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Cover   The discovery that launched the armada of papers on SNAREs and their role in docking and fusion was the presence of VAMP, the first v-SNARE, in synaptic vesicles. It seems logical in retrospect that key elements of membrane fusion would be found in nerve terminals, given their prodigious capacity for rapid and extensive exocytosis. Much of the credit for convincing most, but not all, scientists that nerve terminals are indeed hot beds of exocytosis came from the superb team of Heuser and Reese. In the early 1970s, they used conventional electron microscopy and uptake of extracellular markers to provide compelling evidence that synaptic vesicle membranes recycled into endosomes, from which they returned to the synaptic vesicle pool (Heuser and Reese, 1973). Their superb micrographs and detailed quantitation of membrane flow gave firm anatomical support for the idea of neurotransmitter release by exocytosis. Their work was challenged, however, by two criticisms: they had to stimulate for long periods of time and their chemical fixatives took a long time to act. Spurred by these challenges, Heuser and Reese developed an incredible technology for quick freezing synapses within a few milliseconds of stimulating exocytosis. The upper micrograph on the cover shows a freeze-fracture through frog neuromuscular junctions that had been stimulated 3 ms before freezing. We can see the characteristic double row of particles, which are now believed to be calcium channels, but no signs of exocytotic activity. Two milliseconds later (lower micrograph) the presynaptic plasma membrane was extensively dimpled with openings due to exocytoxic events (square) and regions (asterisks) that were interpreted to be vesicles that have collapsed completely flat after insertion. The number of concepts that are generated by this one picture is large. The first is that the active zone, the region of the nerve terminal at which fusion occurs, is exceedingly narrow. Almost all of the fusion events were within 40 nm of the calcium channel, less than the width of a synaptic vesicle (50 nm). We now know that synaptic vesicle exocytosis is triggered at cytoplasmic calcium levels that are only reached very close to calcium channels. They also showed that exocytosis was random over the active zones, which meant that there were no hot spots of exocytosis. The openings they observed were very large, only slightly less than the diameter of the synaptic vesicle. This is difficult to reconcile with the kiss-and-run model, which is still scientifically fashionable. In the kiss-and-run model only a small pore the size of an ion channel connects the inside of the vesicle to the outside world, quite different from the large holes detected in these micrographs. Finally, the paper linked the morphological observations of exocytosis to the physiological phenomenon of quantal release. From the pioneering studies of Sir Bernard Katz, we had known that neurotransmitter was released from nerve terminals in "packets" or "quanta" of neurotransmitter. In this paper, a group of superb electrophysiologists worked with the Heuser, Reese, and Evans team to show that the number of quanta released equaled the number of exocytotic figures within an accuracy of about 10%. For most scientists, this paper was the death knell for models of transmitter release that did not involve vesicle exocytosis.
  Contemplation of these micrographs still brings its rewards. We are reminded that we do not know what localizes the calcium channels in such distinctive rows, nor the proteins that dock synaptic vesicles so precisely at the active site, nor the molecular bridges that hold the active zones in register with postsynaptic receptors. It also reminds biochemists and molecular biologists that a wonderful way to understand a problem is just to look.Regis B. Kelly

Cover figure reprinted with the permission of the Rockefeller University Press.