Differentiation and Characterization of Excitatory and Inhibitory Synapses by Cryo-electron Tomography and Correlative Microscopy

Abstract

As key functional units in neural circuits, different types of neuronal synapses play distinct roles in brain information processing, learning, and memory. Synaptic abnormalities are believed to underlie various neurological and psychiatric disorders. Here, by combining cryo-electron tomography and cryo-correlative light and electron microscopy, we distinguished intact excitatory and inhibitory synapses of cultured hippocampal neurons, and visualized the in situ 3D organization of synaptic organelles and macromolecules in their native state. Quantitative analyses of >100 synaptic tomograms reveal that excitatory synapses contain a mesh-like postsynaptic density (PSD) with thickness ranging from 20 to 50 nm. In contrast, the PSD in inhibitory synapses assumes a thin sheet-like structure ∼12 nm from the postsynaptic membrane. On the presynaptic side, spherical synaptic vesicles (SVs) of 25–60 nm diameter and discus-shaped ellipsoidal SVs of various sizes coexist in both synaptic types, with more ellipsoidal ones in inhibitory synapses. High-resolution tomograms obtained using a Volta phase plate and electron filtering and counting reveal glutamate receptor-like and GABAA receptor-like structures that interact with putative scaffolding and adhesion molecules, reflecting details of receptor anchoring and PSD organization. These results provide an updated view of the ultrastructure of excitatory and inhibitory synapses, and demonstrate the potential of our approach to gain insight into the organizational principles of cellular architecture underlying distinct synaptic functions.SIGNIFICANCE STATEMENT To understand functional properties of neuronal synapses, it is desirable to analyze their structure at molecular resolution. We have developed an integrative approach combining cryo-electron tomography and correlative fluorescence microscopy to visualize 3D ultrastructural features of intact excitatory and inhibitory synapses in their native state. Our approach shows that inhibitory synapses contain uniform thin sheet-like postsynaptic densities (PSDs), while excitatory synapses contain previously known mesh-like PSDs. We discovered “discus-shaped” ellipsoidal synaptic vesicles, and their distributions along with regular spherical vesicles in synaptic types are characterized. High-resolution tomograms further allowed identification of putative neurotransmitter receptors and their heterogeneous interaction with synaptic scaffolding proteins. The specificity and resolution of our approach enables precise in situ analysis of ultrastructural organization underlying distinct synaptic functions.

Mesophasic organization of GABAAreceptors in hippocampal inhibitory synapse

“…This unique property suggests that the interaction among gephyrin molecules and associated proteins are also semi-ordered, forming flexible networks. Importantly, this network is confined to a two-dimensional sheet parallel to the postsynaptic membrane 7 , probably by the interaction of gephyrin with membrane-bound GABA A Rs. Such a mesophasic assembly exhibits both variability and regularity, demonstrating how ensembles of synaptic molecules acquire great complexity via self-organization.…”

“…We did not observe significant difference among different groups in basic properties of receptor expression and organization and thus pooled the data together for all analyses. For cryo correlative light and electron microscopy (cryoCLEM) experiments, cultures were transfected with mCherry-gephyrin (a gift from Dr. Ann Marie Craig) using Lentivirus at 10 DIV, as described previously 7 . All cultures were used for cryo-electron tomography (cryoET) imaging at 16 days in vitro (DIV).…”

On the cause of sleep: Protein fragments, the concept of sentinels, and links to epilepsy

“…PSDs are located underneath synapse-encompassing patches of the plasma membrane in dendritic spines of postsynaptic neurons. PSDs are rich in scaffold/cytoskeletal proteins (72)(73)(74)(75)(76)(77)(78)(79)(80)(81). Specific scaffold proteins bind to cytosolic domains of glutamate receptors such as AMPAR, NMDAR, and mGluR1, transiently trapping these and other transmembrane proteins within a PSD (80).…”

Auxiliary subunits keep AMPA receptors compact during activation and desensitization

“…The crowded and narrow synaptic cleft is scarcely wider than the receptors are tall 46 themselves and has narrow edges (Zuber et al, 2005;Tao et al, 2018). This 47 observation implies that conformational dynamics of the receptor domains and their 48 relation to synapse dimensions and molecular composition has implications in both 49 health and disease.…”

“…However, 643 long exposures to agonists are at odds with synaptic conditions where AMPA 644 receptors see glutamate on a millisecond timescale, before it is actively cleared by 645 transporters (Clements, 1996). Furthermore, any large structural rearrangements of 646 the extracellular domains would need to be accommodated by the crowded synaptic 647 environment (High et al, 2015;Tao et al, 2018) and potential presynaptic interaction 648 partners (Saglietti et al, 2007) (Elegheert et al, 2016). When in complex with auxiliary 649 subunits, no functional state of AMPA receptors necessitates large domain 650 movements, and compact arrangements of the extracellular layer can sustain the gating process.…”