Doc2α, and not syt7, drives most of the asynchronous glutamate release triggered by a single action potential in cultured hippocampal neurons.

(A) Raw iGluSnFR (A184V) traces from cultured WT mouse hippocampal neurons. A single AP was applied, and images were captured every 10 ms. Peaks that appeared at the first 10 ms timepoint were defined as synchronous release events; peaks that appeared at later time points were defined as asynchronous release events. The vertical magenta line indicates the stimulus, and the vertical dashed lines indicate the imaging frames at 10, 20, 30, 40, 50, 60, and 70 ms.

(B) Three representative traces illustrate synchronous and AR peaks. The horizontal dashed line is five-times the standard deviation of the baseline noise. Individual peaks are indicated by asterisks. Traces showing single synchronous (black) and asynchronous peaks (gray), along with a trace in which both kinds of events occurred (light gray), are shown.

(C) Color coding and binning the temporal changes in iGluSnFR signals within 70 ms after a single stimulus for WT (n=15; from 4 independent litters), Doc2αKO (n=17; from 5 independent litters), syt7KO (n=15; from 4 independent litters), and EGTA-AM treated neurons (n=18; from 4 independent litters). The temporal color code is red (initiation of stimulus) to purple (70 ms after the stimulus); scale bar, 5 µm. Histograms of iGluSnFR (ΔF/F0) peaks were generated using 10 ms binning. Samples were color-coded as follows: WT (gray), Doc2αKO (reddish), syt7KO (cyan), and EGTA-AM (orange) treated neurons. The histograms include donut graph insets to display the fraction of release in each bin.

(D) Violin plot showing the % AR (>10 ms) for each condition. One-way ANOVA, followed by Dunnett’s test, was used to compare mutants against WT. *** indicates p<0.001.

Doc2α, and not syt7, drives most of the AR triggered by a single action potential at Schaffer collaterals.

(A) Schematic of the recording scheme of hippocampal slices showing stimulation of the Schaffer collateral fibers and whole-cell patch clamp recordings of CA1 pyramidal neurons.

(B) Averaged traces of evoked EPSCs recorded from WT (n=18; from 5 independent litters), Doc2αKO (n=15; from 4 independent litters), syt7KO (n=19; from 6 independent litters), and EGTA-AM treated neurons (n=20; from 4 independent litters).

(C) Normalized cumulative EPSC charge transfer over 400 ms.

(D) Bar graphs, from corresponding groups in panels (B-C), summarizing the amplitude, fast and slow EPSC charge, and the fast and slow EPSC t values. One-way ANOVA, followed by Dunnett’s test, was used to compare mutants against WT. **, and *** indicate p< 0.01, and p<0.001.

Both Doc2α and syt7 contribute to asynchronous glutamate release during train stimulation but have opposite effects on short-term plasticity in cultured hippocampal neurons.

(A) Representative iGluSnFR images from trains of 50 APs at 10 Hz (left) or 20 Hz (right). Images on the left, middle and right each show a sum of 20 frames (200 msec) before, during, and after stimulation, respectively; scale bar, 40 µm.

(B) Representative iGluSnFR responses from WT (10 Hz: n=12; from 3 independent litters; 20 Hz: n=17; from 4 independent litters), Doc2αKO (n=14; from 4 independent litters; 20 Hz: n=16; from 4 independent litters), and syt7KO neurons (n=15; from 3 independent litters; 20 Hz: n=12; from 3 independent litters), stimulated at 10 Hz (top) and 20 Hz (bottom).

(C) The AR fraction was defined as iGluSnFR peaks that occurred after the first 10 ms time point (i.e., from the 20 msec time point onward), divided by the total number of peaks that occurred at all time points between each stimulus. These data are plotted as a function of the stimulus number. Mean values (bold lines) ± SEM (shaded regions) are indicated.

(D) Plot of the normalized amplitude of iGluSnFR signals evoked by 50 stimuli at 10 and 20 Hz. Insets show the first three points of each trace on expanded scales.

Both Doc2α and syt7 contribute to AR during train stimulation but have opposite effects on short-term plasticity at Schaffer collaterals.

(A) Representative EPSCs triggered by 20 Hz stimulus trains using WT, Doc2αKO, syt7KO, and EGTA-AM treated neurons. The shaded gray areas indicate the tonic charge component that likely corresponds to AR.

(B) The peak amplitude of each EPSC during the train was normalized to the first EPSC from WT (n=12; from 4 independent litters), Doc2αKO (n=11; from 3 independent litters), syt7KO (n=10; from 3 independent litters), and EGTA-AM treated neurons (n=11, from 3 independent litters).

(C) Bar graph showing the total tonic charge transfer (indicated by the grey areas in panel (A)), as an index of AR, from WT, Doc2 KO, syt7KO, and EGTA-AM treated hippocampal slices.

Doc2α, but not syt7, is required for asynchronous synaptic vesicle fusion triggered by a single action potential in cultured hippocampal neurons.

(A-D) Example transmission electron micrographs of syt7WT (A), syt7KO (B), Doc2αWT (C), and Doc2αKO (D) synapses frozen at the indicated time points after an action potential (AP) or without stimulation (no stim). Arrows indicate pits in the active zone, which are presumed to be synaptic vesicles that have fused with the plasma membrane.

(E) The number of pits in the active zone per synaptic profile (part of the synapse captured in a 2D section) in syt7 wild-type littermate controls and knockouts. Error bars indicate 95% confidence interval of the mean for all data pooled together; each dot indicates the mean from a single biological replicate. Inset: same data from the 11 ms time point from each genotype, with lesser y axis range; lines indicate data are from the same biological replicate (experiment), not that they are paired. P values are from comparisons between wild-type controls (no stim, n = 217; 5 ms, n = 216; 11 ms, n = 217; 14 ms, n = 230 synaptic profiles) and knockouts (no stim, n = 193; 5 ms, n = 215; 11 ms, n = 230; 14 ms, n = 212 synaptic profiles) frozen at the same time point.

(F) same as E, but for Doc2α wild-type controls (no stim, n = 327; 5 ms, n = 229; 11 ms, n = 336; 14 ms, n = 192 synaptic profiles) and knockouts (no stim, n = 330; 5 ms, n = 231; 11 ms, n = 352; 14 ms, n = 321 synaptic profiles).

Number of biological replicates (from separate cultures frozen on different days and analyzed in separate randomized batches): 2 for each of the syt7 time points, 2 for 5 ms and 14 ms Doc2 WT, 2 for 5 ms and 11 ms Doc2 KO, 3 for Doc2 WT no stim and 11 ms, 3 for Doc2 KO no stim and 14 ms. The numbers of pits between wild type and knockout were compared using Brown-Forsythe ANOVAs with post-hoc Games-Howell’s multiple comparisons tests. In pairwise tests, the same time point for wild type vs. knockout as well as each time point within each genotype were compared against each other (only comparisons between the same time point for wild type vs. knockout are shown, see Supplementary Table 1 for all pairwise comparisons). See Source Data 1 for data used to generate the graphs shown. See Fig. S6A for active zone sizes from each condition and Fig. S6B for pit data normalized to the size of active zones.

Syt7 is absolutely required for transient docking, while docked vesicles recover more quickly without Doc2α.

(A-D) Example transmission electron micrographs of syt7WT (A), syt7KO (B), Doc2αWT (C), and Doc2αKO (D) synapses frozen at the indicated time points after an action potential (AP) or without stimulation (no stim). Arrows indicate docked vesicles (synaptic vesicles with no visible distance between their membrane and the plasma membrane).

(E) The number of docked vesicles per synaptic profile (part of the synapse captured in a 2D section) in syt7 wild-type littermate controls and knockouts. Error bars indicate 95% confidence interval of the mean for all data pooled together; each dot indicates the mean from a single biological replicate. P values are from comparisons between wild-type controls (no stim, n = 217; 5 ms, n = 216; 11 ms, n = 217; 14 ms, n = 230 synaptic profiles) and knockouts (no stim, n = 193; 5 ms, n = 215; 11 ms, n = 230; 14 ms, n = 212 synaptic profiles) frozen at the same time point.

(F) same as E, but for Doc2α wild-type controls (no stim, n = 327; 5 ms, n = 229; 11 ms, n = 336; 14 ms, n = 192 synaptic profiles) and knockouts (no stim, n = 330; 5 ms, n = 231; 11 ms, n = 352; 14 ms, n = 321 synaptic profiles).

Number of biological replicates (from separate cultures frozen on different days and analyzed in separate randomized batches): 2 for each of the syt7 time points, 2 for 5 ms and 14 ms Doc2 WT, 2 for 5 ms and 11 ms Doc2 KO, 3 for Doc2 WT no stim and 11 ms, 3 for Doc2 KO no stim and 14 ms. The numbers of pits between wild type and knockout were compared using Brown-Forsythe ANOVAs with post-hoc Games-Howell’s multiple comparisons tests. In pairwise tests, the same time point for wild type vs. knockout as well as each time point within each genotype were compared against each other (only comparisons between the same time point for wild type vs. knockout are shown, see Supplementary Table 2 for all pairwise comparisons). See Source Data 2 for data used to generate the graphs shown. See Fig. S6A for active zone sizes from each condition, Fig. S6C for docked vesicle data normalized to the size of active zones, and Fig. S6D for distribution of undocked vesicles.

A mathematical model in which syt7 catalyzes synaptic vesicle docking and Doc2α promotes fusion can qualitatively explain the observed phenotypes.

(A) Simplified scheme of the model in which syt7 acts as a catalyst of docking (increasing both docking and undocking rates) and Doc2α regulates fusion together with syt1. All proteins only exert their effect when bound to Ca2+. In the model a release site can either be empty or occupied by a tethered or a docked vesicle. For full model scheme see Fig. S7.

(B) Calcium transient used to perform the simulations. The transient corresponds to a signal induced by 10 APs at 20 Hz. Insert shows a zoom-in over the first 5 ms of the transient.

(C) Average number of Ca2+-ions bound to syt1, Doc2α and syt7 over time. Syt1 can maximally bind five Ca2+-ions, Doc2α and syt7 can each bind two Ca2+-ions. Insert shows zoom-in over the first 5 ms of the simulation.

(D) The number of docked vesicles over time for simulations including functionality of all proteins (black, WT), simulations lacking Doc2α (magenta, Doc2α-) and simulations lacking syt7 (cyan, syt7-). Insert shows a zoom-in over the first 15ms of the simulation. (E) Simulated release rates obtained using the full model (WT, left, black), the model lacking Doc2α (Doc2α -, middle, magenta) and the model lacking syt7 (syt7, right, cyan) simulations. Top line shows the entire trace. Bottom panels show a zoom-in to illustrate the asynchronous release component, which is indicated by the gray area.

(F) Cumulative number of asynchronously released vesicles per stimulus. Asynchronous release is quantified as the release between 5ms after the start of the AP and the start of the next AP. Insert shows zoom-in over the first two pulses.

(G) Peak release rates per stimulus for stimulations using the full model (WT, black), the model lacking Doc2α (Doc2α-, magenta) and the model lacking syt7 (syt7-, cyan).

AR in Doc2α/syt7 DKO neurons: non-additive effects on AR indicate function through a common pathway.

All recordings were carried out using acute hippocampal slices, in CA1, as described in Fig. 2 and 4.

(A) Averaged traces of evoked EPSCs recorded from WT (n=18; from 5 independent litters), Doc2αKO (n=15; from 4 independent litters), Doc2α/syt7 DKO (n=14; from 4 independent litters), and EGTA-AM treated neurons (n=20; from 4 independent litters).

(B) Bar graph summarizing the slow EPSC charge values. One-way ANOVA, followed by Dunnett’s test, was used to compare mutants against WT. **, and *** indicate p< 0.01, and p<0.001.

(C) Representative EPSCs triggered by 20 Hz stimulus trains using WT and Doc2α/syt7 DKO (n=15; from 5 independent litters) neurons.

(D) AR in WT, Doc2αKO, syt7KO, Doc2α/syt7 DKO, and EGTA-AM treated neurons was estimated by measuring the total tonic charge transfer during the 20 Hz stimulus train, as described in Fig. 4C.