Scalable Assays for Morphological Analysis of Mammalian Neurons

描述（由申请人提供）：中等和高通量测定（即，筛选）一般不应用于哺乳动物神经元，因为难以大量培养它们，并且因为改变神经元的遗传组成的效率低。此外，由于神经元功能的许多方面只能用电生理学测定来测定，因此难以对筛选命中进行后续分析和验证。我们建议使用自动成像方法来分析突触数量和神经元结构在体外的可扩展的格式。我们已经实施了组织培养和免疫染色方法来监测在多孔板中的神经元上形成的突触的数量和类型。我们将结合慢病毒介导的短发夹RNA的引入来诱导针对神经元中表达的基因的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。该提案代表了几个在神经元功能分析，图像自动分析，病毒介导的基因表达操纵和全基因组转录分析方面具有专业知识的小组之间的重要合作。我们希望，我们的工作将导致，第一次，一个交钥匙和强大的神经元和突触结构的分析方法，适用于可扩展的，全基因组分析。这样的系统将允许对神经精神疾病中涉及的通路进行无偏见和系统的分析，所述神经精神疾病包括神经变性疾病如阿尔茨海默氏症和帕金森氏症以及神经发育障碍如精神发育迟滞和自闭症。

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.