Abstract: Specific visualization and manipulation of neural circuitry has remained a vexing problem in neurobiology. Classical methods rely upon analysis in fixed tissue, preventing characterization of function or behavior. Newer methods allow genetic targeting to specific neuron types and even identify single neurons, but synaptic partners and functional circuits are not accessible by these current methods. A more general related issue is how to induce expression of a transgene in a vertebrate system when two cells make contact. A solution to these issues could have wide applicability, both for experimental studies, as well as for potentially a variety of therapeutic options. My project, Trans-Cellular Activation of Transcriptin to Analyze Dopaminergic Axon Reorganization, describes a novel strategy to analyze vertebrate circuit construction and function. It is the first genetic method for visualizing and driving expression in two cells that make contact, and offers the potential to identify and manipulate neuronal circuits in a vertebrate organism. I designed TCAT (trans-cellular activation of transcription) based on components from the conserved receptor/ligand pair of Notch/Delta. Upon ligand binding to receptor, the intracellular domain of Notch is cleaved and translocates to the nucleus. But by replacing the intracellular domain of Notch with the yeast transcriptional activator Gal4, I can specifically drive expression of transgenes at the Gal4-binding site UAS. TCAT uses the homologs LAG-2 (Delta) and LIN-12 (Notch) from the nematode C. elegans to prevent cross-reactivity with the endogenous zebrafish proteins. Specificity of labeling is provided by restricting expression of LAG-2 or LIN-12 to specific cell types. Expression from the UAS occurs when Gal4 is targeted to the nucleus, and this only occurs in the presence of ligand-receptor (cell-cell) LAG-2 to LIN-12 binding. Critically, this method is more than a means for labeling cells, but allows any inducible form of manipulation to be driven. There has been no other comparable method reported that activates transcription when two cells come into contact. Thus, TCAT will have applicability not only for use in the nervous system, but also in the study of other biological processes. In this proposal I introduce and explain TCAT, and show proof-of-principle that TCAT works in vivo, including when it is expressed by cell-type-specific enhancers. I describe how I will use it in mapping functional neural circuitry, by designing reagents to target TCAT to synapses. Finally, I outline how I will use TCAT to characterize dopamine circuit reorganization following injury, and the relevance of this for both basic and clinical neuroscience. TCAT is a significant technical innovation for mapping and manipulating circuits, but its real power is the ability it provides to create a full and functional understandig of the vertebrate CNS connectome. Public Health Relevance: The project develops a novel method to analyze and understand complex nervous systems, based on genetic approaches in the vertebrate model organism D. rerio. We apply this method to study the molecular mechanisms by which dopamine neurons alter their connections after injury, a clinically important cause of neurological and psychiatric diseases.
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