INDO-1 FLUORESCENCE SIGNALS ELICITED BY MEMBRANE DEPOLARIZATION IN ENZYMATICALLY ISOLATED MOUSE SKELETAL-MUSCLE FIBERS

Indo-1 fluorescence signals were measured from one extremity of enzyma tically isolated skeletal muscle fibers of mice. An original and simpl e method was developed to allow the measurements to be made under volt age-clamp control: the major part of a single fiber was embedded in si licone grease, so that only a short portion of one end of the fiber, f rom which the fluorescence measurements were taken, was in contact wit h the external solution, Membrane potential was held and varied by usi ng a patch-clamp amplifier in whole-cell configuration with a single m icroelectrode, the tip of which was inserted across the silicone greas e within the insulated portion of the fiber, In response to 100-ms dep olarizing command pulses to voltages more positive than -40 mV (from a holding potential of -80 mV), clear changes in fluorescence were qual itatively observed to feature a time course of rise and decay expected from a change in intracellular calcium concentration ([Ca2+](i)) due to voltage-dependent sarcoplasmic reticulum (SR) calcium release, Alth ough the peak [Ca2+](i) elicited by a 100-ms depolarization at 0 or +1 0 mV varied from fiber to fiber, it could clearly reach a value high e nough to saturate indo-1, The overall results show that this method re presents an efficient way of measuring depolarization-induced [Ca2+](i ) changes in enzymatically dissociated skeletal muscle fibers.