Neuroanatomy of melanocortin-4 receptor pathway in the mouse brain

2.1 Animal husbandry

C57/BL6j wild-type male mice (WT, 8–9 weeks old) were obtained from Charles River Laboratories (Beijing, China). The mice (n = 5) were maintained in a normal environment on a 12 h light/dark cycle (lights on 06:00–18:00) with ad libitum access to food and water and were fed a standard diet.

1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals, and has been approved by the Institutional Animal Care and Use Committee (IACUC) of the Inner Mongolia University, in compliance with NIH guidelines (Protocol number – SYCK-002, approved in July 2014).

2.2 Immunofluorescence

Animals were deeply anesthetized and transcardially perfused with ice-cold 0.9% saline, followed by 4% paraformaldehyde-borate fixative (pH 7.4) for 20 min. The mouse brains were removed, then postfixed in the same fixative overnight at 4°C, followed by cryoprotection in 30% sucrose solution at 4°C before being processed for immunocytochemistry. Brains (n = 3) were subsequently sliced as 30 µm sections, encompassing the hypothalamus, with a cryostat microtome (Slee MNT, Mainz, Germany).

The sections were collected into four serially ordered sets of sections in 0.01 M phosphate-buffered saline (PBS). After an initial blocking step (3% normal donkey serum in 0.01 M PBS containing 1% Triton X-100), the sections were incubated with primary antibodies F rabbit anti-MC4R (1:1,000, ab24233, Abcam) overnight at 4°C. Next, the sections were washed and incubated with Alexa Fluor 647 Donkey anti-Rabbit IgG (1:1,000, ab150075, Abcam) for 2 h at about 25°C. After washes, the sections were mounted on frosted gelatin-coated slide and air-dried. All immunofluorescence experiments used the free-floating method under 300 rpm wobbling with an Eppendorf ThermoMixer C. As a negative control, we used the same samples and the same protocol without primary antibody. Fluorescent neuronal cells were visualized and imaged using a Nikon C2 confocal microscope (Japan) with 4× and 10× objectives.

2.3 Western blot

With the remaining animals, those not used for immunofluorescence, brains were removed into ice-cold 0.9% saline and sliced into 200 µm sections (bregma: −4.84 to −7.48 mm) with a vibrating microtome. PVN, VMH, ARC, PBN, NTS, AP, and raphe pallidus (RPa) tissues (n = 2) were collected by capillary glass tube with an aperture of 2 mm under stereomicroscope.

Total protein was extracted for the expression of MC4R. Specifically, brain tissue sections were cut into small pieces and powdered with liquid nitrogen, placed into a homogenization solution containing ice-cold RIPA (2333s, Beijing, China) and protease inhibitors (complete EDTA-free protease inhibitor cocktail; Roche Diagnostics, Alameda, CA), and then briefly sonicated (15 s/time, 90 V, 3 times). Protein concentrations were determined by BCA protein assay (Thermo Scientific, MA, USA), and approximately 30 µg of total protein was electrophoresed through a 12% running and a 5% stacking SDS-PAGE and wet transferred to a nitrocellulose transfer membrane (66485, Pall). Membranes were blocked with 5% nonfat dry milk and then incubated with primary antibodies and the corresponding secondary antibodies: rabbit anti-MC4R (1:1,000, ab24233, Abcam) and rabbit anti-GAPDH (1:10,000, ab181602, Abcam) overnight at 4°C. The membranes were subsequently washed followed by incubation with the corresponding IRDye® 800LT Goat anti-Rabbit (1:10,000; 926-32211, Odyssey) for 1 h at about 25°C. Detection and quantification were performed using an Infrared Imaging System (Odyssey; LI-COR Biosciences). Band intensities were determined using the median background method. The MC4R levels were normalized to GAPDH.

2.4 Statistics

Data analysis was done using Prism 7.0 software (GraphPad, San Diego, CA, USA). Data are presented as means ± standard errors of the means. Comparisons between the expression levels of MC4R in different tissues were made by repeated-measures ANOVA. Results with P values less than 0.05 were considered statistically significant.

3.1 MC4R expression in the DG-mo, CB, hypothalamus, and MY

In sagittal brain sections, a very high density of MC4R-labeled neurons was detected in the dentate gyrus molecular layer (DG-mo; Figure 1a). Other areas of the brain showed moderate signal intensity. The cerebellum (CB; Figure 1b) also showed a very strong MC4R expression. Intense MC4R immunoreactivity was found along the medulla (Figure 1c), an area influencing blood pressure and respiration. Within the region next to the medulla, homogenous MC4R staining was found on the cervical enlargement region (Figure 1d) and hypothalamus (Figure 1h and i). Labeling was also detected within the rhombencephalon, specifically the medulla oblongata (MY; Figure 1j). As expected, we did not observe any staining with the negative control (Figure 1g).

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.

Compared with other hindbrain regions, an abundant MC4R-positive signal was observed in the unique architecture of the IML (Figure 1k) that transmits via parasympathetic preganglionic fibers. MC4R signaling neurons were observed in both the medulla and spinal cord regions. Overall, projections of MC4R-expressing neurons exited the CNS, suggesting that MC4R may be essential in regulating processes of CNS.

3.2 MC4R expression in the MT, VMH, and ARC

Positive MC4R staining was observed in coronal sections, as expected. Strong MC4R immunoreactivity was detected in the medial terminal nucleus layer (Figure 2a–c), and MC4R was homogenously expressed around the third ventricle (3V) throughout all areas of the forebrain. In the subiculum, stellate-like distributions of MC4R immunoreactivity were observed throughout, suggesting that these nerve cells expressed MC4R. Similarly, MC4R staining was intense around the hippocampal formation (Figure 2e); immunopositive neurons were largely confined to ARC areas (Figure 2f and g). Throughout the hypothalamus, a structure central to neuroendocrine function, MC4R immunoreactivity was evident in the ventromedial hypothalamic nucleus (VMH) and ARC.

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.

3.3 MC4R expression in the NTS, PB, and RPa

Within rhombencephalon regions, MC4R expression was prominent in cells surrounding the fourth ventricle (4V) (Figure 3b). MC4R labeling was detected in the NTS (Figure 3c and f) with robust expression surrounding the 4 V. Notably, intense nerve cell MC4R immunoreactivity was seen throughout the parabrachial nucleus (PBN) (Figure 3d and g). Furthermore, compared with other hindbrain regions, a large distribution was observed in the RPa (Figure 3e and h). MC4R staining was not intense in the area postrema, but was evident in a region likely involved in energy intake control. The present data show intense MC4R immunoreactivity throughout the hindbrain.

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.

3.4 MC4R protein expression levels in key nuclei of energy metabolism

MC4R immunoreactivity was detected in the ARC (Figure 4a) and dorsal root ganglion in the same manner as previously reported with MC4R-GFP [11,12]. Protein was isolated from the PVN, VMH, ARC, PBN, NTS, AP, and RPa and separated by SDS-PAGE (Figure 4c). The highest MC4R protein expression was observed in the ARC and VMH, with a significantly lower expression in the PBN and NTS; the lowest amount was observed in the PVN. There was no difference in protein expression between the AP and RPa.

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.

A significant difference was observed between the MC4R protein levels in the ARC and those in the AP, RPa (P = 0.0037 and 0.0030, respectively), and PVN (P < 0.0001). The VMH, compared with the PBN (P = 0.0070) and NTS (P = 0.0239), demonstrated significantly higher MC4R levels than the PVN (P < 0.0001) and the AP and RPa (P = 0.9778). No significant differences were observed between the NTS and PBN (P = 0.9987), or AP and RPa (P = 0.9778, Figure 4b).