ACTIVITY-DEPENDENT RECRUITMENT OF ENDOGENOUS GLUTAMATE FOR EXOCYTOSIS

The Ca2+-dependent release of the neurotransmitter glutamate from purified nerve terminals (synaptosomes) of the rat hippocampus was studied in a rapid perfusion apparatus. The response of the terminals was investigated with respect to the kinitics and duration of the release of endogenous glutamate upon brief and sustained stimulation and upon repetitive stimulation. The synaptosomes were stimulated by sustained chemical depolarization (0.5-3 min 30 mM K+). The cellular levels of glutamate, free Ca2+ and ATP in the nerve terminals were measured. The Ca2+-dependent release of glutamate showed an immediate elevation upon K+-depolarization. When the stimulation was maintained, a prolonged phase of glutamate release was observed. After 3 min, the Ca2+-dependent release stopped, although K+-depolarization was still effective. When synaptosomes were stimulated again after a relatively short stimulation period (30 s), the second response was similar to the previous one. After a longer stimulation period, maintained until termination of release, the second response did not show the immediate initial elevation of Ca2+-dependent glutamate release. Only 30 s after stimulation the release developed with a time profile comparable to the first response. This initial lack of response was not due to low cytosolic levels of glutamate or ATP or to changes in cellular Ca2+-buffering. It can be concluded that the capacity to release glutamate after brief depolarizations is fully restored during the repolarization period. However, if stimulation periods are of long duration (until termination of release), this capacity is no longer fully restored, especially with respect to a fast component of release. New glutamate is recruited only during the subsequent depolarization and with a delay. This points towards both activity- and time-dependent mechanisms. We hypothesize that different populations of synaptic vesicles are involved in the biphasic character of Ca2+-dependent glutamate release and in the activity- and time-dependent recruitment of glutamate: a "fusion ready" population released during a brief stimulation period and a "supplementary" population released during a sustained stimulation period.