Contribution of NMDA and non-NMDA glutamate receptors to locomotor pattern generation in the neonatal rat spinal cord.

The motor programme executed by the spinal cord to generate locomotion involves glutamate-mediated excitatory synaptic transmission. Using the neonatal rat spinal cord as an in vitro model in which the locomotor pattern was evoked by 5-hydroxytryptamine (5-HT), we investigated the role of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in the generation of locomotor patterns recorded electrophysiologically from pairs of ventral roots. In a control solution, 5-HT (2.5-30 microM) elicited persistent alternating activity in left and right lumbar ventral roots. Increasing 5-HT concentration within this range resulted in increased cycle frequency (on average from 8 to 20 cycles min-1). In the presence of NMDA receptor antagonism, persistent alternating activity was still observed as long as 5-HT doses were increased (range 20-40 microM), even if locomotor pattern frequency was lower than in the control solution. In the presence of non-NMDA receptor antagonism, stable locomotor activity (with lower cycle frequency) was also elicited by 5-HT, albeit with doses larger than in the control solution (15-40 microM). When NMDA and non-NMDA receptors were simultaneously blocked, 5-HT (5-120 microM) always failed to elicit locomotor activity. These data show that the operation of one glutamate receptor class was sufficient to express locomotor activity. As locomotor activity developed at a lower frequency than in the control solution after pharmacological block of either NMDA or non-NMDA receptors, it is suggested that both receptor classes were involved in locomotor pattern generation.     

Modeling of slow glutamate diffusion and AMPA receptor activation in the cerebellar glomerulus

Synaptic conductances are influenced markedly by the geometry of the space surrounding the synapse since the transient glutamate concentration in the synaptic cleft is determined by this geometry. Our paper is an attempt to understand the reasons for slow glutamate diffusion in the cerebellar glomerulus, a structure situated around the enlarged mossy fiber terminal in the cerebellum and surrounded by a glial sheath. For this purpose, analytical expressions for glutamate diffusion in the glomerulus were considered in models with two-, three-, and fractional two-three-dimensional (2D-3D) geometry with an absorbing boundary. The time course of average glutamate concentration in the synaptic cleft of the mossy fiber-granule cell connection was calculated for both direct release of glutamate from the same synaptic unit, and for cumulative spillover of glutamate from neighboring release sites. Several kinetic schemes were examined, and the parameters of the diffusion models were estimated by identifying theoretical activation of AMPA receptors with direct release and spillover components of published experimental AMPA receptor-mediated EPSCs. For model selection, the correspondence of simulated paired-pulse ratio and EPSC increase after prevention of desensitization to experimental values were also taken into consideration. Our results suggest at least a 7- to 10-fold lower apparent diffusion coefficient of glutamate in the porous medium of the glomerulus than in water. The modeling of glutamate diffusion in the 2D-3D geometry gives the best fit of experimental EPSCs. We show that it could be only partly explained by normal diffusion of glutamate in the complex geometry of the glomerulus. We assume that anomalous diffusion of glutamate occurs in the glomerulus. A good match of experimental estimations and theoretical parameters, obtained in the simulations that use an approximation of anomalous diffusion by a solution for fractional Brownian motion, confirms our assumption.     

Both NMDA and non-NMDA subtypes of glutamate receptors are concentrated at synapses on cerebral cortical neurons in culture.

N-methyl-D-aspartate (NMDA) and non-NMDA receptors play a key role in synaptic transmission and plasticity in the vertebrate central nervous system. Previous studies have suggested that although both receptor types are present at synapses, the NMDA receptors may be relatively uniformly distributed. We have combined iontophoretic mapping of NMDA and non-NMDA receptors with immunohistochemical localization of synaptic vesicles along dendrites of single neocortical neurons to determine the relationship between NMDA and non-NMDA receptor distribution and the location of synapses. We find that when corrections for glutamate diffusion are made, NMDA responses are concentrated at focal "hot spots" that coincide with non-NMDA hot spots and that there is an excellent correlation between these hot spots and synapses.     

Three-dimensional models of non-NMDA glutamate receptors.

Structural models have been produced for three types of non-NMDA inotropic glutamate receptors: an AMPA receptor, GluR1, a kainate receptor, GluR6; and a low-molecular-weight kainate receptor from goldfish, GFKAR alpha. Modeling was restricted to the domains of the proteins that bind the neurotransmitter glutamate and that form the ion channel. Model building combined homology modeling, distance geometry, molecular mechanics, interactive modeling, and known constraints. The models indicate new potential interactions in the extracellular domain between protein and agonists, and suggest that the transition from the "closed" to the "open" state involves the movement of a conserved positive residue away from, and two conserved negative residues into, the extracellular entrance to the pore upon binding. As a first approximation, the ion channel domain was modeled with a structure comprising a central antiparallel beta-barrel that partially crosses the membrane, and against which alpha-helices from each subunit are packed; a third alpha-helix packs against these two helices in each subunit. Much, but not all, of the available data were consistent with this structure. Modifying the beta-barrel to a loop-like topology produced a model consistent with available data.     

Effects of NMDA and non-NMDA ionotropic glutamate receptors in the medial preoptic area on body temperature in awake rats

Glutamate when microinjected at the medial preoptic area (mPOA) influences brain temperature (T_br) and body temperature (T_b) in rats. Glutamate and its various receptors are present at the mPOA. The aim of this study was to identify the contribution of each of the ionotropic glutamatergic receptors at the mPOA on changes in T_br and T_b in freely moving rats. Adult male Wistar rats (n=40) were implanted with bilateral guide cannula with indwelling styli above the mPOA. A telemetric transmitter was implanted at the peritoneum to record T_b and locomotor activity (LMA). A precalibrated thermocouple wire implanted near the hypothalamus was used to assess T_br. Specific agonist for each ionotropic glutamate receptor was microinjected into the mPOA and its effects on temperature and LMA were measured in the rats. The rats were also microinjected with the respective ionotropic receptor antagonists, 15 min prior to the microinjection of each agonist. Amongst amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-d-aspartate (NMDA) and kainic acid, AMPA increased T_b and LMA when injected at the mPOA. Specific antagonists for AMPA receptors was able to attenuate this increase (p<0.005). Pharmacological blockade of NMDA was able to lower T_br only. Microinjection of kainic acid and its antagonist had no effect on the variables. The finding of the study suggests that activation of the AMPA receptors at the mPOA, leads to the rise in body temperature.     

Reduction in glutamate uptake is associated with extrasynaptic NMDA and metabotropic glutamate receptor activation at the hippocampal CA1 synapse of aged rats

This study aims to determine whether the regulation of extracellular glutamate is altered during aging and its possible consequences on synaptic transmission and plasticity. A decrease in the expression of the glial glutamate transporters GLAST and GLT‐1 and reduced glutamate uptake occur in the aged (24–27 months) Sprague–Dawley rat hippocampus. Glutamatergic excitatory postsynaptic potentials recorded extracellularly in ex vivo hippocampal slices from adult (3–5 months) and aged rats are depressed by DL‐TBOA, an inhibitor of glutamate transporter activity, in an N‐Methyl‐d‐ Aspartate (NMDA)‐receptor‐dependent manner. In aged but not in young rats, part of the depressing effect of DL‐TBOA also involves metabotropic glutamate receptor (mGluRs) activation as it is significantly reduced by the specific mGluR antagonist d‐methyl‐4‐carboxy‐phenylglycine (MCPG). The paired‐pulse facilitation ratio, a functional index of glutamate release, is reduced by MCPG in aged slices to a level comparable to that in young rats both under control conditions and after being enhanced by DL‐TBOA. These results suggest that the age‐associated glutamate uptake deficiency favors presynaptic mGluR activation that lowers glutamate release. In parallel, 2 Hz‐induced long‐term depression is significantly decreased in aged animals and is fully restored by MCPG. All these data indicate a facilitated activation of extrasynaptic NMDAR and mGluRs in aged rats, possibly because of an altered distribution of glutamate in the extrasynaptic space. This in turn affects synaptic transmission and plasticity within the aged hippocampal CA1 network.     

Distinct quantal features of AMPA and NMDA synaptic currents in hippocampal neurons: implication of glutamate spillover and receptor saturation

Excitatory postsynaptic currents (EPSCs) were studied in the CA1 pyramidal cells of rat hippocampal slices. Components mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and by N-methyl-D-aspartate (NMDA) receptors were separated pharmacologically. Quantal parameters of AMPA and NMDA receptor-mediated EPSCs were obtained using both maximal likelihood and autocorrelation techniques. Enhancement of transmitter release with 4-aminopyridine caused a significant increase in quantal size of NMDA EPSC. This was accompanied by a slowing of the EPSC decay. The maximal number of quanta in the NMDA current was unchanged, while the probability of quantal event dramatically enhanced. In contrast, neither the quantal size nor the kinetics of AMPA EPSC was altered by 4-aminopyridine, while the maximal number of quanta increased. These changes in the quantal parameters are consistent with a transition to multivesicular release of the neurotransmitter. Spillover of excessive glutamate on the nonsynaptic areas of dendritic spines causes an increase in the quantal size of NMDA synaptic current. The difference in quantal behavior of AMPA and NMDA EPSCs implies that different mechanisms underlie their quantization: the additive response of nonsaturated AMPA receptors contrasts with the variable involvement of saturated intrasynaptic and nonsaturated extrasynaptic NMDA receptors.     

NMDA和非NMDA受体拮抗剂对伤害性辐射热引起的缩腿反射抑制的比较

我们以前的电生理工作：Ｎ－甲基－Ｄ－门冬氨酸受体主要参与介导皮肤来源的伤害性信息的传入,而非ＮＭＤＡ受体主要参与介导肌肉来源的伤害性信息的传入。为进一步证实这一发现,应用鞘内注射的方法,观察ＮＭＤＡ和非ＮＭＤＡ受体拮抗剂对大鼠伤害性辐射热刺激所引起的缩腿反射潜伏期的影响。     

Vitamin C modulates glutamate transport and NMDA receptor function in the retina

Vitamin C (in the reduced form ascorbate or in the oxidized form dehydroascorbate) is implicated in signaling events throughout the central nervous system (CNS ). In the retina, a high‐affinity transport system for ascorbate has been described and glutamatergic signaling has been reported to control ascorbate release. Here, we investigated the modulatory role played by vitamin C upon glutamate uptake and N ‐methyl‐d ‐aspartate (NMDA ) receptor activation in cultured retinal cells or in intact retinal tissue using biochemical and imaging techniques. We show that both forms of vitamin C, ascorbate or dehydroascorbate, promote an accumulation of extracellular glutamate by a mechanism involving the inhibition of glutamate uptake. This inhibition correlates with the finding that ascorbate promotes a decrease in cell surface levels of the neuronal glutamate transporter excitatory amino acid transporter 3 in retinal neuronal cultures. Interestingly, vitamin C is prone to increase the activity of NMDA receptors but also promotes a decrease in glutamate‐stimulated [³H] MK 801 binding and decreases cell membrane content of NMDA receptor glutamate ionotropic receptor subunit 1 (GluN1) subunits. Both compounds were also able to increase cAMP response element‐binding protein phosphorylation in neuronal nuclei in a glutamate receptor and calcium/calmodulin kinase‐dependent manner. Moreover, the effect of ascorbate is not blocked by sulfinpyrazone and then does not depend on its uptake by retinal cells. Overall, these data indicate a novel molecular and functional target for vitamin C impacting on glutamate signaling in retinal neurons.

     

NMDA as well as non-NMDA receptor antagonists can prevent the phase-shifting effects of light on the circadian system of the golden hamster.

The present experiments were designed to evaluate whether the intraventricular administration of excitatory amino acid (EAA) receptor antagonists would prevent light-induced phase shifts of the circadian rhythm of wheel-running activity in the hamster. Administration of the non-N-methyl-D-aspartate (non-NMDA) antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) blocked light-induced phase advances and delays. Similarly, administration of the competitive NMDA receptor antagonist, 3(2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid (CPP), prevented light-induced phase advances and delays. Neither drug by itself caused any consistent effect on the phase of the rhythm. These data provide further evidence that EAA receptors mediate the effects of light on the circadian system, and suggest that both NMDA and non-NMDA receptor types may be involved.     

Immunochemical characterization of the non-NMDA glutamate receptor using subunit-specific antibodies. Evidence for a hetero-oligomeric structure in rat brain.

Antibodies were made to synthetic peptides corresponding to sequences specific to the glutamate receptor (GluR) subunits, GluR1-4. The specificity of the antibodies was established by Western blotting using membranes of simian kidney cells (COS-7) transfected with GluR subunit DNA. Four antibodies were found to be selective for each of the four GluR subunits, and a fifth antibody recognized both GluR2 and 3. All five antibodies immunoadsorbed Triton X-100-solubilized rat brain [3H]AMPA binding activity and labeled an Mr = 108,000 band in samples of rat brain. The structure of the Triton X-100-solubilized GluR was studied using subunit-specific antibodies covalently attached to protein A-agarose and analyzing GluR subunits bound to the antibodies by Western blotting. Each of the four subunit-specific antibodies immunoadsorbed its respective GluR subunit as well as the other three forms of GluR, showing that the detergent solubilized GluR exists as hetero-oligomers composed of two or more of the four subunits. Evidence supporting a similar structure for membrane bound GluR was obtained using synaptic membranes chemically cross-linked with dithiobis(succinimidylpropionate). GluR was immunoaffinity-purified using the GluR2 and 3-selective antibody. This antibody, covalently attached to protein A-agarose, adsorbed 55% of [3H]AMPA binding activity, and after elution with 1 M KSCN, 22-37% of the binding activity was recovered. Analysis of the purified product showed a major immunoreactive band at Mr = 108,000, and silver staining identified the same major band and no additional polypeptides. The GluR receptor complex, therefore, appears to be made up exclusively of GluR1-4. In the purified GluR preparation, in addition to the Mr = 108,000 band, three higher molecular weight immunoreactive components were also detected. These bands migrated at Mr = 325,000, 470,000, and 590,000. Similar sized proteins were seen in the cross-linked synaptic membrane sample, with the Mr = 590,000 component being substantially enriched after cross-linking. The Mr = 590,000 band is the largest component detected, and it has a size consistent with its being a pentamer of the Mr = 108,000 protein.     

Desensitization of NMDA receptor channels is modulated by glutamate agonists

Two distinct forms of desensitization have been characterized for N-methyl-D-aspartate (NMDA) receptors. One form results from a weakening of agonist affinity when channels are activated whereas the other form of desensitization results when channels enter a long-lived nonconducting state. A weakening of glycine affinity upon NMDA receptor activation has been reported. Cyclic reaction schemes for NMDA receptor activation require that a concomitant affinity shift should be observed for glutamate agonists. In this study, measurements of peak and steady-state NMDA receptor currents yielded EC50 values for glutamate that differed by 1.9-fold, but no differences were found for another agonist, L-cysteine-S-sulfate (LCSS). Simulations show that shifts in EC50 values may be masked by significant degrees of desensitization resulting from channels entering a long-lived nonconducting state. Simulations also show that a decrease in the degree of desensitization with increasing agonist concentration is a good indicator for the existence of desensitization resulting from a weakening of agonist affinity. Both glutamate and LCSS exhibited this trend. An affinity difference of three- to eightfold between high-and low-affinity agonist-binding states was estimated from fitting of dose-response data with models containing both types of desensitization. This indicates that activation of NMDA receptors causes a reduction in both glutamate and glycine affinities.     

Phosphorylation of recombinant non-NMDA glutamate receptors on serine and tyrosine residues

Glutamate receptors are the major excitatory neurotransmitter receptors in the central nervous system. A variety of data has recently suggested that protein phosphorylation of glutamate receptors regulates their function. To examine at a molecular level the role of protein phosphorylation in the modification of glutamate receptors, we have transiently expressed the non-NMDA glutamate receptor subunit GluR1 (flop) in human embryonic kidney 293 cells. Using a polyclonal antipeptide antiserum directed specifically against GluR1, we have immunoprecipitated a 106 kDa phosphoprotein corresponding to the GluR1 subunit. Phosphoamino acid analysis and thermolytic peptide mapping demonstrate that this basal phosphorylation occurs exclusively on serine residues in two phosphopeptides. Application of activators of endogenous cAMP-dependent protein kinase or protein kinase C revealed no consistant changes in the phosphorylation of GluR1. However, coexpression of the GluR1 subunit with the well characterized protein tyrosine kinase v-src results in phosphorylation of GluR1 on tyrosine residues, in a single thermolytic phosphopeptide. These results suggest that GluR1 may be a substrate for protein serine/threonine kinases as well as protein tyrosine kinases in the central nervous system.Abbreviations AMPA -amino-3-hydroxy-5-methyl-4-isoxazolepropionate - CNS central nervous system - NMDA N-methyl-D-aspartate; - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - TBS Tris-buffered saline - TPA phorbol 12-myristate-13-acetate Special issue dedicated to Dr. Paul Greengard.     

Synaptic transmission in the pineal eye of young Xenopus laevis tadpoles: a role for NMDA and non-NMDA glutamate and non-glutaminergic receptors?

The pineal eye of Xenopus laevis tadpoles is directly photosensitive. A sudden reduction in light intensity produces a burst of activity in the pineal ganglion cells, which is closely followed by the onset of swimming. In this paper I present the results of experiments on the effects of agonists and antagonists of candidate pineal transmitters on ganglion cell activity. I found that NMDA and non-NMDA excitatory amino acid (EAA) agonists increased pineal activity, indicating the presence of both types of receptor. Kynurenic acid reduced activity, thus confirming that the photoreceptor transmitter is an EAA. Under physiological conditions, CNQX blocked activity almost completely whilst AP5 had little effect. In Mg²⁺-free saline CNQX had a considerably smaller effect, but joint application of CNQX and AP5 blocked almost all activity; therefore, the NMDA receptors are subject to blockage by Mg²⁺. Although GABA_A and ACh receptors appear to be present, no evidence was found for GABA or ACh as pineal transmitters. In addition, 5-HT had no effect on pineal activity. The main pineal transmitter is an EAA acting on ganglion cells through both NMDA and non-NMDA receptors. Other receptors are present but appear to have no role in controlling pineal activity at this stage. Accepted: 1 March 1997     

The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes.

Polyacrylamide-gel electrophoresis in urea was used to prepare the four molecular species of transferrin:diferric transferrin, apotransferrin and the two monoferric transferrins with either the C-terminal or the N-terminal metal-binding site occupied. The interaction of these 125I-labelled proteins with rabbit reticulocytes was investigated. At 4 degrees C the average value for the association constant for the binding of transferrin to reticulocytes was found to increase with increasing iron content of the protein. The association constant for apotransferrin binding was 4.6 X 10(6)M-1, for monoferric (C-terminal iron) 2.5 X 10(7)M-1, for monoferric (N-terminal iron) 2.8 X 10(7)M-1 and for diferric transferrin, 1.1 X 10(8)M-1. These differences in the association constants did not affect the processing of the transferrin species by the cells at 37 degrees C. Accessibility of the proteins to extracellular proteinase indicated that the transferrin was internalized by the cells regardless of the iron content of the protein, since in each case 70% was inaccessible. Cycling of the cellular receptors may also occur in the absence of bound transferrin.     

Zinc has opposite effects on NMDA and non-NMDA receptors expressed in Xenopus oocytes

Pharmacological characterization of Zn2+ effects on glutamate ionotropic receptors was investigated in Xenopus oocytes injected with rat brain mRNA, using a double microelectrode, voltage-clamp technique. At low concentration, Zn2+ inhibited NMDA currents (IC50 = 42.9 +/- 1.3 microM) and potentiated both AMPA (EC50 = 30.0 +/- 1.2 microM) and desensitized kainate responses (EC50 = 13.0 +/- 0.1 microM). At higher concentrations, Zn2+ inhibited non-NMDA responses with IC50 values of 1.3 +/- 0.1 mM and 1.2 +/- 0.3 mM for AMPA and kainate, respectively. The potentiation of AMPA or quisqualate currents by Zn2+ was more than 2-fold, whereas that of the kainate current was only close to 30%. This potentiating effect of Zn2+ on AMPA current modified neither the affinity of the agonist for its site nor the current-voltage relationship. In addition, 500 microM Zn2+ differentially affected NMDA and non-NMDA components of the glutamate-induced response. The possible physiological relevance of Zn2+ modulation is discussed.     

AMPA and NMDA glutamate receptor trafficking: multiple roads for reaching and leaving the synapse

Glutamate receptor trafficking in and out of synapses is one of the core mechanisms for rapid changes in the number of functional receptors during synaptic plasticity. Recent data have shown that the fast gain and loss of receptors from synaptic sites are accounted for by endocytic/exocytic processes and by their lateral diffusion in the plane of the membrane. These events are interdependent and regulated by neuronal activity and interactions with scaffolding proteins. We review here the main cellular steps for AMPA and NMDA receptor synthesis, traffic within intracellular organelles, membrane exocytosis/endocytosis and surface trafficking. We focus on new findings that shed light on the regulation of receptor cycling events and surface trafficking and the way that this might reshape our thinking about the specific regulation of receptor accumulation at synapses.     

NMDA and non-NMDA receptors mediate responses in the primary gustatory nucleus in goldfish

Primary gustatory afferents from the oropharynx of the goldfish, Carassius auratus, terminate in the vagal lobe, a laminated structure in the dorsal medulla comparable to the gustatory portion of the nucleus of the solitary tract in mammals. We utilized an in vitro brain slice preparation to test the role of different ionotropic glutamate receptor subtypes in synaptic transmission of gustatory information by recording changes in field potentials after application of various glutamate receptor antagonists. Electrical stimulation of the vagus nerve (NX) evokes two short-latency postsynaptic field potentials from sensory layers of the vagal lobe. 6,7-Dinitroquinoxaline-2,3-dione and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, two non-N-methyl-D-aspartate (NMDA) ionotropic receptor antagonists, blocked these short-latency potentials. Slower potentials that were revealed under Mg2+ -free conditions, were abolished by the NMDA receptor antagonist, D(-)-2-amino-5-phosphonovaleric acid (APV). Repetitive stimulation produced short-term facilitation, which was attenuated by application of APV. These results indicate that the synaptic responses in the vagal lobe produced by stimulation of the gustatory roots of the NX involve both NMDA and non-NMDA receptors. An NMDA receptor-mediated facilitation may serve to amplify incoming bursts of primary afferent activity.