- Email: [email protected]
Valproic acid and ethosuximide slow the onset of maximal dentate activation in the rat hippocampus
ELSEVIER
Epilepsy Research 19 (1994) 229-235
Valproic acid and ethosuximide slow the onset of maximal dentate activation in the rat hippocampus Janet L. Stringer * Department of Pharmacology and Division of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 USA
Received 12 April 1994; revised 21 June 1994; accepted 21 June 1994
Abstract Valproic acid and ethosuximide
are commonly
used to treat absence seizures, but their mechanisms of action have not models and different species. We re-examined the effect of valproic acid and ethosuximide in an experimental model of reverberatory seizures in the hippocampus of the urethane-anesthetized rat, maximal dentate activation. Valproic acid, at 450 and 600 mg/kg i.p., caused an increase in the time to onset of seizure discharges, but only the changes with 600 mg/kg reached statistical significance. Both doses of valproic acid shortened the seizure duration. Serum levels of valproic acid were determined and it appears that in this system at least 200-400 pg/ml are needed to produce an effect on seizure onset and duration. Ethosuximide, at 600 mg/kg i.p., delayed the onset of the seizure discharge and blocked the increase in seizure duration. Both ethosuximide and valproic acid delayed the onset of the seizure discharge in this model suggesting that these drugs may help to determine the mechanisms behind seizure initiation in the hippocampal-parahippocampal circuits.
been clearly defined. This is partly due to testing in different experimental
Keywords: Valproic acid; Ethosuximide; Dentate gyms; Seizure initiation; Seizure duration; Antiepileptic drugs
1. Introduction
in the phatmacokinetics
of valproate
between
rodents
have been proposed to explain the higher doses that have to be administered to reach effective brain concentrations in mice or rats than in humans [6]. Valproate is reported to rapidly penetrate the brain [14], but have a short half-life in most species and humans
Valproic acid is one of the commonly used antiepileptic agents. It is effective against both generalized convulsive and nonconvulsive seizures. Experimentally, valproic acid exerts anticonvulsant effects in almost all animal models [6]. However, the potency of valproate varies in the different models, depending on the species, the route of drug administration and the type of seizure induced. Differences
* Corresponding author. Tel.: (713) 7987937; Fax: (713) 7983145; email: [email protected]. 0920-1211/94/$07.00
13761. Ethosuximide, a drug effective against absence seizures, has also been tested in a number of animal models [4,7,9,13,23]. Its effectiveness depends on the type of seizure induced. Whereas both ethosuximide and valproic acid are effective against pentylenetetrazol-induced seizures, ethosuximide is ineffective in other models where valproic acid is
0 1994 Elsevier Science B.V. All rights reserved
SSDI 0920-1211(94)00057-3
efficacious [1,4,7,9,12,15]. The possible mechanisms of action of valproic acid were recently reviewed by L&her [6] and appear to be multiple. The mechanism of action of ethosuximide is presumed to be related to its effect on thalamic calcium currents [Z]. However, valproic acid has no effect on calcium currents in that system. An epileptiform discharge, termed maximal dentate activatian, can be produced in the dentate gyrus of the urethane-anesthetized rat. This discharge is characterized by the appearance of bursts of largeamplitude population spikes in the dentate gyrus associated with a secondary rise in the extracellular potassium and a negative shift of the extracellular DC potential 1201. Maximal dentate activation appears to be a marker for reverberatory seizure activity in the hippocampal-parahippocampal circuit [IS]. Two parameters of maximal dentate activation have been defined. The time to onset of maximal dentate activation appears to be an indication of the ease with which seizures can be initiated in the dentate gyrus. The duration of maximal dentate activation gives an indication of the ability of the brain to terminate the epileptic activity. The effect of a number of drugs on these parameters have been tested 1197. In these earlier studies, valproic acid, at 300 mg/kg, had no effect on the time to onset or duration of maximal dentate activation [19]. Ethosuximide, at 300 mg/kg, had no effect on the duration, but did cause a transient increase in the time to onset. Because more recent work suggests that doses of valproic acid above 300 mg/kg are often needed in rats to see an effect [6], we tested higher doses of valproic acid and ethosuximide in the same experimental system used previously.
2. Methods Adult male Sprague-Dawley rats (190-260 g) were anesthetized with urethane (1.2 g/kg) and placed in a stereotaxic frame. A concentric bipolar electrode fSNEX 100, Rhodes Medical Inst.) was placed in the CA3 region of the left hip~campus at an angle of 10” towards the midline (AP - 3 mm from bregma, 4 mm lateral). A second bipolar stimulating electrode (twisted Teflon-coated stainless steel wire) was lowered into the angular bundle on the
right (AP - 8 mm, lateral 4 mm, depth 3-5 mm). Stimulation through either electrode was controlled by a digital timer led to a constant-cu~ent isolated stimulator. Individual stimuli consisted of 0.3ms biphasic pulses and the intensity varied from 100 to 500 FA. Extracellular field potentials were recorded from stratum granulosum of the dentate gyrus on the right (AP - 3 mm, lateral 1.8 mm) through a glass microelectrode filled with 1% Fast Green in 2 M NaCl. Final placement of the recording electrode was based on responses to angular bundle stimulation. Twenty-hertz stimulus trains were delivered through the CA3 stimulating electrode for a maximum of 30 s and the threshold for maximal dentate activation was determined [20]. Maximal dentate activation was defined by the presence of bursts of 20-40-mV population spikes and a negative shift of the extracellular DC potential (Fig. 1A). Using a stimulus intensity 200 PA above the threshold for maximal dentate activation, a stimulus train was administered every 5 min until an afterdischarge appeared and then every 10 min for at least 5 h. An afterdischarge was defined as at least two bursts of population spikes after the end of a stimulus train. For every train, the stimulus was stopped 2-3 s after the onset of maximal dentate activation. The time to onset of maximal dentate activation was measured from the beginning of the stimulus to the mid-point of the positive to negative DC shift (Fig. 1A). The duration of maximal dentate activation was measured from the mid-point of the positive to negative DC shift to the mid-point of the return of the DC potential to baseline. Using this stimulus protocol, the duration of maximal dentate activation will increase and the time to onset will gradually decrease [20]. After the afterdischarge had begun to lengthen, valproate, ethosuximide or normal saline (0.9%) was administered between the third and fourth stimulus train (Fig. 1B). In order to make comparisons across animals, the measured durations of maximal dentate activation and time to onset were ‘normalized’ by subtracting the duration (or time to onset) in response to the first stimulus from the duration (or time to onset) in response to each subsequent stimulus train. Thus, for individual stimulus trains after the first, a change in duration (or time to onset) was calculated. In this way data from separate animals were averaged and
J.L. Stringer/Epilepsy
Research 19 (1994) 229-235
BM[
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Stimulus Number Fig. 1. Changes in the parameters of maximal dentate activation with repeated elicitation. A: Chart recording during and after stimulation of the left CA3 region at 20 Hz is presented. The time to onset of maximal dentate activation (MDA) was measured from the beginning of the stimulus train to the mid-point of the positive to negative shift of the DC potential. The duration of maximal dentate activation was measured from the mid-point of the shit? of the DC potential to the point where the DC potential had returned halfway to baseline. Calibrations are indicated. B: The time to onset (filled circles) and duration of maximal dentate activation (open circles) during one experiment are graphed. Valproic acid (600 mg/kg) was administered i.p. between stimuli number 3 and 4. The administration of valproic acid shortened the duration of maximal dentate activation and lengthened the time to onset.
comparisons across groups of animals were made [19]. Statistical comparisons were done for stimulus trains number 5, 9 and 15, which are 20, 60, and 120 min after drug administration and are the same time points at which serum levels were determined for valproic acid. At each point the values from the vehicle injected control and the experimental group were compared with a Mann-Whitney U-test with significance defined as P < 0.05.
231
Valproic acid (sodium salt, Sigma) and ethosuximid, &ma) were dissolved in normal saline (0.9%, at 150 mg/ml for ethosuximide and 300 mg/ml for valproic acid) and administered i.p. as a bolus over 2-3 min. Each animal received only one dose of one drug and was followed for at least 4 h after drug administration. Six animals received 600 mg/kg of valproic acid, but this dose proved fatal in two of the animals. The results presented are the data from the remaining four animals. The lower dose of valproic acid and ethosuximide were each tested in five animals. For comparison purposes, the exact procedure was also followed with six animals who received injections of normal saline i.p. (vehicle controls). At the conclusion of each experiment, electrode positions were marked for confirmation histologically. Fast Green was iontophoresed from the recording electrode and current was passed through the stimulating electrodes. The animals were perfused through the heart with 1% potassium ferrocyanide in 10% buffered formalin. The brain was subsequently equilibrated in 30% sucrose in formalin, cut on a sliding microtome and stained with cresyl violet. Serum levels of valproic acid were measured in 11 urethane-anesthetized animals. Blood was drawn by intracardiac puncture at 20 min, 1, 2 and 4 h after i.p. drug administration. Levels were determined by a fluorescent immunoassay in the Special Chemistry Laboratory of the Department of Pathology at Methodist Hospital, Houston, TX.
3. Results The effect of administration of the vehicle, normal saline, on the time to onset and duration of maximal dentate activation was determined in six animals. There was a gradual decrease in the time to onset over the first 15 stimuli and then a plateau. There was also an increase in the duration of maximal dentate activation over the first 15-16 stimuli and then the durations became quite variable. The addition of valproic acid between stimuli 3 and 4 had an effect on both the time to onset and duration (Fig. 1B). There was an increase in the time to onset of maximal dentate activation after 450 mg/kg (n = 5, Fig. 2A) and 600 mg/kg (n = 4) valproic acid that only reached statistical signifi-
232
J.L. Stringer / EpEepsy Research I9
f 1994) 229-235
cance with 600 mg/kg. Both doses of valproic acid decreased the duration of maximal dentate activation below the duration in response to the stimulus just before drug administration (Fig. 2B). Valproic acid, 450 mg/kg, significantly reduced the duration of
‘I I
0
5
10
15
20
25
Stimulus Number
6
0
5
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Fig. 2. Effect of valproic acid on the par~ete~ of maximal dentate activation. The time to onset and duration of maximal dentate activation were measured for each stimulus train. These values were then normalized, averaged and plotted (with SEM) against stimulus number. The dashed line indicates the mean values from the vehicle control animals. Valproic acid was administered ip. between stimuli 3 and 4. The asterisks indicate significant differences behveen the drug group and the vehicle controls. A: The effect of valproic acid at 450 mg/kg (n = 5, open circles) and 600 mg/kg (n = 4, filled circles) on the change in the time to onset of maximal dentate activation. B: The effect of valproic acid at 450 mg/kg (n = 5, filled circles) and 600 mg/kg (n = 4, open circles) on the change in duration of maximal dentate activation.
Fig. 3. Pharmacokinetics of valproic acid in the anesthetized rat. Serum levels of valproic acid were determined from blood drawn at 20 min, 1, 2 and 4 h after administration of 600 mg/kg i.p. The individual serum levels are plotted on a semi-log scale versus the time after drug administration. The line drawn is the calculated linear regression through the data points. The calculated R’ value was 0.9945 and the P value was 0.0028.
maximal dentate activation at 20 and 60 min after the drug dose. After 600 mgfkg valproic acid the reduction in the duration of maximal dentate activation was even longer lasting. Thus, it appears that very high doses of valproic acid can have a long-lasting effect on the onset and duration of seizure activity in the dentate gyrus. Serum levels of valproic acid were determined 20 min and 1, 2 and 4 h after the administration of 600 mg/kg (Fig. 3). The mean level achieved in the serum 20 min after i.p. administration was 936 pg/ml. The serum levels declined over the course of the 4-h period with first-order kinetics. The halflife of valproate was estimated to be about 1 h. Ethosuximide, at 600 mg/kg In = 5, Fig. 4) had a significant effect on the time to onset and duration of maximal dentate activation. Ethosuximide immediately increased the time to onset, an effect that lasted for 30 min. The time to onset then began to gradually decrease, but had not returned to control values by the end of the 4-h recording session. The effect of ethosuximide on the duration of maximal dentate activation was less striking, but still reached statistical significance. Ethosuximide blocked the increase in duration of maximal dentate activation for
J.L. Stringer/Epilepsy
Research 19 (I 994) 229-235
233
30-40 min. Thereafter, the duration of maximal dentate activation increased gradually. 4. Discussion
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Previous experiments, which examined the effect of valproic acid in the same experimental system, showed no effect of the drug on the time to onset or duration of maximal dentate activation at 300 mg/kg [19]. In the present study, 450 mg/kg produced a shortening of the duration of maximal dentate activation suggesting that valproic acid augments the processes involved in seizure termination in hippocampal circuits. At 600 mg/kg, valproic acid also increased the time to onset of maximal dentate activation, thus appearing to also delay seizure initiation. Although doses up to 600 mg/kg have been used in testing the neurochemical effects of valproate [6], the effective doses in this study are higher than those reported to be effective in other models of seizures
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Fig. 4. Effect of ethosuximide on the parameters of maximal dentate activation. The time to onset and duration of maximal dentate activation were measured for each stimulus train. These values were then normalized, averaged and plotted (with SEM) against stimulus number. Ethosuximide was administered i.p. between stimuli 3 and 4. The mean f SEM for the vehicle controls are shown (n = 6, open circles). The asterisks indicate significant differences betweenthe drug group and the vehicle controls. A: The effect of ethosuximide at 600 mg/kg (n = 5, filled circles) on the change in the time to onset of maximal dentate activation. B: The effect of ethosuximide (600 mg/kg, n = 5, filled circles) on the change in duration of maximal dentate activation.
Because 200-400 pg/ml of valproic acid was still an effective plasma level after the administration of 600 mg/kg, it could be postulated that this plasma level is the minimum effective level to produce an effect in this system. However, this conclusion would predict that lower dosages of valproic acid that achieved the same plasma level would also be effective. This was not directly tested in the experiments in this report. Alternatively, the initial higher plasma level achieved after the 600 mg/kg dose may be necessary for the drug effect on the onset and duration of maximal dentate activation. In this case the lower plasma level of 200-400 pug/ml may be able to sustain the effect already initiated by the higher plasma levels. The effective serum levels of valproic acid in the present experiments (200-400 pg/ml) are close to the levels reported to be effective in the rat and dog [lo]. In humans, serum levels between 50 and 100 pg/ml are considered to be in the therapeutic range ill]. It is not clear why the effective concentrations are so widely different across species. It could be related to protein binding in the plasma causing differing brain concentrations. However, studies comparing brain and plasma concentrations of valproic acid have shown a brain/plasma ratio of 0.22-0.35 for rats and mice [14,16], while in humans the same ratio is 0.06-0.28 [22]. These data
23-l
J. L. Stringer / Epilepsy Research
indicate that for a given plasma concentration, the brain concentration is lower in humans than in rodents. Another reason for the increased effective serum level in this model may be an interaction with the anesthetic urethane. There may be a direct interaction with urethane reducing the effectiveness of valproate. In addition, because the animals were anesthetized and the seizures were elicited using stimulus trains that continue until the seizure begins, higher doses of valproic acid may be needed to alter seizure onset in this system than in an awake model. Previously it has been shown that higher doses of convulsants are needed to initiate epileptiform activity in the urethane-anesthetized rat [21]. It is not unreasonable to speculate that higher doses of antiepileptic agents may also be needed to suppress seizure activity once initiated in urethane-anesthetized animals. This point could be addressed by testing the effectiveness of valproic acid on the onset and duration of maximal dentate activation in awake rats. The serum level determinations in these experiments show first-order kinetics of elimination. Previous reports have demonstrated enterohepatic circulation and two phases to the elimination profile [3,5,8,12]. Presumably this was not seen in these experiments because the time course was not followed long enough. Enterohepatic recirculation of valproate appears to take 4-7 h in the rat and dog [lo]. The half-life of valproate in the urethaneanesthetized rat determined in this study was one hour compared to 6-15 h in the human epilepsy patient [ll] and l-2 h in the awake rat 13,131. These data strongly support the statement that valproate has a short half-life in rodents, but do not support the statement that these pharmacokinetic differences explain the higher effective doses that are needed in rodents than humans [6]. However, the kinetics of distribution and elimination of valproic acid and its active metabolites in the brain may differ from the kinetics determined in the plasma. At doses of 300 mg/kg, ethosuximide has previously been shown to transiently increase the time to onset of maximal dentate activation without having any effect on the duration [19]. At 600 mg/kg ethosuximide affects the duration of maximal dentate activation, but the most striking effect remains on the time to onset. Compared to doses used in some
19 (19941 229-235
experimental models, these doses are quite high. Ethosuximide is most effective against pentylenetetrazol-induced seizures with ED,,‘s ranging from 54 to 130 mg/kg [4,9,13,23]. In contrast, ethosuximide is ineffective against maximal electroshock seizures at doses exceeding 1000 mg/kg [4,7]. As discussed for valproic acid, the high doses of ethosuximide that were effective in these experiments may be related to the urethane anesthesia and the fact that the onset of the epileptiform activity is measured during a stimulus train. In order to have an effect on the time to onset of maximal dentate activation, ethosuximide, or any agent, must delay the activation elicited by stimuli being delivered every 50 ms. The findings with valproic acid and ethosuximide suggest that these drugs may be useful in determining the mechanisms behind seizure initiation in the hippocampal-parahippocampal circuits. In contrast to effects on the duration of maximal dentate activation, these two drugs, plus diazepam, are the only antiepileptic drugs that alter the time to onset of maximal dentate activation at doses that have been tested thus far [19]. Seizures are induced in this model by administration of stimulus trains to the CA3 region. Before the onset of maximal dentate activation a late polysynaptic response appears that resembles an angular bundle to dentate gyrus evoked potential [20]. It has been hypothesized that this polysynaptic response represents reverberatory activity through the entorhinal cortex and that this activity is an important step in initiating seizures in these circuits [18]. While neither valproic acid nor ethosuximide prevented the appearance of this potential, both drugs slowed the onset of the seizure activity. Valproic acid and ethosuximide are effective against pentylenetetrazol-induced seizures and it is now known that pentylenetetrazol induces spontaneous activity in these circuits by activation through the entorhinal cortex [17]. This suggests that ethosuximide and valproic acid may slow the onset of seizure activity in the hippocampal circuits by an effect on the entorhinal cortex. If this hypothesis is true, then it would explain both the effectiveness of these drugs against pentylenetetrazol-induced seizures and would support the hypothesis that reverberatory activity through the entorhinal cortex is an important regulator of seizure onset in the hippocampus.
J.L. Stringer/Epilepsy
Acknowledgments This work was supported by NIH NS 28871. The author thanks Michael G. Higgins for technical assistance.
References [l] Clark, C.R. and McMillian, C.L., Comparative anticonvulsant activity of 4-chlorobenzenesulfonamide and prototype antiepileptic drugs in rodents, Epilepsia, 31 (1990) 474-479. [2] Coulter, D.A., Huguenard, J.R. and Prince, D.A., Characterization of ethosuximide reduction of low-threshold calcium current in thalamic neurons, Ann. NeuroZ., 25 (1989) 582593. [3] Dickinson, R.G., Harland, R.C., Ilias, A.M., Rodgers, R.M., Kaufman, S.N., Lynn, R.K. and Gerber, N., Disposition of valproic acid in the rat: Dose-dependent metabolism, distribution, enterohepatic recirculation and choleretic effect, J. Pharmacol. Exp. Ther., 211 (1979) 583-595. [4] Johnson, D.N., White, H.S., Swinyard, EA., Albertson, T.E. and Kilpatrick, B.F., AHR-12245: A potential anti-absence drug, Epilepsy Rex, 8 (1991) 64-70. [5] Liu, M-j., Scott, K.R. and Pollack, G.M., Pharmacokinetics and pharmacodynamics of valproate analogues in rats. I. Spiro[4.6]undecane-Z-carboxylic acid, Epilepsia, 31 (1990) 465-473. [6] Liischer, W., Effects of the antiepileptic drug valproate on metabolism and function of inhibitory and excitatory amino acids in the brain, Neurochem. Rex, 18 (1993) 485-502. [7] Liischer, W., Fassbender, C.P. and Nolting, B., The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. II. Maximal electroshock seizure models, Epilepsy Rex, 8 (1991) 79-94. [8] Liischer, W., Fisher, J.E., Nau, H. and Hiinack, D., Valproic acid in amygdala-kindled rats: Alterations in anticonvulsant efficacy, adverse effects and drug and metabolite levels in various brain regions during chronic treatment, J. Pharmacol. Enp. Ther., 250 (1989) 1067-1078. [9] Liischer, W., H&tack, D., Fassbender, C.P. and Nolting, B., The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. III. Pentylenetetrazol seizure models, Epilepsy Res., 8 (1991) 171-189. [lo] Liischer, W., Honack, D., Nolting, B. and Fassbender, C.P., Trans-2-en-valproate: Reevaluation of its anticonvulsant efficacy in standardized seizure models in mice, rats and dogs, Epilepsy Res., 9 (1991) 195-210.
235
Research 19 (1994) 229-235
ml Macdonald,
R.L. and McLean,
M.J., Anticonvulsant
drugs:
Mechanisms of action. In: A.V. Delgado-Escueta, A.A. Ward, D.M. Woodbury and R.J. Porter (Eds.), Basic Mechanisms of the Epilepsies, Molecular and Cellular Approaches. Advances in Neurology, Vol. 44, Raven Press, New York, 1986, pp. 713-736. WI Mares, P., Maresova, S. and Maresova, D., Antimetrazol action and plasma levels of valproate in developing rats, Physiol. Bohemoslouaca, 38 (1989) 97-107. P., Bonazinga, [131Musolino, R., Gallitto, G., DeDomenico, M.M., Stumiolo, R., Labate, C. and DiPerri, R., Synergistic anticonvulsant effect of valproic acid and ethosuximide on pentylenetetrazole-induced epileptic phenomena in rats, J. Inr. Med. Res., 19 (1991) 55-62. I141Nau, H. and Lijscher, W., Valproic acid: Brain and plasma levels of the drug and its metabolites, anticonvulsant effects and y-aminobutyric acid (GABA) metabolism in the mouse, J. Pharmacol. Exp. Ther., 220 (1982) 654-659. [151Sasa, M., Ohno, Y., Ujihara, Y., Fujita, Y., Yoshimura, M., Takaori, S., Serikawa, T. and Yamada, J., Effects of antiepileptic drugs on absence-like and tonic seizures in the spontaneously epileptic rat, a double mutant rat, Epilepsia, 29 (1988) 505-513. [161Semmes, R.L.O. and Shen, D.D., Comparative pharmacodynamics and brain distribution of E-A’-valproate and valproate in rats, Epilepsia, 32 (1991) 232-241. elicits epileptiform activity [I71Stringer, J.L., Pentylenetetrazol in the dentate gyms of the urethane anesthetized rat by activation of the entorhinal cortex, Brain Res., 636 (1994) 221-226. [181 Stringer, J.L. and Lothman, E.W., Reverberatory seizure discharges in hippocampal-parahippocampal circuits, Exp. NeuroZ., 116 (19921 198-203. [191Stringer, J.L. and Lothman, E.W., Maximal dentate activation: A tool to screen compounds for activity against limbic seizures, Epilepsy Rex, 5 (1990) 169-176. DO1 Stringer, J.L. and Lothman, E.W., Maximal dentate activation: Characteristics and alterations after repeated seizures, J. NeurophysioZ., 62 (1989) 136-143. ml Stringer, J.L. and Sowell, K.L., Kainic acid, bicuculline, pentylenetetrazol and pilocarpine elicit maximal dentate activation in the anesthetized rat, EpiZepsy Res., 18 (1994) 11-21. El Vajda, F.J.E., Donnan, G.A., Phillips, J. and Bladin, P.F., Human brain, plasma, and cerebrospinal fluid concentration of sodium valproate after 72 hours of therapy, Neurology, 31 (1981) 486-487. I., Roztocilova, L., Mares, P., P31 Velisek, L., Kulhankova, Veliskova, J. and Mirvaldova, H., Ethosuximide affects both pentylenetetrazoleand kainate-induced clonic seizures but differentiates between tonic-clonic seizures, Can. J. Physiol. PharmacoZ., 67 (1989) 1357-1361.
Epilepsy Research 19 (1994) 229-235
Valproic acid and ethosuximide slow the onset of maximal dentate activation in the rat hippocampus Janet L. Stringer * Department of Pharmacology and Division of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 USA
Received 12 April 1994; revised 21 June 1994; accepted 21 June 1994
Abstract Valproic acid and ethosuximide
are commonly
used to treat absence seizures, but their mechanisms of action have not models and different species. We re-examined the effect of valproic acid and ethosuximide in an experimental model of reverberatory seizures in the hippocampus of the urethane-anesthetized rat, maximal dentate activation. Valproic acid, at 450 and 600 mg/kg i.p., caused an increase in the time to onset of seizure discharges, but only the changes with 600 mg/kg reached statistical significance. Both doses of valproic acid shortened the seizure duration. Serum levels of valproic acid were determined and it appears that in this system at least 200-400 pg/ml are needed to produce an effect on seizure onset and duration. Ethosuximide, at 600 mg/kg i.p., delayed the onset of the seizure discharge and blocked the increase in seizure duration. Both ethosuximide and valproic acid delayed the onset of the seizure discharge in this model suggesting that these drugs may help to determine the mechanisms behind seizure initiation in the hippocampal-parahippocampal circuits.
been clearly defined. This is partly due to testing in different experimental
Keywords: Valproic acid; Ethosuximide; Dentate gyms; Seizure initiation; Seizure duration; Antiepileptic drugs
1. Introduction
in the phatmacokinetics
of valproate
between
rodents
have been proposed to explain the higher doses that have to be administered to reach effective brain concentrations in mice or rats than in humans [6]. Valproate is reported to rapidly penetrate the brain [14], but have a short half-life in most species and humans
Valproic acid is one of the commonly used antiepileptic agents. It is effective against both generalized convulsive and nonconvulsive seizures. Experimentally, valproic acid exerts anticonvulsant effects in almost all animal models [6]. However, the potency of valproate varies in the different models, depending on the species, the route of drug administration and the type of seizure induced. Differences
* Corresponding author. Tel.: (713) 7987937; Fax: (713) 7983145; email: [email protected]. 0920-1211/94/$07.00
13761. Ethosuximide, a drug effective against absence seizures, has also been tested in a number of animal models [4,7,9,13,23]. Its effectiveness depends on the type of seizure induced. Whereas both ethosuximide and valproic acid are effective against pentylenetetrazol-induced seizures, ethosuximide is ineffective in other models where valproic acid is
0 1994 Elsevier Science B.V. All rights reserved
SSDI 0920-1211(94)00057-3
efficacious [1,4,7,9,12,15]. The possible mechanisms of action of valproic acid were recently reviewed by L&her [6] and appear to be multiple. The mechanism of action of ethosuximide is presumed to be related to its effect on thalamic calcium currents [Z]. However, valproic acid has no effect on calcium currents in that system. An epileptiform discharge, termed maximal dentate activatian, can be produced in the dentate gyrus of the urethane-anesthetized rat. This discharge is characterized by the appearance of bursts of largeamplitude population spikes in the dentate gyrus associated with a secondary rise in the extracellular potassium and a negative shift of the extracellular DC potential 1201. Maximal dentate activation appears to be a marker for reverberatory seizure activity in the hippocampal-parahippocampal circuit [IS]. Two parameters of maximal dentate activation have been defined. The time to onset of maximal dentate activation appears to be an indication of the ease with which seizures can be initiated in the dentate gyrus. The duration of maximal dentate activation gives an indication of the ability of the brain to terminate the epileptic activity. The effect of a number of drugs on these parameters have been tested 1197. In these earlier studies, valproic acid, at 300 mg/kg, had no effect on the time to onset or duration of maximal dentate activation [19]. Ethosuximide, at 300 mg/kg, had no effect on the duration, but did cause a transient increase in the time to onset. Because more recent work suggests that doses of valproic acid above 300 mg/kg are often needed in rats to see an effect [6], we tested higher doses of valproic acid and ethosuximide in the same experimental system used previously.
2. Methods Adult male Sprague-Dawley rats (190-260 g) were anesthetized with urethane (1.2 g/kg) and placed in a stereotaxic frame. A concentric bipolar electrode fSNEX 100, Rhodes Medical Inst.) was placed in the CA3 region of the left hip~campus at an angle of 10” towards the midline (AP - 3 mm from bregma, 4 mm lateral). A second bipolar stimulating electrode (twisted Teflon-coated stainless steel wire) was lowered into the angular bundle on the
right (AP - 8 mm, lateral 4 mm, depth 3-5 mm). Stimulation through either electrode was controlled by a digital timer led to a constant-cu~ent isolated stimulator. Individual stimuli consisted of 0.3ms biphasic pulses and the intensity varied from 100 to 500 FA. Extracellular field potentials were recorded from stratum granulosum of the dentate gyrus on the right (AP - 3 mm, lateral 1.8 mm) through a glass microelectrode filled with 1% Fast Green in 2 M NaCl. Final placement of the recording electrode was based on responses to angular bundle stimulation. Twenty-hertz stimulus trains were delivered through the CA3 stimulating electrode for a maximum of 30 s and the threshold for maximal dentate activation was determined [20]. Maximal dentate activation was defined by the presence of bursts of 20-40-mV population spikes and a negative shift of the extracellular DC potential (Fig. 1A). Using a stimulus intensity 200 PA above the threshold for maximal dentate activation, a stimulus train was administered every 5 min until an afterdischarge appeared and then every 10 min for at least 5 h. An afterdischarge was defined as at least two bursts of population spikes after the end of a stimulus train. For every train, the stimulus was stopped 2-3 s after the onset of maximal dentate activation. The time to onset of maximal dentate activation was measured from the beginning of the stimulus to the mid-point of the positive to negative DC shift (Fig. 1A). The duration of maximal dentate activation was measured from the mid-point of the positive to negative DC shift to the mid-point of the return of the DC potential to baseline. Using this stimulus protocol, the duration of maximal dentate activation will increase and the time to onset will gradually decrease [20]. After the afterdischarge had begun to lengthen, valproate, ethosuximide or normal saline (0.9%) was administered between the third and fourth stimulus train (Fig. 1B). In order to make comparisons across animals, the measured durations of maximal dentate activation and time to onset were ‘normalized’ by subtracting the duration (or time to onset) in response to the first stimulus from the duration (or time to onset) in response to each subsequent stimulus train. Thus, for individual stimulus trains after the first, a change in duration (or time to onset) was calculated. In this way data from separate animals were averaged and
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Research 19 (1994) 229-235
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Stimulus Number Fig. 1. Changes in the parameters of maximal dentate activation with repeated elicitation. A: Chart recording during and after stimulation of the left CA3 region at 20 Hz is presented. The time to onset of maximal dentate activation (MDA) was measured from the beginning of the stimulus train to the mid-point of the positive to negative shift of the DC potential. The duration of maximal dentate activation was measured from the mid-point of the shit? of the DC potential to the point where the DC potential had returned halfway to baseline. Calibrations are indicated. B: The time to onset (filled circles) and duration of maximal dentate activation (open circles) during one experiment are graphed. Valproic acid (600 mg/kg) was administered i.p. between stimuli number 3 and 4. The administration of valproic acid shortened the duration of maximal dentate activation and lengthened the time to onset.
comparisons across groups of animals were made [19]. Statistical comparisons were done for stimulus trains number 5, 9 and 15, which are 20, 60, and 120 min after drug administration and are the same time points at which serum levels were determined for valproic acid. At each point the values from the vehicle injected control and the experimental group were compared with a Mann-Whitney U-test with significance defined as P < 0.05.
231
Valproic acid (sodium salt, Sigma) and ethosuximid, &ma) were dissolved in normal saline (0.9%, at 150 mg/ml for ethosuximide and 300 mg/ml for valproic acid) and administered i.p. as a bolus over 2-3 min. Each animal received only one dose of one drug and was followed for at least 4 h after drug administration. Six animals received 600 mg/kg of valproic acid, but this dose proved fatal in two of the animals. The results presented are the data from the remaining four animals. The lower dose of valproic acid and ethosuximide were each tested in five animals. For comparison purposes, the exact procedure was also followed with six animals who received injections of normal saline i.p. (vehicle controls). At the conclusion of each experiment, electrode positions were marked for confirmation histologically. Fast Green was iontophoresed from the recording electrode and current was passed through the stimulating electrodes. The animals were perfused through the heart with 1% potassium ferrocyanide in 10% buffered formalin. The brain was subsequently equilibrated in 30% sucrose in formalin, cut on a sliding microtome and stained with cresyl violet. Serum levels of valproic acid were measured in 11 urethane-anesthetized animals. Blood was drawn by intracardiac puncture at 20 min, 1, 2 and 4 h after i.p. drug administration. Levels were determined by a fluorescent immunoassay in the Special Chemistry Laboratory of the Department of Pathology at Methodist Hospital, Houston, TX.
3. Results The effect of administration of the vehicle, normal saline, on the time to onset and duration of maximal dentate activation was determined in six animals. There was a gradual decrease in the time to onset over the first 15 stimuli and then a plateau. There was also an increase in the duration of maximal dentate activation over the first 15-16 stimuli and then the durations became quite variable. The addition of valproic acid between stimuli 3 and 4 had an effect on both the time to onset and duration (Fig. 1B). There was an increase in the time to onset of maximal dentate activation after 450 mg/kg (n = 5, Fig. 2A) and 600 mg/kg (n = 4) valproic acid that only reached statistical signifi-
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cance with 600 mg/kg. Both doses of valproic acid decreased the duration of maximal dentate activation below the duration in response to the stimulus just before drug administration (Fig. 2B). Valproic acid, 450 mg/kg, significantly reduced the duration of
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Fig. 2. Effect of valproic acid on the par~ete~ of maximal dentate activation. The time to onset and duration of maximal dentate activation were measured for each stimulus train. These values were then normalized, averaged and plotted (with SEM) against stimulus number. The dashed line indicates the mean values from the vehicle control animals. Valproic acid was administered ip. between stimuli 3 and 4. The asterisks indicate significant differences behveen the drug group and the vehicle controls. A: The effect of valproic acid at 450 mg/kg (n = 5, open circles) and 600 mg/kg (n = 4, filled circles) on the change in the time to onset of maximal dentate activation. B: The effect of valproic acid at 450 mg/kg (n = 5, filled circles) and 600 mg/kg (n = 4, open circles) on the change in duration of maximal dentate activation.
Fig. 3. Pharmacokinetics of valproic acid in the anesthetized rat. Serum levels of valproic acid were determined from blood drawn at 20 min, 1, 2 and 4 h after administration of 600 mg/kg i.p. The individual serum levels are plotted on a semi-log scale versus the time after drug administration. The line drawn is the calculated linear regression through the data points. The calculated R’ value was 0.9945 and the P value was 0.0028.
maximal dentate activation at 20 and 60 min after the drug dose. After 600 mgfkg valproic acid the reduction in the duration of maximal dentate activation was even longer lasting. Thus, it appears that very high doses of valproic acid can have a long-lasting effect on the onset and duration of seizure activity in the dentate gyrus. Serum levels of valproic acid were determined 20 min and 1, 2 and 4 h after the administration of 600 mg/kg (Fig. 3). The mean level achieved in the serum 20 min after i.p. administration was 936 pg/ml. The serum levels declined over the course of the 4-h period with first-order kinetics. The halflife of valproate was estimated to be about 1 h. Ethosuximide, at 600 mg/kg In = 5, Fig. 4) had a significant effect on the time to onset and duration of maximal dentate activation. Ethosuximide immediately increased the time to onset, an effect that lasted for 30 min. The time to onset then began to gradually decrease, but had not returned to control values by the end of the 4-h recording session. The effect of ethosuximide on the duration of maximal dentate activation was less striking, but still reached statistical significance. Ethosuximide blocked the increase in duration of maximal dentate activation for
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30-40 min. Thereafter, the duration of maximal dentate activation increased gradually. 4. Discussion
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Previous experiments, which examined the effect of valproic acid in the same experimental system, showed no effect of the drug on the time to onset or duration of maximal dentate activation at 300 mg/kg [19]. In the present study, 450 mg/kg produced a shortening of the duration of maximal dentate activation suggesting that valproic acid augments the processes involved in seizure termination in hippocampal circuits. At 600 mg/kg, valproic acid also increased the time to onset of maximal dentate activation, thus appearing to also delay seizure initiation. Although doses up to 600 mg/kg have been used in testing the neurochemical effects of valproate [6], the effective doses in this study are higher than those reported to be effective in other models of seizures
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Fig. 4. Effect of ethosuximide on the parameters of maximal dentate activation. The time to onset and duration of maximal dentate activation were measured for each stimulus train. These values were then normalized, averaged and plotted (with SEM) against stimulus number. Ethosuximide was administered i.p. between stimuli 3 and 4. The mean f SEM for the vehicle controls are shown (n = 6, open circles). The asterisks indicate significant differences betweenthe drug group and the vehicle controls. A: The effect of ethosuximide at 600 mg/kg (n = 5, filled circles) on the change in the time to onset of maximal dentate activation. B: The effect of ethosuximide (600 mg/kg, n = 5, filled circles) on the change in duration of maximal dentate activation.
Because 200-400 pg/ml of valproic acid was still an effective plasma level after the administration of 600 mg/kg, it could be postulated that this plasma level is the minimum effective level to produce an effect in this system. However, this conclusion would predict that lower dosages of valproic acid that achieved the same plasma level would also be effective. This was not directly tested in the experiments in this report. Alternatively, the initial higher plasma level achieved after the 600 mg/kg dose may be necessary for the drug effect on the onset and duration of maximal dentate activation. In this case the lower plasma level of 200-400 pug/ml may be able to sustain the effect already initiated by the higher plasma levels. The effective serum levels of valproic acid in the present experiments (200-400 pg/ml) are close to the levels reported to be effective in the rat and dog [lo]. In humans, serum levels between 50 and 100 pg/ml are considered to be in the therapeutic range ill]. It is not clear why the effective concentrations are so widely different across species. It could be related to protein binding in the plasma causing differing brain concentrations. However, studies comparing brain and plasma concentrations of valproic acid have shown a brain/plasma ratio of 0.22-0.35 for rats and mice [14,16], while in humans the same ratio is 0.06-0.28 [22]. These data
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J. L. Stringer / Epilepsy Research
indicate that for a given plasma concentration, the brain concentration is lower in humans than in rodents. Another reason for the increased effective serum level in this model may be an interaction with the anesthetic urethane. There may be a direct interaction with urethane reducing the effectiveness of valproate. In addition, because the animals were anesthetized and the seizures were elicited using stimulus trains that continue until the seizure begins, higher doses of valproic acid may be needed to alter seizure onset in this system than in an awake model. Previously it has been shown that higher doses of convulsants are needed to initiate epileptiform activity in the urethane-anesthetized rat [21]. It is not unreasonable to speculate that higher doses of antiepileptic agents may also be needed to suppress seizure activity once initiated in urethane-anesthetized animals. This point could be addressed by testing the effectiveness of valproic acid on the onset and duration of maximal dentate activation in awake rats. The serum level determinations in these experiments show first-order kinetics of elimination. Previous reports have demonstrated enterohepatic circulation and two phases to the elimination profile [3,5,8,12]. Presumably this was not seen in these experiments because the time course was not followed long enough. Enterohepatic recirculation of valproate appears to take 4-7 h in the rat and dog [lo]. The half-life of valproate in the urethaneanesthetized rat determined in this study was one hour compared to 6-15 h in the human epilepsy patient [ll] and l-2 h in the awake rat 13,131. These data strongly support the statement that valproate has a short half-life in rodents, but do not support the statement that these pharmacokinetic differences explain the higher effective doses that are needed in rodents than humans [6]. However, the kinetics of distribution and elimination of valproic acid and its active metabolites in the brain may differ from the kinetics determined in the plasma. At doses of 300 mg/kg, ethosuximide has previously been shown to transiently increase the time to onset of maximal dentate activation without having any effect on the duration [19]. At 600 mg/kg ethosuximide affects the duration of maximal dentate activation, but the most striking effect remains on the time to onset. Compared to doses used in some
19 (19941 229-235
experimental models, these doses are quite high. Ethosuximide is most effective against pentylenetetrazol-induced seizures with ED,,‘s ranging from 54 to 130 mg/kg [4,9,13,23]. In contrast, ethosuximide is ineffective against maximal electroshock seizures at doses exceeding 1000 mg/kg [4,7]. As discussed for valproic acid, the high doses of ethosuximide that were effective in these experiments may be related to the urethane anesthesia and the fact that the onset of the epileptiform activity is measured during a stimulus train. In order to have an effect on the time to onset of maximal dentate activation, ethosuximide, or any agent, must delay the activation elicited by stimuli being delivered every 50 ms. The findings with valproic acid and ethosuximide suggest that these drugs may be useful in determining the mechanisms behind seizure initiation in the hippocampal-parahippocampal circuits. In contrast to effects on the duration of maximal dentate activation, these two drugs, plus diazepam, are the only antiepileptic drugs that alter the time to onset of maximal dentate activation at doses that have been tested thus far [19]. Seizures are induced in this model by administration of stimulus trains to the CA3 region. Before the onset of maximal dentate activation a late polysynaptic response appears that resembles an angular bundle to dentate gyrus evoked potential [20]. It has been hypothesized that this polysynaptic response represents reverberatory activity through the entorhinal cortex and that this activity is an important step in initiating seizures in these circuits [18]. While neither valproic acid nor ethosuximide prevented the appearance of this potential, both drugs slowed the onset of the seizure activity. Valproic acid and ethosuximide are effective against pentylenetetrazol-induced seizures and it is now known that pentylenetetrazol induces spontaneous activity in these circuits by activation through the entorhinal cortex [17]. This suggests that ethosuximide and valproic acid may slow the onset of seizure activity in the hippocampal circuits by an effect on the entorhinal cortex. If this hypothesis is true, then it would explain both the effectiveness of these drugs against pentylenetetrazol-induced seizures and would support the hypothesis that reverberatory activity through the entorhinal cortex is an important regulator of seizure onset in the hippocampus.
J.L. Stringer/Epilepsy
Acknowledgments This work was supported by NIH NS 28871. The author thanks Michael G. Higgins for technical assistance.
References [l] Clark, C.R. and McMillian, C.L., Comparative anticonvulsant activity of 4-chlorobenzenesulfonamide and prototype antiepileptic drugs in rodents, Epilepsia, 31 (1990) 474-479. [2] Coulter, D.A., Huguenard, J.R. and Prince, D.A., Characterization of ethosuximide reduction of low-threshold calcium current in thalamic neurons, Ann. NeuroZ., 25 (1989) 582593. [3] Dickinson, R.G., Harland, R.C., Ilias, A.M., Rodgers, R.M., Kaufman, S.N., Lynn, R.K. and Gerber, N., Disposition of valproic acid in the rat: Dose-dependent metabolism, distribution, enterohepatic recirculation and choleretic effect, J. Pharmacol. Exp. Ther., 211 (1979) 583-595. [4] Johnson, D.N., White, H.S., Swinyard, EA., Albertson, T.E. and Kilpatrick, B.F., AHR-12245: A potential anti-absence drug, Epilepsy Rex, 8 (1991) 64-70. [5] Liu, M-j., Scott, K.R. and Pollack, G.M., Pharmacokinetics and pharmacodynamics of valproate analogues in rats. I. Spiro[4.6]undecane-Z-carboxylic acid, Epilepsia, 31 (1990) 465-473. [6] Liischer, W., Effects of the antiepileptic drug valproate on metabolism and function of inhibitory and excitatory amino acids in the brain, Neurochem. Rex, 18 (1993) 485-502. [7] Liischer, W., Fassbender, C.P. and Nolting, B., The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. II. Maximal electroshock seizure models, Epilepsy Rex, 8 (1991) 79-94. [8] Liischer, W., Fisher, J.E., Nau, H. and Hiinack, D., Valproic acid in amygdala-kindled rats: Alterations in anticonvulsant efficacy, adverse effects and drug and metabolite levels in various brain regions during chronic treatment, J. Pharmacol. Enp. Ther., 250 (1989) 1067-1078. [9] Liischer, W., H&tack, D., Fassbender, C.P. and Nolting, B., The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. III. Pentylenetetrazol seizure models, Epilepsy Res., 8 (1991) 171-189. [lo] Liischer, W., Honack, D., Nolting, B. and Fassbender, C.P., Trans-2-en-valproate: Reevaluation of its anticonvulsant efficacy in standardized seizure models in mice, rats and dogs, Epilepsy Res., 9 (1991) 195-210.
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Mechanisms of action. In: A.V. Delgado-Escueta, A.A. Ward, D.M. Woodbury and R.J. Porter (Eds.), Basic Mechanisms of the Epilepsies, Molecular and Cellular Approaches. Advances in Neurology, Vol. 44, Raven Press, New York, 1986, pp. 713-736. WI Mares, P., Maresova, S. and Maresova, D., Antimetrazol action and plasma levels of valproate in developing rats, Physiol. Bohemoslouaca, 38 (1989) 97-107. P., Bonazinga, [131Musolino, R., Gallitto, G., DeDomenico, M.M., Stumiolo, R., Labate, C. and DiPerri, R., Synergistic anticonvulsant effect of valproic acid and ethosuximide on pentylenetetrazole-induced epileptic phenomena in rats, J. Inr. Med. Res., 19 (1991) 55-62. I141Nau, H. and Lijscher, W., Valproic acid: Brain and plasma levels of the drug and its metabolites, anticonvulsant effects and y-aminobutyric acid (GABA) metabolism in the mouse, J. Pharmacol. Exp. Ther., 220 (1982) 654-659. [151Sasa, M., Ohno, Y., Ujihara, Y., Fujita, Y., Yoshimura, M., Takaori, S., Serikawa, T. and Yamada, J., Effects of antiepileptic drugs on absence-like and tonic seizures in the spontaneously epileptic rat, a double mutant rat, Epilepsia, 29 (1988) 505-513. [161Semmes, R.L.O. and Shen, D.D., Comparative pharmacodynamics and brain distribution of E-A’-valproate and valproate in rats, Epilepsia, 32 (1991) 232-241. elicits epileptiform activity [I71Stringer, J.L., Pentylenetetrazol in the dentate gyms of the urethane anesthetized rat by activation of the entorhinal cortex, Brain Res., 636 (1994) 221-226. [181 Stringer, J.L. and Lothman, E.W., Reverberatory seizure discharges in hippocampal-parahippocampal circuits, Exp. NeuroZ., 116 (19921 198-203. [191Stringer, J.L. and Lothman, E.W., Maximal dentate activation: A tool to screen compounds for activity against limbic seizures, Epilepsy Rex, 5 (1990) 169-176. DO1 Stringer, J.L. and Lothman, E.W., Maximal dentate activation: Characteristics and alterations after repeated seizures, J. NeurophysioZ., 62 (1989) 136-143. ml Stringer, J.L. and Sowell, K.L., Kainic acid, bicuculline, pentylenetetrazol and pilocarpine elicit maximal dentate activation in the anesthetized rat, EpiZepsy Res., 18 (1994) 11-21. El Vajda, F.J.E., Donnan, G.A., Phillips, J. and Bladin, P.F., Human brain, plasma, and cerebrospinal fluid concentration of sodium valproate after 72 hours of therapy, Neurology, 31 (1981) 486-487. I., Roztocilova, L., Mares, P., P31 Velisek, L., Kulhankova, Veliskova, J. and Mirvaldova, H., Ethosuximide affects both pentylenetetrazoleand kainate-induced clonic seizures but differentiates between tonic-clonic seizures, Can. J. Physiol. PharmacoZ., 67 (1989) 1357-1361.