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.

Maintaining the balance between neuronal excitation and inhibition is essential for proper function of the central nervous system. Inhibitory synaptic transmission plays an important role in maintaining this balance. Although inhibitory transmission has higher kinetic demands compared to excitatory transmission, its properties are poorly understood. In particular, the dynamics and exocytosis of single inhibitory vesicles have not been investigated, due largely to both technical and practical limitations. Using a combination of quantum dots (QDs) conjugated to antibodies against the luminal domain of the vesicular GABA transporter to selectively label GABAergic (i.e., predominantly inhibitory) vesicles together with dual-focus imaging optics, we tracked the real-time three-dimensional position of single GABAergic vesicles up to the moment of exocytosis (i.e., fusion). Using three-dimensional trajectories, we found that GABAergic synaptic vesicles traveled a shorter distance prior to fusion and had a shorter time to fusion compared to synaptotagmin-1 (Syt1)-labeled vesicles, which were mostly from excitatory neurons. Moreover, our analysis revealed that GABAergic synaptic vesicles move more straightly to their release sites than Syt1-labeled vesicles. Finally, we found that GABAergic vesicles have a higher prevalence of kiss-and-run fusion than Syt1-labeled vesicles. These results indicate that inhibitory synaptic vesicles have a unique set of dynamics and exocytosis properties to support rapid synaptic inhibition, thereby maintaining a tightly regulated coordination between excitation and inhibition in the central nervous system.

Excitatory synapses in the mammalian brain exhibit diverse functional properties in transmission and plasticity. Directly visualizing the structural correlates of such functional heterogeneity is often hindered by the diffraction-limited resolution of conventional optical imaging techniques. Here, we used super-resolution stochastic optical reconstruction microscopy (STORM) to resolve structurally distinct excitatory synapses formed on dendritic shafts and spines. The majority of these shaft synapses contained N-methyl-D-aspartate receptors (NMDARs) but not α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), suggesting that they were functionally silent. During development, as more spine synapses formed with increasing sizes and expression of AMPARs and NMDARs, shaft synapses exhibited moderate reduction in density with largely unchanged sizes and receptor expression. Furthermore, upon glycine stimulation to induce chemical long-term potentiation (cLTP), the previously silent shaft synapses became functional shaft synapses by recruiting more AMPARs than did spine synapses. Thus, silent shaft synapse may represent a synaptic state in developing neurons with enhanced capacity of activity-dependent potentiation.

When supramolecular hydrogels are applied as tissue culture scaffolds, their mechanical strength and biocompatibility are the two most important factors that must be considered. However, systematic studies on the structure-mechanical property (or structure-cytotoxicity) relationship of hydrogels are rare. Herein, we rationally designed three hydrogelators and their corresponding phosphate precursors, and systematically studied their self-assembling ability and cytotoxicity. The results indicated that fluorine substitution, but not trifluoromethyl substitution with more fluorine atoms, to the phenylalanine motif enhanced the self-assembling ability and cytotoxicity of the hydrogelators (or precursors). We envision that our preliminary study of hydrogelator fluorination would provide a strategy for the development of supramolecular hydrogels for wider biomedical applications.