摘要：SummaryMost neurological disorders display impaired synaptic connectivity. Hence, modulation of synapse formation may have therapeutic relevance. However, the high density and small size of synapses complicate their quantification. To improve synapse-oriented screens, we analyzed the labeling performance of synapse-targeting antibodies on neuronal cell cultures using segmentation-independent image analysis based on sliding window correlation. When assessing pairwise colocalization, a common readout for mature synapses, overlap was incomplete and confounded by spurious signals. To circumvent this, we implemented a proximity ligation-based approach that only leads to a signal when two markers are sufficiently close. We applied this approach to different marker combinations and demonstrate its utility for detecting synapse density changes in healthy and compromised cultures. Thus, segmentation-independent analysis and exploitation of resident protein proximity increases the sensitivity of synapse quantifications in neuronal cultures and represents a valuable extension to the analytical toolset forin vitrosynapse screens.Graphical AbstractDisplay OmittedHighlights•Synapse antibody library screen using segmentation-independent image analysis•Colocalization of pre- and postsynaptic markers partially reveals mature synapses•Transsynaptic proximity ligation assay enhances sensitivity•Guidelines for synapse-orientedin vitroscreening assaysOptical Imaging; Molecular Biology Experimental Approach; Cellular Neuroscience; Biocomputational Method
