Neuroimmune mechanisms mediating effects of maternal ethanol consumption on offspring’s hypocretin neuron transcriptome and behavior: translation from zebrafish to rodent

神经免疫机制介导母体乙醇消耗对后代下丘脑分泌素神经元转录组和行为的影响：从斑马鱼到啮齿动物的翻译

• 批准号: 10159806
• 负责人: Adam Collier
• 金额: $ 6.86万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10159806
• 关键词: Adolescent; Affect; Alcohol consumption; Anatomy; Animals; Antisense Oligonucleotides; Anxiety; Appetite Stimulants; Area; Behavior; Behavior Control; Behavioral; Bioinformatics; Brain; CRISPR/Cas technology; CXCR4 Receptors; CXCR4 gene; Candidate Disease Gene; Clinical; Consummatory Behavior; Consumption; Data; Dependovirus; Development; Dose; Embryo; Ethanol; Fertility; Flow Cytometry; Genes; Genetic; Genotype; Goals; Human; Hypothalamic structure; Image; Inflammatory; Injections; Knock-out; Label; Laboratory Study; Learning; Life; Ligands; Light; Link; Measurement; Measures; Mediating; Modeling; Molecular; Motor Activity; National Institute on Alcohol Abuse and Alcoholism; Neonatal; Neurobiology; Neuroglia; Neuroimmune; Neuroimmune system; Neuroimmunomodulation; Neurons; Neuropeptides; Operative Surgical Procedures; Optics; Peptides; Pregnancy; Production; Rattus; Reporting; Research; Risk; Rodent; Role; Signal Transduction; Strategic Planning; System; Techniques; Testing; Therapeutic Intervention; Third ventricle structure; Training; Transcript; Transgenic Organisms; Translating; Translations; Zebrafish; adolescent offspring; alcohol behavior; alcohol consumption during pregnancy; alcohol craving; alcohol effect; alcohol exposure; alcohol use disorder; anxiety-related behavior; beta-Chemokines; chemokine; cytokine; density; differential expression; embryonic alcohol exposure; experimental study; hypocretin; insight; knock-down; maternal alcohol use; melanin-concentrating hormone; monocyte chemoattractant protein 1 receptor; neurobehavioral; neurodevelopment; neuron development; novel; offspring; preference; prenatal exposure; receptor; response; small hairpin RNA; targeted treatment; transcriptome; transcriptome sequencing; transcriptomics

Project Summary Compelling evidence suggests that ethanol exposure early in life, even at low levels, disturbs the development of neurons in the brain and increases the risk for developing alcohol use disorder (AUD). Low-dose maternal ethanol (ME) consumption in both zebrafish (ZF) and rats has revealed conserved neurobehavioral effects across species, with ME consumption significantly increasing in the offspring the expression of the hypothalamic orexigenic peptide hypocretin/orexin (Hcrt), a potent stimulator of consummatory behavior, and the consumption of ethanol and anxiety-related behaviors. While there is evidence linking neuroimmune signaling to AUD and showing ethanol in rats to stimulate various inflammatory chemokines and cytokines in glial cells, there are few studies examining neuroimmune factors within neurons and investigating how they coexpress with neuropeptides and affect the development of these neurons and the behaviors they control. Focusing on hypothalamic Hcrt neurons and neuroimmune systems that exist within these neurons, this proposal will use a dual-species approach to test the following hypothesis: Maternal ethanol consumption disturbs specific neuroimmune transcripts in Hcrt neurons of the offspring which, in turn, stimulate the development of these neurons and contribute to an increased propensity for greater ethanol consumption and preference in the offspring. In Aim 1, transgenic Hcrt:EGFP ZF will be used, an advantageous vertebrate due to its external development, genetic tractability, optical transparency and small size, to first evaluate ME’s effects on the well- studied chemokine CXCL12a and its receptor CXCR4b within Hcrt neurons of the offspring. Then, the Hcrt transcriptome will be sequenced using RNA-seq, and bioinformatic analyses will be performed to identify the top differentially expressed neuroimmune gene induced by ethanol exposure. This analysis will be followed by CRISPR/Cas9 gene editing, which will be used to determine in ZF offspring the functional role of the most strongly affected candidate gene as well as CXCL12a and CXCR4b in altering the development of Hcrt neurons, to be examined using quantitative live imaging, and also in stimulating ethanol consumption and related behaviors. In Aim 2, the ZF findings will be translated directly to the rat model, first by confirming results obtained in the ZF and then by knocking down through injection into rat embryo brain of AAV delivered shRNA for the target neuroimmune gene identified in Aim 1. To determine the functional role of this target gene in mediating the effects of ME on neuronal development and behavior, Hcrt neurons will be examined in adolescent rat offspring using iDISCO brain clearing, and ethanol consumption will be measured using the intermittent access two-bottle choice paradigm. With this research being in line with multiple objectives of the NIAAA strategic plan, the proposed studies which will provide extensive training with different techniques should also produce novel insights into how maternal consumption of low ethanol levels acts through the neuroimmune system to affect the development of Hcrt neurons in the offspring and contribute to their increased ethanol intake later in life.

项目摘要 令人信服的证据表明，乙醇在生命的早期，即使在低水平上也扰乱了发展 大脑中神经元的神经元，增加了患酒精疾病障碍（AUD）的风险。低剂量母亲 斑马鱼（ZF）和大鼠的乙醇（ME）消耗均显示出构成神经行为的作用 整个物种，我的消费大大增加了下丘脑的表达 Orexenic Peppeoni/Orexin（HCRT），一种潜在的刺激剂，消费量 乙醇和与焦虑有关的行为。虽然有证据将神经免疫性信号与AUD联系起来 显示大鼠乙醇以刺激神经胶质细胞中各种炎症趋化因子和细胞因子，很少 研究检查神经元内神经免疫因子并研究它们如何与 神经肽并影响这些神经元的发展及其控制的行为。专注于 这些神经元中存在的下丘脑HCRT神经元和神经免疫系统，该建议将使用 测试以下假设的双物种方法：母体乙醇消耗特定于特异性 后代HCRT神经元中的神经免疫性转录本反过来刺激这些神经元的发展 神经元，并有助于增加乙醇消耗和偏好的倾向 后代。在AIM 1中，将使用转基因HCRT：EGFP ZF，由于其外部而导致的有利脊椎动物 开发，遗传障碍性，光学透明度和小规模，首先评估我对井的影响 在后代的HCRT神经元内进行了研究趋化因子CXCL12A及其受体CXCR4B。然后，HCRT 转录组将使用RNA-Seq进行测序，并将执行生物信息学分析以识别顶部 乙醇暴露诱导的差异表达的神经免疫基因。该分析将随后 CRISPR/CAS9基因编辑，将用于确定ZF后代最多的功能作用 强烈影响的候选基因以及CXCL12A和CXCR4B改变了HCRT神经元的发展， 可以使用定量实时成像进行检查，还可以刺激乙醇消耗量和相关 行为。在AIM 2中，ZF的发现将直接转化为大鼠模型，首先是通过确认获得的结果 在ZF中，然后通过注射到AAV的大鼠胚胎大脑中，以 目标1中鉴定出的靶神经免疫性基因。确定该靶基因在介导中的功能作用 我对神经元发展和行为的影响，将在青少年大鼠中检查HCRT神经元 使用IDISCO大脑清除和乙醇消耗的后代将使用间歇性访问来衡量 两瓶选择范式。随着这项研究符合NIAAA战略计划的多个目标， 拟议的研究将提供不同技术的广泛培训，还应产生新颖的研究 了解低乙醇水平的母体消费如何通过神经免疫系统的作用来影响 后代中HCRT神经元的发展，并在生命后期增加了乙醇的摄入量。