A potent antagonist of the strychnine insensitive glycine receptor has anticonvulsant properties

European Journal of Pharmacology, 174 (1989) 197-204 Elsevier

197

EJP 51099

A potent antagonist of the strychnine insensitive glycine receptor has anticonvulsant properties M a l c o l m J. Sheardown, Jorgen Drejer, Leif Helth Jensen, Carsten E. Stidsen 1 a n d Tage H o n o r 6 A / S Ferrosan, CNS Dwtswn, Sydmarken 5, DK-2860 Soeborg, Denmark and 1 Novo -Nordtsk A / S , CNS Dtwswn, Novo AII~, DK-2880 Bagsoaera~ Denmark Recewed 17 May 1989, rewsed MS recewed 5 September 1989, accepted 17 October 1989

5,7-Dlnitro-qumoxahne-2,3-dione (MNQX) displaced [3H]glycme binding to cortical membranes but had no effect on [3H]3-((+)-2-carboxypiperazm-4-yl)-propyl-l-phosphonic acid ([3H]CPP) binding. MNQX potently antagonized N-methyl-D-aspartate (NMDA)-evoked release of [3H]GABA from cultured cortical neurones, NMDA evoked spreading depression and NMDA depolarizations in the rat neo-cortex. All of these responses were reversed by addition of glycine to the perfusion media. These results suggested that MNQX is an antagomst at the strychnine-insensitive glycine receptor associated with the NMDA receptor/ionophore complex. Furthermore the compound was found to antagonise audiogemc selzures in DBA-2 rmce indicating the potential of glyclne antagomsts of tins type in ant~convulsant therapy. MNQX (5,7-dinitro-qumoxaline-2,3-dlone); Glycme antagonist, NMDA (N-methyl-D-aspartate); [ 3H]GABA release; Spreading depression; (Electrophysiology)

1. Introduction The receptors for excitatory amino acids in the central nervous system are divided into three types for which the specific agonists are N-methyl-Daspartate (NMDA), quisqualate and kainate (Watkins and Evans, 1981). Responses to N M D A have recently been shown to be potentiated by glycine (Johnson and Ascher, 1987). This finding led to the further study of glycine binding sites on rat forebrain membranes (Bristow et al., 1986). These sites are distinct from the previously described inhibitory glycine receptors in the spinal cord as they are insensitive to strychnine and activated by glycine concentrations much lower than those required to hyperpolerize spinal cord

Correspondence to M.J. Sheardown, A / S Ferrosan, CNS Division, Sydmarken 5, DK-2860 Soeborg, Denmark

neurones. The strychnine-insensitive glycine binding sites have the same regional distribution as the N M D A receptor-ionophore complex (Bowery, 1987), furthermore the glycine binding is sensitive to glutamate and Mg 2÷ levels (Marviz6n and Skolnick 1988) and glycine binding modulates [3H]MK 801 binding to the N M D A ionophore (Wong et al., 1987). These findings lead one to the conclusion that the glycine binding site is coupled to the N M D A receptor-ionophore complex. It has recently been reported that N M D A blockade produced by the weak antagonist HA-966 (Bonta et al., 1971) can be reversed by glycine (Fletcher and Lodge, 1988; Drejer et al., 1989) indicating that this compound is a glycine antagonist at the N M D A receptor-ionophore complex. In this study using a number of in vitro assays we show that a new qumoxaline dione, 5,7dinitro-quinoxaline-2,3-dione (MNQX) is a potent

0014-2999/89/$03.50 © 1989 Elsewer Science Pubhshers B.V. (Biomedical DiWslon)

198 antagomst at the strychnine-insensitive glycme receptor. Furthermore we show that MNQX has anticonvulsant properties.

2.3. Spreadmg depresston

Binding experiments were performed at 4°C with extensively washed rat cortical membranes (Honor6 and Nielsen, 1985). [3H]Kainate (5 nM) binding in 50 mM Tris-citrate buffer was used to examine kainate receptors (Honor6 et al., 1986), [ 3 H] a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) (5 /~M) binding in 30 mM Tris-HC1 buffer with 2.5 mM CaC12 and 100 mM potassium thiocyanate for quisqualate receptors (Honor6 and Nielsen, 1985) and [3H]3-((+)2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid ([3H]CPP) (5 nM) in 30 nM Tris-HCl buffer with 2.5 mM CaC12 for N M D A receptors (Honor6 et al., 1987). Glycine receptors were studied using [3H]glycine (20 nM) binding in 30 mM Tris-HC1 buffer with 2.5 mM CaC12, Phenylcyclidine sites were studied using [3H]N-(1-(2-thienyl)cyclohexyl)piperidine (TCP) binding in 5 mM Tris-HCl buffer. The IC50 values were calculated using the software 'Kinetic EBDA, Ligand Lowry' from Elsevier-Biosoft.

Spreading depression (S.D.) was studied in chick retina (Martins-Ferreira and Oliveira-Castro, 1966) as detailed by Sheardown (Drejer et al., 1989). In brief the posterior chamber of the eye (vitreous body removed) was initially incubated in a small petri dish at 26°C in mock-CSF (MartlnsFerreira and Ohveira-Castro, 1966) for 30 nun and then transfered to a corresponding medium containing 100 p M NMDA. N M D A triggered waves of S.D. in the chick retina, presenting itself as a milky area spreading slowly across the retina at 2-3 m m / m i n . These changes spread over the entire retina and are easily visible to the unaided eye. Prior to drug test the latency for S.D. to start in each eye cup was measured. After a further 15 rain incubation in normal mock-CSF the eye cups were transfered to normal mock-CSF containing the antagomst with or without 100 ~M glycine and incubated for a further 15 nun. Thereafter the eye cups were transferred to mock-CSF containing 100 /~M N M D A and the same concentration of antagonist with or without 100 /zM glycme. The latency for a S.D. to start was again measured. Recovery from any drug effects was tested for each eye cup. Antagomst effects are expressed as the percentage maximum antagonism of NMDA-evoked S.D., the maximum effect being defined as an increase over the control (NMDA) latency of 30 s.

2.2. GABA release

2.4. Rat neo-cortex shce

Cerebral cortical neurones were cultured from 15 days old mouse embryo cortices as described by Drejer et al. (1987). Excitatory amino acid responses in these cultures were monitored using the continuous [3H]GABA release model described by Drejer et al. (1987). Briefly cells preloaded with [3H]GABA for 30 rain were superfused (2 m l / m i n ) with HEPES-buffered saline (HBS) (see Drejer et al., 1987) for 15 min and then stimulated every 4 min for 30 s by changing the superfusion medxum to 20 /~M N M D A in HBS. When antagonists were tested these substances were added to both the basal and sumulation media.

Rats were killed by decapitation and wedges of cingulate cortex 1.5 mm wide and 500 /xm thick, consisting of cingulate cortex and corpus callosum, were prepared as described by Harrison and Simmonds (1985). The wedges were placed in a two-compartment grease-sealed perfusion chamber in such a way that the cortical tissue was almost entirely contained in one compartment and the corpus callosum entirely contained in the other compartment. Both compartments were perfused (2 m l / m l n ) at 21-24°C with mock-CSF (Sheardown, 1988) equilibrated with 5% CO 2 and 95% 02 . Thirty minutes after the wedge was placed in the chamber, Mg2+-free medium was used in place

2. Materials and methods

2.1. Bmdmg experiments

199

of normal medium in the compartment containing the cortical tissue, since Mg 2+ ions produce a voltage-dependent blockade of NMDA responses. The d.c. potential between the two compartments was recorded using Ag/AgC1 electrodes, amplified and displayed continuously on a chart recorder. After a 2 h stabilization period the agonists were applied to the cortical tissue, contained in 4 ml aliquots of MgZ+-free perfusion medium. After consistent control responses were obtained the compartment containing the cortical tissue was equilibrated with Mg2+-free perfusion medium containing the antagonist, for 30 min, before retesting the agonist responses. 2.5. Audtogentc seizures in DBA-2 mice

Test compounds were given i.p. to groups of 10 male DBA-2 mice (weight 10-12 g) per dose level and 30 min later challenged with a 14 kHz sinusoidal tone at 110 dB. The number of mice with clonic convulsions within the next 2 rnln was noted.

TABLE 1 InlublUon of binding (ICs0) to rat corucal membranes (/~M) Compound

[3H]Glyclne

[3H lTCP

[3H]AMPA

[3HIKinhate

[3HICPP

MNQX CNQX DNQX Glyclne D-Senne HA-966 7-Chlorokynurenate

1.0 14 95 0.16 0 31 15

29 25.9 2.9 > 100 > 100 -

2.4 03 0.5

1.4 15 2.0

> 120 25 40

2.5

6

0.4 /~M. In this model MNQX proved to be a relatively selective antagonist of NMDA response with IC50 values for inhibition of quisqualate and kainate evoked [3H]GABA release of 9.3 and 13 /~M, respectively. The blockade by MNQX of NMDA-evoked [3H]GABA release is glycine-sensitive. Figure 2 shows a concentration-dependent reversal by glycine of the antagonism of NMDA-evoked [3H]GABA release produced by a 1.0 /~M concentration of MNQX.

3. R e s u l t s

3.3. Sprea&ng depresston 3.1. Receptor bm&ng

The IC50 value for MNQX against [3H]glycine binding was 1.0 /tM whereas the compound was inactive as an inhibitor of [3H]CPP binding (IC50 > 120 #M). The IC50 values for displacement of [3H]AMPA and [3H]kainate and [3H]TCP binding were 2.4, 1.4 and 2.9/tM, respectively. For comparison the ICs0s (/tM) of the selective nonNMDA antagonist CNQX were 0.3, 1.7, 28, 23 and 25.9 #M for [3H]AMPA, [3H]kainate, [3H]CPP, [3H]glycine and [3H]TCP, respectively. These values and those for DNQX, glycine, Dserine, HA-966 and 7-choro-kynurenate are shown m table 1.

NMDA produces reproducible S.D. in the chick retina which is easily visible to the unaided eye 100

80

"~

40

a,~

20. o

0.ol

o 1

t

",o0

10

lo0

[UNqX ~ ) ] 3.2. Cultured corttcal neurones

Figure 1, shows that MNQX in the cultured cortical neurones is a potent antagonist of NMDA evoked [3H]GABA release with an IC5o value of

Fig. 1 Inl~bmon of EAA-induced [3H]GABA release from cultured corucal neurons by MNQX. Ordinate, % mlubmon of [3H]GABA release induced by NMDA, 20/~M ( o ) qmsqualate 0.5/~M (e) or kainate, 15/~M (A); abs, ssa, log concentraUon of MNQX. Results are the means± S.E.M. of three mdlwdual experiments performed m duphcatc.

200 100C

80-

L

60

O

100-

/

o

80-

/

'-"

60-

..

40-

0

'~

.1

2o

N

20-

o oo!

0.1 1 10 [ g l y c i n e (/zM)]

(Martins-Ferreira a n d O l i v e i r a - C a s t r o , 1966; L a u r i t z e n et al., in press; D r e j e r et al., 1989). M N Q X a n t a g o n i z e d this S.D. in a c o n c e n t r a t i o n d e p e n d e n t m a n n e r (IC50 v a l u e 0.95 g M ) . T h e IC50 v a l u e s for t h e d e p r e s s i o n o f q u i s q u a l a t e k a i n a t e a n d K C l - e v o k e d S . D . w e r e 0.91, 0.98 a n d 1.01 g M, respectively. Figure 3 shows that the a n t a g o n i s m b y M N Q X o f N M D A - e v o k e d S.D. is r e v e r s e d b y 100 g M glycine. T h e g l y c i n e a n t a g o n i s t

/

100

z *d

,--7,5

......

10

100

Fig. 4 Effect of MNQX on the response of the rat neo-cortex shce to NMDA. Ordinate, % maxamum response of neo-cortex shce to NMDA; absossa, log concentraUon of NMDA. Open circles control; closed circles MNQX 3 /xM; open triangles MNQX 10 ttM Results are the means of 5-10 expenments, S.E.M.s are shown as vertmal bars when extending beyond the symbols.

H A - 9 6 6 ( F l e t c h e r a n d L o d g e , 1988) also b l o c k e d N M D A - e v o k e d S . D . (IC50 2 1 0 / ~ M ) .

3.4. R a t neo-cortex shce Figures 4 and 5 show that MNQX antagonized NMDA- and quisqualate-evoked depolarization of

0

100

0

50

........ 2

[NMDA(~M)l

Fig. 2. Reversal by glycme of MNQX effects on NMDA-mduced [3H]GABA release from cultured cortmal neurons Ordinate, % of control NMDA stlmulatmn in the presence of 1 jaM MNQX; abmssa, log concentratmn of glycme. Results are means of two expenrnents.

03

o

100

°~.&

•

N

o

80

u

60

O

o o la9

T

,o

0

0.1

1.0

10.0

[UNqX (.u)] Fig. 3 Graph showing the concentrataon-dependent block of NMDA-evoked S.D in the clucken retina by MNQX and the reversal of the block by 100 /~M glycme. Ordinate, % maxamum blockade of NMDA-evoked S.D.; abscissa, log concentratmn of MNQX. Open symbols zero glycme, closed symbols 100/~M glycine. Results are the means of SLXexperiments, S.D.s are shown as verlacal bars when extending beyond the symbols.

0

. . . . . . .

2

t

. . . . . . . .

10

,

100

[ q u i s q u a l a t e (~M)] Fig. 5. Effect of MNQX on the response of the rat neo-cortex shce to qu,squalate. Ordinate, % maximum response of rat neo-cortex shce to qmsqualate; absossa, log concentratmn of qmsqualate. Open circles control, closed orcles MNQX 3/~M, open triangles MNQX 10 ~tM. Results are the means of 5-10 experiments, S.E.M.s are shown as vertical bars when extendmg beyond the symbols.

201 100-

~

o

80-

A CONTROL

60~

40-

z

200

QUIS 30 pM

1

. -- . . . . . . . . . . . . . . . 10

100

NMDA 4O HM

B

[uN~ 0,,u)] Fzg 6 Graph showing the concentrataon-related a n t a g o m s m by M N Q X of the response to 40 /~M N M D A m the rat neo-cortex shce, the concentration-response curve to M N Q X is moved to the right m the presence of 100 # M glycme Ordinate, % a n t a g o m s m of the response to 40 p M N M D A ; abscissa, log concentration of M N Q X . Open symbols control, closed symbols plus 100 p M glycme. Results are the means of six experiments, S.E.M.s are shown as vertical bars when extending beyond the symbols

the cortical wedge in a concentration-dependent manner. However in these experiments using full concentration-response curves of NMDA and quisqualate the compound showed little if any selectivity. The NMDA antagonism is reduced by 100 #M glycine as shown in fig. 6. This figure shows the antagonism of the response to 40 #M NMDA by increasing concentrations of MNQX, the IC50 for MNQX in normal medium was 6.5 #M and in the presence of 100 #M glycine 22/xM. The quisqualate antagonism however is not glycine-sensitive as seen in fig. 7 (taken from a single wedge preparation) which shows single responses to quisqualate and NMDA, being reduced by MNQX. The NMDA responses were selectively reversed by 100 #M glycine. The spontaneous potentials obtained in this preparation using Mg/+-free perfusion medium (Harrison and Simmonds, 1985) are blocked by MNQX. Blockade of these 'epileptiform' discharges by 3 #M MNQX was completely reversed and that by 10 #M MNQX partially reversed, by inclusion of 100 #M glycine in the perfusion medium (fig. 8). 3.5. Audtogemc setzures

MNQX was significantly more potent than the

r-'-i NMDA 40 ~M

QUIS 30 p M

MNQX lOpM ÷ g lyc m e 100

~-~

~.i

from

r~t QUIS 30 ~M

r--1 NMDA 40 pM

Fig. 7. Records taken from a single neo-cortex shce experiment showing control responses (A) to 30 # M qmsqualate and 40 # M N M D A . M N Q X 10 # M reduces the responses to both agomsts (B) but the addition of 100 # M glycme (C) reverses only the N M D A blockade havLng no effect on the qmsqualate antagonism. Responses to both agomsts returned to the control levels after 1 h washout of M N Q X (responses not shown).

glycine antagonist HA-966 and the selective nonNMDA receptor antagonists DNQX and CNQX in blocking audiogenic seizures in DBA-2 mice (see table 2). Doses of HA-966 greater than 1 mg/kg showed anticonvulsant activity but the re-

TABLE 2 I n t u b m o n of audlogemc seizures m DBA-2 nuce. Compound

EDs0 ( m g / k g )

MNQX DNQX CNQX HA-966

0.1 25 15 >1

202

A

j

CONTROL

B

MNQX 3 M,M

C

N MNQX 31/.M

+ llly¢Inei00 j~M

lmV

5 m.i.n"

CONTROL

MNQX

MNQX I0/zM

I0/.~M

+ glyclne 100/#.M

Fig 8 Records taken from two neo-cortex shces perfused m Mg2+-free medium taken from the same a m m a l and recorded m parallel Column (A) control records, column (B) 5 n u n after addition of 3/~M (upper line) and 10/~M (lower line), M N Q X , column (C) 5 man after addlUon of 100/~M glycme The blockade of the spontaneous 'epdeptlform' potentials by M N Q X as reversed by 100 ~ M glycine in a m a n n e r related to the M N Q X concentration.

suits were complicated by the toxic effects of the compound at these doses.

4. Discussion

The data presented shows MNQX to be a N M D A antagonist in cultured cortical neurones, the chicken retina S.D. model and the rat neocortex slice (cortical wedge). The finding that N M D A antagonism by MNQX was reversed by the inclusion of glycme m the perfuslon medium, m all of the experimental models studied, suggests that MNQX blocks the glycine binding site at the N M D A receptor complex. This is further supported by the demonstration of MNQX as a potent displacer of [3H]glycine binding without any inhibitory acnvity at the [3H]CPP binding site. The potency of MNQX at the strychnine-insensitive glycine binding site associated with the N M D A receptor/xonophore complex appears to be 15-200 times higher than for HA-966 (Fletcher and Lodge, 1988; Drejer et al., 1989). MNQX also showed affinity to the [3H]kainate and [3H]AMPA binding sites and responses to kainate and quisqualate could be antagonized by MNQX m all the

functional models. This n o n - N M D A antagonism shown by MNQX was however glycine-insensitive. MNQX proved to be a selective antagonist of N M D A as apposed to n o n - N M D A responses in the cultured cortical neurones but showed lower potency in the chick retina and lower potency and little if any selectivaty as a glycine antagonist in the neo-cortex slice. This finding probably reflects the efficient removal and subsequent low levels of glycine present in the cultured cells (Drejer et al., 1989), whereas in the whole tissue preparations the higher level of endogenous glyclne present reduces the potency of the glycine antagonism whilst having no effect on the antagonism of non-NMDA receptor responses. This would also explain why HA-966 is nearly 10 times less potent as a glycine antagonist in the retina and the brain slices than in the cultured neurones (Drejer et al., 1989; Fletcher and Lodge, 1988). The binding data however shows that M N Q X has only a 2.4-fold selectivity for glycine compared with AMPA receptors. Furthermore as the IC50 values for glycine and D-serine in the [3H]glyclne binding assay are very similar to those found in the literature (Kishimoto et al., 1981), it is unlikely that the assay is significantly con-

203 t a m i n a t e d with glycine; so giving an underestim a t e of the potency. H o w e v e r given the very low c o n c e n t r a t i o n of glycine in the c u l t u r e d cortical m t e r n e u r o n e p r e p a r a t i o n ( D r e j e r et al., 1989) it c o u l d b e that o n l y r a p i d l y p e r f u s e d cell culture p r e p a r a t i o n s give a true p i c t u r e of the p o t e n c y a n d selectivity o f glycine a n t a g o n i s t s a n d that the presence of small a m o u n t s of glyclne in m e m b r a n e p r e p a r a t i o n s will reduce the p o t e n c y of the glycine antagonism. It has r e c e n t l y b e e n shown that the N M D A antagonists D-APV, ketamine and phencyclidine are e q u a l l y p o t e n t in b l o c k i n g S.D. evoked b y N M D A , quisqualate, k a i n a t e a n d K C I (Lauritzen et al., in press) the s a m e p a t t e r n of activity as that f o u n d for M N Q X . These findings m a y further s u p p o r t the suggestion that excitation of the N M D A r e c e p t o r / l o n o p h o r c o m p l e x is the final c o m m o n factor in the triggering of S.D. in the chick retina. T h e o b s e r v a t i o n that M N Q X p o t e n t l y inhabits the s p o n t a n e o u s ' e p i l e p t l f o r m ' discharges, e v o k e d b y p e r f u s i o n with Mg2+-free m e d m m in the neocortical slice; suggested that M N Q X m a y have p o t e n t anticonvulsive properties. I n d e e d M N Q X was a relatively p o t e n t a n t a g o n i s t of a u d i o g e n i c seizures in D B A - 2 mice, b e i n g m o r e t h a n 10 times m o r e p o t e n t t h a n HA-966. F u r t h e r m o r e M N Q X was 150 times m o r e p o t e n t t h a n C N Q X a n d 250 times m o r e p o t e n t t h a n D N Q X , two chemically related non-NMDA r e c e p t o r antagonists. A s C N Q X a n d D N Q X are respectively 8 a n d 5 times m o r e p o t e n t t h a n M N Q X as displacers of [ 3 H ] A M P A b i n d i n g ( H o n o r 6 et al., 1988) this i n c r e a s e d a n t i c o n v u l s a n t p o t e n c y of M N Q X is p r o b a b l y r e l a t e d to its higher p o t e n c y as a disp l a c e r of [3H]glycine binding, i n d e e d C N Q X a n d D N Q X are very w e a k o r inactive as glycine a n t a g o n i s t s ( D r e j e r et al., 1989; Birch et al., 1988). T h e i n c r e a s e d a n t i c o n v u l s a n t p o t e n c y of M N Q X c o m p a r e d with D N Q X a n d C N Q X is n o t due to g r e a t e r p e n e t r a t i o n of the b l o o d b r a i n barrier, as all three c o m p o u n d s show similar kinetic p r o p e r ties a n d have p o o r access to the central nervous s y s t e m ( N o r d h o l m , unpublished). Interestingly, C N Q X is also very w e a k as an i n h i b i t o r of s p o n t a n e o u s discharges e v o k e d b y Mg2+-free m e d i u m in the n e o - c o r t i c a l slice (Jensen a n d Sheardown,

1988), suggesting that in vitro b l o c k a d e of epil e p t i f o r m discharges m a y have a role m the e v a l u a t i o n o f potentxal a n t i c o n v u l s a n t c o m pounds. I n c o n c l u s i o n M N Q X is a p o t e n t a n t a g o n i s t at the N M D A r e c e p t o r - a s s o c i a t e d glycine site. This class of c o m p o u n d s a p p e a r s to have p o t e n t m l as a n t i c o n v u l s a n t s a n d in the t r e a t m e n t o f S.D.-associated p h e n o m e n a such as migraine.

References Birch, P.J, C J.S. Grossman and A G. Hayes, 1988, FG 9065 and FG 9041 antagonize responses to NMDA wa an action at the strychmne-lnsensit~ve glyclne receptor, Br. J Pharmacol 97 (Suppl.) 758P. Bonta, I L., C.J. De Vos, H. Gnjsen, F.C Hlllen, E.L. Noach and A W Slm, 1971, 1-Hydroxy-3-armno-pyrrohdone (HA966): a new GABA-hke compound, with potential use m extrapyrarmdal &seases, Br. J Pharmacol. 43, 514. Bowery, N G , 1987, Glyclne binding sites and NMDA receptors m braan, Nature (London) 326, 338 Brlstow, D R, N G. Bowery and G.N Woodruff, 1986, Lght rmcroscoplc autoradlographlc locahzatlon of [3H]glycme and [3H]strychmne binding sites in rat brain, European J. Pharmacol. 126, 303 Drejer, J, T. Honor6 and A Schousboe, 1987, Excitatory amino acid-induced release of 3H-GABA from cultured mouse cerebral cortex lnterneurones, J. NeuroscL 7, 2910. Drejer, J, M J. Sheardown, E O Nielsen and T. Honor& 1989, Glycme reverses the effect of HA-966 on NMDA responses in cultured rat cortical neurones and cluck retina, Neurosc~ Lett 98, 333 Fletcher, E.J and D. Lodge, 1988, Glycme reverses antagomsm of N-methyl-D-aspartate (NMDA) by 1-hydroxy-3-amlnopyrrohdone-2 (HA-966) but not by D-2-amano-5-phosphono-valerate (D-AP5) on rat cortical shces, European J Pharmacol. 151, 161. Harrison, N.L. and M.A Simmonds, 1985, Quantitative studies of some antagomsts of N-methyl-D-aspartate in shces of rat cerebral cortex, Br J. Pharmacol 84, 381. Honorr, T, S.N Davies, J. Drejer, E J. Fletcher, P. Jacobsen, D. Lodge and F.E Nielsen, 1988, Qulnoxahne-dlones. Potent competitive non-NMDA glutamate receptor antagomsts. Science 241, 701. Honorr, T, J. Drejer and M Nielsen, 1986, Calcmm dlscnrmnatlve two [3H]kamate binding sltes with &fferent molecular target s~zes m rat cortex, Neuroscl Lett. 65, 47 Honorr, T., J. Drejer, M. Nielsen, M. Watkms and H.J. Olverman, 1987, Molecular target size of NMDA antagomst binding sites, European J. Pharmacol 136, 137. Honorr, T and M. Nielsen, 1985, Complex structure of qmsqualate-sensitlve glutamate receptors m rat cortex, Neurosci. Lett 54, 27.

204 Jensen, L H. and N.J Sheardown, 1988, Lack of potent anUepdept~c effect of speofic non-NMDA EAA receptor antagomst, m" FronUers m Excitatory Anuno A o d Research, eds. E.A Cavalhelro, J. Lehman and L Turskl (Alan R Llss, Inc., New York) p. 219. Johnson, J.W and P Ascher, 1987, Glycme potenUates the NMDA responses m cultured mouse braan neurones, Nature (London) 325, 529. Kashlmoto, H., J.R. Simon and M.H. Apnson, 1981, Deternunataon of the eqmhbnum dlssocmtlons constants and number of glyeme binding s~te m several areas of the rat central nervous system, using a sodmm independent system, J. Neurochem 37, 1015. Launtzen, M , M Sheardown and A. Hansen, Aspects of calcium ~ons in cort~al spreading depression, m Cerebral Ischerma and Calcmm, eds. A. Hartman and W Kuschmsky (Spnnger Vedag, Berhn) (m press).

Martlns-Ferrelra, H and G Ohvelra-Castro, 1966, Light scattering changes accompanying spreading depression m isolated retina, J Neurophyslol 37, 773 Marwz6n, J.C.G and P Skolmck, 1988, [3H]Glycxne binding is modulated by Mg 2+ and other hgands of the NMDA receptor-caUon channel complex, European J Pharmacol 151,157. Sheardown, M.J., 1988, A new and speofic non-NMDA receptor antagomst FG 9065, blocks L-AP4-evoked depolarization in rat cerebral cortex, European J Pharmacoi. 148, 471 Watkms, J.C and R H Evans, 1981, Excitatory anuno aod transmitters, Ann Rev. Pharmacol. Toxacol. 21, 165. Wong, E.H F , A.R Kmght and R Ransom, 1987, Glycme modulates [3H]MK-801 binding to the NMDA receptor in rat brain, European J. Pharmacol. 142, 487.