Effect of 2-min conditioning voltage pulses of increasing amplitudes on depolarization-evoked cytosolic Ca²⁺ signals. (A) Cytosolic Ca²⁺ changes monitored with Fura-2 (upper traces) in response to 1-s test pulses to +20 mV after conditioning pulses of increasing amplitudes delivered from a holding potential of −80 mV (lower traces) in the same fiber. Double voltage pulses were given every 2 min, and the fluorescence images were collected at a frequency of 1 Hz. (B) Relationship between the mean maximal changes in Fura-2 fluorescence induced by test pulses (measured between the right arrowhead and the dotted line in the example trace) normalized to the value obtained from a holding potential of −80 mV (measured between the left arrowhead and the dotted line) and conditioning voltages (n = 6). The relationship was fitted with a Boltzmann equation with V_1/2 of −51 mV and a slope factor of 5 mV.