Fig. 4: DRG Ca²⁺ signaling amplitude is reduced by MMAE treatment and maintained at normal levels with lithium pretreatment.

a Schematic representation of the perfusion chamber used for the ex vivo Ca²⁺ signaling experiment. DRGs were removed from mice, loaded with 40 µM Fluo-4/AM and placed in a glass coverslip for perfusion with the indicated stimuli. The DRG was attached to the glass coverslip using a harp shaped metal tool with nylon stripes and intracellular Ca²⁺ signaling was visualized with an excitation wavelength of 488 nm. b Representative image of Ca²⁺ signal in ex vivo DRG before and after stimulation with KCl. White arrows indicate cells that responded to KCl stimulus. Scale bar 50 μm. c Amplitude of Ca²⁺ signaling in DRG induced by KCl did not differ among the groups, showing that Ca²⁺ influx from the extracellular medium is not impaired by MMAE (p > 0.05, n = 25–30 cells from 5 mice each group). d Amplitude of Ca²⁺ signaling in DRG induced by carbachol was reduced by MMAE, and the reduction was prevented by lithium pretreatment (p < 0.05, n = 12–16 cells from 5 mice each group). e Representative graphic of the time course of Ca²⁺ signaling upon carbachol stimulation in DRG, showing that MMAE not only reduces the amplitude of Ca²⁺ signal, but it also alters the temporal signaling pattern. f MMAE treatment increases the number of cells responding with an oscillatory pattern and the LiCl+MMAE pretreatment group primarily responds with transient Ca²⁺ increases.