Imaging Synaptic Transmission of Individual Active Zones

Massachusetts Institute Of Technology, Cambridge MA

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Abstract

In the current application, we propose to characterize molecular mechanisms that generate and control presynaptic output strength across several neuronal subtypes using Drosophila as a model system. Synaptic vesicle fusion occurs through a highly probabilistic process, often with only a small percent of action potentials triggering release from individual active zones (AZs). Although AZs largely share the same complement of proteins, release probability (Pr) is highly variable across different neurons and between AZs of the same neuron. Indeed, some AZ-specific proteins are non-uniformly distributed, and the molecular composition of AZs can undergo rapid changes. To date, Ca2+ channel abundance and Ca2+ influx have been most strongly linked to Pr heterogeneity, though other factors are likely to contribute as well. The Drosophila neuromuscular junction (NMJ) has emerged as a robust model system to characterize determinants of Pr. We have developed tools for birth dating and serial in vivo imaging of the same AZ population over a multi-day period beginning shortly after synapse formation. In addition to intravital imaging of AZ development and associated protein content, we developed biosensors that allow quantal imaging of all SV fusion events occurring through both spontaneous and evoked release at single AZs. We also defined the transcriptomes of Drosophila tonic and phasic glutamatergic motoneuron subtypes that display distinct AZ structure, Ca2+ influx and synaptic output. Using these toolkits, we propose to characterize how Ca2+ channels traffic to and accumulate at AZs to regulate presynaptic output. In addition, we will determine the molecular underpinnings that generate distinct synaptic structure and function associated with two closely related glutamatergic cell types. Finally, we will determine how alterations in neuronal activity regulate AZ development and structure. Together, these studies will provide new insights into presynaptic mechanisms that control synaptic communication across several neuronal subtypes.