Endogenous opioid systems play critical roles in analgesia and drug addiction but numerous questions concerning opioid receptor function remain. In this project, we will utilize mice harboring targeted mutations in the DOR1 and MOR1 opioid receptor genes to investigate receptor function in analgesia, development and immune function. We will complement these studies with developmental and genetic analysis of the ORL-1 and orphaninFQ (OFQ) loci that likely interact with classical opioid systems. We will first investigate the pharmacological and behavioral consequences of MOR1 and DOR1 alteration. We will perform receptor binding studies to determine whether the mutations are complete nulls, whether residual binding to morphine, morphine6B-glucuronide (M6G) and receptor subtype-specific ligands can be detected in either mutant. Analgesic assays will be used to determine whether morphine and/or M6G analgesia are absent from the mutant lines, whether pharmacologically-defined subtypes of mu, and delta receptors are coordinately lost, and whether the opioid component of stress-induced analgesia is mediated through MOR1 or DOR1. Confocal microscopy and image analysis will be used to test the hypothesis that receptor alteration leads to abnormalities in neural cell number and morphology at specific sites of normal receptor expression. We will next determine whether a potentially opioid-mediated chemotactic response of macrophages that follows injury to the developing brain is altered in either or both mutants. The expression patterns and levels of non-mutated opioid receptor and opioid peptide genes will be compared in mutant and wild-type mice to establish whether compensation occurs at the transcriptional level. We will then use GTPgS to visualize receptor activation in histological sections and thereby determine when functional receptor coupling begins during development and whether any changes in opioid receptor mRNA in specific mutants extends to the functional level. Finally, we will complete ontogenetic studies of ORL-1 and OFQ expression and then genetically ablate both ORL-1 and OFQ to determine if the early expression of these genes we observe in the nervous system is critical for neuronal differentiation. The phenotypes of these mutants will be compared to evaluate possible functional roles for putative peptides encoded by the OFQ precursor. If viable, these mutants will be tested to resolve conflicting views of OFQ and ORL-1 action in analgesia. Taken together, these studies should resolve multiple uncertanties concerning opioid receptor function in several systems and provide new animal models to investigate analgesia, tolerance, and withdrawal.
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