<p>(A) Model for effects of EphB2 extracellular phosphorylation on the EphB—NMDAR interaction. (B) Experimental approach using intrathecal injection of lentivirus (LV) to avoid effects outside of the spinal cord. Examination of injection site revised transduced neurons within the dorsal horn. (C) Neuronal viral transduction was confirmed with NeuN labeling (marker for neuronal nucleus) surrounded by EphB2- enhanced yellow fluorescent protein (EYFP) wild type (WT). The left panel was stained for EphB2-EYFP with α-GFP. The middle panel was stained for NeuN. The right panel shows the merged image. Control enhanced green fluorescent protein (EGFP), EphB2-EYFP Y504E, and EphB2-EYFP Y504F also show the distribution surrounding NeuN. Scale bar = 50 μm. (D) Distribution of GluN1 and vGlut2 in the dorsal horn of the adult mouse spinal cord, expressing control EGFP, EphB2-EYFP WT, or Y504E or Y504F mutants. The left panel shows GluN1 (cyan). The middle panels show vGlut2 (red) to mark superficial layers of dorsal horn. The right panel shows a merged image of GluN1 and vGlut2 staining. The dashed yellow line indicates superficial layers of dorsal horn. Scale bar = 50 μm. (E) Quantification of the effects of expression of EphB2 WT and Y504E and Y504F mutants on GluN1 intensity in superficial layers of the dorsal horn of the adult mouse spinal cord (*<i>p <</i> 0.05, ***<i>p <</i> 0.05, ANOVA followed by Tukey’s range test, 17 sections from 3 mice for control, 18 sections from 3 mice for EphB2 WT, 22 sections from 4 mice for Y504E mutant, and 24 sections from 4 mice for Y504F mutant). (F) Quantification of the effects of expression of EphB2 WT and Y504E and Y504F mutants on vGlut2 intensity in superficial layers of the dorsal horn of the adult mouse spinal cord. (***<i>p <</i> 0.0005, ****<i>p <</i> 0.0001, ANOVA followed by Tukey’s range test, 17 sections from 3 mice for control, 18 sections from 3 mice for EphB2 WT, 22 sections from 4 mice for Y504E mutant, and 24 sections from 4 mice for Y504F mutant). AU, arbitrary unit.</p
