PROJECT SUMMARY Alternative mRNA splicing (AS) is a fundamental process that regulates the expression of more than 90% of human protein-coding genes. The function of AS in the nervous system is particularly prevalent and has been implicated in multiple neurological disorders that impair learning. Although AS is implicated in activity- dependent gene expression underlying neural plasticity, no systematic analysis on AS has been performed on an in vivo learning model. The complexity of the mammalian brain poses challenges to this type of studies. Thus, we still do not understand (1) to what extent learning engages AS in the nervous system, (2) how AS contributes to learning-induced changes in neuronal gene expression, and (3) how learning modulates AS and splice isoforms of specific genes to generate learned behavior. Here, we propose to address these fundamental questions in C. elegans. The rationale is that the wiring and genetic make-up of the C. elegans nervous system are well characterized, dynamic gene expression can be profiled for the whole brain or individual neurons, functions of genes in learning can be dissected at the cellular resolution with genetic and imaging tools, and the fundamental properties of the development and function of the nervous system are well conserved between C. elegans and more complex animals. In addition, many forms of learning exhibited by C. elegans share similar behavioral characteristics and molecular underpinnings with those displayed by higher organisms. The overall goal of this project is to characterize how AS regulates learning and to provide insights into neurological defects in brain function under many disease conditions. The hypothesis of this project is that AS regulates neuronal gene expression to modulate neural function and produce learning. Specifically, we will first characterize the global patterns of AS network and splice isoforms that are regulated by a learning paradigm well-characterized in our laboratory. We plan to systematically analyze how learning alters splicing or isoform usage of all genes expressed in the C. elegans nervous system. Next, we will use genetic perturbations to address the causal function of learning-regulated splice isoforms of conserved molecules in neural activity and behavior. The grant is exploratory, because it (1) presents the first systematic analysis of AS in an in vivo model of learning and (2) introduces conceptual and technical advances to address causal links between AS and learning behavior. The proposed work is significant, because it (1) tests a highly plausible function of AS, a fundamental gene expression process conserved in eukaryotes, in learning, and (2) characterizes the mechanisms whereby AS of conserved molecules regulates neuronal gene expression and function to produce learning. Meanwhile, our grant is built on a substantial amount of preliminary results that support conceptual and technical productivity. The outcome of this study will provi...