Genetic blockade of NR1 S897 phosphorylation. A, Schematic view of the targeting vector generated for targeting the S897 site of GRIN1 locus of the mouse genome (see Materials and Methods). B, Confirmation of gene targeting in the genomic DNA of the mutant mice by sequencing using a specific primer close to this site. C, Western blot analysis to confirm the absence of phosphorylation at S897 in the S897A mutant animals using the NR1 S897 phosphospecific antibody. Total protein extracts of 50 μg from the frontal cortex (FC), striatum (STR), and hippocampus (HIP) of the WT animals and homozygous mutants were loaded. Top blot, Anti-NR1 S897; middle blot, anti-NR1; bottom blot, anti-actin.
The NR1 S897A mutation depressed synaptic transmission and reduced LTP. A, The top panel shows the representative traces of evoked EPSCs recorded from CA1 neurons in either WT or mutant hippocampal slices. EPSCs at both −60 and +40 mV holding potentials are shown. Calibration: 50 ms, 20 pA. The bottom histogram is the quantification of the ratio of NMDAR- to AMPAR-mediated synaptic transmission for WT and mutant animals (WT, 0.82 ± 0.14, n = 10; mutant mice, 0.31 ± 0.04, n = 12; **p < 0.01, t test). B, Representative traces of AMPAR-mediated mEPSCs recorded from slices of WT (left) or mutant (right) animals. Calibration: 500 ms, 20 pA. Quantification of the amplitude of AMPAR-mediated mEPSCs for WT and mutant animals is described here (WT, 13.5 ± 0.2 pA, n = 1138 events from 10 cells; mutant mice, 12.4 ± 0.2 pA, n = 662 events from 10 cells; **p < 0.01, K–S test). Quantification of the frequency of AMPAR-mediated mEPSCs for WT and mutant animals is described here (WT, 13.8 ± 2.1 per min, n = 10 cells; mutant, 8.5 ± 1.4 per min, n = 10 cells; *p < 0.05, t test). C, Cumulative distribution of the interevent intervals of the mEPSCs from WT and mutant animals (WT, n = 1138 events from 10 cells; mutant mice, n = 662 events from 10 cells; p < 0.01, K–S test). D, Top panel, Representative traces of field EPSPs (fEPSPs) from WT or mutant hippocampal slices. Traces are averaged for time points before (1) and after (2) LTP induction. Calibration: 200 ms, 0.1 mV. Bottom panel, Normalized amplitudes of fEPSPs before and after delivery of the LTP-induction stimuli (arrow). N = 13 for both WT and mutant animals; *p < 0.05. Error bars indicate SEM.
The NR1 S897A mutation decreased synaptic incorporation of glutamate receptors and reduced GluR1 in the synapse. A, Left, Western blot analysis after biochemical fractionation of hippocampal tissues dissected from WT and the homozygous mutant mice. Synaptic membrane-associated proteins (10 μg) were loaded, and the same blot was probed with antibodies against GluR1 (top), NR1 (middle), and PSD-95 (bottom). Right, Quantification of the densities of total synaptic GluR1 (top) and NR1 (bottom) signals from WT (n = 3) and the homozygous mutant (n = 3) mice. Error bars indicate SEM (*p < 0.05, t test). B, Immuno-EM analysis of the CA1 regions of the hippocampus from WT and mutant mice. Left panel, No primary antibody controls; right panels, top, three representative EM fields of the CA1 regions of wild-type animals that were probed with an anti-GluR1 antibody. The dark black staining of GluR1 in postsynaptic areas is the specific GluR1 signal, which is absent in the sections that were probed with the secondary antibody alone (no primary antibody control; on the left). Right panels, Bottom, Three representative EM fields of the CA1 regions of the homozygous mutant mice probed with the anti-GluR1 antibody. The dark black staining shows the mislocalized clusters of GluR1 signal. This signal is specific as it is absent in control sections (probed with the secondary antibody alone; on the left). Quantification of the number of GluR1-positive synapses in each EM field (2 μm2) in the CA1 regions of the wild-type mice and homozygous mutant mice shows a highly significant decrease in the number of GluR1-positive synapses in mutants (p < 0.0001, t test; N = 9 for both groups). Scale bars, 200 nm.
The NR1 S897A mutation causes behavioral deficits. A, B, Social interaction of the experimental mice toward the repetitively presented stimulus mouse (tests 1–4), or a novel stimulus mouse (test 5). Mutant, NR1 S897A homozygous mutant mice; WT, wild-type littermates. A, Quantification of active social investigation: number of sniffs; N = 8 for both groups. One-way repeated-measures ANOVA was used to evaluate recognition memory in WT and mutant mice. Significance (***p < 0.0001; F = 8.3) was further evaluated using Bonferroni's multiple-comparison post hoc test. In test 5, in which a new intruder was introduced, WT mice showed increased recognition memory compared with mutant mice (p < 0.001; t = 5.728). WT mice also showed statistically decreased exploration toward the same intruder through tests 1–4 (p < 0.01, t = 4.226, test 1 vs test 2; p < 0.001, t = 5.449, test 1 vs test 3; and p < 0.001, t = 5.708, test 1 vs test 4), whereas there was no significant difference when compared test 1 (the very first presentation of the intruder, which was presented repeatedly in tests 1–4) with test 5 (the new intruder). In contrary to WT mice, mutant mice did not show recognition memory throughout tests 1–5. B, Quantification of the activity in exploring the cage away from social interest: number of rears (not in relation to the cylinder); N = 8 for both groups. Compared with mutant mice, WT mice littermate exhibited significantly higher exploratory activity in test 1 (p < 0.01; t = 3.4). C, Prepulse inhibition of NR1 S897A phosphomutant mice and WT littermate controls. PPI was expressed as 100 − [(response to startle stimulus after prepulse/response to startle stimulus alone) × 100]. *p < 0.05; N = 10 for both groups. Error bars indicate SEM.