Neurochemical aspects of the N-methyl-d-aspartate receptor complex

Neurochemical aspects of the N-methyl-d-aspartate receptor complex

Neuroscience Research, 10 (1991) 1-33 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0168-0102/91/$03.50 1 N E U R E S 00414 Review Article N...

2MB Sizes 2 Downloads 377 Views

Neuroscience Research, 10 (1991) 1-33 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0168-0102/91/$03.50


N E U R E S 00414

Review Article

Neurochemical aspects of the N-methyl-D-aspartate receptor complex Yukio Yoneda and Kiyokazu Ogita Department of Pharmacology, Setsunan University, Hirakata, Osaka (Japan) (Received 2 July 1990; Accepted 8 September 1990)

Key words: N-Methyl-D-aspartic acid; Receptor ionophore complex; Glycine; Polyamines; Methodological artifacts; Calcium ion

SUMMARY The N-methyl-D-aspartic acid (NMDA)-sensitive subclass of brain excitatory amino acid receptors is supposed to be a receptor-ionophore complex consisting of at least 3 different major domains including an N M D A recognition site, glycine (Gly) recognition site and ion channel site. Biochemical labeling of the N M D A domain using [3H]L-glutamic acid (Glu) as a radioactive ligand often meets with several critical methodological pitfalls and artifacts that cause a serious misinterpretation of the results. Treatment of brain synaptic m e m b r a n e s with a low concentration of Triton X-100 induces a marked disclosure of [3H]Glu binding sensitive to displacement by NMDA with a concomitant removal of other several m e m b r a n o u s constituents with relatively high affinity for the neuroactive amino acid. The N M D A site is also radiolabeled by the competitive antagonist (+)-3-(2-carboxypiperazin-4-yl)propyl-l-phosphonic acid that reveals possible heterogeneity of the site. The Gly domain is sensitive to D-serine and D-alanine but insensitive to strychnine, and this domain seems to be absolutely required for an opening of the N M D A channels by agonists. The ionophore domain is radiolabeled by a non-competitive type of N M D A antagonist that is only able to bind to the open but not closed channels. The binding of these allosteric antagonists is markedly potentiated by N M D A agonists in a m a n n e r sensitive to antagonism by isosteric antagonists in brain synaptic m e m b r a n e s and additionally enhanced by further inclusion of Gly agonists through the Gly domain. Furthermore, physiological and biochemical responses mediated by the N M D A receptor complex are invariably potentiated by several endogenous polyamines, suggesting a novel polyamine site within the complex. At any rate, activation of the N M D A receptor complex results in a marked influx of Ca 2+ as well as Na + ions, which subsequently induces numerous intracellular metabolic alterations that could be associated with neuronal plasticity or excitotoxicity. Therefore, any isosteric and allosteric antagonists would be of great benefit for the therapy and treatment of neurodegenerative disorders with a risk of impairing the acquisition and formation process of memories.


INTRODUCTION ......................................................... METHODOLOGICAL PITFALLS .............................................. A. Radioligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Microbial contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . ~. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 3 4 4 5

Correspondence: Yukio Yoneda, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01, Japan.

D. Membrane



Separation method



111. R E C E P T O R A. NMDA



of QA ........................................................



recognition sites

1. L a b e l i n g b y G l u



2. L a b e l i n g b y a n t a g o n i s t s

7 7 ,9

4. E n d o g e n o u s



.................................................... .............................................


2. A n t a g o n i s t s


3. R e q u i r e m e n t


C . Polyamine sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. P r o f i l e s


2. A n t a g o n i s t s


D . Ion channel sites (ionophore) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. [ 3 H ] T C P b i n d i n g 2. [ 3 H ] M K - 8 0 1



3. Sigma receptors



M o d u l a t o r y cations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. M a g n e s i u m 2. Z i n c



3. S o d i u m


10 10

11 11 12 12 12

13 13 14 15

15 15 16 16

Signal transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


1. C a 2+ i n f l u x



2. C y c l i c G M P



3. Phosphatidylinositol hydrolysis

............................................ 4. A r a c h i d o n a t e release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . W o r k i n g hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. FUNCTIONAL A. Plasticity








B. E x c i t o t o x i c i t y V.


3. G T P e f f e c t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. P r o f i l e s




B. G l y r e c o g n i t i o n s i t e s ( G l y B s i t e s )




............................................................ ...............................................................


17 18 18 20 20 21 21 22 23

I. I N T R O D U C T I O N

Several free acidic amino acids with which the brain is enriched, such as L-glutamic acid (Glu) and L-aspartic acid (Asp), are thought to be excitatory neurotransmitters in the mammalian central nervous system (CNS) 37.54. The prevailing subclassification of synaptic receptors for these excitatory amino acids is made according to differences in the sensitivity to exogenous acidic compounds, such as N-methyl-o-aspartic acid ( N M D A ) , DL-C~-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainic acid (KA) 56.140.225. Although this classification is not based on any physiological responses, evidence is accumulating that the NMDA-sensitive subclass has some physiological a n d / o r pathophysiological significance in numerous central events. For example, the subclass is supposed not only to take part in neuronal plasticity 145, but also to be involved in selective neuronal cell damage in convulsive seizures 35,135,cerebral ischemia 203 and hypoglycemia 232. Significant alterations in the functional activity of the latter

subclass are reported to occur in brains of patients with Alzheimer's 62.65,138.204 and Huntington's 64.265 diseases. Drugs that antagonize the NMDA sites are proposed to be beneficial as adjuvants in the therapy of Parkinson's disease 101. The discovery and subsequent introduction of competitive antagonists highly selective for the NMDA-sensitive receptors have greatly contributed to the progress of these studies. These include D-2-amino-5-phosphonovaleric acid (AP5)38, D-2-amino-7-phosphonoheptanoic acid (AP7) 181, (+).3_(2_carboxypiperazin_4_yl)propyl_l.phosphonic acid (CPP) 440 and cis-4phosphonomethyl-2-piperidine carboxylic acid (CGS 19755) 112. Accordingly, the endogenous acidic amino acids seem to play dual roles as an excitatory neurotransmitter and as an endogenous excitotoxin in the brain at least in part through the NMDA-sensitive receptors. In addition to these competitive antagonists, recent neurophysiological and neurochemical receptor studies have raised the novel concept of a non-competitive type of antagonist. This type of antagonist is an open channel blocker and elicits its blocking action on excitation mediated by the NMDA-sensitive receptors via associating with membrane sites responsible for mediating NMDA responses. These include ketamine 114, phencyclidine (PCP)2, N-[1-(2-thienyl)cyclohexyl]piperidine (TCP)221 and (+)-5methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) 234 The latter non-competitive antagonists are able to associate with the open but not closed NMDA channels and are thereby useful to label biochemically the activated ion channels. The putative inhibitory neurotransmitter in the mammalian lower CNS glycine (Gly) exclusively potentiates in a strychnine-insensitive manner currents caused by the NMDA-sensitive subclass without affecting those mediated by the other subclasses in voltage-clamped cultured neurons 90. In fact, strychnine-insensitive [3H]GIy binding sites are detected in brain sections 20 and synaptic membrane preparations 99 Based on these previous findings, the NMDA-sensitive receptors are supposed to be a receptor-ion channel complex consisting of at least 3 distinct subcomponents: (a) NMDA recognition sites, (b) Gly recognition sites and (c) ion channel sites. Furthermore, a recent binding study has raised the possibility that some endogenous polyamines may physiologically participate in the NMDA-mediated responses through their own binding sites located on the complex 188. The finding that brain synaptic membranes contain specific binding sites of [3H]spermidine (SPD) 248 agrees with this idea. In this review, therefore, the neurochemical aspects of the postulated 4 different subcomponents within the NMDA receptor complex will mainly be outlined. II. METHODOLOGICALPITFALLS Although neurochemical binding techniques have been widely employed to evaluate receptors for neurotransmitters, hormones and autacoids in many laboratories, these studies often meet with various methodological pitfalls a n d / o r artifacts. For instance, early binding studies on excitatory amino acid receptors demonstrated that chloride ions markedly potentiate the binding of [3H]Glu to synaptic membranes 134 with a pharmacological profile different from that known on excitatory amino acid receptors 48. Chloridedependent binding was sensitive to L-2-amino-4-phosphonobutyric acid (AP4) 48.155quisqualic acid (QA) 228 and freezing 120, but insensitive to NMDA, KA and AMPA 75,203. Although these unique properties once supported the idea of a novel subclass identified by sensitivity to L-AP4, subsequent experiments have revealed that the chloride-dependent and AP4-sensitive binding of [3H]Glu is derived from a sequestration process 184.266 a n d / o r exchange system 96 for acidic amino acids into resealed vesicular components in

4 synaptic membrane preparations, rather than attributable to a novel AP4-sensitivc subclass of excitatory amino acid receptors 14o Receptor binding assays using [3H]GIu as a radioligand to label excitatory amino acid receptors hold several other methodological pitfalls as described below, which would bring about a serious misinterpretation of the results. The binding assay is very easy to begin, while it has many pitfalls too difficult for a beginner to overcome by himself. For example, [3H]Glu binding does not always mean the association of a ligand with its recognition sites on the receptors. Sometimes, the observed binding is not net binding of a radioactive ligand to receptors but an adsorption displaceable by Glu to glass fiber filters and microfuge tubes made of potypropylene 86, both of which are routinely used to collect the bound ligand in receptor binding assays. Therefore, experimental conditions should be set up as carefully as possible before starting neurochemical analysis of the receptors. A. Radioligand

When the purity of a commercially obtained batch of [3H]Glu was estimated by cation exchange chromatography using a plastic column packed with Dowex 50W × 8 resin~ some unidentified radioactive material was eluted with water from the column prior to the elution of [3H]Glu 257. This radioactive contaminant accounted for at most 5% of the total radioactivity applied to the column irrespective of commercial sources, and increased in proportion to the duration of storage at 2°C in 0.01 M HCI. The material markedly adsorbed to glass fiber and nitrocellulose membrane filters in a manner not displaceable by excess unlabeled Glu. Extensive rinsing of filters with buffer was ineffective in reducing this adsorption. This non-displaceable adsorption would account for a substantially high non-specific binding determined in the presence of excess unlabeled Glu. The ratio of specific to total binding ought to be over 70% in excitatory amino acid receptor binding assays using radioactive Glu as a ligand. Accordingly, a commercial batch of [3H]GIu should be further purified as mentioned above, and the purified [3H]Glu stocked at - 2 0 ° C after dividing it into small quantities. On the day of the experiment, the necessary amount of frozen [3H]Glu is thawed and diluted with 0.01 M HC1 to make up an appropriate concentration. The diluted endogenous ligand should be used up on the day of the experiment and not be employed any more. B. Microbial contamination

In addition to the radioactive adsorbate described above, the [3H]Gtu binding assay meets with other serious artifacts. Incubation of purified [3H]Glu in the absence of brain membrane preparations occasionally results in a marked retention of radioactivity on the filters after extensive rinsing with buffer in a temperature-dependent fashion 249. The radioactivity is greatly reduced by the inclusion of excess unlabeled Glu in the incubation medium. This pseudo-specific binding is temperature-dependent, structure-selective, stereospecific, semi-reversible and saturable with an apparent K d of 73 _+ 20 nM. The latter displaceable apparent binding is sensitive to displacement by most agonists and antagonists for brain excitatory amino acid receptors except that by N M D A and CPP, and to digestion by various proteases and phospholipase C. However, the pseudo-binding is completely abolished by using incubation buffer after sterilization by boiling for 30 min or by filtration through a nitrocellulose membrane filter with a pore size of 450 nm. These findings clearly show that microbial contamination of incubation buffer is responsible for the displaceable apparent binding of [3H]GIu detected in the absence of brain membrane preparations. The microbial contamination occurs in buffer that is prepared with glassdistilled, deionized and sterilized water, and stored at 2°C for no longer than a week.

Therefore, buffers and any other solutions should be sterilized immediately before each use to avoid artifactual pseudo-binding due to microbial contamination with pharmacological profiles similar to those of excitatory amino acid receptors.

C. Adsorption The afore-mentioned pseudo-binding is attributable to the adsorption of radioactive product to glass fiber filters of [3H]Glu by microbial enzyme with a relatively high affinity for the acidic amino acid. Column chromatography of the incubation media after incubation makes it clear that some unidentified radioactive material is temperature-dependently formed during the incubation of purified [3H]Glu in the buffer in the absence of brain membrane preparations. The radioactive metabolite markedly adsorbs to filters but not to microfuge tubes, and the adsorption is not reduced at all by extensive rinsing of filters. The formation is greatly eliminated by the inclusion of most agonists and antagonists including Glu and QA with a few exceptions as described above. This means that microbial contamination is not noticed as long as a centrifugation method is employed to separate the bound ligand from the the free one. Furthermore, most proposed agonists and antagonists may be not useful for the identification and characterization of a subtype of excitatory amino acid receptors in terms of their selectivity for the receptors. D. Membrane preparation The endogenous ligand [3H]Glu could also bind to substrate recognition sites of some membrane-bound constituents with relatively high affinity and selectivity for the neuroactive amino acid, in addition to associating with receptor sites. For instance, the rodent brain contains at least 3 different transport systems for Glu: (a) Na+-dependent uptake 11,116,120.212,223,(b) ATP-dependent synaptic vesicular sequestration 151,152 and (c) C1--dependent acidic amino acid exchange 6,96,184,266 These membranous components undoubtedly have recognition sites for Glu as a substrate with relatively high affinity. Therefore, [3H]Glu binding often occurs to these substrate recognition sites other than synaptic receptors in brain synaptic membranes. In addition, a membrane-bound enzyme responsible for the metabolism of Glu would in part participate in the observed binding of [3H]Glu to brain membrane preparations. A considerably larger quantity of unidentified radioactive metabolite of [3H]Glu is detected in the incubation media after incubation with brain synaptic membranes extensively washed but not treated with a detergent, which exhibits temperature-dependent [3H]Glu binding sensitive to displacement by QA but not to that by NMDA and KA 156,258. In contrast, treatment of membranes with a low concentration of Triton X-100 drastically discloses NMDA-sensitive [3H]Glu binding with a concomitant elimination of the temperature-dependent formation of radioactive metabolite of [3H]Glu 159. Rat retinal membranes also exhibit temperature-dependent and QA-sensitive binding of [3H]Glu 259, while the QA-sensitive binding appears to be derived from the association with Glu recognition sites of a membrane-bound enzyme having relatively high affinity, judging from the temperature-dependent formation of radioactive metabolite of [3H]Glu during incubation 250. Similar temperature-dependent and QA-sensitive formation of radioactive metabolite is seen with rat peripheral excitable tissues such as the pituitary 244,251 and adrenal 245-247,253. Consequently, the possibility that a membrane-bound enzyme with high affinity for Glu may be in part attributed to the observed binding of [3H]GIu in membrane preparations is not ruled out, unless the incubation is carried out at a temperature as low as possible in membranes treated with a low concentration of a detergent.

E. Separation method Although it is widely accepted that centrifugation is much better than filtration as a termination method for binding assays using ligands with low affinity such as Glu and Gly, the filtration method is also useful to collect accurately and reproducibly a ligand with low affinity bound to its specific binding sites as long as the experimental conditions are carefully controlled in detail 257. One of the most critical disadvantages to the use of the filtration method to collect the bound ligand with low affinity is that the bound ligand would be rapidly dissociated from binding sites during the termination and subsequent rinsing procedures due to its low affinity. However, this weak point could be overcome by the employment of rinsing at low temperature within a very short period of time 257. In contrast, centrifugation often takes a much longer time than filtration and thereby it is difficult to analyze accurately the kinetics of the binding using centrifugation. Specific binding would be contaminated with non-specific sequestration of a radioactive ligand into the extracellular space a n d / o r with non-specific adsorption of the ligand to membranous surfaces, due to insufficient rinsing in the centrifugation method. Furthermore, the centrifugal force for the termination should be equal to that employed for preparing brain synaptic membranes. Otherwise, a large quantity of radioligand bound to membranes would be lost in supernatants during the termination process as a result of insufficient centrifugal force to precipitate whole membrane preparations. Since both methods each have their own advantages and disadvantages, the termination method should be carefully chosen according to the purpose of the experiment.

F. Inadequacy of QA Although QA was once used for the subclassification of excitatory amino acid receptors, this compound is not an agonist selective for the QA-sensitive receptors. The exogenous excitant is less active at the QA-sensitive receptors than AMPA 76 and has relatively high affinity for the specific binding of [3H]Glu 47,136,141,260, [3H]D.AP 5 175 [3H]CP P 148,162~261 and [3H]CGS 19755 149 to the NMDA-sensitive receptors in brain synaptic membranes. The non-selective activity of QA at NMDA-sensitive receptors is also supported by neurochemical investigations employing ligands for ion channels opened through the NMDA-sensitive receptors, including [3H]MK-801 58,133,193.252,262 and [3H]TCP l18A67A68. Furthermore, QA is effective in inhibiting a brain peptidase catalyzing cleavage of N-acetyl-L-Asp-L-Glu194 and an acidic amino acid exchange 266 in the brain. As described above, QA also exhibits considerably high affinity for substrate recognition sites of some membrane-bound enzymes found in the rodent brain 159, retina 250 pituitary 251 and adrenal 253 in addition to a microbial enzyme 44. These findings all clearly indicate that sensitivity to QA does not mean possible involvement of QA-sensitive receptors in neuronal phenomena. Therefore, the subclass formerly referred to as 'QA-sensitive receptors' has recently been termed more precisely as 'AMPA-sensitive receptors'. III. RECEPTOR COMPLEX NMDA-sensitive receptors are able to induce directly an opening of ion channels mainly permeable to Ca 2÷ and Na + ions, and are thus proposed to compose a receptorion channel complex in analogy with the GABAA/benzodiazepine receptor-ion channel complex 174. The N M D A receptor complex comprises several functionally distinct subcomponents with different affinities for the respective endogenous ligands (Table I).


Domains Channels Agonists





Gly B

Glu, Asp, NMDA, QA Gly, D-serine SPD, spermine, BAA *

Glu, Asp NMDA, QA





MK-801, TCP Zn 2+, Mg 2+ D-AP5, D-AP7 CPP, CGS 19755 GTP, Na ÷, 7-C1KYNA DCQX, ifenprodil SL 82.0715?

D-AP5, D-AP7 CPP, CGS 19755 GTP, Na +



[3H]MK-801 [3H]TCP

[ 3 H]GIu

[ 3H]Gly

[ 3H]SPD? [ 3H]ifenprodil?


[ 3H]CPP [3H]CGS 19755

SL 82.0715?

* BAA = bis-(3-aminopropyl)amine.

These include (a) NMDA recognition sites labeled by the agonist Glu or antagonists including CPP and CGS 19755, (b) strychnine-insensitive Gly recognition sites (Gly B sites) labeled by Gly, and (c) ion channel sites labeled by non-competitive antagonists such as MK-801 and TCP. Several independent lines of evidence suggest the possible multiplicity of NMDA recognition sites, and ion channel sites are shown to be modulated by divalent cations such as Mg 2÷ and Zn2÷. In addition, recent neurochemical and neurophysiological studies have raised the possibility that NMDA-mediated responses may be modified through a specific binding site of polyamines located on the complex. Differential modification by polyamines among different central structures is also suggested. A. NMDA recognition sites 1. Labeling by Glu Early studies failed to label the NMDA recognition sites by the radioactive agonist Glu due to several crucial methodological pitfalls mentioned above. Fagg and Matus 47 first reported the successful radioligand labeling of NMDA sites using brain postsynaptic densities. Subsequently, several independent laboratories have succeeded in radiolabeling the sites by [3H]GIu using synaptic plasma membranes prepared by sucrose density gradient centrifugation 136,141 and brain synaptic membranes treated with a low concentration of Triton X-100 159,260. The binding determined under these conditions is sensitive to displacement by NMDA, CPP, D-AP5 and D-AP7 but not to that by AMPA and KA, which is in good agreement with the data provide by electrophysiological analysis. Although each study revealed that the binding sites consist of a single component, both K d and Bmax values are more than 5 times higher in experiments using the centrifugation method 47,136,141 than in those using filtration 159,260. Without the methodological improvements described, the NMDA-sensitive portion of [3H]Glu binding would be masked by the association of [3H]Glu with substrate recognition sites of the afore-mentioned several transport systems in resealed vesicular constituents of synaptic membrane preparations, all of which have lower affinity but much higher capacity than the NMDA recognition

sites. Association of [3H]Glu with membrane-bound enzyme having an ability to metabolize Glu could be also partly responsible for the masking of NMDA-sensitive [3H]Glu binding. Both Gly and o-serine are reported to potentiate NMDA-sensitive [3H]Glu binding via increasing the affinity without altering the density 4~, whereas a recent binding study failed to confirm this modulatory action of Gly on NMDA-sensitive [3H]Glu binding 33. Although this possible interaction between the NMDA recognition sites and Gly B sites is an attractive hypothesis to explain the exclusive potentiation of NMDA-mediated currents by Gly in cultured neurons 90, it does not explain sufficiently the absolute requirement of Gly for the opening of the NMDA channels 100 as described below. Autoradiographic studies using [3H]Glu as a ligand have revealed the presence of at least 3 distinct binding sites in rat brain 63,137. The NMDA-sensitive binding sites are numerous in the stratum radiatum of the hippocampal CA1 field 63,137 the inner portions of the dentate gyrus molecular layer 137 and the granular cell layer of the cerebellum 63 while the NMDA-insensitive but KA-sensitive sites are localized in the stratum lucidum of CA3 and the commissural/associational layer of the dentate gyrus. Sites sensitive to both QA and AMPA are found in the pyramidal cell layer of CA1 and CA3 63,137. In contrast, addition of C1- and Ca 2+ ions discloses pharmacologically and anatomically distinct [3H]Glu binding sites, which are now known to reflect the afore-mentioned ion-sensitive transport systems rather than specific synaptic receptors.

2. Labeling by antagonists A few competitive antagonists highly selective for the NMDA-sensitive receptors are also useful to label NMDA recognition sites located on the complex. These include [3H]CP P 148 and [3H]CGS 19755 149 but not [3H]DL-AP5 57. The binding of both [3H]CPP and [3H]CGS 19755 displays pharmacological profiles similar to electrophysiologically identified NMDA receptors. The filtration method is also applicable mainly to the detection of [3H]CPP binding to the NMDA sites in brain synaptic membranes treated with a low concentration of Triton X-100 162,261, whereas a filtration assay results in straight saturation isotherms of [3H]CGS 19755 binding which are different from the curvilinear isotherms determined by a centrifugation assay 149. Since the elevation of incubation temperature facilitates the dissociation of bound [3H]CPP to a much greater extent than the acceleration of the association 162, the incubation temperature should be maintained as low as possible in order to accurately and reproducibly detect the steady-state level of [3H]CPP binding in synaptic membranes. An autoradiographic study using [3H]CPP as a ligand has demonstrated a central distribution profile similar to that of NMDA-sensitive [3H]Glu binding sites 89 On the other hand, a recent autoradiographic analysis has shown differential distribution profiles in the brain between [3H]CPP binding and NMDA-sensitive [3H]Glu binding 139. Compared with NMDA-sensitive [3H]Glu binding, [3H]CPP binding is poor in the medial striatum and septum but being rich in the thalamus and inner parietal cortex 139. From this standpoint, it is noteworthy that an agonist has higher affinity for NMDA-sensitive [3H]GIu binding but lower affinity for [3H]CPP binding than an antagonist, and vice versa, in brain synaptic membranes treated with Triton X-100 162 Treatment of synaptic membranes with different SH-reactive agents induces a marked inhibition of [3H]CPP binding without significantly affecting NMDA-sensitive [3H]Glu binding 169. The GlyB antagonist with relatively low affinity, 1-hydroxy-3-aminopyrrolidone-2 (HA-966), markedly enhances the binding of [3H]CPP but not of [3H]Glu to the NMDA recognition sites in Triton-treated membranes, while the other Gly B antagonist

with high affinity, 7-Cl-kynurenic acid (7-C1KYNA), invariably inhibits the binding of both ligands 254. Similar differential effects of HA-966 on the binding of [3H]CPP and [3H]Glu have been reported to occur 33. In addition, the molecular target size of [3H]Glu binding sensitive to NMDA is evidently different from that of [3H]CPP binding as revealed by high-energy radiation inactivation analysis 78. However, the possibility that [3H]CPP but not [3H]Glu may additionally label membrane sites other than the NMDA recognition sites cannot be excluded at present. The additional labeling could result in a marked differentiation of distribution profiles, molecular target size or pharmacological properties of [3H]CPP binding from those of NMDA-sensitive [3H]Glu binding as described above.

3. GTP effect Several guanine nucleotides are shown to inhibit temperature-dependent and NMDAinsensitive [3H]Glu binding 200, [3H]DL.AP4 binding 23, [3H]CP P binding 148,261 and NMDA-sensitive [3H]Glu binding 136,142,170. The inhibition, however, appears to be derived from a competitive inhibition of the NMDA recognition sites by guanine nucleotides, rather than from a possible interaction of the GTP-binding proteins with the NMDA recognition sites. Neither cholera nor pertussis toxin affects the inhibitory property of GTP on the binding of [3H]Glu to the NMDA recognition sites in brain synaptic membranes 142. Guanine nucleotides are similarly effective in competitively inhibiting both NMDA-sensitive [3H]Glu binding and [3H]CPP binding, being active as competitive inhibitors of different NMDA-mediated responses in the brain 8. The positive analogs also attenuate the potentiation by Glu of the binding of non-competitive antagonists such as [3H]TCP and [3H]MK-801 263, by reducing the affinity of NMDA recognition sites in a competitive manner. Although the potentiation is a biochemical measure for the open NMDA channels, it is unlikely that GTP attenuates the potentiation by Glu through a molecular mechanism similar to that underlying a direct regulation by the GTP-binding proteins of ion channels associated with neurotransmitter receptors 74.242 The finding that guanine nucleotides potently inhibit the binding of a radioactive ligand to receptor sites does not always mean involvement of the GTP-binding proteins in functional communication between receptors and effectors. A definitive conclusion should not be drawn based only on GTP-induced inhibition in any receptor binding studies. 4. Endogenous ligands L-Homocysteic acid is presumed to be an endogenous agonist more selective for the NMDA-sensitive receptors than the other endogenous ligands Glu and Asp. This amino acid is released by high potassium stimuli in Ca 2+-dependent manner from rat brain slices and electrophysiologically mimics the excitation by NMDA in cat caudate neurons 42 Excitation by homocysteate but not by Glu is antagonized by D-AP5 at concentrations sufficient to prevent NMDA responses in rat neocortical slices 104. In addition, L-homocysteate induces a pattern of chick retinal cytopathology which is similar to neurotoxicity by NMDA but not by KA, and which is blocked by several different types of NMDA antagonists in proportion to their efficacy in preventing NMDA responses 173 Some endogenous tryptophan metabolites are also active at the NMDA-sensitive receptors. Quinolinic acid excites single neurons in rat neocortex with a potency similar to that of Glu and NMDA in a fashion sensitive to antagonism by competitive antagonists such as AP5 and AP7 180. Among the other kynurenines with a convulsant property 106 metabolically related to quinolinate, only kynurenic acid is able to prevent the responses of cortical neurons to iontophoretically applied NMDA, QA and quinolinate without inducing any detectable excitatory activity alone 179. In addition, kynurenate blocks

10 neurotoxicity and seizures induced by the injection of quinolinate in rats 5~. Although these findings argue in favor of the idea that the two metabolically related kynurenines have an opposite quality in regulatory mechanisms of central excitability, their endogenous levels seem to be too low to elicit these effects in the brain in situ 144. In addition, the exact location of the kynurenines in synaptic clefts should be clarified before drawing any conclusions, even if the endogenous levels are elevated in the brain in some particular neurodegenerative disorders 199 In order to interact with the NMDA-sensitive receptors, they must by all means gain access to the NMDA recognition sites which are undoubtedly located at the extracellular surfaces of postsynaptic plasma membranes. In contrast to the above-mentioned two different types of candidates with low endogenous levels, gtutathione (7-L-glutamyl-L-cysteinyl-glycine)is another possible ligand for excitatory amino acid receptors with relatively high endogenous levels in the brain. Both reduced (GSH) and oxidized (GSSG) forms of this tripeptide markedly displace Na+-dependent and -independent binding of [3H]Glu at concentrations that occur in vivo, irrespective of the incubation temperature in brain synaptic membranes extensively washed but not treated with a detergent 165. The tripeptide also inhibits the binding of [3H]G1u 159 and [3H]CPP 261 to the NMDA recognition sites in membranes treated with Triton X-100. However, these two peptides are also effective as displacers of both [3H]AMPA binding to the AMPA-sensitive receptors and [3H]KA binding to the KA-sensitive receptors ~7~. Furthermore, brain synaptic membranes contain temperature-dependent and -independent saturable binding of [3H]GSH that is markedly potentiated selectively by L-cysteine but not by the other endogenous SH-containing amino acids 157,158.160. These previous data, together with the fact that the efflux of intracellular GSH indeed occurs in the brain 132, are favorable to the speculation that glutathione may have some unknown functional significance in the maintenance of central excitability via interacting with excitatory amino acid receptors. B. Gly recognition sites (GlyB sites) 1. Profiles Johnson and Ascher 9o first advocated that Gly exclusively modulates NMDA-mediated currents in a strychnine-insensitive manner without affecting those mediated by the other subclasses in cultured neuronal cells. Prior to the discovery of this modulatory action on the NMDA-sensitive receptors, however, several independent laboratories implied that strychnine may abolish the inhibitory neurotransmission mediated by Gly by associating with sites different from binding sites of Gly interacting with each other in a cooperative m a n n e r 41,264. Indeed, Kishimoto and colleagues have demonstrated the presence of strychnine-insensitive binding sites of [3H]Gly in rat brain structures 99 By analogy with these previous findings, the prevailing concept is that the NMDA-sensitive receptors compose a receptor-ion channel complex with modulatory Gly-binding sites as seen with the genetically related receptor superfamilies such as GABA A and nicotinic receptors 7. These novel [3H]Gly-binding sites insensitive to strychnine are referred to as 'Gly B sites' to differentiate them from classical strychnine-sensitive sites (GlYA sites) t66 The binding of [3H]Gly to GlyB sites is saturable, of relatively high affinity, stereospecific and sensitive to displacement by both D-serine and D-alanine but not by their respective naturally-occurring L-isomers 20,99,209. The afore-mentioned kynurenate is also effective as a displacer of the strychnine-insensitive [3H]Gly binding in the brain 97,143 Sodium ions potentiate [3H]Gly binding in the spinal cord with the binding being unaltered in the cerebral cortex 231, and magnesium ions markedly increase the cortical


binding 124. Treatment with a low concentration of Triton X-100 results in a marked potentiation of [3H]Gly binding in brain synaptic membranes, without significantly affecting that in spinal synaptic membranes 262. Cerebral binding is inversely dependent on the incubation temperature with a complete insensitivity to strychnine, while spinal binding is dependent on the temperature with a partial sensitivity to the alkaloid 262 Preparations after gel filtration of supernatants solubilized from brain membranes by 1% deoxycholic acid exhibit strychnine-insensitive [3H]Gly binding to the Gly B sites 164, in addition to NMDA-sensitive [3H]Glu binding. The preparations contain whole macromolecules consisting of each subcomponent on the NMDA receptor complex 163. Consequently, Gly is able to cause inhibitory postsynaptic potentials (IPSP) due to an opening of anion channels linked to the GlyA sites in a strychnine-sensitive manner in the spinal cord, with eliciting a strychnine-insensitive enhancement of excitatory postsynaptic potentials (EPSP) mediated by an opening of cation channels associated with the NMDA-sensitive subclass through the GlyB sites in the cerebral cortex. Some unidentified regulatory mechanisms for the concentration of Gly in synaptic clefts could be operative in situ in higher central regions as found in the spinal cord.

2. Antagonists Several natural or synthetic compounds are postulated to be competitive antagonists highly selective for the Gly B sites. These include cycloleucine 2o7, kynurenic acid a4, HA-966 53, 6,7-dinitroquinoxaline-2,3-dione (DNQX) as, 6-cyano-7-nitroquinoxaline-2,3dione (CNQX)15, 5,7-dinitroquinoxaline-2,3-dione (MNQX)201, 7-C1KYNA 95, 1aminocyclobutane-l-carboxylic acid 79,226, indole-2-carboxylic acid 84 and 6,7-dichloroquinoxaline-2,3-dione (DCQX)161. Although both CNQX and DNQX are originally proposed to be competitive and specific antagonists for the non-NMDA receptors 77, recent electrophysiological and neurochemical investigations in addition to the literature quoted above have demonstrated that these two quinoxalines have a substantially high affinity for the Gly B sites 98,113,177. In contrast, 1-aminocyclopropane carboxylic acid is shown to have agonistic activity for the Glya sites 125,150. Of the antagonists listed above, DCQX is most useful for elucidating the functional significance of the Gly a sites in the brain. This antagonist potently displaces the binding of [3H]Gly to the Gly B sites without virtually affecting the binding of either [3H]Glu or [3H]CPP to the NMDA recognition sites, whereas the other antagonists have some drawbacks to their use as a specific antagonist of the GlyB sites from the viewpoint of affinity a n d / o r specificity. Both affinity and specificity are definitive criteria in the search for a competitive antagonist of any receptors. 3. Requirement In accord with a previous electrophysiological investigation using Xenopus oocytes injected with rat brain mRNA 100, the specific Gly B antagonist DCQX completely blocks the potentiation of [3H]MK-801 binding mediated by the NMDA recognition sites in brain synaptic membranes treated with Triton X-100 254. Therefore, occupation by Gly of the GlyB sites would be an absolute requirement for the Glu-induced opening of ion channels associated with NMDA recognition sites. In other words, Gly would enable a very small amount of Glu released from nerve terminals to open the NMDA channels. Since Gly occurs in the cerebrospinal fluid in vivo at concentrations that already saturate the GlyB sites 52, extracellular Gly seems to play a functional role as a type of sensitizer in the central excitatory neurotransmission a n d / o r excitotoxicity mediated by NMDA-sensitive receptors. The GlyB sites should play a key role in the functional communication between the NMDA recognition sites and ion channels.

12 C. Polyamine sites 1. Profiles Ransom and Stec first gave support to the proposal that some endogenous polyamines may modify the NMDA-mediated responses via their own binding sites located within the complex 18~. The endogenous factor extracted from rat brains that modulates the NMDA-mediated responses is identified as the polyamine SPD 233. Furthermore, the other endogenous polyamine, spermine, but not SPD is effective in enhancing the binding of [3H]GIy to the Gly~ sites by increasing the affinity in a manner not reversed by antagonists for the NMDA or GlyB sites 197. In fact, brain synaptic membranes are shown to contain specific binding sites of [3H]SPD 248 with a structure selectivity, inverse temperature-dependency, reversibility and saturability 256. A polyamine with a higher affinity for [3H]SPD binding invariably induces a greater potentiation of [3H]MK-801 binding, a biochemical measure for the open NMDA channels, in rat brain membranes 25~, The binding exhibits uneven regional variations in the rodent brain that the medulla-pons has the highest binding with progressively lower binding in the midbrain, striatum, cerebellum, hippocampus, hypothalamus and cerebral cortex 255. However, the distribution profile of [3H]SPD binding seems to correlate negatively with that of [3H]MK-801 binding, NMDA-sensitive [3H]Glu binding and strychnine-insensitive [3H]Gly binding 25~, From this point of view, it is noteworthy that both SPD and spermine in vivo prevent the increase in cerebellar cyclic GMP content induced by an intracerebellar injection of the potent agonist for Gly B sites, D-serine 189. However, the conclusion that endogenous polyamines indeed play a modulatory role in various physiological responses mediated by the NMDA-sensitive receptors in the mammalian brain awaits further confirmation using electrophysiological techniques. Polyamines could only enhance the association of a non-competitive antagonist with open ion channels coupled to the NMDA recognition sites, without altering the opening process of the channels. More recent studies have demonstrated that spermine but not putrescine increases the current induced by saturating concentrations of NMDA and Gly without affecting that induced by KA or QA in voltage-clamped oocytes injected with rat brain mRNA129, and that SPD potentiates whole-cell inward currents induced by NMDA in cultured striatal neurons 211. Although these findings all argue in favor of the idea that some polyamines may really participate in various excitatory neuronal responses mediated by the NMDA-sensitive receptors in the brain, the evidence that polyamines are indeed present in synaptic clefts at sufficiently effective concentrations is lacking at present. Polyamines would be released from neuronal and/or glial cells adjacent to neurons containing the NMDA receptor complex on nerve stimulation to enhance the excitatory responses in some particular situations. 2. Antagonists The anti-ischemic drug, ifenprodil, is supposed to be an antagonist selective for the polyamine sites. This drug and its derivative (+)-c~-(4-chlorophenyl)-4-[(4fluorophenyl)methyl]-l-piperidineethanol (SL 82.0715) prevent the potentiation of [3H]TCP binding by SPD in a competitive manner, without affecting binding determined in the absence of SPD 27. Radiolabeled ifenprodil is shown to be able to bind with high affinity to novel sites in the rat cerebral cortex, which are sensitive to displacement by SPD and spermine but not by putrescine 198. In contrast, both ifenprodil and SL 82.0715 are effective as displacers of the binding of [3H]CPP to the NMDA recognition sites 26, and ifenprodil markedly inhibits the NMDA-mediated binding of [3H]MK-801 in the absence of added polyamines, suggesting that this drug is not a competitive antagonist specific for the polyamine sites 192. More detailed investigations are necessary to identify

13 these two drugs as specific antagonists at the polyamine sites within the NMDA receptor complex. The proposed competitive antagonism between ifenprodil and polyamines might need to be elucidated all over again. In any case, further search for this type of antagonist as well as evaluation of the exact location of polyamine sites within synaptic plasma membranes is mandatory for the clarification of the physiological and pathophysiological significance of the NMDA receptor complex in the vertebrate CNS.

D. Ion channel sites (ionophore) Ion channels opened through the activation of NMDA recognition sites by agonists are neurochemically labeled by non-competitive antagonists such as MK-801 and TCP. This type of antagonist non-competitively prevents the influx of cations permeable to the NMDA channels by associating with sites within the channels responsible for mediating the NMDA responses. Therefore, non-competitive antagonists are useful to label biochemically the activated or open channels.

1. [3H]TCP binding The dissociative anesthetics ketamine and PCP selectively block the excitation of spinal neurons induced by NMDA in a non-competitive manner 2.114. Based on this, the concept that PCP and its analogs may have a functional interaction with the NMDA-sensitive receptors has been introduced 43,70,115,218. Although PCP associates with brain sigma sites as described below in addition to ion channels gated by the NMDA recognition sites, its analog TCP has higher affinity and selectivity for the NMDA channels than PCP 108,221 Indeed, the binding of [3H]TCP is virtually insensitive to displacement by ligands for the sigma sites 46,117, and is markedly potentiated by Glu in a manner sensitive to antagonism by competitive NMDA antagonists 46,117. NMDA-dependent binding is additionally potentiated by Gly in a strychnine-insensitive and AP5-sensitive fashion 18,208, and D-isomers of its analogous amino acids including D-serine and D-alanine are more effective in additionally potentiating the binding than their respective L-isomers 143,167,168,209,216. However, the exact mechanism underlying the potentiation by Glu and Gly has not yet been clarified. Some investigators have demonstrated a marked elevation of the binding at equilibrium by both amino acids 10, while others have shown a drastic facilitation of the initial association rate with the binding at equilibrium being unaltered by these amino acids 102. In brain synaptic membranes treated with a low concentration of Triton X-100 which contain much lower concentrations of the endogenous stimulatory amino acids, Gly as well as Glu drastically accelerates the initial association rate of [3H]TCP binding with a concomitant facilitation of the dissociation of bound [3H]TCP, but fails to elevate the binding at equilibrium 167,168. The binding sites of [3H]TCP are solubilized from rat brain in a manner that keeps a functional coupling to the NMDA recognition sites 1 Autoradiographic studies using [3H]TCP as a radioligand have revealed that the stratum radiatum of the CA1 field of the hippocampus has the highest density of binding sites followed by the dentate gyrus and stratum oriens of the hippocampal CA1 region in decreasing order of binding, with negligible binding in the posterior cingulate gyrus, brainstem and white matter tracts 66,122. Inclusion of Gly in addition to NMDA markedly potentiates the binding of [3H]TCP in the hippocampus with similar distribution profiles 81. These distribution profiles are well consistent with those of NMDA-sensitive [3H]Glu binding 122 and [3H]CPP binding 89. Furthermore, autoradiographic and biochemical binding techniques have suggested the possible multiplicity of binding sites of [3H]TCP in rat brain. The forebrain has high-affinity sites with the hindbrain containing low-affinity sites 67,222, both of which are insensitive to the neuroleptic drug haloperidol

14 but sensitive to inhibition by Ca 2+ ions 67. However, [3H]MK-801 seems to be preferable to [3H]TCP as a radioligand to label the open N M D A channels, as judged from the affinity and specificity described below.

2. [3H]MK-801 binding An anticonvulsant, MK-801, exhibits higher affinity and selectivity for the open N M D A channels than TCP. This compound non-competitively antagonizes depolarizing responses to N M D A but not to QA or KA in rat cortical slices 234, and [3H]MK-801 binds to heat-labile, stereospecific and regionally specific sites with high affinity in brain synaptic membranes 234. The binding is markedly potentiated by compounds with an agonistic activity at the N M D A receptors 58,85, and a clear correlation is seen between the abilities of excitatory amino acid agonists to enhance [3H]MK-801 binding and to displace NMDA-sensitive [3H]Glu binding 58. The binding is virtually insensitive to displacement by ligands selective for sigma sites 236, and the potentiation by Glu is antagonized by the competitive N M D A antagonist D-AP5 103. Amino acids active at the Gly B sites are all effective in additionally potentiating [3H]MK-801 binding found in the presence of Glu alone in a strychnine-insensitive fashion 193,235, which is in good agreement with the data obtained by electrophysiological techniques 9o Treatment with a low concentration of Triton X-100 252,261 as well as extensive washing 58 of brain synaptic membranes markedly reduces [3H]MK-801 binding determined in the absence of added amino acids. The reduction by Triton X-100 seems to be attributable to removal of the endogenous stimulatory amino acids from membrane preparations, since the binding is restored to the level found in membranes not treated with a detergent after the addition of both Glu and Gly 252. The binding does not reach equilibrium within 16 h in the absence of added amino acids in Triton-treated membranes 256, and the addition of Glu alone or G l u / G l y markedly accelerates both the initial association and dissociation rates of [3H]MK-801 binding without significantly affecting the binding at equilibrium in Triton-treated membranes 256. The acceleration by Gly could agree with the electrophysiological finding that Gly markedly increases the frequency of opening of ion channels mediated by the N M D A recognition sites without altering either the amplitude of a single N M D A current or the mean open time of the channels 9o. In contrast, the acceleration by Glu but not by Gly is sensitive to attenuation by SH-reactive agents including N-ethylmaleimide, p-chloromercuribenzoic acid and 5,5'-dithio-bis-(2-nitrobenzoic acid) 255. Successful solubilization of the N M D A receptor complex from rat and porcine brain has been performed using [3H]MK-801 binding as an index. The solubilized binding is sensitive to potentiation by Glu and Gly with pharmacological profiles similar to those in membrane preparations 130, and SPD in addition to the afore-mentioned two stimulatory amino acids potentiates in a concentration-dependent manner the binding of [3H]MK-801 in preparations after gel filtration of supernatants solubilized from rat brain 163 An in vitro quantitative receptor autoradiographic technique has demonstrated that [3H]MK-801 binding sites are unevenly distributed in rat brain, the highest binding being found in the molecular layer of the dentate gyrus 19 The distribution profiles of [3H]MK801 binding are well consistent with those of NMDA-sensitive [3H]GIu and [3H]TCP binding but not with sigma ligand binding, whereas neither Glu nor N M D A potentiates the binding of [3H]MK-801, eliciting a marked reversal of AP5-induced inhibition in these brain thin sections 59 The binding of [3H]MK-801 as well as [3H]TCP is evidently different from a simple association process of a radioactive ligand with sites located on extracellular surfaces of

15 synaptic plasma membranes as seen with the binding of [3H]Glu or [3H]CPP to N M D A recognition sites. The non-competitive antagonists are able to gain access to their binding sites only when the N M D A channels are opened by agonists with the aid of Gly. Therefore, evaluation of [3H]MK-801 binding can better be made according to kinetic analysis rather than to Scatchard analysis which has to be determined at equilibrium. Estimation of the initial association and dissociation rates seems to be highly preferable to the determination of saturation isotherms in the case of neurochemical elucidation of the open N M D A channels using [3H]MK-801 or [3H]TCP. In fact, brain ischemia induces a marked reduction of the initial association rate of [3H]MK-801 binding in the hippocampus of gerbils irrespective of the presence of Glu, Gly or SPD, without significantly affecting the binding measured at equilibrium under the same conditions (unpublished data of the authors).

3. Sigma receptors The sigma sites were at first assumed to be associated with the atypical psychotomimetic and autonomic effects of benzomorphan opiates, particularly N-allynormetazocine (SKF 10,047), and thus the sites were referred to as 'sigma opiate receptors' ~23 However, subsequent radioligand binding studies with [3H]SKF 10,047 identified two mutually related but distinct sites, both of which are virtually insensitive to opiate antagonists, namely PCP receptors and sigma receptors 187,214,224,267. The former PCP receptors are nowadays supposed to be identical to ion channels gated by the N M D A recognition sites as seen with the binding of [3H]MK-801 and [3H]TCP, while the latter sigma receptors are shown to have a clear correlation to novel sites with unique high affinity for both (+)-benzomorphan enantiomers and some neuroleptic drugs 215. In addition to labeling by [3H]SKF 10,047, these sigma receptors are radiolabeled by more selective sigma ligands with no intrinsic activity at PCP receptors (NMDA channels), including [3H] (+)-3-(hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP) 107.108 and [3H]dio-tolylguanidine (DTG)227. The sigma ligand 3-PPP was originally proposed as an agonist selective for dopaminergic autoreceptors 72, whereas recent studies have shown that potent dopaminergic agonists and antagonists are weak inhibitors of [3H]3-PPP binding, with distribution profiles of [3H]3-PPP binding being different from those of dopaminergic n e u r o n s 107,108. Haloperidol has a unique higher affinity for the binding of [3H]3_pp P 107 and [3H]DTG 227 than 3-PPP. Photoaffinity labeling 93 as well as solubilization 94 of sigma receptors has been carried out using [3H]DTG binding as a biochemical index. Since compounds with affinity for the N M D A channels almost invariably have affinity for the sigma receptors, there could be some similarities between these two different receptor molecules in terms of the selectivity for ligands. However, it appears that compounds with higher affinity for the sigma receptors exhibit lower affinity for the N M D A channels and vice versa 167.252

E. Modulatory cations 1. Magnesium Magnesium ions are known to regulate the opening of the N M D A channels in a voltage-dependent m a n n e r 39,127,154. In fact, this divalent cation augments the ability of Glu to potentiate [3H]TCP binding with a concomitant increase in the binding determined in the absence of any added amino acids by increasing the affinity in brain synaptic membranes 91.118. In contrast, Mg 2+ ions in a concentration-dependent manner at concentrations from 10 /~M to 10 mM inhibit [3H]MK-801 binding in brain synaptic membranes not extensively washed, while they potentiate binding in membranes extensively washed at concentrations below 0.3 mM 236. The divalent cation is also shown to

16 invariably inhibit [3H]MK-801 binding irrespective of the presence of two stimulatory amino acids, by inducing a 30-fold more potent inhibition of binding in the presence of both Glu and Gly than that in the absence of added amino acids 190. Inhibition by Mg 2 ions is attributed to the facilitated dissociation of bound [3H]MK-801, and this sort 0t" inhibition seems to be closely related to the mechanisms underlying the inhibition of [3H]MK-801 binding by tricyclic antidepressants and phenothiazine derivatives 191. In solubilized preparations 58, Mg2+ ions markedly enhance [3H]MK-801 binding in the absence of any added amino acids, but inhibit binding in the presence of both Glu and Gly. Finally, Mg 2+ ions are one of the endogenous determinants for NMDA-mediated opening of cation channels permeable to Ca 2+ and Na + ions depending on their concentration around the channels. 2. Zinc

Ion channels gated by the NMDA recognition sites exhibit selective and voltage-independent antagonism by Zn2+ ions through sites different from those responsible for inhibition by Mg 2+ ions 182,229. Zinc ions are shown to be present in the hippocampus and cerebral cortex 71,178 and to be released on nerve stimulation 5,82. Consistent with electrophysiological findings, Zn2+ ions inhibit [3H]MK-801 binding at concentrations above 1 ~M in either the presence or absence of Glu and Gly 190,191. Therefore, Zn 2+ ions could play a role as an allosteric endogenous antagonist after being released along with Glu from presynaptic nerve terminals during the excitation of neurons. Neuronal responses mediated by the NMDA-sensitive receptors appear to be modulated by extracellular concentrations of the afore-mentioned two kinds of divalent cations with different inhibitory properties. 3. Sodium

Sodium ions have an ability to reduce the concentration of excitatory amino acids in synaptic clefts by facilitating their accumulation by the Na+-dependent uptake system into adjacent neuronal a n d / o r glial cells 2. This accumulation plays a role as a termination process of the neurotransmission mediated by excitatory amino acids, In addition, this cation would have a modulatory function by attenuating the association of endogenous ligands with NMDA recognition sites on the NMDA receptor complex. Sodium ions in a concentration-dependent manner at concentrations above 40 mM inhibit binding of both [3H]GIu 260 and [3H]CPP 162 to the NMDA recognition sites in brain synaptic membranes treated with Triton X-100. This monovalent cation also inhibits NMDA-sensitive [3H]Glu binding only at low concentrations in synaptic plasma membranes prepared by sucrose density gradient centrifugation 141, and diminishes NMDA-insensitive and temperature-dependent binding of [3H]Glu 200 and [3H]AP4 22. The latter two binding sites are now proposed to reflect more precisely the cystine-sensitive exchange system for acidic amino acids 6,96,266 than the NMDA recognition sites. At any rate, Na ÷ ions may reduce the affinity of excitatory amino acid receptors for endogenous ligands, and the reduction subsequently assists removal of the ligands bound to receptors by the Na+-dependent uptake system from synaptic clefts. Consequently, Na ÷ ions could accelerate termination of the neurotransmission a n d / o r excitotoxicity mediated by excitatory amino acids by facilitating the dissociation of a ligand bound to receptors in addition to activating its removal from synaptic clefts by the uptake system. F. Signal transduction 1. Ca 2 + influx

The hypothesis that ion channels associated with the NMDA recognition sites have a high permeability to Ca 2+ ions 4.87.126 was directly proved for the first time in cultured

17 spinal cord neurons using the Ca 2+ indicator dye, arsenazo III 121. Excitatory amino acids acting at NMDA-sensitive receptors invariably increase the intracellular free Ca 2+ concentration in a MgE+-sensitive fashion, with the other agonist KA being much less effective in triggering the increase 121. In line with these findings, the activation of NMDA-sensitive receptors markedly stimulates 45Ca2+ influx into cultured cerebellar granule cells 240. In these cells, NMDA, Glu, Asp and KA are able to stimulate the influx of 45Ca2+and the stimulation by N M D A but not by KA is sensitive to inhibition by the non-competitive N M D A antagonist, PCP 240. This stimulatory property of N M D A is further potentiated by the addition of amino acids active at Gly B sites 241, and is more sensitive to the inhibition by ethanol at low concentrations than that of KA 73. NMDA, QA and KC1 are all effective in potentiating both 45Ca2+ uptake into cortical slices and quin-2 fluorescence in cortical synaptosomes, while responses to N M D A only are blocked by non-competitive antagonists such as ketamine, PCP and Mg 2+ as well as the competitive antagonist, AP5 176. In vivo fluorometry using the dye indo-1 has revealed that superfusion of N M D A significantly increases the intracellular free Ca 2+ in a manner sensitive to antagonism by Mg 2+ and AP5 220. A drastic rise in intracellular Ca 2+ ions through the N M D A channels is likely to result in a series of subsequent intracellular metabolic alterations which are connected with neuronal excitement and excitotoxicity. This unique permeability to Ca 2+ ions renders the N M D A receptor complex a subclass of particular physiological and pathophysiological importance among the neurotransmitter receptors.

2. Cyclic GMP accumulation In the immature and adult rat cerebellum, excitatory amino acids markedly elevate endogenous levels of cyclic GMP both in vitro s5 and in vivo 13.237, possibly through the NMDA-sensitive receptors. In immature rat cerebellar slices, N M D A induces a concentration-dependent increase in cyclic G M P levels 60, and this elevation not only is blocked by the competitive antagonist AP5 but is also attenuated by the removal of Ca 2+ ions or the addition of Mg 2+ ions 24 In this in vitro system, (+_)-cis-2,3-piperidine dicarboxylic acid behaves as a partial N M D A agonist in Mg2+-free medium containing the phosphodiesterase inhibitor isobutylmethylxanthine, while its trans-isomer has full agonistic activity in elevating the cerebellar cyclic G M P levels 110. Noradrenaline potently antagonizes in a concentration-dependent fashion at concentrations above 0.1 /~M the stimulatory property of N M D A to elevate cyclic G M P levels in immature rat cerebellar slices, and this antagonism is sensitive to prevention by the Na+,K+-ATPase inhibitor, ouabain, with ouabain alone being able to enhance the effect of N M D A 25. In addition, intracerebellar injection of the Gly B agonist, o-serine, elicits a dose-dependent increase in cerebellar cyclic GMP levels in vivo, which is prevented by the simultaneous injection of CPP, MK-801 or HA-966 238. These in vivo and in vitro effects of N M D A on cerebellar cyclic GMP levels are believed to be mediated by increased intracellular Ca 2+ ions through the N M D A channels 24. Thus, cerebellar cyclic G M P is undoubtedly a good biochemical index of the activity of the N M D A receptor complex in the mammalian CNS. 3. Phosphatidylinositol hydrolysis As a result of the depolarization induced by an NMDA-mediated opening of cationic ion channels, the stimulation of phosphatidylinositol (PI) hydrolysis by muscarinic cholinergic agonists is prevented. Both N M D A and KA are effective in markedly inhibiting the stimulation of PI hydrolysis by carbachol in a non-competitive manner without significantly affecting that by noradrenaline in rat hippocampal slices, and

18 inhibition by NMDA but not by KA is reversed by competitive and non-competitive antagonists selective for the NMDA receptors 9. The inhibitory property seems to be derived from sodium influx through the NMDA channels rather than through the voltage-dependent sodium channels 146. Depolarization by veratridine as well as high potassium stimuli invariably inhibits the stimulation of PI hydrolysis by carbachol in hippocampal slices in a manner not sensitive to antagonism by D-AP5 and CNQX 146. In contrast, both Glu and NMDA facilitate PI hydrolysis through NMDA recognition sites in an AP5-sensitive manner in primary cultures of cerebellar granule cells 153, and Gly additionally facilitates this stimulation of PI hydrolysis induced by NMDA but not that by QA or carbachol 153. Although the mechanisms underlying the stimulation of PI hydrolysis through the NMDA recognition sites are not clear, a likely explanation is thai the NMDA sites may be directly linked to phospholipase C responsible for the hydrolysis of PI in synaptic plasma membranes. Another possibility is that increased influx of Ca 2~ ions through the NMDA channels may induce activation of phospholipase C catalyzing PI hydrolysis. At any rate, depolarization mediated by ionotropic excitatory amino acid receptors could affect PI hydrolysis and not by interacting with a novel hypothetical metabotropic type of receptor. Consequently, NMDA as well as Glu is able to play a dual role in the hydrolysis of PI through the NMDA receptor complex in a manner dependent on the central structures concerned.

4. Arachidonate release In cultured cerebellar granule cells preloaded with [3H]arachidonic acid 109 both Asp and Glu are effective in stimulating the release of [3H]arachidonate with lesser potency by KA, NMDA, QA and carbachol, and stimulation by these agonists is prevented by the inclusion of the non-competitive NMDA antagonist PCP. In primary cultures of mouse striatal neurons devoid of differentiated synapses 44, NMDA as well as Glu markedly stimulates the release of preloaded [3H]arachidonate in a concentration-dependent manner through the NMDA-sensitive receptors, and this stimulation is completely blocked by the phospholipase A 2 inhibitor, mepacrine. Therefore, the afore-mentioned calcium influx through NMDA channels is likely to be responsible for the arachidonate release by activating membranous phospholipase A 2. After all, the ionotropic NMDA receptors could induce different intracellular metabotropic changes in various neuronal cells by way of the well-described calcium influx through the NMDA channels.

G. Working hypothesis Figure 1 shows our proposed model for the NMDA receptor complex. The complex contains at least 6 different modulatory sites for the influx of cations through the channels located within the complex. Three sites are located at extracellular surfaces of synaptic membranes, while the other two sites reside in the intramembranous location. Although the exact position of the last modulatory sites by polyamines is uncertain at present, they are tentatively represented to exist at intracellular surfaces of synaptic membranes judging from the absence of the endogenous ligands at sufficiently effective concentrations from synaptic clefts. The Glu released from presynaptic nerve terminals during ordinal neuronal excitation is able to activate the non-NMDA channels, while unable to open the NMDA channels due to a voltage-dependent inhibition by Mg 2+ ions at concentrations that occur in vivo. However, marked depolarization through the non-NMDA channels by tetanic stimulation of presynaptic neurons a n d / o r by a drastic elevation of the Glu level in synaptic clefts would release the voltage-dependent inhibition of NMDA channels, and thus result in a dramatic influx of Ca 2÷ as well as Na ÷ ions through the open NMDA channels.


NMOA c h a n n ¢ l ~ A F F I N I T ¥ ~

S i g m a r~zc~zplor [+)3 PP~

MK 801, TCP,


SKF 1 0 0 t , 7 , H a l o o g r l d o l . OrG


Memory Learning



[scr~em,a Hypo~lyce mla E plleI~y

Alzhelmcr 'S Hunllngton's Sct~izophr enla

Fig. 1. Proposed model for the N M D A receptor complex. The complex consists of at least 3 different stimulatory domains linked to the N M D A ion channels that have 3 distinct negative allosteric sites. For details, see text.

In addition to inhibition by Mg 2+ ions, the opening of NMDA channels is also diminished in a voltage-independent manner by Zn2+ ions at physiologically reasonable concentrations. Moreover, non-competitive antagonists such as MK-801 and TCP effectively block the influx of Ca 2+ as well as Na ÷ ions by associating with sites responsible for mediating NMDA responses located within the channels. In contrast to these 3 inhibitory sites, the complex has the other 3 distinct stimulatory domains for the opening of channels. Among the 6 different sites on the complex, the Gly B sites seem to play a key role in the opening of NMDA channels by endogenous agonists. Gly is absolutely essential for the Glu-induced potentiation of [3H]MK-801 binding (opening of NMDA channels), and Glu is completely required for the potentiation of [3H]MK-801 binding by SPD. Therefore, modulation by polyamines appears to be dependent on the activity of NMDA recognition sites which absolutely require occupation of the Gly B sites by Gly. Only Glu, but neither Gly nor SPD, is able to open the channels, while both Gly and SPD could modulate the opening process through their own domains. Since there is no evidence for the presence of polyamines at effective concentrations in extracellular synaptic clefts at present, the polyamine domain is tentatively assumed to reside at intracellular surfaces of synaptic membranes. All the polyamine sites could not always be linked to the NMDA receptor complex in the brain, since SPD fails to potentiate [3H]MK-801 binding in both the cerebellum and medulla-pons that have a high density of [3H]SPD binding sites. Accordingly, the NMDA receptor complex consists of at least 4 distinct subcomponents with 6 different modulatory sites. The ion channel subcomponent has 3 negative modulatory sites, while the other 3 subcomponents each have their own

20 positive modulatory sites. The polyamine subcomponent is not always associated with the complex, which suggests possible heterogeneity of the NMDA receptor complex in terms of sensitivity to polyamines. The unique property of N M D A channels to permeate Ca 2 ions is supposed to be responsible for the subsequent intracellular signal transduction which may mediate both neuronal plasticity and excitotoxicity as described below. The fact that a compound with high affinity for NMDA channels invariably exhibits psychotomimetic effects and some affinity for sigma receptors gives support to the proposal that sigma receptors are located close to, but different from, the NMDA channels. It is conceivable that sigma receptors may have some unknown interaction with the NMDA receptor complex. IV. F U N C T I O N A L S I G N I F I C A N C E

The same NMDA receptor complex that mediates excitatory neuronal transmission can also be attributed to neuronal injury. A transient influx of Ca 2+ ions through the NMDA channels could facilitate various intercellular signal communications between neurons including neuronal plasticity 206,239, while prolonged stimulation of the excessive calcium influx would eventually cause necrosis of most central neurons through intracellular Ca2+-dependent mechanisms 28,29,50,88. Moreover, prolonged depolarization induced by the NMDA receptor complex would result in a subsequent passive C1- influx followed by massive water entry which produces osmotic lysis of the cells 172,195. The latter two independent mechanisms may be operative in vivo in the brain neuronal damage found in ischemia, hypoglycemia, epilepsy and several neurodegenerative disorders such as Huntington's disease.

A. Plasticity The NMDA receptor complex is proposed to play a key role in neuronal plasticity mechanisms in the immature as well as mature brain (for review, see Ref. 140). In particular, the complex is responsible for mediating long-term potentiation (LTP) which is a phenomenon supposed to be intimately associated with learning and memory 16. A train of high-frequency stimulation of presynaptic neurons markedly facilitates synaptic efficacy at postsynaptic neurons that may be observed even several days after stimulation. The competitive NMDA antagonist AP5 completely blocks the induction of hippocampal LTP in a reversible manner 32 and a clear correlation is seen between the abilities of these antagonists to block hippocampal LTP and to inhibit the NMDA receptor complex 69. Furthermore, non-competitive antagonists including PCP 213 ~.nd Mg 2+ ions 83 are effective in suppressing the induction of LTP in the hippocampus. The antagonist AP5 also prevents the induction of LTP in rat visual cortex 3, and AP5 much more effectively prevents LTP in the visual cortex of young kittens than of adult cats 219 Indeed, AP5 is able to prevent spatial learning in rats in the water maze test in addition to inhibiting LTP 145, and a significant increase in the density of NMDA-sensitive [3H]GIu binding occurs in chick brain following imprinting that is closely associated with the acquisition process of learning and memory 128. Eventually, the non,NMDA receptor channels would be modified by increased intracellular Ca 2÷ ions flowing in across the NMDA channels through several Ca2+-dependent processes (Fig. 1). This Ca2+-dependent modification could be responsible for the long-lasting facilitation of synaptic efficacy at postsynaptic neurons after a train of ~ . f r e q u e n e y stimulation of presynaptic neurons 3~. However, NMDA antagonists fail to block the induction of LTP in mossy fibers in the guinea-pig hippocampal CA3 region, which strongly suggests the occurrence

21 of LTP mediated by non-NMDA receptors 68. Since evidence is accumulating that LTP is a synaptic model of learning and memory 30.31.217, the NMDA receptor complex undoubtedly participates, at least in part, in the acquisition and formation of memory. Therefore, dysfunction of the complex could give rise to impaired memories observed in various types of dementia and amnesia as described below. The possible involvement of the NMDA receptor complex in decerebrate rigidity has also been suggested 202

B. Excitotoxicity The NMDA receptor complex is proposed to be involved in the neuropathology of brain damage found in ischemia, hypoglycemia, epilepsy and neurodegenerative disorders such as Huntington's, Parkinson's and Alzheimer's diseases. Some of the typical neurological symptoms could result from an impaired ability of the complex to induce neuronal plasticity in central neurons in which the function of the complex is already disturbed by degeneration through some unknown mechanisms, while excessive activation of the complex would result in degeneration of central neurons via its excitotoxic property. Consequently, the NMDA receptor complex may be attributable to the etiological mechanisms underlying neuronal cell death found in some neurodegenerative disorders, in addition to the appearance of neurological symptoms associated with impaired memory such as dementia and amnesia. For instance, transient ischemic brain damage causes an excessive elevation of extracellular Glu concentration a2, which may in turn induce neuronal cell death closely related to the NMDA receptor complex 92,133,196. In fact, ischemia elicits a significant loss of the density of NMDA-sensitive [3H]Glu 34 and [3H]TCP 111 binding sites in the hippocampus that is a central structure vulnerable to deprivation of oxygen and glucose. Competitive and non-competitive antagonists such as AP7 2o3, CPP 17, CGS 19755 17 and MK-801 62 are all effective in protecting against these ischemia-induced hippocampal damages in gerbils. These findings suggest a crucial involvement of the NMDA receptor complex in the etiology of neuronal cell death induced by ischemia, while Buchan and Pulsinelli 2a have recently demonstrated that hypothermia but not NMDA antagonism is responsible for the neuroprotective action of MK-801 against ischemia-induced neuronal damage. At any rate, the possible participation of the complex in the pathological mechanisms underlying neuronal cell death found in the ischemic brain is strongly suggested. In addition to its involvement in the pathogenesis of ischemic neuronal cell death, the NMDA receptor complex is presumed to have intimate correlates with the etiology and pathology of various neurological disorders associated with brain cell damage, such as hypoglycemia 230,232 epilepsy 35,36,131,135,243,aging a83, schizophrenia 239 Alzheimer's disease 61,64,65,138,185,186,204,Huntington's disease 64.265 and Parkinson's disease 101. The complex is also assumed to be involved in the exhibition of some neuropharmacological activities of methamphetamine 210, non-psychotropic cannabinoid 51 and ethanol 105.119.147 V. G E N E R A L CONCLUSION

It thus appears that the NMDA receptor complex consists of at least 3 different stimulatory domains which are linked to cation channels mainly permeable to Ca 2+ as well as Na + ions, with the ion channels containing at least 3 distinct inhibitory sites. Among these 6 positive and negative allosteric modulatory domains within the complex, the Glya sites seem to have critical functional significance in terms of the absolute requirement for the opening of ion channels by NMDA agonists. Occupation of the Gly B

22 sites by Gly may sensitize the endogenous neurotransmitter Glu to open the channels even in a state that is blocked by Mg 2+ a n d / o r Zn 2+ ions. Moreover, the endogenous polyamines SPD and spermine could contribute to the allosteric modulatory mechanisms for the opening of N M D A channels. Excitatory amino acids could play dual roles as excitatory neurotransmitter and excitotoxin, at-least in part through the N M D A receptor complex in the brain. The former role is responsible for inducing central neural plasticity that is closely associated with learning and memory, whereas the latter role gives rise to the neuronal cell death that is intimately related to the etiological mechanisms underlying various neurological disorders. Both roles could be triggered by a marked rise in the concentration of intracellular Ca 2+ ions through the open N M D A channels. Thus, competitive and non-competitive antagonists that eventually block the influx of Ca 2+ ions across the N M D A channels would be beneficial in the therapy of various neurodegenerative diseases without the risk of inhibiting the acquisition and formation process of memory. Therefore, the search for therapeutic drugs acting at the N M D A receptor complex should be carefully carried out as much as possible. The combined use of drugs acting at 2 or 3 different allosteric modulatory domains might be of great benefit to the therapy and treatment of neuropsychiatric disorders associated with neuronal cell death irrespective of the appearance of impaired memory. ABBREVIATIONS AMPA

= a-amino-3-hydroxy-5methylisoxazole4-propionic acid AP4 = 2-amino-4-phosphonobutyric acid AP5 = 2-amino-5-phosphonovaleric acid AP7 = 2-amino-7-phosphonoheptanoic acid Asp = aspartic acid CGS 19755 = cis-4-phosphonomethyl-2piperidine carboxylic acid CNQX = 6-cyano-7-nitroquinoxaline-2,3-dione CNS = central nervous system CPP = 3-(2-carboxypiperazin-4yl)-propyl- 1-phosphonic acid DCQX = 6,7-dichloroquinoxaline2,3-dione DNQX = 6,7-dinitroquinoxaline2,3-dione DTG = di-o-tolylguanidine Glu = glutamic acid Gly = glycine GlyA = strychnine-sensitive glycine-binding sites

strychnine-insensitive glycine-binding sites = reduced glutathione GSH = oxidized glutathione GSSG = 1-hydroxy-3-aminopyrroliHA-966 done-2 = kainic acid KA = long-term potentiation LTP = 5-methyl-10,11-dihydroMK-801 5H-dibenzo[ a, d ]cyclohepten-5,10-imine = 5,7-dinitroquinoxalineMNQX 2,3-dione = N-methyl-D-aspartic acid NMDA = phencyclidine PCP = phosphatidyl inositol PI = quisqualic acid QA 7-C1KYNA = 7-chtorokynurenic acid SKF 10,047 = N-allylnormetazocine SL 82.0715 = ( + )-a-(4-chlorophenyl),4[(4- fluorophenyl)methyl]- 1piperidineethanol = spermidine SPD = N-[1-(2-thienyl)cyclohexyl]TCP piperidine = ( + )- 3-(hydroxyphenyl)-N3-PPP (1-propyl)piperidine Gly~



1 Ambar, I., Kloog, Y. and Sokolovsky, M., Solubilization of rat brain phencyclidine receptors in an active binding form that is sensitive to N-methyl-D-aspartate receptor ligands, J. Neurochem., 51 (1988) 133-140. 2 Anis, N.A., Berry, S.C., Burton, N.R. and Lodge, D., The dissociative anesthetics, ketamine and phencyclidine, selectively reduce excitation of central mammalian neurones by N-methyl-aspartate, Br. J. PharmacoL, 79 (1983) 565-575. 3 Artola, A. and Singer, W., Long-term potentiation and NMDA receptors in rat visual cortex, Nature, 330 (1987) 649-652. 4 Ascher, P. and Nowak, L., Calcium permeability of the channels activated by N-methyl-D-aspartate (NMDA) in isolated mouse central neurons, J. Physiol. (Lond.), 377 (1986) 35P. 5 Assaf, S.Y. and Chung, S., Release of endogenous Zn 2÷ from brain tissue during activity, Nature, 308 (1984) 734-736. 6 Bannai, S., Exchange of cystine and glutamate across plasma membrane of human fibroblasts, J. Biol. Chem., 261 (1986) 2256-2263. 7 Barnard, E.A., Darlison, M.G. and Seeburg, P., Molecular biology of the GABA receptor: the receptor/ channel superfamily, Trends Neurosci., 10 (1987) 502-509. 8 Baron, B.M., Dudley, M.W., McCarty, D.R., Miller, F.P., Reynolds, I.J. and Schmidt, C.J., Guanine nucleotides are competitive inhibitors of N-methyl-D-aspartate at its receptor site both in vitro and in vivo, J. PharmacoL Exp. Ther., 250 (1989) 162-169. 9 Baudry, M., Evans, J. and Lynch, G., Excitatory amino acids inhibit stimulation of phosphatidylinositol metabolism by aminergic agonists in hippocampus, Nature, 319 (1986) 329-331. 10 Benavides, J., Rivy, J.P., Carter, C. and Scatton, B., Differential modulation of [3H]TCP binding to the NMDA receptor by L-glutamate and glycine, Eur. J. PharmacoL, 149 (1988) 67-72. 11 Bennet, Jr., J.P., Logan, W.J. and Snyder, S.H., Amino acids as central nervous transmitters. The influence of ions, amino acid analogues and ontogeny on transport systems for /-glutamic and l-aspartic acids and glycine into central nervous synaptosomes of the rat, J. Neurochem., 21 (1973) 1533-1550. 12 Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem., 43 (1985) 1369-1374. 13 Biggio, G., Brodie, B.B., Costa, E. and Guidotti, A., Mechanisms by which diazepam, muscimol, and other drugs change the content of cGMP in cerebellar cortex, Proc. Natl. Acad. Sci. USA, 74 (1977) 3592-3596. 14 Birch, P.J., Grossman, C.J. and Hayes, A.G., Kynurenic acid antagonises responses to NMDA via an action at the strychnine-insensitive glycine receptor, Eur. J. PharmacoL, 154 (1988) 85-87. 15 Birch, P.J., Grossman, C.J. and Hayes, A.G., 6,7-Dinitro-quinoxaline-2,3-dion and 6-nitro-7-cyanoquinoxaline-2,3-dion antagonise responses to N M D A in the rat spinal cord via an action at the strychnineinsensitive glycine receptor, Eur. J. PharmacoL, 156 (1988) 177-180. 16 Bliss, T.V.P. and Lomo, T., Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path, J. Physiol. (Lond.), 232 (1973) 331-356. 17 Boast, C.A., Gerhardt, S.C., Pastor, G., Lehmann, J., Etienne, P.E. and Liebman, J.M., The N-methyl-Daspartate antagonists CGS 19755 and CPP reduce ischemic brain damage in gerbils, Brain Res., 442 (1988) 345-348. 18 Bonhaus, D.W., Burge, B.C. and McNamara, J.O., Biochemical evidence that glycine allosterically regulates an NMDA receptor-coupled ion channel, Eur. J. Pharmacol., 142 (1987) 489-490. 19 Bowery, N.G., Wong, E.H.F. and Hudson, A.L., Quantitative autoradiography of [3H]-MK-801 binding sites in mammalian brain, Br. J. PharmacoL, 93 (1988) 944-954. 20 Bristow, D.R., Bowery, N.G. and Woodruff, G.N., Light microscopic autoradiographic localisation of [3H]glycine and [3H]strychnine binding sites in rat brain, Eur. J. Pharmacol., 126 (1986) 303-307. 21 Buchan, A. and Pulsinelli, W.A., Hypothermia but not the N-methyl-D-aspartate antagonist, MK-801, attenuates neuronal damage in gerbils subjected to transient global ischemia, J. Neurosci., 10 (1990) 311-316. 22 Butcher, S.P., Roberts, P.J. and Collins, J.F., Ionic regulation of the binding of DL-[3H]2-amino-4-phosphonobutyrate to L-glutamate-sensitive sites on rat brain membranes, J. Neurochem., 43 (1984) 1039-1045. 23 Butcher, S.P., Roberts, P.J. and Collins, J.F., Purine nucleotides inhibit the binding of DL-[aH]2-amino-4phosphonobutyrate (DL-[3H]APB) to L-glutamate-sensitive sites on rat brain membranes, Biochem. PharmacoL, 35 (1986) 991-994. 24 Carter, C.J. and Scatton, B., Ionic mechanisms implicated in the stimulation of cerebeUar cyclic GMP levels by N-methyl-D-aspartate, J. Neurochem., 49 (1987) 195-200.

24 25 Carter, C.J., Gueugnon, J. and Scatton, B., Noradrenatine antagonizes and ouabain potentiates tile effects of N-methyl-D-aspariate on rat cerebellar cyclic GMP production, J. Neurochern.. 51 (1988) 944 949. 26 Carter, C., Benavides, J., Legendre, P., Vincent, J.D., Noel, F., Thuret, F., Lloyd, K.G., Arbilla, S.. Zivkovic, B., MacKenzie, E.T., Scatton, B. and Langer, S.Z., Ifenprodil and SL 82.0715 as cerebral anti-ischemic agents. If. Evidence for N-methyl-D-aspartate receptor antagonist properties, .1. Pharrnac~d. Exp. Ther., 247 (1988) 1222-1232. 27 Carter, C., Rivy, J.-P. and Scatton, B., Ifenprodil and SL 82.0715 are antagonists at the polyamine site ~f the N-methyl-D-aspartate (NMDA) receptor, Eur. J. Pharmacol., 164 (1989) 611 612. 28 Choi, D.W., Glutamate neurotoxicity in cortical cell culture is calcium dependent, Neurosci. Lett., 58 (1985) 293-297. 29 Choi, D.W., Ionic dependence of glutamate neurotoxicity in cortical cell culture..L Neurosci., 7 (1987) 369-379. 30 Collingridge, G., The role of NMDA receptors in learning and memory, Nature, 330 (1987) 604-605. 31 Collingridge, G.L. and Bliss, T.V.P., NMDA receptors - their role in long-term potentiation, Trend~ Neurosci., 10 (1987) 288-293. 32 Collingridge, G.L., Kehl, S.J. and McLennan, H., Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus, J. Physiol. (Lond.), 334 (1983) 33-46. 33 Compton, R.P., Hood, W.F. and Monahan, J.B., Evidence for a functional coupling of the NMDA and glycine recognition sites in synaptic plasma membranes, Eur. J. Pharmacol., 188 (1990) 63-70. 34 Crepel, V., Represa, A. and Ben-Ari, Y., Effect of ischemia and intraamygdaloid kainate injection on the density of NMDA binding sites in the hippocampal CA1 region, Eur. J. Pharmaeol., 151 (1988) 355-356. 35 Croucher, M.J., Collins, J.F. and Meldrum, B.S., Anticonvulsant action of excitatory amino acid antagonists, Science, 216 (1982) 899-901. 36 Croucher, M.J., Bradford, H.F., Sunter, D.C. and Watkins, J.C., Inhibition of the development of electrical kindling of the prepyriform cortex by daily focal injections of excitatory amino acid antagonists, Eur. J. Pharmacol., 152 (1988) 29-38. 37 Curtis, D.R. and Johnston, G.A.R., Amino acid transmitters in the mammalian central nervous system, Ergeb. Physiol., 69 (1974) 94-188. 38 Davies, J. and Watkins, J.C., Action of the D- and L-forms of 2-amino-5-phosphonovalerate and 2-amino4-phosphonobutyrate in the cat spinal cord, Brain Res., 235 (1982) 378-386. 39 Davies, J. and Watkins, J.C., Effect of magnesium ions on the responses of spinal neurons to excitatory amino acids and acetylcholine, Brain Res., 130 (1977) 364-368. 40 Davies, J., Evans, R.H., Herrling, P.L., Jones, A.W., Olverman, H.J., Pook, P. and Watkins, J.C., CPP, a new potent and selective NMDA antagonist. Depression of central neuron responses, affinity for D-[ 3H]AP5 binding sites on brain membranes and anticonvulsant activity, Brain Res., 382 (1986) 169-173. 41 De Feudis, F.V., Oresanz-Mu~.oz, L.M. and Fando, J.L., High affinity giycine binding sites in rat CNS: regional variation and strychnine sensitivity, Gen. Pharmacol., 9 (1978) 171-176. 42 Do, K.Q., Herrling, P.L., Streit, P., Turski, W.A. and Cuenod, M., In vitro release and electrophysiological effects in situ of homocysteic acid, an endogenous N-methyl-(D)-aspartic agonist, in the mammalian striatum, J. Neurosci., 8 (1986) 2226-2234. 43 Duchen, M.R., Burton, N.R. and Biscoe, T.J., An intracellular study of the interactions of N-methyl-oLaspartate with ketamine in the mouse hippocampal slice, Brain Res., 342 (1985) 149-153. 44 Dumuis, A., Sebben, M., Haynes, L., Pin, J.-P. and Bockaert, J., NMDA receptors activate the arachidonic acid cascade system in striatal neurons, Nature, 336 (1988) 68-70. 45 Fadda, E., Danysz, W., Wroblewski, J.T. and Costa, E., Glycine and o-serine increase the affinity of N-methyl-D-aspartate sensitive glutamate binding sites in rat brain synaptic membranes, Neuropharmacology, 27 (1988) 1183-1185. 46 Fagg, G.E., Phencyclidine and related drugs bind to the activated N-methyl-D-aspartate receptor-ion channel complex in rat brain membranes, Neurosci. Lett., 76 (1987) 221-227. 47 Fagg, G.E. and Matus, A., Selective association of N-methyl aspartate and quisqualate types of L-glutamate receptor with brain postsynaptic densities, Proc. Natl. Aead. Sci. USA, 81 (1984) 6876-6880. 48 Fagg, G.E., Foster, A.C., Mena, E.E. and Cotman, C.W., Chloride and calcium ions reveal a pharmacologically distinct population of L-glutamate binding sites in synaptic membranes: correspondence between biochemical and electrophysiological data, J. Neurosei., 2 (1982) 958-965. 49 Fagg, G.E., Mena, E.E., Monaghan, D.T. and Cotman, C.W., Freezing eliminates a specific population of L-glutamate receptors in synaptic membranes, Neurosci. Lett., 38 (1983) 157-162. 50 Farber, J.L., Chieu, K.R. and Mittnacht, S., The pathogenesis of irreversible cell injury in ischemia, Am. J. Pathol., 102 (1981) 271-281.

25 51 Feigenbaum, J.J., Bergmann, F., Richmond, S.A., Mechoulam, R., Nadler, V., Kloog, Y. and Sokolovsky, M., Nonpsychotropic cannabinoid acts as a functional N-methyl-D-aspartate receptor blocker, Proc. Natl. Acad. Sci. USA, 86 (1989) 9584-9587. 52 Ferraro, T.N. and Hare, T.A., Free and conjugated amino acids in human CSF: influence of age and sex, Brain Res., 338 (1985) 53-60. 53 Fletcher, E.J. and Lodge, D., Glycine reverses antagonism of N-methyl-D-aspartate (NMDA) by 1-hydroxy3-aminopyrrolidone-2 (HA-966) but not by D-2-amino-5-phosphonovalerate (D-AP5) on rat cortical slices, Eur. J. Pharmacol., 151 (1988) 161-162. 54 Fonnum, F., Glutamate: a neurotransmitter in mammalian brain, J. Neurochem., 42 (1984) 1-11. 55 Foster, A.C. and Roberts, P.J., Pharmacology of excitatory amino acid receptors mediating the stimulation of rat cerebellar cyclic GMP levels in vitro, Life Sci., 27 (1980) 215-221. 56 Foster, A.C. and Fagg, G.E., Acidic amino acid binding sites in mammalian neuronal membranes: their characteristics and relationship to synaptic receptors, Brain Res. Rev., 7 (1984) 103-164. 57 Foster, A.C. and Fagg, G.E., Comparison of L-[3H]glutamate, D-[3H]aspartate, DL-[3H]AP5 and [3H]NMDA as ligands for NMDA receptors in crude postsynaptic densities from rat brain, Eur. J. Pharmacol., 133 (1987) 291-300. 58 Foster, A.C. and Wong, E.H.F., The novel anticonvulsant MK-801 binds to the activated state of the N-methyl-D-aspartate receptor in rat brain, Br. J. Pharmacol., 91 (1987) 403-409. 59 Foster, A.C., Vezzani, A., French, E.D. and Schwarcz, R., Kynurenic acid blocks neurotoxicity and seizures induced in rats by the related brain metabolite quinolinic acid, Neurosci. Left., 48 (1984) 273-278. 60 Garthwaite, J., Excitatory amino acid receptors and guanosine 3',5'-cyclic monophosphate in incubated slices of immature and adult rat cerebellum, Neuroscience, 7 (1982) 2491-2497. 61 Geddes, J.W., Chang-Chui, H., Cooper, S.M., Lott, I.T. and Cotman, C.W., Density and distribution of NMDA receptors in the human hippocampus in Alzheimer's disease, Brain Res., 399 (1986) 156-161. 62 Gill, R., Foster, A.C. and Woodruff, G.N., Systemic administration of MK-801 protects against ischemiainduced hippocampal neurodegeneration in the gerbil, J. Neurosci., 7 (1987) 3343-3349. 63 Greenamyre, J.T., Olson, J.M.M., Penny, Jr., J.B. and Young, A.B., Autoradiographic characterization of N-methyl-D-aspartate-, quisqualate- and kainate-sensitive glutamate binding sites, J. Pharmacol. Exp. Ther., 233 (1985) 254-263. 64 Greenamyre, J.T., Penny, L.B., Young, A.B., D'Amato, C.J., Hicks, S.P. and Shoulson, I., Alterations in L-glutamate binding in Alzheimer's and Huntington's diseases, Science, 227 (1985) 1496-1499. 65 Greenamyre, J.T., Penny, J.B., D'Amato, C.J. and Young, A.B., Dementia of the Alzheimer's type: changes in hippocampal L-[3H]glutamate binding, J. Neurochem., 48 (1987) 543-551. 66 Gundlach, A.L., Largent, B.L. and Snyder, S.H., Phencyclidine (PCP) receptors: autoradiographic localization in brain with the selective ligand [3H]TCP, Brain Res., 386 (1986) 266-279. 67 Hating, R., Kloog, Y., Kalir, A. and Sokolovsky, M., Binding studies and photoaffinity labeling identify two classes of phencyclidine receptors in rat brain, Biochemistry, 26 (1987) 5854-5861. 68 Harris, E.W. and Cotman, C.W., Long-term potentiation of guinea pig mossy fiber responses is not blocked by N-methyl-D-aspartate antagonists, Neurosci. Lett., 70 (1986) 132-137. 69 Harris, E.W., Ganong, A.H. and Cotman, C.W., Long-term potentiation in the hippocampus involves activation in N-methyl-D-aspartate receptors, Brain Res., 323 (1984) 132-137. 70 Harrison, N.L. and Simmons, M.A., Quantitative studies on some antagonists of N-methyl-D-aspartate in slices of rat cerebral cortex, Br. J. Pharmacol., 84 (1985) 381-391. 71 Haug, F.M.S., Electron microscopic localization of zinc in hippocampal mossy fiber synapses by a modified sulphide-silver procedure, Histochemie, 8 (1967) 355-368. 72 Hjorth, S., Carlsson, A., Wikstrom, H., Lindberg, P., Sanchez, D., Hacksell, U., Arvidsson, L.-E., Svensson, U. and Nilsson, J.L.G., 3-PPP, a new centrally acting DA-autoreceptor agonist with selectivity for autoreceptors, Life Sci., 28 (1981) 1225-1238. 73 Hoffman, P.L., Rabe, C.S., Moses, F. and Tabakoff, B., N-Methyl-o-aspartate receptors and ethanol: inhibition of calcium flux and cyclic GMP production, J. Neurochem., 52 (1989) 1937-1940. 74 Holz, G.G.W., Rane, S.G. and Dunlap, K., GTP-binding proteins mediate neurotransmitter inhibition of voltage-dependent calcium channels, Nature, 319 (1986) 670-672. 75 Honore, T., Lauridsen, J. and Krogsgaard-Larsen, P., Ibotenic acid analogues as inhibitors of [3H]glutamic acid binding to cerebellar membranes, J. Neurochem., 36 (1981) 1302-1304. 76 Honore, T., Lauridsen, J. and Krogsgaard-Larsen, P., The binding of [3H]AMPA, a structural analogue of glutamic acid, to rat brain membranes, J. Neurochem., 38 (1982) 173-178. 77 Honore, T., Davies, S.N., Drejer, J., Fletcher, E.J., Jacobsen, P., Lodge, D. and Nielsen, F.E., Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists, Science, 241 (1988) 701-703.

26 78 Honore, T., Drejer, J., Nielsen, E.O., Watkins, J.C., Olverman, H.J. and Nielsen, M., Molecular target size analyses of the NMDA-receptor complex in rat cortex, Eur. J. Pharrnacol., 172 (1989) 239-247. 79 Hood, W.F., Sun, E.T., Compton, R.P. and Monahan, J.B.. 1-Aminocyclobutane-l-carboxylate (ACBC): a specific antagonist of the N-methyl-D-aspartate receptor coupled glycine receptor, Eur. J. Pharmacol., 161 (1989) 281-282. 80 Hood, W.F., Thomas, J.W., Compton, R.P. and Monahan, J.B., Guanine nucleotide modulation of [3H]TCP binding to the NMDA receptor complex, Eur. J. Pharmacol., 188 (1990) 43-49. 81 Hosford, D.A., Bonhaus, D.W. and McNamara, J.O., Radiohistochemical demonstration of NMDA/glycine-channel activation in rat hippocampus, Eur. J. Pharmacol., 151 (1988) 501-503. 82 Howell, G.A., Welch, M.G. and Fredrichson, C.J., Stimulation induced uptake and release of zinc in hippocampal slices, Nature, 308 (1984) 736-738. 83 Huang, Y.Y., Wigstrom, H. and Gustafsson, B., Facilitated induction of hippocampal long-term potentiation in slices perfused with low concentrations of magnesium, Neuros¢ience, 22 (1987) 9-16. 84 Huettner, J.E., Indole-2-carboxylic acid: a competitive antagonist of potentiation by glycine at the NMDA receptor, Science, 243 (1989) 1611-1613. 85 Huettner, J.E. and Bean, B.P., Block of NMDA-activated current by the anticonvulsant MK-801: selective binding to open channels, Proc. Natl. Acad Sci. USA, 85 (1988) 1307-1311. 86 Ito, M., Periyasamy, S. and Chiu, T.H., Displaceable binding of [3H]L-glutamic acid to non-receptor material, Life Sci., 38 (1986) 1089-1096. 87 Jahr, C.E. and Stevens, C.F., Glutamate activates multiple single channel conductances in hippocampal neurons, Nature, 325 (1987) 522-525. 88 Jancso, G., Karscu, S., Kiraly, E., Szebeni, A., Toth, L., Bacsy, E., Joo, F. and Parducz, A., Neurotoxin induced nerve-cell degeneration: possible involvement of calcium, Brain Res., 295 (1984) 211-216. 89 Jarvis, M.F., Murphy, D.E. and Williams, M., Quantitative autoradiographic localization of NMDA receptors in rat brain using [3H]CPP: comparison with [3H]TCP binding sites, Eur. J. Pharmacol., 141 (1987) 149-152. 90 Johnson, J.W. and Ascher, P., Glycine potentiates the NMDA responses in cultured mouse brain neurons, Nature, 325 (1987) 529-531. 91 Johnson, K.M., Sacaan, A.I. and Snell, L.D., Equilibrium analysis of [3H]TCP binding: effects of glycine, magnesium and N-methyl-D-aspartate agonists, Eur. J. Pharmacol., 152 (1988) 141-146. 92 Jorgensen, M.D. and Diemer, NA., Selective neuron loss after cerebral ischemia in the rat: possible role of transmitter glutamate, Acta Neurol. Scand., 66 (1982) 536-546. 93 Kavanaugh, M.P., Tester, B.C., Scherz, M.W., Keana, J.F.W. and Weber, E., Identification of the binding subunit of the o-type opiate receptor by photoaffinity labeling with 1-(4-azido-2-methyl[6-3H]phenyl)-3-(2methyl[4,6-3H]-phenyl)guanidine, Proc. Natl. Acad Sci. USA, 85 (1988) 2844-2848. 94 Kavanaugh, M.P., Parker, J., Bobker, D.H., Keana, J.F.W. and Weber, E., Solubilization and characterization of o-receptors from guinea-pig brain membranes, J. Neurochem., 53 (1989) 1575-1580. 95 Kemp, J.A., Foster, A.C., Leeson, P.D., Priestley, T., Tridgett, R. and Iversen, L.L., 7-Chlorokynurenic acid is a selective antagonist at the glycine modulatory site of the N-methyl-D-aspartate receptor complex, Proc. Natl. Acad. Sci. USA, 85 (1988) 6547-6550. 96 Kessler, M., Baudry, M. and Lynch, G., Use of cystine to distinguish glutamate binding from glutamate sequestration, Neurosci. Lett., 81 (1987) 221-226. 97 Kessler, M., Terramani, T., Lynch, G. and Baudry, M., A glycine site associated with N-methyl-D-aspartic acid receptors: characterization and identification of a new class of antagonists, J. Neurochem., 52 (1989) 1319-1328. 98 Kessler, M., Baudry, M. and Lynch, G., Quinoxaline derivatives are high-affinity antagonists of the NMDA receptor-associated glycine sites, Brain Res., 489 (1989) 377-382. 99 Kishimoto, H., Simon, J.R. and Aprison, M.H., Determination of the equilibrium dissociation constants and number of glycine binding sites in several areas of the rat central nervous system, J. Neurochem., 37 (1981) 1015-1024. 100 Kleckner, N.W. and Dingledine, R., Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes, Science, 241 (1988) 835-837. 101 Klockgether, T. and Turski, L., Excitatory amino acids and the basal ganglia: implications for the therapy of Parkinson's disease, Trends Neurosci., 12 (1989) 285-286. 102 Kloog, Y., Hating, R. and Sokolovsky, M., Kinetic characterization of the phencyclidine-N-methyl-Daspartate receptor interaction: evidence for a steric blockade of the channel, Biochemistry, 27 (1988) 843-848. 103 Kloog, Y., Nadler, V. and Sokolovsky, M., Mode of binding of [3H]dibenzocycloalkaneimine (MK-801) to the N-methyl-D-aspartate (NMDA) receptor and its therapeutic implication, FEBS Lett.. 230 (t988) 167-170.

27 104 Knopfel, T., Zeise, M.L., Cuenod, M. and Zieglglinsberger, W., L-Homocysteic acid but not L-glutamate is an endogenous N-methyl-D-aspartic acid receptor preferring agonist in rat neocortical neurons in vitro, Neurosci. Left., 81 (1987) 188-192. 105 Kulkarni, S.K., Mehta, A.K. and Ticku, M.K., Comparison of anticonvulsant effect of ethanol against NMDA-, kainic acid- and picrotoxin-induced convulsions in rat, Life Sci., 46 (1990) 481-487. 106 Lapin, I.P., Experimental studies on kynurenines as neuroactive tryptophan metabolites: past, present and future, Trends Pharmacol. Sci., 1 (1980) 410-412. 107 Largent, B.L., Gundlach, A.L. and Snyder, S.H., Psychotomimetic opiate receptors labeled and visualized with (+)-[3H]3-(3-hydr0xyphenyl)-N-(1-propyl)-piperidine, Proc. Natl. Acad. Sci. USA, 81 (1984) 49834987. 108 Largent, B.L., Gundlach, A.L. and Snyder, S.H., Pharmacological and autoradiographic discrimination of sigma and phencyclidine receptor binding sites in brain with (+)-[3H]SKF 10,047, (+)-[3H]-3[3-hydroxyphenyl]-N-(1-propyl)-piperidine and [3H]-l-[1-(2-thienyl)cyclohexyl]piperidine, J. Pharmacol. Exp. Ther., 238 (1986) 739-748. 109 Lazarewicz, J.W., Wroblewski, J.T., Palmer, M.E. and Costa, E., Activation of N-methyl-o-aspartate-sensitive glutamate receptors stimulates arachidonic acid release in primary cultures of cerebellar granule cells, Neuropharmacology, 27 (1988) 765-769. 110 Leach, M.J., Marden, C.M. and Canning, H.M., (+)-cis-Piperidine dicarboxylic acid is a partial N-methylD-aspartate agonist in the in vitro rat cerebellar cGMP model, Eur. J. Pharmacol., 121 (1986) 173-179. 111 Leach, M.J., Hollox, K.J., O'Donnell, R.A. and Miller, A.A., Hippocampal NMDA/phencyclidine receptor binding sites are reduced following forebrain ischemia in the gerbil, Eur. J. Pharmacol., 152 (1988) 189-192. 112 Lehmann, J., Hutchinson, A.J., McPherson, S.E., Mondadori, C., Schmutz, M., Sinton, C.M., Tsai, C., Murphy, D.E., Steel, D.J., Williams, M., Cheney, D.L. and Wood, P.L., CGS 19755, a selective and competitive N-methyl-o-aspartate-type excitatory amino acid receptor antagonist, J. Pharmacol. Exp. Ther., 246 (1988) 65-75. 113 Lester, R.A., Quarum, M.L., Parker, J.D., Weber, E. and Jahr, C.E., Interaction of 6-cyano-7-nitroquinoxaline-2,3-dione with the N-methyl-o-aspartate receptor-associated glycine binding sites, Mol. Pharmacol., 35 (1989) 565-570. 114 Lodge, D., Anis, N.A. and Burton, N.R., Effects of optical isomers of ketamine on excitation of cat and rat spinal neurons by amino-acids and acetylcholine, Neurosci. Lett., 29 (1982) 281-286. 115 Lodge, D. and Johnston, G.A.R., Effect of ketamine on amino acid-evoked release of acetylcholine from rat cerebral cortex in vitro, Neurosci. Lett., 56 (1985) 371-375. 116 Logan, W.J. and Snyder, S.H., Unique high affinity uptake systems for glycine, glutamic and aspartic acids in central nervous tissue of the rat, Nature, 234 (1971) 297-299. 117 Loo, P., Braunwalder, A., Lehmann, J. and Williams, M., Radioligand binding to central phencyclidine recognition sites is dependent on excitatory amino acid receptor agonists, Eur. J. Pharmacol., 123 (1986) 467-468. 118 Loo, P.S., Braunwalder, A.F., Lehmann, J., Williams, M. and Sills, M.A., Interaction of L-glutamate and magnesium with phencyclidine recognition sites in rat brain: evidence for multiple affinity states of the phencyclidine/N-methyl-D-aspartate receptor complex, Mol. Pharmacol., 32 (1987) 820-830. 119 Lovinger, D.M., White, G. and Weight, F.F., Ethanol inhibits NMDA-activated ion current in hippocampal neurons, Science, 243 (1989) 1721-1724. 120 Lund-Karlsen, R. and Fonnum, F., Evidence for glutamate as a neurotransmitter in the corticofugal fibers to the dorsal lateral geniculate body and the superior colliculus in rats, Brain Res., 151 (1978) 457-467. 121 MacDermott, A.B., Mayer, M.L., Westbrook, G.L., Smith, S.J. and Barker, J.L., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurons, Nature, 321 (1986) 519-522. 122 Maragos, W.F., Dorothy, C.M., Chu, J., Greenamyre, T., Penny, J.B. and Young, A.B., High correlation between the localization of [3H]TCP binding and NMDA receptors, Eur. J. Pharmacol., 123 (1986) 173-174. 123 Martin, W.R., Lades, C.G., Thompson, J.A., Huppler, R.E. and Gilbert, P.E., The effects of morphine- and nalorphin-like drugs in the nondependent and morphine-dependent chronic spinal dog, J. Pharmacol. Exp. Ther., 217 (1976) 517-532. 124 Marvizon, J.C.G. and Skolnick, P., [3H]Glycine binding is modulated by Mg 2+ and other ligands of the NMDA receptor-cation channel complex, Eur. J. Pharmacol., 151 (1988) 157-158. 125 Marvizon, J.C.G., Lewin, A.H. and Skolnick, P., 1-Aminocyclopropane carboxylic acid: a potent and selective ligand for the glycine modulatory site of the N-methyl-D-aspartate receptor complex, J. Neurochem., 52 (1989) 992-994.

28 126 Mayer, M.L. and Westbrook, G.L., Permeation and block by divalent cations of N-methyl-D-aspartate receptor channels on cultured mouse central neurons, J. Physiol. (Lond.), 394 (1987) 501-527. 127 Mayer, M.L., Westbrook, G.L. and Guthrie, P.B.,, Voltage-dependent block by Mg 2+ of NMDA responses in spinal cord neurones, Nature, 309 (1984) 261-263. 128 McCabe, B.J. and Horn, G., Learning and memory: regional changes in N-methyl-o-aspartate receptors in the chick brain after imprinting, Proc. Natl. Acad. Sci. USA, 85 (1988) 2849-2853. 129 McGurk, J.F., Zikin, R.S. and Bennett, M.V.L., Effect of polyamines on excitatory amino acid receptor induced currents in oocytes, Neurochem. Int., 16 Suppl. (1990) 53. 130 McKernan, R.M., Castro, S., Poat, J.A. and Wong, E.H.F., Solubilization of the N-methyl-D-aspartate receptor channel complex from rat and porcine brain, ~L Neurochem., 52 (1989) 777-785. 131 McNamara, J.O., Russell, R.D., Rigsbee, L. and Bonhaus, D.W., Anticonvulsant and antiepiteptogenic actions of MK-801 in the kindling and electrical shock models, Neuropharmacology, 27 (1988) 563-568. 132 Meister, A. and Anderson, M.E., Glutathione, Annu. Rev. Biochem., 42 (1983) 711-760. 133 Meldrum, B., Excitatory amino acids and anoxic-ischemic brain damage, Trends Neurosci., 8 (1985) 47-48. 134 Mena, E.E., Fagg, G.E. and Cotman, C.W., Chloride ions enhance L-glutamate binding to rat synaptic membranes, Brain Res., 243 (1982) 378-381. 135 Mody, I. and Heinemann, U., NMDA receptors of dentate gyrus granule cells participate in synaptic neurotransmission following kindling, Nature, 326 (1987) 701-704. 136 Monaghan, D.T. and Cotman, C.W., Identification and properties of N-methyl-o-aspartate receptors in rat brain synaptic membranes, Proc. Natl. Acad. Sci. USA, 83 (1986) 7532-7536. 137 Monaghan, D.T., Holets, V.R., Toy, D.W. and Cotman, C.W., Anatomical distributions of four pharmacologically distinct 3H-glutamate binding sites, Nature, 306 (1984) 176-179. 138 Monaghan, D.T., Geddes, J.W., Yao, D., Chung, C. and Cotman, C.W., [3H]TCP binding sites in Alzheimer's disease, Neurosci. Left., 73 (1987) 197-200. 139 Monaghan, D.T., Olverman, H.J., Nguyen, L., Watkins, J.C. and Cotman, C.W., Two classes of N-methytD-aspartate recognition sites: differential distribution and differential regulation by glycine, Proc. Natl. Acad. Sci. USA, 85 (1988) 9836-9840. 140 Monaghan, D.T., Bridges, R.J. and Cotman, C.W., The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 365-402. 141 Monahan, J.B. and Michel, J., Identification and characterization of an N-methyl-D-aspartate-specific L-[3H]glutamate recognition site in synaptic plasma membranes, J. Neurochem., 48 (1987) 1699-1708. 142 Monahan, J.B., Hood, W.F., Michel, J. and Compton, R.P., Effects of guanine nucleotides on N-methyt-oaspartate receptor-ligand interaction, Mol. Pharmacol., 34 (1988) 111-116. 143 Monahan, J.B., Corpus, V.M.,, Hood, W.F., Thomas, J.W. and Compton, R.P., Characterization of a [3H]glycine recognition site of the N-methyl-D-aspartate receptor complex, J. Neurochem., 53 (1989) 370-375. 144 Moroni, F., Lombardi, G., Moneti, G. and Aldinio, C., The excitotoxin quinolinic acid is present in the brain of several animal species and its cortical content increases during the aging process, Neurosci. Left.. 47 (1984) 51-56. 145 Morris, R.G.M., Anderson, E., Lynch, G.S. and Baudry, M., Selective impairment of learning and blockade of long-term potentiation by N-methyl-o-aspartate receptor antagonist, AP5, Nature, 319 (1986) 774-776. 146 Morrisett, R.A., Chow, C.C., Sakaguchi, T., Shin, C. and McNamara, J.O., Inhibition of muscarinic-coupled phosphoinositide hydrolysis by N-methyl-D-aspartate is dependent on depolarization via channel activation, J. Neurochem., 54 (1990) 1517-1525. 147 Morrisett, R.A., Rezvani, A.H., Overstreet, D., Janowsky, D.S., Wilson, W.A. and Swartzwelder, H.S., MK-801 potently inhibits alcohol withdrawal seizures in rats, Eur. J. Pharmacol., 176 (1990) 103-105. 148 Murphy, D.E., Schneider, J., Boehm, C., Lehmann, J. and Williams, M., Binding of [3H]3-(2-carboxypiperazin-4-yl)propyl-l-phosphonic acid to rat brain membranes: a selective, high-affinity ligand for Nmethyl-D-aspartate receptor, J. Pharmacol. Exp. Ther., 240 (1987) 778-784. 149 Murphy, D.E., Hutchinson, A.J., Hurt, S.D., Williams, M. and Sills, M.A., Characterization of the binding of [3H]-CGS 19755: a novel N-methyl-D-aspartate antagonist with nanomolar affinity in rat brain, Br. J. Pharmacol., 95 (1988) 932-938. 150 Nadler, V., Kloog, Y. and Sokolovsky, M., 1-Aminocyclopropane-l-carboxylic acid (ACC) mimics the effects of glycine on the NMDA receptor ion channel, Eur. J. Pharmacol., 157 (1988) 115-116. 151 Naito, S. and Ueda, T., Adenosine triphosphate-dependent uptake of glutamate into protein I-associated vesicles, J. Biol. Chem., 258 (1983) 696-699.

29 152 Naito, S. and Ueda, T., Characterization of glutamate uptake into synaptic vesicles, J. Neurochem., 44 (1985) 99-109. 153 Nicoletti, F. and Canonico, P.L., Glycine potentiates the stimulation of inositol phospholipid hydrolysis by excitatory amino acids in primary culture of cerebellar neurons, J. Neurochem., 53 (1989) 724-727. 154 Nowak, L., Bregestovski, P., Ascher, P., Herbert, A. and Prochaintz, A., Magnesium gates glutamateactivated channels in mouse central neurones, Nature, 307 (1984) 462-465. 155 Ogita, K. and Yoneda, Y., Differentiation of Ca2+-stimulated binding from the C1--dependent binding of [3H]glutamate in synaptic membranes from rat brain, Neurosci. Res., 4 (1986) 129-142. 156 Ogita, K. and Yoneda, Y., Characterization of Na+-dependent binding sites of [3H]glutamate in synaptic membranes from rat brain, Brain Res., 397 (1986) 137-144. 157 Ogita, K. and Yoneda, Y., Possible presence of [3H]glutathione (GSH) binding sites in synaptic membranes from rat brain, Neurosci. Res., 4 (1987) 486-496. 158 Ogita, K. and Yoneda, Y., Temperature-dependent and -independent apparent binding activities of [3H]glutathione in brain synaptic membranes, Brain Res., 463 (1988) 37-46. 159 Ogita, K. and Yoneda, Y., Disclosure by Triton X-100 of NMDA-sensitive [3H]glutamate binding sites in brain synaptic membranes, Biochem. Biophys. Res. Commun., 153 (1988) 510-517. 160 Ogita, K. and Yoneda, Y., Selective potentiation by L-cysteine of apparent binding activity of [3H]glutathione in synaptic membranes of rat brain, Biochem. Pharmacol., 38 (1989) 1499-1505. 161 Ogita, K. and Yoneda, Y., 6,7-Dichloroquinoxaline-2,3-dione is a competitive antagonist specific to strychnine-insensitive [3H]glycine binding sites on the N-methyl-D-aspartate receptor complex, J. Neurochem., 54 (1990) 699-702. 162 Ogita, K. and Yoneda, Y., Temperature-independent binding of [3H](+)-3-(2-carboxypiperazin-4-yl)propyl-l-phosphonic acid in brain synaptic membranes treated with Triton X-100, Brain Res., 515 (1990) 51-56. 163 Ogita, K. and Yoneda, Y., Solubilization of spermidine-sensitive [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding activity from rat brain, J. Neurochern., 55 (1990) 1515-1520. 164 Ogita, K. and Yoneda, Y., Solubilization of the NMDA receptor ion channel complex from rat brain. In S. Kito, T. Segawa and R. Olsen (Eds.), Neurotransmitter Receptors: Neuroreceptor Mechanisms in Brain, Plenum, New York, 1990, in press. 165 Ogita, K., Kitago, T., Nakamuta, H., Fukuda, Y., Koida, M., Ogawa, Y. and Yoneda, Y., Glutathione-induced inhibition of Na +-independent and -dependent bindings of L-[3H]glutamate in rat brain, Life Sci., 39 (1986) 2411-2418. 166 Ogita, K., Suzuki, T. and Yoneda, Y., Strychnine-insensitive binding of [3H]glycine to synaptic membranes in rat brain, treated with Triton X-100, Neuropharmacology, 28 (1989) 1263-1270. 167 Oglta, K., Nabeshima, T. and Yoneda, Y., [3H]Thienylcyclohexylpiperidine binding activity in brain synaptic membranes treated with Triton X-100, J. Neurochern., 55 (1990) 1639-1646. 168 Ogita, K., Suzuki, T., Enomoto, R., Ohgaki, T., Katagawa, J., Uchida, S., Meguri, H. and Yoneda, Y., Profiles of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine in brain synaptic membranes treated with Triton X-100, Neurosci. Res., 9 (1990) 35-47. 169 Ogita, K., Kouda, T., Suzuki, T., Ohkawara, E., Enomoto, R., Nabeshima, T. and Yoneda, Y., Differential effects of SH-reactive agents on [3H](+)-3-(2-carboxypiperazin-4-yl)propyl-l-phosphonate and [3H]glutamate binding in brain synaptic membranes treated with Triton X-100, Neurochem. Int., 17 (1990) in press. 170 Ohgaki, T., Ogita, K. and Yoneda, Y., Inhibition by GTP of NMDA-sensitive [3H]glutamate binding, Bull. Jpn. Neurochem. Soc., 27 (1988) 402-403. 171 Oja, S.S., Varga, V., Janaky, R., Kontro, P., Aarnio, T. and Marnela, K.M., Glutathione and glutamatergic neurotransmission in the brain. In E.A. Cavalheiro, J. Lehmann and L. Turski (Eds.), Frontiers in Excitatory Amino Acid Research, Alan R. Liss, New York, 1988, pp. 75-78. 172 Olney, J., Price, M., Samson, L. and Labruyere, J., The role of specific ions in glutamate neurotoxicity, Neurosci. Lett., 65 (1986) 65-71. 173 Olney, J.W., Price, M.T., Salles, K.S., Labruyere, J., Ryerson, R., Mahan, K., Frierdich, G. and Samson, L., L-Homocysteic acid: an endogenous excitotoxic ligand of the NMDA receptor, Brain Res. Bull., 19 (1987) 597-602. 174 Olsen, R.W., GABA-benzodiazepine-barbiturate receptor interactions, J. Neurochem., 37 (1981) 1-13. 175 Olverman, H.J., Jones, A.W. and Watkins, J.C., L-Glutamate has higher affinity than other amino acids for [3H]-D-AP5 binding sites in rat brain membranes, Nature, 307 (1984) 460-462. 176 O'Shaughnessy, C.T. and Lodge, D., N-Methyl-D-aspartate receptor-mediated increase in intracellular calcium is reduced by ketamine and phencyclidine, Eur. J. Pharmacol., 153 (1988) 201-209.

30 177 Pellegrini-Giampietro, D., Galli, A., Alesiani, M., Cherici, G. and Moroni, F., Quinoxalines interac~ v, ith the glycine recognition site of NMDA receptors: studies in guinea-pig myenleric plexus and in rat cortic~d membranes, Br. J. Pharmacol., 98 (1989) 1281-1286. 178 Perez-Clausell. J. and Danscher, G., Intravesicular localization of zinc in rat telencephalic boutons. A histochemical study, Brain Res., 337 (1985) 91 98. 179 Perkins, M.N. and Stone, T.W., An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with endogenous excitant quinolinic acid, Brain Res., 247 (1982) 184-187. 180 Perkins, M.N. and Stone, T.W., pharmacology and regional variations of quinolinic acid-evoked excitations in the rat central nervous system, J. PharmacoL Exp. Ther., 226 (1983) 551-557. 181 Perkins, M.N., Stone, T.W., Collins, J.F. and Curry, K., Phosphonate analogues of carboxylic acids as amino acid antagonists on rat cortical neurones, Neurosci. Lett., 23 (1981) 333-336. 182 Peters, S., Koh, J. and Choi, D.W., Zinc selectively blocks the action of N-methyl-D-aspartate on cortical neurons, Science, 236 (1987) 589-593. 183 Peterson, C. and Cotman, C.W., Strain-dependent decrease in glutamate binding to the N-methyl-D-aspartic acid receptor during aging, Neurosci. Lett., 104 (1989) 309-313. 184 Pin, J.P., Bockaert, J. and Recasens, M., The CaZ+/CI dependent e-[3H]glutamate binding: a new receptor or a particular transport process, FEBS Lett., 175 (1984) 31-36. 185 Procter, A.W., Stirling, J.M., Stratmann, G.C., Cross, A.J. and Bowen, D.M., Loss of glycine-dependent radioligand binding to the N-methyl-D-aspartate-phencyclidine receptor complex in patients with Alzheimer's disease, Neurosci. Lett., 101 (1989) 62-66. 186 Procter, A.W., Wong, E.H.F., Stratmann, G.C., Lowe, S.L. and Bowen, D.M., Reduced glycine stimulation of [3H]MK-801 binding in Alzheimer's disease, J. Neuroehem., in press. 187 Quirion, R., Hammer, Jr., R.R., Herkenham, M. and Pert, C.B., Phencyclidine (angel dust)jsigma "opiate" receptor: visualization by tritium-sensitive film, Proc. Natl. Acad. Sci. USA, 78 (1981) 5811-5885. 188 Ransom, R.W. and Stec, N.L., Cooperative modulation of [3H]MK-801 binding to the N-methyl-D-asparrate receptor-ion channel complex by L-ghitamate, glycine and polyamines, J. Neurochem., 51 (1988) 830-836. 189 Rao, T.S., Cler, J.A., Oei, E.J., Emmett, M.R., Mick, S.J., Iyengar, S. and Wood, P.L., The po!yamines, spermine and spermidine, negatively modulate N-methyl-D-aspartate (NMDA) and quisqualate receptor mediated responses in vivo: cerebellar cyclic GMP measurements, Neuroehem. Int., 16 (1990) 199-206. 190 Reynolds, I.J. and Miller, R,J., [3H]MK-801 binding to the NMDA receptor/ionophore complex is regulated by divalent cations: evidence for multiple regulatory sites, Eur. J. Pharmaeol., 151 (1988) 103-112. 191 Reynolds, I.J. and Miller, R.J., [3H]MK-801 binding to the N-methyl-a-aspartate receptor reveals drug interactions with the zinc and magnesium binding sites, J. Pharmacol. Exp. Ther., 247 (1988) 1025-1031. 192 Reynolds, I.J. and Miller, R.J., Ifenprodil is a novel type of N-methyl-D-aspartate receptor antagonist: interaction with polyamines, Mol. Pharmacol., 36 (1989) 758-765. 193 Reynolds, I.J., Murphy, S.N. and Miller, R.J., 3H-labeled MK-801 binding to the excitatory amino acid receptor complex from rat brain is enhanced by glycine, Proc. Natl. Acad. Sci. USA, 84 (1987) 7744-7748. 194 Robinson, M.B., Blakely, R.D. and Coyle, J.T., Quisqualate selectively inhibits a brain peptidase which cleaves N-acetyl-L-aspartyl-L-glutamate in vitro, Eur. J. Pharmacol., 130 (1986) 345-347. 195 Rothman, S.M., The neurotoxicity of excitatory amino acids is produced by passive chloride influx, J. Neurosei., 5 (1985) 1483-1489. 196 Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischemic brain damage, Ann. Neurol., 19 (1986) 105-111. 197 Sacaan, A.I. and Johnson, K.M., Spermine enhances binding to the glycine site associated with the N-methyl-D-aspartate receptor complex, Mol. Pharmacol., 36 (1989) 836-839. 198 Schoemaker, H., Allen, J. and Langer, S.Z., Binding of [3H]ifenprodil, a novel NMDA antagonist, to a polyamine-sensitive site in the rat cerebral cotex, Eur. J. Pharmacol., 176 (1990) 249-250. 199 Schwarcz, R., Foster, A.C., French, E.D., Whetsell, Jr., W.O. and Kohler, C., Excitotoxic models for neurodegenerative disorders, Life Sei., 35 (1984) 19-32. 200 Sharif, N.A. and Roberts, P.J., Regulation of cerebellar L-[3H]glutamate binding: influence of guanine nucleotides and Na + ions, Bioehem. Pharmacol., 30 (1981) 3019-3022. 201 Sheardown, M,J., Drejer, J., Jensen, L.H., Stidsen, C,E. and Honore, T., A potent antagonist of the strychnine insensitive glycine receptor has anticonvulsant properties, Eur. J. Pharmacol., 174 (1989) 197 - 204. 202 Shinozaki, H., Ishida, M. and Goto, Y., Depression of decerebrate rigidity in the rat by antagonists of excitatory amino acids, Neuropharmacology, 28 (1989) 593-598.

31 203 Simon, R.P., Swan, J.H., Griffiths, T. and Meldrum, B.S., Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850-852. 204 Simpson, M.D.C., Royston, M.C., Deakin, J.F.W., Cross, A.J., Mann, D.M.A. and Slater, P., Regional changes in [3H]D-aspartate and [3H]TCP binding sites in Alzheimer's disease brains, Brain Res., 462 (1988) 76-82. 205 Slevin, J., Collins, J.F., Lindsley, K. and Coyle, J.T., Specific binding of [3H]glutamate to cerebellar membranes: evidence for recognition sites heterogeneity, Brain Res., 249 (1982) 353-360. 206 Smith, S.J., Progress on LTP at hippocampal synapses: a post-synaptic Ca trigger for memory storage? Trends Neurosci., 10 (1987) 142-144. 207 Snell, L.D. and Johnson, K.M., Cycloleucine competitively antagonizes the strychnine-insensitive glycine receptor, Eur. J. Pharmacol., 151 (1988) 165-166. 208 Snell, L.D., Morter, R.S. and Johnson, K.M., Glycine potentiates N-methyl-D-aspartate-induced [3H]TCP binding to rat cortical membranes, Neurosci. Lett., 83 (1987) 313-317. 209 Snell, L.D., Morter, R.S. and Johnson, K.M., Structural requirements for activation of the glycine receptor that modulates the N-methyl-D-aspartate operated ion channel, Eur. J. PharmacoL, 156 (1988) 105-110. 210 Sonsalla, P.K., Nicklas, W.J. and Heikkil~i, R.E., Role of excitatory amino acids in methamphetamine-induced nigrostriatal dopaminergic toxicity, Science, 243 (1989) 398-400. 211 Sprosen, T.S. and Woodruff, G.N., Polyamines potentiate NMDA induced whole-cell currents in cultured striatal neurons, Eur. J. PharmacoL, 179 (1990) 477-478. 212 Storm-Mathisen, J. and Iversen, L.L., Uptake of [3H]-glutamic acid in excitatory nerve endings. Light and electron-microscopic observations in the hippocampal formation of the rat, Neuroscience, 4 (1979) 12371253. 213 Stringer, J.L., Greenfield, L.J., Hacket, J.T. and Guyenet, P.G., Blockade of long-term potentiation by phencyclidine and opiates in the hippocampus in vivo, Brain Res., 280 (1983) 127-136. 214 Su, T.P., Evidence for sigma opioid receptor: binding of [3H]SKF 10,047 to etorphine-inaccessible sites in guinea-pig brain, J. Pharmacol. Exp. Ther., 223 (1982) 284-290. 215 Tam, S.W. and Cook, L., o Opiate and certain antipsychotic drugs mutually inhibit (+)-[3H]SKF 10,047 and [3H]haloperidol binding in guinea-pig brain membranes, Proc. Natl. Acad. Sci. USA, 81 (1984) 5618-5621. 216 Thomas, J.W., Hood, W.F., Monahan, J.B., Contreras, P.C. and O'Donohue, T.L., Glycine modulation of the phencyclidine binding site in mammalian brain, Brain Res., 442 (1988) 396-398. 217 Thompson, R.F., The neurobiology of learning and memory, Science, 233 (1986) 941-947. 218 Thomson, A.M., West, D.C. and Lodge, D., An N-methylaspartate receptor-mediated synapse in rat cerebral cortex: a site of action of ketamine, Nature, 313 (1985) 479-481. 219 Tsumoto, T., Hagihara, K., Sato, H. and Hata, Y., NMDA receptors in the visual cortex of young kittens are more effective than those of adult cats, Nature, 327 (1987) 513-514. 220 Uematsu, D., Greenberg, J.H., Reivich, M. and Karp, A., Cytosolic free calcium and N A D / N A D H redox state in the cat cortex during in vivo activation of NMDA receptors, Brain Res., 482 (1989) 129-135. 221 Vignon, J., Chicheportiche, R., Chicheportiche, M., Kamenka, J.-M., Geneste, P. and Lazdunski, M., [3H]TCP: a new tool with high affinity for the PCP receptor in rat brain, Brain Res., 280 (1983) 194-197. 222 Vignon, J., Privat, A., Chaudieu, I., Thierry, A., Kamenka, J.-M. and Chicheportiche, R., [3H]Thienylphencyclidine ([3H]TCP) binds to two different sites in rat brain. Localization by autoradiographic and biochemical techniques, Brain Res., 378 (1986) 133-141. 223 Vincent, S.R. and McGeer, E.G., A comparison of sodium dependent glutamate binding with high affinity glutamate uptake in rat striatum, Brain Res., 184 (1980) 99-105. 224 Vincent, J.P., Kartalorski, B., Geneste, P., Kamenka, J.-M. and Lazdunski, M., Interaction of phencyclidine ('angel dust') with a specific receptor in rat brain membranes, Proc. Natl. Acad. Sci. USA, 76 (1979) 4678-4682. 225 Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Annu. Rev. Pharmacol. Toxicol., 21 (1981) 165-204. 226 Watson, G.B., Bolanowski, M.A., Baganoff, M.P., Deppeler, C.L. and Lanthorn, T.H., Glycine antagonist action of 1-aminocyclobutane-l-carboxylate (ACBA) in Xenopus oocytes injected with rat brain mRNA, Eur. J. Pharmacol., 167 (1989) 291-294. 227 Weber, E., Sonders, M., Quarum, M., McLean, S., Pou, S. and Keana, J.F.W., 1,3-Di-(2-[5-3H]tolyl)guani dine: a selective ligand that labels o-type receptors for psychotomimetic opiates and antipsychotic drugs, Proc. Natl. Acad. Sci. USA, 83 (1986) 8784-8788. 228 Werling, L.L., Doman, A. and Nadler, J.V., L-[3H]glutamate binding to hippocampal synaptic membranes: two binding sites discriminated by their differing affinities for quisqualate, J. Neurochem., 41 (1983) 586-593.

32 229 Westbrook, G.L. and Mayer, M.L., Micromolar concentrations of zinc antagonise NMDA and GABA responses on hippocampal neurons, Nature, 328 (1987) 640-643. 230 Westerberg, E. and Wieloch, T.W., Changes in excitatory amino acid receptor binding in the intact and decorticated rat neostriatum following insulin-induced hypoglycemia, J. Neurochem., 52 (1989) 1340-1347. 231 White, W.F., Brown, K.L. and Frank, D.M., Glycine binding to rat cortex and spinal cord: binding characteristics and pharmacology reveal distinct populations of sites, J. Neurochem., 53 (1989) 503-512. 232 Wieloch, T., Hypoglycemia-induced neuronal damage prevented by an N-methyl-D-aspartate antagonist, Science, 230 (1985) 681-683. 233 Williams, K., Romano, C. and Molinoff, P.B., Effects of polyamines on the binding of [3H]MK-801 to the N-methyl-D-aspartate receptor: pharmacological evidence for the existence of a polyamine recognition site, Mol. Pharmacol., 36 (1989) 575-581. 234 Wong, E.H.F., Kemp, J.A., Priestley, T., Knight, A.R., Woodruff, G.N. and Iversen, L.L., The anti-convulsant MK-801 is a potent N-methyl-D-aspartate antagonist, Proc. Natl. Acad. Sci. USA, 83 (1986) 7104-7108. 235 Wong, E.H.F., Knight, A.R. and Ransom, R., Glycine modulates [3H]MK-801 binding to the NMDA receptor in rat brain, Eur. J. Pharmacol., 142 (1987) 487-488. 236 Wong, E.H.F., Knight, R. and Woodruff, G.N., [3H]MK-801 labels a site on the N-methyt-D-aspartate receptor channel complex in rat brain membranes, J. Neurochem., 50 (1988) 274-281. 237 Wood, P.L., Richard, J.W., Pilapil, C. and Nair, N.P.V., Antagonists of excitatory amino acids and cyclic guanosine monophosphate in cerebellum, Neuropharmacology, 21 (1982) 1235-1238. 238 Wood, P.L., Emmett, M.R., Rao, T.S., Mick, S., Cler, J. and lyengar, S., In vivo modulation of the N-methyl-D-aspartate receptor complex by D-serine: potentiation of ongoing neuronal activity as evidenced by increased cerebellar cyclic GMP, J. Neuroehem., 53 (1989) 979-981. 239 Wroblewski, J.T. and Danysz, W., Modulation of glutamate receptors: molecular mechanisms and functional implications, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 441-474. 240 Wroblewski, J.T., Nicoletti, F., Fadda, E. and Costa, E., Phencyclidine is a negative allosteric modulator of signal transduction at two subclasses of excitatory amino acid receptors, Proc. Natl. Aead. Sci. USA, 84 (1987) 5068-5072. 241 Wroblewski, J.T., Fadda, E., Mazzetta, J., Lazarewicz and Costa, E., Glycine and D-serine act as positive modulators of signal transduction at N-methyl-D-aspartate sensitive glutamate receptors in cultured cerebellar granule cells, Neuropharmaeotogy, 28 (1989) 447-452. 242 Yatani, A., Codina, J., Brown, A.M. and Birnbaumer, L., Direct activation of mammalian atrial muscarinic potassium channels by GTP regulatory protein, GK, Science, 235 (1987) 205-211. 243 Yeh, G.-C., Bonhaus, D.W., Nadler, J.V. and McNamara, J.O., N-Metbyl-D-aspartate receptor plasticity in kindling: quantitative and qualitative alterations in the N-methyl-D-aspartate receptor-channel complex, Proc. Natl. Acad. Sci. USA, 86 (1989) 8157-8160. 244 Yoneda, Y. and Ogita, K., [3H]Glutamate binding sites in the rat pituitary, Neurosci. Res., 3 (1986) 430-435. 245 Yoneda, Y. and Ogita, K., Localization of [3H]glutamate binding sites in rat adrenal medulla, Brain Res.. 383 (1986) 387-391. 246 Yoneda, Y. and Ogita, K., Enhancement of [3H]glutamate binding by N-methyl-D-aspartic acid in rat adrenal, Brain Res., 406 (1987) 24-31. 247 Yoneda, Y. and Oglta, K., Solubilization of novel binding sites for [3H]glutamate in rat adrenal, Biochem. Biophys. Res. Commun., 142 (1987) 609-616. 248 Yoneda, Y. and Ogita, K., Possible role of polyamines as modulators of NMDA receptor complex, Bull. Jpn. Neurochern. Soc., 28 (1989) 136-137. 249 Yoneda, Y. and Ogita, K., Microbial methodological artifacts in [3H]glutamate receptor binding assays, Anal. Biochem., 177 (1989) 250-255. 250 Yoneda, Y. and Ogita, K., Solubilization of quisqualate-sensitive [3H]glutamate binding activity from rat retina, J. Neurochem., 52 (1989) 1501-1507. 251 Yoneda, Y. and Ogita, K., Solubilization of stereospecific and quisqualate-sensitive activity of [3H]glutamate binding in the pituitary of the rat, Neuropharmacology, 28 (1989) 611-616. 252 Yoneda, Y. and Ogita, K., Labeling of NMDA receptor channels by [3H]MK-801 in brain synaptic membranes treated with Triton X-100, Brain Res., 499 (1989) 305-314. 253 Yoneda, Y. and Ogita, K., Characterization of quisqualate-sensitive [3H]glutamate binding activity sotubilized from rat adrenal, Neurochem. Int., 15 (1989) 137-143. 254 Yoneda, Y. and Ogita, K., Abolition of the NMDA-mediated responses by a specific glycine antagonist, 6,7-dichloroquinoxaline-2,3-dione (DCQX), Biochem. Biophys. Res. Commun., 164 (1989) 841-849.

33 255 Yoneda, Y. and Ogita, K., Biochemical and pharmacological properties of the NMDA receptor ion channels in rat brain, Life Sci. Ado. Pharmacol., 1 (1990) in press. 256 Yoneda, Y. and Ogita, K., Novel fourth binding sites of [3H]spermidine within the NMDA receptor complex. In S. Kito, T. Segawa and R. Olsen (Eds.), Neurotransmitter Receptors: Neuroreceptor Mechanisms in Brain, Plenum, New York, 1990, in press. 257 Yoneda, Y. and Ogita, K., Studies on [3H]glutamate binding in nervous tissues. What are the pitfalls? In N.N. Osborne (Ed.), Current Aspects of the Neurosciences, Vol. 3, Macmillan Press, London, 1991, in press. 258 Yoneda, Y., Ogita, K., Nakamuta, H., Koida, M., Ohgaki, T. and Meguri, H., Possible interaction of [3H]glutamate binding sites with anion channels in rat neural tissues, Neurochem. Int., 9 (1986) 521-531. 259 Yoneda, Y., Ogita, K., Nakamuta, H., Fukuda, Y., Koida, M. and Ogawa, Y., Comparative study of [3H]glutamate binding sites in rat retina and cerebral cortex, BiocherrL Pharmacol., 36 (1987) 772-774. 260 Yoneda, Y., Ogita, K., Ohgaki, T., Uchida, S. and Meguri, H., N-Methyl-D-aspartate-sensitive [3H]gluta mate binding sites in brain synaptic membranes treated with Triton X-100, Biochim. Biophys. Acta, 1012 (1989) 74-80. 261 Yoneda, Y., Ogita, K., Kouda, T. and Ogawa, Y., Radioligand labeling of N-methyl-D-aspartic acid (NMDA) receptors by [3H](+)-3-(2-carboxypiperazin-4-yl)propyl-l-phosphonic acid in brain synaptic membranes treated with Triton X-100, Biochem. Pharmacol., 39 (1990) 225-228. 262 Yoneda, Y., Ogita, K. and Suzuki, T., Interaction of strychnine-insensitive glycine binding with MK-801 binding in brain synaptic membranes, J. Neurochem., 55 (1990) 237-244. 263 Yoneda, Y., Ogita, K., Suzuki, T., Enomoto, R. and Zuo, P.P., Competitive inhibition of the NMDA-mediated responses by guanine nucleotides in brain synaptic membranes treated with Triton X-100, Neurosci. Res., 9 (1990) 114-125. 264 Young, A.B. and Snyder, S.H., Strychnine binding in rat spinal cord membranes associated with the synaptic glycine receptor: cooperativity of giycine interactions, MoL Pharmacol., 10 (1974) 790-809. 265 Young, A.B., Greenamyre, J.T., Hollingsworth, Z., Albin, R., D'Amato, C., Shoulson, I. and Penny, J.B., NMDA receptor losses in putamen from patients with Huntington's disease, Science, 241 (1988) 981-983. 266 Zaczek, R., Arlis, S., Markyl, A., Murphy, T., Drucker, H. and Coyle, J.T., Characteristics of chloride-dependent incorporation of glutamate into brain membranes argue against a receptor binding site, Neuropharmacology, 26 (1987) 281-287. 267 Zukin, S.R. and Zukin, R.S., Specific [3H]phencyclidine binding in rat central nervous system, Proc. Natl. Acad. Sci. USA, 76 (1979) 5372-5376.