Genomic Approaches to Deciphering Memory Circuits

描述(申请人提供)：拟议研究的目标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，以确定神经元特性、极性和可塑性的基因组基础。利用从模式生物Aplysia calfornica中研究得很好的撤鳃反射记忆电路，我们的目标是系统地定义组成这一细胞集合的关键神经元的神经突起和单个突触的分子图谱(基因组蓝图)。我们将定义功能回路(细胞和突触)组件(细胞和突触)中的间隔性转录(mRNAs、miRNAs和其他ncRNAs的集合)，这些组件是通过与2-4个特征最好的细胞(L7运动神经元、感觉神经元、刺激和抑制中间神经元)共同培养而在体外重组的。在细胞培养中重建的这种完全可操作的神经回路具有完整回路的许多重要特性，并已成功地用于确定海兔记忆形成的分子基础，其中许多方面在动物界中保守，包括在人脑中。系统生物学方法将被用来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，以获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和系统生物学的方法来探索以下三种基本的大脑机制：(1)神经元识别的分子基础，通过揭示这些神经元或特殊突触所特有或共享的那些转录本；(2)控制细胞极性的分子信号和作为行为基础的精确的相互连接模式的形成，部分由这些细胞的中央和外周隔室中的mRNAs的分布所指导；以及(3)突触特有的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的单个突触内的信使核糖核酸储备。这种结合的方法将利用已经建立的基因组学、生物工程、神经科学和生物信息学方面的专家团队。虽然这些模式将建立在海兔特征良好的大型神经元中，但所揭示的机制和开发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.