Effects of the N-methyl-D-aspartate antagonists on the rise in [Ca2+]i following depolarization in aged rat brain synaptosomes.

The effects of non-competitive NMDA antagonists, MK-801 and dextrorphan in relation to the rise in intracellular Ca2+ concentrations ([Ca2+]i) after stimulation with 15 mM K+ in whole brain synaptosomes from young (3 months old) and aged (24 months old) Fisher344 rats were examined. A fluorescent chelating agent, Rhod-2, was employed to monitor any alterations of K(+)-evoked [Ca2+]i. In young rats, the rise in [Ca2+]i following depolarization was affected by neither dextrorphan (1, 10, 100 microM) nor MK-801 (0.1, 1, 10 microM), while in aged rats, 1 microM dextrorphan and 0.1 microM MK-801 brought about a significant increase in [Ca2+]i following depolarization. In low Mg2+ medium, 10 microM MK-801 and 100 microM dextrorphan significantly inhibited the rise in [Ca2+]i after stimulation with 15 mM K+ in young rats, while neither dextrorphan nor MK-801 could affect the rise in [Ca2+]i significantly in aged rats. When 100 microM NMDA was applied in a medium containing 1.2 mM Mg2+, the rise in [Ca2+]i following depolarization was slightly inhibited by 1 microM MK-801 in young rats, but it was not inhibited significantly by dextrorphan. In aged rats, both 100 microM dextrorphan and 10 microM MK-801 strongly inhibited the rise in [Ca2+]i following depolarization in the presence of 100 microM NMDA. Instead of NMDA, when 100 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a non-NMDA receptor agonist, was applied, dextrorphan did not inhibit the rise in [Ca2+]i. In low Mg2+ medium, 100 microM NMDA potentiated the inhibitory effect of 10 microM dextrorphan in young rats, while 100 microM dextrorphan or MK-801 did not show any further inhibition by adding 100 microM NMDA. The addition of 100 microM AMPA did not affect the effect of dextrorphan in a low Mg2+ medium in young rats. These results suggest that NMDA antagonist-mediated [Ca2+]i homeostatic system may alter through aging. In addition, the findings that NMDA potentiated the inhibitory effect of NMDA antagonist, which being further potentiated by aging or lowered extrasynaptosomal Mg2+, indicate the possibility that the Mg2+ block to NMDA receptors might be attenuated through aging.     

The interactions between plasma membrane depolarization and glutamate receptor activation in the regulation of cytoplasmic free calcium in cultured cerebellar granule cells

The complex modulation of cytoplasmic free calcium concentration ([Ca2+]c) in primary cultures of cerebellar granule cells in response to glutamate receptor agonists has been the subject of several contradictory reports. We here show that 3 components of the [Ca2+]c response can be distinguished: (1) Ca2+ entry through voltage-dependent Ca2+ channels, following KCl- or receptor-evoked depolarization, (2) Ca2+ entry through NMDA receptor channels, and (3) liberation of internal Ca2+ via a metabolotropic receptor. Depolarization with KCl induced a transient [Ca2+]c response (subject to voltage inactivation) decaying to a sustained plateau (largely inhibited by nifedipine). The NMDA response was potentiated by glycine, totally inhibited by (+)5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), and blocked by Mg2+ in a voltage-sensitive manner. Polarized cells displayed small responses to quisqualate (QA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA). Depolarization enhanced a transient response to QA, but not to AMPA. Trans-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD), a selective agonist for the metabolotropic glutamate receptor, caused a transient elevation of [Ca2+]c, which was blocked by prior exposure to QA but not AMPA. The prolonged [Ca2+]c response to kainate (KA) can be resolved into 2 major components: an indirect NMDA receptor-mediated response due to released glutamate and a nifedipine-sensitive component consistent with depolarization-mediated entry via Ca2+ channels. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), QA at greater than 10 microM, and AMPA (but not trans-ACPD) reversed the KA response, consistent with an inactivation of the KA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence measurement of changes in intracellular calcium induced by excitatory amino acids in cultured cortical astrocytes

Population response of [Ca2+]i in cultured cortical astrocytes to excitatory amino acids was measured at room temperature using the calcium-sensitive dye fura-2. Quisqualic acid (QA), glutamate (Glu), and kainic acid (KA) caused a peak increase in [Ca2+]i in the order QA greater than Glu greater than KA. No response to N-methyl-D-aspartic acid (NMDA) was observed whether or not Mg2+ was present externally. Both QA and Glu (100 microM) frequently elicited a decaying oscillatory [Ca2+]i response during sustained agonist application; the period of oscillations initially was 23.5 sec and increased as the response was damped. Comparatively, the [Ca2+]i response to KA was nonoscillatory. Both responses to Glu and KA were reduced slightly by antagonist gamma-D-glutamylaminomethyl-sulfonic acid (1 mM), but virtually were abolished by kynurenic acid (3 mM). Replacement of external Na+ by choline had no significant effect on the Glu response. Removal of external Ca2+ reduced the peak response to QA, Glu, and KA to 40, 34, and 18%, respectively; and markedly reduced the degree of QA- and Glu-induced [Ca2+]i oscillations. Pretreatment with phorbol esters, a potent activator of protein kinase C, blocked the [Ca2+]i response to Glu but not KA. It is concluded that cortical astrocytes express Glu receptors of the non-NMDA type in culture and that receptor activation leads to Ca2+ influx and release of internal Ca2+. Mobilization of Ca2+ apparently occurs via the known Glu-mediated hydrolysis of inositol lipids, which may come under negative-feed-back control by protein kinase C activation. Oscillatory [Ca2+]i signaling offers the possibility of a dynamic population response in an electrically coupled glial network.     

Excitatory amino acid-evoked release of [3H]GABA from hippocampal neurons in primary culture

We investigated the release of gamma-[2,3-3H(N)]aminobutyric acid ([3H]GABA) from hippocampal neurons in primary cell culture. [3H]GABA release was stimulated by the excitatory amino acid neurotransmitter glutamate as well as by N-methyl-D-aspartate (NMDA) and kainate. Cell depolarization induced by raising [K+]o or by veratridine also stimulated [3H]GABA release. NMDA-induced release was completely blocked by 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP+), Mg2+ and Zn2+ whereas the release induced by glutamate and kainate was much less susceptible to inhibition by these substances. Furthermore, removal of external Ca2+ inhibited NMDA-induced release, but not that induced by glutamate, kainate, veratridine or 50 mM K+. Removal of external Na+ reduced [3H]GABA release evoked by all stimuli, but to different extents. All of the excitatory amino acids tested increased [Ca2+]i within hippocampal neurons as assessed by fura-2 based microspectrofluorimetry. This increase in [Ca2+]i was completely dependent on the presence of external Ca2+. These results suggest that Ca2+-dependent and -independent forms of GABA release from hippocampal interneurons may occur. [3H]GABA release evoked by glutamate, kainate, veratridine or 50 mM K+, appeared to be mediated by the reversal of electrogenic, Na+-coupled GABA uptake. Release was inhibited by nipecotic acid, an inhibitor of the Na+-coupled GABA uptake system. However, release induced by NMDA may also include a Ca2+-dependent component.     

Excitatory amino acid-induced alterations of cytoplasmic free Ca2+ in individual cerebellar granule neurons: role in neurotoxicity.

The effects of glutamate on intracellular free Ca2+, [Ca2+]i, and neurotoxicity were compared in cerebellar granule neurons in vitro. [Ca2+]i was measured with fura-2 and digital fluorescence imaging microscopy; neurotoxicity was monitored using a vital dye and colorimetric analysis. Glutamate produced dose-dependent increases in [Ca2+]i, which tended to be transient for glutamate concentrations in a range of 0.01-0.5 microM and sustained for higher levels of glutamate. The ED50 for the [Ca2+]i response to glutamate was 6 microM. The LD50 for glutamate-induced neurotoxicity was similar, i.e., 10 microM. The effect of glutamate on [Ca2+]i was greatly diminished when external Ca2+ was removed and blocked by Mg2+ or N-methyl-D-aspartate (NMDA)-type receptor antagonists. The latter conditions as well as preloading granule neurons with the intracellular Ca2+ chelator quin2 largely prevented glutamate cytotoxicity. The neurotoxic effect of glutamate required incubations with the stimulus for 10-20 min at 25 degrees C. Withdrawal of glutamate after this period was accompanied by a prolonged alteration in [Ca2+]i. Pretreatment of the cells with the ganglioside GM1 reduced this late increase in [Ca2+]i as well as the neurotoxic effects of glutamate. This indicates that glutamate-induced neurotoxicity results from a composite of diverse temporal alterations in Ca2+ homeostasis and that blunting any of these components reduces excitotoxicity.     

Modulatory effects of aluminum, calcium, lithium, magnesium, and zinc ions on [3H]MK-801 binding in human cerebral cortex

The independent and combined effects of Ca2+, Mg2+, Zn2+, Al3+ and Li+ on [3H]MK-801 binding in human cerebral cortical membranes were studied to further characterize the modulatory effects of metal ions on the N-methyl-D-aspartate (NMDA) receptor-ionophore. Glycine, in the presence of glutamate, significantly intensified the Mg2+ inhibition of [3H]MK-801 binding whereas it masked the Ca2+ enhancement and slightly diminished the Zn2+ inhibition. Both Ca2+ and Mg2+ reduced the Zn2+ inhibitory potency. Aluminum demonstrated a potent, relatively glycine-insensitive inhibition of [3H]MK-801 binding as an amorphous Al(OH)3 polymer rather than as the free ion. Cationic modulation of the NMDA receptor-ionophore appears to be regulated at multiple sites which have significant allosteric interactions.     

Modulation of N-methyl-D-aspartate receptor-mediated increases in cytosolic calcium in cultured rat cerebellar granule cells.

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in rat cerebellar granule cells using the fluorescent indicator fura-2. Culturing the cells as monolayers on plastic squares which could be placed into cuvettes allowed measurements of [Ca2+]i to be performed on large and homogeneous populations of CNS neurons. Granule cells so cultured maintained low levels of [Ca2+]i (around 90 nM) which increased promptly upon the addition of various excitatory amino acids including N-methyl-D-aspartate (NMDA). Increases in [Ca2+]i elicited by NMDA were inhibited by Mg2+ (1 mM) and often potentiated by glycine (1 microM). The addition of TTX or strychnine (5 microM each) did not alter responses to NMDA or NMDA plus glycine. Cytosolic Ca2+ responses to NMDA/glycine were dependent on the presence of extracellular Ca2+ and were unaffected by concentrations of nifedipine or verapamil that blocked increases in [Ca2+]i elicited by K+ depolarization. Responses elicited by NMDA/glycine were inhibited competitively by 2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid and non-competitively by MK-801 or Mg2+. HA-966 and 7-chlorokynurenate inhibited responses to NMDA alone and blocked competitively the potentiating effects of glycine. The results demonstrate NMDA-mediated increases in [Ca2+]i in cerebellar granule cells that arise solely from influx of extracellular Ca2+ through dihydropyridine-insensitive channels. The strict dependence of the NMDA-evoked response on extracellular Ca2+ provides little evidence for a coupling of NMDA receptors to inositol phosphate metabolism and mobilization of intracellular Ca2+. The effect of various agents on NMDA/glycine-induced increases in [Ca2+]i parallels their effects on ligand binding to or current flow through the NMDA receptor-channel complex. The measurement of cytosolic Ca2+ in this preparation of neuronal cells thus appears especially well suited for assessing, on a functional level, the regulation of NMDA receptors in the CNS.     

Coupling of glutamatergic receptors to changes in intracellular Ca2+ in rat cerebellar granule cells in primary culture

Changes in cytosolic free Ca2+ concentrations, [Ca2+]i, in response to glutamate and glutamate receptor agonists were measured in rat cerebellar granule cells grown on coverslips. The intracellular Ca2+ as measured with fura-2 increased by applying kainate, N-methyl-D-aspartate (NMDA), quisqualate, and (RS)-d-amino-3-hydroxy-5-methyl-4-isoxazole-propionic (AMPA). When the extracellular Mg2+ was removed, the effects of NMDA and the NMDA receptor agonist cis-(+-)-1-amino-1,3-cyclopentanedicarboxylic acid (cis-ACPD) on intracellular Ca2+ were augmented. Glycine potentiated the effects of NMDA and cis-ACPD if the membrane was depolarized by increasing the extracellular K+ concentration. The NMDA receptor antagonist DL-2-amino-5-phosphonopentanic acid (AP5) abolished and the antagonist 3-([+-]-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) greatly reduced the effect of NMDA in both the normal and the Mg-free media. The dose-response curves of NMDA and, to a lesser extent, of kainate were shifted to the left, and that of quisqualate became biphasic in the Mg-free medium. The increase in [Ca2+]i produced by high quisqualate concentrations in the Mg-free medium was totally abolished by AP5. The results suggest that Ca2+ influx in cerebellar granule cells occurs through both NMDA- and non-NMDA-coupled ion channels. A part of the quisqualate-induced rise in cytosolic Ca2+ seems to be linked to the activation of NMDA receptors.     

Excitatory amino acid regulation of intracellular Ca2+ in isolated catfish cone horizontal cells measured under voltage- and concentration-clamp conditions.

[Ca2+]i was measured using fura-2-loaded isolated catfish horizontal cells in the presence of L-glutamate and the glutamate analogs kainate (KA), quisqualate (QA), and NMDA. Caffeine was used to release Ca2+ from intracellular stores. Cell membrane potential was controlled with a voltage clamp to prevent activation of voltage-dependent Ca2+ channels in the presence of agonist. All excitatory amino acid agonists produced a rapid and sustained rise in [Ca2+]i with the order of potency being QA greater than Glu greater than KA greater than NMDA. The agonist-induced [Ca2+]i increase was blocked in reduced [Ca2+]o and by 6-cyano-7-nitroquinoxaline-2,3-dione and 2-amino-5-phosphonopentanoate, which are specific blockers for QA/KA and NMDA receptors, respectively. The metabotropic receptor agonist trans-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD; 10-200 microM) had no effect on [Ca2+]i. Hill coefficients from curves fitted to concentration-response data suggested an amplification of the Ca2+ signal that was interpreted as calcium-induced calcium release (CICR) from intracellular Ca2+ stores. Caffeine (10 mM) produced a rapid transient rise in [Ca2+]i, confirming the existence of a Ca(2+)-sensitive store. Following caffeine-induced depletion of Ca2+ from intracellular stores, agonists were still able to produce increases in [Ca2+]i, confirming Ca2+ influx through the agonist-gated channel. The agonist-induced increase in [Ca2+]i was decreased following caffeine-induced depletion, confirming a process of CICR. These results are consistent with the hypothesis that excitatory amino acids can produce direct modulation of [Ca2+]i by influx through the agonist-gated channel and by CICR from intracellular stores.     

Redox modulation of NMDA receptor-mediated Ca2+ flux in mammalian central neurons

NMDA receptor-mediated Ca2+ flux was studied in cultured rat retinal ganglion cells and neocortical neurons. Intracellular free calcium ([Ca2+]i was measured with fura-2 fluorescence imaging. Baseline [Ca2+]i was 59 +/- 5 nM. In low [Mg2+]o, 200 microM NMDA reversibly increased [Ca2+]i to 421 +/- 70 nM. This rise in [Ca2+]i was blocked by the NMDA antagonists APV (200 microM) or [Mg2+]o (1 mM), but only slightly inhibited by the non-NMDA antagonist CNQX (10 microM). Chemical reduction with dithiothreitol (DTT) had no effect on resting [Ca2+]i. However, DTT increased the NMDA-induced rise in [Ca2+]i approximately 1.6-fold; the oxidizing agent dithiobisnitrobenzoic acid (DTNB) reversed this effect. In patch-clamp experiments, DTT increased NMDA-activated whole-cell conductance approximately 1.7-fold in low and high [Ca2+]o. The Ca2+/Na+ permeability ratio of approximately 7 for NMDA channels remained unaltered by chemical reduction. Thus, redox modulation of the NMDA receptor/channel complex results in a dramatic alteration in current magnitude but no change in ionic permeabilities.     

HIV-1 envelope protein evokes intracellular calcium oscillations in rat hippocampal neurons

Treatment of single rat hippocampal neurons with 200 pM recombinant HIV-1 envelope glycoprotein, gp120, resulted in large increases in the intracellular free calcium concentration ([Ca2+]i) as measured with indo-1-based microfluorimetry. Three patterns of [Ca2+]i increases were observed: in one pattern the [Ca2+]i rose rapidly and transiently as a single peak, in a second pattern gp120 induced [Ca2+]i oscillations that subsided when the protein was removed, and in a third pattern the oscillations continued long after washout of gp120. Both single peak and oscillatory [Ca2+]i increases were completely blocked by the Ca2+ channel blocker nitrendipine (1 microM). The sustained oscillatory responses were also blocked completely and reversibly by the N-methyl-D-aspartate (NMDA) receptor antagonist CGS19755 (10 microM) and the Na+ channel blocker tetrodotoxin (1 microM). Complete block by antagonists of Ca2+, Na+, and NMDA-gated ion channels suggests that at least two cells are required to maintain the [Ca2+]i oscillations. We hypothesize that gp120 acts as an excitotoxin by increasing synaptic activity in the network of neurons established in primary culture.     

Dexamethasone induces cell death which may be blocked by NMDA receptor antagonists but is insensitive to Mg2+ in cerebellar granule neurons

Since dexamethasone may elevate the Ca2+ influx through NMDA receptors, we have investigated mechanisms of dexamethasone toxicity in rat cerebellar granule neurons. Dexamethasone concentrations over 0.1 microM induced cell death that reached about 20% of the death induced by glutamate. Dexamethasone-induced cell death was reduced by more than 80% by the mineralocorticoid antagonist RU 28318 or the NMDA receptor antagonists MK 801 and CGP 39551, whereas RU 28318 rescued only approximately 30% of cells treated with glutamate, indicating that dexamethasone requires NMDA receptors to induce acute neuronal toxicity and that a fraction of the neurons showed this toxicity. Mg2+ reduced the cell death induced by glutamate at potassium concentrations of 1 mM and 5 mM, but not at 25 mM. In contrast, cell death induced by dexamethasone was not significantly reduced by Mg2+ in any of the potassium concentrations. Both glutamate and dexamethasone induced toxicity with translocation of the apoptosis inducer NGFI-B to the mitochondria seen after 30 min-2 h concomitant with activation of apoptosis inducing factor (AIF) and caspase-3. In conclusion, dexamethasone induces a rapid toxicity which is blocked by NMDA receptor antagonists other than Mg2+, and involves mitochondrial apoptosis inducer NGFI-B.     

Ion Dependence and Receptor Mediation of Glutamate Toxicity in the Immature Rat Hippocampal Slice

Glutamate (glu) is a major excitatory transmitter and a toxin in the brain. In the present study, the immature rat hippocampal slice was used to determine the morphology, topography, ionic mediation and receptor specificity of glu toxicity. Slices were exposed to glu for 30 min, and the damage was evaluated after 3 h of recovery in regular medium. The effects on glu toxicity of changes of [Ca2+], [Cl-] and [Na+] were determined. The receptor preference of glu was assessed by using the N-methyl-D-aspartate (NMDA) antagonist MK-801 and the kainate (KA)/quisqualate (QA) antagonist DNQX, alone or in combination. Further, to see whether glu produces cytotoxicity via osmolysis, the effects of hyperosmolal sucrose on glu toxicity were studied. Glu toxicity was similar to the previously described NMDA toxicity with regard to cytopathology, but differed in some aspects from that caused by KA and QA. The severity of the lesion was determined by the proximity of neurons to the incubation fluid, probably as a consequence of cellular accumulation of the amino acid. Omission of Ca2+ abolished glu toxicity in all neurons except the granule cells of the outer blade. This population was completely protected when Ca2+ was omitted and [Cl-] was reduced. Elevation of [Ca2+] markedly aggravated the lesion caused by glu. Substitution of isethionate for Cl- worsened the glu-induced damage, whilst the amino acid produced qualitatively different neuropathology when choline substituted for Na+. Apparently glu did not damage hippocampal nerve cells through an osmolytic mechanism as medium supplemented with 100 mM sucrose increased the toxicity of glu. Since the lesion produced by glu was more widespread in the presence of high [Ca2+], the effects of receptor antagonists were studied under this condition. MK-801 inhibited glu toxicity whereas DNQX had no effect. Combination of MK-801 and DNQX did not offer better protection than did MK-801 alone. The results suggest that Ca2+ is the main (but not single) determinant of glu toxicity in the immature hippocampal slice. The ionic requirements of glu neurotoxicity are identical to those of NMDA, but differ from those of KA and QA. The notion that glu is a selective NMDA agonist in the present model was confirmed by the protection of MK-801, and by the lack of an effect of DNQX. This is the first report demonstrating that the toxicity of glu is mediated by NMDA receptors in brain tissue which has developed normally. The findings indicate that specific blockade of NMDA receptors may be the most rational strategy in the prevention of glu-related neuronal death occurring in certain neurological anomalies.     

L-[3H]Glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis

The anatomical distribution of L-[3H]glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding of L-[3H]glutamate is accounted for by the presence of 3 distinct binding sites when measured in the absence of Ca2+, Cl- and Na+ ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labelled by D-[3H]2-amino-5-phosphonopentanoate (D-[3H]AP5), [3H]kainate ([3H]KA) and [3H] alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) which are thought to be selective ligands for the N-methyl-D-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively.     

After-hyperpolarizations produced in frog motoneurons by excitatory amino acid analogues

After-hyperpolarizations (AHPs) produced in frog motoneurons by applications of the excitatory amino acid analogues quisqualate (QUIS), N-methyl-D-aspartate (NMDA), and kainate (KA) were studied in the isolated hemisected frog spinal cord using sucrose gap techniques. AHPs were present following 98% of QUIS-induced depolarizations, but were seen in only 35% and 15% of NMDA- and KA-evoked responses respectively. AHPs produced by QUIS are produced both by direct effects of QUIS on motoneuron membranes and by indirect effects mediated through a synaptic process involving interneurons. Thus, application of Mg2+, Mn2+, or tetrodotoxin (TTX) in concentrations sufficient to block synaptic transmission and interneuronal firing, reduced, but did not abolish the AHPs produced by QUIS. In contrast, NMDA- and KA-AHPs appear to be entirely mediated by indirect means as block of synaptic transmission and interneuronal firing eliminated AHPs produced by these substances. Exposure of the cord to Mn2+ after addition of TTX did not affect the size of QUIS-AHPs. In the presence of TTX, QUIS-AHPs were reduced or completely blocked by addition of dinitrophenol (DNP) and sodium cyanide, by dihydro-ouabain, by removal of K+ from the superfusate, by cooling, and by replacement of 50% of the external Na+ with Li+. The results suggest that the QUIS-AHPs are largely the result of the direct effect of the excitatory amino acid agonist on motoneuron membranes and is caused by activation of an electrogenic Na+ pump. AHPs following depolarizations evoked by NMDA and KA are presumably the result of indirect actions of these latter analogues on interneurons.     

NMDA-induced increase in [Ca2+]i and 45Ca2+ uptake in acutely dissociated brain cells derived from adult rats.

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-D-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca2+]i measured with fluo3 and 45Ca2+ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 microM) and N-methyl-DL-aspartate (200 microM) increased [Ca2+]i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated 45Ca2+ uptake about 16-10% in the same regions. The increases in [Ca2+]i and 45Ca2+ uptake were inhibited by 40% in the presence of 1 mM MgCl2 and by 90-50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca2+]i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca2+]i increase in adult age.     

Delayed calcium dysregulation in neurons requires both the NMDA receptor and the reverse Na+/Ca2+ exchanger

Glutamate-induced delayed calcium dysregulation (DCD) is a causal factor leading to neuronal death. The mechanism of DCD is not clear but Ca2+ influx via N-methyl-d-aspartate receptors (NMDAR) and/or the reverse plasmalemmal Na+/Ca2+ exchanger (NCXrev) could be involved in DCD. However, the extent to which NMDAR and NCX(rev) contribute to glutamate-induced DCD is uncertain. Here, we show that both NMDAR and NCX(rev) are critical for DCD in neurons exposed to excitotoxic glutamate. In rat cultured hippocampal neurons, 25 μM glutamate produced DCD accompanied by sustained increase in cytosolic Na+ ([Na+]c) and plasma membrane depolarization. MK801 and memantine, noncompetitive NMDAR inhibitors, added shortly after glutamate, completely prevented DCD whereas AP-5, a competitive NMDAR inhibitor, failed to protect against DCD. None of the tested inhibitors lowered elevated [Na+]c or restored plasma membrane potential. In the experiments with NCX reversal by gramicidin, MK801 and memantine robustly inhibited NCXrev while AP-5 was much less efficacious. In electrophysiological patch-clamp experiments MK801 and memantine inhibited NCXrev-mediated ion currents whereas AP-5 failed. Thus, MK801 and memantine, in addition to NMDAR, inhibited NCXrev. Inhibition of NCXrev either with KB-R7943, or by collapsing Na+ gradient across the plasma membrane, or by inhibiting Na+/H+ exchanger with 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and thus preventing the increase in [Na+]c failed to preclude DCD. However, NCXrev inhibition combined with NMDAR blockade by AP-5 completely prevented DCD. Overall, our data suggest that both NMDAR and NCXrev are essential for DCD in glutamate-exposed neurons and inhibition of individual mechanism is not sufficient to prevent calcium dysregulation.     

Glutamatergic Induction of CREB Phosphorylation and Fos Expression in Primary Cultures of the Suprachiasmatic Hypothalamus In Vitro is Mediated by Co-Ordinate Activity of NMDA and Non-NMDA Receptors

Exposure of Syrian hamsters to light 1 h after lights-off rapidly (10 min) induced nuclear immunoreactivity (–ir) to the phospho-Ser¹³³ form of the Ca²⁺/cAMP response element (CRE) binding protein (pCREB) in the retinorecipient zone of the suprachiasmatic nuclei (SCN). Light also induced nuclear Fos-ir in the same region of the SCN after 1 h. The glutamatergic N-methyl- d -aspartate (NMDA) receptor blocker MK801 attenuated the photic induction of both factors. To investigate glutamatergic regulation of pCREB and Fos further, tissue blocks and primary cultures of neonatal hamster SCN were examined by Western blotting and immunocytochemistry in vitro. On Western blots of SCN tissue, the pCREB-ir signal at 45 kDa was enhanced by glutamate or a mixture of glutamatergic agonists (NMDA, amino-methyl proprionic acid (AMPA), and Kainate (KA)), whereas total CREB did not change. Glutamate or the mixture of agonists also induced a 56 kDa band identified as Fos protein in SCN tissue. In dissociated cultures of SCN, glutamate caused a rapid (15 min) induction of nuclear pCREB-ir and Fos-ir (after 60 min) exclusively in neurones, both GABA-ir and others. Treatment with NMDA alone had no effect on pCREB-ir. AMPA alone caused a slight increase in pCREB-ir. However, kainate alone or in combination with NMDA and AMPA induced nuclear pCREB-ir equal to that induced by glutamate. The effects of glutamate on pCREB-ir and Fos-ir were blocked by antagonists of both NMDA (MK801) and AMPA/KA (NBQX) receptors. In the absence of extracellular Mg²⁺, MK801 blocked glutamatergic induction of Fos-ir. However, the AMPA/KA receptor antagonist was no longer effective at blocking glutamatergic induction of either Fos-ir or pCREB-ir, consistent with the model that glutamate regulates gene expression in the SCN by a co-ordinate action through both NMDA and AMPA/KA receptors. Glutamatergic induction of nuclear pCREB-ir in GABA-ir neurones was blocked by KN-62 an inhibitor of Ca²⁺/Calmodulin (CaM)-dependent kinases, implicating Ca²⁺-dependent signalling pathways in the glutamatergic regulation of gene expression in the SCN.     

Mechanism of the kainate-induced intracellular acidification in leech Retzius neurons

We examined the effect of the glutamatergic agonist kainate on the membrane potential, the intracellular Na+ concentration ([Na+]i), the intracellular-free Ca2+ concentration, and on the intracellular pH of Retzius neurons of the medicinal leech, Hirudo medicinalis, in order to investigate the mechanism responsible for the intracellular acidification caused by glutamatergic stimulation. The recordings were made with Na+- and pH-sensitive microelectrodes and iontophoretically injected Fura-2. Bath application of kainate evoked a marked membrane depolarization, a [Na+]i increase, and an intracellular acidification. The intracellular acidification was unaffected by reversal of the electromotive force for H+, suggesting that an influx of H+ from the interstitial space does not contribute to the acidification. While the Ca2+ channel blockers La3+ and Co2+ had no effect on the kainate-induced intracellular acidification, suggesting that a Ca2+ influx via voltage-dependent Ca2+ channels was not relevant, the acidification was reduced in Ca2+-free saline solution. In Na+-free saline solution the kainate-induced intracellular acidification was absent, suggesting the involvement of Na+ influx in generating the acidification. When injected iontophoretically Na+ induced an intracellular acidification but Li+, K+, Rb+ or Cs+ did not. Furthermore, a [Na+]i increase induced by blocking the Na+/K+ pump also led to an intracellular acidification. We conclude that the [Na+]i increase is the crucial event underlying the kainate-induced intracellular acidification. Possible mechanisms linking the [Na+]i increase to the intracellular acidification are discussed.