Decreased immunoreactivity of the melanocortin neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) after chronic ethanol exposure in Sprague-Dawley rats

Abstract

Background: The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor proopiomelanocortin (POMC). Recent pharmacologic and genetic evidence suggests that MC receptor (MCR) signaling modulates neurobiologic responses to ethanol and ethanol intake. Because ethanol decreases POMC mRNA levels, we determined if exposure to an ethanol-containing diet (ED) would significantly reduce central immunoreactivity of the MC peptide alpha-MSH in rats. We also determined if ethanol exposure would alter the immunoreactivity of agouti-related protein (AgRP), an endogenous MCR antagonist.

Methods: Male Sprague-Dawley rats were given 18 days of access to normal rodent chow or a control diet (CD), or short-term (4 days) or long-term (18 days) access to an ED. At the end of the study, rats were perfused with 4% paraformaldehyde and their brains were sectioned into two sets for processing with alpha-MSH or AgRP immunohistochemistry.

Results: Rats exposed to an ED showed significant reductions of central alpha-MSH immunoreactivity relative to rats exposed to a control diet (CD) or normal rodent chow. Ethanol-induced reductions of alpha-MSH immunoreactivity were site-specific and were noted in regions of the hypothalamus and extended amygdala, as well as the paraventricular nucleus of the thalamus. Because there were no differences in body weights or caloric intake between the CD and ED groups, reductions of alpha-MSH immunoreactivity in ED-treated rats are best explained by ethanol exposure rather than altered energy balance. No significant ethanol-induced alterations in hypothalamic AgRP immunoreactivity were detected.

Conclusions: The present study shows that ethanol site specifically reduces alpha-MSH immunoreactivity in rat brain. These observations, in tandem with recent pharmacologic and genetic studies, suggest that the endogenous MC system modulates neurobiologic responses to ethanol. Thus, compounds which target MCRs may prove to have therapeutic value in the treatment of excessive ethanol consumption and/or the symptoms associated with ethanol withdrawal.

Fig. 1

Quantification of α-melanocyte-stimulating hormone (MSH) immunoreactivity in the arcuate nucleus of the hypothalamus (Arc). Quantification was done by counting α-MSH-positive cell bodies (A) or by measuring the density of α-MSH staining (B) using Image J software, which calculated the percent of the total area examined (% area) that showed signal (cell bodies and processes) relative to a subthreshold background. Groups were given 18-days of access to normal rodent chow (Chow) or an ethanol-free control diet (CD), or an ethanol diet for 4 (ED4) or 18 (ED18) days. Values are represented as mean ± SEM. There are statistical differences between groups that do not share overlapping lettering (a or b; p < 0.05).

Fig. 2

Representative photomicrographs of 40 μm coronal sections showing α-melanocyte-stimulating hormone (MSH) immunoreactivity through the arcuate nucleus of the hypothalamus (A and C) and the lateral nucleus of the hypothalamus (B and D) of rats given 18 days of exposure to the control diet or the ethanol diet (Ethanol Diet 18). Dashed line depicts the region that was selected for quantification. Images were photographed and quantified at a magnification of 10×. Scale bar = 200 μm. α-MSH immunoreactivity in the arcuate nucleus appears in cell bodies, while staining in the lateral hypothalamus is located primarily in cellular processes.

Fig. 3

Quantification of α-melanocyte-stimulating hormone immunoreactivity (% area) in the lateral nucleus of the hypothalamus (LH; A), the dorsomedial nucleus of the hypothalamus (DMH; B), and the paraventricular nucleus of the hypothalamus (PVN; C). Groups were given 18 days of access to normal rodent chow (Chow) or an ethanol-free control diet (CD), or an ethanol diet for 4 (ED4) or 18 (ED18) days. Values are represented as mean ± SEM. There are statistical differences between groups that do not share overlapping lettering (a or b; p < 0.05).

Fig. 4

Quantification of α-melanocyte-stimulating hormone immunoreactivity (% area) in the central nucleus of the amygdala (CeA; A), the bed nucleus of the stria terminalis (BNST; B), the paraventricular nucleus of the thalamus (PVT; C), and the periaqueductal gray (PAG; D). Groups were given 18 days of access to normal rodent chow (Chow) or an ethanol-free control diet (CD), or an ethanol diet for 4 (ED4) or 18 (ED18) days. Values are represented as mean ± SEM. There are statistical differences between groups that do not share overlapping lettering (a or b; p < 0.05).

Fig. 5

Representative photomicrographs of 40 μm coronal sections showing α-melanocyte-stimulating hormone (MSH) immunoreactivity through the central nucleus of the amygdala (A and C) and the bed nucleus of the stria terminalis (B and D) of rats given 18 days of exposure to the control diet or the ethanol diet (Ethanol Diet 18). Dashed line depicts the region that was selected for quantification. Images were photographed and quantified at a magnification of 10×. Scale bar = 200 μm. α-MSH immunoreactivity in these regions is located primarily in cellular processes.

Fig. 6

Representative photomicrographs of 40 μm coronal sections showing α-melanocyte-stimulating hormone (MSH) immunoreactivity through the paraventricular nucleus of the thalamus of rats given 18 days of exposure to the control diet (A) or the ethanol diet (B, Ethanol Diet 18). Dashed line depicts the region that was selected for quantification. Images were photographed and quantified at a magnification of 10×. Scale bar = 200 μm. α-MSH immunoreactivity in this region is located primarily in cellular processes.

Fig. 7

Quantification of AgRP immunoreactivity (% area) in arcuate nucleus of the hypothalamus (Arc). Groups were given 18 days of access to normal rodent chow (Chow) or an ethanol-free control diet (CD), or an ethanol diet for 4 (ED4) or 18 (ED18) days. Values are represented as mean ± SEM.

Fig. 8

Representative photomicrographs of 40 μm coronal sections showing AgRP immunoreactivity through the arcuate nucleus of the hypothalamus of rats given 18 days of exposure ethanol diet (Ethanol Diet 18; A) or to control diet (B). Dashed line depicts the region that was selected for quantification. Images were photographed and quantified at a magnification of 10×. Scale bar = 200 μm. AgRP immunoreactivity in this region is located primarily in cellular processes.