Fig. 1Expression of dopamine D2 receptor (D2R) and melanocortin 4 receptor (MC4R) in the hypothalamus. D2R-enhanced green fluorescent protein (EGFP) mice were sacrificed, and brain samples were prepared as 40-µm-thick coronal cryosections. Brain sections were then subjected to immunofluorescence staining with anti-MC4R antibody. Immunofluorescence revealed D2R and MC4R coexpression in hypothalamic regions, including (A) the paraventricular nucleus (PVN), (B) arcuate nucleus (ARC), and (C) lateral hypothalamus (LH). The left panel shows a low magnification image (scale bar, 200 µm) and the inset shows a high magnification image (scale bar, 20 µm) for D2R (green) and MC4R (red) stained cells, and D2R-MC4R-coexpressing cells (merge). Arrows indicate D2R-MC4R-coexpressing cells. (D) Relative quantification of D2R- and MC4R-positive cells and D2R-MC4R coexpressed cells in each brain region. Relative percentages of D2R- and MC4R-positive cells were normalized with number of total cells counted. D2R-MC4R coexpression was shown as a percentage normalized with number of D2R-positive cells. Quantifications were performed with hemisphere of coronal slices of each brain region, on six slices per mouse (1,730 to 2,094 cells) for ARC and on six slices per mouse (2,743 to 3,664 cells) for LH area. The data for each animal are expressed as the average from three mice. DMH, dorsomedial hypothalamus; VMH, ventromedial hypothalamus; 3V, third ventricle; CPB, cerebral peduncle basal part; PeF, perifornical nucleus.
Fig. 2Expression of dopamine D2 receptor (D2R) and melanocortin 4 receptor (MC4R) in the striatal area, amygdale, and ventral tegmental area (VTA). Fixed brain slices (40-µm thickness) of D2R-enhanced green fluorescent protein (EGFP) mice were prepared, and immunofluorescence staining for MC4R was performed. Representative immunofluorescence images show D2R-MC4R coexpression in (A) the bed nucleus of the stria terminalis (BNST), (B) central amygdala (CeA), and (C) VTA. The left panel shows a low magnification image (scale bar, 200 µm) and the inset shows a high magnification image (scale bar, 20 µm) for D2R (green) and MC4R (red) stained cells, and D2R-MC4R-coexpressing cells (merge). Arrows indicate D2R-MC4R-coexpressing cells. (D) The graphs show the quantification of D2R- and MC4R-positive cells and D2R-MC4R coexpressed cells in each brain region. Relative percentages of D2R- and MC4R-positive cells were normalized with number of total cells counted. Relative ratio of D2R-MC4R coexpressed cells was shown as a percentage normalized with number of D2R-positive cells. Quantifications were performed in hemisphere of serial sections of each region, on four slices per mouse (2,776 to 3,468 cells) for BNST, six slices per mouse (2,229 to 2,844 cells) for CeA, and five slices per mouse (1,085 to 1,803 cells) for VTA area. The data for each animal are expressed as the average from three mice. CPu, caudate putamen; AC, anterior commissure; NAc, nucleus accumbens; BLA, basolateral amygdala; SNC, substantianigra compact part; SNR, substantianigra reticular part.