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

阿片类药物在美国的流行和慢性疼痛的流行产生了对新的 副作用有限的止痛药。由脑干神经组成的疼痛下行控制系统 投射到脊髓的回路，关键地调制脊髓背角的伤害性感受，并可能参与 到慢性疼痛。先前的研究证实，这些下行环路包括几个吻部种群。 延髓腹内侧区(RVM)神经元。RVM神经元最初是根据它们的电生理学进行分类的 属性。具体地说，ON细胞对伤害性刺激表现出更高的活性，并促进脊髓 疼痛传递。相反，RVM外细胞表现出高的基础紧张性放电，在有害的情况下被抑制 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 与伤害性感受器和脊髓神经元相比，在分子水平上研究不足和定义不清，从而防止 针对疼痛管理的下行控制系统。本研究提出将神经网络 电路跟踪、小鼠遗传学和单细胞RNA测序(scRNA-seq)以确定 定义用于功能研究的伤害性RVM神经元类型。目标1将确定分子签名 脊髓投射Rvm→SC On-cell。为了描述Rvm→SC神经元，我们注射了一种逆行报告病毒(AAV- Retro-CAG-GFP)进入背角，并用荧光激活细胞分选(FACS)分离GFP+retro-GFP 标记Rvm的→SC神经元，进行单链RNA序列分析。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。Aim 2将识别RVM神经元 在疼痛时是活跃的。即刻早期基因c-fos的表达已被广泛应用于 中枢神经系统伤害性神经元的标测。为了标记疼痛期间活跃的RVM神经元，我们使用了最近开发的 FosTRAP2小鼠，c-fos启动子联合驱动CreERT2重组酶的表达 带有荧光Cre报告基因的鼠系或病毒。FACS将分离荧光标记的RVM神经元，以及 ScRNA-seq将识别在伤害性刺激过程中激活的神经元类型。最后，RVM经历了 慢性疼痛或阿片类药物暴露时的功能重组。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过渡以及 慢性阿片类药物治疗的副作用。我们将结合使用目标1中描述的方法和 慢性疼痛和阿片类药物暴露模型解决Rvm→SC神经元转录变化的研究 在这些病理性痛觉过敏状态的发展过程中。这项研究将极大地拓宽我们的 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 神经元用于未来对影响痛觉的离散脑干回路的功能识别 和阿片类药物的影响。此外，这项研究可能会在RVM神经元中识别新的分子靶点 开发创新的止痛策略，以调节下行疼痛控制系统的活动。

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.