eSylites: Synthetic Probes for Visualization and Topographic Mapping of Single Excitatory Synapses

The spatiotemporal organization of the postsynaptic density (PSD) is a fundamental determinant of synaptic transmission, information processing, and storage in the brain. The major bottleneck that prevents the direct and precise representation of the nanometer-scaled organization of excitatory glutamatergic synapses is the size of antibodies, nanobodies, and the genetically encoded fluorescent tags. Here, we introduce small, high affinity synthetic probes for simplified, high contrast visualization of excitatory synapses without the limitations of larger biomolecules. In vitro binding quantification together with microscopy-based evaluation identified eSylites, a series of fluorescent bivalent peptides comprising a dye, linker, and sequence composition that show remarkable cellular target selectivity. Applied on primary neurons or brain slices at nanomolar concentrations, eSylites specifically report PSD-95, the key orchestrator of glutamate receptor nanodomains juxtaposed to the presynaptic glutamate release sites that mediate fast synaptic transmission. The eSylite design minimizes a spatial dye offset and thereby enables visualization of PSD-95 with improved localization precision and further time-resolved discrimination. In particular, we find that individual dendritic spines can contain separate nanodomains enriched for either PSD-95 or its closest homologues, PSD-93 or SAP102. Collectively, these data establish eSylites as a broadly applicable tool for simplified excitatory synapse visualization, as well as a high-end microscopy compatible probe for resolving the PSD organization with unprecedented resolution.