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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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Expression of D1 and NMDA receptors in cultured PFC neurons. (A) Localization of D1 receptor and NR2B in cultured PFC neurons. PFC neurons in dissociated culture at 14 DIV were labeled for endogenous D1 and NR2B receptors with double immunofluorescent staining. Lower panel, images at higher magnification showing the colocalization of D1 and NR2B on the dendritic shafts and spines. Scale bars = 10 μm. (B) Analysis by Western blotting shows the protein expression of D1 receptor and NMDA receptor subunits in neuronal lysates. Crude synaptosome (20 μg of protein) from adult rat cortex (lane 1) and 14 DIV cultured PFC neurons (lane 2) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed for NR1, NR2A, NR2B, D1, PSD95, and adaptin. Both adult rat cortex crude synptosome and cultured PFC neurons exhibited similar expressions of NMDA and D1 receptors, as well as synaptic protein, suggesting the validity of the experiments in primary cultured neurons.
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Figure 1: Expression of D1 and NMDA receptors in cultured PFC neurons. (A) Localization of D1 receptor and NR2B in cultured PFC neurons. PFC neurons in dissociated culture at 14 DIV were labeled for endogenous D1 and NR2B receptors with double immunofluorescent staining. Lower panel, images at higher magnification showing the colocalization of D1 and NR2B on the dendritic shafts and spines. Scale bars = 10 μm. (B) Analysis by Western blotting shows the protein expression of D1 receptor and NMDA receptor subunits in neuronal lysates. Crude synaptosome (20 μg of protein) from adult rat cortex (lane 1) and 14 DIV cultured PFC neurons (lane 2) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed for NR1, NR2A, NR2B, D1, PSD95, and adaptin. Both adult rat cortex crude synptosome and cultured PFC neurons exhibited similar expressions of NMDA and D1 receptors, as well as synaptic protein, suggesting the validity of the experiments in primary cultured neurons.

Mentions: To characterize the expression pattern of D1 and NMDA receptors in cultured PFC neurons, we performed double immunofluorescent staining of both D1 and NR2B receptors in low-density cultures at 14 DIV. As shown in Figure 1, both D1 receptors and NR2B subunits exhibited tiny, bright puncta along the dendrites, either appearing to be associated with dendritic spines or located in the dendritic shafts (Figure 1A). The punctate stainings of D1 and NR2B subunits were largely colocalized in cultured PFC neurons. These characteristic patterns of D1 and NR2B distribution in the cultured neurons were further confirmed at the protein levels in cultured PFC neurons at 14 DIV and in adult rat PFC brain homogenate (Figure 1B). The expressions of D1 and NR2B, as well as NR1 and NR2A subunits and PSD95, appeared to be abundant and similar in prefrontal brain crude synaptosome and cultured PFC neurons (Figure 1B). These results provided solid evidence that, despite the less mature phenotype of cultured prefrontal neurons, they contain all of the components and machineries required for D1-NMDA interactions.


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)

Expression of D1 and NMDA receptors in cultured PFC neurons. (A) Localization of D1 receptor and NR2B in cultured PFC neurons. PFC neurons in dissociated culture at 14 DIV were labeled for endogenous D1 and NR2B receptors with double immunofluorescent staining. Lower panel, images at higher magnification showing the colocalization of D1 and NR2B on the dendritic shafts and spines. Scale bars = 10 μm. (B) Analysis by Western blotting shows the protein expression of D1 receptor and NMDA receptor subunits in neuronal lysates. Crude synaptosome (20 μg of protein) from adult rat cortex (lane 1) and 14 DIV cultured PFC neurons (lane 2) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed for NR1, NR2A, NR2B, D1, PSD95, and adaptin. Both adult rat cortex crude synptosome and cultured PFC neurons exhibited similar expressions of NMDA and D1 receptors, as well as synaptic protein, suggesting the validity of the experiments in primary cultured neurons.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902469&req=5

Figure 1: Expression of D1 and NMDA receptors in cultured PFC neurons. (A) Localization of D1 receptor and NR2B in cultured PFC neurons. PFC neurons in dissociated culture at 14 DIV were labeled for endogenous D1 and NR2B receptors with double immunofluorescent staining. Lower panel, images at higher magnification showing the colocalization of D1 and NR2B on the dendritic shafts and spines. Scale bars = 10 μm. (B) Analysis by Western blotting shows the protein expression of D1 receptor and NMDA receptor subunits in neuronal lysates. Crude synaptosome (20 μg of protein) from adult rat cortex (lane 1) and 14 DIV cultured PFC neurons (lane 2) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed for NR1, NR2A, NR2B, D1, PSD95, and adaptin. Both adult rat cortex crude synptosome and cultured PFC neurons exhibited similar expressions of NMDA and D1 receptors, as well as synaptic protein, suggesting the validity of the experiments in primary cultured neurons.
Mentions: To characterize the expression pattern of D1 and NMDA receptors in cultured PFC neurons, we performed double immunofluorescent staining of both D1 and NR2B receptors in low-density cultures at 14 DIV. As shown in Figure 1, both D1 receptors and NR2B subunits exhibited tiny, bright puncta along the dendrites, either appearing to be associated with dendritic spines or located in the dendritic shafts (Figure 1A). The punctate stainings of D1 and NR2B subunits were largely colocalized in cultured PFC neurons. These characteristic patterns of D1 and NR2B distribution in the cultured neurons were further confirmed at the protein levels in cultured PFC neurons at 14 DIV and in adult rat PFC brain homogenate (Figure 1B). The expressions of D1 and NR2B, as well as NR1 and NR2A subunits and PSD95, appeared to be abundant and similar in prefrontal brain crude synaptosome and cultured PFC neurons (Figure 1B). These results provided solid evidence that, despite the less mature phenotype of cultured prefrontal neurons, they contain all of the components and machineries required for D1-NMDA interactions.

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