Scalable Assays for Morphological Analysis of Mammalian Neurons

哺乳动物神经元形态学分析的可扩展测定

基本信息

批准号：
8657123
负责人：
Bernardo L Sabatini
金额：
$ 41.95万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8657123
关键词：
Algorithms Alzheimer&apos s Disease Antibodies Autistic Disorder Axon Benchmarking Biochemical Pathway Biological Assay Cell Line Cell physiology Cells Classification Collaborations Collection Communities Complex Computer software Data Data Analyses Data Set Dendrites Development Disease Ensure Excitatory Synapse Gene Expression Gene Targeting Genes Genetic Genetic Transcription Genome Genomics Goals Image Image Analysis In Vitro Inhibitory Synapse Institutes Label Lead Machine Learning Measurement Mediating Mental Retardation Methods Mining Mitochondria Monitor Morphology Mus Neurobiology Neurodegenerative Disorders Neurodevelopmental Disorder Neuroglia Neurons Organelles Parkinson Disease Pathway Analysis Pathway interactions Phenotype Predisposition Procedures Process Protocols documentation RNA Interference Relative (related person)Structure Subfamily lentivirinae Synapses System Techniques Time Transfection Validation Viral Work base bioimaging brain cell density design fluorescence imaging follow-up genome analysis genome-wide high throughput screening medical schools neuropsychiatry open source prototype scale up screening small hairpin RNA synaptogenesis tissue culture tool

项目摘要

DESCRIPTION (provided by applicant): Medium and high-throughput assays (i.e., screens) have generally not been applied to mammalian neurons because of the difficulties in culturing them in large numbers and because of the low efficiency with which the genetic makeup of neurons can be altered. Furthermore, because many aspects of neuronal function can only be assayed with electrophysiological assays, follow-up analysis and validation of screening hits is difficult. We propose to use automated imaging approaches to analyze synapse number and neuronal structure in vitro in a scalable format. We have implemented tissue culture and immunostaining approaches to monitor the number and types of synapses formed onto neurons in multi-well plates. We will couple this analysis with lentivirus mediated introduction of short-hairpin RNAs to induce RNA interference against genes expressed in neurons. This will be performed in concert with transcriptional analysis of neurons to determine the key changes in gene expression that correlate with structural and synaptic changes. The proposal represents a significant collaboration between several groups with expertise in functional analysis of neurons, automated analysis of images, viral mediated manipulation of gene expression, and whole-genome transcriptional analysis. We hope that our work will lead, for the first time, to a turn-key and robust method of analysis of neuron and synapse structure suitable for scalable, whole-genome analysis. Such a system will permit the unbiased and systematic analysis of pathways involved in neuropsychiatric diseases including neurodegenerative diseases such as Alzheimer's and Parkinson's as well as neurodevelopmental disorders such as mental retardation and autism.

描述（由申请人提供）：中等和高通量测定（即筛选）通常不应用于哺乳动物神经元，因为很难大量培养它们，并且由于可以改变神经元基因组成的效率低。此外，由于神经元功能的许多方面只能通过电生理测定法测定，因此困难的后续分析和筛选验证是困难的。我们建议使用自动成像方法以可扩展格式分析体外的突触数量和神经元结构。我们已经实施了组织培养和免疫染色方法，以监测多孔板中神经元上形成的突触的数量和类型。我们将将这一分析与慢毛病毒介导的短发RNA的引入，以诱导对神经元表达的基因的RNA干扰。这将与神经元的转录分析一起进行，以确定与结构和突触变化相关的基因表达的关键变化。该提案代表了在神经元功能分析，图像自动分析，病毒介导的基因表达的操纵和全基因组转录分析方面具有专业知识的几个小组之间的重要合作。我们希望我们的工作将首次导致神经元和突触结构的分析和强大的分析方法，适用于可扩展的全基因组分析。这样的系统将允许对包括神经退行性疾病（例如阿尔茨海默氏症和帕金森氏症的神经退行性疾病）以及神经退行性疾病以及神经发育疾病（如心理障碍和自闭症）的神经退行性疾病进行公正和系统分析。