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Volume 16, Number 12, Issue of June 15, 1996 pp. 3960-3967
Copyright ©1996 Society for Neuroscience

Synaptic Vesicle Movements Monitored by Fluorescence Recovery after Photobleaching in Nerve Terminals Stained with FM1-43

Received Feb. 5, 1996; revised March 8, 1996; accepted March 12, 1996.

A. W. Henkel1, L. L. Simpson2, R. M. A. P. Ridge3, and W. J. Betz1

1 Department of Physiology, University of Colorado Medical School, Denver, Colorado 80262, 2 Departments of Medicine and Pharmacology, Jefferson Medical College, Philadelphia, Pennsylvania 19107-6799, and 3 Department of Physiology, Bristol University School of Medical Sciences, Bristol BS8 1TD, United Kingdom

We used the fluorescence recovery after photobleaching technique to monitor movements of synaptic vesicles in top views of living frog motor nerve terminals that had been prestained with the fluorescent dye FM1-43. In each experiment, a small portion of a single stained vesicle cluster was bleached with a laser and monitored subsequently for signs of recovery as neighboring, unbleached vesicles moved into the bleached region. In resting terminals, little or no recovery from photobleaching occurred. Repetitive nerve stimulation, which caused all fluorescent spots to grow dim as dye was released from exocytosing vesicles, did not promote recovery from photobleaching. Pretreatment with botulinum toxin (type A, C, or D) blocked exocytosis and destaining, but intense nerve stimulation still did not cause significant recovery in bleached regions. These results suggest that lateral movements of synaptic vesicles are restricted severely in both resting and stimulated nerve terminals.

We tested for laser-induced photodamage in several ways. Bleached regions could be restained fully with FM1-43, and these restained regions could be destained fully by nerve stimulation. Partially bleached regions could be destained, although the rate of destaining was lower than normal. Brisk recovery from photobleaching occurred after treatment with okadaic acid, which disrupts synaptic vesicle clusters and causes vesicles to spread throughout the nerve terminal. These results suggest that vesicle translocation and recycling machinery was intact in photobleached regions.

Key words: photobleach recovery; FPR; FRAP; synaptic vesicles; okadaic acid; FM1-43; exocytosis




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