FLUORESCENCE MICROSCOPY OF CALCIUM AND SYNAPTIC VESICLE DYNAMICS DURING SYNAPSE FORMATION IN TISSUE-CULTURE

The signal transduction process involved in the development of the ner ve terminal is an intriguing question in developmental neurobiology. D uring the formation of the neuromuscular junction, presynaptic develop ment is induced by growth cone's contact with the target muscle cell. Fluorescence microscopy with specific markers has made it possible to follow signalling events during this process. By using fluorescent cal cium indicators, such as fura-2 and fluo-3, we found that a rise in in tracellular calcium is elicited in the growth cone upon its contact wi th a target, and this calcium signal can also be elicited by local app lication of basic fibroblast growth factor. To monitor the clustering of synaptic vesicles in response to target contact, the fluorescent ve sicular probe FMI-43 was used. With this probe, we observed that packe ts of synaptic vesicle are already present along the length of naive n eurite, which has not encountered its synaptic target. The activity-de pendent loading of FMI-43 indicates that these packets can undergo exo cytosis and endocytosis upon depolarization. Time-lapse recording show ed that these packets are quite mobile. Upon target contact, synaptic vesicles become clustered and immobilized at the contact site. The met hodology and instrumentation used in these studies are described in th is article. (C) 1998 Chapman & Hall.