Activity-dependent Fluorescent Staining and Destaining of Living Vertebrate Motor Nerve Terminals

Living motor nerve terminals from several species can be stained in an activity-dependent fashion by certain styryl dyes, such as RH414, RH795, and a new dye, FM1-43, which can be imaged independently of the others. The dyes evidently become trapped within recycled synaptic vesicles. In frog cutaneus pectoris muscle, bright fluorescent spots spaced regularly along the length of the nerve terminals appear after stimulation in the presence of the dye. The spots align well with postsynaptic ACh receptors and are persistent for many hours, unless further stimulation is given, in which case the spots disappear. Destaining, like staining, requires transmitter release and proceeds gradually over several minutes at high stimulus frequencies (e.g., 30 Hz), and fluorescent spots in the same terminal disappear at about the same rate. We suggest that each spot is a cluster of hundred of synaptic vesicles and that the mechanism of staining involves the ability of the dyes to partition reversibly into the outer leaflet of surface membranes, without being able to penetrate the entire membrane thickness. Then, during endocytosis following transmitter release, dye molecules become trapped in recycled synaptic vesicle membranes. The dyes therefore make it possible optically to study vesicle exocytosis and recycling in living nerve terminals in real time, and should be useful for marking terminals in a variety of preparations according to their level of activity.