Effects of Various Antiepileptics on Behavioral and Electroencephalographic Seizures Induced by Maximal Electroshock in Mice

Home , Nefiracetam

Aya Murakami1, Yusuke Watanabe1, Kenshi Takechi1, Akinori Fujiwara1, and Chiaki Kamei1,* 1Department of Medicinal Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, Japan

Received August 30, 2007; Accepted November 12, 2007

Abstract. The changes of electroencephalogram induced by maximal electroshock were studied in comparison with behavioral seizures in mice. After electroshock, mice showed tonic flexor (TF) seizure, tonic extensor (TE) seizure, and clonic (CL) seizure, in this order of occurrence. At the same time, high frequency spike or spike and wave complex in the cortex was observed, and thereafter, the frequency of the spike or spike and wave complex gradually fell. The antiepileptics used in the present study, except for ethosuximide, caused a dose-dependent shortening of the TE seizure duration. In addition, phenobarbital and carbamazepine at relatively high doses also showed a significant shortening of the duration of CL seizure. Although the duration of total electroencephalographic (EEG) seizures was not influenced by all the antiepileptics used in the present study, the duration of high frequency spike or spike and wave complex observed for 7 – 8 s after electroshock was dose-dependently shortened by all the antiepileptics used. From these findings, it may be concluded that high frequency spike or spike and wave complex induced by maximal electroshock is a useful index to assess antiepileptics that are effective in not only tonic-clonic seizures but also absence seizures in humans.

Keywords: maximal electroshock, tonic extensor (TE) seizure, clonic (CL) seizure, electroencephalogram

Introduction encephalographic (EEG) changes were observed not only in generalized seizures but also partial seizures in It is well known that tonic extensor (TE) seizure humans (4, 5); however, there is little information about induced by maximal electroshock is widely used as an the EEG changes induced by maximal electroshock in index of the effect of antiepileptics used for grand mal animals except for a few papers. Stille and Sayers (6) epilepsy (1, 2). We have also reported that TE seizure reported that clinically proven antiepileptic drugs induced by maximal electroshock was selectively (phenytoin, phenobarbital, and carbamazepine) had no inhibited by phenytoin, phenobarbital, topiramate, and influence on EEG convulsive activity in the rat cortex, carbamazepine, which are clinically effective in general- even in doses up to 8-fold the ED50, that showed TE ized tonic-clonic seizures (grand mal) (3). In addition, it seizure. Toman et al. (7) also described that phenytoin has also been demonstrated that electromyographic caused no inhibition on EEG response to supramaximal seizure patterns recorded in the hind legs are more electroshock in the rabbit. Kamei et al. (8) reported reliable and more highly sensitive than TE seizure to previously about the effects of antiepileptics on both assess the potential activity of antiepileptics (3). behavioral and electrographic seizure patterns induced On the other hand, it is well known that electro- by maximal electroshock, but classification of EEG seizure patterns was rather complex, although almost all *Corresponding author. [email protected] antiepileptics showed no influence on electrographic Published online in J-STAGE: January 11, 2008 convulsive activity in rats. There are some reports that doi: 10.1254/jphs.FP0071453 rats were less sensitive than mice in the potency of

78 Electroshock and Electroencephalogram 79 antiepileptics on TE seizure induced by maximal into the following 2 stages: high frequency spike or electroshock (9). In the present study, therefore, the spike and wave complex (6 – 15 Hz, EEG seizures-1) changes of EEG as well as behavioral seizures induced and low frequency spike and wave complex or slow by maximal electroshock were studied using mice. The wave (2 – 6 Hz, EEG seizures-2). effects of certain antiepileptics on both EEG changes and behavioral seizures were also studied. Drugs The following drugs were used: phenobarbital sodium Materials and Methods and phenytoin (Wako, Osaka) and carbamazepine, ethosuximide, and topiramate (Sigma, St. Louis, MO, Animals USA). These drugs were suspended in 0.5% carboxy- Male ddY mice, 4-week-old (body weight, 20 – 24 g) methyl cellulose (CMC) solution and orally admin- were purchased from Japan SLC, Shizuoka. Animals istered 1 h before electrical stimulation at a volume of were maintained in an air-conditioned room with con- 5ml/kg of body weight. Control groups were received trolled temperature (24 ± 2°C) and humidity (55 ± 15%). with 0.5% CMC solution. They were housed in plastic cages with sawdust under a light–dark cycle (lights on from 07:00 to 19:00). The Statistical analyses mice were allowed free access to food and water, except All data are expressed as the means ± S.E.M. The during the experiments. All procedures involving Kruskal-Wallis and Steel tests were used for statistical animals were carried out in accordance with the Guide- analysis of all data, and a P-value less than 0.05 was lines for Animal Experiments at Okayama University considered significant. Advanced Science Research Center. Groups of 8 mice each were used for each dose of the drugs in the study. Results

Experimental procedure Effects of various antiepileptics on the duration of TE For the measurement of EEG, monopolar screw seizure electrodes were implanted in the frontal pole cortex Figure 1 shows the effects of various antiepileptics (FP, area 10), the right occipital cortex (RO, area 17), on the duration of TE seizure. The duration of TE seizure and the left occipital cortex (LO, area 17) under in the control group was 15.5 ± 0.42 s (n = 8). The pentobarbital anesthesia (NembutalR; 50 mg/kg, i.p.). duration of TE seizure induced by maximal electroshock Electroconvulsion was induced by stimulating the was dose-dependently shortened by phenytoin, pheno- animals with 50 Hz, 200 V for 1.0 s (SEN-3301; Nihon barbital, topiramate, and carbamazepine. Significant Kohden, Tokyo) using ear-clip electrodes. EEG was differences were observed with phenytoin (10, 20, and recorded bipolarly (FP-LO and FP-RO) with an electro- 50 mg/kg), phenobarbital (10, 20, and 50 mg/kg), encephalograph (EEG-7314, Nihon Kohden). EEG topiramate (20, 50, and 100 mg/kg), and carbamazepine analysis was done using EEG recordings of FP-LO and (20 and 50 mg/kg). On the other hand, ethosuximide FP-RO. In the present study, EEG changes were divided caused no significant inhibition even at a dose of

Fig. 1. Effects of various antiepileptics on the duration of tonic extensor (TE) seizure. Each column and vertical bar represents the mean ± S.E.M of 8 mice. *, **: Significantly different from the control group at P<0.05 and P<0.01, respectively. 80 A Murakami et al

500 mg/kg. observed immediately after maximal electroshock; thereafter, the frequency of spike or spike and wave Effects of various antiepileptics on the duration of clonic complex was gradually lowered (2 – 6 Hz, for 5.5 s, (CL) seizure EEG seizures-2). Figure 4 shows the relationship CL seizure observed after TE seizure induced by between the duration of TE seizure and EEG changes maximal electroshock was dose-dependently shortened after electroshock. The regression line of the duration of by phenobarbital and carbamazepine. Significant differ- TE seizure and EEG changes was Y = 0.579x + 5.760. ences were observed with phenobarbital (50 mg/kg) and The duration of EEG changes was shorter than that of carbamazepine (50 mg/kg). On the other hand, pheny- TE seizure, and a relatively low correlation coefficient toin, topiramate, and ethosuximide caused no significant (r = 0.730) was observed (A); therefore, we also inhibition even at doses of 50, 100, and 500 mg/kg, analyzed the relationship between the duration of TE respectively (Fig. 2). seizure and EEG seizures-1 or EEG seizures-2. The regression line of the duration of TE seizure and EEG Behavioral and EEG changes induced by maximal seizures-1 was Y = 1.419x + 5.106 (r = 0.892, B). The electroshock regression line of the duration of TE seizure and EEG A representative example of the behavioral and EEG seizures-2 was Y = 0.641x + 8.879 (r = 0.576, C). changes induced by maximal electroshock is shown in Fig. 3. After electroshock, mice showed TF seizure Effects of various antiepileptics on total EEG seizures (1.5 s), TE seizure (14 s), and CL seizure (30 s), in this As shown in Figs. 5 and 6, phenytoin and pheno- order of occurrence. EEG seizure patterns were as barbital caused a dose-dependent shortening of the follows: at first, high frequency spike or spike and wave duration of total EEG seizures, but no significant complex (6 – 15 Hz, for 6 s, EEG seizures-1) was difference was observed even at a dose of 50 mg/kg of

Fig. 2. Effects of various antiepileptics on the duration of clonic (CL) seizure. Each column and vertical bar represents the mean ± S.E.M of 8 mice. *, **: Significantly different from the control group at P<0.05 and P<0.01, respectively.

Fig. 3. Representative example of behavioral and electroencephalographic changes induced by maximal electroshock. E.S., electroshock stimulation; TF, tonic flexor; TE, tonic extensor; CL, clonic convulsion; FP, frontal pole cortex; LO, left occipital cortex; RO, right occipital cortex; EEG seizures-1, high frequency spike or spike and wave complex; EEG seizures-2, low frequency spike and wave complex or slow wave. Electroshock and Electroencephalogram 81

(10, 20, and 50 mg/kg), phenobarbital (10, 20, and 50 mg/kg), topiramate (20, 50, and 100 mg/kg), carbamazepine (20 and 50 mg/kg), and ethosuximide (200 and 500 mg/kg).

Effects of various antiepileptics on EEG seizures-2 The results are shown in Fig. 6. The duration of low frequency spike and wave complex and slow wave was dose-dependently shortened by phenobarbital and ethosuximide, but no significant differences were observed even at doses of 50 and 500 mg/kg, respectively. Phenytoin, topiramate, and carbamazepine also showed no significant inhibition even at doses of 50, 100, and 50 mg/kg, respectively.

Discussion

In the present study, the duration of TE seizure induced by maximal electroshock in mice in the control group was 15.5 ± 0.42 s. Vohora et al. (10) reported that the duration of TE seizure in male Swiss-strain mice was 14.0 ± 0.72 s. Previously, we (11) have also demonstrated that the duration of TE seizure in male ddY mice was 15.2 ± 0.5 s; therefore, we confirmed that the maximal electroshock seizure test used in the present study was a suitable method for estimating TE seizure. In addition, as shown in Fig. 1, all the antiepileptics, except ethosuximide, inhibited TE seizure, and the potency of antiepileptics was almost the same as in our previous work (3). It is well recognized that ethosuximide showed no inhibition of TE seizure induced Fig. 4. Relationship between the durations of tonic extensor by maximal electroshock in mice and rats (9, 12). Next, seizures and EEG seizures. A: Total EEG seizures and tonic extensor as shown in Fig. 2, both phenobarbital and carbam- seizure, B: EEG seizures-1 and tonic extensor seizure, C: EEG azepine also caused an inhibition of CL seizure. The seizures-2 and tonic extensor seizure. TE, tonic extensor; EEG seizures-1, high frequency spike or spike and wave complex; EEG dose that showed a significant difference was relativity seizures-2, low frequency spike and wave complex or slow wave. high in both drugs. Kamei et al. (8) reported that phenobarbital at a dose of 100 mg/kg, p.o. is effective in inhibiting CL seizure induced by maximal electroshock both drugs. Topiramate, carbamazepine, and etho- in rats. As for carbamazepine, Novelli et al. (13) suximide also caused no significant effects even at doses described that the drug at a dose of 10 mg/kg, i.p. of 100, 50, and 500 mg/kg, respectively. As described significantly inhibited CL seizure in mice. On the previously, EEG changes can be divided into 2 stages, other hand, phenytoin showed no significant inhibition that is, high frequency spike or spike and wave complex of CL seizure even at a dose of 50 mg/kg. It is well (6 – 15 Hz, EEG seizures-1) and low frequency spike known that CL seizure induced by maximal electroshock and wave complex or slow wave (2–6Hz, EEG was not inhibited (9) or was rather prolonged by pheny- seizures-2); therefore, we studied the effects of various toin (14). Different from the CL seizure induced by antiepileptics on both EEG seizure patterns. pentetrazol that observed with maximal electroshock was weak and unclear. At the present time, therefore, we Effects of various antiepileptics on EEG seizures-1 assume that CL seizure induced by maximal electro- The results are shown in Fig. 6. The duration of high shock in mice is not a reliable index for estimating the frequency spike or spike and wave complex was dose- potency of antiepileptics. dependently shortened by all drugs used in the test. It was found in the present study that EEG changes, Significant differences were observed with phenytoin that is, spike or spike and wave complex or slow wave 82 A Murakami et al

Fig. 5. Representative example of the effect of phenytoin (50 mg/kg, p.o.) and phenobarbital (50 mg/kg, p.o.) on the behavioral and EEG seizures induced by maximal electroshock. E.S., electroshock stimulation; TE, tonic extensor; CL, clonic convulsion; FP, frontal pole cortex; LO, left occipital cortex; RO, right occipital cortex; EEG seizures-1, high frequency spike or spike and wave complex; EEG seizures-2, low frequency spike and wave complex or slow wave.

Fig. 6. Effects of various antiepileptics on the duration of total EEG seizures, EEG seizures-1, and EEG seizures-2. EEG seizures-1, high frequency spike or spike and wave complex; EEG seizures- 2, low frequency spike and wave complex or slow wave. Each column and vertical bar represents the mean ± S.E.M of 8 mice. *, **: Significantly different from the control group at P<0.05 and P<0.01, respectively. were observed after maximal electroshock. In tonic seizures. In addition, total EEG seizures induced by seizure in humans, Gastaut et al. (15) reported that the maximal electroshock were not inhibited by any anti- EEG shows a rapid synchronization of increasing epileptics used. For these reasons, it can be assumed amplitude (i.e., high frequency spike or spike and wave that the occurrence mechanism between high frequency complex) ending with slow waves (i.e., low frequency EEG seizures and low frequency EEG seizures was spike and wave complex). These EEG changes observed essentially different. When total EEG seizures induced in humans have a striking resemblance to the EEG by maximal electroshock were investigated in detail, it seizures in our animal study. In our study, both TE was found that they were divided into 2 patterns, that is, seizure and EEG changes were observed at the same high frequency spike or spike and wave complex (EEG time; however, no clear relationship was observed seizures-1) and low frequency spike and wave complex between the duration of TE seizure and total EEG or slow wave (EEG seizures-2); therefore, the effects of Electroshock and Electroencephalogram 83 antiepileptics on these 2 patterns of EEG seizures were Pharmacol Exp Ther. 1958;124:161–164. studied. As a result, EEG seizures-1 was significantly 3 Murakami A, Ishikawa T, Takechi K, Ago J, Matsumoto N, inhibited by all antiepileptics used. Previously, Toman Kamei C. Effects of certain antiepileptics on behavioral and et al. 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