METHODS FOR MAPPING GENE EXPRESSION IN THE CNS

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The human brain comprises approximately 100 billion neurons precisely organized into networks that give rise to cognitive phenomena such as emotion, learning and memory, and perception. Unfortunately, there are virtually no unique markers for the many neuronal cell types thought to exist in the CNS. Indeed, we also do not know how many different cell types actually exist. Our long-term goal is to quantitate the diversity of brain regions and neuronal cell types by elucidating region- or cell type- specific patterns of gene expression - the molecular anatomy of the brain. In order to achieve this goal, we aim (1) to develop methods for mapping gene expression within identified brain regions, and (2) to develop methods for defining neuronal identity and marker genes in the vertebrate CNS. The first aim includes improving a novel mRNA amplification procedure we have developed that enables one to construct cDNA libraries from small amounts of tissue, such that the representation of original mRNA lengths is comparable to the best cDNA libraries made from unamplified RNA. Methods will also be developed for mapping gene expression within small regions of the CNS using this improved mRNA amplification procedure in DNA microarray hybridizations. The second aim includes developing methods for defining neuronal cell types and cell-type specific marker genes in heterogeneous neuronal populations. This will be done by using microarrays to characterize gene expression patterns of single cells. Methods will be developed to amplify the mRNA content of single cells to hybridize to microarrays, and statistical methods will be developed for analyzing and comparing single-cell microarray hybridization data from multiple experiments. These latter methods will allow the definition of neuronal cell identities as well as novel marker genes based upon complex gene expression patterns. Our methods to map gene expression in the CNS will enable researchers to identify genes and gene variants that are playing a role in the function of small brain regions in both healthy and diseased tissue. In addition, our methods to identify marker genes will eventually allow individual neuronal types to be isolated or targeted for study.