Mk-801 prevents the decrease in 35S-TBPS binding in the rat cerebral cortex induced by pentylenetetrazol kindling

0361-9230/91 $3.00 + .OO

Brain Research Bulletin, Vol. 21, pp. 835-837. 0 Pergamon Press plc, 1991. Printed in the U.S.A.

RAPID COMMUNICATION

MK-801 Prevents the Decrease in 35S-TBPS Binding in the Rat Cerebral Cortex Induced by Pentylenetetrazol Kindling 0. GIORGI,’

M. ORLANDI,

M. GEIC AND M. G. CORDA

Department of Experimental Biology, Section of Neuroscience,

University

ofCagliari, Italy

Received 6 June 1991 GIORGI, O., M. OIUANDI, M. GEIC AND M. G. COKDA. MK-801 prevents the decrease in 35S-iT3PS binding in the rat cerebral cortex induced by pentyZeneretrazol kindling. BRAIN KES BULL 27(6) 835-837, 1991. -Chemical kindling was induced

in the rat by chronic treatment with pentylenetetrazol (PIZ, 30 mg/kg, IP, three times a week for eight weeks). PI2 kindling was associated with a reduction in central GABAergic function, as reflected by a significant decrease in the density of “S-t-butylbicyclophosphorothionate (3sS-TBPS) binding sites in the cerebral cortex. The pretreatment with the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (1 mg/kg, IP, 40 min before each PTZ injection) prevented the development of kindling as well as the reduction in ?S-TBPS binding. The results suggest that NMDA receptors may play a role in the alterations of GABAergic function observed in PI%kindled rats. Pentylenetetrazol

Kindling

GABA

‘sS-TBPS binding

NMDA receptors

MK-801

METHOD

(PTZ) kindling is a form of chemical kindling in which the repeated administration of this compound causes a progressive increase in the excitability of the central nervous system (CNS), so that the initially subconvulsant doses can produce generalized seizures (1, 4, 9, 13). We have previously reported that PTZ-induced kindling is associated with a reduction in GABA-mediated neurotransmission in the CNS. Thus 35S-t-butylbicyclophosphorothionate (35S-TBPS) binding, and GABA-stimulated %uptake are reduced in the cerebral cortex of PTZ-kindled rats (1,2). In addition, the sensitivity to the convulsant effects of a range of GABA function inhibitors is dramatically increased in rats kindled with PTZ (1,3). Numerous studies have shown that the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors plays an important role in experimental epilepsy and kindling (7, 10, 11, 15, 17, 18, 20). In fact, the noncompetitive NMDA receptor antagonist MK-801 [( +)-S-methyl-lO,l l-dihydro-SH-dibenzo (A,D) cyclohepten-5,10-imine maleate] (19) is able to suppress both the electrophysiological and the behavioral effects induced by the electrical kindling of the amygdala in rats (9,15). Moreover, we have recently shown that MK-801 prevents the development of kindling in rats chronically treated with PTZ (5). It was therefore considered of interest to examine whether MK-801 is also able to prevent the alterations in the biochemical parameters of central GABAergic function induced by the repeated administration of PIZ. PENTYLENETETRAZOL

Eighty male Sprague-Dawley CD rats (Charles River, Como, Italy) weighing 2.50-300 g at the beginning of the chronic treatment were housed five per cage and kept under controlled temperature and lighting (lights on: 8 a.m.-8 p.m.) with food and water freely available. Animals were divided into 4 groups of 20 rats each. One group received PTZ (30 mg/kg, IP) every second day, three times a week for 8 consecutive weeks. A second group received an intraperitoneal injection of MK-80 1 (1 mgkg) 40 min before each PTZ injection throughout the chronic treatment. The third group was treated with MK-801 alone and the last group was injected with PTZ solvent (saline, 2 ml/kg, IP). Rats were observed for one hour after each administration of PTZ, MK-801 or saline and seizures were recorded according to the following scale: 0, no response; 1, ear and facial twitching; 2, one to twenty myoclonic body jerks in ten minutes; 3, more than twenty body jerks in 10 minutes; 4, clonic forelimb convulsions; 5, generalized clonic convulsions with rearing and falling down episodes; 6, generalized convulsions with tonic extension episodes and status epilepticus. Three days after the last drug administration rats were killed by decapitation, the brains were removed, and the cerebral cortex was dissected and used for the biochemical studies. “‘S-TBPS Binding Assay The freshly obtained brain tissue was homogenized with a

‘Requests for mprints should be addressed to Dr. Osvaldo Giorgi, Department of Experimental Biology, Section of Neuroscience, University of Cagliari, Via Palabanda, 12,09123 Cagliari, Italy. 835

836

GIORGI, ORLANDI,

Polytron PTlO (setting 5) for 20 s in 50 volumes of 50 mM Tris-citrate buffer (pH 7.4) containing 100 mM NaCl, centrifuged at 20,000 x g for 20 min and reconstituted in 50 volumes of Tris-citrate buffer containing 200 mM NaCI. 35S-TBPS binding was determined in a final volume of 500 ~1, which consisted of 400 ~1 of tissue (150-200 pg of protein), 50 ~1 of 35S-TBPS, and 50 pl of buffer alone or buffer plus 100 pM picrotoxin to determine nonspecific binding. Saturation analysis of 35S-TBPS binding was performed using 6 different concentrations of ligand, maintaining the concentration of 3sS-TBPS at 2 nM, and varying the concentration of nonradioactive TBPS from 2 to 320 nM. Following a 90-min incubation at 23°C samples were filtered through Whatman GF/B filters using the M-48 Cell Harvester (Brandel Instruments, Gaithersburg, MD). Filters were washed three times with 4 ml of ice-cold buffer, transferred to picovials containing 3.5 ml of Atomlight scintillation fluid and the radioactivity was counted by liquid scintillation spectrometry. All the assays were carried out using triplicate samples. Protein content was measured according to the procedure of Lowry et al. (6).

GEIC AND CORDA

TABLE 1 PREVENTION BY MK-801 OF KINDLING INDUCED BY PENTYLENETETRAZOL (PTZ) IN RATS

Seizure Score Week

Saline

1

0

2 4 6 8

0 0 0 0

PTZ

MK-801

PTZ + MK-801

0

0

0 0 0 0

0 0 ‘t-

0

0.8 2.5 3.4 4.7

& -t 2 t

0.2 0.6 0.5 0.8

0.4 0.7

0.2 0.3

PTZ and MK-801 were administered alone or in combination as described in the Method section. The mean value of the three seizure scores recorded for each rat in the indicated weeks of the chronic treatment was used for statistical analysis. Shown are the mean seizure scores ?SEM of 20 rats per group. A Kruskal-Wallis analysis by ranks revealed a significant difference between the PTZ group and all the other groups @O.O5).

Chemicals “‘S-TBPS (70-90 Ci/mmol) and Atomlight scintillation fluid were obtained from New England Nuclear (Dreieich, Germany). Unlabelled TBPS was kindly provided by Dr. J. E. Casida (Berkeley, CA) and MK-801 was a generous gift from Dr. P. Anderson, Merck Sharp & Dohme, Rahway, NJ. PTZ was purchased from Sigma, St. Louis, MO. Data Analysis The maximum number of binding sites (B,,,) and apparent dissociation constant (Kd) were calculated by linear regression analysis of Scatchard plots of saturation curves. Behavioral data were analyzed using nonparametric procedures (Kruskal-Wallis analysis by ranks) whereas biochemical data were analyzed by ANOVA followed by Tukey’s test.

DISCUSSION

The results of the present study give further experimental support to the view that excitatory amino acid neurotransmission plays an important role in PTZ-induced kindling. Previous behavioral and biochemical studies have shown that the GABAergic neurotransmission is decreased in the CNS of PTZ-kindled rats, as indicated by the increased sensitivity to the convulsant action of several GABA function inhibitors, the decreased density of 35S-TBPS binding sites, and the reduced ability of GABA to stimulate 36C1- uptake in vitro (l-3). The present results confirm our previous report on the ability of MK-801 to prevent PIZ-induced kindling (5) and demonstrate that pretreatment with MK-801 also prevents the effect of chronic PTZ administration on 35S-TBPS binding. TBPS is a cage convulsant that binds with high affinity to a recognition site located in the chloride iono-

RESULTS

The repeated administration of subconvulsant doses of PTZ (30 mg/kg IP) produced a progressive increment in the susceptibility to seizures (chemical kindling). Thus the seizure score increased from 0.8kO.2 in the second week of treatment to 4.7 t 0.8 by the eighth week (Table 1). In addition, the percent of convulsing rats (seizure score equal to or greater than 1) increased from 15% in the second week to 80% by the end of the chronic treatment (not shown). The development of PTZ-induced kindling was prevented by the administration of MK-801 (1 mg/kg IP, 40 min before each dose of PTZ) as indicated by the seizure score (Table 1). Finally, the repeated administration of saline or MK-801 alone failed to induce convulsions throughout the treatment (Table 1). The above results are consistent with previous reports from our laboratory (5). The effects of the repeated administration of PTZ and MK801 on 3sS-TBPS binding were studied 3 days after completion of the chronic treatments. As shown in Fig. 1, the density of 35S-TBPS binding sites was decreased by 22% in the cerebral cortex of rats that had reached a seizure score of at least 4 by the eighth week of treatment with PTZ, whilst the apparent dissociation constant remained unchanged. The repeated administration of MK-801 alone failed to affect 3sS-TBPS binding in the cerebral cortex, whereas the pretreatment with this compound completely prevented the decrease in the density of 35S-TBPS binding sites induced by chronic PTZ (Fig. 1).

.; s h

1.50

! ‘.20 0, f/l 0.90 0 &

0.60 V E 0.30 E m 0.00

mPl-Z+MK

FIG. 1. Decrease in 35S-TBPS binding in the cerebral cortex of rats treated with PTZ: Antagonism by MK-801. Rats were killed three days after the last drug administration and ‘5S-TBPS binding assays were performed as described in the Method section, using pooled tissues from two animals in each experimental group. Shown am the mean&SEM of seven independent experiments for each group. The values of the apparent dissociation constants (Kd, nM) were as follows (mean-C SEM, N=7): saline, 55~2; MK-801, 5623; PTZ, 55-+2; PTZ + MK-801, values: F(3,24) = 6.13, pO.5. *p
837

PENTYLENETETRAZOL KINDLING

phore coupled to the GABA, receptor (16). It appears unlikely that the protective effect of MK-801 on 35S-TBPS binding is due to a direct interaction of this compound with the chloride ionophore. In fact, MK-801 fails to inhibit 35S-TBPS binding in vitro in concentrations up to lop5 M (our unpublished results). Moreover, the results of the present study have shown that the repeated administration of MK-801 alone does not modify the binding parameters of 35S-TBPS in the cerebral cortex. Considered together, the above data suggest that the blockade of NMDA receptors can prevent the alterations in the biochemical parameters of GABAergic transmission elicited by PTZ kindling. In this context, preliminary studies from our laboratory indicate that the pretreatment with MK-801 also prevents the increased susceptibility to the convulsant effects of GABA function inhibitors that has been described in PTZ-kindled rats (Corda et al., in preparation). The mechanism whereby MK-801 prevents the biochemical effects of the repeated administration of PTZ remains to be elucidated. PTZ reduces the chloride current in spinal cord neurons by selectively blocking the GABA, receptor-coupled chloride ionophore (8). In line with this finding, it has been shown that PTZ inhibits in a competitive manner the binding of 35S-TBPS in the rat brain (2,14). Recent findings from our laboratory indicate that the decrease in the density of 35S-TBPS binding sites

induced by the repeated administration of PTZ depends on the development of kindling in a critical manner. In fact, the B,, of 35S-TBPS binding is unchanged in the cerebral cortex of rats that do not sensitize upon chronic treatment with PTZ (Corda et al., in preparation). Therefore, it seems that the decrease in the density of 35S-TBPS binding sites is the consequence rather than the cause of the kindling process. Hence, it may be speculated that MK-801 prevents the alterations in 35S-TBPS binding induced by chronic treatment with PTZ by interfering with the development of kindling. Accordingly, MK-801 and other NMDA receptor antagonists block the development of electrical kindling (7, 12, 15, 18). In conclusion, our results are consistent with the view that an increase in excitatory amino acid-mediated neurotransmission plays an important role in the biochemical and behavioral alterations of GABAergic function in PTZ-kindled rats. In line with this view, it has been shown that kindling induced in vitro in hippocampal slices causes a decrease in GABAergic inhibition that results from stimulus-induced postsynaptic activation of NMDA receptors (17). Finally, these data suggest that NMDA receptor antagonists may have a therapeutic potential in the clinical management of epilepsy.

REFERENCES 1. Corda, M. G.; Giorgi, 0.; Orlandi, M.; Longoni, B.; Biggio, G. Chronic administration of negative modulators produces chemical kindling and GABA, receptor down-regulation. In: Biggio, G.; Costa, E., eds. GABA and benzodiazepine receptor subtypes. New York: Raven Press; 1990:153-166. 2. Corda, M. G.; Grlandi, M.; Lecca, D.; Carboni, G.; Frau, V.; Giorgi, 0. Decrease in the function of the r-aminobutyric acid-coupled chloride channel produced by the repeated administration of pentylenetetrazol to rats. J. Neurochem. 551216-1221; 1990. 3. Corda, M. G.; Grlandi, M.; Lecca, D.; Carboni, G.; Frau, V.; Giorgi, 0. Pentylenetetrazol-induced kindling in rata: Effect of GABA function inhibitors. Pharmacol. B&hem. Behav. 40:329-333; 1991. 4. Diehl, R. G.; Smialowski, A.; Gotwo, T. Development and persistence of kindled seizures after repeated injections of pentylenetetrazol in rats and guinea pigs. Epilepsia 25:506-510; 1984. 5. Giorgi, 0.; Grlandi, M.; Lecca, D.; Corda, M. G. MK-801 prevents chemical kindling induced by pentylenetetrazol in rats. Eur. J. Pharmacol. 193:363-365; 1991. 6. Lowry, 0. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275; 1951. 7. McNamara, J. 0.; Russell, R. D.; Rigsbee, L.; Bonhaus, D. W. Anticonvulsant and antiepileptogenic actions of MK-801 in the kindling and electroshock models. Neuropharmacology 27:563-568; 1988. 8. MacDonald, R. L.; Barker, J. L. Pentylenetetrazol and penicillin are selective antagonists of GABA-mediated postsynaptic inhibition in . ._ cultured mammalian neurones. Nature 267:72&721; 1977. 9. Mason, C. R.; Cooper, R. M. A permanent change in convulsive threshold in normal and brain-damaged rats, with repeated small doses of pentylenetetrazol. Epilepsia 13663474; 1972. 10. Morimoto, K. Seizure-triggering mechanisms in the kindling model of epilepsy: Collapse of GABA-mediated inhibition and activation of NMDA receptors. Neurosci. Biobehav. Rev. 13:253-260; 1989. 11. Morrisett, R. A.; Chow, C.; Nadler, J. V.; McNamara, J. 0. Bio-

12.

13. 14.

15.

16.

17. 18.

19.

20.

chemical evidence for enhanced sensitivity to N-methyl-D-aspartate in the hippocampal formation of kindled rats. Brain Res. 496:2528; 1989. Peterson, D. W.; Collins, J. F.; Bradford, H. F. The kindled amygdala model of epilepsy: anticonvulsant action of amino acid antagonists. Brain Res. 275:169-172; 1983. Pinel, J. P. J.; Cheung, K. F. Controlled demonstration of metrazol kindling. Pharmacol. Biochem. Behav. 6:599X10; 1977. Ramanjaneyulu, R.; Ticku M. J. Interactions of pentamethylenetetrazole and tetrazole analogues with the picrotoxinin site of the benzodiazepine-GABA receptor-ionophore complex. Eur. J. Pharmacol. 98:337-345; 1984. Sato, K.; Morimoto, K.; Okamoto, M. Anticonvulsant action of a noncompetitive antagonist of NMDA receptors (MK-801) in the kindling model of epilepsy. Brain Res. 463:12-20; 1988. Squires, R. F.; Casida, J. E.; Richardson, M.; Saederup, E. [35S]tbutylbicyclophosphorothionate binds with high affinity to brain-specific sites coupled to r-aminobutyric acid-A and ion recognition sites. Mol. Pharmacol. 23:326-336; 1983. Stelzer, A.; Traverse Slater, N.; ten Btuggencate, G. Activation of NMDA receptors blocks GABAergic inhibition in an in vitro model of epilepsy. Nature 326698-701; 1987. Vezzani, A.; Wu, H.-Q.; Moneta, E.; Samanin, R. Role of the N-methyl-D-aspartate-type receptors in the development and maintenance of hippocampal kindling in rata. Neurosci. Lett. 87:6348; 1988. Wong, E. H. F.; Kemp, J. A.; Priestley, T.; Knight, A. R.; Woodruff, G. N.; Iversen, L.L. The anticonvulsant MK-801 is a Potent N-methyl-D-aspartate antagonist. Proc. Natl. Acad. Sci. USA 83: 7104-7107; 1986. Yeh, G.-C.; Bonhaus, D. W.; Nadler, J. V.; McNamara, J. 0. N-methyl-D-aspartate receptor plasticity in kindling: quantitative and qualitative alterations in the N-methyl-D-aspartate receptor-channel complex. Proc. Natl. Acad. Sci. USA 86: 8157-8160; 1989.