Figure 4

VEGF and NMDA administration induced signaling pathways and AMPAR synaptic delivery in hippocampal neurons. (a) Immunoblots of total and phosphorylated forms of CaMKII and PKCγ in PSDs upon treatments. PSD and non-PSD fractions were purified from hippocampal cultures exposed to control, VEGF, NMDA and NMDA+VEGF. (b) Analysis of protein expression levels normalized to control showing the increase in total and active CaMKII (pT286-CaMKII) induced by NMDA and NMDA+VEGF treatments compared with control, which reflects the translocation of these two forms of CaMKII to PSD. Note that the phosphorylated to total PKCγ ratio was also significantly increased in PSDs upon exposition to NMDA+VEGF compared with control. Respectively n=9 independent experiments for CaMKII and n=7 for PKCγ; significant differences between conditions were determined using a Kruskal–Wallis with a post hoc test and are indicated by (*) for P<0.05; (**) for P<0.01. (c) Representative dendritic immunostaining in clusters for GluA1 (green), PSD95 (red) and synapsin (magenta), as well as for GluA1/synapsin and GluA1/PSD95 co-localization (white) (scale bar, 5 μm). (d) Graphs showing the density of GluA1 clusters, respectively, at the dendritic surface and at synaptic sites defined by PSD95 or synapsin labeling. Values were normalized to control and analyzed using a one-way ANOVA and when appropriate a Bonferroni post hoc test. Following NMDA and VEGF treatment, surface GluA1 clusters increased in density compared with control and VEGF alone (with NMDA+VEGF 130±13.8% versus control 100±6.1%, n=10–11, **P<0.01; versus VEGF 90.2±6.2%, n=10–15, **P<0.01) and accumulated at synapses compared with the other conditions (for PSD95 labeled synapses: with NMDA+VEGF 148.9±14.6% versus control 100±7.4%, n=10–11, **P<0.01; versus VEGF 99±9.7%, n=10–15, **P<0.01; versus NMDA 105.5±7.3%, n=10–15, *P<0.05). Data shown are derived from three independent experiments.

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