Mechanisms of Serotonergic Regulation for Connectivity Development

 描述（由申请人提供）：在美国，每年有50万名婴儿早产。早产儿患自闭症谱系障碍（ASD）的风险高出3倍，在大多数早产儿中，ASD的患病率接近25%。研究表明，早产儿在轴突连接形成的发育期间会经历慢性缺氧;早产儿缺氧暴露的程度与ASD的风险相关。患有ASD的前早产儿没有明显的大脑异常，但有证据表明大脑5-羟色胺（5-HT）减少和连接改变。我们的工作解决了理解缺氧破坏轴突连接机制的差距。我们研究了5- HT的一个新作用：感知发育扰动（缺氧），并在响应中改变神经回路。 我们实验室的工作表明，缺氧会破坏脊椎动物的轴突寻路（史蒂文森等人，2012）。缺氧可以破坏5-HT信号传导，最近的体外证据表明，5-HT可以调节轴突导向。我们的假设是发育性缺氧破坏了通过5-羟色胺起作用的轴突寻路。我们开发了一个强大的平台来测试我们的假设。我们使用斑马鱼的研究，结合脊椎动物中枢神经系统结构和基因的相关性，快速和有效地测试分子机制。我们已经产生了独特的和新颖的荧光报告和表达/错误表达线;和能力，以产生大量的动物足够的统计功率。我们的实验结合了基因的联合收割机敲除、敲除和错误表达;药理学操作;以及建立的缺氧模型。 我们的初步数据表明，5-HT 2受体（HTR 2）在连合foxP 2神经元中表达，HTR 2受体的药理学阻断导致中线轴突寻路错误。我们使用foxP 2神经元中htr 2的敲低来确认5-HT在体内轴突引导中的作用（目的1）。我们基因消融5-HT源神经元（中缝核），以证明寻路是由5-HT调节的，我们测试5-HT的效果是否是由foxP 2神经元中表达的指导受体ephrinB 2a介导的。目的二：研究5-HT是否是发育性缺氧的感受器，以调节神经回路和行为发育。已知5-HT回路参与ASD中的焦虑相关行为失调，并且我们的初步工作已经发现缺氧导致5-HT表达的持续降低。我们将通过使用5-HT再摄取抑制剂氟西汀挽救路径发现来表征5- HT在缺氧轴突路径发现错误中的作用。为了确定5-HT回路变化是否调节行为，我们将比较暴露于缺氧或5-HT药理学阻断的对照或实验动物;用氟西汀治疗的缺氧动物;或用foxP 2轴突的2-光子消融的动物的焦虑行为（趋触性/抱壁性）。我们的方法提供了重要的机制见解缺氧伴随早产儿的影响，其参与与自闭症谱系障碍相关的中枢神经系统连接中断。

 DESCRIPTION (provided by applicant): Each year 500,000 infants are born prematurely in the U.S. Premature infants have a 3-times higher risk for developing an autism spectrum disorder (ASD), and the prevalence of ASDs approaches 25% in the very most prematurely-born infants. Premature infants have been shown to experience chronic hypoxia during a period of development when axon connections are forming; and the extent of hypoxic exposure for premature infants correlates with the risk for ASDs. Ex-premature infants who develop ASDs lack conspicuous brain abnormalities, but have evidence of decreased brain serotonin (5-HT) and alterations in connectivity. Our work addresses the gap in understanding the mechanism by which hypoxia disrupts axon connections. We investigate a novel role for 5- HT: sensing a developmental perturbation (hypoxia), and in response altering neural circuitry. Work from our lab has shown that hypoxia disrupts axon pathfinding in vertebrates (Stevenson et al. 2012). Hypoxia can disrupt 5-HT signaling, and recent in vitro evidence demonstrates that 5-HT can modulate axon guidance. Our hypothesis is that developmental hypoxia disrupts axon pathfinding acting through serotonin. We have developed a powerful platform to test our hypothesis. We use studies in zebrafish, combining the relevancy of vertebrate CNS structures and genes, with rapidity and efficiency for testing molecular mechanisms. We have generated unique and novel fluorescent reporter and expression/misexpression lines; and capability to generate large numbers of animals for sufficient statistical power. Our experiments combine knock- down, knock-out, and misexpression of genes; pharmacological manipulations; and an established hypoxia model. Our preliminary data shows that 5-HT2 receptors (htr2) are expressed in commissural foxP2 neurons, and that pharmacological blockade of htr2 receptors causes midline axon pathfinding errors. We use knock-down of htr2 in the foxP2 neurons to confirm the role of 5-HT on axon guidance in vivo (Aim 1). We genetically ablate the 5-HT source neurons (raphe nucleus) to demonstrate that the pathfinding is regulated by 5-HT; and we test whether 5-HT's effect is mediated by the guidance receptor ephrinB2a which is expressed in foxP2 neurons. In Aim 2 we will study whether 5-HT is a sensor for developmental hypoxia to regulate circuit and behavior development. 5-HT circuits are known to be involved in anxiety-related behaviors dysregulated in ASDs, and our preliminary work has found that hypoxia causes a persistent decrease in 5-HT expression. We will characterize 5- HT's role in hypoxia axon pathfinding errors by rescuing pathfinding using the 5-HT reuptake inhibitor fluoxetine. To determine if 5-HT circuitry changes modulate behavior, we will compare anxiety behavior (thigmotaxis/wall- hugging) in control or experimental animals exposed to hypoxia or to 5-HT pharmacological blockade; to hypoxia animals treated with fluoxetine; or to animals with 2-photon ablation of foxP2 axons. Our approach provides important mechanistic insights into the effects of hypoxia accompanying prematurity, and its involvement in disruptions of CNS connectivity associated with autism spectrum disorders.