GENETIC MODIFICATION OF THE CENTRAL NERVOUS SYSTEM

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The central nervous system (CNS) is comprised of numerous pathways and nuclei. Most anatomically definable structures in the CNS are composed of many different cell classes, each of which is presumed to make a specific contribution to the function of the entire structure. A fundamental problem in understanding the role of structures with multiple, interconnected cell populations is that it has seldom been possible, using traditional neurobiological methods, to unequivocally define the role of single populations of neurons in a complex structure. In this application, we propose to use a recently developed technique, genetic ablation, to achieve the selection elimination of a class of neurons from the CNS and subsequently, to study the role of that cell class in CNS structure and function. In particular, we propose to eliminate an identified cell class from the retina. The goals of the proposed experiments are: 1) to identify retinal cell-class specific genes from subtracted retinal cDNA libraries, 2) to isolate the promoter regions of the identified genes, 3) to construct fusion genes by fusing the promoter of the identified gene to an attenuated diphtheria toxin gene, 4) to use this fusion gene to create transgenic mice and 5) to use these transgenic mice in a series of anatomical and neurophysiological assessments designed to characterize the effects of the ablation on the structure and function of the retina. In our preliminary experiments, we have successfully eliminated the rod photoreceptors from the retina, thereby demonstrating that this is a viable approach for the selective ablation of a specific cell population from the CNS. We have chosen the retina as our model system for the application of genetic ablation for a number of reasons. first, it is a laminar structure, with the somata of major, recognized cell classes, and their processes, lying in specific laminae. Second, retinal ganglion cells represent the sole output neurons of the retina and their responses reflect the integration of the activity of the more peripheral retinal elements. These cells, therefore, offer an accessible in vivo assay for the effects of an ablation. Finally, our laboratory has considerable expertise in the analysis of the retina, both anatomically and physiologically. While the experiments described in this application will be performed on neurons in the retina, we fell it is important to emphasize that the approach that we have chosen will be equally powerful in defining the function of any population of cells for which unique genetic material can be identified.