Cellular and molecular analysis of startle modulation

惊吓调节的细胞和分子分析

• 批准号: 10352379
• 负责人: Michael Granato
• 金额: $ 52.89万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-15 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10352379
• 关键词: Acoustics; Adaptor Signaling Protein; Address; Alleles; Animals; Attention deficit hyperactivity disorder; Behavior; Behavioral; Behavioral Assay; Brain; CRISPR/Cas technology; Calcium Channel; Characteristics; Clathrin; Cloning; Cognition Disorders; Cognitive; Collection; Complex; Critical Pathways; Data Set; Defect; Endocytosis; Exhibits; Gene Expression; Gene Mutation; Genes; Genetic; Genetic Models; Genetic Screening; Human; Huntington gene; Image; Impairment; Invertebrates; Knock-out; Larva; Learning; Link; Mammals; Manuals; Measures; Mediating; Metalloproteases; Molecular; Molecular Analysis; Molecular Genetics; Morphology; Nervous system structure; Neurons; Pathway interactions; Pattern; Performance; Pharmacogenetics; Pharmacology; Phenotype; Population; Probability; Process; Proteins; Receptor Signaling; Resolution; Role; Sigma Factor; Signal Pathway; Site; Startle Reaction; Stereotyping; Stimulus; Synapses; System; Transferase; Transgenic Organisms; Vertebrates; Zebrafish; autism spectrum disorder; base; behavioral pharmacology; behavioral phenotyping; behavioral plasticity; behavioral response; bioinformatics pipeline; cell type; experimental study; genetic approach; genome sequencing; habit learning; habituation; hindbrain; human disease; imaging approach; in vivo; learned behavior; molecular modeling; mutant; neural circuit; neuropsychiatric disorder; novel; palmitoylation; prepulse inhibition; receptor; receptor mediated endocytosis; relating to nervous system; response; screening; small molecule libraries; success; synaptic function; tool; voltage; whole genome

Abstract A remarkable feature of the nervous system is its ability to adjust stereotyped behavioral responses in a context dependent manner. In vertebrates, sudden and intense acoustic stimuli evoke an evolutionarily conserved startle response. While the execution of the acoustic startle response is extremely stereotyped, response probability is modulated in a context-dependent manner. For example, repeated presentation of a startling stimulus suppresses a behavioral response, representing a simple form of learning known as habituation. In humans, modulation of startle behavior is impaired in several neuropsychiatric disorders, including in Attention Deficit- Hyperactivity Disorder and autism spectrum disorders. Despite its importance, the molecular mechanisms underlying startle modulation not well understood. Zebrafish show a remarkable behavioral plasticity, and we have previously shown that larvae exhibit modulation of the acoustic startle response- including prepulse inhibition and habituation- with behavioral and pharmacological characteristics similar to those in mammals. We previously conducted the first forward genetic screen in vertebrates to isolate mutants defective in startle modulation, and identified 14 mutants with defects in habituation behavior. None of these 14 mutants exhibit morphological defects or overt defects in startle performance. Importantly, five of the six mutants we cloned so far encode genes previously not implicated in vertebrate habituation. Here we propose to build on our success in using a molecular genetics, phenotype based strategy to decipher the molecular and circuits mechanisms that drive vertebrate habituation behavior. Specifically, rather than focusing on a single habituation gene, our strategy is to continue to use whole genome sequencing to clone six additional mutants from our screen. Combined with the six mutants we have already cloned, this provides an unparalleled toolbox critical to attain a comprehensive model of the molecular-genetic and circuit mechanisms underlying habituation. Simultaneously, we focus on select genes as entry points to further link genetic mutants to behavioral phenotypes and to decipher the molecular and circuit mechanisms that regulate behavior. The experiments in this proposal will: (1) use a molecular genetic approach including transgenic behavioral rescue to identify the neuronal populations in which three genes critical for habituation function; (2) to use molecular and pharmacogenetic approaches in conjunction with a behavioral assay to determine the signaling pathways through which the adaptor protein-2 sigma subunit (AP2s1) critical for receptor endocytosis and the huntingtin interacting gene hip14 promote habituation; and 3) to use an established whole genome sequencing/bioinformatics pipeline to identify the causative gene mutations for six additional habituation mutants isolated from our genetic screen, generate CRISPR/Cas9 alleles to confirm their identity and determine their expression in the brain. Combined this will provide both breadth and depth both at the molecular and at the circuit level critical to comprehensively address fundamental molecular genetic questions in vertebrate startle modulation, also relevant to human disease conditions.

摘要 神经系统的一个显著特征是它能够在特定的环境中调整刻板的行为反应 依赖的态度。在脊椎动物中，突然而强烈的声音刺激会引起进化上保守的惊吓。 回应。虽然声学惊吓反应的执行是非常刻板的，但响应概率是 以上下文相关的方式进行调制。例如，反复呈现令人震惊的刺激 抑制行为反应，代表一种简单的学习形式，称为习惯化。在人类身上， 惊吓行为的调节在几种神经精神障碍中受到损害，包括注意力缺陷- 多动症和自闭症谱系障碍。尽管它很重要，但分子机制 潜在的惊吓调节机制还没有被很好地理解。斑马鱼表现出显著的行为可塑性，我们 先前已经表明，幼虫表现出声学惊吓反应的调制--包括预脉冲 抑制和习惯化--具有与哺乳动物相似的行为和药理学特征。我们 之前在脊椎动物中进行了第一次正向基因筛查，以分离出在惊吓中有缺陷的突变 并鉴定出14个习服行为存在缺陷的突变体。这14个突变体都没有表现出 惊吓表演中的形态缺陷或明显缺陷。重要的是，我们克隆的六个突变体中有五个 FAR编码以前与脊椎动物习惯化无关的基因。在这里，我们建议在我们成功的基础上再接再厉 在使用分子遗传学、基于表型的策略来破译分子和电路机制时， 驱使脊椎动物习服行为。具体地说，我们的策略不是专注于单一的习惯基因，而是 是继续使用全基因组测序从我们的筛查中克隆另外六个突变体。与 我们已经克隆了六个突变体，这提供了一个无与伦比的工具箱，对于实现全面的 习惯化背后的分子遗传和回路机制的模型。同时，我们专注于 选择基因作为切入点，进一步将遗传突变与行为表型联系起来，并破译 调节行为的分子和电路机制。本提案中的实验将：(1)使用 分子遗传学方法，包括转基因行为拯救，以识别其中的神经元群体 对习服功能至关重要的三个基因；(2)结合使用分子和药物遗传学方法 通过行为测试来确定适配器蛋白-2西格玛亚单位通过的信号通路 (AP2s1)受体内吞和亨廷顿蛋白相互作用基因hi14促进习服；以及3) 使用已建立的全基因组测序/生物信息学管道来识别致病基因突变 对于从我们的遗传筛选中分离到的另外六个习惯性突变体，生成CRISPR/Cas9等位基因以确认 他们的身份和决定他们在大脑中的表达。结合起来，这将既提供广度又提供深度 在分子和电路水平上对全面解决基本分子遗传学至关重要 脊椎动物的问题令人震惊，也与人类的疾病状况有关。