Effects of ethanol on Rhod-2 and Fluo-3 fluorescence in cultured neurons with UCP2 overexpression.

<p>Murine neocortical neurons were cultured for 4 days, followed by infection with lentiviral UCP2 or EV expression vector and subsequent further culture for an additional 4 days. Cells were then loaded with either (A) Rhod-2 or (B) Fluo-3, followed by cumulative addition of NMDA at concentrations of 0.1 to 100 µM in either the presence or absence of 250 mM ethanol. Values are percentages over the maximal fluorescence by A23187 in 3–6 independent determinations. ^#P<0.05, ^##P<0.01, significantly different from the value obtained in cells not exposed to ethanol. Statistical significance was determined according to the 2-way ANOVA test analysis.</p

Effects of ethanol at different concentrations on Rhod-2 fluorescence in cultured neurons.

<p>Murine neocortical neurons were cultured for 8 days, followed by loading of Rhod-2 and subsequent cumulative addition of ethanol at different concentrations 3 min after the addition of 10 µM NMDA. Values are percentages over the maximal fluorescence by A23187 in 3 independent determinations. ^#P<0.05, significantly different from the control value obtained in cells before the addition of ethanol. Statistical significance was determined according to the Students’ <i>t</i>-testanalysis.</p

Lentiviral overexpression of UCP2 in cultured neurons.

<p>Murine neocortical neurons were cultured for 4 days, followed by infection with lentiviral UCP2 or EV expression vector at 50 to 200 µl/500 µl and subsequent further culture for an additional 4 days. Cells were then subjected to determination of UCP2 protein expression on (A) immunocytochemistry and (B) immunoblotting analyses. Cells were also loaded with either (C) Rhod-2 or (D) Fluo-3, followed by cumulative addition of NMDA at concentrations of 0.1 to 100 µM. Values are percentages over the maximal fluorescence by A23187 in 6 independent determinations. **P<0.01, significantly different from each control value obtained in cells with <i>EV</i> alone. Statistical significance was determined according to the Students’ <i>t</i>-testanalysis.</p

Proposed model of inhibition by ethanol of mitochondrial Ca²⁺ influx.

<p>Activation of NMDAR would lead to the incorporation of cytosolic Ca²⁺ into mitochondrial matrix in a manner relevant to UCP2 expressed at inner membranes toward the orchestration of mPTP responsible for the leakage of a variety of cytotoxic molecules in neurons. Ethanol could inhibit the mitochondrial Ca²⁺ influx occurred after the interaction between UCP2 and NMDAR channels through an unidentified mechanism.</p

Effects of ethanol on Rhod-2 and Fluo-3 fluorescence in HEK293 cells with GluN1/GluN2B subunits.

<p>HEK293 cells were transfected with expression vectors of GluN1 and GluN2B subunits along with Flag-UCP2, followed by further culture for an additional 24 h and subsequent loading of either (A) Rhod-2 or (B) Fluo-3. Cells were cumulatively exposed to NMDA at 0.1 to 100 µM in either the presence or absence of ethanol at different concentrations. Values are percentages over the maximal fluorescence by A23187 in 3–6 independent determinations. ^#P<0.05, significantly different from the value obtained in cells not exposed to ethanol. Statistical significance was determined according to 2-way ANOVA test analysis.</p

Effects of ethanol on cellular survival in cultured neurons with UCP2 overexpression.

<p>Murine neocortical neurons were cultured for 4 days, followed by infection with lentiviral UCP2 or EV expression vector and subsequent further culture for an additional 4 days. Cells were then exposed to Glu at concentrations of 10 to 100 µM for 1 h in either the presence or absence of 250 mM ethanol, followed by further culture for an additional 24 h and subsequent DNA staining with Hoechst33342 and PI for determination of cellular viability. Values are percentages of PI-positive cells over Hoechst33343-positive cells in 4–7 independent determinations. ^#P<0.05, ^##P<0.01, significantly different from the value obtained in cells not exposed to ethanol. Statistical significance was determined according to the 2-way ANOVA test analysis.</p

Effects of ethanol on cellular survival in HEK293 cells with GluN1/GluN2A subunits.

<p>HEK293 cells were transfected with expression vectors of GluN1 and GluN2A subunits along with Flag-UCP2, followed by further culture for an additional 24 h in either the presence or absence of 250 mM ethanol and subsequent DNA staining with Hoechst33343 and PI for determination of cellular viability. Values are percentages of PI-positive cells over Hoechst33343-positive cells in 4 independent determinations. *P<0.05, significantly different from each control value obtained in cells with <i>EV</i> alone. ^#P<0.05, significantly different from the value obtained in cells not exposed to ethanol. Statistical significance was determined according to the Students’ <i>t</i>-testanalysis.</p

Effects of ethanol on Rhod-2 fluorescence in cultured neurons.

<p>Murine neocortical neurons were cultured for 8 days, followed by loading of Rhod-2 and subsequent cumulative exposure to Glu or NMDA at different concentrations in either the presence or absence of 250 mM ethanol. Values are percentages over the maximal fluorescence by A23187 in 3 independent determinations. ^#P<0.05, significantly different from each control value obtained in cells not exposed to ethanol. Statistical significance was determined according to the Students’ <i>t</i>-testanalysis.</p

Comparison between inhibitions by ethanol on acquired NMDAR channels with GluN2A and GluN2B subunits.

<p>HEK293 cells were transfected with different NMDAR subunits along with Flag-UCP2, followed by further culture for an additional 24 h and subsequent loading of either (A) Rhod-2 or (B) Fluo-3. Cells were cumulatively exposed to NMDA at 0.1 to 100 µM in either the presence or absence of 50 and 250 mM ethanol. Values are percentages of the inhibition by ethanol in 3–6 independent determinations. *P<0.05, **P<0.01, significantly different from each control value obtained in cells with GluN2A subunit. Statistical significance was determined according to the 2-way ANOVA test analysis.</p

Effects of ethanol on Rhod-2 and Fluo-3 fluorescence in HEK293 cells with GluN1/GluN2A subunits.

<p>HEK293 cells were transfected with expression vectors of GluN1 and GluN2A subunits along with Flag-UCP2, followed by further culture for an additional 24 h and subsequent loading of either (A) Rhod-2 or (B) Fluo-3. Cells were cumulatively exposed to NMDA at 0.1 to 100 µM in either the presence or absence of ethanol at different concentrations. Values are percentages over the maximal fluorescence by A23187 in 3–6 independent determinations. ^#P<0.05, significantly different from the value obtained in cells not exposed to ethanol. Statistical significance was determined according to the 2-way ANOVA test analysis.</p