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

Abstract

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 intotranssynaptic 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 3D 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 pre- 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 orassociation 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.SIGNIFICANCEA recent tomographic study uncovered that receptor-like cleft structures align across the synapse. These aligned structures were designated astranssynaptic assembliesand demonstrate the coordinated organization of synaptic transmission molecules between compartments. Our present tomographic study expands upon the definition of transsynaptic assemblies by analyzing the 3D 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.

Cryo-electron tomographic investigation of native hippocampal glutamatergic synapses

“…Subsequent electron microscopy studies, using both cultured neurons and brain tissue-derived samples, together with imaging studies of biochemically isolated synaptosomes, have provided progressively higher resolution visualizations of synapses and their surrounding structures [6][7][8]. More recently, electron microscopy experiments, together with light microscopy studies, suggest that in excitatory, glutamatergic synapses, there is a transsynaptic organization of presynaptic and postsynaptic components that aligns sites of transmitter release and detection [9][10][11][12][13].…”

Activity-dependent diffusion trapping of AMPA receptors as a key step for expression of early LTP

“…This study identified the existence of trans-synaptic nanocolumns, which co-organize the pre-synaptic release machinery in register with post-synaptic AMPARs and improve the efficiency of synaptic transmission [87,88] (figure 1b,c). Interestingly, the recent developments in cryo-electron tomography have allowed the ultrastructural visualization of these protein nanodomains and trans-synaptic assemblies [102][103][104][105][106][107][108]. The burning question that thus arose was: what are the underlying molecular elements that bridge pre-and post-synaptic nanodomains?…”

Modification of the synaptic cleft under excitatory conditions

“…The extracellular space between the pre- and postsynaptic compartments constitutes the synaptic cleft, considered to be the third compartment of the synapse. By electron microscopy (EM), the synaptic cleft is observed to contain electron dense proteinaceous material ( Lucic et al, 2005 ; Zuber et al, 2005 ; Burette et al, 2012 ; High et al, 2015 ; Cole and Reese, 2023 ), including cell adhesion molecules, extracellular proteins and extracellular moieties of various receptors and ion channels ( Missler et al, 2012 ; Loh et al, 2016 ; Cijsouw et al, 2018 ). Trans-synaptic bridges formed through association of cell adhesion molecules from opposite sides of the junction maintain a space of mostly regular width and define the synaptic contact area.…”