Fig 1.
Electron micrograph of an asymmetric synapse from dissociated hippocampal culture labeled for Shank3 with ab2.
Labeling intensity at the PSD is measured by counting all particles within the marked area 120 nm below the postsynaptic membrane, divided by the length of the PSD, and expressed as number of labels/μm PSD. Distance of label from the postsynaptic membrane is measured from the center of the particle to the outer edge of the membrane (white arrow). Scale bar = 0.1 μm.
Fig 2.
(A, B) Western immunoblots with two antibodies for Shank3 (ab1 and ab2) comparing homogenate (H), synaptosome (Syn), and PSD fractions from cerebral cortex of rat show significant enrichment of Shank3 in the PSD fraction. Ten micrograms of protein was loaded into each lane for the homogenate and synaptosome, and five micrograms of protein was loaded for the PSD fraction. (C) Western immunoblots with the two Shank3 antibodies reveal a common set of bands in the PSD fraction. After transfer, the membrane was cut in the middle of the lane to separately probe with the two antibodies.
Fig 3.
Electron micrographs from perfusion fixed-mouse brain showing immunogold labeling for Shank3 using ab1 (A-E) and ab3 (D, inset). PSDs (arrows) were labeled in all regions examined, including hippocampus [stratum radiatum (SR) of the CA1 region (A), mossy fiber synapses in the CA3 region (B)], cerebral cortex (C), and the outer molecular layer (D) and granular layer (E) of the cerebellum. Scale bar = 0.5 μm.
Fig 4.
(A-D) Electron micrographs of asymmetric synapses from dissociated hippocampal cultures labeled for Shank3 using ab2. Scale bars = 0.1 μm (A and B share the same scale bar, C and D share the same scale bar). Labeling intensity increased upon 2 min treatment with either high K+ (B vs. A) or NMDA (D vs. C). (E, F) Bar graphs represent combined data (mean ± SEM) from S1 and S2 Tables. (G-J) Histograms from a typical experiment depicting frequency distribution of gold particles as a function of distance from the postsynaptic membrane. Median distance of label from postsynaptic membrane increased after high K+ (H vs. G) or NMDA treatment (J vs. I), indicating preferential recruitment of Shank3 molecules to the distal layer of the PSD.
Fig 5.
(A-D) Electron micrographs of asymmetric synapses labeled for Shank3 using ab1. Hippocampal cultures were pre-incubated for 1 hr in media with or without zinc and subsequently treated for another 2 min in the same media with or without NMDA, as indicated. After 1 hr zinc incubation (B) or 2 min NMDA treatment (C), labeling intensities were higher than that of control (A). Upon pre-incubation with zinc followed by NMDA treatment (D), labeling intensity was even higher than after either treatment alone. Scale bar = 0.1 μm. (E) Bar graphs represent combined data (mean ± SEM) from S2 Table. (F-I) Median distance of gold particles from the postsynaptic membrane increased upon NMDA treatment, either in the absence (H) or presence of zinc (I), but not in samples incubated with zinc only (G) when compared to control (F).
Fig 6.
(A-D) Electron micrographs of asymmetric synapses labeled for Shank3 using ab2. Hippocampal cultures were pre-incubated for 1 hr in control medium or in media containing zinc or zinc+APV, followed by a 2 min treatment in the same media with or without NMDA, as indicated. Scale bar = 0.1 μm. (E) Bar graphs represent combined data (mean ± SEM) from S5 Table. APV appeared to block NMDA-induced, but not zinc-induced, increase in Shank3 labeling intensity at the PSD. (F-I) Laminar distribution and median distances of gold particles from the postsynaptic membrane were similar in control (F) and zinc-incubated samples (G). APV (I) blocked the increase in median distance in zinc+NMDA samples (H).
Table 1.
Changes in index of curvature of PSDa.
Fig 7.
(A-D) Electron micrographs of asymmetric synapses labeled for CaMKII. Experimental conditions were as described in Fig 5. Label for CaMKII was dispersed in the postsynaptic cytoplasm under basal control conditions (A) or after zinc incubation (C), and the curvature of the PSD was typically flat. After NMDA (B) or zinc+NMDA (D) treatment, label for CaMKII was accumulated at the PSD, and the curvature of PSD was typically arched into the presynaptic terminal. Scale bar = 0.1 μm. (E) Bar graphs represent combined data from S6 Table. Zinc has no effect on the labeling intensity of CaMKII at the PSD either under basal conditions or upon treatment with NMDA.
Fig 8.
(A, B) Electron micrographs of asymmetric synapses labeled for Shank3 with ab2. Hippocampal cultures were pre-incubated for 1 hr with or without zinc, then exposed to NMDA for 2 min. Samples were either fixed immediately or after NMDA washout under two different protocols (30 min in control media or 5 min in EGTA-containing Ca2+-free media). Distribution patterns of label are different in samples that were incubated in the absence (A) or presence (B) of zinc, 30 min after NMDA washout. Scale bar = 0.1 μm. (C) Bar graphs represent combined data (mean ± SEM) from S7 Table. NMDA-induced increase of label for Shank3 is reversed in the absence of zinc, but largely retained in the presence of zinc. (D, E) Laminar distribution and median distances of gold particles from the postsynaptic membrane were also significantly different. NMDA-induced preferential increase of Shank3 in the distal layer of the PSD is reversible after 30 min recovery in control media (D), but maintained in the presence of zinc (E).