Neather, M.; Morgan, J.; Wong, F. K.

Synapses are evolutionarily conserved structures that form the fundamental units of neural communication. In the adult mouse cerebral cortex, most synapses are enveloped by glial protrusions from astrocytes and microglia, forming multi-partite synapses. Despite their prevalence, quantitative tools to systematically analyse these multi-cellular structures are limited to two or at most three markers. Here, we present Synapse Thresholding Image Analyser (SynThIA), an open-source, Python-based pipeline for high-throughput and accurate quantification of synapses, including multi-partite synapses. SynThIA enables multichannel analysis of up to four markers, providing detailed measurements of synaptic composition and distribution. The pipeline features an intuitive graphical interface allowing for users with minimal programming experience and a modular design that allows customization for advanced users. By combining accessibility and precision, SynThIA addresses a key methodological gap in multi-partite synaptic image analysis and provides a robust platform for studying synaptic organization in both in situ and ex situ preparation.