Glutamate Receptor Modulation of Calcium Signaling in Neuropathic Pain

描述(申请人提供)：这个K01职业奖的长期目标是了解导致损伤诱导的中枢敏化的机制，并建立改善慢性疼痛的治疗目标。我的初步数据表明，AMPA受体在脊髓神经元中驱动谷氨酸引起的钙瞬变，并且在神经损伤后谷氨酸引起的钙瞬变被增强。中心假说是，神经损伤通过增加背角GluR2 AMPA受体的磷酸化和转运(目标2)来增强神经元的活性和钙动员(目标1)，并且这种神经病理性疼痛的机制被内源性阿片类药物张力抑制(目标3)。这一假说将被用创新的同步钙成像和全细胞电生理学来验证，以评估成年脊髓脑片背根刺激(DRS)的钙瞬变和神经元活动。目的1.实验1a观察假损伤或备用神经损伤(SNI)对单个AMPA受体钙信号和膜电流的影响。实验1b将评估钙离子动员和膜电流DRS在A？、A？和C-纤维激活强度。目的：虽然AMPA GluR2的磷酸化和转运与炎性疼痛有关，但介导神经损伤诱导的钙水平升高的机制尚不清楚。为了解决这个问题，实验2a将确定总的和磷酸化的GluR2在背角的水平和亚细胞定位。基于炎症诱导的GluR2内化依赖于NMDA受体的研究，实验2b将检验鞘内注射NMDA受体拮抗剂将减少神经损伤诱导的GluR2的磷酸化和内化的假设。目的3.我的新数据表明，阿片类药物在炎症后抑制谷氨酸受体功能，从而掩盖神经病理性疼痛。目标3试图将这一发现扩展到我们的外周神经病理性疼痛模型。实验3a将尝试将钙离子动员与传统SNI和一种可缓解4周以上的改良神经损伤模型后的痛样行为联系起来。建立与行为分解相关的钙动员减少将使我们能够在实验3b中检验我们的假设。实验3b将评估阿片受体拮抗剂对DRS刺激的钙瞬变和神经元活动、GluR2磷酸化和亚细胞定位的影响。我们预计阿片受体拮抗剂将增加钙离子动员、神经元活性、GluR2磷酸化，并将不透钙的GluR2表达从细胞表面转移出去。

DESCRIPTION (provided by applicant): The long term goal of this K01 career award is to understand the mechanisms that lead to injury-induced central sensitization and to establish ameliorative therapeutic targets for chronic pain. My preliminary data suggests that AMPA receptors drive glutamate-evoked Ca2+ transients in spinal cord neurons and that glutamate-evoked Ca2+ transients are potentiated following nerve injury. The central hypothesis is that nerve injury potentiates neuronal activity and Ca2+ mobilization (Aim 1), via increased GluR2 AMPA receptor phosphorylation and trafficking in the dorsal horn (Aim 2), and that this mechanism of neuropathic pain is tonically inhibited by endogenous opioids (Aim 3). This hypothesis will be tested using innovative simultaneous Ca2+ imaging and whole-cell electrophysiology to evaluate dorsal root stimulated (DRS) Ca2+ transients and neuronal activity in dorsal horn of adult spinal cord slices. Aim 1. Experiment 1a will evaluate the effect f sham or spared nerve injury (SNI) on isolated AMPA receptor Ca2+ signals and membrane currents. Experiment 1b will evaluate Ca2+ mobilization and membrane currents DRS at A¿, A¿ and c-fiber activation strength. Aim 2 While AMPA GluR2 phosphorylation and trafficking have been implicated in inflammatory pain, the mechanisms that mediate nerve injury-induced potentiation of Ca2+ levels are not known. To address this question, Experiment 2a will determine levels and subcellular localization of total and phosphorylated GluR2 in dorsal horn. Based on studies indicating inflammation-induced GluR2 internalization is NMDA receptor-dependent, Experiment 2b will test the hypothesis that intrathecal administration of NMDA receptor antagonists will reduce nerve-injury induced phosphorylation and internalization of GluR2. Aim 3. My new data suggest that opioids tonically inhibit glutamate receptor function after inflammation and thus mask neuropathic pain. Aim 3 seeks to extend this discovery to our model of peripheral neuropathic pain. Experiment 3a will attempt to correlate Ca2+ mobilization with pain-like behavior following traditional SNI and a modified nerve injury model that resolves over 4 weeks. Establishing a decrease in Ca2+ mobilization in association with behavioral resolution will allow us to test our hypothesis in Experiment 3b. Experiment 3b will evaluate the effects of opioid receptor antagonists on: DRS-stimulated Ca2+ transients and neuronal activity, and GluR2 phosphorylation and subcellular localization. We expect that opioid receptor antagonists will increase Ca2+ mobilization, neuronal activity, GluR2 phosphorylation and shift Ca2+-impermeable GluR2 expression away from the cell surface.