Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits

The N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) serves critical functions in physiological and pathological processes in the central nervous system, including neuronal development, plasticity and neurodegeneration. Conventional heteromeric NMDARs composed of NR1 and NR2A-D subunits require dual agonists, glutamate and glycine, for activation. They are also highly permeable to Ca2+, and exhibit voltage-dependent inhibition by Mg2+. Coexpression of NR3A with NR1 and NR2 subunits modulates NMDAR activity. Here we report the cloning and characterization of the final member of the NMDAR family, NR3B, which shares high sequence homology with NR3A. From in situ and immunocytochemical analyses, NR3B is expressed predominantly in motor neurons, whereas NR3A is more widely distributed. Remarkably, when co-expressed in Xenopus oocytes, NR3A or NR3B co-assembles with NR1 to form excitatory glycine receptors that are unaffected by glutamate or NMDA, and inhibited by D-serine, a co-activator of conventional NMDARs. Moreover, NR1/NR3A or -3B receptors form relatively Ca2+-impermeable cation channels that are resistant to Mg2+, MK-801, memantine and competitive antagonists. In cerebrocortical neurons containing NR3 family members, glycine triggers a burst of firing, and membrane patches manifest glycine-responsive single channels that are suppressible by D-serine. By itself, glycine is normally thought of as an inhibitory neurotransmitter. In contrast, these NR1/NR3A or -3B 'NMDARs' constitute a type of excitatory glycine receptor.     

Glycine binding primes NMDA receptor internalization

NMDA (N-methyl-d-aspartate) receptors (NMDARs) are a principal subtype of excitatory ligand-gated ion channel with prominent roles in physiological and disease processes in the central nervous system. Recognition that glycine potentiates NMDAR-mediated currents as well as being a requisite co-agonist of the NMDAR subtype of 'glutamate' receptor profoundly changed our understanding of chemical synaptic communication in the central nervous system. The binding of both glycine and glutamate is necessary to cause opening of the NMDAR conductance pore. Although binding of either agonist alone is insufficient to cause current flow through the channel, we report here that stimulation of the glycine site initiates signalling through the NMDAR complex, priming the receptors for clathrin-dependent endocytosis. Glycine binding alone does not cause the receptor to be endocytosed; this requires both glycine and glutamate site activation of NMDARs. The priming effect of glycine is mimicked by the NMDAR glycine site agonist d-serine, and is blocked by competitive glycine site antagonists. Synaptic as well as extrasynaptic NMDARs are primed for internalization by glycine site stimulation. Our results demonstrate transmembrane signal transduction through activating the glycine site of NMDARs, and elucidate a model for modulating cell-cell communication in the central nervous system.     

Subunit arrangement and phenylethanolamine binding in GluN1/GluN2B NMDA receptors

Since it was discovered that the anti-hypertensive agent ifenprodil has neuroprotective activity through its effects on NMDA (N-methyl-D-aspartate) receptors, a determined effort has been made to understand the mechanism of action and to develop improved therapeutic compounds on the basis of this knowledge. Neurotransmission mediated by NMDA receptors is essential for basic brain development and function. These receptors form heteromeric ion channels and become activated after concurrent binding of glycine and glutamate to the GluN1 and GluN2 subunits, respectively. A functional hallmark of NMDA receptors is that their ion-channel activity is allosterically regulated by binding of small compounds to the amino-terminal domain (ATD) in a subtype-specific manner. Ifenprodil and related phenylethanolamine compounds, which specifically inhibit GluN1 and GluN2B NMDA receptors, have been intensely studied for their potential use in the treatment of various neurological disorders and diseases, including depression, Alzheimer's disease and Parkinson's disease. Despite considerable enthusiasm, mechanisms underlying the recognition of phenylethanolamines and ATD-mediated allosteric inhibition remain limited owing to a lack of structural information. Here we report that the GluN1 and GluN2B ATDs form a heterodimer and that phenylethanolamine binds at the interface between GluN1 and GluN2B, rather than within the GluN2B cleft. The crystal structure of the heterodimer formed between the GluN1b ATD from Xenopus laevis and the GluN2B ATD from Rattus norvegicus shows a highly distinct pattern of subunit arrangement that is different from the arrangements observed in homodimeric non-NMDA receptors and reveals the molecular determinants for phenylethanolamine binding. Restriction of domain movement in the bi-lobed structure of the GluN2B ATD, by engineering of an inter-subunit disulphide bond, markedly decreases sensitivity to ifenprodil, indicating that conformational freedom in the GluN2B ATD is essential for ifenprodil-mediated allosteric inhibition of NMDA receptors. These findings pave the way for improving the design of subtype-specific compounds with therapeutic value for neurological disorders and diseases.     

Neuropsin cleaves EphB2 in the amygdala to control anxiety

A minority of individuals experiencing traumatic events develop anxiety disorders. The reason for the lack of correspondence between the prevalence of exposure to psychological trauma and the development of anxiety is unknown. Extracellular proteolysis contributes to fear-associated responses by facilitating neuronal plasticity at the neuron-matrix interface. Here we show in mice that the serine protease neuropsin is critical for stress-related plasticity in the amygdala by regulating the dynamics of the EphB2-NMDA-receptor interaction, the expression of Fkbp5 and anxiety-like behaviour. Stress results in neuropsin-dependent cleavage of EphB2 in the amygdala causing dissociation of EphB2 from the NR1 subunit of the NMDA receptor and promoting membrane turnover of EphB2 receptors. Dynamic EphB2-NR1 interaction enhances NMDA receptor current, induces Fkbp5 gene expression and enhances behavioural signatures of anxiety. On stress, neuropsin-deficient mice do not show EphB2 cleavage and its dissociation from NR1 resulting in a static EphB2-NR1 interaction, attenuated induction of the Fkbp5 gene and low anxiety. The behavioural response to stress can be restored by intra-amygdala injection of neuropsin into neuropsin-deficient mice and disrupted by the injection of either anti-EphB2 antibodies or silencing the Fkbp5 gene in the amygdala of wild-type mice. Our findings establish a novel neuronal pathway linking stress-induced proteolysis of EphB2 in the amygdala to anxiety.     

Reaction mechanism determines NMDA receptor response to repetitive stimulation

At central excitatory synapses, N-methyl-D-aspartate (NMDA) receptors, which have a high affinity for glutamate, produce a slowly rising synaptic current in response to a single transmitter pulse and an additional current after a second, closely timed stimulus. Here we show, by examining the kinetics of transmitter binding and channel gating in single-channel currents from recombinant NR1/NR2A receptors, that the synaptic response to trains of impulses is determined by the molecular reaction mechanism of the receptor. The rate constants estimated for the activation reaction predict that, after binding neurotransmitter, receptors hesitate for approximately 4 ms in a closed high-affinity conformation before they either proceed towards opening or release neurotransmitter, with about equal probabilities. Because only about half of the initially fully occupied receptors become active, repetitive stimulation elicits currents with distinct waveforms depending on pulse frequency. This high-affinity/low-efficiency activation mechanism might serve as a link between stimulation frequency and the directionality of the ensuing synaptic plasticity.     

Multiple-conductance channels activated by excitatory amino acids in cerebellar neurons

In the mammalian central nervous system amino acids such as L-glutamate and L-aspartate are thought to act as fast synaptic transmitters. It has been suggested that at least three pharmacologically-distinguishable types of glutamate receptor occur in central neurons and that these are selectively activated by the glutamate analogues N-methyl-D-aspartate (NMDA), quisqualate and kainate. These three receptor types would be expected to open ion channels with different conductances. Hence if agonists produce similar channel conductances this would suggest they are acting on the same receptor. Another possibility is suggested by experiments on spinal neurons, where GABA (gamma-amino butyric acid) and glycine appear to open different sub-conductance levels of one class of channel while acting on different receptors. By analogy, several types of glutamate receptor could also be linked to a single type of channel with several sub-conductance states. We have examined these possibilities in cerebellar neurons by analysing the single-channel currents activated by L-glutamate, L-aspartate, NMDA, quisqualate and kainate in excised membrane patches. All of these agonists are capable of opening channels with at least five different conductance levels, the largest being about 45-50 pS. NMDA predominantly activated conductance levels above 30 pS while quisqualate and kainate mainly activated ones below 20 pS. The presence of clear transitions between levels favours the idea that the five main levels are all sub-states of the same type of channel.     

NMDA receptors are expressed in oligodendrocytes and activated in ischaemia

Glutamate-mediated damage to oligodendrocytes contributes to mental or physical impairment in periventricular leukomalacia (pre- or perinatal white matter injury leading to cerebral palsy), spinal cord injury, multiple sclerosis and stroke. Unlike neurons, white matter oligodendrocytes reportedly lack NMDA (N-methyl-d-aspartate) receptors. It is believed that glutamate damages oligodendrocytes, especially their precursor cells, by acting on calcium-permeable AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptors alone or by reversing cystine-glutamate exchange and depriving cells of antioxidant protection. Here we show that precursor, immature and mature oligodendrocytes in the white matter of the cerebellum and corpus callosum exhibit NMDA-evoked currents, mediated by receptors that are blocked only weakly by Mg2+ and that may contain NR1, NR2C and NR3 NMDA receptor subunits. NMDA receptors are present in the myelinating processes of oligodendrocytes, where the small intracellular space could lead to a large rise in intracellular ion concentration in response to NMDA receptor activation. Simulating ischaemia led to development of an inward current in oligodendrocytes, which was partly mediated by NMDA receptors. These results point to NMDA receptors of unusual subunit composition as a potential therapeutic target for preventing white matter damage in a variety of diseases.     

Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs.

The glutamate receptor (GluR) channel plays a key part in brain function. Among GluR channel subtypes, the NMDA (N-methyl-D-aspartate) receptor channel which is highly permeable to Ca2+ is essential for the synaptic plasticity underlying memory, learning and development. Furthermore, abnormal activation of the NMDA receptor channel may trigger the neuronal cell death observed in various brain disorders. A complementary DNA encoding a subunit of the rodent NMDA receptor channel (NMDAR1 or zeta 1) has been cloned and its functional properties investigated. Here we report the identification and primary structure of a novel mouse NMDA receptor channel subunit, designated as epsilon 1, after cloning and sequencing the cDNA. The epsilon 1 subunit shows 11-18% amino-acid sequence identity with rodent GluR channel subunits that have been characterized so far and has structural features common to neurotransmitter-gated ion channels. Expression from cloned cDNAs of the epsilon 1 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional GluR channels with high activity and characteristics of the NMDA receptor channel. Furthermore, the heteromeric NMDA receptor channel can be activated by glycine alone.     

Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine

Responses to the excitatory amino acid N-methyl-D-aspartate (NMDA) are markedly potentiated by nanomolar concentrations of glycine. This is due to the action of glycine at a novel strychnine-resistant binding site with an anatomical distribution identical to that for NMDA receptors, suggesting that the NMDA receptor channel complex contains at least two classes of amino-acid recognition site. Antagonists at the glycine-binding site associated with NMDA receptors act as potent non-competitive antagonists, but do not alter the mean open time or conductance, as estimated by fluctuation analysis. The mechanisms by which glycine acts on NMDA receptors are unknown, but single-channel recording experiments show an increase in opening frequency with no change in mean open time or conductance, suggesting that glycine could regulate transitions to states that are intermediate between binding of NMDA receptor agonists and ion-channel gating. It has been suggested that glycine acts as a co-agonist at the NMDA receptor, and that responses to NMDA cannot be obtained in the complete absence of glycine, but in these experiments the response to NMDA was measured at equilibrium, and it is unlikely that sufficient temporal resolution was achieved to detect rapid alterations in receptor gating. Using a fast perfusion system we find that glycine regulates desensitization at NMDA receptors; this has a major effect on the response to NMDA measured at equilibrium, as would occur with slower applications of agonist. Reduction of NMDA receptor desensitization by glycine provides an example of a novel mechanism for regulation of ion-channel activity.     

Proton inhibition of N-methyl-D-aspartate receptors in cerebellar neurons.

Mammalian neurons contain at least three types of excitatory amino-acid receptors, selectively activated by N-methyl-D-aspartate (NMDA) or aspartate, (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate ((S)-AMPA) and kainate. An important aspect of NMDA receptors is their regulation by a variety of factors such as glycine, Mg2+ and Zn2+ that are present in vivo. We show here that NMDA receptor responses are selectively inhibited by protons, with a 50% inhibitory concentration (IC50) that is close to physiological pH, implying that NMDA receptors are not fully active under normal conditions. (S)-AMPA and kainate responses remain unchanged at similar pH levels. Proton inhibition is voltage-insensitive and does not result either from fast channel block, a change in channel conductance, or an increase in the 50% excitatory concentration (EC50) of aspartate/NMDA or glycine. Instead, protons seem to decrease markedly the opening frequency of 30-50 pS NMDA channels, and reduce the relative proportion of longer bursts. This feature of NMDA receptors could be relevant to neurotoxic activation of NMDA receptors during ischaemia, as well as to seizure generation, as extracellular proton changes occur during both of these pathological situations. Furthermore, these results may have implications for normal NMDA receptor function as transient changes in extracellular protons occur during synaptic transmission.     

NMDA receptors mediate calcium accumulation in myelin during chemical ischaemia

Central nervous system myelin is a specialized structure produced by oligodendrocytes that ensheaths axons, allowing rapid and efficient saltatory conduction of action potentials. Many disorders promote damage to and eventual loss of the myelin sheath, which often results in significant neurological morbidity. However, little is known about the fundamental mechanisms that initiate myelin damage, with the assumption being that its fate follows that of the parent oligodendrocyte. Here we show that NMDA (N-methyl-d-aspartate) glutamate receptors mediate Ca2+ accumulation in central myelin in response to chemical ischaemia in vitro. Using two-photon microscopy, we imaged fluorescence of the Ca2+ indicator X-rhod-1 loaded into oligodendrocytes and the cytoplasmic compartment of the myelin sheath in adult rat optic nerves. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor antagonist NBQX completely blocked the ischaemic Ca2+ increase in oligodendroglial cell bodies, but only modestly reduced the Ca2+ increase in myelin. In contrast, the Ca2+ increase in myelin was abolished by broad-spectrum NMDA receptor antagonists (MK-801, 7-chlorokynurenic acid, d-AP5), but not by more selective blockers of NR2A and NR2B subunit-containing receptors (NVP-AAM077 and ifenprodil). In vitro ischaemia causes ultrastructural damage to both axon cylinders and myelin. NMDA receptor antagonism greatly reduced the damage to myelin. NR1, NR2 and NR3 subunits were detected in myelin by immunohistochemistry and immunoprecipitation, indicating that all necessary subunits are present for the formation of functional NMDA receptors. Our data show that the mature myelin sheath can respond independently to injurious stimuli. Given that axons are known to release glutamate, our finding that the Ca2+ increase was mediated in large part by activation of myelinic NMDA receptors suggests a new mechanism of axo-myelinic signalling. Such a mechanism may represent a potentially important therapeutic target in disorders in which demyelination is a prominent feature, such as multiple sclerosis, neurotrauma, infections (for example, HIV encephalomyelopathy) and aspects of ischaemic brain injury.     

Glycine potentiates the NMDA response in cultured mouse brain neurons

Transmitters mediating 'fast' synaptic processes in the vertebrate central nervous system are commonly placed in two separate categories that are believed to exhibit no interaction at the receptor level. The 'inhibitory transmitters' (such as glycine and GABA) are considered to act only on receptors mediating a chloride conductance increase, whereas 'excitatory transmitters' (such as L-glutamate) are considered to activate receptors mediating a cationic conductance increase. The best known excitatory receptor is that specifically activated by N-methyl-D-aspartate (NMDA) which has recently been characterized at the single channel level. The response activated by NMDA agonists is unique in that it exhibits a voltage-dependent Mg block. We report here that this response exhibits another remarkable property: it is dramatically potentiated by glycine. This potentiation is not mediated by the inhibitory strychnine-sensitive glycine receptor, and is detected at a glycine concentration as low as 10 nM. The potentiation can be observed in outside-out patches as an increase in the frequency of opening of the channels activated by NMDA agonists. Thus, in addition to its role as an inhibitory transmitter, glycine may facilitate excitatory transmission in the brain through an allosteric activation of the NMDA receptor.     

Potentiation of NMDA receptor currents by arachidonic acid.

Arachidonic acid is released by phospholipase A2 when activation of N-methyl-D-aspartate (NMDA) receptors by neurotransmitter glutamate raises the calcium concentration in neurons, for example during the initiation of long-term potentiation and during brain anoxia. Here we investigate the effect of arachidonic acid on glutamate-gated ion channels by whole-cell clamping isolated cerebellar granule cells. Arachidonic acid potentiates, and makes more transient, the current through NMDA receptor channels, and slightly reduces the current through non-NMDA receptor channels. Potentiation of the NMDA receptor current results from an increase in channel open probability, with no change in open channel current. We observe potentiation even with saturating levels of agonist at the glutamate- and glycine-binding sites on these channels; it does not result from conversion of arachidonic acid to lipoxygenase or cyclooxygenase derivatives, or from activation of protein kinase C. Arachidonic acid may act by binding to a site on the NMDA receptor, or by modifying the receptor's lipid environment. Our results suggest that arachidonic acid released by activation of NMDA (or other) receptors will potentiate NMDA receptor currents, and thus amplify increases in intracellular calcium concentration caused by glutamate. This may explain why inhibition of phospholipase A2 blocks the induction of long-term potentiation.     

NMDA receptors are expressed in developing oligodendrocyte processes and mediate injury

Injury to oligodendrocyte processes, the structures responsible for myelination, is implicated in many forms of brain disorder. Here we show NMDA (N-methyl-D-aspartate) receptor subunit expression on oligodendrocyte processes, and the presence of NMDA receptor subunit messenger RNA in isolated white matter. NR1, NR2A, NR2B, NR2C, NR2D and NR3A subunits showed clustered expression in cell processes, but NR3B was absent. During modelled ischaemia, NMDA receptor activation resulted in rapid Ca2+-dependent detachment and disintegration of oligodendroglial processes in the white matter of mice expressing green fluorescent protein (GFP) specifically in oligodendrocytes (CNP-GFP mice). This effect occurred at mouse ages corresponding to both the initiation and the conclusion of myelination. NR1 subunits were found mainly in oligodendrocyte processes, whereas AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor subunits were mainly found in the somata. Consistent with this observation, injury to the somata was prevented by blocking AMPA/kainate receptors, and preventing injury to oligodendroglial processes required the blocking of NMDA receptors. The presence of NMDA receptors in oligodendrocyte processes explains why previous studies that have focused on the somata have not detected a role for NMDA receptors in oligodendrocyte injury. These NMDA receptors bestow a high sensitivity to acute injury and represent an important new target for drug development in a variety of brain disorders.     

Mechanism of glutamate receptor desensitization

Ligand-gated ion channels transduce chemical signals into electrical impulses by opening a transmembrane pore in response to binding one or more neurotransmitter molecules. After activation, many ligand-gated ion channels enter a desensitized state in which the neurotransmitter remains bound but the ion channel is closed. Although receptor desensitization is crucial to the functioning of many ligand-gated ion channels in vivo, the molecular basis of this important process has until now defied analysis. Using the GluR2 AMPA-sensitive glutamate receptor, we show here that the ligand-binding cores form dimers and that stabilization of the intradimer interface by either mutations or allosteric modulators reduces desensitization. Perturbations that destabilize the interface enhance desensitization. Receptor activation involves conformational changes within each subunit that result in an increase in the separation of portions of the receptor that are linked to the ion channel. Our analysis defines the dimer interface in the resting and activated state, indicates how ligand binding is coupled to gating, and suggests modes of dimer dimer interaction in the assembled tetramer. Desensitization occurs through rearrangement of the dimer interface, which disengages the agonist-induced conformational change in the ligand-binding core from the ion channel gate.     

Glutamate activates multiple single channel conductances in hippocampal neurons

There is considerable evidence that glutamate is the principal neurotransmitter that mediates fast excitatory synaptic transmission in the vertebrate central nervous system. This single transmitter seems to activate two or three distinct types of receptors, defined by their affinities for three selective structural analogues of glutamate, NMDA (N-methyl-D-aspartate), quisqualate and kainate. All these agonists increase membrane permeability to monovalent cations, but NMDA also activates a conductance that permits significant calcium influx and is blocked in a voltage-dependent manner by extracellular magnesium. Fast synaptic excitation seems to be mediated mainly by kainate/quisqualate receptors, although NMDA receptors are sometimes activated. We have investigated the properties of these conductances using single-channel recording in primary cultures of hippocampal neurons, because the hippocampus contains all subtypes of glutamate receptors and because long-term potentiation of synaptic transmission occurs in this structure. We find that four or more distinct single-channel currents are evoked by applying glutamate to each outside-out membrane patch. These conductances vary in their ionic permeability and in the agonist most effective in causing them to open. Clear transitions between all the conductance levels are observed. Our observations are compatible with the model that all the single channel conductances activated by glutamate reflect the operation of one or two complex molecular entities.     

Interaction with the NMDA receptor locks CaMKII in an active conformation.

Calcium- and calmodulin-dependent protein kinase II (CaMKII) and glutamate receptors are integrally involved in forms of synaptic plasticity that may underlie learning and memory. In the simplest model for long-term potentiation, CaMKII is activated by Ca2+ influx through NMDA (N-methyl-D-aspartate) receptors and then potentiates synaptic efficacy by inducing synaptic insertion and increased single-channel conductance of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Here we show that regulated CaMKII interaction with two sites on the NMDA receptor subunit NR2B provides a mechanism for the glutamate-induced translocation of the kinase to the synapse in hippocampal neurons. This interaction can lead to additional forms of potentiation by: facilitated CaMKII response to synaptic Ca2+; suppression of inhibitory autophosphorylation of CaMKII; and, most notably, direct generation of sustained Ca2+/calmodulin (CaM)-independent (autonomous) kinase activity by a mechanism that is independent of the phosphorylation state. Furthermore, the interaction leads to trapping of CaM that may reduce down-regulation of NMDA receptor activity. CaMKII-NR2B interaction may be prototypical for direct activation of a kinase by its targeting protein.     

Glycine enhances NMDA-receptor mediated synaptic potentials in neocortical slices

One class of excitatory amino-acid receptors, the N-methyl-D-aspartate (NMDA) receptors, mediates transmission at a small, but important, group of synapses in the neocortex. These receptors are implicated in neuronal plasticity during development in young mammals and in memory acquisition in adults. Recently, responses of isolated membrane patches to NMDA were shown to be greatly enhanced by glycine. This, together with the demonstration that the strychnine-insensitive glycine-binding site is distinct from, but linked to, the NMDA receptor has excited intense interest in glycine as a synaptic modulator. Before proposing a physiological function, however, it is important to determine whether glycine could enhance synaptic responses to NMDA receptor activation in intact, adult tissue. An earlier study failed to demonstrate enhancement of NMDA responses when glycine was applied and it was proposed that in intact tissue the high-affinity glycine site was already saturated by endogenous glycine. It remained possible that glycine concentrations can be maintained at low levels close to synaptic receptors. We have examined responses of neurons in slices of adult neocortex to focal applications of excitatory amino acids and glycine and report enhancement by glycine of NMDA receptor-mediated excitatory postsynaptic potentials.     

Cloning of cDNA for the glutamate-binding subunit of an NMDA receptor complex.

The amino acids L-glutamic and L-aspartic acids form the most widespread excitatory transmitter network in mammalian brain. The excitation produced by L-glutamic acid is important in the early development of the nervous system, synaptic plasticity and memory formation, seizures and neuronal degeneration. The receptors activated by L-glutamic acid are a target for therapeutic intervention in neurodegenerative diseases, brain ischaemia and epilepsy. There are two types of receptors for the excitatory amino acids, those that lead to the opening of cation-selective channels and those that activate phospholipase C (ref. 11). The receptors activating ion channels are NMDA (N-methyl-D-aspartate) and kainate/AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-sensitive receptors. The complementary DNAs for the kainate/AMPA receptor and for the metabotropic receptor have been cloned. We report here on the isolation and characterization of a protein complex of four major proteins that represents an intact complex of the NMDA receptor ion channel and on the cloning of the cDNA for one of the subunits of this receptor complex, the glutamate-binding protein.     

NMDA application potentiates synaptic transmission in the hippocampus

The NMDA (N-methyl-D-aspartate) class of glutamate receptor plays a critical role in a variety of forms of synaptic plasticity in the vertebrate central nervous system. One extensively studied example of plasticity is long-term potentiation (LTP), a remarkably long-lasting enhancement of synaptic efficiency induced in the hippocampus by brief, high-frequency stimulation of excitatory synapses. LTP is a strong candidate for a cellular mechanism of learning and memory. The site of LTP induction appears to be the postsynaptic cell and induction requires both activation of NMDA receptors by synaptically released glutamate and depolarization of the postsynaptic membrane. It is proposed that this depolarization relieves a voltage-dependent Mg2+ block of the NMDA receptor channel, resulting in increased calcium influx which is the trigger for the induction of LTP. This model predicts that application of a large depolarizing dose of NMDA should be sufficient to evoke LTP. In agreement with a previous study, we have found that NMDA or glutamate application does potentiate synaptic transmission in the hippocampus. This agonist-induced potentiation is, however, decremental and short-lived, unlike LTP. It is occluded shortly after the induction of LTP and a similar short-term potentiation can be evoked by synaptically released glutamate. We thus propose that LTP has two components, a short-term, decremental component which can be mimicked by NMDA receptor activation, and a long-lasting, non-decremental component which, in addition to requiring activation of NMDA receptors, requires stimulation of presynaptic afferents.