Alterations in Microglial function moderate the development of maladaptive drinking behaviors following early life stress and are exacerbated by ethanol consumption

小胶质细胞功能的改变会减缓早期生活压力后不良饮酒行为的发展，并因乙醇消耗而加剧

• 批准号: 10680078
• 负责人: Stephen Gironda
• 金额: $ 4.77万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680078
• 关键词: 3-Dimensional; Affect; Affinity Chromatography; Alcohol consumption; Alcohols; Behavior; Brain; Brain region; Cessation of life; Darkness; Data; Development; Disease; Early identification; Early treatment; Environmental Exposure; Epidemiology; Ethanol; Event; Exhibits; Exposure to; Functional disorder; Future; Gene Expression; Genetic Transcription; Goals; Grant; Hippocampus; Homeostasis; Investigation; Knock-in Mouse; Label; Lead; Lifestyle-related condition; Light; Limbic System; Link; Literature; Maintenance; Medial; Microglia; Microscopy; Modeling; Morphology; Mus; Outcome; Persons; Phenotype; Prefrontal Cortex; Preventive care; Protocols documentation; RNA; Regulation; Research; Resolution; Rest; Ribosomes; Risk Factors; Rodent Model; Role; Running; Sampling; Societies; Sorting; Stains; Stimulus; Stress; Structure; Synapses; Techniques; Transgenic Mice; Translating; United States; Work; alcohol exposure; alcohol use disorder; cell type; comorbidity; deter alcohol use; drinking; drinking behavior; early life stress; experimental study; insight; mRNA sequencing; nervous system disorder; neuropsychiatric disorder; novel; periadolescent; pre-clinical; prevent; reconstruction; social stress; synaptogenesis

PROJECT SUMMARY Currently 14.5 million people suffer from alcohol use disorder (AUD) in the United States. Identifying early risk factors that increase the likelihood of developing AUD could help elucidate potential models for preventative care and mitigate the impact AUD has on society. One highly studied risk factor associated with developing AUD is early life stress (ELS). The mechanisms that facilitate ELS's impact on the development of AUD is poorly understood. One consistent finding in ELS literature shows a role of microglia in the development of a host of neurological disorders including AUD. Over the past several years, new evidence has been uncovered identifying a role of microglia in the development and maintenance of AUD. To expand this field of work and discover novel targets for treatment, my project seeks to implement a model of early life stress: periadolescent social stress (PSS) and a model to promote voluntary binge-like consumption of ethanol: drinking in the dark. Using these models, I will identify how microglia respond to these factors in isolation and when combined. Over the past two years, I have run experiments to optimize the PSS paradigm and drinking in the dark protocol. I have performed immunohistochemical staining and run qPCR to determine how microglia respond to these stimuli. I have found that combined PSS and binge ethanol consumption decrease microglia number, increase microglial reactivity, and alter microglia morphology in the ventral hippocampus. In addition, markers related to homeostasis are reduced following PSS and binge ethanol consumption. While these results are interesting, evidence suggest that microglia are present heterogeneously throughout the brain and serve a variety of functions. To better characterize the effects of early life stress and ethanol consumption on microglia function, my proposal aims to identify key regions that demonstrate alterations in microglia count, reactivity, and phenotype; and identify how these changes relate to changes in microglial RNA. To determine these key regions, I will use light sheet microscopy. This approach will provide us an opportunity to create an unbiased 3D reconstruction of the whole brain with single microglia resolution. I will use this technique to identify how microglia are distributed throughout the brain and phenotypically altered due to these environmental exposures. Focusing on the regions identified with light sheet microscopy, I will perform targeted purification of polysomal mRNA sequencing (TRAP-seq). Using a CX3CR1CreER transgenic mouse line that primarily labels for microglia in GFP, I will sort microglia from other cell types and identify changes in microglial RNA expression due to PSS and ethanol exposure. Following each of these main experiments, I will determine whether microglia inhibition during PSS will prevent changes in binge-like ethanol consumption and microglial adaptations. This work will mark a significant advancement in the field and will call for the consideration of microglia function as a target for treatment of ELS and prevention of AUD.

项目概要 目前，美国有 1450 万人患有酒精使用障碍 (AUD)。识别早期风险 增加 AUD 发生可能性的因素可能有助于阐明潜在的预防模型 关心并减轻澳元对社会的影响。一项经过深入研究的与发育相关的风险因素 AUD 是早期生活压力 (ELS)。促进 ELS 对 AUD 发展产生影响的机制是 不太了解。 ELS 文献中的一项一致发现表明，小胶质细胞在 包括 AUD 在内的一系列神经系统疾病。过去几年，新的证据不断被发现 确定小胶质细胞在 AUD 的发展和维持中的作用。为了扩大这一工作领域并 为了发现新的治疗目标，我的项目旨在实施一种早期生活压力模型：青春期 社会压力（PSS）和促进自愿狂饮乙醇的模型：在黑暗中饮酒。 使用这些模型，我将确定小胶质细胞如何单独和组合地对这些因素做出反应。超过 在过去的两年里，我进行了实验来优化 PSS 范式和黑暗饮酒协议。我 进行了免疫组织化学染色并运行 qPCR 以确定小胶质细胞如何响应这些 刺激。我发现结合 PSS 和暴饮乙醇消耗会减少小胶质细胞数量，增加 小胶质细胞反应性，并改变腹侧海马的小胶质细胞形态。此外，相关标记 PSS 和暴饮乙醇消耗后体内平衡会降低。虽然这些结果很有趣， 有证据表明，小胶质细胞在整个大脑中存在异质性，并服务于多种功能 功能。为了更好地表征早期生活压力和乙醇消耗对小胶质细胞功能的影响， 我的建议旨在确定显示小胶质细胞数量、反应性和 表型；并确定这些变化与小胶质细胞 RNA 变化的关系。确定这些关键 区域，我将使用光片显微镜。这种方法将为我们提供一个创造公正的机会 以单个小胶质细胞分辨率对整个大脑进行 3D 重建。我将使用这种技术来确定如何 小胶质细胞分布在整个大脑中，并且由于这些环境而发生表型改变 曝光。重点针对光片显微镜识别的区域，进行针对性的纯化 多聚体 mRNA 测序 (TRAP-seq)。使用主要标记的 CX3CR1CreER 转基因小鼠系 对于 GFP 中的小胶质细胞，我将从其他细胞类型中分选小胶质细胞并识别小胶质细胞 RNA 的变化 PSS 和乙醇暴露导致的表达。在每个主要实验之后，我将确定 PSS 期间的小胶质细胞抑制是否会阻止暴饮暴食型乙醇消耗和小胶质细胞的变化 适应。这项工作将标志着该领域的重大进步，并需要考虑 小胶质细胞作为治疗 ELS 和预防 AUD 的靶点。