Anticonvulsant action of a non-competitive antagonist of NMDA receptors (MK-801) in the kindling model of epilepsy

12

Brain Research, 463 (1988) 12-20 Elsevier

BRE 13993

Anticonvulsant action of a non-competitive antagonist of NMDA receptors (MK-801) in the kindling model of epilepsy Keiko Sato, Kiyoshi Morimoto and Motoi Okamoto Department of Neuropsychiatry, Okayama UniversityMedical School, Okayama (Japan) (Accepted 11 May 1988)

Key words: MK-801; N-methyl-D-aspartate receptor; Non-competitive antagonist; Kindling; Rat

Anticonvulsant action of MK-801, a novel non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptors, was investigated in the kindling model of epilepsy in rats. The results obtained were as follows. (1) Both the seizure stage and afterdischarge duration of previously kindled seizures from the amygdala were significantly suppressed following systemic injection of MK-80I (0.25-4 mg/kg) in a dose-dependent manner. The maximum effects were observed between 2 and 4 h after the injection. (2) The MK-801 also showed significant anticonvulsant effects on kindled seizures from the frontal cortex and the ventral and dorsal hippocampus. The efficacy, however, significantly differed between these kindled sites. (3) Daily treatment of MK-801 (0.25 and 1 mg/kg) prior to each electrical stimulation of the amygdala significantly retarded kindling seizure development and increased the total amount of afterdischarge (accumulated AD) required to reach the first stage 5 seizure. During drug sessions of 1 mg/kg MK-801 for 19 days, all rats showed only partial seizures and the growth of afterdischarge was strongly prevented. (4) Pretreatment with reserpine did not antagonize the anticonvulsant effects of MK-801 on previously kindled seizures from the amygdala, suggesting that the effects may not be mediated by catecholaminergic systems. These results indicate that MK-801 has potent anticonvulsant actions on kindled seizures from both limbic and cortical loci, the NMDA system may play a critical role in the seizure-triggering mechanism of kindling. The possible application of NMDA antagonists in clinical epilepsy is suggested.

INTRODUCTION The excitatory amino acids k-glutamate and L-asp a r t a t e are thought to act as principal excitatory neurotransmitters in the m a m m a l i a n brain 17'3°'5°'sl. The receptors mediating their actions are generally divided into 3 m a j o r subtypes of N-methyl-D-aspartate ( N M D A ) , kainate and quisqualate, based on their activation by these selective agonists 1s'16. The N M D A subtype has received a great deal of attention because of its possible involvement in synaptic plasticity 6s'16"35 and in a variety of neuropathologies 16"18"25'37"45. Thus, there is considerable interest in the d e v e l o p m e n t of pharmacological agents that might act to block this receptor 12'14'25. It has recently been emphasized that N M D A receptors in the central nervous system play an essen-

tial excitatory role in the seizure-triggering mechanism in experimentally induced seizures 42. In in vitro studies, selective N M D A r e c e p t o r blockers, such as 2 - a m i n o - 5 - p h o s p h o n o v a l e r a t e ( A P V ) and 2-amino7 - p h o s p h o n o h e p t a n a t e ( A P H ) , reduce epileptiform activity induced by repetitive electrical stimulation 122, chemoconvulsants 2°'21'24'3I'38'41 and ion manipulations 22"2a'49. A l t h o u g h

these

NMDA

com-

pounds do not p e n e t r a t e readily into the central nervous system, they suppress epileptic seizures when a d m i n i s t e r e d directly into the cerebral ventricles 4°'41 or into certain brain sites in vivo j3'33'34. MK-801, [(+)-5-methyl]- 10,11-dibenzo[a,d]-cyclohepten-5,10-imine maleate, is a novel non-competitive antagonist of N M D A receptor, which easily p e n e t r a t e s the b l o o d - b r a i n barrier s2. It has been shown to have an anticonvulsant action on maximum

Correspondence: K. Sato, Department of Neuropsychiatry, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama, 700 Japan. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

13 electroshock seizures and chemically induced seizures when injected systemically5. In the present study, we compared the anticonvulsant action of MK-801 on previously kindled seizures from the amygdala (AM), frontal cortex (FC), ventral and dorsal hippocampus (HIPP). Kindling is an experimental model of human complex partial seizures with secondarily generalization, which results from repeated focal electrical stimulation 19. In addition to the effects on fully kindled responses, we investigated the effects of MK-801 on the development of the kindled state in amygdala-kindled animals. In addition, since MK-801 was reported to act via the central catecholaminergic system -s, we examined the effects of pretreatment of reserpine, a catecholamine-depleting agent, on the anticonvulsant action of MK-801.

zure triggering threshold (GST) was then determined in each rat by application of trains that were increased in intensity at 50-/~A steps at an interval of 20 rain. AM- and HIPP-kindling seizure development was assessed using a modification of Racine's classification ~a, as follows: stage 0, no response or behavior arrest including immobility in HIPP kindling; stage 1, rhythmic mouth and facial movement; stage 2, rhythmic head nodding; stage 3, forelimb clonus; stage 4, rearing and bilateral forelimb clonus; stage 5, rearing and falling. FC kindling was assessed, as follows; stage 0, no response; stage 1, contraversive head turning; stage 2, tonic extension; stage 3, forelimb clonus; stage 4, rearing and bilateral forelimb clonus; stage 5, rearing and falling. The EEG was recorded, between the remaining pole of the tripolar electrode and the skull screw electrode, on a Nihonkoden 5109 electroencephalograph in all trials.

MATERIALS AND METHODS

General procedure Fifty male Sprague-Dawley rats, weighing 250-300 g at the time of surgery, were divided into 4 groups (AM-kindled group, n = 33; FC-kindled group, n = 5; ventral HIPP-kindled group, n = 6; dorsal HIPP-kindled group, n = 6). All rats were housed with a 12-h light-12-h dark cycle and ad libiturn feeding. Under pentobarbital anesthesia (50 mg/kg i.p.), a tripolar electrode was stereotaxically implanted into the left AM (0.6 mm posterior and 4.8 mm lateral from the bregma, and 7.8 mm below the dura), the left FC (1.0 mm anterior, 3.0 mm lateral and 0.3 mm below), the left ventral HIPP (2.6 mm posterior, 5.0 mm lateral and 7.0 mm below) and the left dorsal HIPP (2.6 mm posterior, 3.0 mm lateral and 3.2 mm below). All coordinates were with the incisor bar 5.0 mm above the interaural plane. All tripolar electrodes consisted of 3 twisted Diamel-insulated Nichrome wires (0.18 mm diameter). In addition, a screw electrode was placed in the right frontal skull for recording EEG. After a recovery period of i week, all rats received the kindling stimulus once daily. This consisted of a 1s train of 60-Hz sign-wave pulses delivered at the afterdischarge (AD) threshold intensity. Stimulation was delivered through two poles of the tripolar electrode until at least 5 consecutive generalized convulsions (stage 5 seizure) appeared. The generalized sei-

Experiment 1: anticonvulsant effects of MK-801 on previously kindled seizures Twenty-five previously kindled rats from the AM (n = 8), FC (n = 5), dorsal (n = 6) and ventral HIPP (n = 6) were used. All rats showed stable stage 5 seizures by stimulation at the determined intensity of GST. To examine the time course of the anticonvulsant action of MK-801 in AM-kindled rats, electrical stimulation at the GST was delivered to the AM 24 h before (predrug stimulation) and 0.5, 1, 2, 4, 12, 24, 48 and 72 h after the i.p. injection of MK-801 (2 mg/kg in 0.9% saline). To examine the dose response, AM, FC, ventral and dorsal HIPP-kindled rats received electrical stimulation at the GST 2 h after the administration of saline or MK-801 with various doses (0.25, 0.5, 1, 2 and 4 mg/kg). In these experiments, the sequence of the time and dose was randomly assigned. Each drug or saline trial was separated by at least 48 h. The anticonvulsant effects of MK-801 were assessed in the following parameters: (a) resting EEG and behaviors: (b) kindled seizure stage; (c) AD duration; (d) latency and duration of bilateral forelimb clonus.

Experiment 2: effects of MK-801 on kindling seizure development Twenty rats with AM tripolar electrodes were used. Following 1-2 weeks of a postsurgical recovery period, the kindling train was set at 200/~A peak-to-

14 peak amplitude and was delivered to the AM on day 1. The animals were then divided into 3 groups, matched for the seizure stage and AD duration: 0.25 mg/kg of MK-801 (n = 7); 1 mg/kg (n = 6); saline (n = 7). This dose of MK-801 was selected because it was found in Expt. 1 that 0.25 mg/kg MK-801 significantly reduced the AD duration, whereas 1 mg/kg MK-801 was the dose that completely blocked the bilateral forelimb clonus of previously AM-kindled seizures. Thereafter, at approximately 24-h intervals, electrical stimulation at 200 ~A was delivered 2 h after i.p. administration of MK-801 or saline from kindling day 2 to 20. From day 2 to 15, the stimulus intensity was fixed at 200BA, and, from day 16 to 20, the stimulus intensity was increased to 400,uA only in the drug groups. On day 21, the drug administration was stopped and daily AM stimulation at 200 ~tA was resumed without drug treatment until day 30. GST was then determined as in the previous experiment. Saline-treated animals received stimulation at the intensity of 200 ~tA throughout, and received the GST test on day 21. Kindled seizure development, the growth of AD duration, the seizure stage and AD duration on day 21, and GSTs were measured and compared between the 3 groups.

S. E.M. The AD duration, latency and duration of bilateral forelimb clonus were analyzed by one-way ANOVA, while the seizure stage was by KruskalWallis test. RESULTS

Resting EEG and behaviors After injection of 0.25 to 4 mg/kg of MK-801, there was no change either in the amplitude of awake resting EEG or in the interictal spikes in all rats. However, it caused obvious behavioral changes depending upon the doses. Locomotor activity increased and stereotyped behavior such as bilateral repetitive head turning and sniffing appeared, when MK-801 at doses =< 0.5 mg/kg was administered. MK-801 at doses ->__ 1 mg/kg induced ataxia and hyptonia following the appearance of hyperlocomotion and stereotyped behavior in all rats.

Experiment 1 Time course. Fig. 1 shows the time course of anticonvulsant effects of MK-801 on previously kindled seizures from the AM. Both the seizure stage and A D duration were significantly suppressed 0.5, 2, 4

Experiment 3: effects of reserpine on anticonvulsant effects of MK-801 Five rats previously kindled from the AM were used. Reserpine (Daiichi Seiyaku Co., 3 mg/kg) was administered i.p. 24 h prior to MK-801 treatment (2 mg/kg i.p.). Two hours after the administration of MK-801, electrical stimulation at GST was delivered to the kindled AM. The anticonvulsant action of MK801 alone on previously AM-kindled seizures was compared to that with pretreatment of reserpine in the various seizure parameters as mentioned above.

Seizure Stage

sec 100

AD Duration

Histology For histological e:~amination of the electrode position, the rats were deeply anesthetized and their brains were perfused with 0.9% saline and 10% formalin. The brains were removed, stored in formalin, cornonally sectioned at 10 ~m, stained by hematoxylin eosin and examined under a light microscope.

Statistics All data obtained were expressed as means +

0

pr 0.51 e~ inject

2

4 <; 12

2~,

48

72 hours

Fig. 1. The time course of anticonvulsant effects of MK-80I on kindled seizures from the amygdala. Note that both the AD duration and seizure stage were significantly suppressed 0.5, 2, 4 and 12 h after the injection of 2 mg/kg MK-801, compared with those induced by predrug stimulation. The maximum effects appeared between 2 and 4 h after drug treatment. Value: 2 +_ S.E.M., + + , P < 0.01, Kruskal-Wallis test; *, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA, compared with preinjection.

15 TABLE I

Anticonvulsant effects of MK-801 on previously kindled seizures from the amygdala, frontal cortex, ventral and dorsal hippocampus Values: 2 ± S.E.M.

Dose (mg/kg)

Seizure stage

Amygdala Saline 0.25 0.5 1.0 2.0 4.0 Frontal cortex Saline 0.25 0.5 1.0 2.0 Ventral hippocampus Saline 0.25 0.5 1.0 2.0 Dorsal hippocampus Saline 0.25 0.5 1.0 2.0

AD duration (s)

5.0 2.8 3.5 0.0 ++ 0.3 ++ 0.0 ++

(5-5) (0-5) (0-5) (0-0) (0-2) (0-0)

100.8 46.1 43.2 35.4 37.0 6.7

± ± + ± ± ±

8.6 15.2" 10.0"* 17.4"* 11.4"** 5.6***

5.0 0.8 + 1.4 + 0.2 + 0.4 +

(5-5) (0-1) (1-2) (0-1) (0-1)

5.0 4.8 2.8 + 1.8 ++ 0.4 ++

(5-5) (4-5) (0-3) (0-3) (0-1)

120.3 86.8 64.5 66.3 37.5

± + ± + ±

5.0 2.7 3.8 1.3 ++ 1.3 ++

(5-5) (0-5) (0-5) (0-2) (0-3)

101.2 71.3 76.2 50.3 61.8

± 12.8 ± 14.3 + 15.3 ± 15.5" _+ 13.1

59.8 _+ 10.6 2.4 ± 0.9** 3.2 _+ I).7"* 5 . 0 + 1.4"* 18.4 ± 6.6* 13.8 4.6 13.7" 14.1" 11.8"**

Bilateral forelimb clonus n

Latency (s)

Duration (s)

8/8 4/7 3/6 0/7 0/7 (//3

7.5 ± 1.4 16.5 _+ 5.4 19.6 ± 6.3 -

26.9 11.7 10.0 0.0 0.0 0.0

5/5 0/5 0/5 0/5 0/5

± ± + ± ± +

2.1 4.6* 5.4* 0.0"** 0.0"** 0.0"**

16.6 _+ 3.3 0.0 ± 0.0"** 0.0 ± 0.0"** 0.0+0.0"** 0.0 ± 0.0"**

6/6 5/6 1/6 0/6 0/6

5.2 + 0.6 14.7 ± 3.8* 6.0 -

40.8 29.3 4.6 0.0 0.0

± + ± ± ±

2.9 4.1" 4.3*** 0.0"** 0.0***

6/6 3/6 4/6 0/6 0/6

10.8 ± 2.6 7.3 + 1.3 19.5 ± 5.6 -

30.0 14.1 14.8 0.0 0.0

+ 2.8 ± 6.5 4- 6.5 ± 0.0"** 4- 0.0"**

+P < 0.05; ++P < 0.01; Kruskal-Wallis test, compared with saline. *P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA, compared with saline.

stage 5

SALINE t L-AM " ~

50 sec cut I

MK-801

',

I

~, t I

2 mg/kg

II~,,.,~J,;~ltJ,~J~ I t , i

'11.t~ "Ill llJl,,

q J' 'tlIll ~ ~

li II

J

,ji . ,

I' .,

stage 0

,50pV Isec Fig. 2. An example of afterdischarges recorded from the kindled left amygdala (L-AM) in a previously kindled rat. Arrows indicate the beginning of electrical stimulation. After saline injection a generalized seizure of stage 5 was observed, while the afterdischarge was markedly reduced and the kindled seizure was completely suppressed following 2 mg/kg MK-801 administration.

16 T A B L E II

effect was observed 24, 48 or 72 h after the MK-801 administration. Dose response. As shown in Table I, MK-801 significantly suppressed both the seizure stage and AD duration of AM-kindled seizures in a dose-dependent manner. The appearance of bilateral forelimb clonus was completely suppressed by MK-801 at 1.0, 2.0 and 4.0 mg/kg in all rats. Fig. 2 shows examples of changes in EEG recorded from the kindled AM following MK-801 administration. The 2 mg/kg MK-801 markedly shortened the AD duration and only brief and irregular discharges were induced. Similarly anticonvulsant effects were found in FC, ventral and dorsal HIPP-kindled animals, although the magnitude of the effect differed between the sites (Table I). To clarify these differences, the minimum dose of MK-801 required for completely suppressing kindled generalized seizures (bilateral forelimb clonus), and for reducing A D duration by 50%, is shown in Table II. The minimum dose of MK-801 required to completely suppress generalized seizures in the FC group was significantly lower than that in the AM group. The minimum dose for reducing the AD duration by

The minimum dose of MK-801 requiredfor completely suppressing kindled generalized seizures (bilateralforelimb clonus) and for reducing A D duration by 50% in previously kindled seizures from the amygdala, frontal cortex, ventraland dorsal hippocampus

Values: 2 ± S.E.M.

Kindled sites

Dose of MK-801 Dose of MK-801 for for completely reducing A D duration suppressing by 50% (mg/kg) generalized seizures" (mg/kg)

Amygdala Frontal cortex Ventral hippocampus Dorsal hippocampus

0.61 + 0.13 0.25 ± 0.00

0.43 ± 0.10 0.25 ± 0.00

0.58 ± 0.08

1.73 ± 0.23***

0.54 ± 0.14

1.21 ± 0.31"

*P < 0.05; ***P < 0.00l: one-way ANOVA, compared with the amygdala.

and 12 h after the injection of 2 mg/kg MK-801, compared with those induced by predrug stimulation. The maximum anticonvulsant effects appeared between 2 and 4 h after drug treatment. No significant m N

4 3

i'~

ov

+

+

++

+.

~+

÷+

++

++

+÷

++

++

+÷

÷

2 0

~lr'"=llc~-

~

*~

++

i

,

,

,

o---o Saline

:~

DRUG SESSION

:~

~--aMK-801

0.25 mg/kg

~---~ M K - 8 0 1

1.0

mg/kg

NON DRUG SESSION

"~ 100 ID

z

_o cc

5O

D < 0

5

10

15

ttttt

20

25

30DAYS

Fig. 3. Amygdala kindlin~ seizure development with treatment of MK-801 or saline. MK-801 (0.25 or 1 mg/kg) or saline i.p. was administered from day 2 (~) to 20 (~) 2 h prior to daily electrical stimulation to the amygdala at the intensity of 200 ktA. From day 16 to 20, the intensity of daily stimulation was increased to 400BA ( 1' ), from day 21, drug administration was stopped and the stimulus intensity was decreased to 200 ~tA in the MK-801 groups. Value: 2 + S.E.M., + , P < 0.05; + +, P < 0.01; Kruskal-Wallis test; *, P < 0.05; **, P < 0.01; ** *, P < 0.00l ; one-way AN OVA, compared with the saline group.

17 TABLE III Parameters of amygdala kindling in control and MK-801 (0.25 and 1 mg/kg) treated rats

Values: 5: + S.E.M. 1st Response

21st Day

Seizure stage

Seizure stage

AD duration (s)

Saline 0.3 (0-1) 10.3_+0.7 5.0 (5-5) MK-801 (0.25 mg/kg) 0.1 (0-1) 8.9_+0.9 3.9 (1-5) MK-801 (lmg/kg) 0.2(0-1) 8.2_+0.8 1.5++(1-4)

1st Stage 5 seizure Bilateral forelimb clonus (s)

Total A D duration (s)

GST (/~A)

Generalized seizure

AD duration (s)

AD duration (s)

7/7

91.4_+4.3

69.7_+12.1 31.4_+5.4 400.1_+86.8

114.3+13.5

4/7

61.1+8.4"*

68.0--+7.5

24.9_+2.3 667.6_+82.1

92.9+12.3

1/6

40.0_+10.5"** 74.7_+7.4

22.5_+2.6 694.2_+21.3"

87.5_+9.8

++P < 0.01, Kruskal-Wallis test, compared with saline.

*P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA, compared with saline. 50% required in the F C group was significantly lower and those in the ventral and dorsal H I P P group was significantly higher than that in the A M group. Experiment 2

Fig. 3 shows A M - k i n d l e d seizure d e v e l o p m e n t across the stimulus day with t r e a t m e n t of MK-801 at doses of 0.25 and 1.0 mg/kg. In the saline group, all rats showed progressive increases in both the seizure stage and A D duration, and they reached the first stage 5 seizure by day 17. In the MK-801 groups, however, none of the animals showed a stage 4 or 5 seizure. These rats reached only stage 1 - 3 in the 0.25 mg/kg group and stage 1 - 2 in the i mg/kg group. The A D duration was also significantly reduced from day 8 to 20 in the MK-801 groups. W h e n the stimulus intensity was increased to 400/~A (from 200/~A) from day 16 to 20, no further progression of kindling was observed in the 1 mg/kg group and only a partial increment was seen in the 0.25 mg/kg group. A f t e r the

cessation of MK-801 t r e a t m e n t , an additional 1.6 _+ 0.9 and 5.0 + 0.9 stimulus days were required to produce the first stage 5 seizure in the 0.25 mg/kg and the 1.0 mg/kg groups, respectively. Table III summarizes the effects of MK-801 on A M kindling seizure development. Both the seizure stage and the A D duration on day 21 (the time when the drug was discontinued) were significantly suppressed in the 1 mg/kg MK-801 group c o m p a r e d with the saline group. The total accumulated duration of A D activity required to kindle was significantly longer in the 1 mg/kg MK-801 than that in the saline group. Thus, daily treatment of MK-801 prior to A M stimulation significantly r e t a r d e d A M kindling seizure development. Experiment 3

To investigate mechanisms of the anticonvulsant action of MK-801, rats were p r e t r e a t e d with reserpine prior to MK-801. The results from this experi-

TABLE IV The eff'ects of pretreatment of reserpine on anticonvulsant effects of MK-801 on kindled seizures from the amygdala

Values: 2 _+S.E.M. Seizure stage

A D duration (s)

Bilateral forelimb clonus n

Saline MK-801 (2 mg/kg) Reserpine (3 mg/kg) + MK-801 (2 mg/kg)

5.0 (5-5) 0.0 ++ (0-0)

92.2 _+5.5 55.4 _+8.3*

5/5 0/5

2.2 ++ (1-4)

57.4 _+8.4*

1/5

++P < 0.01; Kruskal-Wallis test, compared with saline. *P < 0.05; ***P < 0.001; one-way ANOVA, compared with saline.

Latency (s)

Duration (s)

6.4 _+0.5

36.8 _+2.0 0.0 _+0.0"**

38.0

1.0 _+0.9***

18 Amygdala AP: + 0 . 6

Frontal Cortex AP: +I.0

dorsal and ventral Hippocampus AP: - 2 , 6

,::..:';ti

Fig. 4. Loci of the centers of the electrode tips estimated from histological examination and plotted on a representative section through the amygdala, frontal cortex, dorsal and ventral hippocampus.

ment are shown in Table IV. Pretreatment of reserpine did not significantly alter the anticonvulsant effects of the MK-801 alone. In only one animal, the anticonvulsant action of MK-801 was partly antagonized by pretreatment of reserpine, and it showed a generalized seizure of stage 4 with a prolonged latency and bilateral forelimb clonus of very short duration.

Histology As shown in Fig. 4, histological examination showed that all electrodes were located within the intended target sites. DISCUSSION The potent anticonvulsant effects of MK-801 on previously kindled seizures in our study are consistent with the previous results of in vivo 40'41 and in v i t r o 1'22A7 studies on anticonvulsant action of competitive antagonist of N M D A receptors, such as APV and APH. The blockade of N M D A receptors by the non-competitive antagonist MK-801 is suggested to be mediated via an interaction with the N M D A receptor-associated ion channels 25. Since high doses of MK-801 markedly shortened the focal self-sustained AD of the kindled sites in our study, the main mechanism of the action may be due to suppressing kindled focal epileptic activity via the block-

ade of the NMDA-related ion channels. As expected, AM-kindled seizure development was quite sensitive to daily pretreatment of MK-801, in which the 1 mg/kg MK-801 almost completely blocked a progressive increase in the AD duration during drug sessions. The accumulated AD duration during kindling has been proposed to be an important determinant of the kindling process 39. In our experiment, the pretreatment of MK-801 markedly prolonged the total accumulated AD duration required to complete kindling. Furthermore, 5 days of stimulation at 400 # A did not increase seizure severity, suggesting that the action of MK-801 was not elevating the threshold of seizure generalization, but served a direct role in the initiation of epileptic activity. It has previously been indicated that N M D A receptors may be activated during kindling and progressive increases in epileptic discharges could be dependent upon this activation 4'23. Recently it was also shown that MK-801 prevented induction of longterm potentiation (LTP) of the hippocampal CA I region 7. Since LTP has been shown to occur during kindling 28'48, MK-801 might prevent development of kindling-induced transsynaptic changes, as well as suppress focal burst activity. Morimoto et al. 32'3a recently acquired that collapse of GABA-mediated inhibition and subsequent activation of the N M D A systems may be the essential

19 seizure-triggering mechanism in kindling. O u r present results support the hypothesis and further imply that N M D A system may play a critical role in the kindled epileptogenesis. A l t h o u g h the m i n i m u m dose of MK-801 required to completely suppress the generalized convulsions did not significantly differ between A M , ventral and dorsal H I P P , a significantly higher dose of MK-801 was n e e d e d to reduce focal A D duration in the ventral and dorsal H I P P than in the A M . This evidence may be related to the highest levels of N M D A receptors in the H I P P 9. In the FC, however, both seizure generalization and focal A D were much m o r e easily suppressed, c o m p a r e d with the limbic foci. A l t h o u g h the reason for these differences remains uncertain, they may be related to the clinical evidence that epileptic seizures which originate from the H | P P are generally intractable 43. In addition, p r e t r e a t m e n t of reserpine, a catecholamine depleting agent, did not antagonize the anticonvulsant action of M K - 8 0 I , suggesting that the anticonvulsant action was not mediated by the catecholaminergic system. In current therapy of intractable epileptic patients, antiepileptics which potentiate the central G A B A e r -

gic system, such as p r o g a b i d e and valproate, have been widely tested 3'1°'27'46. Their efficacy is, however, thought to be insufficient m'j 1,27.46. A recent elec-

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trophysiological study has revealed that, a b n o r m a l cell bursts, similar to those r e c o r d e d in the brain tissue from kindled animals 26'29"36, are o b s e r v e d and prevented by A P V in in vitro slices of epileptic human brain samples excised during surgical treatment 2. Considering the potent anticonvulsant action of MK-801 in kindled seizures, we suggest the clinical utility of N M D A antagonists, providing that the side effects could be minimized.

ACKNOWLEDGEMENTS The authors gratefully thank Dr. P.S. A n d e r s o n of Merck Sharp and D o h m e Research L a b o r a t o r i e s for supplying MK-801. W e also thank Mr. T. M o r i w a k e for his technical assistance, and Prof. Saburo Otsuki and Dr. Kazufumi A k i y a m a for their useful comments on the manuscript. Finally, we wish to express our gratitude to Prof. R.J. Racine of M c M a s t e r University for his critical review of the manuscript.

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