Differential sensitivity of human neurons carrying μ opioid receptor (MOR) N40D variants in response to ethanol

Abstract

The acute and chronic effects of alcohol on the brain and behavior are linked to alterations in inhibitory synaptic transmission. Alcohol's most consistent effect at the synaptic level is probably a facilitation of γ-aminobutyric acid (GABA) release, as seen from several rodent studies. The impact of alcohol on GABAergic neurotransmission in human neurons is unknown, due to a lack of a suitable experimental model. Human neurons can also be used to model effects of genetic variants linked with alcohol use disorders (AUDs). The A118G single nucleotide polymorphism (SNP rs1799971) of the OPRM1 gene encoding the N40D (D40 minor allele) mu-opioid receptor (MOR) variant has been linked with individuals who have an AUD. However, while N40D is clearly associated with other drugs of abuse, involvement with AUDs is controversial. In this study, we employed Ascl1-and Dlx2-induced inhibitory neuronal cells (AD-iNs) generated from human iPS cell lines carrying N40D variants, and investigated the impact of ethanol acutely and chronically on GABAergic synaptic transmission. We found that N40 AD-iNs display a stronger facilitation (versus D40) of spontaneous and miniature inhibitory postsynaptic current frequency in response to acute ethanol application. Quantitative immunocytochemistry of Synapsin 1⁺ synaptic puncta revealed a similar synapse number between N40 and D40 iNs, suggesting an ethanol modulation of presynaptic GABA release without affecting synapse density. Interestingly, D40 iNs exposed to chronic intermittent ethanol application caused a significant increase in mIPSC frequency, with only a modest enhancement observed in N40 iNs. These data suggest that the MOR genotype may confer differential sensitivity to synaptic output, which depends on ethanol exposure time and concentration for AD-iNs and may help explain alcohol dependence in individuals who carry the MOR D40 SNPs. Furthermore, this study supports the use of human neuronal cells carrying risk-associated genetic variants linked to disease, as in vitro models to assay the synaptic actions of alcohol on human neuronal cells.

Keywords: alcohol use disorder (AUD); human disease modeling; induced neuronal cell (iN); induced pluripotent stem (iPS) cell; mu-opioid receptor (MOR); synaptic transmission.