Inhibitory synaptic vesicles have unique dynamics and exocytosis properties

Maintaining the balance between neuronal excitation and inhibition is essential for proper function of the central nervous system, with inhibitory synaptic transmission playing an important role. 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 (VGAT) to selectively label GABAergic (i.e., inhibitory) vesicles together with dual-focus imaging optics, we tracked the real-time three-dimensional position of single inhibitory vesicles up to the moment of exocytosis (i.e., fusion). Using three-dimensional trajectories, we found that inhibitory synaptic vesicles traveled a short 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 a close correlation between the release probability of inhibitory vesicles and both the proximity to their fusion site and the total travel length. Finally, we found that inhibitory vesicles have a higher prevalence of kiss-and-run fusion compared than Syt1-labeled vesicles. These results indicate that inhibitory synaptic vesicles have a unique set of dynamics and fusion properties to support rapid synaptic inhibition, thereby maintaining a tightly regulated balance between excitation and inhibition in the central nervous system. SignificanceDespite playing an important role in maintaining brain function, the dynamics of inhibitory synaptic vesicles are poorly understood. Here, we tracked the three-dimensional position of single inhibitory vesicles up to the moment of exocytosis in real time by loading single inhibitory vesicle with QDs-conjugated to antibodies against the luminal domain of the vesicular GABA transporter (VGAT). We found that inhibitory synaptic vesicles have a smaller total travel length before fusion, a shorter fusion time, and a higher prevalence of kiss-and-run than synaptotagmin-1-lableled vesicles. Our findings provide the first evidence that inhibitory vesicles have a unique set of dynamics and exocytosis properties to support rapid inhibitory synaptic transmission.