TBS-induced LTP and the NMDA- to AMPA-mediated EPSC ratio are reduced in adult <i>NesCreIrs2KO</i> mice.

<p><b>A</b>: In the presence of the GABA_A receptor antagonist SR95531 (6 µM), LTP was reliably induced in control mice (+/+; average EPSP slope change 60 min after induction: 151±6%, N = 4, n = 6) in response to a single theta-burst protocol (TBS). The level of LTP produced under the same conditions, 60 min following TBS, was significantly reduced in <i>NesCreIrs2KO</i> mice (−/−; average EPSP slope change 60 min after induction: 123±6%, N = 4, n = 5, p<0.005). Arrow indicates application of TBS. <b>B</b>: Sample EPSP traces (average of 4 consecutive sweeps) from individual experiments, taken prior to, and 60 min following TBS in control (+/+) and <i>NesCreIrs2KO</i> (−/−) mice. <b>C</b>: In the presence of SR95531 (6 µM), the ratio of NMDA-EPSC amplitude (holding potential: +40 mV) to AMPA-EPSC amplitude (holding potential: −70 mV) was significantly lower in <i>NesCreIrs2KO</i> (−/−; 0.32±0.04, N = 6, n = 11) compared to littermate controls (+/+; 0.44±0.04, N = 5, n = 12, p<0.05). <b>D</b>: EPSC traces (average of 10 consecutive sweeps) from whole-cell patch clamp experiments in <i>NesCreIrs2KO</i> (−/−) and control mice (+/+) illustrating the NMDA-EPSC (measured at a holding potential of +40 mV) and corresponding AMPA-EPSC (measured at a holding potential of −70 mV). NMDA-EPSC amplitude was measured 50 ms following stimulation to minimize contamination from the AMPA-mediated component. <b>E–I</b>: Representative immunoblot images of hippocampal subfield CA1 lysates from control (+/+) and <i>NesCreIrs2KO</i> mice (−/−) probed with antibodies against the total protein or specific phosphorylation sites of the AMPA receptor subunit GluR1 (<b>E–F</b>) and the NMDA subunits NR1 (<b>G</b>), NR2A (<b>H</b>), and NR2B (<b>I</b>). Quantifications of immunoblots showing the protein level or relative proportion of phosphorylated protein over the total are shown in the bar diagrams. <b>E–F</b>: The total level of the AMPA receptor subunit GluR1 was similar in control (100±8%) and <i>NesCreIrs2KO</i> mice (77±9%, n = 10, p = 0.063; <b>upper panels</b>). <b>E</b>: The proportion of GluR1 subunit phosphorylated at Ser845 (p-GluR1-S845) relative to total GluR1 protein was similar in control (100±4%, n = 5) and <i>NesCreIrs2KO</i> mice (93±8%, n = 5, p = 0.46; <b>middle and lower panels</b>). <b>F</b>: The proportion of GluR1 subunit phosphorylated at Ser831 (p-GluR1-S831) relative to total GluR1 protein was similar in control (Ser831: 100±13%; n = 5) and <i>NesCreIrs2KO</i> mice (Ser831: 103±18%, n = 5; p = 0.73; <b>middle and lower panels</b>). <b>G</b>: There was no change in the total level of the NMDA receptor subunit NR1 (control mice: 100±11%; <i>NesCreIrs2KO</i> mice: 93±8%, n = 5, p = 0.46; <b>upper panel</b>). <i>NesCreIrs2KO</i> mice displayed a reduced phosphorylation of NR1 at Ser897 (p-NR1; 45±9%, n = 6) relative to their control littermates (100±9%, n = 6, p<0.001; <b>middle and lower panels</b>). <b>H–I</b>: There were no changes in the total levels of the NMDA receptor subunits NR2A (<b>H</b>; control mice: 100±11%; <i>NesCreIrs2KO</i> mice: 89±11%, n = 7, p = 0.49), and NR2B (<b>I</b>; control mice: 100±17%; <i>NesCreIrs2KO</i> mice: 129±17%, n = 5, p = 0.55). Data are expressed as mean ± SEM. **p<0.01 (unpaired t-test).