Figure 1. SYT7 influences presynaptic neurotransmitter release during short-term synaptic plasticity.

(a) Representative super-folder iGluSnFR S72A (hereon iGluSnFR) traces from single-stimulus experiments. Lighter traces are individual regions of interest (ROIs) and dark bold traces are the average of all light traces from a full field of view (FOV); the single stimulus is denoted with an arrow. Wild-type (WT) are denoted in black and gray, and SYT7KO are represented in red and light red; same scheme applies throughout the figure. (b) Peak iGluSnFR signals between WT (0.203 [95% CI 0.154–0.244] ΔF/F₀) and SYT7KO (0.245 [95% CI 0.160–0.308] ΔF/F₀). Values are medians with 95% CI representing error, Mann-Whitney test, p = 0.4554, each n is a separate FOV (n = 32 (WT) and 34 (SYT7KO) from four independent experiments). (c) Fraction of synchronous release, defined as peak iGluSnFR signals arriving within 10 ms of stimulus from total release of 500 ms following the stimulus, compared between WT (0.9522 [95 % CI 0.902–0.965]) and SYT7KO (0.9808 [95% CI 0.943–0.993]). Data from the same n as in (b). Values are medians with 95% CI representing error, Mann-Whitney test, *p = 0.0326. (d) Average +/- standard deviation traces from paired-pulse ratio (PPR) experiments with four interstimulus intervals compared; n = 14 (WT 20 Hz), 14 (WT 10 Hz), 15 (WT 5 Hz), 13 (WT 2 Hz), 15 (SYT7KO 20 Hz), 13 (SYT7KO 10 Hz), 14 (SYT7KO 5 Hz), 13 (SYT7KO 2 Hz) from three independent experiments. (e) Quantification of PPR (peak iGluSnFR ΔF/F₀) from WT and SYT7KO; values are means +/- SEM. ****p<0.0001, **p = 0.0012, by two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test; full statistics are provided in Figure 1—source data 1. (f) Quantification of fractional active synapses, that is, the number of synapses demonstrating peak release above baseline during the second stimulus relative to the first of a paired pulse. Values are means +/- SEM. **p = 0.0052, **p = 0.0099, and *p = 0.0289, in order from left to right, by two-way ANOVA with Sidak’s multiple comparisons test; full statistics are provided in Figure 1—source data 2. (g) Relative frequency histograms of PPR from all ROIs quantified from PPR trials, 20 Hz, 10 Hz, 5 Hz, 2 Hz, WT, and SYT7KO. Vertical dotted line delineates a PPR of 1.

Figure 1—figure supplement 1. Extended analysis of paired-pulse measurements.

(a) Quantification of iGluSnFR ΔF/F₀ peaks from first stimulation during paired-pulse ratio (PPR) trials. No difference in initial release between any stimulation parameter was noted. Values are means +/- SEM. (b) Quantification of iGluSnFR ΔF/F₀ peaks from second stimulation during PPR trials. Values are means +/- SEM. ****p<0.0001, ***p = 0.0004, by two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test; full statistics are provided in Figure 1—figure supplement 1—source data 1. (c–f) Pie charts showing the percentage of regions of interest (ROIs) that either maintain (gray), activate (green), or deactivate (red) glutamate release in response to an initial stimulus at the labeled stimulation frequencies. Criteria for glutamate release were based on iGluSnFR ΔF/F₀ peak amplitude in relation to baseline noise (>4 SD above noise). (g) Representative images of wild-type (WT) (top) and SYT7KO (bottom) iGluSnFR change in fluorescence in response to two stimuli 50 ms apart (20 Hz). Images are max t-projections of temporally color-coded timeseries. Fluorescence from the first stimulus is coded in green (0–50 ms post-stimulus) and the fluorescence from the second stimulus is coded in purple (50–100 ms post-stimulus). (h) Scatterplot from 20 Hz data of first stimulus peak (X) by second stimulus peak (Y), WT (black), and SYT7KO (red). Population scatter illustrates the difference between WT and SYT7KO ROIs where WT ROIs are grouped predominantly in area (i) because the second stimulus results in facilitation, whereas SYT7KO groups in (ii) because of loss of facilitation.