Transsynaptic Assemblies Link Domains of Presynaptic and Postsynaptic Intracellular Structures across the Synaptic Cleft.

The chemical synapse is a complex machine separated into three parts: presynaptic, postsynaptic, and cleft. Super-resolution light microscopy has revealed alignment of presynaptic vesicle release machinery and postsynaptic neurotransmitter-receptors and scaffolding components in synapse spanning nanocolumns. Cryo-electron tomography confirmed that postsynaptic glutamate receptor-like structures align with presynaptic structures in proximity to synaptic vesicles into transsynaptic assemblies. In our electron tomographic renderings, nearly all transcleft structures visibly connect to intracellular structures through transmembrane structures to form transsynaptic assemblies, potentially providing a structural basis for transsynaptic alignment. Here, we describe the patterns of composition, distribution, and interactions of all assemblies spanning the synapse by producing three-dimensional renderings of all visibly connected structures in excitatory and inhibitory synapses in dissociated rat hippocampal neuronal cultures of both sexes prepared by high-pressure freezing and freeze-substitution. The majority of transcleft structures connect to material in both presynaptic and postsynaptic compartments. We found several instances of assemblies connecting to both synaptic vesicles and postsynaptic density scaffolding. Each excitatory synaptic vesicle within 30 nm of the active zone contacts one or more assembly. Further, intracellular structures were often shared between assemblies, entangling them to form larger complexes or association domains, often in small clusters of vesicles. Our findings suggest that transsynaptic assemblies physically connect the three compartments, allow for coordinated molecular organization, and may combine to form specialized functional association domains, resembling the light-level nanocolumns.SIGNIFICANCE STATEMENT A recent tomographic study uncovered that receptor-like cleft structures align across the synapse. These aligned structures were designated as transsynaptic assemblies and demonstrate the coordinated organization of synaptic transmission molecules between compartments. Our present tomographic study expands on the definition of transsynaptic assemblies by analyzing the three-dimensional distribution and connectivity of all cleft-spanning structures and their connected intracellular structures. While one-to-one component alignment occurs across the synapse, we find that many assemblies share components, leading to a complex entanglement of assemblies, typically around clusters of synaptic vesicles. Transsynaptic assemblies appear to form domains which may be the structural basis for alignment of molecular nanodomains into synapse spanning nanocolumns described by super-resolution light microscopy.

A Network of Three Types of Filaments Organizes Synaptic Vesicles for Storage, Mobilization, and Docking.

Synaptic transmission between neurons requires precise management of synaptic vesicles. While individual molecular components of the presynaptic terminal are well known, exactly how the molecules are organized into a molecular machine serving the storage and mobilization of synaptic vesicles to the active zone remains unclear. Here we report three filament types associated with synaptic vesicles in glutamatergic synapses revealed by electron microscope tomography in unstimulated, dissociated rat hippocampal neurons. One filament type, likely corresponding to the SNAREpin complex, extends from the active zone membrane and surrounds docked vesicles. A second filament type contacts all vesicles throughout the active zone and pairs vesicles together. On the third filament type, vesicles attach to side branches extending from the long filament core and form vesicle clusters that are distributed throughout the vesicle cloud and along the active zone membrane. Detailed analysis of presynaptic structure reveals how each of the three filament types interacts with synaptic vesicles, providing a means to traffic reserved and recycled vesicles from the cloud of vesicles into the docking position at the active zone. SIGNIFICANCE STATEMENT: The formation and release of synaptic vesicles has been extensively investigated. Explanations of the release of synaptic vesicles generally begin with the movement of vesicles from the cloud into the synaptic active zone. However, the presynaptic terminal is filled with filamentous material that would appear to limit vesicular diffusion. Here, we provide a systematic description of three filament types connecting synaptic vesicles. A picture emerges illustrating how the cooperative attachment and release of these three filament types facilitate the movement of vesicles to the active zone to become docked in preparation for release.

Electron microscopic tomography reveals discrete transcleft elements at excitatory and inhibitory synapses.

Electron microscopy has revealed an abundance of material in the clefts of synapses in the mammalian brain, and the biochemical and functional characteristics of proteins occupying synaptic clefts are well documented. However, the detailed spatial organization of the proteins in the synaptic clefts remains unclear. Electron microscope tomography provides a way to delineate and map the proteins spanning the synaptic cleft because freeze substitution preserves molecular details with sufficient contrast to visualize individual cleft proteins. Segmentation and rendering of electron dense material connected across the cleft reveals discrete structural elements that are readily classified into five types at excitatory synapses and four types at inhibitory synapses. Some transcleft elements resemble shapes and sizes of known proteins and could represent single dimers traversing the cleft. Some of the types of cleft elements at inhibitory synapses roughly matched the structure and proportional frequency of cleft elements at excitatory synapses, but the patterns of deployments in the cleft are quite different. Transcleft elements at excitatory synapses were often evenly dispersed in clefts of uniform (18 nm) width but some types show preference for the center or edges of the cleft. Transcleft elements at inhibitory synapses typically were confined to a peripheral region of the cleft where it narrowed to only 6 nm wide. Transcleft elements in both excitatory and inhibitory synapses typically avoid places where synaptic vesicles attach to the presynaptic membrane. These results illustrate that elements spanning synaptic clefts at excitatory and inhibitory synapses consist of distinct structures arranged by type in a specific but different manner at excitatory and inhibitory synapses.

Co-segregation of AMPA receptors with G(M1) ganglioside in synaptosomal membrane subfractions.

Biochemical studies have suggested that certain synaptic proteins associate with lipid rafts to perform key functions within the synapse. However, variability in biochemical preparations raises questions as to which synaptic proteins actually associate with lipid rafts. In the present study, we use both electron microscopy and biochemistry to investigate AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor localization in synaptic membrane subfractions prepared in two different ways, by Triton X-100 detergent treatment or without detergent by sonication at high pH. Immunogold electron microscopy shows that a detergent-resistant synaptosomal membrane subfraction consists of empty vesicles 0.1-1.0 microm in diameter. A subpopulation of these vesicles labelled for glycosphingolipid GM1 ganglioside, a marker of lipid rafts, and 46% of the labelled vesicles also labelled for the AMPA receptor subunit GluR2. This co-segregation into specific vesicles does not depend on effects of detergent because a similar distribution of label was found in vesicles isolated without the use of detergent. Our results suggest that AMPA receptors localize within specific regions of synaptic membranes rich in GM1 ganglioside.