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. 2020 Aug 13;15(1):580-587.
doi: 10.1515/biol-2020-0063. eCollection 2020.

Neuroanatomy of melanocortin-4 receptor pathway in the mouse brain

Kun Wang  1 , Wei Mao  2 , Xiaoyu Zhang  3 , Yufei Zhao  2   4 , Kuikui Fan  2   4 , Deng Pan  2   4 , Haodong Liu  2   4 , Penghui Li  2   4 , Rihan Hai  3 , Chenguang Du  2   3   4
Affiliations

Neuroanatomy of melanocortin-4 receptor pathway in the mouse brain

Kun Wang et al. Open Life Sci. .

Abstract

Objective: Melanocortin-4 receptors (MC4Rs) are key regulators of energy homeostasis and adipose deposition in the central nervous system. Considering that MC4R expression regions and function-related research mainly focus on the paraventricular nucleus (PVN), little is known about their distribution throughout the mouse brain, although its messenger RNA distribution has been analyzed in the rat. Therefore, MC4R protein localization in mouse neurons was the focus of this study.

Methods: MC4R protein distribution was assessed in mice through immunofluorescence and Western blotting.

Results: MC4R was differentially expressed throughout the arcuate nucleus (ARC), nucleus of the solitary tract (NTS), raphe pallidus (RPa), medial cerebellar nucleus, intermediolateral nucleus, and brainstem. The highest MC4R protein levels were found in the ARC and ventromedial hypothalamic nucleus, while they were significantly lower in the parabrachial nucleus and NTS. The lowest MC4R protein levels were found in the PVN; there was no difference in the protein levels between the area postrema and RPa.

Conclusions: These data provide a basic characterization of MC4R-expressing neurons and protein distribution in the mouse brain and may aid further research on its role in energy homeostasis.

Keywords: Western blot; central nervous system; hypothalamus; immunofluorescence; melanocortin-4 receptor.

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Conflict of interest statement

Conflict of interest: The authors state no conflict of interest.

Figures

Figure 1
Figure 1
MC4R expression in the mouse CNS. Representative images of sagittal mouse brain sections showing MC4R (green) expression by immunofluorescence. (a) MC4R signal-labeled neurons were detected in the DG-mo. (b) The CB showed MC4R-positive cells. (c) MC4R labeling in the medulla oblongata is shown. (d) MC4R labeling in the cervical enlargement is shown. (e) Sagittal left-to-right views of the whole brain are shown. (f) The Brain Map–Brain Explorer 2 from the Allen Institute for Brain Science. (g) Negative control. (h and i) MC4R expression in the hypothalamus is shown. (j) MC4R expression in the MY is shown. (k) MC4R expression in the IML is shown. Abbreviations: CB, cerebellum; DG-mo, dentate gyrus molecular layer; IML, intermediolateral nucleus; MC4R, melanocortin-4 receptor; MY, medulla. Scale bars: 100 µm.
Figure 2
Figure 2
MC4R expression in the mouse forebrain. A picture collage of the representative coronal mouse brain sections showing the MC4R distribution within forebrain regions. (a) MC4R expression in the MT is shown. (d) The Brain Map–Brain Explorer 2 from the Allen Institute for Brain Science. (e) MC4R expression in the SUB and HPF is shown. (f) MC4R expression in ARC of the hypothalamus is shown. (b, c and g) Higher magnification of the left images is shown. Abbreviations: ARC, arcuate nucleus; DMH, dorsomedial hypothalamic; HPF, hippocampal formation; MC4R, melanocortin-4 receptor; MT, medial terminal nucleus of the accessory optic tract; SUB, subiculum; VMH, ventromedial hypothalamic nucleus; 3 V, third ventricle. Scale bars: 50 or 100 µm.
Figure 3
Figure 3
MC4R expression in the mouse hindbrain. Representative micrographs showing MC4R expression in the mouse brain in hindbrain regions. (a) The Brain Map–Brain Explorer 2 from the Allen Institute for Brain Science. (b) The coronal–caudal to rostral view of the whole brain. (c) MC4R expression in the NTS is shown. (d) MC4R expression in the PB is shown. (e) MC4R expression in the RPa is shown. (f–h) Higher magnification of the upper images. Abbreviations: AP, area postrema; MC4R, melanocortin-4 receptor; NTS, nucleus of the solitary tract; PB, parabrachial nucleus; RPa, raphe pallidus nucleus; 4V, fourth ventricle. Scale bar: 50 or 100 µm.
Figure 4
Figure 4
MC4R protein expression in the mouse brain. Western blot analysis results of various brain regions. (a) A representative blot of MC4R monomers (37 kDa, upper band) and GAPDH (36 kDa, lower band) proteins in the PVN, VMH, ARC, PBN, NTS, AP, and RPa of the mouse brain is shown. (b) MC4R band intensities were scanned, quantified, and normalized to the corresponding GAPDH level. (c) Materials of brain for Western blotting. *indicates P < 0.05; **indicates P < 0.01; ***indicates P < 0.001; ****indicates P < 0.0001; and ns indicates no significance when compared with the brain regions. Abbreviations: ARC, arcuate nucleus; AP, area postrema; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MC4R, melanocortin-4 receptor; NTS, nucleus of the solitary tract; PBN, parabrachial nucleus; PVN, paraventricular nucleus; RPa, raphe pallidus nucleus; VMH, ventromedial hypothalamus.
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