Three-dimensional architecture of presynaptic terminal cytomatrix

Abstract

Presynaptic terminals are specialized for mediating rapid fusion of synaptic vesicles (SVs) after calcium influx. The regulated trafficking of SVs likely results from a highly organized cytomatrix. How this cytomatrix links SVs, maintains them near the active zones (AZs) of release, and organizes docked SVs at the release sites is not fully understood. To analyze the three-dimensional (3D) architecture of the presynaptic cytomatrix, electron tomography of presynaptic terminals contacting spines was performed in the stratum radiatum of the rat hippocampal CA1 area. To preserve the cytomatrix, hippocampal slices were immobilized using high-pressure freezing, followed by cryosubstitution and embedding. SVs are surrounded by a dense network of filaments. A given vesicle is connected to approximately 1.5 neighboring ones. SVs at the periphery of this network are also linked to the plasma membrane, by longer filaments. More of these filaments are found at the AZ. At the AZ, docked SVs are grouped around presynaptic densities. Filaments with adjacent SVs emerge from these densities. Immunogold localizations revealed that synapsin is located in the presynaptic bouton, whereas Bassoon and CAST (ERC2) are at focal points next to the AZ. In synapsin triple knock-out mice, the number of SVs is reduced by 63%, but the size of the boutons is reduced by only 18%, and the mean distance of SVs to the AZ is unchanged. This 3D analysis reveals the morphological constraints exerted by the presynaptic molecular scaffold. SVs are tightly interconnected in the axonal bouton, and this network is preferentially connected to the AZ.

Figure 1.

Morphology of presynaptic terminals on dendritic spines after HPF. A, Ultrathin 70-nm-thick section. Note the presence of docked SVs (black arrow) and groups of SVs in the axonal terminal (asterisk). Filaments are observed between SVs (white arrowhead). B, C, Virtual sections from the tomographic reconstruction of a synapse. The lipid bilayer of the membranes, the docked SVs (black arrow), and the filaments between the SVs (white arrowhead) are clearly visible. Note the symmetrically arranged filaments on the presynaptic and the postsynaptic sides (black arrowheads). Scale bars, 200 nm.

Figure 2.

Network of filaments linking SVs in the presynaptic terminal. A, The electron-dense materials surrounding the SVs (yellow) are superimposed on a virtual section. B, 3D reconstruction emphasizing that docked (blue) and other (gold) SVs are embedded in a tight meshwork of filaments (pink). C, Higher magnification of a partial reconstruction exemplifying the connections (pink) between neighboring SVs. Scale bar, 200 nm.

Figure 3.

Filaments linking the SVs to the presynaptic membrane. A–E, Example of virtual sections through the same bouton. A, B, Low-power view with SVs either docked (arrow) or at distance (crossed arrow). Filaments interconnect SVs together (white arrowhead) or with the plasma membrane (black arrowhead). Note the short strands (white arrow) between a docked vesicle (black arrow) and the presynaptic membrane. C–E, Sequence of virtual sections (one of six serial sections) establishing the continuity of a filament (arrowheads) linking a vesicle (crossed arrow) to the presynaptic membrane. F–H, Example of a filament contacting several SVs in virtual sections through another bouton. Five SVs are in contact with a filament (arrowheads). I, 3D reconstruction of the filaments (pink) and the adjacent SVs. The SVs numbered in I are those in F–H. Scale bars: A, B, 200 nm; C–H, 100 nm.

Figure 4.

Distribution of the filaments linking SVs to the presynaptic membrane. A–F, 3D reconstruction of the filaments (pink) linking SVs (gold) to the plasma membrane (light pink). Docked SVs are in blue. The limits (arrows) of the synaptic junction are defined by the extension of the cleft (dark blue). B is the same synapse as in A, at an intermediate stage of the reconstruction superimposed on a virtual section. C, D, Note that the filaments are more (C) or less (D) concentrated at the AZ. E, F, Examples of other synapses after removal of the plasma membrane. Scale bar: 200 nm.

Figure 5.

Quantification of the distribution and length of filaments linking the SVs together or to the plasma membrane. A, Distribution of the filaments linking SVs to the plasma membrane. B, Filaments linking SVs to the plasma membrane are longer than those between SVs (mean ± SEM). C, Distribution of the length of the filaments. Black bars, Filaments linking the SVs to the presynaptic membrane in front of the AZ; gray bars, filaments linking the SVs to the perisynaptic membrane; white bars, filaments linking the SVs together in the bouton.

Figure 6.

Distribution of the SVs. A, For 3D rendering, SVs were spotted on individual virtual sections constituting the tomogram. Docked SVs are in purple, and the other ones are in gold. The extension of the synaptic contact is defined by the PSD (green). B, En face view of a synapse from an ultrathin section. The docked SVs (arrows) are not homogeneously distributed at the AZ. C, 3D reconstructions from tomograms of the docked SVs in relation to the PSD. The synapses are ordered according to the number of docked SVs. D, Absence of correlation between the number of docked SVs and the total number of SVs analyzed in 14 synapses. E, Correlation between the number of docked SVs and the size of the PSD in 18 synapses. Scale bar, 200 nm.

Figure 7.

Association of docked SVs with presynaptic densities. A–F, Six virtual sections from a tomogram. The sections are separated by ∼19 nm in the z-axis. The docked SVs (numbered 1–4) are apposed to a presynaptic electron-dense material (asterisk). A filament (arrowhead) emerging from the electron-dense material contacts an SV. G–I, 3D reconstruction of the docked SVs (blue) and the presynaptic electron-dense material (yellow) in front of the PSD (green). G is the same synapse as in A–F. H and I are two other examples. Scale bars: A–F, 50 nm.

Figure 8.

Postembedding immunocytochemistry of presynaptic proteins on ultrathin sections. The immunogold particles are circled in red. The insets show the gold particles. A, B, Synaptophysin. C, D, Synapsin. E, F, Bassoon. G, H, CAST. Histograms, for the indicated antigens, of the percentage of gold particles as a function of their distance to the membrane (bin, 20 nm) are shown. The arrow indicates the position of the presynaptic membrane. n is the number of gold particles analyzed. Scale bars, 200 nm.

Figure 9.

Morphology of presynaptic terminals in synapsin TKO mice. A, B, Ultrathin sections of control (A) and TKO (B) mice. C, The number of SVs is reduced in TKO mice (mean ± SEM; p < 0.01, Student's t test). D, The area of presynaptic profiles is reduced in TKO mice (p < 0.05). E, The mean distance of SVs from the AZ is not changed. F, G, Distribution of SVs spotted on tomograms from sections of control (F) or TKO (G) mice. Scale bars, 200 nm.