7-Chlorokynurenic acid antagonizes the anticonvulsant activity of d -cycloserine in maximal electroshock seizures

73

Epilepsy Research, 13 (1992) 73-8 1 0920-121 l/92/$05.00 0 1992 Elsevier Science Publishers B.V. All rights reserved EPIRES 00512

7Chlorokynurenic acid antagonizes the anticonvulsant activity of D-cycloserine in maximal electroshock seizures

Steven L. Peterson Department

of Medical Pharmacology

and Toxicology,

Texas A&M University,

College Station,

TX 77843-1114.

USA

(Received 13 August 1991; revision received 2 June 1992; accepted 23 June 1992) Key worak: Maximal electroshock; Seizure; o-Cycloserine; L-Cycloserine; ‘I-Chlorokynurenic acid; Glycine receptor

This study evaluated the anticonvulsant activity of D-cycloserine against maximal electroshock seizures in rats. Systemically administered D-cycloserine (i.p.) inhibited maximal electroshock-induced tonic hindlimb extension in a dose-dependent manner with an EDSo of 153 mg/kg. No neurological deficit was detected at any dose of u-cycloserine. In contrast, L-cycloserine had no effect on the maximal electroshock seizures. Administration of the strychnine-insensitive glycine receptor antagonist 7-chlorokynurenic acid (100 nmol, i.c.v.) significantly antagonized the anticonvulsant activity induced by D-cycloserine. Centrally administered D-cycloserine (i.c.v.) induced significant anticonvulsant activity l-2 h after administration with an approximate EDso of 5 pmol. 7-Chlorokynurenic acid (100 nmol, i.c.v.) significantly antagonized the anticonvulsant activity of centrally administered o-cycloserine. L-Cycloserine (icv., 2 h) induced no significant anticonvulsant activity. These results provide evidence that the anticonvulsant activity of o-cycloserine in maximal electroshock seizures may be mediated by strychnine-insensitive glycine receptors.

Introduction

In vitro electrophysiological studies indicate that glycine potentiates the response evoked by N-methyl-D-aspartic acid (NMDA) in neurons cultured from rat cerebral cortex17. Glycine has been proposed to interact with an allosteric recognition site located within the NMDA receptor/ionophore complex2*3, which facilitates opening of the ion channel in a strychnine-insensitive manner17. It has been suggested that glycine is a glutamate coagonist and is an absolute requirement for activation of the NMDA receptor/ionophore complex”.

Correspondence to: S.L. Peterson, Department of Medical Pharmacology and Toxicology, Texas A&M University, College Station, TX 77843-l 114, USA.

Enhancement of ongoing NMDA activity by intracerebellar glycine administration8935953 indicates that the strychnine-insensitive glycine receptors are not saturated in vivo and that exogenous glytine may effect NMDA-mediated neuronal excitability. o-Serine is a strychnine-insensitive glycine receptor agonist that mimics the effects of glycine 19*35F42T53,54, while 7-chlorokynurenic acid (7CLKYNA) is a selective antagonist of the strychnine-insensitive glycine receptor18,20,4’. Based on the role of NMDA receptors in the initiation and propagation of seizures” and the in vitro data demonstrating NMDA potentiation by glytine, it has been proposed that glycine is proconvulsant12. In contrast, systemic administration of glycine to intact animals produces anticonvulsant effects. Glycine alone has anticonvulsant activity in sei-

74

zures induced by hyperbaric oxygen52, 3-mercaptopropionic acid3s, auditory stimuli in DBA/2 mice4’, kynurenine22 and amygdala kindling43.

ture of chloral

Glycine

guide cannulas.

potentiates

y-aminobutyric

the anticonvulsant

acid (GABA)

activity

transaminase

of

inhibi-

tors, a GABA reuptake inhibitor and a GABA agonist in 3-mercaptopropionic acid-induced seizures37,39. Glycine also potentiates the activity of clinically and rat3s33

effective

anticonvulsants

in mouse49

models of epilepsy. Furthermore, the glycine potentiation of anticonvulsant drugs in maximal electroshock is mimicked by D-serine and antagonized by 7-CLKYNA33. Because the effect is stereospecitic and antagonized by a specific antagonist, the results of previous studies indicate that glycine potentiation of anticonvulsant drug activity may be mediated by strychnine-insensitive glycine receptors33. The purpose of the present study was to characterize the anticonvulsant activity of D-cycloserine in maximal electroshock seizures. rr-Cycloserine is reported to be a partial agonist of the strychnineinsensitive glycine receptor’5,24,27*5’ that readily crosses the blood-brain barrier28. In addition, Dcycloserine is a D-amino acid and as such is not involved in mammalian metabolism’3. Thus, any action of D-cycloserine is likely to be mediated by a specific receptor mechanism and not a nonspecific disruption of metabolism. Methods Male Wistar rats, obtained from Harlan, Inc. and initially weighing 76100 g, were used for these experiments. The animals were maintained in a climate controlled vivarium on a 12 h lightdark cycle and were allowed free access to food and water. Maximal electroshock seizures were induced with a 60 cps, 150 mA and 0.2 s duration current generated by a Wahlquist stimulator (Salt Lake City, UT). The stimulus was administered through saline-soaked cornea1 electrodes. The endpoint used to determine the seizure response was the loss of tonic hindlimb extension. This occurred when, after the stimulus, the hindlimbs did not extend beyond a 90” angle to the torso. The rats were anesthetized with Equithesin (mix-

hydrate,

pentobarbital,

magnesium

sulfate and ethanol) for the surgical implantation of the intracerebroventricular (i.c.v.) injection A 22 gauge guide cannula

Products, Inc., Roanoke, mm dorsal to the right

(Plastic

VA) was implanted 0.2 lateral ventricle. A 28

gauge stylet was placed in the cannula to prevent clogging when not in use. Anchor screws were placed in the skull and the entire assembly was secured with dental acrylic cement. All animals were allowed 5-7 days recovery before seizure testing. A 28 gauge injection needle that extended 1 mm beyond the distal end of the guide cannula was inserted into the guide cannula for the i.c.v. drug administration. Each animal was used only once. Verification of the cannula placements was made at the end of the experiments by localization of a dye which had been infused into the lateral ventricles. The rats were anesthetized with Equithesin and infused with 10 ~1 of a saturated Fast Green solution over a 2 min period. The cannula was left in place for 1 min at the end of the infusion to allow diffusion of the solution into the ventricles. The animals were killed 5 min after the start of the infusion, and the brains were dissected for localization of the dye. Only data from animals with dye localized in the lateral ventricles were included in the study. The animals without i.c.v. cannula implants were tested for neurological deficit during the 5 min period preceding the seizure test. A battery of three tests was administered to each animal and failure to pass two of the tests was taken as a determination of neurological deficit or drug-induced neurotoxicity44. The specific tests used were: (1) rotarod; (2) position sense; (3) gait and stance as described previously3’. For systemic administration D-cycloserine and Lcycloserine (Sigma, St. Louis, MO) were dissolved in 0.9% saline and administered by intraperitoneal (i.p.) injection. For i.c.v. administration, 7-CLKYNA (Research Biochemicals, Inc., Natick, MA) was dissolved in 0.5 N NaOH. The solution was adjusted to pH 7.4 with 0.1 M sodium phosphate buffer and brought to final concentration for i.c.v. infusion with 0.9% saline. The 7-CLKYNA vehicle control solution consisted of the same volume of

75

0.5 N NaOH adjusted to pH 7.4 with 0.1 M sodium phosphate buffer and diluted with 0.9% saline. D-Cycloserine was dissolved in 0.9% saline, adjusted to pH 7.4 with 0.1 M sodium phosphate buffer and brought to final concentration for i.c.v. infusion with 0.9% saline. The vehicle control solution consisted of 0.9% saline adjusted to pH 7.4 with 0.1 M sodium phosphate buffer. 10 ~1 of the i.c.v. solutions was infused into the lateral ventricles at the rate of 5 @/min. The cannulas were left in place for 1 min after the end of the infusion to allow diffusion of the solution into the ventricles. After the infusion the protective stylet was replaced and the animal observed for behavioral changes induced by the i.c.v. infusion. The rats were seizure tested 5 min after the start of the infusion unless otherwise indicated. The quanta1 tonic hindlimb extension data were analyzed by probit regression. Estimation of parameters was performed through an iterative process using a maximum likelihood loss function. The significance of addition or deletion of an independent variable (e.g., treatment or treatment dose) was tested by comparing the goodness of tit for models with and without the variable of interest. The result is reported statistically as the incremental maximum likelihood x2 with degrees of freedom and probability level (Complete Statistical System by Statsoft, Inc., Tulsa, OK). The dose response curve and EDse for D-cycloserine were determined by the method of Litchtield and Wilcoxon45.

200

100

D-cycloserine

400

300

500

(mg/kg)

Fig. 1. The anticonvulsant activity of D-cycloserine in maximal electroshock seizures. o-Cycloserine was administered i.p. 1 h prior to the seizure test. Ten rats were tested at each dose. No neurological deficit was detected at any dose.

firmed that a 400 mg/kg dose of D-cycloserine protects all the rats from maximal electroshock-induced tonic hindlimb extension (Fig. 2). In contrast, none of the rats treated with 400 mg/kg Lcycloserine was protected from tonic hindlimb extension (Fig. 2). Central administration (i.c.v.) of 7-CLKYNA inhibited the anticonvulsant activity induced by systemically administered D-cycloserine. As reported previously33, i.c.v. administration of 100 nmol 7CLKYNA 5 min prior to seizure test does not af-

g

z; Et; SW

Systemic administration of D-cycloserine inhibited maximal electroshock-induced tonic hindlimb extension in a dose-dependent manner (Fig. 1). Preliminary experiments determined that the time of peak effect for D-cycloserine after i.p. administration was 1 h (data not shown). The ED5,, for Ddycloserine (i.p., 1 h) was 153 mg/kg and 400 mg/ kg inhibited tonic hindlimb extension in all animals (Fig. 1). No neurological deficit was detected in any of the rats. Systemic administration of L-cycloserine had no anticonvulsant activity in maximal electroshock seizures as determined by the occurrence of tonic hindlimb extension. A second experiment recon-

KJ-

g o& Ez nx

O-

11 r

1

D-cycloserine (400

mg/kg)

L-cycloserine (400

mg/kg)

Fig. 2. L-Cycloserine induces no anticonvulsant activity in maximal electroshock seizures. None of the rats treated with 400 mg/kg L-cycloserine was protected from maximal electroshockinduced tonic hindlimb extension. All of the animals dosed with 400 mg/kg o-cycloserine were protected from tonic hindlimb extension. The drugs were administered i.p. 1 h prior to the seizure test. Eleven rats were tested with each stereoisomer. 1 indicates a significant (2= 30.5, df= 1, P= 0.001) difference between the groups.

76

TABLE

I

Evaluation of the anticonvulsant activity of 7-chlorokynurenic acid and Dcycloserine after i.c.v. administration in maximal electroshock seizures Time of administration indicates

indicates

tonic hindlimb

the number

extension

Drug/Dose

of minutes

as induced

after the beginning

by the maximal

Time of administration

of the i.c.v. infusion

that the rats were seizure tested. THE

electroshock.

(min)

n

Number

with THE

% THE

_

7-CLK YNA 10 nmol

5

5

5

100

50 nmol

5

5

5

100

100 nmol

120

4

4

100

DCycloserine IO pm01

5

7

100

10 pm01

10

3

100

10 pm01

60

I

10 pm01

120

0

0

IO pm01

240

3

100

feet the occurrence of maximal electroshock-induced tonic hindlimb extension. Similarly, 10 or 50 nmol of 7-CLKYNA (i.c.v.) failed to affect the occurrence of tonic hindlimb extension (Table I). A 100 nmol dose of 7-CLKYNA administered 2 h prior to seizure test also had no effect (Table I).

1

vehicle

j

n

control

100 nmole

(I.c.v.)

7.chlorokynurenic

acid (1.c.v.)

16.6

However, as shown in Fig. 3, 100 nmol of 7CLKYNA (i.c.v.) significantly (x2 = 29.1, df = 1, P=O.O02) increased the incidence of tonic hindlimb extension in rats treated with 300 mg/kg Dcycloserine (i.p., 1 h). Central administration of D-cycloserine also inhibited maximal electroshock-induced tonic hindlimb extension, The time of peak effect after i.c.v. administration was 2 h although substantial anticonvulsant activity was observed at 1 h (Table I).

1

II 9

D-cycloserine (300

Fig. 3. 7-CLKYNA systemically ministered

antagonizes

administered 300 mg/kg

i.c.v. infusion

mgikg)

the anticonvulsant

D-cycloserine. n-cycloserine

h and 5 min, respectively,

and i.c.v. solutions

of

were ad-

(i.p.) in addition

of either the vehicle control

tions. The D-cycloserine

activity

All animals

or 7-CLKYNA

to the solu-

were administered

prior to the seizure test. The number

of rats in each group is indicated in the columns. 1 indicates a significant (x2 = 29. 1, df= 1, P = 0.002) difference from the i.c.v. vehicle group.

(pmole, i.c.v., 2 hr)

1 Fig. 4. The anticonvulsant cycloserine administered number

in maximal

activity

electroshock

by i.c.v. infusion of rats in each group

of centrally

administered

seizures. o-Cycloserine

D-

was

2 h prior to the seizure test. The is indicated

in the columns.

77

D-cycloserine (IO pmole,

i.c.v.)

Fig. 5. 7-CLKYNA antagonizes the anticonvulsant activity of centrally administered o-cycloserine. All animals were administered 10 prnol D-cycloserine (i.c.v.) in addition to the i.c.v. infusion of either the vehicle control or 7-CLKYNA solutions. The o-cycloserine was administered 2 h prior to the seizure test while the vehicle or 7-CLKYNA solutions were administered 5 min prior to the seizure test. The number of rats in each group is indicated in the columns. 1 indicates a significant (x2= 17.3, df = 1, P = 0.001) difference from the i.c.v. vehicle group.

The anticonvulsant activity 2 h after i.c.v. administration was dose-dependent with half the animals

_ -

- s

1.0

12

2.5

r

5 *,

5.0

,

4*,

10.0

L-cycloserine (pmole, i.c.v., 2 hr) Fig. 6. Centrally administered L-cycloserine induces nonspecific anticonvulsant activity in maximal electroshock seizures. L-Cycloserine was administered by i.c.v. infusion 2 h prior to the seizure test. The number of rats in each group is indicated in the columns. There were no significant differences in seizure response between the groups treated with 0.1, 1.0 and 2.5 wmol L-cycloserine. The stars indicate that all the animals died before the seizure test.

being protected by 5 pmol D-cycloserine (Fig. 4). The 10 pm01 dose protected all the animals tested while producing only a mild ataxia. 7-CLKYNA (100 nmol, i.c.v.) administered 5 min prior to the seizure test significantly (x2= 17.3, df= 1, P = 0.00 1) antagonized the anticonvulsant activity of 10 pmol D-cycloserine (i.c.v.) administered 2 h prior to the seizure test (Fig. 5). L-Cycloserine appeared to induce nonspecific anticonvulsant activity after central administration. The 1.0 and 2.5 pmol doses (i.c.v.) administered 2 h prior to the seizure test produced nonsignificant anticonvulsant effects (Fig. 6) that were associated with severe neurological deficits (sedation and loss of righting reflex). The 5 and 10 pmol doses of Lcycloserine (i.c.v.), equivalent to the doses of Dcycloserine that were anticonvulsant, resulted in the death of all of the animals within 45 min of administration (Fig. 6). Discussion

D-Cycloserine induced selective anticonvulsant activity in maximal electroshock seizures. Systemically administered (i.p.) D-cycloserine protected all rats from tonic hindlimb extension at doses that were without observable neurological deficit. Centrally administered (i.c.v.) D-cycloserine protected rats from tonic hindlimb extension at doses that induced negligible neurological deficit. Because the D-cycloserine effect as induced by either route of administration was stereospecitic and antagonized by 7-CLKYNA, the results provide evidence that the anticonvulsant activity of D-cycloserine may be mediated by central strychnine-insensitive glycine receptors. D-Cycloserine has been identified as a partial agonist of the strychnine-insensitive glycine receptor on the basis of electrophysiologica124P5’ and receptor binding15,*’ studies. In each of these studies, L-cycloserine induced no activity’5P24,27,5’indicating that the strychnine-insensitive glycine receptor sensitivity is specific for the D-isomer. It may be argued that the partial agonist effects of D-cycloserine explain the mechanism of anticonvulsant action. Theoretically, sufficiently high doses of D-cycloserine as a partial agonist would antagonize the proconvulsant activity of endogenous glycine, a

7x

mechanism of action that has been proposed for the observed anticonvulsant effects of the strychnine-in~nsitive glycine receptor antagonist 7CLKYNA4,2’,40. However, 7-CLKYNA antagonized the anticonvulsant effects of D-cycloserine (Fig. 3) indicating that the anticonvulsant activity was mediated by the agonist properties of D-cycloserine. 7-CLKYNA has been identified as an antagonist of strychnine-insensitive glycine receptors on the basis of electrophysiologica12’ and receptor binding’8’4* studies. 7-CLKYNA has negligible affinity for excitatory amino acid receptors2’ or st~~hnine-sensitive glycine receptors’. 7-CLKYNA acts as an anticonvulsant in mouse models of epilepsy induced by auditory stimuli in DBA/2 mice4’ and NMDA4,2’. In addition, 7-CLKYNA is anticonvulsant in rats with kindled amygdala seizures6T7and seizures induced by bi~uculline infusion into the area tempestas5’. However, 7-CLKYNA does not inhibit the occurrence of tonic hindlimb extension in maximal electroshock seizures in rats ‘6,33.It has been suggested that antagonism of saturated strychnine-in~nsitive glycine receptors is responsible for the anticonvulsant activity of 7CLKYNA7,“. However, evidence that strychnineinsensitive glycine receptors are not saturated under physiological conditionssP35*53may explain the failure of 7-CLKYNA to inhibit the maximal electroshock seizure response. The i.c.v. administration studies provide evidence that the anticonvulsant activity induced by D-cycloserine is mediated by central mechanisms. Centrally administered D-cycloserine induced selective anticonvulsa~t effects while equivalent doses of Lcycloserine were lethal. In addition, centrally administered 7-CLKYNA antagonized the anticonvulsant activity of D-cycloserine, again suggesting a central mechanism of action. The relatively long onset of action of D-cycloserine after i.c.v. administration is similar to that of morphine which also requires l-2 h for peak effect after i.c.v. administration46. The delay in reaching the site of action after administration into the lateral ventricle may be related to the water solubihty of D-cycloserine. In contrast, 7-CLKYNA is poorly soluble in water and apparently reaches the site of action within minutes after i.c.v. administration. The fail-

ure of 7-CLKYNA to inhibit maximal electroshock-induced tonic hindlimb extension does not appear to be related to ineffective ~stribution in the brain as 100 nmol (i.c.v.) tested 2 h post administration also failed to alter the seizure response (Table I). The present data support previous reports that systemic administration of st~chnine-insensitive glycine receptor agonists produce anticonvulsant activity in maximal electroshock seizures3’,33. Although D-cycloserine induced significant anticonv&ant activity by itself, glycine and D-serine have no anticonvuIsant activity when administered by themselves and are most effective when combined with anticonvulsant drugs3’933.A possible explanation for the differences in anticonvulsant activity is that D-cycloserine readily crosses the blood-brain barrier28 while glycine and D-serine do not. Although glycine29 and possibly D-serine’ enter the brain by a specific carrier mechanism, they do so slowly and require large doses to significantly increase brain content38V48.Thus, the lack of significant anticonvulsant activity by glycine or n-serine when administered alone may result from poor penetration of the brain. In addition, because Dcycloserine is a D-amino acid and not involved in mammalian metabolism13, any action of D-cycloserine is likely to be mediated by a specific receptor mechanism and not a nonspecific dis~ption of metabolism as might occur with a large dose of glycine. r,-Cycloserine is a potent anticonvulsant in audiogenie and 3-mercaptopropionic acid-induced seizures of micesa4. The time of peak effect after systemic administration in those models of epilepsy is 3 h which correlates with a maximal inhibition of GABA transaminase and the maximal increase in central GABA content34. L-Cycloserine is not active against tonic convulsions induced by bicucultine, pentyIenetetrazo1 or maxima1 eiectroshock34. The results of the present study demonstrate that L-cycloserine is not active in maximal electroshock seizures when administered in the same dose and with the same time course as D-cycloserine. Thus, the mechanism by which L-cycloserine inhibits convulsions in mice appears to be distinct from the action of D-cycloserine in maximal electroshock seizures of rats.

79

The present results indicate that D-cycloserine is a selective anticonvulsant in maximal electroshock seizures and that the anticonvulsant activity may be mediated by strychnine-insensitive glycine receptors. Although the hypothesis is incongruent with current theory derived from in vitro experimentsi2, it may be that glycine potentiation of NMDA receptor mechanisms in vivo activates endogenous anticonvulsant mechanisms. In this regard, glycine potentiation of NMDA-mediated norepinephrine’ 1723or GABA’4*36 release may result in anticonvulsant activity. Evidence of

NMDA receptor subtypes25V26 suggests multiple functions, some of which could mediate anticonvulsant activity. Finally, evidence of strychnine-insensitive glycine receptor subtypes’ suggests multiple undefined roles for glycine. Acknowledgements The author thanks Laura Hinojosa, Nathan Schwade and Karen Dwyer for their excellent technical assistance. This work was supported by Public Health Service Grant NS24566.

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