THE ACTIVE ZONE. Prof. U.J. McMahan IBRO Lecture December, 2003

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1 THE ACTIVE ZONE In the lecture on the structure of synapses, I pointed out that a major point of interest in the axon terminal is the active zone. It is here that the initial events in synaptic transmission occur. When an impulse arrives at the axon terminal, the depolarization of the presynaptic membrane causes voltage gated calcium channels in the presynaptic membrane to open, permitting calcium ions to enter the terminal s cytoplasm. The influx of calcium, in turn, triggers protein mediated events that cause synaptic vesicles docked at the presynaptic membrane near the calcium channels to fuse with the membrane and release their neurotransmitter into the synaptic cleft by exocytosis. All of this occurs at the active zone marked by its conspicuous aggregate of proteins, the active zone material. In this lecture I will discuss some of the evidence that supports the concepts that the neurotransmitter is released by docked synaptic vesicles, that the release occurs by exocytosis, and that the calcium channels are near the sites on the presynaptic membrane where vesicle docking and exocytosis takes place. I will also introduce the protein interactions that bring about exocytosis and discuss the macromolecular architecture of the active zone material and its role in the docking and fusion of synaptic vesicles with the presynaptic membrane. Much of the evidence was obtained from studies on a particular synapse, the neuromuscular junction. The frog s neuromuscular Junction Nowhere is the functional significance of specific structural specializations better understood than at the neuromuscular junctions in vertebrate skeletal muscles. At a neuromuscular junction the presynaptic component is the axon of a motor neuron, the cell body of which is in the central nervous system. The postsynaptic component is a muscle fiber, a cylindrical multinucleate cell having a diameter many times that of the axon. The neurotransmitter is acetylcholine (ACh). The neuromuscular junction has long been a model synapse for studies on structure/function correlates because of its simple and orderly geometry and because of the ease with which one can make electrophysiological recordings from it. For example, each muscle fiber, unlike nearly all neurons, is innervated by only one axon (in some muscles of submammalian species by a few). The neuromuscular junction usually lies midway along the length of the muscle fiber occupying less than 0.1% of the fibers surface area, and in many living isolated nerve-muscle preparations it can be recognized with a microscope for the accurate placement of electrodes. The neuromuscular junction of the frog has been particularly useful because of all vertebrate neuromuscular junctions so far examined its layout is the most convenient for combined structural and functional studies and isolated amphibian nerve-muscle preparations survive well in simple Ringer s solution. When the preterminal branch of a motor axon reaches the muscle fiber it innervates, it forms an arborization of terminal branches that lie on the surface of the muscle fiber parallel to the fiber s long axis. The terminal branches have varicosities from which neurotransmitter is released, as do the terminal branches of axons in the central nervous system, but the varicosities are less distinct and closer together than those in other axons. Within the varicosities, there is the usual assortment of vesicles focussed on active zone material attached to the presynaptic membrane. Whereas the active zone material at synapses in the central nervous system is in the form of clustered conical patches about 50nm in diameter, those at the frog s neuromuscular junction are in the form of bands, the long axis of which is parallel to the presynaptic membrane and at right angles to the long axis of the axon terminal. Each band is about 100nm across its short axis and it extends about 75nm into the cytoplasm, but it usually extends across the width of the axon terminal, 1-2 micrometers. The bands are about 0.75 micrometers apart and each is flanked by a row of docked vesicles. Thus when one views the terminal branch of a frog s motor axon horizontal to the presynaptic membrane, the orderly arrangement of bands of active zone material looks like the rungs of a ladder with each rung having a row of docked vesicles on each side. Synaptic vesicles fuse with the presynaptic membrane. Evidence that neurotransmitter is released by exocytosis stems from the findings of Fatt and Katz in the 1950 s 1. In their studies on the frog s neuromuscular junction they provided strong evidence that the endplate potential generated in a muscle fiber after a single impulse in the axon terminal was caused by the release of multiple packets or quantal units of the neurotransmitter acetycholine from the terminal. This led them to suggest that each synaptic vesicle contains a quantum (now thought to be 5,000-10,000 molecules) of transmitter. 1

2 However, it was not until 1970 that convincing electron microscope data began to appear in support of the hypothesis: 1. Couteaux and Pécot Dechavassine 2 obtained images showing that synaptic vesicles contacting the presynaptic membrane at the frog s neuromuscular junction were arranged in a row on each flank of the active zone material. Such vesicles are now referred to as being docked at the presynaptic membrane. The also demonstrated that some of these vesicles were fused with the presynaptic membrane presenting an Ω shape. They used this evidence to support the idea that transmitter was released by exocytosis from synaptic vesicles and they suggested further that it was selectively released at active zones. Indeed they coined the term active zone. 2. To test this hypothesis Heuser and Reese 3 in 1973 sought to determine what happened to different membrane compartments of the axon terminal at the frog s neuromuscular junction after prolonged nerve stimulation (20Hz for 20min). For these experiments the nerve-muscle preparation was pinned out in Ringer s solution. If they fixed the preparation immediately after stimulation, they found that the total amount of vesicle membrane in the terminal decreased dramatically and that the decrease was accompanied by a corresponding increase in two other membrane compartments of the terminal: the plasma membrane and the membrane of cisternae/endosomes. If they fixed the preparation at increasing time intervals after stimulation, they found that the total amount of vesicle membrane was gradually restored, and the restoration was accompanied by a sequential decrease first in the amount of plasma membrane and then in the amount of cisternal membrane. These finding would be expected if the synaptic vesicles fused with the presynapic membrane during transmitter release and then a corresponding amount of membrane was gradually retrieved from the presynaptic membrane to form cisternae/endosomes which, in turn, provided the membrane to replenish the synaptic vesicle pool. They, then, stimulated the nerve with the electron dense marker horseradish peroxidase (HRP) in the bath. As expected, when the preparation was fixed at intervals after stimulation ceased, the HRP first began to appear in cisternae and later in the newly formed synaptic vesicles. If they stimulated the nerve in preparations in which the synaptic vesicles had been so loaded with HRP, the HRP was depleted from the vesicle population, which would be expected if the HRP had been released into the synaptic cleft by exocytosis. 3. While the Couteaux and Pécot Dechavassine observation on the relationships of vesicles to the presynaptic membrane at active zones and the Heuser and Reese studies on the fate of vesicle membrane indicated that the synaptic vesicles were involved in exocytosis, evidence was lacking that the timing of vesicle fusion was the same as the release of quantal units of ACh. If they did not agree, then the vesicle fusion with the presynaptic membrane at active zones in response to impulse activity, would not be a structural confirmation for the quantal hypothesis of transmitter release. In order to obtain an estimate of the timing of vesicle fusion with the presynaptic membrane after a single nerve impulse, Heuser, Reese and colleagues 5 used an approach based on rapidly freezing frog nerve-muscle preparations during nerve stimulated transmitter release and then fracturing the muscle for making and viewing replicas of vast areas of the presynaptic membrane. They 4 and others had shown that in replicas of freeze-fractured neuromuscular junctions, it was possible to recognize the portion of the presynaptic membrane underlying entire active zones and points where individual vesicles had fused with the presynaptic membrane. At the active zone, the portion of the presynaptic membrane that underlies the active zone material curves outward forming a ridge. Where the membrane is fractured exposing its interior, the ridge is easily recognized. Moreover, along each slope is a linear array of particles paralleling the long axis of the ridge. Each particle represents a macromolecule. Some of the macromolecules are calcium channels (see below). The points where docked synaptic vesicles are captured undergoing exocytosis are seen as small interruptions in the membrane and they are positioned to each side of the ridge, paralleling the linear array s of particles. Heuser & Reese 5 devised an apparatus on which a nerve in an isolated nervemuscle preparation could be stimulated and then the entire preparation could be frozen as soon as 2 msec later 5. Stimulating the nerve in Ringer s solution resulted in few fusion sites in the replicas of presynaptic membranes, probably because under physiological conditions there are too few to be easily recognized in the small samples provided by freeze fracturing. On the other hand, when they poisoned the axon terminal with 4-aminopyridine, which after a single nerve stimulus increases the number of quanta of acetylcholine released after a single nerve stimulus up to two orders of magnitude above normal depending on the concentration, they saw abundant fusion sites. They then demonstrated that there was a good correlation between the number of quanta released and the number of vesicle fusion sites for a given concentration of 4- aminopyridine. 2

3 Additional evidence that vesicle fusion with the presynaptic membrane accounts for the quantal release of transmitter comes from the use of botulinum toxins and tetanus toxins and knowledge of molecules involved in vesicle docking and fusion as discussed below. But the most direct evidence that vesicle fusion accounts for quantal release of transmitter comes from in vitro studies on chromaffin cells, neuron-like cells in which synaptic vesicles contain a catecholamine neurotransmitter. Almers and his colleagues 6 grew the chromaffin cells on glass coverslips and exposed them to a fluorescent dye that labeled the synaptic vesicles. Then by using a fluorescence microscopy method called evanescent-wave microscopy, which allows one to image only the first 300 nm of the cytoplasm next to the plasma membrane facing the glass coverslip, they could view only those vesicles approaching and docking to the presynaptic membrane. When the vesicles fused with the plasma membrane, they lost fluorescence as the dye was secreted. By using a carbon fiber microelectrode that could detect the release of a single quantum of catecholamine by the current it produces when oxidized in the medium, they observed that whenever a vesicle fused with the plasma membrane a single quantum of catecholamine was released. Location of Calcium Channels Several lines of evidence indicate that the calcium that triggers the protein mediated events leading to the quantal release of transmitter enters the axon terminal through calcium channels that are positioned close to the sites of synaptic vesicle fusion with the presynaptic membrane and the exocytosis of transmitter 7-10 : 1. As indicated previously and discussed again below, calcium triggered fusion of docked synaptic vesicles with the presynaptic membrane is mediated by proteins at or very near the site of fusion. Charlton, Augustine and colleagues used two calcium chelators, BAPTA andedta, which have different binding rates for calcium to obtain an estimate of how far the calcium channels are from the proteins. When they injected BAPTA, a fast calcium chelator, into the presynaptic axon of the squid giant synapse the BAPTA bound the calcium that entered the axon after the arrival of an impulse so rapidly that there was little release of transmitter (as measured by recording from the postsynaptic cell). On the other hand EDTA injection had little effect on the amount of transmitter that was released after the arrival of an impulse. By using the binding rate of calcium to EDTA and the free diffusion rate for calcium Charlton and co-workers determined that the calcium channels must be less than 100nm from the calcium sensing proteins at the vesicle fusion site. 2. Calcium channel immunostaining colocalizes with docked vesicles. At the frog s neuromuscular junction, the active zone material together with docked vesicles that undergo exocytosis during synaptic transmission are positioned at 1μm intervals along the terminal, just opposite and paralleling the mouths of the junctional folds. The mouths of the junctional folds stain with fluorescent markers for acetylcholine receptors such as rhodamine-α-bungarotoxin. In nerve-muscle preparations that are stained with both rhodamine-α-bungarotoxin and flourescent toxin or antibodies to calcium channels, the fluorescence of the calcium channels lines up with the fluorescence of the mouths of the junctional folds and, by inference, the active zone material and docked vesicles. The resolution of fluorescence microscopy is nm, so that these results indicate that the calcium channels and sites of exocytosis are no further apart than that. 3. When antibodies to calcium channels are used to immunoprecipitate the channels from brain fractions, the proteins synaptotagmin and syntaxin accompany the calcium channels, indicating that these proteins are linked to the calcium channels. As discussed below, synaptotagmin is a synaptic vesicle protein and syntaxin is a presynaptic membrane protein. The transmembrane portions of each is thought to be located at or near the sites of vesicle fusion with the presynaptic membrane and both are involved in vesicle fusion. The cytoplasmic domain of each protein, which is linked to the calcium channel is only a few nanometers long. 4. In replicas of freeze-fractured plasma membranes described above, there are rows of large particles (macromolecules) in the membrane parallel the region where the docked vesicles undergo exocytosis. These particles are similar in size to particles in the postsynaptic membrane accepted to be acetylcholine receptors, which are channels, consistent with the possibility that at least some of those in the presynaptic membrane are calcium channels Moreover, using immunogold to label calcium channels in isolated neural membranes from the electric organ of the marine ray and examining the membranes after freeze-fracture, the gold was associated with particles similar to the particles in the presynaptic membrane at the neuromuscular junction. The particles at the neuromuscular junction are 10-20nm from the docked vesicles. In sum, two line of evidence indicate that the calcium channels are less than nm from the sites of exocytosis, and two others indicate the distance may be as little as a few 10s of nanometers. 3

4 Protein Interactions Biochemical and molecular biological studies indicate that synaptic vesicles have many species of transmembrane proteins, and there are many other proteins that are external to but associated with the membrane of the vesicles. As you might imagine much effort has been and is being spent in determining the functional role of each. Two that are thought to be directly involved in vesicle fusion are synaptobrevin (also known as VAMP, vesicle-associated membrane protein) and synaptotagmin. Several lines of evidence indicate that for docked synaptic vesicles the cytoplasmic domain of synaptobrevin interacts with the cytoplasmic domains of two proteins, syntaxin and SNAP-25 (synaptososome-associated protein of 25 kda), in the presynaptic membrane As part of this interaction, the cytoplasmic domains of syntaxin, SNAP-25 and synaptobrevin coil around each other which is thought to bring the vesicle membrane into direct apposition, perhaps even partial fusion, with the presynaptic membrane. Syntaxin, SNAP-25 and synaptobevin are known as SNARE proteins. The synaptotagmin is somehow associated with the SNARE proteins and may be the protein that senses increased calicum levels and triggers the vesicle fusion. One set of studies that provides compelling support for the hypothesis that the SNARE proteins are involved in vesicle fusion is based on the use of botulinum toxins. Botulism is contracted from Clostridium botulinum bacteria 15. The bacterial toxins act by inhibiting exocytosis of acetylcholine at neuromuscular junctions. For humans, depending on the extent of the poisoning and the vigor of the subject, the poisoning can lead to death. The bacteria produce several toxins. The toxins are released from the bacterium as single chain polypeptides of about 150 kda. They are subsequently activated by proteolysis at a single site resulting in the generation of dichain toxins in which the toxigenic light (L) chains (50kDa) remain linked to the heavy (H) chains (100 kda) by way of sulfide bonds. In vivo the toxins selectively attack motor neurons at neuromuscular junctions. The specificity is mediated by the H chain which binds to a receptor in the presynaptic membrane. The toxin then is thought to be internalized by a clatherin coated endocytotic vesicle and the L chain escapes into the cytosol, perhaps through a channel formed by the H chain in the membrane of either the vesicle or an endosome to which the vesicle fuses. Each of the SNARE proteins is recognized by one or more toxins specific for it and is enzymatically degraded by the toxin. In vitro, all neurons are attacked by the toxins with the same consequences as for motor neurons in vivo: SNARE proteins are proteolyzed and exocytosis is blocked. It is this correlation that provides strong support for SNAREs mediating exocytosis at synapses. Results from gene deletion experiments indicate that the absence of synaptotagmin also inhibits the calcium-triggered fusion of synaptic vesicles with the presynaptic membrane 16. Architecture of the active zone material. The proximity of active zone material (AZM) to docked synaptic vesicles and calcium channels makes it reasonable to wonder whether the material is physically connected to one or both structures and what the function of such connections might be. As a step toward resolving these questions, electron microscope tomography (EMT) has been used to examine the structural composition and associations of active zone material in tissue sections from neuromuscular junctions. The electron microscope provides 2D projections of specimens, which are captured on film or CCD. Conventionally, data analysis is done on these projections, but the level of detail is severely restricted by the absence of depth information. In particular, components of the active zone material are indistinct and their relationships are uncertain even in the thinnest sections that can be routinely cut with an ultramicrotome (30-50nm). EMT, which has been developed mostly over the last decade, provides 3D reconstructions of specimens by using a series of 2D projections made at different tilt angles to obtain volumetric data. The increased spatial resolution obtained from 3D reconstructions over 2D projections is more than an order of magnitude along the z-axis. By using using segmentation and surface rendering methods for extracting information from 3D reconstructions of tissue sections, Harlow et al. 17 have shown that AZM at the frog s neuromuscular junction is a highly ordered web of structural components which, in general, are narrow and elongate. Each docked vesicle is contacted by many components, and the points of contact are broadly distributed over the hemisphere of the vesicle that faces the active zone material. Detailed examination of the first 15nm of AZM internal to the presynaptic membrane revealed three topologically distinct components called beams, ribs and pegs. These components have systematic connections: beams are connected to each other and to ribs, ribs are connected to synaptic vesicles and pegs, and pegs are connected to the macromolecules that include calcium channels. These results provide evidence that the AZM helps dock synaptic vesicles at the presynaptic membrane and anchor calcium channels within the membrane, and that the architecture of 4

5 AZM provides a particular spatial relationship and structural linkage between them. The structural linkage may include proteins that mediate the calcium-triggered exocytosis of neurotransmitter during synaptic transmission, such as the cytoplasmic portions of calcium channels and both syntaxin and synaptotagmin, which are thought to be connected to the calcium channels. References 1. Katz, B. The Release of Neural Transmitter Substances. (Thomas, Springfield 1969). 2. Couteaux, R. & Pécot-Dechavassine, M. Vésicules synaptiques et poches au niveau des zones actives de la jonction neuromusculaire. Compt. Rend. 271, (1970). 3. Heuser, J. E. & Reese, T. S. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, (1973). 4. Heuser, J. E., Reese, T. S. & Landis, D. M. D. Functonal changes in frog neuromuscular junctions studied with freeze-fracture. J. Neurocytol. 3, (1974). 5.Heuser, J. E., Reese, T. S., Dennis, M. M., Jan, Y., Jan, L. & Evans, L. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J. Cell Biol. 81, (1979). 6.Steyer, J.A., Horstmann, H. & Almers, W. Transport, docking and exocytosis of single secretory vesicles in live chromaffin cells. Nature 388: Robitalle, R., Alder, E. M. & Charlton, M. P. Strategic location of calcium channels at transmitter release sites of frog neuromuscular synapses. Neuron 5, (1990). 8. Cohen, M. W., Jones, O. T. & Angelides, K. J. Distribution of Ca+2 channels on frog motor nerve terminals revealed by fluorescent omega-conotoxin. J. Neurosci. 11, (1991). 9. Adler, E.M., Augustine, G.J., Duffy, S.N. & Charlton, M.P. Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant synapse. J. Neurosci. 11: (1991). 10. Pumplin, D. W., Reese, T. S. & Llinas, R. Are the presynaptic membrane particles calcium channels? Proc. Natl, Acad. Sci.USA 78, (1981). 11. Weber, T. et al. SNAREpins: Minimal machinery for membrane fusion. Cell 92, (1998). 12. Hazuka, C. D., Foletti, D. L. & Scheller, R., H. in Neurotransmitter Release (ed Bellen, H.J.) (Oxford, Oxford, 1999). 13. Jahn, R. & Sudhof, T.C. Membrane fusion and exocytosis. Ann. Rev. Biochem. 68, (1999). 14. Brunger, A.T. Structural insights into the molecular mechanism of Ca 2+ -dependent exocytosis. Curr. Op. Neurobiol. 10, (2000). 15. Jahn, R. & Niemann, H. Molecular mechanisms of clostridial neurotoxins. Ann. NY Acad. Sci. 773, (1994). 16. Broadie, K., Bellen, H. K., DiAntonio, J., Littleton, J. T. & Schwarz, T. L. Absence of synaptotagmin disrupts excitation-secretion coupling during synaptic transmission. Proc. Natl. Acad. Sci. USA 91, (1994). 17. Harlow, M.L., Ress, D., Stoschek, A., Marshall, R.M. and McMahan, U.J. The architecture of active zone material at the frog s neuromuscular junction. Nature 409: (2001). 5

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