The objectives of the proposed research are the development of new genomic technologies for massive parallel DNA sequencing and large-scale gene expression analysis from single living nerve cells, and applications 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) Massive 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. PERFORMANCE SWE(S) (organization, city, state) Columbia University, New York, NY 10032 The University of Florida, Gainesville, FL 32611 KEY PERSONNEL. See instructions Use continuation pages as needed to provide the required information in the Start with Principal Investigator. List all other key personnel in alphabetical order, last name first Name Organization Ju, Jingyue Columbia University Kandel, Eric Columbia University Moroz, Leonid University of Florida GiUiam, T. Conrad Columbia University Turro, Nicholas J. Columbia University Koberstein, Jeffrey T. Columbia University Leonard, Edward Columbia University Benner, Steven University of Florida Tan, Weihong University of Florida Rzhetsky, Andrey Columbia University Russo, James J. Columbia University Kalachikov, Sergey Columbia University Farmerie, William G. University of Florida Sweedler, Jonathon V. University of Illinois Miller, Mark W. University of Puerto Rico format shown below. Role on Project Director Co-Director Co-Director Professor Professor Professor Professor Professor Associate Professor Assistant Professor Research Scientist Associate Research Scientist Assistant Scientist Professor Professor PHS 398 (Rev, 05/01) Page _, Form Page 2 . Principal KEY PERSONNEL. (Continued from page 2) Name Jeziorski, Michael C. Tzilianos, Eftihia Jockusch, Steffen Li, Zengmin Fan, Qing-Qing Iannuccilli, William J. Sathyanarayanan, Puthanveettil V. Kim, Sobin Seven Graduate Students Five Postdoctoral Scientist PHS 398 (Rev. 05/01) Investigator/Program Oirector (Last, first, middle): Organization University of Florida Columbia University Columbia University Columbia University Columbia University Columbia University Columbia University Columbia University Columbia University Columbia University Page ,_ Ju, Jingyue Role on Project Assistant Scientist Research Scientist Associate Research Scientist Associate Research Scientist Associate Research Scientist Senior Staff Associate Postdoctoral Scientist Postdoctoral Scientist Graduate Students Postdoctoral Scientist Form Page 2 Principal Investigator/Program Director (Last, flrst, middle): Ju, Jingyue The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page .................................................................................................................................................. 1 Description,
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