Novel Split GFP Based Intersynaptic Markers to Study Synaptic Specificity in vivo

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DESCRIPTION (provided by applicant): The nervous system is composed of highly complex circuits that govern thought and behavior. To ensure correct circuit formation, neurons must identify appropriate synaptic targets among the many neurites they contact before forming synaptic connections. Altered synaptogenesis is thought to play a role in disorders such as schizophrenia and autism. Despite its central role in circuit formation, synaptic specificity is poorly understood. I have developed a novel way to visualize synapses between specific neurons in vivo. I propose to use this method and take advantage of the simple, well-characterized nervous system of C. elegans to elucidate molecular mechanisms that underlie this fundamental process. I then propose to extend this my findings to mammalian neurons. This proposal is relevant to the NINDS mission to support basic research in fields related to the causes of neurological disorders, and the NIMH mission to support research on mental disorders and the underlying basic science of brain and behavior. My specific aims are: 1) to visualize changes in synaptic connectivity between a specific pair of pre- and postsynaptic neurons in C. elegans, 2) to discover the molecular determinants of synaptic specificity in this system, and 3) to adapt this new labeling method to mammalian neurons. To visualize changes in synaptic connectivity, I have developed an intersynaptic marker. I have fused complementary fragments of a split GFP to pre- and postsynaptically localized proteins, expressed under cell-specific promoters. I have successfully visualized changes in connectivity in two well-characterized circuits in C. elegans using known synaptic specificity mutants. I am interested in how synaptic specificity is regulated in complex environments, such as nerve bundles, where parallel neurites must distinguish among multiple potential targets to form appropriate connections. Therefore I am now adapting the intersynaptic marker to label synaptic connections between a specific neuron pair in a posterior nerve bundle. I will take an unbiased genetic approach to discover molecular mechanisms guiding synaptic specificity in this system, utilizing this marker to detect defects in connectivity. Finally, I am developing the intersynaptic marker for use in cultured mammalian cortical neurons. Insights gained from this study will aid in understanding synaptic specificity in the complex environments of the vertebrate nervous system. Understanding the mechanisms that regulate circuit formation will bring us closer to understanding and treating neurological diseases.