Gene expression changes in the nucleus accumbens of alcohol-preferring rats following chronic ethanol consumption

Abstract

The objective of this study was to determine the effects of binge-like alcohol drinking on gene expression changes in the nucleus accumbens (ACB) of alcohol-preferring (P) rats. Adult male P rats were given ethanol under multiple scheduled access (MSA; three 1-h dark cycle sessions/day) conditions for 8 weeks. For comparison purposes, a second ethanol drinking group was given continuous/daily alcohol access (CA; 24h/day). A third group was ethanol-naïve (W group). Average ethanol intakes for the CA and MSA groups were approximately 9.5 and 6.5 g/kg/day, respectively. Fifteen hours after the last drinking episode, rats were euthanized, the brains extracted, and the ACB dissected. RNA was extracted and purified for microarray analysis. The only significant differences were between the CA and W groups (p<0.01; Storey false discovery rate=0.15); there were 374 differences in named genes between these 2 groups. There were 20 significant Gene Ontology (GO) categories, which included negative regulation of protein kinase activity, anti-apoptosis, and regulation of G-protein coupled receptor signaling. Ingenuity analysis indicated a network of transcription factors, involving oncogenes (Fos, Jun, Junb had higher expression in the ACB of the CA group), suggesting increased neuronal activity. There were 43 genes located within rat QTLs for alcohol consumption and preference; 4 of these genes (Tgfa, Hspa5, Mtus1 and Creb3l2) are involved in anti-apoptosis and increased transcription, suggesting that they may be contributing to cellular protection and maintaining high alcohol intakes. Overall, these findings suggest that chronic CA drinking results in genomic changes that can be observed during the early acute phase of ethanol withdrawal. Conversely, chronic MSA drinking, with its associated protracted withdrawal periods, results in genomic changes that may be masked by tight regulation of these genes following repeated experiences of ethanol withdrawal.

Fig. 1

The upper panel depicts the 5-day average ethanol intake values (± SEM of the total daily intake indicated in the parentheses) for the first and subsequent 23 hours of the continuous access (CA) P rats and the 5-day average ethanol intake values for the three 1-hour access periods of the multiple scheduled access (MSA) P rats. The box plot in the lower panel indicates that the distributions (CA versus MSA) of drinking scores, for the most part, did not overlap, which includes the most extreme scores. Note: the 5-day averages represent the days that ethanol (15% and 30% concurrent with water) was available to both CA and MSA P rats.

Fig. 2

Abridged Ingenuity® network analysis revealed up-regulation of Fos-related transcription factors. Red indicates up-regulation and green down-regulation of the associated genes. Genes not colored indicate no change in expression but they are highly linked to genes that did change. Solid lines indicate a direct interaction between genes, whereas dashed lines indicate an indirect interaction between the genes. [For interpretation of the references to color in this figure, the reader is referred to the web version of the article] Abbreviations used: Ctgf – connective tissue growth factor; Fos – FBJ osteosarcoma oncogene; Fgf – fibroblast growth factor; Jun – Jun oncogene; Junb -Jun B oncogene; Mmp – matrix metallopeptidase; Plagl1 – pleiomorphic adenoma gene-like 1; Rdbp – RD RNA-binding protein; Rb - retinoblastoma; Spry2 – sprouty homolog 2; Sdc1 – syndecan 1; Tgfa – transforming growth factor alpha.

Fig. 3

Ingenuity® network analysis revealed primarily up-regulation of interacting pathways involved in transcription, calcium signaling, oxidative stress response and glucocorticoid receptor signaling. Red indicates up-regulation and green down-regulation of the associated genes. Genes not colored indicate no change in expression but they are highly linked to genes that did change. Solid lines indicate a direct interaction between genes, whereas dashed lines indicate an indirect interaction between the genes. [For interpretation of the references to color in this figure, the reader is referred to the web version of the article] Abbreviations used: Acta1 – actin alpha 1 skeletal muscle; Actl6a – actin-like 6A; Adam17 – a disintegrin and metallopeptidase domain 17; Adamts1 – a disintegrin-like and metallopeptidase; Apex1 – a purinic/pyrimidinic endonuclease 1; Cdc37l1 – cell division cycle 37 homolog-like 1; Cbx3 – chromobox homolog 3; Cbp/p300 – CREB binding protein; Dr1 – down-regulator of transcription 1; Ep300 – E1A binding protein p300; Gtf2a2 – general transcription factor IIA 2; Hsp70 – heat shock protein 70; Hnrpk – heterogeneous nuclear ribonucleoprotein K; Hdac2 – histone deacetylase 2; Hdac5 – histone deacetylase 5; Irf3 – interferon regulatory factor 3; Kcnq3 – potassium voltage-gated channel subfamily Q member 3; Mxl1 – Max interacting protein 1; Med4 – mediator of RNA polymerase II transcription subunit 4 homolog; Mbd1 – methyl-CpG binding domain protein 1; Mtus1 – mitochondrial tumor suppressor 1; Pi3k – phosphatidylinositol 3-kinase; Pik3c3 – phosphoinositide-3-kinase class 3; Ppp2ca – protein phosphatase 2 catalytic subunit alpha isoform; Rbbp6 – retinoblastoma binding protein 6; Rnf2 – ring finger protein 2; Rbm3 – RNA binding motif protein 3; Tceb3 – transcription elongation factor B polypeptide 3; Tpm3 – tropomyosin 3 gamma.