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的空白。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能提供足够的疗效而没有严重的副作用。长期目标是了解导致损伤性中枢致敏的机制，并为慢性pai建立临床相关的治疗靶点。本应用的目的是评估谷氨酸受体亚型对背根刺激（DRS）在背角诱发的Ca2+瞬态的贡献，并将增强的Ca2+反应与疼痛样行为的程度相关联。基于初步数据表明谷氨酸诱发的小鼠脊髓切片Ca2+反应在神经损伤后增强，主要假设是神经损伤增加背角AMPA受体信号，导致[Ca2+]i增加，导致中枢致敏和神经性疼痛。提出的项目的基本原理是背角神经元中的[Ca2+]i对中枢致敏和疼痛超敏是必不可少的。中心假设将通过追求三个特定目标来验证：AIM 1测试谷氨酸介导的神经元嗜离子性AMPA受体激活驱动Ca2+信号传导的假设。星形胶质细胞的电生理记录和实时荧光标记将用于评估对[Ca2+]i升高的背根刺激（DRS）有反应的细胞类型。其次，相对贡献

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.