Structure of Xenapses - Pure Presynaptic Boutons Induced on Micropatterned Host Substrates

Maintaining of synaptic transmission requires segregation of exo- and endocytic zones. In order to visualize functionally distinct membrane nanodomains by high resolution microscopy we grew hippocampal neurons on microstructured glass coverslips, functionalized with the synaptic cell adhesion protein Neuroligin 1. This triggers formation of presynaptic sites on the microstructured host substrate, which we thus call ‘xenapses’. Ultrastructural analysis of reconstructed serial TEM sections revealed that xenapses are formed exclusively by axons, contain many hundreds of synaptic vesicles, and several mitochondria. At the footprint facing the coverslip tens of physically docked vesicles are found, many of which appear in clusters. Our conditions facilitate growth of both excitatory and inhibitory synapses. Experiments with calcium sensors, FM dyes and endogenously expressed pHluorin constructs have shown, that these xenapses respond to stimulation and are functionally normal. TIRF-dSTORM of various CAZ (cytomatrix of active zone) proteins allows to investigate distribution and interaction of proteins during presynapse formation and function. Xenapses form 5 to 15 active zones, identified by the CAZ protein Bassoon, with diameters of 0.4 - 0.9 µm towards the coverslip, which correlates well with the distribution of docked vesicles seen in EM. Clathrin predominantly localizes to periactive zones, in line with distribution of clathrin-coated structures in EM. Syntaxin is associated with active zones, but not co-localized with Bassoon. Mature (>14 DIV) Neuroligin-induced xenapses lack another CAZ-protein - Piccolo, while in xenapses 3 DIV Piccolo is present and co-localized with Bassoon.We conclude that xenapses allow us to study presynapse formation, organization and maintenance under controlled conditions and in unprecedented detail.