Cellular and Molecular Mechanisms of Forebrain Axon Pathfinding Defects in FASD

项目总结 尽管开展了公众宣传活动，但胎儿酒精谱系障碍(FASD)仍然普遍存在，原因是 怀孕或育龄妇女的饮酒情况。我们都很理解感官 FASD患者的运动信息处理是扭曲的，但酒精对丘脑的影响，这可能是 调节这些功能，还没有得到广泛的研究。产前酒精暴露(1)扰乱轴突 丘脑和皮质之间的联系，这是适当地接受和反应所需的 环境中的感觉提示，(2)损害腹侧端脑(VTEL)，这是一个关键的中间目标 为了这些连接的正确发展，以及(3)改变指导线索和信号通路， 下层细胞和轴突迁移。我们假设适度的酒精暴露会扰乱Sit/Robo 通路，一系列对前脑有直接和间接影响的引导线索和受体 轴突形成。在这项提案中，我们将研究走廊细胞介导的丘脑皮质轴突(TCA)引导 使用FASD的鼠标模型进入VTEL和在VTEL内。我们的实验目的是(1)定义TCA的性质 以及FASD的走廊细胞引导误差；(2)建立Sit和Robo作为FASD的分子靶点 活体神经病理学。为了分析TCA制导误差，我们将使用染料跟踪和免疫染色来 想象这些轴突沿着它们的轨迹。为了评估走廊单元格，我们将计算并确定 免疫组织化学染色观察这些细胞的分布。分析酒精对Sit和Robo的扰动 在体内表达时，我们将对每种异构体进行蛋白质印迹和RT-PCR。我们还将在体外使用 外植体共培养，检测Sit和Robo功能。在FASD大脑中，我们预计TCA会不适当地投射 到中间目标和最终目标，以及要变形的走廊。我们还预测了Sit/Robo表达式 和功能将被抑制。我们的发现将揭示适度酒精暴露对指导的影响 适当的神经系统连接所需的机制，这是目前研究不足的一个领域。这 这项工作还将加强对细胞和分子机制的理解，这些机制可能涉及 在人类FASD中观察到感觉运动加工缺陷。拟议中的实验将在很大程度上进行 由本科生提供指导，为他们提供成功所需的指导和专业知识 在科学事业和研究生工作方面。

PROJECT SUMMARY Despite public awareness campaigns, Fetal Alcohol Spectrum Disorders (FASD) remain prevalent due to alcohol consumption by women who are pregnant or of child-bearing age. It is well appreciated that sensory and motor information processing is distorted in FASD, but alcohol effects on the thalamus, which likely mediates these functions, have not been widely investigated. Prenatal alcohol exposure (1) disrupts axonal connections between the thalamus and cortex which are required to appropriately receive and respond to sensory cues in the environment, (2) damages the ventral telencephalon (vTel), a critical intermediate target for proper development of these connections, and (3) alters guidance cues and signaling pathways that underlie cell and axon migration. We hypothesize that moderate alcohol exposure disrupts the Slit/Robo pathway, a family of guidance cues and receptors that can have both direct and indirect effects on forebrain axon formation. In this proposal we will investigate corridor-cell mediated thalamocortical axon (TCA) guidance into and within the vTel using a mouse model of FASD. Our experiments aim to (1) define the nature of TCA and corridor cell guidance errors in FASD and (2) establish Slit and Robo as molecular targets of FASD neuropathology in vivo. To analyze TCA guidance errors, we will use dye tracing and immunostaining to visualize these axons along their trajectory. To evaluate corridor cells, we will count and determine the distribution of these cells via immunostaining. To analyze alcohol-induced perturbations of Slit and Robo expression in vivo, we will perform western blotting and RT-PCR for each isoform. We will also use in vitro explant co-cultures to test Slit and Robo function. In the FASD brain, we expect TCAs to project inappropriately to intermediate and final targets and the corridor to be malformed. We also predict that Slit/Robo expression and function will be suppressed. Our findings will reveal the impact of moderate alcohol exposure on guidance mechanisms that are required for proper nervous system wiring, an area that is currently understudied. This work will also enhance understanding of cellular and molecular mechanisms that are likely to be involved in sensorimotor processing defects observed in human FASD. The proposed experiments will largely be carried out by undergraduates who will be mentored to provide them the guidance and expertise needed for success in science careers and graduate work.