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.

抽象的 神经系统的一个显着特征是它在上下文中调整刻板印象的行为反应的能力 依赖方式。在脊椎动物中，突然而强烈的声学刺激引起了进化保守的惊吓 回复。虽然表现出惊吓响应的执行极为刻板印象，但响应概率是 以上下文依赖的方式进行调制。例如，重复出现令人震惊的刺激 抑制行为反应，代表一种简单的学习形式，称为习惯。在人类中 在几种神经精神疾病中，包括注意力不足 - 多动障碍和自闭症谱系障碍。尽管其重要性，但分子机制 根本的惊吓调制尚未得到很好的理解。斑马鱼表现出显着的行为可塑性，我们 以前已经表明，幼虫对声音惊吓反应的调制 - 包括预硫 具有行为和药理特征与哺乳动物相似的抑制和习惯。我们 以前在脊椎动物中进行了第一个正向遗传筛选，以分离出惊吓的突变体 调制，并确定了14个在习惯行为中缺陷的突变体。这14个突变体中没有一个 惊吓性能中的形态缺陷或明显的缺陷。重要的是，我们克隆的六个突变体中有五个 远面编码以前不涉及脊椎动物习惯的基因。在这里，我们建议以我们的成功为基础 在使用分子遗传学时，基于表型的策略破译了分子和电路机制 驱动脊椎动物习惯行为。具体而言，我们的策略不是专注于单个习惯基因 是继续使用整个基因组测序，从我们的屏幕上克隆六个其他突变体。与 我们已经克隆的六个突变体，这提供了一个无与伦比的工具箱，至关重要 分子遗传和电路机制的模型。同时，我们专注于 选择基因作为进一步将基因突变体与行为表型联系起来的入口点并破译 调节行为的分子和电路机制。该提案中的实验将：（1）使用 分子遗传学方法包括转基因行为拯救，以鉴定神经元种群 三个对习惯功能至关重要的基因； （2）在结合中使用分子和药物遗传学方法 采用行为测定来确定衔接蛋白-2 Sigma亚基的信号传导途径 （AP2S1）对受体内吞作用和亨廷顿相互作用的基因HIP14至关重要； 3） 使用已建立的整个基因组测序/生物信息学管道来识别因果关系突变 对于从我们的遗传筛选中隔离的六个额外的习惯突变体，生成CRISPR/CAS9等位基因以确认 他们的身份并确定它们在大脑中的表达。结合这将提供既广度和深度 在分子和电路水平上，至关重要地解决基本分子遗传的关键 脊椎动物惊吓调节中的问题，也与人类疾病有关。