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.
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