ModuEation of the NMDA receptor by D-serine in the cortex and the spinal cord, in vitro

European Journal of Pharmacology, 191 (1990) 29-38 Elsevier

29

FJP 51589

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invitro

Felix Brugger, Urs Wicki, Denise Nassenstein-Elton,

Graham

E. Fagg, Hans-Rudolf

Olpe

and Mario F. Pozza Research and Development Deporfment, Pharmaceuticals Division. Ciba-Geigy, ttd,

C&4&X? Base& Switzerland

Received 2!5June 1990, revised MS received 10 August 1990, accepted 28 August 1990

We present a comparative study of the modulation of the N-methyl-D-aspartate (NMDA) receptor at the st~c~ne-insensitive glycine site in the spinal cord and in the cortex. The excitatory effect of NMDA was potentiated by D-serine (a glycine mimetic) in the hemisected rat spinal cord. The non-competitive NMDA antagonists 7-chlorokynurenic acid (7X1 KYNA, 10 FM) and 3-amino-l-hydroxypyrrolid-2-one (HA-966; 100 or 200 +4j antagonized the effect of NMDA in the spinal cord and cortical wedge preparation. The antagonism was reversed by the addition of D-serine. This effect was strychnine-insensitive and hence not related to the inhibitory glycine receptor known to be present in the spinal cord. Our results suggest strongly that glycine positively modulates the NMDA system not only at a supraspinal level but also at the spinal level. As the positive modulation of NMDA responses by D-serine was also seen in the presence of tetrodoto~, we conclude that the NMDA/~y~ine complex is (also) located on motoneurones in addition to the known glycine-mediated inhibitory system. NMDA (N-methyl-D-aspartate); ‘I-Chlorokynurenic acid; HA-966; Glycine: Spinal cord; Cortical wedge; (Electrophysiology)

1. Introduction

It has recently been demonstrated that responses evoked by N-methyl-D-aspartic acid (NMDA) in cultured neurones from mouse cerebral cortex can be potentiated by glycine, previously known primarily as an in~bito~ neurotransmitter in lower brain areas (Johnson et al., 1987). In these expe~ments, the positive modulation of NMDA by glycine and D-serine was shown to be st~c~ne-insensitive, Hence the receptor is unrelated to the well-known inhibitory glycine receptor. The action of glycine seems to be mediated

Correspondence to: F. Brugger, Research and Development Department, Pharmaceuticals Division, Ciba-Geigy Ltd., CH4002 Basel, Switzerland. ~1~2999/90/$03.50

via an allosteric recognition site located on the NMDA receptor ion channel complex since it occurs in isolated membrane patches (Johnson et al., 1987), i.e. in membrane preparations devoid of cellular inte~ty {Reynolds et al., 1987; Ransom and Stec, 1988; Snell et al., 1987) and in solubilized receptor preparations (McKeman et al., 1989; Ikin et al., 1990). The functional significance of the glycine modulatory site has recently been demonstrated in vivo by the ability of glycine or D-serine to promote seizure activity in rats and mice (Larson and Beitz, 1988; Singh et al., 1990) and to antagonize the behavioural effects of phencyclidine (PCP; Toth and Lajtha, 1986; Contreras, 1990). Moreover, the two well-established non-competitive NMDA antagonists 7-chlorokynurenic acid (7-Cl KYNA) and 3-amino-l-hydrox~y~olid-2-one (HA-g%), which exert their

0 1990 Elsevier Science Publishers B.V. ~Biomedic~ Division)

via acti~ti~~t~ ot a st~cbuine-insensitive tor. have been shown to antagonize A-evoked responses in vitro. in a manner can be overcoats by the addition of glycine glycine mimetic D-serine (Fletcher and 88: Kemp et al., 1987; 1988). The tissues mt of the studies investigating the interaction of NMDA and glycine have been of inal origin. Supraspinal areas are known to have a bw density of strychnine-sensitive (Araki et al.. I988~ and a high density of strychnine-insensitive glycine binding sites (B&tow et al., 1986). In contrast. the spinal cord contains the highest concentration of glycine in the central nervous system (Daly and Aprison, 1974) and has a high density of strychnine-sensitive glycine sites which are responsible for the classical inhibitory action of glycine upon motoneurones (Werman et al., 1967: Curtis et al.. 1967). It is not yet known whether a strychnine-insensitive NMDA-coupled gIycine system lie that found in the brain also plays a role in the spinal cord. The aim of the present study was (i) to further characterize the gIycine receptor and the non-compeL~tivc NM3.x antagonists 7-Cl KYNA and HA-966 in receptor birding studies: (ii) to investigate whether modulation of the NMDA receptor at the strychnineinsensitive glycine site also occurs in the spinal cord and to compare these findings with those obtained with cortical tissue by using electrophysiological in vitro techniques. Some of these results have been presented in preliminary form (Brugger et al., 1990).

methods 2. I. Receptor binding

The binding of 2-[3H]glycine to Triton X-100 treated synaptic membranes isolated from the forebrains of adult male rats (Tif: RAI f(SPF)) was determined according to previously published procedures (B&tow et al., 1986; Kessler et al., 1989: Thedinga et al., 1989). In brief, aliquots of 40-50 pg membrane protein were incubated in triplicate at 4OC for 23 min with 20 nM 13H]glycine (plus test inhibitors as appropriate) in

a final volume of 0.5 ml 50 mM Tris-acetate buffer (pH 7.5). Non-specific binding was determined in the presence of 0.5 mM glycine, and membran~bound radioactivity was measured after centrifugation and aspiration of the supematants. All other radioreceptor assays were performed as described previously (e.g. see Fagg et al., 1990). Receptors (and their radioligands) (concentrations are given in table 1B) examined were: GABA, (muscimol), benzodiazepine (flurtitrazepam) and GABA a (3-a~nopropyl-phosp~~c acid: CGP 27492) adenosine (cyclohexyladenosine); muscarinic acetylcholine (dioxalan, quinuclidinylbenzilate: QNB), (it (prazocine), 1y2 ~clo~dine) and /3-adrenergic (dithydroalprenolol, DHA), 5-HT, (5-HT) and S-HT2 (ketanserin), histamine H, (doxepine) and H, (thiotidine), substance P (substance P) and opiate (naloxone).

Experiments were carried out using hemisected spinal cords from 7-15 days old rats (Tif: RAI f(SPF)) essentially as described by Evans (1978). Briefly, under urethane anaesthesia, the spinal cord with attached dorsal and ventral roots (L3-L6) was excised, sagittally hemisected and transferred to a perfusion block. The prep~ation was superfused (1 ml/ruin) with gassed (95% 02, 5% CO,) artificial cerebrospinal fluid (ACSF), which comprised (in mM): NaCl 120, KC1 2.5; KH,PO, 1.25; CaCl, 2.5; MgSO,, 2.0; NaHC4 30 and glucose 10, at 25OC. A dorsal root (L4) was stimulated submaximally using a pair of silver wire electrodes (1 stimulus/t-&) and the resultant dorsal root-ventral root potentials (DR-VRP) were recorded continuously from the corresponding ventral root (together with the spontaneous activity) with a AgfAgCl wick electrode. In some experiments, in order to block synaptic transmission, hemicords were exposed for the first 10 min to ACSF containing 10 PM tetrodotoxin (TTX) and then to ACSF containing 1 PM TTX. Amino acids and other drugs were dissolved in ACSF and applied via the superfusion system. The actions of the test substances on NMDAevoked responses were determined by comparing

31

the amplitudes of NMDA-induced depolarizations before and during drug application. 2.3. Cortical wedge Male rats (Tif: RAI f(SPF)) weighing 150-180 g were used. The rats were killed by decapitation under light h~othane anaesthesia and the brains were rapidly removed. The brains were placed, ventral surface down, in ice-cold ACSF. The ACSF had the following composition (in mM): NaCl 124; KC1 2.5; KH,PO, 1.25; CaCl, 2.5; MgSO, 2.0; NaHCO, 26 and glucose 10 (307 mOsm/kg f 2). Two coronal sections were made: the first at the level of the temporal lobes and the second about 6 mm caudally. The tissue block was further reduced in size by cutting away 1.5 mm horizontally at the ventral surface. The resulting tissue block was then mounted on a tissue chopper (Sorvalle) and 500 pm thick slices were cut. Further cuts with a razor-blade were made to produce a series of wedge shaped pieces of tissue about 2 mm at the pial surface and about 1.5 mm at the corpus callosum. The wedges were transferred to a storage chamber, where they were kept at room temperature in the standard Mg*+-containing ACSF. One slice was transferred to a two-compartment recording chamber, so that most of the cortical tissue was contained in one compartment, and the corpus callosum in the other. A high resistance seal between the two compartments was achieved using a perspex barrier, well greased with a vaseline/paraffin mixture. Each compartment of the chamber was continuously perfused at 2.0 ml/mm via droppers with Mgz+-free ACSF gassed with 95% O,, 5% CO*; drugs dissolved in Mg’+free ACSF were generally applied to the cortical tissue site. The d.c. potential was differentially monitored via Ag/AgCl electrodes and displayed on a chart recorder. Drug effects were normalized to the maximum response obtained in the control period, i.e. the depolarization induced by NMDA (10 @f) equals 100%. Statistical comparisons were made with Student’s t-test. CGP 27492 (3-aminopropyl-phosphinic acid) and CGP 37849 ((DL-(E)-2-amino-4-methyl-5phospbono-3-pentenoic acid) were synthesized in

Ciba-Geigy Laboratories. Other amino acids and drugs were obtained from commercial sources.

3. Results 3. I. Receptor binding In agreement with the results of earlier studies (Bristow et al., 1986; Kessler et al., 1989), the binding of [3H]glycine to rat forebrain synaptic membranes was inhibited by glycine and by the D-isomers of serine and alanine, but not by strychnine (table 1A). This binding site, which has previously been shown to be associated with the NMDA receptor (see Introduction for citations), showed high affinity for 7-Cl KYNA and for HA-966 (table 1A; also see Kemp et al., 1988; Foster and Kemp, 1989). In experiments to examine their selectivity for strychnine-insensitive [ ‘Hjglycine binding sites, 7-CI KYNA and HA-%6 were examined in a number of other receptor binding assays. At a concentration of 100 FM, HA-966 showed negligible interaction with any of the 15 receptor sites analysed (table 1B). However, at this concentration, 7-Cl KYNA inhibited radioligand binding by about 50% at GABA,, adenosine, muscarinic, 5-HT,, histamine Hz and opiate receptors, and by 91% at be~~i~epine binding sites. At 10 PM, 7-Cl KYNA inhibited [3H]benzodiazepine binding by 24%. Thus, although 7-Cl KYNA is clearly highly selective for the NMDA r~eptor-ass~iated glycine binding site, consideration should be given to the possibility that, in functional studies, effects observed at high concentrations may involve non-NMDA receptor m~ha~sms. 3.2. Spinal cord NMDA superfused over a hemicord for 1 min induced short-lasting and concentration-dependent (l-10 FM) depolarizations of the ventral roots (fig. 1). D-se&e was used instead of glycine to study the potential effects on NMDA responses of glycine receptor activation. Glycine is reported to depolarize motoneurones in the hemisected spinal cord preparation of the rat (Evans, 1978). Because

of

probably due to the activasitive glycine sites in the tio of stryc glycine was not suitable for our invese affinity of D-serine (0.38 PM, IC,,; table IA) for the st~~~~insensitive glycine site is only slightly lower than that of glycine (0.20 s actiolip.

TABLE 1 ~~~~~t~~~sof radio&and binding to isoiated braiu membranes by rumpounds active at strychnine-insensitive glycine binding sites_ (A) [‘HjgIycine bmdingz values are ICs,s and are means ~S.E.M. of data from at least three separate experiments. (B) Percent inhibition of radio&and binding by 7-chlorokynurenic acid and HA-966: values are means of triplicate observations. Abbreviations: QNB: quinuclitiylbenzilate; CGP 27492: 3~nop~yl-ph~p~~c acid, DHA: dibydroalprenolol. (A) [SH]Glycine binding Coowound

IC5” WN)

Glycine D-!%rine L-Seti D-Alanine L-Akmine D-Cycloserine L-CycIoserine Kymuenic acid 7-C~o~k~~~~c HA-966

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acid

(B) Other radioligands Radioiigand

0 inhibition by ‘I-Cblorokynurenic acid

Substance P (0.5 nM) Flunitrazepam (1.0 nM) Dixolan (1.0 nM) QNB (0.2 nM) Naloxone (1 .O nM) Muscimol(S.0 nM) CGP 27492 (2.0 nM) Prazocine (0.3 nM) Clonidine (1.0 nMf W-IT, (0.5 nM) Doxepine (OS r&f) DHA (0.4 nM) Cyclohexyl-adenosine (0.5 nM) Ketauscrine (0.4 nM) Thiotidine (2.0 nM)

HA-966 (100 PM)

110$4

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7 24 13 0 0 0 0 0 0 20 0 14

12 91 53 47 47 56 21 0 14 47 26 9

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CM; tabfe IA) and D-serine had no effect on the potentials recorded from the ventral roots in the concentrations tested (10-100 FM). When superfused before and during the application of NMDA (10 FM), D-serine consistently potentiated the NMDA response (21 f 9% at 100 pM D-serine; mean + S.E.M., n = 23). The threshold concentration of D-serine for potentiation of NMDA responses was about 10 PM, with the maximal effect being reached at about IO0 PM. However, the magnitude of the maximal potentiation was not sufficient to make accurate concentration-response curves. After blockade of synaptic transmission with TTX (1 PM), NMDA-evoke ventral root responses were reduced in amplitude (fig. 1 upper trace and inset), and a higher con~ntration of NMDA (30 PM) was required to elicit depolarizations ~mparable in size to those recorded in the absence of TIX. In the presence of TTX, NMDA responses could still be potentiated by D-set&e but to a slightly lesser extent (18 f 68, n = 5) than in its absence. In standard ACSF (no TTX), 7-Ci KYNA (10 PM) weakly but reversibly reduced the DR-VRP and the spontaneous activity. 7-Cl KYNA antagonized the depolarization of the ventral roots induced by NMDA (10 pM) by 44 + 9% (n = 6) in normal ACSF (fig. 1 lower trace) and by 64 & 8% (n = 5) in the presence of TTX (fig. 1 upper trace). Control NMDA responses were restored within about 30 min after 7-Cl KYNA washout. The inhibitory effect of %Cl KYNA could be reversed by D-serine (fig. 1). This effect was st~c~ne-~sensitiv~ (not shown) (but for HA-966 see fig. 2 upper trace), Figure 1 (inset) shows that 10 FM ‘&Cl KYNA largely abolished the NMDA-evoked depolarization in the presence of TTX, while the dose-response curve in the presence of 100 PM D-serine plus 10 FM 7-Cl KYNA was essentially superimposable on the control curve. In our experiments, HA-966 (100 FM) clearly reduced the spontaneous activity and the DR-VRP and antagonized the effect of applied NMDA by 42 f 13% (n = 6) in standard ACSF (no TTX (fig. 2 lower trace)) and by 39 f 23.0% (n = 3) in-the presence of TTX (fig. 2 upper trace). As with 7-Cl KYNA, the i~bition produced by HA-966 was completely overcome by the addition of D-serine (100 PM; fig. 2 inset). In the

33

Fig. 1. Hemisected rat spinal cord. The effect of D-serine and 742 KYNA on NMDA-evoked responses: (upper traces) D-serine (100 FM) increased NMDA responses in the absence and in the presence of TTX. TTX suppressed synaptic activity; the following recordings from a ventral root represent the responses of the montoneurones. 7-C) KYNA reduced the spontaneous activity and the DR-VRP and antagonized the effect of NMDA in the presence or absence (lower traces) of TTX. This antagonism was overcome to a great extent by the addition of D-serine. The inset shows the effect of 743 KYNA (10 @f) with and without D-serine on a NMDA dose-response curve.

presence of TTX, strychnine at concentrations of 1 PM (fig. 2 upper trace) or 10 pM had no marked effect on the NMDA-induced depolarization and did not affect the ability of D-serine to overcome the antagonism of NMDA responses by HA-966. CGP 37849 ((DL-(E)-2-~~4-me~yl-5-phosphono3-pentenoic acid), a novel competitive NMDA receptor antagonist (Fag8 et al., 1990), reduced the DR-VRP and the spontaneous activity of the ventral root in a con~entration-dependent manner (Pozza et al., 1990). CGP 37849 was about 10 times more potent than 7-Cl KYNA and about 100 times more potent than HA-966 in inhibiting the effect of NMDA. At a concentration of 1 PM, CGP 37849 almost completely antagonized NMDA-evoked depolarizations (fig. 3) and in contrast to the experiments with 7-Cl KYNA and HA-966 it was not possible to overcome this blockade with D-serine (100 FM). These results thus suggest that CGP 37849 antagonizes NMDA receptor responses by a rn~h~srn dis-

tinct from that of 7-Cl KYNA and HA-966. Similar observations were made with the non-competitive NMDA receptor blocker, ( + )-5-methyllO,ll-d~ydro-5H-dibe~~a,d]-cy~loh~ten-5,10imine [MI&801; data not shown). 3.3. Cortical wedge As the cortical wedges were bathed in Mg2+-free ACSF, spontaneous depolarization shifts with (only rarely without) rhythmic after-potentials (Burton et al., 1987) were recorded in all experiments. The frequency of these spontaneous events varied from wedge to wedge. NMDA in a concentration range from 3 to 30 PM, bath-applied for 2 mm, induced a concentration-dependent depolarization (not shown)_ 7-Cl KYNA, HA-%6 and the competitive NMDA receptor antagonist CGP 37849 antagonized the effect of applied NMDA (10 PM) in the concentrations tested, as they did in the hemisected rat spinal cord prepara-

34

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Fig. 2. Hemisected rat spinal cord. The effect of HA-946 and D-serine on NMDA-evoked responses: HA-966 reduced the spontaneous activity and the DR-VRP and antagonized the effect of NMDA in the presence (upper traces) and in the absence (lower traces) of ITX. This antagonism was markedly reversed by the addition of D-se&e. The inset shows the effect of HA-966 (100 PM) on a NMDA dose-response curve.

strongly reversed the antagonistic effect of 7-Cl KYNA and completely reversed that of HA-966. It had no effect in conjunction with CGP 37849, demonstrating again that 7-Cl KYNA and HA-966 act at a different site than CGP 37849 (fig. 5). In

tion. 7-Cl KYNA (10 PM) strongly reduced the NMDA-evoked responses by 62 f 8% (n = 4; fig. 5). I-IA-966 (200 pM) by 80 f 8% (n = 10, figs. 4 and 5) and CGP 37849 (0.3 PM) by 69 + 13% (n = 8, fig 5). The co-application of D-serine

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Fig. 3. Hemisected rat spinal cord. The effect of CGP 37849 on NMDA-evoked responses: CGP 37849 (1 PM) reduced the spontaneous activity and the DR-VRP and potently antagonized the effect of NMDA. It was not possible to overcome the antagonism by the addition of D-serine, suggesting that CGP 37849 binds to a different receptor site than 7-Cl KYNA or HA-966.

35

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10 min

Fig. 4. Cortical wedge. Continuous record illustrating the antagonistic effect of 200 FM HA-966 on depolarizations induced by 10 PM NMDA (2 min; short bars) applied to the cortical tissue site for 90 min as indicated by the bar beneath the record (the intervals in the trace represent 40 and 120 min respectively). f&application of 100 FM D-se&e (30 min) reduced the antagonistic action of HA-966. Return to drug-free medium reestablished the full NMDA response as in the control situation. Note that HA-966 reduced the spontaneous depolarization shifts; this effect was only slightly reversed by D-serine but reappeared fully after wa hing out the drug_

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Fig. 5. Cortical wedge. The percentage antagonism of NMDA-induced depolarizations by 10 FM 7-Cl KYNA, 200 pM HA-%6 and the competitive NMDA receptor antagonist CGP 37849 (0.3 FM) and the partial reversal of this effect by D-serine in the case of 7-Cl KYNA and HA-966. It is also demonstrated that, for HA-966, compounds acting at the glycine modulatory site are strychnine-insensitive (the small and not significant decrease of the response elicited by strychnine seemed to be an effect of the relatively high condensation used). The reductions are si~fic~tly different from the control value (P 1 0.001; paired Student’s t-test) and the potentiated responses with D-serine are also significantly different from those with no added D-serine (P > 0 ‘005).The number of experiments ranged between 4 and 10.

preparation, glycine was nearly rtica1 ne in reversing the antagonistic em as effect of 7-Cl KYNA or l-W-966. Neither glycine erine could overcome the antagonistic efCGP 37849 on the NMDA responses. strychnine (I PM) did reduce the NMDA slightly (fig. 5). it had no significant effect on the ~tentiation induced by D-serine or glycine. The rhythmic after-potentials of the spontaneous depolarization shifts were abolished by adding 7-Cl KYNA and HA-966. The amplitude of the initial depolarization shifts was also diminished. This effect was partially overcome by D-serine (For HA-966 see fig. 4).

The main findings of the present study are (1) that the depolarizing action of NMDA can be potentiated by D-serine (an agonist at strychnineinsensitive glycine bmding sites) in the hemisected spinal cord preparation of the immature rat but not in the cortical wedge preparation of the adult rat; (2) NMDA responses in both preparations are suppressed by 7-Cl KYNA, HA-966 MK-801 and CGP 3748 at concentrations which roughly reflect their affinity for the receptors in our radioligand binding assays; and (3) that these four antagonists are qualitatively different, since the effects of 7-Cl KYNA and HA-966 but not of CGP 37849 and MK-801 cau be reversed by D-seriue. In the hemisected spinal cord preparation, Dserine consistently and reversibly potentiated ventral root depolarizations induced by NMDA, thereby suggesting that the modulatory glycine binding site is present not only on NMDA receptors in supraspinal areas, as reported previously, but also at the spinal level. Our data indicate that the glycine site is located on motoneurones, since the ~tentiation of NMDA responses by D-serine was also observed in experiments where synaptic transmission was blocked by TTX. It has been shown earlier that glycine induces a depolarization of motone~ones which can be suppressed by the addition of strychnine (Evans, 1978; Brugger, unpublished) and that glycine is the inhibitory trans-

mitter released from Renshaw-cell synapses to suppress motoneurone firing (Curtis et al., 1976). Thus, our data, together with that of earlier studies on glycine-mediated transmission in the spinal cord, provide the first evidence that this amino acid may bind to distinct recognition sites with essentially opposite physiolo~cal actions on a single neuronal cell type. In contrast to our findings in the spinal cord, neither glycine nor D-serine could augment the NMDA response in the cortical siice preparation. The reason for this discrepancy is unclear. The lack of potentiation of NMDA responses in the cortical wedge preparation is in agreement with earlier studies (Kemp et al., 1988; Fletcher et al., 1988), although this effect has been demonstrated in cortical tissue. For example, glycine potentiates NMDA responses in primary cell cultures of rat cortical neurones, where the extracellular glycine con~ntration can be maintained at very low levels (patch-clamp recordings; Johnson and Ascher, 1987; Kemp et al., 1988). Moreover, with intracellular recordings and local apportion of drugs, glycine has been shown to potentiate NMDA responses in cortical (Thomson et al., 1989) and hippocampal (Minota et al., 1989) slices. The present results thus confirm that, under distinct expe~ental circumstances, it is possible to record a potentiation of NMDA responses by glycine and D-serine in vitro, without the necessity of partially suppressing glycine actions with antagonists. Furthe~ore, our experiments are the first to show such an effect in the spinal cord, a region of the CNS where the highest concentration of glycine is found. In addition, we used extracellular root recording techniques and a simple superfusion system (Evans and Watkins, 1978). These findings thus support the proposal made by Thomson et al. (1989) that the potentiation of NMDA responses by glycine or D-serine may be optimally recorded in tissue preparations which are minimally damaged. The hemisected spinal cord, a relatively large slab of tissue with only one cut surface, may represent such a preparation. One may speculate that, in relatively intact tissue preparations, the levels of transmitters and modulators are regulated by normal release, degradation and uptake mechanisms, thereby maintaining glycine levels in

the synaptic microenvironment below saturating levels. Our receptor binding data confirm that 7-Cl KYNA and HA-946 are selective ligands for the strychnine-insensitive gly”ine modulatory site, although they show that at higher concentrations 7-Cl KYNA may act at other receptor types, notably benzodiazepine binding sites on the GABA receptor. Dir&dine et al. (1990) also have reported that 7-61 KYNA antagonizes kainateevoked responses in Xenopus oocytes at concentrations in the low micromolar range. In cortical tissue, Foster and Kemp (1989) showed that HA-966 has a different profile of antagonism than 7-Cl KYNA and suggested that HA-966 blocks only the component of the NMDA response that is potentiated by glycine whereas 7-Cl KYNA can produce complete flattening of the NMDA concentration-response curve. An alternative possibility, based on our and Dingledine’s observations, is that these differences may result form the nonselectivity of 7-Cl KYNA at higher concentrations. These arguments not ~t~t~~g, we were able to completely reverse the antagonist effects of 10 ;CIM7-Cl KYNA and 100 or 200 PM HA-966 with D-serine in the present experiments. Hence, both antagonists appear to be selective antagonist at the NMDA receptor-associated glycine site in the concentration range used. In summary, we provide the first evidence that the recently described strych~e-~sensitive glycine binding site, which positively modulates the NMDA receptor, is found on spinal motoneurones under in vitro unctions, where glycine inhibitory receptors are also located. Thus, a single substance may play a dual role on this neuronal cell type. Our findings confirm that the potentiation of NMDA by glycine cannot be observed in the cortical wedge preparation. However, in both preparations, 7-Cl KYNA and HA-966 antagonize NMDA-evoked responses to a similar extent, and such antago~sm is reversed by D-serine. Finally, our data show that, as also recently demonstrated in vivo (Smgh et al., 1990; Larson and Beitz, 1988’1,it is possible not only to inhibit but also to potentiate the NMDA receptor via the glycine modulatory site. These findings have profound implications for our understanding the

physiology of excitatory amino acid synaptic transmission and for the development of novel therapeutic agents acting via the NMDA receptor.

Acknowledgements The authorsthankDrs. H. Bittiger and R. Hauser, and M, Benedict and J. Baud for help with receptor bmding assays and also G. Karlsson for reading the manuscript.

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