Article Figures & Data
Figures
- Figure 1.
A, Dissociated rat hippocampal neurons grown for 3 weeks on a layer of glia in the 3 mm gold specimen carrier (inset) used for high-pressure freezing. An area of confluence of fine process from adjacent cell bodies on the specimen carrier (black square) is enclosed in corresponding large square on the right. It is in these areas that numerous synapses are found, as shown in B. Living cells on the gold specimen carrier are visualized by reflection microscopy. B, Examples of synapses with presynaptic vesicles, and postsynaptic densities (arrowheads) from neurons grown on gold specimen carrier and freeze-substituted. Many of these synapses are on spines. Scale bar, 1 μm.
- Figure 2.
A, Synapse from section (80 nm thick) through a hippocampal culture prepared with conventional fixation and staining. Structural details in PSD are obscured. B, Sections (200 nm thick) typical of those used for tomography, through a dendritic spine from hippocampal culture prepared by freeze-substitution. Fine structural details are obscured by overlap within the section. C, Virtual sections (1.4 nm thick) derived from a tomographic reconstruction (tomogram) of synapse shown in B. Many fine structural details become apparent within the PSD, including vertical filaments 5–6 nm in diameter. The asterisk is over a synaptic vesicle. Scale bar, 100 nm. D, Rendering of the fine vertical filaments (red) from the tomogram. In the on-edge view of the PSD, it is apparent that the overlap of individual vertical filaments contributes to the typical thickened appearance of a PSD. Immunolabeling suggests that the vertical filaments contain PSD-95 (see text). E, Face-on view of the postsynaptic membrane (yellow) at a magnification similar to that in D, showing the regular distribution of the vertical filaments (red). F, On-edge view of the PSD at higher magnification in which one type of transmembrane structure at the PSD has been rendered in green on its cleft side and in blue on the cytoplasmic side of the post synaptic membrane. The size and distribution of these transmembrane structures is compatible with that of AMPARs and associated proteins. Most AMPAR-like structures are contacted by one vertical filament (red). G, Second type of transmembrane structure, rendered in gold on its cleft side and azure on its larger cytoplasmic side; this type of transmembrane structure is likely to represent NMDAR. NMDAR-type structures are typically contacted by two vertical filaments (red). H, Cross section of PSD slightly tilted away to reveal its cytoplasmic side. Two types of horizontal filament, rendered in purple and white, contact the vertical filaments (red) that, in turn, contact the two types of transmembrane structures (azure and blue). I, Diagram of the relationships of the two types of transmembrane proteins to the vertical filaments and, in turn, to the two types of horizontal filaments. The azure structures represent the cytoplasmic surfaces of NMDAR-like structures, and dark blue structures represent the AMPAR complexes, whereas the red vertical filaments are predominately PSD-95 family members. These filaments connect receptors to an orthogonal, interlinked scaffold at the core of the PSD. Adapted from Chen et al. (2008a) (with permission).
Tables
High-pressure freezing Method for freezing large volumes of tissue that depends on application of high pressures to prevent formation of ice crystals. Freeze-substitution Ice in frozen tissue is replaced with an organic solvent, thereby introducing stabilizing molecules and heavy metal stains into the tissue. Fiducial marker Gold particles applied to sections to aid fine alignment of a set of tilted images. Projection angle Complement of the angle between the specimen plane and the incoming electron beam. Backprojection Reverse of projection to yield a 3D reconstruction, or tomogram, from a set of tilted images. Missing pyramid Blind spot in two axis tomographic series attributable to limitations in tilt angle, leading to a loss of resolution in the final tomogram. Tomogram Computed 3D volume based on backprojections of all images from a tilt series. Virtual section Any individual section computed from a tomogram whose minimum thickness is one 3D pixel, or voxel.
