Synaptic transmission and plasticity



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


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).



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.