Glutamate Receptor Modulation of Calcium Signaling in Neuropathic Pain

描述（由申请人提供）：该K 01职业奖的长期目标是了解导致损伤诱导的中枢致敏的机制，并建立慢性疼痛的改善治疗靶点。我的初步数据表明，AMPA受体驱动脊髓神经元谷氨酸诱发的Ca 2+瞬变和谷氨酸诱发的Ca 2+瞬变神经损伤后增强。中心假设是神经损伤通过增加背角中GluR 2 AMPA受体磷酸化和运输增强神经元活性和Ca 2+动员（Aim 1）（Aim 2），并且这种神经性疼痛机制受到内源性阿片类药物的张力抑制（Aim 3）。将使用创新的同步Ca 2+成像和全细胞电生理学来测试这一假设，以评估背根刺激（DRS）的Ca 2+瞬变和成人脊髓切片背角中的神经元活动。目标1。实验1a将评估假或备用神经损伤（SNI）对分离的AMPA受体Ca 2+信号和膜电流的影响。实验1b将评价A <$、A <$和c-纤维活化强度下的Ca 2+动员和膜电流DRS。目的2虽然AMPA GluR 2的磷酸化和运输与炎性疼痛有关，但介导神经损伤诱导的Ca 2+水平增强的机制尚不清楚。为了解决这个问题，实验2a将确定背角中总GluR 2和磷酸化GluR 2的水平和亚细胞定位。基于表明炎症诱导的GluR 2内化是NMDA受体依赖性的研究，实验2b将检验以下假设：鞘内施用NMDA受体拮抗剂将减少神经损伤诱导的GluR 2磷酸化和内化。目标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，从而掩盖神经性疼痛。目的3试图将这一发现扩展到我们的外周神经性疼痛模型。实验3a将尝试将传统SNI和4周内消退的改良神经损伤模型后的Ca 2+动员与疼痛样行为相关联。建立与行为分辨率相关的Ca 2+动员减少将使我们能够在实验3b中验证我们的假设。实验3b将评估阿片受体拮抗剂对DRS刺激的Ca 2+瞬变和神经元活性以及GluR 2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Ca 2+动员，神经元活性，GluR 2磷酸化和移动Ca 2 +-不渗透的GluR 2表达远离细胞表面。

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.