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，生命早期接触乙醇，即使是低水平的，也会干扰发育。 并增加患酒精使用障碍(AUD)的风险。低剂量母体 斑马鱼(ZF)和大鼠摄入乙醇(ME)都显示出保守的神经行为效应 跨物种，随着ME的摄入，后代下丘脑的表达显著增加 促食欲素/食欲素(Hcrt)，一种有效的消费行为刺激物，以及 酒精和焦虑相关行为。虽然有证据表明神经免疫信号与AUD和 显示乙醇能刺激大鼠神经胶质细胞中的各种炎性趋化因子和细胞因子，但很少有 研究检测神经元内的神经免疫因子，并研究它们如何与 神经肽，影响这些神经元的发育和它们控制的行为。专注于 下丘脑Hcrt神经元和存在于这些神经元内的神经免疫系统，本提案将使用 双物种方法检验以下假设：母亲饮酒干扰特定的 子代Hcrt神经元中的神经免疫转录，进而刺激这些神经元的发育 神经元，并有助于增加的倾向，更多的乙醇消费和偏爱在 后代。在目标1中，将使用转基因hcrt：egfp zf，这是一种优势脊椎动物，因为其外部 发育、遗传可控性、光学透明度和小尺寸，以首先评估ME对油井的影响 研究了子代Hcrt神经元内趋化因子CXCL12a及其受体CXCR4b。然后，HCRT 转录组将使用rna-seq进行测序，并将进行生物信息学分析以确定顶端。 酒精暴露诱导神经免疫基因的差异表达。这一分析之后将是 CRISPR/Cas9基因编辑，这将用于确定在ZF后代中MOST的功能作用 强烈影响候选基因以及CXCL12a和CXCR4b改变Hcrt神经元的发育， 使用定量活体成像进行检查，并在刺激酒精消费和相关方面 行为。在目标2中，ZF的发现将被直接翻译到大鼠模型，首先通过确认所获得的结果 在ZF中，然后通过将AAV注射到大鼠胚胎脑中击倒，为 AIM中发现的靶神经免疫基因1.确定该靶基因在细胞免疫中的功能 ME对青春期大鼠神经元发育和行为、Hcrt神经元的影响 使用iDISCO大脑清理的后代，以及使用间歇访问的乙醇消耗量将被测量 两瓶选择范式。由于这项研究符合NIAAA战略计划的多个目标， 拟议的研究将用不同的技术提供广泛的培训，也应该产生新的 洞察母亲摄入低酒精水平如何通过神经免疫系统影响 Hcrt神经元在后代中的发育，并有助于他们在以后的生活中增加乙醇摄入量。