Endocytosis and exocytosis were investigated in the synaptic terminal of retinal bipolar cells by monitoring the uptake and loss of the fluorescent dye FM1-43. Depolarization in the presence of Ca2+ stimulated a continuous cycle of exocytosis and endocytosis that was approximately balanced at rates up to 3800 vesicles per s. Vesicles became available for exocytosis within 1 min of endocytosis, and about 700,000 releasable vesicles were specifically localized to a region within 2 microm of the plasma membrane. Release of caged Ca2+ using NP-EGTA while simultaneously monitoring cytosolic Ca2+ with Fura-2 indicated that continuous exocytosis was stimulated by sub-micromolar levels of Ca2+. It has been suggested that the ribbon synapse of bipolar cells only supports transient exocytosis, but our results demonstrate that this synapse is specialized for the continuous secretion of neurotransmitter.

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Ribbon synapses of sensory neurons are able to sustain high rates of exocytosis in response to maintained depolarization, but it is not known how this is achieved. Using the capacitance technique, we have found that Ca 2ϩ regulates the supply of releasable vesicles at the ribbon synapse of depolarizing bipolar cells from the retina of goldfish. Ca 2ϩ had two actions that could be differentiated by introduction of the Ca 2ϩ chelator EGTA; one action stimulated refilling of the rapidly releasable pool of vesicles from a reserve pool, and a second action stimulated recruitment of vesicles to the reserve pool. The capacity of the reserve pool was ϳ3500 vesicles, which is similar to the number that can attach to the ribbons. These results suggest that continuous exocytosis at ribbon synapses is maintained by the Ca 2ϩ -dependent translocation of vesicles from the cytoplasm, through the ribbon, to release sites on the plasma membrane. The processes that make synaptic vesicles available for exocytosis play an important role in determining the efficiency of synaptic transmission during ongoing activity. For instance, many synapses exhibit short-term depression after a period of activity, and this is at least partly because of a decrease in the number of rapidly releasable vesicles docked at the plasma membrane (Del Castillo and Katz, 1954;Betz, 1970;Zucker, 1989;Rosenmund and Stevens, 1996;Dittman and Regehr, 1998). The question of how vesicles become available for exocytosis is of particular importance at ribbon synapses, which can support continuous high rates of exocytosis in response to maintained depolarization (Dowling and Ripps, 1973;Parsons et al., 1994;Lagnado et al., 1996;Rieke and Schwartz, 1996). Ribbon synapses are found in sensory neurons that generate graded voltage signals rather than action potentials, including photoreceptors and bipolar cells in the retina (Gray and Pease, 1971;Rao-Mirotznik et al., 1995;von Gersdorff et al., 1996) and auditory and vestibular hair cells in the ear (Jacobs and Hudspeth, 1990;Lenzi et al., 1999). Electron micrographs of these neurons show that the regions in which synaptic vesicles dock to the plasma membrane are associated with an electron-dense structure, called a ribbon or synaptic body, to which vesicles attach by short filaments. This arrangement suggests that vesicles attached to the ribbon form a reserve pool that supplies the active zone on the plasma membrane (Gray and Pease, 1971), although it has been difficult to test this idea experimentally (Burns and Augustine, 1995;Lenzi et al., 1999). Recently, our knowledge of the structure of ribbon synapses has been complemented by f unctional studies of exocytosis using capacitance and optical techniques. In particular, we have a relatively large amount of information about vesicle cycling at the ribbon synapse of depolarizing bipolar cells from the goldfish retina (Tachibana et al., 1993;Heidelberger et al., 1994;von Gersdorff and Matthews, 1994;Lagnado et al., 1996). An important feature of this synapse is that t...