Figure 6. Mature Motor Neurons are Electrophysiologically Functional and Form Synapses with Myotubes.

(A) A mature sMN marker, choline acetyl transferase (ChAT), was detected in hESC derived-neurons in the long-term culture. Mouse E14.5 spinal cord primary culture (mouse SC) was used as a positive control. White bar indicates 20 µm. (B) A current clamp recording showed repetitive firing of action potentials in response to the injection of a 1-second current pulse (50 pA). (C) A voltage clamp recording showed an inward current in response to the addition of 100 µM glutamate (V_holding = −60 mV). Red line indicates the glutamate application (D and E). A voltage clamp recording (D) and current density-voltage relationship (E) show inward currents in response to depolarizing voltage steps from −60 mV to 40 mV. (F and F') Terminals of βIII-tubulin+ neurons (green) were co-localized with the signals of an acetylcholine receptor marker α-bungarotoxin (red) on C2C12-derived myotubes (arrowheads). High magnification picture (F') indicates the area of open white square in F. (G and G') α-bungarotoxin signals (red, arrowheads) was detected nearby neural synaptic marker, Synapsin (green). Inset in G shows high magnification picture. Bars in F' and G' indicate 20 µm.