A HIGH-THROUGHPUT SYNAPTIC CALCIUM IMAGING ANALYSIS PIPELINE FOR SCREENING NMDAR MODULATORS

Synapses represent the basic unit of information transfer between neurons. Neurons form thousands of these connections with neighboring neurons, and each synapse maintains distinct characteristics, including receptor subtype composition and presynaptic neuron subtype, that collectively control neuronal signaling dynamics. By imaging in a solution containing 0 mM Mg²⁺ and 1 µM TTX, we can visualize spontaneously occurring NMDAR-dependent calcium transients across all visible synapses. Using the open-sourced software Suite2p and a Python analysis pipeline, we routinely analyzed datasets containing tens of thousands of synapses. We verified that these transients are NMDAR-dependent and respond canonically to known agonists and antagonists such as glycine and APV. Moreover, we can visualize and quantify changes in presynaptic vesicle fusion, induced by phorbol esters (indicated by a change in calcium event frequency), postsynaptic function (indicated by a change in calcium event amplitude), and functional synapse number. Overnight application of three different antibodies from patients with anti-NMDAR encephalitis revealed antibody-specific changes in the number of active synapses and synapse-level changes in calcium transient amplitude and frequency, across tens of thousands of individual synapses. Overall, our pipeline provides simultaneous structural and functional readouts to study synapse-specific effects at scale. The ability to detect, track, and quantify activity across tens of thousands of synapses and to record millions of synaptic calcium transients highlights the potential of high-throughput, large-scale synapse analysis to generate new insights into synaptic plasticity and neural network formation in vitro.