Ultrastructural characterization and reconstruction of cultured xenapses

Abstract text (incl. figure legends and references)

In our lab, we have developed a new model to study the neurotransmission process in neurons. Using click-chemistry microstructured glass coverslips were functionalized to grow xenapses - TIRF-amenable presynaptic boutons on the glass surface. The new model system required not only functional but also ultrastructural characterization, and comparison to synapses in conventional neuronal cultures and slices. Given the size of structures of interest, and the resolution required, we chose to perform 3D reconstructions from serial sections of plastic-embedded xenapses.
Mouse neurons, grown on functionalized, carbon-coated coverslips, were fixed by immersion in a mix of 1% glutaraldehyde with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer on ice, additionally post-fixed at room temperature with 1% osmium tetroxide in the same buffer, and en bloc stained with either uranyl or samarium acetate. Samples were dehydrated in an ethanol series and embedded in Epon. The cured Epon blocks had a medium hardness. The region of interest was identified by visual inspection in a light microscope after removal of the glass and then additionally embedded in Epon for stabilization and protection of the sample during sectioning. After trimming the block with a diamond knife to obtain highly polished edges, the serial sections were cut at 40-50 nm and ribbons of 40-50 sections were collected on Formvar-coated slot TEM grids. Imaging was performed with a TEM Philips CM-10 equipped with a bottom-mounted TemCam F-416 camera. Image series were aligned and rendered in Reconstruct. Morphological analysis (size and distribution of synaptic vesicles (SV) within the acquired volume) was performed with MetaMorph software.
Data obtained from EM are in good accordance with our live cell imaging, electrophysiology, and immunofluorescence data and confirmed that xenapses showed all the hallmarks of presynaptic boutons in culture or in brain slices. Xenapses are filled with numerous SVs, analogous in size to SVs in standard cultures; they also harbor other structures typical to presynaptic boutons – like mitochondria, endosomes/cisternae, cytoskeleton elements, multi-vesicular bodies, large dense core and clathrin-coated vesicles. SVs distributed within xenapse's volume may represent the resting and recycling pools of vesicles, whereas the ones at the footprint are analogous to the readily releasable pool of SVs in normal synapses as revealed in experiments with TIRF microscopy.
Taken together our data indicate that the new model shows all structural features of standard presynapses and enables live-cell imaging of single vesicle exo- and endocytosis in space and time.