Molecular profiling of medullary descending pain modulation circuits to discover novel analgesic targets

髓质下行疼痛调制回路的分子分析以发现新的镇痛靶点

• 批准号: 9962357
• 负责人: Gregory Scherrer
• 金额: $ 19.44万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9962357
• 关键词: Action Potentials; Acute; Acute Pain; Afferent Neurons; American; Analgesics; Anatomy; Behavioral; Brain Stem; Cells; Chronic; Classification; Development; Disinhibition; Electrophysiology (science); Exhibits; FOS gene; Fluorescence-Activated Cell Sorting; Fluorescent in Situ Hybridization; Future; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Hyperalgesia; Immediate-Early Genes; Interneurons; Label; Laboratories; Mechanics; Modeling; Molecular; Molecular Profiling; Molecular Target; Mus; Nervous system structure; Neurobiology; Neurons; Nociception; Nociceptors; Opioid; Opioid Analgesics; Pain; Pain management; Pathologic; Pattern; Persistent pain; Pharmaceutical Preparations; Pharmacology Study; Physiological; Population; Prevalence; Property; Reflex action; Reporter; Research; Role; Spinal; Spinal Cord; Spinal Ganglia; Spinal cord posterior horn; Spine pain; Stimulus; System; Systems Development; Tamoxifen; Therapeutic; Tissues; Transgenic Organisms; Trigeminal System; United States; Viral; Virus; Withdrawal; base; behavioral study; cell type; chronic pain; dorsal horn; experimental study; innovation; mouse genetics; mu opioid receptors; neural circuit; neuron development; new therapeutic target; novel; novel marker; opioid epidemic; opioid exposure; opioid use; pain perception; pain relief; prevent; promoter; recombinase; response; side effect; single-cell RNA sequencing; transcriptomics; transmission process

The opioid epidemic and prevalence of chronic pain in the United States have generated a critical need for new pain therapeutics with limited side effects. Pain descending control systems, which consist of brainstem neural circuits projecting to the spinal cord, critically modulate nociception in the spinal dorsal horn and may contribute to chronic pain. Previous studies established that these descending circuits include several populations of rostral ventromedial medulla (RVM) neurons. RVM neurons were originally classified based on their electrophysiological properties. Specifically, ON-cells exhibit increased activity in response to noxious stimuli and facilitate spinal pain transmission. In contrast, RVM OFF-cells display high basal tonic firing that is suppressed following noxious stimulation and exert inhibitory pain control. Despite their importance, RVM nociceptive neurons remain understudied and poorly defined at the molecular level compared to nociceptors and spinal neurons, preventing the targeting of descending control systems for pain management. This research proposes to combine neural circuit tracing, mouse genetics, and single cell RNA-sequencing (scRNA-seq) to identify marker genes that define nociceptive RVM neuronal types for functional studies. Aim 1 will determine the molecular signatures of spinally projecting RVM→SC ON-cells. To profile RVM→SC neurons, we inject a retrograde reporter virus (AAV- retro-CAG-GFP) into the dorsal horn, and use fluorescence-activated cell sorting (FACS) to isolate GFP+ retro- labelled RVM→SC neurons from RVM and perform scRNA-seq. Unbiased clustering of cells will identify numerous RVM neuronal cell types classified by expression of specific marker genes. Aim 2 will identify the RVM neurons that are active during pain. The expression of the immediate early gene c-fos has been used extensively for the mapping of CNS nociceptive neurons. To label RVM neurons active during pain, we use the recently developed FosTRAP2 mice, in which the promoter of c-fos drives the expression of CreERT2 recombinase, in combination with fluorescent Cre-reporter mouse lines or viruses. FACS will isolate fluorescently labeled RVM neurons, and scRNA-seq will identify the neuronal types activated during nociceptive stimulation. Finally, the RVM undergoes functional reorganization during chronic pain or opioid exposure. Aim 3 will thus determine the transcriptional changes in RVM→SC neurons associated with the transition from acute to chronic pain, and the emergence of side effects during chronic opioid treatment. We will use approaches described in Aim 1 in combination with chronic pain and opioid exposure models to resolve the transcriptional changes that occur in RVM→SC neurons during the development of these pathological hyperalgesic states. This research will considerably broaden our understanding of pain neurobiology, by providing a categorization of new markers that define classes of RVM neurons for the future functional identification of the discrete brainstem circuits that influence pain perception and opioids effects. Furthermore, this research may identify novel molecular targets in RVM neurons for the development of innovative analgesic strategies that modulate activity in descending pain control systems.

美国阿片类药物的流行和慢性疼痛的流行迫切需要新的药物 副作用有限的疼痛疗法。疼痛下行控制系统，由脑干神经元组成 投射到脊髓的电路，严格调节脊髓背角的伤害感受，并可能有助于 至慢性疼痛。先前的研究表明，这些下降回路包括几个吻侧种群 腹内侧延髓（RVM）神经元。 RVM 神经元最初是根据其电生理学分类的 特性。具体来说，ON 细胞对有害刺激的反应表现出增强的活性，并促进脊髓 疼痛传递。相比之下，RVM OFF 细胞表现出高基础强直放电，该放电在有害物质后被抑制 刺激并施加抑制性疼痛控制。尽管 RVM 伤害性神经元很重要，但它们仍然存在 与伤害感受器和脊髓神经元相比，在分子水平上研究不足且定义不明确， 以下行控制系统为目标进行疼痛管理。这项研究建议结合神经 电路追踪、小鼠遗传学和单细胞 RNA 测序 (scRNA-seq) 来识别标记基因 定义伤害性 RVM 神经元类型以进行功能研究。目标 1 将确定以下分子特征 脊髓突出的 RVM→SC ON 细胞。为了分析 RVM→SC 神经元，我们注射了逆行报告病毒（AAV- 逆转录-CAG-GFP）进入背角，并使用荧光激活细胞分选（FACS）分离GFP+逆转录- 从 RVM 标记 RVM→SC 神经元并执行 scRNA-seq。细胞的无偏聚类将识别出许多 RVM 神经元细胞类型根据特定标记基因的表达进行分类。目标 2 将识别 RVM 神经元 在疼痛期间活跃。立即早期基因 c-fos 的表达已被广泛用于 中枢神经系统伤害性神经元的绘图。为了标记疼痛期间活跃的 RVM 神经元，我们使用最近开发的 FosTRAP2小鼠，其中c-fos的启动子驱动CreERT2重组酶的表达，组合 带有荧光 Cre 报告小鼠系或病毒。 FACS 将分离荧光标记的 RVM 神经元，并且 scRNA-seq 将识别伤害性刺激期间激活的神经元类型。最后，RVM经历 慢性疼痛或阿片类药物暴露期间的功能重组。因此，目标 3 将决定转录 RVM→SC 神经元的变化与从急性疼痛到慢性疼痛的转变有关，以及 长期阿片类药物治疗期间的副作用。我们将结合使用目标 1 中描述的方法 慢性疼痛和阿片类药物暴露模型可解决 RVM→SC 神经元中发生的转录变化 在这些病理性痛觉过敏状态的发展过程中。这项研究将大大拓宽我们的视野 通过提供定义 RVM 类别的新标记物的分类，了解疼痛神经生物学 神经元用于未来功能识别影响疼痛感知的离散脑干回路 和阿片类药物的影响。此外，这项研究可能会确定 RVM 神经元中的新分子靶点 开发调节下行疼痛控制系统活动的创新镇痛策略。