Thapsigargin

ionomycin calcium salt was purchased from Tocris Bioscience (Minneapolis, MN, USA) and used per the manufacturer’s recommendations. ionomycin was dissolved in DSMO first to make a 2 mM stock concentration. ionomycin was then dissolved in 2 mM Ca²⁺ aCSF at a final concentration of 1, 3, or 5 µM and perfused for 1 hour. Similarly, ionomycin was dissolved in zero Ca²⁺ aCSF for a final concentration of 3 µM and perfused for 1 hour. After 1 hour, the ionomycin buffer was washed out with either zero or normal 2 mM Ca²⁺ aCSF and images were collected every 15 minutes for an additional hour. To inhibit store-operated Ca²⁺ entry (SOCE), we pretreated spinal cords for 30 minutes with YM-58483 (an established blocker of SOCE; 500 nM; Tocris Bioscience) prior to ionomycin perfusion in aCSF. Thapsigargin, an irreversible Sarco-Endoplasmic Reticulum Ca²⁺ ATPase inhibitor, was purchased from AdipoGen Life Sciences (San Diego, CA, USA) and used per the manufacturer’s recommendations. Thapsigargin was dissolved in DMSO to make a 5 mM stock solution and further diluted in 2 mM Ca²⁺ aCSF to make a final concentration of 1 µM to pretreat the spinal cord for 60 minutes to empty calcium stores.