In vivo GCaMP Imaging of Sensory Neurons

For in vivo GCaMP analysis to visualize CIII, CIV, and Ch neurons, 19-12-Gal4 (Class III) and UAS-GCaMP6m were used in combination with ppk1.9-Gal4 (Class IV) or iav-Gal4 (Chordotonal) respectively. in vivo calcium imaging was performed as previously described [26 (link), 59 (link)]. Briefly, third instar larvae were mounted on a microscope slide with minimal water to prevent desiccation and placed on a Peltier stage (Linkam PE120) for time lapse imaging. The following temperature regimen was used during time lapse imaging: 1 minute at 25°C, ramp down to 6°C at 20°C/minute, hold at 6°C for 10 seconds, ramp up to 25°C at 20°C/minute, and hold at 25°C for one minute. Images were recorded at 212.55 μm x 212.55 μm resolution and 307.2 ms per frame. Raw time-lapse files were motion corrected in Fiji using the Stack Reg function. A region of interest was manually drawn around the cell body and mean fluorescence intensity across time was collected. ΔF/F₀ was calculated as previously described [26 (link)].