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Cellular and molecular analysis of startle modulation

惊吓调节的细胞和分子分析

基本信息

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
项目类别：
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

项目摘要

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一定的环境中调整刻板的行为反应依赖的方式。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反应虽然执行的声音惊吓反应是非常刻板，响应概率是以上下文相关的方式调制。例如，重复呈现令人吃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。在人类中，惊吓行为的调节在几种神经精神疾病中受损，包括注意力缺陷，多动症和自闭症谱系障碍。尽管其重要性，潜在的惊吓调节还不太清楚。斑马鱼表现出显著的行为可塑性，我先前已经证明，幼虫表现出声惊吓反应的调制-包括前脉冲抑制和习惯-具有与哺乳动物相似的行为和药理学特征。我们先前在脊椎动物中进行了第一次正向遗传筛选，以分离出惊吓缺陷的突变体调制，并确定了14个突变体的缺陷，在习惯化行为。这14个突变体中没有一个形态缺陷或惊吓性能的明显缺陷。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。在此，我们建议在成功的基础上，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，驱动脊椎动物的习惯行为。具体来说，我们的策略不是专注于单一的习惯基因，是继续使用全基因组测序从我们的筛选中克隆出另外六个突变体。结合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，以进一步将遗传突变体与行为表型联系起来，调节行为的分子和电路机制。该方案的实验将：（1）使用分子遗传学方法，包括转基因行为拯救，以确定神经元群体，其中三个基因对习惯化功能至关重要;（2）结合使用分子和药物遗传学方法通过行为分析来确定衔接蛋白-2 σ亚基（AP 2s 1）对受体内吞作用至关重要，亨廷顿蛋白相互作用基因hip 14促进习惯化;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出的另外六种习惯化突变体，产生CRISPR/Cas9等位基因，以确认他们的身份并决定他们在大脑中的表达。结合起来，这将提供广度和深度在分子和电路水平上，脊椎动物惊吓调节中的问题，也与人类疾病状况有关。