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

项目摘要 令人信服的证据表明，在生命早期接触乙醇，即使是低水平，也会干扰发育。 大脑中的神经元，并增加发展酒精使用障碍（AUD）的风险。低剂量母体 斑马鱼（ZF）和大鼠的乙醇（ME）消耗显示了保守的神经行为效应 在不同物种中，ME消耗显著增加了后代下丘脑的表达， 食欲肽下丘脑分泌素/食欲素（Hcrt），一种有效的消费行为刺激剂， 酒精和焦虑相关的行为。虽然有证据表明神经免疫信号与AUD有关， 显示乙醇在大鼠中刺激神经胶质细胞中的各种炎症趋化因子和细胞因子， 研究检查神经元内的神经免疫因子，并研究它们如何与 神经肽，并影响这些神经元的发育及其控制的行为。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，该提议将使用 双物种的方法来测试以下假设：母亲的乙醇消费干扰特定的 神经免疫转录本在Hcrt神经元的后代，反过来，刺激这些发展， 神经元，并有助于增加的倾向，更大的乙醇消费和偏好， 后代在目的1中，将使用转基因Hcrt：EGFP ZF，其是一种有利的脊椎动物，由于其外部特性， 开发，遗传易处理性，光学透明度和小尺寸，首先评估ME对井的影响- 研究了后代Hcrt神经元内的趋化因子CXCL 12 a及其受体CXCR 4 b。然后，Hcrt 将使用RNA-seq对转录组进行测序，并进行生物信息学分析以确定转录组中的前 乙醇暴露诱导的差异表达的神经免疫基因。分析之后， CRISPR/Cas9基因编辑，这将用于确定在ZF后代中最具功能的作用。 强烈影响候选基因以及CXCL 12 a和CXCR 4 b改变Hcrt神经元的发育， 使用定量活体成像进行检查，以及刺激乙醇消耗和相关 行为。在目标2中，ZF的发现将直接转化为大鼠模型，首先确认所获得的结果 在ZF中，然后通过向大鼠胚胎脑中注射AAV递送的shRNA来敲低， 目的1中鉴定的靶神经免疫基因。为了确定该靶基因在介导 ME对神经元发育和行为的影响，将在青春期大鼠中检查Hcrt神经元 使用iDISCO大脑清洁的后代和乙醇消耗将使用间歇性访问来测量。 两瓶选择模式这项研究符合NIAAA战略计划的多个目标， 拟议的研究将提供广泛的培训与不同的技术，也应该产生新的 深入了解母亲摄入低水平乙醇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。

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.