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. 2012 May 25;287(22):18103-14.
doi: 10.1074/jbc.M112.347427. Epub 2012 Apr 9.

N-methyl-D-aspartate (NMDA) Receptor Composition Modulates Dendritic Spine Morphology in Striatal Medium Spiny Neurons

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N-methyl-D-aspartate (NMDA) Receptor Composition Modulates Dendritic Spine Morphology in Striatal Medium Spiny Neurons

Csaba Vastagh et al. J Biol Chem. .
Free PMC article

Abstract

Dendritic spines of medium spiny neurons represent an essential site of information processing between NMDA and dopamine receptors in striatum. Even if activation of NMDA receptors in the striatum has important implications for synaptic plasticity and disease states, the contribution of specific NMDA receptor subunits still remains to be elucidated. Here, we show that treatment of corticostriatal slices with NR2A antagonist NVP-AAM077 or with NR2A blocking peptide induces a significant increase of spine head width. Sustained treatment with D1 receptor agonist (SKF38393) leads to a significant decrease of NR2A-containing NMDA receptors and to a concomitant increase of spine head width. Interestingly, co-treatment of corticostriatal slices with NR2A antagonist (NVP-AAM077) and D1 receptor agonist augmented the increase of dendritic spine head width as obtained with SKF38393. Conversely, NR2B antagonist (ifenprodil) blocked any morphological effect induced by D1 activation. These results indicate that alteration of NMDA receptor composition at the corticostriatal synapse contributes not only to the clinical features of disease states such as experimental parkinsonism but leads also to a functional and morphological outcome in dendritic spines of medium spiny neurons.

Figures

FIGURE 1.
FIGURE 1.
Effect of NR2A and NR2B antagonists on dendritic spine morphology in MSNs. A, left panel: sample traces of the NMDA isolated currents at +40 mV before and after bath application of the NR2A selective antagonist NVP-AAM077 (300 nm). Right panel: sample traces of the NMDA-isolated currents (+40 mV) before and after the application of the NR2B subunit antagonist ifenprodil (10 μm). Shown is a bar graph showing the effects of NVP-AAM077 300 nm (t test; **, p < 0.01, pre- versus post-application 36.42 ± 5.1%; n = 4) and ifenprodil (t test; ***, p < 0.001, pre- versus post-application, 41.51 ± 6.49%; n = 6) on the NMDA-EPSC amplitude. B, Western blot analysis of NR2A and NR2B subunits and tubulin performed from the TIF obtained from control, NVP-AAM077 (NVP, 300 nm) and ifenprodil-treated (10 μm) corticostriatal slices. The same amount of proteins was loaded in each lane. The graph displays the results of Western blot analysis expressed as control percentage. C, Western blot analysis of D1 receptor and tubulin performed from TIF obtained from control, NVP-AAM077 (NVP, 300 nm) and ifenprodil-treated (10 μm) corticostriatal slices. The same amount of proteins was loaded in each lane. The graph displays the results of Western blot analysis expressed as control percentage. D, diagram showing relative average spines head width (t test; **, p < 0.005, NVP-AAM077 versus control; n > 500 spines from 10 different neurons for each group) of MSNs from control or NVP-AAM077-treated rats. Representative images show dendrites of medium spiny neurons from control or NVP-AAM077-treated corticostriatal slices. E, diagram showing relative average spines head width of medium spiny neurons from control or ifenprodil-treated corticostriatal slices (t test; p > 0.05, ifenprodil versus control, n > 500 spines from 10 different neurons for each group). Representative images show dendrites of MSNs from control or ifenprodil-treated corticostriatal slices.
FIGURE 2.
FIGURE 2.
Effect of the selective NR2A and NR2B uncoupling peptides on dendritic spine morphology of MSNs. A, sample traces of the effect of the NR2A selective uncoupling peptide TAT2A and TAT2B on the NMDA isolated currents evoked at +40 mV and bar graph showing the percentage of the NMDA-EPSC amplitude that is blocked by the drugs, respectively, TAT2A (right panel; t test; *, p < 0.05, pre- versus post-TAT2A application, 64.03 ± 8.81%; n = 4) and TAT2B (right panel; t test; **, p < 0.01, pre- versus post-application, 59.83 ± 2.62%; n = 4). B, Western blot analysis of NR2A and NR2B subunits performed from the TIF obtained from control, TAT2A-treated (300 nm) or TAT2B-treated (300 nm) corticostriatal slices. The same amount of proteins was loaded in each lane. The graph displays the results of Western blot analysis expressed as control percentage (t test; **, p < 0.01). C, diagram showing relative average spines head width (t test; *, p < 0.05; TAT2A versus TAT2A(-SDV); n > 500 spines from 10 different neurons for each group) of MSNs from TAT2A and TAT2A(-SDV)-treated rats. Representative images show dendrites of medium spiny neurons from TAT2A and TAT2A(-SDV)-treated rats. D, diagram showing relative average spines head width of medium spiny neurons from TAT2B and TAT2B(-SDV)-treated rats (t test; n > 500 spines from 10 different neurons for each group). Representative images show dendrites of medium spiny neurons from TAT2B and TAT2B(-SDV)-treated rats.
FIGURE 3.
FIGURE 3.
Localization of NMDA receptor NR2A and NR2B regulatory subunits in D1- and D2-positive striatal MSNs. Immunofluorescence analysis for NR2A (upper panels) and NR2B (lower panels) subunits of NMDA receptor in drd1a-EGFP (A) or drd2-EGFP (B) transgenic mice. Confocal analysis shows a complete overlap between NR2A and NR2B subunits and the two types of dopamine receptors in the transgenic mice, indicating the presence of the two NMDA regulatory subunits in all D1 and D2 containing MSNs. Scale bar, 30 μm.
FIGURE 4.
FIGURE 4.
D1 receptor modulation modifies NR2A/NR2B ratio in corticostriatal slices. A, Western blot (WB) analysis of NR2A and NR2B subunits from the striatal homogenate and TIF fraction obtained from control (C) and SKF38393-treated (10 μm, 45 min) corticostriatal slices. The same amount of protein was loaded in each lane. The bar graph shows the amount of NR2A and NR2B subunits in the homogenate and TIF fraction from SKF38393-treated slices (t test; ***, p < 0.001). B, total homogenate was immunoprecipitated (i.p.) with antibody against D1 receptor and the presence of D1 and NR2A in the immunocomplex was evaluated by Western blot. Treatment with SKF38393 reduces NR2A co-precipitation with D1 (t test; **p < 0.005). C, Western blot of NR2A and NR2B subunits from control and SKF38393-treated corticostriatal slices exposed (+BS3 lanes) or not (−BS3 lanes) to the cross-linking agent BS3. NMDA receptor subunits high-molecular weight complexes that did not enter the gel are not shown (t test; *, p < 0.05, NR2B, SKF38393 versus control). D, Western blot analysis of NR2A and NR2B subunits from the homogenate and TIF fraction obtained from control and SCH23390-treated (SCH, 45 min) corticostriatal slices. The same amount of proteins was loaded in each lane (t test; *, p < 0.05). E, Western blot of GluR1 and GluR2 subunits from control and SKF38393-treated corticostriatal slices exposed (+BS3 lanes) or not (−BS3 lanes) to the cross-linking agent BS3. AMPA receptor subunits high-molecular weight complexes that did not enter the gel are not shown (t test; *, p < 0.05, GluR1, SKF38393 versus control; t test, **, p < 0.01, GluR2, SKF38393 versus control).
FIGURE 5.
FIGURE 5.
Effect of sustained treatment with D1 agonist on NMDA and AMPA currents. A, the bar graph on the left panel shows the effect of 45-min in vitro application of SKF38393 on the NMDAR/AMPAR ratio compared with control condition (**, p < 0.01; control, 0.41 ± 0.056; SKF38393, 0.23 ± 0.026; for each group, n = 10). In the right panel are reported averaged traces of AMPA currents (−70 mV in the presence of picrotoxin) and NMDA-evoked currents (+40 mV, in the presence of picrotoxin and CNQX) in control condition (upper panel) and after in vitro application of SKF38393 (lower panel). B, AMPA-mediated spontaneous excitatory activity. In the bar graphs are reported, respectively, the averaged values of the frequency (left panel) and amplitude (right panel) of spontaneous events AMPA-mediated recorded in MSNs from control slices and after 45 min of SKF38393 application. Representative traces from whole-cell patch clamp experiments showing glutamatergic AMPA-mediated spontaneous EPSCs from MSNs in control condition (upper trace) and in SKF-treated slice (lower trace). C, bar graph showing the effect of SKF incubation on the isolated NMDA-evoked current at +40 mV.
FIGURE 6.
FIGURE 6.
In vivo treatment with D1 receptor agonist SKF38393. A, Western blot analysis of NR2A and NR2B subunits performed in TIF fraction obtained from striatum of control and SKF38393-treated rats (2 mg/ml/kg, 45 min). The same amount of proteins was loaded in each lane (t test; *, p < 0.05). B, the bar graph on the left panel shows the effect of in vivo administration of SKF38393 (2 mg/ml/kg) on the NMDA-AMPA ratio compared with the control condition (**, p < 0.01; control, 0.424 ± 0.188; SKF38393, 0.16 ± 0.066; for each group, n = 10). In the right panel are reported averaged traces of AMPA currents (−70 mV in the presence of picrotoxin) and NMDA-evoked current (+40 mV, in the presence of picrotoxin and CNQX) in control condition (left side) and after in vivo administration of 2 mg/ml/kg SKF (right side). C, the bar graph on the left panel shows the effect of in vivo administration of SKF38393 on the NMDA(NR2A)-AMPA ratio. In the right panel are presented averaged traces of AMPA currents and NMDA(NR2A) currents (+40 mV in the presence of picrotoxin, CNQX, and ifenprodil) in control condition (left side) and after in vivo administration of SKF (right side) (*, p < 0.05; control, 0.146 ± 0.086; SKF38393, 0.063 ± 0.03, respectively, n = 7 and n = 6). D, diagram showing relative average spines head width (t test; n > 380 spines from nine different neurons for each group; ***, p < 0.0001 SKF38393 versus control). F, cumulative frequency plots of spine head width of MSNs from control (blue) or SKF38393-treated (red) rats. E, representative images show dendrites of MSNs from control or SKF38393-treated rats. G, immunohistoschemistry showing colocalization of SP-positive neurons (anti-SP, green) and diolistic labeling (DiL, red).
FIGURE 7.
FIGURE 7.
Effect of the selective NR2A and NR2B antagonists on D1-mediated modification of dendritic spine morphology. A, diagram showing relative average spine head widths (Kruskal-Wallis non-parametric analysis of variance; §, p < 0.0005; SKF38393 (SKF) versus control; ***, p < 0.0001; SKF38393+NVP-AAM077 versus control; **, p < 0.01; SKF38393+NVP-AAM077 versus SKF38393; #, p < 0.005 SKF38393+ifenprodil versus SKF38393; n > 320 spines from eight different neurons for each group). B, cumulative frequency plots of spine head width of medium spiny neurons from control, SKF38393, SKF38393+NVP-AAM077 (NVP), or SKF38393+ifenprodil-treated (IFP) rats. C, representative images show dendrites of medium spiny neurons from control or treated rats.

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