A representative EM image of a synapse at 30,000x reveals the presynaptic terminal, presynaptic vesicles of the active zone, the electron-dense postsynaptic density, and the postsynaptic spine (A). Outlines of the PSD and pre and postsynaptic membranes illustrate bin assignments using SynBin image analysis software (B and C). The postsynaptic spine is divided into 0-30 nm synaptic, 30-60 nm PSD, and nonsynaptic compartments perpendicular to the postsynaptic membrane (B). This analysis reflects the laminar localization of proteins at the synapse. Bins are redesigned to reflect the lateral arrangement of receptor subunits and associated proteins across the synaptic surface (C). The PSD remains divided into 0-30 nm synaptic and 30-60 nm PSD bins, however, the 0-30 nm synaptic bin has been subdivided into middle and lateral 50% bins and an extrasynaptic bin extending 125 nm from either edge of the PSD has also been included. Scale bar = 100 nm.

(A) Electron micrographs show representative NR2A labeling in the synapse (outlined) of a PSD-95^PDZ12 mutant and a wild type littermate. Lateral distribution of NR2A across the synapse is quantified in the adjacent graph where the 0-30 nm synaptic bin is subdivided into 10% bins (inset). For comparison, all bins are normalized to the bin with the highest label. The lateral profile of NR2A in PSD-95^PDZ12 mutants (white diamonds) and wild type littermates (black squares) display no genotype differences, but reveals a fairly uniform distribution across the synapse.
(B) Lateral distribution is further grouped into middle 50% and lateral 50% bins, with a proportional extrasynaptic (ES) 250 nm bin for comparison. All bins are normalized to wild type middle 50% synaptic bins where labeling is highest within experiments and across groups. In PSD-95^PDZ12 mutant mice (white bars), there is no change in NR2A localization to middle, lateral, or extrasynaptic bins compared to wild type littermates (black bars). There is, however, significantly greater localization in both middle and lateral bins compared to extrasynaptic bins (Middle:ES; ***p < 0.0001 for both (−/−) and (wt); Lateral:ES; ***p < 0.0001 (−/−) and **p = 0.003 (wt); ANOVA with post hoc bonferroni test), but no difference between the two bins.
(C) Comparative EM images depict lateral displacement of NR2B in the synapse (outlined) of a PSD-95^PDZ12 mutant compared to a wild type littermate. Quantification of NR2B distribution reveals a significant 16% decrease specifically in the lateral 10-40% of the synapse (*p < 0.05) in PSD-95^PDZ12 mutants (grey diamonds) compared to wild type littermates (black squares). Individually, however, only the lateral 10-20% bin reaches significance (*p < 0.05), with a 15% decrease. NR2B distribution at the synapse is typically dumbbell-shaped, with two peaks on either side of the centerline, but in the absence of PSD-95 this shape is lost.
(D) NR2B is reduced 15% in the lateral 50% of the synapse (**p = 0.012) in PSD-95^PDZ12 mutants (grey bars) compared to wild type littermates (black bars). Comparisons across regions show significantly greater NR2B localization to the middle 50% than to the lateral 50% (Middle:Lateral; **p = 0.003 (−/−) and ***p < 0.0001 (wt)) or the extrasynaptic 250nm bin (Middle:ES; ***p < 0.0001 for both (−/−) and (wt); Lateral:ES; ***p < 0.0001 for both (−/−) and (wt); ANOVA with post hoc bonferroni test).
(E) Comparative EM images illustrate synaptic reduction of NR2A (outlined) in a CaMKII^T305D mutant compared to a wild type littermate. Although significance is only attained in the lateral 0-20% bin (p < 0.05), as a 23% reduction, lateral NR2A distribution reveals a separation in all bins across the synapse between CaMKII^T305D mutant mice (white diamonds) and wild type littermates (black squares). As in PSD-95^PDZ12 mutants, NR2A is evenly distributed across the synapse.
(F) Quantification of NR2A distribution shows a cumulative 16% reduction in the middle 50% of the synapse (*p < 0.05) in CaMKII^T305D mutants (white bars) compared to wild type littermates (black bars). Bin comparisons show NR2A localization is significantly greater in both middle and lateral bins compared to extrasynaptic bins (Middle:ES; ***p < 0.0001 for both (−/−) and (wt); Lateral:ES; ***p < 0.0001 for both (−/−) and (wt); ANOVA with post hoc bonferroni test), however, NR2A is reduced to a greater extent in the lateral 50% compared to the middle 50% bin in CaMKII^T305D mutants (Middle:Lateral; **p = 0.005 (−/−); ANOVA with post hoc bonferroni test).
(G) Representative electron micrographs show similar NR2B localization in the synapse (outlined) of a CaMKII^T305D mutant and wild type littermate that is clustered laterally. Lateral NR2B distributions in synapses of CaMKII^T305D mutant (grey diamonds) and wild type littermates (black squares) exhibit no genotype differences, however, NR2B is highly localized to medial regions of the synapse, rapidly declining laterally, but still retaining the dumbbell-shaped curve characteristic of NR2B distribution. Scale bar = 100 nm.
(H) There is no difference in NR2B localization to middle, lateral, or extrasynaptic bins between CaMKII^T305D mutants (grey bars) and wild type littermates (black bars). There is, however, significant differences in localization between middle, lateral, and extrasynaptic bins, decreasing in that order (Middle:Lateral; ***p < 0.0001 for both (−/−) and (wt); Middle:ES; ***p < 0.0001 for both (−/−) and (wt); Lateral:ES; ***p < 0.0001 for both (−/−) and (wt); ANOVA with post hoc bonferroni test). Error bars represent means ± SEM.

NMDARs are composed of an obligatory NR1 subunit in combination with NR2A, NR2B, or other NR subunit in a heterodimeric, heterotrimeric, or possibly heteromultimeric receptor. NR2A and NR2B-containing NMDARs are interspersed across the synaptic membrane, interacting with a multitude of proteins in the PSD that can stabilize the receptors at the synapse or signal a cascade of events. The PSD is comprised primarily of αCaMKII, PSD-95, and, to a lesser degree, other MAGUKs, depending on developmental stage and synaptic activity (Sans et al., 2000, Chen et al., 2005). They are, therefore, considered important regulators of NMDAR function and expression at the synapse. We propose that αCaMKII is important for synaptic stability of NR2A-containing NMDARs in the absence of activity but that with activity, αCaMKII may increase targeting of NR2A-containing NMDARs to the synapse. This potential interaction may explain the reduced LTP in CaMKII^T305D mutant mice in the absence of αCaMKII (outlined in Red). PSD-95, on the other hand, may be important for the synaptic stability of a subset of NR2B-containing NMDARs laterally at the synapse where PSD-95-dependent regulation is thought to mediate LTD (outlined in Blue). The absence of PSD-95 from the PSD may, therefore, be able to explain the inability of PSD-95^PDZ12 mutant mice to induce LTD. In addition to PSD-95, there exists overlapping, functionally redundant MAGUKs which may be important in stabilizing different populations of NMDARs at the synapse.

(A) Total NR2A (white bars) and NR2B (grey bars) labeling in presynaptic terminals and postsynaptic spines averaged per group, in PSD-95^PDZ12 mutants compared to wild type littermates (black bars). Labeling is normalized to respective wild type postsynaptic bins to demonstrate similarity in labeling between NR2A and NR2B. No gross changes are evident in NR2A or NR2B labeling between mutant and wild type littermates in axons or spines, but there is significantly higher postsynaptic as compared to presynaptic NR2A and NR2B labeling for both mutant and wild type mice (***p < 0.0001, ANOVA with post hoc bonferroni test).
(B) Total NR2A and NR2B labeling in presynaptic terminals and postsynaptic spines averaged per group, in CaMKII^T305D mutants compared to wild type littermates. No genotype differences are observed for NR2B labeling, however, NR2A labeling is reduced 18% in postsynaptic spines of CaMKII^T305D mutant mice compared to wild type littermates (**p = 0.006). Presynaptic labeling is consistently lower for both NR2A and NR2B labeling in both mutant and wild type littermates (***p < 0.0001, ANOVA with post hoc bonferroni test).
(C) The postsynaptic spine is partitioned into a membrane-associated 0-30 nm synaptic PSD bin, a deeper layer, cytoskeletal-associated 30-60 nm PSD bin, and a nonsynaptic, cytoplasmic bin to reflect the functional localization and stability of proteins at the synapse. All bins are normalized to the wild type 0-30 nm synaptic bin where NMDA receptors should be primarily localized. Synaptic localization of NR2A in PSD-95^PDZ12 mutants (white diamonds) is no different than in wild type littermates (315 synapses in n = 3 PSD-95^PDZ12 mice, and 332 synapses in n = 3 wild type littermates). 0-30 nm synaptic : 30-60 nm PSD (SYN:PSD) and 0-30 nm synaptic : nonsynaptic (SYN:NONSYN) bin comparisons show that NR2A subunits are typically localized at or near the synaptic membrane (SYN:PSD; ***p < 0.0001 (−/−) and **p = 0.005 (wt); SYN:NONSYN; **p = 0.004 (−/−) and *p < 0.05 (wt); ANOVA with post hoc bonferroni test). A reference figure illustrating 0-30 nm synaptic, 30-60 nm PSD, and nonsynaptic bins (inset).
(D) NR2A is reduced 16% in the 0-30 nm synaptic bin (***p < 0.001) and 27% in the nonsynaptic, cytoplasmic bin of the spine (**p = 0.006) in CaMKII^T305D mutants (white diamonds) compared to wild type littermates (337 synapses in n = 7 CaMKII^T305D mice, and 286 synapses in n = 6 wild type littermates). NR2A labeling is significantly greater at the synapse than at the PSD (SYN:PSD; ***p < 0.0001 for both (−/−) and (wt); ANOVA with post hoc bonferroni test) but no different than nonsynaptic labeling for both CaMKII^T305D mutants and wild type littermates.
(E) NR2B is reduced 12% in the 0-30 nm synaptic bin (*p < 0.05) in PSD-95^PDZ12 mutants (grey diamonds) compared to wild type littermates (241 synapses in n = 3 PSD-95^PDZ12 mice, and 244 synapses in n = 3 wild type littermates). NR2B distribution is predominantly synaptic (SYN:PSD; ***p < 0.0001 for both (−/−) and (wt); SYN:NONSYN; ***p < 0.0001 for both (−/−) and (wt); ANOVA with post hoc bonferroni test).
(F) There is no change in NR2B localization in synaptic bins between CaMKII^T305D mutant (grey diamonds) and wild type littermates (334 synapses in n = 7 CaMKII^T305D mice, and 278 synapses in n = 6 wild type littermates). NR2B distribution, however, is also highly synaptic in this group (SYN:PSD; ***p < 0.0001 for both (−/−) and (wt); SYN:NONSYN; ***p < 0.0001 for both (−/−) and (wt); ANOVA with post hoc bonferroni test). Error bars represent means ± SEM.

(A) Total αCaMKII (white bars) and PSD-95 (grey bars) localization in presynaptic terminals and postsynaptic spines averaged per group, in PSD-95^PDZ12 mutant and CaMKII^T305D mutant compared to their respective wild type littermates. Labeling is normalized to respective wild type postsynaptic bins for comparison between αCaMKII and PSD-95. There are no genotype differences in αCaMKII or PSD-95 labeling between mutant and wild type littermates, however, postsynaptic labeling is consistently higher than presynaptic labeling (**p = 0.003, ***p < 0.0001; ANOVA with post hoc bonferroni test).
(B) Synaptic bins are normalized to the wild type 0-30 nm synaptic bin, consistent with synaptic NR2 subunit localization. In PSD-95^PDZ12 mutant mice (white diamonds), no change is associated with αCaMKII localization in synaptic bins compared to wild type littermates (black squares), however, αCaMKII labeling reveals a preferentially nonsynaptic arrangement (SYN:NONSYN; **p = 0.007 (wt); ANOVA with post hoc bonferroni test). A reference figure illustrating superficial 0-30 nm synaptic PSD, deeper layer 30-60 nm PSD, and nonsynaptic, cytoplasmic bins (inset).
(C) In CaMKII^T305D mutant mice (grey diamonds), no changes are observed in PSD-95 localization in synaptic bins compared to wild type littermates (black squares). The highly synaptic distribution of PSD-95 resembles that of NR2A and NR2B (SYN:PSD; ***p < 0.0001 for both (−/−) and (wt); SYN:NONSYN; ***p < 0.0001 for both (−/−) and (wt); ANOVA with post hoc bonferroni test).
(D) Electron micrographs show representative αCaMKII labeling in the synapse of a PSD-95^PDZ12 mutant and a wild type littermate that is equally distributed between synaptic (outlined) and nonsynaptic bins of the spine. The lateral profile of αCaMKII reveals a fairly homogeneous, cosine-function-like distribution across the synapse, and there are no genotype differences between PSD-95^PDZ12 mutant mice (white diamonds) and wild type littermates (black squares) in any of the 10% bins. For comparison, all bins are normalized to the bin with highest label. A reference figure corresponding to 10% bins within the 0-30 nm synaptic bin (inset). Scale bar = 100 nm.
(E) There is no difference in αCaMKII localization to middle, lateral, or extrasynaptic bins in PSD-95^PDZ12 mutant mice (white bars) compared to wild type littermates (black bars). Localization to middle and lateral synaptic bins is significantly higher than to extrasynaptic bins (Middle:ES; *p < 0.05 (−/−) and ***p < 0.0001 (wt); Lateral:ES; **p = 0.005 (−/−) and ***p = 0.0001 (wt); ANOVA with post hoc bonferroni test), but αCaMKII is relatively evenly distributed between middle and lateral bins. Bins are normalized to the middle 50% bin, consistent with lateral NR2 subunit localization.
(F) Electron micrographs show representative PSD-95 labeling in the synapse of a CaMKII^T305D mutant and a wild type littermate that is primarily synaptic and closely associated to the synaptic membrane(outlined). The lateral profile of PSD-95 reveals highly synaptic labeling in medial regions of the synapse, in close alignment to that of NR2B. No genotype differences were found for PSD-95 localization between CaMKII^T305D mutant mice (grey diamonds) and wild type littermates (black squares). Scale bar = 100 nm.
(G) No difference in PSD-95 localization to middle, lateral, or extrasynaptic bins in CaMKII^T305D mutants (grey bars) compared to wild type littermates (black bars) is evident. There is, however, significantly higher localization in middle compared to lateral bins and between middle and lateral synaptic bins compared to extrasynaptic bins (Middle:ES; ***p < 0.0001 for both (−/−) and (wt); Lateral:ES; ***p < 0.0001 for both (−/−) and (wt); ANOVA with post hoc bonferroni test). Error bars represent means ± SEM.