Figure 4.

D_2LR protein directly binds 5-HT_1AR and colocalizes with it in serotonergic neurons. A–C, Confocal images showing the localization of D₂R and TPH2 or 5-HT_1AR in the DRN. TPH2 and 5-HT_1AR immunoreactivities almost completely merge. MRN, Medial raphe nucleus; Aq, aqueduct. Scale bar, 400 μm. A, D₂R colocalizes with both TPH2 (B) and 5-HT_1AR (C) in the DRN. D₂R immunoreactivity is not detected in the D₂R-KO DRN samples (C, bottom). Boxed panels are high-magnification images of images in the white rectangles. Blue, DAPI. Scale bars, 10 μm. D, D₂R association with 5-HT_1AR as assessed by dual-recognition PLA. Signals indicative of the D₂R/5-HT_1AR interaction are clearly seen in the WT DRN sections (left). D₂R/5-HT_1AR interaction is undetectable in the D_2LR-KO DRN sections (right). PLA was repeated three times and representative data are shown. Blue, DAPI. Scale bars, 5 μm. E, Top, Representative immunoblot of mouse DRN lysates probed with the indicated antibodies. Bottom, Densitometric analysis of D₂R levels normalized to β-tubulin (A.U., arbitrary units). WT, n = 6 mice; D_2LR-KO, n = 4 mice. Each bar represents the mean ± SEM. **p < 0.01 by two tailed unpaired t test (t = 7.628, DF = 8, p < 0.0001). F, Top, GST pull-down samples with purified recombinant (r)EGFP, EGFP-tagged rD₂R isoforms, or EGFP-tagged rD₁R with recombinant GST-5-HT_1AR protein immunoblotted with GST antibody. Pull-down assay was repeated two times and representative data are shown. Bottom, Mouse DRN lysates were immunoprecipitated (IP) with the indicated antibodies and Western blots were probed with the indicated antibodies. G, Confocal images showing colocalization of EGFP-tagged D₂R isoforms with Halo-tagged 5-HT_1AR. Blue, DAPI. Scale bars, 10 μm. H, BRET saturation curves for the indicated receptor complexes. The fluorescence signal from Venus normalized to the luminescence of Rluc8 is shown on the x-axis. Each bar represents the mean ± SEM.