Rat brain N-methyl-D-aspartate receptors expressed in Xenopus oocytes

N-methyl-D-aspartate (NMDA) activates a class of excitatory amino acid receptor involved in a variety of plastic and pathological processes in the brain. Quantitative study of the NMDA receptor has been difficult in mammalian neurons, because it usually exists with other excitatory amino acid receptors of overlapping pharmacological specificities. Xenopus oocytes injected with messenger RNA isolated from primary cultures of rat brain have now been used to study NMDA receptors. The distinguishing properties of neuronal NMDA receptors have been reproduced in this amphibian cell, including voltage-dependent block by magnesium, block by the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid, and potentiation by glycine. This preparation should facilitate the quantitative study of the regulation of NMDA receptor activation and serve as a tool for purification of the encoding messenger RNA.     

Sustained dendritic gradients of Ca2+ induced by excitatory amino acids in CA1 hippocampal neurons

Spatially resolved measurements of intracellular free calcium and of the changes produced by excitatory amino acids were made in neurons isolated from adult mammalian brain. Extremely long-lasting (minutes) Ca2+ gradients were induced in the apical dendrites of hippocampal CA1 neurons after brief (1 to 3 seconds), local application of either glutamate or N-methyl-D-aspartate (NMDA). These gradients reflect the continuous flux of Ca2+ into the dendrite. The sustained gradients, but not the immediate transient response to the agonists, were prevented by prior treatment with the protein kinase C inhibitor sphingosine. Expression of the long-lasting Ca2+ gradients generally required a priming or conditioning stimulus with the excitatory agonist. The findings demonstrate a coupling between NMDA receptor activation and long-lasting intracellular Ca2+ elevation that could contribute to certain use-dependent modifications of synaptic responses in hippocampal CA1 neurons.     

The role of excitatory amino acids and NMDA receptors in traumatic brain injury

Brain injury induced by fluid percussion in rats caused a marked elevation in extracellular glutamate and aspartate adjacent to the trauma site. This increase in excitatory amino acids was related to the severity of the injury and was associated with a reduction in cellular bioenergetic state and intracellular free magnesium. Treatment with the noncompetitive N-methyl-D-aspartate (NMDA) antagonist dextrophan or the competitive antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid limited the resultant neurological dysfunction; dextrorphan treatment also improved the bioenergetic state after trauma and increased the intracellular free magnesium. Thus, excitatory amino acids contribute to delayed tissue damage after brain trauma; NMDA antagonists may be of benefit in treating acute head injury.     

Hypoglycemia-induced neuronal damage prevented by an N-methyl-D-aspartate antagonist

The possibility that neuronal damage due to hypoglycemia is induced by agonists acting on the N-methyl-D-aspartate (NMDA) receptor was investigated in the rat caudate nucleus. Local injections of an NMDA receptor antagonist, 2-amino-7-phosphonoheptanoic acid, were performed before induction of 30 minutes of reversible, insulin-induced, hypoglycemic coma. Neuronal necrosis in these animals after 1 week of recovery was reduced 90 percent compared to that in saline-injected animals. The results suggest that hypoglycemic neuronal damage is induced by NMDA receptor agonists, such as the excitatory amino acids or related compounds.     

Facilitation of spinal NMDA receptor currents by spillover of synaptically released glycine

In the mammalian CNS, N-methyl-D-aspartate (NMDA) receptors serve prominent roles in many physiological and pathophysiological processes including pain transmission. For full activation, NMDA receptors require the binding of glycine. It is not known whether the brain uses changes in extracellular glycine to modulate synaptic NMDA responses. Here, we show that synaptically released glycine facilitates NMDA receptor currents in the superficial dorsal horn, an area critically involved in pain processing. During high presynaptic activity, glycine released from inhibitory interneurons escapes the synaptic cleft and reaches nearby NMDA receptors by so-called spillover. In vivo, this process may contribute to the development of inflammatory hyperalgesia.     

Contributions of quisqualate and NMDA receptors to the induction and expression of LTP

The contributions of two subclasses of excitatory amino acid transmitter receptors to the induction and expression of long-term potentiation (LTP) were analyzed in hippocampal slices. The quisqualate/kainate receptor antagonist DNQX (6,7-dinitro-quinoxaline-2,3-dione) blocked 85% of the evoked field potential, leaving a small response that was sensitive to D-AP5 (D-2-amino-5-phosphonopentanoate), an N-methyl-D-aspartate (NMDA) receptor blocker. This residual D-AP5-sensitive response was of comparable size in control and previously potentiated inputs. High-frequency stimulation in the presence of DNQX did not result in the development of robust LTP. Washout of the drug, however, revealed the potentiation effect. Thus NMDA-mediated responses can induce, but are not greatly affected by, LTP; non-NMDA receptors, conversely, mediate responses that are not needed to elicit LTP but that are required for its expression.     

Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes

Receptors for N-methyl-D-aspartate (NMDA) are involved in many plastic and pathological processes in the brain. Glycine has been reported to potentiate NMDA responses in neurons and in Xenopus oocytes injected with rat brain messenger RNA. Glycine is now shown to be absolutely required for activation of NMDA receptors in oocytes. In voltage-clamped oocytes, neither perfusion nor rapid pressure application of NMDA onto messenger RNA-injected oocytes caused a distinct ionic current without added glycine. When glycine was added, however, NMDA evoked large inward currents. The concentration of glycine required to produce a half-maximal response was 670 nanomolar, and the glycine dose-response curve extrapolated to zero in the absence of glycine. Several analogs of glycine could substitute for glycine, among which D-serine and D-alanine were the most effective. The observation that D-amino acids are effective will be important in developing drugs targeted at the glycine site.     

Auxiliary subunits assist AMPA-type glutamate receptors

Glutamate, the major excitatory neurotransmitter in the brain, acts primarily on two types of ionotropic receptors: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and N-methyl-d-aspartate (NMDA) receptors. Work over the past decade indicates that regulated changes in the number of synaptic AMPA receptors may serve as a mechanism for information storage. Recent studies demonstrate that a family of small transmembrane AMPA receptor regulatory proteins (TARPs) controls both AMPA receptor trafficking and channel gating. TARPs provide the first example of auxiliary subunits of ionotropic receptors. Here we review the pivotal role that TARPs play in the life cycle of AMPA receptors.     

An excitatory amino acid antagonist blocks cone input to sign-conserving second-order retinal neurons

cis-2,3-Piperidinedicarboxylic acid (PDA), an excitatory amino acid antagonist, reversibly blocked cone input to OFF bipolars and horizontal cells, whereas ON bipolars were relatively unaffected. Kainic acid effects were also blocked, indicating a postsynaptic mechanism of action. The use of PDA helps to characterize one of two classes of excitatory amino acid synaptic receptors that mediate cone influence in the outer retina.     

Pharmacological induction of use-dependent receptive field modifications in the visual cortex

Lasting modifications of the receptive fields of neurons in the visual cortex can be induced by pairing visual stimuli with iontophoretic application of the neuromodulators acetylcholine and noradrenaline or the excitatory amino acids N-methyl-D-aspartate (NMDA) and L-glutamate. The modifications are obtained in less than 1 hour and persist for more than 40 minutes. Thus, acetylcholine and norepinephrine have a permissive role in use-dependent neuronal plasticity. These results support the notion of a postsynaptic threshold for neuronal malleability that differs from that of sodium-dependent action potentials.     

Glutamate, the dominant excitatory transmitter in neuroendocrine regulation

Glutamate has been found to play an unexpectedly important role in neuroendocrine regulation in the hypothalamus, as revealed in converging experiments with ultrastructural immunocytochemistry, optical physiology with a calcium-sensitive dye, and intracellular electrical recording. There were large amounts of glutamate in boutons making synaptic contact with neuroendocrine neurons in the arcuate, paraventricular, and supraoptic nuclei. Almost all medial hypothalamic neurons responded to glutamate and to the glutamate agonists quisqualate and kainate with a consistent increase in intracellular calcium. In all magnocellular and parvocellular neurons of the paraventricular and arcuate nuclei tested, the non-NMDA (non-N-methyl-D-aspartate) glutamate antagonist CNQX (cyano-2,3-dihydroxy-7-nitroquinoxaline) reduced electrically stimulated and spontaneous excitatory postsynaptic potentials, suggesting that the endogenous neurotransmitter is an excitatory amino acid acting primarily on non-NMDA receptors. These results indicate that glutamate plays a major, widespread role in the control of neuroendocrine neurons.     

Dentate gyrus NMDA receptors mediate rapid pattern separation in the hippocampal network

Forming distinct representations of multiple contexts, places, and episodes is a crucial function of the hippocampus. The dentate gyrus subregion has been suggested to fulfill this role. We have tested this hypothesis by generating and analyzing a mouse strain that lacks the gene encoding the essential subunit of the N-methyl-d-aspartate (NMDA) receptor NR1, specifically in dentate gyrus granule cells. The mutant mice performed normally in contextual fear conditioning, but were impaired in the ability to distinguish two similar contexts. A significant reduction in the context-specific modulation of firing rate was observed in the CA3 pyramidal cells when the mutant mice were transferred from one context to another. These results provide evidence that NMDA receptors in the granule cells of the dentate gyrus play a crucial role in the process of pattern separation.     

NMDA receptor losses in putamen from patients with Huntington's disease

N-Methyl-D-aspartate (NMDA), phencyclidine (PCP), and quisqualate receptor binding were compared to benzodiazepine, gamma-aminobutyric acid (GABA), and muscarinic cholinergic receptor binding in the putamen and cerebral cortex of individuals with Huntington's disease (HD). NMDA receptor binding was reduced by 93 percent in putamen from HD brains compared to binding in normal brains. Quisqualate and PCP receptor binding were reduced by 67 percent, and the binding to other receptors was reduced by 55 percent or less. Binding to these receptors in the cerebral cortex was unchanged in HD brains. The results support the hypothesis that NMDA receptor-mediated neurotoxicity plays a role in the pathophysiology of Huntington's disease.     

Molecular cloning and functional expression of glutamate receptor subunit genes

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.     

Role for excitatory amino acids in methamphetamine-induced nigrostriatal dopaminergic toxicity

The systemic administration of either methamphetamine or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to experimental animals produces degenerative changes in nigrostriatal dopaminergic neurons or their axon terminals. This study was conducted to determine if excitatory amino acids, which appear to be involved in various neurodegenerative disorders, might also contribute to the dopaminergic neurotoxicity produced in mice by either methamphetamine or MPTP. MK-801, phencyclidine, and ketamine, noncompetitive antagonists of one subtype of excitatory amino acid receptor, the N-methyl-D-aspartate receptor, provided substantial protection against neurotoxicity produced by methamphetamine but not that produced by MPTP. These findings indicate that excitatory amino acids play an important role in the nigrostriatal dopaminergic damage induced by methamphetamine.     

Beta-N-methylamino-L-alanine neurotoxicity: requirement for bicarbonate as a cofactor

Ingestion of the excitotoxic cycad seed amino acid beta-N-methylamino-L-alanine may be responsible for the neuronal degeneration associated with Guam amyotrophic lateral sclerosis-parkinsonism-dementia in man. However, the basis for the central neurotoxicity of beta-N-methylamino-L-alanine has been unclear, as it lacks the omega acidic (or equivalent electronegative) moiety characteristic of other excitatory amino acids. beta-N-methylamino-L-alanine produced neurotoxic and neuroexcitatory effects in murine cortical cell cultures only when physiological concentrations of bicarbonate were available in the extracellular bathing medium. Bicarbonate may interact noncovalently with beta-N-methylamino-L-alanine to produce, in combination, a molecular configuration that activates glutamate receptors.     

氮素形态对兴安落叶松幼苗的营养效应

采用水培试验方法,以兴安落叶松(Larix gmelinii)幼苗为供试材料,研究兴安落叶松幼苗对氨基酸态氮(甘氨酸、谷氨酸、赖氨酸)、铵态氮(硫酸铵)、硝态氮(硝酸钙)的吸收与响应特性。结果表明:氨基酸态氮和铵态氮对兴安落叶松幼苗的生长及干物质积累的促进作用明显好于硝态氮。氨基酸态氮比无机态氮更能有效提高幼苗体内N、P、K养分质量分数,甘氨酸、谷氨酸对兴安落叶松幼苗铵态氮的供应能力与硫酸铵等效,赖氨酸态氮次之。氨基酸态氮和铵态氮对兴安落叶松幼苗硝态氮的供应无明显效果。供应氨基酸态氮和硝态氮后,营养液pH值显著升高;供应铵态氮,营养液pH值显著下降。     

Corticotropin-releasing factor receptors in rat forebrain: autoradiographic identification

Corticotropin-releasing factor (CRF) receptors were identified in rat forebrain by autoradiography with an iodine-125-labeled analog of ovine CRF substituted with norleucine and tyrosine at amino acid residues 21 and 32, respectively. High-affinity receptors for CRF were found in discrete areas of rat forebrain, including laminae I and IV of the neocortex, the external layer of the medium eminence, the lateral nucleus of the amygdala, and the striatum. These results are consistent with earlier findings on the immunohistochemical distribution of CRF and suggest that endogenous CRF has a physiological role in regulating activity of the central nervous system.     

Effect of serotonergic afferents on quantal release at central inhibitory synapses

Although most examples of modulation of synaptic transmission have been obtained from excitatory rather than from inhibitory connections, serotonin (5HT) is now shown to cause a presynaptic facilitation of release of the inhibitory neurotransmitter glycine. Brief local injections of this amine, or application of a 5HT uptake blocker, produce a long-lasting enhancement of both spontaneous and evoked inhibitory currents in the teleost Mauthner cell. Quantal analysis showed that the probability of release is increased. Focal recording indicated that 5HT acts directly on the inhibitory terminals, possibly reducing potassium conductances. Double staining with specific antibodies demonstrated a morphological substrate for this effect. Nerve endings that contain 5HT contact inhibitory terminals directly apposed to postsynaptic glycine receptors.     

Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists

The N-methyl-D-aspartate (NMDA)-subtype of glutamate receptors has been well described as a result of the early appearance of NMDA antagonists, but no potent antagonist for the "non-NMDA" glutamate receptors has been available. Quinoxalinediones have now been found to be potent and competitive antagonists at non-NMDA glutamate receptors. These compounds will be useful in the determination of the structure-activity relations of quisqualate and kainate receptors and the role of such receptors in synaptic transmission in the mammalian brain.