(A) Representation of all ePNs (magenta) and iPNs (green) labeled by GH146-GAL4 and MZ699-GAL4 using PA-GFP, respectively. Photoactivation of vlPr neurons (orange, MZ699-GAL4) connecting the LH and the vlPr via the plF. The overlay image depicts a pseudo-merge image of the different GAL4-driver lines. (B) Schematic of the olfactory circuit with integrated layout of the transection experiment. After simultaneous Ca2+ imaging of bilateral LHs, the ipsilateral plF and contralateral mACT was transected (red zigzag line) with an infrared laser (dashed red arrow). (C) Projection images of a 7 µm stack of the LH area prior and post transection. Left images, mACT transected; right image, plF transected. The ablated region is indicated by the dashed red arrow. Scale bar, 20 µm. (D) Median time traces displaying percental change of ΔF/F values for indicated ORDs prior to post transection of the mACT (green, left) and the plF (orange, right) for different odorants. Significant changes of odor-evoked Ca2+ signals due to transection are shown in the column SIG difference. Differences were tested with a two-tailed paired Student's t test (p < 0.05). Color codes are indicated by the corresponding scale bar below, n = 4–5. Transecting the mACT eliminates Ca2+ signals in the LH-PM and LH-AM domain, while lesioning the plF significantly abolishes LH-AL responses. Notably, the LH-AL domain is significantly stronger activated after mACT transection following application of 1-octen-3-ol and γ-butyrolactone. (E) Summarized cartoon of the neuron populations contributing to ORD activity prior and post transection of axons of iPNs or vlPr neurons.