Supplementary Figure 8: Synaptic localization of PSD-95-GFP in organotypic brain slices and cultured neurons.
From: Anchoring and synaptic stability of PSD-95 is driven by ephrin-B3
(a) Representative images of cortical neuron apical dendrites in organotypic brain slices. Images show examples of wild type, ephrin-B3 null (Efnb3–/–), Efnb3–/– + flag-ephrin-B3 rescue (flag-eB3) slices, or wild type slices transfected with shRNA targeting ephrin-B3, and shRNA plus flag-eB3 rescue. Neurons were biolistically transfected with the indicated constructs (Transfections) along with PSD-95-GFP and tdTomato. Organotypic slices were fixed 4 days after transfection, re-sectioned, and immunostained for the presynaptic marker vGlut1. Arrows indicate PSD-95-GFP puncta that co-localize with vGlut1 puncta; open arrowheads indicate PSD-95-GFP puncta that do no co-localize with vGlut1 puncta. Scale bar: 2 µm.
(b) Quantification of the fraction of PSD-95-GFP puncta that co-localize with vGlut1 puncta in the transfection conditions indicated in S8a. The fraction of PSD-95-GFP puncta that co-localize with vGlut1 puncta is not significantly different regardless of the presence or absence of ephrin-B3. Data are represented as mean ± s.e.m., p = 0.1522, one-way ANOVA, F (4, 73) = 1.732. Control, n = 14; Control (Efnb3–/–), n = 9; flag-eB3 (Efnb3–/–), n = 16; eB3 shRNA, n = 33; eB3 shRNA + flag-eB3, n = 6 neurons from at least three independent experiments.
(c) PSD-95-GFP appears punctate in DIV0 rat cortical neurons co-transfected with PSD-95-GFP and either control or EphB2 shRNA constructs. At DIV10 neurons were imaged and the density and organization of PSD-95-GFP puncta were analyzed.
(d) Quantification of the density of PSD-95-GFP puncta in neurons transfected with PSD-95-GFP and either control or EphB2 shRNA. EphB2 knockdown caused a significant reduction in the density of PSD-95-GFP puncta. ***p < 0.0001, t = 6.199 df = 37.
(e) Quantification of the intensity of GFP puncta in neurons transfected with PSD-95-GFP and either control or EphB2 shRNA. PSD-95-GFP formed distinct puncta in both control and EphB2 shRNA transfected neurons that did not differ in intensity, p = 0.2059, t = 1.288 df = 37.
(f) Quantification of the intensity of GFP in dendritic shafts of neurons transfected with PSD-95-GFP and either control or EphB2 shRNA. EphB2 shRNA did not cause a detectable increase in GFP fluorescence in dendritic shafts, p = 0.9903, t=0.01230 df = 37.
(g) Quantification of the ratio of the intensity of GFP in dendritic shafts to puncta in neurons transfected with PSD-95-GFP and control or EphB2 shRNA. Ratio of GFP fluorescence in the shaft relative to the GFP fluorescence in puncta was not significantly different between control and EphB2 transfected neurons, p = 0.3736, t = 0.9006 df = 37. For graphs in S8d-g, data are represented as mean ± s.e.m. Control, n = 18 neurons; EphB2 shRNA, n = 21 neurons from two independent biological replicates. Un-paired t-test (two-tailed) was used to determine statistical significance.
