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The unique properties of glutamate receptor channels
Abstract
Rapid excitatory neurotransmission in the central nervous system (CNS) is mediated predominantly by synaptically released L-glutamate which activates cation selective channels with different kinetic and ion conductance properties. Studies with cloned glutamate receptor channels helped delineate the functional properties of channels defined in subunit composition. Moreover, molecular studies have revealed novel genetic mechanisms controlling the expression of important structural channel determinants.
... Mg^"^ and are modulated by glycine (see Sommer and Seeburg, 1992;Sprengel and Seeburg, 1993). However, in depolarised cells the block is released and the NMDA receptor can be activated leading to a large influx of Ca^^ (see Sommer and Seeburg, 1992;Sprengel and Seeburg, 1993). ...
... Mg^"^ and are modulated by glycine (see Sommer and Seeburg, 1992;Sprengel and Seeburg, 1993). However, in depolarised cells the block is released and the NMDA receptor can be activated leading to a large influx of Ca^^ (see Sommer and Seeburg, 1992;Sprengel and Seeburg, 1993). AMPA and kainate receptors may be distinguished by their high affinity for AMPA or kainate respectively. ...
... AMPA and kainate receptors may be distinguished by their high affinity for AMPA or kainate respectively. AMPA receptors exhibit fast kinetics, have low permeability to divalent cations and conduct mainly Na"^ currents (see Sprengel and Seeburg, 1993). Glutamate also activates metabotropic glutamate receptors (mGluR) that are coupled to G-proteins to influence second messenger systems. ...
The impairment of cerebral blood flow during an ischaemic insult restricts the delivery of oxygen and glucose to the brain. The subsequent energy depletion triggers a cascade of events, including release of the excitotoxin, glutamate. In this thesis, the microglial response to pharmacological manipulation of energy metabolism, depletion of oxygen and glucose, and glutamate exposure was explored. Glucose deprivation did not compromise microglial viability. In contrast, glucose deprivation in the presence of the glycolytic inhibitor, 2-deoxyglucose triggered microglial death and induced cytoskeletal changes. Two-deoxyglucose treatment, even in the presence of glucose, strongly activated microglia, attenuated microglial proliferation and/or adhesion and triggered nuclear pyknosis. Microglia were relatively resistant to short exposure to the respiratory chain inhibitor, cyanide, compared with neurons. Oxygen-glucose deprivation was a milder insult for microglia than cyanide or 2-deoxyglucose treatment. It did not trigger cytoskeletal changes or induce nuclear pyknosis. However, both hypoxia and oxygen-glucose deprivation irreversibly compromised microglial proliferation and/or adhesion. Glutamate treatment triggered neuronal but not microglial nuclear pyknosis. Microglial conditioned medium containing soluble factors released from microglia was neuroprotective when added to neurons prior to neuronal exposure to glutamate. However, when microglial conditioned medium was added to untreated neurons, an increase in neuronal calcium levels was triggered, mediated by P₂ purinergic- and NMDA- receptor activation. Taken together these results indicate that microglia are more able to tolerate energy deprivation or glutamate than neurons. Furthermore, the above results demonstrate that microglia may interact with neurons, releasing factors which modulate neuronal signalling and survival. Following a severe metabolic insult, as may occur in the ischaemic core, strong microglial activation is observed. This is associated with microglial death and may be a mechanism of down-regulating the microglial contribution to an inflammatory response, although removal of microglia by these processes may compromise brain repair.
... GIn-s CÉLULA GLIAL Farmacológicamente se han descrito tres tipos de receptores onotrópicos para el glutamato en función de su diferente afinidad por sustancias: los receptores Nmetil-D-aspartato (NMDA), los receptores a-amino -3-hidroxi-5-metil-4-isoxazolpropio-nato (AMPA), y los receptores kainato (Farooqui y Horrocks, 1991;Nakanishi, 1992;Sprengel y Seeburg, 1993). Recientemente se ha descrito la posible existencia de un cuarto tipo de receptores onotrópicos, los receptores delta, basandose en estudios de donación (Petralia y Wenthold, 1996). ...
... Se han descrito sitios de unión específicos para el glutamato o sus agonistas; para glicina; para cationes, en el interior del canal, donde el Mg2 puede unirse y bloquear el flujo de iones; para penciclidinas; para poliaminas; para el Zn2 (distinto del de Mg2'); para H' y un sitio redox (Farooqui y Horrocks, 1991;Nakanishi, 1992;Hollmann y Heinemann, 1994). Los receptores NMDA poseen una elevada permeabilidad para el Ca2', pueden activarse de forma prolongada y pueden ser bloqueados por Mg2' dependiendo del voltaje de la membrana (Nakanishi, 1992;Sprengel y Seeburg, 1993). Sus propiedades indican que el receptor NMDA puede actuar como un detector de coincidencia molecular: su activación depende de la existencia simultánea de actividad presináptica <liberación de glutamato) y postsináptica (despolarización). ...
... Se considera que el receptor AMPA es el responsable de la transmisión excitadora rápida mediada por glutamato y de la activación del receptor NMDA por desbloqueo del canal iónico como consecuencia de la despolarización. Por esto último se comprende que su distribución en el cerebro sea muy similar a la del receptor NMDA: ambos se encuentran en alta concentración en la corteza cerebral, el hipocampo, el estriado y el tálamo (Farooqui y Horrocks, 1991;Sprengel y Seeburg, 1993). ...
El estudio de la interacción entre glutamato/dopamina/GABA en el estriado ha sido el foco de una intensa investigación debido, en parte, a su implicación en el proceso normal de envejecimiento asi como en la patogenia de algunas enfermedades neurodegenerativas, tales como la enfermedad de Parkinson. El objetivo del trabajo de investigación presentado en esta Tesis Doctoral fue el estudio de la interacción entre glutamato, dopamina y GABA en el estriado dorsal y ventral (núcleo accumbens) de la rata despierta, y sus posibles cambios durante el envejecimiento. Para ello se estudió el efecto de un incremento de la concentración extracelular de glutamato endógeno, mediante el bloqueo específico de su recaptura, sobre la concentración extracelular dedopamina y GABA en ratas Wistar macho de diferentes edades: jóvenes (2-4 meses) de edad media (12-14 meses), viejas (27-32 meses) y muy viejas (37 meses). Se utilizaron técnicas de perfusión intracerebral in vivo (microdiálisis) y de HPLC para el análisis de la concentración extracelular de glutamato, dopamina y GABA. Los resultados obtenidos en este trabajo de investigación sugieren la existencia de un gradiente dorsoventral en el estriado en los efectos del envejecimiento sobre la interacción entre glutamato, dopamina y GABA, con cambios relacionados con la edad en el núcleo accumbens pero no en el estriado dorsal. En concreto, el efecto del glutamato sobre la concentración extracelular de dopamina en el núcleo accumbens fue menor en ratas viejas que en ratas jóvenes, mientras que el efecto del glutamato sobre la concentración de GABA tendió a ser mayor en ratas viejas. Es posible que este efecto diferencial del envejecimiento sobre la interacción del neurotransmisores en el estriado y el núcleo accumbens pueda estar en labase de los déficits motores y motivacionales que estas estructuras parecen mediar.
... The iGluR subunits are distinct from subunits of nicotinic acetylcholine receptor, γ-aminobutyric acid receptor A , glycine receptor and serotonin receptor by size, the conserved sequence patterns and by transmembrane topology (1.3.4; reviewed in Sprengel and Seeburg, 1993). The subunits of same subfamily (AMPA, kainate and NMDA) have higher amino acid identity with each other than with subunits of a different subfamily (Fig. 3). ...
... Membrane topology studies have tried to characterize which segments of the receptor proteins are extra-and intracellular and which segments are embedded in the plasma membrane bilayer. Initially, the finding that the subunits contain a N-terminal signal peptide and four suitable length hydrophobic segments led to a suggestion that subunits have four membrane-spanning α-helices like the members of the ligand-gated superfamily (reviewed in Sprengel and Seeburg, 1993). Currently, the revised model suggests three transmembrane (M1, M3 and M4) segments together with one re-entrant membrane segment (M2). ...
... The GluR-B and -D subunits have either 68 or 50 amino acids in their C-terminal domains due to alternative splicing (Köhler et al., 1994; Gallo et al., 1992). The C-terminal domain of kainate receptors are of similar size to GluR-A-D subunits, whereas the C-terminal domain of the NMDA receptor NR2 subunits are significantly larger, ~ 620-640 amino acids (reviewed in Sprengel and Seeburg, 1993). The C-terminal domain is cytoplasmic and has contacts with proteins of the postsynaptic density (PSD). ...
Diss. -- Helsingin yliopisto.
... It quickly became apparent using binding studies that this was an AMPA receptor protein, showing, as with the native AMPA receptors, non-desensitising responses to kainate (see e.g. Sommer et al., 1990) and the name GluR1 and GluR-A became the commonly used names thereafter (Hollmann and Heinemann, 1994; Nakanishi and Masu, 1994; Sprengel and Seeburg, 1993). Following on from the original report from Heinemann's group (Hollmann et al., 1989), cloning by sequence homology revealed further AMPA receptor family members in the next few years. ...
... Cyclothiazide is also inactive on kainate receptors making it also a useful compound for discriminating between AMPA and kainate responses (Woolley and Lodge, unpublished observation). with high affinity and form functional channels activated by glutamate and kainate which are enhanced in the presence of concanavalin A (Hollmann and Heinemann, 1994; Nakanishi and Masu, 1994; Sprengel and Seeburg, 1993). A subunit with even higher kainate affinity, KA1, was cloned by Werner et al. (1991); this was followed by the cloning of a second one variously named KA2 in the rat (Herb et al., 1992) and g in the mouse (Sakimura et al., 1992). ...
In this article, the beginnings of glutamate pharmacology are traced from the early doubts about 'non-specific' excitatory effects, through glutamate- and aspartate-preferring receptors, to NMDA, quisqualate/AMPA and kainate subtypes, and finally to the cloning of genes for these receptor subunits. The development of selective antagonists, crucial to the subtype classification, allowed the fundamental importance of glutamate receptors to synaptic activity throughout the CNS to be realised. The ability to be able to express and manipulate cloned receptor subunits is leading to huge advances in our understanding of these receptors. Similarly the tortuous path of the nomenclature is followed from naming with reference to exogenous agonists, through abortive early attempts at generic schemes, and back to the NC-IUPHAR system based on the natural agonist, the defining exogenous agonist and the gene names.
... Two types of receptors mediate the actions of glutamate: ionotropic and metabotropic receptors (Sprengel and Seeburg, 1993;Hollmann and Heinemann, 1994;Ozawa et al., 1998) (Fig. 2). The ionotropic glutamate receptors can be distinguished by their pharmacological and electrophysiological properties: the N- Fig. 1. ...
... Two types of receptors mediate the depolarizing action of glutamate: ionotropic and metabotropic receptors (Sprengel and Seeburg, 1993;Hollmann and Heinemann, 1994). Table 5 gives data on changes in glutamate receptors during aging. ...
The effects of aging on glutamate neurotransmission in the brain is reviewed and evaluated. Glutamate is the neurotransmitter in most of the excitatory synapses and appears to be involved in functions such as motor behaviour, cognition and emotion, which alter with age. However, relatively few studies have been conducted to study the relationship between glutamate and aging of the brain. The studies presented here indicate the existence of a number of changes in the glutamatergic system during the normal process of aging. First, an age-related decrease of glutamate content in tissue from cerebral cortex and hippocampus has been reported, although it may be mainly a consequence of changes in metabolic activity rather than glutamatergic neurotransmission. On the other hand, studies in vitro and in vivo have shown no changes in glutamate release during aging. Since glutamate sampled in most of these studies is the result of a balance between release and uptake processes, the lack of changes in glutamate release may be due to compensatory changes in glutamate uptake. In fact, a reduced glutamate uptake capacity, as well as a loss in the number of high affinity glutamate transporters in glutamatergic terminals of aged rats, have been described. However, the most significant and consistent finding is the decrease in the density of glutamatergic NMDA receptors with age. A new perspective, in which glutamate interacts with other neurotransmitters to conform the substrates of specific circuits of the brain and its relevance to aging, is included in this review. In particular, studies from our laboratory suggest the existence of age-related changes in the interaction between glutamate and other neurotransmitters, e.g. dopamine and GABA, which are regionally specific.
... Several reviews on the various glutamate receptors in the CNS have been published (Choi, 1988;Sprengel and Seeburg, 1993). Therefore, the following paragraphs will only give a short introduction into their basic features, and attempt a summary of their distribution in white matter, the target tissue of MS. ...
This chapter explores that the exact mechanisms that lead to demyelination, axonal damage, and death of oligodendrocytes in multiple sclerosis (MS) are still unknown. Among the mechanisms implicated are contact with cytotoxic immune cells, antibodies, and soluble mediators especially proinflammatory cytokines. It discusses that in glutamate excitotoxicity, agonist binding to ionotropic glutamate receptors leads to influx of sodium and calcium ions; a cell membrane depolarizing mechanism. Overstimulation, ion influxes, and membrane depolarization is then conducive to the activation of destructive processes, such as interruption of electrolyte and fluid balance, phospholipase and protease activation, and formation of free radicals and activation of cell death pathways. The chapter also reviews the recent findings that expand the mechanisms of glutamate excitotoxicity from gray matter diseases to MS, a white matter disease. For glutamate excitotoxicity in white matter to be a valid mechanism of damage, the presence of glutamate receptors and one or more of these elements are required.
... The vast majority of synapses in the central nervous system use glutamate as a neurotransmitter to produce rapid neuronal excitation (Monaghan et al., 1989) . By cDNA cloning, a number of different glutamate receptor subunits have been identified (for reviews, see Barnes and Henley, 1992 ; Gasic and Hollmann, 1992; Nakanishi, 1992; Sommer and Seeburg, 1992; Sprengel and Seeburg, 1993; Wisden and Seeburg, 1993). Glutamate receptors have been classified into two major categories : ionotropic receptors, which function as ion channels, and metabotropic receptors, which are coupled to second-messenger systems . ...
In order to define the membrane topology of the GluR1 glutamate receptor subunit, we have examined the location of epitopes. Antibodies were produced against peptides corresponding to putative extracellular and intracellular segments of the rat brain GluR1 glutamate receptor subunit. Immunocytochemistry at the electron microscopic level in the dentate gyrus of the hippocampal formation showed that epitopes for the antiserum to the N-terminal part of the subunit are located at the extracellular face of the plasma membrane, whereas the antigenic determinants for the antiserum to the C-terminal part are found at the intracellular face of the postsynaptic membrane. Furthermore, antibodies to the N-terminal residues 253–267 reacted similarly with both intact and permeabilized synaptosomes, whereas the binding of antibodies to the C-terminal residues 877–889 increased about 1.6-fold following permeabilization. Our data suggest that the N- and C-terminal regions are located on the opposite side of the membrane and, therefore, the GluR1 subunit probably has an odd number of membrane spanning segments. The antibody cross-reactivities in different species and their effect on ligand binding activity were also established.
... Assembly of subunits of signaling proteins, such as ionotropic glutamate receptors, into multimers with varying subunit ratios generates diverse functional characteristics (48). Modulation of the subunit composition of CaM kinase II holoenzymes might similarly serve to diversify the functional properties of the enzyme. ...
The assembly of 6–12 subunits of Ca2+/calmodulin-dependent kinase II (CaM kinase II) into holoenzymes is an important structural feature of the enzyme and its
postulated role as a molecular detector of Ca2+ oscillations. Using single cell reverse transcriptase-polymerase chain reaction, we show that α- and β-CaM kinase II mRNAs
are simultaneously present in the majority of hippocampal neurons examined and that co-assembly of their protein products
into heteromers is therefore possible. The subunit composition of CaM kinase II holoenzymes was analyzed by immunoprecipitation
with subunit-specific monoclonal antibodies. Rat forebrain CaM kinase II consists of heteromers composed of α and β subunits
at a ratio of 2:1 and homomers composed of only α subunits. We examined the functional effect of the heteromeric assembly
by analyzing the calmodulin dependence of autophosphorylation. Recombinant homomers of α- or β-CaM kinase II, as well as of
alternatively spliced β isoforms, have distinct calmodulin dependences for autophosphorylation based on differences in their
calmodulin affinities. Half-maximal autophosphorylation of α is achieved at 130 nmcalmodulin, while that for β occurs at 15 nm calmodulin. In CaM kinase II isolated from rat forebrain, however, the calmodulin dependence for autophosphorylation of the
β subunits is shifted toward that of α homomers. This suggests that Thr287 in β subunits is phosphorylated by α subunits present in the same holoenzyme. Once autophosphorylated, β-CaM kinase II traps
calmodulin by reducing the rate of calmodulin dissociation.
... Glutamate receptors mediate excitatory synaptic transmission in the vertebrate central nervous system (1,2). According to their pharmacological properties and gene sequence homology, three subfamilies of ionotropic glutamate receptors have been distinguished: N-methyl-D-aspartate, kainate, and ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) 1 receptors (3,4). ...
The highly negatively charged polysialic acid (PSA) is a carbohydrate predominantly carried by the neural cell adhesion molecule (NCAM) in mammals. NCAM and, in particular, PSA play important roles in cellular and synaptic plasticity. Here we investigated whether PSA modulates the activity of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors (AMPA-Rs). Single channel recordings of affinity-purified AMPA-Rs reconstituted in lipid bilayers revealed that bacterially derived PSA, called colominic acid, prolonged the open channel time of AMPA-R-mediated currents by severalfold and altered the bursting pattern of the receptor channels but did not modify AMPA-R single channel conductance. This effect was reversible, concentration-dependent, and specific, since monomers of sialic acid and another negatively charged carbohydrate, chondroitin sulfate, did not potentiate single channel AMPA-R currents. Recombinant PSA-NCAM also potentiated currents mediated by reconstituted AMPA-Rs. In pyramidal neurons acutely isolated from the CA1 region of the early postnatal hippocampus, l-glutamate or AMPA (applied in the presence of antagonists blocking voltage-gated Na(+) and K(+) currents and N-methyl-d-aspartate and metabotropic glutamate receptors) induced inward currents, which were significantly increased by co-application of colominic acid. Chondroitin sulfate did not affect AMPA-R-mediated currents in CA1 neurons. The effect of colominic acid was age-dependent, since in pyramidal neurons from adult hippocampus, colominic acid failed to potentiate glutamate responses. Thus, our study demonstrates age-dependent potentiation of AMPA receptors by PSA via a mechanism probably involving direct PSA-AMPA-R interactions. This mechanism might amplify AMPA-R-mediated signaling in immature cells, thereby affecting their development.
... Variations in NMDA subunit composition and/or distribution may contribute to the differing effects of thrombin centrally. NMDA receptor complexes are heteromeric structures that can be reconstituted from two subunit types (Sprengel & Seeburg, 1993): NR1 (z1 in mice) plus one of four modulatory NR2 subunits (NR2A–NR2D) (e1–e4 in mice). NR2 subunits differ in anatomical distribution, functional properties, affinity for agonists and antagonists acting at glycine and glutamate sites, and affinities for Mg 2+ . ...
The CNS expresses many components of an extracellular protease signalling system, including the protease-activated receptor-1 (PAR-1) whose tethered ligand is generated by thrombin. Activation of PAR-1 potentiates NMDA receptor activity in hippocampal neurons. Because NMDA activity mediates hyperalgesia, we tested the hypothesis that PAR-1 receptors also regulate pain processing. In contrast to the potentiating effect of thrombin in the hippocampus, NMDA-induced behaviours and the transient mechanical hyperalgesia (von Frey fibres) induced by intrathecally injected NMDA in mice were inhibited by thrombin in a dose-related fashion. This anti-hyperalgesic effect was mimicked by SFLLRN, the natural ligand at PAR-1 binding sites, but not SLIGRL-amide, a PAR-2 agonist. The effects of SFLLRN were less potent and shorter in duration than that of thrombin, consistent with its more transient effect on PAR-1 sites. Both thrombin and SFLLRN inhibited acetic acid-induced abdominal stretch (writhing) behaviours, which were also sensitive to NMDA antagonism, but not hot plate or tail flick latencies, which were insensitive to NMDA antagonists. TFLLR-amide, a selective ligand for PAR-1 sites, mimicked the effects of thrombin while RLLFT-amide, an inactive, reverse peptide sequence, did not. In addition, the effect of TFLLR-amide was prevented by RWJ-56110, a PAR-1 antagonist. Thrombin and TFLLR-amide produced no oedema (Evans Blue extravasation) in the spinal cord that would account for these effects. Based on the reported ability of thrombin to mobilize endothelin-1 from astrocytes, we tested the role of this compound in thrombin's activity. BQ123, an endothelin A receptor antagonist, prevented thrombin's inhibition of writhing and NMDA-induced behaviours while BQ788, an endothelin B receptor antagonist, did not. Thus, activation of PAR-1 sites by thrombin in the CNS appears to inhibit NMDA-mediated nociception by a pathway involving endothelin type A receptors.
... Glutamate acts on neuronal and glial ionotropic receptors (GluRs), coupled to specific ligand-gated cationic channels, and mGluRs, coupled to second messengers (69). AMPA receptor subunits GluR1 to GluR4 are primarily responsible for CNS fast excitatory neurotransmission and the subunit composition determines receptor electrophysiological and pharmacological properties (72). These subunits assemble in vitro to form homomeric or heteromeric configurations permeable to Na + and K + ions, but receptors expressing only GluR1 and GluR3 form homomeric or heteromeric channels showing Ca ++ conductance that is blocked by inclusion of a GluR2 subunit (27). ...
Blockade of receptors for the excitatory neurotransmitter glutamate ameliorates neurological clinical signs in models of the CNS inflammatory demyelinating disease multiple sclerosis (MS). To investigate whether glutamate excitoxicity may play a role in MS pathogenesis, the cellular localization of glutamate and its receptors, transporters and enzymes was examined. Expression of glutamate receptor (GluR) 1, a Ca(++)-permeable ionotropic AMPA receptor subunit, was up-regulated on oligodendrocytes in active MS lesion borders, but Ca(++)-impermeable AMPA GluR2 subunit levels were not increased. Reactive astrocytes in active plaques expressed AMPA GluR3 and metabotropic mGluR1, 2/3 and 5 receptors and the GLT-1 transporter, and a subpopulation was immunostained with glutamate antibodies. Activated microglia and macrophages were immunopositive for GluR2, GluR4 and NMDA receptor subunit 1. Kainate receptor GluR5-7 immunostaining showed endothelial cells and dystrophic axons. Astrocyte and macrophage populations expressed glutamate metabolizing enzymes and unexpectedly the EAAC1 transporter, which may play a role in glutamate uptake in lesions. Thus, reactive astrocytes in MS white matter lesions are equipped for a protective role in sequestering and metabolizing extracellular glutamate. However, they may be unable to maintain glutamate at levels low enough to protect oligodendrocytes rendered vulnerable to excitotoxic damage because of GluR1 up-regulation.
The lateral nucleus of the amygdala (LA) is a critical component of the circuitry through which environmental stimuli are endowed with emotional meaning through association with painful or threatening events. Individual cells in LA receive convergent input from auditory processing areas in the thalamus and cortex, and the excitatory amino-acid l-glutamate (Glu) participates in synaptic transmission in both pathways. Previously, we characterized the ultrastructure of pre- and postsynaptic processes in the thalamo–amygdala pathway, and showed the relation of presynaptic inputs to N-methyl-D-aspartate (NMDA) and α-amino-3-hydoxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits. In the present study, we examined the nature of cortico–amygdala synaptic interactions with Glu receptors in LA and determined whether they are similar or different from those in the thalamo–amygdala pathway. Cortical afferents to the LA were identified by anterograde transport of biotinylated-dextran amine (BDA) and postsynaptic sites were labeled immunocytochemically using antisera directed against the R1 subunit the NMDA receptor, and the R1 and R2/3 subunits of the AMPA receptor. Electron microscopy revealed that the vast majority of cortical afferents (99%) synapse onto distal dendritic processes and most of these processes (62%) contained at least one glutamate receptor subtype. Cortical afferents synapsed on approximately the same proportion of immunoreactive targets for each glutamate receptor subtype examined. These data provide morphological evidence that cortical afferents form direct synaptic contacts with LA neurons that express both NMDA and AMPA receptors and are consistent with recent physiological studies demonstrating the participation of NMDA and AMPA receptors in cortico–amygdala-transmission. These results are nearly identical to those obtained in the studies of the thalamo–amygdala pathway. Synapse 33:218–229, 1999.
Projections from the medial geniculate body (MGB) to the lateral nucleus of the amygdala (LA) have been implicated in the conditioning of emotional reactions to acoustic stimuli. Anatomical and physiological studies indicate that this pathway uses the excitatory amino acid L-glutamate as a transmitter. Recent physiological studies have demonstrated that synaptic transmission in the thalamo-amygdala pathway requires the activation of both N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, two of the major classes of ionotrophic glutamate receptors. In order to characterize the nature of thalamo-amygdala interactions, we examined the synaptic associations between thalamic afferents and amygdala neurons that contain at least one glutamate receptor subtype. Thalamic afferents to the amygdala were identified by lesion-induced anterograde degeneration and anterograde transport of biotinylated dextran-amine, while postsynaptic glutamate receptors were labeled immunocytochemically using antisera directed the R1 subunit of the NMDA receptor and the GluR1 and GluR2/3 subunits of the AMPA receptors. Both methods demonstrated that the majority (77%) of thalamic afferents contact dendritic spines, and most (60%) of these spines express at least one glutamate receptor subtype. To a lesser extent, identified afferents also contacted small and large dendritic shafts, and many of these were immunoreactive. Thalamic afferents terminated on approximately the same proportion (60%) of immunoreactive targets for each glutamate receptor studied. These data provide morphological evidence that thalamic afferents directly synapse onto amygdala neurons that express glutamate receptors and suggest ways in which thalamic afferents activate and influence amygdala circuitry. Synapse 27:106–121, 1997.
Ionotropic glutamate receptors (iGluRs) constitute a family of ligand gated ion channels subdivided in three classes, NMDA, AMPA (iGluA1-4) and KA (1-5) according to the agonists that selectively activate them. iGluRs are tetrameric assemblies of highly homologous receptor subunits. They are critically important for normal brain function and are considered to be involved on neurological disorders and degenerative diseases such as schizophrenia, Alzheimer’s disease, brain damage following stroke and epilepsy. Since the first publication of the structure of recombinant soluble protein of ligand binding domain of GluA2 extensive studies on this group of receptors were performed
and many crystal structures as complexes of GluA2-LBD with agonists, partial agonists and antagonists were obtained. The structural information in combination with functional data makes good platform for consecutive investigation and design of new selective drugs which will be used in treatment of neurodegerative diseases.
Forty years of research into the function of L-glutamic acid as a neurotransmitter in the vertebrate central nervous system (CNS) have uncovered a tremendous complexity in the actions of this excitatory neurotransmitter and an equally great complexity in the molecular structures of the receptors activated by L-glutamate. L-Glutamate is the most widespread excitatory transmitter system in the vertebrate CNS and in addition to its actions as a synaptic transmitter it produces long-lasting changes in neuronal excitability, synaptic structure and function, neuronal migration during development, and neuronal viability. These effects are produced through the activation of two general classes of receptors, those that form ion channels or "ionotropic" and those that are linked to G-proteins or "metabotropic". The pharmacological and physiological characterization of these various forms over the past two decades has led to the definition of three forms of ionotropic receptors, the kainate (KA), AMPA, and NMDA receptors, and three groups of metabotropic receptors. Twenty-seven genes are now identified for specific subunits of these receptors and another five proteins are likely to function as receptor subunits or receptor associated proteins. The regulation of expression of these protein subunits, their localization in neuronal and glial membranes, and their role in determining the physiological properties of glutamate receptors is a fertile field of current investigations into the cell and molecular biology of these receptors. Both ionotropic and metabotropic receptors are linked to multiple intracellular messengers, such as Ca2+, cyclic AMP, reactive oxygen species, and initiate multiple signaling cascades that determine neuronal growth, differentiation and survival. These cascades of complex molecular events are presented in this review.
Cerebellar granule cells maintained in vitro as primary cultures are a relatively homogeneous neuronal population that can be used to evaluate the developmental expression of neurotransmitter receptors and to assess their role in cell survival and degeneration. The toxicity induced by N-methyl-D-aspartate (NMDA) in granule cells maintained under partially depolarizing conditions and in the presence of physiologic extracellular concentrations of Mg2+ was greatest for the neurons maintained for 14 days in vitro (DIV). However, following NMDA receptor activation neurons as young as 5 DIV exhibited increases in the concentration of intracellular free Ca2+ which were as large as those achieved with cells at 8-9 or 13-14 DIV. The less mature neurons exhibited a "down-regulation" of responses to increasing concentrations of NMDA and the more mature cells maintained elevated intracellular Ca2+ levels during the inter-stimulus periods. Immunochemical analyses of the expression of the NMDA receptor-associated proteins NMDAR1 and glutamate-binding protein (GBP) in granule cells indicated a developmental increase in both proteins, albeit the pattern of expression of NMDAR1 was the more complex. No definite correlation has yet been established between toxicity induced by NMDA and the expression of these two proteins. Finally, although the developmental expression of nitric oxide synthase, an enzyme that catalyzes the formation of the potentially neurotoxic radicals nitric oxide and superoxide anion, increased progressively with the maturation of neurons in culture, an inhibitor of this enzyme did not protect neurons from NMDA-induced toxicity. Therefore, the developmental changes in granule cells that lead to increased vulnerability following excessive activation of NMDA receptors are not yet completely defined.
The editing status of mRNA at the Q/R site of the glutamate receptor subtypes -A, -B, -5 and -6 modulates channel conductivity and ion selectivity of glutamate operated ion channels [4,15,26,30]. In order to investigate whether a modification of this editing process may be involved in kindling epileptogenesis, the percentage of edited variant was determined in the hippocampus of kindled rats and compared to the percentage in control animals. In the latter, GluR-A mRNA was detected only in the unedited form (with detection threshold for edited form < 0.7%), whereas GluR-B was completely edited (> 99%). For percentages were not significantly changed in Schaffer collateral/commissural pathway kindled animals that were sacrificed 24 h after the last generalized seizure. It is concluded that the increased sensitivity for the induction of seizures characteristic for Schaffer collateral kindled animals is not related to a less selective or less efficient mRNA editing process of the different glutamate receptor subunits in the hippocampus.
The murine N-methyl D-aspartate receptor subunit NR2C (epsilon-3) is encoded by a unique gene composed of 12 translated and three 5'-untranslated exons that spread over approximately 20 kilobases of genomic sequence. The GC-rich promoter that lacks TATA- and CAAT-positioning elements has two transcriptional start sites separated by 18 base pairs. One of these sites is located in a conserved initiator motif and, together with the first four exons, specifies the 5'-untranslated sequence of 772 nucleotides. In this sequence, two alternative splice variants were detected that show identical expression patterns in adult mouse brain. Comparison of intron positions in genes encoding different members of the glutamate receptor family confirms a close evolutionary relationship of the NR2C and NMDAR1 subunit genes.
Anatomical and physiological studies indicate that the amino acid L-glutamate is the excitatory transmitter in sensory afferent pathways to the amygdala and in intraamygdala circuits involving the lateral and basal nuclei. The regional, cellular, and subcellular immunocytochemical localizations of N-methyl-D-aspartate (NMDA) and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), two major classes of glutamate receptors, were examined in these areas of the amygdala. A monoclonal antibody and a polyclonal antiserum directed against the R1 subunit of the NMDA receptor were used. Each immunoreagent produced distinct distributions of perikaryal and neuropilar staining. Dendritic immunoreactivity was localized primarily to asymmetric (excitatory) synaptic junctions, mostly on spines, consistent with the conventional view of the organization and function of NMDA receptors. Whereas the anti-NMDAR1 antiserum produced sparse presynaptic axon terminal labeling and extensive glial labeling, the anti-NMDAR1 antibody labeled considerably fewer glia and many more presynaptic axon terminals. Labeled presynaptic terminals formed asymmetric and symmetric synapses, suggesting presynaptic regulation of both excitatory and inhibitory transmission. Immunoreactivity for different subunits of the AMPA receptor (GluR1, GluR2/3, and GluR4) was uniquely distributed across neuronal populations, and some receptor subunits were specific to certain cell types. Immunoreactivity for GluR1 and Glu2/3 was predominantely localized to dendritic shafts and was more extensive than that of GluR4 due to heavy labeling of proximal portions of dendrites. The distribution of GluR4 immunoreactivity was similar to NMDAR1: GluR4 was seen in presynaptic terminals, glia, and dendrites and was primarily localized to spines. The presynaptic localization of GluR4 in the absence of GluR2 suggests glutamate-mediated modulation of presynaptic Ca++ concentrations. These data add to our understanding of the morphological basis of pre- and postsynaptic transmission mechanisms and synaptic plasticity in the amygdala.
Ion channels play critical roles in nervous system function, from initiating rapid synaptic activity to propagation of action potentials. Studies have indicated that many of the effects of ethanol on the nervous system are likely caused by the actions of ethanol on ion channels. Ion channels are multimeric structures that gate ions through subtle changes in tertiary structure. Ethanol readily enters molecular sites within multimeric ion channels, modifying intermolecular forces and bonds that are important for the open-close-inactivation kinetic properties of channels. The diversity of channel composition caused by the multimeric structure results in subtypes of channels that have a spectrum of sensitivity to ethanol that translates into brain regional differences in ethanol sensitivity, in part caused by differences in ion channel subunit composition. Ethanol has been shown to affect both receptor-activated ion channels and voltage-gated ion channels. The acute intoxicating and incoordinating effects of ethanol are probably related to inhibition of subtypes of NMDA-glutamate receptor ion channels and potentiation of certain subtypes of GABAA receptor ion channels. Effects on these channels, as well as glycine, nicotinic cholinergic, serotonergic, and other ion channels, likely contribute to the euphoric, sedative, and other acute actions of ethanol. Changes in ion channel subunit composition, density, and properties probably also contribute to ethanol tolerance, dependence, withdrawal hyperexcitability, and neurotoxicity. A substantial number of studies have implicated glutamate NMDA receptor, GABAA, and L-type voltage-gated calcium channels in the adaptive changes in the brain during chronic ethanol exposure. The diversity of ion channels subunits, their prominent role in brain function, and ethanol action are likely to make them important contributors to alcoholism and alcohol abuse.
Zn2+ inhibits NMDA-type excitatory amino acid activity by a non-competitive action. Based on regional differences in the central nervous system (CNS) in binding characteristics of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate ([3H]MK-801) and other non-competitive antagonists of NMDA used to label open channels in the receptor complex, we compared the inhibitory influence of Zn2+ on [3H]MK-801 binding in whole mouse brain and spinal cord membranes. Radioligand binding techniques were used in the presence and absence of maximally effective concentrations of glycine and glutamate. Using extensively washed membranes without exogenous glycine and glutamate, Zn2+ was found to be a weaker inhibitor of the [3H]MK-801-labeled site in the spinal cord than in the whole brain. In contrast, exogenous glycine and glutamate decreased the inhibitory effect of Zn2+ in the brain but dramatically increased the inhibitory effect of Zn2+ in the spinal cord. Thus the inhibitory effect of Zn2+ in the spinal cord appears to be magnified by glutamatergic and glycinergic activity while that in the brain is not. The different actions of Zn2+ may be attributable to the differential distribution of NMDA receptor subunits in the mouse brain and spinal cord.
RED1 is a double-stranded RNA-specific editase characterized in the rat and is implicated in the editing of glutamate receptor subunit pre-mRNAs, particularly in the brain. Starting from human ESTs homologous to the rat RED1 sequence, we have characterized two forms of human RED1 cDNAs, one form coding for a putative peptide of 701 amino acids (similar to the shorter of two rat mRNAs) and a long form coding for a putative protein of 741 amino acids, the extra 120 bp of which are homologous to an AluJ sequence. Both forms were observed at approximately equal levels in cDNA clones and in seven different human tissues tested by RT-PCR. The human and rat short isoforms have 95 and 85% sequence identity at the amino acid and nucleotide levels, respectively. The human sequence (designated ADARB1 by the HGMW Nomenclature Committee) contains two double-stranded RNA-binding domains and a deaminase domain implicated in its editing action. Northern blot analysis detected two transcripts of 8.8 and 4.2 kb strongly expressed in brain and in many human adult and fetal tissues. ADARB1 maps to human chromosome 21q22.3, a region to which several genetic disorders map, including one form of bipolar affective disorder. Recently it was shown that heterozygous mice harboring an editing-incompetent glutamate receptor B allele have early onset fatal epilepsy. Since glutamate receptor channels are essential elements in synaptic function and plasticity and mediate pathology in many neurological disorders, and since RED1 is central in glutamate receptor channel control, ADARB1 is a candidate gene for diseases with neurological symptoms, such as bipolar affective disorder and epilepsy.
Projections from the medial geniculate body (MGB) to the lateral nucleus of the amygdala (LA) have been implicated in the conditioning of emotional reactions to acoustic stimuli. Anatomical and physiological studies indicate that this pathway uses the excitatory amino acid L-glutamate as a transmitter. Recent physiological studies have demonstrated that synaptic transmission in the thalamo-amygdala pathway requires the activation of both N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, two of the major classes of ionotrophic glutamate receptors. In order to characterize the nature of thalamoamygdala interactions, we examined the synaptic associations between thalamic afferents and amygdala neurons that contain at least one glutamate receptor subtype. Thalamic afferents to the amygdala were identified by lesion-induced anterograde degeneration and anterograde transport of biotinylated dextran-amine, while postsynaptic glutamate receptors were labeled immunocytochemically using antisera directed the R1 subunit of the NMDA receptor and the GluR1 and GluR2/3 subunits of the AMPA receptors. Both methods demonstrated that the majority (77%) of thalamic afferents contact dendritic spines, and most (60%) of these spines express at least one glutamate receptor subtype. To a lesser extent, identified afferents also contacted small and large dendritic shafts, and many of these were immunoreactive. Thalamic afferents terminated on approximately the same proportion (60%) of immunoreactive targets for each glutamate receptor studied. These data provide morphological evidence that thalamic afferents directly synapse onto amygdala neurons that express glutamate receptors and suggest ways in which thalamic afferents activate and influence amygdala circuitry.
The basics of neurophysiological, neurochemical and molecular mechanisms of epileptogenesis in early ontogeny are discussed. The role of developmental neuropathology, synaptogenesis; the role of glutamatergic and GABA-ergic transmitter systems are summarized. The candidates of genes in the childhood epilepsies, developmental changes in the expression of genes of voltage-gated ionic channels and receptor genes are reviewed.
We determined the half-lives for two subunits of a complex that functions as a glutamate and N-methyl-D-aspartate (NMDA) receptor-ion channel in synaptic membranes. These two proteins are a 71 kDa glutamate-binding protein (GBP) and an 80 kDa CPP-binding protein (CBP). Seven month-old Fischer 344 rats were injected with L-[14C] leucine. The radioactivity in the two proteins was determined in a crude synaptosomal membrane fraction obtained from the brains of rats sacrificed from 4 hours to 13 days after the injection. The previously reported data on time-dependent appearance and loss of L-[14C] leucine radioactivity in the serum (Ferrington et al., 1997, Biochem. Biophys. Res. Commun. 237, 163-165) was used in the present study to estimate the half-lives of GBP and CBP. Theoretical curves best fit the experimental data obtained for the two proteins assuming apparent half-lives of 14 (+/- 2.4) and 18 (+/- 1.2) hours for CBP and GBP, respectively.
A specific ivermectin-sensitive, glutamate binding site has been identified in the parasitic nematode Haemonchus contortus. Glutamate binding in H. contortus was saturable and occurred in a single class of high-affinity binding sites which appeared to have pharmacological properties different from those of mammalian glutamate receptors. Adult and larval forms of H. contortus had dramatically different glutamate binding kinetics, the larvae showing nearly up to 200-fold higher Bmax values and up to 9-fold increases in Kd values compared to adults. Treatment of adult H. contortus with the anthelmintic, ivermectin, decreased the Bmax value for glutamate binding in the susceptible strain but not in the resistant parasites. Furthermore, selection for ivermectin resistance was associated with a significant increase in Bmax for glutamate binding in adults and a similarly significant increase in glutamate binding affinity in larvae. These results suggest that the H. contortus glutamate binding site identified in this study may be involved in the phenomenon of ivermectin resistance.
The lateral nucleus of the amygdala (LA) is a critical component of the circuitry through which environmental stimuli are endowed with emotional meaning through association with painful or threatening events. Individual cells in LA receive convergent input from auditory processing areas in the thalamus and cortex, and the excitatory amino-acid L-glutamate (Glu) participates in synaptic transmission in both pathways. Previously, we characterized the ultrastructure of pre- and postsynaptic processes in the thalamo-amygdala pathway, and showed the relation of presynaptic inputs to N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydoxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits. In the present study, we examined the nature of cortico-amygdala synaptic interactions with Glu receptors in LA and determined whether they are similar or different from those in the thalamo-amygdala pathway. Cortical afferents to the LA were identified by anterograde transport of biotinylated-dextran amine (BDA) and postsynaptic sites were labeled immunocytochemically using antisera directed against the R1 subunit the NMDA receptor, and the R1 and R2/3 subunits of the AMPA receptor. Electron microscopy revealed that the vast majority of cortical afferents (99%) synapse onto distal dendritic processes and most of these processes (62%) contained at least one glutamate receptor subtype. Cortical afferents synapsed on approximately the same proportion of immunoreactive targets for each glutamate receptor subtype examined. These data provide morphological evidence that cortical afferents form direct synaptic contacts with LA neurons that express both NMDA and AMPA receptors and are consistent with recent physiological studies demonstrating the participation of NMDA and AMPA receptors in cortico-amygdala-transmission. These results are nearly identical to those obtained in the studies of the thalamo-amygdala pathway.
The ionotropic glutamate receptor subunit GluR6 undergoes developmentally and regionally regulated Q/R site RNA editing that reduces the calcium permeability of GluR6-containing kainate receptors. To investigate the functional significance of this editing in vivo, we engineered mice deficient in GluR6 Q/R site editing. In these mutant mice but not in wild types, NMDA receptor-independent long-term potentiation (LTP) could be induced at the medial perforant path-dentate gyrus synapse. This indicates that kainate receptors with unedited GluR6 subunits can mediate LTP. Behavioral analyses revealed no differences from wild types, but mutant mice were more vulnerable to kainate-induced seizures. Together, these results suggest that GluR6 Q/R site RNA editing may modulate synaptic plasticity and seizure vulnerability.
Unlabelled:
It is generally agreed that basal forebrain neuronal circuits contribute to the mediation of the ability to detect, select and discriminate signals, to suppress the processing of irrelevant information, and to allocate processing resources to competing tasks. Rats were trained in a task designed to assess sustained attention, or in a cued discrimination task that did not tax attentional processes. Animals were equipped with guide cannula to infuse bilaterally antisense oligodeoxynucleotides (ODNs) against the N-methyl-D-aspartate (NMDA) NR1 subunits, or missense ODNs, into the substantia innominata of the basal forebrain. Infusions of antisense or missense ODNs did not affect cued visual discrimination performance. Infusions of antisense ODNs dose-dependently impaired sustained attention performance by selectively decreasing the animals' ability to detect signals while their ability to reject nonsignal trials remained unchanged. The detrimental attentional effects of antisense infusions were maximal 24 h after the third and final infusion, and performance returned to baseline 24 h later. Missense infusions did not affect attentional performance. Separate experiments demonstrated extensive suppression of NR1 subunit immunoreactivity in the substantia innominata. Furthermore, infusions of antisense did not produce neurotoxic effects in that region as demonstrated by the Fluoro-Jade
Method:
The present data support the hypothesis that NMDA receptor (NMDAR) stimulation in the basal forebrain, largely via glutamatergic inputs originating in the prefrontal cortex, represents a necessary mechanism to activate the basal forebrain corticopetal system for mediation of attentional performance.
During physiological patterns of activity, synaptic activity is regulated by many forms of short-term plasticity. Here, we compare the functional consequences of such plasticity at the synapse from the climbing fibre to the Purkinje cell in the cerebellum and at the synapse between the retinal ganglion cell and the thalamocortical relay neuron in the lateral geniculate nucleus. Despite superficial similarities between these two powerful synapses, they have distinctive synaptic plasticity. The climbing fibre synapse is highly reliable but accomplishes this through many synaptic specializations. However, the retinogeniculate synapse dynamically regulates the flow of visual information by using two types of receptor that have different types of plasticity. These synapses illustrate the important functional consequences of synaptic plasticity.
Ebola viruses are highly lethal human pathogens that have received considerable attention in recent years due to an increasing re-emergence in Central Africa and a potential for use as a biological weapon. There is no vaccine or treatment licensed for human use. In the past, however, important advances have been made in developing preventive vaccines that are protective in animal models. In this regard, we showed that a single injection of a live-attenuated recombinant vesicular stomatitis virus vector expressing the Ebola virus glycoprotein completely protected rodents and nonhuman primates from lethal Ebola challenge. In contrast, progress in developing therapeutic interventions against Ebola virus infections has been much slower and there is clearly an urgent need to develop effective post-exposure strategies to respond to future outbreaks and acts of bioterrorism, as well as to treat laboratory exposures. Here we tested the efficacy of the vesicular stomatitis virus-based Ebola vaccine vector in post-exposure treatment in three relevant animal models. In the guinea pig and mouse models it was possible to protect 50% and 100% of the animals, respectively, following treatment as late as 24 h after lethal challenge. More important, four out of eight rhesus macaques were protected if treated 20 to 30 min following an otherwise uniformly lethal infection. Currently, this approach provides the most effective post-exposure treatment strategy for Ebola infections and is particularly suited for use in accidentally exposed individuals and in the control of secondary transmission during naturally occurring outbreaks or deliberate release.
A single site in recombinant glutamate receptor channels of the GluR1- GluR4 family has been previously identified as a key regulator of ion permeation. The natural amino acid at this position (arginine in GluR2 but glutamine in GluR1, GluR3, and GluR4) determines both the ability to pass outward current and the divalent cation permeability of kainate- activated receptor channels. By mutagenesis of GluR6, we demonstrated that the same site also controls the ability to pass outward current in another non-NMDA receptor family. Additional mutations at and near this site in GluR3 indicated that the position of the arginine is critical to function, that the ability to pass outward current is not necessarily linked to low barium permeability, and that the size as well as the charge of the side chain at this position influences barium permeation. These results provide evidence that this site forms part of the selectivity filter of glutamate receptor channels.
Transport systems specific for L-glutamate and L-aspartate play an important role in the termination of neurotransmitter signals at excitatory synapses. We describe here the structure and function of a 66-kDa glycoprotein that was purified from rat brain and identified as an L-glutamate/L-aspartate transporter (GLAST). A GLAST-specific cDNA clone was isolated from a rat brain cDNA library. The cDNA insert encodes a polypeptide with 543 amino acid residues (59,697 Da). The amino acid sequence of GLAST suggests a distinctive structure and membrane topology, with some conserved motifs also present in prokaryotic glutamate transporters. The transporter function has been verified by amino acid uptake studies in the Xenopus laevis oocyte system. GLAST is specific for L-glutamate and L-aspartate, shows strict dependence on Na+ ions, and is inhibited by DL-threo-3-hydroxy-aspartate. In situ hybridization reveals a strikingly high density of GLAST mRNA in the Purkinje cell layer of cerebellum, presumably in the Bergmann glia cells, and a less dense distribution throughout the cerebrum. These data suggest that GLAST may be involved in the regulation of neurotransmitter concentration in central nervous system.
A complementary DNA encoding the rat NMDA receptor has been cloned and characterized. The single protein encoded by the cDNA forms a receptor-channel complex that has electrophysiological and pharmacological properties characteristic of the NMDA receptor. This protein has a significant sequence similarity to the AMPA/kainate receptors and contains four putative transmembrane segments following a large extracellular domain. The NMDA receptor messenger RNA is expressed in neuronal cells throughout the brain regions, particularly in the hippocampus, cerebral cortex and cerebellum.
We have isolated a complementary DNA clone by screening a rat brain cDNA library for expression of kainate-gated ion channels in Xenopus oocytes. The cDNA encodes a single protein of relative molecular mass (Mr) 99,800 which on expression in oocytes forms a functional ion channel possessing the electrophysiological and pharmacological properties of the kainate subtype of the glutamate receptor family in the mammalian central nervous system.
The N-methyl d-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent
sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat
brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% ientical in sequence. These are
structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the
NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate-and
NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed
in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger
RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.
Glutamate is the principal excitatory neurotransmitter in the brain. Glutamate activates cation-selective receptor channels carried by nearly every neuron and by glial cells1–4. Bernd Sommer and Peter Seeburg describe how our concepts concerning the molecular and functional design of ionotropic glutamate receptors are rapidly progressing, with the recent discovery of novel receptor properties and new subunits following the landmark cloning of the first receptor subunit by M. Hollmann and his colleagues. New properties currently revealed by the cloned receptor channels may guide physiologists in characterizing the elementary steps in synaptic transmission, help neurologists to define the role of glutamate receptors in acute and chronic neuropathologies, and enlighten all neuroscientists whose models for learning and memory involve the idiosyncracies of particular channel subtypes.
Functionally diverse GIuR channels of the AMPA subtype are generated by the assembly of GIuR-A, -B, -C, and -D subunits into homo- and heteromeric channels. The GIuR-B subunit is dominant in determining functional properties of heteromeric AMPA receptors. This subunit exists in developmentally distinct edited and unedited forms, GIuR-B(R) and GIuR-B(Q), which differ in a single amino acid in transmembrane segment TM2 (Q/R site). Homomeric GIuR-B(R) channels expressed in 293 cells display a low divalent permeability, whereas homomeric GluR-B(Q) and GIuR-D channels exhibit a high divalent permeability. Mutational analysis revealed that both the positive charge and the size of the amino acid side chain located at the Q/R site control the divalent permeability of homomeric channels. Coexpression of Q/R site arginine- and glutamine-containing subunits generates cells with varying divalent permeabilities depending on the amounts of expression vectors used for cell transfection. Intermediate divalent permeabilities were traced to the presence of both divalent permeant homomeric and impermeant heteromeric channels. It is suggested that the positive charge contributed by the arginine of the edited GIuR-B(R) subunit determines low divalent permeability in heteromeric GIuR channels and that changes in GIuR-B(R) expression regulate the AMPA receptor-dependent divalent permeability of a cell.
L-glutamate, the principal excitatory transmitter in the brain, gates ion channels mediating fast neurotrans-mission. Subunit components of two related classes of glutamate receptor channels have been characterized by cDNA cloning and shown to carry either an arginine or a glutamine residue in a defined position of their putative channel-forming segment. The arginine residue in this segment profoundly alters, and dominates, the properties of ion flow, as demonstrated for one channel class. We now show that the genomic DNA sequences encoding the particular channel segment of all subunits harbor a glutamine codon (CAG), even though an arginine codon (CGG) is found in mRNAs of three subunits. Multiple genes and alternative exons were excluded as sources for the arginine codon; hence, we propose that transcripts for three subunits are altered by RNA editing. This process apparently edits subunit transcripts of the two glutamate receptor classes with different efficiency and selectivity.
1. Currents activated by glutamate receptor (GluR) agonists were recorded from outside-out patches isolated from the soma of visually identified pyramidal neurones of the CA3 and CA1 region of rat hippocampal slices. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), L-glutamate (L-Glu), and kainate (KA) were delivered either by bath application through perfusion of the recording chamber or by rapid application via a piezo-driven two-barrelled fast application system. 2. Bath application of each of the three agonists activated inward currents in all patches (n = 134) at holding potentials of -50 or -60 mV. The current amplitude increased in size between 3 to 30 microM-AMPA and 100 microM to 1 mM-KA. With this slow mode of bath application, the responses showed no apparent desensitization even at saturating concentrations of AMPA (30 microM) and KA (1 mM). 3. The ratio of currents activated by 30 microM-AMPA and 300 microM-KA showed a characteristic difference between CA3 and CA1 neurones. The ratio was 0.242 +/- 0.028 (mean +/- S.E.M., n = 16) for CA3 cell patches and 0.097 +/- 0.012 (n = 8) for CA1 cell patches indicating that GluRs in the two cell populations are different. 4. The steady-state current-voltage relations (I-Vs) for AMPA- and KA-activated currents showed pronounced outward rectification for both cell types (when the main cations are Na+ in the bath and Cs+ in the pipette solution). The current reversed close to 0 mV and the ratio of chord conductances 80 mV on either side of the reversal potential was 2.66 for KA-activated currents in CA3 cell patches and 2.60 in CA1 cell patches. AMPA-activated currents showed a time-dependent increase after steps to positive membrane potentials and a decrease after steps to negative voltages, indicating that a gating process is responsible for outward rectification of the steady-state I-V. 5. The permeability (P) of GluR channels was high for Na+ as compared to Cs+ for both cell types (PNa/PCs = 0.88 and 0.84). The permeability was low for N-methyl-D-glucamine+ (PNMG/PCs < or = 0.03) and Ca2+ (PCa/PCs < or = 0.05). 6. The current noise level increased during application of AMPA or KA. Apparent single-channel conductances obtained from fluctuation analysis were higher for AMPA than for KA, but similar for both cell types. In CA3 cell patches, AMPA activated channels with an apparent chord conductance of 7.2 pS, KA of 3.0 pS conductance.(ABSTRACT TRUNCATED AT 400 WORDS)
Glutamate-operated ion channels (GluR channels) of the L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-kainate subtype are found in both neurons and glial cells of the central nervous system. These channels are assembled from the GluR-A, -B, -C, and -D subunits; channels containing a GluR-B subunit show an outwardly rectifying current-voltage relation and low calcium permeability, whereas channels lacking the GluR-B subunit are characterized by a doubly rectifying current-voltage relation and high calcium permeability. Most cell types in the central nervous system coexpress several subunits, including GluR-B. However, Bergmann glia in rat cerebellum do not express GluR-B subunit genes. In a subset of cultured cerebellar glial cells, likely derived from Bergmann glial cells. GluR channels exhibit doubly rectifying current-voltage relations and high calcium permeability, whereas GluR channels of cerebellar neurons have low calcium permeability. Thus, differential expression of the GluR-B subunit gene in neurons and glia is one mechanism by which functional properties of native GluR channels are regulated.
Glutamate receptors, the most abundant excitatory transmitter receptors in the brain, are not restricted to neurons; they
have also been detected on glial cells. Bergmann glial cells in mouse cerebellar slices revealed a kainate-type glutamate
receptor with a sigmoid current-to-voltage relation, as demonstrated with the patch-clamp technique. Calcium was imaged with
fura-2, and a kainate-induced increase in intracellular calcium concentration was observed, which was blocked by the non-N-methyl-D-aspartate
(NMDA) glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and by low concentrations of external calcium,
indicating that there was an influx of calcium through the kainate receptor itself. The entry of calcium led to a marked reduction
in the resting (passive) potassium conductance of the cell. Purkinje cells, which have glutamatergic synapses, are closely
associated with Bergmann glial cells and therefore may provide a functionally important stimulus.
We here report the existence of 6 additional isoforms of the NMDA receptor generated via alternative splicing by molecular analysis of cDNA clones isolated from a rat forebrain cDNA library. These isoforms possess the structures with an insertion at the extracellular amino-terminal region or deletions at two different extracellular carboxyl-terminal regions, or those formed by combinations of the above insertion and deletions. One of the deletions results in the generation of a new carboxyl-terminal sequence. All these isoforms possess the ability to induce electrophysiological responses to NMDA and respond to various antagonists selective to the NMDA receptor in the Xenopus oocyte expression system. In addition, a truncated form of the NMDA receptor also exists that contains only the extreme amino-terminal sequence of this protein molecule. These data indicate that the NMDA receptor consists of heterogeneous molecules that differ in the extracellular sequence of the amino- and carboxyl-terminal regions.
Two novel subunits of the mouse NMDA receptor channel, the epsilon 2 and epsilon 3 subunits, have been identified by cloning and expression of complementary DNAs. The heteromeric epsilon 1/zeta 1, epsilon 2/zeta 1 and epsilon 3/zeta 1 NMDA receptor channels exhibit distinct functional properties in affinities for agonists and sensitivities to competitive antagonists and Mg2+ block. In contrast to the wide distribution of the epsilon 1 and zeta 1 subunit messenger RNAs in the brain, the epsilon 2 subunit mRNA is expressed only in the forebrain and the epsilon 3 subunit mRNA is found predominantly in the cerebellum. The epsilon 1/zeta 1 and epsilon 2/zeta 1 channels expressed in Xenopus oocytes, but not the epsilon 3/zeta 1 channel, are activated by treatment with 12-O-tetradecanoylphorbol 13-acetate. These findings suggest that the molecular diversity of the epsilon subunit family underlies the functional heterogeneity of the NMDA receptor channel.
The N-methyl-D-aspartate (NMDA) receptor forms a cation-selective channel with a high calcium permeability and sensitivity
to channel block by extracellular magnesium. These properties, which are believed to be important for the induction of long-term
changes in synaptic strength, are imparted by asparagine residues in a putative channel-forming segment of the protein, transmembrane
2 (TM2). In the NR1 subunit, replacement of this asparagine by a glutamine residue decreases calcium permeability of the channel
and slightly reduces magnesium block. The same substitution in NR2 subunits strongly reduces magnesium block and increases
the magnesium permeability but barely affects calcium permeability. These asparagines are in a position homologous to the
site in the TM2 region (Q/R site) of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that is occupied
by either glutamine (Q) or arginine (R) and that controls divalent cation permeability of the AMPA receptor channel. Hence
AMPA and NMDA receptor channels contain common structural motifs in their TM2 segments that are responsible for some of their
ion selectivity and conductance properties.
The primary structure of a novel subunit of the mouse NMDA (N-methyl-D-aspartate) receptor channel, designated epsilon 4, has been revealed by cloning and sequencing the cDNA. The epsilon 4 subunit shares high amino acid sequence identity with the epsilon 1, epsilon 2 and epsilon 3 subunits of the mouse NMDA receptor channel, thus constituting the epsilon subfamily of the glutamate receptor channel. Expression from cloned cDNAs of the epsilon 4 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional NMDA receptor channels. The epsilon 4/zeta 1 heteromeric channel exhibits high apparent affinities for agonists and low sensitivities to competitive antagonists. The epsilon 4 subunit is thus distinct in functional properties from the epsilon 1, epsilon 2 and epsilon 3 subunits, and contributes further diversity of the NMDA receptor channel.
Synaptic transmission of most vertebrate synapses is thought to be terminated by rapid transport of the neurotransmitter into presynaptic nerve terminals or neuroglia. L-Glutamate is the major excitatory transmitter in brain and its transport represents the mechanism by which it is removed from the synaptic cleft and kept below toxic levels. Here we use an antibody against a glial L-glutamate transporter from rat brain to isolate a complementary DNA clone encoding this transporter. Expression of this cDNA in transfected HeLa cells indicates that L-glutamate accumulation requires external sodium and internal potassium and transport shows the expected stereospecificity. The cDNA sequence predicts a protein of 573 amino acids with 8-9 putative transmembrane alpha-helices. Database searches indicate that this protein is not homologous to any identified protein of mammalian origin, including the recently described superfamily of neurotransmitter transporters. This protein therefore seems to be a member of a new family of transport molecules.
The neurotransmitter glutamate mediates excitatory synaptic transmission throughout the brain. A family of genes encoding
subunits of the non-N-methyl-D-aspartate (non-NMDA) type of glutamate receptor has been cloned. Some combinations of these
subunits assemble into receptors with a substantial permeability to calcium, whereas others do not. To investigate the structural
features that control ion permeation through these ligand-gated channels, mutant receptor subunits with single-amino acid
changes were constructed. Mutation of a certain amino acid that results in a net charge change (from glutamine to arginine
or vice versa) alters both the current-voltage relation and the calcium permeability of non-NMDA receptors. A site has thus
been identified that regulates the permeation properties of these glutamate receptors.
1. N-methyl-D-aspartate (NMDA)-, quisqualate- and kainate-induced currents were recorded in cultured rat hippocampal neurones using the whole-cell voltage-clamp technique. To isolate the inward currents carried by Ca2+ and other divalent cations (Sr2+, Ba2+, Mn2+ and Mg2+), both Na+ and K+ in the control external solution were replaced with the impermeant cation N-methylglucamine (NMG). 2. Replacement of Na+, K+ and Ca2+ with NMG abolished NMDA-, quisqualate- and kinate-induced inward currents. In Na(+)-, K(+)-free (abbreviated simply as Na(+)-free) solution containing 10 mM-Ca2+ NMDA caused prominent inward currents at -60 mV. In this solution with the internal solution containing 165 mM-Cs+, the reversal potential of the NMDA-induced current was -5.0 +/- 0.7 mV (n = 36), indicating a value of PCa/PCs = 6.2 for the ratio of the permeability coefficients of Ca2+ and Cs+ according to the constant-field equation. 3. NMDA elicited inward current responses at -60 mV in Na(+)-, Ca2(+)-free solution containing 10 mM-Sr2+, Ba2+, or Mn2+, but not in Na(+)-free, 10 mM-Mg2+ solution. On the basis of reversal potential measurements, the permeability sequence of NMDA receptor channels among the divalent cations was determined to be Ba2+ (1.2) greater than Ca2+ (1.0) greater than Sr2+ (0.8) greater than Mn2+ (0.3) much greater than Mg2+ (less than 0.02). 4. The reversal potential of the quisqualate-induced current was more negative than -80 mV in Na(+)-free, 10 mM-Ca2+ solution, indicating a value of PCa/PCs less than 0.18. 5. Kainate-induced current responses were classified into two types. In the type I response the reversal potential of the kainate-induced current was more negative than -80 mV in Na(+)-free, 10 mM-Ca2+ solution, indicating that the Ca2+ permeability of this type of kainate channel is as low as that of the quisqualate channel. In the neurones which showed a type I response, there was a tendency of outward rectification in the current-voltage plots of the kainate response in control solution. 6. In the type II response kainate caused prominent inward currents at -60 mV in Na(+)-free, 10 mM-Ca2+ solution. The reversal potential was -23.3 +/- 5.6 mV (n = 17), indicating a permeability ratio PCa/PCs = 2.3. In the neurones which showed a type II response, a remarkable inward rectification was observed in the current-voltage plots of the kainate response in control solution. 7. Type II kainate channels showed relatively poor selectivity among divalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)
NMDA (N-methyl-D-aspartate) receptors and non-NMDA receptors represent the two major classes of ion channel-linked glutamate
receptors. Unlike the NMDA receptor channels, non-NMDA receptor channels have usually been thought to conduct monovalent cations
only. Non-NMDA receptor ion channels that can be gated by kainic acid (KA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole
propionic acid (AMPA) are formed by the glutamate receptor subunits GluR1, GluR2, and GluR3. These subunits were expressed
in various combinations in Xenopus oocytes so that their permeability to divalent cations could be studied. At physiological
resting potentials, KA and AMPA elicited inward calcium currents in oocytes expressing GluR1, GluR3, and GluR1 plus GluR3.
In contrast, oocytes expressing GluR1 plus GluR2 or GluR3 plus GluR2 showed no such permeability. Thus, in neurons expressing
certain KA-AMPA receptor subunits, glutamate may trigger calcium-dependent intracellular events by activating non-NMDA receptors.
Functional glutamate receptor (GluRs) were transiently expressed in cultured mammalian cells from cloned complementary DNAs
encoding GluR-A, -B, -C, or -D polypeptides. The steady-state current-voltage (I-V) relations of glutamate- and kainate-induced
currents through homomeric channels fell into two classes: channels composed of either the GluR-A, -C, and -D subunits showed
doubly rectifying I-V curves, and channels composed of the GluR-B subunits displayed simple outward rectification. The presence
of GluR-B subunits in heteromeric GluRs determined the I-V behavior of the resulting channels. Site-directed mutagenesis identified
a single amino acid difference (glutamine to arginine) in the putative transmembrane segment TM2 responsible for subunit-specific
I-V relationships. The properties of heteromeric wild-type and mutant GluRs revealed that the dominance of GluR-B is due to
the arginine residue in the TM2 region.
The complementary DNA of a metabotropic glutamate receptor coupled to inositol phosphate/Ca2+ signal transduction has been cloned and characterized. This receptor shows no sequence similarity to conventional G protein-coupled receptors and has a unique structure with large hydrophilic sequences at both sides of seven putative membrane-spanning domains. Abundant expression of this messenger RNA is observed in neuronal cells in hippocampal dentate gyrus and CA2-3 and in cerebellar Purkinje cells, suggesting the importance of this receptor in specific hippocampal and cerebellar functions.
Development of new selective ligands for excitatory amino acid receptors has been fundamental in supporting this rapidly developing field. Some of the most important ligands have come from the laboratories of Jeff Watkins, Povl Krogsgaard-Larsen and Tage Honoré, who collaborate in this double-length review to describe the chemical features and SARs of agonists and antagonists, particularly those features associated with subtype selectivity.
Four cloned cDNAs encoding 900-amino acid putative glutamate receptors with approximately 70 percent sequence identity were
isolated from a rat brain cDNA library. In situ hybridization revealed differential expression patterns of the cognate mRNAs
throughout the brain. Functional expression of the cDNAs in cultured mammalian cells generated receptors displaying alpha-amino-3-hydroxy-5-methyl-4-isoxazole
propionic acid (AMPA)-selective binding pharmacology (AMPA = quisqualate greater than glutamate greater than kainate) as well
as cation channels gated by glutamate, AMPA, and kainate and blocked by 6,7-dinitroquinoxaline-2,3-dione (CNQX).
Three closely related genes, GluR1, GluR2, and GluR3, encode receptor subunits for the excitatory neurotransmitter glutamate.
The proteins encoded by the individual genes form homomeric ion channels in Xenopus oocytes that are sensitive to glutamatergic
agonists such as kainate and quisqualate but not to N-methyl-D-aspartate, indicating that binding sites for kainate and quisqualate
exist on single receptor polypeptides. In addition, kainate-evoked conductances are potentiated in oocytes expressing two
or more of the cloned receptor subunits. Electrophysiological responses obtained with certain subunit combinations show agonist
profiles and current-voltage relations that are similar to those obtained in vivo. Finally, in situ hybridization histochemistry
reveals that these genes are transcribed in shared neuroanatomical loci. Thus, as with gamma-aminobutyric acid, glycine, and
nicotinic acetylcholine receptors, native kainate-quisqualate-sensitive glutamate receptors form a family of heteromeric proteins.
Responses of excitatory amino acid receptors to rapidly applied glutamate were measured in outside-out membrane patches from chick spinal neurons. The peak current varied with glutamate concentration, with a half-maximal response at 510 microM and a Hill coefficient near 2. Currents activated by 1 mM glutamate desensitized and recovered in two phases. The faster time constant was identical to the time constant of decay of synaptic currents, suggesting that glutamatergic synaptic currents are terminated, in part, by receptor desensitization. Steady-state desensitization was evident following application of only 2-3 microM glutamate, concentrations comparable to levels in the extracellular space in the intact brain. Thus, glutamate receptor desensitization can affect synaptic efficacy in two ways: at high concentrations, rapid desensitization of receptors may curtail synaptic currents; at low concentrations, there is a significant reduction in the number of activatable receptors.
1. The after‐effects of repetitive stimulation of the perforant path fibres to the dentate area of the hippocampal formation have been examined with extracellular micro‐electrodes in rabbits anaesthetized with urethane.
2. In fifteen out of eighteen rabbits the population response recorded from granule cells in the dentate area to single perforant path volleys was potentiated for periods ranging from 30 min to 10 hr after one or more conditioning trains at 10–20/sec for 10–15 sec, or 100/sec for 3–4 sec.
3. The population response was analysed in terms of three parameters: the amplitude of the population excitatory post‐synaptic potential (e.p.s.p.), signalling the depolarization of the granule cells, and the amplitude and latency of the population spike, signalling the discharge of the granule cells.
4. All three parameters were potentiated in 29% of the experiments; in other experiments in which long term changes occurred, potentiation was confined to one or two of the three parameters. A reduction in the latency of the population spike was the commonest sign of potentiation, occurring in 57% of all experiments. The amplitude of the population e.p.s.p. was increased in 43%, and of the population spike in 40%, of all experiments.
5. During conditioning at 10–20/sec there was massive potentiation of the population spike (‘frequency potentiation’). The spike was suppressed during stimulation at 100/sec. Both frequencies produced long‐term potentiation.
6. The results suggest that two independent mechanisms are responsible for long‐lasting potentiation: ( a ) an increase in the efficiency of synaptic transmission at the perforant path synapses; ( b ) an increase in the excitability of the granule cell population.
GluR6, a subunit of high affinity kainate receptor channels in the mammalian CNS, carries a glutamine (Q) or arginine (R) residue in a critical position (Q/R site) of the putative channel-forming segment TM2. One form, GluR6(Q), is encoded by the GluR6 gene; the other, GluR6(R), is generated by RNA editing. Further analysis of cloned GluR6 cDNA revealed that two additional positions, located in transmembrane segment TM1, are diversified by RNA editing to generate either isoleucine (I) or valine (V) in one and tyrosine (Y) or cysteine (C) in the other TM1 position. In GluR6 channels, in contrast with AMPA receptor channels, the presence of Q in the TM2 Q/R site determines channels with low Ca2+ permeability, whereas an R determines a higher Ca2+ permeability if TM1 is fully edited. In the TM1 unedited form of GluR6, Ca2+ permeability is less dependent on the presence of either Q or R in TM2. Thus Ca2+ permeability of kainate receptor channels can vary, depending on editing of both TM1 and TM2.
This review focuses on two aspects of synaptic transmission that have recently attracted considerable interest: the quantal release of neurotransmitter at synapses, and a phenomenon, hippocampal long-term potentiation (LTP), that is widely accepted as the substrate for certain forms of memory. The discussion is divided into two main parts. The first presents 'standard views' of synaptic transmission and of LTP, and the second deals with challenges to and uncertainties about these views. The standard views presented are not my own - indeed, I would dispute a variety of their assumptions - but rather they represent classic or popular notions that serve as a framework for the critical discussion in the second part of the presentation. Such a short review of so vast a subject cannot, of course, be comprehensive, and I have selected for discussion several specific topics that currently are being most actively investigated and debated. Each of the sections can be read independently of the others.
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