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: 9927482
• 负责人: Adam Collier
• 金额: $ 6.53万
• 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/9927482
• 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; 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.

项目总结