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Keywords:

  • AMPA receptors;
  • D1 dopamine receptors;
  • GluR1;
  • nucleus accumbens;
  • protein kinase A;
  • receptor trafficking

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

Trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors is an important determinant of synaptic strength. Our prior work suggests that D1 dopamine (DA) receptors regulate AMPA receptor trafficking. This is a possible mechanism by which amphetamine and cocaine, which indirectly stimulate D1 receptors, may alter synaptic strength in addiction-related neuronal circuits. Post-natal rat nucleus accumbens (NAc) cultures were used to study the role of protein kinase A (PKA) in D1 receptor regulation of the surface expression of the AMPA receptor subunit GluR1. Using an immunocytochemical assay that selectively detects newly externalized GluR1, we found that the rate of GluR1 externalization is enhanced by the D1 agonist SKF 81297 (100 nm-1 µm). This was blocked by a D1 receptor antagonist (SCH 23390; 10 µm) and by two different cell-permeable PKA inhibitors, KT5720 (2 and 10 µm) and RpcAMPS (10 µm). Conversely, the PKA activator SpcAMPS increased the rate of GluR1 externalization in a concentration-dependent manner. A maximally effective concentration of SpcAMPS (10 µm) occluded the effect of SKF 81297 (1 µm) on GluR1 externalization. Using similar cultures, we showed previously that D1 receptor stimulation increases GluR1 phosphorylation at the PKA site. Together, our findings suggest that PKA phosphorylation of GluR1 is required for GluR1 externalization in response to D1 receptor stimulation.

Abbreviations used
AMPA

α-amino-3-hydroxy-5-methylisoxazole-4-propionate

DA

dopamine

FBS

fetal bovine serum

LTD

long-term depression

LTP

long-term potentiation

NAc

nucleus accumbens

PBS

phosphate-buffered saline

PKA

protein kinase A

While dopamine (DA) receptors mediate acute effects of amphetamine and cocaine, glutamate transmission plays a critical role in neuroadaptations produced by chronic stimulant administration (Wolf 1998). This suggests that addiction may be a maladaptive form of neuroplasticity that involves glutamate-dependent cellular mechanisms similar to those implicated in long-term potentiation (LTP) and long-term depression (LTD). This hypothesis was first proposed over a decade ago (Karler et al. 1989; Wolf and Khansa 1991), but has received strong support from recent studies showing that LTP and LTD in addiction-related brain regions can be influenced by DA and psychomotor stimulants (Jones et al. 2000; Thomas et al. 2000, 2001; Ungless et al. 2001; Saal et al. 2003).

How do drugs of abuse influence LTP and LTD? Recent studies suggest that trafficking of AMPA receptors in and out of post-synaptic sites is a highly regulated process that is critical for altering the strength of excitatory synapses (Malinow and Malenka 2002). We hypothesized that DA receptors, stimulated during amphetamine or cocaine treatment, influence AMPA receptor trafficking and that this contributes to long-lasting changes in synaptic strength in addiction-related neuronal circuits.

As a first step towards testing this hypothesis, we developed primary cultures of post-natal rat nucleus accumbens (NAc) neurons as a model system for studying DA/AMPA receptor interactions. The NAc is a critical brain region for addiction and is the site of many drug-induced adaptations in glutamate transmission. Neurons in the NAc of the adult rat consist of two major classes (Meredith and Totterdell 1999). The output neurons, medium spiny GABA neurons, receive convergent DA and glutamate inputs and represent 90% of total neurons. The remaining neurons are interneurons, many of which are also GABAergic and regulated by DA and glutamate. Our culture system reproduces many of these features (Chao et al. 2002b). Based on morphological criteria, ∼80% of the neurons are medium spiny neurons and ∼20% are interneurons. Nearly all neurons express glutamic acid decarboxylase, a marker for GABA-containing neurons, consistent with the GABAergic phenotype of medium spiny neurons and most interneurons. Most importantly, all neurons express the AMPA receptor subunit GluR1, ∼80% express D1 receptors, and ∼80% express D2 receptors. These findings imply considerable overlap of DA and AMPA receptor expression at the single cell level, making our cultures a useful model system for studying DA/AMPA receptor interactions.

In the first study to examine AMPA receptor trafficking in neurons of the striatal complex, we found that brief incubation with a D1 agonist increased GluR1 surface expression in cultured NAc neurons (Chao et al. 2002b). The purpose of the present study was to determine whether this effect of D1 receptor stimulation required protein kinase A (PKA) activity. PKA phosphorylation of GluR1 promotes its surface expression in cultured hippocampal and cortical neurons (Ehlers 2000; Esteban et al. 2003) and we have shown previously that D1 receptor stimulation enhances PKA phosphorylation of GluR1 in cultured NAc neurons (Chao et al. 2002a).

To examine the relationship between PKA activity and GluR1 trafficking in NAc neurons, we examined the effect of two PKA inhibitors and a PKA activator on basal and D1 receptor-induced externalization of GluR1. Our results support the hypothesis that phosphorylation of GluR1 at the PKA site promotes its externalization. More generally, they suggest that G protein-coupled receptors linked to the PKA pathway are capable of directly modulating AMPA receptor trafficking and thereby influencing plasticity at glutamate synapses.

Animals

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Chicago Medical School. Pregnant Sprague–Dawley rats (Harlan, Indianapolis, IN, USA; Zivic Miller, Pittsburgh, PA, USA), obtained at 19 days of gestation, were housed individually in breeding cages. One-day-old offspring were used to prepare NAc cultures, and 2- to 3-day-old offspring were used for astrocyte cultures.

Post-natal NAc cultures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

Post-natal NAc cells were prepared according to methods modified from Shi and Rayport (1994). Post-natal (P1) rats were anesthetized by hypothermia on ice. Brains were removed into ice-cold phosphate-buffered saline (PBS). The forebrain was split sagitally at the midline. With the medial surface facing up, a 16G sharp-edged cannula was used to punch out a cylinder of tissue containing the NAc, using the anterior commissure to define its peripheral border (caudal/dorsal). The lateral one-third of the cylinder (cortex) and the medial one-quarter (using the lateral ventricle as a landmark) of the cylinder were removed using a scalpel blade. The middle portion was transferred to ice-cold CMF solution (calcium- and magnesium-free PBS with 0.5% gentamicin, 1% fungizone, and 0.5% glucose) and bubbled with O2 continuously. Tissue was rinsed with cold CMF and dissociated using papain (20–25 units/mL; Worthington, Lakewood, NJ, USA) for 15–20 min at 37°C, followed by trituration with a 22G and a 25G needle. Cells were filtered through 5 mL fetal bovine serum (FBS) in a 15 mL conical tube for 10min at 4°C in a clinical centrifuge (200 g). Viability, determined with 0.4% Trypan blue, ranged from 85 to 95%. NeuroBasal growth media (Gibco, Grand Island, NY, USA; 2 mm glutamine, 0.5% gentamicin, and 25 µm glutamate) was conditioned with astrocyte cultures for 18–24 h. Then, B27 was added (1 : 50; Gibco, Grand Island, NY, USA) and the B27-supplemented NeuroBasal media was used to plate NAc cells at a density of 40 000 cells/mL (12 000 cells/cm2) on poly-d-lysine-coated glass coverslips in 24-well culture plates. Media was replaced with glutamate-free NeuroBasal growth media 24 h later. Then, one-half of the media was replaced every 4 days with glutamate-free NeuroBasal growth media. All experiments were performed using 2- to 3-week-old cultures.

Astrocyte cultures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

The cortex was isolated and cut into 1 mm3 cubes with a scalpel blade. Cells were isolated as described above, except that a longer incubation with papain was performed (30–45 min). After determining viability (95–99%) and density, cells were plated in a poly-d-lysine-coated flask (1000 000 cells/flask) with insulin (1 : 250; Sigma, St Louis, MO, USA) supplemented minimal essential media (MEM; Gibco; 4% FBS, 2 mm glutamine, 0.5% gentamicin, and 0.35% glucose). After 1 h incubation at 37°C, cultures were washed with ice cold MEM to selectively wash off other cell types and leave astrocytes attached to the culture flask. Then, insulin-supplemented MEM was added. Media were replaced every 5–7 days with insulin-supplemented MEM (except, the 4% FBS was decreased to 2% FBS). Astrocyte cultures could be divided 1 : 5 every month up to 3 months.

Immunocytochemistry

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

All experiments were conducted using an immunocytochemical assay that selectively detects newly externalized GluR1 (Lu et al. 2001). First, GluR1 already present on the cell surface was preblocked by incubating 45 min at 4°C with antibody to the extracellular N-terminus of GluR1 (N-GluR1; amino acids 271–285, RTSDSRDHTRVDWKR; Oncogene, Cambridge, MA, USA; 1 : 15), rinsing twice in PBS, and then incubating at 4°C with unlabeled goat anti-rabbit secondary antibody (5 µg/mL; Sigma). Then, cells were incubated at room temperature, in normal media or media containing test drugs, to allow insertion of new GluR1 subunits into the cell membrane. After this incubation, cultures were rinsed, fixed with 4% paraformaldehyde in PBS with 4% sucrose for 15 min at room temperature, and rinsed twice with PBS. To detect the newly externalized GluR1, cultures were incubated overnight at 4°C with N-GluR1 antibody (1 : 15), blocked with 5% donkey serum in PBS for 2 h, and incubated at room temperature for 1 h with Cy3 donkey anti-rabbit secondary antibody (1 : 250, Jackson Immuno Research Laboratories, West Grove, PA, USA). Because cells are not permeabilized, the second round of immunostaining only detects newly externalized GluR1 subunits. After rinsing with PBS, coverslips were mounted with PVA-DABCO on gelatin-coated glass slides. After drying overnight at room temperature, slides were stored at 4°C until image analysis. Experiments were conducted at room temperature because there is considerable constitutive internalization of surface AMPARs at 37°C but relatively little at room temperature (Pickard et al. 2001). Therefore it seemed preferable to test the effects of D1 agonists on GluR1 externalization at room temperature, as there will be less constitutive internalization to produce opposing effects.

Image analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

Images were acquired and analyzed using a Nikon inverted microscope, an ORCA ER digital camera (Hamamatsu Corp., Bridgewater, NJ, USA) and MetaMorph Imaging software (Universal Imaging, West Chester, PA, USA). All experimental groups compared within a figure were run simultaneously using the same culture preparation. For each experimental group, cells from at least four different wells were used and approximately four to six cells from each well were analyzed. Dendritic regions were selected for analysis under phase contrast imaging, and fluorescence images were then collected without further adjustment. This eliminates the possibility of experimenter bias based on the intensity of GluR1 staining. Neurons were divided into two groups based on previously defined morphological criteria (Shi and Rayport 1994; Shetreat et al. 1996). Interneurons were identified by a soma diameter ≥ 15 µm and the presence of extended processes over 10× the length of the soma. Medium spiny neurons were identified by a soma diameter of ∼10 µm, with two to four relatively closer projecting processes. For all experimental groups within each experiment, all images were acquired at identical settings with identical exposure times. Images used for surface GluR1 receptor quantification were acquired with a 40× oil immersion lens. Methods for quantifying surface GluR1 labeling were adapted from those described previously for hippocampal cultures (Carroll et al. 1999a; Liao et al. 1999; Lissin et al. 1999). The number of fluorescent puncta was counted for a fixed length (25 µm for interneurons and 15 µm for medium spiny neurons) of a neuronal process located at least one soma diameter away from the soma. One region was analyzed per cell. Thresholds were set at least two times higher than unlabeled neurites (imaged from cultures incubated with secondary antibody only). Results in figures and text are expressed as percentage of a control group run in the same experiment. For statistical analysis, raw data were analyzed with a Kruskal–Wallis anova on ranks. When a significant group effect was found, specific groups were compared using a Dunn's test (significance set at p < 0.05).

D1 receptor stimulation increases the rate of GluR1 externalization

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

Experiments were conducted using an immunocytochemical assay that selectively detects newly externalized GluR1 (Lu et al. 2001; see Methods). Briefly, GluR1 already present on the cell surface was pre-blocked at 4°C by incubating with primary antibody and unlabeled secondary antibody. Then, cells were brought to room temperature to enable externalization of new GluR1 subunits. A second round of immunostaining, using primary antibody and fluorescent secondary antibody, was performed under non-permeant conditions to selectively detect newly externalized GluR1 subunits. Medium spiny neurons and interneurons were identified based on morphological criteria and analyzed separately (see Methods). We analyzed GluR1 staining on processes rather than the soma because, in the intact striatal complex, virtually all glutamate synapses onto NAc neurons occur on spines (Meredith and Totterdell 1999) and nearly all GluR1 immunostaining is found in spines and dendritic shafts, not in the soma (Chen et al. 1998).

In Fig. 1, results from all experimental groups are normalized to a control group that was never brought to room temperature. To determine the basal rate of GluR1 externalization, cells were brought to room temperature for 15 min in normal media (media group). The 15 min time-point was chosen because preliminary experiments showed that newly externalized GluR1 could be readily measured at this time-point, but that externalization continued to increase at 30- and 60-min time-points (data not shown). Thus, we anticipated that a D1 agonist-induced increase in the rate of GluR1 externalization could be detected at the 15-min time-point. In fact, cells incubated for 15 min in media containing the D1 agonist SKF 81297 (1 µm) exhibited a robust increase in the density of GluR1 puncta and the area of GluR1 puncta (Fig. 1). This was observed for both medium spiny neurons and interneurons. These effects were significantly attenuated when the D1 receptor antagonist SCH 23390 (10 µm) was added 5 min before SKF 81297; an exception was the increase in puncta area in medium spiny neurons, where there was only a trend towards an attenuation of the D1 agonist effect by SCH 23390 (see legend to Fig. 1). When given alone, SCH 23390 had no effect on GluR1 surface expression. Representative images are presented to illustrate these findings for NAc medium spiny neurons and interneurons (Fig. 2). Additional studies demonstrated that the effects of SKF 81297 on GluR1 puncta density and puncta area were concentration-dependent (see left four bars in each graph in Fig. 3). The simplest interpretation of our findings is that SKF 81297 caused the formation of new surface GluR1-containing puncta (reflected by increased puncta density) and also resulted in the addition of GluR1 to existing puncta (reflected by increased puncta area).

image

Figure 1. The D1 receptor agonist SKF 81297 increases the rate of GluR1 externalization and this effect is blocked by the D1 receptor antagonist SCH 23390. Experimental groups: Control – cells incubated in media for 20 min at 4°C, conditions under which GluR1 externalization should be minimal; Media – cells incubated in media for 20 min at room temperature (RT) to define the basal rate of GluR1 externalization; 1 µm SKF – cells incubated at RT for 5 min with media and 15 additional min with 1 µm SKF; 10 µm SCH + SKF – cells incubated at RT for 5 min with 10 µm SCH and 15 additional min with SCH + 1 µm SKF; SCH – cells incubated at RT for 20 min with 10 µm SCH only. Approximately 20 fields (one cell per field) were analyzed for each experimental group (about 50–250 puncta in total, depending on experimental group). Results are presented as the mean number of GluR1 puncta per fixed length of process (a measure of puncta density; left) and the mean area of GluR1 puncta (right), normalized to the Control group (mean ± SEM). Data were analyzed using a Kruskal–Wallis one-way anova on ranks followed by a Dunn's test with significance set at p < 0.05. aSignificantly different from control, media, 10 µm SCH + SKF, and SCH groups. bSignificantly different from Control and 1 µm SKF groups. cSignificantly different from control and media groups.

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image

Figure 2. Representative images illustrating that the D1 receptor agonist SKF 81297 increases the rate of GluR1 externalization in NAc medium spiny neurons (top) and interneurons (bottom). See legend to Fig. 1 for a description of experimental groups and results of quantitative analysis.

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image

Figure 3. The increase in GluR1 externalization produced by the D1 agonist SKF 81297 is concentration-dependent and attenuated by the PKA inhibitor KT5720 (2 µm). Experimental groups: Media – cells incubated in media for 20 min at room temperature (RT) to define the basal rate of GluR1 externalization; 10 nm SKF – cells incubated at RT for 5 min with media and 15 additional min with 10 nm SKF; 100 nm SKF – same protocol as prior SKF group; 1 µm SKF – same protocol as prior SKF groups; 2 µm KT + SKF – cells incubated at RT for 5 min with 2 µm KT5720 and 15 additional min with KT5720 + 1 µm SKF; 2 µm KT – cells incubated at RT for 20 min with 2 µm KT5720 only. Raw data were analyzed using a Kruskal–Wallis one-way anova on ranks followed by a Dunn's test with significance set at p < 0.05. aSignificantly different from media, 2 µm KT, 10 nm SKF, 100 nm SKF and 2 µm KT + SKF groups. bSignificantly different from media, 2 µm KT, 10 nm SKF and 100 nm SKF groups. cSignificantly different from media, 2 µm KT and 10 nm SKF groups. dSignificantly different from media and 2 µm KT groups. eSignificantly different from media and 1 µm SKF groups. Analysis based on 18–21 cells per group.

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GluR1 externalization induced by D1 receptor stimulation requires PKA activity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

The membrane-permeable PKA inhibitors KT5720 and RpcAMPS were selected to test the role of PKA in D1 receptor-induced GluR1 externalization based on their efficacy in similar cell cultures studies (e.g. Ehlers 2000; Crump et al. 2001). In the experiment shown in Fig. 3, cultures were incubated at room temperature for 5 min with 2 µm KT5720 or normal media, and then 1 µm SKF 81297 was added for the final 15 min of the incubation. This dose of KT5720 produced a significant reversal of the D1 agonist-induced increase in interneuron puncta area, but only a trend towards reversal of D1 agonist effects on interneuron puncta density, medium spiny neuron puncta density, and medium spiny neuron puncta area (see legend to Fig. 3). However, a near-complete block of the D1 agonist-induced increase in both puncta area and puncta density was produced by a higher concentration of KT5720 (10 µm) and by a different PKA inhibitor, RpcAMPS (10 µm; Fig. 4). Similar results were obtained in interneurons and medium spiny neurons. Incubation with KT5720 or RpcAMPS alone did not produce any effect on GluR1 externalization (Figs 3 and 4).

image

Figure 4. The increase in GluR1 externalization produced by the D1 agonist SKF 81297 is blocked by the PKA inhibitors KT5720 (10 µm) and RpcAMPS (10 µm). Experimental groups: Media – cells incubated in media for 20 min at room temperature (RT) to define the basal rate of GluR1 externalization; 1 µm SKF – cells incubated at RT for 5 min with media and 15 additional min with 1 µm SKF; 10 µm KT – cells incubated at RT for 20 min with 10 µm KT5720 only; KT + SKF – cells incubated at RT for 5 min with 10 µm KT5720 and 15 additional minutes with 10 µm KT5720 + 1 µm SKF; 10 µm RpcAMPS – cells incubated at RT for 20 min with 10 µm RpcAMPS only; RpcAMPS + SKF – cells incubated at RT for 5 min with 10 µm RpcAMPS and 15 additional min with 10 µm RpcAMPS + 1 µm SKF. Results are presented as the density or area of GluR1 puncta, normalized to the media group. Raw data were analyzed using a Kruskal–Wallis one-way anova on ranks followed by a Dunn's test with significance set at p < 0.05. aSignificantly different from media, 10 µm KT, KT + SKF, 10 µm RpcAMPS and RpcAMPS + SKF groups. bSignificantly different from media, 10 µm KT, 10 µm RpcAMPS and RpcAMPS + SKF groups. Analysis based on 18–23 cells per group.

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SpcAMPS increases GluR1 surface expression

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

Next, we examined the effect of PKA activation on GluR1 externalization using SpcAMPS, a potent membrane-permeable PKA activator. After the pre-blocking step, cells were incubated for 20 min at room temperature in normal media (media group) or in media containing increasing concentrations of SpcAMPS (100 nm, 1 µm, and 10 µm). SpcAMPS produced concentration-dependent effects on GluR1 puncta density and puncta area, with significant increases in both measures produced by the 1 µm and 10 µm concentrations for medium spiny neurons and interneurons (Fig. 5). Representative images illustrating these observations for medium spiny neurons and interneurons are shown in Fig. 6. As PKA inhibitors did not slow basal GluR1 externalization (Figs 3 and 4), whereas PKA activation accelerates basal GluR1 externalization (Figs 5 and 6), it appears that PKA phosphorylation promotes but is not required for the basal level of GluR1 externalization that is occurring in the absence of D1 receptor stimulation.

image

Figure 5. The PKA activator SpcAMPS increases GluR1 externalization in NAc neurons in a concentration-dependent manner. After the preblocking step, cultures were incubated for 20 min at RT in normal media (media group) or media containing 100 nm, 1 µm, and 10 µm SpcAMPS. Results are presented as the density or area of GluR1 puncta, normalized to the media group. Raw data were analyzed using a Kruskal–Wallis one-way anova on ranks followed by a Dunn's test with significance set at p < 0.05. aSignificantly different from media, 100 nm SpcAMPS and 1 µm SpcAMPS groups. bSignificantly different from media and 100 nm SpcAMPS groups. cSignificantly different from media group. Analysis based on 20 cells/group.

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image

Figure 6. Representative images illustrating that the PKA activator SpcAMPS produces a concentration-dependent increase in the rate of GluR1 externalization in NAc medium spiny neurons (top) and interneurons (bottom). See legend to Fig. 5 for a description of experimental groups and results of quantitative analysis.

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SpcAMPS and SKF 81297 work through a common mechanism

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

To further test the hypothesis that the D1 receptor-mediated increase in GluR1 externalization involves the PKA pathway, cultures were incubated with a threshold concentration of SpcAMPS (100 nm) for 5 min and then a threshold concentration of SKF 81297 (100 nm) was added for the final 15 min of the 20-min incubation period. The combined administration of threshold concentrations of SpcAMPS and SKF 81297 produced a significant increase in GluR1 puncta density and puncta area, compared to the media group, both for medium spiny neurons and interneurons (Fig. 7). To further establish a common mechanism of action, we incubated cultures with maximally effective concentrations of SpcAMPS and SKF 81297 (10 µm and 1 µm, respectively). Cells were incubated with SpcAMPS for 5 min and then SKF 81297 was added for the final 15 min of the 20-min incubation period. GluR1 puncta density and puncta area in the SpcAMPS + SKF group did not differ from cells incubated with SpcAMPS alone, indicating that stimulation of PKA occluded the effect of D1 receptor stimulation. This was observed for medium spiny neurons and interneurons (Fig. 8).

image

Figure 7. The combined administration of threshold concentrations of SpcAMPS and SKF 81297 produces a significant increase in GluR1 externalization. Cells were incubated for 5 min at RT in media containing 100 nm SpcAMPS and then 100 nm SKF 81297 was added for the remaining 15 min of the 20-min incubation. Results are presented as the density and area of GluR1 puncta, normalized to the media group. Raw data were analyzed using a Kruskal–Wallis one-way anova on ranks followed by a Dunn's test with significance set at p < 0.05. aSignificantly different from media and 100 nm SpcAMPS groups. bSignificantly different from media and 100 nm SKF groups. cSignificantly different from media group. Analysis based on 18–21 cells/group.

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image

Figure 8. SpcAMPS occludes the effect of SKF 81297 on GluR1 externalization in NAc neurons. Experimental groups: Media – cells incubated in media for 20 min at room temperature (RT) to define the basal rate of GluR1 externalization; 1 µm SKF – cells incubated at RT for 5 min with media and 15 additional min with 1 µm SKF; 10 µm SpcAMPS – cells incubated at RT for 20 min with 10 µm SpcAMPS; SpcAMPS + SKF – cells incubated at RT for 5 min with 10 µm SpcAMPS and 15 additional min with 10 µm SpcAMPS + 1 µm SKF. Results are presented as the density or area of GluR1 puncta, normalized to the media group. Raw data were analyzed using a Kruskal–Wallis one-way anova on ranks followed by a Dunn's test with significance set at p < 0.05. *Significantly different from media group. Analysis based on 20–21 cells per group.

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D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

Our laboratory developed primary cultures of NAc neurons to examine whether D1 receptors, activated during administration of cocaine and amphetamine, modulate cellular mechanisms contributing to LTP and LTD, namely AMPA receptor phosphorylation and trafficking. In prior studies using this culture system, we showed that stimulation of D1 receptors increases GluR1 phosphorylation at the PKA site (Chao et al. 2002a) and increases GluR1 surface expression (Chao et al. 2002b). The purpose of the present study was to examine the relationship between these two events by determining if PKA activation was necessary for the D1 agonist-induced increase in GluR1 surface expression. The relationship between GluR1 phosphorylation and its trafficking is an important issue (Malinow and Malenka 2002) and one that our culture system is well suited to addressing, as we have identified a receptor (D1 DA receptor) that regulates both processes. Furthermore, our system enables us to test the role of signal transduction pathways activated through physiological routes in real neurons. Using an immunocytochemical method that selectively detects newly externalized GluR1, we showed that D1 receptor stimulation increases the rate of GluR1 externalization in medium spiny neurons and interneurons of the NAc. This extends our prior experiments, which showed an increase in total GluR1 surface expression after D1 receptor stimulation, but could not distinguish between an increase in the rate of externalization and a decrease in the rate of internalization (Chao et al. 2002b). The D1 agonist-induced increase in GluR1 externalization was blocked by two different PKA inhibitors, KT5720 and RpcAMPS. Conversely, GluR1 externalization was accelerated by the PKA activator, SpcAMPS. A maximally effective concentration of SpcAMPS occluded D1 agonist-induced GluR1 externalization. In summary, two conclusions can be drawn from these results. First, the basal rate of GluR1 externalization in NAc neurons is increased by PKA activation. Preliminary support for this conclusion was provided by our earlier demonstration that 10 μM forskolin increased total GluR1 surface expression (Chao et al. 2002b). Second, PKA activity is required for GluR1 externalization in response to D1 receptor stimulation.

Several limitations to the interpretation of our findings should be acknowledged. First, the present results do not establish that GluR1 itself is the critical PKA substrate for D1 receptor/PKA-dependent GluR1 externalization in NAc neurons. However, this possibility is strongly supported by the ability of D1 receptors to stimulate PKA phosphorylation of GluR1 in the same NAc culture system (Chao et al. 2002a) and by studies in other brain regions showing that PKA phosphorylation of GluR1 promotes its trafficking to the cell membrane (Ehlers 2000; Esteban et al. 2003). Second, although we have interpreted new GluR1 staining after pre-blocking as reflecting exocytosis of AMPA receptors previously located inside neurons, it is theoretically possible that a portion of the new staining is derived from lateral movement and clustering of AMPA receptors already on the cell surface. The fluorescence signal originating from a single antibody-bound AMPA receptor on the cell surface might be undetectable, but the signal might rise above chosen thresholding limits after summation of fluorescence signals due to clustering of several AMPA receptors. Finally, it is possible that both D1 and D5 DA receptors, the two members of the D1-like receptor family, may have contributed to our results. While the D5 receptor is less abundant than the D1 receptor in the NAc, the D5 receptor nevertheless plays a role in responses to cocaine (see Filip et al. 2000). Like the D1 receptor, the D5 receptor is positively coupled to adenylyl cyclase and PKA (Huff 1997; Sidhu 1998).

Regulation of GluR1 trafficking

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

AMPA receptor trafficking has been studied extensively in the hippocampus, where most AMPA receptors are either GluR1/GluR2 or GluR2/GluR3 hetero-oligomers (Lüscher and Frerking 2001; Malinow and Malenka 2002). Subunits with long carboxy-terminals (GluR1, GluR4) and short carboxy-terminals (GluR2, GluR3) obey different trafficking rules, reflecting different PDZ domain interactions possible for each class (Passafaro et al. 2001; Shi et al. 2001). In hippocampus, GluR2/GluR3-containing receptors undergo constitutive recycling, that is, they are removed from and inserted into the synaptic membrane independent of neuronal activity (Passafaro et al. 2001; Shi et al. 2001). The trafficking of GluR1-containing receptors (e.g. GluR1/GluR2 heteromeric receptors) is regulated very differently. During LTP, GluR1-containing receptors are delivered to synaptic sites through a mechanism known to require CaMKII activation (Shi et al. 1999, 2001; Hayashi et al. 2000; Passafaro et al. 2001). Synaptic delivery of GluR1-containing receptors is a two-stage process, consisting of initial insertion into extrasynaptic sites followed by lateral movement into the synapse (Passafaro et al. 2001). Both steps are regulated by the AMPA receptor-interacting protein stargazin (Chen et al. 2000; Chetkovich et al. 2002; Schnell et al. 2002).

During LTD, there is internalization of GluR1-containing AMPA receptors (e.g. Carroll et al. 1999b; Xiao et al. 2001) as well as AMPA receptors that overlap with the constitutively recycling pool (Lüscher et al. 1999). AMPA receptor internalization is also triggered by activation of NMDA receptors, AMPA receptors, and insulin receptors (Beattie et al. 2000; Ehlers 2000; Lin et al. 2000; Man et al. 2000; Zhou et al. 2001). Our initial report of D1 receptor-mediated increases in GluR1 surface expression was the first to demonstrate AMPA receptor externalization in response to stimulation of a neurotransmitter receptor (Chao et al. 2002b), aside from synaptic insertion triggered by glutamate receptors during LTP (e.g. Lu et al. 2001).

Relationship between GluR1 phosphorylation and its trafficking

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

Supporting a role for PKA phosphorylation of GluR1 in hippocampal synaptic plasticity, GluR1 subunits are dephosphorylated at Ser-845 during LTD (Lee et al. 1998) and phosphorylated at Ser-845 when previously depressed synapses are potentiated (Lee et al. 2000). This could reflect the ability of PKA phosphorylation of GluR1 to increase AMPA receptor currents (Roche et al. 1996) and/or a role for PKA phosphorylation in AMPA receptor insertion into synapses. Ehlers (2000) found that NMDA receptor-mediated GluR1 internalization in dissociated cortical neurons was accompanied by dephosphorylation of GluR1 at the PKA site (Ser845), while subsequent recycling to the cell membrane was accompanied by GluR1 phosphorylation at Ser845. This study supported a role for PKA phosphorylation in GluR1 externalization, but did not examine whether PKA phosphorylation was associated with GluR1 insertion into synaptic sites. A more recent study examined activity-dependent incorporation of GluR1 and GluR4 into synapses in organotypic hippocampal slices (Esteban et al. 2003). PKA phosphorylation of GluR4 was necessary and sufficient to drive GluR4 into synapses, but both PKA phosphorylation and CaMKII activation were necessary for synaptic incorporation of GluR1. GluR4 is expressed early in development while GluR1 is expressed later, suggesting that plasticity mechanisms may change during development (Zhu et al. 2000).

Our results extend these findings in two important ways. First, they indicate that PKA phosphorylation regulates AMPA receptor trafficking in NAc neurons. Prior to our experiments, AMPA receptor surface expression had not been studied in neurons of the striatal complex, despite their critical role in neurological and psychiatric disorders. Second, our results demonstrate that D1 receptors can influence the PKA-dependent mechanisms that normally regulate GluR1 trafficking. Below we discuss the specific implications of this finding for drug addiction. However, it also has general implications for understanding how G protein-coupled receptors might influence synaptic plasticity. For example, it is possible that any receptor positively coupled to PKA may exert effects on AMPA receptors similar to those produced by the D1 receptor, assuming appropriate localization of the two receptors. Supporting this speculation, β1-adrenergic receptors were recently found to stimulate PKA phosphorylation of GluR1 in hippocampus (Vanhoose and Winder 2003); GluR1 trafficking was not examined in that study.

Like the intact NAc, our NAc cultures do not contain glutamate neurons. Therefore a limitation of our study is that we are measuring GluR1 incorporation into extrasynaptic sites. It will be important to determine if the D1/PKA signaling pathway can drive GluR1 into synaptic sites, or whether CaMKII must also be activated, as in hippocampal neurons (Esteban et al. 2003). To address this question, we are developing a co-culture system in which cortical neurons are plated with NAc neurons to restore excitatory synaptic inputs (Sun et al. 2002). Preliminary results in prefrontal cortical neurons plated alone suggest that D1 receptor stimulation increases extrasynaptic GluR1 but is not sufficient to drive GluR1 into synapses (Sun and Wolf 2003). D1 receptor stimulation may render a neuron more likely to undergo LTP by increasing the pool of extrasynaptic AMPA receptors available to enter synaptic sites in response to strong synaptic stimulation.

Regulation of AMPA receptor trafficking is not the same at all synapses (Li et al. 1999; Liu and Cull-Candy 2000). Even within hippocampus, there are differences in AMPA receptor localization between pyramidal neurons and GABA interneurons (Allison et al. 1998). Thus, we anticipated that there might be interesting differences between NAc, a region where principle cells are inhibitory, and hippocampus and cortex, where principal cells are excitatory. So far, however, our results suggest similarities, e.g. AMPA receptor internalization in NAc neurons is induced by glutamate agonists (Chao et al. 2002b; Mangiavacchi and Wolf 2003) and PKA activation promotes GluR1 externalization. In contrast, in the ventral tegmental area, the origin of DA neurons that innervate the NAc, PKA activation produces AMPA receptor internalization and LTD (Gutlerner et al. 2002).

Possible mechanisms underlying DA/glutamate interactions in vivo

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

While the nature of DA/glutamate receptor interactions in striatal neurons depends on many variables, in vivo experiments are consistent with the idea that D1 receptors can enhance excitatory transmission in the intact dorsal striatum and NAc (Gonon and Sundstrom 1996; West and Grace 2002). Our findings suggest two mechanisms that may contribute to this enhancing effect. First, we showed previously that D1 receptor stimulation increases phosphorylation of GluR1 at the PKA site in NAc cultures (Chao et al. 2002a). Although we did not measure AMPA receptor currents in that study, work in dorsal striatum has shown that D1 receptor-mediated phosphorylation of GluR1 at the PKA site enhances AMPA receptor currents by increasing peak open probability of the AMPA receptor channel (Price et al. 1999; Yan et al. 1999; Banke et al. 2000). Thus, D1 receptor stimulation would be predicted to enhance AMPA receptor transmission in the NAc by increasing AMPA receptor currents. Second, results of the present study suggest that D1 receptor-mediated activation of PKA also triggers an increase in the rate of GluR1 externalization. Of course, the extent to which this enhances AMPA receptor transmission depends on whether AMPA receptors enter synaptic sites (see above).

Possible relevance to addiction

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References

As discussed above, our results suggest that a normal function of D1 receptor/PKA signaling is to enhance AMPA receptor transmission, thereby increasing the excitability of NAc neurons and perhaps promoting LTP. This provides a mechanistic framework to explain the ability of psychomotor stimulants, which indirectly stimulate D1 receptors, to facilitate stimulus–reward associations and the attribution of incentive salience, functions that depend in part on the level of NAc glutamate transmission (Berridge and Robinson 1998; Everitt and Wolf 2002). After repeated exposure to psychomotor stimulants, many adaptations in D1 receptor and PKA signaling occur in the NAc (White and Kalivas 1998). We hypothesize that this results in compensatory changes in AMPA receptor trafficking, leading to inappropriate forms of synaptic plasticity, and perhaps to persistent compensatory changes in AMPA receptor expression such as we and others have reported in intact animals following repeated psychostimulant treatment (Wolf 2003). AMPA receptors in the NAc represent a critical point of convergence for excitatory inputs from limbic and cortical regions that trigger drug-seeking behavior (Everitt and Wolf 2002), so abnormal AMPA receptor transmission and plasticity in the NAc could contribute significantly to ‘re-wiring’ of the motivational circuitry in drug addiction.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Post-natal NAc cultures
  6. Astrocyte cultures
  7. Immunocytochemistry
  8. Image analysis
  9. Results
  10. D1 receptor stimulation increases the rate of GluR1 externalization
  11. GluR1 externalization induced by D1 receptor stimulation requires PKA activity
  12. SpcAMPS increases GluR1 surface expression
  13. SpcAMPS and SKF 81297 work through a common mechanism
  14. Discussion
  15. D1 receptor stimulation increases phosphorylation and cell surface expression of GluR1
  16. Regulation of GluR1 trafficking
  17. Relationship between GluR1 phosphorylation and its trafficking
  18. Possible mechanisms underlying DA/glutamate interactions in vivo
  19. Possible relevance to addiction
  20. Acknowledgements
  21. References
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