Novel Molecular Tools for Transsynaptic Circuit Analysis

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DESCRIPTION (Provided by the Applicant): A major goal of neuroscience is to characterize neuronal connectivity in the CNS in order to understand the mechanisms that govern the development and function of neural circuits. This goal requires new tools for the direct labeling of neural circuits, using molecular genetic approaches for trans-synaptic transfer. A further goal is to generate molecular constructs that control gene expression within all the cells of specific neural circuits. This project will advance us toward these goals. There are three specific objectives: First, to generate novel transgenic lines where transsynaptic labeling constructs are expressed in vivo in specific targeted neurons. The constructs are based on both plant lectins (WGA and BL) and the tetanus toxin C chain (TTC), molecules that cross synapses. The carrier proteins will convey various cargos, such as GFP and RFP, across synapses in either a forward or reverse direction. Second, we will identify the essential domains of these carrier proteins through structure-function analysis, generating novel fusion proteins with improved carrier properties. Finally, we will develop a method for controlling gene expression within specific neural circuits, which we term "Inducible Transsynaptic Expression with Amplification" (ITEM). This entails generating molecules for transsynaptic transcriptional activation, based on fusions between known transsynaptic carriers and the GAL4::VP16 transcriptional activator. With ITEM, gene expression in a specific neural circuit is achieved when the transcriptional activator is transferred sequentially from cell to cell. By placing the transsynaptic transcriptional activator under UAS control, the molecule activates its own expression in recipient cells, resulting in amplification of the signal as it passes from cell to cell. The ITEM method will be a powerful tool for mapping neural connectivity, as well as for experimentally controlling the development or function of neural circuits. Although the studies will be pioneered in Drosophila, successful transsynaptic reporter gene activation would provide a proof of principle that could then be adapted for higher systems. Relevance to public health: In this project we will create new tools for looking at how nerve cells form connections with one another. These will help us understand how different patterns of connections give rise to different behaviors, and determine how nerve cells modify their connections in response to experience or to changes in brain function caused by injury or disease.