a, Schematic and representative image depicting direct stimulation of vAB3 neurons by iontophoresis of neurotransmitter onto their dendrites within the VNC. Maximum intensity z-projection (top) and single z-plane (bottom) showing electrode placement in the VNC. The electrode is filled with neurotransmitter and Texas-red dye (red) to enable precise targeting in the Fru+ neuropil (green). b, Representative multi-plane fluorescence increase of Fru+ brain neurons in D. simulans males when the VNC is stimulated with acetylcholine (left), glutamate (middle) and saline (right) iontophoresis. c, To test the necessity of vAB3 in propagating signals from the VNC to the higher brain, we compared response profiles in the brain before severing vAB3 (black), after severing a nearby Fru+ axon (mock control, blue) and then after severing vAB3 axons (orange) in D. melanogaster males (top) and D. simulans males (bottom). Representative multi-plane fluorescence increase of Fru+ neurons (left) shows the loss of evoked functional responses in the brain after severing vAB3. The graph (middle) depicts the relationship between average ΔF/F responses in vAB3 and mAL. Average ΔF/F responses of vAB3 (green) and mAL (red) neurons evoked by vAB3 stimulation, before and after severing vAB3 (right). Responses were lost in both neural populations in both species after vAB3 was severed but not in the mock control. d, The relationship of functional responses in the SEZ and LPC of D. melanogaster and D. simulans males evoked by direct vAB3 stimulation. The dots on the graph represent different stimulation intensities and the trend lines relate the responses of individual D. melanogaster (green) and D. simulans (blue) males. e, Left, the response of vAB3 (top) and mAL (bottom) axonal tracts in response to vAB3 stimulation in D. melanogaster (green) and D. simulans (blue) males. Coloured lines represent single stimulations and black lines represent the average. Right, peak ΔF/F plotted with coloured dots representing the average response per fly and data are mean and s.d. Individual data points are shown. f, g, Comparison of vAB3-evoked responses in D. melanogaster and D. simulans males in vAB3 neurons (f) and P1 neurons (g) before (+) and after (−) mAL severing. Statistical tests used were the Kruskal–Wallis test, in which different letters mark significant differences (c, f, g), and an unpaired t-test (e). Scale bars, 10 μm. See Supplementary Table 1 for details of statistical analyses.