<p>(A) Experimental approach for the figure is illustrated. (B) Mean traces of whole-cell patch-clamp recording at 50 mV show the evoked and miniature excitatory postsynaptic currents (EPSCs) of primary rat cortical day in vitro (DIV) 21–23 neurons expressing enhanced green fluorescent protein (EGFP) without or with EphB2 wild type (WT) and Y504 mutants induced by light stimulation of neurons that express optogenetic light-sensitive channels (channelrhodopsin-2). (C) Effects of overexpression of EphB2 WT and Y504 mutants on the mean amplitude of evoked, spontaneous, and miniature EPSCs (30 milliseconds after the evoked EPSC peak) in mature cultured neurons. Overexpression of EphB2 WT or Y504E significantly increased amplitude of the NMDAR-dependent component of evoked EPSCs compared to the control or the Y504F mutant (****<i>p <</i> 0001, ANOVA followed by Fisher’s exact test, <i>n</i> = 375, 332, 581, and 200 events for control, EphB2 WT, Y504E, and Y504F, respectively). In addition, the amplitude of Y504E-overexpressing neurons was significantly higher than that of WT-overexpressing neurons (**<i>p <</i> 0.02, ANOVA followed by Fisher’s exact test). (D) Representative sample traces of whole-cell patch-clamp recording at 50 mV show that NMDAR-dependent currents in control neurons and neurons expressing EphB2 WT and Y504E, but not Y504F, are greatly reduced by GluN2B-specific antagonist Ro 25–6981 (Ro25). (E) Cumulative probability histogram of miniature EPSC (mESPC) amplitude for Y504E before and after application of Ro25 (2.5 μM). Inset: mean traces of mEPSCs after treatment with Ro25 (<i>p <</i> 0.001, Kolmogorov—Smirnov [K–S] test, <i>n</i> = 720 for Y504E and <i>n</i> = 427 for Y504E + Ro25). Vertical scale bar = 20 picoamperes (pA); horizontal scale bar = 10 milliseconds. (F) Cumulative probability histogram of mEPSC amplitude Y504F as in (E) (<i>p</i> = 0.0775, K–S test, <i>n</i> = 414 for Y504F and <i>n</i> = 404 for Y504F + Ro25). Vertical scale bar = 10 pA; horizontal scale bar = 10 milliseconds. (G) Model depicting 2 scenarios of VGLUT1 (blue), EphB2 (red), and GluN1 (green) localization at dendritic spines. (H) High-contrast images of dendrites of DIV 21 cortical neurons expressing EGFP, EphB2 short hairpin RNA (shRNA), and RNA interference (RNAi)-insensitive FLAG-tagged EphB2 WT, EphB2-Y504E, or EphB2-Y504F. Top panels show confocal EGFP staining (white), second panels show stimulated emission depletion (STED) EphB2 staining (red), third panels show STED GluN1 staining (green), fourth panels show confocal VGLUT1 (presynaptic marker) staining (blue), and bottom panels show merged images. White arrows indicate examples of triple colocalization of EphB2, GluN1, and VGLUT1. Scale bar = 1 μm, 0.5 μm inset. (I) Quantification of the effects of expression of EphB2 Y504 mutants on localization of VGLUT1 to dendritic spines in DIV 21 rat cortical neurons transfected at DIV 14. Graph shows fraction of spines containing VGLUT1 (not significant, ANOVA followed by Fisher’s exact test). (J) Quantification of the effects of expression of EphB2 Y504 mutants on colocalization with GluN1 in dendritic spines in DIV 21 rat cortical neurons transfected at DIV 14. Graph shows percentage of spines containing colocalized EphB2 and GluN1 puncta as defined by Fig 5G (*<i>p <</i> 0.05, ***<i>p <</i> 0.005, ANOVA followed by Fisher’s exact test). (K) Quantification of the effects of expression of EphB2 Y504 mutants on colocalization with GluN1 at synaptic sites in DIV 21 rat cortical neurons transfected at DIV 14. Graph shows percentage of spines containing triple colocalized puncta as defined by Fig 5G (***<i>p <</i> 0.005, ****<i>p <</i> 0.0005, ANOVA followed by Fisher’s exact test).</p
