Model behavior in zebrafish: characterization of the startle response

斑马鱼的模型行为：惊吓反应的表征

• 批准号: 10376471
• 负责人: Joy Hart Meserve
• 金额: $ 3.43万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2021-08-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376471
• 关键词: Acetylcholine; Acoustics; Affect; Alleles; Anatomy; Animal Model; Attention Deficit Disorder; Attention deficit hyperactivity disorder; Auditory; Behavior; Behavior Disorders; Behavioral; Behavioral Symptoms; Biological Assay; Birth; Brain; CRISPR/Cas technology; Cells; Characteristics; Choline; Clinical; Complex; Defect; Development; Disease; Family; Fire - disasters; Foundations; Functional disorder; Genes; Genetic; Genetic Models; Genetic Screening; Head; Human; Image; Individual; Laboratories; Larva; Lesion; Major Depressive Disorder; Membrane Transport Proteins; Mental disorders; Modeling; Molecular; Molecular Cloning; Motor; Motor Neurons; Movement; Mus; Muscle; Mutation; Neurons; Neurophysiology - biologic function; Neurotransmitters; Pathway interactions; Phenotype; Population; Positioning Attribute; Posture; Presynaptic Terminals; Production; Reaction; Reaction Time; Regulation; Response Latencies; Response to stimulus physiology; Role; Schizophrenia; Skeletal Muscle; Speed; Spinal; Spinal Cord; Startle Reaction; Stimulus; System; Tactile; Tail; Techniques; Tissues; Transgenic Organisms; Vertebrates; Zebrafish; auditory stimulus; autism spectrum disorder; behavioral phenotyping; behavioral plasticity; behavioral study; choline transporter; cholinergic; experimental study; gene function; genome sequencing; habituation; hindbrain; human disease; in vivo; insight; member; mutant; nervous system disorder; neural circuit; neurodevelopment; neuronal circuitry; neuropsychiatric disorder; programs; promoter; response; solute; stimulus sensitivity; sustained attention; whole genome

ABSTRACT Although behavioral deficits are common in neurological disorders, the genetic pathways and neural circuits underlying behavior are largely unknown. A behavior that is disrupted in numerous disorders including attention deficit disorder, autism spectrum disorders, and schizophrenia is the startle response. Following an intense auditory stimulus, a short latency response occurs wherein rapid muscle activation produces a defensive posture. Disrupted startle responses likely result from a broad defect in genes or circuits controlling behavior. The genetic contribution to behavioral symptoms in humans has been challenging to uncover, in part because they result from multigenic disorders affecting the vastly complex human brain. Genetically tractable animal models have emerged as a valuable system for investigating gene function in the development and function of neural circuits underlying behavior. The Granato lab performed a genetic screen for regulators of the startle response in the larval zebrafish. Molecular cloning of 15 screen candidates identified a number of genes that modulate the startle response in zebrafish and are implicated in human disease. One such gene is solute carrier family 5 member 7a (slc5a7a), which encodes high affinity choline transporter that is required for synthesis of the neurotransmitter acetylcholine. Mutations in slc5a7 have been implicated in psychiatric disorders including attention deficit disorder and major depression. Mice lacking slc5a7 die immediately after birth, precluding behavioral studies. This proposal aims to determine the contribution of slc5a7a to neural circuit development and/or execution of the zebrafish startle response (Aims 1 and 2). In wild type larval zebrafish, the startle response begins with a sharp turn that initiates at the head and progresses towards the tail. In slc5a7a mutants, the turn initiates randomly along the body axis, resulting in an uncoordinated movement. This phenotype suggests neural circuitry between the hindbrain command neuron that initiates the movement and the downstream spinal motoneurons may be disrupted. Circuit defects in slc5a7a mutants will be investigated using in vivo Ca2+ imaging from individual neurons known to be required for the startle response and whole brain activity mapping of additional required neurons. The developmental stage and anatomical region where slc5a7a is required will be investigated using a combination of transgenic rescue constructs and CRISPR/Cas9 generated mutant alleles. Finally, a number of additional mutants that display uncoordinated or weak startle responses will be sequenced to determine the causative genetic lesion and will be characterized using the same techniques (Aim 3). The results of these proposed experiments will determine how slc5a7a promotes a coordinated startle response. More broadly, the results will elucidate acetylcholine’s role in regulating behavior. Finally, characterization of slca5a7a and genes identified in Aim 3 will contribute to models of the genetic program and neural circuitry underlying vertebrate startle behavior.

摘要 虽然行为缺陷在神经性疾病中很常见，但遗传途径和神经 行为背后的电路在很大程度上是未知的。一种在许多障碍中被破坏的行为，包括 注意力缺陷障碍、自闭症谱系障碍和精神分裂症是令人震惊的反应。在此之后 强烈的听觉刺激，发生一种短潜伏期的反应，其中快速的肌肉激活产生一种防御 姿势。惊吓反应的中断可能是由于控制行为的基因或电路的广泛缺陷造成的。 基因对人类行为症状的影响一直难以发现，部分原因是 它们是由影响极其复杂的人脑的多基因疾病引起的。遗传易驯服的动物 模型已经成为一个有价值的系统，用于研究基因功能在人的发育和功能中的作用 潜伏在行为背后的神经回路。格拉纳托实验室对这种惊吓的调节者进行了基因筛查 斑马鱼幼体的反应。对15个候选筛查对象进行的分子克隆确定了一些基因 调节斑马鱼的惊吓反应，并与人类疾病有关。其中一个这样的基因是溶质载体 家族5成员7a(Slc5a7a)，编码合成所需的高亲和力胆碱转运体 神经递质乙酰胆碱。Slc5a7基因突变与精神疾病有关，包括 注意力缺陷障碍和严重的抑郁症。缺乏slc5a7的小鼠出生后立即死亡，排除了 行为研究。这项建议旨在确定slc5a7a对神经回路发育的贡献。 和/或执行斑马鱼惊吓反应(目标1和2)。 在野生型幼体斑马鱼中，惊吓反应始于头部和头部的急剧转向 向尾巴进发。在slc5a7a突变体中，旋转沿着体轴随机开始，导致 不协调的运动。这种表型表明后脑命令神经元之间存在神经回路。 这启动了运动，下游的脊髓运动神经元可能会被破坏。SLC5a7a中的电路缺陷 突变体将使用体内单个神经元的钙成像进行研究，这些神经元是已知的 惊吓反应和额外所需神经元的全脑活动图。发展阶段和 需要slc5a7a的解剖区域将使用转基因拯救相结合的方法进行研究。 构建和CRISPR/Cas9产生突变等位基因。最后，显示了一些额外的突变体 不协调或弱的惊吓反应将被测序，以确定致病的遗传损害和将 使用相同的技术确定其特征(目标3)。这些拟议实验的结果将决定 Slc5a7a如何促进协调的惊吓反应。更广泛地说，研究结果将阐明乙酰胆碱 在规范行为方面的作用。最后，在目标3中确定的slca5a7a和基因的特征将有助于 脊椎动物惊吓行为背后的遗传程序和神经回路的模型。