Amygdala pain mechanisms

项目摘要 慢性疼痛是一种复杂的多维疾病，仍然是一个主要的卫生保健问题和治疗挑战。的 开发新的和改进的治疗策略需要充分理解慢性疼痛的机制 在所有水平的轴突和所有细胞类型，包括非神经元元素。神经免疫信号已经出现 作为外周和脊髓疼痛机制，但对它的作用、调节和治疗潜力知之甚少。 疼痛背景下大脑中神经元和神经胶质细胞类型之间的分子串扰。处理这一重要 知识差距，我们将建立在我们的国家卫生研究院资助的工作（自1999年以来），通过确定神经可塑性影响该领域 在杏仁核，一个大脑的情绪中心，作为一个关键的机制，情感方面的痛苦和痛苦 调变该项目将测试新的假设，即杏仁核可塑性的慢性化， 神经病理性疼痛行为依赖于神经免疫信号级联，可以靶向减轻 神经性疼痛具体来说，在急性疼痛阶段，某些杏仁核神经元类型的突触驱动增强 激活不同类型的神经胶质，神经胶质衍生因子在不同的杏仁核神经元类型中产生过度兴奋， 慢性阶段以维持神经性疼痛。将采用综合性多学科方法， 整合了最先进的转录组学，生物信息学，行为分析，脑切片电生理学， 药理学、化学遗传学、光遗传学、病毒载体策略、免疫组织化学和分子生物学， 分析杏仁核中不同类型的神经元和神经胶质细胞内部和之间的神经免疫相互作用 输出区（中央核，CeA）在建立良好的脊神经结扎（SNL）大鼠模型的神经性疼痛。 将研究雄性和雌性大鼠。将使用转基因Crh-Cre大鼠模型来研究CeA促肾上腺皮质激素 释放因子（CRF）系统，杏仁核可塑性的重要参与者，及其在神经免疫信号传导中的作用。要求1 将使用细胞特异性批量和单细胞RNA测序（scRNA-Seq）的组合来鉴定单个基因， 与疼痛慢性化相关的分子途径和功能细胞状态。一种新的计算scRNA-Seq- 基于相互作用组的分析将确定神经病理性疼痛的急性和慢性阶段的神经元-胶质细胞相互作用 并确定药物靶向分子因子。目的2确定神经元-胶质细胞-神经元的行为学意义 在神经病理性疼痛的不同阶段，使用不同细胞的化学发生激活和抑制， 类型和药理学（或基于病毒的）工具来调节分子因子。感觉阈值，情绪 将测量反应、非诱发的持续疼痛以及焦虑和抑郁样行为。目标3将决定 在神经病理性疼痛的不同阶段，CeA中神经元-胶质细胞-神经元信号传导的电生理机制， 使用脑切片中不同细胞类型的膜片钳记录，以及化学遗传学和药理学（或病毒 基于）操纵。成功完成这些概念创新的研究将大大推动我们的 神经免疫信号在大脑可塑性和从急性到慢性疼痛的过渡方面的知识，并提供 减轻慢性神经性疼痛的新靶点。

Project Summary Chronic pain, a complex multidimensional disorder, remains a major health care issue and a therapeutic challenge. The development of new and improved therapeutic strategies requires the full understanding of mechanisms of chronic pain at all levels of the neuraxis and for all cell types, including non-neuronal elements. Neuroimmune signaling has emerged as a peripheral and spinal pain mechanism, but little is known about the role, regulation and therapeutic potential of molecular crosstalk between neuronal and glial cell types in the brain in the context of pain. To address this important knowledge gap we will build on our NIH-funded work (since 1999) that impacted the field by identifying neuroplasticity in the amygdala, a brain center for emotions, as a critical mechanism for emotional-affective aspects of pain and pain modulation. The proposed project will test the novel hypothesis that chronification of amygdala plasticity and neuropathic pain behaviors depends on a cascade of neuroimmune signaling that can be targeted to mitigate neuropathic pain. Specifically, enhanced synaptic drive of certain amygdala neuron types at the acute pain stage activates different types of glia, and glia-derived factors generate hyperexcitability in distinct amygdala neuron types at the chronic stage to maintain neuropathic pain. A comprehensive multidisciplinary approach will be used that integrates state-of-the-art transcriptomics, bioinformatics, behavioral assays, brain slice electrophysiology, pharmacology, chemogenetics, optogenetics, viral vector strategies, immunohistochemistry and molecular biology for the analysis of neuroimmune interactions within and between different types of neuronal and glial cells in the amygdala output region (central nucleus, CeA) in the well-established spinal nerve ligation (SNL) rat model of neuropathic pain. Male and female rats will be studied. A transgenic Crh-Cre rat model will be used to study the CeA corticotropin releasing factor (CRF) system, an important player in amygdala plasticity, and its role in neuroimmune signaling. Aim 1 will use a combination of cell-specific bulk and single-cell RNA sequencing (scRNA-Seq) to identify individual genes, molecular pathways and functional cellular states associated with pain chronification. A novel computational scRNA-Seq- based interactome analysis will determine neuron-glia interactions at the acute and chronic stages of neuropathic pain and identify drug-targetable molecular factors. Aim 2 will determine the behavioral significance of neuron-glia-neuron signaling in the CeA at different stages of neuropathic pain, using chemogenetic activation and inhibition of different cell types and pharmacological (or viral based) tools to modulate molecular factors. Sensory thresholds, emotional responses, non-evoked ongoing pain, and anxiety- and depression-like behaviors will be measured. Aim 3 will determine electrophysiological mechanisms of neuron-glia-neuron signaling in the CeA at different stages of neuropathic pain, using patch-clamp recordings of different cell types in brain slices and chemogenetic and pharmacological (or viral based) manipulations. Successful completion of these conceptually innovative studies will significantly advance our knowledge of neuro-immune signaling in brain plasticity and in the transition from acute to chronic pain and provide novel targets to mitigate chronic neuropathic pain.