Fig 2: Functional NMDA receptors are localized on the surface of HPASMCs. Immunofluorescence staining was performed on non-permeabilized HPASMCs. Both GluN1 and GluN2 (2B and 2D) subunits were detected on the surface of HPASMCs (A,B). GluN1 co-localized with GluN2B (A) and GluN2D (B) on the surface of HPASMCs, respectively. Results are representative images taken from at least three separate experiments. Scale bar = 100 µM. (C,D) are representative scatter plots of each fluorescent pixel from confocal images with GluN1 green fluorescent intensity along the x-axis and GluN2 red fluorescent intensity along the y-axis. The shaded area in the upper right quadrant represents colocalized pixels above background with the associated Pearson correlation coefficient indicated for all colocalized pixels. (E) Representative traces for cultured HPASMCs in response to 100 µM NMDA and 10 µM glycine treatment. Cells were clamped at - 50 mV and whole cell recordings were performed. (F) NMDA and glycine treatment evoked an inward whole-cell current of 10.9 ± 1.7 pA (Mean ± SE, **p < 0.01, n = 16). (G) NMDA and glycine treatment evoked increased AUC compared with baseline (*p < 0.05, n = 16).

Fig 3: The GluN2B CaMKII Binding Site Is Dispensable for Theta Burst LTP(A–C) Activity-dependent signaling to ERK1/2 does not require GluN2B CTD-specific sequences. DIV9 cortical neurons of the indicated genotypes were treated with TTX (500 nM), KN-62 (10 µM), or MK-801 (10 µM) for 1 hr, after which protein extracts were made and subjected to western blot analysis for phospho-ERK1/2 levels, normalized to total ERK1/2. (A) shows quantitation and (B) and (C) show example blots.*p < 0.05 two-way ANOVA plus Dunnett’s post hoc test. No genotype-dependent effects were observed (p = 0.39) or genotype-drug interactions (p = 0.16; n = 8 GluN2BWT/WT; n = 4 GluN2B?CaMKII/?CaMKII; n = 4 GluN2B2A(CTR)/2A(CTR)).(D–F) Cumulative probability plots show amplitude (D) and frequency (E) of miniature EPSCs in GluN2BWT/WT and GluN2B?CaMKII/?CaMKII slices. Results are from 11 cells from 3 GluN2BWT/WT mice and 13 cells from 3 GluN2B?CaMKII/?CaMKII mice; p = 0.75 (amplitude); p = 0.94 (frequency); unpaired t test. (F) shows example traces. Scale bar: 10 pA/100 ms.(G) NMDAR:AMPAR EPSC ratios at -80 and +40 mV was measured in GluN2BWT/WT (n = 25; 4 mice) and GluN2B?CaMKII/?CaMKII (n = 30; 4 mice) neurons (unpaired t test; p = 0.95).(H) Weighted time constant (tw) for the decay of EPSCs recorded in (A) at +40 mV was determined using double exponential fits (unpaired t test; p = 0.63).(I) Example traces. Scale bar: 100 pA/20 ms.(J and K) Theta-burst stimulation elicited pathway-specific LTP of synaptic transmission in hippocampal CA1 area. Normalized magnitude of this potentiation 60–65 min after LTP induction did not differ significantly in GluN2BWT/WT mice (29 slices; n = 9) compared to GluN2B?CaMKII/?CaMKII (35 slices; n = 11); p = 0.162 (two-way nested ANOVA). (J) shows quantitation of data and (K) shows example traces before and after LTP induction.(K) Traces show example fEPSP traces immediately before and 1 hr after theta-burst stimulation. Scale bar: 0.5 mV/2 ms.

Fig 4: GluN2A Expression Is Sufficient to Displace GluN2B from NMDARs(A–D) Total GluN2B expression was measured in extracts from the neocortex at the indicated stages for the indicated genotypes (n = 8 per genotype). (A) and (B) show quantitation and example blots, respectively, for GluN2AWT/WT versus GluN2A2B(CTR)/2B(CTR). (C) and (D) show quantitation and example blots, respectively, for GluN2AWT/WT vs. GluN2B?CaMKII/?CaMKII.(E–G) Ectopic GluN2A expression is sufficient to displace GluN2B from NMDARs. Young mouse (E and F) and rat (G) neurons at DIV7 were transfected with a control (ß-globin) or GluN2A-encoding vector and spermine (100 µM) potentiation of NMDAR currents measured 72 hr later. *p < 0.05 (F: n = 10 of both condition; G: n = 8 control; n = 10 GluN2A).(H) Percentage potentiation of NMDAR currents by spermine (200 µM) in DIV15 or DIV16 rat cortical neurons was measured at the indicated stages for GluN2A+/+, GluN2A+/-, and GluN2A-/- genotypes. Person r correlation coefficient: -0.9954; p = 0.031 (one-tailed test); n = 12 cells per genotype.(I) Western blot illustrating GluN2A expression in GluN2A+/+, GluN2A+/–, and GluN2A–/– neurons.(I) A western blot confirming the absence of GluN2A expression in GluN2A-/- and an intermediate expression level in GluN2A+/- neurons.

Fig 5: Reconsolidation-resistant memories created with 10P display reduced plasticity mechanisms in comparison to memories that are reconsolidation-permissive.(A) Animals were trained with either 1P or 10P and were tested the next day. One day later BLA samples were collected and the postsynaptic levels of GluN2B and GluA2 quantified. Left: in comparison with HC controls, GluN2B increases in the 1P group but not in the 10P group (N = 3/6 per group). Right: 1P rats displayed higher GluA2 levels than HC, and 10P pairing displayed higher GluA2 levels than all other groups (N = 6/10 per group). (B) Animals were trained in the 1P protocol and were tested the next day. BLA samples were collected either 1 hr or 1 day after test to probe for postsynaptic and extrasynaptic GluA2 expression. Left: postsynaptic GluA2 is at HC levels 1 hr after test and is increased 24 hr after test (N = 6/11 per group). Right: extrasynaptic GluA2 is increased 1 hr after test and at HC levels 24 hr after test (N = 4/8 per group). (C) Animals were trained in the 10P protocol and were tested the next day. BLA samples were collected either 1 hr or 1 day after test to probe for postsynaptic and extrasynaptic GluA2 expression. Left: postsynaptic GluA2 is increased in comparison to HC at both 1 hr and 24 hr after test (N = 4/6 per group). Right: Extrasynaptic GluA2 is at HC levels at both 1 hr and 24 hr after test (N = 3/7 per group). Graphs show the mean ± s.e.m. Individual values are represented with circles. *p<0.05. The full statistics are available in the Supplementary file 2 and individual values in Figure 3—source data 1.Figure 3—source data 1.Raw data of Figure 3.