Dopamine D1 receptor-mediated NMDA receptor insertion depends on Fyn but not Src kinase pathway in prefrontal cortical neurons
© Hu et al; licensee BioMed Central Ltd. 2010
Received: 1 December 2009
Accepted: 22 June 2010
Published: 22 June 2010
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.
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.
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.
The prefrontal cortex (PFC) plays a well-established role in working memory function [1, 2] and dysfunction of either dopamine D1 receptor or N-methyl-D-aspartic acid (NMDA) receptor in this brain region has long been believed to underlie the symptoms of schizophrenia and other neuropsychiatric disorders [3–6]. Moreover, dopaminergic and glutamatergic afferents, which are from midbrain, thalamus and cortical structures, converge onto the spines of pyramidal neurons in PFC [7, 8], providing the cellular basis for interactions between dopamine and glutamate signaling in the same neuron . In the past decade, extensive studies have focused on the interactions between dopamine and NMDA receptors [3, 6, 9–11]. Studies show that dopamine influences both long-term potentiation and depression in PFC [12, 13]. Moreover, some studies indicate that physical coupling and functional cross-talk may occur between NMDA and D1 receptors [5, 14–16]. Despite the overwhelming evidence of the role of D1 and NMDA receptors in synaptic functions, the molecular mechanisms involved in these interactions, particularly signaling pathways in D1-mediated NMDA receptor trafficking, are still elusive.
Recent evidence indicates that NMDA receptors are not static, as traditionally believed; instead they can move into and out of synapses [17–20]. Indeed, D1 receptor activation led to rapid trafficking of NMDA receptor subunits, with increased expression of NMDA receptor subunits NR1 and NR2B in the dendrites of cultured striatal and prefrontal neurons [21, 22]. The regulation of NMDA receptor trafficking is a dynamic and potentially powerful mechanism for the synaptic plasticity associated with drug addiction, Alzheimer's disease and schizophrenia . Unfortunately, little is known about the signaling pathways implicated in the regulation of NMDA receptor trafficking. Previous studies have emphasized the importance of Src family kinases in this process. Src and Fyn, the major members with the highest degree of primary sequence homology, both exist in the postsynaptic density (PSD)  and likely upregulate NMDA receptor activity in the central nervous system (CNS) [25, 26]. Activation of D1 receptors is known to enhance NMDA receptor functions. Considering the importance of Src and Fyn in NMDA receptor regulation [23, 27–29], we hypothesized that Src family kinases, especially Fyn and Src, mediate D1 receptor activation-induced NMDA receptor increase in PFC. We tested this hypothesis in cultured PFC neurons and found that Fyn, but not Src, is critical in D1 receptor activation-induced NMDA receptor trafficking, particularly in surface insertion.
Cultured prefrontal neurons express both D1 and NMDA receptors
D1 receptor stimulation increases NR2B expression in cultured PFC neurons
Although NMDA receptors are largely composed of NR1 (obligatory subunit), NR2A and NR2B, we focused on the effect of D1 class-mediated regulation in NR2B trafficking to test our hypothesis. This is because NR2B is the major tyrosine-phosphorylated subunit in the post-synaptic density [14, 30, 31] and NR2B subunits are the most dynamic and mobile subunit in the NMDA receptors [19, 32]. In addition, tyrosine phosphorylation of NR2B at Tyr1472 is dependent on Src/Fyn family kinases . Although NR1 is an obligatory subunit of NMDA receptors, it is known to be phosphorylated by serine kinases PKC and PKA but not by tyrosine kinases [30, 33, 34], whereas NR2A seems to be less affected by activation of D1 receptors [21, 22].
Fyn or Src knockdown has no effect on total NR2B expression in cultured PFC neurons
D1 receptor-mediated increases in NR2B expression and surface insertion depend on the Fyn, but not the Src, signaling pathway
Previous studies in other brain regions have demonstrated that activation of Src family kinases leads to phosphorylation of NR2B which appears to be important for surface expression of this subunit [23, 32]. Therefore, we used a specific antibody to look at the phosphorylation state of the NR2B subunit. We found that knockdown of Fyn by siRNA blocked the phosphorylation of NR2B Tyr1472 after D1 activation (vehicle-transfected and non SKF-81297 treatment control: 100%; vehicle-transfected neurons treated with SKF-81297: 163.4% ± 2.4%, p < 0.05; Fyn siRNA, non SKF-81297 treatment: 101.2% ± 2.5%, p > 0.05; Fyn siRNA, SKF-81297: 92.2% ± 2.6%, p > 0.05; n = 3 in each group) (Figure 5C) while siRNA knockdown of Src had no effect on D1-induced NR2B Tyr1472 phosphorylation (vehicle-transfected and non SKF-81297 treatment control: 100%; vehicle-transfected neurons treated with SKF-81297: 156.7% ± 2.5%, p < 0.05; Src siRNA, non SKF-81297 treatment: 99.5% ± 2.5%, p > 0.05; Src siRNA, SKF-81297: 162.2 ± 3.6%, p < 0.05; n = 3 in each group) (Figure 6C). This indicates that under our experimental conditions, Fyn phosphorylates NR2B while Src does not.
We have demonstrated that D1 receptor activation-mediated enhancement of NR2B expression, in the cultured PFC neurons depends on the Fyn but not Src signaling pathway by taking the advantage of siRNA knockdown. Our data provide evidence of a novel molecular mechanism involved in the D1-NMDA interaction in prefrontal neurons.
Interaction of D1 and NMDA receptors in PFC has long been proposed to contribute to synaptic plasticity and to cognitive functions. Pharmacologic blockade of either D1 or NMDA receptor results in many cognitive deficits such as decreases in spatial working memory [1, 36, 37]. Physiologically, D1 receptor stimulation potentiates the NMDA receptor responses in PFC neurons in vitro [5, 9–11, 38]. The D1-dependent NMDA receptor enhancement in PFC appears to be critical both for physiological regulation of synaptic strength in working memory function [6, 39] and for disorders such as drug addiction  and schizophrenia .
NMDA receptors are essential for long-lasting changes in synaptic efficacy such as long-term plasticity. Considerable evidence indicates that NMDA receptors are not static residents in synapses but instead can move in and out , whereby they may regulate receptor function and synaptic plasticity . On the other hand, neuronal activity drives not only local receptor synthesis but also receptor insertion into the plasma membrane, lateral diffusion between synaptic and extrasynaptic sites, and receptor endocytosis [23, 41, 42]. Thus, the notion has been emerging that activity-dependent NMDA receptor trafficking provides a potentially powerful mechanism for the regulation of synaptic efficacy and remodeling. It is increasingly appreciated that NMDA receptor trafficking dysregulation may contribute to neuropsychiatric disorders such as drug addiction , Alzheimer's disease , and schizophrenia . Therefore, understanding the role of D1 receptor stimulation in modulating NMDA receptor trafficking is vital in order to elucidate the mechanisms underlying synaptic plasticity and neuropsychiatric disorders.
Increasing evidence suggests that Src family kinases play essential roles in the regulation of NMDA receptor by D1 receptor activation [46, 47]. Our recent study  shows that D1 receptor-mediated increase in NR2B surface expression and synaptic function in PFC was selectively blocked by Src family kinase inhibitor PP2. The Src family of protein tyrosine kinases expressed in the nervous system includes Src, Fyn, Yes, Lck and Lyn. Because of the high homology between the family members, determining the role of specific members of the Src family kinases in the regulation of NMDA receptor trafficking has been challenging. Whereas Src has been clearly shown to play a role in the regulation of NMDA receptors [28, 48] and in the induction of long-term potentiation in hippocampal CA1 [28, 49], gene-targeted deletion of Src failed to show apparent neurological phenotypes [28, 29]. Several confounding factors may explain these conflicting results. Pharmacological tools for specific blockade of Src or Fyn are lacking. In addition, Src family kinases may have both specific and overlapping functions in various physiological processes . Therefore molecular redundancy among Src family kinases may lead to functional compensation, thus confounding the phenotype of knockout mice. In other words, knockout mice might not be the ideal tools to study the specific functions of Src family members. To determine whether and which specific Src kinases have a role in D1-induced NMDA receptor trafficking, we took the siRNA approach. Indeed, as recent study  has shown, siRNA knockdown approach has some advantages over the gene knockout approach in studying the function of closely related family members such as MAGUKS in glutamate receptor trafficking. Using the siRNA approach, we found that dopamine D1-activation induced NMDA receptor trafficking depends on Fyn, but not Src signaling pathway.
NMDA receptor tyrosine phosphorylation at position 1472 (Tyr1472) might stabilize NMDA receptors on the cell surface, thereby increasing NMDA receptor responses. Accordingly, Src family kinase activation might inhibit NMDA receptor endocytosis [32, 52]. We observed the effect of D1 receptor stimulation on the surface expression of the NR2B subunit in cultured PFC neurons, consistent with previous studies [21, 22]. D1 receptor agonist treatment leads to significant increase in NR2B dendritic localization and colocalization with PSD95. Fyn knockdown effectively blocks the NMDA receptor increase after D1 activation, whereas Src knockdown exhibits no clear effect, suggesting that Fyn, but not Src, is involved in D1 receptor modulation of NMDA receptor expression. Our data agree with numerous recent studies in which Tyr1472 phosphorylation was found to be required for proper NR2B localization at synapses in the striatum [21, 46], hippocampus , amygdala , and PFC . It should be mentioned that although we focused on the NR2B subunit in this study, we found that D1 also increased NR1 subunits (Li et al., unpublished observations). This is important as there are functional differences among NMDA receptors as recent work has demonstrated .
Our RNAi efficiency is similar to that of others as reported in neurons . The reason why that the protein level of Src or Fyn is reduced to ~50% of the control is a matter of speculation. Due to the variance in protein stability and cell systems, the efficacy of siRNA also varies. As for why the remaining Fyn is insufficient for enhancing NMDA receptor trafficking after D1 activation, we speculate that Fyn might have some important functions in cell survival and cell physiology , and for the economy of cells, it is not unreasonable to think that cell survival has a higher priority than the receptor trafficking.
Our findings demonstrate that dopamine receptors may regulate synaptic plasticity by modulating NMDA receptor synaptic expression through a Fyn-dependent signaling pathway. Considering that NMDA receptor activity alterations are involved in the clinical features of schizophrenia and drug abuse, our study not only provides insight into the roles of Src family kinases in NMDA receptor trafficking but also offers the possibility of generating new pharmacologic reagents targeting Fyn kinase signaling pathway. This intervention could be promising for psychiatric disorders involving D1-NMDA interaction, such as schizophrenia and drug addiction.
Chemicals and antibodies
The chemicals used for treatment of PFC neurons were purchased from the following sources: selective D1 agonist SKF-81297, D1 antagonist SCH-23390, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies used for immunofluorescence and Western blotting included mouse anti-NR2B (1:2000, Millipore, Billerica, MA), rabbit phospho-Y1472 NR2B (1:500, PhosphoSolutions, Aurora, CO), rabbit anti-PSD95 (1:1000, Frontier Science Co., Hokkaido, Japan), rat anti-D1 (1:1000, Sigma), rabbit anti-Fyn (1:1000, Millipore), mouse anti-Src (1:1000, Millipore), and mouse antitubulin (1:10000, Millipore). The primary antibody dilutions listed above were for Western blotting. For immunofluorescence staining, dilutions were all 1:800. The following reagents were obtained from Jackson ImmunoResearch (West Grove, PA): Cy3-conjugated donkey antimouse secondary antibody (1:800), FITC-conjugated donkey anti-mouse secondary antibody (1: 800), Cy3-conjugated donkey anti-rabbit secondary antibody (1:800), FITC-conjugated donkey anti-rabbit secondary antibody (1:800), peroxidase-conjugated goat anti-mouse secondary antibody (1:1000), and peroxidase-conjugated goat anti-rabbit secondary antibody (1:1000).
Timed pregnant rats and adult rats (3 months old) were purchased from Marshall Farms (New York, NY) and the animal procedures were in strict accordance with the National Institute of Health (NIH) animal use guideline. The experimental protocols were approved by the Institutional Animal Care and Use Committee at Drexel University College of Medicine. Primary neuron cultures were prepared from embryonic day 20 rat PFC. Briefly, PFC tissues were isolated and dissociated with trypsin (Gibco, Carlsbad, CA) at 37°C, filtered by cell filter (BD Falcon, San Jose, CA), rinsed with Neurobasal medium (Gibco, Carlsbad, CA) supplemented with B27and L-glutamine(Gibco, Carlsbad, CA) and 5% fetal bovine serum (FBS). Cultures were plated at 2 × 105 cells per well on poly-l-lysine coated glass coverslips in 6-well plates (BD Falcon, San Jose, CA) and kept in Neurobasal medium supplemented with B27, L-glutamine, and 5% FBS at 37°C/5% CO2 for 4 h. Cells were then maintained in Neurobasal medium supplemented with B27, and L-glutamine without FBS at 37°C/5% CO2. Cells were fed 2 times per week, with one third of the media changed each time. Cultured PFC neurons were used between 14 and 18 days in vitro (DIV) for the studies described.
Src and Fyn siRNAs were purchased from Millipore, including pKD-Fyn-v6 (mammalian Fyn siRNA expression plasmid) and pKD-Src-v2 (mammalian Src siRNA expression plasmid).
Src siRNA sequence:
Fyn siRNA sequence:
The siRNAs were used to transfect PFC neurons (750 ng per well) with GeneSilencer siRNA transfection reagent from Genlantis (San Diego, CA) when cultures reached 70% confluence. Neurons in the control conditions were transfected with the same volume of transfection reagent without siRNA. After 4 h, neurons were washed with serum-free Neurobasal medium, and neurons were maintained in Neurobasal medium supplemented with B27 and L-glutamine at 37°C/5% CO2 for 48 to 72 h to allow for detectable knockdown prior to the treatment with other pharmacologic reagents. The efficiencies of siRNA transfection were assessed by both immunostaining and Western blotting with antibodies against Fyn or Src, whereas the antibody specificities were demonstrated by re-probing the Src and Fyn, respectively.
Pharmacologic treatments of PFC neurons
To assess the roles of Fyn and Src in D1 receptor-mediated modulation of NMDA receptor, PFC neurons with or without transfection were treated with the selective D1 receptor agonist SKF-81297 (10 μM) for 10 min or pretreated with selective D1 receptor antagonist SCH-23390 (15 μM) followed by SKF-81297 (10 μM) for 10 min. The cultured neurons were then restored to normal medium at 37°C/5% CO2 for another 15 min to allow the trafficking of NMDA receptors. For control, the same volume of DMSO (0.1%) was added as vehicle and the cultured neurons were subjected to the same conditions. Precautions were taken to protect samples from light exposure and oxidation. The treated PFC neurons were used for both immunofluorescent staining and Western blotting.
Immunofluorescent staining of PFC neurons
PFC neurons were fixed by methanol at -20 °C for 5 min and rinsed with 0.2% Triton X-100 (Dow Chemical, Midland, MI) in phosphate-buffered saline (PBS) to permeabilize the plasma membrane. Coverslips were blocked with 10% bovine serum albumin in PBS for 1 h at room temperature. Double immunofluorescent stainings were conducted with antibodies against NMDA receptors (NR2B), D1 receptor, Fyn, Src, or postsynaptic marker PSD95, followed by appropriate secondary antibodies. Images were acquired using a Zeiss Axiovert 200 M inverted microscope with Axiovision software (Zeiss Microscopy, Jena, Germany). All analysis and quantifications were performed using the NIH Image J software. The dendritic segments in neurons (40 μm) were randomly selected for puncta analysis. The average intensity of fluorescence staining and the punctate number of NR2B/Fyn/Src were quantified using the NIH Image J software and were blindly confirmed by other researchers in the laboratory. Results were presented as the mean number of total puncta or mean intensity of fluorescence ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey multiple-comparison tests for several experimental groups. Images were prepared for printing with Adobe Photoshop (San Jose, CA) and Canvas (ACD Systems, Ltd., Victoria, BC, Canada).
The biotinylation was performed as described with minor modification . After transfection with Fyn or Src siRNA, neurons were treated with the pharmacologic agents as described above. For cell surface receptor biotinylation, neurons were rinsed twice with ice-cold 0.1 M PBS for 1 min each and were then incubated with 1.5 mg/mL sulfo-NHS-SS-Biotin (Pierce, Rockford, IL, USA) in 0.1 M PBS for 20 min at 4 °C. The sulfo-NHS biotin was quenched with PBS containing 50 mM glycine. After washing twice with ice-cold 0.1 M PBS (5 min each), neurons were then lysed in radioimmunoprecipitation assay (RIPA) buffer. The homogenates were centrifuged at 14,000 g for 10 min at 4 °C. The resulting supernatant volume was measured and 20% of it separated as the total (T) protein. The remaining 80% of the supernatant was incubated with NeutrAvidin agarose beads (Pierce) overnight at 4 °C. NeutrAvidin agarose beads were washed with RIPA buffer and then spun down for 2 min at 5000 rpm, which was repeated 3 times. Finally, the biotinylated proteins were eluted from the NeutrAvidin by incubation with 2 × sample SDS-PAGE buffer at 95°C for 5 min and used as surface (S) proteins. Western blotting was performed using antibody against NR2B. Data were quantified by comparing the ratio of surface biotinylated to total input protein and normalized to control group as percentages.
Crude Synaptosome (P2) Preparation
Crude synaptosome (P2) was prepared from rat frontal cortex as previously described [58, 59] with minor modification. Cerebral cortices were collected and homogenized in 40 ml buffered sucrose (0.32 M Sucrose/1 mM NaHCO3). The homogenate was centrifuged at 1,400 g for 10 min, then the pellet (P1) was discarded, while the supernatant (S1) was saved and centrifuged at 13,800 g for 10 min. The resulting pellet (P2) was crude synaptosome, which was resuspended by RIPA buffer and resolved by electrophoresis. The crude synaptosome was used in Figure 1B, whereas homogenates were used in all other experiments for the data exhibited in Figures 2, 3, 4, 5, 6 and 7.
Proteins were isolated from adult rat PFC or high-density prefrontal neuronal cultures with RIPA buffer. After centrifugation at 14,000 rpm at 4 °C for 15 min, the supernatants were resolved by electrophoresis on 7.5% polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked in 5% nonfat milk in TBS for 1 h and were incubated with the following antibodies overnight at 4 °C: mouse anti-NR2B, rabbi anti-PSD95, rat anti-D1, rabbit anti-Fyn, mouse anti-Src, and mouse antitubulin. After incubation with appropriate horseradish peroxidase-conjugated secondary antibodies, antigens were identified by enhanced chemiluminescence reagents . Blots were scanned and quantified using Image J software. Control protein levels were set at 100% after being normalized to loading control tubulin. All other values were normalized as percentages of control. Each set of experiments was repeated at least 3 times to reduce interblot variability. Images were prepared for printing with Adobe Photoshop and Canvas.
The authors thank Dr. Gianluca Gallo (Drexel University College of Medicine) for comments on the manuscript. Supported by NARSAD Young Investigator Award (Yue-Qiao Huang) and Drexel University College of Medicine.
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