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Seizures and wet-dog shakes induced by 4-aminopyridine, and their potentiation by nifedipine
European Journal of Pharmacology, 178 (1990) 275-284
275
Elsevier EJP 51219
Seizures and wet-dog shakes induced by 4-aminopyridine, and their potentiation by nifedipine Jorge Fragoso-Veloz, Lourdes Massieu, Rafil Alvarado and Ricardo Tapia Departamento de Neurociencias, Instituto de Fisiologla Celular, Universidad Nacional A utbnoma de M~xico, and lnstituto Nacional de Neurologla y Neurocirugla, Secretarla de Salud, M~xico D.F., M~xieo
Received 18 September 1989, revised MS received 1 December 1989, accepted 2 January 1990
The behavioral and electrographic effects of 4-aminopyridine (4-AP) administered i.p. or microinjected into the hippocampal CA1 region (i.h.) were studied in rats. The modification of such effects by the systemic administration of the Ca 2+ antagonist dihydropyridine, nifedipine, was also studied. 4-AP i.p. (5 mg/kg) induced generalized tonic convulsions in 74% of the animals and death in 13%. Convulsions were characterized by electrical discharges of relatively short duration in all structures studied (frontal cortex, amygdala, dorsal hippocampus and dorsal raphe). Limbic seizures and frequent wet-dog shakes were observed when 4-AP was administered i.h. (2-4 nmol) and this behavior was correlated with hippocampal discharges, which rapidly propagated to the other structures. Pretreatment with nifedipine (7.5-50 m g / k g s.c.) markedly potentiated the effects of 4-AP. The percentage of rats that died during generalized convulsion after i.p. 4-AP increased to 56-87% and the frequency of wet-dog shakes increased after i.h. microinjection of 4-AP. Moreover, nifedipine-treated rats showed long-lasting ( > 60 min) continuous discharges in all structures studied (status epilepticus). These results are discussed in the light of the possible participation of Ca 2+ channels in the convuisant effect of 4-AP and its potentiation by nifedipine. 4-Aminopyridine; Dihydropyridines; Ca2+; Hippocampus; Limbic seizures; Wet-dog shakes
1. Introduction T h e systemic administration of 4-aminopyridine (4-AP) induces convulsions in a variety of species (Schafer et al., 1973), including m a n (Spyker et al., 1980). A l t h o u g h its mechanism of action is not well understood, it has been demonstrated that 4-AP enhances neurotransmitter release, leading to facilitated neural transmission. In the neuromuscular junction, 4-AP increases the quantal release of acetylcholine ( L u n d h and Thesleft, 1977; Molg6 et al., 1979), and in the central
Correspondence to: R. Tapia, Departamento de Neurociencias, Instituto de Fisiologla Celular, Universidad Nacional Autrnoma de M~xico, Apartado Postal 70-600, 04510-Mrxico, D.F. Mrxico.
nervous system it increases the amplitude of both excitatory and inhibitory postsynaptic potentials (Jankowska et al., 1977; Buckle and Haas, 1982). This effect might be explained by blockade of the delayed potassium current, which leads to prolongation of the action potential, as d e m o n s t r a t e d in the squid giant axon (Yeh et al., 1976; Meves and Pichon, 1977). However, in isolated nerve endings (Tapia and Sitges, 1982) and rat striatal slices (DoleZal and TuEek, 1983) it has been shown that 4-AP stimulates the release of neurotransmitters in the absence of depolarization in a Ca 2 +-dependent manner. Moreover, this effect is antagonized by Ca 2+ blockers like l a n t h a n u m and ruthenium red (Tapia et al., 1985); conversely, ruthenium red-induced paralysis in vivo is antagonized by 4-AP (Tapia, 1982). F r o m these findings it has been
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
276
suggested that 4-AP facilitates the action of C a 2+ on neurotransmitter release. The participation of Ca 2+ in convulsive activity has been suggested since Ca 2 + influx precedes the paroxysmal depolarization shifts during epileptic events (Pumain et al., 1983) and seems to underlie the synchronization and propagation of epileptic activity (Heinemann and Pumain, 1981). This hypothesis is supported by the anticonvulsant action of some Ca 2+ channel antagonists of the dihydropyridine type (Meyer et al., 1986a, b; Dolin et al., 1988; Vezzani et al., 1988), as well as by the convulsant effect of the intracerebroventricular (i.c.v.) injection of BAY-K-8644, a dihydropyridine Ca 2+ channel agonist (Shelton et al., 1987). To study the possible role of Ca 2+ in the convulsant effect of 4-AP, we characterized, both behaviorally and electrographically, the seizures induced by the i.p. and intrahippocampal (i.h.) administration of this drug. We also tested the effect of nifedipine and other dihydropyridine calcium channel antagonists on the epileptic activity induced by 4-AP.
2. Materials and methods
2.1. Behavioral observations Adult male Wistar rats (195-327 g) were used in all the experiments. 4-AP was dissolved in distilled water and administered i.p. (5 mg/kg) to control animals and to rats that had been injected i.p. or s.c. 30 min before with the doses of nifedipine indicated under Results. The solution of nifedipine was prepared by appropriate dilution in 20% Tween 80 of the content of capsules of Adalat (Bayer). Both the dilutions and the injections were carried out under subdued lighting. Nisoldipine and nitrendipine were dissolved in 'Adalat placebo solution' (60 g glycerine, 100 g H20, 1129 g polyethylene glycol 400; Schramm et al., 1983) and then diluted in 20% Tween 80 to reach the appropriate concentration. Control Animals were injected with vehicle. Animals were observed until they had recovered (2-3 h) or had died during convulsion.
For i.h. administration of 4-AP, rats were anesthetized with halothane and placed in a Kopf stereotaxic frame. 4-AP (2.1 nmol) was injected (0.1 ~tl) during anesthesia into the CA1 region of the right hippocampus, using a 1 /~1 Hamilton syringe mounted on a manual Kopf injector. The solution also contained 6 m g / m l of Direct blue 15 to enable location of the site of injection. This dye did not produce any effect per se nor did it potentiate the action of the drugs used. The stereotaxic coordinates used were according to the Atlas of Paxinos and Watson (1986): P 3.3, L 2.0 and V 3.0 from the bregma. Some rats were injected s.c. with nifedipine (20 mg/kg) 30 min before the i.h. injection of 4-AP and observed until they had recovered (about 2 h).
2.2. Electrographic observations 2.2.1. Systemic administration of 4-AP To study the pattern and spread of the epileptic discharges induced by systemically administered 4-AP, rats were stereotaxically implanted under ketamine anesthesia with stainless steel dipolar electrodes (0.2 mm diameter, 0.5 mm tip, 0.5 mm interelectrode distance). The electrode leads were attached to a multipin socket, and the electrodes were anchored to the skull with miniature screws and dental acrylic cement. Signals were amplified on a Grass model 78D polygraph. Recording sites included the frontal cortex (A 2.0, L 2.0, V 1.0), left amygdala (P 0.0, L 5.0, V 7.0), right dorsal hippocampus (P 3.3, L 2.0, V 2.6) and dorsal raphe (P 6.0, L 0.0, V 6.0), according to the Atlas of Pellegrino and Cushman (1967). After at least one week of postoperative recovery, 4-AP was injected i.p. (6 mg/kg) and the animals were recorded for about 2 h or until they died during convulsion. It was necessary to inject a slightly higher dose of 4-AP to implanted animals to induce the seizure (6 m g / k g compared to 5 m g / k g for the non-implanted rats). When the effect of nifedipine was tested, it was injected s.c. (20 m g / k g ) 30 min before 4-AP. 2.2.2. Intrahippocampal microinjection of 4-AP For i.h. injections of 4-AP, a cannula guide (stainless steel, 20-gauge) was implanted in
277 a d d i t i o n to the electrodes. The c a n n u l a was sealed with a d u m m y injection c a n n u l a until the time of drug a d m i n i s t r a t i o n . After at least one weak of postoperative recovery, 4 - A P (4.2 n m o l in 0.2 ~1) was injected through a n injection needle (27gauge), which was attached to a 1 /~1 H a m i l t o n syringe a n d aimed at the right area CA1 of the h i p p o c a m p u s , according to the coordinates indicated above; the d e p t h of p e n e t r a t i o n was controlled by m e a n s of a stopper on the injection needle. P r o b a b l y because of the tissue response to the i m p l a n t e d electrodes, a higher dose of 4-AP (4.2 n m o l c o m p a r e d to 2.1 n m o l used for the n o n - i m p l a n t e d rats) was necessary to induce similar behavioral changes. The 4-AP solution contained Direct blue 15, as indicated above. A n i m a l s were recorded for a b o u t 2 h, when electrical activity r e t u r n e d to n o r m a l . W h e n the effect of nifedip i n e was tested, it was injected s.c. (20 m g / k g ) 30 rain before 4-AP.
2.3. Histology A t the e n d of the experiments, the a n i m a l s were anesthetized with k e t a m i n e a n d the b r a i n was perfused with 10% f o r m a l i n solution through the left cardiac ventricle. The b r a i n was removed a n d 40-80 # m thick c o r o n a l sections were sliced in a cryostat. Both the location of the electrodes a n d
the site of injection in the h i p p o c a m p u s were verified histologically.
2.4. Drugs 4-AP, T w e e n 80 (polyoxyethylene s o r b i t a n monooleate), Direct blue 15 a n d p o l y e t h y l e n e glycol 400 were o b t a i n e d from Sigma Chemical Co. N i f e d i p i n e was purchased as A d a l a t (Bayer), a n d nisoldipine a n d n i t r e n d i p i n e were k i n d l y provided by Dr. A. Scriabine (Miles Laboratories, N e w Haven, CT, U.S.A.).
3. R e s u l t s
3.1. Behavioral observations 3.1.1. Systemic 4-AP The i.p. a d m i n i s t r a t i o n of 4-AP p r o d u c e d generalized tonic c o n v u l s i o n ( G T C ) in 74% of the a n i m a l s a p p r o x i m a t e l y 30 m i n after the injection (table 1). All the a n i m a l s that did n o t die (87%) recovered completely 2-3 h after drug a d m i n i s t r a tion. Most a n i m a l s had o n l y one convulsion, although some a n i m a l s had two G T C s . All a n i m a l s showed a series of preconvulsive s y m p t o m s before the seizure, i n c l u d i n g hyperexcitability, tremors, head n o d d i n g , forepaw tremor, salivation, sniffing,
TABLE 1 Effect of dihydropyridines on seizures induced by the i.p. injection of 4-AP (5 mg/kg). Treatment
Dihydropyridine dose (mg/kg) a
Total number of generalized tonic convulsions (GTC)/ number of rats treated
% of rats showing GTC
% of rats dying during GTC b
4-AP 4-AP + nifedipine 4-AP + nifedipine 4-AP + nifedipine 4-AP+nifedipine 4-AP + nifedipine 4-AP + nifedipine 4-AP + nifedipine 4-AP + nisoldipine 4-AP + nitrendipine
-
103/119 = 0.8 c 11/15 = 0.7 26/30 = 0.8 8/7 = 1.1 16/8 = 2 22/16 = 1.3 14/8 = 1.7 7/4 = 1.7 10/6 = 1.6 8/5 = 1.6
73.9 66.1 70 100 100 100 100 100 83.3 80
12.6 20.0 16.0 28.5 75.0 d 56.2 a 87.5 a 75.0 d 83.3 d 60.0 a
0.1 0.25 5 7.5 10 20 50 20 20
a The dihydropyridines were administered s.c., except the two lower doses, which were injected i.p., 30 rain before the administration of 4-AP. b The time to death was equally variable in the dihydropyridine-treated and the control animals (20-78 min after 4-AP injection), c First GTC occurred at 32.8+ 1.6 min after 4-AP injection, o p < 0.005, as compared to 4-AP alone (chi-square test).
278
rearing, chewing, grooming and myoclonus. One or more of these symptoms first appeared at 4.68 + 0.3 min (n = 119) after 4-AP administration. Low doses of nifedipine (0.1-5.0 m g / k g ) had no effect on seizures induced by 4-AP. However, as shown in table 1, at higher doses of nifedipine or of the other dihydropyridines tested (7.5-50.0 m g / k g ) an increase in co:vulsive behavior was observed. This potentiating effect was evident from the number of rats showing G T C (100% in nifedipine-treated vs. 74% in controls), from the number of convulsions per rat (1.3-2.0 in dihydropyridine-treated vs. 0.8 in controls) and from the number of rats that died during G T C (56-87% in dihydropyridine-treated vs. 13% in controls). Neither the latency to the initial symptoms nor the symptoms themselves were affected by nifedipine at any of the doses tested. The latency to death was as variable in the dihydropyridine-treated animals as in those injected with only 4-AP, and there was no correlation between the dose of nifedipine tested and the latency to death.
ing, rearing and forepaw tremor. The most evident sign, however, was the occurrence of 'wet-dog shakes' (WDS). These motor signs began to occur immediately after the animals recovered from anesthesia. The frequency of W D S increased progressively, reached a plateau (20-25/5 min) between 25 and 60 min after 4-AP microinjection and decreased slowly thereafter (fig. 1). As with systemically administered 4-AP, nifedipine seemed to potentiate the effects of the i.h. injection. Nifedipine-pretreated animals showed a significant increase in the frequency of W D S during the 10-30 min period after 4-AP injection (fig. 1). The frequency of WDS during this 20 min period was 10-15/5 min for the control group and 20-45/5 min for the nifedipinetreated group. This effect was no longer significant during the decay phase of the action of 4-AP (fig. 1), and there was also no significant change in the total number of WDS observed (control, 305 + 32.3; nifedipine-treated, 314 + 45.3; mean + S.E.),
3.1.2. Intrahippocampal 4-AP In contrast to the effects of i.p. injected 4-AP, the microinjection of 4-AP into the CA1 region of the hippocampus induced 'limbic seizures'. The motor signs that characterized these seizures were analogous to those described for the i.p. administration of kainic acid (Ben-Ari et al., 1981). These included sniffing, masticatory movements, groom-
3.2. Electrographic observations
50
3.2.1. Systemic 4-AP The epileptiform activity induced by 4-AP was characterized electrographically by the simultaneous appearance of discharges in all the structures studied (fig. 2), which occurred at the time of occurrence of the G T C (19.1 + 3.9 min, n = 8).
(d) NO NIFEDIPINE
40
(a) (b)
I
NIFEDIPINE
,o t 0
20
4
'o
60 PERIOD
80
I00
120
( MIN )
Fig. 1. Effect of nifedipine (20 m g / k g s.c.) on the occurrence of WDS induced by the administration of 4-AP (2.1 nmol) into the C A I hippocampal region. Nifedipine was injected 30 min before 4-AP. Values are mean numbers of W D S / 5 m i n i S . E , for nine (no nifedipine) and eight (nifedipine) rats. a p < 0.05, b p < 0.02, c p < 0.01, d p < 0.001, as compared with the corresponding rats not treated with nifedipine.
279 i
? 31 mln
CONTROL
,
? 31
mln
31 mln
15 8
30 8
?
? 57 rain i,~l lllll !, I~11 I Ill I
43
mln
I
? 43 min T 30 a
44
mln
Fig. 2. Electrographic record of the effect of i.p. administered 4-AP (6 mg/kg). In this and the following figures the abbreviations are: CX, frontal cortex; AMG, left amygdala; HPC, fight dorsal hippocampus; DR, dorsal raphe. This rat had two generalized tonic convulsions, at 31 and 43 min (arrows), and died at 45 min. Similar recordings were obtained for eight rats. Horizontal bar = 1 s; vertical bars = 0.5 inV.
These discharges were characterized spikes, p o l y - s p i k e s , a n d s p i k e - w a v e S p i k e s w e r e o f h i g h a m p l i t u d e a n d the d i s c h a r g e w a s 28.2 _+ 5.3 s. I n g e n e r a l ,
by isolated complexes. duration of the animals
h a d o n l y o n e G T C . It w a s d i f f i c u l t to e s t a b l i s h a b e h a v i o r a l - e l e c t r o g r a p h i c c o r r e l a t i o n since s o m e e p i l e p t i f o r m p a t t e r n s w e r e r e c o r d e d w i t h o u t evid e n t m o t o r signs.
280 A n interesting preconvulsive electrographic feature was observed some min after the injection of 4-AP: a theta r h y t h m with high regularity appeared. The frequency and amplitude of theta r h y t h m increased progressively in all structures studied, especially in the h i p p o c a m p u s (data not shown). This finding was observed in all treated rats and was not related to b o d y m o v e m e n t s or l o c o m o t o r activity. Six rats were treated with nifedipine (20 m g / k g s.c.) 30 rain before 4-AP. As described above for the non-implanted rats, the n u m b e r of G T C per
rat increased in the nifedipine-pretreated rats. These animals had two and sometimes three convulsions, in contrast to the nifedipine-untreated rats, which generally had only one GTC. The characteristics of the electrographic discharges p r o d u c e d by 4-AP were not modified by nifedipine pretreatment and, although their m e a n duration increased from 28.2 + 5.3 to 47.7 + 8.7 s, this difference was not significant. W h e n nifedipine was injected alone it had no effect on behavior nor on the electrical activity of the animals at any of the doses tested.
CX ~
A M O ~'-~- -
~
~
~
30 s
CONTROL
I t
5 min
34
tmln
I ! ,fi!,,,'l!j:i
9 rain
t
7 I rnln
Fig. 3. Electrographic record of the effect of i.h. administered 4-AP (4.2 nmol). In this rat the first rhythmic spikes appeared in the hippocampus at 30 s, and at 9 rain (arrows) the discharge propagated to the cortex. At 34 rain the epileptic activity became generalized, hut at 71 rain spikes were observed only in the hippocampus. These spikes continued for about 3 h and then disappeared. Similar recordings were obtained for four rats. Horizontal bar ~ 1 s; vertical bars ~ 0.5 mV.
281
3.2.2. Lh. 4 - A P T h e a d m i n i s t r a t i o n of 4-AP into the CA1 region of the h i p p o c a m p u s i n d u c e d r h y t h m i c high a m p l i t u d e spikes, which initially appeared exclusively in this structure, with a m e a n latency of 48.7 + 18.7 s (n = 4) (fig. 3). This activity t e n d e d to increase progressively in frequency a n d ampli-
tude, a n d after a few m i n (5.5 + 2.3) the epileptic activity p r o p a g a t e d to the other structures studied a n d b e c a m e generalized after 16.1 + 6.5 min. After a b o u t 1 h the discharges in the cortex, a m y g d a l a a n d raphe decreased in a m p l i t u d e a n d frequency, b u t in the h i p p o c a m p u s they c o n t i n u e d for a b o u t 3 h. I m p l a n t e d a n i m a l s showed fewer W D S (47.5
CX ~ . , ~ , ~ , ; ' . ~ < , ~ w ~ , ~ , , . ~ , ~ , ~ v ~ AMO " , ~ v ~ c , ~ ' ~ r ~ ' ~ ¢ % ~ HPC ~,,,,~dW-V~
DR ~¢ ~ . ~ ¢ ~ ~ . . . ~ , ~ ~ ' ~
t
4 rain
CONTROL
,
6 mln
,
!/ 12 min
I
3 5 mln
134
5'
rain
Fig. 4. Electrographic record of the status epilepticus induced by 4-AP in rats pretreated with nifedipine (20 mg/kg s.c.). In this rat the first spikes appeared at 4 rain in the hippocampus and propagated to the cortex and the amygdala at 6 rain (arrows). The continuous generalized discharge characteristic of status epilepticus appeared at 12 rain and lasted for more than 1 h. Similar recordings were obtained for six rats. Horizontal bar = 1 s; vertical bars = 0.5 mV for AMG and HPC, and 0.05 mV for CX and DR.
282 + 14.7 per h) than the non-implanted 4-AP-treated rats (see fig. 1); W D S frequently appeared after the discharges. Six rats were treated with nifedipine (20 m g / k g s.c.) 30 min before the i.h. injection of 4-AP. They showed an increased latency of appearance of rhythmic spikes (139.6 _+ 48.1 s) compared to the untreated animals (48.7_+ 18.7 s), although this difference was not significant. As in control rats, the discharge occurred initially in the hippocampus, rapidly propagated to the other structures (4.9 + 0.6 min) and subsequently became generalized (6.5 _+ 1.1 min). The most interesting effect of nifedipine was the induction of a status epilepticus, which was observed in all animals pretreated with this drug and was characterized by continuous discharges of very high amplitude and frequency in all structures studied (fig. 4). The discharges appeared 13.1 + 2.4 min after microinjection of 4-AP and lasted in all cases for more than 1 h (the mean time of disappearance of this status was 92.0_+ 29.9 min after 4-AP injection). As a consequence, a marked increase in the percentage of time spent in discharge was observed (60.6 + 11.3 min in nifedipine-treated animals vs. 12.6 + 1.5 rain in controls). The time to achieve recovery was also increased considerably (135.8 _+ 26.4 vs. 71.8 _+ 8.8 min in controls). There was no significant difference in the number of W D S per hour between the nifedipine-treated animals (66.9 _+ 21.4) and the untreated group (47.5 _+ 14.7). As already mentioned, nifedipine per se did not produce any significant behavioral or electrographic alteration.
4. Discussion
The convulsant action of systemically administered 4-AP has long been recognized (Schafer et al., 1973). However, the effects of i.h. administered 4-AP, as well as the electrographic correlates of both routes of administration, have not been studied previously. The main effect of systemic 4-AP was to induce convulsive activity, including G T C and electrographic discharges in all structures studied. In contrast, microinjection of 4-AP into the CA1
region of the hippocampus resulted in limbic-type seizures accompanied by frequent WDS. Since an increased entry of Ca z+ into neuronal somas or dendrites has been implicated in epileptic discharges (Wong and Prince, 1978; Heinem a n n and Pumain, 1981; Pumain et al., 1983), a possible action of 4-AP could be to directly facilitate Ca 2+ influx through somatic voltage-dependent Ca 2+ channels. Such an effect of 4-AP, independent of its action as a K + channel blocker, has been demonstrated in cultured spinal cord neurons (Rogawski and Barker, 1983). Contrary to what we expected, nifedipine and the other dihydropyridines used clearly enhanced the convulsant effects of 4-AP. The potentiation of the convulsions produced by i.p. 4-AP was manifest both behaviorally and electrographically, since the number of G T C per rat doubled, the percentage of animals that died during G T C increased 5- to 7-fold, and electrographically there was a 70% increase in the mean duration of the discharges. The nifedipine potentiation was even more evident when 4-AP was administered into the CA1 region of the hippocampus. Behaviorally, there was a notable increase in the number of W D S during the first 30 min, and electrographically the epileptic discharge in all areas studied was continuous during at least 1 h in all animals pretreated with nifedipine. It is unlikely that this facilitatory effect of nifedipine occurred as a consequence of its cardiovascular actions, since it has been shown that, in the rat, a dose of 100 m g / k g of nitrendipine, which is 5- to 10-fold higher than the effective doses used in the present experiments, causes only a slight decrease (25%) in blood pressure (Knorr and Garthoff, 1984). It also seems improbable that the potentiation was due to an action of nifedipine on presynaptic calcium channels, since we have previously demonstrated that the dihydropyridines do not affect the Ca2+-dependent stimulation of transmitter release produced by 4-AP in synaptosomes (Massieu and Tapia, 1988). The potentiating action of nifedipine was surprising in view of previous reports indicating that the Ca 2+ antagonist dihydropyridines possess anticonvulsant properties. These dihydropyridines have been shown to be effective against seizures
283 induced by ischemia, pentylenetetrazole, bicucull i n e a n d e l e c t r o s h o c k in t h e r a b b i t ( M e y e r et al., 1986a, b), a n d b y p e n t y l e n e t e t r a z o l e , h i g h p r e s s u r e ( D o l i n et al., 1988), k i n d l i n g ( V e z z a n i et al., 1988) a n d s o m e n e u r o t o x i n s a n d 4 - A P itself ( G a n d o l f o et al., 1989) in t h e rat. F u r t h e r m o r e , the i.c.v. a d m i n i s t r a t i o n of t h e C a 2÷ a g o n i s t , d i h y d r o p y r i d i n e B A Y - K - 8 6 4 4 , p r o d u c e s seizures t h a t are a n t a g o n i z e d b y s y s t e m i c n i f e d i p i n e ( S h e l t o n et al., 1987). T h e d i s c r e p a n c y b e t w e e n t h e s e o b s e r v a t i o n s a n d t h o s e o f the p r e s e n t p a p e r , h o w e v e r , are m o r e a p p a r e n t t h a n real, since, b e s i d e s i m p o r t a n t d i f f e r e n c e s in the r o u t e o f a d m i n i s t r a t i o n o f t h e d i h y d r o p y r i d i n e s used, the s t u d i e s m e n t i o n e d a b o v e s h o w e d t h a t the a n t i c o n v u l s a n t a c t i o n o f d i h y d r o p y r i d i n e s w a s r e s t r i c t e d to c e r t a i n t y p e s o f convulsions and that these drugs even potentiated o t h e r t y p e o f seizures. T h u s , D o l i n et al. (1988) found that nitrendipine and nimodipine were not e f f e c t i v e a g a i n s t s t r y c h n i n e , a n d i n c r e a s e d t h e incidence of clonic convulsions and mortality prod u c e d b y N - m e t h y l - a s p a r t a t e . F u r t h e r m o r e , Vezz a n i et al. (1988) o b s e r v e d s o m e p o t e n t i a t i o n b y nifedipine of kainate- and quinolinate-induced seizures. I n this r e s p e c t , it h a s b e e n r e p o r t e d t h a t n i f e d i p i n e i n c r e a s e s C a 2÷ u p t a k e in b r a i n c o r t e x slices ( R i v e r o s a n d O r r e g o , 1986). T h i s s u g g e s t s that, u n d e r c e r t a i n c o n d i t i o n s , the d i h y d r o p y i r i d i n e s g e n e r a l l y c o n s i d e r e d as C a 2÷ a n t a g o n i s t s b e h a v e as p a r t i a l a g o n i s t s , as has also b e e n s h o w n f o r n i t r e n d i p i n e in c a r d i a c m u s c l e cells ( H e s s et al., 1984) a n d in c o r o n a r y a r t e r y rings ( M i w a a n d S c h w a r t z , 1987).
Acknowledgements The authors wish to thank Mr, Joaquin Manjarrez for his expert assistance in the electrode implantation. This work was supported in part by the Consejo Nacional de Ciencia y Tecnologia, M6xico, D.F. (project P228CCOX-880370).
References Ben-Aft, Y., E. Tremblay, D. Riche, G. Ghilini and R. Naquet, 1981, Electrographic, clinical and pathological alterations following systemic administration of kainic acid, bicucul-
line or pentetrazole: metabolic mapping using the deoxyglycose method with special reference to the pathology of epilepsy, Neuroscience 6, 1361. Buckle, P.J. and H.L. Haas, 1982, Enhancement of synaptic transmission by 4-aminopyridine in hippocampal slices of the rat, J. Physiol. (London) 326, 109. Dole~al, V. and S. Tu6ek, 1983, The effects of 4-aminopyridine and tetrodotoxin on the release of acetylcholine from rat striatal slices, Naunyn-Schmiedeb. Arch. Pharmacol. 323, 90. Dolin, S.J., A.B. Hunter, M.J. Halsey and H.J. Little, 1988, Anticonvulsant profile of the dihydropyridine calcium channel antagonists, nitrendipine and nimodipine, European J. Pharmacol. 152, 19. Gandolfo, G., C. Gottesmann, J.N. Bidard and M. Lazdunski, 1989, Ca 2÷ channel blockers prevent seizures induced by a class of K ÷ channel inhibitors, European J. Pharmacol. 160, 173. Heinemann, U. and R. Pumain, 1981, Effects of tetrodotoxin on changes in extracellular free calcium induced by repetitive electrical simulation and iontophoretic application of excitatory amino acids in the sensorimotor cortex of cats, Neurosci. Lett. 21, 87. Hess, P., J.B. Lansman and R.W. Tsien, 1984, Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists, Nature 311,538. Jankowska, E., A. Lundberg, P. Rudomin and E. Sykova, 1977, Effects of 4-aminopyridine on transmission in excitatory and inhibitory synapses in the spinal cord, Brain Res. 136, 387. Knorr, A. and B. Garthoff, 1984, Differential influence of the calcium antagonist nitrendipine and the vasodilator hydralazine on normal and elevated blood pressure, Arch. Int. Pharmacodyn. 269, 316. Lundh, H. and S. Thesleff, 1977, The mode of action of 4-aminopyridine and guanidine on transmitter release from motor nerve terminals, European J. Pharmacol. 42, 411. Massieu, L. and R. Tapia, 1988, Relationship of dihydropyridine binding sites with calcium-dependent neurotransmitter release in synaptosomes, J. Neurochem. 51, 1184. Meves, H. and Y. Pichon, 1977, The effect of internal and external 4-aminopyridine on the potassium currents in intracellularly perfused squid giant axons, J. Physiol. (London) 268, 511. Meyer, F.B., R.E. Anderson, T.M. Sundt and F.W. Sharbrough, 1986a, Selective central nervous system calcium channel blockers - a new class of anticonvulsant agents, Mayo Clin. Proc. 61,239. Meyer, F.B., P.W. Tally, R.E. Anderson, T.M. Sundt, T.L. Yaksh and F.W. Sharbrough, 1986b, Inhibition of electrically induced seizures by a dihydropyridine calcium channel blocker, Brain Res. 384, 180. Miwa, K. and A. Schwartz, 1987, Paradoxical augmentation of ( - ) B A Y K 8644-induced calcium influx by nitrendipine, Biochem. Biophys. Res. Commun. 148, 1. Molg6, J., M. Lemeignan and P. Lechat, 1979, Analysis of the action of 4-aminopyridine during repetitive stimulation at
284 the neuromuscular junction, European J. Pharmacol. 53, 307. Paxinos, G. and C. Watson, 1986, The Rat Brain in Stereotaxic Coordinates (Academic Press, Sydney). Pellegrino, L.J. and A.J. Cushman, 1967, A Stereotaxic Atlas of the Rat Brain (Appleton-Century-Crofts, New York). Pumain, R., I. Kurcewicz and J. Louvel, 1983, Fast extracellular calcium transients: Involvement in epileptic processes, Science 222, 177. Riveros, N. and F. Orrego, 1986, N-Methylaspartate-activated calcium channels in rat brain cortex slices. Effect of calcium channel blockers and of inhibitory and depressant substances, Neuroscience 17, 541. Rogawski, M.A. and J.L. Barker, 1983, Effects of 4-aminopyridine on calcium action potentials and calcium current under voltage clamp in spinal neurons, Brain Res. 280, 180. Schafer, E.W., R.B. Brunton and D.J. Cunningham, 1973, A summary of the acute toxicity of 4-aminopyridine to birds and mammals, Toxicol. Appl. Pharmacol. 26, 532. Schramm, M., G. Thomas, R. Towart and G. Franckowiak, 1983, Activation of calcium channels by novel 1,4-dihydropyridines, Arzneim. Forsch. 33, 1268. Shelton, R.C., J.A. Grebb and W.J. Freed, 1987, Induction of seizures in mice by intracerebroventricular administration
of the calcium channel agonist BAY k 8644, Brain Res. 402, 399. Spyker, D.A., C. Lynch, J. Shabanowitz and J.A. Sinn, 1980, Poisoning with 4-aminopyridine: Report of three cases, Clin. Toxicol. 16, 487. Tapia, R., 1982, Antagonism of the ruthenium red-induced paralysis in mice by 4-aminopyridine, guanidine and lanthanum, Neurosci. Left. 30, 73. Tapia, R. and M. Sitges, 1982, Effect of 4-aminopyridine on transmitter release in synaptosomes, Brain Res. 250, 291. Tapia, R., M. Sitges and E. Morales, 1985, Mechanism of the calcium-dependent stimulation of transmitter release by 4-aminopyridine in synaptosomes, Brain Res. 361, 373. Vezzani, A., H.Q. Wu, M.A. Stasi, P. Angelico and R. Samanin, • 1988, Effect of various calcium channel blockers on three different models of limbic seizures in rats, Neuropharmacology 27, 451. Wong, R.K.S. and D.A. Prince, 1978, Participation of calcium spikes during intrinsic burst firing in hippocampal neurons, Brain Res. 159, 385. Yeh, J.Z., G.S. Oxford, C.H. Wu and T. Narahashi, 1976, Interactions of aminopyridines with potassium channels of squid axon membranes, Biophys. J. 16, 77.
275
Elsevier EJP 51219
Seizures and wet-dog shakes induced by 4-aminopyridine, and their potentiation by nifedipine Jorge Fragoso-Veloz, Lourdes Massieu, Rafil Alvarado and Ricardo Tapia Departamento de Neurociencias, Instituto de Fisiologla Celular, Universidad Nacional A utbnoma de M~xico, and lnstituto Nacional de Neurologla y Neurocirugla, Secretarla de Salud, M~xico D.F., M~xieo
Received 18 September 1989, revised MS received 1 December 1989, accepted 2 January 1990
The behavioral and electrographic effects of 4-aminopyridine (4-AP) administered i.p. or microinjected into the hippocampal CA1 region (i.h.) were studied in rats. The modification of such effects by the systemic administration of the Ca 2+ antagonist dihydropyridine, nifedipine, was also studied. 4-AP i.p. (5 mg/kg) induced generalized tonic convulsions in 74% of the animals and death in 13%. Convulsions were characterized by electrical discharges of relatively short duration in all structures studied (frontal cortex, amygdala, dorsal hippocampus and dorsal raphe). Limbic seizures and frequent wet-dog shakes were observed when 4-AP was administered i.h. (2-4 nmol) and this behavior was correlated with hippocampal discharges, which rapidly propagated to the other structures. Pretreatment with nifedipine (7.5-50 m g / k g s.c.) markedly potentiated the effects of 4-AP. The percentage of rats that died during generalized convulsion after i.p. 4-AP increased to 56-87% and the frequency of wet-dog shakes increased after i.h. microinjection of 4-AP. Moreover, nifedipine-treated rats showed long-lasting ( > 60 min) continuous discharges in all structures studied (status epilepticus). These results are discussed in the light of the possible participation of Ca 2+ channels in the convuisant effect of 4-AP and its potentiation by nifedipine. 4-Aminopyridine; Dihydropyridines; Ca2+; Hippocampus; Limbic seizures; Wet-dog shakes
1. Introduction T h e systemic administration of 4-aminopyridine (4-AP) induces convulsions in a variety of species (Schafer et al., 1973), including m a n (Spyker et al., 1980). A l t h o u g h its mechanism of action is not well understood, it has been demonstrated that 4-AP enhances neurotransmitter release, leading to facilitated neural transmission. In the neuromuscular junction, 4-AP increases the quantal release of acetylcholine ( L u n d h and Thesleft, 1977; Molg6 et al., 1979), and in the central
Correspondence to: R. Tapia, Departamento de Neurociencias, Instituto de Fisiologla Celular, Universidad Nacional Autrnoma de M~xico, Apartado Postal 70-600, 04510-Mrxico, D.F. Mrxico.
nervous system it increases the amplitude of both excitatory and inhibitory postsynaptic potentials (Jankowska et al., 1977; Buckle and Haas, 1982). This effect might be explained by blockade of the delayed potassium current, which leads to prolongation of the action potential, as d e m o n s t r a t e d in the squid giant axon (Yeh et al., 1976; Meves and Pichon, 1977). However, in isolated nerve endings (Tapia and Sitges, 1982) and rat striatal slices (DoleZal and TuEek, 1983) it has been shown that 4-AP stimulates the release of neurotransmitters in the absence of depolarization in a Ca 2 +-dependent manner. Moreover, this effect is antagonized by Ca 2+ blockers like l a n t h a n u m and ruthenium red (Tapia et al., 1985); conversely, ruthenium red-induced paralysis in vivo is antagonized by 4-AP (Tapia, 1982). F r o m these findings it has been
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
276
suggested that 4-AP facilitates the action of C a 2+ on neurotransmitter release. The participation of Ca 2+ in convulsive activity has been suggested since Ca 2 + influx precedes the paroxysmal depolarization shifts during epileptic events (Pumain et al., 1983) and seems to underlie the synchronization and propagation of epileptic activity (Heinemann and Pumain, 1981). This hypothesis is supported by the anticonvulsant action of some Ca 2+ channel antagonists of the dihydropyridine type (Meyer et al., 1986a, b; Dolin et al., 1988; Vezzani et al., 1988), as well as by the convulsant effect of the intracerebroventricular (i.c.v.) injection of BAY-K-8644, a dihydropyridine Ca 2+ channel agonist (Shelton et al., 1987). To study the possible role of Ca 2+ in the convulsant effect of 4-AP, we characterized, both behaviorally and electrographically, the seizures induced by the i.p. and intrahippocampal (i.h.) administration of this drug. We also tested the effect of nifedipine and other dihydropyridine calcium channel antagonists on the epileptic activity induced by 4-AP.
2. Materials and methods
2.1. Behavioral observations Adult male Wistar rats (195-327 g) were used in all the experiments. 4-AP was dissolved in distilled water and administered i.p. (5 mg/kg) to control animals and to rats that had been injected i.p. or s.c. 30 min before with the doses of nifedipine indicated under Results. The solution of nifedipine was prepared by appropriate dilution in 20% Tween 80 of the content of capsules of Adalat (Bayer). Both the dilutions and the injections were carried out under subdued lighting. Nisoldipine and nitrendipine were dissolved in 'Adalat placebo solution' (60 g glycerine, 100 g H20, 1129 g polyethylene glycol 400; Schramm et al., 1983) and then diluted in 20% Tween 80 to reach the appropriate concentration. Control Animals were injected with vehicle. Animals were observed until they had recovered (2-3 h) or had died during convulsion.
For i.h. administration of 4-AP, rats were anesthetized with halothane and placed in a Kopf stereotaxic frame. 4-AP (2.1 nmol) was injected (0.1 ~tl) during anesthesia into the CA1 region of the right hippocampus, using a 1 /~1 Hamilton syringe mounted on a manual Kopf injector. The solution also contained 6 m g / m l of Direct blue 15 to enable location of the site of injection. This dye did not produce any effect per se nor did it potentiate the action of the drugs used. The stereotaxic coordinates used were according to the Atlas of Paxinos and Watson (1986): P 3.3, L 2.0 and V 3.0 from the bregma. Some rats were injected s.c. with nifedipine (20 mg/kg) 30 min before the i.h. injection of 4-AP and observed until they had recovered (about 2 h).
2.2. Electrographic observations 2.2.1. Systemic administration of 4-AP To study the pattern and spread of the epileptic discharges induced by systemically administered 4-AP, rats were stereotaxically implanted under ketamine anesthesia with stainless steel dipolar electrodes (0.2 mm diameter, 0.5 mm tip, 0.5 mm interelectrode distance). The electrode leads were attached to a multipin socket, and the electrodes were anchored to the skull with miniature screws and dental acrylic cement. Signals were amplified on a Grass model 78D polygraph. Recording sites included the frontal cortex (A 2.0, L 2.0, V 1.0), left amygdala (P 0.0, L 5.0, V 7.0), right dorsal hippocampus (P 3.3, L 2.0, V 2.6) and dorsal raphe (P 6.0, L 0.0, V 6.0), according to the Atlas of Pellegrino and Cushman (1967). After at least one week of postoperative recovery, 4-AP was injected i.p. (6 mg/kg) and the animals were recorded for about 2 h or until they died during convulsion. It was necessary to inject a slightly higher dose of 4-AP to implanted animals to induce the seizure (6 m g / k g compared to 5 m g / k g for the non-implanted rats). When the effect of nifedipine was tested, it was injected s.c. (20 m g / k g ) 30 min before 4-AP. 2.2.2. Intrahippocampal microinjection of 4-AP For i.h. injections of 4-AP, a cannula guide (stainless steel, 20-gauge) was implanted in
277 a d d i t i o n to the electrodes. The c a n n u l a was sealed with a d u m m y injection c a n n u l a until the time of drug a d m i n i s t r a t i o n . After at least one weak of postoperative recovery, 4 - A P (4.2 n m o l in 0.2 ~1) was injected through a n injection needle (27gauge), which was attached to a 1 /~1 H a m i l t o n syringe a n d aimed at the right area CA1 of the h i p p o c a m p u s , according to the coordinates indicated above; the d e p t h of p e n e t r a t i o n was controlled by m e a n s of a stopper on the injection needle. P r o b a b l y because of the tissue response to the i m p l a n t e d electrodes, a higher dose of 4-AP (4.2 n m o l c o m p a r e d to 2.1 n m o l used for the n o n - i m p l a n t e d rats) was necessary to induce similar behavioral changes. The 4-AP solution contained Direct blue 15, as indicated above. A n i m a l s were recorded for a b o u t 2 h, when electrical activity r e t u r n e d to n o r m a l . W h e n the effect of nifedip i n e was tested, it was injected s.c. (20 m g / k g ) 30 rain before 4-AP.
2.3. Histology A t the e n d of the experiments, the a n i m a l s were anesthetized with k e t a m i n e a n d the b r a i n was perfused with 10% f o r m a l i n solution through the left cardiac ventricle. The b r a i n was removed a n d 40-80 # m thick c o r o n a l sections were sliced in a cryostat. Both the location of the electrodes a n d
the site of injection in the h i p p o c a m p u s were verified histologically.
2.4. Drugs 4-AP, T w e e n 80 (polyoxyethylene s o r b i t a n monooleate), Direct blue 15 a n d p o l y e t h y l e n e glycol 400 were o b t a i n e d from Sigma Chemical Co. N i f e d i p i n e was purchased as A d a l a t (Bayer), a n d nisoldipine a n d n i t r e n d i p i n e were k i n d l y provided by Dr. A. Scriabine (Miles Laboratories, N e w Haven, CT, U.S.A.).
3. R e s u l t s
3.1. Behavioral observations 3.1.1. Systemic 4-AP The i.p. a d m i n i s t r a t i o n of 4-AP p r o d u c e d generalized tonic c o n v u l s i o n ( G T C ) in 74% of the a n i m a l s a p p r o x i m a t e l y 30 m i n after the injection (table 1). All the a n i m a l s that did n o t die (87%) recovered completely 2-3 h after drug a d m i n i s t r a tion. Most a n i m a l s had o n l y one convulsion, although some a n i m a l s had two G T C s . All a n i m a l s showed a series of preconvulsive s y m p t o m s before the seizure, i n c l u d i n g hyperexcitability, tremors, head n o d d i n g , forepaw tremor, salivation, sniffing,
TABLE 1 Effect of dihydropyridines on seizures induced by the i.p. injection of 4-AP (5 mg/kg). Treatment
Dihydropyridine dose (mg/kg) a
Total number of generalized tonic convulsions (GTC)/ number of rats treated
% of rats showing GTC
% of rats dying during GTC b
4-AP 4-AP + nifedipine 4-AP + nifedipine 4-AP + nifedipine 4-AP+nifedipine 4-AP + nifedipine 4-AP + nifedipine 4-AP + nifedipine 4-AP + nisoldipine 4-AP + nitrendipine
-
103/119 = 0.8 c 11/15 = 0.7 26/30 = 0.8 8/7 = 1.1 16/8 = 2 22/16 = 1.3 14/8 = 1.7 7/4 = 1.7 10/6 = 1.6 8/5 = 1.6
73.9 66.1 70 100 100 100 100 100 83.3 80
12.6 20.0 16.0 28.5 75.0 d 56.2 a 87.5 a 75.0 d 83.3 d 60.0 a
0.1 0.25 5 7.5 10 20 50 20 20
a The dihydropyridines were administered s.c., except the two lower doses, which were injected i.p., 30 rain before the administration of 4-AP. b The time to death was equally variable in the dihydropyridine-treated and the control animals (20-78 min after 4-AP injection), c First GTC occurred at 32.8+ 1.6 min after 4-AP injection, o p < 0.005, as compared to 4-AP alone (chi-square test).
278
rearing, chewing, grooming and myoclonus. One or more of these symptoms first appeared at 4.68 + 0.3 min (n = 119) after 4-AP administration. Low doses of nifedipine (0.1-5.0 m g / k g ) had no effect on seizures induced by 4-AP. However, as shown in table 1, at higher doses of nifedipine or of the other dihydropyridines tested (7.5-50.0 m g / k g ) an increase in co:vulsive behavior was observed. This potentiating effect was evident from the number of rats showing G T C (100% in nifedipine-treated vs. 74% in controls), from the number of convulsions per rat (1.3-2.0 in dihydropyridine-treated vs. 0.8 in controls) and from the number of rats that died during G T C (56-87% in dihydropyridine-treated vs. 13% in controls). Neither the latency to the initial symptoms nor the symptoms themselves were affected by nifedipine at any of the doses tested. The latency to death was as variable in the dihydropyridine-treated animals as in those injected with only 4-AP, and there was no correlation between the dose of nifedipine tested and the latency to death.
ing, rearing and forepaw tremor. The most evident sign, however, was the occurrence of 'wet-dog shakes' (WDS). These motor signs began to occur immediately after the animals recovered from anesthesia. The frequency of W D S increased progressively, reached a plateau (20-25/5 min) between 25 and 60 min after 4-AP microinjection and decreased slowly thereafter (fig. 1). As with systemically administered 4-AP, nifedipine seemed to potentiate the effects of the i.h. injection. Nifedipine-pretreated animals showed a significant increase in the frequency of W D S during the 10-30 min period after 4-AP injection (fig. 1). The frequency of WDS during this 20 min period was 10-15/5 min for the control group and 20-45/5 min for the nifedipinetreated group. This effect was no longer significant during the decay phase of the action of 4-AP (fig. 1), and there was also no significant change in the total number of WDS observed (control, 305 + 32.3; nifedipine-treated, 314 + 45.3; mean + S.E.),
3.1.2. Intrahippocampal 4-AP In contrast to the effects of i.p. injected 4-AP, the microinjection of 4-AP into the CA1 region of the hippocampus induced 'limbic seizures'. The motor signs that characterized these seizures were analogous to those described for the i.p. administration of kainic acid (Ben-Ari et al., 1981). These included sniffing, masticatory movements, groom-
3.2. Electrographic observations
50
3.2.1. Systemic 4-AP The epileptiform activity induced by 4-AP was characterized electrographically by the simultaneous appearance of discharges in all the structures studied (fig. 2), which occurred at the time of occurrence of the G T C (19.1 + 3.9 min, n = 8).
(d) NO NIFEDIPINE
40
(a) (b)
I
NIFEDIPINE
,o t 0
20
4
'o
60 PERIOD
80
I00
120
( MIN )
Fig. 1. Effect of nifedipine (20 m g / k g s.c.) on the occurrence of WDS induced by the administration of 4-AP (2.1 nmol) into the C A I hippocampal region. Nifedipine was injected 30 min before 4-AP. Values are mean numbers of W D S / 5 m i n i S . E , for nine (no nifedipine) and eight (nifedipine) rats. a p < 0.05, b p < 0.02, c p < 0.01, d p < 0.001, as compared with the corresponding rats not treated with nifedipine.
279 i
? 31 mln
CONTROL
,
? 31
mln
31 mln
15 8
30 8
?
? 57 rain i,~l lllll !, I~11 I Ill I
43
mln
I
? 43 min T 30 a
44
mln
Fig. 2. Electrographic record of the effect of i.p. administered 4-AP (6 mg/kg). In this and the following figures the abbreviations are: CX, frontal cortex; AMG, left amygdala; HPC, fight dorsal hippocampus; DR, dorsal raphe. This rat had two generalized tonic convulsions, at 31 and 43 min (arrows), and died at 45 min. Similar recordings were obtained for eight rats. Horizontal bar = 1 s; vertical bars = 0.5 inV.
These discharges were characterized spikes, p o l y - s p i k e s , a n d s p i k e - w a v e S p i k e s w e r e o f h i g h a m p l i t u d e a n d the d i s c h a r g e w a s 28.2 _+ 5.3 s. I n g e n e r a l ,
by isolated complexes. duration of the animals
h a d o n l y o n e G T C . It w a s d i f f i c u l t to e s t a b l i s h a b e h a v i o r a l - e l e c t r o g r a p h i c c o r r e l a t i o n since s o m e e p i l e p t i f o r m p a t t e r n s w e r e r e c o r d e d w i t h o u t evid e n t m o t o r signs.
280 A n interesting preconvulsive electrographic feature was observed some min after the injection of 4-AP: a theta r h y t h m with high regularity appeared. The frequency and amplitude of theta r h y t h m increased progressively in all structures studied, especially in the h i p p o c a m p u s (data not shown). This finding was observed in all treated rats and was not related to b o d y m o v e m e n t s or l o c o m o t o r activity. Six rats were treated with nifedipine (20 m g / k g s.c.) 30 rain before 4-AP. As described above for the non-implanted rats, the n u m b e r of G T C per
rat increased in the nifedipine-pretreated rats. These animals had two and sometimes three convulsions, in contrast to the nifedipine-untreated rats, which generally had only one GTC. The characteristics of the electrographic discharges p r o d u c e d by 4-AP were not modified by nifedipine pretreatment and, although their m e a n duration increased from 28.2 + 5.3 to 47.7 + 8.7 s, this difference was not significant. W h e n nifedipine was injected alone it had no effect on behavior nor on the electrical activity of the animals at any of the doses tested.
CX ~
A M O ~'-~- -
~
~
~
30 s
CONTROL
I t
5 min
34
tmln
I ! ,fi!,,,'l!j:i
9 rain
t
7 I rnln
Fig. 3. Electrographic record of the effect of i.h. administered 4-AP (4.2 nmol). In this rat the first rhythmic spikes appeared in the hippocampus at 30 s, and at 9 rain (arrows) the discharge propagated to the cortex. At 34 rain the epileptic activity became generalized, hut at 71 rain spikes were observed only in the hippocampus. These spikes continued for about 3 h and then disappeared. Similar recordings were obtained for four rats. Horizontal bar ~ 1 s; vertical bars ~ 0.5 mV.
281
3.2.2. Lh. 4 - A P T h e a d m i n i s t r a t i o n of 4-AP into the CA1 region of the h i p p o c a m p u s i n d u c e d r h y t h m i c high a m p l i t u d e spikes, which initially appeared exclusively in this structure, with a m e a n latency of 48.7 + 18.7 s (n = 4) (fig. 3). This activity t e n d e d to increase progressively in frequency a n d ampli-
tude, a n d after a few m i n (5.5 + 2.3) the epileptic activity p r o p a g a t e d to the other structures studied a n d b e c a m e generalized after 16.1 + 6.5 min. After a b o u t 1 h the discharges in the cortex, a m y g d a l a a n d raphe decreased in a m p l i t u d e a n d frequency, b u t in the h i p p o c a m p u s they c o n t i n u e d for a b o u t 3 h. I m p l a n t e d a n i m a l s showed fewer W D S (47.5
CX ~ . , ~ , ~ , ; ' . ~ < , ~ w ~ , ~ , , . ~ , ~ , ~ v ~ AMO " , ~ v ~ c , ~ ' ~ r ~ ' ~ ¢ % ~ HPC ~,,,,~dW-V~
DR ~¢ ~ . ~ ¢ ~ ~ . . . ~ , ~ ~ ' ~
t
4 rain
CONTROL
,
6 mln
,
!/ 12 min
I
3 5 mln
134
5'
rain
Fig. 4. Electrographic record of the status epilepticus induced by 4-AP in rats pretreated with nifedipine (20 mg/kg s.c.). In this rat the first spikes appeared at 4 rain in the hippocampus and propagated to the cortex and the amygdala at 6 rain (arrows). The continuous generalized discharge characteristic of status epilepticus appeared at 12 rain and lasted for more than 1 h. Similar recordings were obtained for six rats. Horizontal bar = 1 s; vertical bars = 0.5 mV for AMG and HPC, and 0.05 mV for CX and DR.
282 + 14.7 per h) than the non-implanted 4-AP-treated rats (see fig. 1); W D S frequently appeared after the discharges. Six rats were treated with nifedipine (20 m g / k g s.c.) 30 min before the i.h. injection of 4-AP. They showed an increased latency of appearance of rhythmic spikes (139.6 _+ 48.1 s) compared to the untreated animals (48.7_+ 18.7 s), although this difference was not significant. As in control rats, the discharge occurred initially in the hippocampus, rapidly propagated to the other structures (4.9 + 0.6 min) and subsequently became generalized (6.5 _+ 1.1 min). The most interesting effect of nifedipine was the induction of a status epilepticus, which was observed in all animals pretreated with this drug and was characterized by continuous discharges of very high amplitude and frequency in all structures studied (fig. 4). The discharges appeared 13.1 + 2.4 min after microinjection of 4-AP and lasted in all cases for more than 1 h (the mean time of disappearance of this status was 92.0_+ 29.9 min after 4-AP injection). As a consequence, a marked increase in the percentage of time spent in discharge was observed (60.6 + 11.3 min in nifedipine-treated animals vs. 12.6 + 1.5 rain in controls). The time to achieve recovery was also increased considerably (135.8 _+ 26.4 vs. 71.8 _+ 8.8 min in controls). There was no significant difference in the number of W D S per hour between the nifedipine-treated animals (66.9 _+ 21.4) and the untreated group (47.5 _+ 14.7). As already mentioned, nifedipine per se did not produce any significant behavioral or electrographic alteration.
4. Discussion
The convulsant action of systemically administered 4-AP has long been recognized (Schafer et al., 1973). However, the effects of i.h. administered 4-AP, as well as the electrographic correlates of both routes of administration, have not been studied previously. The main effect of systemic 4-AP was to induce convulsive activity, including G T C and electrographic discharges in all structures studied. In contrast, microinjection of 4-AP into the CA1
region of the hippocampus resulted in limbic-type seizures accompanied by frequent WDS. Since an increased entry of Ca z+ into neuronal somas or dendrites has been implicated in epileptic discharges (Wong and Prince, 1978; Heinem a n n and Pumain, 1981; Pumain et al., 1983), a possible action of 4-AP could be to directly facilitate Ca 2+ influx through somatic voltage-dependent Ca 2+ channels. Such an effect of 4-AP, independent of its action as a K + channel blocker, has been demonstrated in cultured spinal cord neurons (Rogawski and Barker, 1983). Contrary to what we expected, nifedipine and the other dihydropyridines used clearly enhanced the convulsant effects of 4-AP. The potentiation of the convulsions produced by i.p. 4-AP was manifest both behaviorally and electrographically, since the number of G T C per rat doubled, the percentage of animals that died during G T C increased 5- to 7-fold, and electrographically there was a 70% increase in the mean duration of the discharges. The nifedipine potentiation was even more evident when 4-AP was administered into the CA1 region of the hippocampus. Behaviorally, there was a notable increase in the number of W D S during the first 30 min, and electrographically the epileptic discharge in all areas studied was continuous during at least 1 h in all animals pretreated with nifedipine. It is unlikely that this facilitatory effect of nifedipine occurred as a consequence of its cardiovascular actions, since it has been shown that, in the rat, a dose of 100 m g / k g of nitrendipine, which is 5- to 10-fold higher than the effective doses used in the present experiments, causes only a slight decrease (25%) in blood pressure (Knorr and Garthoff, 1984). It also seems improbable that the potentiation was due to an action of nifedipine on presynaptic calcium channels, since we have previously demonstrated that the dihydropyridines do not affect the Ca2+-dependent stimulation of transmitter release produced by 4-AP in synaptosomes (Massieu and Tapia, 1988). The potentiating action of nifedipine was surprising in view of previous reports indicating that the Ca 2+ antagonist dihydropyridines possess anticonvulsant properties. These dihydropyridines have been shown to be effective against seizures
283 induced by ischemia, pentylenetetrazole, bicucull i n e a n d e l e c t r o s h o c k in t h e r a b b i t ( M e y e r et al., 1986a, b), a n d b y p e n t y l e n e t e t r a z o l e , h i g h p r e s s u r e ( D o l i n et al., 1988), k i n d l i n g ( V e z z a n i et al., 1988) a n d s o m e n e u r o t o x i n s a n d 4 - A P itself ( G a n d o l f o et al., 1989) in t h e rat. F u r t h e r m o r e , the i.c.v. a d m i n i s t r a t i o n of t h e C a 2÷ a g o n i s t , d i h y d r o p y r i d i n e B A Y - K - 8 6 4 4 , p r o d u c e s seizures t h a t are a n t a g o n i z e d b y s y s t e m i c n i f e d i p i n e ( S h e l t o n et al., 1987). T h e d i s c r e p a n c y b e t w e e n t h e s e o b s e r v a t i o n s a n d t h o s e o f the p r e s e n t p a p e r , h o w e v e r , are m o r e a p p a r e n t t h a n real, since, b e s i d e s i m p o r t a n t d i f f e r e n c e s in the r o u t e o f a d m i n i s t r a t i o n o f t h e d i h y d r o p y r i d i n e s used, the s t u d i e s m e n t i o n e d a b o v e s h o w e d t h a t the a n t i c o n v u l s a n t a c t i o n o f d i h y d r o p y r i d i n e s w a s r e s t r i c t e d to c e r t a i n t y p e s o f convulsions and that these drugs even potentiated o t h e r t y p e o f seizures. T h u s , D o l i n et al. (1988) found that nitrendipine and nimodipine were not e f f e c t i v e a g a i n s t s t r y c h n i n e , a n d i n c r e a s e d t h e incidence of clonic convulsions and mortality prod u c e d b y N - m e t h y l - a s p a r t a t e . F u r t h e r m o r e , Vezz a n i et al. (1988) o b s e r v e d s o m e p o t e n t i a t i o n b y nifedipine of kainate- and quinolinate-induced seizures. I n this r e s p e c t , it h a s b e e n r e p o r t e d t h a t n i f e d i p i n e i n c r e a s e s C a 2÷ u p t a k e in b r a i n c o r t e x slices ( R i v e r o s a n d O r r e g o , 1986). T h i s s u g g e s t s that, u n d e r c e r t a i n c o n d i t i o n s , the d i h y d r o p y i r i d i n e s g e n e r a l l y c o n s i d e r e d as C a 2÷ a n t a g o n i s t s b e h a v e as p a r t i a l a g o n i s t s , as has also b e e n s h o w n f o r n i t r e n d i p i n e in c a r d i a c m u s c l e cells ( H e s s et al., 1984) a n d in c o r o n a r y a r t e r y rings ( M i w a a n d S c h w a r t z , 1987).
Acknowledgements The authors wish to thank Mr, Joaquin Manjarrez for his expert assistance in the electrode implantation. This work was supported in part by the Consejo Nacional de Ciencia y Tecnologia, M6xico, D.F. (project P228CCOX-880370).
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