Examining the effects of alcohol on GABA receptor mRNA expression and function in neural cultures generated from control and alcohol dependent donor induced pluripotent stem cells.
- Authors
- Lieberman, Richard; Kranzler, Henry R; Levine, Eric S; Covault, Jonathan
- Year
- 2018
- Journal
- Alcohol (Fayetteville, N.Y.)
- PMID
- 29156239
- DOI
- 10.1016/j.alcohol.2017.08.005
- PMCID
- PMC5743620
Factors influencing the development of alcohol-use disorder (AUD) are complex and heterogeneous. While animal models have been crucial to identifying actions of alcohol on neural cells, human-derived in vitro systems that reflect an individual's genetic background hold promise in furthering our understanding of the molecular and functional effects of alcohol exposure and the pathophysiology of AUD. In this report, we utilized induced pluripotent stem cell (iPSCs)-derived neural cell cultures obtained from healthy individuals (CTLs) and those with alcohol dependence (ADs) to 1) examine the effect of 21-day alcohol exposure on mRNA expression of three genes encoding GABA receptor subunits (GABRA1, GABRG2, and GABRD) using quantitative PCR, and 2) examine the effect of acute and chronic alcohol exposure on GABA-evoked currents using whole-cell patch-clamp electrophysiology. iPSCs from CTLs and ADs were differentiated into neural cultures enriched for forebrain-type excitatory glutamate neurons. Following 21-day alcohol exposure, significant treatment effects were observed in GABRA1, GABRG2, and GABRD mRNA expression. A modestly significant interaction between treatment and donor phenotype was observed for GABRD, which was increased in cell cultures derived from ADs. No effect of acute or chronic alcohol was observed on GABA-evoked currents in neurons from either CTLs or ADs. This work extends findings examining the effects of alcohol on the GABA receptor in human cell in vitro model systems.
Effect of 21-day alcohol exposure on GABAA subunit mRNA expression12–14 week old neural cultures derived from controls and alcoholics were characterized by immunocytochemistry. (A) Representative image from a control subject indicating iPSCs differentiate into mixed neural cultures containing MAP2-postive neurons and GFAP-positive astrocytes. (B) Representative image from a control subject indicating that cultures are enriched for TBR1+ forebrain-type glutamate neurons. (C) No difference was observed in the number of TBR1+ neurons between neural cultures derived from controls and alcoholics. 10,255 DAPI+ cells derived from 6 control and 6 alcoholic neural cell lines were analyzed. (D, E, F) 12-week old neural cell lines (13 control and 9 alcoholic) were treated daily with neural media supplemented with 50 mM alcohol. (D) A significant effect of alcohol treatment was observed for GABRA1 gene expression. (E) A significant effect of alcohol treatment was observed for GABRG2 gene expression. (F) A significant effect of alcohol treatment was observed for GABRD gene expression. A modestly significant interaction between donor status (alcoholic vs. control) and alcohol treatment was observed. (Two-way repeated measures ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001 for alcohol treatment; #p = 0.054 for interaction between donor status and alcohol treatment)
Effect of acute and chronic alcohol on GABA-evoked currentsMature neurons (22–36 weeks post plating) derived from 6 control and 7 alcoholic lines were used for electrophysiological recordings. (A) Example trace of a GABA-evoked current in response to brief pressure application of 50 μM GABA. (B) Example recording of a neuron from a control subject. Each dot represents a GABA-evoked response, which was evoked every 30-seconds. Following a 5-minute baseline recording, aCSF containing 50 mM alcohol was perfused for a total of 15 minutes, which was subsequently washed out for an additional 15 minutes. Boxes indicate responses within 5-minute bins that were averaged for analysis. (C) No significant effect of acute alcohol was observed on the amplitude of GABA-evoked responses in neurons derived from 6 control or 7 alcoholic iPSC lines. Washout contains data from a subset of 46 control neurons and 32 alcoholic neurons. (D) Example trace of a GABA response in a control neuron evoked by bath perfusion of aCSF supplemented with 10 μM GABA. Bath perfusion of GABA evoked a reversible current that plateaued after 30–40 seconds. (E) No difference in maximum current density of GABA-evoked current was observed following 9–21 days of exposure to 50 mM alcohol in neurons derived from 4 control or 5 alcoholic iPSC lines. (F and G) No significant difference was observed in membrane capacitance (an indicator of cell size) or resting membrane potential in non-alcohol treated neurons derived from the 6 control and 7 alcoholic iPSC lines. (Numbers within bars indicate the total patched neurons).
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