(A) mCD8:GFP driven by 412-Gal4, labeled with anti-GFP (green). (A') Cha3.3kb-Gal80 reduces expression of mCD8:GFP (anti-GFP, green) in DnB neurons. (B) DnB neuron (anti-GFP, green); (C') Boxed region: DnB cell body (arrowhead) shows overlap with ChAT (anti-ChAT; dsRed), (C'') ChAT; dsRed channel only. (C) DnB neuron (anti-GFP, green); (B') Boxed region: DnB cell body (arrowhead) shows no overlap with vGLUT (anti-vGLUT; dsRed); (B'') VGLUT; dsRed channel only. (D) DnB neuron (anti-GFP, green); (D') Boxed region: DnB cell body (arrowhead) shows no overlap with GABA (anti-GABA; dsRed), (D'') GABA; dsRed channel only. (E) tdTomato driven by 412-Gal4 labeling DnB neuron (anti-dsRed, red) (D') Single section showing DnB cell body overlapping with ChAT-eGFP (anti-GFP, green) (top: overlap, middle: tdTomato, red channel only, bottom: ChAT-eGFP cell body) (D''–D''') Single sections showing DnB axons; left panels DnB axon and ChAT-eGFP puncta overlap; middle: DnB axons, tdTomato channel; right panels: ChAT-eGFP puncta, GFP channel only. (F) Electron micrograph of DnB synapse showing dense core vesicles (arrowheads) indicative of peptidergic or aminergic release. (G) Neuropeptide precursor, PreproANF fused with emerald GFP (anti-GFP, magenta) is processed and expressed in DnB axons (arrowhead; anti-dsRed, green), suggesting that DnBs are peptidergic. Genotypes: (A,B,C) 412-Gal4, UAS-mCD8:GFP /+ (A') 412-Gal4, UAS-mCD8:GFP/Cha3.3Kb-Gal80 (D–D''') 412-Gal4, UAS-TdTomato/Chat-EGFP-Flash-Tev3xFlag. (G) UAS-Prepro:ANF-EMD;;412-Gal4,UAS-TdTomato/+. Scale bars = 50 µm (A– A') 20 µm (B,C,D,G).