Induction of Hippocampal Synapses on Functionalized Micropatterns

We developed a novel presynaptic neuronal preparation in culture. Using microstructured coverslips functionalized with protein domains of synaptic cell adhesion molecules (SCAMs) as artificial postsynapse we are able to induce hippocampal neurons to form hybrid synapses, i.e. purely presynaptic structures ‘en face’ directly onto the coverslip.Axons growing over such micropatterns, functionalized with SCAM domains by click chemistry, form large flat varicosities. 4Pi microscopy revealed the presence of several presynaptic markers like the synaptic vesicle protein Synaptophysin1 or the active zone scaffold proteins RIM1/2, while postsynaptic staining against e.g. PSD 95, HOMER and the AMPA receptor was absent. Serial section transmission electron microscopy (TEM) as well as focused ion beam scanning electron microscopy (FIB-SEM) confirmed that these varicosities harbor a few hundred synaptic vesicles in several clusters near and at the bottom membrane and show typical active zone hallmarks at the bottom membrane. We found that isolated presynaptic varicosities are maintained and stable for at least two weeks allowing transfection and expression of fluorescent probes such as fusion constructs of the pH sensitive pHluorin protein. PHluorin is quenched at the acidic pH within vesicles and becomes fully fluorescent upon fusion. Neurons transfected with synaptophysin-pHluorin could be imaged after ten to 14 days in vitro by total internal reflection fluorescence microscopy (TIRFM). Where varicosities were formed on the functionalized micropatterns, we observed single fusion events, visible as diffraction-limited spots, on stimulation with single action potentials.Thus, this novel purely presynaptic preparation allows us to monitor the dynamics of presynaptic exo- and endocytosis in unprecedented detail.