Binge-Like Ethanol Drinking Increases Otx2, Wnt1, and Mdk Gene Expression in the Ventral Tegmental Area of Adult Mice.

Abstract

Alcohol use disorder is associated with pathophysiological changes in the dopaminergic system. Orthodenticle homeobox 2 (OTX2) is a transcription factor important for the development of dopaminergic neurons residing in the ventral tegmental area (VTA), a critical region of the brain involved in drug reinforcement. Previous studies have demonstrated that ethanol exposure during embryonic development reduces Otx2 mRNA levels in the central nervous system. We hypothesized that levels of OTX2 would be altered by binge-like ethanol consumption in adult animals. To test this, Otx2 mRNA and protein levels in the mouse VTA were measured by quantitative real-time PCR and western blotting, respectively, after mice drank ethanol for 4 days in a procedure that elicits binge levels of ethanol consumption (drinking in the dark). Expression of known and putative OTX2 transcriptional target genes (Sema3c, Wnt1, and Mdk) were also measured in the VTA after ethanol drinking. Otx2 mRNA and protein levels were elevated in the VTA 24 hours after the fourth drinking session and there was a corresponding increase in the expression of Mdk transcript. Interestingly, Wnt1 transcript was elevated in the VTA immediately after the fourth drinking session but returned to control levels 24 hours later. We next investigated if viral-mediated reduction of Otx2 in the mouse VTA would alter ethanol or sucrose intake. Lentiviral vectors expressing a shRNA targeting Otx2 or a control shRNA were injected into the VTA and mice were tested in the drinking in the dark protocol for ethanol and sucrose drinking. Reducing levels of OTX2 in the VTA did not alter ethanol or sucrose consumption. One limitation is that the extent of OTX2 reduction may not have been sufficient. Although OTX2 in the VTA may not play a role in binge-like drinking in adult mice, OTX2 could contribute to ethanol-induced transcriptional changes in this region.