Staged 3rd instar larvae (96h AEL±3h AEL) were pinned on a Sylgard (Dow Corning) plate and partially dissected in physiological saline21 (link) to expose the VNC. A08n or DP-ilp7 neuron somata or C4da neuron axon terminals expressing GCaMP6m were live imaged by confocal microscopy with a 40× water objective (Olympus FV1000MP). Activation of sensory neurons was achieved either by class specific expression of CsChrimson (625 nm/5 s, 0.2 mW/mm2) or providing a mechano-nociceptive cue using a micromanipulator mounted von Frey filament (45 mN) and stimulation of mid-abdominal segments (a3–5). Baseline (F0) and relative maximum intensity change (Fmax) of GCaMP6m fluorescence was analyzed. Fmax/F0 values were represented as box-plots with whiskers, with the centerline representing median values, upper and lower edges of the boxes representing the 25th and 75th percentiles of the sample data, respectively. The short line within the box represents the mean of the data. Upper and lower whiskers represent the 5th and 95th percentile of the sample data, respectively. Statistical significance was analyzed using Mann-Whitney U-test with Bonferroni correction for multiple comparisons.