Calcium imaging
Changes in depolarization-induced calcium influx in rat DRG neurons were determined by loading neurons with 3 mM Fura-2AM for 30 minutes at 37°C (Cat# F1221; Thermo Fisher Scientific, Waltham, MA, stock solution prepared at 1 mM in DMSO, 0.02% pluronic acid, Cat# P-3000MP; Life Technologies, Carlsbad, CA) as previously described (Bellampalli et al., 2019). DRG neurons were incubated overnight with 20 µM of test compounds. A standard bath solution containing 139 mM NaCl, 3 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 10 mM Na-HEPES, 5 mM glucose, pH 7.4, was used. Depolarization was evoked with a 10 sec pulse of 90 mM KCl. Fluorescence imaging was achieved with an inverted microscope, Nikon Eclipse TE2000-U, using an objective Nikon Super Fluor 4X and a Photometrics-cooled CCD camera CoolSNAPHQ (Roper Scientific, Tucson, AZ) controlled by Nis Elements software (version 4.20; Nikon Instruments, Melville, NY). The excitation light was delivered by a Lambda-LS system (Sutter Instruments, Novato, CA). The excitation filters (340 ± 5 nm and 380 ± 7 nm) were controlled by a Lambda 10 to 2 optical filter change (Sutter Instruments, Novato, CA). Fluorescence was recorded through a 505-nm dichroic mirror at 535 ± 25 nm. Images were taken every ~2.4 seconds during the time course of the experiment to minimize photobleaching and phototoxicity. To provide acceptable image quality, a minimal exposure time that provided acceptable image quality was used. Changes in [Ca2+]c were monitored following a ratio of F340/F380, calculated after subtracting the background from both channels.