Direct imaging of rapid tethering of synaptic vesicles accompanying exocytosis at a fast central synapse

Abstract

A high rate of synaptic vesicle (SV) release is required at cerebellar mossy fiber terminals for rapid information processing. As the number of release sites is limited, fast SV reloading is necessary to achieve sustained release. However, rapid reloading has not been observed directly. Here, we visualize SV movements near presynaptic membrane using total internal reflection fluorescence (TIRF) microscopy. Upon stimulation, SVs appeared in the TIRF-field and became tethered to the presynaptic membrane with unexpectedly rapid time course, almost as fast as SVs disappeared due to release. However, such stimulus-induced tethering was abolished by inhibiting exocytosis, suggesting that the tethering is tightly coupled to preceding exocytosis. The newly tethered vesicles became fusion competent not immediately but only 300 ms to 400 ms after tethering. Together with model simulations, we propose that rapid tethering leads to an immediate filling of vacated spaces and release sites within <100 nm of the active zone by SVs, which serve as precursors of readily releasable vesicles, thereby shortening delays during sustained activity.

Keywords: presynapse; synapse; synaptic vesicle; vesicle recruitment.

Fig. 1.

Rapid vesicle tethering upon a depolarization pulse and APs at dissociated cMF terminals. (A) An example of transmitted light image of a dissociated cMF terminal. The expanded image was obtained using TIRF microscopy. SVs were labeled by FM1-43. (B) Representative traces of 100-ms depolarization-induced Ca²⁺ current (I_Ca) and capacitance jump (C_m). The slope of the capacitance increase after the depolarization pulse was fitted with a line shown in red. (C) The amount of capacitance increase by a 0-mV depolarization pulse for 100 ms was plotted against peak Ca²⁺ current, basal membrane capacitance of the recorded terminals (n = 58), and the slope of the capacitance increase after the depolarization pulse (n = 56). Data from individual terminals were plotted by open circles. These plots were fitted with a line shown in red. (D) We observed “vanish” and “tethering” types of vesicle movements in TIRF image. (Top) Examples of the two types of events in TIRF image. (Bottom) Averages of the normalized fluorescent intensity were plotted against time for 347-nm-diameter circle centered on the spots (filled squares) and the concentric annulus around the circles (outer diameter of 867 nm; open squares). Error bars show SEM (n = 35, and 31 for vanish and tethering, respectively). “Vanish” events correspond to vesicles moving out from the TIRF field or vesicles undergoing exocytosis. “Tethering” events correspond to vesicles recruiting to the TIRF field. (Scale bars, 500 nm.) (E) Using whole-cell mode, Ca²⁺ current and capacitance change upon a depolarization pulse were recorded. (F) Two types of events were observed by simultaneous TIRF imaging. Peristimulus time histograms for the two types of events and cumulative number of the events from 56 terminals are shown. Fits consist of the sum of a double exponential function and a line for “vanish” events, and the sum of a single exponential function and a line for “tethering” events. According to the result of the different pulse duration experiments (SI Appendix, Fig. S1B), τ₁ for “vanish” events was fixed at 10 ms. The fit has a ratio of the amount of first to second component of 0.06. The 100-ms depolarization pulse was applied from 0 s to 0.1 s. The depolarizing period is indicated by the gray color (the same for subsequent figures). (G) In cell-attached mode, APs were evoked by 2-ms depolarization pulses at 50 Hz for 1 s. (H) Peristimulus time histograms and cumulative number of events detected by simultaneous TIRF imaging from 43 terminals. The time of the train is indicated by the gray color. The cumulative numbers of the events were fitted with a single exponential curve (red).

Fig. 2.

The effects of TeNT on “tethering” and “vanish” events. (A) TeNT (500 nM) was applied by adding to the internal solution for a patch pipette. Example traces were recorded at 5 and 10 min after break-in (black, and blue, respectively). (B) (Top) Peristimulus time histograms for “vanish” and “tethering” events detected by TIRF imaging at the TeNT-treated terminals (n = 26 terminals). A 100-ms depolarization pulse was applied from 0 s to 0.1 s. The stimulation time period is indicated by the gray color. (Bottom) The cumulative numbers of each event are shown. Gray dashed lines show fits of the cumulative number of events between −0.5 s and 0 s, representing basal “vanish” and “tethering” events. (C) (Top Left) An example of the normalized fluorescent intensity for 347-nm-diameter circle centered on the spot in “tethering−vanish” events (filled circles). Open circles show the times of occurrence of the bright spot appearing (“tethering”) and disappearing (“vanish”). Dwell time is defined as the time difference between the times of occurrence of “tethering” and “vanish.” (Bottom Left) Average dwell time of “tethering−vanish” events in various experimental conditions and a simulation for the TeNT experiments (n = 32, 49, 55, and 48 for control, AP, high-temperature, and TeNT experiments, respectively). Individual data are shown by open circles. Error bars show SEM. **P < 0.01, one-way ANOVA followed by Tukey’s test. (Top Right) Distributions of the dwell times of “tethering−vanish” events in all control, AP, and high-temperature experiments, and (Middle Right) in the TeNT experiment. (Bottom Right) Corresponding cumulative distributions of dwell time for control (black) and TeNT (blue). **P < 0.01, Kolmogorov−Smirnov test.

Fig. 3.

Newly tethered events contribute to the sustained release at cMF terminals. (A) Representative traces for Ca²⁺ current and capacitance in double pulse experiments. Two 0-mV depolarization pulses for 30 ms were applied with an interstimulus interval of 100 ms. (B) Ca²⁺ currents and capacitance jumps by the first and the second depolarization pulses, and the slope of the capacitance increase after the pulses were measured (n = 32; mean ± SEM). ***P < 0.001, Student’s t test. (C) (Top) Peristimulus time histograms for “vanish” and “tethering” events observed by TIRF imaging from 80 terminals. The stimulation periods of the pulses are indicated by gray. (Bottom) Cumulative numbers of events for the first response were fitted with a single exponential curve (red). Cumulative numbers for the second response were fitted with an exponential curve plus a line (blue). (D) Comparison of the time course of fluorescent spots disappearing between total (Top) “vanish” events after the second pulse and (Bottom) “tethering−vanish” events. In “tethering−vanish” events, the events visibly appearing between 0 ms and 130 ms were analyzed. The cumulative number of vanish events that tether between pulses was fitted with a single exponential curve (red). (E) (Left) Representative and (Right) averaged (n = 7) time courses of the fluorescent intensity within a center (347 nm diameter; filled squares) and concentric annulus (347 nm inner and 867 nm outer diameter; open squares) for “diffuse” events that are tethered between pulses. Average images are taken at the times indicated by numbers. Error bars show SEM. (Scale bar, 500 nm.)

Fig. 4.

Model simulation reveals the SV pool dynamics within the TIRF field. (A) A sequential two-pool model for SV movement within TIRF field. One pool (TIRF-PRP) is for SVs tethered from outside of the TIRF field, and the other pool (RRP) is for readily releasable vesicles. (B) Monte Carlo simulation for the dwell time of the “tethering−vanish” events in the TeNT experiments provide the rate constant of 6 s⁻¹ for SV recruitment to the TIRF field (yellow arrow). Since TeNT blocked exocytosis, the vanish and tethering events in the TeNT experiments correspond to SV movements between the outside and inside of the TIRF field to maintain the equilibrium of the occupancy of the pool for tethered SVs. The dashed lines show the corresponding results of the model simulation. (C) In the control condition, stimulus-induced tethering events are forced to take place with a time constant of 200 ms in the model to follow the experimental data. Readily releasable vesicles are released with a time constant of 10 ms during stimulation (SI Appendix, Fig. S1). The sustained release continues for 300 ms, as we observed in the capacitance increase after stimulation. The best fit of the capacitance increase with the model determines a rate constant of 3 s⁻¹ for priming to the RRP (dashed curve in red). The time course of the vanish events representing the sum of release and untethering events is well reproduced by the model (dashed curve in purple). To see changes of the sustained release, the stimulus induced tethering events are abolished in the model (dashed curves in light colors). (D) The probabilities of occupancies for the two pools in the TIRF field and schematics of the various phases of the pools (for the data shown in C). In the resting state, a space 3 times larger is occupied by the RRP. Once release starts, the space for the RRP immediately becomes close to 0 (see a schema numbered 2). The TIRF-PRP is then overfilled and occupies up to 70 to 80% of the space by rapid tethering (see a schema numbered 3). The newly tethering SVs undergo a SV docking/priming step for 300 ms, in order to be readily releasable for the following sustained release. The stimulation time period is indicated by the gray color in B–D.