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.     

Glutamate stimulation of tyrosine hydroxylase is mediated by NMDA receptors in the rat striatum.

We have studied the action of glutamate on striatal tyrosine hydroxylase activity and determined which type of glutamate receptors are involved. Glutamate stimulated (EC50 = 4 +/- 2 microM) the activity of tyrosine hydroxylase in slices of rat neostriatum. The selective N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (10 microM) blocked the stimulation; however, both the non-NMDA receptor antagonist glutamate diethyl ester (10 microM) and the general excitatory amino acid antagonist kynurenate (10 microM) had no effect. NMDA was even more potent than glutamate in stimulating tyrosine hydroxylase activity. Quisqualate (100 microM) only slightly stimulated the enzyme, and kainate had practically no effect. Omission of Mg2+ from the incubation medium potentiated the glutamate stimulation. Neither tetrodotoxin nor atropine prevented the stimulation. These results suggest that glutamate stimulates striatal tyrosine hydroxylase activity via NMDA receptors. The lack of effect of tetrodotoxin and atropine suggests that glutamate acts on NMDA receptors located on the dopaminergic nigrostriatal terminal. The stimulation may involve the entry of Ca2+ into the terminal through the NMDA receptor ionophore, since a Ca(2+)-free medium or cadmium totally blocked the stimulation of the enzyme by glutamate.     

N-methyl-D-aspartate enhancement of the glycine response in the rat sacral dorsal commissural neurons

The effect of N-methyl-D-aspartate (NMDA) on the glycine (Gly) response was examined in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. The application of 100 microM NMDA to SDCN neurons reversibly potentiated Gly-activated Cl- currents (IGly) without affecting the Gly binding affinity and the reversal potential of IGly. A selective NMDA receptor antagonist, APV (100 microM), blocked the NMDA-induced potentiation of IGly, whereas 50 microM CNQX, a non-NMDA receptor antagonist, did not. The potentiation effect was reduced when NMDA was applied in a Ca2+-free extracellular solution or in the presence of BAPTA AM, and was independent of the activation of voltage-dependent Ca2+ channels. Pretreatment with KN-62, a selective Ca2+-calmodulin-dependent protein kinase II (CaMKII) inhibitor, abolished the NMDA action. Inhibition of calcineurin (CaN) further enhanced the NMDA-induced potentiation of IGly. In addition, the GABAA receptor-mediated currents were suppressed by NMDA receptor activation in the SDCN neurons. The present results show that Ca2+ entry through NMDA receptors modulates the Gly receptor function via coactivation of CaMKII and CaN in the rat SDCN neurons. This interaction may represent one of the important regulatory mechanisms of spinal nociception. The results also suggest that GABAA and Gly receptors may be subject to different intracellular modulatory pathways.     

Inhibiting neuronal migration by blocking NMDA receptors in the embryonic rat cerebral cortex: a tissue culture study

To investigate the role of the NMDA receptor on neuronal migration in the cerebral cortex, we performed a tissue culture study using embryonic rat brain. After we labeled progenitor cells in the ventricular zone of E16 cerebral cortex explants by [3H]thymidine, the explants were cultured for 48 h. Then distribution of labeled cells was evaluated autoradiographically. Blocking NMDA receptors by adding the NMDA receptor antagonist, (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801: 1 or 10 microM) or d(-)-2-amino-5-phosphonopentanoic acid (d-AP5: 100 microM) to the culture medium, caused significantly decreased distribution of labeled cells in the outer intermediate zone (control 14.2+/-5.5%, 1 microM MK-801 5.8+/-7.2%, 10 microM MK-801 3.6+/-1.4%, and d-AP5 8.6+/-4.0%; mean+/-S.D.). This suggests that blocking NMDA receptors inhibits neuronal migration in the cerebral cortex. Furthermore, the influence of decreased intracellular Ca2+ concentration on neuronal migration was examined by adding intracellular Ca2+ chelator, 1, 2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM: 5 or 25 microM). This also resulted in inhibited neuronal migration. Therefore, it seems that neuronal migration in the cerebral cortex is regulated by intracellular Ca2+ concentration, which the NMDA receptor may influence.     

Exposure to N-methyl-D-aspartate increases release of arachidonic acid in primary cultures of rat hippocampal neurons and not in astrocytes

The release of [3H]arachidonic acid (ARA) was investigated from prelabelled primary cultures of hippocampal neurons and astroglial cells. The activation of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors resulted in a dose-dependent stimulation of [3H]ARA release. The half maximal effect was obtained at about 15 microM NMDA, whereas the maximum concentration (50 microM NMDA) produced about a 2-fold increase in 7-day-old cultures. This elevation in [3H]ARA release was blocked in a dose-related manner by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV), and by Mg2+ which blocks NMDA receptor-linked Ca2+ ion channels. The removal of external Ca2+ inhibited NMDA-induced release, whereas treatment with calcimycin (A 23187, a Ca2+ ionophore) greatly increased the [3H]ARA release. The inhibitors of phospholipase A2, nordihydroguaiaretic acid and mepacrine, decreased the NMDA-dependent [3H]ARA release in a dose-related manner, maximum inhibition reaching to about 90% at high doses. Entry of Ca2+ brought about by opening the voltage-sensitive channels by high K+ had no effect on the release of [3H]ARA, indicating that NMDA gated channels are situated in a part of the neuron where Ca2+ entry through this route is more efficiently coupled to the activation of phospholipase A2. Treatment with NMDA had no significant effect on [3H]ARA release in hippocampal astroglial cells as opposed to neurons. This was not due to inability of astrocytes to release ARA, for ATP still evoked [3H]ARA release, and this was markedly inhibited by mepacrine. It is suggested that ARA act as both intracellular and intercellular messengers in the functioning of NMDA receptors in synaptic transmission and plasticity in the hippocampus.     

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.     

Characterization of l-2-Amino-4-Phosphonobutanoate Action Following Sensitization by Quisqualate in Rat Hippocampal Slice Cultures

An excitatory action of l-2-amino-4-phosphonobutanoate (l-AP4), a glutamate analogue, is observed following pre-exposure of tissue to quisqualate. We have studied the mechanism of sensitization of l-AP4 responses by quisqualate in voltage-clamped CA3 pyramidal cells in rat hippocampal slice cultures in the presence of tetrodotoxin. Prior to quisqualate addition, CA3 cells did not respond to l-AP4 (50 - 1000 microM). Following brief application of quisqualate (500 nM for 30 s), l-AP4 (50 - 200 microM) induced a complex excitatory response which could be obtained for >1 h. l-AP4 caused an ionotropic inward current associated with a conductance increase. This response was in part sensitive to 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and in part sensitive to d-2-amino-5-phosphonovalerate (d-AP5) and Mg2+ ions. At depolarizing potentials, in the presence of CNQX and d-AP5, l-AP4 caused excitation by depressing K+ currents, mimicking the metabotropic action of glutamate. This indicates that the action of l-AP4 is mediated by three different receptor types: N-methyl-d-aspartate (NMDA) receptors, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptors, and glutamatergic metabotropic receptors. The l-AP4 response persisted in solutions containing low Ca2+ and high Mg2+ concentrations or 100 - 200 microM Cd2+, suggesting that it is independent of extracellular Ca2+. We were unable to identify any substance other than quisqualate capable of sensitizing the l-AP4 action. This effect also occurred when quisqualate was applied in Ca2+-free solution or in solutions containing low concentrations of Na+ or Cl-. Sensitization of l-AP4 responses by quisqualate was not observed in acutely dissociated pyramidal cells recorded by means of the whole-cell recording mode, although ionotropic quisqualate responses were present. Sensitization was readily reversed by short applications of the endogenous excitatory amino acids glutamate, aspartate and homocysteate at concentrations of 10 - 100 microM. Our data are consistent with the hypothesis that the excitatory action of l-AP4 results from a Ca2+-independent release of endogenous excitatory amino acids from some presynaptic neuronal or glial site.     

Caffeine-dependent stimulus-triggered oscillations in the CA3 region of hippocampal slices from rats chronically exposed to lead

Yoshimura et al. [Yoshimura, H., Sugai, T., Onoda, N., Segami, N., Kato, N., 2002. Age-dependent occurrence of synchronized population oscillation suggestive of a developing functional coupling between NMDA and ryanodine receptors in the neocortex. Dev. Brain Res., 136, 63-68.] have shown that caffeine can elicit synchronized oscillations (10-12 Hz) dependent on calcium-induced calcium release in rat neocortex neurons. In the present work, synchronized oscillations in the CA3 region of rat hippocampus were studied by recording field excitatory postsynaptic potentials (fEPSPs) in vitro. In the presence of 0.1 mM caffeine, in CA3 of 44 of 45 (97.8%) slices from chronic lead-exposed rats, single electrical stimuli triggered a burst of high-frequency oscillations (approximately 230 Hz), whereas in CA3 of caffeine-treated slices from control rats, such oscillations could be elicited in only 2 of 24 (8.3%) slices. The complete (but fully reversible) block of caffeine-dependent oscillations by 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 20 microM) indicates that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors are necessary for the high-frequency synchronized oscillations. 2-Amino-5-phosphonopentanoate (AP-5; 50 micoM) partially reduced the amplitude of caffeine-dependent oscillations without significantly altering their frequency. Caffeine-dependent oscillations could be abolished by application of AP-5 and 3 mM Mg2+ during the initial period of bursting, indicating that N-methyl-D-aspartate (NMDA) receptors play an important role in the generation of oscillations. The Ca2+ chelator ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 5 mM) added in standard artificial cerebrospinal fluid (ACSF) containing 0.1 mM caffeine fully blocked the oscillations. Caffeine-dependent oscillations are insensitive to an antagonist of gamma-aminobutyric acid (GABAA) receptors (10 microM bicuculline), L-type Ca2+ channels (10 muM nicardipine), L-type and N-type voltage-dependent calcium channels (100 microM Cd2)), and T-type Ca2+ channels (100 microM Ni2+). Previous studies have demonstrated that expression and function of NMDA and AMPA receptors are altered in the hippocampus of chronic lead-exposed rats. We propose that caffeine-dependent stimulus-induced oscillations in CA3 area of hippocampus from chronic lead-exposed rats are mainly mediated by the entry of extracellular Ca2+ through NMDA and non-NMDA receptors, without participation of GABAA receptors. Additionally, the underlying mechanisms are also discussed.     

Transient Ca2+-permeable AMPA receptors in postnatal rat primary auditory neurons

Fast excitatory transmission in the nervous system is mostly mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors whose subunit composition governs physiological characteristics such as ligand affinity and ion conductance properties. Here, we report that AMPA receptors at inner hair cell (IHC) synapses lack the GluR2 subunit and are transiently Ca2+-permeable before hearing onset as evidenced using agonist-induced Co2+ accumulation, Western blots and GluR2 confocal microscopy in the rat cochlea. AMPA (100 microM) induced Co2+ accumulation in primary auditory neurons until postnatal day (PND) 10. This accumulation was concentration-dependent, strengthened by cyclothiazide (50 microM) and blocked by GYKI 52466 (80 microM) and Joro spider toxin (1 microM). It was unaffected by D-AP5 (50 microM), and it could not be elicited by 56 mM K+ or 1 mM NMDA + 10 microM glycine. Western blots showed that GluR1 immunoreactivity, present in homogenates of immature cochleas, had disappeared by PND12. GluR2 immunoreactivity was not detected until PND10 and GluR3 and GluR4 immunoreactivities were detected at all the ages examined. Confocal microscopy confirmed that the GluR2 immunofluorescence was not located postsynaptically to IHCs before PND10. In conclusion, AMPA receptors on maturing primary auditory neurons differ from those on adult neurons. They are probably composed of GluR1, GluR3 and GluR4 subunits and have a high Ca2+ permeability. The postsynaptic expression of GluR2 subunits may be continuously regulated by the presynaptic activity allowing for variations in the Ca2+ permeability and physiological properties of the receptor.     

Protection by ethanol of cortical neurons from N-methyl-D-aspartate-induced neurotoxicity is associated with blocking calcium influx

Effect of ethanol on N-methyl-D-aspartate (NMDA)-induced neurotoxicity in rat dissociated cortical cells (8-12 day cultures) was studied. Treatment of cells with NMDA (50 and 500 microM) for 15 min caused cytotoxic effects on the cells, as examined by microscopic observations and lactate dehydrogenase release from cells 18 h after the treatment. Ca2+ is essential for these effects in medium during treatment. Presence of ethanol (50-300 mM) simultaneously with NMDA protected cells from the cytotoxicity depending on the concentration of ethanol. Calcium accumulation in cells on addition of NMDA, as monitored by fluorescence ratio (F405/F485) of Indo-1-preloaded cortical cells, was also decreased depending on the concentration of added ethanol. APV (200 microM) and ketamine (100 microM) blocked both the cytotoxicity and cellular calcium accumulation due to NMDA. These results suggest that ethanol effects its protection of neurons from NMDA-induced cytotoxicity by blocking the receptor-mediated calcium influx.     

N-methyl-D-aspartate autoreceptors respond to low and high agonist concentrations by facilitating, respectively, exocytosis and carrier-mediated release of glutamate in rat hippocampus

Presynaptic NMDA autoreceptors regulating glutamate release have rarely been investigated. High-micromolar N-methyl-D-aspartate (NMDA) was reported to elicit glutamate release from hippocampal synaptosomes in a Ca(2+)-independent manner by reversal of excitatory amino acid transporters. The aim of this work was to characterize excitatory amino acid release evoked by low-micromolar NMDA from glutamatergic axon terminals. Purified rat hippocampal synaptosomes were prelabelled with [(3)H]D-aspartate ([(3)H]D-ASP) and exposed in superfusion to varying concentrations of NMDA in the presence of 1 microM glycine. The release of [(3)H]D-ASP and also that of endogenous glutamate provoked by 10 microM NMDA were external Ca(2+) dependent and sensitive to the NMDA channel blocker MK-801 but insensitive to the glutamate transporter inhibitor DL-TBOA, which, on the contrary, prevented the Ca(2+)-independent release evoked by 100 microM NMDA. The NMDA (10 microM) response was blocked by 1 nM Zn(2+) and 1 microM ifenprodil, compatible with the involvement of a NR1/NR2A/NR2B assembly, although the presence of two separate receptor populations, i.e., NR1/NR2A and NR1/NR2B, cannot be excluded. This response was strongly antagonized by submicromolar (0.01-1 microM) concentrations of kynurenic acid and was mimicked by quinolinic acid (1-100 microM) plus 1 microM glycine. Finally, the HIV-1 protein gp120 potently mimicked the NMDA co-agonists glycine and D-serine, being significantly effective at 30 pM. In conclusion, glutamatergic nerve terminals possess NMDA autoreceptors mediating different types of release when activated by different agonist concentrations: low-micromolar glutamate would potentiate glutamate exocytosis, whereas higher glutamate concentrations would also provoke carrier-mediated release.     

Agmatine induces glutamate release and cell death in cultured rat cerebellar granule neurons

We investigated the effect of agmatine on cell viability of rat cerebellar granule neurons in a high-K+ (27.5 mM) medium. Exposure of cultured rat cerebellar granule neurons to agmatine (200-800 microM) resulted in a significant decrease in cell viability. Agmatine-induced neuronal death began to occur 6-12 h after addition, and gradually progressed. The agmatine neurotoxicity was attenuated by N-methyl-D-aspartate (NMDA) receptor antagonists and by enzymatic degradation of L-glutamate with glutamic pyruvic transaminase. Furthermore, a significant increase in extracellular L-glutamate concentration was detected before cell death occurred. In addition, agmatine-induced glutamate release and cell death were both blocked by pretreatment with botulinum toxin C, which is known to specifically inhibit the exocytosis. The agmatine neurotoxicity was not observed when extracellular K+ concentration was lower (10 mM). These results suggest that agmatine induces glutamate release through the exocytosis and thereby causes NMDA receptor-mediated neuronal death in conditions in which extracellular K+ concentrations are elevated.     

Two distinct mechanisms, differentially affected by excitatory amino acids, trigger GABA release from fetal mouse striatal neurons in primary culture

The mechanisms leading to Ca2+-dependent and Ca2+-independent GABA release were studied on highly purified striatal neurons developed in primary culture. Ca2+-dependent GABA release, which represents about 75% of the 56 mM K+ effect was totally inhibited when striatal neurons were first exposed to tetanus toxin (TnTx) (10 micrograms/ml) for 24 hr. The K+ effect was potentiated when 1 mM nipecotic acid (an inhibitor of the GABA uptake system) was added during the stimulation period or when Na+ was replaced by Li+. However, no difference in the GABA release measured under high-K+ conditions was observed after a 22 min preincubation of the neurons in a medium containing nipecotic acid or Li+. Replacement of Cl- ions by SO4(2-) did not modify K+-evoked GABA release. Ca2+-independent GABA release was stimulated by veratridine (20 microM), ouabain (3 mM), and monensin (20 microM), as well as the excitatory amino acids glutamate (100 microM), N-methyl-D-aspartate (100 microM), quisqualate (10 microM), and kainate (1 mM), drugs known to increase intracellular Na+ concentration. The veratridine- or glutamate-evoked GABA release was neither inhibited when intracellular Ca2+ content was reduced by more than 90% nor by treatment of the neurons to TnTx. However, the Ca2+-independent GABA release elicited by veratridine was inhibited by preincubation of the neurons in a medium containing 1 mM nipectotic acid and in a medium containing Li+ instead of Na+ or SO4(2-) instead of Cl-. These results strongly suggest that 2 different GABA release mechanisms exist in striatal neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuation of Zn2+ neurotoxicity by aspirin: role of N-type Ca2+ channel and the carboxyl acid group

Synaptically released Zn2+ ions enter into neurons primarily through voltage-gated Ca2+ channels (VGCC) or N-methyl-d-aspartate (NMDA) receptors, which can mediate pathological neuronal death. We studied the possibility (and underlying mechanisms) that aspirin, known to prevent NMDA neurotoxicity, would also attenuate Zn2+ neurotoxicity. Administration of 3 to 10 mM aspirin, in cortical cell cultures, attenuated the evolution of neuronal death following exposure to 300 microM Zn2+ for 30 min. This neuroprotective effect of aspirin was attributable to the prevention of Zn2+ ion entry. Aspirin interfered with inward currents and an increase in [Ca2+]i through VGCC and selective binding of omega-conotoxin, sensitive to N-type Ca2+ channel. The omega-conotoxins GVIA or MVIIC, the selective inhibitors of N-type Ca2+ channels, attenuated Zn2+ neurotoxicity. Aspirin derivatives lacking the carboxyl acid group did not reduce Zn2+ neurotoxicity. The present findings suggest that aspirin prevents Zn2+-mediated neuronal death by interfering with VGCC, and its action specifically requires the carboxyl acid group.     

Routes of zinc entry in mouse cortical neurons: role in zinc-induced neurotoxicity

Exposure of central neurons to Zn2+ triggers neuronal death. The routes of Zn2+ entry were investigated in living cortical neurons from the mouse using the specific Zn2+ fluorescent dye N-(6-methoxy-8-quinolyl)-p-toluene sulphonamide (TSQ), which preferentially detects membrane-bound Zn2+. Exposure of cortical neurons to increasing concentrations of Zn2+ (1-100 microM) induced a progressive increase in the fluorescence of TSQ. This fluorescence signal was not attenuated by the permeation of plasma membrane with digitonin. Accordingly, the major part of TSQ fluorescence (two-thirds) was associated to the particulate fraction of cortical neurons exposed to Zn2+. These results suggest that Zn2+ detected with TSQ in neurons is mainly bound to membranes. TSQ fluorescence measured in neurons exposed to 3 microM Zn2+ was enhanced by Na+-pyrithione, a Zn2+ ionophore, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartate (NMDA) or KCl-induced depolarization. However, in the absence of any treatment, TSQ labelling of neurons exposed to 3 microM Zn2+ was only decreased by NMDA receptor antagonists, whereas it remained unaltered in the presence of antagonists of AMPA receptors or L-type voltage-gated Ca2+ channels. Zn2+ entry through NMDA receptors did not contribute to Zn2+-induced neuronal death, as it was prevented by antagonists of NMDA receptors only when they were added after the Zn2+ exposure. Finally, Zn2+ induced a delayed accumulation of extracellular glutamate which might be responsible for the delayed NMDA receptor activation that leads to neuronal death.     

Dopamine release in the prefrontal cortex during stress is reduced by the local activation of glutamate receptors

Using microdialysis, we investigated the effects of the ionotropic glutamatergic agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on the stress-induced dopamine release in the prefrontal cortex of the freely moving rat. Handling-stress during 40 min increased extracellular dopamine by 195% and dopamine metabolites dihydroxyphenilacetic acid (DOPAC) by 120% and homovallinic acid (HVA) by 155% of baseline, but it did not modify extracellular glutamate, in the prefrontal cortex. Both NMDA (100 microM) and AMPA (20 microM), perfused through the microdialysis probe in the prefrontal cortex simultaneously to stress, significantly reduced the stress-induced dopamine release. These same doses or lower doses of NMDA (20 and 100 microM) and AMPA (1 and 20 microM) did not significantly modify basal dopamine release in the prefrontal cortex, but higher doses of these glutamatergic agonists significantly decreased (NMDA 500 microM) or increased (AMPA 100 microM) basal dopamine release in this area of the brain. These results show that the local activation of prefrontal glutamatergic ionotropic receptors reduces the stress-induced dopamine release in the prefrontal cortex of the rat.     

Intracellular cAMP regulates the response to NMDA in hippocampal neurons.

We have studied the effects of intracellular cyclic AMP (cAMP) on the response to N-methyl-D-aspartate (NMDA) in hippocampal cultured neurons by loading them with 2'-o-dibutyryladenosine 3', 5'-cyclic monophosphate (dcAMP) and have obtained evidence for regulation of Ca2+ release from intracellular stores by cAMP. Extracellular Ringer's solution was either Ca(2+)-containing or Ca(2+)-free. A brief initial stimulation with NMDA (10-100 microM, 12 s) was required before the neurons were loaded with dcAMP to potentiate the changes in intracellular Ca2+ concentration induced by the second stimulus of NMDA. Forskolin (10 microM) mimicked dcAMP, but to a lesser extent. A phorbol ester 12-o-tetradecanoylphorbol-13-acetate (100 nM) inhibited the effect of dcAMP.     

Spontaneous and beta-adrenergic receptor-mediated taurine release from astroglial cells are independent of manipulations of intracellular calcium

Stimulation of beta-adrenergic receptors on LRM55 astroglial cells results in cAMP-dependent release of taurine. We have previously demonstrated that extracellular Ca2+ is not required for either spontaneous or receptor-mediated taurine release (Martin et al., 1988b). In the present series of experiments we investigated the relationship between changes in intracellular free Ca2+ ([Ca2+]i) and taurine release. [Ca2+]i was measured using the fluorescent probe fura-2 and was manipulated by changing the concentration of Ca2+ in the incubation medium and by using the Ca2+ ionophore ionomycin. [Ca2+]i was reduced from 150 +/- 95 nM (n = 46) in control medium (containing 1.1 mM CaCl2) to 46 +/- 10 nM (n = 43) in saline containing no CaCl2 and 10 microM EGTA. [Ca2+]i was rapidly elevated to greater than or equal to 1 microM in medium containing 100 microM CaCl2 and 10 microM ionomycin. Taurine release, either spontaneous or stimulated by isoproterenol, was not significantly affected by these manipulations of [Ca2+]i. [Ca2+]i did not change when cells were stimulated with 100 nM isoproterenol in either control saline containing 1.1 mM CaCl2 or in CaCl2-free saline containing 10 microM EGTA. Other secretogogs (serotonin and ethanol) did not cause changes in [Ca2+]i. These data indicate that neither spontaneous or receptor-mediated taurine release from astroglial cells is Ca2+ dependent. However, when cells were preloaded with Ca2+, allowed to recover briefly, and then stimulated with isoproterenol, it was possible to demonstrate transient increases in Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium mobilization elicited by two types of nicotinic acetylcholine receptors in mouse substantia nigra pars compacta

Nicotinic acetylcholine receptors (nAChRs) are expressed in the midbrain ascending dopaminergic system, a target of many addictive drugs. Here we assessed the intracellular Ca2+ level by imaging fura-2-loaded cells in substantia nigra pars compacta in mouse brain slices, and we examined the influence on this level of prolonged exposures to nicotine using mice lacking the nAChR beta2-subunit. In control cells, superfusion with nicotine (10-100 microM) caused a long-lasting rise of intracellular Ca2+ level which depended on extracellular Ca2+. This nicotinic response was almost completely absent in beta2-/- mutant mice, leaving a small residual response to a high concentration (100 microM) of nicotine which was inhibited by the alpha7-subunit-selective antagonist, methyllycaconitine. Conversely, the alpha7-subunit-selective agonist choline (10 mM) caused a methyllycaconitine-sensitive increase in intracellular Ca2+ level both in wild-type and beta2-/- mutant mice. Nicotine-elicited Ca2+ mobilization was reduced by the Na+ channel blocker tetrodotoxin (TTX) and by T-type Ca2+ channel blocking agents, whereas the choline-elicited Ca2+ increase was insensitive to TTX. Neither nicotine nor choline produced Ca2+ increase following inhibition of the release of Ca2+ from intracellular stores by dantrolene. These results demonstrate that in nigral dopaminergic neurons, nicotine can elicit Ca2+ mobilization via activation of two distinct nAChR subtypes: that of beta2-subunit-containing nAChR followed by activation of Na+ channel and T-type Ca2+ channels, and/or activation of alpha7-subunit-containing nAChR. The Ca2+ influx due to nAChR activation is subsequently amplified by the recruitment of intracellular Ca2+ stores. This Ca2+ mobilization may possibly contribute to the long-term effects of nicotine on the dopaminergic system.     

Regulation of CCK release in cerebral cortex by N-methyl-D-aspartate receptors: sensitivity to APV, MK-801, kynurenate, magnesium and zinc ions.

Cholecystokinin octapeptide (CCK) is an abundant neuropeptide of cerebral cortex but its function in this region is largely unknown. In this study we have examined the effect of glutamate receptor activation on the depolarisation-induced release of CCK-like immunoreactivity from tissue slices of rat cerebral cortex. The K(+)-evoked release of cholecystokinin was found to be significantly increased by the three excitatory amino acid agonists effective against glutamate receptor subtypes. N-methyl-D-aspartate (NMDA), kainic acid and quisqualate. The facilitation of CCK release by NMDA was examined in more detail and shown to be significantly attenuated by Mg2+ (2.5 mM), Zn2+ (50 microM), MK-801 (0.1 and 0.3 microM), aminophosphonovaleric acid (100 microM) and kynurenic acid (100 microM and 300 microM). These results support the conclusion that release of CCK in cerebral cortex is modulated by the NMDA receptor.