Novel Molecular Tools for Imaging Synaptic Dynamics

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DESCRIPTION (provided by applicant): The goal of this project is to analyze the dynamic changes that occur at a cellular and molecular level during synaptic development and plasticity. These events will be examined in a well characterized genetic model system, the Drosophila neuromuscular junction (NMJ). We will generate a toolset of photoactivatable green fluorescent protein (PA-GFP) fusions made to a panel of pre- and postsynaptic proteins. Multiple transgenic lines will be made, including both promoter fusions and UAS effector lines. The molecular probes include PA- GFP fusions of the vesicular SNARE protein synaptotagmin, the PSD95/MAGUK adaptor protein disks large (Dlg) and the glutamate receptor subunit dGluRIIA. In addition, we will make fusion constructs of molecular components of the TGF-beta signaling cascade that is believed to be involved in the retrograde control of synaptic development. These include the Smad protein Mad, and the type II BMP receptor wishful thinking (wit). Extensive genetic and physiological validation experiments will be performed to demonstrate that the PA- GFP fusion reporters faithfully localize to the correct synaptic sites, are functional and can rescue mutant phenotypes, and do not confer dominant phenotypes when expressed in vivo. In the second specific aim, we propose to use the photoactivatable probes to investigate several outstanding questions in synaptic development and plasticity. The experiments include an analysis of activity-dependent growth of the synapse, the trafficking of retrograde growth factors involved in NMJ growth, the translocation of vesicular components between release sites, and the tagging of synapses undergoing plasticity. The proposed experiments were selected both for the importance of the question and for their potential for follow-on studies. The lines expressing the molecular probes will be of widespread use to researchers studying synaptic development and plasticity, and will be freely shared. The problems we will investigate are of universal interest to researchers studying synaptic development and plasticity, and the results will be of broad relevance. PUBLIC HEALTH RELEVANCE: In this project we will generate new molecular tools for examining how synapses modify their structures and deploy molecular components as a function of neural activity. We will make available to basic researchers a variety of valuable tools for imaging neuronal connections as they develop and as they are modified as a function of prior experience. These tools will allow researchers to study how specific proteins are deployed and regulated. As there are numerous neurological disorders that involve defects in synaptic growth, maintenance, and/or plasticity, these studies will contribute to a rational analysis of these disorders of the nervous system.