Deciphering the Nanoscale Architecture of Presynaptic Actin Using a Micropatterned Presynapse-on-Glass Model

Chemical synapses are fundamental units for the transmission of information throughout the nervous system. The cytoskeleton allows to build, maintain, and transform both pre- and postsynaptic contacts, yet its organization and the role of its unique synaptic nanostructures are still poorly understood. Here we present a presynapse-on-glass model based on cultured neurons from rat pups of either sex. Presynaptic specializations are robustly induced along axons by micropatterned dots of neuroligin, allowing the controlled orientation and easy optical visualization of functional induced presynapses. We demonstrate the relevance and usefulness of this presynapse-on-glass model for the study of presynaptic actin architecture, showing that a majority of induced presynapses are enriched in actin, with this enrichment being correlated to higher synaptic cycling activity. We confirm our previous results on bead-induced presynapses by identifying distinct actin nanostructures within presynapses: corrals, rails, and mesh. Furthermore, we leverage the controlled orientation of the presynapse-on-glass model, visualizing the arrangement of these actin structures relative to the active zone nanoclusters using multicolor 3D single-molecule localization microscopy (SMLM) and relative to the subdiffractive localization exocytic events using a correlative live-cell and SMLM approach.