Presynaptic NMDAR Regulation of Substance P Release in the RVM After Injury

描述（由申请人提供）：慢性疼痛是一个重大的卫生保健问题，对全球数百万人的生活质量产生不利影响，每年因生产力损失造成数十亿美元的损失。在过去的十年中进行的研究表明，脑干中的疼痛易化神经元在外周损伤后慢性疼痛的发展，特别是维持中起着重要作用。然而，确切的机制仍然难以捉摸。本实验室和其他实验室最近的行为学研究表明，在延髓头端腹内侧（RVM）释放P物质介导后爪炎症损伤后的热和机械超敏反应。因此，RVM中神经激肽-1受体的局部拮抗作用足以逆转完全弗氏佐剂（CFA）诱导的痛觉过敏和超敏反应。全细胞膜片钳记录进一步表明，促进脊髓投射RVM神经元观察CFA处理的大鼠是依赖于P物质的释放。最后，突触后N-甲基-D-天冬氨酸受体（NMDAR）的药理拮抗作用是没有效果的，而细胞外应用的NMDAR拮抗剂防止促进。这些数据导致的假设，NMDARs位于突触前的P物质传入RVM神经元的功能，以促进P物质的释放，这些受体的上调后，炎症损伤设置在运动中的正前馈机制，以维持活动在RVM的疼痛易化神经元。提出了三组互补的研究来开始检验这一假设。首先，谷氨酸或NMDA促进P物质释放的能力将在来自大鼠RVM的分离的突触前末梢（突触体）中进行研究，所述大鼠在4小时、4天或2周前接受了盐水或CFA的足底注射。将使用亚单位特异性拮抗剂确定NMDA受体的亚型。其次，使用定量真实的时间PCR和蛋白质印迹法，用亚基特异性抗体、磷酸化特异性抗体和生物素化探针，在RVM的突触体中评估NMDA受体亚基转录、翻译、表面表达和磷酸化状态的炎症诱导的变化。最后，免疫组织化学方法将被用来确认CFA治疗后RVM中P物质免疫反应性突触前结构中NMDAR亚基表达的变化。从这些研究中获得的结果将进一步了解脊髓上核的功能，以维持损伤后的慢性疼痛的机制，以及探测突触前NMDAR的活动依赖性可塑性。这些信息可以帮助开发用于治疗持续性疼痛状态的靶向、中枢活性疗法。选择测试这一假设的方法将为申请人提供神经化学，药理学，分子和免疫组织化学方法方面的广泛培训，为独立研究者的职业生涯做准备。 公共卫生相关性：本项目旨在进一步阐明外周炎症损伤后脑干神经元启动并维持慢性疼痛的机制。由于慢性疼痛仅在美国就影响着数百万人，因此找到更好的治疗方法符合公众的利益。这项提议的结果可能有助于理解和开发用于治疗慢性疼痛的新型中枢作用疗法。

DESCRIPTION (provided by applicant): Chronic pain is a significant health care problem, adversely affecting the quality of life of millions of people worldwide and costing billions of dollars each year in lost productivity. Research conducted in the past decade has revealed that pain facilitatory neurons in the brainstem play an important role in the development and particularly the maintenance of chronic pain after peripheral injury. However, the exact mechanisms remain elusive. Recent behavioral studies by this and other laboratories indicate that release of substance P in the rostral ventromedial medulla (RVM) mediates the thermal and mechanical hypersensitivity that follow inflammatory injury of the hindpaw. Thus, local antagonism of neurokinin-1 receptors in the RVM was sufficient to reversal hyperalgesia and hypersensitivity induced by complete Freund's adjuvant (CFA). Whole cell patch clamp recordings further revealed that the facilitation of glutamatergic inputs to spinally projecting RVM neurons observed in CFA-treated rats was dependent on release of substance P. Finally, pharmacological antagonism of postsynaptic N-methyl-D-aspartate receptors (NMDAR) was without effect, whereas extracellular application of a NMDAR antagonist prevented the facilitation. These data lead to the hypothesis that NMDARs situated presynaptically on the terminals of substance P afferents to RVM neurons function to promote the release of substance P, and that the up-regulation of these receptors after inflammatory injury sets in motion a positive feed-forward mechanism to sustain activity in pain facilitatory neurons in the RVM. Three complementary sets of studies are proposed to begin to test this hypothesis. First, the ability of glutamate or NMDA to promote the release of substance P will be investigated in isolated presynaptic terminals (synaptosomes) from the RVM of rats that received an intraplantar injection of saline- or CFA four hrs, four days or two weeks earlier. The subtype of NMDA receptor will be determined using subunit specific antagonists. Second, inflammation-induced changes in NMDA receptor subunit transcription, translation, surface expression and phosphorylation state will be assessed in synaptosomes of the RVM using quantitative real time PCR and western blotting with subunit specific antibodies, phospho-specific antibodies and biotinylation probes. Lastly, immunohistochemical methods will be used to confirm changes in the expression of NMDAR subunits in substance P-immunoreactive presynaptic structures in the RVM after CFA-treatment. Results obtained from these studies will further our understanding of the mechanism by which supraspinal nuclei function to maintain chronic pain after injury, as well as probe the activity-dependent plasticity of presynaptic NMDAR. This information could aid the development of targeted, centrally active therapeutics for the treatment of persistent pain states. The approaches selected to test this hypothesis will provide the applicant with broad-based training in neurochemical, pharmacological, molecular and immunohistochemical approaches in preparation for a career as an independent investigator. PUBLIC HEALTH RELEVANCE: This project seeks to further elucidate the mechanisms by which brainstem neurons function to initiate and then maintain chronic pain after peripheral inflammatory injury. As chronic pain affects millions of people in the USA alone, it is in the public's interest to find better ways to treat this condition. Results from this proposal could potentially aid in understanding and developing novel, centrally acting therapeutics for the treatment of chronic pain.