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Effect of nifedipine and anticonvulsants of kainic acid-induced seizures in mice
Brain Research, 533 (1990) 157-160
157
Elsevier
BRES 24377
Effect of nifedipine and anticonvulsants on kainic acid-induced seizures in mice* D a l e E. B r a u n 1 a n d William J. F r e e d 2 1Department of Neurosurgery, Naval Hospital Bethesda, Bethesda, MD 20814 (U.S.A.) and ZPreclinical Neurosciences Section, Neuropsychiatry Branch, NIMH Neuroscience Center at St. Elizabeths, Washington, DC 20032 (U.S.A.)
(Accepted 31 July 1990) Key words: Nifedipine; Kainic acid; Excitatory amino acid; Anticonvulsant; Seizure; Calcium channel inhibitor
The calcium-channel inhibitor nifedipine and several anticonvulsant drugs were evaluated for effects on seizures induced by intracerebroventricular injection of 0.14/~g of kainic acid. These seizures were markedly exacerbated by valproic acid and moderately inhibited by diazepam. Nifedipine decreased the duration of each individual seizure episode, but did not block the development of seizures. It is concluded that nifedipine prevents the maintenance or propagation of kainate-induced seizures.
Kainic acid, an agonist of one type of excitatory amino acid receptor 12'22, induces seizures when administered to animals either intracerebrally 2 or systemically21. Intracerebral kainic acid produces seizures which begin as electroencephalographic changes in the limbic system, particularly the hippocampus 2'13. Because of their electroencephalographic properties, kainic acid-induced seizures have been proposed as a model of partial complex, or limbic, seizures in man 2'~3. Only a few studies have examined effects of anticonvulsants on seizures induced by kainic acid. Stone and Javid 21 found that seizures induced by intraperitoneal kainic acid were attenuated by phenobarbital, trimethadione, and chlordiazepoxide, but not by ethosuximide or phenytoin. Fuller and Olney 9 induced seizures by 12 mg/kg kainic acid administered subcutaneously, and found that these seizures were attenuated by diazepam and phenobarbital, but not phenytoin or valproic acid. The only study to examine effects of anticonvulsants on seizures induced by intracerebral kainic acid was that of Ault and coworkers 1, who found that baclofen (5 mg/kg) and phenobarbital (40 mg/kg) both attenuated seizures induced by intracerebroventricular kainic acid in a dosage of 0.2/tg. As the excitatory amino acids are major regulators of excitatory transmission in the CNS 12'22, it is reasonable to suggest that the excitatory amino acids may have some role in epilepsy 6,16. There is also evidence that excitatory amino acids are involved in neurodegenerative disorders
of various types, such as hypoxia/ischemia and CNS trauma, and the neurotoxic effects of certain food poisons 7'1°'14'2°. Investigation of the functional role of kainic acid in the generation of seizures has been hampered, however, because of the paucity of specific antagonists. In the present experiment, we have taken the opposite approach; that is, we have determined whether seizures induced by kainic acid can be blocked by commonly-used anticonvulsant compounds, and by the calcium channel inhibitor nifedipine. Several recent studies suggest that some forms of seizures can be blocked by calcium channel inhibitors, such as nifedipine 8'1~'1s. Adult female Swiss-Webster mice received intracerebroventricular injections of kainic acid (Sigma) by the method of Clark and coworkers 5. Animals first received kainic acid in a range of dosages from 0.05 to 2/~g in a volume of 5/~1 per mouse in normal saline. A dosage of 0.14/tg was selected, because it induced easily-quantifiable seizures lasting for about one-half of the observation period in 100% of the animals, with minimal mortality. This dosage of kainic acid produced either tonic seizures, with raised forearms and rigidity, or tonic-opisthotonic episodes. Animals that showed a rigid posture and a lack of responsiveness to tactile stimuli were considered to be undergoing tonic seizures. Usually the first seizure observed was tonic-opisthotonic, and later episodes were of the tonic variety. Animals were also hyperreactive to stimuli after kainic acid administration. Animals were pretreated with one of the various test
* These data were presented at the 19th Annual Meeting of the Society for Neuroscience, Phoenix, AZ, November, 1989. Correspondence: W. Freed, NIMH Neuroscience Center at St. Elizabeths, Washington, DC 20032, U.S.A.
158 drugs in a vehicle of 10% Tween 80/90% normal saline, in a volume of 10 mi/kg i.p. Thirty min after injection, each animal received 0.14 g g of kainic acid intracerebroventricularly. The time of onset of the first seizure and the time of onset and termination of each succeeding seizure was r e c o r d e d to the nearest 0.1 rain. Animals were observed for 60 min and then euthanized. D a t a r e c o r d e d were: (i) time to onset of the first seizure (latency), (ii) total time elapsed from the start of the first seizure to the end of the last seizure (duration), and (iii) the total length of seizures during the observation period (sum of the duration of each individual seizure episode = total seizure time). For each of these three measures, d a t a were analyzed by a one-way analysis of variance followed by Fisher P L S D tests to c o m p a r e each individual drug dosage to vehicle.
In general, baclofen, p h e n o b a r b i t a l , and L-glutamic acid diethyl ester HCI had no effect on kainate-induced seizures (Table I). C a r b a m a z e p i n e had only a slight effect of significantly decreasing seizure duration. Latency to seizure onset and total seizure time, however, were not altered. The largest dosage of phenytoin, 100 mg/kg, significantly e x a c e r b a t e d the seizures, increasing both seizure duration and total seizure time. T h e smaller dosage of p h e n y t o i n (50 mg/kg) increased latency to seizure onset, but did not alter duration or total seizure time (Table I). K a i n a t e - i n d u c e d seizures were m a r k e d l y worsened by valproic acid at both dosage levels; animals p r e t r e a t e d with v a l p r o a t e showed increased seizure duration, seizure time, and mortality, as well as a decreased latency to seizure onset (Table I). All animals treated with valproic acid exhibited severe tonic-opisthotonic
TABLE I Effects o f anticonvulsants and other compounds on kainic acid-induced seizures
Data shown are means ± S.E.M. Compound (dose)
Latency to seizure onset ~ Duration b
Total seizure time c
Mortality
Number o f mice
Vehicle
1.9 ± 0.24
33,3 + 2.2
29.9 ± 2.1
1
28
Phenytoin 50 mg/kg 100 mg/kg
4.3±1.2" 1.2±0.41
34.1±3.7 51.0±3.9"
31.5±3.3 51.0±3.9'
0 0
9 7
Carbamazepine 50 mg/kg
1.1±0.31
24.9±2.5*
24.9±2.5
0
9
Baclofen 5 mg/kg 10 mg/kg
1.2±0.31 1.1±0.40
30.6±3.6 34.5±2.5
29.8±3.6 34.5±2.5
1 0
10 8
Phenobarbital 40 mg/kg 80 mg/kg
0.81±0.08 0.72±0.13
29.3±1.7 26.7±1.1
28.8±1.8 26.7±1.1
0 0
10 10
Vaiproic acid 125 mg/kg 250 mg/kg
0.79±0.10 1.14±0.21
53.6±2.9* 58.8±0.26*
53.5±2.8* 58.8±0.26*
4 8
10 10
Diazepam 1 mg/kg 2 mg/kg
0.68±0.10" 1.08±0.21
18.3±1.6" 17.5±0.86"
18.3±1.6" 17.0±0.80"
0 0
10 13
Glutamic acid diethyl ester 480mg/kg 960 mg/kg
1.1±0.23 1.4±0.29
25.7±2.7 32.9±2.5
25.7±2.7 32.6±2.5
0 0
9 9
Nifedipine 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg
2.1 + 4.0 + 4.8 + 4.4 + 2.0 +
23.3 + 1.5* 27.4 + 1.8 26.6 + 2.5* 25.7 + 0.8 19.8 + 13.4"
21.4 _ 0.68* 14.5 + 0.64* 12.3 + 1.2" 10.6 + 2.8* 19.8 ± 13.4"
0 1 2 1 5
5 4 18 3 8
*
0.45 0.29 1.1" 0.28 0.62
Indicates significantly different from vehicle (P <~0.05) by Fisher PLSD test.
a F~s,z~ = 5.94, P < 1.0 × 10-4; b F~sA6a= 23.38, P < 1.0 x 10-4; c F1~,166= 15,31, P < 1.0 x 10-4.
159 4O Durltion I
o
(¢)
30
D a
0'
2 "5
5 0'
'
t00
Dose Img/kgl
Fig. 1. Dose-response curve for the effect of nifedipine on seizure duration (time from beginning of first to end of last seizure; open circles) and total seizure time (sum of the duration of all seizure episodes; closed triangles) following intracerebral injection of 0.14 /~g kainic acid. The highest dosage (200 mg/kg) is not shown (see Table I). Data shown are means + S.E.M. Note that seizure time is decreased to a much greater extent than seizure duration; this was because nifedipine decreased the duration of each individual seizure episode.
seizures and most animals that received the larger dosage expired. Diazepam produced a modest decrease in kainateinduced seizures. The latency to seizure onset was not increased, but there were significant parallel decreases in seizure duration and total seizure time (Table I). Both dosages, and particularly the largest dosage, 2 mg/kg, had a substantial sedative effect. Nifedipine, on the other hand, caused a significant dose-dependent decrease in total seizure time (Fig. 1). A significant 28% decrease in total seizure time was seen even at the lowest dosage, 12.5 mg/kg. On the other hand, seizure duration was not greatly decreased. It was noted that the animals treated with nifedipine had similar numbers of seizures, but recovered more rapidly from each individual seizure. Between seizure episodes, the nifedipine-treated animals were relatively normal in appearance, were responsive to tactile stimulation, and did not show evidence of continued tonus or rigidity. Thus the effect of nifedipine was primarily to induce a more rapid recovery from each individual seizure episode. There was also a minor tendency for an increased latency to seizure onset, which was significant only at 50 mg/kg. Therefore, most anticonvulsants did not decrease seizures induced by intracerebral kainic acid. In fact, the present data contrast with previous reports that phenobarbital and baclofen suppress kainate-induced seizures L 9.21. The reason for this difference is unknown. These seizures were somewhat exacerbated by phenytoin and markedly exacerbated by valproic acid, which is consis-
tent with previous reports that both agents failed to inhibit kainate-induced seizures 9'21. As has previously been reported 9, diazepam inhibited kainate°induced seizures, but also had a pronounced sedative effect, while carbamazepine had only a slight effect (Table I). Quisqualate-induced seizures, by comparison, are inhibited by both diazepam and valproic acid 17. The only agent which was found to markedly inhibit kainic acid-induced seizures was nifedipine. This compound did not, however, prevent the development of seizures, and had only a minor effect on the total time from the onset to the end of seizures (i.e., seizure duration). The primary effect of nifedipine was apparently to cause each individual seizure episode to terminate more rapidly. The reasons for this particular mode of action of nifedipine are not known. Nifedipine has recently been reported to block the neurotoxic actions of kainic acid, as well as other excitatory amino acids including N-methyl-o-aspartate and a-amino-3-hydroxy-5-methyl-4-isoxazole propionate ~. In that the excitatory effects of kainate on neurons was not antagonized by nifedipine 2a, it appears likely that the anti-neurotoxic effect of nifedipine was mediated by its primary mode of action, blockade of voltage-gated calcium channels. Therefore, influx of calcium into neurons is probably a secondary event which is required for the manifestation of kainate-induced neurotoxicity~. The nature of the nifedipine effect seen in the present study suggests that similar calcium-mediated events may be involved in propagating or maintaining kainateinduced seizures. Rather than preventing seizures, nifedipine decreased the duration of seizure episodes, so that the animals recovered more rapidly from each individual seizure. Therefore, influx of calcium into neurons via dihydropyridine-sensitive channels may play an essential role in sustaining kainate-induced seizures. One possible application for nifedipine is as a treatment for the seizures or neuronal damage induced by neurotoxins which act at non-NMDA receptors, including fl-N-oxalylaminoalanin~4,2°, or the shellfish contaminant domoic acid 3'7,10,15. Domoic acid in particular is very similar in structure to kainic acid 7"19, and has caused numerous cases of poisoning in Canada 1°. Although relatively high concentrations of nifedipine were required to block neurotoxicity in vitro 23, the present data suggest that effective concentrations can also be achieved in vivo by systemic injection, and that nifedipine is also effective in blocking the seizures produced by kainic acid. These data suggest that anticonvulsant-resistant seizures are the most likely candidate for potential clinical applications of kainic acid antagonists as anticonvulsant drugs. The present data also suggest that calcium channel antagonists might be useful as a treatment modality for
160 certain seizure types that are resistant to classical anticonvulsants. Especially, calcium channel inhibitors might be useful for treating conditions related to excess acti-
vation of the kainic acid class of excitatory amino acid receptor.
1 Ault, B., Gruenthal, M., Armstrong, D.R. and Nadler, J.V., Efficacy of baclofen and phenobarbital against the kainic acid limbic seizure-brain damage syndrome, J. Pharmacol. Exp. Ther., 239 (1986) 612-617. 2 Ben-Ari, Y., Limbic seizures and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy, Neuroscience, 14 (1985) 375-403. 3 Bose, R., Glavin, G.V. and Pinsky, C., Neurotoxicity and lethality of toxic extracts from Atlantic coast shellfish, Prog. Neuro-Psychopharmacol. Biol. Psychiat., 13 (1989)559-562. 4 Bridges, R.J., Stevens, D.R., Kahle, J.S., Nunn, P.B., Kadri, M. and Cotman, C.W., Structure-function studies on N-oxalyldiaminodicarboxylic acids and excitatory amino acid receptors: evidence that fl-L-ODAP is a selective non-NMDA agonist, J. Neurosci., 9 (1989) 2073-2079. 5 Clark, W.G., Vivonia, C.A. and Baxter, C.F., Accurate freehand injections into the lateral brain ventricle of the conscious mouse, J. Appl. Physiol., 25 (1968) 319-321. 6 Croucher, M.J., Collins, J.F. and Meldrum, B.S., Anticonvulsant action of excitatory amino acid antagonists, Science, 216 (1982) 899-901. 7 Debonnel, G., Beauchesne, L. and deMontigny, C., Domoic acid, the alleged 'mussel toxin', might produce its neurotoxic effect through kainate receptor activation: an electrophysiological study in the dorsal hippocampus, Can. J. PhysioL Pharmacol., 67 (1989) 29-33. 8 Desmendt, L.K.C., Niemegeers, C.J.E. and Janssen, P.A.J., Anticonvulsant properties of cinnarizine and flunarizine in rats and mice, Arzneim.-Forsch. (Drug Res.), 25 (1975) 1408-1413. 9 Fuller, T.A, and Olney, J.W., Only certain anticonvulsants protect against kainate neurotoxicity, Neurobehav. Toxicol. Teratol., 3 (1981) 355-361. 10 Glavin, G.V., Pinsky, C. and Bose, R., Mussel poisoning and excitatory amino acid receptors, Trends Physiol. Sci., 10 (1989) 15-16. 11 Meyer, F.B., Anderson, F.E., Sundt, T.M. and Sharbrough, F.W., Selective central nervous system calcium channel blockers - - a new class of anticonvulsant agents, Mayo Clin. Proc., 61
(1986) 239-247. 12 Monaghan, D.T., Bridges, R.J. and Cotman, C.W., The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 365-402. 13 Nadler, J.V., Kainic acid as a tool for the study of temporal lobe epilepsy, Life Sci., 29 (1981) 2031-2042. 14 Olney, J.W., Excitatory amino acids and neuropsychiatric disorders, Biol. Psychiat., 26 (1989) 505-525. 15 Pinsky, C., Glavin, G.B. and Bose, R., Kynurenic acid protects against neurotoxicity and lethality of toxic extracts from contaminated Atlantic coast mussels, Prog. Neuro-Psychopharrnacol. Biol. Psychiat., 13 (1989) 595-598. 16 Schwarcz, R. and Meldrum, B., Excitatory amino acid antagonists provide a therapeutic approach to neurological disorders, Lancet, 2 (1985) 140-143. 17 Schwarz, S.S. and Freed, W.J., Inhibition of quisqualateinduced seizures by glutamic acid diethyl ester and anti-epileptic drugs, J. Neural Transm., 67 (1986) 191-203. 18 Shelton, R.C., Grebb, J.A. and Freed, W.J., Induction of seizures by intracerebroventricular administration of the calcium channel agonist BAY k8644, Brain Research, 402 (1987) 399402. 19 Shinozaki, H. and Shibuya, I., Effects of kainic acid analogues on crayfish opener muscle, Neuropharmacology, 15 (1976) 145-147. 20 Spencer, P.S., Ludolph, A., Dwivedi, M.P., Roy, D.N., Hugen, J. and Sehaumburg, H.H., Lathyrism: evidence for a role of the neuroexcitatory amino acid BOAA, Lancet, ii (1986) 1066--1067. 21 Stone, W.E. and Javid, M.L, Effects of anticonvulsants and glutamate antagonists on the convulsive action of kainic acid, Arch. Int. Pharmacodyn., 23 (1980) 56-65. 22 Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Annu. Rev. Pharmacol. Toxicol., 21 (1981) 165-204. 23 Weiss, J.H., Hartley, D.M., Koh, J. and Choi, D.W., The calcium channel blocker nifedipine attenuates slow excitatory amino acid neurotoxicity, Science, 247 (1990) 1474-1477.
157
Elsevier
BRES 24377
Effect of nifedipine and anticonvulsants on kainic acid-induced seizures in mice* D a l e E. B r a u n 1 a n d William J. F r e e d 2 1Department of Neurosurgery, Naval Hospital Bethesda, Bethesda, MD 20814 (U.S.A.) and ZPreclinical Neurosciences Section, Neuropsychiatry Branch, NIMH Neuroscience Center at St. Elizabeths, Washington, DC 20032 (U.S.A.)
(Accepted 31 July 1990) Key words: Nifedipine; Kainic acid; Excitatory amino acid; Anticonvulsant; Seizure; Calcium channel inhibitor
The calcium-channel inhibitor nifedipine and several anticonvulsant drugs were evaluated for effects on seizures induced by intracerebroventricular injection of 0.14/~g of kainic acid. These seizures were markedly exacerbated by valproic acid and moderately inhibited by diazepam. Nifedipine decreased the duration of each individual seizure episode, but did not block the development of seizures. It is concluded that nifedipine prevents the maintenance or propagation of kainate-induced seizures.
Kainic acid, an agonist of one type of excitatory amino acid receptor 12'22, induces seizures when administered to animals either intracerebrally 2 or systemically21. Intracerebral kainic acid produces seizures which begin as electroencephalographic changes in the limbic system, particularly the hippocampus 2'13. Because of their electroencephalographic properties, kainic acid-induced seizures have been proposed as a model of partial complex, or limbic, seizures in man 2'~3. Only a few studies have examined effects of anticonvulsants on seizures induced by kainic acid. Stone and Javid 21 found that seizures induced by intraperitoneal kainic acid were attenuated by phenobarbital, trimethadione, and chlordiazepoxide, but not by ethosuximide or phenytoin. Fuller and Olney 9 induced seizures by 12 mg/kg kainic acid administered subcutaneously, and found that these seizures were attenuated by diazepam and phenobarbital, but not phenytoin or valproic acid. The only study to examine effects of anticonvulsants on seizures induced by intracerebral kainic acid was that of Ault and coworkers 1, who found that baclofen (5 mg/kg) and phenobarbital (40 mg/kg) both attenuated seizures induced by intracerebroventricular kainic acid in a dosage of 0.2/tg. As the excitatory amino acids are major regulators of excitatory transmission in the CNS 12'22, it is reasonable to suggest that the excitatory amino acids may have some role in epilepsy 6,16. There is also evidence that excitatory amino acids are involved in neurodegenerative disorders
of various types, such as hypoxia/ischemia and CNS trauma, and the neurotoxic effects of certain food poisons 7'1°'14'2°. Investigation of the functional role of kainic acid in the generation of seizures has been hampered, however, because of the paucity of specific antagonists. In the present experiment, we have taken the opposite approach; that is, we have determined whether seizures induced by kainic acid can be blocked by commonly-used anticonvulsant compounds, and by the calcium channel inhibitor nifedipine. Several recent studies suggest that some forms of seizures can be blocked by calcium channel inhibitors, such as nifedipine 8'1~'1s. Adult female Swiss-Webster mice received intracerebroventricular injections of kainic acid (Sigma) by the method of Clark and coworkers 5. Animals first received kainic acid in a range of dosages from 0.05 to 2/~g in a volume of 5/~1 per mouse in normal saline. A dosage of 0.14/tg was selected, because it induced easily-quantifiable seizures lasting for about one-half of the observation period in 100% of the animals, with minimal mortality. This dosage of kainic acid produced either tonic seizures, with raised forearms and rigidity, or tonic-opisthotonic episodes. Animals that showed a rigid posture and a lack of responsiveness to tactile stimuli were considered to be undergoing tonic seizures. Usually the first seizure observed was tonic-opisthotonic, and later episodes were of the tonic variety. Animals were also hyperreactive to stimuli after kainic acid administration. Animals were pretreated with one of the various test
* These data were presented at the 19th Annual Meeting of the Society for Neuroscience, Phoenix, AZ, November, 1989. Correspondence: W. Freed, NIMH Neuroscience Center at St. Elizabeths, Washington, DC 20032, U.S.A.
158 drugs in a vehicle of 10% Tween 80/90% normal saline, in a volume of 10 mi/kg i.p. Thirty min after injection, each animal received 0.14 g g of kainic acid intracerebroventricularly. The time of onset of the first seizure and the time of onset and termination of each succeeding seizure was r e c o r d e d to the nearest 0.1 rain. Animals were observed for 60 min and then euthanized. D a t a r e c o r d e d were: (i) time to onset of the first seizure (latency), (ii) total time elapsed from the start of the first seizure to the end of the last seizure (duration), and (iii) the total length of seizures during the observation period (sum of the duration of each individual seizure episode = total seizure time). For each of these three measures, d a t a were analyzed by a one-way analysis of variance followed by Fisher P L S D tests to c o m p a r e each individual drug dosage to vehicle.
In general, baclofen, p h e n o b a r b i t a l , and L-glutamic acid diethyl ester HCI had no effect on kainate-induced seizures (Table I). C a r b a m a z e p i n e had only a slight effect of significantly decreasing seizure duration. Latency to seizure onset and total seizure time, however, were not altered. The largest dosage of phenytoin, 100 mg/kg, significantly e x a c e r b a t e d the seizures, increasing both seizure duration and total seizure time. T h e smaller dosage of p h e n y t o i n (50 mg/kg) increased latency to seizure onset, but did not alter duration or total seizure time (Table I). K a i n a t e - i n d u c e d seizures were m a r k e d l y worsened by valproic acid at both dosage levels; animals p r e t r e a t e d with v a l p r o a t e showed increased seizure duration, seizure time, and mortality, as well as a decreased latency to seizure onset (Table I). All animals treated with valproic acid exhibited severe tonic-opisthotonic
TABLE I Effects o f anticonvulsants and other compounds on kainic acid-induced seizures
Data shown are means ± S.E.M. Compound (dose)
Latency to seizure onset ~ Duration b
Total seizure time c
Mortality
Number o f mice
Vehicle
1.9 ± 0.24
33,3 + 2.2
29.9 ± 2.1
1
28
Phenytoin 50 mg/kg 100 mg/kg
4.3±1.2" 1.2±0.41
34.1±3.7 51.0±3.9"
31.5±3.3 51.0±3.9'
0 0
9 7
Carbamazepine 50 mg/kg
1.1±0.31
24.9±2.5*
24.9±2.5
0
9
Baclofen 5 mg/kg 10 mg/kg
1.2±0.31 1.1±0.40
30.6±3.6 34.5±2.5
29.8±3.6 34.5±2.5
1 0
10 8
Phenobarbital 40 mg/kg 80 mg/kg
0.81±0.08 0.72±0.13
29.3±1.7 26.7±1.1
28.8±1.8 26.7±1.1
0 0
10 10
Vaiproic acid 125 mg/kg 250 mg/kg
0.79±0.10 1.14±0.21
53.6±2.9* 58.8±0.26*
53.5±2.8* 58.8±0.26*
4 8
10 10
Diazepam 1 mg/kg 2 mg/kg
0.68±0.10" 1.08±0.21
18.3±1.6" 17.5±0.86"
18.3±1.6" 17.0±0.80"
0 0
10 13
Glutamic acid diethyl ester 480mg/kg 960 mg/kg
1.1±0.23 1.4±0.29
25.7±2.7 32.9±2.5
25.7±2.7 32.6±2.5
0 0
9 9
Nifedipine 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg
2.1 + 4.0 + 4.8 + 4.4 + 2.0 +
23.3 + 1.5* 27.4 + 1.8 26.6 + 2.5* 25.7 + 0.8 19.8 + 13.4"
21.4 _ 0.68* 14.5 + 0.64* 12.3 + 1.2" 10.6 + 2.8* 19.8 ± 13.4"
0 1 2 1 5
5 4 18 3 8
*
0.45 0.29 1.1" 0.28 0.62
Indicates significantly different from vehicle (P <~0.05) by Fisher PLSD test.
a F~s,z~ = 5.94, P < 1.0 × 10-4; b F~sA6a= 23.38, P < 1.0 x 10-4; c F1~,166= 15,31, P < 1.0 x 10-4.
159 4O Durltion I
o
(¢)
30
D a
0'
2 "5
5 0'
'
t00
Dose Img/kgl
Fig. 1. Dose-response curve for the effect of nifedipine on seizure duration (time from beginning of first to end of last seizure; open circles) and total seizure time (sum of the duration of all seizure episodes; closed triangles) following intracerebral injection of 0.14 /~g kainic acid. The highest dosage (200 mg/kg) is not shown (see Table I). Data shown are means + S.E.M. Note that seizure time is decreased to a much greater extent than seizure duration; this was because nifedipine decreased the duration of each individual seizure episode.
seizures and most animals that received the larger dosage expired. Diazepam produced a modest decrease in kainateinduced seizures. The latency to seizure onset was not increased, but there were significant parallel decreases in seizure duration and total seizure time (Table I). Both dosages, and particularly the largest dosage, 2 mg/kg, had a substantial sedative effect. Nifedipine, on the other hand, caused a significant dose-dependent decrease in total seizure time (Fig. 1). A significant 28% decrease in total seizure time was seen even at the lowest dosage, 12.5 mg/kg. On the other hand, seizure duration was not greatly decreased. It was noted that the animals treated with nifedipine had similar numbers of seizures, but recovered more rapidly from each individual seizure. Between seizure episodes, the nifedipine-treated animals were relatively normal in appearance, were responsive to tactile stimulation, and did not show evidence of continued tonus or rigidity. Thus the effect of nifedipine was primarily to induce a more rapid recovery from each individual seizure episode. There was also a minor tendency for an increased latency to seizure onset, which was significant only at 50 mg/kg. Therefore, most anticonvulsants did not decrease seizures induced by intracerebral kainic acid. In fact, the present data contrast with previous reports that phenobarbital and baclofen suppress kainate-induced seizures L 9.21. The reason for this difference is unknown. These seizures were somewhat exacerbated by phenytoin and markedly exacerbated by valproic acid, which is consis-
tent with previous reports that both agents failed to inhibit kainate-induced seizures 9'21. As has previously been reported 9, diazepam inhibited kainate°induced seizures, but also had a pronounced sedative effect, while carbamazepine had only a slight effect (Table I). Quisqualate-induced seizures, by comparison, are inhibited by both diazepam and valproic acid 17. The only agent which was found to markedly inhibit kainic acid-induced seizures was nifedipine. This compound did not, however, prevent the development of seizures, and had only a minor effect on the total time from the onset to the end of seizures (i.e., seizure duration). The primary effect of nifedipine was apparently to cause each individual seizure episode to terminate more rapidly. The reasons for this particular mode of action of nifedipine are not known. Nifedipine has recently been reported to block the neurotoxic actions of kainic acid, as well as other excitatory amino acids including N-methyl-o-aspartate and a-amino-3-hydroxy-5-methyl-4-isoxazole propionate ~. In that the excitatory effects of kainate on neurons was not antagonized by nifedipine 2a, it appears likely that the anti-neurotoxic effect of nifedipine was mediated by its primary mode of action, blockade of voltage-gated calcium channels. Therefore, influx of calcium into neurons is probably a secondary event which is required for the manifestation of kainate-induced neurotoxicity~. The nature of the nifedipine effect seen in the present study suggests that similar calcium-mediated events may be involved in propagating or maintaining kainateinduced seizures. Rather than preventing seizures, nifedipine decreased the duration of seizure episodes, so that the animals recovered more rapidly from each individual seizure. Therefore, influx of calcium into neurons via dihydropyridine-sensitive channels may play an essential role in sustaining kainate-induced seizures. One possible application for nifedipine is as a treatment for the seizures or neuronal damage induced by neurotoxins which act at non-NMDA receptors, including fl-N-oxalylaminoalanin~4,2°, or the shellfish contaminant domoic acid 3'7,10,15. Domoic acid in particular is very similar in structure to kainic acid 7"19, and has caused numerous cases of poisoning in Canada 1°. Although relatively high concentrations of nifedipine were required to block neurotoxicity in vitro 23, the present data suggest that effective concentrations can also be achieved in vivo by systemic injection, and that nifedipine is also effective in blocking the seizures produced by kainic acid. These data suggest that anticonvulsant-resistant seizures are the most likely candidate for potential clinical applications of kainic acid antagonists as anticonvulsant drugs. The present data also suggest that calcium channel antagonists might be useful as a treatment modality for
160 certain seizure types that are resistant to classical anticonvulsants. Especially, calcium channel inhibitors might be useful for treating conditions related to excess acti-
vation of the kainic acid class of excitatory amino acid receptor.
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