NOVEL METHODS FOR SINGLE NEURON GENE/FUNCTION STUDIES

目前的建议是进一步开发一种新的和潜在的最佳制备，用于分析单个脑神经元的基因表达和生理功能。同时，该项目将进一步开发单个神经元中mRNA信息的比例扩增方法，以便使用实时96孔PCR或cDNA微阵列技术进行大规模的多基因表达研究。另一个重点将是确保这些技术与老年动物神经元研究的兼容性。我们研究部分分离的海马切片（通常被称为拉链切片，因为它倾向于沿着主要细胞体层逐渐打开或解开）已经大约5年了。拉链切片提供了无与伦比的访问和最小的创伤，成年和老年动物的神经元单通道补丁记录。此外，拉链在所有制备方法中是独一无二的，因为它允许在完整的过程中从整个神经元的切片中提取。我们已经使用这些非中断的单个神经元来开发单细胞- PCR的半定量方法。此外，随着多基因表达谱新技术的出现，提取的完整神经元似乎非常适合按比例扩增单个神经元的mRNA库，以充分进行大规模谱分析。因此，本项目提出1)优化拉链片用于单细胞mRNA分析；2)开发单细胞蛋白质含量的伴随检测；3)通过开发在开始解压缩程序之前研究功能的方法，大大扩展了切片的生理多功能性；4)建立分离根尖树突mRNA的研究技术；5)进一步开发和验证扩增单个神经元mRNA库的方法，以允许大规模表达谱分析。这些技术将首先在大鼠身上进行优化，然后扩展到年轻和年老的老鼠身上。总体目标将是开发一系列系统的程序，以促进单个脑神经元的生理研究，收集整个神经元及其全套mRNA转录物和蛋白质，以及随后对该神经元的大规模表达谱。因此，这些研究应该大大提高这种有价值的制剂的可及性和更广泛的使用。

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.