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病理性疼痛的细胞机制方面存在着根本的差距。这一差距是一个重要的问题，因为目前的止痛药很少能在没有严重副作用的情况下提供足够的疗效。长期目标是了解导致损伤诱导的中枢敏化的机制，并为慢性PAI建立临床相关的治疗靶点。本研究的目的是评估谷氨酸受体亚型在背根刺激(DRS)诱发的背角钙瞬变中的作用，并将增强的钙反应与痛样行为的程度相关联。根据初步数据显示，神经损伤后小鼠脊髓脑片谷氨酸诱发的钙反应增强，中心假设是神经损伤增加了背角AMPA受体信号，导致[Ca+]i增加，从而导致中枢敏化和神经病理性疼痛。该项目的基本原理是，背角神经元中的[Ca~(2+)]i对中枢敏感化和疼痛超敏是必不可少的。中心假说将通过追求三个具体目标来检验：Aim 1检验谷氨酸介导的神经元离子型AMPA受体激活驱动钙信号传导的假说。电生理记录和星形胶质细胞的实时荧光标记将被用来评估响应背根刺激(DRS)的细胞类型随着[Ca~(2+)]i的升高。 谷氨酸受体亚型将通过在选择性拮抗剂存在的情况下量化DRS诱发的[Ca~(2+)]i瞬变来确定。目的2验证周围神经损伤增强DRS诱发的钙反应的假说，这将与痛觉过敏的程度相关。为了进行行为和[Ca~(2+)]i之间的相关性分析，已经建立了一种不同的神经损伤模型，该模型在1周内逐渐引起强烈的异位痛觉，然后在4周内消失。分别于伤后7天、14天和21天处死动物，观察其行为痛敏反应，并与DRS诱发的[Ca~(2+)]i进行比较。目的3验证PKM介导SNI引起的痛觉过敏、钙信号和AP频率增加的假说。我们将给Sham和SNI小鼠应用多种PKM抑制剂，并测量疼痛行为、钙瞬变和/或AP频率。根据我们的初步结果，我们预测，阻断PKM将逆转损伤引起的痛敏，使[Ca~(2+)]i和AP频率增加。该项目采用了创新的广域钙成像，同时从成年小鼠的脊髓切片中提取大量细胞。这项研究意义重大，因为它揭示了调节DRS诱发的钙瞬变的钙通道，并且是理解神经损伤诱导的神经元钙信号增强的关键的第一步。最终，这些知识将建立临床上相关的治疗目标，以缓解慢性疼痛。

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.