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The Timing of Synaptic Vesicle Endocytosis (FM 1-43/Confocal Microscopy/Recycling) TIMOTHY A

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The Timing of Synaptic Vesicle Endocytosis (FM 1-43/Confocal Microscopy/Recycling) TIMOTHY A 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) ABSTRACT Alternative models to describe the endocyto- longed stimulation and that this slowing results from a main- sis phase of synaptic vesicle recycling are associated with time tained increase in intracellular Ca2l ([Ca2+]). scales of vesicle recovery ranging from milliseconds to tens of Details of the physiology and biochemistry of endocytosis seconds. There have been suggestions that one of the major remain scarce, because direct measurements of endocytosis models, envisioned as a slow process that occurs only after have proven more difficult to obtain than those of exocytosis. complete fusion of the vesicle membrane with the neuro- Unfortunately, although measurements of membrane capaci- lemma, might be applicable only under conditions of heavy, tance have been a useful probe of endocytosis in many systems nonphysiological stimulation. Using FM 1-43 and similar (9, 10), they are not readily applicable to typical fast synapses; fluorescent probes to label recycling synaptic vesicles in rat the distance of the release site from the cell body, the size of hippocampal neurons, we have measured the kinetics of the small clear synaptic vesicles (equivalent capacitance of 0.07 endocytosis with a wide range of action-potential-driven exo- fF per vesicle), as well as the low average number of vesicles cytotic loads. Our results indicate that when either 5% or 25% per bouton ("200) (11) make such measurements at these of the vesicle is vesicles are recovered with a nerve terminals problematic. Here we make use of the optical pool used, tracer method, pioneered in elegant imaging studies by Betz and half-time on the order of20 s (24°C). This endocytosis rate was Bewick (12, 13), to measure the residence time ofsynapticvesicles not influenced by operations designed to alter intracellular in the plasma membrane after action-potential-stimulated exo- Ca2l during membrane retrieval, suggesting that residual cytosis in synapses of cultured hippocampal neurons. Ca2+ after strong stimuli probably does not greatly retard In previous studies (14, 15), we obtained estimates of the endocytosis. Finally, we have shown that vesicle-destaining time course of endocytosis after large, potentially nonphysi- kinetics are not strongly influenced by the substantially ological stimuli. The results presented here extend these differing rates at which two marker dyes tested dissociate from measurements to stimuli one order of magnitude smaller than membranes. This observation suggests that vesicles remain those previously measured. We investigated the kinetics of open long enough for essentially complete dissociation of even membrane recovery under stimulation conditions that use as the slower dye (a few seconds) or, alternatively, that both dyes little as 5% of the available vesicle pool and compared it with readily escape vesicle membrane by lateral diffusion through the recovery of heavier exocytotic loads. Our data indicate that any exocytotic opening. These data seem most consistent with even after such stimuli, endocytosis proceeds with a slow applicability of the slow-endocytosis, complete-fusion model half-time (20 s). We have found also that endocytosis at these at low as well as high levels of exocytosis. synapses is independent of extracellular Ca2+ over a wide concentration range. Finally, we have measured the impact of The recapture of synaptic vesicle membrane by endocytosis is the tracer's dissociation rate upon the tracer's ability to escape an important step in the recycling of synaptic vesicles and is from the vesicle membrane after exocytosis and before recap- necessary, along with vesicle repriming, for the maintenance of ture. All of these measurements seem to favor the slower, a releasable pool during physiological neurotransmitter re- complete-fusion model of endocytosis at hippocampal syn- lease. The mechanisms responsible for this recapture are apses, even at action potential frequencies as low as 1 Hz. largely unknown, but two distinct models have arisen out of unresolved differences in interpretation among the classic MATERIALS AND METHODS release. ultrastructural studies of vesicular neurotransmitter Hippocampal CA1-CA3 regions were dissected from 3- to Heuser, Reese, and colleagues (1-3) proposed a model in 5-day-old Sprague-Dawley rats, dissociated, and plated onto which all recycling vesicles undergo complete fusion with the coverslips coated with Matrigel (Collaborative Research) in- plasmalemma after releasing their contents, followed by a side microwells formed by sealing a 6-mm-diameter glass selective but rather slow (tens of seconds) endocytic retrieval cylinder (Bellco Glass) onto the coverslip using silicone sealant of vesicle membrane at sites distinct from the active zone for (Dow-Corning). Most details are as described (15). Culture release. Ceccarelli and colleagues (4), on the other hand, media consisted of minimal essential media (GIBCO), 0.6% proposed that vesicles are physiologically retrieved intact at the glucose, 0.1 g/l of bovine transferrin (Calbiochem), 0.25 g/l of active zone, after a very brief (<1 s) and reversible opening insulin (Sigma), 0.3 g/l of glutamine, 5-10% fetal calf serum event. Models of this type have come to be known colloquially (HyClone), 2% B-27 (GIBCO), and 8 ,uM cytosine ,B-D- as "kiss-and-run." It has been suggested that the slower, arabinofuranoside. Cultures were maintained at 37°C in a 95% complete-fusion model is only manifest under heavier non- air/5% CO2 humidified incubator, and culture media were physiological stimulation (5, 6). These suggestions have been replaced every 3 days. supported by recent measurements from tonic retinal synapses Cells were used 3-5 weeks after plating, and the coverslip (7, 8), which indicate that endocytosis is slowed under pro- was mounted in a low volume (75 ,ul) laminar superfusion microscope chamber and superfused at a rate of 1.5 ml/min. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" inL Abbreviation: [Ca +]i, intracellular Ca +. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5567 Downloaded by guest on September 26, 2021 5568 Neurobiology: Ryan et aL Proc. Natl. Acad. Sci. USA 93 (1996) The chamber, with agar bridges and Ag-AgCl electrodes fixed on opposite sides, was mounted on the stage ofthe microscope. Unless otherwise noted, cells were continuously superfused in a saline solution consisting of 119 mM NaCl, 2.5 mM KCI, 2 mM CaCl2, 2 mM MgCl2, 25 mM Hepes (buffered to pH 7.4), 30 mM glucose, and 10 ,M 6-cyano-7-nitroquinoxaline-2,3- dione (Research Biochemicals, Natick, MA). Action potentials were stimulated by passing 1-ms current pulses yielding fields of -10 V/cm through the chamber. Cells were labeled with 15 ,uM FM 1-43, 2 ,uM FM 1-84, or 1.5 mM FM 2-10 as indicated; stimulated electrically to fire a defined train of action potentials; and then rinsed for 5-10 min in saline before destaining measurements. Changes in [Ca2+], were measured using the fluorescent Ca2+ indicator fluo-3 1.25, AM. All measurements were performed at room temperature U, 3 1.00 (-24°C). Scanning fluorescence and differential interference 0 20H contrast images were acquired at a rate of 1 image every 2.2 s cU) 0.75 using a modified Bio-Rad MRC 500 laser scanning unit CD coupled to a Zeiss IM-35 inverted microscope and a Nikon 0 x40 1.3 NA Fluor objective and stored digitally. Fluorescence 'L 0.25 was excited using the 488-nm line of an argon laser, and _ 0.500 D emissions were detected through a 515-nm-long pass filter. 0'P0.00 Digital time-lapse sequences were analyzed as described ULa (15) with the following modifications. Estimates of vesicular IL. -0.25 release of fluorescence from a given bouton (AF; see Fig. 2A) 0 20 40 60 80 100 were obtained by calculating the magnitude of the difference time (sec) in the fluorescence intensity averaged over the first 10 time FIG. 1. (A) A network of hippocampal axons and dendrites typical points (before electrical stimulation) with that averaged over of those used in these experiments, imaged by scanning Nomarski the asymptotic phase of release (the last five time points) of a differential interference contrast microscopy optics. (B) The localiza- given run. Errors given are computed SEM. tion of many individual presynaptic boutons is revealed after the cells have been bathed in saline containing 15 mM FM 1-43, stimulated for 20 s at 20 Hz, and rinsed for 5 min. Staining patterns such as these have RESULTS previously been shown to require the presence of extracellular Ca2+ Fig. 1 illustrates the imaging procedures used in the present and to colocalize with markers of presynaptic proteins (14). (C) The same field of view as in B after 100 s of stimulation at 20 Hz. The loss study. When action potentials are fired in the presence of FM of fluorescence from individual boutons, typically >90% of the 1-43, membrane endocytosed during vesicle recycling becomes original intensity, is interpreted as the result of release of the dye to labeled and provides a means of visualizing active synapses the extracellular milieu during the exocytosis of labeled synaptic after rinsing away the excess dye (Fig. 1B). This result allows vesicles. (Bar = 10 jum.) (D) The kinetics of the loss of fluorescence for later measurement of exocytosis, because vesicles then vs.
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