(A-C), (E-H) Confocal z-projections, dorsal view. (A) R72A10 > UAS-myr::GFP (green) showing the three OLPs (neuropil marker: ChAT, red) and close-up on the cells bodies (box) showing that one OLP is glutamatergic (Aiii) and at least another one is clearly cholinergic (Aiv). B: SS01724 >UAS-myr::GFP (green) showing a lOLP with dense arborization within the LON, reduced projections, and which is glutamatergic (DVGluT in red) (Bi), (Bii) close-up of the cell body (box)). (C) R72A10 > UAS-myr::GFP (green) close up on the three OLPs cell bodies where we can observe the strongly cholinergic cell (ChAT, red) sending its axon towards the LON via a different path (arrow) than the glutamatergic cell (DVGluT, blue) and the third cell (arrowhead). (D) 3D reconstruction of the three OLPs in the left hemisphere of the ssTEM dataset where we could observe two cells sending their axons together to the LON (arrowhead), whereas the third one takes a separate path (arrow). Comparing (C) and (D) and based on their anatomy, the three OLPs can be distinguished in the ssTEM dataset (D): the projection OLP (pOLP, blue) and the glutamatergic lOLP (glu-lOLP, green) axons fasciculate together (arrowhead) but not with the cholinergic lOLP (cha-lOLP, light green, arrow). (E) SS01745 >UAS-myr::GFP (green) showing the pOLP anatomy with weak arborization within the LON and deep projection into the neuropil. (E’, E’’) Close up on pOLP cell body showing a weak but clear cholinergic cell (arrowhead, ChAT in red/white). (F) R20C08 > UAS-myr::GFP (green) showing a single looping neuron with a strong overlap in the LON and having its cell body situated postero-ventro-laterally, corresponding to PVL09 (neuropil marker ChAT in red). (F’), (F’’) Close up on a PVL09 cell body (SS0671 >UAS-H2B-RFP, RFP in blue) that is cholinergic (ChAT, red, arrowhead). Single section. (G) SS01777 >UAS-myr::GFP (green) showing a stochastic single clone of the third-order neuron VPLN that appears glutamatergic (arrowhead, DVGluT in red/white, (G’), (G’’) close up). (H) R19C05 > UAS-myr::GFP (green) showing three cells among which only one is PER-positive (5th-LaN, white arrow) and two are PER-negatives (nc-LaNs, magenta arrow) (H’), PER in blue/white). All three cells are cholinergic (H’’), ChAT in red/white). Four additional cells weakly covered by the Gal4 line and expressing PER correspond to the Pdf-LaNs (H’), arrowheads). Scale bars: A, B, E, F, G, H: 10 μm. C: 5 μm. (I) Electron microscopy view of a bouton rich in dense-core vesicles and clear vesicles in the Pdf-LaN numbered 2 of the left hemisphere.