Fig. 6
NMDA-CB2R heteromer levels and functionality in adult APPSw/Ind mice brain slices. A–F Expression of NMDA-CB2R heteromers in adult brain (brain cortex) slices from control (B, C) and APPSw/Ind transgenic mice (D, E) as determined by PLA (see the “Materials and Methods” section) using specific primary antibodies against the GluN1 subunit and against the CB2R receptor. For negative control, only the anti-GluN1 antibody was used (A). Confocal microscopy images (stacks of 3 consecutive panels) show heteroreceptor complexes as red clusters; nuclei were stained with Hoechst (blue). Three independent experiments were performed using, for each condition, 5 preparations per session. Scale bar: 40 μm. Bar graphs (F) show the amount of red dots/cell in APPSw/Ind mice and control animals (*p < 0.05; One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test were used for statistical analysis). MAPK phosphorylation assays were performed in control (G) and APPSw/Ind (H) transgenic mice brain slices. Slices were treated with selective agonists (15 μM NMDA, and/or 100 nM JWH-133). When indicated, slices were pretreated with the CB2R selective receptor antagonist (1 μM SR-144528). Results are expressed as a percentage over basal and are the extracellular signal regulated (ERK) 1/2 phosphorylation mean ± SEM signals of three independent experiments performed in triplicates. ANOVA summary: F F: 10.4, p<0.001, G F: 7.7, p<0.001, H F: 1.2 , n.s