In vivo calcium imaging of ADL sensory neurons was performed essentially as described previously (7, 40). Worms expressing yellow cameleon 3.60 (YC3.60) driven by the sre-1p promoter [sre-1p::yc3.60 (pTOM63)] were used for in vivo calcium imaging of ADL neurons. Animals were cultured under well-fed conditions on 2% (w/v) agar NGM plates containing E. coli OP50 in 6.0-cm-diameter plastic dishes. Two or three well-fed adult(s) (P0) were placed onto NGM plates at 25° or 15°C, and their progenies were incubated at 25° or 15°C from egg to adulthood for 3 or 6 days, respectively. Adult animals were glued onto a 2% (w/v) agar pad on a glass slide and immersed in M9 buffer under a glass coverslip. Sample preparation was completed within 3 min. Samples were then placed onto an indium tin oxide glass-based thermocontroller (Tokai Hit Co. Ltd., Fujinomiya, Japan) mounted on the stage of an Olympus IX81 microscope (Olympus Corporation, Tokyo, Japan) and equilibrated at the initial imaging temperature for a few minutes. Fluorescence was observed using a Dual-View (Molecular Devices, USA) optical system. YC3.60 donor and acceptor fluorescent signals were captured simultaneously using an electron multiplying charge-coupled device camera (Evolve 512, Photometrics, USA). Images were taken with 30-ms exposure times and 1 × 1 binning. Temperature on the agar pad was monitored using a thermometer system (MATS-5500RA-KY, Tokai Hit). For each imaging experiment, fluorescence intensities were acquired and processed using MetaMorph (Molecular Devices) image analysis software. Relative changes in intracellular calcium concentrations were measured as changes in the acceptor/donor fluorescence ratio of YC3.60. Band-pass filters for all YC3.60 experiments were as described in a previous report (40).

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