Modulation by magnesium of N-methyl-D-aspartate receptors in developing human brain

AIM: To investigate age related alterations in glutamate N-methyl-D-aspartate (NMDA) receptor binding produced by the modulatory compounds glutamate, glycine, and magnesium (Mg2+) sulphate. METHODS: The effects produced by glutamate plus glycine, and Mg2+ on the binding of [3H]MK-801, a ligand for the N-methyl-D-aspartate ion channel phencyclidine site, were measured in membrane preparations made from prefrontal cortex from human neonate (n = 5), infant (n = 6), and adult (n = 6) necropsy brains. RESULTS: Neonatal brains had the least [3H]MK-801 binding, suggesting either a low density of NMDA receptors or a more restricted access of [3H]MK-801 to cation channel sites. Infant brains had the most [3H]MK-801 binding which was stimulated to a greater extent by L-glutamate (100 microM) and glycine (10 microM) than in neonatal and adult brains. MG2+ invariably inhibited [3H]MK-801 binding. However, the Mg2+ IC50 value was higher in neonatal brain (3.6 mM) than infant (1.4 mM) and adult (0.87 mM) brains. CONCLUSION: Infant brain may have excess NMDA receptors which are hyper responsive to glutamate and glycine. The lower potency of Mg2+ to inhibit [3H]MK-801 binding in neonatal cortex may be because newborn babies have NMDA receptors without the normal complement of Mg2+ sites. The findings suggest that therapeutic NMDA receptor block in neonates requires higher concentrations of magnesium sulphate in brain tissue.     

Long-term adrenalectomy decreases NMDA receptors in rat hippocampus

The effect of long-term adrenalectomy on NMDA receptors in the rat hippocampus was studied. Hippocampal sections of control and adrenalectomized rats were incubated with [3H]MK-801, a radiolabeled non-competitive inhibitor of the NMDA receptor. Analysis by in vitro autoradiography showed a significant decrease in [3H]MK-801 binding in the dentate gyrus, CA1 and CA4 areas, as well as the temporal cortex. Results of this study suggest that glucocorticoids are vital for the regulation of the NMDA receptors.     

Perivascular localization of nitric oxide synthase in the rat adenohypophysis: potential implications for function and cell-cell interaction

Effects of activation of protein kinase C (PKC) on N-methyl-D-aspartate) NMDA receptor function were analyzed by quantitative autoradiography using [3H]MK-801 in rat brain slices. The density of [3H]MK-801 binding was highest in hippocampus and high levels were found in cortex, striatum and thalamus. Levels in brainstem and molecular layer of cerebellum were low. The receptor binding was markedly decreased in almost all areas by addition of 2. 5 mM Mg2+. After activation of PKC by 100 nM phorbol-12, 13-dibutyrate (PDBu), [3H]MK-801 binding was increased in most areas, but binding levels were not changed in brainstem and cerebellum. The elevated [3H]MK-801 binding produced by PDBu was significantly inhibited by addition of Mg2+ except in inferior colliculus and cerebellum. These results suggest that activation of PKC potentiates NMDA receptor function in a region-specific manner in the rat brain.     

Binding characteristics of a potent AMPA receptor antagonist [3H]Ro 48-8587 in rat brain

A new AMPA receptor antagonist, Ro 48-8587, was characterized pharmacologically in vitro. It is highly potent and selective for AMPA receptors as shown by its effects on [3H]AMPA, [3H] kainate, and [3H] MK-801 binding to rat brain membranes and on AMPA- or NMDA-induced depolarization in rat cortical wedges. [3H]Ro 48-8587 bound with a high affinity (KD = 3 nM) to a single population of binding sites with a Bmax of 1 pmol/mg of protein in rat whole brain membranes. [3H]Ro 48-8587 binding to rat whole brain membranes was inhibited by several compounds with the following rank order of potency: Ro 48-8587 > 6-nitro-7-sulphamoylbenzo[f] quinoxaline-2,3-dione (NBQX) > YM 90K > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > quisqualate > AMPA > glutamate > kainate > NMDA. The distribution and abundance of specific binding sites (approximately 95% of total) in sections of rat CNS, revealed by quantitative receptor radioautography and image analysis, indicated a very discrete localization. Highest binding values were observed in cortical layers (binding in layers 1 and 2 > binding in layers 3-6), hippocampal formation, striatum, dorsal septum, reticular thalamic nucleus, cerebellar molecular layer, and spinal cord dorsal horn. At 1 nM, the values for specific binding were highest in the cortical layers 1 and 2 and lowest in the brainstem (approximately 2.6 and 0.4 pmol/mg of protein, respectively). Ro 48-8587 is a potent and selective AMPA receptor antagonist with improved binding characteristics (higher affinity, selectivity, and specific binding) compared with those previously reported.     

Effect of spinal cord transection on N-methyl-D-aspartate receptors in the cord

Spinal cord injury can lead to an exaggeration of transmission through spinal pathways, resulting in muscle spasticity, chronic pain, and abnormal control of blood pressure and bladder function. These conditions are mediated, in part, by N-methyl-D-aspartate (NMDA) receptors on spinal neurons, but the effects of cord injury on the expression or function of these receptors is unknown. Therefore, antibodies to the NMDA-R1 receptor subunit and binding of [3H]MK-801 were used to assess NMDA receptors in the spinal cord. Receptor density in rats with intact spinal cords was compared to that in rats 1 and 2 weeks after spinal cord transection (SCT) at the mid-thoracic level. At 1 and 2 weeks after SCT, [3H]MK-801 binding was reduced in most laminae in cord segments caudal to the injury, whereas no decrease in amount of R1 subunit immunoreactivity was observed. No significant changes in [3H]MK-801 binding and NMDA-R1 immunoreactivity could be seen rostral to the transection. Since [3H]MK-801 binding requires an open ion channel, the discrepancy between [3H]MK-801 binding and immunocytochemistry may indicate a loss of functional receptors without a consistent change in their total number. Therefore, the exaggerated reflexes that are well established in rats 2 weeks after cord injury must be mediated by a mechanism that withstands attenuation of NMDA receptor function.     

Activation of PKC reverses apparent NMDA receptor reduction in ALS

The binding of [3H]MK-801 to NMDA receptors was reduced by 40-45% in the dorsal and ventral horns of spinal cords from patients who died with amyotrophic lateral sclerosis (ALS) compared with controls. These results reflect either neurone death with concomitant receptor loss or regulation-related receptor decreases independent of motoneurone degeneration. To distinguish between these possibilities we explored aspects of NMDA receptor regulation using phorbol ester to activate protein kinase C (PKC). Spinal cord sections were exposed to phorbol ester before incubation with [3H]MK-801 to determine levels of NMDA binding. Phorbol ester treatment increased [3H]MK-801 binding in both ALS and control tissue to almost identical levels of specific binding for both groups. The increased [3H]MK-801 binding could be completely blocked by concurrent exposure of spinal cord sections to H-7, a general protein kinase inhibitor. These results suggest that NMDA receptors in ALS spinal cord are decreased as a result of abnormal enzyme activity independent of motoneurone degeneration.     

Role of NMDA receptors in pentobarbital tolerance/dependence

Effects of continuous pentobarbital administration on binding characteristics of [3H]MK-801 in the rat brain were examined by autoradiography. Animals were rendered tolerant to pentobarbital using i.c.v. infusion of pentobarbital (300 micrograms/10 microliters/hr for 7 days) by osmotic minipumps and dependent by abrupt withdrawal from pentobarbital. The levels of [3H]MK-801 binding were elevated in rats 24-hr after withdrawal from pentobarbital while there were no changes except in septum and anterior ventral nuclei in tolerant rats. For assessing the role of NMDA receptor in barbiturate action, an NMDA receptor antagonist (MK-801, 2.7 femto g/10 microliters/hr) was co-infused with pentobarbital. The pentobarbital-infused group had a shorter duration of pentobarbital-induced loss of righting reflex (sleeping time) than that of the control group, and MK-801 alone did not affect the righting reflex. However, co-infusion of MK-801 blocked hyperthermia, and prolonged the onset of convulsions induced by t-butylbicyclophosphorothionate (TBPS) in pentobarbital withdrawal rats. In addition, elevated [35S]TBPS binding was significantly attenuated by co-infusion with MK-801. These results suggest the involvement of NMDA receptor up-regulation in pentobarbital withdrawal and that the development of dependence can be attenuated by the treatment of subtoxic dose of MK-801.     

Differential effects of aging on binding sites of the activated NMDA receptor complex in mice

The NMDA receptor site has been shown to be vulnerable to the effects of aging. Decreases in binding to the receptor site of up to 50% have been reported in aged animals. The present study was designed to quantitate and compare the effects of aging on multiple binding sites of the NMDA receptor complex in various brain regions. Autoradiography with [3H]glutamate, [3H]CPP, [3H]glycine, [3H]MK801 and [3H]TCP was performed on brain sections from 3, 10 and 28-30 month old C57B1/6 mice. The percent declines between 3 and 28-30 months of age in [3H]-glutamate (15-35% declines) and [3H]CPP (20-42% declines) binding were similar within most cortical regions and the caudate nucleus but [3H]glutamate binding showed less change (0-11% declines) than [3H]CPP (13-27% declines) in the occipital/temporal cortex and hippocampal regions. [3H]MK801 and [3H]TCP binding, stimulated by 10 microM glutamate, exhibited intermediate aging changes between the glycine and NMDA sites, both in percent decline (3-28% and 0-26%, respectively) and in the number of brain regions involved. [3H]Glycine binding, stimulated by 10 microM glutamate, showed no significant overall effect of age (declines ranged from 0-34%). [3H]CPP binding was significantly more affected than [3H]glycine binding in many regions. These results suggest that aging has heterogeneous effects on different sites on the NMDA receptor complex throughout the brain and on NMDA receptor agonist versus antagonist binding in selected brain regions.     

Research on factors affecting neonatal tetanus and its prevention through immunization

This study examined [3H]MK-801 binding to the N-methyl-D-aspartate (NMDA) receptor in membranes prepared from cerebral cortex, hippocampus and corpus striatum of 3 week old rats exposed to 10 weeks of intermittent hypobaric hypoxia (4300 m; 450 Torr) and compared results with those of normoxic controls. The cortex, hippocampus and striatum of hypoxic animals had a 36, 35 and 31% reduction in binding sites (Bmax) and a 29, 32 and 17% decrease (reflecting increased affinity) in the dissociation constant (Kd) when compared to controls. In the cerebral cortex, both glutamate (100 microM) and glycine (10 microM) enhanced 3[H]MK-801 binding by two to 3-fold. Coagonist glutamate, however, had a higher EC50 (0.44 microM) in the hypoxic cortical membranes when compared to controls (0.28 microM). No significant differences were found in the EC50 of glycine. The results show that the NMDA receptor is altered in several brain regions of rats developing in a hypoxic environment.     

Unchanged [3H]MK-801 binding and increased [3H]flunitrazepam binding in turtle forebrain during anoxia

In order to determine if functional changes in N-methyl-D-aspartate receptors and GABAA receptors play a role in the remarkable anoxia tolerance of freshwater turtle brain, we used autoradiographic techniques to assay [3H]MK-801 and [3H]flunitrazepam binding in turtle forebrain after turtles had been subjected to anoxia for 2 or 6 h. The effects of glutamate, glycine, competitive N-methyl-D-aspartate antagonists, glycine antagonists, polyamines, magnesium, and zinc on [3H]MK-801 binding were the same in anoxic and control turtle forebrains. These results indicate that NMDA receptor regulation plays no role in the adaptive responses to anoxia in turtle brain. In contrast, [3H]flunitrazepam binding was significantly increased in the anoxic dorsal cortex and striatum. The most parsimonious explanation for elevated benzodiazepine receptor binding is that the rise in extracellular GABA levels known to accompany anoxia enhances benzodiazepine receptor affinity. It is possible, however, that GABAA receptor upregulation during anoxia increases the effectiveness of the inhibitory action of released GABA and contributes to the anoxia tolerance of turtles.     

Global cerebral ischemia and reperfusion alters NMDA receptor binding in canine brain

We employed a canine model to test whether binding to the N-methyl-D-aspartate (NMDA) class of glutamate receptor channels is altered by global cerebral ischemia and/or reperfusion. Ischemia was induced by 10-min cardiac arrest, followed by restoration of spontaneous circulation for periods of 0, 0.5, 2, 4, and 24 h. In vitro autoradiography was performed on frozen brain sections with three radioligands: [3H]glutamate (under conditions to label the NMDA site), [3H]glycine, and [3H]MK-801. Modest decreases in [3H]glutamate and [3H]MK-801 binding were seen in several regions of hippocampus, and parietal and temporal cortex at early times after reperfusion, with values returning toward control by 24 h. In the striatum, a different pattern was seen: [3H]glutamate and [3H]MK-801 binding increased 50-200% at 0.5-4 h after the start of reperfusion, returning toward control levels by 24 h. These increases correlate with findings of increased sensitivity to NMDA-stimulated release of dopamine from striatal tissue in the same model (Werling et al., 1993), and suggest that changes in tissue receptors may contribute to the selective vulnerability to ischemic damage during the first hours following reperfusion.     

Chronic ethanol increases N-methyl-D-aspartate-stimulated nitric oxide formation but not receptor density in cultured cortical neurons

The effects of prolonged ethanol exposure on excitatory amino acid receptor stimulated nitric oxide (NO) formation were examined in primary rat cortical neuronal cultures. Chronic ethanol (4 days, 100 mM) potentiated N-methyl-D-aspartate (NMDA)-stimulated NO formation as determined by measuring the conversion of [3H]arginine to [3H]citrulline. In contrast, chronic ethanol had no effect on NO formation stimulated by kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxalonepropionic acid, or the calcium ionophore ionomycin. Potassium chloride-stimulated NO formation was also enhanced by chronic ethanol treatment, but this effect was not seen in the presence of the ionotropic glutamate receptor antagonists MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione. Immunoblot analysis of expression of NR1, NR2A, and NR2B receptor subunits showed no difference between control and chronic ethanol-treated cultures. In support of this apparent lack of change in receptor density, there was no difference in the specific binding of 125I-MK-801 between control and chronic ethanol-treated groups. These results demonstrate that prolonged ethanol exposure selectively enhanced NMDA receptor-stimulated NO formation, which may play an important role in alcohol dependence, withdrawal, and alcohol-associated brain damage. These results also suggest that chronic ethanol-induced increases in NMDA receptor function may not be due to a simple increase in the number of NMDA receptors or change in NMDA receptor subunit composition but may instead reflect more complicated and subtle changes.     

Aromatic analogs of arcaine inhibit MK-801 binding to the NMDA receptor

Aromatic analogs of arcaine were shown to have inhibitory effects on the binding of the channel blocking drug [3H]MK-801 to the NMDA receptor complex. The most potent compound of the series was an N,N'-bis(propyl)guanidinium which inhibited [3H]MK-801 binding with an IC50 of 0.58 microM and an IC50 of 12.17 microM upon addition of 100 microM spermidine. The increase in IC50 upon addition of spermidine suggests competitive antagonism between the inhibitor and spermidine at the arcaine-sensitive polyamine site of the NMDA receptor complex.     

Chronic treatment with MK-801 decreases D2 dopamine receptor function in rat striatum

Present results show that a single treatment with dizocilpine (MK-801, 0.25 mg/kg IP) failed to modify the specific binding to D1 or D2 DA receptors. In contrast, repeated administrations for 3 weeks resulted in a statistically significant decrease of [3H]Spiroperidol binding to cortical or striatal membranes but did not change the number or the apparent affinity of [3H]MK-801 binding in well-washed cortical membranes. Consistent reduction in specific D2 receptor mediated behavior was obtained. The data suggest that the changes in DAergic function following repeated administrations with MK-801 could be suggestive of potential therapeutic uses of negative allosteric drugs in some DA related dysfunctions.     

Up-regulation of brain N-methyl-D-aspartate receptors following multiple intracerebroventricular injections of [D-Pen2, D-Pen5] enkephalin and [D-Ala2, Glu4]deltorphin II in mice

The effects of chronic administration of [D-Pen2, D-Pen5]enkephalin and [D-Ala2, Glu4]deltorphin II, the selective agonists of the delta 1- and delta 2-opioid receptors, on the binding of [3H]MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [D-Pen2, D-Pen5]enkephalin or [D-Ala2, Glu4]deltorphin II (20 micrograms/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [3H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM-concentration, the binding of [3H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [D-Pen2, D-Pen5]enkephalin treated mice. In [D-Ala2, Glu4]deltorphin II treated mice, the binding of [3H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the Bmax value of [3H]MK-801. It is concluded that tolerance to delta 1- and delta 2-opioid receptor agonists is associated with up-regulation of brain N-methyl-D-aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N-methyl-D-aspartate receptors antagonists block tolerance to the analgesic action of delta 1- and delta 2-opioid receptor agonists.     

Ischemia-induced neuronal cell loss is associated with loss of atypical angiotensin type-1 receptor expression in the gerbil hippocampal formation

The hippocampal formation of Mongolian gerbils expresses high amounts of atypical angiotensin II type-1 receptors. We studied the expression of these receptors by in situ hybridization using specific [35S]-labeled riboprobes and by receptor autoradiography using [125I]Sarcosine1-angiotensin II. Angiotensin II receptor mRNA was found in the pyramidal cell layer of the CA1, CA2 and CA3 subfields, with the highest expression in the CA2 subfield, and in the granular cell layer of the dentate gyrus. Angiotensin II binding was detected in the stratum oriens and stratum radiatum of the CA1 and CA2 subfields, in the stratum oriens of the CA3 subfield, and in the molecular layer of the dentate gyrus. We then studied the effect of ischemia on hippocampal angiotensin II receptor expression, 1, 4 and 15 days after bilateral occlusion of the common carotid arteries for 5 min. No changes in angiotensin II receptor mRNA or binding were detected 1 day after ischemia. Delayed, progressive loss of angiotensin II mRNA and binding occurred 4 and 15 days after ischemia, in the CA1, CA2 and CA3 subfields. The decline was faster in the CA1 subfield, and paralleled the loss of neurons after ischemia. In the dentate gyrus, angiotensin II receptor mRNA and angiotensin II binding were not changed when compared to sham operated controls. The decrease of angiotensin II receptor expression may reflect the loss of angiotensin II receptor-producing neurons rather than a down-regulation of receptor expression.     

Chronic ethanol-mediated up-regulation of the N-methyl-D-aspartate receptor polypeptide subunits in mouse cortical neurons in culture

The goal of this study was to determine whether chronic ethanol-mediated up-regulation of the N-methyl-D-aspartate receptors (NMDAR) was associated with an augmentation of the NMDAR polypeptide subunits in the mammalian cortical neurons. The results show that chronic ethanol treatment produced an increase in the R1 and R2B polypeptide subunits. The R2A subunit was not expressed in these neurons. Chronic NMDAR antagonist ((+)-3-2-(carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP)) treatment also increased the R1 and R2B polypeptide subunits. A similar increase was observed when ethanol and CPP were used in combination. Binding studies using [3H]MK-801 ((+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclophenptan-5,10-imine maleate), a noncompetitive NMDAR antagonist, confirmed that concomitant exposure of ethanol and CPP up-regulated the NMDAR. Our results demonstrate for the first time that chronic ethanol treatment increased the NMDA receptor polypeptide subunit synthesis and that it was associated with an increase in [3H]MK-801 binding sites.     

Structure-activity studies of conantokins as human N-methyl-D-aspartate receptor modulators

The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [3H]MK-801 binding to human glutamate-N-methyl-D-aspartate (NMDA) receptors, and their structures have been examined using CD and 1H NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [3H]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8, D17], and con-G[A7] have similar conformations in the presence of Ca2+. This consists of a helix for residues 2-16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7-10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [3H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca2+ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca2+-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.     

Regulation of cerebral microvessels by glutamatergic mechanisms

We studied changes in glutamate receptors, expression of immediate early genes, and AP-1 DNA binding activity in the brains of phenobarbital (PB)-dependent and -withdrawn rats to investigate the possible involvement of activation of glutamate receptors in PB withdrawal syndrome. PB-dependent rats were prepared by feeding drug-admixed food for 5 weeks. Autoradiographic analysis showed that binding of [3H(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imin e (MK-801), an antagonist of N-methyl-D-aspartic acid (NMDA) receptors, increased significantly in the cerebral cortices of PB-dependent and 24-h-withdrawn rats. However, [3H]MK-801 binding in the hippocampus and [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [3H]kainic acid binding in the hippocampus and cerebral cortex were essentially unchanged in both groups. PB withdrawal seizures were followed by increased expression of c-fos mRNA in the hippocampus and cerebral cortex and of c-jun mRNA in the cerebral cortex. The induction of c-fos and c-jun mRNA was suppressed by administration of MK-801. Furthermore, PB withdrawal enhanced AP-1 DNA binding activity in the brain. The present findings suggest functional enhancement of glutamatergic neurotransmission during the development of PB withdrawal syndrome.     

Characterization of ionotropic glutamate receptor-mediated nitric oxide production in vivo in rats

BACKGROUND AND PURPOSE: Glutamate receptor activation can stimulate nitric oxide (NO) production and possibly play a role in long-term potentiation and excitotoxic-mediated injury. We studied the differential effect of agonist-induced activation of ion channel-linked N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subtypes on NO production in vivo in rat hippocampus. We also studied whether dantrolene, a ryanodine calcium channel inhibitor previously shown to attenuate metabotropic glutamate receptor stimulation of NO production, also attenuated ionotropic glutamate receptor-mediated stimulation of NO production. METHODS: Microdialysis probes were placed bilaterally into the CA3 region of the hippocampus of pentobarbital-anesthetized adult Sprague-Dawley rats and were perfused for 5 hours with artificial cerebrospinal fluid (CSF) containing 3 mumol/L [14C]L-arginine. Recovery of [14C]L-citrulline in the effluent was used as a marker of NO production. In 13 groups of rats, increases in [14C]L-citrulline recovery were compared between right- and left-sided probes perfused with no additional drugs versus combinations of NMDA, AMPA, the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), the non-competitive glutamate receptor blocker MK-801, the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and dantrolene. RESULTS: Recovery of [14C]L-citrulline during perfusion with artificial CSF progressively increased to 272 +/- 73 fmol/min (+/-SEM) over 5 hours. Contralateral perfusion with 1 mmol/L L-NAME inhibited [14C]L-citrulline recovery. Perfusion with 1 mmol/L MK-801 or 1 mmol/L CNQX reduced [14C]L-citrulline recovery compared with contralateral perfusion with CSF alone. Perfusion with 1 mmol/L NMDA enhanced [14C]L-citrulline recovery, and this enhancement was attenuated by L-NAME, MK-801, and CNQX but not by dantrolene. Perfusion with 1 mmol/L AMPA enhanced [14C]L-citrulline recovery, and this enhancement was also attenuated by L-NAME, MK-801, and CNQX but not by dantrolene. CONCLUSIONS: Through an indirect method of assessing NO production in vivo, results with MK-801 and CNQX indicate that NMDA and AMPA receptor activation contribute to basal NO production in the rat hippocampus. Enhanced NO production with NMDA and AMPA agonists appears to involve a complex neuronal interaction because the effect of NMDA was attenuated by both MK-801 and CNQX and because the effect of AMPA was attenuated by both CNQX and MK-801. In contrast to metabotropic glutamate receptor activation, release of calcium from intracellular ryanodine calcium channels does not appear to be a prominent mediator of ionotropic glutamate receptor stimulation of NO production.