Calcium imaging in live rat optic nerve myelinated axons in vitro using confocal laser microscopy

Intracellular Ca2+ plays a major role in the physiological responses of exc itable cells, and excessive accumulation of internal Ca2+ is a key determin ant of cell injury and death. Many studies have been carried out on the int ernal Ca2+ dynamics in neurons. In constrast, there is virtually no such in formation for mammalian central myelinated axons, due in large part to tech nical difficulty with dye loading and imaging such fine myelinated structur es. We developed a technique to allow imaging of ionized Ca2+ in live rat o ptic nerve axons with simultaneous electrophysiological recording in vitro at 37 degreesC using confocal microscopy. The K+ salt of the Ca2+-sensitive indicator Oregon Green 488 BAPTA-2 and the Ca2+-insensitive reference dye Sulforhodamine 101 were loaded together into rat optic nerves using a low-C a2+/low-Na+ solution. Axonal profiles, confirmed immunohistochemically by d ouble staining with neurofilament-160 antibodies, were clearly visualized b y S101 fluorescence up to 800 mum from the cut ends. The Ca2+ signal was ve ry low at rest, just above the background fluorescence intensity, indicatin g healthy tissue, and increased significantly after caffeine (20 mM) exposu re designed to release internal Ca2+ stores. The health of imaged regions w as further confirmed by a virtual absence of spectrin breakdown, which is i nduced by calpain activation in damaged CNS tissue. Red and green fluoresce nce decayed to no less than 70% of control after 60 min of recording at 37 degreesC, with the green:red fluorescence ratio increasing slightly by 21% after 60 min. Electrophysiological responses recorded simultaneously with c onfocal images remained largely stable as well. (C) 2000 Elsevier Science B .V. All rights reserved.