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Dopamine D1 receptor-mediated NMDA receptor insertion depends on Fyn but not Src kinase pathway in prefrontal cortical neurons.

Hu JL, Liu G, Li YC, Gao WJ, Huang YQ - Mol Brain (2010)

Bottom Line: Activation of D1 receptor by selective agonist SKF-81297 significantly increased the expression of NR2B subunits.Under control conditions, neither Fyn nor Src knockdown exhibited significant effect on basal NR2B expression.D1 stimulation significantly enhanced NR2B insertion into plasma membrane in cultured PFC neurons, a process obstructed by Fyn, but not Src, knockdown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA 19129, USA. jh356@drexel.edu

ABSTRACT

Background: Interactions between dopamine and glutamate in the prefrontal cortex are essential for cognitive functions such as working memory. Modulation of N-methyl-D-aspartic acid (NMDA) receptor functions by dopamine D1 receptor is believed to play a critical role in these functions. The aim of the work reported here is to explore the signaling pathway underlying D1 receptor-mediated trafficking of NMDA receptors in cultured rat prefrontal cortical neurons.

Results: Activation of D1 receptor by selective agonist SKF-81297 significantly increased the expression of NR2B subunits. This effect was completely blocked by small interfering RNA knockdown of Fyn, but not Src. Under control conditions, neither Fyn nor Src knockdown exhibited significant effect on basal NR2B expression. D1 stimulation significantly enhanced NR2B insertion into plasma membrane in cultured PFC neurons, a process obstructed by Fyn, but not Src, knockdown.

Conclusions: Dopamine D1 receptor-mediated increase of NMDA receptors is thus Fyn kinase dependent. Targeting this signaling pathway may be useful in treating drug addiction and schizophrenia.

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Related in: MedlinePlus

D1 receptor agonist SKF-81297 enhances the expression and clustering of NR2B subunit. (A) PFC neurons at 16 DIV were treated with vehicle (a, d, g, j), SKF-81297 (10 μM; b, e, h, k), or SKF in the presence of SCH (15 μM; c, f, i, l), respectively, and double labeled for endogenous NR2B (green) and PSD95 (red). Panel j, k and l are merged from respective NR2B (green) and PSD-95(red) staining. Scale bars = 10 μm. (B) Quantification of total NR2B immunofluorescence staining. White bars: NR2B puncta number; black bars: NR2B fluorescence intensity. SKF-81297 significantly increased total NR2B puncta number and immunofluorescence intensity compared with control. Results are presented as mean number and fluorescence intensity of total NR2B puncta. Statistical analysis was performed using ANOVA followed by Tukey multiple comparison test (n = 20, ** p < 0.01, *** p < 0.001). Data represent mean ± SEM. (C) Proteins were isolated at 16 DIV from cultured high-density PFC neurons treated with DMSO, D1 receptor agonist SKF-81297, or SKF-81297 + SCH-23390. The proteins were resolved on SDS-PAGE and immunoblotted for NR2B and reprobed with tubulin. (D) Quantification of NR2B protein expression in PFC neurons. SKF-81297 significantly increased NR2B expression, which was completely blocked by pretreatment with SCH-23390 (n = 4, ** p < 0.01, *** p < 0.001). Integrated intensity was measured using Image J and control protein level of NR2B was set to 100% after being normalized to tubulin. These results indicate that D1 receptor stimulation does increase NR2B expression, which may eventually increase NMDA receptor trafficking to membrane surface.
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Figure 2: D1 receptor agonist SKF-81297 enhances the expression and clustering of NR2B subunit. (A) PFC neurons at 16 DIV were treated with vehicle (a, d, g, j), SKF-81297 (10 μM; b, e, h, k), or SKF in the presence of SCH (15 μM; c, f, i, l), respectively, and double labeled for endogenous NR2B (green) and PSD95 (red). Panel j, k and l are merged from respective NR2B (green) and PSD-95(red) staining. Scale bars = 10 μm. (B) Quantification of total NR2B immunofluorescence staining. White bars: NR2B puncta number; black bars: NR2B fluorescence intensity. SKF-81297 significantly increased total NR2B puncta number and immunofluorescence intensity compared with control. Results are presented as mean number and fluorescence intensity of total NR2B puncta. Statistical analysis was performed using ANOVA followed by Tukey multiple comparison test (n = 20, ** p < 0.01, *** p < 0.001). Data represent mean ± SEM. (C) Proteins were isolated at 16 DIV from cultured high-density PFC neurons treated with DMSO, D1 receptor agonist SKF-81297, or SKF-81297 + SCH-23390. The proteins were resolved on SDS-PAGE and immunoblotted for NR2B and reprobed with tubulin. (D) Quantification of NR2B protein expression in PFC neurons. SKF-81297 significantly increased NR2B expression, which was completely blocked by pretreatment with SCH-23390 (n = 4, ** p < 0.01, *** p < 0.001). Integrated intensity was measured using Image J and control protein level of NR2B was set to 100% after being normalized to tubulin. These results indicate that D1 receptor stimulation does increase NR2B expression, which may eventually increase NMDA receptor trafficking to membrane surface.

Mentions: To study D1 receptor activation-mediated NR2B expression in cultured PFC neurons, the cultures were treated for 10 min with DMSO (0.1%) as vehicle control, the selective dopamine D1 receptor agonist SKF-81297 (10 μM), or the dopamine D1 receptor antagonist SCH-23390 (15 μM) followed by SKF-81297 (10 μM) for 10 min. The cultures were then restored to 37°C/5% CO2 in normal medium for another 15 min to allow the receptor trafficking. When the PFC neurons were treated with the D1 agonist SKF-81297 alone, expression of NR2B subunits in cultured PFC neurons was significantly enhanced compared with control, as shown in Figure 2 (puncta number: 23.8 ± 0.93 in control vs 35.0 ± 1.09 in SKF, n = 20, p < 0.001; fluorescence intensity: 33,875 ± 2439 in control vs 56,497 ± 3397 in SKF, n = 20, p < 0.01; Figure 2A and 2B). However, when cultured PFC neurons were pretreated by D1 antagonist SCH-23390 followed by SKF-81297 administration, the enhancement in NR2B expression was significantly attenuated (puncta number: 21.8 ± 1.10 in SKF + SCH, n = 25, p < 0.001; fluorescence intensity: 30,544 ± 2504 in SKF + SCH, n = 25, p < 0.001; Figure 2A and 2B). These results were further confirmed using Western blot analysis. After treatment, the homogenates from high-density cultured PFC neurons were prepared and changes in NR2B protein expression were probed by immunoblotting. SKF-81297 treatment increased NR2B protein by 91.1% ± 14.51% compared with control (n = 4; p < 0.001; Figure 2C) and the increase was specifically blocked by pretreatment of SCH-23390 (Figure 2C and 2D). In contrast, D1 receptor stimulation did not show any detectable effect on PSD95 expression (Figure 2A). Since the D1-activation induced an increase in total NR2B subunit staining, we asked whether this increase is sensitive to the protein synthesis inhibitor anisomycin (see Additional File 1). These results indicate that D1 receptor stimulation does increase NR2B expression, which may eventually increase the NMDA receptor trafficking to membrane surface.


Dopamine D1 receptor-mediated NMDA receptor insertion depends on Fyn but not Src kinase pathway in prefrontal cortical neurons.

Hu JL, Liu G, Li YC, Gao WJ, Huang YQ - Mol Brain (2010)

D1 receptor agonist SKF-81297 enhances the expression and clustering of NR2B subunit. (A) PFC neurons at 16 DIV were treated with vehicle (a, d, g, j), SKF-81297 (10 μM; b, e, h, k), or SKF in the presence of SCH (15 μM; c, f, i, l), respectively, and double labeled for endogenous NR2B (green) and PSD95 (red). Panel j, k and l are merged from respective NR2B (green) and PSD-95(red) staining. Scale bars = 10 μm. (B) Quantification of total NR2B immunofluorescence staining. White bars: NR2B puncta number; black bars: NR2B fluorescence intensity. SKF-81297 significantly increased total NR2B puncta number and immunofluorescence intensity compared with control. Results are presented as mean number and fluorescence intensity of total NR2B puncta. Statistical analysis was performed using ANOVA followed by Tukey multiple comparison test (n = 20, ** p < 0.01, *** p < 0.001). Data represent mean ± SEM. (C) Proteins were isolated at 16 DIV from cultured high-density PFC neurons treated with DMSO, D1 receptor agonist SKF-81297, or SKF-81297 + SCH-23390. The proteins were resolved on SDS-PAGE and immunoblotted for NR2B and reprobed with tubulin. (D) Quantification of NR2B protein expression in PFC neurons. SKF-81297 significantly increased NR2B expression, which was completely blocked by pretreatment with SCH-23390 (n = 4, ** p < 0.01, *** p < 0.001). Integrated intensity was measured using Image J and control protein level of NR2B was set to 100% after being normalized to tubulin. These results indicate that D1 receptor stimulation does increase NR2B expression, which may eventually increase NMDA receptor trafficking to membrane surface.
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Figure 2: D1 receptor agonist SKF-81297 enhances the expression and clustering of NR2B subunit. (A) PFC neurons at 16 DIV were treated with vehicle (a, d, g, j), SKF-81297 (10 μM; b, e, h, k), or SKF in the presence of SCH (15 μM; c, f, i, l), respectively, and double labeled for endogenous NR2B (green) and PSD95 (red). Panel j, k and l are merged from respective NR2B (green) and PSD-95(red) staining. Scale bars = 10 μm. (B) Quantification of total NR2B immunofluorescence staining. White bars: NR2B puncta number; black bars: NR2B fluorescence intensity. SKF-81297 significantly increased total NR2B puncta number and immunofluorescence intensity compared with control. Results are presented as mean number and fluorescence intensity of total NR2B puncta. Statistical analysis was performed using ANOVA followed by Tukey multiple comparison test (n = 20, ** p < 0.01, *** p < 0.001). Data represent mean ± SEM. (C) Proteins were isolated at 16 DIV from cultured high-density PFC neurons treated with DMSO, D1 receptor agonist SKF-81297, or SKF-81297 + SCH-23390. The proteins were resolved on SDS-PAGE and immunoblotted for NR2B and reprobed with tubulin. (D) Quantification of NR2B protein expression in PFC neurons. SKF-81297 significantly increased NR2B expression, which was completely blocked by pretreatment with SCH-23390 (n = 4, ** p < 0.01, *** p < 0.001). Integrated intensity was measured using Image J and control protein level of NR2B was set to 100% after being normalized to tubulin. These results indicate that D1 receptor stimulation does increase NR2B expression, which may eventually increase NMDA receptor trafficking to membrane surface.
Mentions: To study D1 receptor activation-mediated NR2B expression in cultured PFC neurons, the cultures were treated for 10 min with DMSO (0.1%) as vehicle control, the selective dopamine D1 receptor agonist SKF-81297 (10 μM), or the dopamine D1 receptor antagonist SCH-23390 (15 μM) followed by SKF-81297 (10 μM) for 10 min. The cultures were then restored to 37°C/5% CO2 in normal medium for another 15 min to allow the receptor trafficking. When the PFC neurons were treated with the D1 agonist SKF-81297 alone, expression of NR2B subunits in cultured PFC neurons was significantly enhanced compared with control, as shown in Figure 2 (puncta number: 23.8 ± 0.93 in control vs 35.0 ± 1.09 in SKF, n = 20, p < 0.001; fluorescence intensity: 33,875 ± 2439 in control vs 56,497 ± 3397 in SKF, n = 20, p < 0.01; Figure 2A and 2B). However, when cultured PFC neurons were pretreated by D1 antagonist SCH-23390 followed by SKF-81297 administration, the enhancement in NR2B expression was significantly attenuated (puncta number: 21.8 ± 1.10 in SKF + SCH, n = 25, p < 0.001; fluorescence intensity: 30,544 ± 2504 in SKF + SCH, n = 25, p < 0.001; Figure 2A and 2B). These results were further confirmed using Western blot analysis. After treatment, the homogenates from high-density cultured PFC neurons were prepared and changes in NR2B protein expression were probed by immunoblotting. SKF-81297 treatment increased NR2B protein by 91.1% ± 14.51% compared with control (n = 4; p < 0.001; Figure 2C) and the increase was specifically blocked by pretreatment of SCH-23390 (Figure 2C and 2D). In contrast, D1 receptor stimulation did not show any detectable effect on PSD95 expression (Figure 2A). Since the D1-activation induced an increase in total NR2B subunit staining, we asked whether this increase is sensitive to the protein synthesis inhibitor anisomycin (see Additional File 1). These results indicate that D1 receptor stimulation does increase NR2B expression, which may eventually increase the NMDA receptor trafficking to membrane surface.

Bottom Line: Activation of D1 receptor by selective agonist SKF-81297 significantly increased the expression of NR2B subunits.Under control conditions, neither Fyn nor Src knockdown exhibited significant effect on basal NR2B expression.D1 stimulation significantly enhanced NR2B insertion into plasma membrane in cultured PFC neurons, a process obstructed by Fyn, but not Src, knockdown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA 19129, USA. jh356@drexel.edu

ABSTRACT

Background: Interactions between dopamine and glutamate in the prefrontal cortex are essential for cognitive functions such as working memory. Modulation of N-methyl-D-aspartic acid (NMDA) receptor functions by dopamine D1 receptor is believed to play a critical role in these functions. The aim of the work reported here is to explore the signaling pathway underlying D1 receptor-mediated trafficking of NMDA receptors in cultured rat prefrontal cortical neurons.

Results: Activation of D1 receptor by selective agonist SKF-81297 significantly increased the expression of NR2B subunits. This effect was completely blocked by small interfering RNA knockdown of Fyn, but not Src. Under control conditions, neither Fyn nor Src knockdown exhibited significant effect on basal NR2B expression. D1 stimulation significantly enhanced NR2B insertion into plasma membrane in cultured PFC neurons, a process obstructed by Fyn, but not Src, knockdown.

Conclusions: Dopamine D1 receptor-mediated increase of NMDA receptors is thus Fyn kinase dependent. Targeting this signaling pathway may be useful in treating drug addiction and schizophrenia.

Show MeSH
Related in: MedlinePlus