Enhanced NMDA receptor-mediated synaptic transmission, enhanced long-term potentiation, and impaired learning and memory in mice lacking IRSp53

Abstract

IRSp53 is an adaptor protein that acts downstream of Rac and Cdc42 small GTPases and is implicated in the regulation of membrane deformation and actin filament assembly. In neurons, IRSp53 is an abundant postsynaptic protein and regulates actin-rich dendritic spines; however, its in vivo functions have not been explored. We characterized transgenic mice deficient of IRSp53 expression. Unexpectedly, IRSp53(-/-) neurons do not show significant changes in the density and ultrastructural morphologies of dendritic spines. Instead, IRSp53(-/-) neurons exhibit reduced AMPA/NMDA ratio of excitatory synaptic transmission and a selective increase in NMDA but not AMPA receptor-mediated transmission. IRSp53(-/-) hippocampal slices show a markedly enhanced long-term potentiation (LTP) with no changes in long-term depression. LTP-inducing theta burst stimulation enhances NMDA receptor-mediated transmission. Spatial learning and novel object recognition are impaired in IRSp53(-/-) mice. These results suggest that IRSp53 is involved in the regulation of NMDA receptor-mediated excitatory synaptic transmission, LTP, and learning and memory behaviors.

Figure 1.

Generation and characterization of IRSp53^−/− mice. A, Diagram depicting the structure of the IRSp53 gene harboring exons and introns, and the insertion site of the genetrap in the intron between exons 3 and 4. B, Identification of the trapped IRSp53 gene by PCR genotyping. C, Undetectable expression of IRSp53 proteins in IRSp53^−/− mice. Total brain homogenates from WT, IRSp53^+/−, and IRSp53^−/− mice (4 weeks) were immunoblotted with IRSp53 antibodies. D, Unchanged expression levels of other synaptic proteins and IRSp53-interacting proteins (PSD-95, Mena, and Eps8) in whole brain and hippocampal extracts of IRSp53^−/− mice. E, Normal gross morphology of IRSp53^−/− brain (7 weeks) revealed by Nissl staining. Scale bars: top; 150μm; bottom, 40 μm.

Figure 2.

Expression patterns of IRSp53 proteins revealed by X-gal staining of IRSp53^+/− brain slices. Coronal (A) and sagittal (B) brain slices from IRSp53^+/− mice (8 weeks) were incubated with X-gal to visualize brain regions expressing IRSp53-β-geo fusion proteins. Brain regions with relatively high IRSp53-β-geo expression include the cortex (C), hippocampus (D), striatum (E), and cerebellum (F). Scale bars: A, B, 150 μm; C–F, 30 μm.

Figure 3.

Normal spine density and ultrastructure in IRSp53^−/− CA1 pyramidal neurons. A, Representative electron micrographs of the stratum radiatum of the hippocampal CA1 region from WT and IRSp53^−/− mice (4 weeks). Normal and perforated PSDs are indicated by arrow and arrowheads, respectively. Scale bar, 0.5 μm. B, Quantitative analysis of the density of total and perforated spines. Bar graph represents mean ± SEM (total area of the brain region analyzed, 943 μm² per mouse; n = 3 mice for each genotype). C, Head area of dendritic spines (total, normal/nonperforated, and perforated). Bar graph represents mean ± SEM (total number of spines analyzed, 1648 for WT and 1535 for KO; n = 3).

Figure 4.

Normal basal synaptic transmission in IRSp53^−/− mice. A, Normal basal transmission at IRSp53^−/− SC–CA1 synapses. The input–output relationship of basal synaptic transmission was determined by plotting the slopes of fEPSPs against fiber volley amplitudes. The insets show sample traces of fEPSPs. Data represent mean ± SEM (n = 60–62 slices from 10–11 mice, 4–6 weeks). B, Normal paired-pulse facilitation ratio at IRSp53^−/− SC–CA1 synapses. Facilitation ratios were plotted against interstimulus intervals. The insets show sample traces of fEPSPs. Mean ± SEM (n = 13 slices from 4 mice for both WT and KO mice, 5–8 weeks). C–E, Normal amplitude and frequency of mEPSCs. Bar graph represents mean ± SEM [amplitude, WT, 26.89 ± 1.47; KO, 29.06 ± 1.63 pA; frequency, WT, 0.115 ± 0.034; KO, 0.107 ± 0.028 Hz; n = 17–18 cells from 3 mice at postnatal day 16 (P16)–P21].

Figure 5.

Reduced AMPA/NMDA ratio of excitatory transmission in IRSp53^−/− mice. A–C, Decreased AMPA/NMDA ratio of eEPSCs at IRSp53^−/− SC–CA1 synapses, compared with WT mice. A, Mean eEPSC traces mediated by AMPAR (downward) and NMDAR (upward) recorded at holding potentials of −70 and +40 mV, respectively. The gray and black lines indicate current traces from WT and KO mice, respectively. Amplitudes of AMPA and NMDA receptor-mediated EPSCs from WT and KO neurons were plotted for comparison in B and C. Each open or gray symbol represents a pair of recordings. The filled circle or square represents mean ± SEM. D, Reduced AMPA/NMDA ratio of fEPSP slopes at IRSp53^−/− SC–CA1 synapses, compared with WT mice. Sample traces of fEPSPs in the absence and presence of NBQX (an AMPA receptor antagonist) are shown (left). Summary of AMPAR- and NMDAR-mediated fEPSP slopes (right, n = 18 slices from 6 mice for WT and 17 slices from 6 mice for KO; 5–8 weeks; *p < 0.05, Student's t test). E, Normal fiber volley amplitudes against stimulation intensities in IRSp53^−/− Schaffer collaterals, measured by field recordings. F, G, Normal decay kinetics of NMDA receptor eEPSCs. F, AMPAR- and NMDAR-mediated eEPSCs shown in A were scaled to the same maximum for comparison of decay phases (WT, gray; KO, black). G, Fast and slow decay kinetics of NMDA receptor eEPSCs was not significantly different between WT and KO mice. The decay kinetics of AMPA receptor eEPSCs was analyzed in parallel for control.

Figure 6.

Enhanced NMDA receptor-mediated synaptic transmission in IRSp53^−/− mice. A–C, Sample traces (A) showing that the amplitude (B), but not frequency (C), of NMDA receptor-mediated mEPSCs is enhanced at IRSp53^−/− SC–CA1 synapses. In control recordings, NMDA receptor mEPSCs were abolished in the presence of 1.3 mm Mg²⁺ and 50 μm APV (an NMDA receptor antagonist). Bar graph represents mean ± SEM (amplitude, WT, 26.14 ± 1.20; KO, 32.43 ± 1.28 pA; frequency, WT, 0.13 ± 0.01; KO, 0.14 ± 0.01 Hz; n = 15 cells from 3 mice at P21–P27; **p < 0.01, Student's t test).

Figure 7.

Selective enhancement of LTP at IRSp53^−/− SC–CA1 synapses. A, Enhanced LTP. LTP was induced by theta burst stimulation. Shown is mean ± SEM (n = 22–23 slices from 13–14 mice). The gray and black traces were taken at 0 min and at the end of recording, respectively. *p < 0.001 compared to baseline. B, Unchanged LTD. LTD was induced by paired-pulse low frequency stimulation (900 paired pulses at 1 Hz; interstimulus interval, 40 ms). Shown is mean ± SEM (n = 9–14 slices from 7 mice). Calibration: 10 ms, 0.5 mV.

Figure 8.

Enhanced NMDA receptor-mediated synaptic transmission during the delivery of TBS. A, Mean traces of baseline AMPA-EPSCs and NMDA-EPSCs. Baseline AMPA-EPSCs and NMDA-EPSCs were induced by paired-pulse stimulation (100 ms apart) at the holding potentials of −70 and +40 mV, respectively. B, Mean traces of NMDA-EPSCs during the first episode (10 stimulus trains) of TBS. The inset shows NMDA-EPSCs during the first train of TBS (boxed). Calibration (inset): 20 ms, 200 pA. C, Enhanced NMDA-EPSCs during TBS. Cumulative NMDA-EPSCs during TBS were normalized to baseline AMPA-EPSCs. n = 13 cells for WT and 12 for KO from 6 mice; *p < 0.05 compared to WT, Student's t test. D, NMDA-EPSCs during TBS are not changed when normalized to baseline NMDA-EPSCs. E, AMPA-EPSC slopes correlate well with AMPA-EPSC amplitudes. F, NMDA-EPSC slopes correlate well with NMDA-EPSC amplitudes. G, Similar levels of paired-pulse facilitations are observed when NMDA-EPSCs are analyzed by EPSC slopes and amplitudes. Error bars indicate SEM. H, The ratio of AMPA/NMDA transmission is reduced in IRSp53^−/− mice when analyzed by EPSC slopes.

Figure 9.

Impaired learning and memory in IRSp53^−/− mice. A, Impaired spatial learning of IRSp53^−/− mice in the hidden-platform Morris water maze test. The latency to find a hidden platform was plotted against the days of training. Data represent mean ± SEM (n = 5 for WT and 6 for KO; *p < 0.05, Student's t test). B, Decreased target quadrant occupancy of IRSp53^−/− mice in the probe test. The percentage of time spent in each quadrant is shown. The target quadrant is the area of the pool where the platform was located during training. T, Target; L, left; R, right; O, opposite. Bar graph represents mean ± SEM (WT, 43.5 ± 4.2%; KO, 29.5 ± 2.2% in the target quadrant; n = 5 for WT and 6 for KO; *p < 0.05, Student's t test). C, D, Reduced novel object recognition in IRSp53^−/− mice. C, During training, mice were presented with objects A and B for 10 min, which are equally explored by WT and IRSp53^−/− mice. D, Twenty-four hours after the training, the mice were allowed to explore objects A and C (a new object). Object preference indicates the percentage amount of time spent on each objects during the training or test periods. Bar graph represents mean ± SEM (n = 10 mice for WT and 8 for KO; *p < 0.05, Student's t test).