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

描述(申请人提供)：慢性疼痛是一个严重的医疗保健问题，对全球数百万人的生活质量产生不利影响，并每年造成数十亿美元的生产力损失。过去十年的研究表明，脑干中的疼痛易感神经元在周围损伤后慢性疼痛的发生，特别是维持过程中发挥着重要作用。然而，确切的机制仍然难以捉摸。该实验室和其他实验室最近的行为学研究表明，P物质在延髓头端腹内侧(RVM)的释放介导了后爪炎性损伤后的热和机械超敏反应。因此，神经激肽-1受体在右室的局部拮抗足以逆转完全弗氏佐剂(CFA)引起的痛觉过敏和超敏反应。全细胞膜片钳记录进一步显示，在CFA处理的大鼠，谷氨酸能传入神经元对脊髓投射的RVM神经元的促进作用依赖于P物质的释放。最后，突触后N-甲基-D-天冬氨酸受体(NMDAR)的药理拮抗作用没有作用，而细胞外应用NMDAR拮抗剂则阻止了这种促进作用。这些数据导致假设，位于RVM神经元P物质传入终末的NMDAR具有促进P物质释放的功能，炎性损伤后这些受体的上调启动了一种正反馈机制，以维持RVM内疼痛易化神经元的活动。三组互补的研究被提出来开始检验这一假说。首先，在四小时、四天或两周前接受足底注射生理盐水或CFA的大鼠的RVM中，将在分离的突触前终末(突触体)中研究谷氨酸或NMDA促进P物质释放的能力。NMDA受体的亚型将使用亚基特异性拮抗剂来确定。其次，炎症诱导的NMDA受体亚单位转录、翻译、表面表达和磷酸化状态的变化将使用实时定量聚合酶链式反应和免疫印迹技术对亚单位特异性抗体、磷酸化特异性抗体和生物素化探针在RVM突触体内进行评估。最后，用免疫组织化学方法证实CFA处理后RVM内P物质免疫反应阳性突触前结构中NMDAR亚单位表达的变化。这些研究结果将进一步加深我们对脊髓上核维持损伤后慢性疼痛的机制的理解，以及探索突触前NMDAR的活动依赖性可塑性。这些信息可能有助于开发有针对性的、中心活跃的疗法来治疗持续性疼痛状态。被选来检验这一假设的方法将为申请者提供神经化学、药理学、分子和免疫组织化学方法方面的广泛培训，为作为独立研究人员的职业生涯做准备。

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.