Probabilistic Analysis of Confocally Imaged Synaptic Calcium Activity (PACISCA)

Calcium imaging is a widely used approach in neuroscience to analyze neuronal activity patterns in neural networks to correlate animal behavior with neural activity. Confocal microscopy has been brought up as an option for synaptic imaging in the past (as compared to 2-photon microscopy), but one of its main drawbacks is the lack of options for processing, particularly in the context of synaptic imaging. Most existing pipelines, such as CaImAn, EZCalcium, etc. are designed for one/two photon microscopy and detect/analyze neuronal regions of interest (ROIs). Compared to neuronal calcium imaging, quantitative tools to analyze synaptic calcium signals in an automated manner are lacking due to the nanoscopic organization of synapses. To address this technical challenge, we developed a novel pipeline that analyzes synaptic ROIs to map the probability of synaptic transmission at the resolution of individual active zones: PACISCA. The pipeline has four main components: synapse annotation/image motion correction, flash identification, fluorescence tracing, and probability mapping. Our work provides an efficient computational framework to analyze synaptic calcium imaging signals from neuroscience experiments studying synaptic functions. Improving the reliability of our analytic tool on larger datasets will be the next steps of this work.