What anatomical features of the cell might account for the quantum of transmitter? The physiological observations indicating that transmitter is released in fixed quanta coincided with the discovery, through electron microscopy, of accumulations of small clear vesicles in the presynaptic terminal. Del Castillo and Katz speculated that the vesicles are organelles for the storage of transmitter, each vesicle stores one quantum of transmitter (amounting to several thousand molecules), and each vesicle releases its entire contents into the synaptic cleft in an all-or-none manner at sites specialized for release.
The sites of release, the active zones, contain a cloud of synaptic vesicles that cluster above a fuzzy electron-dense material attached to the internal face of the presynaptic membrane (see Figure 12–4A). At all rapid synapses the vesicles are typically clear, small, and ovoid, with a diameter of approximately 40 nm. Although most synaptic vesicles do not contact the active zone, some are physically bound. These are called the docked vesicles and are thought to be the ones immediately available for release. At the neuromuscular junction the active zones are linear structures (see Figure 12–4), whereas in central synapses they are disc-shaped structures approximately 0.1 μm2 in area with dense projections pointing into the cytoplasm. Active zones are always precisely apposed to the postsynaptic membrane patches that contain the neurotransmitter receptors. Thus presynaptic and postsynaptic specializations are functionally and morphologically attuned to each other. As we shall learn later, several key active zone proteins involved in transmitter release have now been identified and characterized.
Quantal transmission has been demonstrated at all chemical synapses so far examined with only one exception: at the synapse between photoreceptors and bipolar neurons in the retina (Chapter 26). Nevertheless, the efficacy of transmitter release from a single presynaptic cell onto a single postsynaptic cell varies widely in the nervous system and depends on several factors: (1) the number of synapses between a pair of presynaptic and postsynaptic cells (ie, the number of presynaptic boutons that contact the postsynaptic cell); (2) the number of active zones in an individual synaptic terminal; and (3) the probability that a presynaptic action potential will trigger release of one or more quanta of transmitter at an active zone (Box 12–1).
In the central nervous system most presynaptic terminals have only a single active zone where an action potential usually releases at most a single quantum of transmitter in an all-or-none manner. However at some central synapses, such as the calyx of Held, the presynaptic terminal may contain many active zones and thus can release a large number of quanta in response to a single presynaptic action potential. Central neurons also differ in the number of synapses that a typical presynaptic cell makes with a typical postsynaptic cell. Whereas most central neurons form only a few synapses with any one postsynaptic cell, a single climbing fiber forms up to 10,000 terminals on a single Purkinje neuron in the cerebellum! Finally, the mean probability of transmitter release from a single active zone also varies widely among different presynaptic terminals, from less than 0.1 (that is, a 10% chance that a presynaptic action potential will trigger release of a vesicle) to greater than 0.9. This wide range of probabilities can even be seen among the individual boutons at different synapses between one specific type of presynaptic cell and one specific type of postsynaptic cell.
Thus central neurons vary widely in the efficacy and reliability of synaptic transmission. Synaptic reliability is defined as the probability that an action potential in a presynaptic cell leads to some measurable response in the postsynaptic cell—that is, the probability that a presynaptic action potential releases one or more quanta of transmitter. Efficacy refers to the mean amplitude of the synaptic response, which depends on both the reliability of synaptic transmission and on the mean size of the response when synaptic transmission does occur.
Most central neurons communicate at synapses that have a low probability of transmitter release. The high failure rate of release at most central synapses (ie, their low release probability) is not a design defect but serves a purpose. As we discuss below, this feature allows transmitter release to be regulated over a wide dynamic range, which is important for learning and memory. In synaptic connections where a low probability of release is deleterious for function, this limitation is overcome by simply having many active zones in one synapse, as is the case at the calyx of Held and the nerve-muscle synapse. Both contain hundreds of independent active zones, so an action potential reliably releases 150 to 250 quanta, ensuring that a presynaptic signal is always followed by a postsynaptic action potential. Reliable transmission at the neuromuscular junction is essential for survival. An animal would not survive if its ability to move away from a predator was hampered by a low-probability response.
Not all chemical signaling between neurons depends on synapses. Some substances, such as certain lipid metabolites and the gas nitric oxide (see Chapter 11), can diffuse across the lipid bilayer of the membrane. Others can be moved out of nerve endings by carrier proteins if their intracellular concentration is sufficiently high. Plasma membrane transporters for glutamate or γ-aminobutyric acid (GABA) normally take up transmitter into a cell from the synaptic cleft following a presynaptic action potential (see Chapter 13). However, in certain glial cells of the retina, the direction of glutamate transport can be reversed under certain conditions, causing glutamate to leave the cell through the transporter into the synaptic cleft. Still other substances simply leak out of nerve terminals at a low rate. Surprisingly, approximately 90% of the ACh that leaves the presynaptic terminals at the neuromuscular junction does so through continuous leakage. This leakage is ineffective, because it is diffuse and not targeted to receptors at the end-plate region, and because it is continuous and low level rather than synchronous and concentrated.
Synaptic Vesicles Discharge Transmitter by Exocytosis and Are Recycled by Endocytosis
The quantal hypothesis of del Castillo and Katz has been amply confirmed by direct experimental evidence that synaptic vesicles do indeed package neurotransmitter and that they release their contents by directly fusing with the presynaptic membrane, a process termed exocytosis.
Forty years ago Victor Whittaker discovered that the synaptic vesicles in the motor nerve terminals of the electric organ of the fish Torpedo contain a high concentration of ACh. Later, Thomas Reese and John Heuser and their colleagues obtained electron micrographs that caught vesicles in the act of exocytosis. To observe the brief exocytotic event, they rapidly froze the nerve-muscle synapse by immersing it in liquid helium at precisely defined intervals after the presynaptic nerve was stimulated. In addition, they increased the number of quanta of transmitter discharged with each nerve impulse by applying the drug 4-aminopyridine, a compound that blocks certain voltage-gated K+ channels, which increases the duration of the action potential, thereby enhancing Ca2+ influx.
These techniques provided clear images of synaptic vesicles at the active zone during exocytosis. Using a technique called freeze-fracture electron microscopy, Reese and Heuser noted deformations of the presynaptic membrane along the active zone immediately after synaptic activity, which they interpreted as invaginations of the cell membrane caused by fusion of synaptic vesicles. These deformations lay along one or two rows of unusually large intramembranous particles, visible along both margins of the presynaptic density and now thought to be the voltage-gated Ca2+ channels (Figure 12–7). The particle density (approximately 1,500 per μm2) is approximately that of the Ca2+ channels essential for transmitter release. Moreover, the proximity of the particles to the release site is consistent with the short time interval between the onset of the Ca2+ current and the release of transmitter.
Synaptic vesicles release transmitter by exocytosis and are retrieved by endocytosis.
The images on the left are freeze-fracture electron micrographs at a neuromuscular junction. The views shown are of the cytoplasmic leaflet of the presynaptic membrane looking up from the synaptic cleft. The images on the right are thin-section electron micrographs showing cross-section views of the presynaptic terminal, synaptic cleft, and postsynaptic muscle membrane. (Reproduced, with permission, from Heuser and Reese 1981.)
A. Parallel rows of intramembranous particles arrayed on either side of an active zone are thought to be the voltage-gated Ca2+ channels essential for transmitter release. The thin-section image at right shows the synaptic vesicles adjacent to the active zone.
B. Synaptic vesicles release transmitter by fusing with the plasma membrane (exocytosis). Here synaptic vesicles are caught in the act of fusing with the plasma membrane by rapid freezing of the tissue within 5 ms after a depolarizing stimulus. Each depression in the plasma membrane represents the fusion of one synaptic vesicle. In the micrograph at right vesicle fusion is observed as Ω-shaped structures.
C. After exocytosis, synaptic vesicle membrane is retrieved by endocytosis. Within approximately 10 seconds after fusion of the vesicles with the presynaptic membrane, coated pits form. After another 10 seconds the coated pits begin to pinch off by endocytosis to form coated vesicles. These vesicles include the membrane proteins of the original synaptic vesicle and also molecules captured from the external medium. The vesicles are recycled at the terminals or are transported to the cell body, where the membrane constituents are degraded or recycled (see Chapter 4).
Finally, Heuser and Reese found that these deformations are transient; they occur only when vesicles are discharged and do not persist after transmitter has been released. Thin-section electron micrographs revealed a number of omega-shaped (Ω) structures that have the appearance of synaptic vesicles that have just fused with the membrane, prior to the complete collapse of the vesicle membrane into the plasma membrane (Figure 12–7B). Heuser and Reese confirmed this idea by showing that the number of Ω-shaped structures is directly correlated with the size of the EPSP when they varied the concentration of 4-aminopyridine to alter the amount of transmitter release. These morphological studies provide striking evidence that transmitter is released from synaptic vesicles by means of exocytosis.
Following exocytosis, the excess membrane added to the presynaptic terminal is retrieved. When Heuser and Reese obtained images of presynaptic terminals 10 to 20 seconds after stimulation, they observed new structures at the plasma membrane, the coated pits, which represent membrane retrieval through the process of endocytosis (Figure 12–7C). Several seconds later the coated pits are seen to pinch off from the membrane and appear as coated vesicles in the cytoplasm. As we will see below, endocytosis through coated pit formation represents one of several means of vesicle membrane retrieval.
Capacitance Measurements Provide Insight into the Kinetics of Exocytosis and Endocytosis
In certain neurons with large presynaptic terminals the increase in surface area of the plasma membrane during exocytosis can be detected in electrical measurements as increases in membrane capacitance. As we saw in Chapter 6, the capacitance of the membrane is proportional to its surface area. Neher discovered that one could use measurements of capacitance to monitor exocytosis in secretory cells.
In adrenal chromaffin cells (which release epinephrine and norepinephrine) and in mast cells of the rat peritoneum (which release histamine and serotonin) individual dense-core vesicles are large enough to permit measurement of the increase in capacitance associated with fusion of a single vesicle. Release of transmitter in these cells is accompanied by stepwise increases in capacitance, followed somewhat later by stepwise decreases, which reflect the retrieval and recycling of the excess membrane (Figure 12–8).
Changes in capacitance reveal the time course of exocytosis and endocytosis.
A. Electron micrographs show a mast cell before (left) and after (right) inducing exocytosis. Mast cells are secretory cells of the immune system that contain large dense-core vesicles filled with the transmitter histamine. Exocytosis of the secretory vesicles is normally triggered by the binding of antigen complexed to an immunoglobulin (IgE). Under experimental conditions massive exocytosis can be triggered by the inclusion of a nonhydrolyzable analog of guanosine triphosphate (GTP) in an intracellular recording electrode. (Reproduced, with permission, from Lawson et al. 1977.)
B. Stepwise increases in capacitance reflect the successive fusion of individual secretory vesicles with the mast cell membrane. The step increases are unequal because of variability in the membrane area of the vesicles. After exocytosis the membrane added through fusion is retrieved through endocytosis. Endocytosis of individual vesicles gives rise to the stepwise decreases in membrane capacitance. In this way the cell maintains a constant size. (The units are in femtofarads, fF, where 1 fF 0.1 μm2 of membrane area.) (Adapted, with permission, from Fernandez, Neher, and Gomperts 1984.)
C. The giant presynaptic terminals of bipolar neurons in the retina are more than 5 μm in diameter, permitting direct patch-clamp recordings of membrane capacitance and Ca2+ current. A brief depolarizing voltage-clamp step in membrane potential (Vm) elicits a large sustained Ca2+ current (ICa) and a rise in the cytoplasmic Ca2+ concentration, [Ca]i. This results in the fusion of several thousand small synaptic vesicles with the cell membrane, leading to an increase in total membrane capacitance. The increments in capacitance caused by fusion of individual vesicles are too small to resolve. As the internal Ca2+ concentration falls back to its resting level upon repolarization, the extra membrane area is retrieved and capacitance returns to its baseline value. The increases in capacitance and Ca2+ concentration outlast the brief depolarization and Ca2+ current (note different time scales) because of the relative slowness of endocytosis and Ca2+ metabolism. (Micrograph reproduced, with permission, from Zenisek et al. 2004; traces adapted, with permission, from von Gersdorff and Matthews 1994.)
In neurons the changes in capacitance caused by fusion of single, small synaptic vesicles are usually too low to resolve. In certain favorable synaptic preparations that release large numbers of vesicles (such as the giant presynaptic terminals of bipolar neurons in the retina), membrane depolarization triggers a transient smooth rise in the total capacitance of the terminal as a result of the exocytosis and retrieval of the membrane from hundreds of individual synaptic vesicles (Figure 12–8C). These results provide direct measurements of the rates of membrane fusion and retrieval.
Exocytosis Involves the Formation of a Temporary Fusion Pore
Morphological studies of mast cells using rapid freezing suggest that exocytosis depends on the formation of a temporary fusion pore that spans the membranes of the vesicle and plasma membranes. In electrophysiological studies of capacitance increases in mast cells, a channel-like fusion pore was detected in the electrophysiological recordings prior to complete fusion of vesicles and cell membranes. This fusion pore starts out with a single-channel conductance of approximately 200 pS, similar to that of gap-junction channels, which also bridge two membranes. During exocytosis the pore rapidly dilates, probably from around 5 to 50 nm in diameter, and the conductance increases dramatically (Figure 12–9A).
Some transmitter is released through temporary fusion pores.
A. A patch clamp is used to record membrane current associated with the opening of a fusion pore. As a vesicle fuses with the plasma membrane, the capacitance of the vesicle (Cg) is initially connected to the capacitance of the rest of the cell membrane (Cm) through the high resistance of the fusion pore (rp) . Because the membrane potential of the vesicle (lumenal side negative) is normally much more negative than the membrane potential of the cell, charge flows from the vesicle to the cell membrane during fusion. This transient current (I,) is associated with the increase in membrane capacitance (Cm) . The magnitude of the conductance of the fusion pore (gp) can be calculated from the time constant of the transient current according to τ = Cg rp = Cg,/gp. The pore diameter can be calculated from the pore conductance, assuming that the pore spans two lipid bilayers and is filled with a solution whose resistivity is equal to that of the cytoplasm. In the plot on the right the pore shows an initial conductance of approximately 200 pS, similar to the conductance of a gap-junction channel, corresponding to a pore diameter of approximately 2 nm. The poor diameter and conductance rapidly increase as the pore dilates to approximately 7 to 8 nm in 10 ms (filled circles). (Adapted, with permission, from Monck and Fernandez 1992; Spruce et al. 1990.)
B. Transmitter release is measured by amperometry. A cell is voltage-clamped with an intracellular patch electrode while an extracellular carbon fiber is pressed against the cell surface. A large voltage applied to the tip of the carbon electrode oxidizes certain amine transmitters (such as serotonin or norepinephrine). This oxidation of one molecule generates one or more free electrons, which results in an electrical current that is proportional to the amount of transmitter release. The current can be recorded through an amplifier (A2) connected to the carbon electrode. Membrane current and capacitance are recorded through the patch electrode amplifier (A1) . Recordings of transmitter release and capacitance measurements from mast cell secretory vesicles are shown at the right. These records indicate that serotonin may be released through the reversible opening and closing of the fusion pore prior to full fusion or by reversible fusion pore opening and closing through the fusion pore alone, that is without full fusion. During the brief fusion pore openings small amounts of transmitter escape through the pore, resulting in a low-level signal (a foot) that precedes a large spike of transmitter release upon full fusion (see inset for illustration). During the foot the cell membrane capacitance (proportional to cell surface area) undergoes reversible step-like changes as the fusion pore opens and closes. Sometimes the reversible opening and closing is not followed by full fusion, such that transmitter is released through the fusion pore alone. (Adapted, with permission, from Neher 1993.)
The fusion pore is not just an intermediate structure leading to exocytosis of transmitter, as transmitter can be released through the pore prior to full fusion. This was first shown by amperometry, a method that uses an extracellular carbon-fiber electrode to detect certain amine neurotransmitters, such as serotonin, based on an electrochemical reaction between the transmitter and the electrode that generates an electrical current proportional to the local transmitter concentration. Firing of an action potential in serotonergic cells leads to a large transient increase in electrode current, corresponding to the exocytosis of the contents of a single dense-core vesicle. In some instances these large transient increases are preceded by smaller, longer-lasting current signals that reflect leakage of transmitter through a fusion pore that flickers open and closed several times prior to complete fusion (Figure 12–9B).
Transmitter can also be released solely through transient fusion pores, that is, without full collapse of the vesicle membrane into the plasma membrane. Capacitance measurements for exocytosis of both large dense-core vesicles and small clear vesicles in neuroendocrine cells show that the fusion pore can open and close rapidly and reversibly. The reversible opening and closing of a fusion pore represents a very rapid method of membrane retrieval. The circumstances under which the small clear vesicles at fast synapses discharge transmitter through a fusion pore, as opposed to full membrane collapse, are uncertain.
The Synaptic Vesicle Cycle Involves Several Steps
When firing at high frequency, a typical neuron is able to maintain a high rate of transmitter release. This can result in the exocytosis of a large number of vesicles, more than the number originally present within the presynaptic terminal. To prevent the supply of vesicles from being rapidly depleted, used vesicles are rapidly retrieved and recycled. Because nerve terminals are usually some distance from the cell body, replenishing vesicles by synthesis in the cell body and transport to the terminals would be too slow to be practical.
Synaptic vesicles are released and reused in a simple cycle (Figure 12–10A). Vesicles fill with neurotransmitter and cluster in the nerve terminal. They then dock at the active zone where they undergo a complex priming process that makes vesicles competent to respond to the Ca2+ signal that triggers the fusion process.
The synaptic vesicle cycle.
A. Synaptic vesicles are filled with neurotransmitters by active transport (step 1) and join the vesicle cluster that may represent a reserve pool (step 2). Filled vesicles dock at the active zone (step 3) where they undergo an ATP-dependent priming reaction (step 4) that makes them competent for calcium-triggered fusion (step 5). After discharging their contents, synaptic vesicles are recycled through one of several routes (see part B). In one common route, vesicle membrane is retrieved via clathrin-mediated endocytosis (step 6) and recycled directly (step 7) or by endosomes (step 8).
B. Retrieval of vesicles after transmitter discharge is thought to occur via three mechanisms, each with distinct kinetics. 1. A reversible fusion pore is the most rapid mechanism for reusing vesicles. The vesicle membrane does not completely fuse with the plasma membrane and transmitter is released through the fusion pore. Vesicle retrieval requires only the closure of the fusion pore and thus can occur rapidly, in tens to hundreds of milliseconds. This pathway may predominate at lower to normal release rates. The spent vesicle may either remain at the membrane (kiss-and-stay) or relocate from the membrane to the reserve pool of vesicles (kiss-and-run). 2. In the classical pathway excess membrane is retrieved through endocytosis by means of clathrin-coated pits. These pits are found throughout the axon terminal except at the active zones. This pathway may be important at normal to high rates of release. 3. In the bulk retrieval pathway, excess membrane reenters the terminal by budding from uncoated pits. These uncoated cisternae are formed primarily at the active zones. This pathway may be used only after high rates of release and not during the usual functioning of the synapse. (Adapted, with permission, from Schweizer, Betz, and Augustine 1995; Südhof 2004.)
Three mechanisms exist for retrieving the synaptic vesicle membrane following exocytosis and each has a distinct time course. The first, most rapid mechanism involves the reversible opening and closing of the fusion pore, without the full collapse of the vesicle membrane into the plasma membrane (Figure 12–10B1). In the kiss-and-stay pathway the vesicle remains at the active zone after the fusion pore closes, ready for a second release event. In the kiss-and-run pathway the vesicle leaves the active zone after the fusion pore closes, but is competent for rapid rerelease. Vesicles are thought to be preferentially recycled through these pathways during stimulation at low frequencies.
Stimulation at higher frequencies recruits a second, slower recycling pathway that uses clathrin to retrieve the vesicle membrane after fusion with the plasma membrane (see Figure 12–10B2). (The clathrin-coated vesicle membranes are the coated pits observed by Heuser and Reese.) In this pathway the retrieved vesicular membrane must be recycled through an endosomal compartment before the vesicles can be reused. Clathrin-mediated recycling requires up to a minute for completion, and appears to shift from the active zone to the membrane surrounding the active zone (see Figure 12–7).
A third mechanism operates after prolonged high-frequency stimulation. Under these conditions large membranous invaginations into the presynaptic terminal are visible, which are thought to reflect membrane recycling through a process called bulk retrieval (Figure 12–10B3).