N-methyl-D-aspartate Receptor-channel Complex Research Articles

The Anesthetic Cascade

The Anesthetic Cascade

Selective interaction of nitric oxide synthase inhibition with phencyclidine: behavioural and NMDA receptor binding studies in the rat

The psychotomimetic drugs, phencyclidine (PCP) and MK-801, are non-competitive antagonists of the N-methyl- d-aspartate (NMDA) receptor and used as pharmacological tools to mimic a possible NMDA receptor hypofunction in schizophrenia. These drugs were tested in two behavioural paradigms in the present study: prepulse inhibition (PPI) of acoustic startle and locomotor activity (LMA) in an open field. Recent studies show that several behavioural and biochemical effects of PCP are blocked by nitric oxide synthase (NOS) inhibition. Hence, it is likely that some effects of PCP are mediated via an increase in NO production, an assumption not in accordance with the NMDA receptor antagonistic effect of PCP. Experiments were conducted in rats to further elucidate the involvement of NO-dependent mechanisms in the effects of PCP and MK-801, and how these effects may involve the NMDA receptor. The NOS inhibitor N G-nitro- l-arginine methyl ester ( l-NAME) (10 mg/kg) normalised the disruptive effect of PCP (2 mg/kg) on PPI and the stimulatory effect of PCP (4 mg/kg) on LMA. In contrast to these observations, the deficit in PPI induced by MK-801 (0.1 mg/kg) was not affected by l-NAME (10, 20 or 40 mg/kg). MK-801 (0.15 mg/kg)-induced hyperlocomotion was not affected by l-NAME (10 mg/kg), but attenuated by l-NAME (40 mg/kg). Furthermore, receptor binding studies aimed at investigating the influence of l-NAME on the binding of PCP to the MK-801-sensitive NMDA receptor binding site failed to show such an influence. These results suggest that the NO-sensitive effects of PCP are not sufficiently explained by its antagonistic effect at the NMDA receptor channel complex.

Removal of NMDA receptor Mg(2+) block extends the action of NT-3 on synaptic transmission in neonatal rat motoneurons.

NT-3 has previously been reported to enhance AMPA/kainate receptor-mediated synaptic responses in motoneurons via an effect on the N-methyl-D-aspartate (NMDA) receptor. To investigate neurotrophin-3 (NT-3) action further, we measured the NMDA receptor (NMDAR)-mediated synaptic response directly by intracellular recording in motoneurons after blocking AMPA/kainate, GABA(A), GABA(B) and glycine receptor-mediated responses pharmacologically. Two pathways were stimulated, the segmental dorsal root (DR) and the descending ventrolateral fasciculus (VLF). The DR-evoked NMDAR-mediated response in motoneurons of rats younger than 1 wk has two components, the initial one of which is generated monosynaptically. NT-3 strongly potentiated both NMDA components in a rapidly reversible manner. No NMDAR-mediated responses were present at VLF connections and at DR connections in older (1- to 2-wk-old) neonates. Bath-applied NT-3-induced potentiation of the AMPA/kainate receptor-mediated response occurred only at connections that exhibit a synaptic NMDA receptor-mediated response. Reducing Mg(2+) concentration in the bathing solution restored the NMDAR-mediated response elicited by DR stimulation in older neonates and by VLF throughout the neonatal period (0-2 wk). In low-Mg(2+), NT-3 enhanced AMPA/kainate receptor-mediated responses elicited by inputs normally not influenced by NT-3. Thus a major reason for the loss of NT-3 action on AMPA/kainate synaptic responses is the reduced activity of the NMDA receptor due to developing Mg(2+) block of NMDA receptor-channel complex as the animal matures, and both can be re-established by reducing Mg(2+) concentration in fluid bathing the spinal cord.

Developmental maturation of the N-methyl-d-aspartic acid receptor channel complex in postnatal rat brain

The N-methyl-d-aspartate (NMDA) receptor plays an important role in developmental plasticity. Previous studies have reported differences between the NMDA receptor-channel complex in the rat pup brain and the adult brain. In the present study, modulation of the NMDA channel complex as a function of age was measured to determine when the temporal switching of the NMDA receptor from the immature form to the adult mature form takes place. [3H]MK-801 binding was measured in the rat forebrain from postnatal day 1 to day 21. Our data suggest the presence of two types of NMDA receptors — an immature type and a mature type. The immature NMDA receptor, seen during the early postnatal period (day 1–day 14) is highly sensitive to spermidine, l-glutamate alone potentiates [3H]MK-801 binding, and glycine failed to potentiate an l-glutamate-induced increase in [3H]MK-801 binding. During the late postnatal period (after day 14) spermidine alone did not increase [3H]MK-801 binding as potently as it did during the early postnatal period, high-affinity [3H]MK-801 binding was not seen in the presence of l-glutamate alone, and l-glutamate and glycine or l-glutamate and spermidine or l-glutamate, glycine and spermidine together, significantly increased [3H]MK-801 binding in a manner similar to that reported in the adult brain. Together, the pharmacology of the NMDA receptor during the early postnatal period differs from the adult-like receptor seen during the late postnatal period, and that in rats the apparent switching of the NMDA receptor from the immature type to the mature type takes place after the second postnatal week.

NMDA receptor subunits in the postsynaptic density of rat brain: expression and phosphorylation by endogenous protein kinases

N-methyl- d-aspartate (NMDA) receptors (NRs) play critical roles in diverse synaptic processes in the brain. However, subcellular distribution, spatiotemporal expression and regulation of NR subunits in brain synapses are unknown. We report that NR1 and NR2A–2C subunits are all enriched in the postsynaptic density (PSD), which plays critical roles in trophin-mediated synaptic plasticity. Significant expression of NRs was observed the first two weeks after birth, during synaptogenesis, and in adulthood. Functional diversity of NRs, resulting from heterogeneous composition, was supported by the finding that different NR2 subunits were associated in a region-specific manner with NR1. Phosphorylation of NR1, a key subunit of the NMDA receptor-channel complex, was significantly enhanced by activators of calmodulin (CaM) kinases (CKs) or protein kinase C (PKC), but not by those of PKA. Co-immunoprecipitation studies revealed that NR1 was physically associated with functionally active PKCγ and the major PSD protein (mPSDp) through noncovalent interactions. Our results suggest that NMDA receptors play roles in postsynaptic mechanisms in a subunit-, composition-, brain region- and developmental-specific manner. Our findings also indicate that the PSD is a coherent functional unit containing protein kinases that potentially regulate NMDA receptor function via phosphorylation.

Regulation of NMDA-induced [3H] dopamine release from rat hippocampal slices through sigma-1 binding sites

To examine the interaction between ionotropic glutamate receptors and s binding sites, we made use of [3H] dopamine release from rat hippocampal slices. Agonists for ionotropic glutamate receptors such as N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate evoked release of [3H] dopamine from rat hippocampal slices, in a dose-dependent manner. (+ )-Pentazocine, a prototype s1 agonist, attenuated the NMDA-induced [3H] dopamine release dose-dependently and significantly as did non-competitive NMDA antagonists such as 5-methyl-10,11-dihydro-5H-dibe-nzo(a,b)cyclohepten-5,10-imine maleate (MK-801) and phencyclidine. In contrast, (+)-pentazocine had no effect on AMPA- or on kainate-induced [3H] dopamine release. Sigma-l receptor antagonists including N,N-dipropyl-2- 4-methoxy-3-(2-phen-ylethoxy)phenyl ethylamine monohydrochloride (NE-100), 1(cyclopropylmethyl)-4-(2’-(4”fluorophenyl)-2’-oxoethylpiperidine (DuP734) and 1-(cyclopropylmethyl)-4-(2’,4” -cianophenyl)-2’-oxoethyl)-piperidine hydrobromide (XJ448) prevented significantly the inhibitory effect of (+)-pentazocine on NMDA-induced [3H] dopamine release, without affecting the release of [3H] dopamine evoked by NMDA. The inhibitory effect of (+)-pentazocine on [3H] dopamine release was preserved even in the presence of tetrodotoxin. These results suggest that s1 binding sites selectively interact with the NMDA receptor channel complex among ionotropic glutamate receptors, and that s1 binding sites may be involved in modulating the release of dopamine in the rat hippocampus by interacting with the NMDA receptor on dopaminergic nerve terminal. © 1998 Elsevier Science Ltd. All rights reserved.

3H]5,7-Dichlorokynurenic Acid Recognizes two Binding Sites in Rat Cerebral Cortex Membranes

Binding of [3H]5,7-dichlorokynurenic acid ([3H]DCKA), a competitive antagonist of the strychnine-insensitive glycine site of the N-methyl-D-aspartate (NMDA) receptor channel complex, was characterized in synaptic plasma membranes from rat cerebral cortex. Non linear curve fitting of [3H]DCKA saturation and homologous displacement isotherms indicated the existence of two binding sites: a specific, saturable, high affinity site, with a pKD value of 7.24 (KD = 57.5 nmol/1) and a maximum binding value (Bmax) of 6.9 pmol/mg of protein and a second site, with micromolar affinity. The pharmacological profile of both binding components was determined by studying the effect on [3H]DCKA and [3H]glycine binding of a series of compounds known to interact with different excitatory and inhibitory amino acid receptors. These studies confirmed the identity of the high affinity site of [3H]DCKA binding with the strychnine-insensitive glycine site of the NMDA receptor channel complex. 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt (GV 150526A), a new, high affinity, selective glycine site antagonist (1), was the most potent inhibitor of this component of binding (pKi = 8.24, Ki = 5.6 nmol/1). The low affinity component of [3H]DCKA binding was insensitive to the agonists glycine and D-serine and the partial agonist (±)-3-amino-1-hydroxy-2-pyrrolidone (HA 966), though recognised by glycine site antagonists. The precise nature of this second, low affinity [3H]DCKA binding site remains to be elucidated.

Altered NMDA Sensitivity and Learning Following Chronic Developmental NMDA Antagonism

We have previously shown that chronic developmental administration of N-methyl- D-aspartate (NMDA) antagonists reduces synaptic development; however, on withdrawal from NMDA antagonism, there is a rebound period during which synaptogenesis exceeds control levels. The current research was undertaken to explore this period of withdrawal, using the noncompetitive antagonist phencyclidine (PCP), examining 2 behavioral measures in which the NMDA receptor is implicated: 1. NMDA-induced seizures, and 2. learning and memory in the Morris water maze. Using a protocol identical to that previously used to examine synaptic development, male Long-Evans rats were given 1 daily SC injection of either 10 mg/kg PCP or its physiological saline vehicle for a period of 15 days, beginning on postnatal Day 5 (P5) and ending on P20. Animals were then assessed for either sensitivity to NMDA-induced seizures on P21, P26, P36, or P56, or they were assessed for their acquisition performance and initial heading in the Morris water maze on P23, P26, P30, P38, and P75. Chronic treatment with PCP resulted in greater behavioral ratings of seizure activity after NMDA administration, observed 1 (P21), 5 (P26), and 15 (P36) days after the last injection of PCP, indicating increased sensitivity of the NMDA receptor/channel complex during this period after withdrawal from developmental NMDA antagonism. PCP-treated animals also required significantly more trials to reach criterion in the Morris water maze on P23, P26, and P30, and displayed significantly less accurate initial swim headings on all test days. The results are discussed in terms of the role of the NMDA receptor-channel complex in development and learning/memory processes.

Competitive NMDA and Strychnine-Insensitive Glycine-Site Antagonists Disrupt Prepulse Inhibition

Prepulse inhibition (PPI) is thought to reflect the operation of a sensorimotor gating system in the brain. Sensorimotor gating abnormalities have been identified in schizophrenic patients, and various neural systems are involved in this function. To study the modulation of the sensorimotor gating system by the N-methyl- d-aspartate (NMDA) receptor–channel complex, the effects of noncompetitive and competitive NMDA antagonists on PPI were examined in rats. PPI was not disrupted by CGS 19755, a competitive NMDA antagonist, at 30 min after subcutaneous (SC) administration. However, CGS 19755 (40 mg/kg SC) decreased PPI at 120 min after administration with a marked decrease of startle amplitude. Late onset of the effect of CGS 19755 was also observed in the increase of spontaneous locomotor activity (SLA). On the other hand, phencyclidine, a noncompetitive NMDA antagonist, disrupted PPI at 30 min after administration and increased SLA from 20 min after administration. PPI was also disrupted by bilateral intracerebroventricular administration of 5,7-dichlorokynurenate (10 and 20 μg/side × 2), an antagonist at the strychnine-insensitive glycine receptor, which is an allosteric binding site in the NMDA receptor–channel complex. It is concluded that the NMDA receptor–channel complex plays an important role in regulation of PPI.

The influence of nimodipine and MK-801 on the brain free arachidonic acid level and the learning ability in hypoxia-exposed rats

1. 1. The influence of voltage dependent calcium channel blocker (VDCC), nimodipine and N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 on the brain free arachidonic acid (FAA) level and on the learning ability in hypoxia-exposed rats was examined. 2. 2. Some animals were decapitated after cerebral hypoxia had been obtained and the brain FAA level was determined by gas chromatography.The other animals were trained in a passive avoidance procedure and were exposed to hypoxic conditions immediately after the learning trial response had been acquired. A passive avoidance retention test was performed 24 hours later. 3. 3. Various doses of nimodipine (0.03;0.1;0.3 and 1.0 mg/kg) and MK-801 (0.03;0.1 and 0.3 mg/kg) had been injected 30 minutes before biochemical or behavioral procedures started. 4. 4. It was found that hypoxia strongly increased the brain FAA level and impaired the retention of the passive avoidance response. 5. 5. Pretreatment with 0.3 mg/kg and 1.0 mg/kg of nimodipine prevented the brain FAA accumulation. It has also been shown that all tested doses of nimodipine significantly improved the retention deficit in the animals exposed to hypoxia. 6. 6. Neither the one of tested doses of MK-801 influenced significantly the increase of the brain FAA level and/or passive avoidance behavior in hypoxic animals. 7. 7. These results confirm the hypothesis that the brain FAA accumulation and cognitive impairment, caused by hypoxia, are maybe associated with disturbances in calcium homeostasis and that nimodipine may be useful in ameliorating the hypoxia-induced brain tissue damage. Blocade of NMDA receptor-channel complex by MK-801 was not sufficient to prevent hypoxia-induced neuronal damage.

Regionally different N-methyl-D-aspartate receptors distinguished by ligand binding and quantitative autoradiography of [3H]-CGP 39653 in rat brain.

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.

Neuroprotective effects of RPR 104632, a novel antagonist at the glycine site of the NMDA receptor, in vitro

The NMDA antagonist and neuroprotective effects of RPR 104632 (2 H-1,2,4-benzothiadiazine-1-dioxide-3-carboxylic acid), a new benzothiadiazine derivative, with affinity for the glycine site of the NMDA receptor-channel complex are described. RPR 104632 antagonized the binding of [ 3H]5,7-dichlorokynurenic acid to the rat cerebral cortex, with a K i of 4.9 nM. This effect was stereospecific, since the (-)-isomer was 500-fold more potent than the (+)-isomer. The potent affinity of RPR 104632 for the glycine site was confirmed by the observation that RPR 104632 inhibited [ 3H] N-(1-(2-thienyl)cyclohexyl]-3,4-piperidine ([ 3H]TCP) binding in the presence of N-methyl- d-aspartate (NMDA) (IC 50 = 55 nM), whereas it had no effect on the competitive NMDA site or on the dissociative anaesthetic site. RPR 104632 inhibited the NMDA-evoked increase in guanosine 3′,5′-cyclic monophosphate (cGMP) levels of neonatal rat cerebellar slices (IC 50 = 890 nM) in a non-competitive manner and markedly reduced NMDA-induced neurotoxicity in rat hippocampal slices and in cortical primary cell cultures. These results suggest that RPR 104632 is a high-affinity specific antagonist of the glycine site coupled to the NMDA receptor channel with potent neuroprotective properties in vitro.

Blockade by sigma site ligands of N‐methyl‐D‐aspartate‐evoked responses in rat and mouse cultured hippocampal pyramidal neurones

1. The effects of a range of structurally-dissimilar compounds which possess affinity for sigma binding sites were examined on the responses of cultured hippocampal pyramidal neurones to the excitatory amino acid analogues N-methyl-D-aspartate (NMDA), kainate and (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). 2. In mouse hippocampal neurones under whole-cell voltage-clamp, the compounds tested reversibly attenuated NMDA-, but not kainate- or AMPA-, evoked currents with a rank order potency (IC50 values in microM): ifenprodil (0.8) > (+)-N-allylnormetazocine (1.1) > dextromethorphan (1.8) = haloperidol (1.9) > (+)-pentazocine (7.2) > 1S,2R-(-)-cis-N-methyl-N-[2-(3, 4-dichlorophenyl) ethyl]-2-(1-pyrrolidinyl)cyclohexylamine (17) = rimcazole (18) > 1,3-di(2-tolyl)guanidine (37) > opipramol (96) > caramiphen (110) = carbetapentane (112) > > (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (485). 3. The attenuation of NMDA-evoked responses was not mediated through interactions with the agonist, glycine (except haloperidol) or polyamine (except ifenprodil) binding sites on the NMDA receptor-channel complex but, in the light of the voltage- and, in some cases, use-dependent nature of their antagonism, an interaction with the ion channel appears to be a likely mechanism of action for many of the compounds. 4. Micromolar concentrations of selected sigma site ligands also reduced NMDA-evoked rises in intracellular free calcium concentration in Fura-2-loaded cultured hippocampal neurones of the rat with the same rank order potency as observed in the electrophysiological studies. 5. The data indicate that, at micromolar concentrations, the sigma site ligands tested act as NMDA receptor antagonists, an action which does not appear to be mediated by high-affinity sigma binding site(s). The functional effects of micromolar concentrations of sigma site ligands cannot, therefore, be attributed exclusively to interactions with high-affinity sigma binding sites.

The actions of L-687,384, a σ receptor ligand, on NMDA-induced currents in cultured rat hippocampal pyramidal neurons

The actions of the σ receptor ligand L-687,384 were studied on N- methyl- d - aspartate (NMDA)-induced currents recorded from outside-out patches obtained from cultured rat hippocampal pyramidal neurons and on NMDA-evoked rises in [Ca 2+] i in the same preparation. L-687,384 did not change the magnitudes of unitary NMDA currents or frequency of channel-openings but diminished channel-open probability by decreasing mean open time. This action was consistent with a voltage-dependent open-channel block of the NMDA channel by L-687,384 with a blocking rate constant of 5.9 × 10 6 M −1s −1 at −80 mV. L-687,384 also reduced NMDA-evoked rises in [Ca 2+] i in Fura-2-loaded neurons with an apparent IC 50 value of 49±8 μM. The results demonstrate that L-687,384 acts as an antagonist at the NMDA receptor-channel complex.

Patch clamp studies on the kinetics and selectivity of [formula omitted] receptor antagonism by memantine (1-amino-3,5-dimethyladamantan)

Memantine (1-amino-3,5-dimethyladamantan) was tested as an antagonist of N- methyl- d-aspartate (NMDA) receptors on cultured superior collicular and hippocampal neurones using the patch clamp technique and its actions were compared to those of Mg 2+ ions, ketamine, dextrorphan, dextromethorphan, phencyclidine and dizocilpine (MK-801). Memantine (2–33 μM) concentration-dependently antagonized responses to NMDA 100 μM with an IC 50 of 2.92 ± 0.05 μM. In contrast, current responses to (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( l-AMPA 50–100 μM) and γ-amino butyric acid (GABA 10 μM) were unaffected by Memantine 8 μM. Memantine 8 μM caused a non-parallel shift of the NMDA concentration-response curve to the right in a manner indicative of uncompetitive open channel block. The effects of memantine were similar to ketamine in that both antagonists were weakly use- and strongly voltage-dependent. In contrast, MK-801, phencyclidine and dextrorphan showed much slower kinetics that was reflected in their marked use- and weaker voltage-dependency. The antagonistic effects of memantine were not reversed by increasing concentrations of glycine (0.1–100 μM) ruling out the possibility of an interaction of memantine with the strychnine-insensitive glycine modulatory site associated with the NMDA receptor-channel complex. Memantine (1–100 μM) also selectively antagonized responses to NMDA (40 μM) in the cortical wedge preparation with IC 50 of 12.9 ± 1.5 μM.

Antisense oligodeoxynucleotides to NMDA-R1 receptor channel protect cortical neurons from excitotoxicity and reduce focal ischaemic infarctions.

The excitatory amino acid, L-glutamate, acting through its N-methyl-D-aspartate (NMDA) receptor, may contribute to neuronal death following cerebral vascular occlusion. In support of this hypothesis, NMDA receptor antagonists reduce the volume of infarction produced by occlusion of the middle cerebral artery in vivo and attenuate Ca2+ influx and neuronal death elicited by L-glutamate or NMDA in vitro. A complementary DNA coding for a major component of the NMDA receptor channel complex, a single protein of M(r) 105.5K (NMDA-R1), has been isolated from rat brain. Here we demonstrate that inhibition of the synthesis of NMDA-R1 by treatment with antisense oligodeoxynucleotides selectively reduces the expression of NMDA receptors, prevents the neurotoxicity elicited by NMDA in vitro and reduces the volume of the focal ischaemic infarction produced by occlusion of the middle cerebral artery in the rat.

Haloperidol interacts with the strychnine-insensitive glycine site at the NMDA receptor in cultured mouse hippocampal neurones

N-Methyl-D-aspartate (NMDA)-evoked responses in voltage-clamped hippocampal neurones in culture were reversibly, but not completely, attenuated on superfusion with micromolar concentrations (0.1–100 μM) of haloperidol with an IC 50 (± S.E.M.) value of 1.9 ± 0.2 μM (n = 7). In contrast, kainate- and (RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-evoked responses were relatively unaffected on application of 50 μM haloperidol. The NMDA receptor antagonist action of haloperidol was neither competitive in nature nor voltage-dependent but was reduced upon elevation of the extracellular concentration of glycine. Furthermore, in the absence of added glycine haloperidol (at 0.1 μM) frequently potentiated NMDA-evoked responses. Haloperidol thus appears to be a partial agonist for the strychnine-insensitive glycine site associated with the NMDA receptor-channel complex.

Ketamine-induced anesthesia involves the [formula omitted] receptor-channel complex in mice

The role of the N- methyl- d-aspartate (NMDA) receptor-channel complex in ketamine-induced anesthesia was examined in mice. General anesthetic potencies were evaluated on a rating scale, which provided the data for anesthetic scores, loss of righting reflex, sleeping time and recovery time. All drugs were administered intraperitoneally. NMDA (60–300 mg/kg), an NMDA receptor agonist, dose-dependently antagonized the general anesthetic potencies of ketamine at a dose of 100 mg/kg which produced loss of righting reflex in more than 90% of the mice. On the other hand, a high dose of N- methyl- l-aspartate (400 mg/kg), a stereoisomer of NMDA, did not. A dose of 300 mg/kg of NMDA significantly shifted the dose-response curve of ketamine for loss of righting reflex to the right. A high dose of d-cycloserine (200 mg/kg), an agonist at the glycine site on the NMDA receptor complex, slightly but significantly shortened the sleeping time caused by ketamine (100 mg/kg). However, neither a critical subconvulsive dose of kainate (15 mg/kg), a kainate receptor agonist, nor a subsconvulsive dose of quisqualate (120 mg/kg), an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor agonist, reversed general anesthesia induced by 100 mg/kg of ketamine. Both the competitive NMDA antagonist, (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; 10–30 mg/kg), and the non-competitive NMDA antagonist, MgCl 2 (30–120 mg/kg), dose-dependently potentiated the anesthetic actions induced by a lower dose of ketamine (70 mg/kg) which produced loss of righting reflex in less than 20% of the mice, but (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine hydrogen maleate (MK-801; 0.3–3.0 mg/kg), another potent non-competitive NMDA antagonist, did not. However, pretreatment with a low dose of haloperidol (0.1 mg/kg), a dopamine receptor antagonist, along with 0.3 mg/kg of MK-801 (which in itself elicited only stimulant actions), significantly increased the anesthetic potencies of ketamine at 70 mg/kg. Pharmacokinetic studies revealed that NMDA (300 mg/kg) and CPP (30 mg/kg) did not significantly alter the level of ketamine in the brain. These results suggest that ketamine-induced anesthesia is mediated, at least in part, by the NMDA receptor-channel complex.

Equilibrium and kinetic study of glycine action on the N-methyl-D-aspartate receptor in cultured mouse brain neurons.

1. The characteristics of the activation of the N-methyl-D-aspartate (NMDA) response by glycine were studied using whole-cell and outside-out patch clamp recording techniques. 2. Glycine concentration-response (C-R) curves were measured in the presence of 10 microM-NMDA and fitted with the Hill equation modified to account for the response to NMDA observed in the absence of added glycine. The mean value of the apparent dissociation constant (KD) was 150 nM, and the mean value of the Hill coefficient (nH) was 1.1. When the KD was corrected for the concentration of contaminating glycine in nominally glycine-free solutions, estimated assuming that there is no response in the absence of glycine, the value was 130 nM. 3. The question of how many glycine binding sites there are on each NMDA receptor-channel complex was addressed by examining the curvature at the foot of the glycine C-R curve. An equation that allowed estimation of both the concentration of contaminating glycine and of the value of nH was fitted to glycine C-R data up to 50 nM. The mean value of nH was found to be 1.0, consistent with the idea that there is one glycine binding site. 4. The kinetics of the interaction of glycine with the NMDA receptor were measured by fitting single exponential curves to the current relaxation following a jump in glycine concentration in the presence of 10 microM-NMDA. The plot of the inverse of the relaxation time constant as a function of glycine concentration after the concentration jump was linear. The association rate constant was estimated from these data as 1.2 x 10(7) M-1 s-1 and the dissociation rate as 1.0 s-1. 5. Experiments were devised to allow the evaluation of the KD and dissociation rates of glycine in the absence of NMDA. They led to a value for KD of 80 nM, slightly but significantly lower than the value of 150 nM estimated in the presence of 10 microM-NMDA. The glycine dissociation rate in the absence of NMDA was found to be 0.7 s-1, not significantly different from that measured in the presence of 10 microM-NMDA. 6. The results are consistent with a model of the NMDA receptor with a single glycine binding site. The characteristics of glycine binding are similar in the absence and the presence of 10 microM-NMDA, although NMDA binding may cause a small increase in the glycine KD.(ABSTRACT TRUNCATED AT 250 WORDS)

Redox modulation of NMDA receptor-mediated synaptic activity in the hippocampus.

The sulfhydryl reducing agent dithiothreitol (DTT) and the oxidizing agent 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) reversibly modulate the component of synaptic potentials mediated by N-methyl-D-aspartate (NMDA) receptors in slices of hippocampal area CA1. DTT (1 mM) reversibly potentiates NMDA receptor-mediated synaptic potentials while DTNB (1 mM) has the opposite effect. However, treatment of the slices with the irreversible sulfhydryl alkylating agent N-ethylmaleimide (300 microM) prevents DTNB from reversing the potentiation induced by DTT. These results further implicate the redox modulatory site as a regulator of the NMDA receptor-channel complex in vivo.