Mechanisms in nociceptors driving ongoing activity and ongoing pain.

项目摘要 持续疼痛的管理不仅改善了患者的舒适度，还加速了术后的恢复和 减少发生慢性疼痛的可能性，最终降低护理成本。多种类型的疼痛 休息(自发或持续性疼痛)可能是由伤害性感受器的持续活动(OA)驱动的，这种活动发生在 缺乏不连续的刺激。在神经病理性疼痛模型中，伤害性骨性关节炎是由长时间的 静息膜电位去极化，动作电位阈值降低，An增加 发生膜电位的大幅去极化自发波动(DSF)。一种低剂量的 炎症介质5-羟色胺(5-羟色胺)，当与人工去极化结合时，强烈增强 在未受伤的大鼠可能的伤害性感受器中产生大的DSFs和OA，表明DSFs也可以 极大地增强了促进办公自动化的能力。多种5-羟色胺受体在感觉神经元中表达，它是 尚不清楚这些受体类型和下游通路中哪些对DSFs的5-羟色胺增强有重要作用。 以往的研究表明，AKAP支架上的PKA活性对维持骨关节炎具有重要意义。 脊髓损伤后的伤害性感受器T型电压门控钙通道Cav3.2受PKA和 GS偶联的5HT7受体活性和依赖PKA的Slack KNA通道内化诱导伤害性感受器 过度兴奋。这些和其他观察结果导致假设外周5-羟色胺调节特异性 通过PKA的CA2(T型)和K(KNA)电导产生大的DSF，促进伤害性感受器中的OA 最终会带来持续的痛苦。这一假设将在三个具体目标上得到检验。在目标1中，我们将确定 DSFs和OA中5-羟色胺增强的受体和细胞信号机制。使用可用的 药理工具以及膜片钳和高含量显微镜技术，我们将测试 假说DSFs的5-羟色胺增强主要由Gs偶联的5-羟色胺受体下游的PKA激活所介导。 羟色胺受体。在目标2中，对于5-羟色胺依赖的大DSFs的产生和 将对办公自动化进行定义。我们将利用膜片钳记录和药理学来探讨其必要性和充分性。 T型钙通道电导和TRPC通道电导的增加以及KNA通道电导的抑制 大型DSF的生成。我们用于增强大型DSF和OA生成的机制模型可能 尤其与深部组织切开疼痛有关，在深组织切开疼痛中，血小板聚集物在A组及附近释放5-HT。 伤口。深部组织切开已被证明在伤害性感受器和自发性疼痛行为中都能诱发骨性关节炎。 受伤后的几天。目前尚不清楚5-羟色胺或切口处或附近的PKA是否在骨性关节炎中起作用。 以及切割后持续的疼痛。在目标3中，我们将确定外周5-羟色胺和PKA是否参与 术后疼痛模型中的疼痛行为。这项拟议的研究将为分子生物学提供新的见解。 痛觉感受器痛相关持续活动的机制及外周5-羟色胺的作用 在术后疼痛模型中，PKA与持续的和诱发的疼痛有关。

Project Summary Management of ongoing pain not only improves patient comfort but also accelerates postoperative recovery and diminishes the likelihood of developing chronic pain, ultimately reducing the cost of care. Many types of pain at rest (spontaneous or ongoing pain) are likely driven by ongoing activity (OA) in nociceptors that occurs in the absence of a discrete stimulus. In a model of neuropathic pain, nociceptive OA is produced by a prolonged depolarization of resting membrane potential, reduction of the action potential threshold, and an increased incidence of large depolarizing spontaneous fluctuations (DSFs) of the membrane potential. A low dose of the inflammatory mediator serotonin (5-HT), when combined with artificial depolarization, strongly potentiates the generation of large DSFs and OA in probable nociceptors from uninjured rats, showing that DSFs can also be enhanced acutely to promote OA. A variety of 5-HT receptors are expressed in sensory neurons, and it is unknown which of these receptor types and downstream pathways are important for 5-HT potentiation of DSFs. Previous studies demonstrate that AKAP-scaffolded PKA activity is important for maintenance of OA in nociceptors after spinal cord injury. The T-type voltage-gated Ca2+ channel Cav3.2 is stimulated by PKA and by Gs-coupled 5HT7 receptor activity, and PKA-dependent internalization of Slack KNa channels induces nociceptor hyperexcitability. These and other observations led to the hypothesis that peripheral 5-HT modulates specific Ca2+ (T-type), and K+ (KNa) conductances via PKA to generate large DSFs that promote OA in nociceptors and ultimately ongoing pain. This hypothesis will be tested in three specific aims. In Aim 1, we will determine the receptors and cell signaling mechanisms mediating 5-HT potentiation of DSFs and OA. Using available pharmacological tools along with patch clamp and high content microscopy techniques, we will test the hypothesis that 5-HT enhancement of DSFs is mediated largely by PKA activation downstream of Gs-coupled 5- HT receptors. In Aim 2, the specific conductances important for 5-HT-dependent generation of large DSFs and OA will be defined. Using patch clamp recording and pharmacology, we will probe the necessity and sufficiency of increased T-type Ca2+ and TRPC channel conductances and inhibition of KNa channel conductance for generation of large DSFs. Our mechanistic model for enhancement of the generation of large DSFs and OA may be particularly relevant to deep tissue incision pain in which platelet aggregates release 5-HT at and near a wound. Deep tissue incision has been shown to induce both OA in nociceptors and spontaneous pain behavior for a few days after injury. It is unknown whether 5-HT or PKA at or near the incision site plays a role in the OA and ongoing pain following incision. In Aim 3, we will determine whether peripheral 5-HT and PKA contribute to pain behavior in a model of postoperative pain. This proposed study will contribute new insight into the molecular mechanisms underlying pain-related ongoing activity in nociceptors and the contributions of peripheral serotonin and PKA to ongoing and evoked pain in a postoperative pain model.