Synaptic activity-dependent changes in the spatio-temporal distribution of calcium ions regulate important neuronal functions such as dendritic integration and synaptic plasticity, but the processes that terminate the free Ca 2ϩ transients associated with these changes remain unclear. We have characterized at the electron microscopic level the intracellular compartments involved in buffering free Ca 2ϩ transients in dendritic cytoplasm of CA3 neurons by measuring the larger changes in the concentrations of total Ca that persist for several minutes after neuronal activity. Quantitative energy-dispersive x-ray microanalysis of cryosections from hippocampal slice cultures rapidly frozen 3 min after afferent synaptic activity identified a subset of dendritic endoplasmic reticulum (ER) as a highcapacity Ca 2ϩ buffer. Calcium sequestration by cisterns of this subset of ER was graded, reversible, and dependent on a thapsigargin-sensitive Ca 2ϩ -ATPase. Sequestration was so robust that after repetitive high-frequency stimulation the Ca content of responsive ER cisterns increased as much as 20-fold. These results demonstrate that a subpopulation of ER is the major dendritic Ca sequestration compartment in the minutes after neuronal activity.

It is generally accepted that Ca is essentially involved in regulated secretion, but the role of this cation, as well as others such as Na, is not well understood. An illustrative example occurs in neurohypophysial secretion, where an experimentally induced increase in the cytosolic concentration of Na ؉ can induce continuous neuropeptide release. In contrast, an increase in cytosolic Ca 2؉ will have only a transient stimulatory effect. The secretion-promoting targets for Ca 2؉ are not known; they may be cytosolic, as is usually assumed, but they may also be intravesicular, especially in view of evidence that Ca-rich secretory vesicles are preferentially secreted. In the present work, we have investigated the movements of these cations into and out of secretory vesicles during stimulus-secretion coupling. Isolated rat neurohypophysial nerve endings were stimulated by potassium (55 mM) depolarization, and at 6 min (peak secretion) and 20 min after the onset of stimulation, the elemental content of individual secretory vesicles was measured by quantitative x-ray microanalysis. A depolarization-induced transient increase in intravesicular Na ؉ concentration was found to coincide with the onset of secretion. Moreover, only a predicted small fraction of peripheral vesicles-presumably the docked ones-were Na ؉ -loaded. The low sulfur concentration of Na ؉ -rich vesicles most likely resulted from vesicle swelling. The results suggest that high intravesicular Na ؉ concentrations in docked vesicles, occurring by Na ؉ ͞Ca 2؉ exchange or by transient fusion pore opening, is a proximal event in exocytosis. Ca 2ϩplays an essential role in several stimulatory, and perhaps also inhibitory, steps of regulated exocytosis but, despite much recent progress, its precise targets are still largely a matter of debate (1-3). In neurohypophysial secretion particularly, it is clear that Ca 2ϩ cannot be the ultimate and sufficient trigger for neurosecretion, because the obligatory depolarizationinduced increase in cytosolic Ca 2ϩ concentration stimulates release for only a few minutes (4, 5), even when depolarization is maintained. This behavior contrasts with the known but mechanistically obscure secretagogue effect of Na ϩ (6-8), wherein an artificially induced increase in cytosolic Na ϩ concentration can induce a sustained and continuous secretion (8). In previous work on neurohypophysial nerve endings, one of our laboratories reported that the Ca content of the secretory vesicles increased upon secretory stimulation, and that Ca-rich vesicles accumulated when secretion was blocked (9), suggesting a role of intravesicular ion concentration in the secretory mechanism. This hypothesis is further supported by observation that in insulin-secreting cells, Ca 2ϩ depletion from secretory granules inhibits exocytosis (10). Because the swelling of some granule matrices is inhibited by Ca 2ϩ and promoted by Na ϩ (11, 12), we have investigated in isolated and stimulated rat neurohypophysial nerve endings possible changes in the concentra...