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（由申请人提供）：美国每年有 500,000 名早产婴儿。早产儿患自闭症谱系障碍 (ASD) 的风险高出 3 倍，并且在最早产儿中，ASD 的患病率接近 25%。早产儿已被证明在轴突连接形成的发育时期会经历慢性缺氧，以及早产儿缺氧的程度；患有 ASD 的早产儿缺乏明显的大脑异常，但有大脑血清素 (5-HT) 和连通性改变的证据，我们的工作解决了对缺氧破坏轴突机制的理解空白。我们研究了 5-HT 的一个新作用：感知发育扰动（缺氧），并据此改变神经回路。我们实验室的工作表明了这一点。缺氧会破坏脊椎动物的轴突寻路（Stevenson et al. 2012），缺氧会破坏 5-HT 信号传导，最近的体外证据表明，5-HT 可以调节轴突引导。我们的假设是，发育性缺氧会破坏通过血清素发挥作用的轴突寻路。我们开发了一个强大的平台来检验我们的假设，我们利用斑马鱼的研究，结合了脊椎动物中枢神经系统结构的相关性和相关性。我们已经生成了独特且新颖的荧光报告基因和表达/错误表达系；并且能够产生大量动物以获得足够的统计能力。基因的错误表达；以及已建立的缺氧模型，表明 5-HT2 受体 (htr2) 在连合的 FoxP2 神经元中表达，并且 htr2 受体的药理学阻断会导致中线轴突寻路。我们使用 FoxP2 神经元中 htr2 的敲低来确认 5-HT 在体内轴突引导中的作用（目标 1）我们通过基因消融 5-HT 源神经元（中缝核）来证明寻路是有效的。受 5-HT 调节；我们测试 5-HT 的作用是否由 FoxP2 神经元中表达的指导受体 ephrinB2a 介导。 5-HT 是发育性缺氧的传感器，可调节回路和行为发育，已知 5-HT 回路参与 ASD 中焦虑相关行为的失调，我们的初步工作发现缺氧会导致 5-HT 持续减少。我们将通过使用 5-HT 再摄取抑制剂氟西汀挽救寻路来表征 5-HT 在缺氧轴突寻路错误中的作用，以确定 5-HT 电路是否发生变化。行为，我们将比较暴露于缺氧或 5-HT 药物阻断的对照或实验动物的焦虑行为（趋触性/抱壁）；与接受氟西汀治疗的缺氧动物；或与进行了 FoxP2 轴突 2 光子消融的动物。该方法为早产儿缺氧的影响及其与自闭症谱系障碍相关的中枢神经系统连接破坏提供了重要的机制见解。