Fig. 1: Cryo-CLEM and cryo-ET of cortico-striatal and dopaminergic synaptosomes.
A Left, schematic of the expected organization between DA varicosities (magenta) and GLU synapses (green), forming DHSs. Right, fluorescence of a sagittal slice of VGLUT1venus*DAT-Cre*Ai14tdTomato mouse. CS synapses and DA projections generate intense fluorescence signals in the striatum (Str, dotted line). Hpc Hippocampus, SN Substancia Nigra; Scale bar: 3 mm. B Diagram of synaptosome preparation from fresh striatum obtained after differential centrifugations. The fractions (H, S1, P2, B, see Methods) are increasingly enriched in synaptosomes. C A suspension of synaptosomes mixed with fluorescent and gold fiducial beads is layered on the grid, blotted and plunge-frozen in liquid ethane. D Example of a cryo-fluorescence microscopy image of a single grid square from a VGLUT1venus x DAT-Cre x Ai14tdTomato with four channels: brightfield, fluorescence of tdTomato, Venus and blue fiducial beads. These images were used to select synaptosomes of interest, green (VGLUT1venus) or magenta (DAT+), next to several blue beads, for observation with electron microscopy. Scale bar 10 µm. E Left cryo-EM image of the same square as displayed in (D). Scale bar 10 µm. Right, subset of cryo-fluorescence (top) and EM (bottom) of the same portion of the grid with three fiducial blue beads visible in both modalities (yellow circles) for fine registration. Scale bar: 3 µm. Synaptosomes with clearly visible beads and good ice quality were further imaged with high-magnification tilted series. F Reconstructed tomograms of fluorescent synaptosomes obtained by cryo-electron tomography. Left, overlay of fluorescence and brightfield channels showing tagged synaptosomes of interest and fiducial beads. Scale bars: 5 µm. Right, representative single tomographic slice of 1.558 nm of thickness showing a clear structure outline and intracellular organelles. Magenta or green cross marks point to the registered center of the fluorescent tag (GLU or DA synaptosome), Scale bars: 500 nm. G 3D models from the synaptosomes displayed in (F) were obtained by segmentation of membranes. The display of 3D models are as follows: plasma membrane of synaptosomes in green for GLU or magenta for DA; PSE in gray; active zone (AZ) in red; Small (synaptic, <80 nm) vesicles in light blue; large endosome-like organelles (>80 nm) in yellow; elongated endoplasmic-reticulum-like structures in orange; mitochondrion in pink; multivesicular bodies in dark blue (see Fig. 2). For both GLU synaptosomes, a clear synaptic cleft was detected, with either a closed post-synaptic element (PSE) in gray (top), or an opened post-synaptic membrane (bottom). Both of them exhibit a post-synaptic density. The presynaptic GLU active zones are shown in red. DA synaptosomes originate from 16 different preparations, GLU synaptosomes from 14 preparations, and DHS from 10 preparations, see Table S1. Scale bar: 500 nm.
