Figure 1.
Dopamine D1R and D2R colocalized in dual phenotype GABA/glutamate-expressing MSNs in cultured neonatal striatal neurons.
Confocal images revealed D1R and D2R colocalization with the GABA neuronal marker, GAD67 (top row), and the glutamate markers, VGLUT1 and VGLUT2 in striatal neurons cultured 7–10 days. Scale bar 10 µm.
Figure 2.
Dopamine D1R and D2R colocalized in GABA/glutamate-coexpressing MSNs in adult rat NAc.
(A) Confocal images revealed D1R and D2R colocalization with GAD67 (top row) and VGLUT1 and VGLUT2 in NAc core. GAD67 was also shown to colocalize with VGLUT1 and VGLUT2 in these neurons (bottom row). (B) Colocalization of the D1R and D2R with VGLUT1 (white arrows) and in the absence of VGLUT1 (yellow arrows) in NAc shell. Scale bar 10 µm.
Figure 3.
Dopamine D1R and D2R colocalized in GABA/glutamate-coexpressing MSNs in adult rat CP.
(A) Confocal images showing D1R and D2R colocalization with GAD67 (top row), and VGLUT1 and VGLUT2 in CP. GAD67 also colocalized with VGLUT1 and VGLUT2 in CP (bottom row). Note the high levels of dendritic staining for the D1R in this region. (B) A neuron showing colocalization of the D1R and D2R in with in the absence of VGLUT2. Scale bar 10 µm.
Figure 4.
Enhanced BDNF and GAD67, and reduced VGLUT1/2 expression following D1R–D2R heteromer activation.
(A) Representative blots depicting the effects treatment of striatal neuronal cultures with vehicle or the D1R–D2R heteromer-selective agonist SKF 83959 (100 nM) for 30, 60 or 120 min on BDNF, GAD67, VGLUT1 and VGLUT2 expression. (B) Treatment of the neuronal cultures with SKF 83959 for 120 min increased the expression of BDNF and GAD67. In contrast, the expression of VGLUT1 and VGLUT2 was reduced with a 30 min treatment of SKF 83959. Values shown are mean ± S.D.
Figure 5.
D1R–D2R heteromer discretely regulates the expression of proteins involved in GABA or glutamate activity.
(A) Representative blots depicting the effects of a single injection of the D1R–D2R heteromer agonist SKF 83959 (1.5 mg/kg, sc) on BDNF, GAD67, VGAT, VGLUT1 and VGLUT2 expression in NAc, CP, VTA and SN (n = 8–9 rats/group). GAPDH was used as a loading control. (B, C) SKF 83959 increased expression of BDNF and GAD67 in NAc and VTA. No drug effects were observed on VGAT, VGLUT1 or VGLUT2 levels in NAc, while a significant increase in VGLUT2 only was seen in VTA. (D, E) SKF 83959 had no effect on BDNF and GAD67 expression in CP, but diminished expression in SN. Increased levels of VGLUT2 were also evident in response to SKF 83959 in CP, with both VGLUT1 and VGLUT2 being elevated in SN. Bars shown represent means ± s.e.m. and are expressed as a percentage of saline controls. *P<0.05, ** P<0.01.
Figure 6.
Region-specific regulation of pCaMKII, pERK, GABA and glutamate expression by the dopamine D1R–D2R heteromer.
(A) Representative blots depicting the effects of a single systemic injection of SKF 83959 (1.5 mg/kg, sc) on pCaMKII and pERK expression in NAc, CP, VTA and SN (n = 8 rats/group). GAPDH was used as a loading control. (B–E) SKF 83959 increased CaMKII phosphorylation in NAc, had no effects in VTA and CP, and reduced pCaMKII levels in SN. Phosphorylation of ERK42 and ERK44 was elevated in both VTA and SN, and pERK44 levels were increased in CP. Data are expressed as a percentage of saline controls. (F–I) In the presence of SKF 83959 D1R−/− mice exhibited significantly lower levels of pCaMKII than D5R−/− mice in NAc and VTA. There were no changes in pERK42 or pERK44 expression by SKF 83959 in either gene-deleted strain in these regions. SKF 83959 did not alter pCaMKII expression in either the D1R−/− or D5R−/− mice in CP or SN. However, SKF 83959 induced a significant increase in SN pERK expression in D5R−/− mice, but not D1R−/− mice. (J) Representative dot blots showing effects of SKF 83959 on total GABA and glutamate levels in NAc (left panels) and CP (right panels). (K) SKF 83959 increased GABA levels, relative to glutamate, in NAc, but reduced levels in CP. No drug effects were observed in VTA or SN (N = 8–9 rats/group). Bars shown represent means ± s.e.m. *P<0.05, ** P<0.01, *** P<0.001, #P<0.05 compared to D5R−/− mice.
Figure 7.
NAc core versus shell activation of dopamine D1R–D2R heteromer differentially regulates protein expression in VTA and SN.
(A) Representative blots depicting the effects of a single intra-NAc core or shell injection of SKF 83959 (0.75 µg/0.5 µl unilateral) on BDNF, GAD67, VGLUT1 and VGLUT2 expression in VTA and SN (n = 9 rats/group). GAPDH was used as a loading control. (B) There was no effect of an intra-NAc core injection of SKF 83959 on protein expression in VTA (left panel) or SN (right panel). (C) SKF 83959 administration into NAc shell induced a significant increase in GAD67 expression in VTA, but had no effect on BDNF or VGLUT expression (left panel). In contrast, SN showed elevated levels of BDNF, GAD67, VGLUT1 and VGLUT2 (right panel). Bars shown represent means ± s.e.m. and are expressed as a percentage of controls. *P<0.05, ** P<0.01.
Figure 8.
Grooming induced by SKF 83959 is mediated by the dopamine D1R–D2R heteromer.
A single systemic injection of SKF 83959 (1.5 mg/kg, sc) induced a significant elevation in the amount of time spent grooming (n = 7 mice/group). This effect was also present in mice gene-deleted for the D5R (D5R−/−) but absent in mice gene-deleted for the D1R (D1R−/−). D1R−/− mice, but not D5R−/− mice, also exhibited reduced basal levels of grooming compared to wildtype (WT). (Strain {F(2,36) = 12.1, P<0.001}; Drug {F(1,36} = 17.8, P<0.001}; Strain x Drug {F(2,36) = 4.0, P<0.03}). Bars shown represent means ± s.e.m. and are expressed in seconds (s). *P<0.05, ** P<0.01 compared to basal levels within the same strain. #P = 0.025 compared to basal wildtype.