Genomic Approaches to Deciphering Memory Circuits

描述（由申请人提供）：拟研究的目的是通过应用高通量基因组技术进行彻底的单细胞和细胞室基因表达研究，以确定神经元身份，极性和可塑性的基因组基础。利用模型生物加利福尼亚海桃的鳃退缩反射记忆回路，我们的目标是系统地定义构成这个细胞集合的神经突和关键神经元的单个突触的分子库（基因组蓝图）。我们将定义功能回路组件（细胞和突触）内的区室转录组（mRNAs， miRNAs和其他ncRNAs的集合），通过体外共培养2-4个最具特征的细胞（L7运动神经元，感觉神经元，刺激和抑制性中间神经元）来重建。这种在细胞培养中重建的完全可操作的神经回路具有完整回路的许多重要特性，并已被成功地用于确定澳大利亚记忆形成的分子基础，其中许多方面在动物王国中是保守的，包括在人类大脑中。系统生物学方法将应用于揭示基因调控网络及其在建立和维持长期记忆中的潜在作用，以习得性恐惧为实验范式，重点关注长期促进（LTF）和抑郁（LTD）的突触机制。我们将使用这种基因组学和系统生物学方法来探索以下三个基本的大脑机制：(1)神经元身份的分子基础，通过揭示这些神经元或专门突触之间独特或共享的转录本；(2)控制细胞极性和构成行为基础的精确互连模式的分子信号，部分受这些细胞中央和外周区室mrna分布的指导；(3)突触特异性神经元可塑性和神经元生长的分子基础，特别关注突触前和突触后神经元对之间连接处单个突触内的mRNA库。这种结合的方法将利用基因组学、生物工程、神经科学和生物信息学方面已经建立的专家团队。虽然这些模式将在大的、特征良好的澳大利亚神经元中建立，但其揭示的机制和发展的技术将对任何具有不对称rna和蛋白质分布的极化细胞类型的生物学产生广泛的影响。

DESCRIPTION (provided by applicant): The objective of the proposed research is to conduct a thorough single-cell and cell-compartment gene expression study through the application of high throughput genomic technologies to identify the genomic bases of neuronal identity, polarity and plasticity. Utilizing the well-studied gill withdrawal reflex memory circuit from the model organism Aplysia californica, our goal is to define systematically the molecular repertoire (genomic blueprint) of the neurites and individual synapses of the key neurons that make up this cellular ensemble. We will define the compartmental transcriptomes (the sets of mRNAs, miRNAs and other ncRNAs) within the components of the functional circuit (cells and synapses), which are reconstituted in vitro by co- culture of 2-4 of its best characterized cells (L7 motor neuron, sensory neuron, stimulatory and inhibitory interneurons). This fully operational neural circuit reconstructed in cell culture bears many important properties of the intact circuit, and has been used with great success to ascertain the molecular underpinnings of memory formation in Aplysia, numerous aspects of which are conserved within the animal kingdom, including in the human brain. The systems biology approach will be applied to reveal gene regulatory networks and their potential role in the establishment and maintenance of long-term memory using learned fear as an experimental paradigm, focusing on synaptic mechanisms of long-term facilitation (LTF) and depression (LTD). We will use this genomic and systems biology approach to explore the following three fundamental brain mechanisms: (1) the molecular basis of neuronal identity, by revealing those transcripts that are unique to or shared among these neurons or specialized synapses; (2) the molecular signals controlling cellular polarity and the formation of the precise pattern of interconnections which underlie behavior, in part directed by the distribution of mRNAs in the central and peripheral compartments of these cells; and (3) the molecular basis of synapse-specific neuronal plasticity and neuronal growth, with special attention paid to the mRNA repertoire within the individual synapses at the junctions between pairs of pre- and post-synaptic neurons. The combined approach will take advantage of an already established team of experts in genomics, bioengineering, neuroscience, and bioinformatics. Though these paradigms will be established in the large well-characterized neurons of Aplysia, the mechanisms revealed and the technologies developed will have a broad impact in the biology of any polarized cell type with asymmetric distribution of RNAs and proteins.