Differential inhibition by ferrous ions of [H]MK-801 binding to native N-methyl- -aspartate channel in neonatal and adult rat brains

In vitro addition or pretreatment with ≥1 μM ferrous chloride markedly inhibited in a concentration-dependent manner [³H]dizocilpine (MK-801) binding to an open ion channel associated with the N-methyl- -aspartate (NMDA) receptor in rat brain synaptic membranes. The addition of NMDA agonists invariably attenuated the inhibition of [³H]MK-801 binding in hippocampal synaptic membranes previously treated with ferrous chloride, without significantly affecting that in cerebellar synaptic membranes. In the absence of spermidine, ferrous chloride was more potent in inhibiting binding in the cerebral cortex and hippocampus in adult rats than in those in rats at 3 days after birth, while in the striatum [³H]MK-801 binding was 10 times more sensitive to inhibition by added ferrous chloride in neonatal rats than in adult rats. Addition of spermidine significantly attenuated the potency of ferrous chloride to inhibit binding in the cerebral cortex of adult rats, with facilitation of the inhibition in newborn rats. Moreover, spermidine significantly reduced the inhibitory potency of ferrous chloride in neonatal rat striatum, without markedly affecting that in adult rat striatum. These results suggest that ferrous ions may interfere with opening processes of the native NMDA channel through molecular mechanisms peculiar to neuronal development in a manner associated with the polyamine recognition domain.     

Ontogeny of glycine-enhanced [3H]MK-801 binding to N-methyl-D-aspartate receptor-coupled ion channels.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is thought to play a critical role in neuronal development, differentiation and plasticity. A number of studies have shown an enhanced sensitivity to NMDA receptor ligands in neonatal animals. This study examined the ontogenetic changes in the glycinergic modulation of NMDA-coupled cation channels in the developing central nervous system of rat pups. The nonequilibrium binding of the specific channel ligand [3H]MK-801 was used as a measure of NMDA channel access. Glycine (10(-5) M) enhancement of [3H]MK-801 binding at 2 h in forebrain membranes from adult rats was significantly greater than that observed in tissues from 8- to 28-day-old rat pups. This difference was due to changes in the efficacy, but not potency of glycine. The observed ontogenetic changes in the efficacy of glycine-enhanced [3H]MK-801 binding were attributable to developmental changes in receptor site density, as determined by equilibrium [3H]MK-801 saturation isotherms. Kinetic studies revealed that glycine increased the association rate constants of [3H]MK-801 in 8-day and adult membranes by a similar magnitude (0.111 +/- 0.021 vs 0.094 +/- 0.009 nM-1 h-1, respectively). Similarly, the fractional amount of [3H]MK-801 bound (i.e., amount bound at time t normalized to amount bound at equilibrium) in the presence of glycine was relatively constant throughout neonatal development. These findings suggest that the allosteric modulation of the NMDA ionophore by glycine is similar in postnatal and adult rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

The N-methyl- -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, -glutamate alone potentiates [³H]MK-801 binding, and glycine failed to potentiate an -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 -glutamate alone, and -glutamate and glycine or -glutamate and spermidine or -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.     

Polyamine effects upon N-methyl-D-aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists.

Polyamines such as spermidine potentiate activation of the N-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. The goal of the present study was to investigate interactions between the putative polyamine binding site and previously described sites for glutamate and glycine. Binding of the high-potency PCP receptor ligand [3H]MK-801 to well-washed rat brain membranes was used as an in vitro probe of NMDA receptor activation. Spermidine concentration-response studies were performed in the absence and presence of both glutamate and glycine, with and without D-(-)-2-amino-5-phosphonovaleric acid (D(-)-AP-5) or 7-chlorokynurenic acid (7Cl-KYN). Incubation in the presence of spermidine alone induced a 20.4-fold increase in [3H]MK-801 binding with an EC50 value of 13.3 microM. The mean concentration of spermidine which induced maximal stimulation of binding was 130 microM (n = 10, S.E.M. = 24.66, range = 25-250 microM). Glutamate (10 microM) decreased the EC50 value for spermidine-induced stimulation of [3H]MK-801 binding to 3.4 microM. Glycine (10 microM) did not significantly alter either maximum spermidine-induced [3H]MK-801 binding or the EC50 value for spermidine-induced stimulation of [3H]MK-801 binding. Incubation in the presence of the specific glutamate antagonist D(-)AP-5 attenuated [3H]MK-801 binding in a glutamate-reversible fashion. The competitive glycine antagonist 7Cl-KYN decreased maximum spermidine-induced [3H]MK-801 binding in a glycine-reversible fashion. In addition, 7Cl-KYN increased the EC50 value for spermidine-induced stimulation of [3H]MK-801 binding while D(-)AP-5 was without effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

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. [(3)H]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 [(3)H]MK-801 binding, and glycine failed to potentiate an L-glutamate-induced increase in [(3)H]MK-801 binding. During the late postnatal period (after day 14) spermidine alone did not increase [(3)H]MK-801 binding as potently as it did during the early postnatal period, high-affinity [(3)H]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 [(3)H]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.     

Radioligand binding studies reveal agmatine is a more selective antagonist for a polyamine-site on the NMDA receptor than arcaine or ifenprodil

Ifenprodil, arcaine and agmatine have all been reported to inhibit the NMDA receptor by actions at polyamine-sites, however the specific sites with which these compounds interact is unknown. Here we used radioligand binding of [3H]MK-801 to a membrane preparation from rat cerebral cortex to investigate the interactions of these compounds with the NMDA receptor complex. In the absence of exogenous polyamines, agmatine reduced [3H]MK-801 binding only at concentrations over 500 micro M, as opposed to the putative polyamine-site antagonists arcaine and ifenprodil which directly reduce ligand binding at much lower concentrations (5 micro M) in the absence of polyamines. In our studies, all three compounds significantly reduced spermidine-potentiated [3H]MK-801 binding, however agmatine was the only compound effective at concentrations below those that produced direct inhibition of [3H]MK-801 binding. Under these conditions, agmatine had a K(i)=14.8 micro M for spermidine-potentiated [3H]MK-801 binding and displayed characteristics of a competitive antagonist. Agmatine, as well as ifenprodil and arcaine, also displaced [3H]spermidine from rat cortical membranes at concentrations similar to those that were effective at reducing spermidine-potentiated [3H]MK-801 binding. In conclusion, these data suggest that agmatine reduces the potentiating effects of polyamines by competitive antagonism at a specific site on the NMDA receptor complex, and that these actions of agmatine differ from those of ifenprodil and arcaine.     

Chronic treatment with the uncompetitive NMDA receptor antagonist memantine influences the polyamine and glycine binding sites of the NMDA receptor complex in aged rats

Summary Receptor binding studies on rat cortical membranes were used to characterize the NMDA receptor in aged rats (22 months) treated for 20 months with a memantine containing diet delivering 30 mg/kg/day in comparison to aged and young/adult rats treated with control-diet. Spatial memory impairing effects of (+)-MK-801 (0.16 mg/kg) in the radial maze was not altered within the course of memantine-treatment (up to 16 months). However, chronic memantine-treatment significantly increased the number of [³H]MK-801 binding sites and the affinity of [³H]glycine. A non-significant trend to such changes was also seen in aged-control rats. Glycine-dependent [³H]MK-801 binding (functional binding under non-equilibrium conditions at a fixed L-glutamate concentration) revealed that a decreased ability of glycine to stimulate channel opening in aged rats was partially attenuated by the long-term memantine treatment. Furthermore, an increased ability of spermidine to enhance [³H]MK-801 binding in aged-control rats was even more pronounced in the aged memantine-treated group. Together these findings may indicate that changes in functional receptor-channel properties during the process of aging occur prior to a detectable loss of binding sites and that memantine enhances an endogenous compensatory mechanism triggered by glutamatergic hypofunction which is suggested to take place in aging.     

Postnatal stress selectively upregulates striatal N-methyl-D-aspartate receptors in male rats

Early life events have been thought to contribute towards vulnerability to drug addiction later in life. In the present investigation, the effect of daily neonatal maternal isolation stress on NMDA channel activity was studied. [3H]MK-801 binding was measured in several brain regions from neonatally isolated (ISO) and nonhandled (NH) adult male and female rats. Maximal [3H]MK-801 binding in the caudate-putamen of male ISO rats was 58% higher compared to same sex NH rats. Unlike male rats, maximal [3H]MK-801 binding in the caudate-putamen of female ISO rats was lower than female NH rats. No other brain region showed any significant difference in maximal [3H]MK-801 binding between ISO and NH male and female rats, respectively. There was no effect of pup isolation on the binding affinity (K(d) value) in either sex. Repeated maternal isolation is associated with alterations in the NMDA channel activity in the caudate-putamen of adult rats, and may be responsible for the augmentation in the addictive behavior reported.     

Polyamine effects uponN-methyl-D-aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists

Polyamines such as spermidine potentiate activation of theN-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. The goal of the present study was to investigate interactions between the putative polyamine binding site and previously described sites for glutamate and glycine. Binding of the high-potency PCP receptor ligand [³H]MK-801 to well-washed rat brain membranes was used as an in vitro probe of NMDA receptor activation. Spermidine concentration-response studies were performed in the absence and presence of both glutamate and glycine, with and withoutD-(−)-2-amino-5-phosphonovaleric acid (D(−)AP-5) or 7-chlorokynurenic acid (7Cl-KYN). Incubation in the presence of spermidine alone induced a 20.4-fold increase in [³H]MK-801 binding with an EC₅₀ value of 13.3 μM. The mean concentration of spermidine which induced maximal stimulation of binding was 130 μM (n = 10,S.E.M.= 24.66,range= 25–250 μM). Glutamate (10 μM) decreased the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding to 3.4 μM. Glycine (10 μM) did not significantly alter either maximum spermidine-induced [³H]MK-801 binding or the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding. Incubation in the presence of the specific glutamate antagonistD(−)AP-5 attenuated [³H]MK-801 binding in a glutamate-reversible fashion. The competitive glycine antagonist 7Cl-KYN decreased maximum spermidine-induced [³H]MK-801 binding in a glycine-reversible fashion. In addition, 7Cl-KYN increased the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding whileD(−)AP-5 was without effect. These findings suggest that glutamate and glycine regulate the polyamine binding site differentially. PCP-like agents induce a psychotomimetic state closely resembling schizophrenia by inhibiting NMDA receptor-mediated neurotransmission. The ability of polyamines to modulate NMDA receptor functioning suggests a potential site for pharmacological intervention.     

Search for novel ligands selective at a polyamine recognition domain on theN-methyl-d-aspartate receptor complex using membrane binding techniques

Among over 60 polyamine derivatives tested, onlyN-(3-aminopropyl)octanediamine and bis-(3-aminopropyl)nonanediamine (TE393) markedly inhibited [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding at equilibrium in the presence of added spermidine (SPD) in “non-washed” rat brain synaptic membranes, without affecting that in the absence of added SPD. Although TE393 significantly potentiated [³H]MK-801 binding before equilibrium in the presence ofl-glutamic acid (Glu) alone or both Glu and glycine (Gly) added in “Triton-treated” membranes, the putative polyamine antagonists 1,10-decanediamine (DA10) and arcaine invariably inhibited binding irrespective of the addition of agonists. In the absence of added SPD, in addition, TE393 markedly enhanced abilities of both Glu and Gly to potentiate [³H]MK-801 binding before equilibrium. However, TE393 induced a rightward shift of the concentration-response curve of SPD for [³H]MK-801 binding before equilibrium. Moreover, TE393 was effective in potentiating binding of an antagonist but not an agonist radioligand to the NMDA domain and in inhibiting binding of an antagonist but not an agonist radioligand to the Gly domain. The potentiation of NMDA antagonist binding by TE393 occurred in a manner sensitive to prevention by arcaine but not by DA10. These results suggest that TE393 may be a novel ligand at the polyamine domain with an ability to interact with both the NMDA and Gly recognition domains in antagonist-preferring forms.     

Modulatory effects of aluminum, calcium, lithium, magnesium, and zinc ions on [3H]MK-801 binding in human cerebral cortex

The independent and combined effects of Ca2+, Mg2+, Zn2+, Al3+ and Li+ on [3H]MK-801 binding in human cerebral cortical membranes were studied to further characterize the modulatory effects of metal ions on the N-methyl-D-aspartate (NMDA) receptor-ionophore. Glycine, in the presence of glutamate, significantly intensified the Mg2+ inhibition of [3H]MK-801 binding whereas it masked the Ca2+ enhancement and slightly diminished the Zn2+ inhibition. Both Ca2+ and Mg2+ reduced the Zn2+ inhibitory potency. Aluminum demonstrated a potent, relatively glycine-insensitive inhibition of [3H]MK-801 binding as an amorphous Al(OH)3 polymer rather than as the free ion. Cationic modulation of the NMDA receptor-ionophore appears to be regulated at multiple sites which have significant allosteric interactions.     

3H]MK-801 binding to the NMDA receptor complex, and its modulation in human frontal cortex during development and aging.

[3H]MK-801 binding was found to decline with age in well washed membranes from human frontal cortex taken from an age series from 24 weeks gestation to 100 years old. The decline was significant under basal conditions (no added modulators) (P less than 0.01), and highly significant under stimulation with glutamate, glycine and spermidine alone and in combination (P less than 0.001). Scatchard analysis in the presence of glutamate and glycine showed this decline was due to a loss in the number of [3H]MK-801 binding sites rather than a change in the affinity of the binding site. There was a highly significant age related reduction in the attenuation of [3H]MK-801 binding by zinc (P less than 0.001). In foetal and neonatal cases up to 7 weeks of age spermidine behaved in an antagonistic manner, inhibiting rather than stimulating [3H]MK-801 binding, when alone or in the presence of glutamate and glycine. The changes in influence of glutamate, glycine, spermidine and zinc on [3H]MK-801 binding during development and aging were not due to other pre- or postmortem factors. The reverse effect of spermidine in the foetal and neonatal cases has therapeutic implications in the treatment of neonates with antiischaemic agents whose action involves the polyamine site.     

Inhibition of N-methyl-D-aspartate evoked sodium flux by MK-801

The inhibition of N-methyl-D-aspartate (NMDA) stimulated 22Na+ efflux from rat hippocampal slices was studied using competitive and non-competitive receptor antagonists. There was a good correlation between the abilities of the competitive antagonists to block NMDA evoked 22Na+ efflux and their potencies as inhibitors of L-[3H]glutamate binding. The recently reported novel NMDA receptor antagonist, (+)-5-methyl-16,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) was shown to non-competitively inhibit NMDA stimulated 22Na+ efflux with an IC50 value of 0.4 microM. Relatively high (10 microM) concentrations of MK-801 had no effects on quisqualic acid, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), or kainic acid stimulated efflux. However, MK-801 was able to block 22Na+ efflux induced by ibotenic acid and L-homocysteic acid, amino acids that act as NMDA receptor agonists. MK-801, (-)-MK-801, and non-competitive NMDA receptor antagonists of the arylcyclohexylamine and dioxolane classes inhibited NMDA stimulated 22Na+ efflux with potencies that reflected their abilities to compete for [3H]MK-801 binding sites in rat cortical membranes. These results indicate the utility of the 22Na+ efflux assay in studying the properties of NMDA receptors and confirm the nature and selectivity of the inhibition of NMDA receptor linked ion channel activation by MK-801.     

Postnatal stress selectively upregulates striatal N-methyl- -aspartate receptors in male rats

Early life events have been thought to contribute towards vulnerability to drug addiction later in life. In the present investigation, the effect of daily neonatal maternal isolation stress on NMDA channel activity was studied. [³H]MK-801 binding was measured in several brain regions from neonatally isolated (ISO) and nonhandled (NH) adult male and female rats. Maximal [³H]MK-801 binding in the caudate–putamen of male ISO rats was 58% higher compared to same sex NH rats. Unlike male rats, maximal [³H]MK-801 binding in the caudate–putamen of female ISO rats was lower than female NH rats. No other brain region showed any significant difference in maximal [³H]MK-801 binding between ISO and NH male and female rats, respectively. There was no effect of pup isolation on the binding affinity (K_d value) in either sex. Repeated maternal isolation is associated with alterations in the NMDA channel activity in the caudate–putamen of adult rats, and may be responsible for the augmentation in the addictive behavior reported.     

Supporting evidence for negative modulation by protons of an ion channel associated with theN-methyl-D-aspartate receptor complex in rat brain using ligand binding techniques

The addition ofL-glutamic acid (Glu) alone, both Glu and glycine (Gly) or Glu/Gly/spermidine (SPD) was effective in potentiating[³H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding before equilibrium to an ion channel associated with theN-methyl-D-aspartate (NMDA) receptor complex in brain synaptic membranes extensively washed and treated with Triton X-100. The binding dependent on Glu almost linearly increased in proportion to decreasing proton concentrations at a pH range of 6.0 to 9.0 in external incubation medium, while a Gly-dependent portion of the binding increased with decreasing proton concentrations up to a pH of 7.5 with a plateau thereafter. In contrast, the SPD-dependent binding increased in proportion to decreasing proton concentrations up to a pH of 7.0 with a gradual decline thereafter. Similar profiles were also obtained with [³H]MK-801 binding at equilibrium, with an exception that significant binding of [³H]MK-801 was detected in the absence of any added agonists. The potency of SPD to potentiate [³H]MK-801 binding before equilibrium increased in proportion to decreasing proton concentrations, with those of both Glu and Gly being unchanged. In contrast, the ability of (+)MK-801 to displace [³H]MK-801 binding at equilibrium was not significantly affected by a decrement of external proton concentrations from pH 7.5 to pH 8.5 in the presence of Glu/Gly and Glu/Gly/SPD added. However, similar changes in external proton concentrations did not similarly affect binding of several radioligands for the NMDA and Gly domains on the receptor complex. Decreasing proton concentrations were effective in exponentially potentiating binding of [³H]SPD at a pH range of 6.0 to 9.0 without virtually altering [³H]D, L-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid binding. In addition, [³H]kainic acid binding markedly decreased with decreasing proton concentrations only in the presence of Ca²⁺ ions. These results suggest that protons negatively modulate neuronal responses mediated by the NMDA receptor ionophore complex through interference with opening mechanisms of the channel domain without disturbing association processes of the endogenous agonists with the respective recognition domains in rat brain. Moreover, possible modulation by protons of responses mediated by the kainate receptor in the presence of Ca²⁺ ions at concentrations that occur in vivo is also suggested.     

[H]MK-801 binding to the NMDA receptor complex, and its modulation in human frontal cortex during development and aging

[³H]MK-801 binding was found to decline with age in well washed membranes from human frontal cortex taken from an age series from 24 weeks gestation to 100 years old. The decline was significant under basal conditions (no added modulators) (P < 0.01), and highly significant under stimulation with glutamate, glycine and spermidine alone and in combination (P < 0.001). Scatchard analysis in the presence of glutamate and glycine showed this decline was due to a loss in the number [³H]MK-801 binding sites rather than a change in the affinity of the binding site. There was a highly significant age related reduction in the attenuation of [³H]MK-801 binding by zinc (P < 0.001). In foetal and neonatal cases up to 7 weeks of age spermidine behaved in an antagonistic manner, inhibiting rather than stimulating [³H]MK-801 binding, when alone or in the presence of glutamate and glycine. The changes in influence of glutamate, glycine, spermidine and zinc on [³H]MK-801 binding during development and aging were not due to other pre- or postmortem factors. The reverse effect of spermidine in the foetal and neonatal cases has therapeutic implications in the treatment of neonates with antiischaemic agents whose action involves the polyamine site.     

Washable endogenous substances and regional heterogeneity in agonist enhanced [H]MK-801 binding in rat brain

NMDA receptor/ion channel function is modulated through a number of distinct sites that regulate channel opening. Published studies report widely varying results in modulatory site agonist effects due to assay conditions and technique. Also, NMDA receptor regulation at these sites by endogenous substances remains poorly characterized. The objectives of the present study in Sprague-Dawley rat forebrain sections were: (i) determine the contribution of various prewash variables on agonist stimulation of the NMDA receptor, (ii) compare regional differences in functional glycine, spermidine and NMDA binding sites under optimized prewash conditions, and (iii) define the influence of endogenous substances at each modulatory site by analyzing changes in binding at different prewash durations. We demonstrate that prewash conditions have a critical influence on [³H]MK-801 binding in rat tissue sections and that this effect was differentially expressed across brain regions. An extended prewash duration caused a regionally specificdecrease in unenhanced [³H]MK-801 binding, while a short prewash caused a regionally specific biphasic effect on enhanced [³H]MK-801 binding. After prolonged prewash, binding was restored to previous (unwashed) binding levels with exogenously added glycine, NMDA, or spermidine alone or combinations of agonists. These data suggest that washable endogenous substances contribute to the full functionality of the NMDA receptor and the regional heterogeneity in [³H]MK-801 binding is dependent on the interaction of receptor protein subtypes and the presence of one or more endogenous substances.     

Endogenous indoles as novel polyamine site ligands at the N-methyl-D-aspartate receptor complex

High-throughput ligand displacement screens of a series of endogenous indoles revealed that tryptamine, serotonin and 5-methoxytryptamine readily displace [3H]spermidine and [3H]MK-801 from their respective binding sites in rat brain homogenate. These data, coupled with their potent inhibition of spermidine-potentiated [3H]MK-801 binding, suggest that certain endogenous indoles may act as ligands to one or more polyamine binding sites in the brain, including those on the N-methyl-D-aspartate receptor complex.     

Nicotine reduces the binding of [H]MK-801 to brain membranes, but not via the stimulation of high-affinity nicotinic receptors

Nicotine (10 and 100 μM) inhibited [³H]MK-801 binding to rat cerebral cortical membranes and this effect was not blocked by dihydro-β-erythroidine, (+)-tubocurarine or mecamylamine. Cytisine, muscarine, mecamylamine and (+)-tubocurarine also inhibited [³H]MK-801 binding. Neither raising the MK-801 concentration, nor the addition ofn-methyl-D-aspartate (NMDA) receptor agonists altered the effects of nicotine. Hence this response is not mediated via high-affinity nicotinic receptor stimulation, competition for MK-801 binding sites or require NMDA receptor activation.     

Autoradiographical analysis of excitatory amino acid binding sites in rat hippocampus during the development of hippocampal kindling

Binding sites for excitatory amino acids have been determined by autoradiographical procedures in the rat hippocampus and striatum during hippocampal kindling. The binding sites measured were the N-methyl-D-aspartate (NMDA)-sensitive sites for L-[3H]glutamate and [3H]MK-801 sites (transmitter recognition site and ion channel of the NMDA receptor, respectively), [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) sites (quisqualate receptor), [3H]kainate sites (kainate receptor) and NMDA-insensitive sites for L-[3H]glutamate. In general, little change was apparent in the hippocampus or striatum for any of these binding sites when assessed 48 h after attaining stages 1/2, 3 or 5 of kindling. These results suggest that hippocampal kindling does not bring about a change in the excitatory amino acid receptor binding sites examined, and that the appearance of an NMDA receptor-mediated component to synaptic responses in the hippocampus produced by kindling, cannot be explained on this basis.