Intracellular Ca(2+) plays a major role in the physiological responses of excitable cells, and excessive accumulation of internal Ca(2+) is a key determinant of cell injury and death. Many studies have been carried out on the internal Ca(2+) dynamics in neurons. In constrast, there is virtually no such information for mammalian central myelinated axons, due in large part to technical difficulty with dye loading and imaging such fine myelinated structures. We developed a technique to allow imaging of ionized Ca(2+) in live rat optic nerve axons with simultaneous electrophysiological recording in vitro at 37 degrees C using confocal microscopy. The K(+) salt of the Ca(2+)-sensitive indicator Oregon Green 488 BAPTA-2 and the Ca(2+)-insensitive reference dye Sulforhodamine 101 were loaded together into rat optic nerves using a low-Ca(2+)/low-Na(+) solution. Axonal profiles, confirmed immunohistochemically by double staining with neurofilament-160 antibodies, were clearly visualized by S101 fluorescence up to 800 microm from the cut ends. The Ca(2+) signal was very low at rest, just above the background fluorescence intensity, indicating healthy tissue, and increased significantly after caffeine (20 mM) exposure designed to release internal Ca(2+) stores. The health of imaged regions was further confirmed by a virtual absence of spectrin breakdown, which is induced by calpain activation in damaged CNS tissue. Red and green fluorescence decayed to no less than 70% of control after 60 min of recording at 37 degrees C, with the green:red fluorescence ratio increasing slightly by 21% after 60 min. Electrophysiological responses recorded simultaneously with confocal images remained largely stable as well.