The effect of eugenol on GABA-induced currents in HEK 293 cells with GABA receptors.

<p>Eugenol (500 μM) blocked the GABA (20 μM)-induced current. This blocking was observed in both the L- and S-forms of the GABA_A receptor (*p < 0.05). Data are expressed as mean ± SEM.</p

Dose-response curve for eugenol action on GABA-induced currents in HEK 293 cells with GABA receptors.

<p>Eugenol (50, 100, 300, 500, 1000, and 2000 μM) blocked the GABA (20 μM)-induced current in a dose-dependent manner. This blocking effect of eugenol on GABA-induced current was observed in HEK 293 cells transfected with the L- or S-form of the GABA_A receptor. Data are expressed as mean 00B1 SEM.</p

The effect of eugenol on GABA-induced currents in trigeminal ganglion neurons.

<p>(A) Eugenol (1 mM) abolished the GABA (500 μM)-induced current in TG neurons; the current recovered to the control value after washout of eugenol (*p < 0.05). (B) The γ2 subunit of the GABA_A receptor was expressed in TG neurons. Data are expressed as mean ± SEM.</p

The effect of eugenol on GABA-induced currents in HEK 293 cells expressing GABA receptors was independent of G-protein activation.

<p>Eugenol (1 mM) blocked the GABA (20 μM)-induced current in HEK 293 cells transfected with the L- or S-form of the GABA_A receptors in the presence of 100 μM GDP-βS (*p < 0.05). Data are expressed as mean ± SEM.</p

Non-competitive blocking effect of eugenol on GABA-induced currents in HEK 293 cells expressing GABA receptors.

<p>Eugenol (500 μM) blocked the GABA (20, 200, 1000 μM)-induced current. This blocking was similar for L- and S-forms of the GABA_A receptor (p < 0.05 compared to each GABA concentration). Data are expressed as mean ± SEM.</p

MNIM-EOH cells can trigger AChR clustering and form functional connections with cocultured myotubes.

<p>(<b>A</b>) Fluorescence images of MNIM-EOH cells at 1 day after cocultured with C2C12 myotubes. α-BTX staining revealed AChR clustering on C2C12 muscle fibers. Confocal Z-stack imaging, in a line through the region of apparent colocalization, confirmed EGFP+ axons in close proximity to AChRs (arrows). Scale bars: 10 µm. (<b>B</b>) End-plate currents (EPCs) were recorded from myotubes located in close proximity to MNIM-EOH cells. EPCs were blocked from the same cell after application of 15 µM pancuronium. However, after washing out of pancuronium, EPCs could be recorded again. The bars represent mean±SEM. * <i>P</i><0.05. The significance was determined by Student’s <i>t</i> test.</p

Increased expression of mature neuronal/motoneuronal markers and cell-cycle arrest occur in MNIM-treated EOH

<p><b>cells.</b> (<b>A</b>) Immunocytochemistry analysis of neuronal and MN-specific markers in uninduced EOH cells, MNIM-EOH cells, and MNIM-EOH cells further cultured in growth medium for 2 days after complete induction. Each row represents NeuN (red), NF-M (red), ChAT (red), and Islet-1 (red) staining with EGFP (green). Scale bar: 50 µm. (<b>B</b>) Quantitative analysis of NeuN, NF-M, ChAT, and Islet-1 expression. Fifty to 150 cells per group were analyzed in randomly chosen fields. The bars represent mean±SEM of at least three independent experiments. * <i>P</i><0.05, ** <i>P</i><0.001 versus GM. † <i>P</i><0.05, †† <i>P</i><0.001 versus MNIM. The significance was determined by ANOVA followed by Fisher’s LSD <i>post hoc</i> test. (<b>C</b>) Immunocytochemistry of BrdU incorporation (red) and EGFP (green) in EOH cells cultured in GM, MNIM, and MNIM+GM. (<b>D</b>) Quantitative analysis of BrdU incorporation. Fifty to 150 cells per group were analyzed in randomly chosen fields. The bars represent mean±SEM of at least three independent experiments. * <i>P</i><0.05 versus GM. The significance was determined by ANOVA followed by Fisher’s LSD <i>post hoc</i> test.</p

Excitable properties of MNIM-treated EOH cells.

<p>Representative perforated patch clamp recordings from uninduced EOH cells (<b>A</b>) or MNIM-EOH cells (<b>B</b>). The cells were held at −60 mV and currents elicited by stepping from −90 to +30 mV in 10 mV steps. MNIM-EOH cells express robust outward K+ currents but very small inward Na+ currents (filled upright triangle). Current injection (200 pA, 100 ms) induced action potential in MNIM-EOH cells, not in uninduced EOH cells. (<b>C</b>) Resting membrane potential in uninduced EOH cells and MNIM-EOH cells. The bars represent mean ± SEM. * <i>P</i><0.05. The significance was determined by Student’s <i>t</i> test.</p

Quantitative analysis of cell morphology (% of total cells).

<p>The values are expressed as the mean±SEM.</p

Polymerase chain reaction primer pairs.

<p>Polymerase chain reaction primer pairs.</p