Colocalization of synapse marker proteins evaluated by STED-microscopy reveals patterns of neuronal synapse distribution in vitro

Quantification of synapses and their morphological analysis are extensively used in network development and connectivity studies, drug screening and other areas of neuroscience. Thus, a number of quantitative approaches were introduced so far. However, most of the available methods are highly tailored to specific applications and have limitations for widespread use. We present a new plugin for the open-source software ImageJ to provide a modifiable, high-throughput and easy to use method for synaptic puncta analysis. Our approach is based on colocalization of pre- and postsynaptic protein markers. Structurally completed glutamatergic and GABAergic synapses were identified by VGLUT1-PSD95 and VGAT-gephyrin colocalization, respectively. By combining conventional confocal microscopy with stimulated emission depletion (STED) imaging, we propose a method to quantify the number of scaffolding protein clusters, recruited to a single postsynaptic density. In a proof-of-concept study, we reveal the differential distribution of glutamatergic and GABAergic synapse density with reference to perineuronal net (PNN) expression. Using super-resolution STED imaging, we demonstrate that postsynaptic puncta of completed synapses are composed of significantly more protein clusters, compared to uncompleted synapses. Our Synapse Counter plugin for ImageJ offers a rapid and unbiased research tool for a broad spectrum of neuroscientists. The proposed method of synaptic protein clusters quantification exploits super-resolution imaging to provide a comprehensive approach to the analysis of postsynaptic density composition. Our results strongly substantiate the benefits of colocalization-based synapse detection.