Src-induced potentiation of NMDA receptor-mediated EPSCs is abolished in medium spiny neurons in acute striatal slices from YF/YF mice. (A) Examples of NMDA receptor-mediated EPSCs. (B) Src-dependent change in the peak amplitude of NMDA receptor-mediated EPSCs in medium spiny neurons from WT/WT and YF/YF mice. WT/WT, n=15; YF/YF, n=11; ^*P=0.036, paired t-test.

Normal amounts of monoamines, their metabolites, and amino acids in the striatum of YF/YF mice. (A–F) Biopsies of the striatum were obtained from frozen brain sections and monoamines and their metabolites were extracted and analysed. (A) The amount of dopamine (DA). (B) The amounts of DOPAC and HVA. (C) DA turnover. (D) The amount of serotonin (5-HT). (E) The amount of 5-HIAA. (F) 5-HT turnover. (G, H) Biopsies of the striatum were obtained from frozen brain sections and amino acids were derivatized with OPA and analysed. (G) The amount of GABA. (H) The amount of glutamate. No significant differences were observed between WT/WT and YF/YF mice for any of the measurements (WT/WT, n=6; YF/YF, n=6, P>0.05, one-way ANOVA). NS: not significant.

Increased DARPP-32 phosphorylation at Thr 34 and decreased calcineurin activity in YF/YF mice. (A) Increased DARPP-32 phosphorylation at Thr 34 in YF/YF mice. Striata from WT/WT and YF/YF mice were dissected and homogenized in TNE buffer. Then striatal lysates from WT/WT and YF/YF mice were subjected to immunoblotting with the anti-pThr 34 antibody, followed by a re-blot using the anti-DARPP-32 antibody. A representative blot is shown on the left (^*P<0.05, n=3, Student's t-test). (B) Increased ERK activity in YF/YF mice. Striatal lysates from WT/WT and YF/YF mice were subjected to immunoblotting with the anti-pERK (pT185 and pY187) antibody followed by re-blot with anti-ERK2 antibody. A representative blot is shown on the left (^*P<0.05, n=3, Student's t-test). (C) Decreased calcineurin activity in YF/YF mice. Calcineurin activity in the striatum from WT/WT and YF/YF mice was analysed by measuring free phosphate released from the synthetic RII phosphopeptide substrate as described in the Materials and methods (^*P<0.05, n=3, Student's t-test). (D) Normal CaMKII activity in YF/YF mice. Striatal lysates from WT/WT and YF/YF mice were subjected to immunoblotting with the anti-phospho-CaMKII (pT286) antibody followed by re-blot with anti-CaMKII antibody. A representative blot is shown on the left (P>0.05, n=3, Student's t-test). NS: not significant.

Increased Tyr 1325 phosphorylation during the forced swim test and the tail suspension test. (A) The level of Tyr 1325 phosphorylation in the striatum was increased during the forced swim test. Equal amounts of NR2A immunoprecipitates from lysates of the forced swim test-exposed mice and control mice were probed using the anti-Tyr 1325 antibody followed by a re-blot with the anti-NR2A antibody. ^*P<0.05, n=3, Student's t-test. The levels of Tyr 1472 phosphorylation on NR2B and Ser 896 phosphorylation on NR1 were unchanged after the forced swim test. Equal amounts of striatal lysates from the forced swim test-exposed mice and control mice were subjected to immunoblotting with antibodies against pY1472/NR2B, NR2B, pS896/NR1, NR1, pY418/Src kinases, and Src kinases. (P>0.05, n=3, Student's t-test). It was noteworthy that Src was activated after the forced swim test. (^*P<0.05, n=3, Student's t test). FST, the forced swim test. (B) The level of Tyr 1325 phosphorylation in the striatum was increased during the tail suspension test. Equal amounts of NR2A immunoprecipitates from lysates of the tail suspension test-exposed mice and control mice were probed using the anti-Tyr 1325 antibody followed by re-blot using the anti-NR2A antibody. (^*P<0.05, n=5, Student's t-test). The level of Tyr 1472 phosphorylation on NR2B was unchanged by the tail suspension test. (P>0.05, n=5, Student's t-test). TST: the tail suspension test; NS: not significant.

A proposed model for the role of Tyr 1325 phosphorylation on NR2A subunit in depression-related behaviour. (left) In WT/WT mice, Src-mediated Tyr 1325 phosphorylation increases the Ca²⁺ influx through the NR2A subunit-containing NMDA receptor channels, and thereby activates a Ca²⁺-dependent protein phosphatase, calcineurin. Activated calcineurin then de-phosphorylates Thr 34 of DARPP-32, leading to the generation of active protein phosphatase-1 (PP-1). Consequently, the phosphorylation levels of PP-1 substrates, including ERK, are decreased. (right) In YF/YF mice, Ca²⁺ influx through the NR2A subunit-containing NMDA receptor channels is decreased in the absence of Tyr 1325 phosphorylation, resulting in decreased calcineurin activity and increased phosphorylation of Thr 34 on DARPP-32. Phosphorylation of Thr 34 on DARPP-32 converts DARPP-32 into a potent inhibitor of PP-1, and thereby increases the phosphorylation level of PP-1 substrates including ERK. The antidepressant-like phenotype of YF/YF mice may be attributable partly to the increased Thr 34 phosphorylation of PP-1 substrates including ERK.

Generation of mice with a mutation of Tyr 1325 phosphorylation site on NR2A. (A) Schematic representations of the structures of wild-type, targeting vector, and targeted and Neo-targeted NR2A alleles. TK, thymidine kinase gene; neo, neomycin resistance gene. (B) Absence of Tyr 1325 phosphorylation in homozygous YF/YF mice. Equal amounts of brain lysates from WT/WT and YF/YF mice were probed with the anti-pY1325 antibody followed by a re-blot using the anti-NR2A antibody. (C) NR2A tyrosine-phosphorylation in YF/YF mice. Equal amounts of NR2A immunoprecipitates from striatal lysates of WT/WT and YF/YF mice were probed with the anti-phosphotyrosine (4G10) antibody followed by a re-blot with the anti-NR2A antibody. A representative blot is shown on the left (^*P<0.01, n=3, Student's t-test). (D) Nissl-stained coronal sections of central nervous system structures including striatum from WT/WT and YF/YF mice. Cpu: Caudata-putamen.

Reduced depression-related behaviour in YF/YF mice. (A) Percent immobility of WT/WT and YF/YF mice in the tail suspension test. (B) Summary of immobility in the tail suspension test during the last 4 min of the 6-min test session. WT/WT, n=13; YF/YF, n=11; F_(1,22)=6.00, ^*P=0.02, one-way ANOVA. (C) Percent immobility of WT/WT and YF/YF mice in the forced swim test. (D) Summary of immobility in the forced swim test during the last 7 min of the 10-min test session. WT/WT, n=12; YF/YF, n=12; F_(1,22)=4.69, ^*P=0.04, one-way ANOVA. (E) Normal locomotor activity and anxiety-like behaviour of YF/YF mice in the open field test. WT/WT, n=13; YF/YF, n=12, P>0.05, one-way ANOVA. (F) Normal anxiety-like behaviour of YF/YF mice in the light–dark emergence test. WT/WT, n=13; YF/YF, n=12, P>0.05, one-way ANOVA. (G) Normal behaviour of YF/YF mice in the social interaction test. WT/WT, n=11; YF/YF, n=11, P>0.05, one-way ANOVA. (H) Normal anxiety-like behaviour of YF/YF mice in the elevated plus maze test. The time spent on open arms and the number of entries into open arms: WT/WT, n=13; YF/YF, n=12, P>0.05, one-way ANOVA. Number of head dips: WT/WT, n=10; YF/YF, n=10, P>0.05, one-way ANOVA. NS: not significant.

Identification of Tyr 1325 as one of the principal Src family kinase-mediated phosphorylation sites of the NR2A subunit. (A) Schematic diagram of GST fusion proteins containing the intracellular C-terminal region of the NR2A subunit. (B) Two-dimensional tryptic phosphopeptide maps of GST–C1, GST–C2, GST–C2–Y1325F, and GST–C3. The dot in each map shows the origin of electrophoresis. Note that P7 (Tyr 1325) was most prominently phosphorylated in vitro. (C) Identification of Tyr 1325 as one of the principal phosphorylation sites in HEK 293T cells. HEK293T cells were transfected with combinations of expression plasmids for the NR2A subunit, NR2A Y1325F mutants, and active Src (SrcYF) or inactive Src (SrcKM). NR2A immunoprecipitates from cell lysates were subjected to immunoblotting using the anti-pY antibody (4G10) (top), followed by a re-blot using the anti-NR2A antibody (middle). The expression levels of Src were confirmed by immunoblotting (bottom). ^*P<0.001, n=4, Student's t-test. Note that all lanes are from the same gel, but lanes originally present between lanes 2 and 3 have been omitted for brevity.

Point mutation at Tyr 1325 on the NR2A subunit blocks Src-mediated potentiation of recombinant NMDA receptor currents in HEK 293T cells. (A) Application of Src differentially potentiated NMDA-activated currents mediated by recombinant NR1–wild-type (WT) NR2A channels (upper), but not those mediated by recombinant NR1–Y1325F NR2A channels (lower). Peak currents evoked by NMDA applications (100 μM NMDA and 10 μM glycine: 0.5 s) at 30-s intervals were normalized to the first responses at 3 min after establishing whole-cell configuration (time: 0 min; holding potential: −60 mV). Src (30 U/ml) was added to the internal pipette solution. WT NR2A (control), n=5; WT NR2A (+Src), n=6; Y1325F NR2A (control), n=4; Y1325F NR2A (+Src), n=5. Representative averaged whole-cell currents evoked at −60 mV by the NMDA application are shown in the left panel and the traces recorded at 3 min (grey) and 20 min (black) are superimposed. Scale bars: 0.2 s; 100 pA. (B) Src-dependent increment of free calcium levels in cells expressing NR1/wild-type NR2A, but not NR1/Y1325F–NR2A channel. Intracellular free calcium levels, before and after NMDA stimulation in fura-2/AM-loaded cells, were monitored by spectrofluorometry. Increase in the ratio of the fluorescence intensities at excitation wavelengths of 340 nm (Fex340) and 380 nm (Fex380) after NMDA stimulation are shown (n=4, ^*P<0.05, Student's t-test). NS: not significant.