Effects of phenytoin, carbamazepine, and clonazepam on cortex- and amygdala-evoked potentials in partially kindled rats

EXPERIMENTAL

NEUROLOGY

106,150-155

(1989)

Effects of Phenytoin, Carbamazepine, and Clonazepam on Cortex- and Amygdala-Evoked Potentials in Partially Kindled Rats W. M. BURNHAM,*

R. J. RACINE,~ N. W. MILGRAM,~ AND P. S. ALBRIGHT*

*Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5S lA8; *Department of Psychology, University of Toronto, Scarborough College, West Hill, Ontario, Canada MlC lA4; and TDepartment of Psychology, MeMaster University, Hamilton, Ontario, Canada L.&f3lB9

The kindling technique has been reported to produce a long-lasting enhancement in both the early and late phases of evoked potentials triggered from the kindled focus. It also alters paired-pulse facilitation and depression in the pathways which support these phenomena. The present experiment was designed to determine whether the drugs which antagonize secondary generalization in the kindling model also antagonize kindling-enhanced excitation in the pathways leading out of the focus. Multiple doses of phenytoin, carbamazepine, and clonazepam were therefore tested against single- and double-pulse evoked potentials triggered from the focus in rats that had been subjected to partial kindling from either the amygdala or the cortex. Responses were recorded in monosynaptic sites and in the mesencephalic reticular formation-a polysynaptic site thought to play an important role in secondary generalization. No drug-related effects were found on early evoked potential components, either in the single-pulse or the double-pulse paradigm. Kindling-enhanced late components (“late waves”), however, were clearly and selectively antagonized by clonazepam. o 1999 Academic Press.

Inc.

INTRODUCTION

In the kindling model, the repeated elicitation of focal discharge leads to the gradual development of secondary generalization and the eventual onset of behavioral convulsions. This effect appears to be based on permanent changes in the brain, since it does not regress even when stimulation is withdrawn for many months (see (13) for review). Electrophysiological studies of kindled brains, performed during the interictal period, have revealed a number of characteristic and long-lasting electrographic abnormalities. Prominent among these are changes in the evoked potentials observed in pathways leading out of the kindled focus. Three different types of evoked-potential change have been reported: (1) an increase in the amplitude of the early or “primary” peak of the poten0014-4886/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

150

tials (e.g., (16)); (2) the development of entirely new components which follow the primary peak (“late waves”) (e.g., (14,16)); and (3) an enhancement of double-pulse facilitation/depression in pathways which support these phenomena (e.g., (8, 17)). Several of these changes are excitatory in nature, and-separately or in combination-might contribute to the enhancement of secondary generalization which is the hallmark of the “kindled state.” A number of anticonvulsant drugs-including phenytoin, carbamazepine, clonazepam, and diazepamhave been shown to antagonize secondary generalization in the kindling model (1). One of these, diazepam, has also been shown to block late waves in kindling-enhanced evoked potentials (15). It would be of interest to know (a) whether all of the drugs which block kindled seizures also normalize kindling-enhanced evoked potentials and (b) whether these two effects occur at similar doses. Such a finding would suggest (a) that enhanced focal/extra-focal transmission is crucially related to secondary generalization in kindled subjects and (b) that anticonvulsant drugs block kindled seizures by antagonizing such enhanced transmission. A previous study by Albright and Burnham (2) has shown that anticonvulsants have no dramatic effect on normal (nonenhanced) evoked potentials. The present study, therefore, was designed to examine the effects of carbamazepine, phenytoin, and clonazepam on evoked potentials enhanced by kindling. Stimulation was applied to either the kindled amygdala or the kindled cortex, and single- and double-pulse evoked responses were recorded in (a) the contralateral cortex (monosynaptically related to the cortical focus (7)); (b) the entorhinal cortex (monosynaptically related to the amygdaloid focus (12)); or (3) the mesencephalic reticular formation or MRF (polysynaptically related to both cortex and amygdala and thought to be crucially involved in the generalization of kindled convulsions (4, 6)). Separate measurements were made of drug effects on the early and late phases of the kindling-enhanced potentials. Similar sites and drugs were utilized pre-

ANTICONVULSANTS

TABLE Experimental Implantation Characterization Kindling Characterization

Design

of electrodes of prekindling

2

Week

3

Week

4

Perfusion

viously kindled

potentials

of postkindling

potentials Stability test Vehicle test Drug No. 1 Dose 1 2 3 Drug No. 2 Dose 1 2 3 Drug No. 3 Dose 1 2 3

actions

on non-

METHODS The overall design of the present lined in Table 1.

experiments

151

POTENTIALS

Characterization

and histology

in our study of anticonvulsant evoked potentials (2).

KINDLED

Three doses of each drug (plus vehicle control) were administered. Doses were as follows: phenytoin-12.5, 50, and 150 mg/kg; carbamazepine-3.0,lO and 50 mg/ kg; clonazepam-0.05, 0.5 and 5.0 mg/kg. These doses were chosen to span the anticonvulsant and low toxic ranges for each drug, as determined by previous studies involving kindled convulsions (1). They also matched the doses previously employed in our study of nonkindled evoked potentials (2).

1

Drug tests Week 1 Week

AND

is out-

Subjects and Surgery Concentric electrodes were implanted in 22 male Royal Victoria hooded rats (250-300 g) using pentobarbital anesthesia and standard stereotaxic techniques. In 11 of the animals, electrodes were placed in the anterior neocortex, the homologous contralateral cortex, and the MRF. In the other 11 rats, electrodes were implanted into the amygdala, entorhinal cortex, and the MRF. Coordinates and details of electrode construction have been reported previously (2). Subjects were allowed at least 2 weeks of postoperative recovery and handling before experimental procedures were begun.

Drugs Carbamazepine (Tegretol, Ciba-Geigy Canada, Ltd.) and clonazepam (Rivotril, Hoffman-LaRoche, Ltd.) were obtained in pure substance form from their manufacturers. Phenytoin (Dilantin) was obtained in injectable form from Parke-Davis. All drugs were mixed (or diluted) fresh on the day of use, and were injected ip in a standard volume of 1 ml/kg. (Larger volumes of 1.7 and 3 ml/kg were required for the highest doses of carbamazepine and phenytoin, respectively). Vehicles for drug injection consisted of either propylene glycol (carbamazepine and clonazepam) or 40% propylene glycol, 10% ethanol, and 50% water (phenytoin). A standard injection-to-test interval of 30 min was used for all drugs.

of Prekindling

Potentials

In initial screening trials, the cortex or the amygdala was stimulated and prekindling single- and double-pulse responses were measured at each secondary site. Evoked responses were elicited by biphasic symmetrical squarewave pulses with a duration of 0.1 ms, an interpulse interval of 0.25 ms, and a frequency of 0.5 Hz. At least 16 responses were averaged on each trial using a Fabritek computer. To characterize the single-pulse responses, current intensity was increased in small increments from subthreshold (no response) to “maximum” intensity (no further increase in response). Intensities ranged from 100-1700 PA (peak-to-peak). To characterize the double-pulse responses, two pulses at 80% of maximum intensity were applied and the interval between them was increased in increments from 20 to 300 ms.

Kindling

Procedure

After characterization of prekindling potentials, all animals were subjected to daily kindling stimulation of the cortex or amygdala using a l-s train of 60-Hz biphasic symmetrical square-wave pulses, with a pulse duration of 1 ms and an interpulse interval of 0.5 ms. Standard stimulation intensities were set at 500 and 800 PA for the amygdala (AMYG) and cortex (CX), respectively (peak-to-peak values). Different intensities were required for cortex and amygdala because of differences in cortex and amygdala afterdischarge thresholds (3). Full kindling, which requires different numbers of stimulations for each subject and produces long-lasting inhibitory effects (see (13)), was not attempted. Instead, animals were stimulated for 8 days in the amygdala group and for 27 days in the cortex group. These values represent approximately two-thirds of the stimulations normally required for kindling (3) and are sufficient to produce long-lasting potentiation of evoked responses (Ratine, unpublished data).

Characterization Establishment

of Postkindling Potentials/ of Test Parameters

One week after the last kindling session, evoked responses were remeasured in all sites, using the same procedure which had been used for prekindling characterization. Standard stimulation parameters-calculated

152

BURNHAM

separately for each subject-were then established: (1) drug-test intensity, which was determined as threshold intensity plus 80%; and (2) double-pulse interval, which was determined as the interval producing the greatest effect on the second response (usually in the range of 3050 ms). The stability of these responses over time and after vehicle injection was then assessedas described previously (2). Only “stable” animals were utilized in subsequent drug tests.

Drug Tests Drug testing was done over a 3-week period. A different drug was tested each week, the three dosesbeing administered with a drug-free day interposed between each dose. Drug and dose order were randomized across animals. During each drug test, the cortex or amygdala was stimulated, and single- and double-pulse responses were recorded in the secondary sites. Stimulation was administered first under the predrug condition (to establish a predrug baseline) and then again 30 min after drug injection.

Scoring and Data Analysis Three basic measurements were derived from each set of data: Amplitude of the primary peak-Single-pulse paradigm. The amplitude of the primary or “early” component of the evoked response was measured peak-to-peak in microvolts. The single-pulse data were used for this purpose. During drug tests, the change in amplitude under drug was calculated as a percentage of the baseline (predrug) value for that day. (Note: Amplitude of the primary peak was measured-rather than duration or “area under the curve”-because the appearance of late waves artificially inflates measurements of duration and area. See below.) Amplitude of the primary peak-double-pulse paradigm. The degree of facilitation or depression observed in the paired-pulse paradigm (R2/Rl) was calculated as a ratio of the amplitude of the second response (primary component, peak to peak) to the amplitude of the first response (primary component, peak to peak). During drug tests, the change in R2/Rl in the presence of drug was expressed as a percentage of the baseline (predrug) value for that day. Late waves. A late wave was defined as a component of the evoked response which occurred after the primary peak and which appeared in the records only after kindling. It was found that the latency and amplitude of late waves were variable and that they were often partially or totally merged with the primary component. (Total merging resulted in an apparent increase in the duration of the primary peak. See Fig. 1.) Because of this, no at-

ET AL.

tempt was made to obtain exact measurements of latewave parameters. Rather, the data were presented to two judges (W.M.B. and R.J.R.) who scored them “blind,” determining only whether late waves were present or absent. Late-wave analysis was done on a reduced number of subjects (seven cortical, eight amygdala), whose records were judged to be clear and complete enough for unambiguous determinations. Statistical analysis of the amplitude data was done by analysis of variance, each of the major parameters being subjected to a 2 X 3 X 3 analysis (factors are stimulation site, drug, and dose), with repeated measures on the drug and dose factors (18). Significant interactions (P > 0.05) were further analyzed by a postiori “simple effect” tests (18). Statistical analysis of the late-wave data was done by the chi-square test (18).

Perfusion and Histology Upon completion of the experiment, subjects were killed with an overdose of pentobarbital and perfused through the heart with 0.9% saline followed by a 10% formalin solution. The brains were frozen, sliced in 30PM sections, and stained with thionin. All animals had correctly positioned electrodes. RESULTS

Table 2 presents numerical data related to the three basic evoked-potential parameters which were measured. Figure 1 presents representative evoked potentials before and after kindling.

Primary

Peak-Single

Pulse

The configuration of prekindled evoked potentials in these pathways has been described in a previous paper (2). Briefly, cortical stimulation produces a short-latency, high-amplitude, monophasic response in the contralateral cortex and a longer latency, low-amplitude monophasic response in the MRF. Amygdaloid stimulation produces a short-latency, high-amplitude, often biphasic response in the ipsilateral entorhinal cortex (ENT) and a longer latency, moderate amplitude, bi- or polyphasic response in the MRF. These configurations were unchanged after kindling, except for the addition of late components. Column 1 of Table 2 presents amplitudes (mean + SE) for the primary peaks observed in each recording site before and after kindling. As indicated, mean amplitude was decreased by kindling in three instances and little changed in the fourth. Statistical analysis showed that one of the decreases (CX-MRF) reached significance (P < 0.05). Thus, no kindling-induced potentiation of the primary peak was observed in the present experiment, and a significant depression was actually seen in the one pathway.

ANTICONVULSANTS

TABLE

AND

2

Evoked Potential Parameters Amplitude of primary component ( pV f SE) cx-cx Before partial kindling After partial kindling Pbenytoin (High) Carbamazepine (High) Clonazepam Wish) CX-MRF Before partial kindling After partial kindling Phenytoin Carbamazepine Clonazepam AMYG-ENT Before partial kindling After partial kindling Phenytoin Carbamazepine Clonazepam AMYG-MRF Before partial kindling After partial kindling Phenytoin Carbamazepine Clonazepam

(N=

11)

R2/Rl (double pulse)

Late

(N = 11)

waves

(N=

758 + 131

1.69 -t 0.11

O/7

806 + 142

1.74 + 0.10

5/7

978 f 213

1.51 + 0.09

5/7

834 + 168

1.69 f 0.12

5/7

766 + 119

1.73 f 0.11

O/7

7)

KINDLED

CX-MRF and AMYG-MRF pathways (P < 0.05). Since postkindling potentiation of the primary peak was not observed, no statements can be made concerning drug reversal of that phenomenon. No drug reversal was observed in the case of the one significant postkindling change which did occur, CX-MRF depression. This effect was exacerbated by clonazepam. Primary Peak-Double-Pulse Column 2 of Table 2 presents pre- and postkindling means (&SE) for the ratios related to double-pulse effects on the primary peak. As indicated, before kindling three pathways showed double-pulse facilitation (CX-CX, CX-MRF, and AMYG-ENT), and one pathBEFORE

(N=

11)

(N=

292 +

26

1.36 f 0.15

199f 163 f 162 + 108 f

31* 33 22 16**

2.08 2.27 2.22 2.32

(N=

11)

f + + +

(N=

11)

0.13* 0.45 0.22 0.46

6/7+ 617 6/7 o/7**

11)

(N=8)

1.35 f 0.14

f-‘/f3

822 889 844 839

1.16 1.12 1.28 1.02

5/8* 5/8 5/8 O/8**

246 256 233 264

(N = 11)

& i f +

(N=

0.34 0.29 0.47 0.56 11)

45

0.72 + 0.20

O/8

289f 334& 349+ 213 f

31 64 54 71**

0.85 0.63 0.51 2.10

318 3/8 3/8

0.29 0.18 0.10 O-80**

Cortex-Contralateral

AFTER

KINDLING

Cx

Cortex-MRF

(N=8)

370+

f f f +

KINDLING

(N=7)

928 + 173 + k 2 +

153

POTENTIALS

‘W

Note. Abbreviations:

AMYG, amygdala; CX, cortex; ENT, entorhinal cortex; MRF, mesencephalic reticular formation. Drug data indicate effects observed at highest dose: 150 mg/kg phenytoin, 50 mg/kg carbamazepine, 5 mg/kg clonazepam. * Differs from prekindhng values. P < 0.05. ** Differs from vehicle control values taken on the same day. P < 0.05.

Column 1 of Table 2 also indicates amplitudes (mean + SE) of the postkindling primary peaks measured in the presence of the three test drugs (highest dose). As indicated, phenytoin and carbamazepine had no significant effects, whereas clonazepam tended to reduce amplitudes. These reductions reached significance in the

n

IA-

2ooL.

FIG. 1. Representative evoked potentials, pre- and postkindling. In the cortex-cortex potential, the postkindling “late wave” may be seen as a new component added to the primary peak. This added component is reduced in the double-pulse paradigm. In the cortex-MRF potential, the primary peak and late wave are merged, kindling-induced enhancement is seen as an apparent “broadening” of the primary peak. In the lowest set of traces (marked with arrows), a further series of long-latency late waves is illustrated. These waves, which occurred well after the primary peak, could be observed only when extended sweeps were used. No attempt was made to analyze these longlatency waves in the present experiments.

154

BURNHAM

way showed double-pulse depression (AMY G-MRF) . After kindling, a significant enhancement of doublepulse facilitation was observed in the CX-MRF pathway (P > 0.05). Changes in the other pathways failed to reach significance. Column 2 of Table 2 also indicates postkindling means (*SE) for double-pulse ratios measured in the presence of the three test drugs (highest dose). As indicated, phenytoin and carbamazepine had no significant effects. Clonazepam greatly increased double-pulse facilitation in the AMYG-MRF pathway-an effect previously reported in nonkindled animals by (2). The kindling-induced enhancement of double-pulse facilitation which was observed in the CX-MRF pathway was not affected by any of the drugs.

Late Waves Column 3 of Table 2 indicates the number of subjects that displayed clear-cut late waves in each pathway before and after kindling. (By definition, prekindling values are 0.) As indicated, late waves were seen in every pathway after kindling, although they were not observed in every subject. (The small number of late waves observed in the AMYG-MRF pathway may be an artifact of scoring. The bi- or polyphasic potentials normally observed in this path may have masked kindling-induced changes in the latter part of the potential.) Late waves were often diminished or absent in the second response of the paired-pulse paradigm (Fig. 1). Column 3 of Table 2 also indicates the number of postkindling subjects that displayed late waves in the presence of the three test drugs (highest dose), As indicated, neither phenytoin nor carbamazepine had any significant effect. Clonazepam, however, suppressed late waves in every subject tested. Examination of individual records indicated that these effects were generally seen at a dose of 0.5 mg/kg or lower. DISCUSSION The present study was designed to investigate the effects of three drugs which suppress kindled seizurescarbamazepine, phenytoin, and clonazepam-or enhanced evoked responses in the pathways leading out of the kindled focus. Three types of kindling-induced enhancement were studied, enhancement of the singlepulse primary peak, enhancement of double-pulse facilitation, and the development of late waves. Enhancement of the amplitude of the primary peak in postkindling potentials was first described by Racine et al. (14). It is now believed that this effect of kindling is a form of long-term potentiation (16). This type of postkindling potentiation was not observed in the present study, possibly because of the time of observation. Ratine et aZ. (16) have reported that enhancement of the

ET

AL.

primary component is maximal early in the kindling process, returning later to near- or subbaseline levels. Since no enhancement was observed in the present data, no conclusions can be made regarding drug effects on this form of kindling-induced potentiation. In passing, it is interesting to note that a long-lasting depression of the primary peak was observed in the CX-MRF pathway. To our knowledge, this phenomenon has not been reported before. This long-lasting effect of the kindling procedure was not reversed by any of the drugs studied. Enhancement of double-pulse facilitation by the kindling technique was first reported by King et al. (11). While this effect has been little studied, it appears to be long-lasting and, theoretically, might contribute to the “kindled state.” In the present study, one clear-cut example of kindling-enhanced double-pulse facilitation was observed, the postkindling increase of about 50% observed in the CX-MRF pathway. None of the drugs reversed this effect at any dose tested. Thus, this sort of postkindling potentiation resists all of the drugs which suppress kindled seizures. Late waves were observed in the earliest studies of kindling-enhanced evoked potentials (14). They were not clearly distinguished from potentiation of the primary peak, however, until further studies had been completed (16). It is currently believed that the late wave is an epileptiform response, akin to the interictal spike (10). Late waves, like interictal spikes, are very long lasting (Racine et al., 1983), and might contribute to the “kindled state.” In the present study, late waves were observed in every pathway studied. They were unaffected by phenytoin and carbamazepine, but were suppressed in a dose-related fashion by clonazepam. Clonazepam effects were generally observed at doses of 0.5 mg/kg or lower. These doses are similar to the low, nontoxic doses of clonazepam which suppress secondary generalization in kindled subjects. (Albright and Burnham (1) report ED5,, values of 0.07 and 0.3 mg/kg for amygdala- and cortex-generalized convulsions). Diazepam, another benzodiazepine, has also been reported to antagonize kindling-induced late waves (Racine and Milgram, 1980). A major goal of the present study was to test the hypothesis that the anticonvulsants which suppress secondary generalization in kindled subjects do so by antagonizing the kindling-induced potentiation in pathways leading out of the kindled focus. The data derived from the present study do not support this hypothesis-at least in its general form. Two anticonvulsants, phenytoin and carbamazepine, had no effect on potentiated responses, and the third, clonazepam, antagonized only late waves. It is interesting to note, however, that there was a good correlation between the dose of clonazepam required to suppress late waves and the previously reported EDSO for clonazepam suppression of kindled convulsions. These

ANTICONVULSANTS

AND KINDLED

data suggest a more limited hypothesis-the hypothesis that one type of anticonvulsants, the benzodiazepines, suppress kindled seizures by antagonizing one sort of potentiated response, the late wave. Experiments designed to test this hypothesis are now in progress. If the hypothesis can be substantiated-and possibly extended to the other anticonvulsants which (like the benzodiazepines) enhance GABAergic activity-the results may have considerable relevance to the GABA hypothesis of kindling (5). Besides providing a test of our initial hypothesis, the present data provide insights into both the kindling processand mechanisms of anticonvulsant action. With regard to kindling, the present data provide a clear-cut distinction between three types of postkindling potentiation: enhancement of the primary peak, which was not observed in any pathway at this period of observation, enhancement of double-pulse potentiation, which was observed only in the CX-MRF pathway and which resisted all drugs, and the development of late waves, which occurred in all pathways and was responsive to clonazepam. It is clear that the kindling process potentiates neural pathways in several distinct and separate fashions. With regard to anticonvulsant mechanisms, the present data reinforce previous studies which show sharply differing profiles of action for the benzodiazepines and the more conventional anticonvulsants, such as phenytoin and carbamazepine (see ( 1,9)). ACKNOWLEDGMENTS This work was supported by Grant MA 5611 from the Medical Research Council of Canada. The authors thank Mr. Jerome Cheng for technical assistance, and Ciba-Geigy Canada, Ltd., and HoffmanLaRoche, Ltd., for their kind donations of (respectively) carbamazepine and clonazepam in pure substance form. Dr. Albright’s present address is Parke-Davis Canada, Inc., 2200 Eglinton Avenue East, Scarborough, Ontario, Canada MlL 2N3. REFERENCES 1. ALBRIGHT, P. S., AND W. M. BURNHAM. 1980. Development of a new pharmacological seizure model: Effects of anticonvulsants on cortical- and amygdala-kindled seizures in the rat. Epilepsia 21: 681-689. 2. ALBRIGHT, P. S., AND W. M. BURNHAM. 1983. The effects of phenytoin, carbamazepine and clonazepam on cortex- and amygdala-evoked potentials. Exp. Neurol. 81: 308-319.

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15. RACINE, R. J., AND N. W. MILGRAM. 1980. Post-activation potentiation and epilepsy. Pages 166-173 in V. M. OKUJAVA, Ed., Neurophysiological Mechanisms of Epilepsy. Metsniereba, Tbilisi. 16. RACINE, R. J., N. W. MILGRAM, AND S. HAFNER. 1983. Longterm potentiation phenomena in the rat limbic forebrain. Brain Res. 260: 217-231. 17. TUFF, L. P., R. J. RACINE, AND H. ADAMEC. 1983. The effects of kindling on GABA-mediated inhibition in the dentate gyrus of the rat. I. Paired-pulse depression. Brain Res. 277: 79-90. 18. WINER, B. J. 1971. Statistical Principles in Experimental Design. McGraw-Hill, New York.