Resolving synaptic events using subsynaptically targeted GCaMP8 variants

While genetically encoded Ca 2+ indicators are valuable for visualizing neural activity, their speed and sensitivity have had limited performance when compared to chemical dyes and electrophysiology, particularly at synaptic compartments. We addressed these limitations by engineering a suite of next-generation GCaMP8-based indicators, targeted to presynaptic boutons, active zones, and postsynaptic compartments at the Drosophila neuromuscular junction. We first validated these sensors to be superior to previous versions and synthetic dyes. Next, we developed a Python-based analysis program, CaFire , which enables the automated quantification of evoked and spontaneous Ca² + signals. Using CaFire , we show a ratiometric presynaptic GCaMP8m sensor accurately captures physiologically relevant presynaptic Ca 2+ changes with superior sensitivity and similar kinetics compared to chemical dyes. Moreover, we test the ability of an active zone-targeted, ratiometric GCaMP8m sensor to report differences in Ca² + between release sites. Finally, a newly engineered postsynaptic GCaMP8m, positioned near glutamate receptors, detects quantal events with temporal and signal resolution comparable to electrophysiological recordings. These next-generation indicators and analytical methods demonstrate that GCaMP8 sensors, targeted to synaptic compartments, can now achieve the speed and sensitivity necessary to resolve Ca 2+ dynamics at levels previously only attainable with chemical dyes or electrophysiology.