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 sub-diffractive localization exocytic events using a correlative live-cell and SMLM approach. Significance statement The actin cytoskeleton plays important but poorly understood roles at presynapses, fundamental compartments for communication in the nervous system. We developed a presynapse-on-glass model to induce isolated, optically accessible presynaptic specializations along the axon of cultured neurons. This model recapitulates the presynaptic actin enrichment and distinct nanostructures we previously uncovered using presynapses induction by large beads. The controlled orientation of presynapses in our new model allows to go further: we visualized the nanoscale arrangement of actin and presynaptic components by multicolor nanoscopy, and could link actin nanostructures to the precise location of synaptic vesicle release thanks to a correlative live-cell/super-resolution microscopy approach. This demonstrates the relevance of our model for deciphering the nano-architecture of presynapses and understand their molecular functioning.
Tumminia et al. (Thu,) studied this question.