Figure 2. Elevated intracellular Ca²⁺ and Ca²⁺-dependent signaling in the GluN3A KO brain.

Intracellular free Ca²⁺ concentration [Ca²⁺]_i and Ca²⁺-dependent signaling proteins were measured in the hippocampus and cortex of WT and GluN3A KO mice of 6–12-month old. A. In situ Ca²⁺ imaging using Fura-2AM was performed under a two-photon microscope. Images show Fura-2AM labeled CA1 and DG neurons in brain slices from WT and GluN3A KO mice. B. The Fura-2AM ratio measurements of [Ca²⁺]_i in WT and GluN3A KO neurons at 6–7 month and 10–12-month old n=4 and 5 for WT and KO mice of 6–7-months group, respectively; n=4 animals for both WT and KO mice of 10–12-months group; *P<0.05 vs. WT; Mann-Whitney U test). C. The percentage of CA1 and DG cells with [Ca²⁺]_i above and below 200 nM in total cells measured. CA1 neurons of 6–7-months old GluN3A KO mice showed a strong trend of greater percentage of high Ca²⁺ neurons compared to WT cells (P=0.05). At the age of 10–12-month old, the percentage of high Ca²⁺ cells was significantly larger than that in WT mice (animal numbers were the same as in B; *P<0.05 vs. WT; Chi-Squared test). D. Western blotting of hippocampus and cortex tissues from aging WT and GluN3A KO mice (10–12-month old). E and F. Summary bar graphs of Western blot data. In the hippocampus, the expression of pCaMKII and PKC-α increased significantly in GluN3A KO mice while CaMKII and calpain showed trends of increase (E). In the cortex of GluN3A KO mice, the expression of CaMKII and pCaMKII was significantly higher while PKC-α remained tentatively higher compared to levels in WT (n=6 animals per group; *P<0.05 vs. WT, **P<0.01 vs. WT; Two-way ANOVA).