Calcium imaging of dorsal root ganglion neurons

A total of 16 rats, killed by CO₂ overdose, were used for the calcium imaging studies. Spinal columns were removed and dorsal root ganglia (DRGs) neurons were collected [20]; cell preparation and culture were as described previously [21, 22].

Cells were washed three times with calcium imaging buffer (NaCl, 145 mM; KCl, 5 mM; CaCl₂, 2 mM; MgSO₄.7H₂O, 1 mM; HEPES, 10 mM; glucose, 10 mM; pH, 7.4). Then cells were loaded with 5 μl of Fura2-AM in 895 μl of calcium buffer with 100 μl of fetal calf serum (FCS) and incubated for 30 min in the dark. Cells were washed with calcium buffer and left for at least 15 min prior to imaging. [Ca²⁺]_i was measured as the ratio of peak fluorescence emission intensities (measured at 500 nm) at excitation wavelengths of 340 nm and 380 nm, respectively, using an Andor IQ imaging system and expressed as changes in relative fluorescence units (ΔRU). Coverslips were fixed to a Perspex chamber using vacuum grease, and the DRG neurons were suprafused with calcium buffer at a rate of 2 ml/min. All drugs were applied by suprafusion in calcium buffer.