Impairment of auditory fear conditioning in YF/YF mice. (A) Freezing responses of WT/WT and YF/YF mice in auditory fear conditioning on the conditioning day (at 3 min; WT/WT, 5.88±1.43, n=17; YF/YF, 5.69±1.41, n=17; P>0.9, ANOVA). 170 s after mice were placed in the conditioning chamber, a tone was presented for 10 s (solid line); at the end of the tone mice were given a footshock (arrow). (B) Freezing responses 24 h after conditioning. At 3 min after mice were placed into a testing chamber with novel contexts, the tone was presented (solid line). WT/WT, n=17; YF/YF, n=17; ^*P< 0.03, ANOVA. (C, D) Summary of freezing responses 24 h after conditioning (C) and 1 h after conditioning (D). Freezing in the absence of the tone (Pre-CS) and in the presence of the tone (CS) was calculated as the mean freezing response from 2 to 3 min and from 3 to 4 min, respectively, after placement in the testing chamber. (24 h, ^*P< 0.03, ANOVA; 1 h, WT/WT, n=20; YF/YF, n=10; ^*P<0.04, ANOVA).

Normal basic properties of synaptic transmission in YF/YF mice. (A) Sample traces (average of 10 traces) of evoked AMPAR-mediated (downward traces) and NMDAR-mediated (upward traces) EPSCs in WT/WT and YF/YF mice (left). Ratio of amplitudes of NMDAR-mediated EPSCs to those of AMPAR-mediated EPSCs (right) (WT/WT mice, n=9; YF/YF mice, n=12). The ratio was calculated for each cell, and the values were then averaged for all cells. (B) Unaltered AMPAR-mediated mESPCs in YF/YF mice. Sample traces showing multiple events on a slower time scale (top). Averaged traces of mEPSCs recorded from single neurons (consecutive 100 events are averaged for WT/WT mice; consecutive 82 events for YF/YF mice) (bottom, left). Summary of the mean amplitude of AMPAR-mediated mEPSCs (bottom, right) in WT/WT (n=23) and YF/YF mice (n=20). Horizontal bars indicate the means. (C) Sample traces of AMPAR-mediated mEPSCs (faster traces) and AMPAR-plus NMDAR-mediated mEPSCs (slower traces) in WT/WT (average of 28 traces for AMPA and NMDA mEPSCs; average of 50 traces for AMPA mEPSCs) and YF/YF (average of 53 traces for both AMPA and NMDA mEPSCs and AMPA mEPSCs) mice (left). Ratio of the mean amplitude of NMDAR-mediated mEPSCs (AMPA mEPSCs were digitally subtracted from AMPA plus NMDA mEPSCs) to that of AMPAR-mediated mEPSCs (WT/WT, n=9; YF/YF, n=7). Horizontal bars indicate the means. (D) Current–voltage relationships of evoked NMDA synaptic currents recorded in the presence of 10 μM CNQX in WT/WT (n=5) and YF/YF (n=5) mice. The current values were normalized to the value at +34 mV in each cell, and the values were then averaged for all cells. The liquid junction potential was compensated.

Improper localization of the NR2B subunit in synaptic and perisynaptic regions in YF/YF mice. (A) Postembedding immunogold staining of NR2B subunits at asymmetrical axo-spinous synapses in the LA of WT/WT (WT/WT1 in B) and YF/YF (YF/YF1 in B) mice. NT, nerve terminal; Sp, spine. The calibration bar, 50 nm. (B–D) Tangential distribution of the NR2B subunit (WT/WT, 88 synapses; YF/YF, 102 synapses). (B) Histograms for the tangential distribution of NR2B subunits at synapses of WT/WT and YF/YF mice. Arrows indicate a boundary of the PSD. (C) Ratio of the number of immunogold particles for the NR2B subunit in the central region to that in the peripheral region. ^*P<0.01, Student's t-test. (D) The number of immunogold particles for the NR2B subunit in the perisynaptic regions. Results are expressed as percent of the total number of immunogold particles at synapses. *P<0.03, Student's t-test. (E) Histograms for the perpendicular distribution of NR2B subunits at synapses of WT/WT (66 synapses) and YF/YF mice (72 synapses). (F) The number of immunogold particles for the NR2B subunit per profile of synapses in the LA of the WT/WT (447 synapses) and YF/YF (451 synapses) mice. P>0.7, Student's t-test.

Generation of mice with a mutation of the NR2B tyrosine phosphorylation site. (A) Schematic representations of the structures of WT, targeting vector, and targeted and floxed-targeted NR2B alleles with relevant restriction sites. The probe for Southern blotting is indicated. pBKS, pBluescriptIIKS plasmid; DTA, diphtheria toxin gene; neo, neomycin-resistance gene. (B) The Southern blot analysis of HindIII-digested genomic DNAs from WT/WT, WT/YF and YF/YF mice. (C) The absence of Tyr-1472 phosphorylation in homozygous YF/YF mice (upper). Equal amounts of amygdaloid lysates from WT/WT and YF/YF mice were probed with the anti-pY1472 antibody. (D) A reduced level of NR2B tyrosine-phosphorylation in YF/YF mice. Equal amounts of NR2B immunoprecipitates from amygdaloid lysates of WT/WT and YF/YF mice were probed with the anti-pY (4G10) mAb. A representative blot is shown in the inset. All data are presented as the mean±s.e.m. (Figures 1, 2, 3, 4, 5, 6 and 7 and Supplementary Figures 1–7). ^*P<0.001, Student's t-test.

Impaired amygdaloid LTP in YF/YF mice. (A) The averaged time course of LTP in WT/WT (n=17) and YF/YF (n=16) mice. Maximal initial EPSP slopes were normalized in each experiment to the averaged maximal initial EPSP slope during the control period (−10 to 0 min). LTP was induced by ‘pairing' at time 0. Sample traces of EPSPs (average of 10 consecutive responses) in WT/WT and YF/YF mice recorded at the times indicated by the numbers are shown in the inset. (B) Summary of LTP (WT/WT, n=17; YF/YF, n=16; P<0.02, Student's t-test) expressed as percent of the mean EPSP slope from 40 to 50 min after the induction relative to the mean EPSP slope during the control period (−10 to 0 min). Horizontal bars indicate the means. (C) Sample traces showing the temporal summation of NMDAR-mediated EPSCs elicited by the same stimulation protocol used for the LTP induction. Fifteen traces evoked by 30-Hz stimulation at 10-s intervals and a single EPSC trace (average of 10 traces) are superimposed. (D) (left) The level of Tyr-1472 phosphorylation is increased after electrical stimulation in the LA. Coronal sections including the LA were stained with the anti-phospho-Tyr1472 antibody. La, lateral amygdala. (right) Quantification of Tyr-1472 phosphorylation in the LA. ^*P<0.004, Student's t-test, n=3. The calibration bar, 500 μm.

Impaired NMDAR-mediated CaMKII signaling in YF/YF mice. (A) Interaction of CaMKII with the WT NR2B, but very little interaction with Y1472FNR2B. NR2B immunoprecipitates (top) and lysates (bottom) were probed with the anti-CaMKII mAb. (B) Impaired glutamate-induced upregulation of CaMKII in YF/YF mice. Stimulation of coronal sections including the LA with glutamate was performed as described in Materials and methods. (C) Quantification of phospho-CaMKII in WT/WT (n=6 from 3 mice) and YF/YF mice (n=6 from 3 mice). *P<0.01, Student's t-test.

Improper localization of NR1 but normal localization of NR2A in synaptic and perisynaptic regions in YF/YF mice. (A) Postembedding immunogold staining of the NR1 subunit at asymmetrical axo-spinous synapses in the LA of WT/WT (WT/WT1 in Supplementary Figure 4A) and YF/YF (YF/YF1 in Supplementary Figure 4A) mice. NT, nerve terminal; Sp, spine. The calibration bar, 50 nm. (B, C) Tangential distribution of the NR1 subunit (WT/WT, 81 synapses; YF/YF, 84 synapses). (B) Ratio of the number of immunogold particles for the NR1 subunit in the central region to that in the peripheral region (WT/WT, 2.70±0.88; YF/YF, 0.79±0.14; ^*P<0.02, Student's t-test). (C) The number of immunogold particles for the NR1 subunit in the perisynaptic regions. Results are expressed as percent of the total number of immunogold particles at synapses (WT/WT, 2.27±1.63%; YF/YF, 8.47±3.89%; ^*P<0.03, Student's t-test). (D) The number of immunogold particles for the NR1 subunit per profile of synapses in the LA of WT/WT (2.45±0.43, 100 synapses) and YF/YF (2.07±0.20, 100 synapses; P>0.2, Student's t-test) mice. (E) Postembedding immunogold staining of the NR2A subunit at asymmetrical axo-spinous synapses in the LA of WT/WT (WT/WT1 in Supplementary Figure 4C) and YF/YF (YF/YF1 in Supplementary Figure 4C) mice. The calibration bar, 50 nm. (F, G) Tangential distribution of the NR2A subunit (WT/WT, 70 synapses; YF/YF, 55 synapses). (F) Ratio of the number of immunogold particles for the NR2A subunit in the central region to that in the peripheral region (WT/WT, 1.69±0.36; YF/YF, 1.59±0.25; P>0.7, Student's t-test). (G) The number of immunogold particles for the NR2A subunit in the perisynaptic regions (WT/WT, 5.38±3.15%; YF/YF, 3.55±1.51%; P>0.2, Student's t-test). (H) The number of immunogold particles for the NR2A subunit per profile of synapses in the LA of WT/WT (2.32±0.15, 70 synapses) and YF/YF (2.35±0.16, 55 synapses; P>0.7, Student's t-test) mice.