Identification of novel analgesic targets in ascending spinal projection neurons

项目摘要/摘要 虽然慢性疼痛是一个巨大的公共卫生问题，其经济成本令人震惊 仅在美国，每年就有5600-6350亿美元，驱动神经元过度兴奋的分子机制 在伤害性反应的通路中，人们仍然不完全了解。在以下方面取得了重大进展： 初级感觉神经元的遗传异质性及其在神经损伤后的可塑性 或组织损伤。然而，人们对这些神经元的全面分子图谱知之甚少。 它们将伤害性信息从脊髓传递到大脑，尽管它们对疼痛显然很重要 感知力。更好地了解脊髓投射神经元内基因表达的完整模式 可以揭示新的基于证据的策略来选择性地抑制脊髓伤害性网络的输出 作为缓解慢性疼痛的一种手段。长期目标是更好地了解神经和组织损伤是如何 改变中枢神经系统伤害性感受回路的功能。该应用程序的目标是识别由伤害引起的 慢性疼痛增强脊髓投射神经元内基因表达的变化 条件。中心假设是上升的脊髓投射神经元表现出一种独特的分子 外周损伤显著调节的表型，以促进膜的超兴奋性。这个 这项拟议工作的基本原理是，识别在脊髓中优先表达的基因 投射神经元将产生新的药理学方法来抑制伤害性感受器的上行流动 信息传递到大脑，同时最大限度地减少对全局感觉运动处理的有害干扰 脊髓。中心假设将通过追求以下具体目标来检验：(1)识别基因 丰富的成人脊髓内的上升投射神经元；以及(2)阐明 慢性疼痛条件下投射神经元的基因表达增加膜兴奋性。 这些目标将通过翻译核糖体亲和纯化(TRAP)和下一代来实现 RNA测序技术与生物信息学、电生理、免疫组织化学相结合 和原位杂交法。这项拟议的工作具有创新性，因为它将首次揭示 连接脊髓伤害性感觉回路和小鼠大脑的神经元的遗传表型 严重参与神经病理性和炎症性疼痛的产生，以及阐明 这一群体的分子特征在慢性疼痛状态下发生了变化。这些措施的结果 研究将发现脊髓投射神经元的新的细胞类型特异性标记物和 周围损伤可放大伤害性“获得”的潜在遗传机制的识别 在脊髓中的传播。因此，这项拟议的研究具有重要意义，因为它将揭示新奇 分子靶点可以被操纵来选择性地沉默上升的脊髓投射神经元 为了唤起安全和有效的止痛，同时最大限度地减少不良副作用。

Project Summary/Abstract While chronic pain represents a massive public health problem with a staggering economic cost of $560-$635 billion each year in the U.S. alone, the molecular mechanisms driving neuronal hyperexcitability within nociceptive pathways remain incompletely understood. Significant progress has been made towards elucidating the genetic heterogeneity of primary sensory neurons and their plasticity in the aftermath of nerve or tissue damage. However, much less is known about the comprehensive molecular profile of those neurons that convey nociceptive information from the spinal cord to the brain, despite their clear importance for pain perception. A better understanding of the complete pattern of gene expression within spinal projection neurons could reveal new evidence-based strategies to selectively dampen the output of the spinal nociceptive network as a means to alleviate chronic pain. The long-term goal is to better understand how nerve and tissue damage alter the function of nociceptive circuits in the CNS. The objective of this application is to identify injury-evoked changes in gene expression within spinal projection neurons that enhance their firing under chronic pain conditions. The central hypothesis is that ascending spinal projection neurons exhibit a unique molecular phenotype that is significantly modulated by peripheral injury to promote membrane hyperexcitability. The rationale for the proposed work is that the identification of genes that are preferentially expressed in spinal projection neurons will yield new pharmacological approaches to suppress the ascending flow of nociceptive information to the brain, while minimizing unwanted disruptions to global sensorimotor processing within the spinal cord. The central hypothesis will be tested by pursuing the following specific aims: (1) Identify genes that are enriched in ascending projection neurons within the adult spinal cord; and (2) Elucidate changes in gene expression in projection neurons under chronic pain conditions that increase membrane excitability. These aims will be accomplished by using translating ribosome affinity purification (TRAP) and next generation RNA sequencing techniques in combination with bioinformatics, electrophysiological, immunohistochemical and in situ hybridization approaches. The proposed work is innovative because it will reveal, for the first time, the genetic phenotype of those neurons connecting the spinal nociceptive circuit to the mouse brain that are critically involved in the generation of neuropathic and inflammatory pain, as well as elucidate how the molecular signature of this population changes during the chronic pain state. The outcome of these investigations will be the discovery of new, cell type-specific markers of spinal projection neurons and the identification of potential genetic mechanisms by which peripheral injuries can amplify the “gain” of nociceptive transmission in the spinal cord. As a result, the proposed research is significant because it will reveal novel molecular targets which could be manipulated to selectively silence ascending spinal projection neurons after injury, in order to evoke safe and effective analgesia while minimizing undesirable side effects.