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）：初级传入和背角神经元之间的突触是疼痛调节的关键部位，因为它们确定了进入脊髓的伤害性信号的强度。 NMDA受体（NMDAR）由初级传入表示，并存在于其中央和外围末端。虽然对 它们在疼痛中的作用，中央终端中的NMDAR可能参与这些突触的长期增强，从而有助于中央敏化。该项目基于我们的结果，表明这些NMDAR通常处于非功能状态，并在诱导慢性疼痛期间起作用。这解决了一个持续的争议：发现的初始报告 NMDA向大鼠注射NMDA会导致主要传入释放P的物质P，但无法通过包括我们自己的（我们自己的）（包括我们自己的）的后期研究来复制它。现在，我们发现鞘内NMDA之前是脑衍生的神经营养因子（BDNF）引起了大量物质P释放。 SRC家族激酶（SFK）抑制剂与BDNF共同注射抑制了NMDA的作用，这表明BDNF诱导了NMDAR的SFK磷酸化。除BDNF外，Ephrinb2还可以使NMDA诱导的物质P释放。重要的是，我们发现在神经损伤模型中，这些NMDAR的功能持续了3天，这与神经损伤诱导BDNF从活化的小胶质细胞中释放并激活Ephrinb/Ephb受体系统的证据一致。因此，该项目将检验以下假设：1）具有功能性，主要传入中的NMDAR需要其NR2B亚基的磷酸化，这取决于SFK和蛋白质酪氨酸磷酸酶的活性平衡，2）sfk phossent and by biage in by and bdrins and bdnf的NMDARS磷酸化是bdnf ins in bdnf ins of bdnf ins of bdnf ins of nr2b subunit。在正常条件下，末端是非功能（即未磷酸化的），并且在某些慢性疼痛状态下磷酸化。具体目的是：1）研究SFK磷酸化在调节初级传入中的NMDAR中的作用，2）确定这些NMDAR的SFK磷酸化上游的信号，以及3）研究NMDAR的参与慢性疼痛。该方法包括使用两种在背根神经节（DRG）中选择性敲低NMDAR的转基因小鼠菌株，以确定诱导物质P释放的NMDAR是否释放（以神经蛋白1受体内在化测量）并导致神经性和内脏疼痛有助于基本亲戚。磷酸化这些NMDAR的SFK将通过在五个神经元SFK中的每一个中的DRG中选择性小干扰RNA（siRNA）敲低来识别。 DRG提取物的蛋白质印迹将用于测量用BDNF和Ephrinb处理后NR2B亚基的酪氨酸磷酸化。培养的DRG神经元中的斑块钳记录将研究添加BDNF和Ephrinb后NMDARS电流的变化。使用啮齿动物模型，我们将测试一个预测，即神经性疼痛和内脏疼痛会增加NMDA诱导的SP释放，并在主要传入中NMDAR的选择性敲低后减少。 公共卫生相关性：神经性疼痛包括自发疼痛或疼痛强度的增加，源于神经或中枢神经系统的病变，并影响大量人群（美国200万例）。炎症疼痛，肠易激综合征和其他形式的慢性疼痛也是重大的健康问题。该项目将研究慢性疼痛期间疼痛纤维传递信息到脊髓的机制的变化，重点关注NMDA受体和调节活性的信号分子。