Endogenous opioid peptides are believed to modulate the physiological response to stress, regulating pain sensation, reward pathways and behavioral responses to the environment. This proposal will assess the mediating role of the endogenous kappa opioid system in responses to a mild stressor, the forced swim test. Mice will be exposed to forced swimming to induce the release of endogenous opioid peptides known to activate kappa opioid receptors. Initial results show that the mild stress induced by repeated forced swim testing increased behavioral immobility and subsequent tail flick latency through a mechanism sensitive to the kappa opioid antagonist, nor-BNI or dynorphin gene disruption. Further assessment of the endogenous activation of kappa opioid receptors is possible with a novel antibody probe (KOR-P) that can distinguish the agonist-activated form of the receptor. When opioid receptors are activated by applied agonist or endogenously released opioid peptide, G-protein receptor kinase phosphorylates the serine-369 residue on the kappa opioid receptor to initiate the desensitization process. Preliminary data suggest that the KOR-P antibody can distinguish the phosphorylated kappa opioid receptor from the basal state, and thus may be used as a probe in Western blot and immunocytochemical analyses to detect prior activation of the kappa opioid system. Pilot Western blot results demonstrate that the forced-swim stress induced an increase in KOR-P antibody labeling of brain protein isolated from tested mice. The increase in specific labeling was blocked by administration of nor-BNI prior to swim testing. Future studies are planned to determine the endogenous opioid peptides that are producing the kappa opioid receptor activation in response to the swim stressor, starting with examination of dynorphin knockout mice and their wild-type littermates in the forced swim test. In addition, studies will be extended to refine immunocytochemical methods and determine where in the brain kappa opioid receptor activation occurs in response to environmental stress. It is expected that this data set would define a functional neural circuit activated by behavioral stress and regulated by the action of the endogenous kappa opioid system. A better understanding of the relationship between stress and endogenous kappa systems may lead to new insights into such disorders as depression and relapse of drug abuse, possibly providing new therapeutic approaches in these syndromes.
