Topiramate reduces abnormally high extracellular levels of glutamate and aspartate in the hippocampus of spontaneously epileptic rats (SER)

Life Sciences, Vol. 59, No. 19, pp. 1607-1616, 19% Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights resewed 0024-3205/% $15.00 + .@I

PI1 SOO24-3205(96)00492-4

ELSEVIER

TOPIRAMATE

REDUCES ABNORMALLY

GLUTAMATE

AND ASPARTATE

HIGH EXTRACELLULAR

IN THE HIPPOCAMPUS

LEVELS OF

OF SPONTANEOUSLY

EPILEPTIC RATS (SER) Tomoyuki Kanda, Masako Kurokawa, Sachiko Tamura, Joji Nakamura. Akio Ishii, Yoshihisa Kuwana, Tadao Serikawa”. Junzo Yamada”, Kumatoshi Ishiharab. Masashi Sass”. Pharmaceutical

Research Laboratories.

Kyowa Hakko Kogyo Co. Ltd., Shizuoka 411, Japan.

“Institute of Laboratory Animals, Faculty of Medicine, Kyoto University, “Department of Pharmacology,

Kyoto 606, Japan.

Hiroshima University School of Medicine. Hiroshima 734, Japan. (Received in

final

form August 27, 19%)

Summary ‘l‘he spontaneously

epileptic rat (SER), a double mutant. manifests both tonic and

absence-like

seizures. The effect of topiramate,

extracellular

levels of excitatory amino acids (EAA) in the hippocampus

investigated

using in vivo microdialysis.

in dialysates of hippocampus

a novel antiepileptic

drug, on the of SER was

The basal levels of glutamate and aspartate

in SER were 2- to 3-fold higher than those in normal

Wistar rats. Both the dose-response relationship and the time course ofthe suppression oftonic seizures by topiramate were similarto the attenuation ofglutamate level in SER. l’opiramate

(40 mgikg

i.p.) significantly

(PcO.05) reduced both glutamate

and

aspartate lebels in SER while showing no effect on normal Wistar rats. These findings suggest that topiramate reduces abnormally high extracellular levels of glutamate and aspartate in ihe hippocampus anticonvulsant

of SER. This effect may. at least in part, be related to the

activity of topiramate.

Key Words: spontaneously epileptic rat, in viva microdialysis, excitatory amino acids, glutamate, tonic seizure, topiramate Topiramate

(2,3:4,5-bis-O-(

novel antiepileptic

Address correspondence Pharmaceutical Nagaizumi-cho.

1-methylethylidene)&D-fructopyranose

drug that inhibits

electroshock-induced

seizures

sulfamate] (Fig. 1) is a in rats and mice, sound-

to: Yoshihisa Kuwana, Ph.D.

Research Laboratories. Sunto-gun,

Kyowa Hakko Kogyo Co. Ltd.. 1188. Shimotogari

Shizuoka, 411, Japan. Tel. 81. 559. 89. 2013 Fax. 81. 559. 86. 7430.

Topiramate Reduces EAA Levels in SER

1608

induced

seizures

in DBA/2

pentylenetetrazol-,

mice

and absense-like

picrotoxin- and bicuculline-induced

Vol. 59, No. 19,1996

seizures

in SER, but does not affect

seizures ( l-4). In clinical studies, topiramate

has been found to be effective in treating refractory partial seizures with or without secondary generalization

(5 8). The mechanism underlying

FH,OSO,NH, I 0 CH,

0

0

OR’or

X

unclear. Previous

glutamate

receptors, (NMDA),

including

N-methyl-D-

DL-u-amino-3-hydroxy-5-

methyl-4-isoxazole-propionate

(AMPA)

and

kainate (KA) receptor subtypes, y-aminobutyric acid

(GABA),

benzodiazepine

receptors (1). Biochemical

t-

channels Fig. 1.

(9). However,

in electrophysiological

studies, topiramate reduced both the duration and

Chemical structure of topiramate.

spike frequency hippocampal

frequency of action potentials

elicited by depolarizing

of epileptiform-like

activity of

neurons in culture and reduced the

electrical currents (10). On the other hand,

enhanced the activity of GABA at some types of GABA,

electrophysiological

or adrenergic

studies also indicated

that topiramate does not block calcium or sodium

CH3

topiramate

remains

studies indicate that topiramate does not bind to aspartate

CH,

6;

H3C

action of topiramate

the anticonvulsive

receptors (11). Moreover.

studies suggested that topiramate is a weak antagonist of AMPA/KA-sensitive

receptors (10). An abnormal release of excitatory amino acids (EAA) is involved in the generation and expression of some epileptic seizures (12- 16). In addition, a change in the number of quisqualate-sensitive glutamate receptor sites has been found during the kindling process (17). Therefore, some forms of epilepsy may result from a breakdown in the regulation of EAA neurotransmitters. The spontaneously

epileptic rat (SER) is a double mutant rat (zi/zi, t&m)

obtained by mating the

tremor heterozygous rat (tm/+) with the zitter homozygous rat (zi/zi) (18,19). SER exhibit both tonic seizures and absence-like

seizures without external stimuli. Tonic seizures are also induced by mild

stimuli such as tapping or sound (20,21). Although the pathological basis for these epileptic seizures is not yet fully revealed, the SER appears to be a useful model for some forms of human epilepsy (2 l23). There is no change in the density of phencyclidine

binding sites of glutamate receptors in SER

brain (24). In this study we demonstrate that the extracellular levels of glutamate and aspartate are abnormally high in dialysates from the hippocampus these EAAs.

of SER and that topiramate at anticonvulsant

doses reduces

Topiramate Reduces BAA Levels in SER

Vol. 59, No. 19, 1996

1609

Materials and Methods Animals

Male and female SER and male Wistar rats (17-I 8 weeks old) were used. SER were bred

in our institute and kept individually

in pail-type cages in the animal quarters. Male Wistar rats were

bought from Charles River (Tokyo, Japan). Commercial food pellets (F-2, Funabashi Farm, Japan) and water were given ad libitum. Room temperature and relative humidity were kept at 23-25 ’ C and 50-60%, respectively. In viva microdialysis

The room was illuminated

from 7:00 to 19:O0.

Under anesthesia with chloral hydrate (300 mgikg i.p.) or sodium pentobarbital

(40 mg/kg i.p.), the animals wereplaced in a stereotaxic frame (Narishige, Japan). Each microdialysis probe (Eicom, Japan) with a cylindrical dialysis membrane 2 mm long and 0.24 mm outer diameter (BDP-I-8-02)

was tested with a standard solution for recovery of chemicals in the dialysate at room

temperature before implantation.

The probe was then held in a micromanipulator,

implanted into the

hippocampus (coordinates ofthe target location: A -5.8 mm from Bregma, L 4.5 mm, depth 6.5 mm from the brain surface ), according to the atlas of the rat brain (25) and fixed on the skull with dental cement. At least 24 hours after implantation out under unanesthetized

and unrestrained

of the probe. the microdialysis

experiment was carried

conditions. Ringer’s solution (147 mM NaCI, 4 mM KCI.

2.97 mM CaCl,) was perfused at a flow rate of2 ulimin. Dialysates were collected in microtest tubes containing

10 pl of 40 pmol homoserine in 0.1 N perchloric acid as internal standard. Sampling was

made every 10 min for analysis of amino acids by a microfraction Medicine AB, Sweden).

After being allowed to equilibrate

collector (CMA 140, Carnegie

( 120 min), 9 samples were collected

before and 8 samples after drug administration. Aliquots

(20 ul) of dialysates

chromatography derivatization

with

were analyzed

a fluorescence

for amino acids using high-performance

detector

following

(26). The sample was derivatized

0-phthalaldehyde

using a computer-controlled

(OPA)

liquid reagent

autoinjector

with

column switching (CMA 200/240, Carnegie Medicine AB, Sweden). Each sample was automatically added to 10 ul of OPA reagent from the stock solution and mixed for 60 sec. Amino acids were separated on an ODS reversed-phase

column (Bio Phase ODS AA. BAS, Japan). The column

temperature was kept at 35 o C. The mobile phase was 0.1 M acetic acid trihydrate buffer, pH 6.00, containing

0.5 mM EDTA 2Na, 9% acetonitrile,

3% tetrahydrofuran.

Detection was made using a

fluorescence detector at 345 nm and 440 nm wavelengths for excitation and emission, respectively. Measurement spontaneous

of tonic seizure duration in SER Tonic seizures, both of tactile stimulus-induced seizures, were monitored continuously

beginning 90 min before drug administration.

for 170 min during the microdialysis

and

period,

The duration of each seizure was measured, and total

seizure duration was summed at 10 min intervals. To trigger seizures, a mild and momentary tactile stimulation

was applied to the back of the rat as specified in the figures.

Chemicals

Topiramate was obtained from R. W. Johnson Pharmaceutical

House, PA, USA). 0-phthaldialdehyde,

Research Institute (Spring

homoserine, glutamate and aspartate were purchased from

1610

Topiramate Reduces EAA Levels in SER

Vol. 59, No. 19, 1996

Sigma Chemical Co. (St. Louis, MO, USA). Topiramate was dissolved in a mixture of ethylalcohol, propylene glycol and distilled water at the ratio of 2.1: 8: 9.9 (V/V). Other reagents used were of analytical grade. To prepare the OPA reagent, 5.4 mg of 0-phthaldialdehyde ~1ofethylalcohol

andthenmixedwith

5 pl of2-mercaptoethanol

was dissolved in 200

anddilutedwitho.

1 Mborate buffer,

pH 9.1. This reagent mixture, once prepared, was never used for longer than a 3-day period. Statistical analysis

Significant

differences of the basal levels of glutamate and aspartate between

SER and Wistar rats were determined with the Mann-Whitney’s

U-test. Significant

differences of

parameters between the vehicle- and drug-treated groups were determined with the Mann-Whitney’s U-test (Fig. 5) or with the Kruskal-Wallis

test followed by Scheffe-type non-parametric

test (Fig.

3 and 4). P values smaller than 0.05 were considered significant. Histological examination

At the termination

of the experiments,

anesthesia with an overdose of sodium pentobarbital,

rats were decapitated under deep

and the brain removed was fixed with 10%

formalin in 0.1 M sodium phosphate buffer, pH 7.2. Then, the coronal section ofthe brain was stained with hematoxylin

and eosin. Location of the implantation

tissue damage near the site of implantation

site of the dialysis probe and the extent of

were examined.

A -5.h

Fig. 2. Position of the tip of dialysis probe. (A): representative

coronal

hippocampus.

The number at the upper left

section

through

the

corner indicates the distance (mm) from bregma according to a stereotaxic atlas (Paxinos and Watson, 1986). Dotted line of dialysis probe indicates the membrane portion ofeach dialysis probe.

HPC: hippocampus.

(B): a coronal

section stained with hematoxylin and eosin. (C): larger magnification of a part of B. The arrow in the figure indicates the lesion made by removal of the implanted probe.

Results Location of the microdialysis ~.~_

probe ~~ in_ the hjppocampus.

In all 25 rats examined, the tip of the

Topiramate Reduces EAA Levels in SER

Vol. 59, No. 19, 1996

microdialysis

probe was properly located in the hippocampal

1611

CAI-3 area (Fig. 2A and B). The

lesions were less than 250 pm in diameter (Fig. 2C). There were no remarkable tissue changes, such as glial response, at the termination

of the experiment.

Effect of topiramate on tonic seizure in SER. The mean total durations of the tonic seizure induced by tactile stimulationand (n= 8), respectively dependently

spontaneous convulsions were45.8*

(the pretreatment

5.0 (S.E.M.) and 6.8h2.1 sec/lOmin

values in Fig. 3 VEH). The tonic seizures in SER were dose

suppressed by topiramate at doses of 10.20 and 40 mg/kg i.p. (Fig. 3). The vehicle did

not affect the tonic seizures (Fig. 3 VEH).

TOP 20 mg/kg

VEH 70 80

1.1

11

111

11

11

11

D

I

11

40

0

-30

30

60

~90

(mln)

Tame

~30

40

*2*

*

1

90

0

-o&-o 30

Time

TOP 1 Omg/kg

11

-

60

(md

TOP 40 mg/kg

11

80

z-70 5

60

i

50

111 11

11

h: 40

2

$ 30 P 20 : e 10 2 90

-60

-30

0 Time

30

60

0 -90

-60

30

(mm)

0 Time

30

60 (m1n)

FigJ

The time course of the

vehicle-treated

inhibitory effects of topiramate on the total duration of tonic seizures in SER. VEH:

group. TOP 10, 20 and 40 mg/kg : topiramate IO, 20 and 40 mgikg i.p.-treated group,

respectively. The abscissa represents the time after administration. The mean of the total duration of tonic seizures was calculated at 10 min interval. Mild tactile stimuli were applied twice 5 min-interval at the time indicated by the arrow. Each point and bar represents the mean and S.E.M. obtained from 6-8 ani mals, respectively. Arrow indicatesthetimeofthestimulusapplication with the vehicle-treated group.

tothebackoftheanimal.

*P40.05 ascompared

Vol. 59, No. 19, 1996

Topiramate Reduces JZAA Levels in SER

1612

Effect oftopiramate onthe level of amino acids indialysates

from the hippocampusof

SERand _-._._ Wistar _

rats. Topiramate caused a significant reduction of glutamate levels in SER dose dependently

(Fig.

4). The maximum decrease in glutamate level occurred within 20-40 min after drug administration, and the return of glutamate to basal level occurred more slowly after higher dosages of topiramate (Fig. 4). The mean basal level of glutamate and aspartate in the hippocampal dialysates of SER were 2 to 3-fold higher than those observed in the Wistar rats. The mean basal level of glutamate and aspartate ofthevehicle-treatedgroupinFig.

5 was5.29~0.77and3.79~0.37pmole/lOmin(n=8),respectively.

On the other hand, those of Wistar rats were 2.46 * 0.33 and 1.39 * 0.30 pmole/lO were statistically

180

1

1601 Q,

140-

t;;

5 ZG 5 z b 4

120loo80 *

76

z

60

fi ?I s0

40 20 -90

I

I

/

I

I

-60

-30

0

30

60

Time

(min)

Effects oftopiramate on hippocampal glutamate level in SER. The abscissa represents the time after administration. The ordinate indicates the values calculated as % of basal level at 10 min interval. Basal value calculated average of four points, -80 to -60 and -40 to -20 min (before drug treatment and without tactile stimulation points). Each point and bar represents the mean and S.E.M. obtained from 5-8 animals, respectively. Open circle indicates the vehicle-treated group. Closed symbols indicate the topiramate-treated groups (circle, triangle and square: IO,20 and 40 mg/kg i.p., respectively), Arrow indicates the time of stimulus application to the backofthe animal. *P
1613

Topiramate Reduces E!.AALevels in SER

Vol. 59, No. 19, 1996

significant (P
(Fig. 4, 5). Topiramate

of

(40 mg/kg i.p.) caused a

significant reduction of glutamate and aspartate levels in SER while causing no change in normal Wistar rats (Fig. 5). The glutamate level in SER was reduced by topiramate (40 mg/kg i.p.) to that in normal Wistar rats untreated with the drug.

Wistar

SER

(A) 12

111 11

11

1

04

11

”

-90

-60

-30

0

30

T1m.E

60

0 0

-60

-30

0

30

60

-90

“‘,“*,-“““‘( -60

-30

0

30

60

Time

(mid

Time

(mm)

Time

(min)

(mm)

Fig.. The effect oftopiramate (40 mg/kg i.p.) on concentrations of glutamate and aspartate in dialysates of SER and Wistar rats hippocampus. Each figure represents (A) glutamate, (B) aspartate in SER and (C) glutamate, (D) aspartate in Wistar rats. The abscissa represents the time after administration. Open and closed circles indicate the vehicle and topiramate-treated

groups, respectively. Each point and bar represents the mean and S.E.M.

obtained from 5-8 animals, respectively. Arrow indicates the time of stimulus application to the back of the animal. *PCO.O5as compared with the vehicle-treated group.

Discussion ~__ The basal extracellular

levels of glutamate and aspartate as determined by microdialysis

to 3-fold higher in the hippocampus

were 2-

of SER as compared to those of normal Wistar rats, despite the

fact that the SER were nearly seizure free (for example, see the -90 to -80 min-values

in Fig. 5).

1614

Topiramate

Abnormal extracellular

Reduces EAA Levels in SER

Vol. 59, No. 19, 1996

levels of excitatory amino acids (EAA) have also been observed in other

animal models of epilepsy. For instance, in kindled animals. a decrease in GABA levels and an increase of EAA levels in hippocampus has been reported (27 -30). A microdialysis animals has demonstrated during establishment spontaneous

that extracellular

of kindling

and maintained

nor tactile stimulus-induced

study in kindled

levels of glutamate in the rat hippocampus

increased

there after (31). On the other hand, neither

seizures affected EAA levels in the SER. The change in

EAA levels could not be detected probably because the tonic seizures of SER are milder than those ofelectroconvulsive

shock-induced

seizures. Therefore. the high extracellular levels ofEAA in SER

may not have been induced by seizures, but rather may be one of the causal factors in the induction of convulsive seizures in SER. although a decreased content of dopamine may also contribute to the occurrence of the seizures(32). The causes of increased levels

of EAAs

in hippocampus

in SER are

presently not clear. but they may possibly involve an increase in their synthesis (and/or release) or decrease in metabolism

(and/or reuptake of the EAAs to glial cells or neurons).

In the present study. topiramate

reduced extracellular

hippocampus of SER. The topiramate-induced

levels of glutamate and aspartate in the

reduction oftonic seizures corresponded well with the

decrease of glutamate levels in SER. Especially. during 60-80 min after topiramate treatment in Fig. 3 and 4, the time course and dose-dependence

of reduction of tonic seizures by topiramate matched

well with those of the decreased

glutamate

levels in SER. It seems likely, therefore. that the

anticonvulsant

in SER is due, at least in part, to reduction of an abnormally high

extracellular

effect oftopiramate

level of EAA. Brown et al. (11) reported that topiramate enhanced GABA-mediated

ofmouse cerebellar

chloride flux in a primary culture reduced kainate-induced

granule cells. On the other hand, topiramate

inward currents in slices of normal rat hippocampus,

although the drug has

no affinity for glutamate receptors (3). These findings suggested that topiramate may have enhancing cl’fects on GABAergic

inhibitory

transmission

and/or a suppressing

effect on glutamatergic

excitatory transmission,

which could result in the decrease of extraccllular levels of these EAAs in

SLR. In conclusion, higher levels of EAAs were found in the hippocampus of SER than Wistar rats, and topiramate reduces these EAA levels at anticonvulsant

doses.

Acknowledgements We wish to thank Dr. Richard P. Shank for helpful discussions. and Ms. Toyoko Kashiwagi for histological

We also thank Mr. Jun-ichi Sano

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