NMDA Receptors in Primary Afferents

初级传入神经中的 NMDA 受体

• 批准号: 8401725
• 负责人: JAMES A MCROBERTS
• 金额: $ 27.72万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8401725
• 关键词: Affect; Aftercare; Autoreceptors; Binding; Brain-Derived Neurotrophic Factor; C Fiber; Chronic; Ephrin B Receptor; Ephrins; Equilibrium; Fiber; Health; Immunoprecipitation; Inflammation; Inflammatory; Injection of therapeutic agent; Intrathecal Injections; Irritable Bowel Syndrome; Lesion; Long-Term Potentiation; Measures; Microglia; Modeling; Mus; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; Nerve; Neuraxis; Neurons; Neurostimulation procedures of spinal cord tissue; Neurotrophic Tyrosine Kinase Receptor Type 2; Nociception; Pain; Pain intensity; Peripheral; Phosphorylation; Population; Posterior Horn Cells; Protein Tyrosine Phosphatase; RNA Interference; Rattus; Receptor Signaling; Reporting; Rodent Model; Role; Signal Transduction; Signaling Molecule; Site; Slice; Spinal Cord; Spinal Ganglia; Substance P; Substance P Receptor; Synapses; System; Testing; Transgenic Mice; Tyrosine Phosphorylation; United States; Visceral; Visceral pain; Western Blotting; base; central sensitization; chronic pain; dorsal horn; inhibitor/antagonist; kinase inhibitor; nerve injury; neurophysiology; painful neuropathy; patch clamp; presynaptic; receptor function; receptor internalization; research study; spinal nerve posterior root; spontaneous pain; src-Family Kinases

DESCRIPTION (provided by applicant): Synapses between primary afferents and dorsal horn neurons are key sites for pain modulation, because they determine the intensity of nociceptive signals entering the spinal cord. NMDA receptors (NMDARs) are expressed by primary afferents and are present in their central and peripheral terminals. Although little is known about their role in pain, NMDARs in the central terminals may be involved in long-term potentiation of these synapses, thus contributing to central sensitization. This project is based on our results indicating that these NMDARs are normally in a non-functional state and become functional during the induction of chronic pain. This resolves an ongoing controversy: an initial report found that intrathecal injections of NMDA to rats induced substance P release from primary afferents, but it could not be replicated by later studies including our own. We now find that intrathecal NMDA preceded by brain-derived neurotrophic factor (BDNF) elicited a substantial amount of substance P release. Co-injection of a Src family kinase (SFK) inhibitor with BDNF suppressed the effect of NMDA, suggesting that BDNF induces the SFK phosphorylation of NMDARs. In addition to BDNF, ephrinB2 also appears to enable NMDA-induced substance P release. Importantly, we found that in a nerve injury model these NMDARs become functional for 3 days, consistent with evidence that nerve injury induces BDNF release from activated microglia and activates the ephrinB/EphB receptor system. Accordingly, this project will test the following hypotheses: 1) To be functional, NMDARs in primary afferents require Tyr-phosphorylation of their NR2B subunit, which is determined by the balance of activities of SFKs and protein tyrosine phosphatases, 2) SFK phosphorylation of NMDARs in primary afferents is initiated by BDNF and ephrins, and 3) NMDARs in primary afferent terminals are non-functional (i.e. not phosphorylated) under normal conditions and become phosphorylated in some chronic pain states. The Specific Aims are: 1) study the role of SFK phosphorylation in modulating NMDARs in primary afferents, 2) identify the signals upstream of SFK phosphorylation of these NMDARs, and 3) investigate the involvement of NMDARs in primary afferents in chronic pain. The approach includes the use of two strains of transgenic mice with selective knockdown of NMDARs in dorsal root ganglia (DRG) to determine whether the NMDARs that induce substance P release (measured as neurokinin 1 receptor internalization) and contribute to neuropathic and visceral pain are located in primary afferents. The SFK that phosphorylates these NMDARs will be identified by selective small interference RNA (siRNA) knockdown in DRG of each of the five neuronal SFKs. Western blots of DRG extracts will serve to measure tyrosine phosphorylation of the NR2B subunit after treatment with BDNF and ephrinB. Patch-clamp recordings in cultured DRG neurons will study changes in NMDARs currents after adding BDNF and ephrinB. Using rodent models, we will test the predictions that neuropathic and visceral pain increase NMDA-induced SP release, and decrease after selective knockdown of NMDARs in primary afferents. PUBLIC HEALTH RELEVANCE: Neuropathic pain consists in spontaneous pain or increases in pain intensity originating with lesions in nerves or the central nervous system, and affects a large sector of the population (two million cases in the United States). Inflammation pain, irritable bowel syndrome and other forms of chronic pain are also significant health concerns. This project will study changes that occur during chronic pain in the mechanisms by which pain fibers deliver their messages to the spinal cord, focusing on the NMDA receptors and the signaling molecules that modulate their activity.

描述（由申请人提供）：初级传入神经元和背角神经元之间的突触是疼痛调节的关键位点，因为它们决定进入脊髓的伤害性信号的强度。 NMDA 受体 (NMDAR) 由初级传入神经表达，存在于其中枢和外周末梢。虽然人们知之甚少 考虑到它们在疼痛中的作用，中枢末端的 NMDAR 可能参与这些突触的长期增强，从而有助于中枢敏化。该项目基于我们的结果，表明这些 NMDAR 通常处于非功能状态，但在诱发慢性疼痛期间变得有功能。这解决了一个持续存在的争议：初步报告发现 向大鼠鞘内注射 NMDA 会诱导初级传入神经释放 P 物质，但后来的研究（包括我们自己的研究）无法复制这一结果。我们现在发现鞘内注射 NMDA 后加入脑源性神经营养因子 (BDNF) 会引发大量 P 物质释放。将 Src 家族激酶 (SFK) 抑制剂与 BDNF 共同注射可抑制 NMDA 的作用，表明 BDNF 诱导 NMDAR 的 SFK 磷酸化。除了 BDNF 之外，ephrinB2 似乎也能够促进 NMDA 诱导的 P 物质释放。重要的是，我们发现在神经损伤模型中，这些 NMDAR 可以发挥作用 3 天，这与神经损伤诱导激活的小胶质细胞释放 BDNF 并激活 ephrinB/EphB 受体系统的证据一致。因此，该项目将测试以下假设：1) 为了发挥功能，初级传入神经中的 NMDAR 需要其 NR2B 亚基的酪氨酸磷酸化，这是由 SFK 和蛋白酪氨酸磷酸酶的活性平衡决定的，2) 初级传入神经中 NMDAR 的 SFK 磷酸化是由 BDNF 和肝配蛋白启动的，3) 初级传入神经中的 NMDAR 末端在正常情况下是无功能的（即未磷酸化），但在某些慢性疼痛状态下会被磷酸化。具体目标是：1) 研究 SFK 磷酸化在调节初级传入神经中 NMDAR 中的作用，2) 识别这些 NMDAR SFK 磷酸化上游的信号，以及 3) 研究慢性疼痛中 NMDAR 在初级传入神经中的参与情况。该方法包括使用两种转基因小鼠品系，在背根神经节 (DRG) 中选择性敲低 NMDAR，以确定诱导 P 物质释放（以神经激肽 1 受体内化测量）并导致神经病理性和内脏疼痛的 NMDAR 是否位于初级传入神经中。磷酸化这些 NMDAR 的 SFK 将通过 5 个神经元 SFK 中每一个的 DRG 中的选择性小干扰 RNA (siRNA) 敲低来鉴定。 DRG 提取物的蛋白质印迹将用于测量 BDNF 和 ephrinB 处理后 NR2B 亚基的酪氨酸磷酸化。培养的 DRG 神经元中的膜片钳记录将研究添加 BDNF 和 ephrinB 后 NMDAR 电流的变化。使用啮齿动物模型，我们将测试以下预测：神经性疼痛和内脏疼痛会增加 NMDA 诱导的 SP 释放，并在初级传入神经中选择性敲除 NMDAR 后减少 SP 释放。 公共卫生相关性：神经性疼痛包括自发性疼痛或源自神经或中枢神经系统损伤的疼痛强度增加，影响很大一部分人群（美国有 200 万例）。炎症疼痛、肠易激综合症和其他形式的慢性疼痛也是重要的健康问题。该项目将研究慢性疼痛期间疼痛纤维向脊髓传递信息的机制发生的变化，重点关注 NMDA 受体和调节其活动的信号分子。