The specific aim of this proposal is to create cDNA libraries from neuronal processes in order to study the localization of mRNAs to dendrites and/or axons. A number of studies have indicated that localization of mRNAs to neuronal processes and regulated translation of these mRNAs serves as an important mechanism of gene expression in neurons. To begin to study this form of gene expression, we will create preparations of isolated neuronal processes, develop methodologies for creating cDNA libraries from the severely limited amount of mRNA collectable from these preparations, and develop methodologies for analyzing the representation and accumulation of mRNA species in neuronal processes in both basal and stimulated states. We will use cultured Aplysia sensory neurons and cultured mouse hippocampal neurons. The unique advantage of Aplysia neurons is that cell bodies and glia can be manually removed from cultured neurons, leaving pure neuronal processes as a starting material for cDNA library construction. Rodent hippocampal neurons can be cultured on membranes such that the neuronal axons and dendrites can penetrate through the pores in the membrane and can therefore be separated from cell bodies. Using these preparations we will: 1. Develop preparations of pure neuronal processes 2. Develop methodologies for creating cDNA libraries from these neuronal processes 3. Develop methodologies to test the representation and localized accumulation of transcripts in unstimulated and stimulated neuronal processes.
| Zhao, Yali; Hegde, Ashok N; Martin, Kelsey C (2003) The ubiquitin proteasome system functions as an inhibitory constraint on synaptic strengthening. Curr Biol 13:887-98 |
