Dopamine receptors regulate NMDA receptor surface expression in prefrontal cortex neurons

Abstract

Interactions between dopamine (DA) and glutamate systems in the prefrontal cortex (PFC) are important in addiction and other psychiatric disorders. Here, we examined DA receptor regulation of NMDA receptor surface expression in postnatal rat PFC neuronal cultures. Immunocytochemical analysis demonstrated that surface expression (synaptic and non-synaptic) of NR1 and NR2B on PFC pyramidal neurons was increased by the D1 receptor agonist SKF 81297 (1 microM, 5 min). Activation of protein kinase A (PKA) did not alter NR1 distribution, indicating that PKA does not mediate the effect of D1 receptor stimulation. However, the tyrosine kinase inhibitor genistein (50 microM, 30 min) completely blocked the effect of SKF 81297 on NR1 and NR2B surface expression. Protein cross-linking studies confirmed that SKF 81297 (1 microM, 5 min) increased NR1 and NR2B surface expression, and further showed that NR2A surface expression was not affected. Genistein blocked the effect of SKF 81297 on NR1 and NR2B. Surface-expressed immunoreactivity detected with a phospho-specific antibody to tyrosine 1472 of NR2B also increased after D1 agonist treatment. Our results show that tyrosine phosphorylation plays an important role in the trafficking of NR2B-containing NMDA receptors in PFC neurons and the regulation of their trafficking by DA receptors.

Fig. 1

D1 receptor stimulation increases synaptic and non-synaptic NR1 surface expression in cultured PFC pyramidal neurons, while D2 receptor stimulation has the opposite effect. (a) Neurons were treated with medium, the D1 agonist SKF 81297 (SKF, 1 µM, 5 min), the D1 receptor antagonist SCH 23390 (SCH, 10 µM, 5 min), or SCH+SKF (SCH was added 5 min before SKF). SKF 81297 significantly increased total surface NR1 expression including both synaptic and non-synaptic NR1 area (n = 23–51, Dunn’s test, *p < 0.05). (b) Neurons were treated with medium, the D2 receptor agonist quinpirole (Quin, 1 µM, 5 min), the D2 receptor antagonist raclopride (Rac, 10 µM, 5 min), or Rac+Quin (Rac was added 5 min before Quin). Quinpirole significantly decreased total surface NR1 expression including both synaptic and non-synaptic NR1 area (n = 20–48, Dunn’s test, *p < 0.05). Values are mean ± SEM. (c) Representative images of surface expression of NR1 (green), synaptophysin (red), and overlay (yellow). Lower panels show higher resolution of boxed regions. Scale bars: 20 µm for upper panels, 5 µm for lowest panels.

Fig. 2

Protein tyrosine kinase inhibition blocks the effect of D1 receptor stimulation on NR2B surface expression in cultured PFC pyramidal neurons. (a) Neurons were treated with vehicle (0.2% DMSO), SKF 81297 (SKF, 1 µM), the tyrosine kinase inhibitor genistein (Gen, 50 µM), or Gen+SKF. Total incubation time was 30 min, with SKF 81297 added to vehicle or genistein treated cultures for the last 5 min. Genistein blocked the effect of SKF 81297 on surface NR2B expression, synaptic NR2B area, and non-synaptic NR2B area (n = 19–26, Dunn’s test, *p < 0.05). Values are mean ± SEM. (b) Representative images of NR2B (red), synaptobrevin (green), and overlay (yellow). Lower panels show higher resolution of boxed regions. Scale bars: 20 µm for upper panels, 5 µm for lower panels.

Fig. 3

Protein tyrosine kinase inhibition blocks the effect of D1 receptor stimulation on NR1 surface expression in cultured PFC pyramidal neurons. (a) Neurons were treated with vehicle (0.2% DMSO), SKF 81297 (SKF, 1 µM), the tyrosine kinase inhibitor genistein (Gen, 50 µM), or Gen+SKF. Total incubation time was 30 min, with SKF 81297 added to vehicle or genistein treated cultures for the last 5 min. Genistein blocked the effect of SKF 81297 on total surface NR1 expression, synaptic NR1 area, and non-synaptic NR1 area (n = 19–26, Dunn’s test, *p < 0.05). (b) Neurons were treated with medium, SKF 81297 (SKF, 1 µM), the tyrosine phosphatase inhibitor pervanadate (Per, 200 µM), or Per+SKF. Total incubation time was 30 min, with SKF 81297 added to vehicle or pervanadate treated cultures for the last 5 min. Pervanadate alone produced a trend towards increased total surface NR1 expression, synaptic NR1 area, and non-synaptic NR1 area, but did not alter the effect of SKF (n = 18–28, Dunn’s test, *p < 0.05). Values are mean ± SEM.

Fig. 4

D1 receptor stimulation increases NR1 and NR2B surface expression in PFC cultures, measured by BS³ cross-linking. PFC neurons were treated with medium (control), the D1 agonist SKF 81297 (SKF, 1 µM, 5 min), or the D2 agonist quinpirole (Quin, 1 µM, 5 min). After this incubation, cells were incubated with the membrane impermeant protein cross-linking reagent BS³ for 10 min and then harvested in lysis buffer. Some control cultures were treated identically but in the absence of BS³ (Non-X-link lanes). Protein (15–20 µg/ lane) was analyzed by SDS-PAGE and western blotting. Both high (surface-expressed, cross-linked; S) and predicted (intracellular, unmodified; I) molecular weight bands were detected in tissue from the cross-linked cultures (first 3 lanes in panel a) whereas only the predicted molecular weight bands were detected in non-cross-linked cells (Non-X-link lanes). The S band is estimated to be ~400–600 kDa while I bands for NR1, NR2A and NR2B are 130, 170 and 180 kDa, respectively. SKF 81297 significantly increased surface (S) levels of NR1 (b) and NR2B (d), but not NR2A (c) (n = 6–9, Dunn’s test, *p < 0.05); SKF 81297 had no significant effect on intracellular (I) levels of NR1, NR2A, or NR2B (b–d, n = 6–9, ANOVA, p > 0.05). Values are mean ± SEM.

Fig. 5

Protein tyrosine kinase inhibition blocks the effect of D1 receptor stimulation on NMDA receptor surface expression in PFC cultures, measured by BS³ cross-linking. Neurons were treated with vehicle (0.2% DMSO), SKF 81297 (SKF, 1 µM), the tyrosine kinase inhibitor genistein (Gen, 50 µM), or Gen+SKF. Total incubation time was 30 min, with SKF 81297 added to vehicle or genistein treated cultures for the last 5 min. After 10 min of cross-linking with BS³, the cells were harvested in lysis buffer. Western analysis of surface bands was conducted as described in the legend to Fig. 4. Genistein blocked the effect of SKF 81297 on surface levels of both NR1 (a) and NR2B (b) (n = 7, Dunn’s test, *p < 0.05). Values are mean ± SEM.

Fig. 6

D1 receptor stimulation increases surface-expressed immunoreactivity detected with a phospho-specific antibody to NR2B, measured by BS³ cross-linking. Neurons were treated as described in the legend to Fig. 5. SKF 81297 increased immunoreactivity detected in the cell surface band with NR2B-Tyr1472 phospho-specific antibody. Genistein blocked the effect of SKF 81297 (n = 3–5, Dunn’s test, *p < 0.05). Values are mean ± SEM.