Ethanol inhibition of NMDA mediated depolarizations is increased in the presence of Mg

The inhibitory potency of ethanol upon excitatory amino acid induced depolarizations of rat hippocampal CA1 pyramidal cells was assessed in the presence and absence of magnesium (Mg²⁺) using the grease-gap technique. Ethanol shifted theN-methyl-d-aspartate (NMDA) dose-response curves to the right in a non-parallel manner. In the presence of Mg²⁺, ethanol appeared to be a more effective NMDA antagonist (IC₅₀ 47 mM) than in the absence of Mg²⁺ (IC₅₀ 107 mM). The IC₅₀ for ethanol upon non-NMDA mediated CA1 pyramidal cell depolarizations was in excess of 170 mM. These results strongly suggest a preferential inhibitory action of ethanol against NMDA, rather than non-NMDA, mediated responses. Experiments in which ethanol and Mg²⁺ were covaried indicated that these substances act by two distinct mechanisms to antagonize the action of NMDA. These effects of ethanol, at concentrations which elicit intoxication(< 50mM) but not anesthesia, suggest that the NMDA receptor complex may play an important role in the acute effects of ethanol.     

Trigeminal inputs to reticulospinal neurones in lampreys are mediated by excitatory and inhibitory amino acids

Reticulospinal (RS) neurones integrate sensory inputs from several modalities to generate appropriate motor commands for maintaining body orientation and initiation of locomotion in lampreys. As in other vertebrates, trigeminal afferents convey sensory inputs from the head region. The in vitro brainstem/spinal cord preparation of the lamprey was used for characterizing trigeminal inputs to RS neurones as well as the transmitter systems involved. The trigeminal nerve on each side was electrically stimulated and synaptic responses, which consisted of mixed excitation and inhibition, were recorded intracellularly in the middle and posterior rhombencephalic reticular nuclei. The EPSPs were mediated by activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors. An increase in the late phase of the excitatory response occurred when Mg²⁺ ions were removed from the Ringer's solution. This effect was antagonized by 2-amino-5-phosphonopentanoate (2-AP5) or reversed by restoring Mg²⁺ ions to the perfusate suggesting the activation of N-methyl-d-aspartate (NMDA) receptors. IPSPs were mediated by glycine. These findings are similar to those reported for other types of sensory inputs conveyed to RS neurones, where excitatory and inhibitory amino acid transmission is also involved.     

Evidence forN-methyl-d-aspartic acid receptor-mediated modulation of the commissural input to central vestibular neurons of the frog

We have investigated the role ofN-methyl-d-asparte (NMDA) receptors in the excitatory synaptic transmission to central vestibular neurons in the isolated superfused brainstem of the frog. In superfusate containing 1 mM Mg²⁺ field potentials in the vestibular nuclei evoked by electrical stimulation of either the ipsi- or the contralateral VIIIth nerve were not affected by bath-appliedd-2-amino-5-phosphonovaleric acid (D-APV, 25–50 μM), a selective NMDA antagonist. In a low Mg²⁺ solution postsynaptic field potential components were larger than control but still unaffected by D-APV. Ipsi- and contralaterally evoked excitatory postsynaptic potentials (EPSPs) differed in their shape parameters as well as their pharmacological sensitivity. Ipsilaterally evoked EPSPs were not affected by D-APV and had a rise time that was faster than that of contralaterally evoked EPSPs. The peak amplitude of the latter was reduced by D-APV (25–50 μM) to about 65% of the control value in the presence of 1 mM Mg²⁺. During bath application of NMDA (100 μM) an increased input resistance and repetitive de- and hyperpolarizing membrane potential shifts were observed. Similar events were observed during a reduction of the Mg²⁺ concentration. Bath application of NMDA (0.1–1 μM) resulted in an enhanced size of the recorded EPSPs. Dendritic and somatic EPSPs were stimulated on a computer with the assumption of a constant NMDA receptor activation and a pulse-like non-NMDA receptor activation. The results of these stimulations are consistent with the hypothesis that the efficacy of non-NMDA-mediated vestibular commissural synaptic transmission is modulated through tonically activated NMDA receptors.     

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.     

Potentiation of excitatory postsynaptic potentials by a metabotropic glutamate receptor agonist (1S,3R-ACPD) in frog spinal motoneurons

We conducted intracellular recordings of lumbar motoneurons in the arterially-perfused frog spinal cord and investigated the effects of a metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), on excitatory postsynaptic potentials evoked by stimulation of the descending lateral column fibers (LC-EPSPs). In the absence of Mg²⁺, ACPD reversibly potentiated the amplitude of monosynaptic LC-EPSPs by more than 15% in 15 of 19 cells with 5 μM ACPD and in 7 of 12 cells with 0.5 μM ACPD. The EPSP amplitudes with 5 and 0.5 μM ACPD were 142±10% (mean±S.E.M., n=19) and 130±13% (n=12) of the controls. The potentiation was seen without a decrease in the input conductance. Glutamate-induced depolarizations in the absence and the presence of 0.5 μM ACPD were not significantly different in cells perfused with the low Ca²⁺-high Mg²⁺ solution which eliminated chemical transmission. Paired pulse facilitation of LC-EPSPs was reversibly decreased in association with the potentiation. ACPD-induced potentiation of monosynaptic LC-EPSPs was seen in 5 of 6 cells in the presence ofd-(−)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist. ACPD occasionally activated polysynaptic components of LC-EPSPs which were mediated mainly via NMDA receptors. On the other hand, ACPD-induced potentiation of EPSPs was inhibited by extracellular Mg²⁺. Five μM ACPD potentiated monosynaptic EPSPs in 4 of 6 cells with 1 mM Mg²⁺ in the solution and in 2 of 17 cells with 4 mM Mg²⁺, and the EPSP amplitude was 123±9% (n=6) and 98±3% (n=17) of those before application of ACPD, respectively. These results suggest that activation of metabotropic glutamate receptors potentiates LC-EPSPs via mechanisms sensitive to Mg²⁺ and may work as a positive feedback mechanism at the excitatory amino acid-mediated synapses between the descending fibers and lumbar spinal motoneurons.     

Voltage Oscillations in Xenopus Spinal Cord Neurons: Developmental Onset and Dependence on Co-activation of NMDA and 5HT Receptors

The development of intrinsic, N-methyl-D-aspartate (NMDA) receptor-mediated voltage oscillations and their dependence on co-activation of 5-hydroxytryptamine (5HT) receptors was explored in motor neurons of late embryonic and early larval Xenopus laevis. Under tetrodotoxin, 100 μM NMDA elicited a membrane depolarization of around 20 mV, but did not lead to voltage oscillations. However, following the addition of 2–5 μM 5HT, oscillations were observed in 12% of embryonic and 70% of larval motor neurons. The voltage oscillations depended upon co-activation of NMDA and 5HT receptors since they were curtailed by selectively blocking NMDA receptors with D-2-amino-5-phosphonovaleric acid (APV) or by excluding Mg²⁺ from the experimental saline. 5HT applied in the absence of NMDA also failed to elicit oscillations. Oscillations could be induced by the non-selective 5HT_1a receptor agonist, 5-carboxamidotryptamine (5CT) and both 5HT- and 5CT-induced oscillations were abolished by pindobind-5HT₁, a selective 5HT_1a receptor antagonist. To test whether 5HT enables voltage oscillations by modulating the voltage-dependent block of NMDA channels by Mg²⁺, membrane conductance was monitored under tetrodotoxin. Although 5HT caused membrane hyperpolarization of 4–8 mV, there was little detectable change in conductance. NMDA application caused an approximate 20 mV depolarization and an ‘apparent’ decrease in conductance, presumably due to the conductance pulse bringing the membrane into a voltage region where Mg²⁺ blocks the NMDA ionophore. 5HT further decreased conductance, which we propose is due to its enhancement of the voltage-dependent Mg²⁺ block. When the membrane potential was depolarized by ～20 mV via depolarizing current injection (to mimic the NMDA-induced depolarization), 5HT increased rather than decreased membrane conductance. Furthermore, 5HT did not affect the increase in membrane conductance following NMDA applications in zero Mg²⁺ saline. The results suggest that intrinsic, NMDA receptor-mediated voltage oscillations develop in a brief period after hatching, and that they depend upon the co-activation of 5HT and NMDA receptors. The enabling function of 5HT may involve the facilitation of the voltage-dependent block of the NMDA ionophore by Mg²⁺ through activation of receptors with 5HT_1a-like pharmacology.     

Functional and Morphological Changes Induced by Transient in Vivo Ischemia

Brief transient ischemia causes a delayed neuronal death of pyramidal neurons in the CA1 area of hippocampus after a period of hyperexcitability. We have previously shown that the hyperexcitability is due to an increase in an N -methyl- -aspartate (NMDA) component of the response. In the present study, we recorded intracellularly from pyramidal neurons in CA1 and find that there is little change in membrane potential or input resistance at this point in time. The dramatic increase in the NMDA component of the synaptic response is a result of a significant reduction in the ability of Mg²⁺ to induce a normal voltage-dependent blockade of the response. In spite of the relatively normal membrane properties, there is at this time a significant reduction in the amplitude of the population excitatory potential and a near total loss of long-term potentiation. In contrast, post-tetanic potentiation is unchanged in magnitude and character. These observations suggest more severe damage to the neuron than indicated by the membrane potential and resistance. When single neurons were injected with horseradish peroxidase and visualized after the electrophysiological recording, we found extensive beading of the dendrites in both the apical and basal regions, presumably reflecting a disproportionate damage to the dendritic areas, which are the primary sites of the excitatory amino acid synapses onto the neuron. These observations are consistent with the hypothesis that transient ischemia causes a fundamental change in the NMDA-activated ion channel such that Mg²⁺ is no longer able to block the response, resulting in increased entry of calcium into synaptic regions, which causes dendritic damage that progresses to neuronal cell death.     

Intracellular calcium elevation during plateau potentials mediated by extrasynaptic NMDA receptor activation in rat hippocampal CA1 pyramidal neurons is primarily due to calcium entry through voltage‐gated calcium channels

We reported previously that plateau potentials mediated by extrasynaptic N‐methyl‐d ‐aspartate receptors (NMDARs) can be induced either by synaptic stimulation in the presence of glutamate transporter antagonist or by iontophoresis of NMDA in rat hippocampal CA1 pyramidal neurons. To examine whether the plateau potentials are accompanied by an elevation of intracellular Ca²⁺ and to determine the source of Ca²⁺ elevation, we performed Ca²⁺ imaging during the plateau potential. Neurons were loaded with Ca²⁺ indicator fluo‐4, and the plateau potentials were generated either synaptically in the presence of glutamate transporter antagonist or by iontophoretically applying NMDA. We have found that a transient elevation in intracellular Ca²⁺ accompanies the plateau potential. The synaptically induced plateau potential and the Ca²⁺ elevation were blocked by 5,7‐dichlorokynurenic acid (5,7‐dCK), an antagonist for the glycine‐binding sites of NMDAR. A mixture of Cd²⁺ and tetrodotoxin did not block NMDA‐induced plateau potentials, but completely abolished the accompanying Ca²⁺ elevation in both the presence and absence of Mg²⁺ ions in the bathing solution. The NMDA‐induced plateau potential was blocked by further adding 5,7‐dCK. Our results show that the NMDAR‐mediated plateau potential is accompanied by elevation of intracellular Ca²⁺ that is primarily caused by the influx of Ca²⁺ through voltage‐gated Ca²⁺ channels.     

A presynaptic N‐methyl‐d‐aspartate autoreceptor in rat hippocampus modulating amino acid release from a cytoplasmic pool

A possible role of the N-methyl-d -aspartate receptor (NMDA-R) as a presynaptic autoreceptor was investigated using Percoll-purified hippocampus nerve terminals (synaptosomes). This preparation contained only a neglectable amount of postsynaptic structures. Two main effects of NMDA were observed. First, NMDA dose-dependently (10–100 μm ) and in the absence of Mg²⁺, stimulated basal release of aspartate and glutamate, but not of GABA. MK801 (10 μm ), an open NMDA-R-channel blocker, reduced this effect even below control levels, indicating endogenous NMDA-R activation. By superfusing synaptosomes, which prevents a tonic receptor occupation, also basal GABA release was stimulated by NMDA. The NMDA-induced potentiation of amino acid superfusate levels was blocked both by MK801 and Mg²⁺ (1 m m ), was slow in onset and returned to baseline after NMDA-removal. The NMDA-effect was also found in the absence of extracellular Ca²⁺, suggesting that amino acids were released from a non-vesicular (cytoplasmic) pool. Secondly, in KCl-depolarized synaptosomes exposed to 1 m m Mg²⁺, NMDA did not affect the release of the amino acids. MK801, however, reduced the KCl-evoked Ca²⁺-independent release of aspartate and glutamate, but not of GABA. l -trans-PDC, the selective inhibitor of the glutamate/aspartate transporter, prevented this MK801-effect, suggesting a coupling between NMDA-Rs and these transporters. These data provide evidence for a presynaptic NMDA autoreceptor in rat hippocampus. We speculate on the role of this NMDA-R to depolarize the presynaptic membrane by Na⁺-entry, which may induce reversal of amino acid transporters and thereby releasing amino acids from a cytoplasmic pool.     

Supraoptic Oxytocin and Vasopressin Neurones Show Differential Sensitivity to the Neurosteroid Pregnenolone Sulphate

The neurosteroid pregnenolone sulphate (PS) interacts allosterically with ionotropic glutamate receptors and thereby could be an important modulator of activity within the hypothalamic magnocellular nuclei. The present in-vitro study therefore examined the effect of perifusion of PS (100 μM) on activity of supraoptic oxytocin (OT) and vasopressin (VP) neurones, in which firing was stimulated by local application of glutamate, NMDA or AMPA. In the presence of locally applied glutamate, PS significantly potentiated firing in putative VP neurones, but had little effect on putative OT neurones. In both cell types, PS increased firing in the presence of NMDA and depressed firing in the presence of AMPA. The action of PS on glutamate- and NMDA-stimulated firing was unaffected by addition of the GABA_A receptor antagonist, picrotoxin (50 μM). The suppressive action of PS on AMPA-stimulated firing was, however, reversed by picrotoxin and therefore probably requires intact GABAergic transmission for its expression. When putative VP neurones were stimulated by local application of K⁺, in the presence of picrotoxin, PS evoked a small increase in the ongoing activity, although this did not reach significance. When the glutamate receptor antagonists, NBQX (20 μM) and AP5 (40 μM), were included in the medium, no change in K⁺–stimulated firing was observed. Hence PS has no effect on activity of putative VP neurones in the absence of exogenous and endogenous glutamate excitation. In conclusion, PS selectively potentiates glutamate-stimulated activity in putative VP neurones, probably via NMDA receptors, thus providing a mechanism whereby this neurosteroid might exert rapid non-genomic effects on VP secretion. The lack of effect of PS in putative OT neurones probably relates to the relatively small involvement of NMDA receptors in mediating glutamate excitation in this cell type.     

Crocin antagonizes ethanol inhibition of NMDA receptor-mediated responses in rat hippocampal neurons

We have previously found that crocin (crocetin di-gentiobiose ester) antagonizes the inhibitory effect of ethanol on long-term potentiation in the rat hippocampus in vivo and in vitro. To explore mechanisms underlying the antagonism of crocin against ethanol, we investigated the effects of ethanol and crocin on synaptic potentials mediated by N-methyl-d-aspartate (NMDA) receptors in the dentate gyrus of rat hippocampal slices. Synaptic potential mediated by non-NMDA receptors was recorded in normal medium (1.3 mM Mg²⁺), while NMDA receptor-mediated synaptic potential was isolated in low (0.13 mM) Mg²⁺ medium containing the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 μM). Crocin (10 μM) alone did not affect synaptic potentials mediated by non-NMDA nor NMDA receptors. Non-NMDA response was slightly inhibited by 100 mM ethanol, while NMDA response was selectively inhibited by lower concentrations (10–50 mM) of ethanol. Crocin (10 μM) did not affect the inhibition of non-NMDA response by 100 mM ethanol, but significantly blocked the inhibition of NMDA response by 10–50 mM ethanol. In addition, we performed whole-cell patch recording with primary cultured rat hippocampal neurons, and confirmed that crocin blocked ethanol inhibition of inward currents evoked by application of NMDA. These results suggest that crocin specifically antagonizes the inhibitory effect of ethanol on NMDA receptor-mediated responses in hippocampal neurons.     

Cytotoxic actions and effects on intracellular Ca2+ and cGMP concentrations of sulphur-containing excitatory amino acids in cultured cerebral cortical neurons

Effects of the sulphur-containing acidic amino acids (SAAs) cysteic acid (CA), homocysteic acid (HCA), cysteine sulphinic acid (CSA), homocysteine sulphinic acid (HCSA), and S-sulphocysteine (SC) on intracellular concentrations of Ca²⁺ ([Ca²⁺]_i) and cGMP ([cGMP]_i) as well as their cytotoxic actions were investigated in cultured cerebral cortical neurons. The glutamate receptor subtype selective antagonists APV (D-(?)-2-amino-5-phosphonopentanoate) acting on N-methyl-D-aspartate (NMDA) receptors and DNQX (6,7-dinitroquinoxaline-2,3-dione) acting on non-NMDA receptors were employed to obtain information about the involvement of glutamate receptor subtypes in these actions of the SAAs. It was found that all SAAs exerted a cytotoxic action on the neurons. The ED₅₀ values for CSA, CA, HCSA, and HCA were around 30 to 50 μM and that for SC was about 150 μM. The glutamate transport blocker L-aspartate-β-hydroxamate increased the efficacy of CSA and CA but had no effect on the cytotoxic actions of the remaining SAAs. In case of CA, HCA, and SC the cytotoxicity could be prevented by APV alone and for HCSA, DNQX could block the toxic action. DNQX reduced the toxicity of HCA somewhat but the presence of APV was required for complete protection. CSA toxicity could only be blocked by the combination of APV and DNQX. All SAAs induced an increase in [cGMP]_i and [Ca²⁺]_i and with regard to [Ca²⁺]_i SC was the most potent and CA the least potent SAA. The effect of all SAAs on [cGMP]_i could be blocked by APV alone whereas DNQX had no effect except in the case of HCSA where the response was blocked completely and HCA where the response was inhibited by 75%. The SAA-induced increase in [Ca²⁺]_i could in all cases be significantly reduced by 0.6 mM Mg²⁺ and in the presence of Mg²⁺, APV dose dependently blocked the remaining SAA induced increase in [Ca²⁺]_i completely. Under these conditions DNQX was also found to block the SAA-induced increase in [Ca²⁺]_i dose dependently. In the absence of Mg²⁺, DNQX (25 μM) inhibited the response of the SAAs only by 65–75%. Under these conditions all SAA responses except that to SC could be fully antagonized by 300 μM APV. The SC-induced increase in [Ca²⁺]_i was inhibited by 60% by APV. The results show that no simple correlation exists between SAA-induced cytotoxicity and their ability to increase intracellular levels of Ca²⁺ and cGMP. However, when both NMDA and non-NMDA receptors were antagonized no toxicity or changes in calcium or cGMP were observed. © 1993 Wiley-Liss, Inc.     

Protective effect of MgSO4 infusion on NMDA receptor binding characteristics during cerebral cortical hypoxia in the newborn piglet

This study tests the hypothesis that magnesium, a selective non-competitive antagonist of the NMDA receptor, will attenuate hypoxia-induced alteration in NMDA receptors and preserve MK-801 binding characteristics during cerebral hypoxia in vivo. Anesthetized, ventilated and instrumented newborn piglets were divided into three groups: normoxic controls were compared to untreated hypoxic and Mg²⁺-treated hypoxic piglets. Cerebral hypoxia was induced by lowering the FiO₂ to 5–7% and confirmed biochemically by a decrease in the levels of phosphocreatine (82% lower than control). The Mg²⁺-treated group received MgSO₄ 600 mg/kg over 30 min followed by 300 mg/kg administered during 60 min of hypoxia. Plasma Mg²⁺ concentrations increased from1.6 ± 0.1mg/dl to17.7 ± 3.3mg/dl.³H-MK-801 binding was used as an index of NMDA receptor modification. TheB_max in control, hypoxic and Mg²⁺-treated hypoxic piglets was1.09 ± 0.17, 0.70 ± 0.25and0.96 ± 0.14pmoles/mg protein, respectively. TheK_d for the same groups were10.02 ± 2.04, 4.88 ± 1.43and8.71 ± 2.23nM, respectively. TheB_max andK_d in the hypoxic group were significantly lower compared to the control and Mg²⁺-treated hypoxic groups, indicating a preservation of NMDA receptor number and affinity for MK-801 during hypoxia with Mg²⁺. The activity of Na⁺, K⁺ ATPase, a marker of neuronal membrane function, was lower in the hypoxic group compared to the control and Mg²⁺-treated hypoxic groups. These findings show that MgSO₄ prevents the hypoxia-induced modification of the NMDA receptor and attenuates neuronal membrane dysfunction. We suggest that the administration of Mg²⁺ prior to and during hypoxia may be neuroprotective in vivo, possibly by reducing the NMDA receptor-mediated influx of calcium.     

Electrophysiological studies of NMDA receptors

Recent electrophysiological studies of NMDA receptors and of the associated ion channels (NMDA channels) have revealed five properties of this system: (1) the NMDA channels are blocked by Mg²⁺ in a voltage-dependent way; (2) the NMDA channels are permeable to Ca²⁺ as well as to Na⁺ and K⁺; (3) the NMDA channels may adopt multiple conductance states; some of the minor states (small conductances) resemble the major conductance states opened by non-NMDA agonists; (4) continued exposure to NMDA agonists produces short-term and long-term decreases in the sensitivity of the NMDA system; and (5) glycine potentiates the response to NMDA.     

Stimulation of the N-methyl-d-aspartate receptor promotes the biochemical differentiation of cerebellar granule neurons and not astrocytes

Cerebellar granule cells are believed to be glutamatergic, but, as they receive excitatory amino acidergic input from mossy fibers, they also possess N-methyl-d-aspartate (NMDA) receptors. The possible involvement of these NMDA receptors in the biochemical differentiation of cultured granule neurons was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 3 days (i.e. between 2 and 5 days in vitro), it elevated specific activity of glutaminase in the dose- and time-dependent manners. The half-maximal effect was obtained at about 10 μM NMDA, whereas the maximum concentration, which produced about a 2.7-fold increase in 5-day-old cultures, was about 50 μM NMDA. This increase in glutaminase was completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and by the NMDA receptor-linked Ca²⁺ ion channel blockers, MK 801 and Mg²⁺. The effect of NMDA was not related to the survival of the granule cells, as the experiments were carried out before the dependence on high K⁺ for the survival of granule cells develops in culture, and during the period of investigation none of the compounds used compromised the survival of these cells. The enhancement of glutaminase activity was due to an induction in enzyme protein, since it was completely blocked by cycloheximide and actinomycin D. In contrast to granule neurons, the treatment with NMDA had no significant effect on the activity of glutaminase and glutamine synthetase in cultured cerebellar astroglial cells. Our present results on glutaminase enzyme would indicate that an increase in the cellular concentration of free Ca²⁺ mediated through the NMDA induced increase in Ca²⁺ conductance, leads to long term changes in differentiating cerebellar granule neurons, and it is possible that this kind of physiological stimulation of granule cells is normally provided in vivo by the presynaptic glutamatergic mossy fibers.     

Modulatory effects of aluminum, calcium, lithium, magnesium, and zinc ions on [ H]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.     

Ca2+ influx into leech glial cells and neurones caused by pharmacologically distinct glutamate receptors

The effect of glutamatergic agonists on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of neuropile glial cells and Retzius neurones in intact segmental ganglia of the medicinal leech Hirudo medicinalis was investigated by using iontophoretically injected fura-2. In physiological Ringer solution the [Ca²⁺]_i levels of both cell types were almost the ssame (glial cells: 58 ± 30 nM, n = 51; Retzius neurones: 61 ± 27 nM, n = 64). In both cell types glutamate, kainate, and quisqualate induced an increase in [Ca²⁺]_i which was inhibited by 6,7-dinitroquinoxaline-2,3-dione (DNQX). This increase was caused by a Ca²⁺ influx from the extracellular space because the response was greatly diminished upon removal of extracellular Ca²⁺. The glutamate receptors of neuropile glial cells and Retzius neurones differed with respect to the relative effectiveness of the agonists used, as well as with regard to the inhibitory strenght of DNQX. In Retzius neurones the agonist-induced [Ca²⁺]_i increase was abolished after replacing extracellular Na⁺ by organic cations or by mM amounts of Ni²⁺, whereas in glial cells the [Ca²⁺]_i increase was largely preserved under both conditions. It is concluded that in Retzius neurones the Ca²⁺ influx is predominantly mediated by voltage-dependent Ca²⁺ channels, whereas in neuropile glial cells the major influx occurs via the ion channels that are associated with the glutamate receptors.     

Suppression of long-term potentiation in hippocampal slices by copper

Cu²⁺-ions are known to interfere with γ-aminobutyric acid (GABA)- and glutamate-operated ion channels from experiments with isolated neurons. Such actions are likely involved in the pathophysiology of Wilson's disease. We have now studied the effects of Cu²⁺ in the CA 1 region of hippocampal slices. Field excitatory postsynaptic potential (EPSP) slopes in the CA1 region were unaffected by 1 μM Cu²⁺ but were depressed by 10 μM (to 85%) and 100 μM (to 50%). A paired-pulse test revealed no difference in facilitation in the presence or absence of Cu²⁺, indicating a postsynaptic action. A late component of intracellularly registered EPSPs in Mg²⁺-free solution was also reduced by Cu²⁺. The N-methyl-D-aspartate (NMDA) component of the field EPSP, isolated by adding CNQX and bicuculline in Mg²⁺-free solution, was reduced to 69% of control by 1 μM and to 50% of control by 10 μM Cu²⁺. Long-term potentiation, evoked by 3 × 50 pulses at 100 Hz, 20 s interval amounted to 132 ± 11% 90 min after tetanization under control conditions but was absent in the presence of 1 μM Cu²⁺ in the bath. Thus low concentrations of copper can selectively reduce NMDA-mediated potentials and synaptic plasticity. Hippocampus 1997;7:666–669. © 1997 Wiley-Liss, Inc.     

Depolarization triggers intracellular magnesium surge in cultured dorsal root ganglion neurons

Intracellular magnesium concentration ([Mg²⁺]_i) of cultured dorsal root ganglion (DRG) neurons was measured using the magnesium indicator Mag-Fura-2/AM. [Mg²⁺]_i was 0.48±0.08 mM (mean±SEM, n=23) at rest, and it increased 3-fold by depolarization with a 60-mM K⁺ solution. The [Mg²⁺]_i increase was observed in the absence of extracellular Mg²⁺, but the increase disappeared in the absence of extracellular Ca²⁺. 50 μM cadmium or 100 μM verapamil, a Ca²⁺ channel blocker, also diminished the rise of [Mg²⁺]_i. The additional measurement of an intracellular Ca²⁺ concentration ([Ca²⁺]_i) indicated that the [Mg²⁺]_i rise requires a threshold concentration of [Ca²⁺]_i to be reached; above 60 nM. The present results indicate that depolarization induces a Ca²⁺-influx through voltage dependent Ca channels and this causes the release of Mg²⁺ from intracellular stores into the cytoplasm.     

Magnesium ion augmentation of inhibitory effects of adenosine on dopamine release in the rat striatum

Abstract The effects of adenosine and magnesium ion (Mg²⁺) on striatal dopamine release were studied in awake rats by in vivo microdialysis. The mean striatal basal levels of dopamine release at Mg²⁺ free perfusate were 56.95 ± 5.30 fmol/sample (for 20 min). By varying the Mg²⁺ levels in perfusate from 0 mmol/L to 1, 10 or 40 mmol/L, the dopamine release was inhibited by Mg²⁺ in a level-dependent manner. Perfusion with modified Ringer's solution containing zero Mg²⁺ and from 5 to 50 μmol/L adenosine, non-selective adenosine agonist, as well as 0.1 μmol/L 2-chloro-N⁶-cyclopentyladenosine (CCPA), selective adenosine Al agonist, showed no effect on dopamine release. However, from 5 to 50 μmol/L adenosine and from 0.1 to 1 μmol/L CCPA plus Mg²⁺ (1 and 40 μmol/L) perfusion decreased the dopamine release. This inhibitory effect of adenosine and CCPA on striatal dopamine release was enhanced by an increase in extracellular Mg²⁺ levels. Levels of 50 μmol/L of 8-cyclopentyl-l,3-dimethylxanthine (CPT), a selective adenosine Al receptor antagonist, in perfusate increased the dopamine release under conditions both with and without Mg²⁺. This stimulatory effect of CPT on striatal dopamine release was reduced by an increase in extracellular Mg²⁺ levels. As a result, CPT antagonized the inhibitory effects of adenosine and CCPA on dopamine release under conditions of the presence and absence of Mg²⁺. These results suggest that the inhibition of striatal dopamine release by adenosine was mediated by adenosine Al receptor. This inhibition was intensified by Mg²⁺. This study also revealed that the concentrations of Mg²⁺, which ranged from physiological to supraphysiological, reduced the striatal dopamine release; furthermore it was found that the physiological concentration of Mg²⁺ potentiated the effects of adenosine agonists, but inhibited adenosine antagonist. Thus, the present study, using in vivo microdialysis preparations, suggests Mg²⁺ inhibits the calcium ion channels and enhances the adenosinergic function in the central nervous system.