Endogenous Opioid Mechanisms Modulating Stress

描述（由申请人提供）：内源性阿片肽被认为调节对应激的生理反应，调节疼痛感觉、奖赏途径和对环境的行为反应。本研究将评估内源性κ阿片系统在轻度应激（强迫游泳试验）中的介导作用。 小鼠将暴露于强迫游泳以诱导已知激活κ阿片受体的内源性阿片肽的释放。初步结果表明，由重复强迫游泳测试引起的轻度应激通过对κ阿片拮抗剂、nor-BNI或强啡肽基因破坏敏感的机制增加了行为不动性和随后的甩尾潜伏期。κ阿片受体的内源性激活的进一步评估是可能的，与一种新的抗体探针（KOR-P），可以区分激动剂激活形式的受体。当阿片受体被应用的激动剂或内源性释放的阿片肽激活时，G蛋白受体激酶磷酸化κ阿片受体上的丝氨酸-369残基以启动脱敏过程。初步数据表明，KOR-P抗体可以区分磷酸化κ阿片受体的基础状态，因此可以用作探针在蛋白质印迹和免疫细胞化学分析，以检测之前激活的κ阿片系统。初步Western印迹结果表明，强迫游泳应激诱导从测试小鼠分离的脑蛋白的KOR-P抗体标记增加。特异性标记的增加在游泳测试之前通过给予nor-BNI来阻断。未来的研究计划确定内源性阿片肽，产生κ阿片受体激活响应于游泳应激源，开始检查强啡肽敲除小鼠及其野生型同窝仔在强迫游泳试验。此外，研究将扩展到完善免疫细胞化学方法，并确定在大脑中的κ阿片受体激活发生在响应环境压力。预计该数据集将定义由行为应激激活并由内源性κ阿片样物质系统的作用调节的功能性神经回路。更好地了解压力和内源性kappa系统之间的关系，可能会导致新的见解，如抑郁症和药物滥用复发等疾病，可能提供新的治疗方法，在这些综合征。

DESCRIPTION (provided by applicant): 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.