Molecular Engineering Approach to Study Long Term Synaptic Plasticity

描述（由研究者提供）：本研究的目标是开发新的分子工程技术，用于单个神经元的大规模基因表达分析，并应用这些技术识别和表征参与长期突触可塑性和生长的基因。我们将结合化学，工程和生物学的研究专长，致力于以下新的分子工程方法的研究和发展：(i)大规模并行DNA测序芯片系统，用于从单细胞和细胞区室进行数字基因表达分析；（ii）用于实时监测活神经元和特定细胞微域中多种mRNA物种的新型分子探针。这些技术中的每一项都将使用Aplysia（一种独特的神经生物学模型生物）更简单的记忆形成网络进行严格的测试和验证。作为“概念验证”，我们将重点使用这些方法来识别学习诱导的突触生长背后的基因调控网络。具体来说，我们将描述由血清素诱导的分子级联事件，导致新突触的形成和突触强度的长期增强，也被称为学习和记忆机制的细胞表现。该项目的长期目标是利用这些新技术来探索两个基本的大脑机制：(1)神经元生长的分子基础；(2)控制突触特异性神经元可塑性的分子信号。我们将以猕猴桃神经网络中的感觉神经元为实验模型，研究mRNA不对称分布在真核细胞综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，其中平行基因组和功能分析将根据以下方案进行：单个神经元->单个轴突->单个突触。在5-HT诱导的突触生长的不同阶段，基因表达谱将通过一组互补方法进行验证，这些方法与功能表征神经元和突触末端的选定mrna的功能成像相关。基于化学、工程和神经科学的综合方法将用于理解神经元和突触在学习和记忆的背景下是如何运作的。在该项目中开发的技术和生物学发现将对破译神经疾病的分子机制产生广泛的影响。

DESCRIPTION (provided by investigator): The objectives of the proposed research are the development of new molecular engineering technologies for large-scale gene expression analysis from single neurons, and applications of these technologies to identify and characterize genes that are involved in long-term synaptic plasticity and growth. We will combine research expertise in Chemistry, Engineering and Biology to pursue the research and development of the following new molecular engineering approaches: (i) Massive Parallel DNA Sequencing Chip System for digital gene expression analysis from single cells and cell compartments; and (ii) Novel Molecular Probes for Real-time monitoring of multiple mRNA species in living neurons and defined cellular microdomains. Each of these technologies will be rigorously tested and validated using the simpler memory-forming network of Aplysia, a unique model organism for neurobiology. As a "proof-of concept", we will focus on using these approaches for the identification of gene-regulatory networks underlying the learning-induced synaptic growth. Specifically, we will characterize a molecular cascade of events induced by serotonin, leading to the formation of new synapses and a long-term enhancement of synaptic strength also known as cellular manifestations of learning and memory mechanisms. The long-term goal of this project is to implement these new technologies to explore two fundamental brain mechanisms: (1) the molecular basis of neuronal growth; (2) the molecular signals controlling synapse-specific neuronal plasticity. Using the sensory neurons of the neuronal networks in Aplysia as an experimental model, 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 will be performed according to the following scheme: single neuron->single axon->single synapse. The gene expression profiling will be validated using a set of complementary approaches, correlated with functional imaging of selected mRNAs at functionally characterized neurons and synaptic terminals during various stages of 5-HT induced synaptic growth. The combined approach based on Chemistry, Engineering, 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 a broad impact in deciphering the molecular mechanisms of neurological disorders.