Idebenone attenuates neuronal degeneration induced by intrastriatal injection of excitotoxins

EXPERIMENTAL

NEUROLOGY

108,38-45

(1990)

ldebenone Attenuates Neuronal Degeneration Induced by lntrastriatal Injection of Excitotoxins MASAOMI

MIYAMOTO

AND JOSEPH T. COYLE

Departments of Psychiatry, Neuroscience, and Pharmacology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205

each of these receptors through direct intracerebral injection of their agonists results in a selective pattern of neuronal degeneration that affects neurons in proximity to the injection site but spares axons of passage and of termination from distant neurons as well as nonneuronal elements (37,51). On the basis of structural activity studies carried out with the systemic administration of glutamate receptor agonists and their analogs, Olney et al. (29) classified these agents as excitotoxins and proposed that their mechanism of neurotoxicity resulted from their depolarizing action. Nevertheless, structural activity studies demonstrate a poor correlation between excitatory potency and neurotoxic action, both across receptor subtypes (50,51) and within a specific receptor subtype (21). Furthermore, recent studies carried out in primary cultures have distinguished two forms of glutamate-induced neuronal degeneration, one with a rapid onset that is sodium chloride dependent and a delayed form which appears to be mediated by NMDA receptors and is Ca2+ dependent (8,33). The latter mechanism has been implicated in the neuronal degeneration that occurs as a consequence of hypoxia, ischemia, and hypoglycemia since competitive and noncompetitive antagonists of NMDA receptors attenuate the neuronal degeneration induced by these insults (32,33,40,47). In spite of these advances, the postreceptor mechanisms accounting for neuronal degeneration induced by glutamate and its analogs remain poorly understood. In an attempt to define these postreceptor mechanisms, we exploited a neuronal-like cell line, the N18-RE-105, which is depolarized by pressure application of glutamate or quisqualate, but not NMDA, and undergoes a delayed form of Ca’+-dependent degeneration when incubated with glutamate, quisqualate, or ibotenate, but not NMDA or kainate (25,28). Subsequent studies (26) revealed that glutamate, quisqualate, and ibotenate are competitive inhibitors of a high affinity cystine antiporter, which appears to be selectively expressed in neurons (19,49). Inhibition of cystine transport results in a progressive reduction in cellular glutathione levels, the nadir of which is temporally associated with the onset of cytolysis. Since cellular oxidants were demonstrated to increase as a consequence of glutathione depletion, and since the addition of antioxidants to the culture media

‘Previous studies with the NlS-RE105 neuronal-like cell line and primary cortical cultures demonstrate that glutamate can produce a calcium-dependent, delayed form of neuronal degeneration that results from its competitive inhibition of cystine transport, which leads to cellular glutathione depletion and death by oxidative stress. Idebenone, a centrally active antioxidant used to treat multiinfarct dementia, protects cells from this form of glutamate-induced cytotoxicity in vitro. In the present study, we have examined the effects of systemic treatment with idebenone on the neurotoxic consequences of intrastriatal injection of kainic acid, quisqualic acid, or quinolinic acid, an NMDA receptor agonist, on neuronal degeneration. Striatal damage was assessed by quantitative neurochemistry with measurement of choline acetyltransferase activity and glutamate decarboxylase activity, by histochemical analysis for acetylcholinesterase and NADPH diaphorase staining and by behavioral assessment of circling produced by systemic apomorphine treatment 10 days after the unilateral lesion. The results indicate that treatment with idebenone provides significant protection against the neuronal degeneration induced by intrastriatal injection of kainic acid and quisqualic acid, but not the NMDA receptor agonist, quinolinic acid. The results suggest that oxidative stress may contribute to the proximate cause of neuronal degeneration induced by quisqualate and by kainate receptor agonists and that the mechanisms of neuronal degeneration caused by quisqualate/kainate receptor agonists differ from those associated with NMDA receptor agonists. o 1990 Academic Press,

Inc.

INTRODUCTION The physiologic effects of glutamate are mediated by at least three distinct receptors named after potent, conformationally restricted, or synthetic analogs that serve as specific agonists: kainic acid, quisqualic acid, and Nmethyl-D-aspartate (NMDA) receptors (13,31,46). The distinct nature of these receptors has been demonstrated by receptor ligand binding studies, sensitivity to antagonists, biophysical characteristics, and expression in the Xenopus oocytes (10, 15, 44). Persistent activation of 0014-4636/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

38 Inc. reserved.

IDEBENONE

PROTECTS

AGAINST

prevented the glutamate-induced cell degeneration, oxidative stress appeared to be the proximate cause of the degenerative process. Recently, we have demonstrated that idebenone, a drug which is used to treat prophylactically multiinfarct dementia (38) and which is a centrally active free radical scavenger (42), is effective in the low micromolar range in preventing the glutamate-induced degeneration of the N18-RE-105 cells (24). Thus, idebenone would appear to be a useful pharmacologic probe for assessing the role of oxidative stress in the neurodegeneration produced by intracerebral injection of the glutamate receptor subtype agonists: kainic acid, quisqualic acid, and quinolinic acid. METHODS

OF PROCEDURE

Excitotoxin lesions. After anesthesia with pentobarbitol, Sprague-Dawley male rats (160 g) were positioned in a David Kopf small animal stereotaxic apparatus, and a 0.3-mm Hamilton cannula was inserted into the right striatum through a burr hole in the calvarium (coordinates: 7.9 mm A; 2.6 mm L; 4.8 mm V) as previously described (35). Excitatory amino acid analogs were dissolved in phosphate-buffered normal saline (pH 7.2) and infused over a period of 5 min in a total volume of 0.5 ~1. The injected doses for quisqualic acid, kainic acid or quinolinic acid were 150, 1.5, or 150 nmol, respectively. Sham-operated, control rats were infused with 0.5 ~1 of the phosphate-buffered saline, to which was added an amount of sodium chloride to achieve equal osmolarity with the excitatory amino acid analog. Idebenone (Takeda Chemical Industries) was administered by intraperitoneal injection 0.5 h before and 4,12,24,36, and 48 h after the excitatory amino acid analog injection. Synaptic neurochemistry. Ten days after the excitotoxin injection, rats were sacrificed by decapitation and the striata were dissected at 5”C, according to the method of Glowinski and Iversen (14). Injected, contralateral, and control striata were homogenized in 20 vol (w/v) of 0.05 M Tris-HCl, pH 7.4, containing 0.2% (v/ v) Triton X-100. The activity of choline acetyltransferase was measured by the method of Bull and OderfeldNowak (7) and the activity of glutamic acid decarboxylase was measured by the method of Wilson et al. (48). Protein content was measured by the method of Lowry et al. (22) with crystalline bovine serum albumin as the standard. Histochemistry. Rats were anesthetized with pentobarbital 10 days after the intrastriatal injection of 100 nmol of quisqualic acid and perfused transcardially through the ascending aorta with 50 mh4 phosphatebuffered normal saline, followed by 10% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed, postfixed for 90 min, and then stored overnight in 0.1 Mphosphate buffer containing 20% (w/v) sucrose.

EXCITOTOXIC

DAMAGE

39

The forebrain was sectioned at 40 pm on a frozen stage microtome. Acetylcholinesterase histochemical staining was performed by the method of Hardy et al. (18) and histochemical staining for NADPH diaphorase was performed by the method of Vincent et al. (45). Behavioral analyses. One week after the unilateral, intrastriatal injection of the excitotoxins, the rats were given a single subcutaneous injection of apomorphine at a dose of 1 mg/kg. They were placed in a circular observation cage, 90 cm in diameter and 30 cm in height. The total number of circlings occurring during lo-min time bins were recorded for a period of 60 min by an evaluator blind to the treatment condition (36,43). Statistics. All results are presented as a mean f the standard error of the mean. Statistical analysis of the data was carried out by the use of analysis of variance followed by the Newman-Keuls test. A P-value of less than 0.05 was the threshold for statistical significance. RESULTS

Synaptic neurochemistry. Consistent with previous reports, kainic acid proved to be approximately loo-fold more potent than quinolinate, resulting in approximately 50% decrements for the presynaptic markers for striatal cholinergic and GABAergic neurons (Tables 1 & 2), choline acetyltransferase, and glutamic acid decarboxylase at 10 days after injection (13,51). The 150 nmol dose of quisqualate produced somewhat more modest, 30% declines in these two presynaptic markers. Systemic treatment with idebenone at the time of excitotoxin injection provided significant protection against the reduction in choline acetyltransferase and glutamic acid decarboxylase activities measured 10 days following the intrastriatal injection of quisqualate or kainate. Notably, the specific activity of glutamate decarboxylase, following quisqualate and kainate injection in idebenone-treated rats, did not differ significantly from that in sham-lesion controls. In contrast, treatment with idebenone at the time of quinolinate injection resulted in only small and insignificant (less than 5%) increments in the specific activities of choline acetyltransferase and glutamic acid decarboxylase, 10 days after injection. Histologic analysis of the effects of idebenone on the striatal toxicity of quisquakzte. Examination of Nisslstained sections through the quisqualate-injected striaturn 10 days after the lesion revealed a spherical area of neuronal cell loss with a diameter of approximately 3 mm at its maximum extent. Treatment of rats with idebenone at the time of quisqualate injection resulted in an approximately 53 f 6% reduction in the diameter of the lesion. The degree of protection is more clearly demonstrated in tissue sections histochemically stained for acetylcholinesterase, a presynaptic marker highly associated with striatal cholinergic neurons. As shown in Fig. 1, the quisqualate injection results in a spherical area of

40

MIYAMOTO

AND

TABLE Effects

of Idebenone

on Reduced

Striatal of Excitatory

CAT

1

Activity Produced by Unilateral Amino Acids in Rats CAT

Lesion Sham Quisqualate

Sham Kainate Sham Quinolinate

(150 nmol)

(1.5 nmol)

(150 nmol)

“w Saline Saline Idebenone Saline Saline Idebone Saline Saline Idebenone

COYLE

Dose bdk) 3 10 10 10

Control 169.1 176.2 161.3 173.5 164.7 152.2 168.1 176.3 184.9 172.3

activity side z!z 7.7 + 7.0 k 9.2 + 7.8 + 6.4 +- 11.5 + 14.9 + 5.7 f 7.4 * 8.2

(nmol/Rg

Striatal

protein

h-i)

Lesioned

side

170.0 121.2 125.3 146.6 160.5 80.8 115.9 174.0 70.8 76.3

zk f + + + * f + f. +

Injection

% Control

6.3 8.6** 4.9 9.37 7.3 11.1** 16.3tt 8.1 11.4** 11.5

side

101.1+ 3.9 69.4 k 4.4** 78.3 + 4.2 84.4 + 4.4t 97.7 + 4.2 54.3+4.4** 71.7 + 7.1t 98.7 If: 2.9 39.3 t-6.7** 43.9 f 5.6

(6) (7)

(6) (7)

(6) (6) (‘3 (6) (7) (7)

Note. Each value is the mean + SEM of CAT activity and of the percentage of CAT activity in the lesioned side compared to that in the control side. The number of rats used are shown in parentheses. **P < 0.01, compared with respective sham controls; tP < 0.05, ftP < 0.01, compared with respective lesion controls (Newman-Keuls test). Quisqualate lesion: Two-way ANOVA showed significant effects of the lesion and group X lesion interaction, F(1,22) = 55.03, P < 0.01 and F(3,22) + 8.07, P < 0.01, respectively. One-way ANOVA also showed a significant difference among groups in the bilateral comparison, F(3,22) = 9.62, P < 0.01. Follow-up comparisons revealed that the decrease in CAT activity caused by quisqualate injection was significantly prevented by the idebenone treatment. Kainate lesion: There were significant effects of the group, lesion, and group X lesion interaction, F(2,15) = 4.32, P < 0.05; F(1,15) = 57.17, P < 0.01; and F(2,15) = 13.51, P < 0.01, respectively. Newman-Keuls comparison revealed that the treatment with idebenone also significantly reversed the decreased CAT activity. Quinolinate lesion: ANOVA showed significant effects of the group, lesion, and group X lesion interaction, F(2,17) = 15.99, P < 0.01; F(1,17) = 124.1, P < 0.01; and F(2,17) = 26.84, P < 0.01, respectively. The decreased CAT activity by quinolinate was not reversed by the idebenone treatment.

loss of acetylcholinesterase staining, which closely approximates the region of neuronal cell loss observed in Nissl-stained sections. In the idebenone-treated rats, the volume of the lesion, as ascertained in the reduction in diameter at its largest extent, was decreased by 81 & 10%. NADPH diaphorase histochemical staining is localized to a subpopulation of striatal neurons that contain somatostatin (45). As previous studies indicate, the NADPH diaphorase-positive neurons are vulnerable to quisqualate lesions (4, 20). However, idebenone treatment resulted in marked protection against quisqualate neurotoxicity to NAPHD diaphorase neurons (Fig. 1). Behavioral effects of idebenone treatment with striatal ercitotoxin lesions. Previous studies have demonstrated that unilateral excitotoxin lesions of the striaturn result in circling behavior in animals challenged with apomorphine, and the intensity of circling correlates with the extent of the striatal lesion (36). As demonstrated in Fig. 2, the striatal lesions with quisqualate, kainate, and quinolinate resulted in striking rotatory behavior with apomorphine challenge that peaked 20-30 min after administration of the dopamine receptor agonist. The intensity of circling was significantly greater in the quinolinate-lesioned animals consistent with the somewhat greater reduction in presynaptic markers for striatal cholinergic and GABAergic neurons with this lesion as compared to the kainate and quisqualate lesions. Low dose treatment with idebenone (3 mg/kg) at the time of quisqualate injection produced a modest but significant reduction in rotatory behavior. However, the 10

mg/kg dose of idebenone completely prevented motor asymmetry in response to apomorphine challenge in animals receiving unilateral striatal injections of either quisqualate acid or kainate acid. In contrast, idebenone treatment at the time of injection produced only a minimal but nonsignificant decrease in apomorphine-induced rotatory behavior in the rats subjected to unilateral quinolinate lesions. DISCUSSION Previous research in this laboratory has demonstrated in the NlB-RE-105 retinal-hybridoma cell line that glutamate, quisqualate, and ibotenate caused cytolysis when present in the culture medium for 8 to 12 h, whereas NMDA and kainic acid were devoid of toxic effects at concentrations as high as 10 mM (25). While glutamate and quisqualate do produce depolarizing but rapidly desensitizing responses in these cells as assessed by intracellular recording techniques, cytotoxicity is unrelated to depolarization since persistent depolarization with elevated potassium, veratridine, or ouabain did not result in cytotoxicity (28). These cells express a quisqualate-sensitive high affinity, chloride-dependent glutamate carrier site similar to that which has been localized to brain neurons (19,49). Recent evidence indicates that this sequestration site represents a cystine antiporter at which glutamate, quisqualate, and ibotenate are potent competitive inhibitors (26). Since the cytotoxic potency of glutamate and quisqualate is inversely proportionate

IDEBENONE

PROTECTS

AGAINST

EXCITOTOXIC

TABLE Effects of Idebenone

on Reduced

Striatal of Excitatory

GAD

2

Activity Produced Amino Acids in Rats GAD (nmol/pg

Lesion Sham Quisqualate

Sham Kainate Sham Quinolinate

(150 nmol)

(1.5 nmol)

(150 nmol)

Drug Saline Saline Idebenone Saline Saline Idebone Saline Saline Idebenone

Dose b-&g)

3 10 10 10

41

DAMAGE

Control

by Unilateral

activity protein

Striatal

Injection

h-*) Lesioned side

% Control

29.0 +- 1.0

29.0 + 0.9

100.2 f

2.1

(6)

30.6 27.9 28.5 28.6

21.9 + 24.4 f 27.0f 28.5 +

5.9** 4.at 5.9t 7.2 4.2** lO.lt 5.2

(7)

side

t f f +

1.4 0.8 0.5 1.4

28.0 f 2.7

26.0 k 1.8tt 29.3 + 2.2

71.8f 87.7 + 94.8 k lOl.l+ 59.3 + 88.9 f 106.5 +

29.2 f 1.3

16.2 + 1.8**

55.1 f

5.2**

28.7 k 2.2

17.8 f 1.6

63.9 f

7.6

31.3 + 1.8 30.1 zk 1.7

1.8** 1.5 1.77-f 0.9

side

18.3 + l.l**

(6) (7)

(6) (f-3) (6) (6) (7) (7)

Note. Each value is the mean k SEM of GAD activity and of the percentage of GAD activity in the lesioned side compared to that in the control side. The number of rats used are shown in parentheses. **P < 0.01, compared with respective sham controls; tP < 0.05, ttP < 0.01, compared with respective lesion controls (Newman-Keuls test). Quisqualate lesion: Two-way ANOVA showed significant effects of the lesion and group X lesion interaction, F (1,22) = 20.77, P < 0.01 and F(3.22) = 6.20, P < 0.01, respectively. One-way ANOVA also revealed a significant difference among groups in the bilateral comparison, F(3,22) = 5.90, P < 0.01. Follow-up comparisons indicated that the idebenone treatment significantly reversed the decreased GAD activity. Kainate lesion: There were significant effects of the group, lesion, and group X lesion interaction, F(2,15) = 5.27, P < 0.05; F(1,15) = 17.32, P < 0.01; and F(2,15) = 7.55, P < 0.01, respectively. The treatment with idebenone significantly protected against the reduction of GAD activity. Quinolinate lesion: Significant effects of the lesion and group X lesion interaction were noted by ANOVA, F(1,17) = 49.01, P < 0.01 and F(2,17) = 11.60, P c 0.01, respectively. However, the idebenone treatment failed to reverse the reduced GAD activity.

to the concentration of cystine in the culture medium, there is compelling evidence that the cytotoxicity results from competitive inhibition of cystine transport into the cells. Inhibition of cystine transport deprives the cells of an essential amino acid in the synthesis of the important intracellular reductant, glutathione (1, 2). Accordingly, reduction in medium concentration of cystine or the addition of the competitive inhibitors of its transport results in a time-dependent reduction of cellular glutathione levels. Coincident with the reduction of cellular glutathione, there is a marked increase in intracellular oxidants as assessedby the oxidizable fluorescent probe, dichlorylfluorescin diacetate. The sequence of cytopathologic alterations, including cellular membrane blebbing, fragmentation of neuritic processes, perikaryal granulation, and cell lysis, is reminiscent of the evolution of histologic changes associated with excitotoxininduced neuronal degeneration, both in vivo and in vitro (8,9,29). They are also consistent with the cytopathology associated with a variety of toxins that act through oxidative stress (16). Consistent with this inference, the addition of antioxidants to the tissue culture media prevented the cytotoxic effects of glutamate (26). Of all the antioxidants examined, idebenone, a drug used to treat multiinfarct dementia, proved to be the most potent, providing protection in the low micromolar range. In fact, idebenone was 30- to loo-fold more potent than (Ytocopherol(24). However, idebenone does not alter cys-

tine transport (24), nor does it at 100 @4 inhibit ligand binding to either quisqualate or kainate receptors in brain membrane preparation (unpublished observations) . The oxidative stress mechanism that involves glutamatelquisqualate inhibition of cystine transport has been extended to neurons in studies of primary cultures of fetal mouse cerebral cortex (27). Cultured neurons exhibit the same structural activity relation profile with vulnerability to glutamate, quisqualate, and ibotenate, but not kainate or NMDA, 2 days after plating. Notably, glial cells remain viable under conditions of glutamate exposure that result in widespread neuronal degeneration. This may reflect the high ambient levels of glutathione and, possibly, its slower turnover in glia as compared to neurons (41). Addition of antioxidants to the culture medium, including idebenone, protected against this form of glutamate/quisqualate-induced neurotoxicity. Since idebenone is a potent antioxidant, which readily crosses the blood-brain barrier (42), it might serve as a useful pharmacologic probe to assessthe potential contribution of oxidative stress in the neurotoxic action of intracerebrally injected glutamate analogs. Consistent with the findings with the NWRE-105 cell line and primary cortical cultures (24, 27), systemic administration of idebenone at the time of intracerebral injection of quisqualic acid provided significant reversal of striatal neuronal degeneration as demonstrated by quantitative

42

MIYAMOTO

AND

COYLE

Quisqualate Sham Lesion (Saline) Lesion (Ide 3) Lesion (Ide 10)

10 20 30 Time after apomorphine

40 50 injection (min)

60

Kainate

B m-C,+

0

10 20 30 Time after apomorphine

40 50 injection (min)

Sham Lesion (Saline) Lesion (Ide 10)

60

Quinolinate 70 -

C Sham Lesion (Saline) Lesion (Ide 10)

50 30 10 0 -10;.

I 0

” *

I

” .I.,

10 20 30 Time after apomorphine

”

” I,.

40 50 injection (min)

0 ,

60

FIG. 1. Effects of idebenone on circling behavior induced by apomorphine in rats with unilateral striatal lesions by glutamate receptor agonists. Each value shows the mean + SEM of the number of ipsilateral circlings, every lo-min period. (A) Effects in the rats lesioned with quisqualate. ANOVA showed a significant difference among groups, F(3,22) + 12.49, P < 0.01. The lesion control (n = 7) showed a significant increase in the number of circlings at lo- to 50-min periods (P < O.Ol), compared with the sham-operated control (n = 6). Repeated administration of idebenone suppressed the circling in a dose-dependent manner, both groups treated with 3 mg/kg (Ide 3, n = 6) and 10 mg/kg (Ide 10, n = 7) significantly suppressed the circling behavior at lo- to 50-min periods (P < 0.05 or P < 0.01). (B) Effects in rats with striatal lesions by kainate. There was a significant difference among groups, F(2,15) = 131.8, P < 0.01. Kainate-induced lesions (n = 6) also resulted in a significant increase in the number of circlings compared with the sham control (n = 6). The circling was almost completely blocked by 10 mg/kg of idebenone (Ide 10, n = 6) at lo- to 50-min periods (P < 0.01). (C) Effect in rats lesioned with quinolinate. A significant group effect was noted, F(2,17) = 17.92, P < 0.01. Although the lesion control (n = 7) exhibited marked circling behavior compared with the sham control (n = 6) at lo- to 50-min periods, the idebenone treatment failed to show a significant effect on the circling.

IDEBENONE

FIG. 2. Effects of idebenone on histochemical diaphorase neurons in the striatum injected with injected with quisqualate accompanied by systemic

PROTECTS

AGAINST

EXCITOTOXIC

changes induced by quisqualate injection phosphate-buffered saline (A, D), striatum treatment with idebenone (C, F).

synaptic neurochemical analyses for presynaptic markers of the striatal cholinergic and GABAergic neurons, histochemical analyses of acetylcholinesterase and NADPH diaphorase-reactive neurons, and behavioral analysis of the motor asymmetry that results from unilateral striatal lesion. In contrast, idebenone treatment provided minimal and statistically insignificant protection against the neurotoxic action of the NMDA receptor agonist, quinolinic acid, as measured by quantitative synaptic chemistry or rotatory behavior induced by apomorphine challenge.

DAMAGE

into the striatum. AChE injected with quisqualate

43

activity and NADPH (B, E), and striatum

An unanticipated finding was that idebenone treatment provided virtually complete protection against the neurotoxic action of intrastriatally injected kainic acid as demonstrated by quantitative neurochemistry as well as the rotatory responses to apomorphine challenge. This protective effect of idebenone against kainic acid neurotoxicity appears to be inconsistent with the observation that kainic acid does not inhibit the cystine antiporter nor exert neurotoxic effects against the NlSRE105 cell line or immature primary cortical neuronal cultures (26). Nevertheless, Dykens et al. (11) have pre-

44

MIYAMOTO

viously demonstrated that free radical scavengers protect against kainate toxicity in mature primary cerebellar cultures. The results suggest dissociation between mechanisms involved in NMDA receptor-mediated and non-NMDA receptor (quisqualate and kainate)-mediated neuronal degeneration, with the former not directly involving oxidative stress as a proximate cause of neuronal cell death. This hypothesis is consistent with the results of Beal et al. (3), who have shown that a variety of systemically administered antioxidants do not affect neuronal degeneration induced by quinolinic acid, an NMDA receptor agonist. These findings, however, do not discount the important role of depolarization related to activation of kainate or quisqualate receptors in the degenerative phenomenon. Depolarization is associated with an increase in neuronal metabolic activity, which results in an increased level of cellular oxidants due to activation of the mitochondrial oxidant cascade (16,17). But neuronal activity per se would not appear to be sufficient cause for a neuronal degeneration. In this context, it is reasonable to speculate that intracerebral injection of quisqualic acid directly and potently inhibits the neuronal cystine transport process resulting in the impaired synthesis of neuronal glutathione, thereby rendering neurons vulnerable to oxidative stress. Persistent depolarization due to activation of a quisqualate subtype of receptors, as well as secondary increases in intracellular calcium (30), which result in activation of enzymatic processes yielding cellular oxidants (34), creates a scenario that may account for quisqualate-induced idebenone-reversible neuronal degeneration. The mechanism for kainate-induced neuronal degeneration as demonstrated by its requirement for intact, presumably glutamatergic innervation (6) is indirect. Kainate has direct depolarizing effects on neurons (23, 39) and secondarily may increase calcium influx (30). Together, these two effects would result in an enhanced generation of interneuronal oxidants. While kainate does not directly inhibit the cystine carrier (26), it does act presynaptically to markedly increase the release of endogenous glutamate (12), a potent inhibitor of the cystine carrier. Thus, the idebenone-reversible action of the neurotoxic effects of kainic acid may reflect the wellestablished indirect mechanism of neurotoxicity of kainit acid (6), although other mechanisms of protective interaction cannot be precluded at present. In summary, the present in uiuo results are consistent with previous findings that distinguish the neurophysiologic and neurotoxic actions of NMDA as compared to non-NMDA receptor agonists. The efficacy of idebenone in protecting against the neurotoxic actions of intracerebrally injected kainic acid and quisqualic acid supports the hypothesis that oxidative stress is involved in the final common pathway of neuronal degeneration induced by kainate and quisqualate receptor agonists. Since idebenone has proved to be effective in preventing

AND COYLE

the neuronal degeneration associated with multiinfarct dementia (38), these results point to an alternative pharmacologic strategy for preventing, or at least attenuating, neuronal degeneration associated with glutamatergic mechanisms in the brain (5). ACKNOWLEDGMENTS The authors thank Alice Trawinski for her assistance in preparing the manuscript. This research was supported by USPHS Grant NS 13584 and a grant from the McKnight Foundation.

REFERENCES 1. BANNAI, S. 1986. Exchange of cystine and glutamate across plasma membrane of human fibroblasts. J. Biol. Chem. 261: 2256-2263. 2. BANNAI, S., AND E. KITAMURA. 1980. Transport interaction of Lcystine and L-glutamate in human diploid fibroblasts in culture. J. Biol.

Chem.

255:

2312-2376.

3. BEAL, M. F., N. W. KOWALL, K. J. SWARTZ, R. J. FERRANTE, AND J. B. MARTIN. 1988. Systemic approaches to modifying quinolinic acid striatal lesion in rats. J. Neurosci. 8: 3901-3908. 4. BEAL, M. F., N. W. KOWALL, D. W. ELLISONS, M. F. MAZURAK, K. SWARTZ, AND J. B. MARTIN. 1986. Replication of the neurochemical characteristics of Huntington’s disease by quinolinic acid. Nature (London) 321: 168-171. 5. BENVENISTE, H., J. DREJER, A. SCHOUSBOE, AND N. H. DIEMER. 1984. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem. 43: 1369-1374. 6. BIZIERE, K., AND J. T. COYLE. 1979. Effects of cortical ablation on the neurotoxicity and receptor binding of kainic acid in striaturn. J. Neurosci. Res. 4: 383-398. I. BULL, G., AND B. ODERFELD-NOWAK. 1971. Standardization of a radiochemical assay of choline acetyltransferase and a study of the activation of the enzyme in rabbit brain. J. Neurochem. 19: 935-947. 8. CHOI, D. W., M. A. MAULUCCI-GEDDE, AND A. R. KRIEGSTEIN. 1987. Glutamate neurotoxicity in cortical cell culture. J. Neurosci.

7: 357-368.

9. COYLE, J. T., M. E. MOLLIVER, AND M. J. KUHAR. 1978. In situ injection of kainic acid: A new method for selectively lesioning neuronal cell bodies while sparing axons of passage. J. Comp. Neurol.180:301-323. 10. CULL-CANDY, S. G., AND M. M. USOWICZ. 1987. Multiple conductance channels activated by excitatory amino acids in cerebellar neurons. Nature (London) 326: 525-528. 11. DYKENS, J. A., A. STERN, AND E. TREKNER. 1987. Mechanism of kainate toxicity to cerebellar neurons in vitro is analogous to reperfusion injury. J. Neurochem. 49: 1222-1228. 12. FERKANY, J. W., R. ZACZEK, AND J. T. COYLE. 1982. Kainic acid stimulates excitatory amino acid neurotransmitter release at presynaptic receptors in the cerebellum. Nature (London) 298: 757-759.

13. FOSTER, A. C., J. F. COLLINS, AND R. SCHWARCZ. 1983. On the excitotoxic properties of quinolinic acid, 2,3-piperidine dicarboxylic acids and structurally related compounds. Neuropharmacology 22: 1331-1342. 14. GLOWINSKI, J., AND L. L. IVERSEN. 1966. Regional studies of catecholamines in rat brain. I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]DOPA in various regions of the brain. J. Neurochem. 13: 655-669.

IDEBENONE

PROTECTS

AGAINST

15. GREENAMYRE, J. T., J. M. OLSEN, J. B. PENNEY, AND A. B. YOUNG. 1985. Autoradiographic characterization of N-methyl-Daspartate, quisqualate and kainate sensitive glutamate binding sites. J. Pharmacol. Exp. Ther. 233: 254-263. 16. HALLIWELL, B. 1987. Oxidants and human disease: Some new concepts. FASEB J. 1: 358-364. 17. HALLIWELL, B., J. R. HOULT, AND D. R. BLAKE. 1988. Oxidants, inflammation and anti-inflammatory drugs. J. FASEB 2: 28672873. 18. HARDY, H., L. HEIMER, R. SWITZER, AND D. WATKINS. 1976. Simultaneous demonstrations of HRP and acetylcholinesterase. Neurosci. Lett. 3:1-5. 19. KESSLER, M., M. BAUDRY, AND G. LYNCH. 1987. Use of cystine to distinguish glutamate binding from glutamate sequestration. Neurosci. Lett. 81: 221-226. 20. KOH, J., AND CHOI, D. W. 1988. Vulnerability of cultured cortical neurons to damage by excitotoxins: Differential susceptibility of neurons containing NADPH-diaphorase. J. Neurosci. 8: 21532163. 21. LEHMANN, J., J. W. FERKANY, P. SCHAEFFER, AND J. T. COYLE. 1985. Dissociation between the excitatory and “excitotoxic” effects of quinolinic acid analogs on the striatal cholinergic interneuron. J. Phurmacol. Exp. Ther. 232: 873-882. 22. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265-275. 23. MCLENNAN, H. 1980. The effect of decortication on the excitatory amino acid sensitivity of striatal neurons. Neurosci. L&t. 18: 313-316. 24. MIYAMOTO, M., T. H. MURPHY, R. L. SCHNAAR, AND J. T. COYLE. 1989. Antioxidants protect against glutamate-induced cytotoxicity in a neurona cell line. J. Pharmacol. Exp. Ther. 260: 1132-1140. 25. MURPHY, T. H., A. T. MALOUF, A. SASTRE, R. L. SCHNAAR, AND J. T. COYLE. 1988a. Calcium-dependent glutamate cytotoxicity in a neuronal cell line. Brain Res. 444: 325-332. 26. MURPHY, T. H., M. MIYAMOTO, A. SASTRE, R. L. SCHNAAR, AND J. T. COYLE. 1989. Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neuron 2: 1547-1558. 27. MURPHY, T. H., R. L. SCHNAAR, AND J. T. COYLE. 1989. Immature cortical neurons in culture are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake. FASEB J, in press. 28. MURPHY, T. H., R. L. SCHNAAR, J. T. COYLE, AND A. SASTRE. 1988b. Glutamate cytotoxicity in a neuronal cell line is blocked by membrane depolarization. Brain Res. 460: 155-160. 29. OLNEY, J. W., 0. Ho, AND V. RHEE. 1972. Cytotoxic effects of acidic and sulphur-containing amino acids on the infant mouse central nervous system. Exp. Brain Res. 14: 61-76. 30. RETZ, K. C., AND J. T. COYLE. 1984. The differential effects of excitatory amino acids on “CaC12 by slices from mouse striatum. Neuropharmacology

31.

23:

89-94.

ROBINSON, M. B., AND J. T. COYLE. 1988. Glutamate and related acidic excitatory neurotransmitters: From basic science to clinical application. FASEB 1: 446-455. 32. ROTHMAN, S. 1984. Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death. J. Neurosci. 4: 1884-1891. 33. ROTHMAN, S. M., AND J. W. OLNEY. 1986. Glutamate and the pathophysiology of hypoxic-ischemic brain damage. Ann. Neurol. 19: 105-111.

EXCITOTOXIC

DAMAGE

45

34. SCHANNE, F. A., A. B. KANE, E. E. YOUNG, AND J. C. FARBER. 1979. Calcium dependence of toxic cell death: A common pathway. Science 206: 700-702. 35. SCHWARCZ, R., AND J. T. COYLE. 1977. Striatal lesions with kainit acid: Neurochemical characteristics. Brain Res. 127: 235249. 36. SCHWARCZ, R., K. FUXES, L. F. AGNATI, T. HOKFELT, AND J. T. COYLE. 1979. Rotational behavior in rats with unilateral striatal kainic acid lesions: A behavioral model for studies on intact dopamine receptors. Brain Res. 170: 485-495. 37. SCHWARCZ, R., D. SCHOLZ, AND J. T. COYLE. 1978. Structureactivity relations for the neurotoxicity of kainic acid derivatives and glutamate analogues. Neurophurmacology 17: 145-151. 38. SEKIMOTO, H., I. NAKADA, T. NAKANO, N. FUSE, K. HASEDA, K. YASUMOTO, T. SHINAGAWA, T. HAGAI, T. OHKA, S. UCHIYAMA, AND T. TAKEGOSHI. 1985. Efficacy and safety of CV-2619(idebenone) in multiple cerebral infarction, cerebrovascular dementia and senile dementia. Ther. Res. 2: 957-972. SHINOZAKI, H., AND S. KONISHI. 1970. Action of several antihel39. minthics and insecticides on rat cortical neurons. Brain Res. 24: 368-371. 40. SIMON, R. P., J. H. SWAN, T. GRIFFITHS, AND B. S. MELDRUM. 1984. Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in brain. Science 226: 850-852. 41. SLIVKA, A., M. B. SPINA, H. I. CALVIN, AND G. COHEN. 1988. Depletion of brain glutathione in preweanling mice by L-buthionine sulfoxime. J. Neurochem. 50: 1391-1393. 42. SUNO, M., AND A, NAGAOKA. 1984. Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition. Biochem. Btiphys. Res. Commun. 125: 1046-1052. 43. UNGERSTEDT, U., AND G. W. ARBUTHNOTT. 1970. Quantitative recording of rotational behavior in rats after 6-hydroxydopamine lesions of the nigrostriatal dopamine system. Brain Res. 24: 485493.

44. VERDOORN, T. A., AND DINGELDINE, R. 1988. Excitatory amino acid receptors expressed in Xenopus oocytes: Agonist pharmacology. Mol. Pharmacol. 34: 298-307. 45. VINCENT, S. R., 0. JOHANSSON, T. HOKFELT, L. SKIRBOLL, R. P. ELDE, L. TERENIUS, J. KIMMEL, AND M. GOLDSTEIN. 1983. NADPH-diaphorase: A selective histochemical marker for striatal neurons containing both somatostatin and avidin pancreatic polypeptide (APP)-like immunoreactivity. J. Comp. Neural. 217:252-263. 46. WATKINS, J., AND R. H. EVANS. 1981. Excitatory amino acid transmitters. Annu. Rev. Pharmacol. Toxicol. 21: 165-204. 47. WIELOCH, T. 1985. Hypoglycemia-induced neuronal damage prevented by an N-methyl-D-aspartate antagonist. Science 230: 681-683. 48. WILSON, S. H., B. K. SCHRIER, J. L. FARBER, E. J. TOMPSON, R. N. ROSENBERG, A. J. BLUME, AND M. W. NIRENBERG. 1972. Markers for gene expression in cultured cells from nervous system. J. Biol. Chem. 247: 3159-3160. 49. ZACZEK, R., M. BALM, S. ARLIS, H. DRUCKER, AND J. T. COYLE. 1987. Quisqualate-sensitive, chloride dependent transport of glutamate into rat brain synaptosomes. J. Neurosci. Res. 18: 425431. 50. ZACZEK, R., J. COLLINS, AND J. T. COYLE. 1981. N-Methyl Daspartic acid: A potent convulsant with weak neurotoxic properties. Neurosci. Lett. 24: 181-186. 51. ZACZEK, R., AND J. T. COYLE. 1982. Excitatory amino acid analogues: Neurotoxicity and seizures. Neuropharmacology 21: 15-26.