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The objectives of
the proposed research are the development of new genomic technologies
for massively parallel DNA sequencing and large-scale gene expression
analysis from single living nerve cells, and application 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) Massively 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.
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