FIGURE 6.

Methadone attenuates the developmental increase of non-inactivating K⁺ currents. (A) K⁺ current traces recorded from a representative neuron at the edge of 16-week-old organoids without methadone treatment. Left panels, the overall potassium current (I_K) recorded at voltages from –80 to +80 mV. Middle panels, non-inactivating delayed-rectifier K+ current (I_KD) preceded by a prepulse to +20 mV. Right panels, transient A-type current (I_KA) obtained by subtracting the I_KD. (B) Neuronal K⁺ current traces (depolarized at +20 mV from holding potential of –80 mV) recorded from another neuron before and after the application of 5 mM 4-AP. (C) Plot of I_KD size as a function of voltage determined from neurons of cortical organoids at indicated ages and methadone treatments. (D,E) Scatter plot showing the peak I_KD size (D) and density (E) of neurons from human cortical organoids with indicated ages and methadone treatments. (F) Plot of I_KA size as a function of voltage determined from neurons of cortical organoids at indicated ages and methadone treatments. (G,H) Scatter plot showing the peak I_KA size (G) and density (H) of neurons from human cortical organoids with indicated ages and methadone treatments. Graph of scatter plots display mean and SEM. n = 8–19 neurons (6–12 organoids). Two-way ANOVA followed by Tukey’s multiple comparisons test: (D) F_{(6, 162)} = 10.80, p < 0.0001; (E) F_{(6, 162)} = 3.80, p < 0.01; (G) F_{(6, 162)} = 1.34, p = 0.24; (H) F_{(6, 162)} = 0.45, p = 0.84. Control groups at 16-week, 20-week, 24-week were compared to 12-week group, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001. Each methadone-treated groups were compared to the control group at the same age, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.