Mechanisms underlying diverse effects of low-dose embryonic ethanol on development and function of hypocretin/orexin neurons

项目总结 酒精使用障碍(AUD)是一种慢性精神障碍，具有严重的社会后果 美国有超过1500万成年人，全球有7600万成年人。在生命早期，特别是在子宫中接触乙醇， 显著改变胎儿大脑发育，增加患AUD的风险。即使暴露在低浓度的 酒精可以产生最普遍、最轻微的胎儿酒精谱系障碍。我们最近在中国的研究 这只大鼠证明，产前接触低水平的乙醇，除了增加乙醇消耗量 以及后代的焦虑，刺激神经再生，而细胞毒性效应可以忽略不计。有趣的是，产前 乙醇刺激促食欲神经肽Hcrt表达的神经元密度 几乎完全位于下丘脑，在促进AUD相关行为方面发挥着重要作用。这些 在我们的啮齿动物模型中观察到的令人信服的效果促使我们寻找一种更简单的脊椎动物模型来进行真实研究- 时间，在细胞和解剖学水平上，乙醇对胚胎发育影响的机制 Hcrt神经元。斑马鱼(ZF)是我们研究的理想脊椎动物模型系统，由于其光学特性 可获得性和外部发展，其体积小和成本低，以及早期开发的可比CNS 并迅速地与相对复杂的行为剧目一起。我们最近的出版物显示出 为了保护乙醇对物种的影响，我们在实验室建立了多种技术 特别适合于对ZF脑的全面研究，包括延时实时成像、光遗传学、 钙成像和体内靶向激光消融，并获得了初步结果，显示出稳健和 Hcrt神经元和神经免疫系统的意外变化可能导致特定的 Hcrt亚群不对称。基于这些新发现，我们建议检验以下假设： 胚胎暴露于低剂量乙醇对Hcrt神经元的发育有不同的影响，这些神经元是 由局部炎性趋化因子系统的动态变化介导，并有助于 不对称的、位置异常的Hcrt亚群，表现出神经活动的改变，并有因果关系 对酒精消费和相关行为的干扰。在三个具体目标中，我们对ZF进行了初步的规划 暴露在低剂量乙醇中：1)彻底描述Hcrt神经元的发育和行为， 在正常条件下和酒精暴露后，直接检测这些神经元的行为功能； 2)在单细胞水平上精确地确定特定亚群中的Hcrt神经元是否在其出生时是独一无二的 日期、起源地点、迁徙路径和信号活动，以及它们是否与行为相关 以及3)测试胚胎乙醇对Hcrt神经元发育和 行为由局部神经免疫系统介导，特别是CXCL12a/CXCR4b/CXCR7b和 CCL2/CCR2。本研究旨在深入阐明动态多样的神经肽、神经免疫和 子宫内低水平酒精暴露导致的行为变化增加了患AUD的风险。

PROJECT SUMMARY Alcohol use disorder (AUD) is a chronic psychiatric disorder with severe societal consequences that impacts over 15 million adults in the USA and 76 million worldwide. Ethanol exposure early in life, particularly in utero, strikingly alters fetal brain development and increases the risk for AUD. Even exposure to low concentrations of ethanol can produce the most prevalent, milder forms of fetal alcohol spectrum disorder. Our recent studies in the rat demonstrate that prenatal exposure to low levels of ethanol, in addition to increasing ethanol consumption and anxiety in the offspring, stimulates neurogenesis with negligible cytotoxic effects. Interestingly, prenatal ethanol stimulates the density of neurons expressing hypocretin/orexin (Hcrt), an orexigenic neuropeptide located almost exclusively in the hypothalamus that has a major role in promoting AUD-related behaviors. These compelling effects observed in our rodent model led us to seek a simpler vertebrate model to investigate in real- time, at cellular and anatomical levels, the mechanisms involved in ethanol’s effects on embryonic development of Hcrt neurons. The zebrafish (ZF) is a perfect vertebrate model system for our studies, due to its optical accessibility and external development, its small size and low cost, and its comparable CNS that develops early and rapidly alongside a relatively sophisticated behavioral repertoire. With our recent publications showing a conservation of ethanol’s effects across species, we established in our laboratory a variety of techniques particularly suited for comprehensive studies of the ZF brain, including time-lapse live imaging, optogenetics, calcium imaging, and in vivo targeted laser ablation, and obtained preliminary results revealing robust and unexpected changes in Hcrt neurons and neuroimmune systems that may lead to the formation of specific asymmetric Hcrt subpopulations. Based on these new findings, we propose to test the following hypothesis: Embryonic exposure to ethanol at low doses has diverse effects on the development of Hcrt neurons, which are mediated by dynamic changes in local inflammatory chemokine systems and contribute to the formation of asymmetric, abnormally-located Hcrt subpopulations that exhibit altered neural activity and are causally related to disturbances in ethanol consumption and associated behaviors. In 3 specific aims, we plan in ZF embryonically exposed to low-dose ethanol: 1) to thoroughly characterize Hcrt neuronal development along with behavior, under normal conditions and after ethanol exposure, and directly test the behavioral functions of these neurons; 2) to precisely determine, at a single-cell level, if Hcrt neurons in specific subpopulations are unique in their birth date, site of origin, migratory path, and signaling activity, and if they are causally related to behavioral disturbances; and 3) to test the possibility that effects of embryonic ethanol on Hcrt neuronal development and behavior are mediated by local neuroimmune systems, specifically CXCL12a/CXCR4b/CXCR7b and CCL2/CCR2. This research aims to elucidate in depth the dynamic and diverse neuropeptide, neuroimmune and behavioral changes caused by low levels of in utero ethanol exposure that increase the risk for AUD.