Visualizing Synapses between Specific Neurons In vivo with Light Microscopy

Specific synaptic connections underlie the ability of our nervous systems to perform the complex computations that account for our daily perception and behavior. How these connections arise during development is a central question in neuroscience. Due to the cellular complexity of the central nervous system (CNS) and the small size of synapses, it is difficult to efficiently visualize synapses of identified neurons in vivo, and this has become a major obstacle to studying mechanisms of synapse formation and synaptic specificity. Synapses are traditionally visualized with Electron Microscopy (EM), which can generate comprehensive and accurate synaptic connectivity maps. However, EM techniques are time consuming and labor intensive, making it difficult to study the dynamic process of synaptic development and identify molecular pathways involved in synapse formation using this method. Therefore, my thesis research has focused on developing techniques facilitating synapse visualization in vivo with light microscopy. I first adapted a technique originally developed in C elegans called GFP reconstitution across synaptic partners (GRASP) to the nervous system of Drosophila melanogaster. I showed that adapted GRASP effectively detected synapses between known synaptic partner neurons within the fly visual system. It also successfully mapped neuronal connections within the neural circuit that underlies fly mating behavior. Due to some caveats in the design, in certain cell types GRASP failed to distinguish synapses from general cell-cell contacts. Inspired by GRASP and the prospect of addressing its limitations, I designed a new method called Synaptic Tagging with Recombination (STaR), which labels endogenous presynaptic and postsynaptic proteins in a cell-type-specific fashion. I modified genomic loci encoding synaptic proteins within bacterial artificial chromosomes such that these proteins, expressed at endogenous levels and with normal spatiotemporal patterns, were labeled in an inducible fashion in specific neurons through targeted expression of site-specific recombinases. Within the fly visual system, the number and distribution of synapses labeled with STaR correlate with EM studies. Using two different recombination systems, presynaptic and postsynaptic specializations of synaptic pairs can be co-labeled, and synapses between specific partners can be identified by assessing the apposition of these specializations. With STaR, I characterized synaptic development in photoreceptor neurons and uncovered a novel transformation phase of growth cones to synaptic terminals. This has led to the generation of gene expression profiles before, during and after the transformation phase and an in vivo RNAi screen to identify genes regulating photoreceptor synaptogenesis using the STaR markers. Furthermore, combining STaR with two-photon microscopy allowed visualization of synapse formation in live animals. In principle, STaR can be adapted to the mammalian nervous system. Both GRASP and STaR will facilitate our future investigation of key questions in synapse biology.