Integrative single-cell spatial transcriptomic, anatomical, and functional profiling of brain-wide ensembles engaged by opioid relapse

项目摘要 阿片类药物滥用正在破坏美国各地的社区，并造成了难以言状的痛苦。的 合成阿片类芬太尼，无论是由于处方或非法使用，涉及近一半的报告阿片类药物， 相关死亡。与海洛因和吗啡不同，芬太尼通常通过非静脉途径给药， 但我们对芬太尼的使用和滥用脆弱性的机械理解有限。一个主要的假设 在脆弱的个体中，从使用到滥用的转变是药物诱导的基因表达的招募 在反复接触药物后某些脑回路和细胞的变化。然而，识别这种电路- 和细胞类型特异性群体，结合其潜在的遗传和功能适应， 通常受到当前分析的分辨率、通量和数据配准的限制。本项目的总体目标 是（i）生成口服和静脉内芬太尼自我给药的机器引导的行为表征 和恢复在男性和女性小鼠，捕捉个人的风险脆弱性，和（ii）执行全脑 通过整合新的使能技术平台，包括单细胞空间转录组学（Pixel- seq）、全脑细胞类型特异性电路连接性和功能性分布的全脑神经活动 记录（Neuropixel 2.0），最近由我们团队的PI开发。为了实现分子和 功能性脑映射和数据配准，我们有三个具体目标：1）执行单细胞空间 在具有不同程度芬太尼寻求行为的小鼠中，具有回路特异性的转录组学分析; 2） 进行单细胞全脑活动映射，细胞类型和回路特异性，在小鼠中， 芬太尼寻求行为的程度; 3）进行全脑分布的大规模电生理检查 在口服芬太尼寻找过程中的记录，并将空间分辨的转录组数据与记录数据整合。 在第一个目标中，Pixel-seq将用于生成细胞图谱，分析药物和行为相关的空间 保守的基因表达，并通过与逆行病毒追踪偶联来绘制神经元连接。在 第二个目标是，我们将立即进行基于基因的早期全脑活动映射， 芬太尼自我给药和恢复后的类型特异性传入连接。在第三个目标中，我们将 首先对全脑分布的感兴趣区域进行Neuropixel2.0电生理记录， 然后将Neuropixel2.0和Pixel-seq数据整合并对齐到称为NeuroPixel-seq（NP-seq）的新检测中。的 拟议的项目是创新的，因为我们的综合方法将首次产生空间多模态 在类阿片复吸风险背景下，数据的深度和分辨率前所未有。这很重要，因为我们的 数据将为神经科学提供急需的可获得的口服芬太尼自我给药模型 社区，与第一个芬太尼细胞分辨率图谱配对，允许非专业实验室访问 滩头阵地参与确定芬太尼使用和复发的机制基础。

PROJECT SUMMARY Opioid abuse is devastating communities across the United States and is responsible for untold suffering. The synthetic opioid fentanyl, whether due to prescription or illicit use, is involved in nearly half of reported opioid- related deaths. Unlike heroin and morphine, fentanyl is commonly administered through non-intravenous routes, but we have a limited mechanistic understanding of fentanyl use and abuse vulnerability. A leading hypothesis for the transition from use to abuse, in vulnerable individuals, is the recruitment of drug-induced gene expression changes in certain brain circuits and cells following repeated drug exposure. However, identifying such circuit- and cell-type specific populations, in combination with their underlying genetic and functional adaptations, is often limited by the resolution, throughput, and data registration of current assays. The overall goal of this project is to (i) generate machine--guided behavioral characterization of oral and intravenous fentanyl self-administration and reinstatement in male and female mice that captures individual risk vulnerability, and (ii) perform brain-wide mapping by integrating new enabling technological platforms including single-cell spatial transcriptomics (Pixel- seq), whole brain cell type specific circuit connectivity, and functional distributed brain-wide neural activity recordings (Neuropixel 2.0), recently developed by PIs in our team. To realize the potential for the molecular and functional brain mapping and data registration, we have three Specific Aims: 1) perform single-cell spatial transcriptomic profiling, with circuit-specificity, in mice with varying degrees of fentanyl-seeking behavior; 2) perform single-cell whole-brain activity mapping, with cell-type and circuit-specificity, in mice with varying degrees of fentanyl-seeking behavior; 3) perform brain-wide distributed large-scale electrophysiological recordings during oral fentanyl-seeking and integrate spatially resolved transcriptomic data with recording data. In the first Aim, Pixel-seq will be used to generate cell atlases, analyze drug- and behavior-associated spatially conserved gene expression, and map neuronal connectivity by coupling with retrograde viral tracing. In the second Aim, we will perform immediate early gene-based whole-brain activity mapping, contextualized by cell type-specific afferent connectivity, after fentanyl self-administration and reinstatement. In the third Aim, we will first perform Neuropixel2.0 electrophysiological recording of the brain-wide distributed regions of interest, and then integrate and align Neuropixel2.0 and Pixel-seq data in a new assay called NeuroPixel-seq (NP-seq). The proposed project is innovative as our integrative approaches will, for the first time, generate spatial multimodal data of unprecedented depth and resolution within the context of opioid relapse risk. It is significant because our data will provide a much-needed accessible oral fentanyl self-administration model for the neuroscience community, paired with the first fentanyl cellular-resolution atlas, allowing non-specialized labs an accessible beachhead for participating in the identification of the mechanistic basis for fentanyl use and relapse in mice.