pore structure allows neurotransmitter efflux while partition- ing vesicle membrane from the neurolemma. Such a protein- aceous pore structure might well act as ...
Proc. Natl. Acad. Sci. USA Vol. 93, pp. 5567-5571, May 1996 Neurobiology
The timing of synaptic vesicle endocytosis (FM 1-43/confocal microscopy/recycling)
TIMOTHY A. RYAN*t, STEPHEN J SMITH*, AND HARALD REUTERt *Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305; and tDepartment of Pharmacology, University of Bern, CH-3010 Bern, Switzerland
Communicated by Charles F. Stevens, The Salk Institute for Biological Studies, San Diego, CA, January 17, 1996 (received for review October 2, 1995)
longed stimulation and that this slowing results from a maintained increase in intracellular Ca2l ([Ca2+]). Details of the physiology and biochemistry of endocytosis remain scarce, because direct measurements of endocytosis have proven more difficult to obtain than those of exocytosis. Unfortunately, although measurements of membrane capacitance have been a useful probe of endocytosis in many systems (9, 10), they are not readily applicable to typical fast synapses; the distance of the release site from the cell body, the size of the small clear synaptic vesicles (equivalent capacitance of 0.07 fF per vesicle), as well as the low average number of vesicles per bouton ("200) (11) make such measurements at these nerve terminals problematic. Here we make use of the optical tracer method, pioneered in elegant imaging studies by Betz and Bewick (12, 13), to measure the residence time of synaptic vesicles in the plasma membrane after action-potential-stimulated exocytosis in synapses of cultured hippocampal neurons. In previous studies (14, 15), we obtained estimates of the time course of endocytosis after large, potentially nonphysiological stimuli. The results presented here extend these measurements to stimuli one order of magnitude smaller than those previously measured. We investigated the kinetics of membrane recovery under stimulation conditions that use as little as 5% of the available vesicle pool and compared it with the recovery of heavier exocytotic loads. Our data indicate that even after such stimuli, endocytosis proceeds with a slow half-time (20 s). We have found also that endocytosis at these synapses is independent of extracellular Ca2+ over a wide concentration range. Finally, we have measured the impact of the tracer's dissociation rate upon the tracer's ability to escape from the vesicle membrane after exocytosis and before recapture. All of these measurements seem to favor the slower, complete-fusion model of endocytosis at hippocampal synapses, even at action potential frequencies as low as 1 Hz.
Alternative models to describe the endocytoABSTRACT sis phase of synaptic vesicle recycling are associated with time scales of vesicle recovery ranging from milliseconds to tens of seconds. There have been suggestions that one of the major models, envisioned as a slow process that occurs only after complete fusion of the vesicle membrane with the neurolemma, might be applicable only under conditions of heavy, nonphysiological stimulation. Using FM 1-43 and similar fluorescent probes to label recycling synaptic vesicles in rat hippocampal neurons, we have measured the kinetics of endocytosis with a wide range of action-potential-driven exocytotic loads. Our results indicate that when either 5% or 25% of the vesicle pool is used, vesicles are recovered with a half-time on the order of20 s (24°C). This endocytosis rate was not influenced by operations designed to alter intracellular Ca2l during membrane retrieval, suggesting that residual Ca2+ after strong stimuli probably does not greatly retard endocytosis. Finally, we have shown that vesicle-destaining kinetics are not strongly influenced by the substantially differing rates at which two marker dyes tested dissociate from membranes. This observation suggests that vesicles remain open long enough for essentially complete dissociation of even the slower dye (a few seconds) or, alternatively, that both dyes readily escape vesicle membrane by lateral diffusion through any exocytotic opening. These data seem most consistent with applicability of the slow-endocytosis, complete-fusion model at low as well as high levels of exocytosis.
The recapture of synaptic vesicle membrane by endocytosis is an important step in the recycling of synaptic vesicles and is necessary, along with vesicle repriming, for the maintenance of a releasable pool during physiological neurotransmitter release. The mechanisms responsible for this recapture are largely unknown, but two distinct models have arisen out of unresolved differences in interpretation among the classic ultrastructural studies of vesicular neurotransmitter release. Heuser, Reese, and colleagues (1-3) proposed a model in which all recycling vesicles undergo complete fusion with the plasmalemma after releasing their contents, followed by a selective but rather slow (tens of seconds) endocytic retrieval of vesicle membrane at sites distinct from the active zone for release. Ceccarelli and colleagues (4), on the other hand, proposed that vesicles are physiologically retrieved intact at the active zone, after a very brief (90% of the original intensity, is interpreted as the result of release of the dye to the extracellular milieu during the exocytosis of labeled synaptic vesicles. (Bar = 10 jum.) (D) The kinetics of the loss of fluorescence vs. time measured from 20 individual fluorescent puncta like those shown in B during a train of action potentials at 20 Hz. The initial slope computed from the first 5 s after the beginning of the electrical stimulation (dashed line, slope 4.4%/s) gives an estimate of the relative impact of a train of action potentials at 20 Hz on the use of the entire, releasable pool of vesicles as a function of time. This has previously been determined by comparing the amount of dye incorporated as a function of stimulus length with the rate of destaining during electrical stimulation (14).
after the beginning of endocytosis were -2-3 times above background in the case of the 100 AP load and represent a signal from potentially as little as 2.5% of the vesicle pool. Fig. 2A shows a series of the dye release curves used to measure a set of endocytosis time points after a 20 AP train. The measurements were made from the same bouton, using repeated loadings at different At after the same stimulus train. Dye uptake time course curves compiled from many such measurements are illustrated in Fig. 2B. One curve shows dye uptake after a 20 AP train, whereas the other depicts uptake after a 100 AP train, both at 20 Hz. The two curves must reflect the time course of vesicle membrane becoming inaccessible to extracellular dye, and should thus represent accurately the time course of endocytosis. The data for each time point is normalized to the uptake occurring when dye is present during, as well as after, stimulation. For example, the first data point on the 20 AP load curve shows that only -10% of the total amount of endocytosis takes place during the time of stimulation and the following 5 s (Fig. 2B, dashed line). This data, therefore, set a very restrictive upper limit on the magnitude of any very fast component (subsecond) of endocytosis. Consequently, these data strongly suggest that most or all of the
Proc. Natl. Acad. Sci. USA 93 (1996)
Neurobiology: Ryan et al.
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