Fig. 4: Pan-neuronal calcium dynamics in the song motor circuit during the singing of pulse and sine song.

a, Schematic of the wing-related neuropils. b, Schematic of the region imaged. For each fly, images (10 z-planes at a 4-μm step) were obtained from either a dorsal or medial part of the VNC. CsChrimson and GCaMP were expressed using the song and pan-neuronal drivers, respectively. c, Left, the sound data from an example recording. Bars at the top indicate periods during which example calcium imaging frames, shown on the right, are presented. The shaded area represents the period during which laser stimulation was applied. Right, example frames. For each period, images were averaged across z-planes and time. Blue boxes highlight the region that showed strong song-selective signals. Scale bar, 50 μm. d, Population-averaged ΔF/F for the difference between pulse and sine song around song-type transitions for the recordings from the dorsal part of the VNC (n = 9 flies). Scale bar, 50 μm. e, Histogram of the mean difference in ΔF/F between pulse and sine song during song-type transitions for the voxels that changed ΔF/F depending on song type (n = 9 flies; P < 0.001, two-sided permutation test). To obtain a histogram expected by chance, the same analysis was conducted after randomizing the relationship between song and calcium signals (‘Trial shuffle’; Methods). f,g, Same as d (f) and e (g) but for the recordings from the medial part of the VNC (n = 3 flies). See also Extended Data Fig. 6. LTct, lower tectulum; IntTct, intermediate tectulum; WTct, wing tectulum; D, dorsal; V, ventral.