Functional and genetic characterization of human DRG and spinal cord at single cell resolution

具体目标：数千万美国人遭受慢性疼痛。不幸的是，这些患者 在治疗方面我们所能提供的很少。阿片类药物是治疗疼痛的主要工具，但它们的使用 在慢性疼痛的情况下，患者的证据基础较差，并存在上瘾的固有风险。当前的危机 与阿片类药物有关的死亡人数的增加突显了与广泛使用阿片类药物有关的风险。慢性病的平行危机 与疼痛和阿片类药物相关的死亡导致了NIH发起了Heave倡议，主要目标之一是 确定用于治疗疼痛的新型止痛药，重点放在不会上瘾的药物上。这个 精密人类疼痛网络寻求提供有关细胞类型多样性的基础数据 构成了痛性神经轴。我们提议的精密人类疼痛网络U19中心的目标包括 利用原代人体细胞和组织建立优化的实验方案和功能分析 (包括背根神经节神经元和脊髓切片)，并阐明了 分子/细胞表型是人类疼痛转导、传递和处理的基础 不同的疼痛状况。这个项目旨在增加我们对心脏的生理特性的了解。 人类痛觉神经轴中的神经元。这里的三个目标将开始编目功能配置文件和 有或无疼痛或阿片类药物病史的供者的背根节和脊髓神经元的转录组特征 使用。我们将继续努力优化人类尖端功能和基因研究的方案 神经系统组织。我们将与其他中心合作，优化方案，以评估生理， 人背根神经节和脊髓神经元在细胞分辨率下的转录和形态特征 从有疼痛病史的患者恢复的组织中，这些特性是如何受到影响的。因为我们的捐赠者 人群还包括滥用药物和滥用药物的患者，在某些情况下，这与慢性 疼痛条件下，我们也将表征类似的特性的背根节和脊髓神经元的患者和 没有最近的阿片类药物使用史。这些研究将提供关于人类神经元的基础知识 通过将人背根神经节和脊髓中神经元的电生理特征映射到 人类背根节基因定义的亚群或脊髓神经元的形态类别。这项研究将 还分析了来自有或没有慢性疼痛或阿片类药物使用/滥用史的捐赠者的组织，提供 对疼痛和物质使用对人类疼痛中这些神经元的影响进行新理解的机会 神经轴。本研究的另一项主要成果将是为 拟议分析的关键方面，包括膜片钳神经元的RNA测序方案 (PATCH-SEQ)以及人脊髓切片的准备、维护和记录。

Specific Aims: Tens of millions of Americans suffer from chronic pain. Unfortunately for these patients, there is very little we can offer in the way of treatments. Opioids represent the main tool for treating pain, but their use in chronic pain conditions suffers from a poor evidence base and the inherent risk of addiction. The current crisis of opioid-related deaths highlights the risk associated with widespread opioid use. The parallel crises of chronic pain and opioid-related deaths has led the NIH to launch the HEAL Initiative, with one of the major goals being the identification of novel analgesics for the treatment of pain, with a focus on drugs that are non-addicting. The PRECISION Human Pain Network seeks to provide foundational data on the diversity among cell types that comprise the pain neuraxis. Goals for our proposed PRECISION Human Pain Network U19 center include the development of optimized experimental protocols and functional assays utilizing primary human cells and tissue (including dorsal root ganglion neurons and spinal cord slices), and the elucidation of comprehensive molecular/cellular phenotypes that underlie human pain transduction, transmission, and processing under different pain conditions. This project seeks to increase our understanding of the physiological properties of human neurons in the pain neuraxis. The three aims here will begin to catalog the functional profiles and transcriptinoal signatures of DRG and spinal neurons from donors with and without a history of pain or opioid use. We will continue our work to optimize protocols for cutting edge functional and genetic studies in human nervous system tissue. We will work with other centers to optimize protocols to assess physiological, transcriptional, and morphological features of human DRG and spinal cord neurons at cellular resolution, and how these properties are impacted in tissues recovered from patients with a history of pain. Because our donor population also includes patients with substance use and misuse, and in some cases this overlaps with chronic pain conditions, we will also characterize similar properties of DRG and spinal cord neurons in patients with and without a recent history of opioid use. These studies will provide foundational knowledge of human neurons in the pain neuraxis by mapping electrophysiological features of neurons in the human DRG and spinal cord onto genetically-defined subpopulations of human DRG or morphological classes of spinal neurons. The study will also profile tissues derived from donors with and without a history of chronic pain or opioid use/misuse, providing opportunity for new understanding of the impact of pain and substance use on these neurons in the human pain neuraxis. Another major deliverable from the present study will be the development of optimized protocols for key aspects of the proposed analyses, including protocols for RNA sequencing from patch clamped neurons (Patch-seq) and for the preparation, maintenance, and recording form human spinal cord slices.