Extended Data Fig. 6. Testing the connectome-based prediction for stand-alone DNs that behaviors depend weakly on downstream DNs.

(a-d)(first column) The morphology of tested DNs in the adult female brain connectome. DNs are color-coded based on their somata localization within the cerebral ganglia (purple) or gnathal ganglia (orange). The number of DNs is indicated. (second column) A network schematic of direct connections from tested to downstream DNs. Edge widths reflect the number of synapses and is consistent across plots. Edge colors denote excitatory (red), inhibitory (blue), or glutamatergic (pink) which can be excitatory or inhibitory depending on receptor type⁶⁰. (third column) Quantitative analyses of optogenetically-driven behaviors and movements in intact (black traces) and headless animals (blue traces). The number of flies for each condition are indicated. Each fly is optogenetically stimulated ten times. Thus, the average and 95% confidence interval of the mean for a total of n*10 trials is shown. (fourth column) Identical behavioral analysis for control flies without DN opsin expression. Note that controls for different parameters include the same five animals. Two-sided Mann-Whitney U tests comparing the trial mean of intact and headless animals (black bars, above each plot) and comparing headless experimental with headless control flies (blue, in between experimental and control plots) are shown (*** means p < 0.001, ** means p < 0.01, * means p < 0.05, n.s. means p≥0.05; for exact p-values see Supplementary Table 5). (fifth column) An illustration of the behavioral parameter(s) being quantified. (a) oviDNs have four direct downstream partners and trigger curling of the abdomen in both intact and headless animals. This movement is quantified as a change in the vertical positioning of the ovum during optogenetic stimulation. (b) All together, six DNg11 neurons have four downstream partners and trigger foreleg rubbing²¹. This movement is quantified by the angle drawn by the axis between the coxa and front legs’ tibia-tarsus joint, and the coxa-neck axis. This metric allows to compare positions across flies. (c) The DN ‘Mute’ has no monosynaptic connections to other DNs and triggers ovipositor extension in both intact and headless animals. This movement is quantified as a change in the horizontal position of the ovipositor relative to the 1 s prior to stimulus onset. (d) DNg14 has no monosynaptic connections to other DNs and triggers abdominal dipping and vibration in both intact and headless animals. This movement is quantified as a change in the vertical position of the anal plate relative to 1 s before stimulus onset. These are the same data as in Fig. 5d–f).