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

描述（由申请人提供）：慢性疼痛是一个重要的卫生保健问题，对全世界数百万人的生活质量产生不利影响，每年造成数十亿美元的生产力损失。近十年来的研究表明，脑干中的疼痛促进神经元在外周损伤后慢性疼痛的发生和维持中起着重要作用。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物质在吻侧腹内侧髓质（RVM）的释放介导了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显示，在cfa处理的大鼠中，谷氨酸能对脊髓突起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）的药理拮抗作用无效，而NMDAR拮抗剂的细胞外应用阻止了这种促进作用。这些数据导致假设NMDARs位于P物质传入RVM神经元的突触前末端，其功能是促进P物质的释放，并且这些受体在炎症损伤后的上调启动了一种积极的前馈机制，以维持RVM疼痛促进神经元的活动。提出了三组互补的研究来开始检验这一假设。首先，在4小时、4天或2周前接受足底注射生理盐水或CFA的大鼠RVM的分离突触前终末（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确定。其次，利用实时定量PCR和western blotting，结合亚基特异性抗体、磷酸化特异性抗体和生物素化探针，评估RVM突触体中NMDA受体亚基转录、翻译、表面表达和磷酸化状态在炎症诱导下的变化。最后，采用免疫组织化学方法确认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。这些研究结果将有助于我们进一步了解棘上核维持损伤后慢性疼痛的机制，并探讨突触前NMDAR的活动依赖性可塑性。这一信息有助于开发针对性的、集中主动的治疗方法来治疗持续性疼痛状态。为检验这一假设而选择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。

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.