Imaging odor-evoked activity in larval olfactory neurons. (a) Schematic of the larval imaging preparation showing head dissection (left) and mounting of inverted sample for G-CaMP imaging (right). (b) Whole-mount immunofluorescence staining of G-CaMP in terminals of Or35a and Or42a OSNs (anti-GFP; green) counterstained with the neuropil marker nc82 (magenta). Confocal image is a flattened z-stack of 7 × 7.2 μm optical slices that covers the anterior portion of the larval brain neuropil oriented with anterior at bottom. Scale bar = 50 μm. Genotypes for this and all other strains used in the paper are listed in the Additional data file 1. (c) Schematic of the larval olfactory circuit of the animal in (b). Olfactory sensory neuron (OSN) activity is imaged at axon terminals in the antennal lobe (blue box). Glomeruli also receive input from local interneurons (LNs) and projection neurons (PNs). Intrinsic G-CaMP fluorescence of OSN axon termini viewed in the imaging setup (right). (d) Calcium dynamics of Or35a and Or42a OSNs in a single animal in response to three odorants (10-2 odor dilution) and paraffin oil (solvent). For each stimulus, raw gray-scale fluorescent images presented at 600-ms intervals are shown at the top and false color-coded time traces represented by ΔF/F (%) (scale at right) are shown at the bottom. Odor presentation (1 s) is indicated in magenta on the gray time axis at the bottom.