Medical Physiology, 3rd Edition

Fast Amino Acid–Mediated Synapses in the CNS

Fast amino acid–mediated synapses account for most of the neural activity that we associate with specific information processing in the brain: events directly responsible for sensory perception, motor control, and cognition, for example. Glutamate-mediated excitation and GABA-mediated inhibition have been intensively studied. In physiological terms, these are also the best understood of the brain's synapses, and this section describes their function.

As a rule, postsynaptic events are more easily measured than presynaptic events; thus, we know more about them. Of course, by measuring postsynaptic events, we also have a window onto the functions of the presynaptic terminal, and this is often the best view we can get of presynaptic functions. For this reason, we begin our description with the downstream, postsynaptic side of the synapse and then work backward to the presynaptic side.

Most EPSPs in the brain are mediated by two types of glutamate-gated channels

Most glutamate-mediated synapses generate an EPSP with two distinct components, one much faster than the other. Both are triggered by the same presynaptic terminal releasing a single bolus of transmitter, but the two EPSP components are generated by different types of ion channels that are gated by distinct postsynaptic receptors—a case of transmitter divergence. The behavior of these channels helps in understanding the characteristics of the EPSP.

Glutamate can act on two major classes of receptors: GPCRs or metabotropic receptors, and ion channels or ionotropic receptors. As noted above, metabotropic glutamate receptors (mGluRs—the m stands for metabotropic) have seven membrane-spanning segments and are linked to heterotrimeric G proteins (Fig. 13-15A). At least eight metabotropic receptors have been identified, and comparisons of their primary structure have been used to infer the evolutionary relationships among receptor subunits (see Fig. 13-15B). The mGluRs form three groups that differ in their sequence similarity, pharmacology, and associated signal-transduction systems.

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FIGURE 13-15 Comparison of ionotropic and metabotropic glutamate receptors. In C, the inset shows a prototypic subunit, with a large extracellular glutamate-binding domain, a membrane-spanning segment, a short loop that partially re-enters the membrane from the cytosolic side, and two more membrane-spanning segments. Four of these subunits appear to come together to form a single channel/receptor with a central pore.

The three classes of ionotropic glutamate receptors are the AMPA, NMDA, and kainate receptors (Table 13-2). By definition, each is activated by binding glutamate, but their pharmacology and functions differ. The receptor names are derived from their relatively specific agonists: AMPA stands for α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid. NMDA stands for N-methyl-D-aspartate. The kainate receptor is named for one of its agonists, kainic acid, and it can also be activated by domoic acid. The three ionotropic glutamate receptors can also be distinguished by their selective antagonists. AMPA and kainate receptors, but not NMDA receptors, are blocked by drugs such as CNQX (6-cyano-7-nitroquinoxaline-2,3-dione). Moreover, AMPA receptors can be specifically antagonized by 2,3-benzodiazepine derivatives, such as GYKI 53655. NMDA receptors, but not AMPA and kainate receptors, are blocked by APV (2-amino-5-phosphonovaleric acid). Selective antagonists of kainate receptors have also been discovered.

TABLE 13-2

Ionotropic Glutamate Receptors

CLASS OF RECEPTOR

AGONIST

ANTAGONIST

KINETICS

PERMEABILITY

AMPA
Genes: GRIA
Proteins: GluA

α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid

CNQX (6-cyano-7-nitroquinoxaline-2,3-dione)
GYKI 53655 (2,3-benzodiazepine derivatives)

Fast

Na+, K+ (Ca2+ in a few cases)

Kainate
Genes: GRIK
Proteins: GluK

Kainic acid
Domoic acid

CNQX
UBP 296 [(RS)-1-(2-amino-2-carboxyethyl)-3-(2-carboxybenzyl)pyrimidine-2,4-dione]

Fast

Na+, K+

NMDA
Genes: GRIN
Proteins: GluN

N-methyl-D-aspartate

APV (2-amino-5-phosphonovaleric acid)

Slow

Na+, K+, Ca2+

For nomenclature of ionotropic glutamate receptor genes and proteins, see Table 6-2, family No. 12.

Ionotropic glutamate receptors are constructed from ~14 different subunits. Each of these has a large extracellular glutamate-binding domain, followed by a transmembrane segment, a loop that partially enters the membrane from the cytosolic side, and then two more transmembrane segments (see Fig. 13-15C). The loop appears to line the channel pore and may be important for ion selectivity. Kinetic and structural studies indicate that the receptors are heterotetramers, with four subunits arranged around a central channel. Comparisons of primary structures can be used to infer evolutionary relationships among receptor monomeric subunits. Figure 13-15D shows a hypothesized phylogenetic tree for the three classes of ionotropic glutamate receptors, with the major subtypes clustered together. Note that the various NMDA receptor subunits (e.g., GluN1, GluN2A through GluN2D) that combine to make the NMDA receptors are more closely related to each other than to the subunits (e.g., GluK1 through GluK5) that combine to make the kainate receptors or to the subunits (e.g., GluA1 through GluA4) that combine to make the AMPA receptors. The metabotropic and ionotropic glutamate receptors have separate family trees because, although both receptor types bind glutamate, they are so different in structure that they almost certainly evolved from different ancestral protein lines.

As noted above, most glutamate-mediated synapses generate an EPSP with two temporal components (Fig. 13-16A, C). The two phases of the glutamate-mediated EPSP have different pharmacological profiles, kinetics, voltage dependencies, ion dependencies, and permeabilities, and most important, they serve distinct functions in the brain. Pharmacological analysis reveals that the faster phase is mediated by an AMPA-type glutamate receptor and the slower phase by an NMDA-type glutamate receptor. These glutamate-gated channels have been extensively studied with single-channel recording methods. Both AMPA and NMDA receptors have nearly equal permeability to Na+ and K+, but they differ in several ways.

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FIGURE 13-16 Glutamate-gated channels. A, At most glutamate-mediated synapses, the EPSP (red curve) is the sum of two components: (1) a rapid component that is mediated by an AMPA receptor (green curve) and (2) a slow component that is mediated by an NMDA receptor (orange curve). In this example, in which the postsynaptic Vm is relatively negative, the contribution of the NMDA receptor is very small. B, At a relatively negative initial Vm in the postsynaptic cell, as in A, the NMDA receptor does not open. The AMPA receptor, which is independent of postsynaptic Vm, opens. The result is a fast depolarization. C, In this example, in which the postsynaptic Vm is relatively positive, the contribution of the NMDA receptor is fairly large. D, At a relatively positive initial Vm in the postsynaptic cell, as in C, glutamate activates both the AMPA and the NMDA receptors. The recruitment of the NMDA receptors is important because, unlike most AMPA receptors, they allow the entry of Ca2+ and have slower kinetics.

AMPA-gated channels are found in most excitatory synapses in the brain, and they mediate fast excitation, with most types of AMPA channels normally letting very little Ca2+ into cells. Their single-channel conductance is relatively low, ~15 picosiemens (pS), and they show little voltage dependence.

NMDA-gated channels have more complex behavior. Each has a higher conductance, ~50 pS, and much slower kinetics. The ion selectivity of NMDA channels is the key to their functions: permeability to Na+and K+ causes depolarization and thus excitation of a cell, but their high permeability to Ca2+ allows them to influence [Ca2+]i significantly. It is difficult to overstate the importance of intracellular [Ca2+]. Ca2+ can activate many enzymes, regulate the opening of a variety of channels, and affect the expression of genes. Excess Ca2+ can even precipitate the death of a cell.

The gating of NMDA channels is unusual: at normal resting voltage (about −70 mV), the channel is clogged by Mg2+, and few ions pass through it; the Mg2+ pops out only when the membrane is depolarized above about −60 mV. Thus, the NMDA channel is voltage dependent in addition to being ligand gated; both glutamate and a relatively positive Vm are necessary for the channel to open. How do the NMDA-gated channels open? NMDA-gated channels coexist with AMPA-gated channels in many synapses of the brain. When the postsynaptic cell is at a relatively negative resting potential (see Fig. 13-16A, B), the glutamate released from a synaptic terminal can open the AMPA-gated channel, which is voltage independent, but not the NMDA-gated channel. However, when the postsynaptic cell is more depolarized because of the action of other synapses (see Fig. 13-16C, D), the larger depolarization of the postsynaptic membrane allows the NMDA-gated channel to open by relieving its Mg2+ block. Indeed, under natural conditions, the slower NMDA channels open only after the membrane has been sufficiently depolarized by the action of the faster AMPA channels from many simultaneously active synapses. imageN13-4

N13-4

Differential Ca2+ Permeabilities of AMPA- and NMDA-Type Glutamate Receptors

Contributed by Barry W. Connors

If the AMPA-type and NMDA-type glutamate receptor channels are so closely related phylogenetically (see Fig. 13-15D), how is it that they have such different Ca2+ permeabilities? Most of the AMPA-type glutamate receptor channels have a relatively low Ca2+ permeability because they include at least one GluA2 subunit. GluA2 (but not GluA1, GluA3, or GluA4) has a positively charge arginine residue at a particular site within the channel-forming domain. The arginine in GluA2 is critical for the low Ca2+ permeability of the native AMPA-type receptor channel. GluA1, GluA3, and GluA4 all have a neutral glutamine in place of the arginine. Indeed, if one constructs a complete AMPA-gated channel in which none of the four subunits has the arginine at the critical site, this channel will have an unnaturally high Ca2+ permeability. If one starts from such a channel lacking the critically placed arginines, reintroducing a single arginine into any of the four subunits restores the low Ca2+ permeability.

Because the NMDA-gated channel is naturally permeable to Ca2+, one can guess that it follows the same structural rules as the AMPA-gated channel. Indeed, if one locates the homologous amino-acid residue where the neutral arginine would be in the AMPA-gated channel, one will find a neutral asparagine in all subunits of the NMDA-gated channel. Predictably these neutral asparagines—like glutamines at the homologous sites in the mutant subunits of the AMPA-gated channel (see previous paragraph)—allow Ca2+ to pass through the pore of NMDA-gated channels.

The physiological function of kainate-gated channels is still largely a mystery, although recent evidence suggests that they may contribute to some glutamate-mediated EPSPs in specific neuron types. The kainate receptor channels also exist on presynaptic GABAergic and glutamatergic terminals, where they regulate release of the inhibitory and excitatory transmitters (i.e., GABA and glutamate).

Most IPSPs in the brain are mediated by the GABAA receptor, which is activated by several classes of drugs

GABA mediates the bulk of fast synaptic inhibition in the CNS, and glycine mediates most of the rest. Both the GABAA receptor and the glycine receptor are ionotropic receptors that are, in fact, Cl-selective channels. Note that GABA can also activate the relatively common GABAB receptor, which is a GPCR or metabotropic receptor that is linked to either the opening of K+ channels or the suppression of Ca2+channels. Finally, GABA can activate the ionotropic GABAC receptor, found primarily in neurons of the retina.

Synaptic inhibition must be tightly regulated in the brain. Too much inhibition causes sedation, loss of consciousness, and coma, whereas too little leads to anxiety, hyperexcitability, and seizures. The need to control inhibition may explain why the GABAA receptor channel has, in addition to its GABA binding site, several other sites where chemicals can bind and thus dramatically modulate the function of the GABAA receptor channel. For example, two classes of drugs, benzodiazepines (one of which is the tranquilizer diazepam [Valium]) and barbiturates (e.g., the sedative phenobarbital), each bind to their own specific sites on the outside face of the GABAA receptor channel. By themselves, these drugs do very little to the channel's activity. However, when GABA is around, benzodiazepines increase the frequency of channel opening, whereas barbiturates increase the duration of channel opening. In addition, the benzodiazepines can increase Cl conductance of the GABAA receptor channel. Figure 13-17A to D shows the effects of barbiturates on both the IPSP and the single-channel currents. The result, for both the benzodiazepines and the barbiturates, is more inhibitory Cl current, stronger IPSPs, and the behavioral consequences of enhanced inhibition.

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FIGURE 13-17 Physiology and structure of the GABAA receptor channel. A, When a pulse of GABA is released from a synapse, it elicits a small IPSP. B, In the presence of a low dose of pentobarbital, the pulse of synaptic GABA elicits a much larger IPSP. Thus, the barbiturate enhances inhibition. C, At the single-channel level, GABA by itself elicits brief channel openings. D, A barbiturate (50 µM pentobarbital, in this case) does not by itself activate the GABAA receptor channel but increases the channel open time when GABA is present. E, The channel receptor is a heteropentamer. It has not only a pore for Cl but also separate binding sites for GABA and several classes of channel modulators. The inset shows the presumed structure of one of the five monomers. The M2 domain of each of the five subunits presumably lines the central channel pore. (C and D, Data from Puia G, Santi MR, Vicini S, et al: Neurosteroids act on recombinant human GABAA receptors. Neuron 4:759–765, 1990.)

Surely, however, the GABAA receptor did not evolve specialized binding sites just for the benefit of our modern drugs. This sort of logic has motivated research to find endogenous ligands, or natural chemicals that bind to the benzodiazepine and barbiturate sites and serve as regulators of inhibition. Figure 13-17E shows some of the binding sites on the GABAA receptor. Among the potential natural modulators of the GABAA receptor may be various metabolites of the steroid hormones progesterone, corticosterone, and testosterone. Some of these hormones increase the lifetime of GABA-activated single-channel currents or the opening frequency of these channels and may thus enhance inhibition. The steroid effect is unlike the usual genomic mechanisms of steroid hormones (see pp. 71–72). Instead, steroids modulate the GABAA receptor in a manner similar to barbiturates—directly, through binding sites that are distinct from the other drug-binding sites on the GABAA receptor. Thus, these steroids are not the natural agonists of the benzodiazepine and barbiturate binding sites. The GABAA receptor is also subject to modulation by the effects of phosphorylation triggered by second-messenger signaling pathways within neurons.

The ionotropic receptors for ACh, serotonin, GABA, and glycine belong to the superfamily of ligand-gated/pentameric channels

We now know the amino-acid sequences of all major ligand-gated ion channels in the brain. Even though the receptors for ACh, serotonin, GABA, and glycine are gated by such different ligands and have such different permeabilities, they have the same overall structure: five protein subunits, with each subunit being made up of four membrane-spanning segments (as shown for the GABAA receptor channel in Fig. 13-17E and inset). For example, inhibitory GABAA and glycine receptors have structures very similar to those of excitatory nicotinic ACh receptors, even though the first two are selective for anions and the last is selective for cations. For both the glycine and nicotinic ACh receptors, the transmitters bind only to the α subunits, whereas for the GABAA receptor, the transmitter GABA binds to a site at the interface of the α and β subunits.

The primary structures of the many subunit types are remarkably similar, particularly within the amino-acid sequences of the hydrophobic membrane-spanning segments. One such stretch, called the M2 domain, tends to have repeating sequences of the polar amino acids threonine and serine. Each of the five subunits that constitute a channel contributes one M2 domain, and the set of five combines to form the water-lined pore through which ions can flow (see Fig. 13-17E). For the channels gated by GABA and glycine, selectivity for Cl may be determined by positively charged arginines and lysines near the mouth of the pore.

Not quite all ligand-gated channels belong to the same superfamily. We have already seen that the family of ionotropic glutamate receptors is distinct from the family of ligand-gated/pentameric channels. Extensive evidence also indicates that ATP is a synaptic transmitter between certain neurons and at neuron–smooth-muscle cell synapses, with rapid actions similar to those of glutamate and ACh. One of the purinergic receptors or purinoceptors, called P2X, is an ATP-gated cation channel with relatively high Ca2+ permeability. The sequence of this receptor bears little resemblance to those of either the ionotropic glutamate receptor family or the ligand-gated/pentameric channel superfamily. Instead, each subunit appears to have only two membrane-spanning segments, and a full channel comprises just three subunits. Functionally, the ATP-gated channel closely resembles the nicotinic ACh receptor; structurally, it is much more akin to the channel superfamily that includes voltage-gated Na+ and K+ channels (see pp. 182–183) and to some mechanosensitive channels. This similarity appears to be a case of convergent evolution among ion channels.

Most neuronal synapses release a very small number of transmitter quanta with each action potential

A single neuromuscular junction has ~1000 active zones (see p. 210). A single presynaptic impulse releases 100 to 200 quanta of transmitter molecules (i.e., ACh), which generates an EPSP of >40 mV in the muscle cell. This is excitation with a vengeance, because the total number of quanta is far more than necessary to cause the muscle cell to fire an action potential and generate a brief contraction. Evolution has designed a neuromuscular junction that works every time, with a large margin of excess for safety. Synapses in the brain are quite different. A typical glutamatergic synapse, which has as few as one active zone, generates EPSPs of only 10 to 1000 µV. In most neurons, one EPSP is rarely enough to cause a postsynaptic cell to fire an action potential.

The basis for the small effect of central synapses has been explored by quantal analysis, with refinements of methods originally applied to the neuromuscular junction. In this approach, a single presynaptic axon is stimulated repeatedly while the postsynaptic response is recorded under voltage-clamp conditions. The frequency distribution of amplitudes of excitatory postsynaptic currents (EPSCs) is analyzed, as described for the neuromuscular junction (see Fig. 8-12B). Recall that according to standard quantal theory:

image

(13-1)

Here, m is the total number of quanta released, n is the maximal number of releasable quanta (perhaps equivalent to the number of active zones), and p is the average probability of release. Measurement of these parameters is very difficult. Only in rare cases in central neurons is it possible to find amplitude distributions of EPSCs with clearly separate peaks that may correspond to quantal increments of transmitter. In most cases, EPSC distributions are smooth and broad, which makes quantal analysis difficult to interpret. The analysis is hampered because EPSCs are small, it is extremely difficult to identify each small synapse, the dendrites electrically filter the recorded synaptic signals, noise arises from numerous sources (including the ion channels themselves), and synapses exhibit considerable variability. Nevertheless, what is clear is that most synaptic terminals in the CNS release only a small number of transmitter molecules per impulse, often just those contained in a single quantum (e.g., 1000 to 5000 glutamate molecules). Furthermore, the probability of release of that single quantum is often substantially less than 1; in other words, a presynaptic action potential often results in the release of no transmitter at all. When a quantum of transmitter molecules is released, only a limited number of postsynaptic receptors is available for the transmitter to bind to, usually not more than 100. In addition, because not all the receptors open their channels during each response, only 10 to 40 channels contribute current to each postsynaptic response, compared with the thousands of channels opening in concert during each neuromuscular EPSP.

Because most glutamatergic synapses in the brain contribute such a weak excitatory effect, it may require the nearly simultaneous action of many synapses (and the summation of their EPSPs) to bring the postsynaptic membrane potential above the threshold for an action potential. The threshold number of synapses varies greatly among neurons, but it is roughly in the range of 10 to 100.

Some exceptions to the rule of small synaptic strengths in the CNS may be noted. One of the strongest connections in the CNS is the one between the climbing fibers and Purkinje cells of the cerebellum (see Fig. 13-1B). Climbing fibers are glutamatergic axons arising from cells in the inferior olivary nucleus, and they are a critical input to the cerebellum. Climbing fibers and Purkinje cells have a dedicated, one-to-one relationship. The climbing fiber branches extensively and winds intimately around each Purkinje cell, making numerous synaptic contacts. When the climbing fiber fires, it generates a massive EPSP (~40 mV, similar to the neuromuscular EPSP) that evokes a burst of spikes in the Purkinje cell. Like the neuromuscular junction, the climbing fiber–Purkinje cell relationship seems to be designed to deliver a suprathreshold response every time it is activated. It achieves this strength in the standard way: each climbing fiber makes ~200 synaptic contacts with each Purkinje cell.

When multiple transmitters colocalize to the same synapse, the exocytosis of large vesicles requires high-frequency stimulation

As we mentioned previously, some presynaptic terminals have two or more transmitters colocalized within them. In these cases, the small transmitters are packaged into relatively small vesicles (~40 nm in diameter), whereas neuropeptides are in larger dense-core vesicles (100 to 200 nm in diameter), as noted above. This dual-packaging scheme allows the neuron some control over the relative release rates of its two types of transmitters (Fig. 13-18A). In general, low-frequency stimulation of the presynaptic terminal triggers the release of only the small transmitter (see Fig. 13-18B); co-release of both transmitters requires bursts of high-frequency stimulation (see Fig. 13-18C). This frequency sensitivity may result from the size and spatial profile of presynaptic [Ca2+]i levels achieved by the different patterns of stimulation. Presynaptic Ca2+ channels are located close to the vesicle fusion sites. Low frequencies of activation yield only localized elevations of [Ca2+]i, an amount sufficient to trigger the exocytosis of small vesicles near active zones. Larger peptide-filled vesicles are farther from active zones, and high-frequency stimulation may be necessary to achieve higher, more distributed elevations of [Ca2+]i. With this arrangement, it is obvious that the synaptic effect (resulting from the mixture of transmitters released) depends strongly on the way that the synapse is activated.

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FIGURE 13-18 Selective release of colocalized small transmitters and neuroactive peptides. A, The presynaptic terminal at rest is filled with small vesicles (containing small transmitter molecules) and large dense-core vesicles (containing neuroactive peptides). B, Fusion of small vesicles containing small transmitters. C, Fusion of large, dense-core vesicles.