Mechanisms of Serotonergic Regulation for Connectivity Development

连接发展的血清素调节机制

• 批准号: 8889940
• 负责人: Josh Leitch Bonkowsky
• 金额: $ 22.35万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8889940
• 关键词: Ablation; Address; Animal Model; Animals; Anxiety; Axon; Behavior; Brain; Chronic; Commissure; Control Animal; Cues; Data; Development; Enhancers; Fluoxetine; Gene Structure; Genes; Genetic; Hypoxia; Imagery; In Vitro; Infant; Invertebrates; Knock-out; Lead; Link; Mediating; Metronidazole; Modeling; Molecular; Neurons; Nitroreductases; Play; Premature Infant; Prevalence; Reporter; Risk; Role; Serotonin; Signal Transduction; Source; Stimulus; System; Testing; Time; Vertebrates; Work; Zebrafish; anxiety-related behavior; autism spectrum disorder; axon guidance; behavioral study; experience; high risk; human NTN1 protein; in vivo; inhibitor/antagonist; insight; knock-down; mutant; netrin-1; neural circuit; neuronal circuitry; novel; premature; public health relevance; raphe nuclei; receptor; research study; response; reuptake; sensor; serotonergic regulation; two-photon

 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.

 描述（由适用提供）：每年有500,000名婴儿在美国早产婴儿中过早出生，患有自闭症谱系障碍（ASD）的风险更高，而ASD的患病率在最早出生的婴儿中接近25％。在形成轴突连接时，已经证明早产婴儿会经历慢性缺氧。低氧前母亲的程度与ASD的风险相关。发展ASD的前婴儿缺乏明显的脑异常，但有证据表明脑血清素（5-HT）和连通性的改变。我们的工作解决了理解缺氧破坏轴突连接的机制的差距。我们研究了5-HT的新作用：感知发育扰动（缺氧），以及改变神经回路的反应。我们实验室的工作表明，缺氧会破坏脊椎动物中的轴突途径（Stevenson等，2012）。缺氧会破坏5-HT信号，最近的体外证据表明5-HT可以调节轴突的指导。我们的假设是，发育缺氧会破坏通过血清素作用的轴突探索。我们已经开发了一个强大的平台来检验我们的假设。我们在斑马鱼中使用研究，结合了脊椎动物CNS结构和基因的相关性，以及测试分子机制的速度和效率。我们已经产生了独特而新颖的荧光报告基因和表达/misexpression系列。以及能够产生大量动物以获得足够的统计能力。我们的实验结合了基因的敲除，敲除和束缚。药物操纵；和已建立的缺氧模型。我们的初步数据表明，5-HT2受体（HTR2）在合并FOXP2神经元中表达，HTR2受体的药理学阻断会导致中线轴突示意性误差。我们在FOXP2神经元中使用HTR2的敲除，以确认5-HT在体内轴突引导中的作用（AIM 1）。我们通常消除5-HT源神经元（Raphe Nucleus），以证明该途径受到5-HT的调节。我们测试了5-HT的效应是否是由FOXP2神经元中表达的引导受体ephrinb2a介导的。在AIM 2中，我们将研究5-HT是否是发育缺氧调节电路和行为发展的传感器。已知5-HT电路参与与ASD的焦虑相关行为，我们的初步工作发现缺氧会导致5-HT表达持续下降。我们将通过使用5-HT再摄取抑制剂氟西汀来挽救路线来表征5-HT在缺氧轴突探索错误中的作用。为了确定5-HT电路是否改变了调制行为，我们将比较暴露于缺氧或5-HT药物阻滞的对照或实验动物中的动画行为（thigmotaxis/壁挂）；用氟西汀治疗的低氧动物；或用于FOXP2轴突2光子消融的动物。我们的方法为缺氧参与早产的影响及其参与与自闭症谱系障碍相关的中枢神经系统连通性的中断提供了重要的机械见解。