Syt7 – 3 Processes
SYT7 influences presynaptic neurotransmitter release during short-term synaptic plasticity.
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
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.
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).