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产生了对新药物的迫切需求。 副作用有限的疼痛疗法。疼痛下行控制系统，由脑干神经元 投射到脊髓的回路，关键地调节脊髓背角中的伤害性感受， 慢性疼痛。以前的研究表明，这些下行回路包括几个种群的喙 延髓腹内侧（RVM）神经元。RVM神经元最初根据其电生理学进行分类 特性.具体地，ON细胞在对伤害性刺激的反应中表现出增加的活性，并促进脊髓损伤。 疼痛传递相反，RVM OFF-细胞显示高基础紧张性放电，其在有害性刺激后被抑制。 刺激和施加抑制性疼痛控制。尽管RVM伤害感受神经元很重要， 与伤害感受器和脊髓神经元相比， 针对疼痛管理的下行控制系统。本研究提出将联合收割机神经 电路追踪，小鼠遗传学和单细胞RNA测序（scRNA-seq），以确定标记基因， 定义伤害感受RVM神经元类型用于功能研究。Aim 1将确定 脊髓投射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-100）。 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-CAG-GFP）。 标记的RVM→SC神经元，并进行scRNA-seq。细胞的无偏聚类将识别出许多 RVM神经元细胞类型按特定标记基因的表达分类。目标2将识别RVM神经元 在疼痛时活跃。即早基因c-fos的表达已被广泛应用于 CNS伤害感受神经元的定位。为了标记疼痛期间活动的RVM神经元，我们使用最近开发的 FosTRAP 2小鼠，其中c-fos的启动子驱动CreERT 2重组酶的表达， 用荧光Cre-reporter小鼠系或病毒。FACS将分离荧光标记的RVM神经元，并且 scRNA-seq将鉴定在伤害性刺激期间激活的神经元类型。最后，RVM经历了 慢性疼痛或阿片类药物暴露期间的功能重组。因此，Aim 3将决定转录水平。 RVM→SC神经元的变化与从急性疼痛到慢性疼痛的转变有关， 慢性阿片类药物治疗的副作用我们将使用目标1中描述的方法， 慢性疼痛和阿片类药物暴露模型，以解决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 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 神经元的未来功能识别的离散脑干电路，影响疼痛的感觉 阿片类药物的作用。此外，这项研究可能会在RVM神经元中确定新的分子靶点， 开发创新的镇痛策略，调节下行疼痛控制系统的活动。