Figure 2. All JO neurons, except EDM, increase their activity during flight, and this increase is caused by the movements of the ipsilateral antenna. Left of the dotted vertical line, Average changes in the fluorescence of GCaMP3 (left column) or EGFP (middle column) relative to its baseline value (ΔF/F) during flight for ROIs containing each JO neuron class. Right columns, ΔF/F of GCaMP3 from flies whose second and third segments of the ipsilateral antenna are glued together. In these experiments, we used JO1-GAL4 or F-GAL4 to express GCaMP3 or EGFP in all JO neuron classes, respectively. Circles represent the average ΔF/F during the entire flight trial for each JO neuron class in a single fly. Gray shaded boxes and black horizontal lines represent interquartile ranges and the median values, respectively, of the ΔF/F for each JO neuron class. One outlier for JO-A neuron whose average ΔF/F during flight was 12.22 is not shown on the plot but was included in the analysis. Black horizontal brackets indicate groups within each JO neuron class that were significantly different from each other: *p < 0.05; **p < 0.01 (Tukey–Kramer post hoc comparison of the medians). Black horizontal dotted line indicates ΔF/F = 0. All JO neuron classes, except EDM, significantly increased the GCaMP3 fluorescence compared with the EGFP fluorescence during flight, suggesting an increase in the activity. Gluing the ipsilateral antenna eliminated this increase in the activity. Inset, Example image of JO neuron terminals shown in Figure 1D (JO-B neurons cannot be seen in this example). For each JO neuron class, the number of flies used for measuring ΔF/F during flight for GCaMP3, EGFP, and GCaMP3 of the ipsilateral antenna glued flies are as follows: JO-A, 22, 11, and 14; JO-B, 26, 10, and 20; JO-C, 29, 12, and 21; JO-D, 27, 12, and 21; JO-EDM, 26, 12, and 20; JO-EVM, 26, 12, and 20. Right of the dotted vertical line, The same as the panel on the left side, except that we used more specific GAL4 drivers JO-AB-GAL4 or JO-CE-GAL4 to express GCaMP3 in JO-A/B or JO-C–E neurons, respectively. We found similar pattern of increase in GCaMP fluorescence during flight using these GAL4 drivers. Asterisks indicate JO neuron class that showed significantly larger ΔF/F during flight compared with EGFP control: **p < 0.01 (Wilcoxon rank sum test with Bonferroni correction). Number of flies for each JO neuron class are as follows: JO-A, 10; JO-B, 10; JO-C, 15; JO-EDM, 16; JO-EVM, 16.