Synaptic vesicles transiently dock to refill release sites

Abstract

Synaptic vesicles fuse with the plasma membrane to release neurotransmitter following an action potential, after which new vesicles must ‘dock’ to refill vacated release sites. To capture synaptic vesicle exocytosis at cultured mouse hippocampal synapses, we induced single action potentials by electrical field stimulation, then subjected neurons to high-pressure freezing to examine their morphology by electron microscopy. During synchronous release, multiple vesicles can fuse at a single active zone. Fusions during synchronous release are distributed throughout the active zone, whereas fusions during asynchronous release are biased toward the center of the active zone. After stimulation, the total number of docked vesicles across all synapses decreases by ~40%. Within 14 ms, new vesicles are recruited and fully replenish the docked pool, but this docking is transient and they either undock or fuse within 100 ms. These results demonstrate that the recruitment of synaptic vesicles to release sites is rapid and reversible.

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Fig. 1: Zap-and-freeze captures synaptic vesicle fusion.
Fig. 2: Multiple fusion events at single active zones after a single action potential.
Fig. 3: Vesicle fusion during the first 14 ms after an action potential.
Fig. 4: Fusions captured at 5 and 11 ms after an action potential represent synchronous and asynchronous release.
Fig. 5: Transient docking refills the docked vesicle pool within milliseconds.

Data availability

Full data tables underlying the figures are available at https://figshare.com/authors/Shigeki_Watanabe/910686 and in the source data. Raw images and image analysis files are available upon request. Source data are provided with this paper.

Code availability

Custom R, Matlab and Fiji scripts are available at https://github.com/shigekiwatanabe/SynapsEM and are the subject of a manuscript currently in preparation.

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Acknowledgements

We are indebted to S. Li, Q. Gan, K. Itoh, D. Lubsanjav, C. Zhang and S. Markert for help with cell culture and freezing and for stimulating discussions. We also thank M. Delanoy and B. Smith for technical assistance with electron microscopy and K. T. DiNapoli for developing R code to randomize images. We thank P. Wurzinger and C. Tomova at Leica for the design and manufacture of the middle plate, M. A. Herman for initial tests of using a capacitor for field stimulation, H. Goldschmidt for help validating the stimulation device using pHluorin imaging, and N. Livingston for help with voltage imaging. We also thank the Marine Biological Laboratory and their neurobiology course for supporting the initial set of experiments (course supported by National Institutes of Health grant R25NS063307). S.W. and this work were supported by start-up funds from the Johns Hopkins University School of Medicine, Johns Hopkins Discovery funds and the National Science Foundation (1727260), and the National Institutes of Health (1DP2 NS111133-01 and 1R01 NS105810-01A1) awarded to S.W. S.W. is an Alfred P. Sloan fellow, a McKnight Foundation Scholar and a Klingenstein and Simons Foundation scholar. E.M.J. is an Investigator of the Howard Hughes Medical Institute. G.F.K. was supported by a grant from the National Institutes of Health to the Biochemistry, Cellular and Molecular Biology program of the Johns Hopkins University School of Medicine (T32 GM007445) and is a National Science Foundation Graduate Research Fellow (2016217537). The EM ICE high-pressure freezer was purchased partly with funds from an equipment grant from the National Institutes of Health (S10RR026445) awarded to S. C. Kuo.

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Authors

Contributions

M.W.D., S.W. and E.M.J. conceived the zap-and-freeze technique. G.F.K. and S.W. designed the experiments and analyzed the data. S.W., G.F.K. and E.M.J. wrote the manuscript. G.F.K. performed all freezing experiments and single-section electron microscopy sample preparation, imaging and analyses, and FM dye uptake experiments, with technical assistance from S.W., with the exception of the first replicate of the 105-ms time point experiment (which was performed by S.W.) and the 1-s and 10-s time point experiments (which were performed by S.R.). M.C. and G.F.K. performed the serial sectioning 3D reconstruction electron microscopy imaging and analyses. S.W. and M.C. developed the Matlab code for image analyses. K.P.A., E.J.H., K.L., S.R. and T.V. performed pilot zap-and-freeze experiments and electron microscopy sample preparation, imaging and analyses. M.W.D. designed the prototype zap-and-freeze stimulation device. S.W. funded and oversaw the research.

Corresponding author

Correspondence to Shigeki Watanabe.

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Peer review information Nature Neuroscience thanks Erwin Neher and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Schematic of events at the active zone of a hippocampal bouton within the first 15 ms after an action potential.

Vesicles close to the active zone are proposed to transit between docked and undocked states, with the on- and off-rates resulting in a certain number of vesicles docked and ready to fuse at any given time. Synchronous fusion, often of multiple vesicles, begins throughout the active zone within hundreds of microseconds, and the vesicles finish collapsing into the plasma membrane by 11 ms (note that the high local calcium shown only lasts ~ 100 microseconds). Between 5 and 11 ms, residual calcium triggers asynchronous fusion, preferentially toward the center of the active zone. Although shown here as taking place in the same active zone, the degree to which synchronous and asynchronous release may occur at the same active zone after a single action potential is unknown. By 14 ms, the vesicles from the peak of asynchronous fusion, which can continue for tens to hundreds of milliseconds, have fully collapsed into the plasma membrane, and new docked vesicles, which may start to be recruited in less than 10 ms, have fully replaced the vesicles used for fusion. These vesicles then undock or fuse within 100 ms. Whether these new vesicles dock at the same sites vacated by the fused vesicles, and whether newly docked vesicles contributed to synchronous and asynchronous fusion during the first 11 ms, remains to be tested.

Extended Data Fig. 2 Examples of pits in the active zone compared to pits outside the active zone or features not quantified as pits.

a, Examples of pits in the active zone 5 ms after stimulation, indicated by black arrows. b, Examples of features not counted as pits, for instance because the curvature in the presynaptic membrane is mirrored by the postsynaptic membrane, indicated by white arrows. Scale bar: 100 nm c, Full fields-of-view of micrographs from the FM dye experiment described in Fig. 4b. Images from experiments described throughout the manuscript (N = 10 freezes from separate cultures on separate days). Cultured mouse hippocampal neurons were pre-incubated in 30 µM Pitstop 2 in physiological saline (1 mM Ca2+) for 2 min, then either not stimulated or subjected to 10x 1 ms pulses at 20 Hz, at 37 °C in FM 1-43FX, followed by washing and fixation. Note that puncta along the processes became apparent after, suggesting that the zap-and-freeze device induces synaptic vesicle fusion and endocytosis. These images were collected randomly throughout the sapphire disks. Scale bar: 20 microns N = 1 experiment.

Extended Data Fig. 3 Multiple fusion events in serial-sectioning reconstructions of active zones from neurons frozen 5 ms after an action potential.

a, Example transmission electron micrographs from serial sections of active zones from neurons frozen 5 ms after an action potential in 1.2 mM, 2 mM, and 4 mM extracellular Ca2+ (from the same experiments described in Fig. 2). Scale bar: 100 nm. PSD: post-synaptic density. AP: action potential. Arrows indicate “pits” in the active zone (opposite the post-synaptic density), which are presumed to be vesicles fusing with the plasma membrane. Note that pits within the same active zone are often different depths. All data are from two experiments from separate cultures frozen on different days.; experiments in 1.2 mM Ca2+ were performed on separate days from a separate culture from the experiments in 2 mM and 4 mM Ca2+.

Extended Data Fig. 4 Representations of serial-sectioning reconstructions of active zones with exocytic pits.

a, Graphical depictions of serial-sectioned active zones containing exocytic pits, from the experiments described in Fig. 2. b, Sizes of active zones from the data set shown in Fig. 2. Area was calculated as the product of the longest 2-D length of active zone in a 2-D profile from that synapse, the number of sections containing the active zone, and section thickness (that is, as the area of a rectangle). 1.2 mM, no stim, n = 62; 1.2 mM, stim, n = 68; 2 mM, no stim, n = 64; 2 mM, stim, n = 66; 4 mM, no stim, n = 65; 4 mM, stim, n = 64 reconstructed active zones. Error bars indicate median and interquartile range. c, Density of docked vesicles in the active zone, from the experiments shown in Fig. 2. Calculated as the total number of docked vesicles from a given sample x10000, divided by the sum of the total area of active zones from that sample. d, Density of exocytic pits in the active zone, from the experiments shown in Fig. 2. Calculated as the total number of pits from a given sample x10000, divided by the sum of the total area of active zones from that sample. e, Same data shown in b, sorted by whether the active zone contained an exocytic pit or not. See Supplementary Table 1 for full pairwise comparisons and summary statistics. Source data

Extended Data Fig. 5 Fusion intermediates at multiple time points during the first 14 ms after an action potential.

a-b, Example transmission electron micrographs of synapses from neurons frozen without stimulation or 5, 8, 11, or 14 ms after an action potential (these are other examples from the same experiments shown in Fig. 3). c, Cumulative relative frequency of locations of docked vesicles within the active zone, normalized to the size of the active zone (no stim, n = 447; 5 ms, n = 300; 8 ms, n = 348; 11 ms, n = 188; 14 ms, n = 306 docked vesicles). d, Same data as in c, showing only vesicles from synaptic profiles that contain pits. e, Same data as in c, showing only vesicles from synaptic profiles that contain do not contain pits. f, Size of active zones from data in Fig. 3. Tukey boxplot shown; center: median, lower bound of box: 25th percentile, upper bound of box: 75th percentile, lower whisker: 25th percentile minus 1.5x interquartile range, upper whisker: 75th percentile plus 1.5x interquartile range; dots indicate values outside this range. No stim, n = 274; 5 ms, n = 315; 8 ms, n = 343; 11 ms, n = 192; 14 ms, n = 211; TTX, no stim, n = 121; and TTX, 5 ms, n = 255 synaptic profiles. g, Density of pits in the active zone, from the experiments shown in Fig. 3. Calculated as the total number of pits from a given sample x100, divided by the sum of the total length of active zones from that sample. h, Density of docked vesicles in the active zone, from the experiments shown in Fig. 3. Calculated as the total number of docked vesicles from a given sample x100, divided by the sum of the total length of active zones from that sample. Scale bar: 100 nm. PSD: post-synaptic density. AP: action potential. Arrows indicate “pits” in the active zone (opposite the post-synaptic density), which are presumed to be vesicles fusing with the plasma membrane. All data are from two experiments from separate cultures frozen on different days, except for the data from TTX treatment without stimulation, which are from a single experiment, and data from 5 and 8 ms, which are from three experiments. See Supplementary Table 1 for full pairwise comparisons and summary statistics. Source data

Extended Data Fig. 6 Chelating residual Ca2+ blocks fusion intermediates at 11 ms but not 5 ms after an action potential.

a-b, Example transmission electron micrographs of synapses from neurons pre-treated with a 0.25% DMSO or b 25 μM EGTA-AM and frozen either without stimulation, 5 ms after stimulation, or 11 ms after stimulation (these are other examples from the same experiments shown in Fig. 4). Scale bar: 100 nm. PSD: post-synaptic density. AP: action potential. Arrows indicate “pits” in the active zone (opposite the post-synaptic density), which are presumed to be vesicles fusing with the plasma membrane. c, Size of active zones from data in Fig. 4. Tukey boxplot shown; center: median, lower bound of box: 25th percentile, upper bound of box: 75th percentile, lower whisker: 25th percentile minus 1.5x interquartile range, upper whisker: 75th percentile plus 1.5x interquartile range; dots indicate values outside this range. See Supplementary Table 1 for full pairwise comparisons and summary statistics. d, Distances of synaptic vesicles from the plasma membrane at the active zone, including both vesicles that were annotated as docked and not docked, from data in Fig. 3. No stim, n = 274; 5 ms, n = 315; 8 ms, n = 343; 11 ms, n = 192; 14 ms, n = 211; TTX, no stim, n = 121; and TTX, 5 ms, n = 255 synaptic profiles. Distances are binned in 2-nm increments, except for “0”, which indicates vesicles ~0 nm from the active zone membrane (“2” indicates vesicles 0.1-2 nm from the membrane, “4” indicates 3-4 nm, etc.). Error bars indicate standard error of the mean. e, Number of pits in the active zone per 100 nm of active zone, from data in Fig. 4. f, Number of docked per 100 nm of active zone, from data in Fig. 4. All data from the experiments described in Fig. 4 are from 4 experiments for no stim and 5 ms time points, 3 experiments for 11 ms, and 2 experiments for 14 ms, from separate cultures frozen on different days (See Supplementary Table 2 for count data from each experiment). Source data

Supplementary information

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Supplementary Tables

Full summary statistics and hypothesis testing performed (Supplementary Table 1) and per-replicate mean/median count data (Supplementary Table 2).

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Source Data Extended Data Fig. 5

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Kusick, G.F., Chin, M., Raychaudhuri, S. et al. Synaptic vesicles transiently dock to refill release sites. Nat Neurosci (2020). https://doi.org/10.1038/s41593-020-00716-1

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