Neuroseienee Letters, 143 (1992) 177 180 177 ~', 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

NSL 08885

Quisqualic acid-induced neurotoxicity is protected by NMDA and non-NMDA receptor antagonists

Karni re Sadashiv Pai and Vijayalakshmi R a v i n d r a n a t h

Department q/'Neurochemistr T, National lnstitule { { l l Mental Hetlllh and Neuroscietwes, Ban galore : Italia:

(Received 20 May 1992: Accepted 25 May 1992)

Key words." Quisqualic acid: NMDA receptor: MK-801: Brain slice: Quinoxalinedione

Quisqualic acid-mediated excitotoxicity has been attributed essentially to the activation of non-N-lnethyl-i>-aspartatc (non-NMI)A) rcccptors. In the present study we demonstrate the possible involvement of both NMDA and non-NMDA receptors in quisqualic acid-induced toxicity in mouse brain slices, in vitro. Incubation of mouse brain sagittal slices with various concentrations of quisqualic acid resulted in significant increase in the leakage of lactate dehydrogenase and potassium from the slices into the medium. Prior incubation of mouse brain slices with N M DA (M K-S01 or AP7) or non-NMDA receptor antagonists (GDEE or quinoxalinediones) protected against quisqualic acid-mediated toxicity. Slices prcpared from animals pretreated in vivo with MK-801 (5 mg/kg b.wt.) were also resistant to the toxic effects of quisqualic acid, indicating the possible involvement of NMDA receptors in quisqualic acid toxicity.

The excitotoxic effect of glutamic acid has been attri- buted to excessive stimulation of glutamate receptors [16]. Various glutamate receptors have been classified on the basis of the preferred agonists, namely N-methyl-D- aspartate (NMDA), kainate or ~-amino-3-hydroxy-5- methylisoxazole-4-propionate (AMPA)/quisqualate [4, 17]. Quisqualic acid is known to induce neuronal damage via non-NMDA receptors and non-NMDA receptor an- tagonists offer protection against quisqualic acid-in- duced toxicity [10]. Autoradiographic studies have shown that quisqualic acid has weak affinity for NMDA receptors [5]. Electrophysiological studies have also den> onstrated the activation of NMDA receptor channels by quisqualic acid [7]. The present study was carried out to determine the effects of NMDA and non-NMDA recep- tor antagonists on quisqualic acid-induced toxicity, using sagittal slices of mouse brain, in vitro.

Swiss Albino mice (2-3 months old) obtained from Central Animal Research Facility of the Institute were used for all experiments. Animals had free access to pe- letted diet (Lipton India Ltd., Calcutta, India) and water ad libitum. Mice were sacrificed and sagittal slices of brain were prepared using an indigenous slicer as de- scribed [14]. Slices were incubated in artificial cerebrospi-

Correspondence: V. Ravindranath. Department of Neurochemistry, NIMHANS, Hosur Road, Bangalore 560029, India. Fax: (91)(812) 643130.

nal fluid (ACSF) [14] with various concentrations of quisqualic acid for 1 h at 37°C in an atmosphere of oxy- gen. Slices incubated in ACSF alone served as controls. Following incubation, leakage of the cytosolic enzyme lactate dehydrogenase and potassium (from the slice into the medium) was monitored as a measure of cell death [13]. Brain slices were preincubated with NMDA recep- tor antagonists (namely, 5-methyl-10,1 l-dehydro-5H- benzo(cc6)cyclohepten-5-10-imine maleate (MK-801, 100 nM), 2-amino-7-phosphono-heptanoic acid (AP7. 100#M) or non-NMDA receptor antagonists (glutamate diethyl ester (GDEE, 50/aM) or 6,7-dinitroquinoxaline- 2,3-dione (DNQX, 1 /aM)) for 30 min at 37°C. Quis- qualic acid (1 or 100 nM) was added to the incubation medium and the incubations were continued for 1 h. 6- Cyano-7-nitroquinoxaline-2,3-dione (CNQX, 30 mg/kg b.wt.), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)qui- noxaline (NBQX, 30 mg/kg b.wt.) or MK-801 (5 mg/kg b.wt.) were injected subcutaneously to mice and the ani- mals were sacrificed 4 h later. Brain slices were prepared from the treated animals and incubated with and without quisqualic acid (1 nM) for 1 h at 37°C. LDH activity and potassium concentration were measured in the medium following incubation. Statistical analysis was carried out using Student's t-test and one-way analysis of variance (ANOVA) with Duncan's test where appropriate.

Incubation of brain slices with various concentrations ofquisqualic acid (1 pM, 1 nM, 100 nM and 1 /aM) re-

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Fig. 1. Effect of quisqualic acid on lactate dehydrogenase (LDH) and potassium leakage from the slice into the medium. Mouse brain slices were incubated with various concentrations of quisqualic acid (1 pM to 1 ,aM) for 1 h at 37°C. LDH (A) and potassium (B) leakage were monitored in the

medium following incubation. Values are mean _+ S.D. (n=6 8). Asterisks represent values significantly different from controls, P<0.05.

suited in an increase in the leakage of lactate dehydroge- nase (LDH) and potassium from the slice into the me- dium (Fig. 1). Significant leakage was observed when slices were incubated with 1 nM quisqualic acid.

Preincubation of mouse brain slices with NMDA re- ceptor antagonists, MK-801 or APT protected the slices against quisqualic acid-induced leakage of LDH and potassium from the slice into the medium. Non-NMDA receptor antagonists GDEE or DNQX also protected the slices against quisqualic acid-induced toxicity in vitro. Brain slices prepared from mice pretreated with CNQX or NBQX were resistant to quisqualic acid-in- duced toxicity (Fig. 2). No protection was seen when the quinoxalinediones (CNQX or NBQX) were added in vitro to the slices (data not shown).

Mice were injected with MK-801, subcutaneously, and sacrificed 4 h later. Brain slices prepared from MK-801- treated animals were incubated with and without quis- qualic acid for 1 h at 37°C. Prior administration of MK- 801, in vivo, completely ameliorated quisqualic acid-in- duced LDH and potassium leakage from the slices into the medium (Fig. 2).

Quisqualic acid (1 nM) caused significant leakage of LDH and potassium from the slices into the medium. However, LDH and potassium leakage did not increase in a dose-dependent manner with the increase in quis- qualic acid concentration in incubation medium (Fig. 1). This may be due to the selective vulnerability of specific neuronal cell populations to the toxic effects of excita-

tory amino acids [11]. Measurement of LDH and potas- sium leakage has been shown to be a sensitive index of monitoring excitatory amino acid mediated neuronal damage [9, 13].

Quisqualic acid-induced toxocity is abolished by non- specific non-NMDA receptor antagonist, namely GDEE (Fig. 2). Quinoxalinediones [8], namely DNQX and CNQX, also protected slices against quisqualic acid-me- diated LDH and potassium leakage, when added to the incubation medium in vitro or administered in vwo. re- spectively (Fig. 2). Quinoxalinediones (DNQX and CNQX) have also been shown to act as NMDA antago- nist by displacing glycine from modulatory site on NMDA receptor complex [2]. However. DNQX at 1 ~M concentration (used in the present study) is unlikely to act via NMDA receptors since earlier in vitro studies have shown that quinoxalinediones are effective in blocking NMDA responses only when used in higher concentration (10 ~M or higher) [2]. In the present study, the highly selective AMPA receptor antagonist NBQX [15] completely abolished quisqualic acid-induced toxic- ity. NBQX has been shown to have very low affinity for kainate receptor and does not act on the glycine site of NMDA receptor. Administration of NBQX in vivo to the animals has been shown to protect against ischemic injury by acting via non-NMDA receptors [15].

Selective NMDA receptor antagonists [17], namely MK-801 and APT, protected the slices against quisqualic acid-mediated toxicity when added in vitro (Fig. 2). Ex-

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Fig. 2. Effect of glutamate receptor antagonists on quisqualic acid-induced LDH and potassium leakage. Mouse brain slices were incubated with glutamate receptor antagonists namely, MK-801 (100 nM), AP7 (100 p M I, GDEE (50 #M) or DNQX (1 #M) for 30 rain at 37°(7. Quisqualic acid (QA, 1 or 100 nM) was then added to the incubation medium and the incubation was continued for 1 h. CNQX (30 mg/kg b.wt.), NBQX (30 mg/kg b.wt.) or MK801" (5 mg/kg b.wt.) were injected subcutaneously to mice and the animals were sacrificed 4 h later. Brain slices were prepared from treated animals and incubated with and without quisqualic acid (QA, 1 nM) for 1 h at 37°C. LDH activity and potassium concentration were measured in the medium at the end of incubation period. Values are mean ?- S.D. (n-4 6). The open bars ([::3) represent LDH activity while bars with transverse lines (k~) represent potassium concentration. The values are expressed as percent of control incubations, which did not contain any

quisqualic acid. Asterisks represent values which differed significantly from corresponding controls (P<0.05).

periments were carried out to determine if similar effects were observed when MK-801 was administered in vivo. The slices prepared from MK-801-pretreated animals were resistant to quisqualic acid toxicity. LDH activity and potassium concentration in the medium were similar in both quisqualic acid-treated and untreated control slices (Fig. 2).

Quisqualic acid displaces radiolabelled glutamate, NMDA and non-NMDA antagonists from their respec- tive binding sites [5, 12]. In the rat central nervous sys- tem, quisqualic acid binds to two distinct high- and low- affinity sites. The binding of quisqualate to NMDA re- ceptor may be mediated by a low-affinity binding site [5, 6]. However, in the present study the NMDA antagonists MK-801 and AP7 protected the slices from quisqualic acid-induced toxicity when both high (100 nM) and low (1 nM) concentrations of quisqualic acid were added to the medium (Fig. 2).

Quisqualic acid activates channel openings with con- ductances of 8 15 pS in size. In addition, quisqualic acid produces channel openings with conductances similar in size (40 50 pS) to those activated by NMDA agonists [1,

7]. NMDA antagonists have been shown to prevent quis- qualic acid-mediated 40-50 pS single-channel events [7] indicating the ability of quisqualic acid to open NMDA channels following binding to NMDA receptors. NMDA receptor-mediated toxicity has been shown to be protected by substitution of choline for sodium in the extracellular medium [3]. Similar manipulation of ex- tracellular medium also blocked quisqualic acid-medi- ated neuronal swellings [10] indicating that the toxic ac- tion of quisqualic acid may be mediated by NMDA re- ceptors.

Earlier studies have indicated that quisqualic acid could presumptively act via NMDA receptor in addition to mediating excitotoxic events via the quisqualic acid/ AMPA receptor. The direct evidence for the above, via study of the effects of variety of receptor specific antago- nists has not been demonstrated before. It is conceivable that the interaction of quisqualic acid at the non-NMDA receptor could possibly lead to enhanced release of gluta- mate from the terminal, which could then act on NMDA receptor. Thus, NMDA receptor may not be the primary site of action of quisqualic acid, but could be a secondary

180

site of action. Nevertheless, the present study provides evidence that both N M D A and non-NMDA receptor antagonists are protectors of quisqualic acid-induced toxicity, in vitro.

We thank Novo Nordisk, Denmark, for the kind gift of NBQX. K.S.P. also thanks the CSIR, Government of India, for the award of a Senior Research Fellowship.

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