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Selective Depression of the Spinal Polysynaptic Reflex by the NMDA Receptor Antagonists in an Isolated Spinal Cord in vitro
Gen. Pharmac. Vol. 29, No. 4, pp. 645–649, 1997 Copyright 1997 Elsevier Science Inc. Printed in the USA.
ISSN 0306-3623/97 $17.00 1 .00 PII S0306-3623(96)00514-9 All rights reserved
Selective Depression of the Spinal Polysynaptic Reflex by the NMDA Receptor Antagonists in an Isolated Spinal Cord in vitro Yoshimi Maruoka, Yukihiro Ohno,* Hiroyasu Tanaka, Hirokazu Yasuda, Ken-Ichi Ohtani, Hideki Sakamoto, Akiko Kawabe, Chika Tamamura and Mitsutaka Nakamura Discovery Research Laboratories II, Research Center, Sumitomo Pharmaceuticals Co., Ltd., 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554, Japan [Tel: (81) 6 466 5215; Fax: (81) 6 466 5218] ABSTRACT. 1. The effects of N-methyl-D-aspartate (NMDA) receptor glycine-binding site antagonists 7-chlorokynurenate (7-Clkyn) and (6)-3-amino-1-hydroxy-2-pyrrolidone (HA-966) on spinal reflexes in an isolated spinal cord that was maintained in Mg21-free medium in vitro were examined. The actions of 7-Clkyn and HA-966 were compared with those of the channel-site antagonist (i.e., dizocilpine) and NMDA-binding site antagonists—that is, 3-[(6)-2-carboxypiperazin-4-yl]-propyl-1phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV). 2. 7-Clkyn and HA-966 produced a selective depression of the polysynaptic reflex (PSR) while negligibly affecting the activity of the monosynaptic reflex (MSR). The PSR was also differentially suppressed by dizocilpine, CPP and APV. The PSR inhibitory activity of the NMDA antagonists was in the following order: dizocilpine.CPP.APV57-Clkyn.HA-966. 3. The inhibitory effects of 7-Clkyn on PSR were markedly antagonized by the simultaneous application of D-serine, an agonist for the NMDA receptor glycine-binding sites. However, PSR inhibition by dizocilpine and CPP was unaffected. 4. Inhibition of the PSR by 7-Clkyn persisted in the presence of strychnine, which markedly increased the PSR activity by itself. 5. These findings suggest that the NMDA receptor glycine-binding sites play a role in generating the NMDA receptor-mediated PSR in the spinal cord in vitro. gen pharmac 29;4:645–649, 1997. 1997 Elsevier Science Inc. KEY WORDS. NMDA receptor, glycine-binding site, 7-chlorokynurenate, HA-966, d-serine, spinal reflex, isolated spinal cord INTRODUCTION N-Methyl-d-aspartate (NMDA) receptors mediate the excitatory neurotransmission that has been implicated in various pathophysiological conditions including epilepsy, pain processing and hypoxicischemic brain injury (Meldrum, 1994; Muir and Lees, 1995; Yoneda and Ogita, 1991). The NMDA receptors are considered to be heteromeric assemblies consisting of a certain combination of the NMDA receptor subunits (e.g., NMDAR1 and NMDAR2A–D), which form the Ca21 -permeable ion-channel complex (Meguro et al., 1992; Monyer et al., 1992). The NMDA receptors also contain strychnine-insensitive glycine-binding sites, where endogenous glycine seems to act as agonist to activate the receptor function (Kemp et al., 1988; Kleckner and Dingledine, 1988). To date, several antagonists specific for the multiple regulatory sites of the NMDA receptors have been developed. Dizocilpine [(1)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801)] and phencyclidine (PCP) are known to bind to the ion-channel sites (Kemp et al., 1987; Wong et al., 1986), dl-2-amino-5-phosphono valerate (APV) and 3-[(6)-2-carboxypiperazin-4-yl]propyl1-phosphonate (CPP) are the antagonists for the NMDA-binding *To whom all correspondence should be addressed. Received 4 June 1996; revised 24 September 1996; accepted 18 October 1996.
sites (Davies et al., 1986; Lehmann et al., 1987), and 7-chlorokynurenate (7-Clkyn) and (6)-3-amino-1-hydroxy-2-pyrrolidone (HA966) are the antagonists for the glycine-binding sites at the NMDA receptor/ion-channel complex (Foster and Kemp, 1989; Kemp et al., 1988). Although the actions of these agents have been widely studied, the role of the respective regulatory site in the NMDA receptor functions still remains to be defined. In previous studies, Ohno and Warnick (1988, 1989, 1990) showed that PCP analogs [e.g., PCP, thienylcyclohexylpiperizine (TCP), m-amino PCP and m-nitro PCP] inhibited the spinal dorsal root–ventral root reflexes through two distinct mechanisms in isolated spinal cord preparations in vitro. These agents, at low concentrations (nanomolar range), selectively suppressed the Mg21 -sensitive polysynaptic reflex (PSR) by blocking the NMDA receptors, whereas higher concentrations (micromolar range) inhibited the monosynaptic reflex (MSR) by activating the spinal serotonergic system (Ohno and Warnick, 1989; 1990). These findings, consistent with previous in vivo studies (Block and Schwarz, 1994; Polc, 1985; Turski et al., 1990), suggested that the NMDA receptors take part in the generation of a segmental PSR in the spinal cord. To explore the role of the glycine-binding sites at the NMDA receptors in modulating the Mg21 -sensitive PSR in vitro, we extended the evaluation to the selective glycine-binding site antagonists 7-Clkyn and HA-966 and compared their actions with those of the ion-
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Y. Maruoka et al. oscilloscope and stored on FM tapes. Acquired responses were signal averaged with on-line computer assistance (ATAC-3300, Nihon Koden) and analyzed for amplitude, area and latency. The mean value of area of the reflex activities was obtained from ten successive responses in each experiment before the drug application and after the effects of the test drug reached a plateau.
Solutions and drugs
FIGURE 1. Effects of 7-Clkyn, dizocilpine and CPP on monosynaptic and polysynaptic reflexes in spinal-cord preparations of neonatal rats in vitro. The spinal cords were superfused with a Mg21-free physiological solution throughout the experiment. After the control recordings were obtained, the spinal cord was exposed to either 7-Clkyn (3 mM), dizocilpine (0.1 mM) or CPP (0.3 mM) until the response reached a plateau. The traces are signal-averaged records of ten successive responses before and after the drug application (at 15 min for 7-Clkyn and CPP and at 120 min for dizocilpine). The traces in each column were obtained in the same preparation. The calibrations are 2 mV and 5 msec.
channel site (i.e., dizocilpine) and the NMDA-binding site (i.e., CPP and APV) antagonists. MATERIALS AND METHODS
Preparation of spinal cord and recording of reflex activity The isolation of the spinal cord and recording of the ventral root potentials were performed as described previously (Ohno and Warnick, 1990). Briefly, the spinal cords were dissected from male Sprague-Dawley rats (6–9 days old) under ether anesthesia, hemisected longitudinally and placed in a recording chamber, where they were superfused with Mg21-free physiological solution at a flow rate of 1.5–2.5 ml/min. The superfusion medium was bubbled continuously with 95% O2, 5% CO2, and the temperature was kept at 25.060.58C. Test stimuli (0.15-msec duration and 5–30 V intensity) were applied to the L4-6 dorsal root through a suction electrode every 10 sec. The discharge of the corresponding ventral root was recorded with a glass suction electrode, amplified, monitored on an
The Mg21 -free physiological solution contained (in millimolar): NaCl, 116.4; KCl, 5.4; CaCl2, 2.5; NaH2PO4, 0.92; NaHCO3, 26.2 and glucose, 11.0. When bubbled with 95% O2, 5% CO2, the solution had a pH of 7.3. 7-Clkyn, dizocilpine and CPP were synthesized in our laboratories. Other drugs used were as follows: strychnine and APV (Sigma), HA-966 (Res. Biochem. Inc.) and d-serine (Aldrich).
Statistical procedure Reflex activities were expressed as a percentage of the pre-drug control. The IC50 value, which produces 50% inhibition of the reflex activity, for each drug was determined by the Litchfield and Wilcoxon method. Statistical significance of the data was determined by Student’s t-test. A P value of less than 0.05 was considered statistically significant. RESULTS
Effects of 7-Clkyn, HA-966 and other NMDA antagonists on the spinal reflex Stimulation of an L4-6 dorsal root in the absence of Mg21 ions evoked two temporally distinct reflex potentials in the corresponding ventral root (Fig. 1). The potentials with short and long latencies are apparently of monosynaptic and polysynaptic origin, respectively. The mean amplitude and latency of the MSR were 3.5260.16 mV and 6.4360.21 msec, and those for the PSR were 1.2460.09 mV and 11.860.3 msec (N529), respectively. Bath application of 7-Clkyn (3 mM) markedly inhibited the PSR while negligibly affecting the MSR (see Fig. 1). The activity of PSR was depressed in a concentration-dependent manner by 0.3–10.0 mM of 7-Clkyn, but the MSR activity was only slightly reduced (about 25%) at 10 mM (Fig. 2). The IC50 value of 7-Clkyn for the inhibition of PSR was 1.7 mM. Similarly, application of HA-966 at 3–100 mM also produced a selective depression of PSR with an IC50 value of 40 mM (see Fig. 2). The inhibitory effects of 7-Clkyn and
FIGURE 2. Concentration-response curves for the depression of MSR (open circles) and PSR (closed circles) by 7-Clkyn and HA-966 in spinal-cord preparations of neonatal rats in vitro. After the control recordings were obtained, the spinal cord was exposed to either 7-Clkyn or HA-966 until the response reached a plateau. Each point indicates the mean6SEM of three separate experiments.
NMDA Receptors and Spinal Reflex in vitro
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FIGURE 3. Concentration-response curves for the depression of MSR (open circles) and PSR (closed circles) by dizocilpine, CPP and APV in spinal-cord preparations of neonatal rats in vitro. After the control recordings were obtained, the spinal cord was exposed to either dizocilpine, CPP or APV until the response reached a plateau. Each point indicates the mean6SEM of three or four separate experiments.
HA-966 on the PSR usually appeared 1–3 min after the application, reached a plateau at 15–20 min and recovered to the control level in 10–20 min after cessation of the application. On the other hand, the NMDA receptor ion-channel site antagonist dizocilpine and the NMDA-binding site antagonists CPP and APV also showed a preferential depression of the PSR (see Fig. 1). Dizocilpine (0.003–0.1 mM), CPP (0.03–1.0 mM) and APV (0.1–10.0 mM), all reduced the PSR activity in a concentration-dependent manner with an IC50 value of 0.012, 0.15 and 1.2 mM, respectively (Fig. 3). Thus the order of the inhibitory activity of the NMDA antagonists tested was as follows: dizocilpine.CPP.APV57-Clkyn.HA-966. The timecourse of the inhibitory actions of CPP and APV was similar to that of 7-Clkyn or HA-966. The depression of PSR by dizocilpine progressed very gradually with a plateau time of 60–120 min after the application, and its effects persisted for more than 60 min after washout. The magnitude of MSR was only slightly reduced (about 10–30%) by application of either dizocilpine, CPP or APV (see Fig. 3).
mM). Strychnine by itself markedly enhanced the PSR activity by about 135% without affecting the MSR (Fig. 5). However, the simultaneous application of 7-Clkyn and strychnine markedly suppressed the PSR in a concentration-dependent manner (see Fig. 5). The MSR activity was also slightly reduced (about 25%) by high concentrations of 7-Clkyn. DISCUSSION The present study demonstrated that NMDA receptor glycine-binding site antagonists 7-Clkyn and HA-966 produce a selective depression of the spinal PSR in an isolated spinal cord maintained in a Mg21-free medium in vitro. The differential suppression of the PSR was also obtained by the channel-site antagonist dizocilpine and the NMDA-binding site antagonists CPP and APV. The PSR inhibitory activity of these drugs was in the following order: dizocilpine.CPP.APV57-Clkyn.HA-966. In addition, the inhibitory action of 7-Clkyn on PSR could be differentiated from those of the
Effects of D -serine on 7-Clkyn-, dizocilpine- and CPP-induced inhibition of PSR We next examined the effects of d-serine, an agonist for glycinebinding sites of NMDA receptors, on the 7-Clkyn-, dizocilpine- and CPP-induced inhibition of PSR. The spinal cords were first exposed to d-serine (100 mM) for 15 min and then to 7-Clkyn (3 mM), dizocilpine (0.03 mM) or CPP (0.3 mM) in the presence of d-serine (100 mM). Under these conditions, the inhibitory effects of 7-Clkyn were markedly antagonized by the simultaneous application of d-serine (Fig. 4). The magnitude of the PSR was reduced to 1667% of the control by 3 mM 7-Clkyn, but the value significantly increased to 8761% in the presence of d-serine. In contrast, the inhibition of the PSR either by dizocilpine or by CPP was unaffected by the d-serine application (see Fig. 4). Pretreatment of the spinal cord with d-serine (100 mM) alone only slightly increased the magnitude of the PSR, by about 8%, without affecting the MSR activity (data not shown).
Comparison of the effects of strychnine and 7-Clkyn on PSR Because the spinal cord contains a high density of the strychninesensitive glycine receptors (Snyder et al., 1973), we compared the effects of strychnine on the PSR with those of 7-Clkyn. The spinal cords were first exposed to 1 mM of strychnine for 50 to 70 min, and continuously for another 15 min in the presence of 7-Clkyn (1–30
FIGURE 4. Effects of D-serine on the 7-Clkyn-, dizocilpine- and CPP-induced depression of the PSR in spinal cord preparations of neonatal rats in vitro. The spinal cord was exposed first to D-serine at 100 mM for 15 min and then to 7-Clkyn (at 3 mM for 15 min), dizocilpine (at 0.03 mM for 120 min) or CPP (0.3 mM for 15 min) in the presence of D-serine. Each column with vertical bar indicates the mean6SEM of three to six separate experiments. *P,0.01, statistically different from the value without D-serine (Student’s t-test).
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FIGURE 5. Effects of 7-Clkyn on the strychnine-induced enhancement of MSR (open columns) and PSR (closed columns). The spinal cord was exposed first to strychnine at 1 mM for 50–70 min and continuously for another 15 min in the presence of 7-Clkyn. Each column with vertical bar indicates the mean6SEM of three separate experiments.
other types of NMDA antagonists by its response to d-serine, an agonist for the glycine-binding sites of NMDA receptors (Kleckner and Dingledine, 1988); that is, d-serine largely diminished the 7-Clkyn-induced inhibition of PSR without affecting the inhibitory actions of dizocilpine and CPP, indicating the selectivity of the action of 7-Clkyn for the NMDA receptor glycine-binding sites. In addition, both glycine-binding site antagonists, like the channel-site and NMDA-binding site antagonists, could completely block PSR activity. This is in agreement with previous findings that NMDA receptors can be activated only in the presence of glycine (Kleckner and Dingledine, 1988; Lerma et al., 1990). Thus our findings support the idea (Block and Schwartz, 1994; Jahr and Yoshioka, 1986; Ohno and Warnick, 1990; Polc, 1995; Turski et al., 1990) that the segmental PSR is mediated by NMDA receptors and suggest that the glycine-binding sites play a crucial role in activation of NMDA receptors in the spinal cord. In this study, exogenously applied d-serine only slightly increased the PSR activity by itself. Thus our spinal-cord preparation probably contained a sufficient amount of endogenous glycine for the generation of the PSR under the present experimental conditions. In this study, dizocilpine, unlike other types of NMDA receptor antagonists, caused a progressive and long-lasting inhibition of the PSR, and the inhibitory effects of dizocilpine usually required from 60 to 120 min to reach a plateau. These characteristics are consistent with the previous observation in patch-clamp studies (Huettner and Bean, 1988) that dizocilpine exerts its blocking action at NMDA receptors only when the receptors are activated by the agonists. Because the PSR is evoked by temporal activation of NMDA receptors upon electrical stimulation, dizocilpine seemed to exert its inhibitory effects gradually on the transsynaptically activated NMDA receptors. In contrast with the PSR, only a small fraction of the MSR was inhibited by the NMDA antagonists under the Mg21-free condition. The activity of the MSR was reduced to a similar extent (about 10– 30%) by any type of NMDA antagonist examined in this study. These findings are consistent with previous studies (Jahr and Yoshioka, 1986; Turski et al., 1990) that have suggested that the MSR is mediated by non-NMDA receptors. We have previously shown that
Y. Maruoka et al. MSR activity in the presence of Mg21 ions increased by about 20% after Mg21 withdrawal, which could be blocked by PCP analogs or a Mg21 supplement (Ohno and Warnick, 1990). Thus a small part of the MSR activity in the absence of Mg21 may be mediated by NMDA receptors. Glycine also acts as an inhibitory neurotransmitter at strychninesensitive glycine receptors, which exist in the spinal cord with a relatively high density (Snyder et al., 1973). We therefore compared the effects of strychnine on the spinal reflexes with those of 7-Clkyn. The application of strychnine alone markedly potentiated the magnitude of the PSR, suggesting that endogenous glycine negatively regulates the PSR through activating the strychnine-sensitive glycine receptors. However, 7-Clkyn markedly inhibited the PSR activity even in the presence of strychnine. The contrasting effects of 7-Clkyn and strychnine on the PSR exclude the possibility that 7-Clkyn acts on the strychnine-sensitive glycine receptors. In addition, these findings further suggest that the NMDA receptor glycine-binding sites play a role in the generation of the PSR. It is of interest that endogenous glycine seems to exert multiple actions in regulating PSR activity in the spinal cord—that is, as an agonist at the glycine-binding sites of NMDA receptors to generate the PSR and as an inhibitory neurotransmitter at the strychnine-sensitive glycine receptors to negatively regulate the PSR. However, the precise mechanism and the origin of glycine for the activation of NMDA receptors are still uncertain. Endogenous substances other than glycine also may act as agonists at the NMDA receptor glycine-binding sites, because recent studies have shown that the brain tissue contains a significant amount of endogenous d-serine (Hashimoto et al., 1992; Nagata et al., 1994; Schell et al., 1995). In summary, the NMDA receptor glycine-binding site antagonists 7-Clkyn and HA-966 produced a selective depression of PSR while negligibly affecting the activity of MSR. The PSR was also differentially suppressed by the channel-site (i.e., dizocilpine) and the NMDA-binding–site (i.e., CPP and APV) antagonists. The PSR inhibition by 7-Clkyn, but not by dizocilpine and CPP, was markedly antagonized by the simultaneous application of d-serine, an agonist for the glycine-binding sites of NMDA receptors, and persisted in the presence of strychnine. These findings suggest that the NMDA receptor glycine-binding sites play a role in the generation of the NMDA receptor-mediated PSR in the spinal cord in vitro. Our results, however, do not rule out the possibility that other receptor systems (e.g., non-NMDA receptors) also are involved in the generation of PSR (Farkas and Ono, 1995). Further studies are required to define the role of the NMDA receptor glycine-binding sites in the spinal cord. References Block F. and Schwarz M. (1994) The depressant effect of GYKI 52466 on spinal reflex transmission in rats is mediated via non-NMDA and benzodiazepine receptors. Eur. J. Pharmac. 256, 149–153. Davies J., Evans R. H., Herrling P. L., Jones A. W., Olverman H. J., Pook P. and Watkins J. C. (1986) CPP, a new potent and selective NMDA antagonist: depression of central neuron responses, affinity for [3H]d-AP5 binding sites on brain membranes and anticonvulsant activity. Brain Res. 382, 169–173. Farkas S. and Ono H. (1995) Participation of NMDA and non-NMDA excitatory amino acid receptors in the mediation of spinal reflex potentials in rats: an in vivo study. Br. J. Pharmac. 114, 1193–1205. Foster A. C. and Kemp J. A. (1989) HA-966 antagonizes N-methyl-d-aspartate receptors through a selective interaction with the glycine modulatory site. J. Neurosci. 9, 2191–2196. Hashimoto A., Nishikawa T., Hayashi T., Fujii N., Harada K., Oka T. and Takahashi K. (1992) The presence of free d-serine in rat brain. FEBS Lett. 296, 33–36. Huettner J. E. and Bean B. P. (1988) Block of N-methyl-d-aspartate-acti-
NMDA Receptors and Spinal Reflex in vitro vated current by the anticonvulsant MK-801: selective binding to open channels. Proc. Natl. Acad. Sci. USA 85, 1307–1311. Jahr C. E. and Yoshioka K. (1986) Ia afferent excitation of motoneurones in the in vitro new-born rat spinal cord is selectively antagonized by kynurenate. J. Physiol. 370, 515–530. Kemp J. A., Foster A. C., Leeson P. D., Priestley T., Tridgett R., Iversen L. L. and Woodruff G. N. (1988) 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, 6547–6550. Kemp J. A., Foster A. C. and Wong E. H. F. (1987) Non-competitive antagonists of excitatory amino acid receptors. Trends Neurosci. 10, 294–298. Kleckner N. W. and Dingledine R. (1988) Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes. Science 241, 835–837. Lehmann J., Schneider J., McPherson S., Murphy D. E., Bernard P., Tsai C., Bennett D. A., Pastor G., Steel D. J., Boehm C, Cheney D. L., Liebman J. M., Williams M. and Wood P. L. (1987) CPP, a selective N-methyld-aspartate (NMDA)-type receptor antagonist: characterization in vitro and in vivo. J. Pharmac. Exp. Ther. 240, 737–746. Lerma J., Zukin R. S. and Bennett M. V. L. (1990) Glycine decreases desensitization of N-methyl-d-aspartate (NMDA) receptors expressed in Xenopus oocytes and is required for NMDA responses. Proc. Natl. Acad. Sci. USA 87, 2354–2358. Meguro H., Mori H., Araki K., Kushiya E., Kutsuwada T., Yamazaki M., Kumanishi T., Arakawa M., Sakimura K. and Mishina M. (1992) Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs. Nature 357, 70–74. Meldrum B. S. (1994) The role of glutamate in epilepsy and other CNS disorders. Neurology 44(Suppl. 8), S14–S23. Monyer H., Sprengel R., Schoepfer R., Herb A., Higuchi M., Lomeli H., Burnashev N., Sakmann B. and Seeburg P. H. (1992) Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 256, 1217–1221.
649 Muir K. W. and Lees K. R. (1995) Clinical experience with excitatory amino acid antagonist drugs. Stroke 26, 503–513. Nagata Y., Horiike K. and Maeda T. (1994) Distribution of free d-serine in vertebrate brains. Brain Res. 634, 291–295. Ohno Y. and Warnick J. E. (1988) Effects of thyrotropin-releasing hormone on phencyclidine- and ketamine-induced spinal depression in the neonatal rats. Neuropharmacology 27, 1013–1018. Ohno Y. and Warnick J. E. (1989) Presynaptic activation of the spinal serotonergic system in the rat by phencyclidine in vitro. J. Pharmac. Exp. Ther. 250, 177–183. Ohno Y. and Warnick J. E. (1990) Selective depression of the segmental polysynaptic reflex by phencyclidine and its analogs in the rat in vitro: interaction with N-methyl-d-aspartate receptors. J. Pharmac. Exp. Ther. 252, 246–252. Polc P. (1985) 2-Amino-7-phosphonoheptanoic acid depresses g-motoneurons and polysynaptic reflex in the cat spinal cord. Eur. J. Pharmac. 117, 387–389. Schell M. J., Molliver M. E. and Snyder S. H. (1995) d-Serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release. Proc. Natl. Acad. Sci. USA 92, 3948–3952. Snyder S. H., Young A. B., Bennett J. P. and Mulder A. H. (1973) Synaptic biochemistry of amino acids. Fed. Proc. 32, 2039–2047. Turski L., Bressler K., Klockgether T. and Stephens D. N. (1990) Differential effects of the excitatory amino acid antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 3-((6)-2-carboxypiperazin- 4-yl)propyl-1-phophonic acid (CPP), on spinal reflex activity in mice. Neurosci. Lett. 113, 66–71. Wong E. H. F., Kemp J. A., Priestley T, Knight A. R., Woodruff G. N. and Iversen L. L. (1986) The anticonvulsant MK-801 is a potent N-methyld-aspartate antagonist. Proc. Natl. Acad. Sci. USA 83, 7104–7108. Yoneda Y. and Ogita K. (1991) Neurochemical aspects of N-methyl-d-aspartate receptor complex. Neurosci. Res. 10, 1–33.
ISSN 0306-3623/97 $17.00 1 .00 PII S0306-3623(96)00514-9 All rights reserved
Selective Depression of the Spinal Polysynaptic Reflex by the NMDA Receptor Antagonists in an Isolated Spinal Cord in vitro Yoshimi Maruoka, Yukihiro Ohno,* Hiroyasu Tanaka, Hirokazu Yasuda, Ken-Ichi Ohtani, Hideki Sakamoto, Akiko Kawabe, Chika Tamamura and Mitsutaka Nakamura Discovery Research Laboratories II, Research Center, Sumitomo Pharmaceuticals Co., Ltd., 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554, Japan [Tel: (81) 6 466 5215; Fax: (81) 6 466 5218] ABSTRACT. 1. The effects of N-methyl-D-aspartate (NMDA) receptor glycine-binding site antagonists 7-chlorokynurenate (7-Clkyn) and (6)-3-amino-1-hydroxy-2-pyrrolidone (HA-966) on spinal reflexes in an isolated spinal cord that was maintained in Mg21-free medium in vitro were examined. The actions of 7-Clkyn and HA-966 were compared with those of the channel-site antagonist (i.e., dizocilpine) and NMDA-binding site antagonists—that is, 3-[(6)-2-carboxypiperazin-4-yl]-propyl-1phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV). 2. 7-Clkyn and HA-966 produced a selective depression of the polysynaptic reflex (PSR) while negligibly affecting the activity of the monosynaptic reflex (MSR). The PSR was also differentially suppressed by dizocilpine, CPP and APV. The PSR inhibitory activity of the NMDA antagonists was in the following order: dizocilpine.CPP.APV57-Clkyn.HA-966. 3. The inhibitory effects of 7-Clkyn on PSR were markedly antagonized by the simultaneous application of D-serine, an agonist for the NMDA receptor glycine-binding sites. However, PSR inhibition by dizocilpine and CPP was unaffected. 4. Inhibition of the PSR by 7-Clkyn persisted in the presence of strychnine, which markedly increased the PSR activity by itself. 5. These findings suggest that the NMDA receptor glycine-binding sites play a role in generating the NMDA receptor-mediated PSR in the spinal cord in vitro. gen pharmac 29;4:645–649, 1997. 1997 Elsevier Science Inc. KEY WORDS. NMDA receptor, glycine-binding site, 7-chlorokynurenate, HA-966, d-serine, spinal reflex, isolated spinal cord INTRODUCTION N-Methyl-d-aspartate (NMDA) receptors mediate the excitatory neurotransmission that has been implicated in various pathophysiological conditions including epilepsy, pain processing and hypoxicischemic brain injury (Meldrum, 1994; Muir and Lees, 1995; Yoneda and Ogita, 1991). The NMDA receptors are considered to be heteromeric assemblies consisting of a certain combination of the NMDA receptor subunits (e.g., NMDAR1 and NMDAR2A–D), which form the Ca21 -permeable ion-channel complex (Meguro et al., 1992; Monyer et al., 1992). The NMDA receptors also contain strychnine-insensitive glycine-binding sites, where endogenous glycine seems to act as agonist to activate the receptor function (Kemp et al., 1988; Kleckner and Dingledine, 1988). To date, several antagonists specific for the multiple regulatory sites of the NMDA receptors have been developed. Dizocilpine [(1)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801)] and phencyclidine (PCP) are known to bind to the ion-channel sites (Kemp et al., 1987; Wong et al., 1986), dl-2-amino-5-phosphono valerate (APV) and 3-[(6)-2-carboxypiperazin-4-yl]propyl1-phosphonate (CPP) are the antagonists for the NMDA-binding *To whom all correspondence should be addressed. Received 4 June 1996; revised 24 September 1996; accepted 18 October 1996.
sites (Davies et al., 1986; Lehmann et al., 1987), and 7-chlorokynurenate (7-Clkyn) and (6)-3-amino-1-hydroxy-2-pyrrolidone (HA966) are the antagonists for the glycine-binding sites at the NMDA receptor/ion-channel complex (Foster and Kemp, 1989; Kemp et al., 1988). Although the actions of these agents have been widely studied, the role of the respective regulatory site in the NMDA receptor functions still remains to be defined. In previous studies, Ohno and Warnick (1988, 1989, 1990) showed that PCP analogs [e.g., PCP, thienylcyclohexylpiperizine (TCP), m-amino PCP and m-nitro PCP] inhibited the spinal dorsal root–ventral root reflexes through two distinct mechanisms in isolated spinal cord preparations in vitro. These agents, at low concentrations (nanomolar range), selectively suppressed the Mg21 -sensitive polysynaptic reflex (PSR) by blocking the NMDA receptors, whereas higher concentrations (micromolar range) inhibited the monosynaptic reflex (MSR) by activating the spinal serotonergic system (Ohno and Warnick, 1989; 1990). These findings, consistent with previous in vivo studies (Block and Schwarz, 1994; Polc, 1985; Turski et al., 1990), suggested that the NMDA receptors take part in the generation of a segmental PSR in the spinal cord. To explore the role of the glycine-binding sites at the NMDA receptors in modulating the Mg21 -sensitive PSR in vitro, we extended the evaluation to the selective glycine-binding site antagonists 7-Clkyn and HA-966 and compared their actions with those of the ion-
646
Y. Maruoka et al. oscilloscope and stored on FM tapes. Acquired responses were signal averaged with on-line computer assistance (ATAC-3300, Nihon Koden) and analyzed for amplitude, area and latency. The mean value of area of the reflex activities was obtained from ten successive responses in each experiment before the drug application and after the effects of the test drug reached a plateau.
Solutions and drugs
FIGURE 1. Effects of 7-Clkyn, dizocilpine and CPP on monosynaptic and polysynaptic reflexes in spinal-cord preparations of neonatal rats in vitro. The spinal cords were superfused with a Mg21-free physiological solution throughout the experiment. After the control recordings were obtained, the spinal cord was exposed to either 7-Clkyn (3 mM), dizocilpine (0.1 mM) or CPP (0.3 mM) until the response reached a plateau. The traces are signal-averaged records of ten successive responses before and after the drug application (at 15 min for 7-Clkyn and CPP and at 120 min for dizocilpine). The traces in each column were obtained in the same preparation. The calibrations are 2 mV and 5 msec.
channel site (i.e., dizocilpine) and the NMDA-binding site (i.e., CPP and APV) antagonists. MATERIALS AND METHODS
Preparation of spinal cord and recording of reflex activity The isolation of the spinal cord and recording of the ventral root potentials were performed as described previously (Ohno and Warnick, 1990). Briefly, the spinal cords were dissected from male Sprague-Dawley rats (6–9 days old) under ether anesthesia, hemisected longitudinally and placed in a recording chamber, where they were superfused with Mg21-free physiological solution at a flow rate of 1.5–2.5 ml/min. The superfusion medium was bubbled continuously with 95% O2, 5% CO2, and the temperature was kept at 25.060.58C. Test stimuli (0.15-msec duration and 5–30 V intensity) were applied to the L4-6 dorsal root through a suction electrode every 10 sec. The discharge of the corresponding ventral root was recorded with a glass suction electrode, amplified, monitored on an
The Mg21 -free physiological solution contained (in millimolar): NaCl, 116.4; KCl, 5.4; CaCl2, 2.5; NaH2PO4, 0.92; NaHCO3, 26.2 and glucose, 11.0. When bubbled with 95% O2, 5% CO2, the solution had a pH of 7.3. 7-Clkyn, dizocilpine and CPP were synthesized in our laboratories. Other drugs used were as follows: strychnine and APV (Sigma), HA-966 (Res. Biochem. Inc.) and d-serine (Aldrich).
Statistical procedure Reflex activities were expressed as a percentage of the pre-drug control. The IC50 value, which produces 50% inhibition of the reflex activity, for each drug was determined by the Litchfield and Wilcoxon method. Statistical significance of the data was determined by Student’s t-test. A P value of less than 0.05 was considered statistically significant. RESULTS
Effects of 7-Clkyn, HA-966 and other NMDA antagonists on the spinal reflex Stimulation of an L4-6 dorsal root in the absence of Mg21 ions evoked two temporally distinct reflex potentials in the corresponding ventral root (Fig. 1). The potentials with short and long latencies are apparently of monosynaptic and polysynaptic origin, respectively. The mean amplitude and latency of the MSR were 3.5260.16 mV and 6.4360.21 msec, and those for the PSR were 1.2460.09 mV and 11.860.3 msec (N529), respectively. Bath application of 7-Clkyn (3 mM) markedly inhibited the PSR while negligibly affecting the MSR (see Fig. 1). The activity of PSR was depressed in a concentration-dependent manner by 0.3–10.0 mM of 7-Clkyn, but the MSR activity was only slightly reduced (about 25%) at 10 mM (Fig. 2). The IC50 value of 7-Clkyn for the inhibition of PSR was 1.7 mM. Similarly, application of HA-966 at 3–100 mM also produced a selective depression of PSR with an IC50 value of 40 mM (see Fig. 2). The inhibitory effects of 7-Clkyn and
FIGURE 2. Concentration-response curves for the depression of MSR (open circles) and PSR (closed circles) by 7-Clkyn and HA-966 in spinal-cord preparations of neonatal rats in vitro. After the control recordings were obtained, the spinal cord was exposed to either 7-Clkyn or HA-966 until the response reached a plateau. Each point indicates the mean6SEM of three separate experiments.
NMDA Receptors and Spinal Reflex in vitro
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FIGURE 3. Concentration-response curves for the depression of MSR (open circles) and PSR (closed circles) by dizocilpine, CPP and APV in spinal-cord preparations of neonatal rats in vitro. After the control recordings were obtained, the spinal cord was exposed to either dizocilpine, CPP or APV until the response reached a plateau. Each point indicates the mean6SEM of three or four separate experiments.
HA-966 on the PSR usually appeared 1–3 min after the application, reached a plateau at 15–20 min and recovered to the control level in 10–20 min after cessation of the application. On the other hand, the NMDA receptor ion-channel site antagonist dizocilpine and the NMDA-binding site antagonists CPP and APV also showed a preferential depression of the PSR (see Fig. 1). Dizocilpine (0.003–0.1 mM), CPP (0.03–1.0 mM) and APV (0.1–10.0 mM), all reduced the PSR activity in a concentration-dependent manner with an IC50 value of 0.012, 0.15 and 1.2 mM, respectively (Fig. 3). Thus the order of the inhibitory activity of the NMDA antagonists tested was as follows: dizocilpine.CPP.APV57-Clkyn.HA-966. The timecourse of the inhibitory actions of CPP and APV was similar to that of 7-Clkyn or HA-966. The depression of PSR by dizocilpine progressed very gradually with a plateau time of 60–120 min after the application, and its effects persisted for more than 60 min after washout. The magnitude of MSR was only slightly reduced (about 10–30%) by application of either dizocilpine, CPP or APV (see Fig. 3).
mM). Strychnine by itself markedly enhanced the PSR activity by about 135% without affecting the MSR (Fig. 5). However, the simultaneous application of 7-Clkyn and strychnine markedly suppressed the PSR in a concentration-dependent manner (see Fig. 5). The MSR activity was also slightly reduced (about 25%) by high concentrations of 7-Clkyn. DISCUSSION The present study demonstrated that NMDA receptor glycine-binding site antagonists 7-Clkyn and HA-966 produce a selective depression of the spinal PSR in an isolated spinal cord maintained in a Mg21-free medium in vitro. The differential suppression of the PSR was also obtained by the channel-site antagonist dizocilpine and the NMDA-binding site antagonists CPP and APV. The PSR inhibitory activity of these drugs was in the following order: dizocilpine.CPP.APV57-Clkyn.HA-966. In addition, the inhibitory action of 7-Clkyn on PSR could be differentiated from those of the
Effects of D -serine on 7-Clkyn-, dizocilpine- and CPP-induced inhibition of PSR We next examined the effects of d-serine, an agonist for glycinebinding sites of NMDA receptors, on the 7-Clkyn-, dizocilpine- and CPP-induced inhibition of PSR. The spinal cords were first exposed to d-serine (100 mM) for 15 min and then to 7-Clkyn (3 mM), dizocilpine (0.03 mM) or CPP (0.3 mM) in the presence of d-serine (100 mM). Under these conditions, the inhibitory effects of 7-Clkyn were markedly antagonized by the simultaneous application of d-serine (Fig. 4). The magnitude of the PSR was reduced to 1667% of the control by 3 mM 7-Clkyn, but the value significantly increased to 8761% in the presence of d-serine. In contrast, the inhibition of the PSR either by dizocilpine or by CPP was unaffected by the d-serine application (see Fig. 4). Pretreatment of the spinal cord with d-serine (100 mM) alone only slightly increased the magnitude of the PSR, by about 8%, without affecting the MSR activity (data not shown).
Comparison of the effects of strychnine and 7-Clkyn on PSR Because the spinal cord contains a high density of the strychninesensitive glycine receptors (Snyder et al., 1973), we compared the effects of strychnine on the PSR with those of 7-Clkyn. The spinal cords were first exposed to 1 mM of strychnine for 50 to 70 min, and continuously for another 15 min in the presence of 7-Clkyn (1–30
FIGURE 4. Effects of D-serine on the 7-Clkyn-, dizocilpine- and CPP-induced depression of the PSR in spinal cord preparations of neonatal rats in vitro. The spinal cord was exposed first to D-serine at 100 mM for 15 min and then to 7-Clkyn (at 3 mM for 15 min), dizocilpine (at 0.03 mM for 120 min) or CPP (0.3 mM for 15 min) in the presence of D-serine. Each column with vertical bar indicates the mean6SEM of three to six separate experiments. *P,0.01, statistically different from the value without D-serine (Student’s t-test).
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FIGURE 5. Effects of 7-Clkyn on the strychnine-induced enhancement of MSR (open columns) and PSR (closed columns). The spinal cord was exposed first to strychnine at 1 mM for 50–70 min and continuously for another 15 min in the presence of 7-Clkyn. Each column with vertical bar indicates the mean6SEM of three separate experiments.
other types of NMDA antagonists by its response to d-serine, an agonist for the glycine-binding sites of NMDA receptors (Kleckner and Dingledine, 1988); that is, d-serine largely diminished the 7-Clkyn-induced inhibition of PSR without affecting the inhibitory actions of dizocilpine and CPP, indicating the selectivity of the action of 7-Clkyn for the NMDA receptor glycine-binding sites. In addition, both glycine-binding site antagonists, like the channel-site and NMDA-binding site antagonists, could completely block PSR activity. This is in agreement with previous findings that NMDA receptors can be activated only in the presence of glycine (Kleckner and Dingledine, 1988; Lerma et al., 1990). Thus our findings support the idea (Block and Schwartz, 1994; Jahr and Yoshioka, 1986; Ohno and Warnick, 1990; Polc, 1995; Turski et al., 1990) that the segmental PSR is mediated by NMDA receptors and suggest that the glycine-binding sites play a crucial role in activation of NMDA receptors in the spinal cord. In this study, exogenously applied d-serine only slightly increased the PSR activity by itself. Thus our spinal-cord preparation probably contained a sufficient amount of endogenous glycine for the generation of the PSR under the present experimental conditions. In this study, dizocilpine, unlike other types of NMDA receptor antagonists, caused a progressive and long-lasting inhibition of the PSR, and the inhibitory effects of dizocilpine usually required from 60 to 120 min to reach a plateau. These characteristics are consistent with the previous observation in patch-clamp studies (Huettner and Bean, 1988) that dizocilpine exerts its blocking action at NMDA receptors only when the receptors are activated by the agonists. Because the PSR is evoked by temporal activation of NMDA receptors upon electrical stimulation, dizocilpine seemed to exert its inhibitory effects gradually on the transsynaptically activated NMDA receptors. In contrast with the PSR, only a small fraction of the MSR was inhibited by the NMDA antagonists under the Mg21-free condition. The activity of the MSR was reduced to a similar extent (about 10– 30%) by any type of NMDA antagonist examined in this study. These findings are consistent with previous studies (Jahr and Yoshioka, 1986; Turski et al., 1990) that have suggested that the MSR is mediated by non-NMDA receptors. We have previously shown that
Y. Maruoka et al. MSR activity in the presence of Mg21 ions increased by about 20% after Mg21 withdrawal, which could be blocked by PCP analogs or a Mg21 supplement (Ohno and Warnick, 1990). Thus a small part of the MSR activity in the absence of Mg21 may be mediated by NMDA receptors. Glycine also acts as an inhibitory neurotransmitter at strychninesensitive glycine receptors, which exist in the spinal cord with a relatively high density (Snyder et al., 1973). We therefore compared the effects of strychnine on the spinal reflexes with those of 7-Clkyn. The application of strychnine alone markedly potentiated the magnitude of the PSR, suggesting that endogenous glycine negatively regulates the PSR through activating the strychnine-sensitive glycine receptors. However, 7-Clkyn markedly inhibited the PSR activity even in the presence of strychnine. The contrasting effects of 7-Clkyn and strychnine on the PSR exclude the possibility that 7-Clkyn acts on the strychnine-sensitive glycine receptors. In addition, these findings further suggest that the NMDA receptor glycine-binding sites play a role in the generation of the PSR. It is of interest that endogenous glycine seems to exert multiple actions in regulating PSR activity in the spinal cord—that is, as an agonist at the glycine-binding sites of NMDA receptors to generate the PSR and as an inhibitory neurotransmitter at the strychnine-sensitive glycine receptors to negatively regulate the PSR. However, the precise mechanism and the origin of glycine for the activation of NMDA receptors are still uncertain. Endogenous substances other than glycine also may act as agonists at the NMDA receptor glycine-binding sites, because recent studies have shown that the brain tissue contains a significant amount of endogenous d-serine (Hashimoto et al., 1992; Nagata et al., 1994; Schell et al., 1995). In summary, the NMDA receptor glycine-binding site antagonists 7-Clkyn and HA-966 produced a selective depression of PSR while negligibly affecting the activity of MSR. The PSR was also differentially suppressed by the channel-site (i.e., dizocilpine) and the NMDA-binding–site (i.e., CPP and APV) antagonists. The PSR inhibition by 7-Clkyn, but not by dizocilpine and CPP, was markedly antagonized by the simultaneous application of d-serine, an agonist for the glycine-binding sites of NMDA receptors, and persisted in the presence of strychnine. These findings suggest that the NMDA receptor glycine-binding sites play a role in the generation of the NMDA receptor-mediated PSR in the spinal cord in vitro. Our results, however, do not rule out the possibility that other receptor systems (e.g., non-NMDA receptors) also are involved in the generation of PSR (Farkas and Ono, 1995). Further studies are required to define the role of the NMDA receptor glycine-binding sites in the spinal cord. References Block F. and Schwarz M. (1994) The depressant effect of GYKI 52466 on spinal reflex transmission in rats is mediated via non-NMDA and benzodiazepine receptors. Eur. J. Pharmac. 256, 149–153. Davies J., Evans R. H., Herrling P. L., Jones A. W., Olverman H. J., Pook P. and Watkins J. C. (1986) CPP, a new potent and selective NMDA antagonist: depression of central neuron responses, affinity for [3H]d-AP5 binding sites on brain membranes and anticonvulsant activity. Brain Res. 382, 169–173. Farkas S. and Ono H. (1995) Participation of NMDA and non-NMDA excitatory amino acid receptors in the mediation of spinal reflex potentials in rats: an in vivo study. Br. J. Pharmac. 114, 1193–1205. Foster A. C. and Kemp J. A. (1989) HA-966 antagonizes N-methyl-d-aspartate receptors through a selective interaction with the glycine modulatory site. J. Neurosci. 9, 2191–2196. Hashimoto A., Nishikawa T., Hayashi T., Fujii N., Harada K., Oka T. and Takahashi K. (1992) The presence of free d-serine in rat brain. FEBS Lett. 296, 33–36. Huettner J. E. and Bean B. P. (1988) Block of N-methyl-d-aspartate-acti-
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