Extended Data Figure 8 |. Simultaneous imaging of the protocerebral bridge and ellipsoid body for each cell type separately and dual-colour imaging of GCaMP6f and jRGECO1a in E-PGs in the ellipsoid body.

a, We imaged the bridge and ellipsoid body in the same fly, at the same time, using a tall z-stack that encompassed both structures, to determine the relationship between the signals measured in each structure. b, Phase-nulled P-EN1 signals measured in the bridge (orange) and ellipsoid body (grey). The signals measured in the bridge were replotted onto the ellipsoid body using the P-EN projection pattern. c, d, Same as b but for P-EN2 signals from VT020739-Gal4 (c) and 12D09-Gal4 (d). e, As in a but for imaging E-PGs, with the bridge in blue. f, Same as b for E-PGs, with the left and right bridge in blue. In b–d and f, the mean and s.e.m. across flies are shown (in f, the s.e.m. for the bridge curves (blue) are omitted for clarity). Both the bridge and ellipsoid body signals were nulled using the ellipsoid body phase. Note that the positions of the left- and right-bridge peaks are inverted between P-EN1 and P-EN2. These results are consistent with the dual-imaging experiments in Fig. 3, and support the idea that the results in Fig. 3 were not due to crosstalk between the red and green channels. g, Schematic illustrating imaging from the ellipsoid body. h–j, Phase-nulled ellipsoid body signals of GCaMP6f and jRGECO1a co-expressed in E-PGs, computed for when the fly turned left (h, –300°/s), walked straight (i, 0°/s) or turned right (j, +300°/s), 300 ms before the calcium signal, as in Fig. 3k-p. The mean and s.e.m. across flies are shown. We observed no consistent, strong asymmetries in the jRGECO1a and GCaMP6f signals during left or right turns when both indicators were expressed in E-PGs. These data argue that the asymmetries we observed in dual imaging of P-ENs and E-PGs in the ellipsoid body (Fig. 3m–p) were not an artefact of indicator kinetics. Data are averaged over bar and dark conditions.