Spatiotemporal Regulation of Synaptic Vesicle Fusion Sites in Central Synapses  Dario Maschi, Vitaly A. Klyachko  Neuron  Volume 94, Issue 1, Pages 65-73.e3 (April 2017) DOI: 10.1016/j.neuron.2017.03.006 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Robust Detection of Individual Vesicle Fusion Events in Hippocampal Synapses (A) Robust vGlut1-pHluorin responses to single stimuli. Average fluorescence changes from 71 boutons and corresponding ΔF evoked by 1 Hz stimulation. Average decay time of individual responses is 1.1 ± 0.44 s. (B) Examples of fail and single-vesicle fusion event evoked by 1 AP from the same bouton. Images are background subtracted. Corresponding raw images are shown in Figure S1A. (C) Examples of one- and two-vesicle fusion events evoked by 1 AP. Images are background subtracted. (D) Examples of fluorescence intensity traces of one- and two-vesicle fusion events evoked by 1 AP showing that boutons respond in quantal steps. (E) Histogram of integrated fluorescence changes evoked by a single AP stimulation at 1 Hz, plotted for detected events (gray) and failures (black). n = 690 boutons from >20 coverslips from 3 independent cultures. (F–H) Same as (C)–(E) for spontaneous fusion events. (I) Fluorescence increase evoked by 1 Hz stimulation (black; average of 563 boutons) does not recover in the presence of 5 μM bafilomycin (red; average of 1,429 boutons) (left). The first responses from the left traces are enlarged for comparison (right). (J) Left: sample images and Gaussian fits of a stationary green fluorescent 40 nm bead (top) and a vesicle fusion event (bottom). Right: average width of the PSF δ (half-width) obtained from Gaussian fits for beads or fusion events. ns, not significant. (K) Release site localization precision calculated as described in Thompson et al. (2002). Scale bars represent 1 μm. Neuron 2017 94, 65-73.e3DOI: (10.1016/j.neuron.2017.03.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Vesicle Fusion Events Are Widely Distributed across the Individual Active Zones (A) Sample map of ten subsequent vesicle fusion events within a single hippocampal bouton. For each fusion event, one frame preceding (left) and one frame following (right) the stimulus are shown. Numbers correspond to the temporal order of the events. Red dots show sub-pixel event localization in the corresponding frame. (B) Median distance between consecutive fusion events in individual boutons. n = 2,049 events from 16 coverslips from 5 independent cultures. (C) Distance between peripheral fusion events and the center of the fusion area in individual boutons (same as in B) approximated as a center of mass of the convex envelope (convex hull) of all fusion events in each bouton. Data are plotted as median ± SD. (D) Left: sample release event distribution for a single bouton evoked by 1 Hz stimulation. Shaded area represents a convex hull of all release events used to estimate the size of the release area. Right: sample release areas of 28 different boutons. (E) Distribution of AZ sizes calculated as in (D). Scale bar represents 1 μm. All data are from at least five independent cultures. Neuron 2017 94, 65-73.e3DOI: (10.1016/j.neuron.2017.03.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Multiple Distinct Vesicle Release Sites in Central Synapses (A) Sample map of vesicle fusion events within a single hippocampal bouton. Hierarchical cluster analysis was used to cluster fusion locations (various colors) into individual release sites (crossed circles) with a clustering diameter of 50 nm. Events clustered into the same release site are shown by the same color. (B) Average number of clusters detected in individual terminals as a function of the number of release events observed. n = 178 boutons from 16 coverslips from 5 independent cultures. Note that a small subset of data for boutons with 20 or more detected events is not shown because of its sparsity but give a very similar estimate of 9.8 ± 1.0 clusters per bouton. (C) Time between two subsequent fusion events at the same release site. (D) Distribution of distances between all clusters within each bouton. (E) Distribution of distances from clusters to the center of release area within each bouton. In (B)–(E), n = 178 boutons from 16 coverslips from 5 independent cultures. Only boutons with at least 10 detected release events were analyzed. Neuron 2017 94, 65-73.e3DOI: (10.1016/j.neuron.2017.03.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Activity Dependence of Release Site Properties (A) Examples of consecutive vesicle fusion events evoked by 10 Hz stimulation in the same terminal. (B) Distribution of distances of release events to AZ center evoked at 1 Hz or 10 Hz. p = 2.5E−6, two-sample K-S test. Inset: the mean distance of release events to the AZ center per bouton at 1 Hz or 10 Hz. ∗p = 0.0011, t test. 1 Hz: n = 178 boutons from 16 coverslips from 5 independent cultures. 10 Hz: n = 230 boutons from 14 coverslips from 4 independent cultures. (C) Probability that release site used at 1 Hz is reused with subsequent 1 Hz or 10 Hz stimulation (bottom). Stimulation paradigms (top): 20 APs at 1 Hz followed by 20 APs at 1 Hz (black) or 20 APs at 1 Hz followed by 20 APs at 10 Hz (the same total recording time and number of stimuli per frequency). ns, not significant (t test). n = 507 boutons from 13 coverslips and 1,070 boutons from 18 coverslips, respectively, from 3 independent cultures each. (D) Proportion of release sites that are reused at different stimulation frequencies normalized to the value at 1 Hz and the number of detected events in each condition (bottom). Stimulation paradigm (top). 1 Hz: 120 APs; 10 Hz: 6 bursts of 20 APs, repeated at 0.05 Hz (the same total recording time and number of stimuli per frequency). ∗p < 0.05, chi-square test. Same dataset as in (B). (E) Proportion of release sites that are reused at different inter pulse times (1,000 ms; 100 ms) normalized to the value at 1,000 ms. n = 712, 386 pairs from 13 coverslips each, from 3 independent cultures each. Scale bars represent 1 μm. Neuron 2017 94, 65-73.e3DOI: (10.1016/j.neuron.2017.03.006) Copyright © 2017 Elsevier Inc. Terms and Conditions