptides including opioids. We will first determine the fetal expression patterns for novel carboxypeptidase-like and prohormone convertase genes. We will also compare the prenatal expression of PC2 and its chaperone 7B2, combined with analysis of PC2 maturation by immunoblotting, to define the relationship between 7B2 expression and the maturation of PC2 activity. Morphologic and genetic approaches will be used to elucidate specific aspects of PAM function. The relationship between sites of PAM expression and a specific substrate/receptor system in the developing nervous system will be examined; then gene-targeted mice deficient in PAM will be produced to identify required developmental roles for this locus. We will continue to examine the role of endogenous opioid peptides in three paradigms. First, we will determine whether sensitivity to exogenous opioid ligands is altered in enkephalin and beta-EP/enk KO mice and whether there are accompanying changes in the expression or activity of opioid system components. We will evaluate the contributions of enkephalin and beta-EP/enk KO mice p-EP peptides to stress-induced analgesia by analyzing single- and double-KO mice. Finally, we will produce gene-targeted mice null for dynorphin, which will be examined for development abnormalities, changes in analgesic responses, and alterations in opioid system gene expression. Our last goal will be to investigate genetically the role of the D2 and D3 receptors in pituitary and brain development. We will determine whether prenatal changes in D3 expression accompany a gene-targeted mutation of D2 and whether the D2 mutant mice, alone or in combination with D3 mutations, exhibit alterations in intermediate pituitary (IL) differentiation and maturation. Solution and in situ hybridization will determine whether changes in IL POMC expression accompany either KO and whether the change in neonatal IL glucocorticoid response, thought to be dopamine mediated, is altered in D2 or D2/D3 mutants. Taken together, these studies will elucidate multiple aspects of fetal prohormone processing while using and establishing genetic models to investigate the roles of endogenous opioid ligands in analgesia and mechanisms of plasticity that underlie drug abuse.
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