in vivo calcium imaging

Calcium imaging experiments were performed essentially as described previously (Takeishi et al., 2016). AWC^ON and AWC^OFF neurons were identified via expression of mScarlet driven under srsx-3 regulatory sequences. Growth-synchronized L4 larval animals were cultivated overnight with ample OP50 at 20°C. Young adult animals were starved by placing them on unseeded NGM agar plates and re-cultivating at 20°C for 3 hr prior to imaging.

Individual animals were glued (WormGlu, GluStitch Inc) to an NGM agar pad on a cover glass, bathed in M9, and mounted under a second cover glass for imaging. For imaging of AFD and AIY, 5–10 fed or starved young adult worms were picked into 10 µM levamisole diluted in M9 on a 5% agarose pad and immobilized between two coverslips. The edges of the cover glass sandwich were sealed with a mixture of paraffin wax (Fisher Scientific), and Vaseline, and the sandwich was transferred to a slide placed on a Peltier device on the microscope stage. Animals were imaged within 3 min of being removed from their cultivation temperature.

Animals were subjected to linear temperature ramps rising at 0.05 °C/s unless noted otherwise, via temperature-regulated feedback using LabView (National Instruments) and a T-type thermocouple (McShane Inc). The slope of the temperature stimulus was selected to align with the temperature changes experienced by animals navigating the short thermal gradient. Based on average worm forward velocity of ~0.15 mm/s, animals are expected to experience temperature differences of 0.01 °C/s on the short thermal gradient. Since we observed few if any temperature responses when using a linear temperature ramp of 0.01 °C/s, we elected to use ramps rising at the rate of 0.05 °C/s to enable higher throughput analyses of responses. Individual animals were imaged for four mins at a rate of 2 Hz. Images were captured using a Zeiss 40X air objective (NA 0.9) or a Zeiss 10X air objective (NA 0.3) (for AFD and AIY imaging) on a Zeiss Axioskop2 Plus microscope, using a Hamamatsu Orca digital camera (Hamamatsu), and MetaMorph software (Molecular Devices). Data were analyzed using custom scripts in MATLAB (Mathworks) (Takeishi et al., 2016).

Each calcium trace was defined as the percent change in the relative fluorescence of the neuron from its baseline fluorescence level (average fluorescence of first 10 frames of each image) following background subtraction for all neurons with the exception of AFD. A fluorescence change of >10% in each neuron was considered a response, and the duration of calcium events was calculated as the sum of all events in each animal. Baseline fluorescence was set to zero to offset fluorescence change caused by photobleaching or movement artifacts. Calcium transients were imaged in the soma of AFD, AWC, AIB, and in the neurites of AIY, AIA and AIZ. T*_AFD was calculated as described previously (Takeishi et al., 2016).

For calculation of AIY response penetrance, ΔF/F traces during the ‘peri-T_c’ temperature range (18–22°C) were detrended using the MATLAB detrend function and plotted. Responses were identified manually, assisted by an overlaid plot of the first derivative of the detrended trace. Fluorescence traces were analyzed using MATLAB code (https://github.com/wyartlab/Cantaut-Belarif-et-al.-2020) (Sternberg et al., 2018; Cantaut-Belarif, 2020).

Analysis code can be found at: https://github.com/SenguptaLab/Starvation_ttx.