Single cell analysis of functional and transcriptional changes in somatosensory neurons after peripheral nerve injury

周围神经损伤后体感神经元功能和转录变化的单细胞分析

• 批准号: 9258783
• 负责人: Ivan Tochitsky
• 金额: $ 6.02万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9258783
• 关键词: Afferent Neurons; Animal Model; Bioinformatics; Calcium; Cells; Data; Data Set; Development; Disease; Electrophysiology (science); Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Harvest; Heterogeneity; Image; In Vitro; Individual; Injury; Ion Channel; Lead; Masks; Measures; Mechanoreceptors; Messenger RNA; Molecular; Molecular Profiling; Mus; Neurons; Neurotransmitter Receptor; Pain; Pain Disorder; Pathologic; Peripheral; Peripheral Nervous System; Peripheral nerve injury; Population; Potassium; RNA; Sodium; Spinal Ganglia; Stimulus; Therapeutic; Transgenic Mice; Transgenic Organisms; activating transcription factor 3; base; calcium indicator; chronic neuropathic pain; chronic pain; improved; injured; nerve injury; new therapeutic target; painful neuropathy; patch clamp; single cell analysis; somatosensory; spared nerve; spontaneous pain

Project Summary/Abstract Neuropathic pain is associated with the spontaneous activity and hyperexcitability of a small subset of somatosensory neurons. Changes in somatosensory neuron gene expression also follow nerve injury in animal models of neuropathic pain. To date, gene expression studies of somatosensory neurons have analyzed changes in bulk populations of cells. However, bulk studies mask the functional and transcriptional heterogeneity of somatosensory neurons. Thus, it is still unclear which subpopulation(s) of somatosensory neurons are the primary drivers of neuropathic pain, nor is it known what transcriptional changes lead to ectopic activity of individual somatosensory neurons after nerve injury. A detailed single cell characterization of electrophysiological and transcriptional changes in somatosensory neurons following nerve injury will greatly expand our understanding of chronic pain and may identify novel drug targets for the treatment of chronic and neuropathic pain. I propose to use calcium imaging in dorsal root ganglion (DRG) neurons from transgenic mice expressing the genetically encoded calcium indicator (GCaMP6f) in vitro to identify ectopically active neurons after nerve injury. After identifying spontaneously active neurons, I will perform electrophysiological recordings on these neurons to confirm the presence of ectopic activity. I will also compare the general excitability of injured and uninjured DRG neurons, and measure their sodium and potassium currents to determine whether nerve injury indeed makes somatosensory neurons hyperexcitable. After characterizing the electrophysiological excitability of individual DRG neurons, I will harvest each cell separately, isolate its mRNA and perform massively parallel single cell qPCR to measure the expression of all known ion channels and neurotransmitter receptors. In addition, I will use transcriptional markers to classify individual DRG neurons into separate subtypes. After obtaining this dataset, I will identify changes in ion channel expression associated with neuronal hyperexcitability after injury and determine in which subtype(s) of sensory neurons these changes occur. This analysis should help identify the cellular and molecular drivers of injury-induced neuropathic pain in the peripheral nervous system.

项目总结/摘要 神经病理性疼痛与神经系统的自发活动和过度兴奋有关。 躯体感觉神经元的一小部分。体感神经元基因的变化 在神经病理性疼痛的动物模型中，表达也跟随神经损伤。迄今为止， 对躯体感觉神经元表达的研究分析了 细胞群。然而，大量的研究掩盖了功能和转录 躯体感觉神经元的异质性。因此，尚不清楚哪些亚群 的躯体感觉神经元是神经性疼痛的主要驱动因素，也不知道 是什么样的转录变化导致了个体躯体感觉的异位活动 神经损伤后的神经元详细的单细胞电生理特性 神经损伤后体感神经元的转录变化将大大增加 扩大我们对慢性疼痛的理解，并可能为慢性疼痛确定新的药物靶点。 治疗慢性和神经性疼痛。 我建议在转基因小鼠背根神经节（DRG）神经元中使用钙成像， 在体外表达遗传编码的钙指示剂（GCaMP6f）的小鼠， 神经损伤后异位活跃的神经元。在识别出自发活动后 神经元，我将对这些神经元进行电生理记录，以确认 存在异位活动。我还将比较受伤者的一般兴奋性， 未受伤的背根神经节神经元，并测量它们的钠电流和钾电流， 确定神经损伤是否确实使躯体感觉神经元过度兴奋。 在描述单个DRG神经元的电生理兴奋性之后，我将 分别收获每个细胞，分离其mRNA并进行大规模平行单细胞培养， qPCR测量所有已知离子通道和神经递质的表达 受体。此外，我将使用转录标记对单个DRG进行分类 神经元分成不同的亚型。在获得此数据集后，我将确定 损伤后与神经元过度兴奋相关的离子通道表达， 确定这些变化发生在感觉神经元的哪些亚型中。该分析 应该有助于识别损伤诱导的神经病理性疼痛的细胞和分子驱动因素 在周围神经系统中。