Calcium Imaging of C4da Neuron Terminals

Calcium imaging of C4da axon terminals was performed as previously described (Figure 4—figure supplement 1; Hu et al., 2017 (link)). Briefly, staged third instar larvae (96 ± 3 hr AEL) were pinned on a Sylgard (Dow Corning) plate and partially dissected in physiological saline to expose the ventral nerve cord (VNC). C4da neuron axon terminals expressing GCaMP6m were live-imaged by confocal microscopy with a 40 × water objective (Olympus FV1000MP) with 3x zoom to image at least four segments ensuring the calcium response could be detected. Activation of sensory neurons was achieved by providing a mechanonociceptive cue using a micromanipulator-mounted von Frey filament (45 mN) for stimulation of midabdominal segments (A3–A5). The most robust responses to local von Frey filament stimulation are restricted to a single VNC hemisegment corresponding to the stimulation site on the body wall although the adjacent segment(s) could also be slightly activated. The transient dip in fluorescence intensity (Figure 4B) is due to the larval brain moving out of focus briefly during and after mechanical stimulation. Baseline (F₀) and relative maximum intensity change (ΔF_max) of GCaMP6m fluorescence were analysed.