Dissecting the functional heterogeneity of glutamatergic synapses with high-throughput optical physiology

Fluorescent reporters for glutamate release and postsynaptic Ca²⁺ signaling are essential tools for quantifying synapse functional heterogeneity across neurons and circuits. However, leveraging these probes for neuroscience requires scalable experimental frameworks. Here, we devised a high-throughput approach to efficiently collect and analyze hundreds of optical recordings of glutamate release activity at presynaptic boutons in cultured rat hippocampal neurons. Boutons exhibited remarkable functional heterogeneity and could be separated into multiple functional classes based on their iGluSnFR3 responses to single action potentials, paired stimuli, and synaptic parameters derived from mean-variance analysis. Finally, we developed a novel all-optical assay of pre- and postsynaptic glutamatergic synapse function. We deployed iGluSnFR3 with a red-shifted, postsynaptically-targeted Ca²⁺ sensor, enabling direct imaging and analysis of NMDA receptor-mediated synaptic transmission at large numbers of dendritic spines. This work enables direct observation of the flow of information at single synapses and should speed detailed investigations of synaptic functional heterogeneity.