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Fig. 4 Live Ca2+ imaging shows that impulses propagate the Drosophila tracheal network. Btl:GCamp3 embryos were imaged in 3D+time (3 sec time resolution) from stage 13 to 16 using two-photon light-sheet microscopy. (A,B) Time-lapse imaging of two control embryos at stage 16 reveal Ca2+ pulses propagating through electrically coupled cells once the tracheal network has fused, such as between adjacent transverse connectives via the dorsal trunk (A), or bidirectionally (B) (follow arrows in numbered sequence). The time points shown are (A) 21 and (B) 9 sec apart. Scale bars: 50 µm. (C) At 20 ms time resolution, a typical Ca2+ spike shows a fast upstroke and slower decay. (D) The mean Ca2+ spiking frequency and SEM for control, CPA-treated, and CPA+PMA-treated embryos at each stage are plotted (n>3; mean±s.e.m.). Spike frequency increases with embryo age. Compared to controls, the frequency of Ca2+ impulses at later stages is diminished by SERCA blockade, even in the presence of the PKC activator PMA. (E) Histograms of Ca2+ spike duration at stages 13-14 (left) and 15-16 (right) for embryos treated with DMSO (green), CPA (red) and CPA+PMA (yellow). In contrast to DMSO controls, embryos treated with CPA±PMA feature two types of Ca2+ pulse: (1) normal duration (clustered around 18 sec) and (2) prolonged with slow decay (see Movie 7).