Neurons receive an enormous variety of inputs that are often segregated in discrete domains on their extracellular surface. This heterogeneity of distribution is reflected in the quantities and kinds of proteins that are targeted to different synapses around the cell. Synaptic heterogeneity is also reflected in the differential distributions of selected mRNAs that are transported into dendrites. It is thought that some mRNAs localized within dendrites play a role in synaptic plasticity. The purpose of the experiments described in this proposal is to provide support for the following general hypothesis: Local electrical signals affect the distribution and utilization of mRNAs in neuronal dendrites via cis-acting mRNA sequences, and this level of control is required to provide a means of adapting the cell to changing patterns and levels of electrical stimulation. This proposal will take advantage of the unique properties of a Purkinje cell-specific mRNA, Pcp-2(L7), and of the cerebellar system to investigate the molecular and cellular mechanisms of dendritic routing of mRNAs. A combined in vivo (transgenic mice) and in vitro (direct injection of labeled mRNAs in cerebellar slices) approach will be used to map RNA routing signals (Aim 1). In addition, dynamic aspects of mRNA trafficking and effects due to electrical activity on mRNA routing, localization and utilization will be examined, and these studies will be facilitated by some novel means of analyzing newly synthesized Pcp-2(L7) mRNA (Aim 2). To test the functional relevance of dendritic mRNA a variant form of mRNA that encodes Pcp-2(L7) protein but is not routed into dendrites will be compared to a normally routed mRNA in behavioral rescue experiments in Pcp-2(L7) null mutant mice (Aim 3). Thus, the molecular analysis in Aim 1 will provide tools to study the functional significance of mRNA routing in Aim 3 as well as an assay (direct mRNA injection) for studying extracellular signals that influence intradendritic localization in Aim 2. Likewise, establishment of inducible expression of L7 mRNA and protein in an L7 null mutant background in Aim 3 will enable an additional tool to be used to study stimulatory effects on newly synthesized mRNA and protein in Aim 2.
