NOVEL METHODS FOR SINGLE NEURON GENE/FUNCTION STUDIES

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The present proposal is to further develop a novel and potentially optimal preparation for the analysis of gene expression and physiological function in single brain neurons. Concomitantly this project will further develop methods for the proportional amplification of mRNA messages in a single neuron, to allow for large scale multi-gene expression studies, using either real-time 96-well PCR or cDNA microarray technologies. An additional focus will be to ensure compatibility of these techniques with studies of neurons in aged animals. We have been working with the partially-dissociated hippocampal slice (often termed the zipper slice for its tendency to gradually open or unzip along the major cell body layers) for approximately 5 years. The zipper slice provides unparalleled access and minimal trauma to neurons of adult and aged animals for single channel patch recording. In addition, however, the zipper is unique among all preparations in that it permits the ready extraction from the slice of an entire neuron with its processes intact. We have used these non-disrupted single neurons to develop semi-quantitative approaches for single cell- PCR. Moreover, with the advent of new technologies for multiple gene expression profiling, the extracted intact neurons seem to be ideally suited for attempts to proportionally amplify the mRNA pool of a single neuron sufficiently to allow large scale profiling. Thus, this project proposes to 1) optimize the zipper slice s use for single cell mRNA analyses; 2) develop concomitant assays for single cell protein content; 3) greatly extend the slice s physiological versatility by developing methods to study function before beginning the unzipping procedure; 4) develop techniques for studying mRNA in isolated apical dendrites; and 5) further develop and validate methods for amplifying the mRNA pool of a single neuron sufficiently to permit large-scale expression profiling. These techniques will be optimized first in rats, and then extended to young and aged mice. The overall goal will be to develop a systematic series of procedures that facilitate physiological studies of a single brain neuron, collection of the entire neuron with its full set of mRNA transcripts and protein, and subsequent large-scale expression profiling of that neuron. Thus, these studies should substantially enhance the accessibility and wider use of this valuable preparation.