Endocytosis at Nerve Terminals: Timing Is Everything
Authors: Timothy A Ryan
Journal: Neuron (1996)

The nervous system has evolved to make use of a variety of mechanisms that allow information to flow and be processed among a large collection of individual cells. Perhaps one of the most elegant forms of this communication occurs at chemical synapses, where the exocytosis of synaptic vesicles is regulated to provide rapid (< ms) secretion of a few thousand molecules of neurotransmitter on demand. The orchestration of this cellular process is initiated by a rise in intracellular Ca2+ (25Zucker R.S Neuron. 1996; 17 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, this issue of Neuron) and is thought to be critically dependent upon the correct functioning of specific proteins on both the synaptic vesicle and plasma membranes (16Scheller R.H Neuron. 1995; 14: 893-897Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 17Südhof T.C Nature. 1995; 375: 645-653Crossref PubMed Scopus (1766) Google Scholar). Equally intriguing is the subsequent retrieval of synaptic vesicle components, which is accomplished by a remarkably efficient recycling system that operates using local endocytic machinery in the presynaptic terminal (3De Camilli P Takei K Neuron. 1996; 16: 481-486Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). At synapses where the action of the secreted neurotransmitter is terminated promptly (within milliseconds, such as for the fast acting neurotransmitters acetylcholine, glutamate, GABA, and glycine), synaptic transmission can in principle occur at high frequency. Unless the recycling of synaptic vesicles occurs with speeds similar to, or faster than, exocytosis, the maintenance of synaptic transmission during repetitive stimulation will be limited by the size of the releasable pool and the relative balance of the rates of recycling and release. Thus, obtaining good estimates of all of these parameters is crucial to understanding the efficacy of fast synapses over a full range of physiological stimuli. In this minireview, I shall focus on recent measurements of the speed of the first step in the recycling process, endocytosis. The job of endocytosis is to recover synaptic vesicle components after the synaptic vesicle has fused with the plasma membrane. The efficiency of this recovery process is considerable; integral membrane proteins of synaptic vesicles are only transiently accessible to labeling with specific antibodies following nerve stimulation (19Valtorta F Jahn R Fesce R Greengard P Ceccarelli B J. Cell Biol. 1988; 107: 2717-2727Crossref PubMed Scopus (104) Google Scholar, 9Kraszewski K Mundigl O Daniell L Verderio C Matteoli M De Camilli P J. Neurosci. 1995; 15: 4328-4342Crossref PubMed Google Scholar). This indicates that although these proteins become part of the plasma membrane, their residence in that membrane is short lived. The recovered components are then reused locally in the assembly of new vesicles. This distributed recycling system thus maintains the supply of vesicles at sites typically at great distances from the cell soma, the traditional location of organelle assembly and protein synthesis. Recently, two different techniques have brought this branch of the synaptic vesicle cycle into sharper focus: whole-cell capacitance measurements and fluorescent optical tracers. Application of these techniques has led to quantitative estimates of the time scale of the membrane recovery process as well as provided clues to possible modulation of this pathway. The capacitance change associated with the addition of a single small, clear synaptic vesicle to the plasma membrane (∼30–70 aF) is beyond the current sensitivity range of capacitance measurements. However, in two different types of presynaptic terminals, it has been possible to measure the simultaneous fusion of vesicles from a large number of release sites acting in parallel. In both hair cells of the frog sacculus (11Parsons T.D Lenzi D Almers W Roberts W.M Neuron. 1994; 13: 875-883Abstract Full Text PDF PubMed Scopus (216) Google Scholar) and giant terminals of goldfish retinal bipolar cells (20von Gersdorff H Matthews G Nature. 1994; 367: 735-739Crossref PubMed Scopus (384) Google Scholar), synaptic vesicles fuse with the plasma membrane at multiple specialized release sites (∼20 dense body synpases in hair cells, ∼60 ribbon synapses in bipolar cells) within a single terminal. The synchronous fusion increases the surface membrane area in the entire terminal and, therefore, provides a measurable change in capacitance. The development of vital probes of membrane recycling, in particular the amphipathic fluorescent probe FM1-43 (1Betz W.J Bewick G.S Science. 1992; 255: 200-203Crossref PubMed Scopus (649) Google Scholar), has permitted measurements of many steps of synaptic vesicle recycling at more conventional fast synapses, where capacitance measurements have so far proven impractical. These probes are used as tracers of membrane traffic that can be applied or washed away at specific times with respect to the exocytotic fusion of the synaptic vesicle with the plasma membrane. The approach is similar to the original tracer studies used with electron microscopy, which were central to identifying synaptic vesicle recycling (7Heuser J.E Cell Biol. Int. Reports. 1989; 13: 1063-1076Crossref PubMed Scopus (79) Google Scholar). With the new vital fluorescent probes, measurements in living tissue have successfully been applied to the study of synaptic vesicle recycling at the frog neuromuscular junction (NMJ) and synapses formed between hippocampal neurons from rat or mouse grown in culture. The earliest estimates of the kinetics of endocytosis came from ultrastructural observations of the uptake of extracellular tracers (horseradish peroxidase, HRP) at varied times after a burst of exocytosis (reviewed by7Heuser J.E Cell Biol. Int. Reports. 1989; 13: 1063-1076Crossref PubMed Scopus (79) Google Scholar). These studies indicated that endocytosis proceeded rather slowly, over the course of many tens of seconds. Measurable uptake of HRP was only obtained using rather intense, nonphysiological stimulation. This was thought to provide a possible explanation for the discrepancy between the time scale of endocytosis measured with this method and more recent observations of much faster, but stimulus intensity-dependent, membrane retrieval. Capacitance measurements in retinal bipolar cells (20von Gersdorff H Matthews G Nature. 1994; 367: 735-739Crossref PubMed Scopus (384) Google Scholar) indicate that for brief membrane depolarizations, the newly exocytosed membrane is recovered in 1–2 s. Recent studies using FM1-43 to measure the time course of endocytosis at the same synapse for which the original EM studies were performed, i.e., the frog neuromuscular junction (23Wu L.-G Betz W.J Neuron. 1996; 17: 769-779Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), indicate that the recovery process occurs with a time constant of ∼20 s for the lightest stimuli used (although see below). This is in agreement with measurements from both hippocampal synapses (14Ryan T.A Smith S.J Reuter H Proc. Natl. Acad. Sci. USA. 1996; 93: 5567-5571Crossref PubMed Scopus (204) Google Scholar) and frog sacculus hair cells (11Parsons T.D Lenzi D Almers W Roberts W.M Neuron. 1994; 13: 875-883Abstract Full Text PDF PubMed Scopus (216) Google Scholar). Thus, at all of the fast synapses studied, endocytosis follows a schedule of events three to five orders of magnitude slower than exocytosis, and under conditions where exocytosis might exhaust the available supply of vesicles membrane, recovery appears to proceed more slowly, not more rapidly, in several nerve terminal types (see below). Although there is general agreement that endocytosis proceeds much more slowly than exocytosis at fast synapses, the modulation of this pathway appears to vary considerably among different preparations. One form of modulation appears to occur in response to prolonged stimulation of the terminal in question. In frog NMJ (23Wu L.-G Betz W.J Neuron. 1996; 17: 769-779Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) as well as retinal bipolar cells (20von Gersdorff H Matthews G Nature. 1994; 367: 735-739Crossref PubMed Scopus (384) Google Scholar), prolonging the stimulus gives rise to a gradual lengthening of the time course of endocytosis. In contrast, at hippocampal synapses, the time course of recovery appears relatively insensitive to the length of the stimulus train (14Ryan T.A Smith S.J Reuter H Proc. Natl. Acad. Sci. USA. 1996; 93: 5567-5571Crossref PubMed Scopus (204) Google Scholar, 13Ryan T.A Smith S.J Neuron. 1995; 14: 983-989Abstract Full Text PDF PubMed Scopus (244) Google Scholar). At least two different variables that might be important in endocytosis are altered during such manipulations: the total amount of Ca2+ flowing in to the terminal, and the amount of membrane in need of recovery by the endocytic machinery. Several groups have investigated the possible role of Ca2+ in endocytosis by either direct or indirect manipulation of cytosolic Ca2+ levels. Although the rate of membrane recovery is enhanced by steep elevations in intracellular Ca2+ in neuroendocrine cells (18Thomas P Lee A.K Wong J.G Almers W.J J. Cell Biol. 1994; 124: 667-675Crossref PubMed Scopus (158) Google Scholar, 5Heineman C Chow R.H Neher E Zucker R.S Biophys. J. 1994; 67: 2546-2557Abstract Full Text PDF PubMed Scopus (293) Google Scholar), it does not appear to be dictated by the instantaneous intracellular Ca2+ levels in the frog NMJ (23Wu L.-G Betz W.J Neuron. 1996; 17: 769-779Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) or hippocampal synapses (14Ryan T.A Smith S.J Reuter H Proc. Natl. Acad. Sci. USA. 1996; 93: 5567-5571Crossref PubMed Scopus (204) Google Scholar, 12Reuter H Porzig H Neuron. 1995; 15: 1077-1084Abstract Full Text PDF PubMed Scopus (113) Google Scholar). However, an extreme form of modulation was uncovered in the retinal bipolar terminals: when the presynaptic terminal cytosol is dialysed by whole-cell patch clamping with a solution buffered to 1 μM Ca2+ or higher, endocytosis is completely inhibited (21von Gersdorff H Matthews G Nature. 1994; 370: 652-655Crossref PubMed Scopus (227) Google Scholar). Thus, although there does appear to be a Ca2+-sensitive element capable of blocking endocytosis at these ribbon synapses, it is either not present or not engaged by the transient rises in intracellular Ca2+ evoked during action potential stimuli at the frog NMJ or in cultured hippocampal synapses. If elevations in Ca2+ cannot account for all of the prolongation of endocytosis with stimulus length, what slows things down? One possibility is that the endocytic machinery is limited in capacity and becomes saturated with the continued addition of new membrane during prolonged stimulation. At hippocampal synapses, which have the smallest total pool of recycling vesicles per cluster (average ∼200), a very slight increase in endocytic rate is seen with increasing numbers of action potentials. This suggests that the endocytosis never becomes saturated, even with longer stimulation. At these synapses, however, the entire pool of vesicles is released with a time constant of ∼20 s (at 10 action potentials/s13Ryan T.A Smith S.J Neuron. 1995; 14: 983-989Abstract Full Text PDF PubMed Scopus (244) Google Scholar), so prolonging the stimulus beyond this point has little impact on the total vesicle load for endocytosis. At the frog NMJ, a total recycling pool of ∼1000 vesicles/cluster is depleted with a time constant of ∼60 s (at 30 action potentials/s2Betz W.J Bewick G.S J. Physiol. 1993; 460: 287-309PubMed Google Scholar), so varying the stimulus between 10 s and 300 s (as in 23Wu L.-G Betz W.J Neuron. 1996; 17: 769-779Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) increases the load for endocytosis. For the lightest stimuli, from which membrane is recovered in ∼20 s, the approximate vesicle load was ∼10 vesicles per active zone at the hippocampal synapses and 80 vesicles per active zone at the frog NMJ (23Wu L.-G Betz W.J Neuron. 1996; 17: 769-779Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In retinal bipolar terminals a stimulus resulting in fusion of ∼125 vesicles per ribbon (22von Gersdorff H Vardi E Matthews G Sterling P Neuron. 1996; 16: 1221-1227Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar) triggers a retrieval process lasting only 1–2 s. Thus, even for comparable or smaller vesicle loads, both the frog NMJ and hippocampal synapses appear to recover membrane ∼10-fold more slowly than in retinal bipolar cell terminals. It is not clear whether this reflects true differences in the physiology of these synapses or differences in experimental methodology. Clearly, direct comparisons of these two methods at the same synapse will help settle this issue. Finally, there are hints that a profound increase in endocytic rate might occur under certain conditions. At the frog NMJ, bath application of the kinase inhibitor staurosporine drastically reduces the ability of FM1-43-stained vesicles to release dye during stimulation, while release of neurotransmitter and the ability to acquire dye appears intact (6Henkel A.W Betz W.J J. Neurosci. 1995; 15: 8246-8258PubMed Google Scholar). Although this drug appears to act differently in hippocampal synapses (10Kraszewski K Daniell L Mundigl O De Camilli P J. Neurosci. 1996; 16: 5905-5913PubMed Google Scholar), the data at the frog NMJ implies that under these conditions the total exposure time of the vesicle lumen following fusion might only be on the order of 1 s. This interpretation also requires that a barrier to lateral diffusion at the fusion pore is maintained between the time of fusion and fission of the vesicle. This barrier would prevent mixing of vesicle membrane and plasma membrane components (including FM1-43). Any differences between the fate of vesicles retrieved in this manner compared to the slower internalization route, remain unknown. The possible physiological trigger of such a switch to a rapid, intact vesicle retrieval, also remains to be discovered. The rather slow endocytic rates observed for prolonged stimuli in four different types of fast synapses suggests that during high frequency release of neurotransmitter, the availability of synaptic vesicles may well become limiting in maintaining synaptic transmission. This appears to be particularly true at cultured hippocampal synapses. Here, you find the compromising combination of a small total recycling pool, a fast pool depletion rate, and a relatively slow rate of endocytosis (13Ryan T.A Smith S.J Neuron. 1995; 14: 983-989Abstract Full Text PDF PubMed Scopus (244) Google Scholar). In addition, the complete and functional recycling of vesicles will also require at least one additional step following endocytosis during which vesicles must be filled with neurotransmitter. It thus appears likely that the total pool of releasable vesicles will be significantly reduced under certain stimuli, and hence understanding the relationship between the remaining pool size and the probability of release will be important. Two studies suggest that the total releasable pool size and the probability of release are closely related. In the shibire mutant of Drosophila, a temperature sensitive block of endocytosis allowed the acute systematic reduction in the available pool during nerve stimulation. Commensurate with this decrease in pool was a decrease in the amount of transmitter release (8Koenig J.H Kosaka T Ikeda K J. Neurosci. 1989; 9: 1937-1942Crossref PubMed Google Scholar). Similarly, in mouse hippocampal synapses, a chronic reduction in the total pool size, resulting from the deletion of the synapsin I gene, resulted in a similar decrease in release probability (15Ryan T.A Li L Chin L.-S Greengard P Smith S.J J. Cell Biol. 1996; 134: 1219-1227Crossref PubMed Scopus (145) Google Scholar). The underlying physiological processes for this intimate relationship between pool size and release probability remains to be explored. Nonetheless, these data suggest that as the pool of vesicles diminishes during high frequency stimulation, the release probability decreases in proportion, thus compromising the efficacy of synaptic transmission. This dynamic underscores the importance of the recycling pathway in replenishing the vesicle pool. It is clear that endocytosis is a critically important physiological process for the local replenishment of synaptic vesicles. One of the next frontiers in developing a better understanding of this process will be in fully determining the molecular substrates of membrane recovery. At present, the only molecule identified whose correct functioning is absolutely necessary is the GTPase dynamin (3De Camilli P Takei K Neuron. 1996; 16: 481-486Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Defects in vesicle recycling resulting from mutations in the gene encoding synaptotagmin in C. elegans (4Jorgensen E.M Hartwieg E Schuske K Nonet M.L Jin Y Horvitz H.R Nature. 1995; 378: 196-199Crossref PubMed Scopus (265) Google Scholar), as well as the ability of synaptotagmin to bind the clathrin adaptor AP-2 (24Zhang J.Z Davletov B.A Südhof T.C Anderson R.G Cell. 1994; 78: 751-760Abstract Full Text PDF PubMed Scopus (433) Google Scholar), suggest a role for this protein in the recovery process as well. No doubt the identification of additional components of this machinery will emerge, and the combination of molecular approaches and biophysical techniques will help illuminate their cellular roles. In the near future the emphasis will shift away from the question of timing to determining what transpires in the time between exocytosis and the reinternalization of vesicle membrane. At this point, two central questions move to the forefront. First, what signal is delivered during exocytosis to trigger endocytosis? Second, how are vesicle components sorted out from others in the plasma membrane during recapture?