Action Of Glu Research Articles

Metabotropic glutamate receptor genetic variants and peripheral receptor expression affects trait scores of autistic probands

Glutamate (Glu) is important for memory and learning. Hence, Glu imbalance is speculated to affect autism spectrum disorder (ASD) pathophysiology. The action of Glu is mediated through receptors and we analyzed four metabotropic Glu receptors (mGluR/GRM) in Indo-Caucasoid families with ASD probands and controls. The trait scores of the ASD probands were assessed using the Childhood Autism Rating Scale2-ST. Peripheral blood was collected, genomic DNA isolated, and GRM5 rs905646, GRM6 rs762724 & rs2067011, and GRM7 rs3792452 were analyzed by PCR/RFLP or Taqman assay. Expression of mGluRs was measured in the peripheral blood by qPCR. Significantly higher frequencies of rs2067011 ‘A’ allele/ AA’ genotype were detected in the probands. rs905646 ‘A ‘exhibited significantly higher parental transmission. Genetic variants showed independent as well as interactive effects in the probands. Receptor expression was down-regulated in the probands, especially in the presence of rs905646 ‘AA’, rs762724 ‘TT’, rs2067011 ‘GG’, and rs3792452 ‘CC’. Trait scores were higher in the presence of rs762724 ‘T’ and rs2067011 ‘G’. Therefore, in the presence of risk genetic variants, down-regulated mGluR expression may increase autistic trait scores. Since our investigation was confined to the peripheral system, in-depth exploration involving peripheral as well as central nervous systems may validate our observation.

Indirect determination of Escherichia coli based on β-D-glucuronidase activity and the voltammetric oxidation of phenolphthalein at graphite screen-printed electrodes

Abstract Of the methods available for the determination of Escherichia coli (E. coli), an index of fecal contamination in aquatic systems, electrochemical methods based on the activity of β-D-glucuronidase (GLU) offer distinct advantages in terms of portability and instrumentation simplicity. GLU is an enzyme marker of E. coli and its activity can be measured by the voltammetric determination of various electroactive compounds released upon the action of GLU on specific substrates. In this work, we report for the first time on the development of graphite screen-printed electrodes (SPEs) and electrochemical 3-electrode cells (SPCs) that offer highly sensitive determination of phenolphthalein (Phe) by differential pulse (DP) voltammetry. DP voltammograms of Phe possess an oxidation peak at 0.69 V (SPE vs. Ag/AgCl reference electrode) or at 0.53 V (SPC vs. pseudoreference Ag printed electrode). Oxidation currents are related linearly with the concentration of Phe over the concentration range 10–1000 and 25–600 nM Phe, while for a signal-to-noise ratio of 3 the limits of detection were 5 and 10 nM Phe, respectively. SPCs were further used for the indirect determination of E. coli, via the electrochemical oxidation of the enzymatically liberated Phe upon the action of GLU on phenolphthalein β-D-glucuronide. Various experimental variables regarding the induction of GLU, the permeabilization of the cells, the preconcentration of GLU in the cell extract as well as the electrochemical determination of Phe were optimized, and the method was successfully used for the determination of 100 cfu/mL E. coli within 4 h.

Влияние ингибирования поли (АДФ-рибозо) полимеразы-1 на уровень внутриклеточного кальция, АТФ и выживаемость культивируемых нейронов мозжечка крысы при гиперстимуляции глутаматных рецепторов

Актуальность. Изучение механизмов действия ингибиторов ядерного фермента поли (АТФ-рибозо) полимеразы-1 (PARP-1) на динамику изменения внутриклеточной концентрации Ca2+ ([Ca2+]i), содержание АТФ в нейронах и их выживаемость при избыточной стимуляции глутаматных рецепторов способствует пониманию биохимических процессов при инсультах мозга и подбору препаратов для терапии последствий. Цель. Определить изменения [Ca2+]i, концентрации ATФ ([ATФ]) и выживаемости клеток при одновременном действии токсических концентраций глутамата (Glu) и ингибиторов PARP-1, и выяснить возможность участия нейрональной NO-синтазы (nNOS) в активации PARP-1. Методы и материалы. Исследование выполнено с использованием 7-8 дневных первичных культур нейронов мозжечка крысы. Изменения [Ca2+]i определяли методом флуоресцентной микроскопии с помощью Fura-FF. Cодержание [АТФ] измеряли с использованием люциферин-люциферазной биолюминесценции. Выживаемость нейронов оценивали с помощью МТТ-теста по восстановлению соли тетразолия до формазана. Активность PARP-1 и nNOS контролировали с помощью ингибиторов. Результаты. Показано, что ингибиторы PARP-1 миноциклин (0,2 мкМ) и бензамид 1 мМ) отдаляют наступление индуцированной глутаматом отсроченной кальциевой дизрегуляции, уменьшают понижение внутриклеточной [АТФ] и увеличивают выживаемость культивируемых нейронов. Ингибитор nNOS 7-нитроиндазол (100мкМ) оказывал на [ATФ] и выживаемость нейронов эффекты, аналогичные ингибиторам PARP-1. Выводы. Положительныое действие ингибиторов PARP-1 и nNOS на выживаемость клеток, подвергнутых токсическому действию Glu, обусловлено, вероятно, тем, что ингибиторы поддерживают более высокую [ATФ] во время и после прекращения воздействия Glu, позволяя большей доле нейронов в культуре восстановить ионный гомеостаз. Background. Studying mechanisms of the nuclear enzyme, poly (ADP-ribose) polymerase-1 (PARP-1), inhibition on changes in intracellular Ca2+ ([Ca2+]i and ATP [ATP] concentrations in neurons provides understanding of biochemical processes in stroke and helps selection of drugs for the treatment of consequences of glutamate hyperexcitability. Aim. To determine changes in [Ca2+]i, [ATP], and cell survival under a simultaneous action of toxic concentrations of glutamate (Glu) and PARP-1 inhibitors, and to elucidate a possibility of neuronal NO synthase (nNOS) contribution to the PARP-1 activation. Methods and materials. Changes in [Ca2+]i were studied using fluorescence microscopy. The [ATP] was measured using luciferin-luciferase bioluminescence. Survival of neurons was assessed using the MTT assay based on reduction of a tetrazolium salt into formazan. Results. The PARP-1 inhibitors, minocycline (0.2 μM) and benzamide (1 mM), delayed the onset of the secondary rise of [Ca2+]i (delayed calcium dysregulation), limited the Glu-induced decrease in intracellular [ATP], and improved survival of cultured neurons. The nNOS inhibitor, 7-nitroindazole (100 μM), exerted effect on [ATP] and neuron survival similar to those of PARP-1 inhibitors. Conclusion. The positive effects of PARP-1 nNOS inhibitors on cell survival were presumably due to the fact that the inhibitors maintained a higher [ATP] during and after the end of the toxic Glu action, which allowed a greater proportion of cultured neurons to restore the ion homeostasis.

Study of the Mechanism of the Neuron Sensitization to the Repeated Glutamate Challenge

Exposure of cultured neurons to high concentrations of Glu leads to a strong depolarization of mitochondria, which develops synchronously with the secondary rise in the intracellular Ca2+ concentration (delayed calcium deregulation, DCD). In this study, using the primary culture of rat cerebellar neurons, we investigated the mechanism of neuronal sensitization, which manifests itself in the reduction of latent periods of DCD during repeated exposures to Glu. It was shown that the most likely cause of sensitization is the inability of mitochondria to maintain a high transmembrane potential (ΔΨm) as a result of an increase in the proton conductivity of the internal mitochondrial membrane, but not the opening of the mitochondrial permeability transition pore in the inner mitochondrial membrane. Mitochondrial dysfunction reduces the production of ATP, leading to the inability of neurons to quickly restore the concentration of Na+, ATP, and NADH in the intervals between successive Glu administrations. One of the reasons that aggravate the dysfunction of mitochondria and contribute to the sensitization of neurons to the repeated action of Glu is Ca2+ accumulated in the mitochondria during the first glutamate impact.

Sugar based N,N\u2032-didodecyl-N,N\u2032digluconamideethylenediamine gemini surfactant as corrosion inhibitor for mild steel in 3.5% NaCl solution-effect of synergistic KI additive

The inhibitory behaviour of non-ionic sugar based N,N′-didodecyl-N,N′-digluconamideethylenediamine gemini surfactant, designated as Glu(12)-2-Glu(12) on mild steel (MS) corrosion in 3.5% NaCl at 30–60 °C was explored using weight loss, PDP, EIS and SEM/EDAX/AFM techniques. The compound inhibited the corrosion of mild steel in 3.5% NaCl and the extent of inhibition was dependent on concentration and temperature. The inhibiting action of Glu(12)-2-Glu(12) is synergistically enhanced on addition of potassium iodide (KI) at all concentrations and temperatures. The inhibiting formulation comprising of 2.5 × 10−3 mM of Glu(12)-2-Glu(12) and 10 mM of KI exhibits an inhibition efficiency of 96.9% at 60 °C. Quantum chemical calculations and MD simulation were applied to analyze the experimental data and elucidate the adsorption behaviour and inhibition mechanism of inhibitors. MD simulation showed a nearly parallel or flat disposition for Glu(12)-2-Glu(12) molecules on the MS surface providing larger blocking area to prevent the metal surface from corrosion.

Elevated excitatory neurotransmitter levels in the fibromyalgia brain

Consistent brain imaging findings demonstrate that neurobiological factors may contribute to the pathology of 'central' pain states such as fibromyalgia (FM). Studies using proton magnetic resonance spectroscopy suggest that glutamate (Glu), a key excitatory neurotransmitter, may be present in higher concentrations within the brains of FM patients. This neurotransmitter imbalance is present in multiple brain regions that have been implicated in processing pain information. However, it is unknown if elevated Glu is acting at the synapse. New investigations are needed to investigate the molecular action of Glu in FM and to investigate these findings during treatment that modulates glutamatergic neurotransmission.

Role of glutamate in the rostral ventrolateral medulla in acupuncture-related modulation of visceral reflex sympathoexcitation

Visceral sympathoexcitatory reflexes induced by stimulation of the gallbladder with bradykinin (BK) are attenuated by electroacupuncture (EA) at Neiguan-Jianshi (P5-6) acupoints located over the median nerve. Previous studies have shown that neurons in the rostral ventrolateral medulla (rVLM) receive convergent input from visceral organs and somatic nerves (activated by EA). Glutamate (Glu), an important excitatory neurotransmitter in the rVLM, processes visceral sympathoexcitatory cardiovascular reflexes. In the present study, we determined the relation between EA-mediated opioidergic modulation of visceral cardiovascular responses and Glu. Reflex cardiovascular responses were evoked by application of BK to the gallbladder before and after EA in anesthetized cats. Glu concentrations ([Glu]) were measured by HPLC from samples collected by microdialysis probe(s) inserted unilaterally or bilaterally into the rVLM. BK-induced reflex responses and [Glu] were attenuated by 45% and 70%, respectively, after 30 min of EA (n = 6). EA alone did not change [Glu] in the rVLM (n = 6, P > 0.05). However, microdialysis of naloxone (100 mM) into the rVLM reversed EA-related inhibition of blood pressure and [Glu] (n = 5). Immunohistochemical visualization showed that delta-opioid receptors colocalized with, and were in close apposition to, vesicular Glu transporter 3- and c-Fos-double-labeled perikarya and processes of rVLM neurons after gallbladder stimulation with BK. These data suggest that EA attenuates BK-induced visceral sympathoexcitatory reflexes through opioid-mediated inhibition of Glu's action in the rVLM.

Glutamatergic innervation in bone.

Bone is highly innervated, and evidence for a regulation of bone metabolism by nerve fibers has been suggested by many clinical and experimental studies. However, the nature of the neuromediators involved in these processes has not been well documented. Glutamate (Glu), a major neuromediator of the central nervous system (CNS), was recently identified in nerve fibers running in bone marrow in close contact with bone cells, suggesting that Glu may also act as a neuromediator in this tissue. During the last few years, all the machinery required for glutamate signalling in the CNS was demonstrated in bone. Osteoblasts and osteoclasts express ionotropic Glu receptors (iGluR) (NMDA, AMPA, and Kainate) and metabotropic Glu receptors (mGluR) as well as Glu transporters. Electrophysiological studies have demonstrated that NMDA receptors (NMDAR) and mGluR are functional on bone cells. NMDAR are involved in osteoclast formation and bone resorption and preliminary studies suggest that they may also participate in mechanisms underlying osteoblast proliferation or differentiation, providing evidence for a direct action of Glu on bone cells. The bone loss induced in a model of sciatic neurectomy in growing rats is associated with a decrease of glutamatergic innervation, suggesting that Glu released by nerve fibers may contribute to the regulation of bone remodeling. The manipulation of Glu action in bone may, therefore, represent a new therapeutic target for pathologies associated with modifications of bone remodeling.

The antioxidant enzymatic blood profile in Alzheimer's and vascular diseases. Their association and a possible assay to differentiate demented subjects and controls

A study of several elements of the antioxidative system: Cu-Zn Superoxide dismutase (SOD), catalase (CAT), glutathione system (GLU), chemiluminescence (CHE), and antioxidant capacity (AOX), was conducted in 20 demented probable Alzheimer's (DAT), and 15 vascular demented (VD) patients, 19 control (C) subjects, and 11 relatives (F) of one DAT patient. A significant association was found between the variables of the antioxidant system, measured in blood samples, and the neurological pathologies VD and DAT: Kruskal-Wallis test; p = 0.0006 ( p = 0.014 when the analysis did not include SOD). This demonstrated that VD and DAT diseases are accompanied by oxidative disorders. The VD and DAT diseases are differentially distinguishable by changes in blood profiles. A graphical method for classification, the Principal Components Analysis (PCA), distinguished between demented and non-demented subjects on the basis of their laboratory variables. A numerical method, Discriminant Functions (DF), constructed to separate the clinical groups on the basis of the same variables, obtained relatively high percentages of success: 92% of demented were detected against healthy subjects; of the latter 82% have been correctly identified as non-demented. Discrimination between VD and DAT patients was achieved for 100% of VD and 86% of DAT patients. DF were similarly successful in detecting the healthy condition of DAT relatives. Possible different mechanisms involved in H 2O 2 elimination in DAT and VD patients are proposed, where CAT is the responsible enzyme of this reaction in DAT patients, while in VD this function would be achieved mainly through the action of GLU. It seems that SOD levels are stable, at least, within one year. Variations appear to be linked with clinical changes.

Bepridil exacerbates glutamate-induced deterioration of calcium homeostasis and cultured nerve cell injury.

Application of 50 microM bepridil (BPD) to cultured nerve cells did not greatly affect the resting cytoplasmic Ca2+ concentration ([Ca2+]i) but caused its pronounced increase both during prolonged glutamate (GLU, 100 microM) treatment and, especially, in the postglutamate period in case of partial [Ca2+]i recovery. In contrast, in cells exhibiting a high [Ca2+]i plateau in the postglutamate period, BPD application either did not cause any additional elevation of [Ca2+]i or caused a very small increase. Under identical conditions replacement of external Na+ by Li+ or N-methyl-D-glucamine (NMDG) either did not change [Ca2+]i or produced a very small increase, strongly indicating that the BPD-evoked Ca2+ responses could not be explained solely by Na+/Ca2+ exchange inhibition but resulted from some other BPD effects. Indeed, in experiments with Rhodamine 123-loaded neurons it has been shown that 50 microM BPD induced prominent mitochondrial depolarization which is known to abolish the mitochondrial Ca2+ uptake. Finally it was revealed that BPD application to the cell culture either in the period of a prolonged (15 min) GLU action or, especially, in the postglutamate period greatly exacerbated delayed neuronal death, apparently due to a complex inhibitory action of the drug on both Ca2+ buffering and Ca2+ extrusion systems.

Differential L-glutamate responsiveness among superficial dorsal horn neurons

1. Intracellular recordings were made from 128 superficial dorsal horn (laminae I and II) neurons in slice preparations of the lumbosacral spinal cord obtained from young hamsters. Stimulation of the segmental dorsal root evoked postsynaptic potentials in all neurons. The average transmembrane resting potential was -61 +/- 1 mV (mean +/- SE; n = 123). The mean action potential amplitude was 75 +/- 1 mV (n = 105) with a duration at half peak of 1.1 +/- 0.1 ms (n = 102). The mean input resistance of these neurons was 72 +/- 4 M omega (n = 125). These values are comparable to those reported in other studies on neurons of this region using penetrating microelectrodes. 2. Bath application of N-methyl-D-aspartate (NMDA; 50 microM) depolarized 67 of 71 (94%) of the tested neurons. Superfusion with the non-NMDA amino acid agonists DL-alpha-amino-3-hydroxy-5-methyl-4- isoxazole propionic acid (AMPA; 20 microM) and kainate (KA; 50 microM) depolarized all tested neurons by > 10 mV. On the other hand, only 13 of 67 (19%) tested neurons were depolarized > 4 mV by superfusion solutions containing 3 mM L-glutamate (Glu). L-Aspartate at 3 mM depolarized three out of seven neurons by > 4 mV and appeared to be equally as effective as Glu. 3. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) substantially attenuated the AMPA- and KA-induced depolarizations and partially attenuated the NMDA-induced depolarizations. The NMDA antagonist 3 [(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP; 50 microM) reversibly blocked the NMDA-induced depolarization in all tested neurons. Glu-induced depolarization was unaffected by CNQX but was attenuated by CPP in three of three tested neurons. These observations indicate that some of the Glu-induced depolarization was mediated by NMDA receptors. 4. CNQX reversibly attenuated excitatory postsynaptic potentials (EPSPs) produced by primary afferent activity in A delta- and C-fibers whereas CPP suppressed only the late EPSP components. Therefore in the neurons sampled, synaptic responses evoked from primary afferent fibers appear to be mediated by both non-NMDA and NMDA receptors. 5. The glutamate uptake inhibitors, L-trans-pyrrolidine-2,4-dicarboxylate (L-trans PDC; 50 microM; n = 6) and threo-3-hydroxy-D-aspartate (1 mM; n = 1) did not have a consistent effect upon Glu action background discharge, RN or Vm in Glu-unresponsive neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for a kynurenate-insensitive glutamate receptor in nucleus tractus solitarii.

Previous studies have shown that pharmacological blockade of ionotropic excitatory amino acid (EAA) receptors in the nucleus tractus solitarii (NTS) with kynurenate (Kyn) abolishes baroreceptor reflexes but fails to affect cardiovascular responses evoked by microinjections of L-glutamate (Glu) into the NTS. These observations have raised doubts as to whether Glu is a neurotransmitter of baroreceptor information in the NTS because the pharmacological actions of exogenously administered Glu are not identical to those of the neurotransmitter released in the NTS coincident with baroreceptor activation. One possible explanation for these results is that exogenously administered Glu might act at receptors that are not blocked by Kyn and are not accessible to synaptically released Glu in the NTS baroreflex pathway. The purpose of this study was to determine if Kyn-insensitive Glu receptors are present in the NTS. One candidate for this Kyn-insensitive receptor is the metabotropic EAA receptor that is selectively activated by trans-DL-1-amino-1,3-cyclopentane-dicarboxylic acid (ACPD). Microinjections of ACPD into the NTS of anesthetized rats produced dose-related depressor responses that were not reduced by Kyn or by pretreatment with the putative ACPD receptor antagonist L-2-amino-3-phosphonopropionate (L-AP-3). Similarly, depressor responses produced by Glu also were not affected by Kyn or by L-AP-3. These data demonstrate the presence of a Kyn-insensitive Glu receptor in the NTS. Moreover, they suggest that the failure of Kyn to reduce cardiovascular responses evoked by Glu injections into the NTS can be explained by an action of Glu at Kyn-insensitive ACPD receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

A Specific Trarisduction Mechanism for the Glutamate Action on Phosphoinositide Metabolism via the Quisqualate Metabotropic Receptor in Rat Brain Synaptoneurosomes: I. External Na+ Requirement

The characteristics of the transduction mechanism(s) activated by glutamate (Glu) via the quisqualate metabotropic receptor, as well as by depolarizing agents, to trigger formation of inositol phosphates (IPs) were investigated in 8-day-old rat forebrain synaptoneurosomes. The replacement of external Na+ by various compounds (Li+, Tris+, N-methyl-D-glucamine+, and sucrose) induces an increase in basal accumulation of IPs and depolarizes synaptoneurosome membranes. Under these conditions, Glu- and K(+)-induced accumulations of IPs are inhibited, whereas the carbachol (Carb)-elicited response of IPs parallels the basal one. Agents increasing Na+ influx, such as veratridine and monensin, depolarize synaptoneurosomes and stimulate formation of IPs. These stimulations are not additive with responses of IPs elicited by Glu or K+. These data suggest that (a) Glu activates phosphoinositide metabolism via a specific mechanism (distinct from that of cholinergic agonists), (b) depolarizing agents and Glu share at least one common intermediate step in their mechanisms of activation of the metabolism of IPs, and (c) the depolarization may correspond to this common step. In addition, Na+ seems to be required for Glu stimulation of metabolism of IPs. The depolarization associated with the action of Glu on formation of IPs results neither from an influx via tetrodotoxin-sensitive voltage-dependent Na+ channels nor from an entry via the classically characterized Na+/Ca2+ or Na+/H+ exchangers. In fact, tetrodotoxin (2 microM) has no effect on the Glu- or K(+)-elicited response of IPs. Amiloride (greater than 50 microM) and some of its derivatives similarly inhibit not only Glu- and K(+)- but also Carb-evoked formation of IPs.

Glutamate cytotoxicity in a neuronal cell line is blocked by membrane depolarization

To understand better the proximate mechanism involved in the excitotoxic response to l-glutamate (Glu), we have exploited the Glu receptor present in the N18-RE-105 neuroblastoma-embryonic retinal hybrid cell line. These cells undergo lysis dependent on extracellular Ca 2+ when exposed to Glu. We now report that the depolarizing action of Glu is not responsible for its cytotoxic effects. Furthermore, depolarization of these cells with elevated K +, ouabain or veratridine does not cause cytotoxicity but rather protects against the cytotoxic effects of Glu. Our results may implicate a role for voltage-sensitive Ca 2+ channels (VSCCs) in cytotoxicity, and depolarization-induced inactivation of VSCCs ( Nature (Lond.), 316 (1985) 440–443.) as a protection against Glu receptor agonists. Our findings demonstrate a clear dissociation between depolarization and the neuronal degeneration caused by Glu.

Increase in glutamate sensitivity of subthalamic nucleus neurons following bilateral decortication: a microiontophoretic study in the rat

Responses of subthalamic neurons (STN) to the iontophoretic application of glutamate (Glu), acetylcholine (ACh) and GABA were studied in rats 2 or 3 weeks following bilateral decortication, and were compared to those obtained in normal animals. All the cells recorded in lesioned rats showed a decrease in their spontaneous firing rate. They also proved to be significantly more sensitive to the excitatory action of Glu, although their responsiveness to ACh or GABA was not modified.

Electrophysiological and pharmacological actions of N-acetylaspartylglutamate intracellularly studied in cultured chick cerebellar neurons

The electrophysiological and pharmacological actions of N-acetylaspartylglutamate (NAAG) in cultured chick cerebellar neurons were intracellularly investigated in comparison with l-aspartate (ASP) and l-glutamate (GLU). Iontophoretically applied NAAG dose-dependently induced depolarizations associated with increases in spike discharge and changes in membrane conductance. Relative excitatory potencies seemed to beGLU>ASP⩾NAAG. The voltage-dependent increase in input resistance observable in the presence of Mg ions was most notable for ASP, moderate for NAAG and least for GLU. The reversal potential of NAAG-induced depolarization was at about 0 mV and similar to that for ASP or GLU, indicating primary concern of Na +/K +-conductances to the NAAG action. Mg ions depressed the actions of ASP and NAAG more strongly than the GLU action. 2-Amino-5-phosphonovalerate (APV) and d-α-aminoadipate antagonized the actions of ASP and NAAG more effectively than the GLU action. 2-Amino-4-phosphonobutyrate (APB) and glutamic acid diethylester showed rather non-selective antagonism to NAAG, ASP and GLU. These results suggest that NAAG is excitatory to cultured chick cerebellar neurons functionally resembles ASP or is intermediate between ASP and GLU, and may also support the suggested candidacy of NAAG for a neurotransmitter in the CNS.

The role of specific ions in glutamate neurotoxicity

When the chick embryo retina is incubated in balanced salt solution containing glutamate (Glu) in 1 mM concentration, a neurodegenerative reaction occurs within 30 min. Here we report that the neurotoxic action of Glu on retinal neurons is dependent on the presence of Na + and Cl −, but not Ca 2+, in the incubation medium. Also, we report that depolarizing concentrations of K + can induce a severe cytotoxic reaction in chick retina which, like the depolarization-linked neurotoxicity of Glu, is a Cl −-dependent phenomenon.

Calcium influx accompanies but does not cause excitotoxin-induced neuronal necrosis in retina

Several authors have recently proposed that excessive calcium (Ca ++) influx into postsynaptic cells may be the mechanism by which excitotoxins such as glutamate (Glu), N-methylaspartate (NMA) and kainic acid (KA) cause neuronal necrosis. Here we have undertaken both in vivo and in vitro studies to explore this hypothesis. Our findings indicate that Ca ++ does accumulate selectively in neural elements undergoing degeneration in the in vivo mouse hypothalamus following subcutaneous administration of NMA. However, pretreatment with the putative Ca ++ channel blocker nimodipine resulted in augmentation rather than suppression of the toxic action of NMA and Glu on the mouse hypothalamus and eliminating Ca ++ from the incubation medium did not interfere with the toxic action of Glu, NMA or KA on the chick embryo retina in vitro. We conclude, therefore, that Ca ++ influx is an unlikely explanation for excitotoxin-induced degeneration of retinal or hypothalamic neurons.

The effect of glutamate on the frog semicircular canal

l-glutamate (Glu) has at least two sites of action in the frog semicircular canal: the hair cell (presynaptic) and the primary afferent nerve fibres (postsynaptic). Glu's action on the hair cell results in an increased release of the natural transmitter which is responsible for a substantial increase in the frequency of firing in primary afferents. The presynaptic action of Glu is antagonized by d-alpha-aminoadipate ( dαAA). Glu produces a long-lasting depolarization on the afferent nerve fibres which does not by itself elicit any afferentv discharge of impulses when the release of the natural transmitter is prevented. Glu-induced nerve depolarization is only partially antagonized by dαAA. The difficulty of reconciling some of the observations made of the effects of Glu in semicircular canals with its presumed role as an afferent transmitter in this organ is discussed, but this role is not definitely rejected.

Effects of L-glutamate on the anomalous rectifier potassium current in horizontal cells of Carassius auratus retina.

The effects of externally applied L-glutamate (Glu) on K currents through the anomalous rectifier were studied in solitary horizontal cells dissociated from goldfish retinae under whole-cell voltage-clamp or cell-attached patch-clamp conditions using 'giga-seal' suction pipettes. In the whole-cell clamp experiments, hyperpolarization of the membrane below the resting potential (ca. -57 mV) induced a large voltage-dependent inward current which has been identified as the K current through the anomalous rectifier (Ianomal.). Application of Glu to the external medium reduced Ianomal.. Reduction of the inward current was not seen in preparations in which Ianomal. has been blocked by an application of Cs or Ba ions to the external medium. Single-channel currents through the anomalous rectifier were recorded under cell-attached patch-clamp conditions. The current showed an inward rectification; its amplitude increased with hyperpolarization of the patch membrane, and became below the noise level near the equilibrium potential of K ions (EK). No polarity reversal was observed even by a strong membrane depolarization. The patch membrane potential at which the current amplitude became undetectable shifted in parallel to the shift of EK. The open probability changed little with polarization of the patch membrane. When Glu (greater than 100 microM) was applied to the outside of the patch membrane, the number of available Ianomal. channels was decreased, but neither the single-channel conductance, open or closed time constants, nor the open probability changed significantly. Removal of Glu produced the opposite sequence; i.e. the number of available Ianomal. channels increased with time. It was concluded that the reduction of the Glu-induced current at hyperpolarized potentials in the whole-cell recording configuration is due to the blocking action of Glu on Ianomal..