Genomic Approaches to Neuronal Diversity and Plasticity

拟议研究的目标是开发新的基因组技术，用于大规模并行DNA测序和单个活神经细胞的大规模基因表达分析，并应用这些技术研究神经功能。来自哥伦比亚大学和佛罗里达大学的研究团队将紧密合作，开发三项创新的基因组技术：（i）大规模并行DNA测序芯片系统，用于对神经元SAGE文库进行测序;（ii）纳米级DNA阵列，用于在单个细胞和亚细胞区室水平上进行全球基因表达谱分析，和（iii）以高空间分辨率和快速时间分辨率实时监测活神经元和限定的细胞微区中的多种mRNA种类。这些技术中的每一项都将通过使用Anglasia的模型记忆形成网络进行严格的测试和验证。然后，将实施这些技术来探索三种基本的大脑机制：（1）神经元身份的分子基础，（2）控制相互连接的精确模式形成的分子信号，这是行为的基础，（3）突触特异性神经元可塑性和神经元生长的分子基础。我们将利用已鉴定的神经元作为实验模型，研究mRNA的不对称分布在真核细胞整合功能和表型中的作用。我们将使用分层设计以降序方式实现单细胞分析的结构分辨率，其中将在相同的记忆形成网络内进行平行的基因组和功能分析：单神经元到单轴突到单突触。在记忆巩固过程中，基因表达谱将与简单网络中功能特征神经元和突触末梢的功能成像相关。基于基因组学，光化学，纳米科学和工程，生物化学和神经科学的综合方法将用于了解神经元和突触在学习和记忆的背景下如何运作。该项目开发的技术和生物学发现将产生广泛的影响和应用，以研究基因如何调节细胞和生物体行为，从无脊椎动物的简单神经系统到人类大脑。

The objectives of the proposed research are the development of new genomic technologies for massively parallel DNA sequencing and large-scale gene expression analysis from single living nerve cells, and application of these technologies to study neural functions. The research teams from Columbia University and the University of Florida will closely interact to develop three innovative genomic technologies:(i) Massively Parallel DNA sequencing Chip System for sequencing SAGE library from neurons; (ii) Nanoscopic DNA Arrays for global gene expression profiling at the level of individual cells and subcellular compartments, and (iii) Real-time monitoring of multiple mRNA species in living neurons and defined cellular microdomains with high spatial resolution and fast temporal resolution. Each of these technologies will be rigorously tested and validated using a model memory-forming network of Aplysia. The technologies will then be implemented to explore three fundamental brain mechanisms: (1) the molecular basis of neuronal identity, (2) the molecular signals controlling the formation of the precise pattern of interconnections, which underlie behavior and, (3) the molecular basis of synapse-specific neuronal plasticity and neuronal growth. Using identified neurons in networks of Aplysia as experimental models we will study the role of asymmetric mRNA distribution in integrative functions and phenotypes of eukaryotic cells. We will use a hierarchical design to achieve structural resolution of single-cell profiling in a descending fashion, where a parallel genomic and functional analysis within the same memory-forming networks will be performed in the scheme: single neuron to single axon to single synapse. The gene expression profiling will be correlated with functional imaging at functionally characterized neurons and synaptic terminals in a simple network during the memory consolidation. The combined approach based on genomics, photochemistry, nanoscience and engineering, biochemistry, and neuroscience will be used to understand how neurons and synapses operate in the context of learning and memory. The technologies developed and the biological discoveries made in the project will have broad impact and applications to study how genes regulate cellular and organism behavior on the scale from simpler nervous systems in invertebrates to the human brain.