The Vesicle Hypothesis of Quantal Transmitter Release

To understand the basis of the packaging of ACh in quanta, it is necessary to look at the structure of the synaptic terminal, which is shown schematically in Figure 8-9. The terminal contains a large number of tiny, membrane-bound structures called synaptic vesicles. These vesicles contain ACh, and it is

Figure 8-8 Spontaneous miniature end-plate potentials recorded from the end-plate region of a muscle cell. These randomly occurring small depolarizations of the muscle cell are caused by spontaneous release of single quanta of ACh from the synaptic terminal of the motor neuron. (a) Four 5-sec samples of muscle cell fm, measured via an intracellular microelectrode. The spontaneous depolarizations occur at a rate of approximately one per second. (b) Spontaneous miniature end-plate potentials viewed on an expanded time-scale to show the shape of the events more clearly.

Figure 8-8 Spontaneous miniature end-plate potentials recorded from the end-plate region of a muscle cell. These randomly occurring small depolarizations of the muscle cell are caused by spontaneous release of single quanta of ACh from the synaptic terminal of the motor neuron. (a) Four 5-sec samples of muscle cell fm, measured via an intracellular microelectrode. The spontaneous depolarizations occur at a rate of approximately one per second. (b) Spontaneous miniature end-plate potentials viewed on an expanded time-scale to show the shape of the events more clearly.

natural to assume that they represent the packets of ACh that are released in response to a presynaptic action potential. Indeed, these vesicles are depleted by any manipulation, such as prolonged depolarization or firing of large numbers of action potentials, which causes release of large amounts of ACh. It is now generally accepted that release of ACh is accomplished by the fusion of the vesicle membrane with the plasma membrane of the terminal, so that the contents of the vesicle are dumped into the extracellular space between the terminal and the muscle cell. The vesicles do not fuse with the plasma membrane just anywhere; rather, they apparently fuse only at specialized membrane regions, called release sites or active zones, that are found only on the membrane face opposite the postsynaptic muscle cell. Thus, quanta of ACh are released only into the narrow space, the synaptic cleft, separating the pre- and postsynaptic cells. With freeze-fracture electron microscopy, the active zone of

Synaptic vesicle

Mitochondrion

Synaptic terminal

Synaptic terminal

Synaptic cleft

Muscle cell y r_x

Synaptic cleft

Muscle cell

Synaptic terminal

Release site (active zone)

Plasma membrane of synaptic terminal Release site icle

particle Synaptic

ACh vesicle

Plasma membrane of synaptic terminal Release site icle particle Synaptic

ACh vesicle

End-plate membrane

ACh receptor molecule

End-plate membrane

ACh receptor molecule

Muscle fiber

Figure 8-9 A schematic diagram of synaptic vesicles fusing with the plasma membrane to release ACh at the neuromuscular junction. Release occurs at specialized active zones in the presynaptic terminal. (Animation available at www.blackwellscience.com)

the presynaptic terminal appears as a double row of large membrane particles, which are probably membrane proteins involved in the fusion between the membrane of the synaptic vesicle and the presynaptic plasma membrane. Examples of these active zone particles can be seen in Figure 8-10.

Figure 8-10 Electronmicrographs of the freeze-fractured face of a presynaptic terminal at the neuromuscular junction. (a) An unstimulated nerve terminal. Note the double row of particles defining a presynaptic release site or active zone (az). The arrow points to what appears to be a synaptic vesicle spontaneously fusing with the presynaptic membrane. Such spontaneous fusions presumably underlie the spontaneous miniature end-plate potentials shown in Figure 8-8. The arrowhead at the left points to a synaptic vesicle visible in a region where the membrane fractured all the way through to reveal a portion of the intracellular fluid. (b) A higher-power view of an active zone of a nerve terminal frozen during release of ACh stimulated by presynaptic action potentials. The ice-filled depressions arrayed along either side of the active zone correspond to regions where synaptic vesicles are in the process of fusing with the presynaptic membrane. (Reproduced from C.-P. Ko, Regeneration of the active zone at the frog neuromuscular junction. Journal of Cell Biology 1984;98:1685-1695; by copyright permission of the Rockefeller University Press.)

Figure 8-10 Electronmicrographs of the freeze-fractured face of a presynaptic terminal at the neuromuscular junction. (a) An unstimulated nerve terminal. Note the double row of particles defining a presynaptic release site or active zone (az). The arrow points to what appears to be a synaptic vesicle spontaneously fusing with the presynaptic membrane. Such spontaneous fusions presumably underlie the spontaneous miniature end-plate potentials shown in Figure 8-8. The arrowhead at the left points to a synaptic vesicle visible in a region where the membrane fractured all the way through to reveal a portion of the intracellular fluid. (b) A higher-power view of an active zone of a nerve terminal frozen during release of ACh stimulated by presynaptic action potentials. The ice-filled depressions arrayed along either side of the active zone correspond to regions where synaptic vesicles are in the process of fusing with the presynaptic membrane. (Reproduced from C.-P. Ko, Regeneration of the active zone at the frog neuromuscular junction. Journal of Cell Biology 1984;98:1685-1695; by copyright permission of the Rockefeller University Press.)

Anatomical evidence supporting vesicle exocytosis as the mechanism of ACh release was provided by freeze-fracture electron microscopy. In these experiments, a muscle and its attached nerve were placed in an apparatus that could very rapidly freeze the nerve and muscle. Then, the release process was literally frozen at the instant just after arrival of an action potential in the synaptic terminal, when ACh was being released. At this stage of transmission, synaptic vesicles can be seen in the process of fusing with the plasma membrane, as shown in Figure 8-10. The fusing vesicles appear as ice-filled pits or depressions in the presynaptic membrane, lined up along the pre-synaptic release sites. Fusing vesicles were observed only when ACh release should have been occurring, not before or after the action potential in the terminal. Further, the fusion occurred only when calcium was present in the ECF, which we have seen is prerequisite for release to occur.

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