Serotonin Modulation of Trigeminal Nociceptors

描述(由申请人提供)：本研究培训计划的长期目标是作为一种培训机制，发展和磨练作为戴娜·R·洛伊德博士的神经科学家进行翻译研究所需的关键技能。利用博士后研究奖学金和结构化临床调查硕士(MSCI)计划作为正式培训工具，Loyd博士将开展一个关键研究项目，利用基础科学和临床研究方法回答有关颅面部疼痛外围机制的重要问题。目前F32应用的总体方法是表征5-羟色胺(5-HT)受体在大鼠和人三叉神经辣椒素敏感伤害性感受器(传递头面部疼痛的感觉神经元)上的分布和5-羟色胺的整体综合作用。瞬时受体电位V1通道(TRPV1)在热和辣椒素(辣椒中的刺激性成分)的刺激下，在三叉神经伤害性感受器上高度表达，在痛觉过敏的形成中起关键作用。5-羟色胺可能通过三叉神经伤害性感受器增加TRPV1的活性，作为痛觉诱发的机制。这项提案中概述的实验旨在检验这一假设，即作用于外周5HT受体的5HT增加了辣椒素敏感的三叉神经感觉神经元的活性。具体目的1将利用荧光原位杂交和免疫组织化学相结合的方法，表征口腔面部炎症大鼠三叉神经感觉神经元中表达TRPV1的5HT受体的表达。具体目标2将使用细胞信号(细胞内钙积累)和功能(促炎性肽释放)分析来确定5-羟色胺是否激活和/或敏化原代培养的大鼠辣椒素敏感感觉神经元。特殊目的3将使用荧光免疫组织化学方法分析正常牙髓和炎症牙髓中表达TRPV1的人伤害性感受器上5HT受体的表达。此外，Aim 3还将在体外检测5-羟色胺对辣椒素敏感的人三叉神经伤害性感受器释放促炎性多肽的能力。这项拟议的研究结果对于理解外周5-羟色胺如何参与调节大鼠和人类三叉神经伤害性感受器至关重要。长期以来，5-羟色胺一直被认为是一种由血小板、内皮细胞和免疫细胞在损伤部位释放的促炎介质，然而，5-羟色胺作用于感觉神经元调节疼痛的机制(S)尚不清楚。这些知识将提供对外周颅面疼痛机制的洞察，这可能会发现科学和医学以前未知的新的治疗靶点。我们的初步数据为这一假说提供了强有力的支持，其结果将对翻译产生重大影响。 公共卫生相关性：头面部疼痛，如偏头痛，代表着一个普遍且昂贵的医疗保健问题。该项目将评估5-羟色胺对大鼠和人三叉神经感觉神经元的潜在调制作用。如果是这样，那么针对外周5-羟色胺受体的新型药物可能在治疗颅面部疼痛方面非常有效。

DESCRIPTION (provided by applicant): The long-term objectives of this research training plan are to serve as a training mechanism to develop and hone the critical skills necessary for conducting translational research as a neuroscientist for Dr. Dayna R. Loyd. Using a postdoctoral research fellowship and a structured Masters in Clinical Investigation (MSCI) program as a formal training vehicle, Dr. Loyd will conduct a critical research project utilizing both basic science and clinical research methodologies to answer important questions on the peripheral mechanisms of craniofacial pain. The overall approach of the present F32 application is to characterize the distribution of serotonin (5HT) receptors and overall integrated actions of 5HT on rat and human trigeminal capsaicin- sensitive nociceptors (craniofacial pain transmitting sensory neurons). The transient receptor potential V1 channel (TRPV1), gaited by heat and capsaicin (the pungent ingredient in chili peppers), is highly expressed on trigeminal nociceptors and is critical to the development of hypersensitivity to pain. 5HT may increase the activity of TRPV1 as a mechanism to evoke pain via trigeminal nociceptors. Experiments outlined in this proposal aim to test the hypothesis that 5HT, acting at peripheral 5HT receptors, increases the activity of capsaicin-sensitive trigeminal sensory neurons. Specific Aim 1 will characterize the expression of 5HT receptors, utilizing a combination of florescent in situ hybridization and immunohistochemistry, in TRPV1-expressing trigeminal sensory neurons from rats with orofacial inflammation. Specific Aim 2 will determine whether 5HT activates and/or sensitizes rat capsaicin-sensitive sensory neurons in primary culture using cell signaling (intracellular calcium accumulation) and functional (proinflammatory peptide release) assays. Specific Aim 3 will analyze 5HT receptor expression on human TRPV1-expressing nociceptors in normal versus inflamed dental pulp using fluorescent immunohistochemistry. In addition, aim 3 will examine the ability of 5HT to increase proinflammatory peptide release from isolated capsaicin-sensitive human trigeminal nociceptors in vitro. The results of the proposed research are critical to understanding how peripheral serotonin is involved in modulating both rat and human trigeminal nociceptors. Serotonin has long been recognized as a pro- inflammatory mediator released at the site of injury by platelets, endothelial cells, and immune cells, however, the mechanism(s) by which serotonin acts on sensory neurons to modulate pain are unknown. This knowledge will provide insight on peripheral craniofacial pain mechanisms, which may uncover novel therapeutic targets previously unknown to science and medicine. Our preliminary data provide strong support for the hypothesis and the results will have a significant translational impact. PUBLIC HEALTH RELEVANCE: Craniofacial pain, such as migraine, represents a prevalent and expensive healthcare problem. This project will evaluate the potential modulation of rat and human trigeminal sensory neurons by serotonin. If so, then novel drugs targeting peripheral serotonin receptors may be highly effective at treating craniofacial pain.