Odor responses of larval projection neurons. (a) Schematic for measuring functional activation of larval PNs in Or35a-, Or42a-, or Or42b-functional larvae at axon terminals in the mushroom body (blue box). Intrinsic G-CaMP fluorescence of the mushroom body, with subdomains 1–4 and sample orientation indicated (bottom). (b) Confocal image (flattened z-stack of 2 × 1.2 μm optical slices) of PN cell bodies stained to reveal G-CaMP (anti-GFP antibody, green) and Drosophila choline acetyltransferase (anti-ChAT, magenta). Scale bar = 20 μm. (c) Representative G-CaMP activity in PN terminals in mushroom body elicited by three odorants (10-2 dilution) and paraffin oil (solvent) in (left to right): Or35a-, Or42a-, and Or42b-functional larvae. Top row shows intrinsic mushroom body G-CaMP fluorescence and bottom four rows show false color-coded image of mushroom body taken 600 ms after stimulus onset, and represented as %ΔF/F (scale at the right). (d) Responses of PNs of single-functional larvae in (b) to eight odorants (10-2 dilution except as indicated) and paraffin oil (solvent) represented as false color-coded time traces (%ΔF/F; scale at bottom right). Traces from n = 11–14 animals per stimulus are stacked. Region of analysis is from major subdomain 1–4, as indicated in (a-b). (e) Responses of major subdomains 1–4 of PN axon termini in mushroom body of Or35a-, Or42a-, and Or42b-functional larvae to an ethyl butyrate concentration series and paraffin oil (solvent) represented as ΔF/F (%) (scale at right). Traces from n = 8 animals per genotype and stimulus are stacked.