Presence Of MK-801 Research Articles

Modulation of cellular differentiation by N-methyl-D-aspartate receptors in osteoblasts.

N-methyl-D-aspartate (NMDA) receptors for the central neurotransmitter l-glutamate (Glu) have been shown to be present in both osteoblasts and osteoclasts. Sustained exposure to the NMDA channel antagonist dizocilpine (MK-801) significantly prevented increases in both alkaline phosphatase activity and Ca2+ accumulation in a concentration-dependent manner in osteoblasts cultured for 7-28 days in vitro (DIV), without significantly affecting cell survivability. Osteocalcin expression was markedly reduced in the presence of MK-801 in osteoblasts cultured for 28 DIV. Both an NMDA domain antagonist and a glycine domain antagonist similarly prevented Ca2+ accumulation in osteoblasts exposed for 28 consecutive DIV. MK-801 was effective in significantly inhibiting Ca2+ accumulation determined at 28 DIV in osteoblasts exposed before 7 DIV but was ineffective in cells exposed after 11-21 DIV. Sustained exposure to MK-801 significantly inhibited DNA binding activity and expression of core binding factor alpha-1 (CBFA1) in osteoblasts exposed after 7 DIV up to 28 DIV, but not in those exposed before 7 DIV. These results suggest that heteromeric NMDA receptor channels may be functionally expressed to regulate mechanisms underlying cellular differentiation rather than proliferation and/or maturation through modulation of expression of CBFA1 in cultured rat calvarial osteoblasts.

Modulation of AMPA receptors by cAMP‐dependent protein kinase in PreBötzinger complex inspiratory neurons regulates respiratory rhythm in the rat

We hypothesize that phosphorylation of AMPA receptors or associated synaptic proteins modulates the excitability of respiratory neurons in the preBötzinger Complex (preBötC), affecting respiratory rhythm. Using neonatal rat medullary slices that spontaneously generate respiratory rhythm, we examined the role of the cAMP-PKA pathway (PKA: cAMP-dependent protein kinase) in modulating glutamatergic synaptic transmission, the excitability of inspiratory neurons in the preBötC and respiratory rhythm. Microinjection of forskolin, an activator of adenylate cyclase, into the preBötC with or without the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), decreased the period (increased the frequency) of respiratory-related rhythmic motor output in the hypoglossal nerve (XIIn) to 84 % (without IBMX) and to 72 % (with IBMX) of the pre-injection baseline. In the presence of MK-801, a non-competitive NMDA receptor antagonist, microinjection of forskolin plus IBMX decreased the period to 66 % of baseline levels. Microinjection of Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), a PKA inhibitor, increased the period to 145 % of baseline levels. Concurrent microinjection of Rp-cAMPS and forskolin had no effect on the period. Bath application of 7beta-deacetyl-7beta-[gamma-(morpholino)butyryl]-forskolin hydrochloride (7Db-forskolin, a water-soluble derivative of forskolin): (1) decreased the period to 67 % of baseline levels without affecting the amplitude of integrated XIIn inspiratory discharge, (2) induced a tonic inward current of 29 pA and enhanced inspiratory drive current (the amplitude increased to 183 % and the integral increased to 184 % of baseline) in voltage-clamped (holding potential = -60 mV) preBötC inspiratory neurons and (3) increased the frequency to 195 % and amplitude to 118 % of spontaneous excitatory postsynaptic currents (sEPSCs) during expiratory periods. Dideoxy-forskolin did not have these effects. Intracellular perfusion with the catalytic subunit of PKA (cPKA) into preBötC inspiratory neurons progressively enhanced inspiratory drive currents and, in the presence of TTX, increased the inward currents induced by local ejection of AMPA; the latter currents were blocked by 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulphonamide (NBQX, an AMPA/kainate receptor antagonist). The effects of cPKA were blocked by co-application of PKA inhibitor (6-22) amide (PKI). These results suggest that phosphorylation of postsynaptic AMPA receptors through the cAMP-PKA pathway modulates both tonic and phasic excitatory amino acid synaptic transmission and excitability of inspiratory neurons in the preBötC and, therefore, regulates respiratory rhythm. Moreover, the basal level of endogenous PKA activity appears to be a determinant of resting respiratory frequency.

Age dependent NMDA contribution to impaired hypotensive cerebral hemodynamics following brain injury

Previous studies have observed that fluid percussion brain injury (FPI) impaired NMDA induced pial artery dilation (PAD) in an age dependent manner. Unrelated studies observed a similar age dependent impairment of hypotensive cerebral autoregulation after FPI. This study was designed to test the hypothesis that NMDA receptor activation contributes to impairment of cerebral autoregulation during hypotension after FPI in an age dependent manner. Therefore, the role of NMDA in impaired hypotensive cerebrovascular regulation after FPI was compared in newborn and juvenile pigs equipped with a closed cranial window. Ten minutes of hypotension (10–15 ml blood/kg) decreased mean arterial blood pressure uniformly in both groups (≈44%). In the newborn, hypotensive PAD was blunted within 1 h of FPI but partially protected by pretreatment with the NMDA antagonist MK801 (1 mg/kg i.v.) (34±1 vs. 8±1 vs. 25±2% for sham control, FPI, and FPI-MK801, respectively). Cerebral blood flow (CBF) was reduced during normotension by FPI, further reduced by hypotension, but both were partially protected by MK801 in the newborn (56±5, 35±2, and 16±1 vs. 62±6, 45±3, and 30±2 ml/min 100 g for normotension, normotension-FPI, and hypotension-FPI in the absence and presence of MK801, respectively). In contrast, blunted hypotensive PAD was protected significantly less by MK801 in the juvenile (32±2 vs. 7±2 vs. 16±2% for sham control, FPI, and FPI-MK801, respectively). Similarly, MK801 had less protective effect on normotensive and hypotensive CBF values post FPI in the juvenile. These data indicate that NMDA receptor activation contributes to impaired hypotensive cerebral hemodynamics following brain injury in an age dependent manner.

Specific inhibition of N-methyl-D-aspartate receptor function in rat hippocampal neurons by L-phenylalanine at concentrations observed during phenylketonuria.

Hippocampal N-methyl-D-aspartate receptors (NMDARs) are thought to be involved in the regulation of memory formation and learning. Investigation of NMDAR function during experimental conditions known to be associated with impaired cognition in vivo may provide new insights into the role of NMDARs in learning and memory. Specifically, the mechanism whereby high concentrations of L-phenylalanine (L-Phe) during phenylketonuria (>1.2 mM) cause mental retardation remains unknown. Therefore, the effects of L-Phe on NMDA-activated currents (I(NMDA)) were studied in cultured hippocampal neurons from newborn rats using the patch-clamp technique. L-Phe specifically and reversibly attenuated I(NMDA) in a concentration-dependent manner (IC(50) = 1.71 +/- 0.24 mM). In contrast, L-tyrosine (L-Tyr), an amino acid synthesized from L-Phe in normal subjects, did not significantly change I(NMDA). Although the L-Phe-I(NMDA) concentration-response relationship was independent of the concentration of NMDA, it was shifted rightward by increasing the concentration of glycine. Consistent with an effect of L-Phe on the NMDAR glycine-binding site, L-Phe (1 mM) did not attenuate I(NMDA) in the presence of D-alanine (10 microM). Furthermore, L-Phe significantly attenuated neither glutamate-activated current in the presence of MK-801, nor current activated by AMPA. The finding that L-Phe inhibits specifically NMDAR current in hippocampal neurons by competing for the glycine-binding site suggests a role for impaired NMDAR function in the development of mental retardation during phenylketonuria and accordingly an important role for NMDARs in memory formation and learning.

Blockade of Ca2+-Permeable AMPA/Kainate Channels Decreases Oxygen–Glucose Deprivation-Induced Zn2+Accumulation and Neuronal Loss in Hippocampal Pyramidal Neurons

Synaptic release of Zn2+ and its translocation into postsynaptic neurons probably contribute to neuronal injury after ischemia or epilepsy. Studies in cultured neurons have revealed that of the three major routes of divalent cation entry, NMDA channels, voltage-sensitive Ca2+ channels (VSCCs), and Ca2+-permeable AMPA/kainate (Ca-A/K) channels, Ca-A/K channels exhibit the highest permeability to exogenously applied Zn2+. However, routes through which synaptically released Zn2+ gains entry to postsynaptic neurons have not been characterized in vivo. To model ischemia-induced Zn2+ movement in a system approximating the in vivo situation, we subjected mouse hippocampal slice preparations to controlled periods of oxygen and glucose deprivation (OGD). Timm's staining revealed little reactive Zn2+ in CA1 and CA3 pyramidal neurons of slices exposed in the presence of O2 and glucose. However, 15 min of OGD resulted in marked labeling in both regions. Whereas strong Zn2+ labeling persisted if both the NMDA antagonist MK-801 and the VSCC blocker Gd3+ were present during OGD, the presence of either the Ca-A/K channel blocker 1-naphthyl acetyl spermine (NAS) or the extracellular Zn2+ chelator Ca2+ EDTA substantially decreased Zn2+ accumulation in pyramidal neurons of both subregions. In parallel experiments, slices were subjected to 5 min OGD exposures as described above, followed 4 hr later by staining with the cell-death marker propidium iodide. As in the Timm's staining experiments, substantial CA1 or CA3 pyramidal neuronal damage occurred despite the presence of MK-801 and Gd3+, whereas injury was decreased by NAS or by Ca2+ EDTA (in CA1).

Survival promotion of mesencephalic dopaminergic neurons by depolarizing concentrations of K+ requires concurrent inactivation of NMDA or AMPA/kainate receptors.

The death of dopaminergic neurons that occurs spontaneously in mesencephalic cultures was prevented by depolarizing concentrations of K+ (20-50 mM). However, unlike that observed previously in other neuronal populations of the PNS or CNS, promotion of survival required concurrent blockade of either NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors by the specific antagonists, MK-801 and GYKI-52466, respectively. Rescued neurons appeared to be healthy and functional because the same treatment also dramatically enhanced their capacity to accumulate dopamine. The effects on survival and uptake were rather specific to dopaminergic neurons, rapidly reversible and still observed when treatment was delayed after plating. Glutamate release increased substantially in the presence of elevated concentrations of K+, and chronic treatment with glutamate induced a loss of dopaminergic neurons that was prevented by MK-801 or GYKI-52466 suggesting that an excitotoxic process interfered with survival when only the depolarizing treatment was applied. The effects of the depolarizing stimulus in the presence of MK-801 were mimicked by BAY K-8644 and abolished by nifedipine, suggesting that neuroprotection resulted from Ca(2+) influx through L-type calcium channels. Measurement of intracellular calcium revealed that MK-801 or GYKI-52466 were required to maintain Ca(2+) levels within a trophic range, thus preventing K+-induced excitotoxic stress and Ca(2+) overload. Altogether, our results suggest that dopaminergic neurons may require a finely tuned interplay between glutamatergic receptors and calcium channels for their development and maturation.

MK-801 prevents dopamine D1 but not serotonin 2A stimulation of striatal preprotachykinin mRNA expression.

We examined dopamine (DA) and serotonin (5-HT) receptor-mediated influences on striatal preprotachykinin (PPT, tachykinin precursor) mRNA regulation in organotypic slice cultures. A 3 h exposure to SKF-38393 (10 microM, DA D1 agonist) or DOI (10 microM, 5-HT2 agonist) increased PPT mRNA levels to 196.4% and 154.0%, respectively. Responses to SKF-38393 were prevented by SCH-23390 (10 microM, D1 antagonist) whereas DOI-stimulated increases were prevented by ketanserin (10 microM, 5-HT2A antagonist). Since striatal tachykinin neurons also possess NMDA receptors that regulate gene expression, stimulation of PPT message levels was examined in the presence of MK-801, a non-competitive NMDA antagonist. Alone, MK-801 (10 nM) did not significantly alter basal PPT message levels. However, MK-801 prevented SKF-38393-stimulated increases in PPT mRNA expression while DOI-induced expression was not affected. These results provide evidence that D1 regulation of striatal tachykinin expression is dependent on NMDA-type glutamate neurotransmission while 5-HT2A regulation appears independent.

Behavioral interactions caused by combined administration of morphine and MK-801 in rats.

The NMDA antagonist MK-801 reportedly blocks experience-dependent changes in sensitivity to morphine, including tolerance to its analgesic actions and sensitization to its locomotor-stimulating effects. However, evidence in the existing literature suggests that some of MK-801's effects are additive (or synergistic) with those of morphine. Experiments were conducted to characterize the effects of acute and repeated administration of the combination of MK-801 and morphine on analgesia, locomotor activity, and drug discrimination in rats. In each experiment, rats were first tested repeatedly after treatment with the combination of MK-801 and morphine, and then after treatment with either drug alone. The analgesic effects of MK-801 combined with morphine were greater than those of morphine alone, but tolerance to the combination of drugs developed at a similar rate as to morphine alone. The locomotor-stimulating effects of MK-801 combined with morphine were also greater than those of either drug alone, and locomotor sensitization developed to the combination of drugs but not to either drug alone at the low doses used. Rats learned to discriminate a combination of MK-801 and morphine from vehicle as quickly as they learned to discriminate morphine alone from vehicle, but those trained with the combination of MK-801 and morphine responded primarily at the vehicle-appropriate lever when given either drug alone. Since behavioral adaptations readily occur in the presence of MK-801, it appears that NMDA antagonists fail to invariably block the cellular plasticity that underlies such adaptations. Rather, the expression of adaptations in drug sensitivity appears related, at least in part, to the continued presence of the discriminative stimulus cues that are present during conditioning. Although NMDA receptors are important for some forms of cellular plasticity, the present studies illustrate the difficulty in interpreting behavioral studies in which MK-801 is given with morphine.

Characterization of iodoacetate-mediated neurotoxicity in vitro using primary cultures of rat cerebellar granule cells

The neuroprotective efficacy of antioxidant molecules against iodoacetate (IAA) neurotoxicity in rat cerebellar granule cell (CGC) cultures was investigated. Transient exposure to IAA caused a concentration-dependent decrease in cell viability (ED50 = 9.8μM). Dizocilpine maleate (MK-801), and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), failed to prevent IAA toxicity. Certain antioxidant molecules were shown to be neuroprotective against IAA when combined with MK-801 but were ineffective when administered alone. (S)-(-)-Trolox, butylated hydroxytoluene (BHT), and U-83836E exhibited EC50 values of 78, 5.9, and 0.25 μM, respectively, in the presence of 10 μM MK-801. IAA also induced an increase in intracellular oxidative stress, which was quenched by the antioxidants (in the presence of MK-801) in cultures loaded with the oxidant sensitive dye 2′7′-dichlorodihydrofluorescein diacetate (DCFH-DA).

Involvement of the N-methyl-D-aspartate receptor in neuronal cell death induced by cytotoxic T cell-derived secretory granules.

The mechanisms underlying neurotoxicity mediated by cytotoxic T lymphocytes (CTL) and their secretory granule proteins perforin and granzymes remain unclear. We evaluated the possible role of the neurotransmitter glutamate in cell death observed in differentiated neurons exposed to CTL-derived granules. Excitotoxicity induced by excessive concentrations of extracellular glutamate is associated with a rise in intracellular calcium and can lead to generation of NO through the activation of glutamatergic N-methyl-D-aspartate (NMDA) receptors. Consistent with an involvement of glutamate, we found that cell death in mature cerebral granule cells was inhibited by 65-80% by two NMDA receptor blockers (MK-801 and D-2-amino-5-phosphonovaleric acid) or a NO synthase blocker (N(G)-nitro-L-arginine methylester). Furthermore, neurons treated with secretory granules responded with a biphasic rise in the intracellular calcium concentration ([Ca2+]i). Whereas MK-801 did not interfere with the immediate rise of [Ca2+]i, the second wave of calcium accumulation starting at 40 min was delayed by 20 min and reduced in amplitude in the presence of MK-801. In immature, NMDA receptor-negative neurons, MK-801 prevented neither the cytotoxicity nor the calcium influx observed 5 min after addition of cytotoxic granules. The demonstration that NMDA receptors and NO are involved in granule-mediated killing of mature neurons opens new avenues in the treatment of neuronal cell death in CTL-mediated diseases such as viral encephalitis.

Glutamate release is involved in PAF-increased cyclic GMP levels in hippocampus.

The involvement of glutamate in PAF-increased cyclic GMP levels was studied. Glutamate treatment caused a dose-response increase of cyclic GMP levels in hippocampal slices. The presence of 1 mM glutamate did not modify the effect caused by 10(-7)M PAF. To elucidate the involvement of glutamate in this action, slices were treated with PAF in the presence of MK-801, a NMDA receptor antagonist. Results indicate that PAF-increased cyclic GMP levels were obtained by NMDA receptors activation. Finally, results obtained from the experiments performed with PAF in the presence of riluzole, to inhibit the glutamate release, demonstrated that glutamate release is a stage in the PAF-induced increase of cyclic GMP levels in hippocampus.

Antioxidative and proapoptotic effects of riluzole on cultured cortical neurons.

Riluzole is used clinically in patients with amyotrophic lateral sclerosis. As oxidative stress, in addition to excitotoxicity, may be a major mechanism of motoneuron degeneration in patients with amyotrophic lateral sclerosis, we examined whether riluzole protects against nonexcitotoxic oxidative injury. Probably reflecting its weak antiexcitotoxic effects, riluzole (1-30 microM) attenuated submaximal neuronal death induced by 24-h exposure to 30 microM kainate or NMDA, but not that by 100 microM NMDA, in cortical cultures. Riluzole also attenuated nonexcitotoxic oxidative injury induced by exposure to FeCl3 in the presence of MK-801 and CNQX. Consistent with its antioxidative effects, riluzole reduced Fe3+-induced lipid peroxidation, and inhibited cytosolic phospholipase A2. By contrast, riluzole did not attenuate neuronal apoptosis induced by staurosporine. Rather unexpectedly, 24-48-h exposure to 100-300 microM riluzole induced neuronal death accompanied by nuclear and DNA fragmentations, which was attenuated by caspase inhibitor carbobenzyloxy-Val-Ala-Asp-fluoromethyl ketone but not by protein synthesis inhibitor cycloheximide. The present study demonstrates that riluzole has direct antioxidative actions, perhaps in part by inhibiting phospholipase A2. However, in the same neurons, riluzole paradoxically induces neuronal apoptosis in a caspase-sensitive manner. Considering current clinical use of riluzole, further studies are warranted to investigate its potential cytolethal effects.

Role of desensitization and subunit expression for kainate receptor-mediated neurotoxicity in murine neocortical cultures.

The neurotoxic actions of kainate and domoate were studied in cultured murine neocortical neurons at various days in culture and found to be developmentally regulated involving three components of neurotoxicity: (1) toxicity via indirect activation of N-methyl-D-aspartate (NMDA) receptors, (2) toxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and (3) toxicity that can be mediated by kainate receptors when desensitization of the receptors is blocked. The indirect action at NMDA receptors was discovered because (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine (MK-801), an NMDA receptor antagonist, was able to block part of the toxicity. The activation of NMDA receptors is most likely a secondary effect resulting from glutamate release upon kainate or domoate stimulation. 1-(4-Aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-ethyle nedioxy-5H-2,3-benzodiazepine (GYKI 53655), a selective AMPA receptor antagonist, abolished the remaining toxicity. These results indicated that kainate- and domoate-mediated toxicity involves both the NMDA and the AMPA receptors. Pretreatment of the cultures with concanavalin A to prevent desensitization of kainate receptors led to an increased neurotoxicity upon stimulation with kainate or domoate. In neurons cultured for 12 days in vitro a small but significant neurotoxic effect was observed when stimulated with agonist in the presence of MK-801 and GYKI 53655. This indicates that the toxicity is produced by kainate receptors in mature cultures. Examining the subunit expression of the kainate receptor subunits GluR6/7 and KA2 did, however, not reveal any major change during development of the cultures.

Potent ς 1 -Receptor Ligand 4-Phenyl-1-(4-Phenylbutyl) Piperidine Modulates Basal and N -Methyl- d -Aspartate–Evoked Nitric Oxide Production In Vivo

final sigma-Receptor ligands ameliorate ischemic neuronal injury and modulate neuronal responses to N-methyl-D-aspartate (NMDA) receptor stimulation. Because NMDA-evoked synthesis of nitric oxide (NO) may play an important role in excitotoxic-mediated injury, we tested the hypothesis that final sigma-receptor ligands attenuate basal and NMDA-evoked NO production in the striatum in vivo. Microdialysis probes were placed bilaterally into the striatum of halothane-anesthetized adult Wistar rats. Rats were divided into 7 treatment groups and perfused with artificial cerebrospinal fluid (aCSF) containing 3 micromol/L [14C]L-arginine for 2 to 3 hours followed by NMDA in various combinations with the following drugs: L-nitroarginine (L-NNA); the final sigma1-receptor ligand 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP); the selective final sigma1-receptor antagonist 1-(cyclopropylmethyl)-4-(2'-oxoethyl) piperidine hydrobromide (DuP 734); and the noncompetitive NMDA receptor blocker MK-801 in aCSF. Right-left differences between [14C]L-citrulline in the effluent from rats treated with different drug combinations were assumed to reflect differences in NO production. After a 3-hour loading period with [14C]L-arginine, addition of 1 mmol/L NMDA increased [14C]L-citrulline recovery compared with aCSF alone. This NMDA-evoked increase was inhibited by 1 mmol/L of L-NNA and PPBP. Perfusion of 1 mmol/L of the final sigma1-receptor antagonist DuP 734 with 1 mmol/L PPBP augmented NMDA-evoked [14C]L-citrulline recovery compared with perfusion with PPBP and NMDA. MK-801 attenuated the basal as well as NMDA-evoked [14C]L-citrulline recovery. PPBP did not cause any further attenuation in the basal and NMDA-evoked [14C]L-citrulline recovery in the presence of MK-801. These data indicate that a final sigma1-receptor ligand attenuates basal as well as NMDA-evoked NO production. Because the attenuated NO production was reversed by DuP 734, PPBP appears to act as an agonist at the final sigma1-receptor. Attenuated NO production by final sigma1-receptor agonists provides one possible mechanism for focal ischemic neuroprotection.

Discrepant intracellular pH changes following intracellular Ca 2+ increases induced by glutamate and Ca 2+ ionophores in rat hippocampal neurons

We investigated changes in intracellular pH (pHi) in relation to intracellular Ca 2+ concentration ([Ca 2+]i) in primary cultured hippocampal neurons treated with glutamate. [Ca 2+]i and pHi were imaged with fluorescent dyes and confocal microscopy. Exposure to 1 mM glutamate for 10 min increased [Ca 2+]i and evoked acidosis. These changes persisted for at least 60 min, even after removal of glutamate. The increase in [Ca 2+]i and the acidosis were not observed in Ca 2+-free solution and were attenuated in the presence of MK-801, an NMDA receptor antagonist. We also found that the increase in [Ca 2+]i and acidosis could be induced by addition of Ca 2+ to the extracellular solution in the cells pretreated with glutamate in Ca 2+-free solution, even if glutamate did not exist in the extracellular solution. On the other hand, ionomycin and Br-A23187, calcium ionophores, increased [Ca 2+]i to almost the same level as glutamate and increased pHi. Extracellular Ca 2+ was also indispensable for the increase in [Ca 2+]i and the alkalosis. These results suggest the followings: 1) intracellular acidosis by glutamate is dependent on the presence of extracellular Ca 2+; 2) the acidosis does not result from only the increase in [Ca 2+]i; and 3) glutamate induces the irreversible disorder of regulatory mechanisms of [Ca 2+]i not only by Ca 2+-dependent process, but also by Ca 2+-independent process.

Excitatory amino acid receptor antagonists decrease hypoxia induced increase in extracellular dopamine in striatum of newborn piglets

The present study tested the hypothesis that the increase in extracellular striatal dopamine during hypoxia is least partly associated with activation of N-methyl-D-aspartate (NMDA) and/or non-NMDA excitatory amino acid receptors. Studies were performed in anesthetized and mechanically ventilated 2–3 days old piglets. Hypoxic insult was induced by decreasing the oxygen fraction in inspired gas (FiO 2) from 22 to 7% for 1 h, followed by 1 h reoxygenation at 22%. Cortical oxygen pressure was measured optically by oxygen dependent quenching of phosphorescence, and extracellular striatal dopamine was measured using in vivo microdialysis. The microdialysis probes were perfused with Ringer solution ±50 μM (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) or 50 μM 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX). One hour of hypoxia decreased the cortical oxygen pressure from 46±3 Torr to 10±1.8 Torr. In striatum perfused with Ringer, statistically significant increase in extracellular dopamine, to 1050±310% of control, was observed after 20 min of hypoxia. By 40 min of hypoxia the extracellular level of dopamine increased to 4730±900% of control; by the end of the hypoxic period the values increased to 18 451±1670% of control. The presence of MK-801 in the perfusate significantly decreased the levels of extracellular dopamine during hypoxia. At 20, 40 and 60 min of hypoxia extracellular level of dopamine increased to 278±94% of control, 1530±339% of control and 14 709±1095 of control, respectively. The presence of NBQX caused a statistically significant decrease, by about 30% in the extracellular dopamine compared to control, only at the end of the hypoxic period. It can be concluded that in striatum of newborn piglets, the excitatory NMDA receptors but not the non-NMDA receptors may be modulating the changes in extracellular levels of dopamine. The NMDA receptor antagonist, MK-801, may exert part of its reported neuroprotective effect to hypoxic stress in striatum by decreasing the levels of extracellular dopamine.

Prenatal ethanol exposure enhances glutamate release stimulated by quisqualate in rat cerebellar granule cell cultures.

Effects of prenatal ethanol exposure on extracellular glutamate accumulation stimulated by glutamate receptor agonists were studied in rat cerebellar granule cell cultures. The prenatal exposure to ethanol was achieved via maternal consumption of a Sustacal liquid diet containing either 5% ethanol or isocaloric sucrose (pair-fed) substituted for ethanol from gestation d 11 until the day of parturition. Neither the basal level of extracellular glutamate nor the increased accumulation of glutamate stimulated by KCl (40 mM) or by ionotropic glutamate receptor agonists, N-methyl-D-aspartate (NMDA) or kainate (KA) (100 microM each), in cells prepared from the ethanol-fed group was significantly different from that in cells prepared from the pair-fed group. Glutamate accumulation stimulated by quisqualate (QA, 100 microM) or by trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD, 250 microM) in the ethanol-fed group was higher than that in the pair-fed group by 116 and 36%, respectively. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 100 microM), an ionotropic QA receptor antagonist, the QA-induced accumulation of glutamate in the ethanol-fed group was still higher than that in the pair-fed group. In the presence of MK-801 (5 microM), an antagonist of the NMDA receptor, the enhanced accumulation of glutamate stimulated by either QA or t-ACPD was still observable in the ethanol-fed group as compared to the pair-fed group. Addition of (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 500 microM), a selective antagonist of the metabotropic glutamate receptor, abolished the enhanced accumulation of glutamate stimulated by either QA or t-ACPD in the ethanol-fed group. Although immunoblotting of mGluR1 and mGluR2/3 did not show apparent differences between the pair-fed and the ethanol-fed groups, the overall results suggest that the effect of prenatal ethanol exposure was selectively through a pathway mediated by the metabotropic glutamate receptor.

Neonatal hypothalamic c-fos expression in an excitotoxicity-induced model of precocious puberty

We have used immunocytochemical detection of c-fos expression (FOS-like immunoreactivity, FLI) to establish the site of action of monosodium glutamate (MSG) in neonatal rats in a model of lesion-induced precocious puberty. The primary target appears to be the hypothalamic arcuate nucleus (ARC) but other circumventricular organs (CVO) are also affected (e.g. subfornical organ). Single injections of MSG (1–4 mg/g single dose, postnatal day 2 (P2)) which result in precocious puberty induce an area of edema, surrounded by a ring of FLI in the basal hypothalamus. In contrast, a maximal sub-lethal dose of NMDA ( N-methyl- d-aspartate; 3 mg/kg) which produces a different pattern of ARC FLI, without edema, has no effect on puberty. Multiple doses of MSG (4 mg/g P2, P4, P6, P8), consistent with severe ARC damage and resultant sterility, markedly attenuates the FLI response by P8, with no visible edematous reaction following the final injection. In efforts to block the effect of MSG, pretreatment with the NMDA receptor-specific antagonist MK-801 (dizocilpine maleate) prevented the appearance of edema, as well as the onset of precocious puberty. However, MK-801 did not completely eliminate the FLI, but transformed the pattern of staining so that the original edematous area now contained many FOS-positive cells. This remaining MSG-induced FLI could not be eliminated by higher doses of MK-801 or by the non-NMDA antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione). The combination of MK-801 and DNQX was also ineffective. MK-801 or DNQX had no effect on FLI when injected alone (i.e., without MSG) or in combination. The receptor which mediates MSG-induced c-fos expression, in the presence of MK-801 or DNQX, needs to be identified. We conclude, in conjunction with our previous work, that MSG induces precocious sexual maturation via an MK-801-sensitive mechanism associated with an edematous response of the basal hypothalamus. Whether the appearance of edema is indicative of an excitotoxic action of MSG, resulting in the removal of neurons inhibitory to sexual maturation, remains to be established.

Mitochondrial Depolarization in Glutamate-Stimulated Neurons: An Early Signal Specific to Excitotoxin Exposure

A brief exposure to high concentrations of glutamate kills cultured forebrain neurons by an excitotoxic process that is dependent on Ca2+ influx through the NMDA receptor. In this study, we have measured striking changes in mitochondrial function during and immediately after intense glutamate receptor activation. Using indo-1 microfluorometry and a specific inhibitor of the mitochondrial Na+/Ca2+ exchanger, CGP-37157, we have demonstrated that mitochondria accumulate large quantities of Ca2+ during a toxic glutamate stimulus and further that Ca2+ efflux from mitochondria contributes to the prolonged [Ca2+]i elevation after glutamate removal. We then used JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine+ ++ iodide), a ratiometric indicator of mitochondrial membrane potential (delta psi), to show that Ca2+ accumulation within the organelle dissipates delta psi. The abrupt loss of delta psi after glutamate stimulation did not occur in the presence of MK801 or in the absence of extracellular Ca2+. The mitochondrial depolarization was also cyclosporin A-sensitive, indicating a probable role for the permeability transition pore. Hence mitochondrial Ca2+ accumulation and the subsequent permeability transition may be a critical early event specific to the NMDA receptor-mediated excitotoxic cascade.

Mechanism of Calcium Influx in Cortical Synaptosomes of the Guinea Pig Fetus. 257

Calcium ion flux through the N-methyl-D-aspartate (NMDA) receptor ion-channel has been proposed as a mechanism of hypoxia-induced neuronal injury in the newborn and fetal brain. The present study tests the hypothesis that increased free intrasynaptosomal calcium ([Ca2+]i) during hypoxia is mediated by an influx of calcium via the NMDA receptor and is associated with the release of calcium from intracellular stores. Studies were conducted in 12 term guinea pig fetuses, 6 normoxic and 6 whose mothers were exposed to an FiO2 of 7% for 1 hr. Tissue hypoxia was confirmed by decreased ATP and phosphocreatine levels. Cortical synaptosomes were prepared and[Ca2+]i was measured fluorometrically with Fura-2 after NMDA receptor activation by 100μM NMDA and 100μM glutamate in the presence of 100μM glucine. The relative contribution of the NMDA receptor and intrasynaptosomal stores to increased [Ca2+]i was assessed by determining the effect of MK-801, a non-competitive NMDA receptor antagonist, and thapsigardin, an intracellular Ca2+-ATPase inhibitor. For the normoxic group, the increase in [Ca2+]i after NMDA addition was 79 ± 39 nM for untreated synaptosomes (U), 32 ± 38 nM in the presence of MK-801(M), p<0.05, and 21 ± 11 nM in the presence of thapsigardin (T), p<0.01. The increase in [Ca2+]i after the addition of glutamate was 55 ± 32 nM (U), 15 ± 16 nM (M), p<0.05, and 17 ± 18 nM (T), p<0.01. Similarly, in the hypoxic group, the increase in [Ca2+]i after NMDA addition was 110± 38 nM (U), 26 ± 13 nM (M), p<0.05 and 13 ±24 nM (T), p<0.01. The increase in [Ca2+]i after glutamate addition was 52 ± 22 nM (U), 15 ± 16 nM (M), p<0.05 and 17 ± 18 nM(T), p<0.01. The results show that both MK-801 and thapsigardin inhibit the increase in [Ca2+]i during normoxia and hypoxia, demonstrating that the increase in [Ca2+]i during cerebral hypoxia is both NMDA receptor-mediated and Ca2+-ATPase-dependent. We speculate that hypoxia-induced modification of the NMDA receptor leading to a calcium influx may initiate the inositol phosphate pathway and the release of Ca2+ from intrasynaptosomal stores. (Funded NIH # HD-20337)