Alcohol consumption induces global gene expression changes in VTA dopaminergic neurons

Abstract

Alcoholism is associated with dysregulation in the neural circuitry that mediates motivated and goal-directed behaviors. The dopaminergic (DA) connection between the ventral tegmental area (VTA) and the nucleus accumbens is viewed as a critical component of the neurocircuitry mediating alcohol's rewarding and behavioral effects. We sought to determine the effects of binge alcohol drinking on global gene expression in VTA DA neurons. Alcohol-preferring C57BL/6J × FVB/NJ F1 hybrid female mice were exposed to a modified drinking in the dark (DID) procedure for 3 weeks, while control animals had access to water only. Global gene expression of laser-captured tyrosine hydroxylase (TH)-positive VTA DA neurons was measured using microarrays. A total of 644 transcripts were differentially expressed between the drinking and nondrinking mice, and 930 transcripts correlated with alcohol intake during the last 2 days of drinking in the alcohol group. Bioinformatics analysis of alcohol-responsive genes identified molecular pathways and networks perturbed in DA neurons by alcohol consumption, which included neuroimmune and epigenetic functions, alcohol metabolism and brain disorders. The majority of genes with high and specific expression in DA neurons were downregulated by or negatively correlated with alcohol consumption, suggesting a decreased activity of DA neurons in high drinking animals. These changes in the DA transcriptome provide a foundation for alcohol-induced neuroadaptations that may play a crucial role in the transition to addiction.

Keywords: Alcohol consumption; dopaminergic neurons; gene expression; laser capture microdissection; microarrays; neuroadaptation; ventral tegmental area.

Figure 1. Laser-capture microdissection and validation of tyrosine hydroxylase enrichment in VTA DA neurons

Frozen 12µm coronal brain sections of the VTA were fixed in acetone and stained with an antibody against tyrosine hydroxylase (TH) to label DA neurons (see methods for details). (a) A section of VTA stained for TH-positive neurons, with several neurons being removed using laser-assisted microdissection. (b). Laser captured neurons are catapulted onto P.A.L.M Adhesive Cap for collection (c). Results of RT-PCR to measure Th abundance using RNA extracted from 300 neurons or whole VTA (n=3 samples per group). A 40-fold enrichment was obtained in captured neurons vs. whole VTA tissue (two-tailed t-test p<0.01).

Figure 2. Drinking behavior and global gene expression

Female B6 × FVB F1 hybrid mice were subjected to a 2-bottle choice DID paradigm (20% ethanol, 3 hours daily, 20 days). Data for alcohol preference (a) and intake (b) were averaged for three periods: days 1–6, 7–12 and 13–20. Asterisk indicates a significant difference from 0.5 no preference level (one sample t-test adjusted p<0.01). (c). Numbers of genes (transcripts) differentially expressed between alcohol and control groups (DEGs) or correlated with either preference (Pref) or intake in the alcohol group averaged during three time periods (all 20 days, last 8 days or last 2 days) of the procedure. Horizontal line at ~500 gene level represents the 5% chance. Numbers of regulated genes in three gene lists were significantly greater than chance (adjusted χ² p values: * = p<0.01; ** = p<0.0001). (d). DA-enriched differentially expressed gene, cadherin, Celsr3 (* = p<0.01). (e). Three DA-enriched genes significantly correlated with last two days of ethanol intake (glucose transporter, Slc2a6, R=−0.87; proto-oncogene, Ret, R=−0.63; vesicle amine transporter, Vat1, R=−0.78; all p<0.05).

Figure 3. Top gene networks identified using Ingenuity Pathway Analysis (IPA)

IPA generates networks based on the Ingenuity Knowledge Base that relies on known biological relationships among genes. Shown are top gene networks for DEGs (a): Neurological Disease, Psychological Disorders and Post Translational Modifications (IPA Score = 51) and ACGs (b): Lipid metabolism, Nucleic acid metabolism, Small Molecular Biochemistry (IPA Score = 51). Red represents molecules that are upregulated in alcohol group or positively correlated with alcohol intake, while green represents those that are downregulated or negatively correlated. Solid lines represent direct interactions while dashed lines represent indirect interactions between molecules.