Role of Orphanin/FQ in the Behavioral and Neuroinflammatory Response to Stress

慢性应激可激活大脑中的神经炎症系统，包括小胶质细胞 然后可能产生促炎细胞因子白细胞介素-1 β（IL-1 β）、IL-6和肿瘤坏死因子-α（TNF α）。这种炎症反应的一种可能的调节剂是孤啡肽FQ/痛敏肽（OFQ/N），其是一种有助于多种生理调节过程的神经肽，包括抑制致焦虑和/或抑郁样行为。事实上，OFQ/N与全身免疫和炎症过程的调节有关，初步数据显示，在缺乏OFQ/N前体多肽（ppOFQ/N）的小鼠中，14天的慢性不可预测应激导致海马IL-1 β显著升高，但TNF α未升高。因此，我们假设OFQ/N是减弱应激诱导的脑神经炎症系统激活所必需的，包括小胶质细胞激活和促炎细胞因子（如IL-1 β）的合成和释放。两个具体目标将检验这一假设。在具体目标1中，将对ppOFQ/N或OFQ/N受体NOP-1缺陷的小鼠施加慢性应激。将野生型小鼠（应激和非应激）用作对照。将在前额皮质、海马、杏仁核和下丘脑中获得IL-1 β、IL-6和TNF α以及抗炎细胞因子IL-10和TGF β的蛋白质和mRNA测量值。此外，免疫组织化学将确定是否存在基因型依赖性激活和细胞因子定位于小胶质细胞，以及NOP-1表达的应激依赖性变化。最后，将对在小胶质细胞中表达eGFP的OFQ/N和NOP-1 KO和WT小鼠进行小胶质细胞活性的双光子离体海马切片成像，以评估对小胶质细胞形态和动力学的基因型和/或应激依赖性影响。这些生物化学和细胞观察将在具体目标2中通过行为分析进行补充。将ppOFQ/N和NOP-1 KO和WT小鼠暴露于慢性应激，然后进行行为测试以评估学习和记忆以及焦虑样行为。一个主要的焦点是使用工作记忆版本的视标依赖的T-迷宫空间学习任务。在应激KO小鼠中这些行为的过度缺陷，而不是暴露于应激的WT小鼠，这表明OFQ/N限制了应激期间的神经炎症反应，以保持行为稳定。如果数据导致这种解释，则将在应激和非应激KO和WT小鼠中使用IL-1受体拮抗剂给药检测IL-1 β的作用。这些实验将确立OFQ/N配体-受体系统作为在面对持续压力时维持行为弹性的抗炎机制的重要性。此外，这将在理解与压力相关的心理健康障碍的原因和治疗方面取得新的进展，并表明矫正系统（如OFQ/N）的失败可能导致对慢性压力的适应能力受损。

Exposure to chronic stress can activate the neuroinflammatory system in the brain, including microglial cell that may then produce the proinflammatory cytokines interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-alpha (TNFα). One possible regulator of this inflammatory response is Orphanin FQ/nociceptin (OFQ/N), a neuropeptide that contributes to a variety of physiological regulatory processes, including inhibition of anxiogenic and/or depressive-like behaviors. Indeed, OFQ/N has been implicated in the regulation of systemic immunological and inflammatory processes, and in preliminary data, it was found that 14 days of chronic unpredictable stress produced a significant and augmented elevation in hippocampal IL-1β, but not TNFα, in mice lacking the precursor polypeptide for OFQ/N (ppOFQ/N). We therefore hypothesize that OFQ/N is necessary to attenuate stressor-induced activation of the brain neuroinflammatory system, including microglial activation and the synthesis and release of proinflammatory cytokines, such as IL-1β. Two Specific Aims will test this hypothesis. In Specific Aim 1, chronic stress will be imposed on mice deficient for either ppOFQ/N or the OFQ/N receptor, NOP-1. Wildtype mice (stressed and non-stressed) will be used as controls. Measures of protein and mRNA for IL-1β, IL-6, and TNFα, as well as the anti-inflammatory cytokines IL-10 and TGFβ will be obtained in the prefrontal cortex, hippocampus, amygdala and hypothalamus. In addition, immunohistochemistry will determine whether there is genotype-dependent activation and cytokine localization to microglial cells, as well as stress-dependent changes in NOP-1 expression. Finally, 2-photon ex vivo hippocampal slice imaging of microglial activity will be performed on OFQ/N and NOP-1 KO and WT mice expressing eGFP in microglia to assess genotype- and/or stress-dependent effects on microglial morphology and dynamics. These biochemical and cellular observations will be complemented by behavioral analysis in Specific Aim 2. The ppOFQ/N and NOP-1 KO and WT mice will be exposed to chronic stress and then subjected to behavioral testing to assess learning and memory, as well as anxiety-like behavior. A major focus is to use a working memory version of the hippocampal-dependent T-maze spatial learning task. Excessive deficits in these behaviors in stressed KO mice, but not WT mice exposed to stress, will suggest that OFQ/N limits the neuroinflammatory response during stress in order to maintain behavioral stability. If the data lead to this interpretation, the role of IL-1β will be tested using IL-1 receptor antagonist administration in stressed and non-stressed KO and WT mice. These experiments will establish the importance of the OFQ/N ligand-receptor system as an anti-inflammatory mechanism for maintaining behavioral resilience in the face of persistent stress. Further, this will create new developments in understanding the cause and treatment of stress-related mental health disorders, and indicate that the failure of correction systems (such as OFQ/N) may result in impaired adaptation to chronic stress.