Extended Data Fig. 10. (related to Fig. 6) Identification of driver lines for EM-reconstructed Feedback neurons.

We were able to generate Split-GAL4 lines that drive expression in a single pair of neurons, or in very few cell types, for three different pairs of FBNs or FB2Ns that target VL DANs (a-c). We used these lines to optogenetically activate these neurons instead of a US during an associative learning paradigm (Fig. 6). i) Skeletons of specific feedback neurons reconstructed in the EM. Red dots, presynaptic sites. Blue dots, postsynaptic sites. Grey, mushroom body vertical lobe (MB vl) for reference. d, dendritic arbor. ii) Maximum intensity projections of confocal stacks of larval brains showing the same neurons visualized with reporters targeted using specific Split-GAL4 lines. For some lines multicolor flp-outs were used to visualize each neuron in a different color to facilitate identification and comparison with EM (N=1). Grey, neuropil visualized with N-cad. Dashed line, brain outline. iii) Maximum intensity projections of confocal stacks of the entire nervous system showing the complete expression pattern of each line revealed by driving UAS-myr-GFP. Grey, neuropil visualized with N-cad. Dashed line, nervous system outline. Representative image from N=3. a, The SS02401-Split-GAL4 line drives expression in FB2N-19 (i) in the brain (ii), and very weakly and stochastically (not reproducibly in all samples) in a few ascending neurons and ensheathing glia in the nerve chord (iii). b, The SS02108-Split-GAL4 line drives expression in FAN-7 and MB2ON-86 (i) in the brain visualized with multicolor flp-outs in (ii). Complete expression pattern of SS02108-Split-GAL4 visualized with UAS-myr-GFP shows additional expression in a few somatosensory interneurons in the nerve cord, called ladders, that mediate avoidance behavior and are hence unlikely to have a positive valence and evoke the appetitive memory observed in Fig.6b. We identified the SS04330-Split-GAL4 line as driving expression specifically in the MB2ON-86 neuron and used it as an additional control in Fig.6b. c, The SS01778-Split-GAL4 line drives expression in both FB2N-18 and FB2N-11, which have very similar morphology and very similar connectivity (Supplementary Figures 3 and 4b-d). The SS02181-Split-GAL4 line (ii shows multi-color flp-outs) drives expression in FB2N-18 and in MB2IN-207, one of the weakly connected pre-modulatory neurons from lineage DAMv12. Notice the ventrally projecting dendrite (d), a distinctive feature of MB2IN-207 neuron (i). UAS-myr-GFP expression patterns of the two lines show that they do not drive expression in any other neurons in the nerve cord (iii).