The Major Excitatory Neurotransmitter
Long before glutamate’s role in neurotransmission was established, investigators observed that glutamate excites most neurons in the CNS. In fact, it is responsible for most neurotransmitter action at excitatory amino acid receptors. Glutamate is present in high concentrations in the adult CNS and is released in a Ca2+-dependent manner by electrical stimulation. Enzymes responsible for glutamate synthesis and degradation are located in both neurons and glial cells, as are high-affinity glutamate receptors and excitatory amino acid reuptake transporters (EAATs), which terminate the synaptic actions of glutamate. Many of these reuptake and receptor proteins also respond to aspartate, which may mediate transmission at a small number of central excitatory synapses.
Synthetic and Degradative Pathways
Glutamate and aspartate are charged amino acids and therefore do not cross the blood–brain barrier. As a result, they must be synthesized in the brain from glucose and a variety of other precursors. Glutamate, which is the reduced form of glutamic acid, is in a metabolic pool with α-oxoglutaric acid and glutamine. It is packaged into presynaptic vesicles via several steps 5–1. After action potential–driven release, glutamate is primarily taken up by glial cells, where it is converted into glutamine by glutamine synthetase. The resulting glutamine is transported out of glia by system N-1 (SN1), a Na+- and H+-dependent pump that is homologous to the vesicular GABA transporter (VGAT). Glutamine is subsequently taken up by neurons, by means of a transport process that remains poorly described, and is converted back into glutamate by glutaminase. As discussed later in this chapter, glutamine also replenishes the transmitter pool of GABA.
Synthesis and degradation of the neurotransmitter pool of glutamate. In the mitochondrial compartment of glutamatergic nerve terminals, glutamine (gln) is converted to glutamate (glut) by the enzyme glutaminase. Alternatively, glutamate is synthesized by the transamination of aspartate (asp) by transaminase. Newly synthesized glutamate is packaged into synaptic vesicles by means of a Mg2+- and ATP-dependent proton gradient–coupled uptake process. After it is released from nerve terminals, most of it is taken up into glial cells, where it is converted into glutamine by glutamine synthetase. Glutamine is then pumped out of the glia by SN1 and taken up by nerve terminals, where it is converted back to glutamate to replenish the transmitter pool. Uptake of glutamate into glial (and, to a lesser extent, neural) compartments is achieved by the Na+-dependent glutamate transporters EAAT1 to 3.
Synaptic vesicles actively accumulate glutamate through a Mg2+- and adenosine triphosphate (ATP)-dependent uptake process that is driven by an electrical gradient across their membranes. During uptake, concentrations of glutamate in these vesicles likely exceed 20 mM. Substances that destroy the electrochemical gradient across vesicular membranes prevent the transporters from concentrating this amino acid. The three isoforms of the vesicular glutamate transporter, VGluT1 to 3, are highly selective with a high affinity for glutamate and a low affinity for aspartate (Chapter 3).
After release from presynaptic terminals, the reuptake of glutamate and aspartate serves to control the extracellular concentrations of these amino acids in the CNS. Na+-dependent glutamate plasma membrane transporters are coupled to electrochemical gradients for Na+, K+, and H+ 5–1. Because such coupling permits the transport of glutamate and aspartate against their concentration gradients, glutamate transporters are capable of decreasing extracellular glutamate concentrations to submicromolar levels. These transporters take up glutamate and aspartate with similar affinity and maximum velocity (Vmax). Glutamate transport is electrogenic, resulting in the net inward movement of positive charge during each transport cycle.
Five principal members of the high-affinity Na+-dependent family of glutamate plasma membrane transporters have been cloned: EAAT1 (also known as glutamate–aspartate transporter [GLAST]), EAAT2 (glutamate transporter-1 [GLT-1]), EAAT3 (excitatory amino acid carrier-1 [EAAC1]), and EAAT4 and 5. Each of these transporters belongs to a large superfamily whose members transport a wide range of neurotransmitters and related substances (Chapter 3). EAAT1 and 2, which are expressed by glial cells, appear to be responsible for the majority of glutamate reuptake in the CNS (see 5–1).
Several pathologic conditions, such as ischemia, can lead to the accumulation of glutamate or aspartate in extracellular spaces, which results in the excessive activation of glutamate receptors (Chapters 18 and 20). Such activation can in turn cause a variety of pathologic changes and can, in its extreme form, result in cell death. Glutamate transporters limit the concentrations of free glutamate and aspartate in extracellular spaces, and thereby prevent the excessive stimulation of glutamate receptors. Consequently, agents capable of facilitating transporter function might limit the damage caused by ischemia and other neurologic insults that cause extracellular glutamate to increase. However, most progress to date has involved developing inhibitors of glutamate transporters. Two such inhibitors are D,L-threo-3-hydroxyaspartate (THA), a broad-spectrum antagonist of glutamate and aspartate transport, and dihydrokainate (DHK), an inhibitor selective for the glial EAAT2 transporter. Although these drugs are useful experimental tools, they have no apparent clinical use.
Glutamate receptors comprise two large families: the ionotropic and the metabotropic receptors. Ionotropic glutamate receptors contain associated ion channels that are gated by agonist binding. Three classes of ionotropic glutamate receptors, N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and kainate receptors, were originally named based on the ability of these drugs to serve as selective agonists 5–1.
5–1Glutamate Receptors ||Download (.pdf) 5–1 Glutamate Receptors
|Functional Classes ||Gene Families ||Agonists ||Antagonists|
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Metabotropic glutamate receptors (mGluRs) belong to the large superfamily of G protein–coupled receptors. These receptors, which are characterized by seven transmembrane domains (TMs), couple to G proteins and in turn mediate the biologic effects of receptor activation (Chapter 4). The term metabotropic was used to indicate that these receptors affect cellular biochemical (“metabolic”) processes, and do not form ion channels. However, mGluRs, like other G protein–coupled receptors, can exert profound effects on neuronal function through the regulation of other ion channels, second messenger cascades, and protein phosphorylation.
Mediation of fast excitatory transmission
Synaptically released glutamate interacts with postsynaptic receptors located on dendrites or nerve terminals of immediately adjacent cells or on the nerve terminals from which glutamate is released. The binding of glutamate to AMPA and NMDA receptors opens postsynaptic cation channels and initiates a two-component excitatory postsynaptic current (EPSC) at most central synapses 5–2. Considerable evidence suggests that AMPA and NMDA receptors colocalize at most functional excitatory synapses. However, the ratio of AMPA to NMDA receptors at individual synapses can vary greatly; indeed, some synapses may contain only NMDA or AMPA receptors. In contrast, smaller numbers of kainate receptors are present in most CNS regions.
Synaptically released glutamate interacts with postsynaptic AMPA and NMDA receptors. A. Excitatory postsynaptic currents (EPSCs) caused by glutamate in a hippocampal neuron. When glutamate binds to NMDA and AMPA receptors, postsynaptic cation channels open, initiating a two-component EPSC. These components can be pharmacologically separated. Blockade of NMDA receptors by the antagonist D-APV (left) reveals a rapid AMPA receptor–mediated component of the EPSC. Conversely, blockade of AMPA receptors by CNQX (right) reveals a slowly rising and decaying NMDA receptor–mediated component of the EPSC at positive voltages. In contrast, application of D-APV at negative voltages (lower left) reveals little NMDA receptor contribution to the EPSC due to the profound block of these channels by Mg2+ ions. B. The current–voltage relationship for AMPA receptors. This relationship is roughly linear and reverses at approximately 0 mV, indicating that the channels do not discriminate well between Na+ and K+ ions. C. The current–voltage relationship for NMDA receptors. The concentration of Mg2+ in the extracellular fluid of the brain (approximately 1 mM) is sufficient to virtually abolish ion flux through NMDA receptor channels at membrane potentials close to resting potential (approximately −70 mV). As the membrane potential becomes less negative or even positive, the affinity of Mg2+ for its binding site decreases, permitting the passage of ionic current. Removal of extracellular Mg2+ linearizes the current–voltage relationship and permits significant current flow at negative voltages.
The activation of an AMPA receptor mediates a synaptic current that has a rapid onset and decay, whereas the current mediated by an NMDA receptor has a slower onset and a decay that lasts as long as several hundred milliseconds (see 5–2). The decay time of the NMDA receptor–mediated current is approximately 100 times longer than the mean open time of its channel. Such prolonged activation is believed to be caused by glutamate’s high affinity (Kd = 3–8 μM) for and consequent slow dissociation from these receptors. In contrast, glutamate has a much lower affinity for AMPA receptors (Kd = 200 μM), from which it rapidly dissociates.
AMPA receptors respond to single vesicles of glutamate. NMDA receptors, however, because of their special voltage dependence (described later in this chapter), commonly require coordinated input from many synapses for substantial activation. This allows NMDA receptors to act as coincidence detectors that can sense the activity of many independent synaptic inputs converging on the same cell 5–3.
The NMDA glutamate receptor as a coincidence detector. A. A single synaptic input results in the generation of a short-lasting excitatory postsynaptic potential (EPSP) that is mediated entirely by AMPA receptors. B. When multiple inputs occur simultaneously, the same single synaptic input generates a longer-lasting EPSP that is mediated by both AMPA and NMDA receptors. Thus, the NMDA receptor can “sense” the activity in adjacent inputs.
Current is carried through AMPA receptors primarily by the movement of Na+ from the extracellular space into the intracellular compartment. However, because the reversal potential of current (the membrane potential at which net current flow is zero; see Chapter 2) through AMPA channels is close to 0 mV, an outward current carried by K+ must counterbalance the inward flow of Na+ ions. The resulting current–voltage relationship for these AMPA receptors is roughly linear (see 5–2).
Some AMPA receptors, on both neurons and astrocytes, are also permeable to Ca2+. The translocation of Ca2+ from the extracellular space to the intracellular compartment plays a key role in the regulation of several second messenger systems. Thus, the permeability of some AMPA receptors to Ca2+ may have great functional importance, particularly in cells that do not contain NMDA receptors (which can always flux Ca2+). AMPA receptor channels that are permeable to Ca2+ lack the GluA2 subunit (see below) and exhibit an inwardly rectifying type of current–voltage relationship; they pass current readily in the inward direction but not in the outward direction. This occurs because intracellular polyamines such as spermine and spermidine, which associate with the channel, prohibit outward current from passing through. A spermine analogue, N(1)-acetylspermine (NASPM), a selective antagonist of GluA2-lacking AMPA receptors, is a valuable tool to study the influence of such receptors in brain function and plasticity.
All AMPA receptors can be blocked selectively by certain quinoxaline diones, the most notable of which is 6-nitro-7-sulfamobenzo-quinoxaline 2,3-dione (NBQX; see 5–1). NBQX is a potent competitive antagonist of AMPA receptors, which is reasonably selective for AMPA receptors over kainate receptors. Drugs in the 2,3-benzodiazepine class, such as GYKI 53655, are noncompetitive, selective antagonists of AMPA receptors and are being explored as neuroprotective drugs for the treatment of stroke. Because it has minimal effects on kainate or NMDA receptors, this class of drug permits unequivocal separation of the AMPA receptors from other categories of glutamate receptors.
AMPA and kainite receptors desensitize within milliseconds of exposure to agonist and can be reliably distinguished from one another by their response to two drugs, cyclothiazide and the lectin concanavalin A. Cyclothiazide relieves AMPA receptor desensitization but does not affect kainate receptors. In contrast, concanavalin A relieves the desensitization of kainate receptors, most likely by interacting with surface sugar chains, but does not have significant effects on AMPA receptors.
The possibility that enhancement of AMPA receptor function may be beneficial therapeutically has received increased attention with the development of positive allosteric modulators (PAMs) of AMPA receptors, also termed AMPAkines or AMPA potentiators. These drugs enhance AMPA receptor–mediated excitatory synaptic transmission by slowing deactivation or desensitization of the receptors, with the former mechanism appearing to be more functionally important. Clinical trials are currently being conducted in a wide range of neuropsychiatric disorders (eg, Alzheimer disease, depression, and attention deficit hyperactivity disorder [ADHD]) to determine whether this class of compound may be clinically useful.
Molecular composition of AMPA receptors
Three families of ionotropic glutamate receptor subunits, encoded by at least 16 genes, assemble to form functional AMPA, NMDA, or kainate receptors (see 5–1). Within each family there is greater than 80% identity at the amino acid level over membrane-spanning domains. Between families, a lower degree of identity exists (~50%). Ionotropic glutamate receptors are believed to exist as tetramers. Different subunit combinations produce functionally different glutamate receptors. Moreover, there are striking regional differences in the expression of genes that encode these subunits.
Four subunits, termed GluA1 to GluA4 (also known as GluR1–GluR4), coassemble to form AMPA receptors. Each of these subunits is encoded by a distinct gene, and each exists in two forms, termed flip and flop. The flip and flop forms, which are generated by alternative splicing, exhibit region-specific patterns of expression in the brain, and give rise to receptors that differ in desensitization rates. The best characterized AMPA receptor subunit is GluA1, which is predicted to be 889 amino acids long; other ionotropic receptor subunits, such as those of nicotinic, GABAA, or glycine receptors, are approximately 420 amino acids long. The extra length of GluRs results from unusually large N-terminal extracellular domains.
AMPA and kainate glutamate receptor families have a topology that differs from that of certain other ligand-gated ion channels, such as GABAA and glycine receptors 5–4. Glutamate receptor subunits possess only three transmembrane-spanning domains. What appears to be an abridged TM, located between TM2 and TM3 in glutamate subunits, is a reentrant loop whose both ends face the cytoplasm. Homology mapping of glutamate receptors suggests that agonist binding requires portions of both the large N terminus and the short region between TM1 and TM2 (see 5–4).
Membrane topology of AMPA glutamate, GABAA, and glycine receptors. A. AMPA glutamate receptor subunits (GluA1–4) possess only three transmembrane-spanning domains. The channel-lining domain between TM1 and TM2 is a reentrant loop with both ends facing the cytoplasm. The Q/R site, which controls the Ca2+ permeability of AMPA receptor subunits, is located in this loop. The flip–flop site, located extracellularly between TM2 and TM3, yields two splice variants of each subunit. The glutamate-binding site of AMPA receptors is formed by several amino acids in the N-terminal and extracellular loop. B. In contrast to AMPA receptors, GABAA and glycine receptors possess four putative membrane-spanning domains.
As stated earlier, AMPA receptors lacking the GluA2 subunit are highly permeable to Ca2+ ions and are commonly found on GABAergic inhibitory interneurons throughout the brain. This Ca2+ permeability has been traced to a single amino acid in the reentrant loop of GluA2, which is known as the Q/R site (see 5–4). In GluA1, GluA3, and GluA4, a glutamine (Q) resides at this position, but in GluA2 an arginine (R) is present. When heteromeric receptors contain GluA2, they are relatively impermeable to Ca2+, most likely because the positive charge of the arginine residue repels Ca2+ from the channel pore. The replacement of glutamine with arginine in GluA2 occurs because of a process called RNA editing: the gene for GluA2 encodes a glutamine, and the unedited form of GluA2 is expressed in various regions of the brain during development. However, virtually all of the GluA2 mRNA present in adult mammalian brain is edited to a codon that encodes arginine at this site. This editing, which is catalyzed by adenosine deaminases acting on RNA (ADARs), changes a single base within GluA2 transcripts. In addition to Ca2+ permeability, the Q/R site of GluA2 influences the single channel conductance properties of associated AMPA receptors and the sensitivity of the receptor complex to blockage by polyamine spider toxins and polyamines (eg, NASPM). AMPA receptors that do not contain edited GluA2 show greater overall conductance. In addition, there is voltage-dependent inhibition of outward current flow by endogenous intracellular polyamines at positive membrane potentials giving rise to the inward rectification that is characteristic of GluA2-lacking AMPA receptors. A deficiency in GluA2 editing has been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS), acting perhaps by increasing Ca2+ flux into motor neurons and promoting excitotoxicity (Chapter 18).
The flip and flop splice variants of receptor subunits GluA1 to GluA4, together with the products of Q/R editing, yield a wide range of AMPA receptor subunit proteins. This diversity provides neurons with an extraordinary degree of flexibility in the construction of AMPA receptors. The degree of AMPA receptor heterogeneity employed by neurons in vivo remains unknown. Ca2+-permeable AMPA receptors lacking the GluA2 subunit are consistently found at excitatory synapses on GABAergic inhibitory interneurons, whereas most principal cells (ie, projection neurons) express Ca2+-impermeable AMPA receptors that contain the edited form of GluA2. There is growing evidence that the number of GluA2-lacking AMPA receptors at particular synapses is subject to dynamic regulation and may be an important mechanism of neural and behavioral plasticity.
Kainate receptors, like AMPA receptors, are cation-selective ligand-gated ion channels that strongly depolarize neurons when activated. They are found on presynaptic terminals, of both excitatory and inhibitory synapses, where their activation can modify neurotransmitter release, both because of their depolarizing actions and because, depending on their molecular composition, they are permeable to Ca2+. Depending on the synapse and the degree of activation, presynaptic kainate receptors can either facilitate or depress transmitter release. Kainate receptors are also found postsynaptically on certain neurons, where they normally generate slow, small, but functionally important, postsynaptic potentials. In the hippocampus and cortex, they may be particularly important in the early development of neural circuits. Recent data suggest that kainate receptors can also be metabotropic, initiating a G protein signaling cascade independent of their ionotropic signaling.
Kainate receptors are composed of a distinct array of subunits. The subunits GluK1 to GluK3 coassemble with GluK4 or GluK5 subunits to form functional kainate receptors. Homomeric GluK1, GluK2, and GluK3 receptors expressed in mammalian cell lines bind kainate with an affinity of approximately 80 to 100 nM. These homomeric receptors may correspond to low-affinity kainate binding sites previously identified in membrane fractions of the brain. In contrast, homomeric GluK4 receptors bind kainate with an affinity of 4 nM and may correspond to high-affinity kainate binding sites in the brain. However, when they are expressed alone, GluK4 and GluK5 are virtually inactive because they lack functional channels. Consequently, it is believed that they serve as modulatory subunits that confer high-affinity kainate binding on channels formed by GluK1 to GluK3. GluK1 and GluK2 exist in several splice variants, and also undergo Q/R editing, meaning that kainate channels can vary in their Ca2+ permeability.
Selective kainate receptor antagonists are under development; those currently available, such as LY382884 5–1, are primarily selective for the GluK1 subunit. LU97175 generally antagonizes kainate receptors, although it is most potent for GluK3-containing receptors. SYM 2081 is potent agonist for GluK1- and GluK2-containing kainate receptors, and rapidly desensitizes the receptors; it can therefore be used as an antagonist. However, GYKI 53655 remains the most valuable agent for determining whether a given synaptic current is mediated by AMPA or kainate receptors, as stated earlier. Domoic acid, derived from red algae and implicated as a cause of shellfish poisoning, acts as a neurotoxin via activation of kainate receptors.
Kainate receptors are believed to contribute to the development of temporal lobe epilepsy, and genetic studies have linked alleles of GluK2 to Huntington disease (in which it might be a disease modifier). However, the role of kainate receptors in normal neuronal function, let alone in the development of complex neuropathologies, remains uncertain.
NMDA receptors have several properties that set them apart from other ligand-gated receptors. At membrane potentials more negative than approximately −50 mV, the Mg2+ in the extracellular fluid of the brain virtually abolishes ion flux through NMDA receptor channels, even in the presence of glutamate. Thus, at the resting membrane potentials that are typical of most neurons, approximately −60 to −70 mV, the activation of these receptors results in little current flow. This is because the entry of Mg2+ into the channel pore blocks the movement of monovalent ions across the channel.
In the presence of Mg2+ ions, the receptor’s current–voltage relationship has a region of slope negativity that produces a characteristic J shape when plotted (see 5–2). As the receptor’s membrane potential becomes less negative (more depolarized), the affinity of Mg2+ for its binding site decreases, and the blocking action of Mg2+ becomes ineffective; consequently, ionic current can pass through the channel as the cell membrane depolarizes.
As previously mentioned, NMDA receptors can be thought of as coincidence detectors capable of sensing simultaneous activity at a number of adjacent synapses (see 5–3). They possess this capability because they function (ie, pass current) only when they are stimulated by presynaptically released glutamate at a time when the postsynaptic cell is depolarized. Repetitive stimulation is often required because the depolarization produced by single inputs is not sufficient to relieve the blockage of the NMDA receptor channel by Mg2+.
The activation of NMDA receptors, like that of AMPA receptors, produces a nonspecific increase in permeability to the monovalent cations Na+ and K+. However, unlike most AMPA and kainate receptors in the adult CNS, NMDA receptors are highly permeable to Ca2+. Although their activation results in appreciable current and tends to depolarize the cell membrane toward the threshold for action potential firing, such activity is unlikely to represent the primary function of these receptors. Instead, NMDA receptors likely provide one of the most significant mechanisms by which synaptic activity can increase the level of intracellular Ca2+ at individual synapses. The major endogenous agonist for NMDA receptors is glutamate itself, although there is some evidence that aspartate can also activate this receptor. Numerous competitive antagonists of the agonist recognition site are available, notably D-2-amino-5-phosphonopentanoic acid (AP-5 or APV) and 3-(2-carboxypiperazin-4-yl)-1-propenyl-1-phosphonic acid (CPP). These compounds are not useful clinically because they are polar and penetrate the blood–brain barrier poorly.
The NMDA receptor is unique among all neurotransmitter receptors in that its activation requires the simultaneous binding of two different agonists. In addition to the binding of glutamate at the conventional agonist-binding site, the binding of glycine appears to be required for receptor activation 5–5. Because neither of these agonists alone can open this ion channel, glutamate and glycine are referred to as coagonists of the NMDA receptor. The physiologic significance of the glycine-binding site is unclear; however, there is some evidence to suggest that D-serine may be the endogenous agonist for this site. D-Serine, made through the conversion of L-serine by serine racemase, is subject to regulated release and specific reuptake primarily from glial cells. Thus, the glial environment of neurons may have a critical influence on NMDA receptor synaptic function.
Pharmacologic binding sites of the NMDA receptor. Sites for drugs that promote receptor function (Na+ and Ca2+ influx) appear in green, and sites for drugs that inhibit receptor function appear in red. 5,7-DCK, 5,7-dichlorokynurenic acid.
Regardless of its physiologic role, the glycine site on the NMDA receptor may prove to be an important drug target. Cycloserine, originally developed as an antitubercular drug, is a weak partial agonist at this site and thus can modulate NMDA receptor function in vitro and in vivo. Cycloserine is reported to enhance the effects of antipsychotic drugs in patients with schizophrenia. More specific agonists, such as HA966, have been used in laboratory animals but are not yet available for clinical investigation. Derivatives of kynurenic acid, such as 5,7-dichlorokynurenic acid (5,7-DCK) and quinolinecarboxylic acid, are also competitive antagonists at the glycine site. It is important to note that the glycine site on NMDA receptors is distinct from the strychnine-sensitive glycine receptor, which mediates the independent neurotransmitter functions of glycine (see below).
Another important site on the NMDA glutamate receptor binds phencyclidine (PCP) and related drugs such as MK801 and ketamine 5–5. These drugs, which bind at or near the Mg2+-binding site, occlude the NMDA receptor channel. Thus, they act as noncompetitive receptor antagonists, and their actions, like those of Mg2+, are somewhat voltage dependent. These drugs exert potent effects on the brain. At higher concentrations they are psychotomimetic and produce effects, such as cognitive impairment, hallucinations, and delusions that are similar to some of the symptoms of schizophrenia (Chapter 17). These effects have led to the hypothesis that schizophrenia may involve hypofunction of NMDA receptors, which has in turn prompted the development of drugs that block the reuptake of glycine for the treatment of schizophrenia. The idea is that glycine transporter-1 (GlyT1) inhibitors would increase levels of extracellular glycine in forebrain and thereby facilitate activation of NMDA receptors. At still higher doses, drugs such as PCP and ketamine are dissociative anesthetics, used primarily in veterinary and pediatric practice. Interestingly, doses of ethanol associated with the upper range of intoxication in humans exert effects on NMDA receptors that are similar to those produced by PCP, ketamine, and related drugs. More recent research has shown that lower doses of ketamine, devoid of most psychotomimetic effects, exert rapidly acting antidepressant effects in a subset of individuals with treatment-resistant depression (Chapter 15).
NMDA receptors also have one or more modulatory sites that bind polyamines. The occupancy of one of these sites relieves tonic proton block and thereby potentiates NMDA receptor activation. At higher concentrations, however, polyamines act on an extracellular site to produce a voltage-dependent block of the ion channel and consequently inhibit receptor activation. Many additional drugs, such as amantadine, dextromethorphan, and memantine (Chapter 18), are weak antagonists of NMDA receptors, although it remains uncertain as to whether such actions contribute to the clinical utility of these medications.
Because they mediate a large number of important physiologic functions, and also contribute to cell damage and death when they are overactivated (Chapters 18 and 20), NMDA receptors have been a prominent target for therapeutic drugs. Many drugs that target these receptors have been developed and tested in clinical trials for conditions such as stroke or head trauma in which excitotoxicity may play a significant role. However, compounds with clear efficacy and tolerable side effects have yet to be identified. The large number of modulatory sites on the NMDA receptor increases the likelihood that clinically useful compounds will be discovered.
Molecular composition of NMDA receptors
Two families of NMDA receptor subunits have been identified. One family is represented by a single gene (GluN1) that encodes proteins composed of approximately 900 amino acids; the other is represented by four genes (GluN2A–2D; see 5–1) that encode proteins composed of approximately 1450 amino acids. Although homomeric GluN1 receptors appear to possess all of the pharmacologic features characteristic of bona fide NMDA receptors, the physiologically relevant glutamate-binding site is probably located on the GluN2 subunit. Moreover, the very small currents supported by homomeric GluN1 receptors increase by more than 100-fold when such receptors are coexpressed with GluN2 subunits. It is therefore believed that NMDA receptors in the brain exist as GluN1–N2 heteromeric complexes. Some neurons also express the GluN3A or GluN3B subunit, which modulates NMDA receptor function, decreasing both the conductance and the Ca2+ permeability of the channel. The physiologic significance of the GluN3 subunit is not understood.
More than nine splice variants of GluN1 have been cloned. These variants differ with regard to their regional patterns of expression in the CNS, their regulation (eg, by phosphorylation, polyamines, and Zn2+), the electrophysiologic properties of channels they form, and their affinity for elements of the neuronal cytoskeleton. Such distinguishing features suggest that these variants may be involved in different components of the synapse.
The subtype of GluN2 subunit that combines with GluN1 subunits influences the biophysical and pharmacologic properties of endogenous NMDA receptors. During early postnatal development, many synaptic NMDA receptors are composed of GluN1 and GluN2B. This subunit combination yields a receptor that produces very long-lasting synaptic responses and one that is strongly inhibited by the NMDA receptor antagonist ifenprodil. Gradually during the first few weeks of brain development, it is thought that the GluN2B subunit is largely replaced by GluN2A (and perhaps GluN2C), yielding a receptor that produces shorter synaptic currents and that is no longer sensitive to ifenprodil. There is also evidence that different synapses in the adult brain preferentially express GluN1–N2A versus GluN1–N2B NMDA receptors, which makes the development of selective agonists and antagonists a high priority. Indeed, selective antagonists of GluN2B-containing NMDA receptors are being evaluated for the treatment of several neuropsychiatric disorders.
Metabotropic Glutamate Receptors
Eight mGluRs, termed mGlu1 to mGlu8, have been cloned (see 5–1). mGluRs are considerably larger than other G protein–coupled receptors, and a comparison of their amino acid sequences with those of other receptors reveals little homology or common features. mGluRs are therefore considered to constitute a separate family of receptors. Like other G protein–coupled receptors, mGluRs contain seven membrane-spanning domains; however, like the ionotropic receptors, they also possess an unusually large N-terminal extracellular domain that precedes the membrane-spanning segments 5–6.
Proposed membrane topology of mGluRs and GABAB receptors. mGluRs and GABAB receptors contain seven transmembrane domains and are members of the G protein–coupled receptor superfamily. These receptors can be distinguished from most other G protein–coupled receptors by their unusually large extracellular N-terminal domains. In mGluRs, the second intracellular loop and the intracellular C-terminal domain determine the specificity of G protein coupling.
There are three functional groups of mGluRs based on amino acid sequence homology, agonist pharmacology, and the signal transduction pathways to which they are coupled. Group I includes mGlu1 and mGlu5, which are generally found on postsynaptic neurons adjacent to excitatory synapses. Group II is composed of mGlu2 and mGlu3, while group III comprises mGlu4, mGlu6, mGlu7, and mGlu8. Group II and III mGluRs are often found on presynaptic terminals where they modulate transmitter release. Members of each group share approximately 70% sequence homology, with approximately 45% homology exhibited between groups. Alternatively spliced variants also have been described for mGlu1, mGlu4, mGlu5, and mGlu7.
The classification of mGluRs into three groups is supported by their signal transduction mechanisms. Group I mGluRs stimulate phospholipase C activity by means of the G protein Gq, and thereby release Ca2+ from cytoplasmic stores through IP3 (Chapter 4). Yet group I mGluRs vary in their ability to increase intracellular Ca2+ levels, most likely because each receptor has a different affinity for Gq. Activation of phospholipase C leads to the formation of not only IP3 but also diacylglycerol, which, in conjunction with increases in intracellular Ca2+, activates protein kinase C. In contrast to group I mGluRs, group II and III mGluRs inhibit adenylyl cyclase and regulate specific K+ and Ca2+ ion channels, actions believed to be mediated via coupling to Gi/o.
Glutamate activates all of the mGluRs, with potencies that range from 2 nM for mGlu8 to 1 μM for mGlu7. Highly selective agonists for each of the three groups also have been identified 5–1. 3,5-Dihydroxyphenylglycine (DHPG) appears to be a selective group I agonist; LY354740 is a highly selective agonist for group II mGluRs, and L-amino-4-phosphonobutyrate (L-AP4) is a selective agonist of the group III mGluRs. mGluR antagonists have been developed for the various subtypes, but few antagonize a whole group. LY341495 at low concentrations will inhibit group II mGluRs, but at high concentrations will inhibit all mGluRs. 2-Methyl-6-(phenylethynyl)-pyridine (MPEP) is a somewhat selective group I antagonist. Positive and negative allosteric modulators of specific mGluRs (termed mGluR PAMs or NAMs) are also in development.
Modulation of ion channel activity
mGluRs located on the postsynaptic membrane modulate a variety of ligand- and voltage-gated ion channels expressed on central neurons 5–7. The activation of each of the three groups of mGluRs has been found to inhibit L-type voltage-gated Ca2+ channels, and groups I and II are capable of inhibiting N-type Ca2+ channels. Additionally, mGluR activation can close voltage-gated K+ channels, resulting in a slow depolarization and neuronal excitation. The exact mechanism by which mGluRs inhibit K+ currents is not yet clear. In cerebellar granule cells, mGluRs increase the activity of Ca2+-dependent K+ channels, termed BK channels, and thereby reduce cell excitability. In some cells, mGluRs also activate G protein–coupled, inwardly rectifying K+ (GIRK) channels. Thus, postsynaptic mGluRs can have a wide range of effects, depending on the cell type involved and the mGluR subtype that is activated.
Typical excitatory (glutamate) synapse. AMPA and NMDA receptors (which are ionotropic) and mGluRs (which are G protein–coupled) are all localized in the postsynaptic spine but have different subcellular locations (not depicted in figure). mGluRs are also located on the presynaptic terminal, where they act as inhibitory autoreceptors, in part by reducing Ca2+ influx. Not shown are kainate receptors, which can have either postsynaptic or presynaptic locations, depending on the synapse. Prototypical drugs that affect the functioning of excitatory synapses are shown in blue boxes; green lines depict activators, and red lines depict inhibitors.
mGluR-mediated presynaptic inhibition at excitatory synapses
Several types of mGluRs are located on the presynaptic terminals of central neurons, and the activation of presynaptic mGluRs blocks both excitatory glutamatergic and inhibitory GABAergic synaptic transmission in many CNS regions 5–7. In the hippocampus, mossy fiber-evoked EPSPs on CA3 pyramidal neurons are blocked by DCG-IV, which acts on presynaptic group II mGluRs located on granule cell terminals. Similar effects have been observed throughout the cerebral neocortex. In contrast, synaptic transmission between Schaeffer collaterals and CA1 pyramidal cells is resistant to DCG-IV but is reduced by L-AP4, a group III mGluRs agonist. One mechanism by which activation of mGluRs decreases neurotransmitter release may involve the inhibition of voltage-gated Ca2+ channels on the presynaptic nerve terminal membrane. Such activity has been observed directly at certain glutamatergic synapses, where mGluR agonists suppress voltage-gated P/Q-type Ca2+ channels and thereby inhibit transmitter release. Accordingly, mGluRs most likely function as inhibitory autoreceptors at many glutamatergic nerve terminals.
The numerous effects that mGluRs can have on both postsynaptic cells and presynaptic terminals are confusing, especially because the conditions under which these receptors are physiologically activated are not always clear. Yet the diversity that characterizes mGluRs and their actions promises to further the development of subtype-specific drugs for the treatment of neuropsychiatric disorders. Several drugs that target specific mGluRs are under evaluation for the treatment of a variety of brain disorders; however, none have yet proved clearly efficacious in large clinical trials.
Synaptic Clustering of Glutamate Receptors
Effective synaptic communication requires the precise localization of high concentrations of appropriate presynaptic and postsynaptic proteins at the synapse. On the postsynaptic side of the synapse, neurons must be able to target glutamate receptors to excitatory but not inhibitory synapses and must ensure that the receptors are appropriately clustered opposite presynaptic terminals that release glutamate. Several members of the protein family involved in this clustering contain single or multiple copies of an amino acid sequence termed the PDZ domain, which is important for mediating many important protein–protein interactions 5–1. An evolving hypothesis is that each subtype of glutamate receptor interacts with distinct proteins at the synapse. Accordingly, the subcellular localization of each type of receptor may be independently regulated under physiologic conditions. Although these mechanisms remain poorly understood, they appear to be important for certain forms of neural plasticity.
5–1 PDZ Proteins and Synaptic Clustering of Glutamate Receptors
Immunocytochemical and ultrastructural studies have revealed that individual excitatory synapses can contain profoundly different densities of AMPA, NMDA, kainite, and metabotropic glutamate receptors. Furthermore, their localization in dendritic spines usually differs. The ionotropic receptors (AMPA and NMDA) are located in the central part of the postsynaptic density opposite presynaptic release sites. In contrast, mGluRs are located at the periphery of synapses. This subsynaptic segregation of ionotropic and metabotropic receptors may permit the differential activation of these receptors based on patterns of presynaptic activity. A series of proteins have been identified in recent years that assist in clustering glutamate receptors at synapses. Most of these contain several PDZ domains, structures known to mediate many intracellular protein–protein interactions.
The first of these proteins to be isolated was postsynaptic density protein of 95 kDa (PSD-95); as its name implies, this protein occurs in high concentrations in the postsynaptic density, the most prominent structural specialization of the postsynaptic membrane of excitatory synapses. PSD-95 is a member of a family of related scaffolding proteins, including PSD-93, SAP-97, and SAP-102. These proteins all contain three PDZ domains, two of which can interact with the C-terminus of NMDA receptors. PSD-95 and related family members are believed to be critical components of the molecular mechanism responsible for the clustering of NMDA and AMPA receptors at synapses (see figure). PSD-95 also binds neuroligins, which—with neurexins—control the formation of synapses.
In contrast to NMDA receptors, AMPA receptors and mGluRs do not bind directly to PSD-95 but instead bind to their cognate PDZ-containing proteins. AMPA receptors bind to a member of a protein family known as transmembrane AMPA receptor regulatory proteins (TARPs). TARPs link AMPA receptors to PSD-95, and regulate the trafficking of AMPA receptors to the cell surface and the synapse. This trafficking has been shown to be critical for certain forms of synaptic plasticity, such as long-term potentiation (LTP) 5–8. Other proteins regulate AMPA receptor trafficking, such as glutamate receptor interacting protein-1 (GRIP1). Homer, a protein that binds to the C-terminus of group I mGluRs, contains a single PDZ-like domain. Interestingly, expression of some splice variants of Homer can be regulated by neuronal activity; thus, nerve impulses may influence the efficacy of mGluR synaptic transmission by regulating receptor clustering at physiologic sites. Interactions between mGluRs and NMDA receptors are mediated via still additional linker proteins, such as shank proteins and guanylate kinase–associated protein (GKAP). Mutations in several of these postsynaptic proteins have been implicated in autism (Chapter 14) and schizophrenia and bipolar disorder (Chapter 17).
Role of Glutamate in Neural Plasticity
The high permeability of NMDA receptor channels to divalent cations, especially to Ca2+, has many implications for cell function. The concentration of Ca2+ in the cell interior typically is heavily buffered to approximately 100 nM and is tightly regulated by several mechanisms, including storage in intracellular organelles. Ca2+ entry through NMDA receptor channels can lead to a transient increase in intracellular Ca2+ concentrations to the micromolar range. Such an increase can in turn result in the activation of many Ca2+-dependent enzymes, including Ca2+/calmodulin-dependent protein kinases (CaM-kinases), calcineurin (protein phosphatase 2B or PPP3), protein kinase C, phospholipase A2, phospholipase C, nitric oxide synthase, and several proteases (Chapter 4).
One of the most important consequences of NMDA receptor activation is the generation of often long-lasting changes in synaptic function termed synaptic plasticity. Many forms of synaptic plasticity have been discovered in the mammalian CNS, but long-term potentiation (LTP) and long-term depression (LTD) of excitatory synaptic responses in CA1 pyramidal cells in the hippocampus have been the most extensively characterized. LTP and LTD are activity-dependent alterations in synaptic efficacy that can last weeks or months in vivo. Changes in receptor number (eg, via altered receptor trafficking 5–1 or transcription) or changes in receptor function (eg, due to phosphorylation 5–2) contribute to these changes in synaptic strength. The bidirectional control of synaptic strength by LTP and LTD is believed to be important for many forms of experience-dependent plasticity including some forms of learning and memory in the mammalian brain.
5–2 Modulation of Glutamate and GABA Receptors by Phosphorylation
Among synapses in the brain, the most ubiquitous are excitatory synapses that utilize glutamate and inhibitory synapses that utilize GABA. Consequently, the modulation of glutamate and GABA receptors by intracellular second messenger cascades is believed to be of paramount importance for a host of normal brain functions.
The functioning of all subtypes of ionotropic glutamate receptors, including that of AMPA, NMDA, and kainate receptors, can be altered by phosphorylation. The AMPA receptor subunit GluA1 is phosphorylated by three major protein serine–threonine kinases: protein kinase A, protein kinase C, and Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II). All three kinases increase the current elicited by agonist activation of GluA1-containing AMPA receptors by phosphorylating distinct residues in their intracellular C-terminus. Similarly, when the kainate receptor subunit GluK2 is phosphorylated by protein kinase A, these receptors become more responsive.
Phosphorylation/dephosphorylation of these receptors or closely associated proteins (see 5–1) also can dramatically influence their trafficking to synapses or their removal from synapses. Such modulation may underlie activity-dependent forms of synaptic plasticity believed to be involved in learning and memory 5–8.
The modulation of NMDA receptor function appears to involve both serine–threonine kinases and protein tyrosine kinases. Activation of the tyrosine kinase Src, for example, causes an increase in NMDA-induced currents. The biochemical mechanism responsible for this process is unclear but may involve phosphorylation of the intracellular C-terminus of GluN2A (and perhaps GluN2B). Protein kinase C also has been found to enhance NMDA receptor function, and to disrupt the clustering of NMDA receptors, perhaps by interfering with the interaction between cytoskeletal elements and the C-terminus of GluN1. Protein kinase A, on the other hand, can affect the Ca2+ permeability of NMDA receptors.
GABAA receptors are phosphorylated by at least two different protein serine–threonine kinases: protein kinase C and protein kinase A. In most studies, protein kinase C inhibits GABAA receptor function, in part by phosphorylating serine residues on β1 and γ2 subunits. The effects of protein kinase A on GABAA receptors are more variable. Protein kinase A can phosphorylate the same serine on the β1 subunit as protein kinase C and thereby attenuate GABAA receptor–mediated currents; however, the biophysical consequences of protein kinase A phosphorylation depend on the exact subunit composition of the GABAA receptor. In certain cell types, such as cerebellar Purkinje and retinal bipolar cells, the activation of protein kinase A potentiates GABA-mediated responses.
Because G protein–coupled receptors are linked to the activation or inhibition of protein kinases, many types of neurotransmitters, such as acetylcholine and several neuropeptides, can modulate glutamate and GABA receptor function. Similarly, changes in the amount of intracellular Ca2+ can regulate protein phosphorylation cascades and thereby modify receptor function. Furthermore, the phosphorylation states of these receptors are controlled by protein phosphatases, the inhibition of which often mimics the effects of increased protein kinase activity.
The best understood form of long-lasting synaptic plasticity is NMDA receptor–dependent LTP, which is found in many regions of the mammalian brain but has been most extensively studied in the hippocampus because of its key role in learning and memory (Chapter 14). The triggering of this form of LTP requires activation of NMDA receptors by synaptically released glutamate when the postsynaptic membrane is already strongly depolarized. This depolarization relieves the voltage-dependent block of the NMDA receptor channel by Mg2+, and allows Ca2+ to enter the postsynaptic dendritic spine when the receptor is activated by glutamate. The rise in postsynaptic Ca2+ concentration, the critical trigger for LTP, activates complex intracellular signaling cascades that include several protein kinases, most notably CaM-kinase II 5–8A. The primary mechanism underlying the increase in synaptic strength during LTP is a change in AMPA receptor trafficking that results in an increased number of AMPA receptors in the postsynaptic plasma membrane. Within a few hours, the maintenance of LTP requires protein synthesis and there is good evidence that LTP is accompanied by observable enlargements of dendritic spines. Such structural changes may be essential to cement the information storage process initiated at synapses on LTP induction.
Major forms of LTP and LTD. Highly simplified diagrams of the induction and expression mechanisms thought to underlie some major forms of LTP and LTD. A. NMDA receptor–dependent LTP is dependent on postsynaptic NMDA receptor activation leading to a rise in Ca2+ and activation of CaM-kinase II (CaMKII) for its initiation. AMPA receptor insertion into the postsynaptic membrane is a major mechanism underlying LTP expression. B. NMDA receptor–dependent LTD is triggered by a modest Ca2+ entry through postsynaptic NMDA receptor channels leading to increases in the activity of the protein phosphatases calcineurin and protein phosphatase 1 (PPP1). The primary expression mechanism involves internalization of postsynaptic AMPARs. C. mGluR-dependent LTD is due to activation of postsynaptic mGluR1/5 leading to the internalization of postsynaptic AMPARs, a process that under some conditions requires protein synthesis. D. Presynaptic LTP is triggered by a large rise in presynaptic Ca2+, which activates Ca2+-sensitive adenylyl cyclases (AC) leading to a rise in cAMP and activation of PKA. This in turn modifies the functions of unknown proteins causing a long-lasting increase in glutamate release. E. Endocannabinoid (eCB) LTD is commonly triggered by increases in intracellular Ca2+ levels, either by mGluR1/5-mediated activation of phospholipase C (PLC) or by activation of voltage-gated Ca2+ channels, in the postsynaptic neuron. This triggers the synthesis and release of an eCB that travels retrogradely to bind to presynaptic cannabinoid 1 receptors (CB1). This prolonged activation of CB1 depresses neurotransmitter release via unknown mechanisms (Chapter 8). (Adapted with permission from Kauer JA, Malenka RC. Synaptic plasticity and addiction. Nature Rev Neurosci. 2007;8(11):844–858.)
At many excitatory synapses throughout the brain, weaker activation of NMDA receptors can elicit the opposite phenomenon, NMDA receptor–dependent LTD, which is thought to result from a smaller rise in postsynaptic Ca2+ than is required for LTP 5–8B. This more modest change in postsynaptic Ca2+ activates serine/threonine phosphatases, which dephosphorylate critical synaptic substrates including AMPA receptors themselves. The depression of synaptic strength during NMDA receptor–dependent LTD is due to the removal of synaptic AMPA receptors via dynamin- and clathrin-dependent endocytosis. An intriguing feature of NMDA receptor–dependent LTD is that NMDA receptor–mediated synaptic responses are also depressed via mechanisms distinct from those responsible for the LTD of AMPA receptor–mediated responses.
Surprisingly, activation of postsynaptic mGluRs can also lead to a form of LTD that was first described in the cerebellum but also occurs in the hippocampus and neocortex. At the cerebellar parallel fiber synapse, LTD requires both postsynaptic Ca2+ influx through voltage-gated Ca2+ channels and postsynaptic group I mGluR activation, while at other synapses activation of postsynaptic mGluRs alone appears to be sufficient. In most cases, however, this form of LTD is mediated by clathrin-dependent endocytosis of synaptic AMPA receptors 5–8C. At certain developmental stages, rapid protein synthesis is required for both mGluR-triggered AMPA receptor endocytosis and LTD. There is evidence from mouse models that this form of LTD may be relevant to fragile X mental retardation syndrome. Fragile X mental retardation protein (FMRP), which is deficient in fragile X syndrome due to abnormal methylation of the FMRP gene promoter, normally opposes group I mGluR-mediated induction of local protein synthesis in dendrites and the resulting LTD. Accordingly, it is proposed that fragile X symptoms arise in part through excessive dendritic protein synthesis and LTD. Consistent with this hypothesis are the recent findings that many of the fragile X–like symptoms exhibited in mouse models can be reversed by MPEP, a group I mGluR antagonist. This raises the possibility of novel treatments for fragile X syndrome in humans.
The aforementioned forms of synaptic plasticity are all triggered by activation of postsynaptic glutamate receptors and involve changes in the number of AMPA receptors at synapses. In contrast, there are also forms of LTP and LTD that involve long-lasting presynaptic changes in the release of glutamate. A presynaptic form of LTP was first described in the CA3 region of the hippocampus and is now known to occur in the striatum and cerebellum as well. This form of LTP does not require NMDA receptors and appears to be initiated by an activity-dependent rise in intracellular Ca2+ within the presynaptic terminals 5–8D. The Ca2+ rise may activate particular isoforms of adenylyl cyclases to produce cAMP, with subsequent activation of cAMP-dependent protein kinase (PKA) (Chapter 4). Via unknown mechanisms, this in turn leads to a persistent increase in the amount of glutamate released each time an action potential enters the nerve terminal. A potentially important form of presynaptic LTD may also occur at certain glutamatergic as well as at some inhibitory GABAergic synapses. This is commonly due to postsynaptic activation of mGluRs or voltage-gated Ca2+ channels, which triggers the synthesis of endocannabinoids, lipophilic molecules that are released by postsynaptic cells and can travel retrogradely across the synapse to bind to presynaptic cannabinoid receptors (discussed further in Chapter 8). Depending on the specific synapse, these endocannabinoids can either transiently depress neurotransmitter release for a period of many seconds or cause an LTD mediated by a long-lasting depression of transmitter release 5–8E. Why endocannabinoid release produces only a transient synaptic depression at some synapses, but more persistent LTD at others, is not fully understood.