CHAPTER 3: Synaptic Transmission

• Synaptic transmission is a signal transduction process that begins with the action potential–dependent release of a neurotransmitter from a presynaptic terminal. The neurotransmitter then binds to and activates postsynaptic receptors that modify the electrical and biochemical properties of the postsynaptic cell.

• The major classes of neurotransmitters are amino acid transmitters, such as glutamate and GABA; monoamines, including dopamine, norepinephrine, and serotonin; acetylcholine; peptides; diffusible gases, such as nitric oxide; lipid-derived molecules, such as endocannabinoids; and nucleosides and derivatives, such as adenosine and ATP.

• Neurotransmitters are stored in small organelles called synaptic vesicles that fuse with the presynaptic terminal membrane and release their contents when an action potential invades the terminal and causes a rise in calcium due to activation of voltage-dependent calcium channels.

• A single neurotransmitter typically activates several different subtypes of receptors.

• Neurotransmitter receptors are classified as ligand-gated ion channels or G protein–coupled receptors.

• After being released, most neurotransmitters are transported back into the presynaptic terminal or into glia by specialized proteins called plasma membrane transporters. A different family of transporters is responsible for pumping neurotransmitter into synaptic vesicles.

• Neurotransmitter transporters are important targets of many antidepressant medications and psychostimulant drugs such as cocaine and amphetamines.

• The proteins that are responsible for the fusion of synaptic vesicles with the presynaptic plasma membrane, a process known as exocytosis, have been identified and extensively characterized. Some of these proteins are the targets of bacterial toxins (eg, tetanus and botulinum toxin) and black widow spider venom (α-latrotoxin).

• After exocytosis, synaptic vesicles are recycled and used again by repackaging them with neurotransmitter.

When we think, feel, or move, information passes rapidly between neurons across specialized gaps called synapses. When we learn and remember, synapses undergo significant activity-dependent alterations. Given the centrality of synaptic transmission to the function of the nervous system, it is not surprising that the large majority of drugs used to treat neuropsychiatric illnesses act on different protein components of specific synapses. This chapter explores the biochemical basis of synaptic transmission and explains how this process is regulated.

Neurons are morphologically specialized to receive, process, and send information 3–1. As discussed in Chapter 2, the key structure across which information is transferred, by use of chemical neurotransmitters, is the synapse. Synaptic transmission is a result of three types of processes that convert electrical information into a chemical signal and then back again: (1) electrical information in the axon of a presynaptic neuron is converted to a chemical signal in its nerve terminal, (2) this chemical signal is transmitted to another cell across a synapse, and (3) the chemical message received by the postsynaptic cell is converted into an electrical signal plus a range of chemical signals. It should be noted that a small percentage of synapses are gap junctions, morphologic connections between two neurons that permit the direct flow of electrical current from cell to cell (Chapter 2). These electrical junctions are morphologically distinct from chemical synapses. Because virtually all pharmacologic agents act at chemical synapses rather than at gap junctions, chemical synapses are the sole focus of this chapter.

Most types of communication between neurons must be precise and rapid. Fast and accurate information exchange is partly ensured by restricting the process within a synapse. The synapse was originally defined by three distinct elements: a presynaptic nerve terminal or bouton, its postsynaptic target, and the space between them known as the synaptic cleft. Over the past several decades, extensive investigation has revealed additional details, including presynaptic structures specialized to release chemical neurotransmitters from the nerve terminal, and postsynaptic structures involved in processing these signals 3–1 and 3–2. On the presynaptic side of the synapse, in the active zone of the nerve terminal, neurotransmitter is released from vesicles that cluster near the plasma membrane. On the other side of the synapse are postsynaptic specializations, the composition of which varies with the type of neurotransmitter released from the presynaptic terminal. The postsynaptic density, which is detected at excitatory synapses, is the best-characterized postsynaptic specialization. It is easily identified in electron micrographs 3–2 as a proteinaceous matrix of fine filaments just under the plasma membrane of postsynaptic dendritic spines. The proteins that comprise this structure, such as postsynaptic density protein 95 (PSD95), act as a scaffold to cluster and organize molecules involved in postsynaptic signaling. The synaptic cleft itself is bound by extracellular matrix proteins and cell adhesion molecules that may function to strengthen or weaken synaptic contacts.

Neurotransmitters

Neurotransmitters are often classified by their chemical makeup, although there is only a rough correlation between these classes and neurotransmitter function 3–1. The major chemical groupings are amino acid transmitters, such as glutamate and γ-aminobutyric acid (GABA); monoamines, including dopamine, norepinephrine, and serotonin; peptides; diffusible gases, such as nitric oxide; nucleosides and derivatives, such as adenosine and adenosine triphosphate (ATP); and lipid-derived signaling molecules, such as endogenous cannabinoids (endocannabinoids). Historically, it was believed that only one type of transmitter could be synthesized and released from a cell, but we now know that a single neuron may utilize multiple neurotransmitters. Examples include corelease of monoamines such as norepinephrine with peptides such as neuropeptide Y, but some neurons also release more than one small-molecule neurotransmitter, and some neurons release multiple neuropeptides (Chapter 7). The biosynthesis, metabolism, and functions of the different types of neurotransmitters, as well as the receptors that mediate their signaling, are discussed extensively in subsequent chapters. This chapter focuses predominantly on the mechanisms underlying neurotransmitter release.

Storage and Release of Neurotransmitters

With the exception of the diffusible gases and lipid-derived signaling molecules, most chemical transmitters are stored in secretory vesicles. Like other cellular organelles, these vesicles have a limiting membrane composed of a phospholipid bilayer and an aqueous lumen 3–1. The lumen is filled with thousands of transmitter molecules that sometimes reach near-molar concentrations. During synaptic transmission, secretory vesicles fuse briefly with the plasma membrane, allowing communication between the aqueous interior of the vesicle and the extracellular space. The most abundant form of secretory vesicle in the central nervous system (CNS) is the so-called small synaptic vesicle. These vesicles cluster in the active zone of the nerve terminal and normally release all or most of their contents rapidly when the nerve terminal depolarizes.

The amount of transmitter in each vesicle is called a quantum. Because transmitter release proceeds through the fusion of individual vesicles with the presynaptic terminal plasma membrane, the release process is known as quantal release.

Postsynaptic Signaling

After a neurotransmitter diffuses across the synaptic cleft to the postsynaptic neuron, receptors embedded in the postsynaptic membrane bind to the neurotransmitter and cause an electrical or biochemical alteration in the postsynaptic cell. Excitatory signals cause the membrane to depolarize so that positive charge flows into the cell, and inhibitory signals cause the membrane to hyperpolarize, whereby positive charge flows out of the cell or negative charge flows into the cell. These electrical signals are integrated in the dendrites and cell body and, if depolarization is sufficient, an all-or-none action potential fires and transmits an electrical impulse along the axon (Chapter 2). Whether synaptic transmission is fast, occurring within several milliseconds, or slow—taking place in seconds or minutes—depends on the type of receptor targeted by the neurotransmitter 3–3.

Fast Synaptic Transmission

Fast synaptic transmission is mediated by transmitter- or ligand-gated ion channels, also called ionotropic receptors. The most extensively studied of these is the nicotinic acetylcholine receptor, which is composed of five subunits that enclose a central aqueous pore. When acetylcholine binds to this receptor, the pore opens transiently (1–10 ms) and allows the passage of approximately 20,000 positively charged Na⁺ ions. The vast majority of excitatory and inhibitory synapses in the brain utilize ionotropic receptors for the amino acids glutamate and GABA (Chapter 5). Molecular cloning has revealed that the basic design of most, if not all, ligand-gated channels is similar to that of voltage-gated channels (Chapters 2 and 5).

Slow Synaptic Transmission

Many of the neurotransmitters involved in fast synaptic transmission also induce slower synaptic responses when they activate receptors coupled to heterotrimeric guanine nucleotide–binding proteins (G proteins). These receptors are often termed metabotropic receptors. Most other types of neurotransmitters, including the monoamines and neuropeptides, also activate G protein–coupled receptors. When a neurotransmitter binds to a G protein–coupled receptor, the receptor activates an associated G protein. The α subunit of the G protein subsequently dissociates from its βγ subunits, with both α and βγ subunits then activating downstream molecules, including specific types of ion channels (Chapter 4). These biochemical steps mediate slow synaptic transmission because they generally require more time to develop compared with the opening of a ligand-gated channel. Stimulation of G protein–coupled receptors does not always produce excitatory or inhibitory transmission; in some cases it modulates the actions of other neurotransmitters, such as glutamate.

In addition to regulating ion channels, G protein α and βγ subunits activate several enzymes that regulate the formation of second messengers such as cAMP, cGMP, and diacylglycerol (DAG). G proteins also regulate the flow of the second messenger Ca²⁺ into neurons. Once formed, second messengers stimulate or inhibit protein kinases or protein phosphatases, whose subsequent phosphorylation or dephosphorylation of ion channels results in the generation of postsynaptic electrical signals. Some second messengers, such as cAMP and cGMP, and G protein subunits (α and βγ) can directly bind to and modify the activity of specific ion channels. Moreover, these second messenger cascades regulate gene transcription and protein synthesis, which may alter the types and amounts of ion channels expressed by a neuron. Such activities can have long-lasting effects on neuronal function (Chapter 4). Thus, the involvement of G protein–coupled receptors greatly increases the complexity and flexibility of the types of electrical signals that can be generated by neurotransmitters.

Receptor Subtypes

Ligand-gated ion channels and G protein–coupled receptors each comprise multiple subtypes. A single neurotransmitter can activate a variety of these receptor subtypes, and several members of the same receptor family can colocalize at a single synapse; consequently, the signal received by a postsynaptic neuron can vary considerably and may be quite complex. Ultimately, the actions of any individual neurotransmitter are dictated by the receptor subtype to which it binds and by the subsynaptic localization of that receptor.

Before the advent of molecular cloning techniques, receptor subtypes were grouped according to their pharmacologic profiles, including their responses to particular agonists and antagonists. More recently, cloned receptors have been grouped more precisely on the basis of structural and functional similarities and according to their cellular localization. Some receptor subtypes tend to be located in particular cell types or subcellular regions. Presynaptic localization does not exclude postsynaptic localization, and the presence of some receptor subtypes—for example, the serotonin 5HT_1A receptor—in both presynaptic terminals and postsynaptic cells further increases the complexity of synaptic communication. The activation of receptors located in the presynaptic bouton typically modifies the release of neurotransmitter. Presynaptic receptors that respond to the transmitter released by that terminal are called autoreceptors; presynaptic receptors that respond to other transmitters are sometimes called heteroreceptors. Variations in signaling pathways activated by different receptor subtypes underscore the central role of receptors in dictating the actions of the transmitters that bind to them.

The Role of Calcium Ions

Our current understanding of synaptic transmission is based on work performed in the 1950s, which uncovered the role of Ca²⁺ in neurotransmitter release at the neuromuscular junction (NMJ). The NMJ comprises a presynaptic motor nerve terminal and a postsynaptic specialization on the muscle cell known as an end plate. Although the NMJ differs significantly from a synapse in the CNS, it is similar enough to serve as a model. When the motor nerve of the NMJ is electrically stimulated, it releases a chemical message that produces an electrical signal in the muscle cell, called the excitatory postsynaptic current, which in turn causes contraction of the muscle. When this contractile process is paralyzed, the relationship between chemical transmission and the electrical response can be studied in isolation. The chemical transmitter at the vertebrate NMJ is acetylcholine, identified in 1921 as the first known chemical neurotransmitter.

Early studies of the NMJ found that the action potential per se was not required for acetylcholine release. Rather, the action potential caused the nerve terminal to depolarize, which in turn caused the release of transmitter. This was demonstrated by eliminating the action potential with tetrodotoxin, which blocks voltage-gated Na⁺ channels (Chapter 2), and changing the voltage in the nerve terminal with an electrode, which makes it possible to depolarize the terminal and induce transmitter release directly. It was subsequently discovered that eliminating extracellular Ca²⁺ prevented the release of neurotransmitter; it was therefore concluded that the entry of Ca²⁺ into the nerve terminal triggered transmitter release. We now know that the opening of voltage-gated Ca²⁺ channels in response to depolarization (Chapter 2) causes a rapid increase in the intracellular concentration of Ca²⁺, which in turn causes the release of neurotransmitter.

Quantal Release

Related studies of the NMJ revealed that, in the absence of presynaptic stimulation, the postsynaptic cell exhibited small, spontaneous electrical responses. Equally important was the discovery that these miniature end plate potentials (MEPPs or minis) were all approximately the same size. Indeed, when the nerve was stimulated to evoke larger postsynaptic currents, the resulting end plate potentials were always integral sums of the MEPPs 3–4. These findings led to the hypothesis that a single MEPP represents the postsynaptic response to the smallest amount of transmitter released by a presynaptic cell, thus giving birth to the idea of quantal release.

The precise physical equivalent of a quantum remained to be determined, but two pieces of evidence demonstrated that this elemental unit represented the transmitter contained in a single synaptic vesicle. First, electron micrographs revealed clusters of regular round vesicles lined up at the presynaptic membrane (see 3–2). Second, these vesicles sometimes appeared to be fused with the plasma membrane and, in the process of opening their lumen to the extracellular space, such vesicles look like the Greek letter Ω and are called omega profiles. Thus, small vesicles at the site of transmitter release appeared capable of releasing their contents into the synaptic cleft by fusing with the plasma membrane. Whether these synaptic vesicles contained neurotransmitter was unclear; however, biochemical studies demonstrated that synaptic vesicles at the NMJ contained acetylcholine. These findings validated the theory that a synaptic vesicle represents the anatomic equivalent of electrophysiologically defined quanta. Fittingly, the process of transmitter release was subsequently referred to as exocytosis.

Although morphologic studies indicate that there are tens to hundreds of synaptic vesicles in each nerve terminal, neurotransmitter is not released every time an action potential invades this structure. Instead, neurotransmitter release is a stochastic or probabilistic process: in response to an action potential it sometimes occurs and sometimes fails to occur. The probability of transmitter release can vary widely from synapse to synapse and can be modified by the activation of presynaptic neurotransmitter receptors, by drugs, and by recent synaptic activity.

The quantal hypothesis has stood the test of time. With few exceptions, studies have confirmed that neurotransmitter is released from the nerve terminal in synaptic vesicles. Whether these packets of neurotransmitter are always the same size has been technically difficult to determine; however, variations among quanta most likely exist. Indeed, some of the first synaptic vesicles isolated from the NMJ revealed cholinergic vesicles that contained varying amounts of transmitter. In the CNS, quantal size may vary even more than it does at the NMJ. Variations in the postsynaptic response to each quantum and evidence indicating that different populations of synaptic vesicles may differ in the amount of neurotransmitter they store strongly support this theory. Although investigators have yet to determine the mechanisms underlying variations in synaptic vesicle content, they most likely involve the proteins that synthesize neurotransmitters and transport them into synaptic vesicles.

Packaging and Transport of Neurotransmitters

Small-molecule neurotransmitters such as the monoamines and amino acid transmitters are synthesized in the cytoplasm of the neuron; the enzymes that convert precursors into mature neurotransmitters are discussed in subsequent chapters on individual neurotransmitters. Because they are synthesized in the cytoplasm, these neurotransmitters must be transported across the synaptic vesicle membrane into its lumen. The process of vesicular transport serves to package neurotransmitters for regulated exocytotic release.

Purified secretory vesicles express distinct proteins called vesicular transporters that mediate the pumping or transport of monoamines, such as dopamine, norepinephrine, and serotonin; acetylcholine; GABA and glycine; and glutamate into the vesicles 3–5 and 3–2. Vesicular transport of all monoamines is mediated by a single protein, termed vesicular monoamine transporter 2 (VMAT2). The active transport of monoamines and acetylcholine is driven by the pH gradient (ΔpH) across the vesicle membrane, which propels the exchange of two luminal protons for one cytoplasmic transmitter molecule. In contrast, glutamate transport depends primarily on an electrochemical gradient (ΔΨ), and GABA transport depends on both ΔpH and ΔΨ. A vesicular nucleotide transporter (VNuT) that concentrates ATP within synaptic vesicles has been identified recently. Drugs that inhibit vesicular transport have significant behavioral effects. Reserpine inhibits the vesicular transport of monoamines and causes their depletion at the synapse (Chapter 6). As a result, reserpine can decrease blood pressure, but its side effects, including depression in some patients, limited its clinical use. Amphetamines, which act on both vesicular and plasma membrane transporters (see below), release vesicular monoamine stores, first into the cytoplasm and then into the synapse (Chapter 6), resulting in potent psychostimulant effects. Because of their central role in synaptic transmission, vesicular transporters are potential targets for the development of novel psychoactive drugs.

Termination of Neurotransmitter Action

After exocytosis, neurotransmitter is inactivated, either by enzymatic degradation or by active transport out of the synaptic cleft. The latter process may include transport across the plasma membrane of the presynaptic terminal, postsynaptic neurons, or neighboring glia 3–5. Like vesicular transport, plasma membrane transport requires specific transport proteins. However, unlike vesicular transport, it depends on energy provided by the Na⁺ gradient across the plasma membrane. These transport activities have been important targets of therapeutic drugs since the early 1960s, when it was discovered that cocaine and many antidepressants act initially by inhibiting the plasma membrane transport of monoamines such as dopamine, norepinephrine, or serotonin (Chapters 6, 15, and 16).

Most of the proteins responsible for plasma membrane transport of neurotransmitter have been identified through molecular cloning and belong to one of two families. One family includes transporters for GABA and for monoamine transmitters. Transporters of dopamine, norepinephrine, or serotonin are expressed only in their specific monoaminergic cell populations. Plasma membrane transporters of both monoamines and GABA use the cotransport of Na⁺ and Cl⁻ ions to drive transmitter into the cytosol. A second family of plasma membrane transporters includes at least four structurally related glutamate transporters. In contrast to monoamine and GABA transporters, glutamate transporters drive transmitter uptake through cotransport of Na⁺ and H⁺ into the cell and the countertransport of K⁺ out of the cell. In addition, glutamate transporters may function as Cl⁻ channels, an activity that appears to be uncoupled from transmitter transport.

Differences in the subcellular localization of plasma membrane transporters have important functional ramifications (see 3–5). The monoamine transporters are concentrated in the plasma membranes of presynaptic terminals; thus, in addition to terminating synaptic transmission, plasma membrane transport of monoamines also serves to recycle them for another round of exocytosis. Four isoforms of the GABA transporter have been identified, and at least 1 (GAT-1) is found in the presynaptic terminals of GABAergic neurons. However, other GAT isoforms are located on glia and possibly postsynaptic neurons, which suggests that a portion of exocytosed GABA is not recycled. Likewise, glutamate transporters are located primarily on glia and to a lesser extent on postsynaptic neurons and perhaps presynaptic nerve terminals; thus, the bulk of exocytosed glutamate is not recycled; rather, each round of glutamatergic transmission uses predominantly transmitter synthesized de novo.

Degradative enzymes play an important role in the termination of synaptic transmission as well. Enzymes that inactivate monoamines in this manner are located in the extracellular space of the synaptic cleft, as well as intracellularly. At the NMJ and in the CNS, acetylcholine is rapidly inactivated by acetylcholinesterases in the synaptic cleft; the metabolite choline is subsequently transported into the presynaptic terminal by a plasma membrane choline transporter for recycling. Acetylcholine itself is not transported across the plasma membrane (see 3–5).

The fate of exocytosed transmitter also may include diffusion out of the synaptic cleft. Electrophysiologic studies suggest that glutamate may diffuse out of the synaptic cleft and bind to receptors at adjacent synapses in a process that may serve to alter the spatial specificity of synaptic transmission. In contrast, monoamine plasma membrane transporters can be concentrated at the boundaries of the active zone of a presynaptic nerve terminal and may act to restrict diffusion of transmitter to adjacent synapses.

Synaptic Vesicles and Large Dense Core Vesicles

Many neurons express a distinct class of secretory vesicles called large dense core vesicles (LDCVs), which release neurotransmitter but differ from small synaptic vesicles. Synaptic vesicles are smaller (approximately 40 nm in diameter) and more abundant and cluster at active zones in the nerve terminal. In contrast, LDCVs are slightly larger (80–120 nm) and contain neuropeptides (Chapter 7). In the adrenal medulla and certain cultured cells, LDCVs also contain monoamine transmitters in their lumen, although in the CNS most synaptically released monoamine is derived from small synaptic vesicles. LDCVs also contain aggregates of soluble proteins that appear as a dense core in electron micrographs—hence, their name. Unlike synaptic vesicles, LDCVs are not closely linked to the active zone and are released from other sites in the cell. Although both types of vesicles use similar machinery for exocytosis, the release of neurotransmitter from LDCVs occurs in response to different concentrations of intracellular Ca²⁺ and to different patterns of presynaptic activity compared with release from small synaptic vesicles.

The cornerstone of presynaptic function is the quantal release of transmitter through exocytosis. The quantal release of transmitter must be localized and rapid, repeatable at high frequencies, and amenable to upregulation or downregulation over time. Rapid release is necessary to allow communication between neurons within milliseconds, and temporal precision is necessary to preserve the timing of the electrical information encoded in an action potential. Thus, exocytosis must be precisely coordinated with the influx of Ca²⁺ induced by depolarization of the nerve terminal. Terminals must be capable of sustained firing and neurotransmitter release because communication between neurons often involves repeated trains of stimuli. Finally, exocytosis must be a highly regulated process to accommodate the neural plasticity that underlies learning and memory. These requirements are satisfied by the following five steps in the cycle of exocytotic release 3–6:

1. Docking. Synaptic vesicles release neurotransmitter only at the active zone of a nerve terminal, exactly opposite the signal transduction machinery of the postsynaptic cell. This arrangement minimizes the time required for transmitter to reach the postsynaptic cell and also enhances the precision of communication between the two cells. Because the active zone occupies a relatively restricted area along the plasma membrane of the nerve terminal, vesicles must be specifically targeted to this region through a process referred to as docking.

2. Priming. Morphologic analyses indicate that approximately 10 to 30 vesicles are docked at the active zone of CNS synapses. However, most of these vesicles are not capable of Ca²⁺-induced release and must undergo an active maturation process known as priming, which requires ATP hydrolysis. Primed vesicles release neurotransmitter within a millisecond of Ca²⁺ influx into the cell. The speed of this release suggests that priming may initiate a partial fusion of the synaptic vesicle with the plasma membrane. Moreover, the energy obtained from ATP hydrolysis may be used to alter the conformation of proteins involved in exocytosis.

3. Fusion/Exocytosis. Fusion of the synaptic vesicle with the presynaptic plasma membrane requires deformation of the two apposing membranes. Because this event is tightly linked to the influx of Ca²⁺, the underlying protein machinery involves a Ca²⁺ sensor. Fusion of the membranes allows exocytosis: extrusion of vesicle contents into the synaptic cleft.

4. Endocytosis. After the synaptic vesicle fuses with the plasma membrane and releases its contents, the vesicle membrane is recycled by a process of internalization known as endocytosis. As with other endocytotic processes, the internalization of synaptic vesicles is facilitated by a protein lattice that coats the internal face of the plasma membrane.

5. Recycling. This mechanism conserves synaptic vesicle membrane and helps to maintain a pool of release-competent vesicles. Two major models of synaptic vesicle recycling have been proposed. In the first, vesicles fuse with larger endosomes after endocytosis; subsequently, synaptic vesicles bud from these endosomes to engage in another round of exocytosis. According to the second model, synaptic vesicles bud directly from deep invaginations of the plasma membrane that are distinct from endosomes. Although synaptic vesicles recycle according to the first model in some neuroendocrine cells in culture, the latter process is believed to predominate in the CNS. A third process, which may occur intermittently at individual presynaptic terminals, is “kiss and run” exocytosis during which vesicles never completely fuse with the plasma membrane; in this model only a portion of a vesicle’s stored transmitter leaks out through a pore that opens between the lumen of the vesicle and the extracellular space.

The entire exocytotic cycle lasts approximately 30 to 60 seconds. Exocytosis occurs within 1 ms and endocytosis occurs within 5 seconds; the other steps of the cycle occupy the remaining time. The speed of exocytosis, which is truly breathtaking, is required for the overall efficiency of information processing in the nervous system.

Proteins Involved in the Exocytotic Cycle

Until the 1990s, the mechanisms underlying vesicle release and recycling remained obscure, partly because the proteins involved in these processes were not known. During the past 20 years many of these proteins have been identified. Thus, the component of synaptic transmission that was for decades represented by a black box, namely, the triggering of presynaptic vesicle exocytosis by Ca²⁺ entry, has been replaced by a series of precisely defined molecular events. It should be noted that the SNARE proteins that mediate exocytosis are also localized postsynaptically, where they regulate other aspects of membrane function, which are not discussed in this chapter.

The Function of SNARES: Vesicle Docking or Fusion?

The morphologic differences between vesicles docked at the active zone of a nerve terminal and those that are undocked suggest that neurons contain mechanisms for segregating these pools and for targeting vesicles to the active zone. Initial experiments suggested that a group of proteins known as SNAREs might be involved in this process. SNAREs include three synaptic proteins that were first isolated biochemically and have since become integral to our understanding of vesicle fusion and transmitter release: synaptobrevin, also known as vesicle-associated membrane protein (VAMP); syntaxin; and synaptosomal-associated protein of 25 kDa (SNAP-25), each of which have several isoforms. Synaptobrevins span the vesicle membrane, with one transmembrane domain near the C terminus. Syntaxins are also integral membrane proteins. In contrast, SNAP-25 is anchored to the membrane by lipid groups (long-chain fatty acids) attached to central cysteine residues. At the active zone, syntaxin and SNAP-25 localize in the plasma membrane and are often known as t-SNAREs (t for target membranes). In contrast, synaptobrevin is localized primarily in the vesicular membrane and thus has been labeled a v-SNARE, or vesicle SNARE.

Three important findings led to the hypothesis that these proteins are involved in exocytosis at the presynaptic terminal. First, several bacterial toxins known to cause neurologic symptoms were proven to inhibit exocytosis and to cleave specific SNARE proteins 3–2. Likewise, the black widow spider toxin (α-latrotoxin) affects transmitter release by acting on proteins in the presynaptic nerve terminal. Second, mutation of yeast homologs of the SNAREs causes defects in membrane trafficking. These two lines of investigation implicated the SNAREs in synaptic transmission, but did not address the mechanism through which they might promote exocytosis. The third finding, which emerged from in vitro biochemical studies, was that syntaxin, synaptobrevin, and SNAP-25 spontaneously form a thermodynamically stable 1:1:1 complex. Because syntaxin and SNAP-25 are located primarily in the plasma membrane, and synaptobrevin is located primarily in the vesicle membrane, it was proposed that the association of these proteins might serve to target synaptic vesicles to the plasma membrane during the docking step of the exocytotic cycle. This postulate, known as the SNARE hypothesis, has been influential in the study of membrane trafficking in general as well as the molecular mechanisms underlying synaptic transmission.

Subsequent evidence has indicated that SNAREs play a direct role in membrane fusion, with other molecules facilitating docking. Genetic deletion of syntaxin in Drosophila decreases exocytosis, but does not decrease the number of docked vesicles in nerve terminals. Similarly, the injection of tetanus toxin (see 3–2) or fragments of SNARE proteins into the presynaptic terminal of the giant axon of the squid leads to an increased number of docked vesicles. A decrease in the number of docked vesicles would be expected if the SNAREs were required for docking. These findings therefore suggest that the SNAREs may function predominantly at a later step in the exocytotic cycle, namely, fusion.

This hypothesis is supported by several lines of evidence. SNAREs that have been reconstituted into lipid vesicles have been assayed for vesicle fusion with the use of fluorescent dyes. Vesicles containing equimolar amounts of syntaxin and SNAP-25 fused with vesicles containing synaptobrevin, indicating that v-SNAREs and t-SNAREs alone can generate fusion between lipid bilayers. The mechanism for fusion has been suggested by in vitro experiments in which SNAREs bind to each other in parallel with the C terminus of each SNARE similarly oriented. Thus, like viral hairpin proteins, which mediate the entry of viruses into cells, the SNAREs (or SNAREpins) may change their conformation or binding properties to allow fusion between opposing bilayers 3–7 and 3–8. The favorable thermodynamics of SNARE complex formation may drive the fusion of two such bilayers over the energy barrier that normally prevents the intermingling of bilayers.

Disassembly of the SNAREs by NSF

Because association of the SNAREs appears to drive fusion of the vesicle membrane with the plasma membrane, dissociation of the SNAREs must occur before the vesicle can be reclaimed from the plasma membrane to embark on a new exocytotic cycle. N-Ethylmaleimide sensitive factor (NSF) and the soluble NSF attachment proteins (SNAPs) may perform this function (see 3–7).¹ NSF is a soluble tetramer and requires SNAPs to bind to vesicle membranes and participate in exocytosis. A key finding in the search for the molecular composition of the exocytotic apparatus was the discovery that both NSF and the SNAPs also bind with high affinity to the SNAREs, including syntaxin, synaptobrevin, and SNAP-25. Remarkably, the aggregate composed of these proteins can be dissociated into its component proteins by the addition of ATP. This occurs because NSF is an ATPase; by hydrolyzing ATP, NSF generates the energy to dissociate the thermodynamically stable complex of NSF, SNAPs, and SNARES.

Disassembly is likely to occur when SNAREs occupy the same membrane. Indeed, t-SNAREs and v-SNAREs have been found to coexist on synaptic vesicle membranes. These observations suggest the following model: SNAREs induce fusion between a vesicle membrane and a plasma membrane by fastening together in parallel; to allow another round of exocytosis, NSF uses energy obtained from ATP hydrolysis to unzip the stable SNAREs 3–7 and 3–8.

Synaptotagmin: The Ca²⁺ Sensor for Transmitter Release

Unlike most membrane trafficking events, which appear to proceed constitutively, neurotransmitter release is triggered by Ca²⁺. Thus, an important goal in the study of synaptic transmission has been to identify the mechanism by which exocytotic machinery senses Ca²⁺ influx. Several lines of evidence suggest that the 65-kDa protein synaptotagmin is the primary mediator of this process. Investigators first proposed this possibility after the elucidation of synaptotagmin’s primary structure revealed so-called C2 domains similar to those found in protein kinase C, a Ca²⁺ and phospholipid-dependent protein kinase (Chapter 4). Synaptotagmin contains two C2 domains, C2A and C2B 3–9, each of which binds two Ca²⁺ atoms. Extensive NMR and crystallographic analyses reveal that these domains form a novel Ca²⁺-binding motif composed primarily of β sheets. Such binding does not induce a conformational change but is believed to induce a significant shift in electrostatic potential, or charge, in the Ca²⁺-binding domain, which may function to regulate the interaction of synaptotagmin with other molecules involved in vesicle fusion.

Because there are many different forms of synaptotagmin, a variety of methods have been used to determine whether the predominant form, synaptotagmin I, is a Ca²⁺ sensor in vivo. Most convincingly, studies of synaptotagmin mutations in Drosophila, C. elegans, and mice have confirmed that synaptotagmin I plays an important role in exocytosis. Knockout and knockin mice have provided particularly useful information. In synaptotagmin I knockouts, fast Ca²⁺-dependent transmitter release is strongly suppressed, while a second, slower phase of transmitter release, which is also Ca²⁺-dependent, is not affected. This second phase of release may be mediated by other synaptotagmin isoforms. Furthermore, genetically modifying the affinity of synaptotagmin I for Ca²⁺ has an exactly parallel effect on the Ca²⁺ sensitivity of transmitter release. Other parameters of transmitter release remain unchanged in these mice, thus verifying that synaptotagmin I regulates only one step of the exocytotic cycle: fast Ca²⁺-triggered fusion.

Additional Proteins Involved in Neurotransmitter Release

Thus far we have only mentioned a small number of the 100 or so proteins that are involved in neurotransmitter release and its regulation. The active zone, the site at which vesicles dock and fuse with the presynaptic plasma membrane, is a highly organized macromolecular complex containing so-called “scaffolding” proteins, which, via their multiple protein–protein interaction domains, are critical for the appropriate positioning of the proteins that are key components of the vesicle cycling and fusion machinery. These include families of proteins termed RIMs (the most common variants being RIM1α and RIM2α) and Munc-13s as well as the extraordinarily large proteins bassoon and piccolo. RIMs appear to be particularly important for the localization of Ca²⁺ channels near the active zone so that the entry of Ca²⁺ occurs immediately next to the docked synaptic vesicles. Furthermore, individual presynaptic terminals express different complements of these active zone proteins and this likely contributes to significant differences in their functions.

Rab proteins are a large family of small GTP-binding proteins that are crucial in regulating many types of intracellular membrane transport (Chapter 4). Rab3a plays a particularly important role in regulating synaptic vesicle trafficking and exocytosis. It undergoes a cycle of association with and dissociation from synaptic vesicles in parallel with their exocytosis and endocytosis. It appears to function at a late stage in synaptic vesicle exocytosis after docking. Importantly, it directly interacts with RIM1α, and both Rab3a and RIM1α are required for the long-lasting, activity-dependent enhancement of transmitter release (termed presynaptic long-term potentiation [LTP]) that occurs at some presynaptic terminals following their repetitive activation. In addition to Rab3a, presynaptic terminals contain one or more of three other Rab3 isoforms termed Rab3b, c, and d, the exact functions of which are unknown.

Although we have emphasized the central function of the SNAREs, synaptobrevin, syntaxin, and SNAP-25, in mediating vesicle exocytosis, this process is highly regulated due to the participation of several additional proteins. Among the most important of these are complexins, which bind to SNARE complexes and are critical for the function of synaptotagmin I in triggering Ca²⁺-dependent synaptic vesicle fusion. Another important protein is Munc-18, which binds to syntaxin and thereby regulates the SNARE-dependent fusion of synaptic vesicles.

The number of docked synaptic vesicles at individual presynaptic terminals is small, on the order of 3 to 10. Thus, during prolonged bursts of activity there are mechanisms in place to replenish these vesicles from another pool of vesicles called the “reserve” pool. Synapsins are a family of proteins that bind to synaptic vesicles and tether them to the presynaptic actin cytoskeleton. They are believed to be important for regulating the trafficking of vesicles between the “reserve” pool and the “readily releasable” pool of vesicles that are docked at the presynaptic plasma membrane. When phosphorylated by protein kinases such as protein kinase A, synapsins dissociate from synaptic vesicles, a biochemical change that contributes to short-term, activity-dependent changes in neurotransmitter release.

Endocytosis and Recycling

Clathrin-mediated endocytosis

After fusion and exocytosis, synaptic vesicle proteins are reclaimed from the plasma membrane through clathrin-mediated endocytosis 3–10. Clathrin is composed of two highly conserved subunits that self-assemble into structures called triskelions, each of which consists of three light-chain and three heavy-chain subunits. It forms a proteinaceous coat that helps shape the membrane into a bud that is subsequently pulled away from the inner surface of the plasma membrane. Clathrin is linked to membranes through heteromeric adapter proteins (APs), each of which comprises a large central domain and two projecting ears. At least four types of adapters are known to exist, and each localizes in a different subcellular organelle; the adapter at the plasma membrane is called AP-2. Each type of AP may contain cell type–specific subunits. A neuron-specific subunit known as β3b, or β-NAP, was originally isolated as the antigen responsible for a so-called paraneoplastic syndrome, or tumor-induced autoimmunity syndrome, which results in cerebellar degeneration.

To ensure that other types of proteins are not mistakenly recruited into synaptic vesicles, adapters bind to highly specific proteins at the plasma membrane. The only synaptic vesicle protein known to bind AP-2 is synaptotagmin. Thus, this protein may play a dual role: it may serve as a Ca²⁺ sensor during exocytosis and as a tag during endocytosis. However, different isoforms of synaptotagmin may be involved in these processes.

After endocytosis, the clathrin coat is removed from the membrane to allow further trafficking and recycling to the existing pool of synaptic vesicles. The molecular chaperone heat shock protein 70c (hsp70c) likely is involved in disassembling clathrin, with the assistance of additional tissue-specific cofactors.

Endocytic machinery

An important component of the biochemical machinery underlying endocytosis is the protein dynamin. This protein cycles between phosphorylated and dephosphorylated states during the exocytotic cycle. Biochemical assays have revealed that dynamin encodes a GTPase activity that was linked to endocytosis after the discovery of a mutation in the Drosophila dynamin gene known as shibere. Flies containing this mutation were isolated more than 25 years ago and display defects in cell signaling. Electron micrographs of the nerve terminals of mutant flies revealed hundreds of vesicles attached to each plasma membrane by short ring-coated necks. Discovery of the shibere phenotype suggested that these rings, which are oligomers of shibere protein, may function to pinch off the neck of a vesicle as it buds from the plasma membrane. Recent findings strongly suggest that dynamin is intimately involved in the endocytosis of synaptic vesicles and that GTP hydrolysis helps to drive this process. Thus, as originally suggested by the shibere mutant, dynamin, with the assistance of proteins that are located in the necks of budding vesicles, appears to function as a “pinchase” to sever or pinch off the necks of coated vesicles as they bud from the plasma membrane. Dynamin-mediated endocytosis has also been implicated in the downregulation of G protein–coupled receptors in response to sustained receptor activation (Chapters 4 and 6).

Vesicle recycling

After endocytosis, synaptic vesicles are recycled. As mentioned previously, the biogenesis and recycling of synaptic vesicles may involve budding from both plasma membrane invaginations and endosomal compartments discrete from the plasma membrane. Although the contribution of each pathway to vesicle formation in neurons remains unclear, some of the molecular components of the endosomal pathway have been determined with the use of model neuroendocrine cells. Like endocytosis at the plasma membrane, the budding of synaptic vesicles from endosomes involves protein coats formed from heteromeric APs. However, endosomal budding employs a different AP protein (AP-3) and does not require clathrin. Interestingly, mutations in one of the large subunits of AP-3 (δ3) may disrupt vesicle trafficking in both mice and humans. The phenotypes of mocha, a δ3 mutation in mice, include an increase in baseline electrical activity in the brain and a unique hypersynchronized 6- to 7-Hz EEG pattern. In humans, a similar mutation results in a form of Hermansky–Pudlak syndrome (HPS), which is notable for defects in storage granules. Moreover, tumor-related antibodies to a neuronally expressed β subunit of AP-3 (also known as β3b or β-NAP) induce cerebellar degeneration. Such findings suggest that defects in secretory vesicle biogenesis and its regulation may underlie other neuropsychiatric syndromes.

Complexity and Specialization

During the past two decades, many of the molecules responsible for the quantal release of neurotransmitter have been identified, and some of the protein–protein interactions underlying this complex process and its intricate regulation have been teased apart. Yet, this chapter presents a simplified view. Neurons express multiple isoforms of many of the proteins described here; for example, 16 isoforms of synaptotagmin have been isolated, and each may regulate a different aspect of synaptic transmission. Isoforms of other synaptic proteins exhibit striking cell type–specific distributions in the brain. Furthermore, almost all of the molecular understanding of the process of synaptic vesicle exocyotsis and endocytosis derives from the study of excitatory and inhibitory synapses that utilize the neurotransmitters glutamate and GABA, respectively. Whether the presynaptic release of major modulatory transmitters such as monoamines involves identical molecular machinery remains to be determined.

Given that most, if not all, neuropsychiatric disorders can be conceptualized as being due to dysfunctions in synaptic transmission, elucidating the detailed molecular mechanisms underlying this fundamental signal transduction process is likely to reveal a great deal about the pathophysiology, and perhaps even the pathogenesis, of such brain disorders. Furthermore, it is anticipated that several proteins involved in neurotransmitter release or the termination of transmitter action will be important targets of therapeutic agents. Indeed, the many isoforms of these proteins should facilitate the design of drugs that act on specific neuronal cell types or synaptic processes.