Cytosolic and SR Ca²⁺ decline during long-lasting trains of action potentials or voltage pulses. (A) Means and SEM of cytosolic Ca²⁺ changes monitored with Fura-2 in response to trains of action potentials of 1-min duration induced by bursts of supraliminal current pulses of 0.5-ms duration at a frequency of 50 Hz in current-clamp conditions (n = 3). In each cell, fluorescence values were normalized to the maximal change in fluorescence obtained during the pulse. The frequency of image capture was 1 Hz. (B) Means and SEM of SR Ca²⁺ content changes monitored with Fluo-5N in response to trains of action potentials of 1-min duration obtained in the same conditions as in A (n = 5). In each cell, fluorescence values were normalized to the maximal change in fluorescence obtained during the pulse. Frequency of image capture was 1 Hz. (C) SR Ca²⁺ content changes monitored with Fluo-5N (upper traces) in response to 30-s, 50-Hz trains of voltage pulses of increasing duration delivered to +20 mV from a holding potential of −80 mV (indicated by the middle and lower traces) in the same fiber in voltage-clamp conditions. For each train, the frequency of fluorescence image capture was 20 Hz until the end of the first second of the train, and then it was switched to 0.2 Hz. The time interval between the two sampling frequencies is 5 s. Trains were applied every 2 min. The vertical dotted line indicates the time at which the train ended. (D) Relationship between the mean Fluo-5N fluorescence decay at the end of the train (measured between the right arrowhead and the dotted line in the example trace) normalized to maximal Fluo-5N fluorescence change during the train (measured between the left arrowhead and the horizontal dotted line) and pulse duration (n = 9). The relationship was fitted with a Boltzmann equation with t_1/2 of 16 ms (indicated by the dotted lines) and a slope factor of 1.4 ms.