Scalable Assays for Morphological Analysis of Mammalian Neurons

描述（由申请人提供）：中通量和高通量测定（即筛选）通常未应用于哺乳动物神经元，因为大量培养它们很困难，而且神经元的遗传组成可以改变的效率很低。此外，由于神经元功能的许多方面只能通过电生理分析来分析，因此很难对筛选结果进行后续分析和验证。我们建议使用自动成像方法来分析体外突触数量和神经元结构，以可扩展的格式。我们已经实施了组织培养和免疫染色方法来监测多孔板中神经元上形成的突触的数量和类型。我们将把这一分析与慢病毒介导的短发夹RNA的引入结合起来，诱导RNA干扰神经元中表达的基因。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首次为神经元和突触结构的分析提供一种交钥匙和强大的方法，适用于可扩展的全基因组分析。这样一个系统将允许对涉及神经精神疾病的途径进行公正和系统的分析，包括神经退行性疾病，如阿尔茨海默氏症和帕金森氏症，以及神经发育障碍，如智力迟钝和自闭症。

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.