Fig. 5. AQP4 knockout alters spontaneous Ca²⁺ oscillations and suppresses high concentration KCl-induced Ca²⁺ transient in ANSCs. (A) Double immunocytochemistry of nestin (green) and AQP4 (red) in cells migrated out of adherent neurospheres 24 hours after plating. (B) Spontaneous Ca²⁺ oscillations recorded from three Aqp4^+/+ and Aqp4^–/– ANSCs (the oscillations of each cell are represented by a different color) loaded with fluo-3 AM. (C) Statistical analysis of data obtained from three independent experiments indicated that the mean amplitude (F/F0) of spontaneous Ca²⁺ oscillations in Aqp4^–/– ANSCs was significantly lower than that of Aqp4^+/+ control (^*P<0.01). The frequency of spontaneous Ca²⁺ oscillations recorded from Aqp4^–/– ANSCs was significantly enhanced compared with that of Aqp4^+/+ control (^*P<0.01). (D) Sequence of images recorded over 90 seconds from Aqp4^+/+ and Aqp4^–/– ANSCs loaded with fluo-3 AM following KCl-induced depolarization. (E) Fluo-3 fluorescence expressed as F/F0; increased fluorescence indicates elevated [Ca²⁺]_i. There was a significant inhibition of peak amplitudes and no significant sustained phase durations in Aqp4^–/– ANSCs compared with Aqp4^+/+ control. (F) Peak amplitudes of the KCl-induced Ca²⁺ transient show a significant decrease in Aqp4^–/– ANSCs compared with that in the Aqp4^+/+ control (^*P<0.001). Scale bars: 50 µm.