Membrane recycling due to low and high rates of nerve stimulation at release sites in the amphibian (Bufo marinus) neuromuscular junction

The activity-dependent labelling of motor nerve terminals with the dye FM1- 43 has been used to estimate the relative levels of membrane recycling (due to synaptic vesicle exocytosis and recovery) at release sites in response to 1,200 nerve stimulations delivered at either low (0.5 Hz) or high (30 Hz ) frequency. Dye in terminals appears as fluorescent spots distributed alon g the terminal branches; each spot is thought to be a cluster of labelled v esicles associated with a release site. Relative fluorescence in spots was quantified from images obtained with a confocal microscope. Spot intensitie s varied widely within branches following labelling at both frequencies, bu t the distribution was highly skewed towards lower intensities at low frequ ency stimulation; at high frequency, more spots had stronger fluorescence. Both weak and strongly stained spots were uniformly distributed along the l ength of terminal branches after low frequency stimulation; however, there was a gradual decline in all spot intensities towards the distal end of bra nches loaded with dye at high frequency stimulation. Antibody staining for synaptic vesicles was, on average, uniformly distributed along the branches . The increase in number of more strongly FM 1-43-labelled spots in termina l branches stimulated at high compared with low frequency suggests that mor e release sites are active at high rates of nerve stimulation. This "recrui tment" of release sites at high frequency stimulation occurs mostly in the proximal half of terminal branches and is not related to the abundance of s ynaptic vesicles in the terminal. Synapse 32:110-118, 1999. (C) 1999 Wiley- Liss, Inc.