Glutamate-evoked calcium signaling in spinal cord after nerve injury

描述（由申请人提供）：在理解引发和维持神经性疼痛的细胞机制方面存在根本性差距。这一差距代表了一个重要的问题，因为目前的镇痛药物很少提供足够的疗效而没有严重的副作用。长期目标是了解损伤诱导的中枢致敏机制，并建立慢性派相关的临床治疗靶点。本申请的目的是评估谷氨酸受体亚型对背根刺激（DRS）诱发的背角Ca 2+瞬变的贡献，并将增强的Ca 2+反应与疼痛样行为的幅度相关联。基于表明神经损伤后小鼠脊髓切片中谷氨酸诱发的Ca 2+反应增强的初步数据，中心假设是神经损伤增加了背角中的AMPA受体信号传导，导致[Ca 2 +]i增加，从而导致中枢敏化和神经性疼痛。该项目的基本原理是背角神经元中的[Ca 2 +]i对于中枢敏化和疼痛超敏反应是必不可少的。中心假设将通过追求三个具体目标进行测试：AIM 1测试的假设，谷氨酸介导的激活神经元离子型AMPA受体驱动Ca 2+信号。星形胶质细胞的电生理记录和实时荧光标记将用于评价响应背根刺激（DRS）的[Ca 2 +]i升高的细胞类型。其次，相对贡献 谷氨酸受体亚型将通过在选择性拮抗剂存在下定量DRS诱发的[Ca 2 +]i瞬变来确定。目的2：验证周围神经损伤可增强背根反射诱发的Ca ~（2+）反应，并与痛觉过敏程度相关的假说。为了允许行为和[Ca 2 +]i之间的相关性分析，已经开发了神经损伤的变体模型，其在1周内逐渐诱发强烈的异常性疼痛，然后在4周内消退。将评价行为痛觉过敏，并将其与来自在损伤后7、14和21天处死的假手术、传统和变异神经损伤动物的脊髓切片中的DRS诱发的[Ca 2 +]i进行比较。目的3检验PKM？介导SNI诱导的痛觉过敏、Ca 2+信号和背角AP频率增加的假设。我们将给予假手术和SNI小鼠多种PKM抑制剂，并测量疼痛样行为、Ca 2+瞬变和/或AP频率。基于我们的初步结果，我们预测PKM？阻断将逆转损伤诱导的痛觉过敏，[Ca 2 +]i和AP频率的增加。 该项目采用了创新的宽场钙成像，同时从成年小鼠脊髓切片的许多细胞。这项研究具有重要意义，因为它揭示了调节DRS诱发的Ca 2+瞬变的Ca 2+通道，并且是理解神经损伤诱导的神经元Ca 2+信号增强的关键第一步。最终，这些知识将建立临床相关的治疗目标，以减轻慢性疼痛。 公共卫生相关性：慢性疼痛管理是一项重大的科学和卫生保健挑战，因为目前的镇痛药物很少在没有严重副作用的情况下提供足够的疗效。该项目与NINDS的使命相关，因为它将1）确定导致损伤诱导的中枢致敏的机制，2）建立缓解慢性疼痛的临床相关治疗靶点。这项研究计划采用了高度创新的宽场钙成像技术，从成年小鼠的单个脊髓切片中的许多细胞中获得。

DESCRIPTION (provided by applicant): A fundamental gap exists in understanding the cellular mechanisms that initiate and maintain neuropathic pain. This gap represents an important problem because current analgesic drugs rarely provide sufficient efficacy without serious side effects. The long-term goal is to understand the mechanisms that lead to injury-induced central sensitization and establish clinically relevant therapeutic targets for chronic pai. The objective in this application is to evaluate the contribution glutamate receptor subtypes to dorsal root stimulation (DRS)-evoked Ca2+ transients in the dorsal horn, and correlate enhanced Ca2+ responses with the magnitude of pain-like behavior. Based on preliminary data suggesting that glutamate-evoked Ca2+ responses in mouse spinal cord slices are potentiated after nerve injury, the central hypothesis is that nerve injury increases AMPA receptor signaling in the dorsal horn, leading to increases in [Ca2+]i that results in central sensitization and neuropathic pain. The rationale for the proposed project is that [Ca2+]i in dorsal horn neurons is essential for central sensitization and pain hypersensitivity. The central hypothesis will be teste by pursuing three specific aims: AIM 1 tests the hypothesis that glutamate-mediated activation of neuronal ionotropic AMPA receptors drives Ca2+ signaling. Electrophysiological recordings and real-time fluorescent labeling of astrocytes will be used to evaluate the cell types that respond to dorsal root stimulation (DRS) with a rise in [Ca2+]i. Next, the relative contribution of glutamate receptor subtypes will be determined by quantifying DRS-evoked [Ca2+]i transients in the presence of selective antagonists. AIM 2 tests the hypothesis that peripheral nerve injury potentiates DRS-evoked Ca2+ responses, and this will correlate with the magnitude of hyperalgesia. To allow for a correlation analysis between behavior and [Ca2+]i, a variant model of nerve injury has been developed that gradually elicits robust allodynia in 1 week and then resolves in 4 weeks. Behavioral hyperalgesia will be evaluated and compared to DRS-evoked [Ca2+]i in spinal cord slices from sham, traditional and variant nerve injured animals sacrificed at 7, 14 and 21 d after injury. AIM 3 tests the hypothesis that PKM¿ mediates SNI-induced increases in hyperalgesia, Ca2+ signaling and AP frequency in dorsal horn. We will administer multiple PKM¿ inhibitors to sham and SNI mice and measure pain- like behavior, Ca2+ transients and/or AP frequency. Based on our preliminary results, we predict that PKM¿ blockade will reverse injury-induced hyperalgesia, increases in [Ca2+]i and AP frequency. This project employs innovative wide-field calcium imaging simultaneously from numerous cells in spinal cord slices from adult mice. The proposed research is significant because it reveals the Ca2+ channels that regulate DRS-evoked Ca2+ transients, and is a critical first step in understanding nerve injury-induced potentiation of neuronal Ca2+ signaling. Ultimately, this knowledge will establish clinically relevant therapeutic targets for alleviating chronic pain. PUBLIC HEALTH RELEVANCE: Chronic pain management is a major scientific and health care challenge, as current analgesic drugs rarely provide sufficient efficacy in the absence of serious side effects. This project is relevant to NINDS's mission because it will 1) determine mechanisms that lead to injury-induced central sensitization and 2) establish clinically relevant therapeutic targets for alleviating chronic pain. This Research Plan employs highly innovative wide-field calcium imaging from numerous cells in a single spinal cord slice from adult mice.