Figure 2.

Electrophysiologic Characterization of Maturation of hiPSC-Derived DA Neurons

(A) Quantification of resting membrane potential in hiPSC-derived DA neurons cultured in differentiation media for 10, 15, and 20 days. Numbers in the bars represent the numbers of recorded cells (n = 3 independent experiments; mean ± SEM).

(B) Representative traces of membrane potential responding to step depolarization by current injections ranging from −10 pA to 90 pA at 20-pA increments. Membrane potential was current-clamped at the resting membrane potential.

(C) Quantification of Na⁺ and K⁺ currents recorded from DA neurons at indicated days after differentiation. (n = 3 independent experiments with 13–16 cells per group in Na⁺ currents record, 15 cells per group in K⁺ currents record; mean ± SEM).

(D) Representative traces of Na⁺ currents (upper panel) and K⁺ currents (lower panel) in voltage-clamp mode; cells were held at −70 mV; step depolarization from −70 mV to +50 mV at 10-mV increments were delivered (lower panel). Na⁺ currents and K⁺ currents recorded from DA neurons were blocked by 0.5 μM tetrodotoxin (TTX) and 10 mM tetraethylammonium (TEA), respectively. Both currents recovered following TTX or TEA washout.

(E) TTX-sensitive spontaneous action potentials recorded from a DA neuron 20 days after differentiation. No current injection was applied.

(F) Immunocytochemistry of synaptophysin (SYN, marker for synaptic terminals, green) and TH (red). Scale bars, 20 μm.

(G) Quantification of numbers of SYN-positive clusters along the branches of hiPSC-derived DA neurons (TH-positive dendrites) cultured in differentiation media for 10, 15, and 20 days (n = 3 independent experiments; mean ± SEM, with 4 cells quantified per experiment).

(H) Analysis of western blots for Syn protein expression. Quantification of SYN expression level is shown in the upper panel. Data are normalized to the expression level of internal control β-actin (n = 3 independent experiments; mean ± SEM). Representative images are shown for SYN and β-actin in the lower panel.

(I) Representative spontaneous postsynaptic currents (sPSCs) recorded from hiPSC-derived DA neurons cultured in differentiation media for 20 days.

Statistical analysis was performed using StatView version 5.0.1. For (A), (G), and (H), one-way ANOVA was used for repeated-measures analysis, followed by Fisher's protected least significant difference for post hoc comparisons. Data are presented as mean ± SEM. Three independent experiments in (A), (C), (G), and (H); n = 32–37 cells per group in (A). In (C), n = 13–16 cells per group in Na⁺ currents record, and n = 15 cells per group in K⁺ currents record. In (G), n = 4 cells per group per experiment.