Estrogen-Dependent Upregulation of Adcyap1r1 Expression in Nucleus Accumbens Is Associated With Genetic Predisposition of Sex-Specific QTL for Alcohol Consumption on Rat Chromosome 4.

John Paul Spence,Tamara Graves,Kent E Williams,Bin Qiu,Weidong Yong,Yi Zou,Dawn K Garcia,Paula J Bice,Hao Gu,Lingling Zhang,Jill L Reiter,Zhao Lai,Tiebing Liang

doi:10.3389/fgene.2018.00513

Abstract

Humans show sex differences related to alcohol use disorders (AUD). Animal model research has the potential to provide important insight into how sex differences affect alcohol consumption, particularly because female animals frequently drink more than males. In previous work, inbred strains of the selectively bred alcohol-preferring (P) and non-preferring (NP) rat lines revealed a highly significant quantitative trait locus (QTL) on rat chromosome 4, with a logarithm of the odds score of 9.2 for alcohol consumption. Recently, interval-specific congenic strains (ISCS) were developed by backcrossing the congenic P.NP line to inbred P (iP) rats to further refine the chromosome 4 QTL region. Two ISCS sub-strains, ISCS-A and ISCS-B, were obtained with a narrowed QTL, where the smallest region of overlap consisted of 8.9 Mb in ISCS-B. Interestingly, we found that females from both ISCS lines consumed significantly less alcohol than female iP controls (p < 0.05), while no differences in alcohol consumption were observed between male ISCS and iP controls. RNA-sequencing was performed on the nucleus accumbens of alcohol-naïve female ISCS-B and iP rats, which revealed differentially expressed genes (DEG) with greater than 2-fold change and that were functionally relevant to behavior. These DEGs included down-regulation of Oxt, Asb4, Gabre, Gabrq, Chat, Slc5a7, Slc18a8, Slc10a4, and Ngfr, and up-regulation of Ttr, Msln, Mpzl2, Wnt6, Slc17a7, Aldh1a2, and Gstm2. Pathway analysis identified significant alterations in gene networks controlling nervous system development and function, as well as cell signaling, GABA and serotonin receptor signaling and G-protein coupled receptor signaling. In addition, β-estradiol was identified as the most significant upstream regulator. The expression levels of estrogen-responsive genes that mapped to the QTL interval and have been previously associated with alcohol consumption were measured using RT-qPCR. We found that expression of the Adcyap1r1 gene, encoding the pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor, was upregulated in female ISCS-B compared to female iP controls, while no differences were exhibited in males. In addition, sequence variants in the Adcyap1r1 promoter region showed a differential response to estrogen stimulation in vitro. These findings demonstrate that rat chromosome 4 QTL contains genetic variants that respond to estrogen and are associated with female alcohol consumption.

Highlights

The development of alcohol use disorders has a strong genetic component
We examined whether the narrowed chromosome 4 (Chr4) QTL in the ISCSA and interval-specific congenic strains (ISCS)-B sub-strains affected the drinking phenotype by testing alcohol consumption
When ISCS alcohol consumption was compared to the inbred alcohol-preferring (iP) background strain, both female ISCS-A and ISCS-B rats consumed 20–30% less alcohol (p < 0.05), while no differences were observed in the male rats (Figure 1)

Summary

Introduction

The development of alcohol use disorders has a strong genetic component. Genetic factors account for more than 50% of the variance in developing alcoholism (Heath et al, 1997; Ducci and Goldman, 2008), and several specific genetic variants are associated with an increased risk for alcoholism (Koss and Goldman, 2000; Dick et al, 2006; Gatti et al, 2010). Sex-based differences in drug and alcohol use, abuse, and dependence are supported by epidemiological and clinical research (Prescott, 2002; Nolen-Hoeksema, 2004; Ceylan-Isik et al, 2010). Sexspecific quantitative trait loci (QTLs) have been reported for alcohol preference, ethanol sensitivity, and ethanol locomotor activation in mice and ethanol drinking in rats; specific genes mediating these effects have yet to be identified (Melo et al, 1996; Gill et al, 1998; Peirce et al, 1998; Radcliffe et al, 2000; Bice et al, 2006; Vendruscolo et al, 2006; Chesler et al, 2012; DuBose et al, 2013; Vanderlinden et al, 2015)

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Results

Conclusion