Effect of 2-min conditioning voltage pulses of increasing amplitudes on depolarization-evoked SR Ca²⁺ signals. (A) SR Ca²⁺ content changes monitored with Fluo-5N (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. For each double pulse, the frequency of fluorescence image capture was 0.2 Hz before and during the conditioning pulses, and then it was switched to 15 Hz just before delivering the test pulse. The time interval between the two sampling frequencies is 5 s. (B) Relationship between the mean changes in Fluo-5N fluorescence induced by test pulses (measured between the two arrowheads in the example trace) normalized relative to the total Fluo-5N fluorescence decrease induced by the conditioning pulse plus the test pulse (measured between the right arrowhead and the dotted line) and conditioning voltages (n = 9). The relationship was fitted with a Boltzmann equation with V_1/2 of −51 mV and a slope factor of 5 mV.