Ionic changes induced by excitatory amino acids in the rat cerebral cortex

The ionic mechanisms underlying the action of excitatory amino acids were investigated in the rat motor cortex. Ion-selective microelectrodes were attached to micropipettes such that their tips were very close and local changes in extracellular concentration of sodium, calcium, and potassium ions elicited through ionophoretic applications of glutamate (Glu) and of its agonists N-methyl-D-aspartate (NMDA), quisqualate (Quis), and kainate (Ka) were measured. These agents produced moderate increases in [K+]o (up to 13 mM) but, in contrast, substantial tetrodotoxin-insensitive decreases in [Na+]o (maximally of 60 mM). NMDA-induced sodium responses could be blocked by manganese, while the Quis- and Ka-induced responses were not. Quis and Ka produced increases in [Ca2+]o or biphasic responses while NMDA, even with small doses, induced each time drastic decreases in [Ca2+]o (maximally of 1.15 mM), which could be attenuated or blocked by manganese but not by organic calcium channel blockers. NMDA responses could be abolished by reduced doses of 2-amino-phosphonovalerate. The largest Glu- and NMDA-induced calcium responses were observed in the superficial cortical layers, but such maxima disappeared after selective degeneration of pyramidal tract neurons. All amino acids produced sizeable reductions in the extracellular space volume. The following can be concluded. (i) All the excitatory amino acids tested induce an increased permeability to sodium and potassium ions. (ii) In addition, the NMDA-operated channels have specifically a large permeability for calcium, although calcium ions contribute only by less than 10% to the NMDA-induced inward currents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of excitatory amino acids on mesencephalic trigeminal neurons

Mesencephalic trigeminal (MeV) neurons are primary sensory neurons of which the cell soma is located within the brainstem, and is associated with synaptic contacts. In previous studies it has been reported that these cells are resistant to kainic acid excitotoxicity, and have little or no responsiveness to exogenously applied glutamate or selective agonists. In an in vitro slice preparation with intracellular recording, we have found that these cells respond to pressure-applied glutamate, N-methyl-D-aspartic acid (NMDA), kainate (KA), and (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). The kainate and AMPA responses appear to be mediated by different receptors, at least in part, since they exhibit differing sensitivity to an AMPA receptor selective antagonist. The agonists generally evoke larger responses than glutamate and exhibit a long-duration desensitization requiring approximately 10 min for full recovery. Some cross-desensitization between the glutamate agonists is also observed. Mesencephalic trigeminal neurons exhibit high-frequency oscillatory activity during depolarizations that approach threshold potentials, and these could combine with transmitter-induced depolarizations to enhance the excitability of these cells. Previous reports of nonsensitivity to glutamate and to kainate excitotoxicity are attributable to relatively small responses, and to the desensitization expressed by these neurons.     

Interaction of excitatory and depressant amino acids in the frog retina

(1) Application of excitatory or depressant amino acids (concentrations from 10(-4) to 10(-2) M) could modify response patterns of the retinal ganglion cells to photic stimulus. Excitatory amino acids gave rise to spontaneous discharge while depressant amino acids inhibited spike discharge in response to test flashes. (2) Application of excitatory amino acids of more than 10(-3) M resulted in irreversible termination of spike discharges while recovery was always observed in the case of depressant amino acids even when the concentration of the applied solution was as high as 10(-2) M. No effect was observed when one exciting and one depressant amino acid were properly combined. (3) There is a mixture of four amino acids (two excitatory and two depressant) which could enhance the spike discharge in response to test flashes without giving rise to spontaneous firing. (4) It is implied that proper balance of excitatory and depressant amino acids is important in regulating the excitability of a number of neurons.     

Effects of excitatory amino acids on inositol phosphate accumulation in slices of the cerebral cortex of young and aged rats

The effects of glutamate, NMDA and quisqualate on carbachol-and norepinephrine-elicited formation of inositol phosphate (IP) were evaluated in slices prepared from the cerebral cortex of 3-and 24-month Sprague-Dawley rats. Glutamate, NMDA, and quisqualate antagonized the IP response to carbachol in a concentration-dependent fashion. This antagonism was more pronounced in aged than in young rats, both for glutamate (IC5O 0.114 and 0.210 mM) and NMDA (IC5O 0.0029 and 0.127 mM), but not for quisqualate. Glutamate (but not NMDA) also antagonized in a concentration-dependent fashion the IP response to norepinephrine, IC50s were 0.061 and 0.126 mM for aged and young rats, respectively; quisqualate had an inhibitory effect only at 1 mM concentration in the two age-groups, while in aged rats some stimulatory effect was present at 0.1 mM concentration. Glutamate, NMDA and quisqualate (1 mM) did not affect basal IP accumulation in either young or aged rats; quisqualate, however, at 0.1 mM concentration had some stimulatory effect, more pronounced in aged rats. This effect was probably responsible for the biphasic effect of quisqualate in this age-group. The most important finding consists of the demonstration of an age-related increase in the inhibitory effects of NMDA on carbachol-induced IP accumulation. This implies an altered modulation of cholinergic post-receptor mechanisms by glutamatergic mechanisms.     

重复经颅磁刺激对创伤性颅脑损伤患者脑脊液中兴奋性氨基酸含量的影响

目的：探讨重复经颅磁刺激（rTMS）对急性颅脑损伤患者脑脊液中兴奋性氨基酸（EAA）含量的影响。方法：30例创伤性颅脑损伤（TBI）病人按格拉斯哥昏迷评分分为轻型组（rTMS3）、中型组（rTMS2）、重型组（rTMS1）,每组10例,各组病人分别随机分为rTMS对照亚组（A组）及治疗亚组（B组）,每亚组5例。于TBI后第15天行腰椎穿刺采集脑脊液,采用高效液相色谱法测定脑脊液中谷氨酸（ASP）及门冬氨酸（GLU）含量。结果：脑脊液ASP和GLU水平随着脑损伤程度的加重而升高,各rTMS治疗组与相应各对照组的EAA相比,rTMS治疗组EAA的水平均低于相应对照组。结论：rTMS可通过降低TBI后脑脊液EAA水平发挥脑保护作用。脑脊液EAA的含量变化可作为TBI严重程度的生化指标。     

Action of excitatory amino acids and their antagonists on hippocampal neurons

Intracellular recordings were obtained from guinea pig hippocampal neurons maintained in vitro. Current- and voltage-clamp techniques were used to study the effect of microiontophoresis of excitatory amino acid agonists. Modification of agonist responses by bath application of known concentrations of antagonist agents was also examined. All agonists used, glutamate, aspartate, N-methyl-D-aspartic acid (NMDA), and quisqualate, depolarized hippocampal neurons and caused repetitive firing. NMDA was also noted to induce burst-firing in some neurons. Quisqualate and NMDA were more potent than glutamate or aspartate. In slices perfused with a nominally calcium-free saline containing tetrodotoxin and manganese, quisqualate application produced a depolarization associated with a conductance increase. Under those conditions, NMDA-induced depolarizations caused apparent decreases as well as increases in conductance. The apparent decreases in conductance were observed in the voltage range of -40 to -70 mV, whereas increases in conductance were observed at membrane potentials more positive than -35 mV. Under voltage-clamp conditions, quisqualate produced an inward current whose amplitude increased with hyperpolarization and decreased upon depolarization, reversing near 0 mV. The conductance change induced by quisqualate was independent of voltage. NMDA application resulted in an inward current that was maximal around the resting potential and decreased with both hyperpolarization and depolarization. Response reversal was not observed with hyperpolarization to -100 mV but was apparent with depolarization beyond 0 mV. Conductance changes induced by NMDA were voltage dependent, and the application of this agent was associated with the appearance of a region of negative slope conductance in the current-voltage relationship. Apparent decreases in conductance in response to NMDA were reduced when the extracellular magnesium concentration was lowered. Response amplitudes were not affected. The NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (DL-APV) was a potent and selective blocker of NMDA responses, whereas the antagonist DL-2-amino-4-phosphonobutyric acid (DL-APB) was less potent and did not select between NMDA and quisqualate responses. Analysis of iontophoretic dose-response curves indicated that DL-APV was a competitive antagonist. The results of these experiments indicate that hippocampal CA1 pyramidal neurons possess separate receptors for quisqualate and NMDA, with different pharmacological and electrophysiological profiles.     

重复经颅磁刺激对创伤性颅脑损伤患者脑脊液中兴奋性氨基酸 含量的影响

目的:探讨重复经颅磁刺激(rTMS)对急性颅脑损伤患者脑脊液中兴奋性氨基酸(EAA)含量的影响。方法:30例创伤性颅脑损伤(TBI)病人按格拉斯哥昏迷评分分为轻型组(rTMS3)、中型组(rTMS2)、重型组(rTMS1),每组10例,各组病人分别随机分为rTMS对照亚组(A组)及治疗亚组(B组),每亚组5例。于TBI后第15天行腰椎穿刺采集脑脊液,采用高效液相色谱法测定脑脊液中谷氨酸(ASP)及门冬氨酸(GLU)含量。结果:脑脊液ASP和GLU水平随着脑损伤程度的加重而升高,各rTMS治疗组与相应各对照组的EAA相比,rTMS治疗组EAA的水平均低于相应对照组。结论:rTMS可通过降低TBI后脑脊液EAA水平发挥脑保护作用。脑脊液EAA的含量变化可作为TBI严重程度的生化指标。     

Role of excitatory amino acids in hypoxic preconditioning.

We examined the effects of the extrinsic ionotropic NMDA receptor agonist (aspartate) and antagonist (ketamine) on the hypoxic preconditioning of mice and the concentration changes of intrinsic excitatory amino acids (EAAs), aspartate and glutamate, in the whole brain and different brain regions during preconditioning by an HPLC method. Our results showed that aspartate and ketamine significantly prolonged and shortened the standard tolerance time of mice during preconditioning and survival time in hypobaric chambers, respectively. After the 1st exposure, EAA concentrations in the whole brain and brain regions were increased. After run 4, they were decreased or maintained. It is suggested that the activation and suppression of ionotropic NMDA receptors is harmful and beneficial to hypoxic preconditioning, respectively. Degradation and/or inactivation of EAAs might be beneficial to the tolerance of mice to hypoxia.     

The role of excitatory amino acids in synaptic transmission in the hippocampus

We have highlighted some aspects of the action of excitatory amino acid transmission in the hippocampus. Fast epsps can be blocked by CNQX to reveal a component of synaptic transmission which is mediated by NMDA receptors. Extracellular recordings of ionic activities show that NMDA and non-NMDA ionophores are permeable to the major monovalent cations, while NMDA ionophores also appear to be permeable to Ca2+. Interactions of agonists applied by iontophoresis may be correlates of phenomena such as LTP, which can be evoked by appropriate synaptic stimulation.     

Ultrastructural immunocytochemical localization of excitatory amino acids in the somatosensory system.

We studied the ultrastructure and the synaptic arrangement of glutamate-immunoreactive terminals in rats, in the superficial laminae of the spinal cord, the brainstem cuneate nucleus, and the thalamic ventroposterolateral nucleus, where a role for glutamate as neurotransmitter has been suggested by biochemical, physiological and pharmacological approaches. The antiserum employed was raised against glutaramate conjugated to keyhole limpet hemocyanin with glutaraldehyde, and was used for pre-embedding staining with an avidin-biotin-peroxidase method and for post-embedding staining with an immunogold procedure. Both methods yielded similar results, consisting of labeling of selected terminals in all the areas examined. Double immunogold labeling on the same thin section using antisera against gamma-amino-butyric acid (GABA) or substance P (SP), in combination with the anti-glutamate serum, showed that staining for glutamate and GABA was present in different terminals in all the regions examined; glutamate and SP were co-localized in a few terminals only in the superficial laminae of the spinal cord. By performing immunogold staining in combination with anterograde tracing, glutamate immunoreactivity could be localized in identified primary afferents to the dorsal spinal cord and cuneate nucleus, and in lemniscal afferents to the thalamus.     

Inhibition by excitatory sulphur amino acids of the high-affinityl-glutamate transporter in synaptosomes and in primary cultures of cortical astrocytes and cerebellar neurons

A detailed kinetic study of the inhibitory effects ofl- andd-enantiomers of cysteate, cysteine sulphinate, homocysteine sulphinate, homocysteate, and S-sulpho-cysteine on the neuronal, astroglial and synaptosomal high-affinity glutamate transport system was undertaken.d-[³H] Aspartate was used as the transport substrate. Kinetic characterisation of uptake in the absence of sulphur compounds confirmed the high-affinity nature of the transport systems, the Michaelis constant (K _m) ford-aspartate uptake being 6 M, 21 M and 84 M, respectively, in rat brain cortical synaptosomes and primary cultures of mouse cerebellar granule cells and cortical astrocytes. In those cases where significant effects could be demonstrated, the nature of the inhibition was competitive irrespective of the neuronal versus glial systems. The rank order of inhibition was essentially similar in synaptosomes, neurons and astrocytes. Potent inhibition (K _iK _m) of transport in each system was exhibited byl-cysteate, andl- andd-cysteine sulphinate whereas substantially weaker inhibitory effects (K _i>10–1000 times the appropriateK _m value) were exhibited by the remaining sulphur amino acids. In general, inhibition: (i) was markedly stereospecific in favor of thel-enantiomers (except for cysteine sulphinate) and (ii) was found to decrease with increasing chain length. Computer-assisted molecular modelling studies, in which volume contour maps of the sulphur compounds were superimposed on those ofd-aspartate andl-glutamate, demonstrated an order of inhibitory potency which was, qualitatively, in agreement with that obtained quantitatively by in vitro kinetic studies.Special issue dedicated to Dr. Elling Kvamme     

Local sensitivity to excitatory and inhibitory amino acids in spinal interneurons of the lamprey

Sensitivity to glutamate, aspartate, glycine and GABA was examined in giant interneurons of the lamprey spinal cord.1. The membrane potentials evoked by iontophoretic application decayed with varied time constants specific to amino acids: 2.5 sec for glutamate, 6.3 sec for glycine and 10.3 sec for GABA. li|2. Bath-applied amino acids reduced the input resistance by varying degrees; when glutamate effect was taken as 1, relative effects of aspartate, glycine and GABA were 0.28, 40.5 and 12.3, respectively.3. Glutamate sensitivity was fairly uniform in both the soma and the dendrites. Glycine sensitivity, as well as GABA, was high in the soma and declined steeply along the dendrites by iontophoresis.     

Role of excitatory amino acids in the regulation of dopamine synthesis and release in the neostriatum

Summary We have explored the role of excitatory amino acids in the increased dopamine (DA) release that occurs in the neostriatum during stress-induced behavioral activation. Studies were performed in awake, freely moving rats, usingin vivo microdialysis. Extracellular DA was used as a measure of DA release; extracellular 3,4-dihydroxyphenylalanine (DOPA) after inhibition of DOPA decarboxylase provided a measure of apparent DA synthesis. Mild stress increased the synthesis and release of DA in striatum. DA synthesis and release also were enhanced by the intra-striatal infusion of N-methyl-D-aspartate (NMDA), an agonist at NMDA receptors, and kainic acid, an agonist at the DL-a-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA)/kainate site. Stress-induced increase in DAsynthesis was attenuated by co-infusion of 2-amino-5-phosphonovalerate (APV) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), antagonists of NMDA and AMPA/kainate receptors, respectively. In contrast, intrastriatal APV, CNQX, or kynurenic acid (a non-selective ionotropic glutamate receptor antagonist) did not block the stress-induced increase in DArelease. Stress-induced increase in DA release was, however, blocked by administration of tetrodotoxin along the nigrostriatal DA projection. It also was attenuated when APV was infused into substantia nigra. Thus, glutamate may act via ionotropic receptors within striatum to regulate DA synthesis, whereas glutamate may influence DA release via an action on receptors in substantia nigra. However, our method for monitoring DA synthesis lowers extracellular DA and this may permit the appearance of an intra-striatal glutamatergic influence by reducing a local inhibitory influence of DA. If so, under conditions of low extracellular DA glutamate may influence DA release, as well as DA synthesis, by an intrastriatal action. Such conditions might occur during prolonged severe stress and/or DA neuron degeneration. These results may have implications for the impact of glutamate antagonists on the ability of patients with Parkinson's disease to tolerate stress.     

兴奋性氨基酸介导脊髓伤害性信息传递

ＮＭＤＡ和非ＮＭＤＡ受体广泛存在于猫脊髓背角神经元上,并参与介导伤害性信息传递;ＮＭＤＡ受体主要介导皮肤的伤害性传入,非ＮＭＤＡ受体则主要介导肌肉和内脏的伤害性传入;皮肤和肌肉的伤害性传入分别诱发释放更多的门冬氨酸和谷氨酸可能是这种差别的主要原因之一;ＮＭＤＡ受体的不同调节位点在伤害性信息传递中有密切的协同作用;兴奋性氨基酸和Ｐ物质及其受体在介导和调制伤害性信息传递中的相互作用可以分别发生在神经元