Czondor_2025_Methods.Mol.Biol_2910_3

One of the difficulties for studying the mechanisms of synaptogenesis stems from the spatial unpredictability of contact formation between neurons, and the involvement of many parallel adhesive pathways mediating axon-dendrite recognition. To circumvent these limitations, we describe here a method allowing for the investigation of biomimetic synaptic contacts at controlled locations with high precision and statistics. Specifically, primary neurons are cultured on micropatterned substrates comprising arrays of micron-scale dots coated with purified synaptogenic adhesion molecules. Coating the substrates with the homophilic adhesion molecule SynCAM1 triggers the formation of functional presynaptic structures in axons, while neurexin-1beta elicits postsynapses in dendrites from neurons expressing the counter receptor neuroligin-1. This assay can be combined with various optical imaging techniques, including immunocytochemistry to screen the accumulation of synaptic components, long-term live cell recordings to probe the kinetics of neurite growth and synapse differentiation, as well as high-resolution single-molecule tracking.