Chronic alcohol consumption from adolescence-to-adulthood in mice--hypothalamic gene expression changes in the dilated cardiomyopathy signaling pathway

Abstract

Background: Adolescence is a developmental stage vulnerable to alcohol drinking-related problems and the onset of alcoholism. Hypothalamus is a key brain region for food and water intake regulation, and is one of the alcohol-sensitive brain regions. However, it is not known what would be the alcohol effect on hypothalamus following adolescent alcohol intake, chronically over the adolescent development, at moderate levels.

Results: We employed a paradigm of chronic moderate alcohol intake from adolescence-to-adulthood in mice, and analyzed the alcohol effect on both behavioral and hypothalamic gene expression changes. A total of 751 genes were found and subjected to pathway analysis. The dilated cardiomyopathy (DCM) pathway was identified. The changes of ten genes under this pathway were further verified using RT-PCR. Chronic alcohol consumption during adolescence, even at moderate levels, led to a decrease of motor activity in mice, and also a concerted down regulation of signaling pathway initiating factor (SPIF) genes in the DCM signaling pathway, including β1-adrenergic receptor (Adrb1), Gs protein (Gnas), adenylyl cyclase 1 (Adcy1), and dihydropyridine receptor/L-type calcium channel (Cacna1d).

Conclusions: These findings suggest that adolescent alcohol intake may trigger gene expression changes in the CNS that parallel those found in the dilated cardiomyopathy signaling pathway. If such effects also take place in humans, our findings would serve as a warning against alcohol intake in youth, such as by teens and/or college students.

Figure 1

Chronic self-administration of alcohol in mice during adolescence. (A) Adolescent mice (3 weeks old) had free access to water and alcohol solutions. Two alcohol groups were used; one group had 5% alcohol (filled triangles), and the other had 10% alcohol (open triangles). Data are shown for daily alcohol consumption (grams of alcohol per kilogram mouse body weight per day, average over 5 days). Data are expressed as mean ± S.E.M. (n = 18 for each group). There was no statistically significant difference between the two groups at any time point (p > 0.05, two-way ANOVA with Bonferroni post-test). (B) Alcohol preference over water (% v/v), from the same mouse alcohol groups as in (A), showing that mice from the 5% alcohol group consumed higher volume of alcohol solution than those from the 10% alcohol group. The average amounts of alcohol consumed by the 5% vs. 10% groups were not significantly different. (C) Alcohol consumption data of the mice used for microarray gene expression study. Mice used for the gene expression study were selected from the 5% (n = 9) and 10% (n = 10) alcohol group, respectively. Data are expressed as mean ± S.E.M. (n = 19). A steady increase of alcohol consumption was present, as the alcohol consumption data from later time points (day 40 and later) are significantly different from the data at the beginning (p < 0.05, Repeated measures ANOVA with post test of Dunnett's multiple comparison test).

Figure 2

Chronic alcohol consumption leads to a decrease of motor activity in mice. Upper panels (A and B): locomotion; lower panels (C and D): rearing. Time course of locomotion (A) and rearing (C) are shown as beam breaks per 5 min. Cumulative locomotion (B) and rearing (D) for the entire period (0–60 min) are shown. Data are expressed as mean ± S.E.M. (n = 19 for the alcohol group, n = 8 for the water-only control group). *, significant difference compared with the water-only control group (p < 0.05, unpaired t-test).

Figure 3

Principal component analysis (PCA) plot of microarray gene expression study. Twelve samples were used: C1 – C3 for water control mice (open circles), and A1 – A9 for alcohol consumption mice (filled squares). All probe sets that passed the filters for high-quality array data (see Methods) were used for the analysis. Component A is plotted on the horizontal axis and component B is plotted on the vertical axis.

Figure 4

Clustering display of differentially expressed genes using the unsupervised hierarchical clustering method. C1-C3 represent water-only control samples; A1-A9 are for chronic alcohol samples.

Figure 5

Hypothesis of adolescent chronic alcohol consumption effect on SPIF gene down regulation in the dilated cardiomyopathy signaling pathway. Diagram was adapted from KEGG pathway (http://www.genome.jp/kegg/) [92] for dilated cardiomyopathy (DCM), showing the β1AR-DHPR-Ca²⁺ branch of the DCM pathway. Adolescent chronic alcohol consumption is hypothesized to result in the down regulation of SPIF genes (marked with grey color and solid down arrows), including β1-adrenergic receptor (Adrb1), Gs protein (Gnas), adenylyl cyclase 1 (Adcy1), and dihydropyridine receptor/L-type calcium channel (Cacna1d). The coordinated down regulation of these SPIF genes leads to impaired intracellular Ca²⁺ homeostasis at the cellular level.