NMDA Receptors in Primary Afferents

描述(申请人提供)：初级传入神经元和背角神经元之间的突触是痛觉调制的关键部位，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，存在于中枢和外周终末。尽管人们对此知之甚少 它们在疼痛中的作用，中央终末的NMDAR可能参与了这些突触的长期增强，从而有助于中枢敏化。这个项目是基于我们的结果，表明这些NMDAR通常处于非功能状态，并在慢性疼痛诱导期间变得有功能。这解决了一个正在进行的争议：一份初步报告发现 鞘内注射NMDA可诱导初级传入细胞释放P物质，但这一结果不能被包括我们自己在内的后续研究所复制。我们现在发现，在鞘内注射NMDA之前，脑源性神经营养因子(BDNF)可以引起大量的P物质释放。联合注射Src家族激酶(SFK)抑制剂和BDNF可抑制NMDA的作用，提示BDNF可诱导NMDARs的SFK磷酸化。除了脑源性神经营养因子外，ewitinB2似乎还能使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可以在3天内发挥作用，这与神经损伤诱导激活的小胶质细胞释放BDNF并激活ePhinB/EphB受体系统的证据一致。因此，本项目将检验以下假设：1)初级传入中的NMDAR需要其NR2B亚单位的酪氨酸磷酸化，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磷酸化是由BDNF和ephins启动的；以及3)初级传入终末中的NMDAR在正常情况下是无功能的(即未被磷酸化)，并在某些慢性疼痛状态下变得磷酸化。其具体目的是：1)研究SFK磷酸化在调节初级传入NMDAR中的作用；2)确定这些NMDAR的SFK磷酸化上游信号；3)研究NMDAR在慢性疼痛初级传入中的作用。该方法包括使用两个品系的转基因小鼠选择性地敲除背根神经节(DRG)中的NMDAR，以确定诱导P物质释放(测量为神经激肽1受体内化)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。将通过在五个神经元SFK的DRG中选择性地敲除小干扰RNA(SiRNA)来识别使这些NMDAR磷酸化的SFK。背根神经节提取物的Western blotting将用于测量在BDNF和ewitinB处理后NR2B亚单位的酪氨酸磷酸化。培养的背根节神经元的膜片钳记录将研究加入BDNF和ewitinB后NMDARs电流的变化。使用啮齿动物模型，我们将检验神经病理性和内脏痛增加NMDA诱导的SP释放，并在选择性地敲除NMDARs后减少初级传入的预测。

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.