Potentiation of N-methyl-D-aspartate-induced currents by the nootropic drug nefiracetam in rat cortical neurons

Nefiracetam is a new pyrrolidone nootropic drug being developed for the treatment of Alzheimer's type and post-stroke vascular-type dementia. In the brain of Alzheimer's disease patients, down-regulation of both cholinergic and glutamatergic systems has been found and is thought to play an important role in impairment of cognition, learning and memory. We have previously shown that the activity of neuronal nicotinic acetylcholine receptors is potently augmented by nefiracetam. The present study was undertaken to elucidate the mechanism of action of nefiracetam on glutamatergic receptors. Currents were recorded from rat cortical neurons in long-term primary culture using the whole-cell patch-clamp technique at a holding potential of -70 mV in Mg2+-free solutions. N-Methyl-D-aspartate (NMDA)-evoked currents were greatly and reversibly potentiated by bath application of nefiracetam resulting in a bell-shaped dose-response curve. The minimum effective nefiracetam concentration was 1 nM, and the maximum potentiation to 170% of the control was produced at 10 nM. Nefiracetam potentiation occurred at high NMDA concentrations that evoked the saturated response, and in a manner independent of NMDA concentrations ranging from 3 to 1,000 microM. Glycine at 3 microM potentiated NMDA currents but this effect was attenuated with an increasing concentration of nefiracetam from 1 to 10,000 nM. 7-Chlorokynurenic acid at 1 microM prevented nefiracetam from potentiating NMDA currents. Nefiracetam at 10 nM shifted the dose-response relationship for the 7-chlorokynurenic acid inhibition of NMDA currents in the direction of higher concentrations. Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid- and kainate-induced currents were not significantly affected by application of 10 nM nefiracetam. It was concluded that nefiracetam potentiated NMDA currents through interactions with the glycine binding site of the NMDA receptor.     

Modulation of N-methyl-D-aspartate receptors by donepezil in rat cortical neurons

Nicotinic acetylcholine receptors and N-methyl-D-aspartate (NMDA) receptors are known to be down-regulated in the brain of patients with Alzheimer's disease. It was previously shown that the nootropic drugs nefiracetam and galantamine potentiate the activity of both nicotinic and NMDA receptors. We hypothesized that donepezil, a nootropic with a potent anticholinesterase activity, might also affect the NMDA system. NMDA-induced currents were recorded from rat cortical neurons in primary culture using the whole-cell patch-clamp technique at a holding potential of -70 mV in Mg2+-free solutions. In multipolar neurons, NMDA currents were decreased by bath and U-tube applications of 1 to 10 microM donepezil but were increased by 30 to 100 microM donepezil. Donepezil suppression occurred in a manner independent of NMDA concentrations ranging from 3 to 1000 microM. The donepezil suppression of NMDA currents was prevented by inhibition of protein kinase C (PKC) but unaffected by protein kinase A (PKA) and G proteins. In bipolar neurons, however, NMDA currents were potently augmented by bath and U-tube applications of 0.01 to 100 microM donepezil. Donepezil potentiation occurred at high NMDA concentrations that evoked the saturating responses and in a manner independent of NMDA concentrations ranging from 3 to 1000 microM. The potentiation of NMDA currents by donepezil was decreased by inhibition of PKC and abolished by modulation of G proteins but not by PKA inhibition. It was concluded that donepezil at low therapeutic concentrations (0.01-1 microM) potentiated the activity of the NMDA system and that this action together with cholinesterase inhibition would contribute to the improvement of learning, memory, and cognition in patients with Alzheimer's disease.     

Electrophysiological evidence for expression of glycine receptors in freshly isolated neurons from nucleus accumbens

In the course of studying N-methyl-D-aspartate (NMDA) receptors of the nucleus accumbens (NAcc), we found that 20% of freshly isolated medium spiny neurons, as well as all interneurons, responded in an unexpected way to long (5-s) coapplication of NMDA and glycine, the coagonist of NMDA receptors. Whereas the reversal potential of the peak NMDA current of this subset of neurons was still around 0 mV, the desensitizing current became outward at hyperpolarized potentials around -30 mV. A Cl(-)-free solution shifted the equilibrium potentials of the desensitized currents to around 0 mV. This outward current was not blocked by a Ca(2+)-free, Ba(2+)-containing solution, suggesting that the anionic conductance was not activated by Ca(2+) influx through NMDA receptor channels. Interestingly, glycine alone also evoked a current with a similar hyperpolarized reversal potential in this subset of neurons. The glycine current reversed around -50 mV, rectified outwardly, and inactivated strongly. Its desensitization was best fitted with a double exponential. Only the slow desensitization showed clear voltage dependence. The glycine current was not blocked by 200 microM picrotoxin and 10 microM zinc, was weakly antagonized by 1 microM strychnine, and was not enhanced by 1 microM zinc. In addition, 1 mM taurine, but not GABA, inactivated glycine currents, and 1 mM glycine occluded 10 mM taurine-mediated currents. These data indicate that a subset of nucleus accumbens neurons expresses glycine receptors and that either glycine or taurine could be an endogenous agonist for these receptors.     

Electrophysiological characterization of N-methyl-D-aspartate receptors in rat dorsal root ganglia neurons

In the peripheral nervous system, N-methyl-D-aspartate receptors (NMDAR) expressed on the central and peripheral terminals of primary afferent neurons are involved in nociception. We used single cell imaging of intracellular calcium concentration ([Ca2+]i) and patch clamp techniques to characterize the functional properties of NMDARs on adult rat dorsal root ganglia (DRG) neurons in primary culture and selectively on those innervating the distal colon. In Mg2+-free extracellular solution, rapid perfusion of DRG neurons with 250 microM NMDA and 10 microM glycine caused a significant increase in [Ca2+]i, and elicited inward currents in whole cell patch clamp recordings when the holding potential was -60 mV. Both effects were reversibly inhibited by 200 microM ketamine in a use-dependent manner. The EC50 values for NMDA and glycine were 64 and 1.9 microM with Hill slope coefficients of 1.4 and 1.3, respectively. At negative potentials, extracellular Mg2+ blocked currents in a concentration- and voltage-dependent manner. The IC50 for Mg2+ at a holding potential of -100 mV was 2.0 microM. The NMDAR subtype-selective antagonist, ifenprodil, inhibited 94% of the NMDA and glycine-induced current with an IC50 of 2.6 microM. There was no evidence of multiple binding sites for ifenprodil. There was no significant difference in the NMDAR current density on DRG neurons that had innervated the colon, nor was there a difference in the EC50 for ifenprodil. These results demonstrate that functional NMDARs expressed by DRG neurons innervating both somatic and visceral tissues of adult rats are composed predominantly of NR2B subunits.     

Ontogeny of N-methyl-D-aspartate-induced current in cultured hippocampal neurons.

The ontogeny of the N-methyl-D-aspartate (NMDA) subtype of glutamatergic receptor/ion channel was studied by examining whole cell currents evoked by NMDA in cultured hippocampal neurons 1 to 30 days after plating of cells from 18- to 20-day-gestation rat fetuses. We observed a maturation-dependent increase in conductance, compatible with an increased density of NMDA receptors, which is in agreement with previous binding data. The whole cell currents evoked by NMDA (10-100 microM) in the presence of glycine (1-100 microM) had two components that contributed to the peak amplitude. The first was a rapidly decaying current (fast component) and the second a slowly decaying current (slow component), their ratio depending upon glycine concentration. The EC50 values for glycine were 1.8 and 0.3 microM for the fast and slow components of the current, respectively. The quantitative analysis of these components indicated the existence of two distinct glycine sites, which differ in their affinity for glycine. The fast component originates from the action of glycine at the site with lower affinity. Moreover, the ratio of the fast to the slow component was also dependent on the time lapsed after plating of the fetal hippocampal neurons. The slow component became more predominant and the fast component less predominant along with cell maturation in culture, a phenomenon which reflects a change in the ratio of high- to low-affinity glycine binding sites. In addition, studies on Zn2+ gave further evidence of a change in the NMDA receptor/channel properties related to maturation of the cultured neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by intracellular Mg(2+) of recombinant N-methyl-D-aspartate receptors expressed in Chinese hamster ovary cells

Intracellular Mg(2+) (Mg(i)(2+)) inhibits the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors in cultured cortical neurons. To examine the effects of Mg(i)(2+) on recombinant NMDA receptors composed of subunit combinations found in cortical neurons, we expressed heteromeric receptors composed of NR1/NR2A and of NR1/NR2B subunits in Chinese hamster ovary (CHO) cells. We recorded whole-cell currents from the recombinant receptors in the absence and presence of Mg(i)(2+). The voltage dependence of control (0 Mg(i)(2+)) NMDA-activated currents obtained from CHO cells transfected with NR1/NR2A and with NR1/NR2B receptors showed outward rectification, a property that has been observed previously in native cortical NMDA receptors. The magnitude and voltage dependence of inhibition by Mg(i)(2+) of NMDA-activated currents were similar in CHO cells transfected with NR1/NR2A receptors, CHO cells transfected with NR1/NR2B receptors, and in cultured neurons expressing native NMDA receptors. These observations suggest that Mg(i)(2+) has uniform effects on the native NMDA receptors expressed in cortical neurons. Furthermore, inhibition by Mg(i)(2+) must not depend on intracellular factors or post-translational receptor modifications that are specific to neurons. Finally, the results indicate that the previously observed differences between whole-cell and outside-out patch measurements of Mg(i)(2+) inhibition could not result from poor control of voltage or Mg(i)(2+) concentration in the dendrites of neurons. The most likely alternative explanation is that patch excision causes an alteration in NMDA receptors that results in more effective inhibition by Mg(i)(2+).     

Competitive antagonism between the nicotinic allosteric potentiating ligand galantamine and kynurenic acid at alpha7* nicotinic receptors

Galantamine, a drug used to treat Alzheimer's disease, is a nicotinic allosteric potentiating ligand, and kynurenic acid (KYNA), a neuroactive metabolite of the kynurenine pathway, is an endogenous noncompetitive inhibitor of alpha7* nicotinic receptors (nAChRs) [the asterisk next to the nAChR subunit is intended to indicate that the exact subunit composition of the receptor is not known (Pharmacol Rev 51:397-401, 1999)]. Here, possible interactions between KYNA and galantamine at alpha7* nAChRs were examined in vitro and in vivo. In the presence of tetrodotoxin (TTX), approximately 85% of cultured hippocampal neurons responded to choline (0.3-30 mM) with alpha7* nAChR-subserved whole-cell (type IA) currents. In the absence of TTX and in the presence of glutamate receptor antagonists, choline triggered inhibitory postsynaptic currents (IPSCs) by activating alpha7* nAChRs on GABAergic neurons synapsing onto the neurons under study. Galantamine (1-10 microM) potentiated, whereas KYNA (10 nM-1 mM) inhibited, choline-triggered responses. Galantamine (1 microM), applied before KYNA, shifted to the right the concentration-response relationship for KYNA to inhibit type IA currents, increasing the IC(50) of KYNA from 13.9 +/- 8.3 to 271 +/- 131 microM. Galantamine, applied before or after KYNA, antagonized inhibition of choline-triggered IPSCs by KYNA. Local infusion of KYNA (100 nM) in the rat striatum reduced extracellular dopamine levels in vivo. This effect resulted from alpha7* nAChR inhibition and was blocked by coapplied galantamine (1-5 microM). It is concluded that galantamine competitively antagonizes the actions of KYNA on alpha7* nAChRs. Reducing alpha7* nAChR inhibition by endogenous KYNA may be an important determinant of the effectiveness of galantamine in neurological and psychiatric disorders associated with decreased alpha7* nAChR activity in the brain.     

Ethanol suppresses fast potentiation of glycine currents by glutamate

Excitatory (glutamate) and inhibitory (GABA(A) and glycine) receptor/channels coexist in many neurons. To assess effects of ethanol on the interaction of glutamate and glycine receptors, glycine-induced current (I(Gly)) was recorded by a whole-cell patch-clamp technique from neurons freshly dissociated from the ventral tegmental area of rats. A conditioning prepulse of glutamate (1-3 s, 1 mM) significantly and reversibly potentiated responses to a pulse of glycine. This potentiation was increased when extracellular calcium was raised to 12 mM and reduced by including 10 mM 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the internal recording medium. It was not affected by 5 microM 1-N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a selective inhibitor of calcium/calmodulin-dependent protein kinase II. In a concentration-response analysis, a conditioning pulse of glutamate significantly lowered the EC(50) for glycine and increased the maximal I(Gly). Kinetic analysis of the currents indicated that glutamate slowed deactivation of glycine-gated chloride channels; therefore, glutamate may increase the affinity of glycine receptors for glycine. When coapplied with glycine, ethanol (10 mM) potentiated I(Gly) in 35% of neurons from the ventral tegmental area. In contrast, when coapplied with glutamate and glycine, ethanol suppressed the glutamate-induced potentiation of I(Gly) in these neurons. This suppression was also observed when ethanol and glycine were coapplied after a glutamate prepulse. A similar effect was observed when ethanol alone did not potentiate I(Gly). These findings suggest that glutamate-induced calcium influx modulates glycine receptors by a mechanism that can be blocked by ethanol.     

Ethanol potentiation of glycine-induced responses in dissociated neurons of rat ventral tegmental area

The potentiation of glycine-induced responses by ethanol (EtOH) was studied in neurons freshly dissociated from the ventral tegmental area (VTA) of 5- to 14-day-old postnatal rats using whole-cell and gramicidin-perforated patch-clamp techniques. Under current-clamp conditions, EtOH increased glycine-induced membrane depolarization and action potential firing. Under voltage-clamp conditions, EtOH (0. 1-40 mM) alone did not elicit a current. When coapplied with glycine, EtOH enhanced the glycine-induced current in 35% (180 of 474) of the neurons. The EtOH-induced enhancement of glycine current was independent of membrane potential (between -60 and +60 mV); the reversal potential was not changed. Concentration-response analysis showed that in the presence of EtOH (10 mM), the EC(50) for glycine decreased from 25 +/- 4 to 14 +/- 3 microM; the Hill coefficient increased from 1.5 +/- 0.2 to 1.9 +/- 0.3. Kinetic analysis of glycine currents indicated that EtOH decreased the time constant of activation and increased the time constant of deactivation of glycine-gated chloride channels. EtOH may accelerate glycine association with its receptor at the agonist binding site and increase the apparent agonist affinity. Our observations suggest that, at pharmacologically relevant concentrations, EtOH alters the function of glycine receptors and thus the excitability of neonatal VTA neurons. This action of EtOH may contribute to the neurobehavioral disturbances associated with fetal alcohol syndrome.     

In vitro and in vivo characterization of conantokin-R, a selective NMDA receptor antagonist isolated from the venom of the fish-hunting snail Conus radiatus

The purification, characterization, and synthesis of conantokin-R (Con-R), an N-methyl-D-aspartate (NMDA) receptor peptide antagonist from the venom of Conus radiatus, are described. With the use of well defined animal seizure models, Con-R was found to possess an anticonvulsant profile superior to that of ifenprodil and dizocilpine (MK-801). With voltage-clamp recording of Xenopus oocytes expressing heteromeric NMDA receptors from cloned NR1 and NR2 subunit RNAs, Con-R exhibited the following order of preference for NR2 subunits: NR2B approximately NR2A > NR2C > NR2D. Con-R was without effect on oocytes expressing the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR1 or the kainate receptor subunit GluR6. In mouse cortical neurons voltage-clamped at -60 mV, Con-R application produced a slowly developing block of inward currents evoked by 10 microM NMDA and 1 microM glycine (IC(50) = 350 nM). At 3 microM, Con-R did not affect gamma-aminobutyric acid- or kainate-evoked currents. Con-R prevented sound-induced tonic extension seizures in the Frings audiogenic seizure-susceptible mice at i.c.v. doses below toxic levels. It was also effective at nontoxic doses in CF#1 mice against tonic extension seizures induced by threshold (15 mA) and maximal (50 mA) stimulation, and it partially blocked clonic seizures induced by s.c. pentylenetetrazol. In contrast, MK-801 and ifenprodil were effective only at doses approaching (audiogenic seizures) or exceeding (electrical and pentylenetetrazol seizures) those required to produce significant behavioral impairment. These results indicate that the subtype selectivity and other properties of Con-R afford a distinct advantage over the noncompetitive NMDA antagonists MK-801 and ifenprodil. Con-R is a useful new pharmacological agent for differentiation between the anticonvulsant and toxic effects of NMDA antagonists.     

Protein kinase C modulation of ethanol inhibition of glycine-activated current in dissociated neurons of rat ventral tegmental area

The brain is particularly sensitive to alcohol during its growth spurt period. To better understand the mechanism(s) involved, we studied the effects of ethanol on neurons freshly dissociated from the ventral tegmental area (VTA) in neonatal rats. Ethanol enhanced (35%) or depressed (45%) glycine-induced responses in VTA neurons (Ye et al., 2001a, 2001b). In this report, we investigated the role of protein kinase C (PKC) and protein kinase A (PKA) in ethanol-induced inhibition of glycine-activated current, using whole-cell patch-clamp technique. Ethanol inhibited glycine-activated current when it was coapplied with the agonist. This inhibition was enhanced when neurons were pretreated with ethanol before the subsequent coapplication of ethanol and glycine. Ethanol's inhibition of glycine-activated currents increased with the length of ethanol pretreatment time (ranging from 1 to 30 s), and reached the maximum at 30 s. However, this enhanced inhibition was not seen in the absence of internal ATP. In addition, phorbol-12-myristate-13-acetate (PMA, 100 nM), a PKC activator, markedly inhibited glycine-activated current. Blockade of PKC by chelerythrine or by PKC inhibitor peptide significantly attenuated ethanol-induced inhibition. Although partial increase of PKC activity by 1 nM PMA enhanced ethanol inhibition, pretreatment of ethanol did not increase ethanol inhibition after the neurons were treated with 100 nM PMA. These data suggest that ethanol and PKC share the same pathway to suppress glycine receptors. H-89 (1 microM), a selective PKA inhibitor, did not alter glycine-activated current or ethanol inhibition. Our observations suggest that activation of PKC (but not PKA) contributes to ethanol-induced inhibition of glycine receptors.     

Ethanol directly depresses AMPA and NMDA glutamate currents in spinal cord motor neurons independent of actions on GABAA or glycine receptors.

Ethanol is a general anesthetic agent as defined by abolition of movement in response to noxious stimulation. This anesthetic endpoint is due to spinal anesthetic actions. This study was designed to test the hypothesis that ethanol acts directly on motor neurons to inhibit excitatory synaptic transmission at glutamate receptors. Whole cell recordings were made in visually identified motor neurons in spinal cord slices from 14- to 23-day-old rats. Currents were evoked by stimulating a dorsal root fragment or by brief pulses of glutamate. Ethanol at general anesthetic concentrations (50-200 mM) depressed both responses. Ethanol also depressed glutamate-evoked responses in the presence of tetrodotoxin (300 nM), showing that its actions are postsynaptic. Block of inhibitory gamma-aminobutyric acidA and glycine receptors by bicuculline (50 microM) and strychnine (5 microM), respectively, did not significantly reduce the effects of ethanol on glutamate currents. Ethanol also depressed glutamate-evoked currents when the inhibitory receptors were blocked and either D, L-2-amino-5-phosphonopentanoic acid (40 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione disodium (10 microM) were applied to block N-methyl-D-aspartate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors, respectively. The results show that ethanol exerts direct depressant effects on both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate glutamate currents in motor neurons. Enhancement of gamma-aminobutyric acidA and glycine inhibition is not required for this effect. Direct depression of glutamatergic excitatory transmission by a postsynaptic action on motor neurons thus may contribute to general anesthesia as defined by immobility in response to a noxious stimulus.     

Stimulatory and inhibitory properties of aminoglycoside antibiotics at N-methyl-D-aspartate receptors.

The effects of aminoglycoside antibiotics on N-methyl-D-aspartate (NMDA) receptors were studied using voltage-clamp recording of recombinant NMDA receptors expressed in Xenopus oocytes. A number of aminoglycosides were found to potentiate macroscopic currents at heteromeric NR1A/NR2B receptors, but not at NR1A/NR2A, NR1A/NR2C, NR1A/NR2D, or NR1B/NR2B receptors. The degree of potentiation had a rank order neomycin B > paromomycin > gentamicin C > geneticin > kanamycin A > streptomycin. Potentiation was not seen with kasugamycin and spectinomycin. The degree of stimulation paralleled the number of the amino groups in the aminoglycosides. The stimulatory effects of aminoglycosides were more pronounced at subsaturating concentrations of glycine and at acidic pH, similar to the stimulatory effects of spermine. We measured the effects of aminoglycosides at mutant NMDA receptors to determine which amino acid residues in NMDA receptor subunits are involved in stimulation. Mutations that reduced or abolished spermine stimulation also reduced stimulation by aminoglycosides. Several aminoglycosides produced a weak voltage-dependent block of NMDA receptors, but the degree of inhibition did not appear to correlate with the number of amino groups in the molecule. The results suggest that aminoglycosides having more than three amino groups have stimulatory effects that are mediated through the spermine-binding site on NMDA receptors.     

Increased C-fiber nociceptive input potentiates inhibitory glycinergic transmission in the spinal dorsal horn

Glycine is an important inhibitory neurotransmitter in the spinal cord, but it also acts as a coagonist at the glycine site of N-methyl-d-aspartate (NMDA) receptors to potentiate nociceptive transmission. However, little is known about how increased nociceptive inflow alters synaptic glycine release in the spinal dorsal horn and its functional significance. In this study, we performed whole-cell recordings in rat lamina II neurons to record glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs). The transient receptor potential vanilloid receptor 1 agonist capsaicin caused a prolonged increase in the frequency of sIPSCs in 17 of 25 (68%) neurons tested. The potentiating effect of capsaicin on sIPSCs was blocked by ionotropic glutamate receptor antagonists or tetrodotoxin in most lamina II neurons examined. In contrast, the P2X agonist alphabeta-methylene-ATP increased sIPSCs in only two of 16 (12.5%) neurons. The glutamate transporter inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid either increased or reduced the basal frequency of sIPSCs but did not significantly alter the potentiating effect of capsaicin on sIPSCs. Furthermore, the groups II and III metabotropic glutamate receptor antagonists had no significant effect on the capsaicin-induced increase in the sIPSC frequency. Although capsaicin reduced the amplitude of evoked excitatory postsynaptic currents at high stimulation currents, it did not change the ratio of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/NMDA currents. This study provides the important new information that increased nociceptive inflow augments synaptic glycine release to spinal dorsal horn neurons through endogenous glutamate release. Potentiation of inhibitory glycinergic tone by stimulation of nociceptive primary afferents may function as a negative feedback mechanism to attenuate nociceptive transmission at the spinal level.     

A comparison of N-methyl-D-aspartate-evoked release of adenosine and [3H]norepinephrine from rat cortical slices

Tetrodotoxin reduced N-methyl-D-aspartate (NMDA)-evoked release of adenosine by 35% but virtually abolished [3H]norepinephrine release. Although [3H]norepinephrine release from rat cortical slices evoked by 500 microM NMDA was abolished by 1.2 mM Mg++, which produces a voltage-sensitive, uncompetitive block of NMDA-channels, adenosine release was increased in the presence of Mg++. Partial depolarization with 12 mM K+ relieved the Mg++ block of 500 microM NMDA-evoked [3H]norepinephrine release but did not affect adenosine release, indicating that a Mg++ requirement for the adenosine release process per se cannot account for this discrepancy. NMDA was 33 times more potent in releasing adenosine than [3H]norepinephrine. At submaximal concentrations of NMDA (10 and 20 microM), adenosine release was augmented in Mg+(+)-free medium. Although a high concentration of the uncompetitive NMDA antagonist MK-801 [(+)-5-methyl-10,11,dihydro-5H-dibenzo[a,d]cyclohepten-5-10-imine maleate] (3 microM) blocked NMDA-evoked release of [3H]norepinephrine and adenosine, a lower concentration (300 nM) decreased NMDA-evoked [3H]norepinephrine release by 66% without affecting adenosine release. These findings suggest that maximal adenosine release occurs when relatively few NMDA receptors are activated, raising the possibility that spare receptors exist for NMDA-evoked adenosine release. Rather than acting as a protectant against excessive NMDA excitation, released adenosine might provide an inhibitory threshold which must be overcome for NMDA-mediated neurotransmission to proceed.     

Characterization of two novel N-methyl-D-aspartate antagonists: EAA-090 (2-[8,9-dioxo-2,6-diazabicyclo [5.2.0]non-1(7)-en2-yl]ethylphosphonic acid) and EAB-318 (R-alpha-amino-5-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid hydrochloride)

Two novel N-methyl-d-aspartate (NMDA) antagonists with unique chemical structures, EAA-090 (2-[8,9-dioxo-2, 6-diazabicyclo[5.2.0]non-1(7)-en2-yl]ethylphosphonic acid) and EAB-318 (R-alpha-amino-5-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid hydrochloride), were compared with CGS-19755 (Selfotel) in ligand binding, electrophysiology, and neuroprotection assays. CGS-19755, EAA-090 and EAB-318 inhibited [(3)H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid binding to NMDA receptors with IC(50) values of 55, 28, and 7.9 nM, respectively. All three compounds decreased the duration of spontaneous synaptic currents and inhibited NMDA-activated currents in rat hippocampal neurons. IC(50) values for inhibition of current induced by 10 microM NMDA were 795, 477, and 69 nM for CGS-19755, EAA-090, and EAB-318, respectively. The NMDA antagonists protected chick embryo retina slices and cultured rat hippocampal and cortical neurons from glutamate- and NMDA-induced neurotoxicity. In experiments in which different NMDA receptor splice variants and subtypes were expressed in Xenopus oocytes, all three antagonists preferentially blocked NMDA-elicited currents mediated by N-methyl-d-aspartate receptor (NR)1 splice variants containing the N-terminal insertion. They also favored NR2A-versus NR2B- or NR2C-containing NMDA receptors, with EAA-090 showing the greatest selectivity. EAA-090 was 10 times more potent at blocking NR2A-versus NR2B- or NR2C-containing NMDA receptors. In addition to being the most potent NMDA antagonist, EAB-318 inhibited alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors. The combination of NMDA and AMPA/kainate block enabled EAB-318 to protect neurons against ischemia induced cell death.     

A further evaluation of the effects of K+ depolarization on glutamate-evoked [3H]dopamine release from striatal slices.

Exogenous glutamate will evoke dopamine (DA) release from striatal slices in vitro. To further characterize glutamate-evoked DA release from striatal slices, experiments were designed to: 1) determine if sufficient endogenous glutamate can be released in vitro to presynaptically mediate [3H]DA release in the absence of Mg++ and 2) reevaluate how K+ depolarization affects glutamate-evoked [3H]DA release. Removal of Mg++ to potentiate N-methyl-D-aspartate (NMDA) receptor-mediated DA release increased 15 mM K(+)-evoked [3H]DA release to about 200% of control. The potentiation of this release was probably not mediated by NMDA receptors because it was not blocked by the glutamate receptor antagonists MK-801, 6,7-dinitroquinoxalinedione (DNQX) or kynurenate. Furthermore, the removal of Mg++ increased DA release substantially (200%) in the presence of 5 microM sulpiride and 10 microM nomifensine, indicating that DA reuptake and DA D2 autoreceptors are not primarily responsible for increased DA release. In the absence of Mg++, depolarization produced by 20 mM or greater [K+] inhibited DA released by exogenous glutamate, whereas a much higher [K+] was necessary to evoke endogenous glutamate release. In the presence of 1.5 mM Mg++, a reduction of the "Mg++ blockade" of NMDA receptors by 15 mM K+ depolarization during glutamate-evoked DA release was evaluated with and without the DA reuptake inhibitor nomifensine and the DA D2 antagonist sulpiride. DA released by K+ depolarization (Mg++ present) was markedly increased by 1 mM glutamate, but this effect was only partially reversed by kynurenate or high concentrations of either MK-801 (25 microM) or DNQX (100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Memantine blocks alpha7* nicotinic acetylcholine receptors more potently than n-methyl-D-aspartate receptors in rat hippocampal neurons

The N-methyl-d-aspartate (NMDA) receptor antagonist memantine is an approved drug for treatment of Alzheimer's disease (AD). Other such treatments are cholinesterase inhibitors and nicotinic acetylcholine receptor (nAChR)-sensitizing agents such as galantamine. The present study was designed to test whether memantine exerts any effect on the cholinergic system, in particular the Ca(2+)-conducting alpha7(*) nAChR, in cultured hippocampal neurons. Memantine caused a concentration-dependent reduction of the amplitudes of whole-cell currents evoked by the alpha7(*) nAChR-selective agonist choline (10 mM) or by N-methyl-d-aspartate (NMDA) (50 muM) plus glycine (10 muM). It also inhibited tonically activated NMDA receptors. Memantine was more potent in inhibiting alpha7(*) nAChRs than NMDA receptors; at -60 mV, the IC(50) values for memantine were 0.34 and 5.1 muM, respectively. Consistent with an open-channel blocking mechanism, memantine-induced NMDA receptor inhibition was voltage and use-dependent; the Hill coefficient (n(H)) was approximately 1. Memantine-induced alpha7(*) nAChR inhibition had an n(H) < 1 and showed a variable voltage dependence; the effect was voltage-independent at 0.1 muM, becoming voltage-dependent at >/=1 muM. Thus, memantine interacts with more than one class of sites on the alpha7(*) nAChRs. One is voltage-sensitive and therefore likely to be within the receptor channel. The other is voltage-insensitive and therefore likely to be in the extracellular domain of the receptor. It is suggested that blockade of alpha7(*) nAChRs by memantine could decrease its effectiveness for treatment of AD, particularly at early stages when the degrees of nAChR dysfunction and of cognitive decline correlate well.     

Divalent cations modulate N-methyl-D-aspartate receptor function at the glycine site.

The modulation of the N-methyl-D-aspartate (NMDA) receptor (NMDAR) by divalent cations was examined using (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten 5,10-imine maleate ([(3)H]MK-801) binding as a functional indicator of NMDAR function. Ca(2+) and Mg(2+) produce a biphasic effect on the binding of [(3)H]MK-801 to the NMDAR channel in extensively washed adult rat brain membranes. Concentrations of Ca(2+) and Mg(2+) between 1 and 600 microM potentiate binding, but higher concentrations inhibit binding. The potentiating effect of Ca(2+) and Mg(2+) on [(3)H]MK-801 binding is due to an increase in the maximal number of binding sites (B(max)) with no effect on binding affinity (K(d)). Ca(2+)- and Mg(2+)induced potentiation is the result of an apparent increase in the affinity of the NMDAR for glycine. The ontogeny of NMDAR potentiation by Ca(2+) and Mg(2+) was also investigated. The number of [(3)H]MK-801 binding sites associated with divalent cation potentiation are present at low levels shortly after birth, and increase to peak level at 17 days of age before declining to adult levels. The potency of Ca(2+) and Mg(2+) to stimulate [(3)H]MK-801 binding did not change as a function of age. Lead (Pb(2+)) and zinc (Zn(2+)), potent inhibitors of the NMDAR, antagonize NMDAR potentiation by Ca(2+) and Mg(2+). These findings indicate that divalent cations differentially regulate NMDAR function by modulation of the glycine site. The NMDAR glycine site may be important in the regulation of glutamatergic neurotransmission by physiologically and toxicologically relevant cations.