Mechanisms in Primary Nociceptors that Drive Ongoing Activity and Ongoing Pain

项目摘要 这个项目的长期目标是发现新的、高度有针对性的方法来治疗正在进行的 通过定义驱动疼痛的主要伤害性感受器中正在进行的活动(OA)的关键机制来实现疼痛。近期 研究发现，OA在可能的伤害性感受器中自发产生，与持续的疼痛有关 脊髓损伤(SCI)后与所有三种电生理改变有关，原则上可以 推广办公自动化。这些是静息膜电位(RMP)的去极化，降低的电压阈值 动作电位(AP)和大的、瞬时的、去极化的自发波动的频率增加 (DSF)。与炎症有关的两种外源性介质，5-羟色胺(5-羟色胺)和辣椒素(模拟 内源性TRPV1激活剂)也促进骨关节炎，在很大程度上是通过增强DSF来实现的。几乎什么都不知道 关于大型DSF背后的机制。三个特定的目标将检验关于DSF生成和 增强，使用全细胞膜片记录，通过钙离子去激活，药理和 转基因方法、活体记录和行为测试。目标1将定义离子电导和电池 信号(Ca~(2+)和cAMP)在大型DSFs的急性生成中发挥作用，利用这一能力 使用幼稚的大鼠和转基因小鼠，使用5-羟色胺、Forsklin和辣椒素来快速刺激大型DSFs。这个 重点将包括HCN通道、T-型钙通道和Nav1.8通道。我们将特别注意 TRPC4/5频道，这对办公自动化很重要，并且具有独特的特性 大型DSF的数量。目标2将定义离子电导和细胞信号，以促进在 慢性脊髓损伤和亚急性外周炎症模型(后爪注射完全弗氏佐剂 -终审法院)。将测试目标1中发现的对大型DSF重要的通道是否有更改的贡献和 在每个模型中都有表达。促进办公自动化的变更预计将在这些模型中共享(因此 可能会导致多种形式的持续性疼痛)。目标3将测试联合干预措施的预测 选择性地阻断较大的DSF和提高AP阈值将减少持续的疼痛。一种新型止痛药 将测试一种策略，该策略将一种防止大量DSF产生的药物(TRPC4/5阻滞剂)与 选择性提高伤害性感受器AP阈值的药物(Nav1.8阻滞剂)。组合应该是 以低于观察所需的剂量有效地抑制伤害性感受器OA和随之而来的持续性疼痛 单独服用任何一种药物对持续疼痛的影响。这一预测将在体内通过记录的C-纤维OA进行验证 从麻醉大鼠的背根和脊髓损伤大鼠以及大鼠和小鼠CFA模型中观察其对持续性疼痛的影响。这 有针对性的方法可以为严重持续疼痛的新治疗奠定基础，这些疼痛相对较少 副作用，并提供阿片类药物的替代品，其风险也随之而来。

Project Summary The long-term objective of this project is to discover novel, highly targeted approaches for treating ongoing pain by defining critical mechanisms of ongoing activity (OA) in primary nociceptors that drive this pain. Recent discoveries revealed that the OA generated spontaneously in probable nociceptors and linked to ongoing pain after spinal cord injury (SCI) is associated with all three electrophysiological alterations that, in principle, can promote OA. These are depolarization of resting membrane potential (RMP), reduced voltage threshold for action potentials (APs), and increased frequency of large, transient, depolarizing spontaneous fluctuations (DSFs). Two extrinsic mediators related to inflammation, serotonin (5-HT) and capsaicin (mimicking endogenous TRPV1 activators), also promote OA, in large part by enhancing DSFs. Virtually nothing is known about mechanisms underlying large DSFs. Three specific aims will test hypotheses about DSF generation and potentiation, employing whole cell patch recording, stimulation by Ca2+ uncaging, pharmacological and transgenic approaches, in vivo recording, and behavioral tests. Aim 1 will define ion conductance and cell signaling (Ca2+ and cAMP) contributions to the acute generation of large DSFs, taking advantage of the ability of 5-HT, forskolin, and capsaicin to rapidly stimulate large DSFs, using naïve rats and transgenic mice. The focus will include HCN channels, T-type Ca2+ channels, and Nav1.8 channels. Special attention will be paid to TRPC4/5 channels, which are important for OA and have unusual properties that account for unique features of large DSFs. Aim 2 will define ion conductances and cell signals that promote large DSF generation in chronic SCI and in a subacute peripheral inflammation model (hindpaw injection of complete Freund's adjuvant - CFA). The channels found in Aim 1 to be important for large DSFs will be tested for altered contributions and expression in each model. Alterations promoting OA are predicted to be shared in these models (and thus to potentially drive many forms of ongoing pain). Aim 3 will test the prediction that combined interventions selectively blocking large DSFs and elevating AP threshold will reduce ongoing pain. A novel analgesic strategy will be tested, which combines a drug that prevents large DSF generation (a TRPC4/5 blocker) with a drug that selectively elevates AP threshold in nociceptors (a Nav1.8 blocker). The combination should efficiently suppress nociceptor OA and consequent ongoing pain at doses lower than required to observe any effect on ongoing pain from either drug alone. This prediction will be tested in vivo both on C-fiber OA recorded from dorsal roots of anesthetized rats and on ongoing pain in SCI rats and in rat and mouse CFA models. This targeted approach could lay the foundation for new treatments for severe ongoing pain that have relatively few side effects and provide an alternative to opioids, with their attendant risks.