Abstract
Ultrastructural observations made in the study of the frog neuromuscular junction (NMJ) almost three decades ago showed that synaptic vesicle cycling functions through a slow pathway, requiring the use of clathrin-coated vesicles and an endosomal compartment. Simultaneously, a conceptually simpler model emerged, postulating rapid retrieval of vesicle membrane through a mechanism similar to a reversal of vesicle fusion. With the advent of fluorescence imaging which allows the investigator to monitor recycling in living nerve-muscle preparations, new data appeared which reconcile at least in part the two models, indicating that both may be important at this synapse. Two different synaptic vesicle pools can be defined, a readily releasable pool (RRP), consisting of quanta that are immediately available for release, and a reserve pool (RP) that is exocytosed only after prolonged stimulation. Vesicles in the RRP recycle through a fast endocytic pathway, which does not rely on an endosomal compartment, while vesicles in the RP cycle more slowly through formation of infoldings and endosomes and their subsequent severance into vesicles. The two pools mix slowly, and their recycling may be regulated by different mechanisms.
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BAIN, J., MCLAUCHLAN, H., ELLIOT, M. & COHEN, P. (2003) The specificities of protein kinase inhibitors; an update. Biochem J., 2003, Jan 21, In press (manuscript BJ20021535).
BETZ, W. J. & BEWICK, G. S. (1992) Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science 255, 20–203.
BETZ, W. J., MAO, F. & BEWICK, G. S. (1992) Activity dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J. Neurosci. 12, 36–375.
BETZ, W. J., MAO, F. & SMITH, C. B. (1996) Imaging exocytosis and endocytosis. Curr Opin. Neurobiol. 6(3), 36–371.
BIRKS, R. & MACINTOSH, F. C. (1961) Acetylcholine metabolism of a sympathetic ganglion. Canad. J. Biochem. Physiol. 39, 78–827.
BRODIN, L., LOW, P. & SHUPLIAKOV, O. (2000) Sequential steps in clathrin-mediated synaptic vesicle endocytosis. Curr. Opin. Neurobiol. 10(3), 31–320.
CECCARELLI, B., HURLBUT, W. P. & MAURO, A. (1973) Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J. Cell. Biol. 57(2) 49– 524.
CECCARELLI, B. & HURLBUT, W. P. (1980) Vesicle hypothesis of the release of quanta of acetylcholine. Physiol. Rev. 60, 39–441.
COCHILLA, A. J., ANGLESON, J. K. & BETZ, W. J. (1999) Monitoring secretory membrane with FM1-43 fluorescence. Annu. Rev. Neurosci. 22, –10.
COUSIN, M. A. & ROBINSON, P. J. (1999) Mechanisms of synaptic vesicle recycling illuminated by fluorescent dyes. J. Neurochem. 73, 222–2239.
CREMONA, O. & DE CAMILLI, P. (1997) Synaptic vesicle endocytosis. Curr Opin. Neurobiol. 7, 32–330.
CREMONA, O. & DE CAMILLI, P. (2001) Phosphoinositides in membrane traffic at the synapse. J. Cell. Sci. 114, 104–1052.
DOMIN, J., GAIDAROV, I., SMITH, M. E., KEEN, J. H. & WATERFIELD, M. D. (2000) The class II phosphoinositide 3-kinase PI3K-C2alpha is concentrated in the trans-Golgi network and present in clathrin-coated vesicles. J. Biol. Chem. 275, 1194–11950.
ELMQUIST, D. & QUASTEL, D. M. J. (1965) Aquantitative study of end-plate potentials in isolated human muscle. J. Physiol. 178, 50–529.
FESCE, R., GROHOVAZ, F., VALTORTA, F. & MELDOLESI, J. (1994) Neurotransmitter release: Fusion or 'kiss-and-run'? Trends Cell Biol. 4(1), –4.
GAIDAROV, I., CHEN, Q., FALCK, J. R., REDDY, K. K. & KEEN, J. H. (1996) A functional phosphatidylinositol 3,4,5-trisphosphate/phosphoinositide binding domain in the clathrin adaptor AP-2 alpha subunit. Implications for the endocytic pathway. J. Biol. Chem. 271, 2092–20929.
GAIDAROV, I., SMITH, M. E., DOMIN, J. & KEEN, J. H. (2001) The class II phosphoinositide 3-kinase C2alpha is activated by clathrin and regulates clathrin mediated membrane trafficking. Mol. Cell. 7, 44–449.
GENNARO, J. F., NASTUK, W. L. & RUTHERFORD, D. T. (1978) Reversible depletion of synaptic vesicles induced by application of high external potassium to the frog neuromuscular junction. J. Physiol. 280, 23–247.
GREENGARD, P., VALTORTA, F., CZERNIK, A. J. & BENFENATI, F. (1993) Synaptic vesicle phosphoproteins and regulation of synaptic function. Science 259, 78–785.
HAO, W., TAN, Z., PRASAD, K., REDDY, K. K., CHEN, J., PRESTWICH, G. D., FALCK, J. R., SHEARS, S. B. & LAFER, E. M. (1997) Regulation of AP-3 function by inositides. Identification of phosphatidylinositol 3,4,5-trisphosphate as a potent ligand. J. Biol. Chem. 272, 639–6398.
HENKEL, A. W., LUBKE, J. & BETZ, W. J. (1996a) FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals. Proc. Natl. Acad. Sci. USA 93(5), 191–1923.
HENKEL, A. W., SIMPSON, L. L., RIDGE, R. M. & BETZ, W. J. (1996b) Synaptic vesicle movements monitored by fluorescence recovery after photobleaching in nerve terminals stained with FM1-43. J. Neurosci. 16, 396–3967.
HEUSER, J. E. & REESE, T. S. (1973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 31–344.
HILFIKER, S., SCHWEIZER, F. E., KAO, H. T., CZERNIK, A. J., GREENGARD, P. & AUGUSTINE, G. J. (1998) Two sites of action for synapsin domain E in regulating neurotransmitter release. Nat. Neurosci. 1(1), 2–35.
HILFIKER, S., PIERIBONE, V. A., CZERNIK, A. J., KAO, H. T., AUGUSTINE, G. J. & GREENGARD, P. (1999) Synapsins as regulators of neurotransmitter release. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 354(1381), 26–279.
HUMEAU, Y., DOUSSAU, F., VITIELLO, F., GREENGARD, P., BENFENATI, F. & POULAIN, B. (2001) Synapsin controls both reserve and releasable synaptic vesicle pools during neuronal activity and short-term plasticity in Aplysia. J. Neurosci. 21, 419–4206.
KLINGAUF, J., KAVALALI, E. T. & TSIEN, R. W. (1998). Kinetics and regulation of fast endocytosis at hippocampal synapses. Nature 394, 58–585.
KOENIG, J. H. & IKEDA, K. (1996) Synaptic vesicles have two distinct recycling pathways. J. Cell Biol. 135, 79–808.
KOENIG, J. H. & IKEDA, K. (1999) Contribution of active zone subpopulation of vesicles to evoked and spontaneous release. J. Neurophysiol. 81, 149–1505.
KRASZEWSKI, K., DANIELL, L., MUNDIGL, O. & DE CAMILLI, P. (1996) Mobility of synaptic vesicles in nerve endings monitored by recovery from photobleaching of synaptic vesicle-associated fluorescence. J. Neurosci. 16, 590–5913.
KUROMI, H. & KIDOKORO, Y. (1998) Two distinct pools of synaptic vesicles in single presynaptic boutons in a temperature-sensitive Drosophila mutant, shibire. Neuron 20, 91–925.
KUROMI, H. & KIDOKORO, Y. (2000) Tetanic stimulation recruits vesicles from reserve pool via a cAMP-mediated process in Drosophila synapses. Neuron 27, 13–143.
KUROMI, H. & KIDOKORO, Y. (2002) Selective replenishment of two vesicle pools depends on the source of Ca2+ at the Drosophila synapse. Neuron 35(2), 33–343.
LI, L., CHIN, L. S., SHUPLIAKOV, O., BRODIN, L., SIHRA, T. S., HVALBY, O., JENSEN, V., ZHENG, D., MCNAMARA, J. O. & GREENGARD, P. (1995) Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice. Proc. Natl. Acad. Sci. USA 26(92), 923–9239.
LICHTMAN, J. W., WILKINSON, R. S. & RICH, M. M. (1985) Multiple innervation of tonic endplates revealed by activity-dependent uptake of fluorescent probes. Nature 314, 35–359.
LU, B., CZERNIK, A. J., POPOV, S., WANG, T., POO, M. M. & GREENGARD, P. (1996) Expression of synapsin I correlates with maturation of the neuromuscular synapse. Neuroscience 74(4), 108–1097.
LU, B., GREENGARD, P. & POO, M. M. (1992) Exogenous synapsin Ipromotes functional maturation of developing neuromuscular synapses. Neuron 8(3), 52–529.
MILLER, T. M. & HEUSER, J. E. (1984) Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction. J. Cell Biol. 98(2), 68–698.
MOCHIDA, S., KOBAYASHI, H., MATSUDA, Y., YUDA, Y., MURAMOTO, K. & NONOMURA, Y. (1994) Myosin II is involved in transmitter release at synapses formed between rat sympathetic neurons in culture. Neuron 13, 113–11342.
MURTHY, V. N. & STEVENS, C. F. (1998) Synaptic vesicles retain their identity through the endocytic cycle. Nature 392, 49–501.
NEVES, G. & LAGNADO, L. (1999) The kinetics of exocytosis and endocytosis in the synaptic terminal of goldfish retinal bipolar cells. J. Physiol. 515, 18–202.
NORRIS, F. A., UNGEWICKELL, E. & MAJERUS, P. W. (1995) Inositol hexakisphosphate binds to clathrin assembly protein 3 (AP-3/AP180) and inhibits clathrin cage assembly in vitro. J. Biol. Chem. 270, 21–217.
ROTHENFLUH, S., CZERNIK, A. J. & GREENGARD, P. (1995) Distinct pools of synaptic vesicles in neurotransmitter release. Nature 375, 49–497.
RENTERIA, E. S. & TSIEN, R. W. (2000) Rapid reuse of readily releasable pool vesicles at hippocampal synapses. Neuron 28, 22–231.
RICHARDS, D. A. & BETZ, W. J. (2000) A possible link between phosphatidyl-inositol signalling and the actin cytoskeleton in synaptic vesicle cycling at the frog neuromuscular junction. Society for Neuroscience Abstracts 333.15.
RICHARDS, D. A., GUATIMOSIM, C. & BETZ, W. J. (2000) Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals. Neuron 27, 55–559.
RIZZOLI, S. O. & BETZ, W. J. (2002) Effects of 2-(4-Morpholinyl)-8-Phenyl-4H-1-Benzopyran-4-One on Synaptic Vesicle Cycling at the Frog Neuromuscular Junction. J. Neurosci. 22, 1068–10689.
RODESCH, C. K. & BROADIE, K. (2000) Genetic studies in Drosophila: Vesicle pools and cytoskeleton-based regulation of synaptic transmission. Neuroreport 11, R4–R53.
ROSENMUND, C. & STEVENS, C. F. (1996) Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16, 119–1207.
RYAN, T. A. (1999) Inhibitors of myosin light chain kinase block synaptic vesicle pool mobilization during action potential firing. J. Neurosci. 19, 131–1323.
RYAN, T. A. & SMITH, S. J. (1995) Vesicle pool mobilization during action potential firing at hippocampal synapses. Neuron 14, 98–989.
RYAN, T. A., SMITH, S. J. & REUTER, H. (1996) The timing of synaptic vesicle endocytosis. Proc. Natl. Acad. Sci. USA 93(11), 556–5571.
SCHIKORSKI, T. & STEVENS, C. F. (2001) Morphological correlates of functionally defined synaptic vesicle populations. Nat. Neurosci. 4, 39–395.
SCHNEGGENBURGER, R., SAKABA, T. & NEHER, E. (2002) Vesicle pools and short-term synaptic depression: Lessons from a large synapse. Trends Neurosci. 25, 20–212.
STEVENS, C. F. & SULLIVAN, J. M. (1998) Regulation of the readily releasable vesicle pool by protein kinase C. Neuron 21(4), 88–893.
STEVENS, C. F. & TSUJIMOTO, T. (1995) Estimates for the pool size of releasable quanta at a single central synapse and for the time required to refill the pool. Proc. Natl. Acad. Sci. USA 92, 84–849.
STEVENS, C. F. & WILLIAMS, J. H. (2000) "Kiss and run" exocytosis at hippocampal synapses. Proc. Natl. Acad. Sci. USA 97, 1282–12833.
TAKEI, K. & HAUCKE, V. (2001) Clathrin-mediated endocytosis: Membrane factors pull the trigger. Trends Cell Biol. 11(9), 38–391.
TAKEI, K., MUNDIGL, O., DANIELL, L. & DE CAMILLI, P. (1996) The synaptic vesicle cycle: A single vesicle budding step involving clathrin and dynamin. J. Cell Biol. 133, 123–1250.
TARASKA, J. W., PERRAIS, D., OHARA-IMAIZUMI, M., NAGAMATSU, S. & ALMERS, W. (2003) Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells. Proc. Natl. Acad. Sci. USA 100, 207–2075.
TENG, H., COLE, J. C., ROBERTS, R. L. & WILKINSON, R. S. (1999) Endocytic active zones: Hot spots for endocytosis in vertebrate neuromuscular terminals. J. Neurosci. 19, 485–4866.
TORRI TARELLI, F., BOSSI, M., FESCE, R., GREENGARD, P. & VALTORTA, F. (1992) Synapsin I partially dissociates from synaptic vesicles during exocytosis induced by electrical stimulation. Neuron 9(6), 114–1153.
VALTORTA, F., IEZZI, N., BENFENATI, F., LU, B., POO, M. M. & GREENGARD, P. (1995) Accelerated structural maturation induced by synapsin I at developing neuromuscular synapses of Xenopus laevis. Eur J. Neurosci 7(2), 26–270.
VALTORTA, F., MELDOLESI, J. & FESCE, R. (2001) Synaptic vesicles: Is kissing a matter of competence? Trends Cell Biol. 11, 32–328.
VLAHOS, C. J., MATTER, W. F., HUI, K. Y. & BROWN, R. F. (1994) A specific inhibitor of phosphatidylinositol Vesicle cycling at frog motor nerve terminals 549 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269, 524–5248.
VON GERSDORFF, H., VARDI, E., MATTHEWS, G. & STERLING, P. (1996) Evidence that vesicles on the synaptic ribbon of retinal bipolar neurons can be rapidly released. Neuron 16, 122–1227.
VON GERSDORFF, H. & MATTHEWS, G. (1997) Depletion and replenishment of vesicle pools at a ribbon-type synaptic terminal. J. Neurosci. 17, 191–1927.
WILKINSON, R. S. & COLE, J. C. (2001) Resolving the Heuser-Ceccarelli debate. Trends Neurosci. 24, 19–197.
WU, L. G. & BETZ, W. J. (1996) Nerve activity but not intracellular calcium determines the time course of endocytosis at the frog neuromuscular junction. Neuron 17, 76–779.
WU, L. G. & BETZ, W. J. (1998) Kinetics of synaptic depression and vesicle recycling after tetanic stimulation of frog motor nerve terminals. Biophys. J. 74(6), 300–3009.
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Rizzoli, S.O., Richards, D.A. & Betz, W.J. Monitoring synaptic vesicle recycling in frog motor nerve terminals with FM dyes. J Neurocytol 32, 539–549 (2003). https://doi.org/10.1023/B:NEUR.0000020609.19873.e8
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DOI: https://doi.org/10.1023/B:NEUR.0000020609.19873.e8