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

• 批准号: 8192511
• 负责人: Bernardo L Sabatini
• 金额: $ 42.25万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8192511
• 关键词: Algorithms; Alzheimer' 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); Screening procedure; Structure; Subfamily lentivirinae; Synapses; System; Techniques; Time; Transfection; Validation; Viral; Work; base; bioimaging; brain cell; density; design; fluorescence imaging; follow-up; genome-wide; high throughput screening; medical schools; neuropsychiatry; open source; prototype; scale up; small hairpin RNA; synaptogenesis; tissue culture; tool

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. PUBLIC HEALTH RELEVANCE: Massively parallel analysis of cells in many conditions has allowed the discovery of key pathways that control cell function. Unfortunately, these techniques have not been applied to neurons due to difficulties in handling, manipulating, and analyzing large numbers of brain cells. We propose to develop imaging-based techniques to analyze neurons in dishes at a high throughput in order to find pathways that control their development and susceptibility to disease.

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。 公共卫生相关性：在许多条件下对细胞的大规模平行分析允许发现控制细胞功能的关键途径。不幸的是，由于处理，操纵和分析大量脑细胞的困难，这些技术尚未应用于神经元。我们建议开发基于成像的技术来分析高通量的菜肴中的神经元，以找到控制其发育和疾病易感性的途径。