NMDA Receptors in Primary Afferents

描述（由申请人提供）：初级传入神经和背角神经元之间的突触是疼痛调制的关键部位，因为它们决定了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并存在于其中枢和外周终末。尽管我们对 虽然它们在疼痛中的作用，但中枢终末的NMDAR可能参与这些突触的长时程增强，从而有助于中枢敏化。这个项目是基于我们的研究结果，表明这些NMDAR通常处于非功能状态，并在慢性疼痛诱导过程中发挥作用。这解决了一个持续的争议：一份初步报告发现， 大鼠鞘内注射NMDA可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）未能重复这一结果。我们现在发现，鞘内注射NMDA之前，脑源性神经营养因子（BDNF）引起了大量的P物质释放。共同注射Src家族激酶（SFK）抑制剂与BDNF抑制NMDA的作用，表明BDNF诱导NMDAR的SFK磷酸化。除了BDNF，ephrinB 2似乎也能使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在3天内变得有功能，这与神经损伤诱导BDNF从活化的小胶质细胞释放并活化ephrinB/EphB受体系统的证据一致。因此，本项目将检验以下假设：1）为了发挥功能，初级传入中的NMDAR需要其NR 2B亚基的酪氨酸磷酸化，这由SFK和蛋白酪氨酸磷酸酶的活性平衡决定，2）初级传入中NMDAR的SFK磷酸化由BDNF和肝配蛋白启动，和3）初级传入终末中的NMDAR在正常条件下是无功能的（即不磷酸化），而在某些慢性疼痛状态下变为磷酸化。具体目标是：1）研究SFK磷酸化在调节初级传入中的NMDAR中的作用，2）鉴定这些NMDAR的SFK磷酸化的上游信号，和3）研究慢性疼痛中初级传入中NMDAR的参与。该方法包括使用两种选择性敲低背根神经节（DRG）中NMDAR的转基因小鼠品系，以确定诱导P物质释放（以神经激肽1受体内化测量）并导致神经性疼痛和内脏疼痛的NMDAR是否位于初级传入神经中。将通过在五种神经元SFK中的每一种的DRG中的选择性小干扰RNA（siRNA）敲低来鉴定使这些NMDAR磷酸化的SFK。DRG提取物的Western印迹将用于测量用BDNF和ephrinB处理后NR 2B亚基的酪氨酸磷酸化。在培养的DRG神经元中的膜片钳记录将研究添加BDNF和ephrinB后NMDARs电流的变化。使用啮齿动物模型，我们将测试神经性疼痛和内脏痛增加NMDA诱导的SP释放的预测，并在初级传入中选择性敲低NMDAR后减少。

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.