Unique dynamics and exocytosis properties of GABAergic synaptic vesicles revealed by three-dimensional single vesicle tracking Chungwon Parka, Xingxiang Chena, Chong-Li Tianb,c, Gyu Nam Parkd,e, Nicolas Chenouardf, Hunki Leea,g, Xin Yi Yeoh,i, Sangyong Jungh,j, Richard W. Tsienk,1, Guo-Qiang Bib,c, and Hyokeun Parka,l,m,1 aDivision of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, 999077 Kowloon, Hong Kong; bSchool of Life Sciences, University of Science and Technology of China, Hefei, 230052 Anhui, China; cChinese Academy of Sciences, Key Laboratory of Brain Function and Disease, University of Science and Technology of China, Hefei, 230052 Anhui, China; dDepartment of Chemistry, Chung-Ang University, 06974 Seoul, Korea; eCreative Research Initiative Center for Chemical Dynamics in Living Cells, Chung-Ang University, 06974 Seoul, Korea; fCNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, University of Bordeaux, F-33000 Bordeaux, France; gInstitute of Medical Physics and Biophysics, University of Münster, 48149 Münster, Germany; hSingapore Bioimaging Consortium, Agency for Science, Technology, and Research, 138667 Singapore; iDepartment of Psychological Medicine, Yong Loo Lin School of Medicine, National University of Singapore, 117597 Singapore; jDepartment of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 117597 Singapore; kDepartment of Neuroscience and Physiology, New York University Neuroscience Institute, New York University, New York, NY 10016; lDepartment of Physics, The Hong Kong University of Science and Technology, Clear Water Bay, 999077 Kowloon, Hong Kong; and mState Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay, 999077 Kowloon, Hong Kong Contributed by Richard W. Tsien, December 8, 2020 (sent for review October 26, 2020; reviewed by Ege T. Kavalali and Yulong Li) Maintaining the balance between neuronal excitation and inhibi- synchronization of the neuronal network, thereby regulating tion is essential for proper function of the central nervous system. neuronal excitability (8, 9). Thus, maintaining a tightly regulated Inhibitory synaptic transmission plays an important role in main- coordination between excitatory and inhibitory neurotransmis- taining this balance. Although inhibitory transmission has higher sion is essential for proper brain function. kinetic demands compared to excitatory transmission, its proper- An extensive analysis of the components and molecular events ties are poorly understood. In particular, the dynamics and exocy- involved in vesicle fusion and neurotransmitter release has yielded tosis of single inhibitory vesicles have not been investigated, due general models describing the organization and functional NEUROSCIENCE largely to both technical and practical limitations. Using a combi- properties of both presynaptic and postsynaptic components nation of quantum dots (QDs) conjugated to antibodies against (10–12). On the presynaptic side, a transient increase in local the luminal domain of the vesicular GABA transporter to selec- Ca2+ concentration due to activation of voltage-gated Ca2+ chan- tively label GABAergic (i.e., predominantly inhibitory) vesicles nels triggers the localized buckling of the plasma membrane via a together with dual-focus imaging optics, we tracked the real- direct interaction between the C2B domain in the protein time three-dimensional position of single GABAergic vesicles up synaptotagmin-1 (Syt1) and lipids in the membrane (13–15). This to the moment of exocytosis (i.e., fusion). Using three-dimensional leads to the synchronous fusion between the synaptic vesicles and the trajectories, we found that GABAergic synaptic vesicles traveled a plasma membrane and release of the vesicle’s contents into the syn- shorter distance prior to fusion and had a shorter time to fusion aptic cleft (16), enabling excitatory and inhibitory neurotransmitters to 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 Significance release sites than Syt1-labeled vesicles. Finally, we found that GABAergic vesicles have a higher prevalence of kiss-and-run fu- Despite playing an important role in maintaining brain func- sion than Syt1-labeled vesicles. These results indicate that inhibi- tion, the dynamics and exocytosis of inhibitory synaptic vesi- tory synaptic vesicles have a unique set of dynamics and exocytosis cles are poorly understood. Here, we tracked the three- properties to support rapid synaptic inhibition, thereby maintain- dimensional position of single GABAergic vesicles up to the ing a tightly regulated coordination between excitation and inhi- moment of exocytosis in real time by loading single GABAergic bition in the central nervous system. vesicles with quantum dots conjugated to antibodies against the luminal domain of the vesicular GABA transporter. We synaptic vesicles | dynamics | exocytosis | three-dimensional tracking | found that GABAergic synaptic vesicles have a smaller total inhibitory synaptic transmission travel length before fusion, a shorter fusion latency, and a higher prevalence of kiss-and-run fusion than synaptotagmin- 1–labeled vesicles. Our findings provide evidence that inhibi- eurons communicate with other neurons by releasing neu- tory vesicles have a unique set of dynamics and exocytosis rotransmitters from their presynaptic terminals via the exocy- N properties to support rapid inhibitory synaptic transmission. tosis (i.e., fusion) of synaptic vesicles at the presynaptic membrane, subsequently activating postsynaptic receptors to mediate down- Author contributions: C.P. and H.P. designed research; C.P., X.C., C.-L.T., X.Y.Y., and S.J. stream effects (1–4). Synapses in the central nervous system can performed research; C.P. and H.P. contributed new reagents/analytic tools; C.P., X.C., be broadly classified as either excitatory or inhibitory, depending C.-L.T., G.N.P., N.C., and H.L. analyzed data; C.P., X.C., N.C., X.Y.Y., S.J., R.W.T., G.-Q.B., and on the type of neurotransmitters that they release and the effects H.P. wrote the paper; and S.J., G.-Q.B., and H.P. supervised researching graduate students. Reviewers: E.T.K., Vanderbilt University; and Y.L., Peking-Tsinghua Center for of those neurotransmitters. While excitatory synapses cause the Life Sciences. generation, propagation, and potentiation of neuronal responses The authors declare no competing interest. for processing information (5), inhibitory synapses play an es- Published under the PNAS license. sential role in feedback and feedforward inhibition in order to 1To whom correspondence may be addressed. Email: [email protected] or control neural excitability (6) and in the control of brain rhythms [email protected]. (7). In the central nervous system, inhibitory synaptic transmis- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ sion is mediated primarily by release of the neurotransmitter doi:10.1073/pnas.2022133118/-/DCSupplemental. GABA and serves to coordinate the pattern of excitation and the Published February 23, 2021. PNAS 2021 Vol. 118 No. 9 e2022133118 https://doi.org/10.1073/pnas.2022133118 | 1of10 Downloaded by guest on September 24, 2021 diffuse to and act upon postsynaptic glutamate and GABA receptors, with VGAT–CypHer5E were used for studying GABAergic respectively. vesicle dynamics and exocytosis. Importantly, our general understanding of neurotransmitter In our experiments, exocytosis of a QD-loaded vesicle is release stems from studying excitatory neurotransmission and is reflected by a sudden, irreversible drop in QD fluorescence currently unable to adequately explain the distinct features as- caused by quenching with extracellular Trypan blue (23). To sociated with inhibitory synaptic transmission. For example, the measure the position of single QD-loaded GABAergic synaptic size of the readily releasable pool (RRP) of synaptic vesicles in vesicles in the x-y plane, we used fluorescence imaging with one- striatal inhibitory GABAergic neurons—probed by a hypertonic nanometer accuracy (FIONA) (23, 25, 26); the vesicle’s z-position sucrose solution—is three times larger than the RRP in excit- was localized using dual-focus imaging (23, 27). Fig. 1C shows the atory hippocampal glutamatergic neurons (17). Furthermore, real-time three-dimensional position of a single QD-loaded inhibitory neurons have both higher average vesicular release GABAergic synaptic vesicle in a cultured neuron stimulated at x y probability (Pr) and more release sites compared to excitatory 10 Hz up to the time of exocytosis; the - plane projection is neurons (17–19). These results suggest that quantitative differ- overlaid on the fluorescence image of a GABAergic presynaptic ences exist between inhibitory and excitatory synapses with re- terminal labeled with VGAT–CypHer5E, showing that the spect to vesicle dynamics, release, and recycling. In addition, GABAergic vesicle remained near the edge of the presynaptic disproportionate effects between inhibitory and excitatory syn- terminal before undergoing exocytosis. Fluorescence images of apses following knockout of some or all synapsin genes provide a the QD-loaded GABAergic vesicle just before and after exocy- molecular perspective on the putative differences between these tosis show near-complete, irreversible quenching