Synaptic transmission and plasticity


Syt7 – 3 Processes



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.


SYT7 counteracts depression and promotes asynchronous release during sustained stimulation.


(a) Representative traces of iGluSnFR ΔF/F0 signals (single regions of interest (ROIs) A-E), from one full field of view (FOV) during high-frequency stimulation (HFS) of wild-type (WT) (i) and SYT7KO (ii) neuronal preparations. Samples were field stimulated with a frequency of 20 Hz for 2.5 s (50 action potentials (APs)). (b) Average iGluSnFR ΔF/F0 traces during high-frequency stimulation (HFS) for WT (black, n = 17) and SYT7KO (red, n = 16), from three independent experiments (same source data for b–f). (c) Fraction of active synapses, defined as synapses releasing peak glutamate above baseline, >4 SD above noise, as a function of stimulation number during HFS. Values are means (lines) +/- SEM (lighter shade error), ****p<0.0001 by two-way analysis of variance (ANOVA) comparing genotypes. (d) Plot of the average cumulative iGluSnFR ΔF/F0 signal from WT (black) and SYT7KO (red) neurons vs time. Dotted lines represent SEM and gray (WT) and light red (SYT7KO) linear lines represent linear fits to the last 1.5 s of the train. (e) Synaptic vesicle (SV) replenishment rates were calculated from slopes of linear regressions from individual traces used in panel (d). Values are means +/- SEM, WT (0.077 +/- 0.009) and SYT7KO (0.042 +/- 0.004); **p = 0.0019 using unpaired two-tailed t-test. (f) Fraction of synchronous release, defined as peak iGluSnFR ΔF/F0 within 10 ms of each stimulus from the total interstimulus interval, as a function of stimulation number during HFS. Values are means (bold lines) +/- SEM (lighter shade fill); ****p<0.0001 by two-way ANOVA comparing genotypes. (g) Quantal analysis using all detected iGluSnFR peaks (n>6000) from the first two stimuli of a 20 Hz train from WT neurons binned into 0.02 ΔF/F0. (h) Quantal analysis using all detected iGluSnFR peaks (n>10,000) from the last five stimuli of a 2.5-s 20 Hz train from WT neurons. (i) Quantal analysis using asynchronous iGluSnFR peaks (n = 254) from the first two stimuli of a train from WT neurons. (j) Quantal analysis using asynchronous iGluSnFR peaks (n = 156) from the first two stimuli of a train from S7KO neurons (asynchronous is defined as iGluSnFR peaks that occur more than 10 ms after a stimulus, but before the proceeding stimulus). Gaussian distributions were generated with no restrictions in panels (g) and (h). In panels (i) and (j), 1q and 2q labels were added based on the mean values from panels (g) and (h). From panel (g), mean (2q) = 0.31 [95% CI 0.30–0.32] and from panel (h), mean (1q) = 0.14 [95% CI 0.14–0.15]. WT asynchronous vs S7KO asynchronous distributions in panels (i) and (j) are different by Kolmogorov-Smirnov test; approximate p-value = 0.005 with K-S D = 0.1760.


Syt7 and Doc2




Syt7 transiently docks synaptic vesicles, while Doc2a directly triggers asynchronous neurotransmitter release.


The arrival of a single action potential (AP) triggers asynchronous release from 11 ms onward (left panel). Also, it activates a transient docking (TD) step in which vesicles re-attach to the active zone (solid gray line) in a syt7 (granite green) dependent way. Loss of syt7 (Syt7tm1Nan/tm1Nan) abolishes TD (middle up panel). Doc2a (lilac) directly mediates synaptic vesicle fusion, captured via ‘Zap-and-Freeze’ electron microscope and shown as the fusion pit (black arrow; right up panel) at 11 ms. Doc2a knockout synapse (Doc2aem1Smoc/em1Smoc) abolishes fusion pits at 11 ms (right bottom panel).




Doc2a triggers asynchronous release (AR) in response to single action potentials; during repetitive stimulation, syt7 mediates transient docking to feed docked vesicles to Doc2a for ongoing AR.


At rest, SVs are docked at the active zone (solid gray line) in a dynamic equilibrium indicated by the antiparallel black arrows (far left panel). Under these conditions, docking is not affected by syt7. A single action potential triggers both undocking (UD) and synchronous release (SR), resulting in a 40% reduction in docked vesicles in ~5 ms. This is followed by a transient docking (TD) step in which vesicles re-attach to the active zone via a process that takes 14 msec and is mediated by Ca2+ and syt7 (granite green). The same single AP also triggers AR mediated directly by Doc2a (lilac), at the 11 ms time point. Syt7 plays no role in release during the first action potential, again because it does not regulate docking in the resting state. However, loss of syt7 abolishes activity-dependent TD, which decreases the availability of docked vesicles during subsequent (i.e., two or more) action potentials, indirectly reducing Doc2a-triggered AR during ongoing activity. As a test of this model, an epistasis experiment revealed that the Doc2a/syt7 DKO AR phenotype was non-additive. This validates a sequential, two-step, model, in which—during ongoing activity—syt7 feeds docked vesicles to Doc2a, which directly mediates AR. Hence, syt7 acts upstream of Doc2a, as a docking factor, while Doc2a functions as the proximal Ca2+ sensor for AR in hippocampal synapses. This model can account for why only Doc2a drives AR in response to a single action potential, while Doc2a and syt7 contribute equally to AR during subsequent action potentials.




Synaptotagmin 9 (SYT9) is a regulator of substance P (SP) release from striatal neurons.

a) Repetitive electrical stimulation of cultured striatal neurons expressing SP-pHluorin (SP-pH) causes exocytosis of SP-pH-bearing dense core vesicles (DCVs), resulting in an increase in fluorescence. NH4Cl application dequenches all pHluorin fluorescence and allows visualization of the total pool of SP-pH-bearing DCVs. b) Representative traces of individual SP-pH DCV fusion events; the stimulation paradigm is overlaid in red, and alkalinization of DCVs with NH4Cl is shown in green. c) The released fraction of SP-pH DCVs is reduced in Syt9 KO neurons compared to WT. d) DCV pool size per neuron was unchanged in Syt9 KO neurons.

Figure modified from:

Seibert MJ, Evans CS, Stanley KS, Wu Z, Briguglio JS, Chapman ER. Synaptotagmin 9 modulates spontaneous neurotransmitter release in striatal neurons by regulating substance P secretion. bioRxiv, 2022.2004.2018.488681, doi:10.1101/2022.04.18.488681 (2022).



Example timelapse of stimulated SP-pH release and subsequent NH4Cl perfusion of a cultured striatal neuron





Ruhl et al. Nat. Commun. 2019 Fig. 5
Syt-17 regulates the early secretory pathway and interacts with Golgi resident proteins. a VSVG-YFP-2xUVR8 aggregates in the ER in the dark (top), disaggregates upon illumination at 300 nm, and is trafficked to the Golgi (bottom). b Representative images of uncaging and accumulation in WT and KO neurons. c Syt-17 KOs exhibit a slower time to peak Golgi fluorescence (t12 = 2.425, p = 0.03, r2 = 0.329, meanwt = 18 ± 1.84 s, meanko = 23 ± 1.33, Nwt = 7 and Nko = 7 neurons). d Time course for each cell. In KO neurons (orange) cargo accumulates more slowly. Measurements made from three independent preparations. All error bars indicate S.E.M.s. e Representative electron micrograph of the somatic Golgi complex in WT (left) and KO (right) neurons, demonstrating vesicle accumulation in the KO. Scale bar indicates 200 nm. f Histogram of vesicle diameters quantified across four fields of view from two litters of mice. Error bars indicate S.E.M.s. g Result of a DEEPN analysis for syt-17 interactors. Plotted is the ratio of each gene abundance in the selected (-His grown) sub-population vs. the non-selected (+His) subpopulation (see Methods section). Genes for which the majority of plasmids were in the proper reading frame with respect to the Gal4 activation domain (i.e., potential interactors) are shown in blue. Two Golgi proteins among the top hits, GOLGA6A and ICA1, are indicated. h Binary yeast two-hybrid assays showing interaction of full-length (WT) syt-17, and the indicated syt-17 mutants, with Golgi proteins GOLGA6A and ICA1. Incubations with 1 mM and 10 mM of 3-aminitriazole (see Methods) are shown on right




Ruhl et al. Nat. Commun. 2019 Fig. 7
Alterations in endocytic recycling associated with accumulation of postsynaptic AMPA receptors and defective synaptic plasticity, in syt-17 KOs. a Colocalization of syt17 (Halo fusion construct, magenta), early endosomes (Rab5-GFP, green), and AMPA receptors (anti-GluR, blue) in dendritic spines of a 14 DIV hippocampal neuron. The traces to the right represent normalized fluorescence from the indicated 3 µm linescan. b Left: L-Glu was pressure-applied to apical dendrites. Middle: Representative traces of AMPAR Glu response at −70 and +40 mV in WT, KO, and KO+rescue neurons. Right: I–V plot showing current as a function of holding voltage. The amplitude of postsynaptic responses in syt-17 KO neurons was uniformly increased (except near the reversal potential), and this effect was rescued by expression of exogenous syt-17. c Left: GluR2-pHluorin was exogenously expressed in WT and KO neurons. Right: Spine fluorescence of GluR2-pHluorin was quantified in ACSF of pH 7.4 (extracellular pH) and 5.5 (vesicular pH), and in the presence of NH4Cl to unquench all pHluorin. Traces show intensity at indicated linescans. d Surface expression of GluR2-pHluorin was increased in syt-17 KO (t21 = 2.417, p = 0.02, r2 = 0.218, meanwt = 51.95 ± 3.27% surface fraction, meanko = 64.01 ± 3.81, Nwt = 12 and Nko = 11 neurons). e Rab5-GFP was expressed in WT and KO neurons. f KO neurons had significantly fewer early endosomes per unit dendrite (t35 = 4.529, p < 0.001, r2 = 0.37, meanwt = 5.75 ± 0.43 early endosomes per 10 μm dendrite, meanko = 3.42 ± 0.29, Nwt = 18 and Nko = 19 neurons). Scale bar indicates 10 μm. All experiments were performed on 3–4 independent preparations of animals. g Chemical long-term depression (LTD) along the Shaffer collaterals in hippocampal slices. Right: Representative field excitatory postsynaptic potentials (fEPSPs, 50% of maximal) before (solid lines) and after (dashed lines) five-minute NMDA application. h KO slices fail to exhibit LTD following the induction protocol (p = .012 two-sample t-test post-induction). Experiments were performed on slices from five animals per genotype. All error bars indicate S.E.M.s


Ca2+ sensors for minis

In this work, we discovered that spontaneous glutamate release (mEPSCs) was disrupted by the genetic loss of Doc2α while spontaneous GABA release (mIPSCs) was selectively reduced by the loss of Doc2β. This cell-type specific phenotype was due to deferential expression patterns of these two isoforms, as demonstrated by RNAScope in situ hybridization (A). Furthermore, mutant Doc2α and β constructs that were unable to bind calcium, were also unable to rescue spontaneous release (B). Together, this work demonstrates that Doc2 promotes spontaneous fusion via calcium binding, and that Doc2 isoforms act in a cell-type dependent manner (C). In contrast, syt-1 only promoted spontaneous GABA, and not glutamate, release in a Ca2+-depending manner.



Fusion Clamp

In this work, we discovered that C2B domain of syntapotagmin (syt) 1, in constructs that lack the C2A domain, generates a “superclamped” phenotype that greatly impedes action-potential dependent (A) and action-potential independent synaptic vesicle fusion. Excitingly, this property of the C2B domain of syt1 was independent of complexin; KD of complexin had no effect on this “superclamped” phenotype (B and C).





Xue et al. PNAS 2018 Fig. 2.
Ca2+•Doc2β mediates munc13-1 translocation to the plasma membrane to drive augmentation. (A) WT-Doc2β, Doc2βclm in which two acidic Ca2+ ligands were neutralized to disrupt Ca2+ binding to the C2B domain (clm; Ca2+ ligand mutant), and Doc2βMID-scrm in which the MID domain was scrambled, were expressed in neurons. (B) Upon depolarization with 60 mM KCl, both munc13-1–mCherry (magenta) and WT Doc2β-GFP (green) translocated to the plasma membrane. (Scale bar: 10 μm.) (C) Magnified images are shown. (D) The ratio of fluorescence intensity (plasma membrane/cytosol) was quantified and normalized to baseline, as detailed in SI Appendix, Fig. S3, and plotted versus time. (E) Upon depolarization, Doc2βclm-GFP neither translocates to the plasma membrane nor recruits munc13-1–mCherry (Upper); Doc2βMID-scrm-GFP translocates but was also unable to recruit munc13-1–mCherry (Lower). (F) Translocation data from E were quantified and plotted. (G) Normalized peak amplitudes of EPSCs before and after the augmentation protocol, as described in , recorded from Doc2α/β DKO neurons expressing Doc2βclm (Upper) or Doc2βMID-scrm (Lower) are plotted as mean ± SEM versus time. Data from WT and Doc2α/β DKO neurons () are shown again as controls. Both Doc2β mutants failed to rescue synaptic augmentation.