Fig. 3: Ethanol (230 mg/dL, 6 h) induces apoptosis in neurons but not astrocytes in 2-month cerebral organoids from iPSC line 1.

A NeuN and TUNEL co-stained fluorescent images of cerebral treated with or without 50 mM ethanol for 6 h. In order to identify whether neurons in organoids undergo apoptosis following ethanol exposure, the brain section was co-stained with TUNEL (detection of the DNA fragmentation caused by apoptotic signaling cascades) and neuronal nuclear antigen (NeuN; a neuron marker). Cell nuclei were stained with Hoechst 33342. Blue, green, and red represent cell nuclei, NeuN, and TUNEL signals, respectively. These images include either individual channel or overlaid images, showing that ethanol treatment induces apoptosis in neurons as evidenced by co-localized TUNEL and NeuN signals in the same neurons. Three representative NeuN and TUNEL double-positive apoptotic neurons are indicated by pink, white, and green arrows. Scale bar = 20 µm. B Fluorescence images S100β (an astrocyte marker) and TUNEL co-stained organoids. Blue, green, and red represent cell nuclei, S100β, and TUNEL, respectively. The images showed that TUNEL and S100β double-positive apoptotic astrocytes were not increased in ethanol-treated organoids, indicating that ethanol does not cause astrocyte apoptosis. Scale bar = 20 µm.