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Correlation between electrocorticographic and motor phenomena induced by pentamethylenetetrazol during ontogenesis in rats
EXPERIMENTAL
NEUROLOGY
84,
153-l 64 ( 1984)
Correlation between Electrocorticographic and Motor Phenomena Induced by Pentamethylenetetrazol during Ontogenesis in Rats RENBE SCHICKEROVA, PAVEL MARES, AND STANISLAV TROJAN’ Institute of Physiology, Faculty of Medicine, Charles University, and Institute of Physiology, Czechoslovak Academy of Sciences. Prague, Czechoslovakia Received August 9, 1983; revision received November 3. 1983 Motor and electrocorticographic (EGG) phenomena induced by pentamethylenetetrazol (PTZ) were directly compared in adult as well as in young rats (age groups of 7, 12, and 18 days). The technique for implantation of cortical electrodes was elaborated in young rats. Myoclonic jerks were elicited by PTZ in all age groups studied. In adult animals they were accompanied by isolated spikes and/or spike-andwave complexes, whereas in 7day-old rats there was a poor correlation between ECoG abnormalities and myoclonic jerks. With increasing age an improved correlation between ECoG abnormalities and behavioral changes were noted. AAer further injections of PTZ, rats had generalized tonic-clonic motor seizures. Ictal ECoG activity usually started in one cortical region in 7-day-old rats and became progressively generalized whereas in older rats generalized electrical discharges accompanied the motor seizures from onset. The clonic phase of motor seizures was not always accompanied by specific ECoCi changes in young rats. At all ages the correlation between ECoG activity and individual phases of the generalized tonic-clonic seizures was poor.
INTRODUCTION Minimal Metrazol seizures in rodents are used for screening anticonvulsant action of drugs; efficacy against this model predicts clinical usefulness against primary generalized seizures of the absence type ( 13). This type of seizure is age specific, with its incidence highest in children of preschool and elementary school age (11). Abbreviations: ECoG-electrocorticogram, PTZ-pentamethylenetetrazol. I Please address reprint requests to: Dr. P. Ma&, Institute of Physiology, Czechoslovak Academy of Sciences, Videiiska 1083, CS-142 20 Prague 4, Czechoslovakia. 153 00 14-4886/84 $3.00 Copyright 0 1984 by Academic Press. Inc. All rigbu of reproduction in any form resewed
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With higher doses of Metrazol [pentamethylenetetrazol (PTZ)], maximal or generalized tonic-clonic seizures can be induced (15). During the ontogenetic development, the PTZ-induced generalized motor seizures can be provoked at any age whereas minimal Metrazol seizures cannot be induced until the third postnatal week (2, 7, 14). However, little research has been done to date correlating electroencephalographic and clinical features in young rats. The purpose of this study was to evaluate the ontogenesis of ECoG and behavior changes after injection of PTZ in rats of various ages. The results were published in a preliminary form (12). METHODS Experiments were carried on 7-(N = 13) 12-(N = 12) and 18-day-old (N = 20) rats as well as on 12 adult rats. These age groups were selected on the basis of our previous results (7). Albino rats of the Wistar strain were bred under barrier (i.e., nearly specific pathogen free) conditions. Litters were reduced to eight pups; the day of birth was taken as 0. Cortical recording electrodes were implanted in adult rats under Nembutal anesthesia. The ECoG recordings were not made until at least 1 week after implantation. The immature rats were implanted semichronically using our original method. Under ether anesthesia four incisions in the skull were made and a flat arm of an L-shape silver electrode was introduced into each incision under the bone (Figs. 1 and 2). The four cortical electrodes and a reference one on the nasal bone were cemented together by a thin layer of dental acrylic. All electrodes were directly connected to wires leading to the EEG apparatus. The entire surgical preparation lasted 40 to 50 min. During this period as well as during subsequent recuperation the rats were placed on a heating pad (30 to 33°C). The recuperation period lasted 2 to 3 h and rats were artificially fed (a drop of 10% solution of glucose was put into their mouths each 20 to 30 min). The recording was always preceded by a neurologic examination according to Bureg et al. (1). One animal showing signs of abnormalities was omitted from the group and examination of the brain revealed a subdural hemorrhage due to a broken electrode. Initially a 30min period of spontaneous ECoG activity was registered (time constant0.3 s, filters of high frequencies-70 Hz) followed by a subcutaneous injection of a 10% solution of PTZ at a dose of 50 mg/kg. During the next 20 min the ECoG activity was recorded and motor behavior of the animals was observed. A 20-mg/kg dose of PTZ was then given and ECoG recordings continued for another 20 min. When the second dose did not lead to generalized seizures, another 20-mg/kg dose was injected. In 13 of 20 18-dayold rats, in which we tried to induce rapidly generalized seizures, we started with a 70-mg/kg dose and then proceeded with 20-mg/kg incremental doses.
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FIG. I. Schematic drawing of the placement of the electrodes in young rats.
The ECoG recordings as well as PTZ applications were conducted in a similar manner in the adult animals. RESULTS Interictal Behavior and Electrocorticographic Findings. Seven-day-old rats: A discontinuous ECoG was recorded. Rare, isolated large delta waves (amplitude up to 100 PV) did not correlate with motor phenomena. The animals were motionless during a large portion of the time; however, occasional jerks of the head and/or the limbs as well as short periods of searching activity were seen, Twelve-day-old rats: The ECoG activity was continuous and was occasionally interrupted by short low-voltage periods. A dominant, rhythmic background activity was not present. Fast activity of low amplitude (up to 20 rV) combined with large waves (up to 100 pV) occurred individually or in groups. Behaviorally, the animals exhibited periods of orienting activity
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FIG. 2. Technique of implantation in a 12day-old rat. An indifferent electrode is cemented to the skull; electrodes in both frontal regions are prepared to be cemented.
as well as periods of rest. Occasionally, isolated myoclonic jerks and trembling were seen. It was possible to differentiate sleep from the awake state. An increase in the amount and amplitude of isolated delta, eventually theta, waves as well as periods of rhythmic activity with a frequency of 13 to 16 Hz were observed during sleep. In some animals periods of low-voltage ECoG activity were recorded during sleep. Eighteen-day-old rats: Background rhythm with a dominant frequency of 5 to 6 Hz was present in occipital regions. A decrease in frequency and increase in amplitude accompanied the transition from wakefulness to sleep. These animals exhibited marked motor activity predominantly of the orienting type. Their movements were smooth and well coordinated without jerks and trembling. Adult rats: The ECoG demonstrated a background dominant frequency of about 5 Hz which was recorded symmetrically from the occipital regions; this rhythm could be blocked with strong stimuli. The adult rats spent nearly the entire observation period in relaxed wakefulness. Behaviorally, a short period of orienting reaction took place and then the animals sat motionless in one comer of the box.
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Changes Induced by Pentamethylenetetrazol. Seven-day-old rats: Restlessness and an increase in locomotor activity formed the first behavioral signs of the action of PTZ. The ECoG activity became continuous and the amplitude of individual waves increased. Periods of flattening of the ECoG waves accompanied periods of relative rest. Isolated sharp waves (or sharp and slow wave complexes) of high amplitude were accompanied in 20% of the events by myoclonic jerks of the whole body (Fig. 3). With an additional dose of PTZ both motor and ECoG signs progressively intensified. In eight animals isolated jerks became repetitive and progressed into a short phase of wild running. This phase was followed by tonic seizures consisting of opisthotonus, tonic flexion, and extension of the forelimbs, and flexion of the hind limbs (Fig. 4). The tonic phase of the hind limbs was shorter than the tonic phase of the forelimbs and occasionally it failed to appear. The tonic phase was replaced by a clonic one consisting of the animals laying on
J
J
::
FIG. 3. Electrocorticographic (E&G) correlates of jerks induced by metrazol. From top to bottom: ECoG activity from a 7day-old, a 12day-old, and an 1l-day-old rat. Individual leads: RF-right frontal, RO-right occipital, LF-left frontal, and LO-let? occipital cortical areas. Amplitude callibration 0.5 mV, time mark 1 s. The letter ‘J” under the recording denotes wholebody jerk. In the recording from a 12day-old rat there are three similar sharp waves in the ECoC but only two of them are accompanied by motor phenomena.
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FIG. 4. Motor seizure in a ‘I-day-old rat-tonic
phase.
their side or back and exhibiting poorly coordinated movements of all four limbs. This phase was the longest one. After some minutes the movements slowed and, finally, only isolated jerks were present. Electroencephalographically generalized seizures were preceded by sharp delta waves (equaling 1 Hz) of high amplitude (to 2 mV) with superimposed low-amplitude fast activity. Ictal activity started in one or two cortical regions and slowly generalized. Motor seizures always appeared at the moment when ictal activity became generalized. Little synchronization of sharp delta waves among various cortical regions occurred. The superimposed activity progressively decreased whereas the frequency of high waves increased. From the ECoG point of view it was impossible to delineate sharply individual phases of generalized seizures (Fig. 5). During the clonic phase of motor seizures sharp delta waves became intermittent with short periods of low-amplitude activity or electrocerebral inactivity. During the postictal depression, isolated high-amplitude waves (1 to 2 mV) accompanied myoclonic jerks. Twelve-day-old rats: The first signs of action of PTZ were similar to those described in 7day-old animals; i.e., restlessness and increased orienting and locomotor activity accompanied by irregular hyperventilation. The ECoG demonstrated sharp delta and/or theta waves which, at first, were isolated and then occurred bifrontally sometimes associated with a following slow wave. During the preictal phase there was a progressive augmentation of delta and theta activity. In nearly half of the animals isolated sharp waves were accompanied by myoclonic jerks of the whole body (Fig. 3). On occasion rhythmic activity occurred (Fig. 6).
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FIG.5. The ECoG recordings of seizures in a 7&y-old rat with a description of motor behavior. The number between sections denotes the length of the omitted recording in seconds. Other details as in Fig. 3.
Motor seizures started again with wild running followed by the tonic and clonic phases. The only difference in comparison with 7day-old animals was the increased speed of all movements. ECoG ictal activity began with highamplitude delta waves (to 2 to 3 mV) often with superimposed fast activity. As a rule, this activity started in one cortical region and quickly became generalized. The duration of these single delta waves progressively shortened so that clear cut spikes or spike-and-wave complexes were recorded. There
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FIG. 6. Rhythmic ECoG activity in a 12day-old (top), in an lSday-old (middle), and in a 90-day-old rat (bottom). Details as in Fig. 3.
was no clear correlation between the motor tonic and clonic phase and ECoG patterns (Fig. 7). During the clonic phase high-amplitude sharp elements faded so that an unchanged motor pattern could be accompanied by dysrhythmic activity without specific epileptiform activity. Motor seizures were interrupted by periods of rest in some animals. When second paroxysm started, ECoG activity was always better organized and specific epileptic features were better defined than at the beginning of the first seizure. Generally, all ECoG activities were better expressed in frontal than in occipital leads. Eighteen-day-old rats: The first motor signs of the action of PTZ were the same as in younger animals. There were no ECoG correlates of the increased motor activity. Isolated myoclonic jerks correlated with isolated spikes and/ or spike-and-wave complexes in more than 60% of the cases (Fig. 3). A new phenomenon appeared in approximately one-half of the rats in this group: short periods of freezing (animals sat still with only small jerks of the head) accompanied by periods of rhythmic spike activity in the ECoG (3 to 5 Hz). This activity was formed by spikes or spike-and-wave rhythms with the amplitude exceeding that of the background activity (Fig. 6). Only generalized tonic-clonic seizures were recorded in our group due to a rapid increase in dosage of PTZ. Motor patterns of the major seizures exhibited all three phases: in contrast to younger animals, tonic hind limb
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FIG. 7. The ECoG recordings of seizures in a 12day-old rat. Details as in Fig. 5.
extension was present and the clonic seizures were fast and well organized. The ECoG started with irregular high-amplitude spikes. During the tonic phase this spike activity became regular and was followed by slow waves forming spike-and-wave complexes (I; = 3 to 6 Hz) which continued into the clonic phase. There was not a distinct ECoG change in the transition from the tonic to clonic phase. During the clonic phase the ECoG activity often changed to a dysrhythmic mixture in a broad frequency spectrum not associated with changing motor patterns. The ECoG later returned to spike-
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and-wave rhythm (Fig. 8). A low-voltage recording with isolated sharp elements corresponding to jerks appeared toward the end of seizures. When seizures lasted for several minutes, serrated waves were also recorded. Adult rats: Restlessness was not as marked as in young animals. The first ECoG signs consisted of periods of theta waves (or spike-and-wave complexes of ~-HZ frequency) lasting 2 to 10 s and accompanied by freezing of the animal with minute jerks of the head musculature (e.g., rhythmic jerks of whiskers or ears). Rarely, isolated spikes appeared in the ECoG and were nearly always accompanied by body myoclonic jerks. With additional doses Ch
FIG.
18.6
8. The ECoG recordings of seizures in an 18-day-old rat. Details as in Fig. 5.
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of PTZ two types of motor seizures could be elicited. Minimal Metrazol seizures correlated with the ECoG spike activity which progressively changed into spike-and-wave rhythm. Major, generalized tonic-clonic seizures exhibited a similar ECoG pattern as in the 18-day-old rats with the exception of an absence of dysrhythmia during the clonic phase in adults. The beginning as well as the end of the ictal activity was abrupt during both minimal and major seizures. DISCUSSION Technique of Implantation. Secure implantation of electrodes in young rats is a difficult technical problem. Jouvet-Mounier (6) and Gramsbergen (4) only slightly modified the technique used in adult rats. Studies of kindling in immature rats (3, 5, 9, 10) led to an increased interest in this problem. Our method is based on that published by Gilbert and Cain (3) who used wire hooks cemented to the skull. We also used hooks and applied them to cortical electrodes. Two pairs of Gshape electrodes placed in opposition on two hemispheres are sufficient for fixation of the whole assembly. Even during wild running there were no artifacts in our recordings. This method can also be used for simultaneous implantation of subcortical electrodes (unpublished results). Ontogenetic Development of Epileptic Manifestations. The development of ECoG phenomena demonstrated the same principles as in our previous papers (8, 16); i.e., an increase in frequency of individual wave forms with maturation, progressive generalization of ictal activity, and episodes of rhythmic theta (and/or delta) waves as an age-specific phenomenon clearly visible for the first time in 18day-old rats. Correlation between EIectrocorticographic and Motor Phenomena. In general, the correlation between ECoG and motor phenomena induced by PTZ progressively increased with maturation. Myoclonic jerks which accompanied only approximately 20% of isolated sharp waves in 7day-old rats, exhibited nearly complete synchronization with isolated spikes and/or spike-and-wave complexes in adult animals. Only localized jerks restricted to limbs did not have ECoG counterparts. In 7- and 12-day-old rats only generalized tonic-clonic seizures could be induced by PTZ. Minimal Metrazol seizures represented an age-dependent phenomenon which could be elicited from the third week of postnatal life (2, 7). Due to a rapid increase in PTZ doses we did not observe this type of seizures in the present group of 18-day-old rats. In adult animals, both types of seizures were recorded and they did not differ in their ECoG pattern. ECoG ictal activity started in a restricted cortical region in 7day-old rats. The tendency to generalization markedly matured until the age of 18 days. At that age only generalized ECoG seizures were seen. There were no consistent
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correlations between ECoG patterns and phases of motor seizures in any age group. Even in adult animals the transition between the tonic and clonic phases was not punctually accompanied by a change in ECoG recording. Wave forms increased progressively forming a continuous transition from pure spike activity to spike-and-wave rhythm. We never saw motor seizures without accompanying ECoG changes in mature animals whereas this situation was often recorded in the younger rats. The absence of specific epileptic activity in the cerebral cortex during motor seizures in young rats indicates that the cerebral cortex is not necessary for the generation of generalized motor seizures. Structures governing this motor activity must, therefore, be subcortical. The correlation between ECoG and motor seizures in adult animals can be interpreted as a sign of well functioning cortical afferent pathways from the generator structure. REFERENCES 1. BURE& J., 0. BURESOVA, AND J. HUSTON. 1976. Techniques and Basic Experiments fir the Study ofBrain and Behavior. Elsevier, Amsterdam. 2. DE CASRILEVITZ, M., E. ENGELHARDT, AND C. A. ESB~RARD. 1971. Maturation of convulsogenic activity induced by leptazol in the albino rat. Br. J. Pharmacol. 42: 31-42. 3. GILBERT, M. E., AND D. P. CAIN. 1980. Electrode implantation in infant rats for kindling and chronic brain recording. Behav. Bruin Res 1: 553-555. 4. GRAMSBERGEN, A. 1976. EEG development in normal and undernourished rats. Brain Res. 105: 287-308. 5. HOLMES, G. L. 6. 7.
8.
9. 10. 11. 12. 13. 14. 15. 16.
1983. Effect of serial seizures on subsequent kindling in the immature brain. Dev. Brain Res. 6: 190-192. JOU~ET-MOUNTER, D. 1968. Ontogent%e des &tats de vigilance chez quelques mammtj&es, pp. 39-5 I. Tixier and Fils, Lyon. MARES, P., AND R. SCHICKEROVL. 1980. Seizures elicited by subcutaneous injection of metrazol during ontogenesis in rats. Activ. Nerv. Super. 22: 264-268. MARES, P., AND L. VEL~SEK. 1982. Electrocorticographic effectsof metrazol and ethosuximide during ontogenesis in rats. Physiol. Bohemoslov. 31: 455. MOSH& S. L. 1981. The effect of age on the kindling phenomenon. Dev. Psychobiol. 14: 75-81. MOSHB, S. L. 198 1. The kindling phenomenon and its possible relevance to febrile seizures. Pages 59-63 in K. H. NELSON AND J. H. ELLENBERG, Eds., Febrile Seizures. Raven Press, New York. O’DONOHOE, N. V. 1981. Epilepsies of Childhood, pp. 25-28. Butterworths, London. SCHICKEROV& R., P. MARES, AND S. TROJAN. 1982. Correlations entre le comportement et EEG pendant les crises epileptiques induites par metrazol au cows de l’ontogen& chez le rat. .I. Physiol. (Paris) 78: 46A. SWINYARD,E. A. 1973. Assay of antiepileptic drug activity in experimental animals. Stand& tests.Pages47-65 in J. MERCIER, Ed., Anticonvulsant Drugs, Int. Encycl. Pharma&Ther., Vol. 1, Sect. 19. Pergamon Press, Oxford. VERNADAKIS,A., AND D. M. WOODBURY. 1969. The developing animal as a model. Epilepsia lo: 163-178. VIDA, J. A. 1977. Anticonvulsants. Medicinal Chemistry: A Series ofMonographs, Vol. 15. Academic Press, New York. ZOUHAR, A., P. MARES, AND G. BROZEK. 1980. Electrocorticographic activity elicited by metrazol during ontogenesis in rats. Arch. Int. Pharmacodyn. Ther. 248: 280-288.
NEUROLOGY
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153-l 64 ( 1984)
Correlation between Electrocorticographic and Motor Phenomena Induced by Pentamethylenetetrazol during Ontogenesis in Rats RENBE SCHICKEROVA, PAVEL MARES, AND STANISLAV TROJAN’ Institute of Physiology, Faculty of Medicine, Charles University, and Institute of Physiology, Czechoslovak Academy of Sciences. Prague, Czechoslovakia Received August 9, 1983; revision received November 3. 1983 Motor and electrocorticographic (EGG) phenomena induced by pentamethylenetetrazol (PTZ) were directly compared in adult as well as in young rats (age groups of 7, 12, and 18 days). The technique for implantation of cortical electrodes was elaborated in young rats. Myoclonic jerks were elicited by PTZ in all age groups studied. In adult animals they were accompanied by isolated spikes and/or spike-andwave complexes, whereas in 7day-old rats there was a poor correlation between ECoG abnormalities and myoclonic jerks. With increasing age an improved correlation between ECoG abnormalities and behavioral changes were noted. AAer further injections of PTZ, rats had generalized tonic-clonic motor seizures. Ictal ECoG activity usually started in one cortical region in 7-day-old rats and became progressively generalized whereas in older rats generalized electrical discharges accompanied the motor seizures from onset. The clonic phase of motor seizures was not always accompanied by specific ECoCi changes in young rats. At all ages the correlation between ECoG activity and individual phases of the generalized tonic-clonic seizures was poor.
INTRODUCTION Minimal Metrazol seizures in rodents are used for screening anticonvulsant action of drugs; efficacy against this model predicts clinical usefulness against primary generalized seizures of the absence type ( 13). This type of seizure is age specific, with its incidence highest in children of preschool and elementary school age (11). Abbreviations: ECoG-electrocorticogram, PTZ-pentamethylenetetrazol. I Please address reprint requests to: Dr. P. Ma&, Institute of Physiology, Czechoslovak Academy of Sciences, Videiiska 1083, CS-142 20 Prague 4, Czechoslovakia. 153 00 14-4886/84 $3.00 Copyright 0 1984 by Academic Press. Inc. All rigbu of reproduction in any form resewed
154
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MARES, AND TROJAN
With higher doses of Metrazol [pentamethylenetetrazol (PTZ)], maximal or generalized tonic-clonic seizures can be induced (15). During the ontogenetic development, the PTZ-induced generalized motor seizures can be provoked at any age whereas minimal Metrazol seizures cannot be induced until the third postnatal week (2, 7, 14). However, little research has been done to date correlating electroencephalographic and clinical features in young rats. The purpose of this study was to evaluate the ontogenesis of ECoG and behavior changes after injection of PTZ in rats of various ages. The results were published in a preliminary form (12). METHODS Experiments were carried on 7-(N = 13) 12-(N = 12) and 18-day-old (N = 20) rats as well as on 12 adult rats. These age groups were selected on the basis of our previous results (7). Albino rats of the Wistar strain were bred under barrier (i.e., nearly specific pathogen free) conditions. Litters were reduced to eight pups; the day of birth was taken as 0. Cortical recording electrodes were implanted in adult rats under Nembutal anesthesia. The ECoG recordings were not made until at least 1 week after implantation. The immature rats were implanted semichronically using our original method. Under ether anesthesia four incisions in the skull were made and a flat arm of an L-shape silver electrode was introduced into each incision under the bone (Figs. 1 and 2). The four cortical electrodes and a reference one on the nasal bone were cemented together by a thin layer of dental acrylic. All electrodes were directly connected to wires leading to the EEG apparatus. The entire surgical preparation lasted 40 to 50 min. During this period as well as during subsequent recuperation the rats were placed on a heating pad (30 to 33°C). The recuperation period lasted 2 to 3 h and rats were artificially fed (a drop of 10% solution of glucose was put into their mouths each 20 to 30 min). The recording was always preceded by a neurologic examination according to Bureg et al. (1). One animal showing signs of abnormalities was omitted from the group and examination of the brain revealed a subdural hemorrhage due to a broken electrode. Initially a 30min period of spontaneous ECoG activity was registered (time constant0.3 s, filters of high frequencies-70 Hz) followed by a subcutaneous injection of a 10% solution of PTZ at a dose of 50 mg/kg. During the next 20 min the ECoG activity was recorded and motor behavior of the animals was observed. A 20-mg/kg dose of PTZ was then given and ECoG recordings continued for another 20 min. When the second dose did not lead to generalized seizures, another 20-mg/kg dose was injected. In 13 of 20 18-dayold rats, in which we tried to induce rapidly generalized seizures, we started with a 70-mg/kg dose and then proceeded with 20-mg/kg incremental doses.
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FIG. I. Schematic drawing of the placement of the electrodes in young rats.
The ECoG recordings as well as PTZ applications were conducted in a similar manner in the adult animals. RESULTS Interictal Behavior and Electrocorticographic Findings. Seven-day-old rats: A discontinuous ECoG was recorded. Rare, isolated large delta waves (amplitude up to 100 PV) did not correlate with motor phenomena. The animals were motionless during a large portion of the time; however, occasional jerks of the head and/or the limbs as well as short periods of searching activity were seen, Twelve-day-old rats: The ECoG activity was continuous and was occasionally interrupted by short low-voltage periods. A dominant, rhythmic background activity was not present. Fast activity of low amplitude (up to 20 rV) combined with large waves (up to 100 pV) occurred individually or in groups. Behaviorally, the animals exhibited periods of orienting activity
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FIG. 2. Technique of implantation in a 12day-old rat. An indifferent electrode is cemented to the skull; electrodes in both frontal regions are prepared to be cemented.
as well as periods of rest. Occasionally, isolated myoclonic jerks and trembling were seen. It was possible to differentiate sleep from the awake state. An increase in the amount and amplitude of isolated delta, eventually theta, waves as well as periods of rhythmic activity with a frequency of 13 to 16 Hz were observed during sleep. In some animals periods of low-voltage ECoG activity were recorded during sleep. Eighteen-day-old rats: Background rhythm with a dominant frequency of 5 to 6 Hz was present in occipital regions. A decrease in frequency and increase in amplitude accompanied the transition from wakefulness to sleep. These animals exhibited marked motor activity predominantly of the orienting type. Their movements were smooth and well coordinated without jerks and trembling. Adult rats: The ECoG demonstrated a background dominant frequency of about 5 Hz which was recorded symmetrically from the occipital regions; this rhythm could be blocked with strong stimuli. The adult rats spent nearly the entire observation period in relaxed wakefulness. Behaviorally, a short period of orienting reaction took place and then the animals sat motionless in one comer of the box.
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Changes Induced by Pentamethylenetetrazol. Seven-day-old rats: Restlessness and an increase in locomotor activity formed the first behavioral signs of the action of PTZ. The ECoG activity became continuous and the amplitude of individual waves increased. Periods of flattening of the ECoG waves accompanied periods of relative rest. Isolated sharp waves (or sharp and slow wave complexes) of high amplitude were accompanied in 20% of the events by myoclonic jerks of the whole body (Fig. 3). With an additional dose of PTZ both motor and ECoG signs progressively intensified. In eight animals isolated jerks became repetitive and progressed into a short phase of wild running. This phase was followed by tonic seizures consisting of opisthotonus, tonic flexion, and extension of the forelimbs, and flexion of the hind limbs (Fig. 4). The tonic phase of the hind limbs was shorter than the tonic phase of the forelimbs and occasionally it failed to appear. The tonic phase was replaced by a clonic one consisting of the animals laying on
J
J
::
FIG. 3. Electrocorticographic (E&G) correlates of jerks induced by metrazol. From top to bottom: ECoG activity from a 7day-old, a 12day-old, and an 1l-day-old rat. Individual leads: RF-right frontal, RO-right occipital, LF-left frontal, and LO-let? occipital cortical areas. Amplitude callibration 0.5 mV, time mark 1 s. The letter ‘J” under the recording denotes wholebody jerk. In the recording from a 12day-old rat there are three similar sharp waves in the ECoC but only two of them are accompanied by motor phenomena.
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FIG. 4. Motor seizure in a ‘I-day-old rat-tonic
phase.
their side or back and exhibiting poorly coordinated movements of all four limbs. This phase was the longest one. After some minutes the movements slowed and, finally, only isolated jerks were present. Electroencephalographically generalized seizures were preceded by sharp delta waves (equaling 1 Hz) of high amplitude (to 2 mV) with superimposed low-amplitude fast activity. Ictal activity started in one or two cortical regions and slowly generalized. Motor seizures always appeared at the moment when ictal activity became generalized. Little synchronization of sharp delta waves among various cortical regions occurred. The superimposed activity progressively decreased whereas the frequency of high waves increased. From the ECoG point of view it was impossible to delineate sharply individual phases of generalized seizures (Fig. 5). During the clonic phase of motor seizures sharp delta waves became intermittent with short periods of low-amplitude activity or electrocerebral inactivity. During the postictal depression, isolated high-amplitude waves (1 to 2 mV) accompanied myoclonic jerks. Twelve-day-old rats: The first signs of action of PTZ were similar to those described in 7day-old animals; i.e., restlessness and increased orienting and locomotor activity accompanied by irregular hyperventilation. The ECoG demonstrated sharp delta and/or theta waves which, at first, were isolated and then occurred bifrontally sometimes associated with a following slow wave. During the preictal phase there was a progressive augmentation of delta and theta activity. In nearly half of the animals isolated sharp waves were accompanied by myoclonic jerks of the whole body (Fig. 3). On occasion rhythmic activity occurred (Fig. 6).
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FIG.5. The ECoG recordings of seizures in a 7&y-old rat with a description of motor behavior. The number between sections denotes the length of the omitted recording in seconds. Other details as in Fig. 3.
Motor seizures started again with wild running followed by the tonic and clonic phases. The only difference in comparison with 7day-old animals was the increased speed of all movements. ECoG ictal activity began with highamplitude delta waves (to 2 to 3 mV) often with superimposed fast activity. As a rule, this activity started in one cortical region and quickly became generalized. The duration of these single delta waves progressively shortened so that clear cut spikes or spike-and-wave complexes were recorded. There
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FIG. 6. Rhythmic ECoG activity in a 12day-old (top), in an lSday-old (middle), and in a 90-day-old rat (bottom). Details as in Fig. 3.
was no clear correlation between the motor tonic and clonic phase and ECoG patterns (Fig. 7). During the clonic phase high-amplitude sharp elements faded so that an unchanged motor pattern could be accompanied by dysrhythmic activity without specific epileptiform activity. Motor seizures were interrupted by periods of rest in some animals. When second paroxysm started, ECoG activity was always better organized and specific epileptic features were better defined than at the beginning of the first seizure. Generally, all ECoG activities were better expressed in frontal than in occipital leads. Eighteen-day-old rats: The first motor signs of the action of PTZ were the same as in younger animals. There were no ECoG correlates of the increased motor activity. Isolated myoclonic jerks correlated with isolated spikes and/ or spike-and-wave complexes in more than 60% of the cases (Fig. 3). A new phenomenon appeared in approximately one-half of the rats in this group: short periods of freezing (animals sat still with only small jerks of the head) accompanied by periods of rhythmic spike activity in the ECoG (3 to 5 Hz). This activity was formed by spikes or spike-and-wave rhythms with the amplitude exceeding that of the background activity (Fig. 6). Only generalized tonic-clonic seizures were recorded in our group due to a rapid increase in dosage of PTZ. Motor patterns of the major seizures exhibited all three phases: in contrast to younger animals, tonic hind limb
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IN YOUNG
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FIG. 7. The ECoG recordings of seizures in a 12day-old rat. Details as in Fig. 5.
extension was present and the clonic seizures were fast and well organized. The ECoG started with irregular high-amplitude spikes. During the tonic phase this spike activity became regular and was followed by slow waves forming spike-and-wave complexes (I; = 3 to 6 Hz) which continued into the clonic phase. There was not a distinct ECoG change in the transition from the tonic to clonic phase. During the clonic phase the ECoG activity often changed to a dysrhythmic mixture in a broad frequency spectrum not associated with changing motor patterns. The ECoG later returned to spike-
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and-wave rhythm (Fig. 8). A low-voltage recording with isolated sharp elements corresponding to jerks appeared toward the end of seizures. When seizures lasted for several minutes, serrated waves were also recorded. Adult rats: Restlessness was not as marked as in young animals. The first ECoG signs consisted of periods of theta waves (or spike-and-wave complexes of ~-HZ frequency) lasting 2 to 10 s and accompanied by freezing of the animal with minute jerks of the head musculature (e.g., rhythmic jerks of whiskers or ears). Rarely, isolated spikes appeared in the ECoG and were nearly always accompanied by body myoclonic jerks. With additional doses Ch
FIG.
18.6
8. The ECoG recordings of seizures in an 18-day-old rat. Details as in Fig. 5.
METRAZOL
IN
YOUNG
RATS
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of PTZ two types of motor seizures could be elicited. Minimal Metrazol seizures correlated with the ECoG spike activity which progressively changed into spike-and-wave rhythm. Major, generalized tonic-clonic seizures exhibited a similar ECoG pattern as in the 18-day-old rats with the exception of an absence of dysrhythmia during the clonic phase in adults. The beginning as well as the end of the ictal activity was abrupt during both minimal and major seizures. DISCUSSION Technique of Implantation. Secure implantation of electrodes in young rats is a difficult technical problem. Jouvet-Mounier (6) and Gramsbergen (4) only slightly modified the technique used in adult rats. Studies of kindling in immature rats (3, 5, 9, 10) led to an increased interest in this problem. Our method is based on that published by Gilbert and Cain (3) who used wire hooks cemented to the skull. We also used hooks and applied them to cortical electrodes. Two pairs of Gshape electrodes placed in opposition on two hemispheres are sufficient for fixation of the whole assembly. Even during wild running there were no artifacts in our recordings. This method can also be used for simultaneous implantation of subcortical electrodes (unpublished results). Ontogenetic Development of Epileptic Manifestations. The development of ECoG phenomena demonstrated the same principles as in our previous papers (8, 16); i.e., an increase in frequency of individual wave forms with maturation, progressive generalization of ictal activity, and episodes of rhythmic theta (and/or delta) waves as an age-specific phenomenon clearly visible for the first time in 18day-old rats. Correlation between EIectrocorticographic and Motor Phenomena. In general, the correlation between ECoG and motor phenomena induced by PTZ progressively increased with maturation. Myoclonic jerks which accompanied only approximately 20% of isolated sharp waves in 7day-old rats, exhibited nearly complete synchronization with isolated spikes and/or spike-and-wave complexes in adult animals. Only localized jerks restricted to limbs did not have ECoG counterparts. In 7- and 12-day-old rats only generalized tonic-clonic seizures could be induced by PTZ. Minimal Metrazol seizures represented an age-dependent phenomenon which could be elicited from the third week of postnatal life (2, 7). Due to a rapid increase in PTZ doses we did not observe this type of seizures in the present group of 18-day-old rats. In adult animals, both types of seizures were recorded and they did not differ in their ECoG pattern. ECoG ictal activity started in a restricted cortical region in 7day-old rats. The tendency to generalization markedly matured until the age of 18 days. At that age only generalized ECoG seizures were seen. There were no consistent
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correlations between ECoG patterns and phases of motor seizures in any age group. Even in adult animals the transition between the tonic and clonic phases was not punctually accompanied by a change in ECoG recording. Wave forms increased progressively forming a continuous transition from pure spike activity to spike-and-wave rhythm. We never saw motor seizures without accompanying ECoG changes in mature animals whereas this situation was often recorded in the younger rats. The absence of specific epileptic activity in the cerebral cortex during motor seizures in young rats indicates that the cerebral cortex is not necessary for the generation of generalized motor seizures. Structures governing this motor activity must, therefore, be subcortical. The correlation between ECoG and motor seizures in adult animals can be interpreted as a sign of well functioning cortical afferent pathways from the generator structure. REFERENCES 1. BURE& J., 0. BURESOVA, AND J. HUSTON. 1976. Techniques and Basic Experiments fir the Study ofBrain and Behavior. Elsevier, Amsterdam. 2. DE CASRILEVITZ, M., E. ENGELHARDT, AND C. A. ESB~RARD. 1971. Maturation of convulsogenic activity induced by leptazol in the albino rat. Br. J. Pharmacol. 42: 31-42. 3. GILBERT, M. E., AND D. P. CAIN. 1980. Electrode implantation in infant rats for kindling and chronic brain recording. Behav. Bruin Res 1: 553-555. 4. GRAMSBERGEN, A. 1976. EEG development in normal and undernourished rats. Brain Res. 105: 287-308. 5. HOLMES, G. L. 6. 7.
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1983. Effect of serial seizures on subsequent kindling in the immature brain. Dev. Brain Res. 6: 190-192. JOU~ET-MOUNTER, D. 1968. Ontogent%e des &tats de vigilance chez quelques mammtj&es, pp. 39-5 I. Tixier and Fils, Lyon. MARES, P., AND R. SCHICKEROVL. 1980. Seizures elicited by subcutaneous injection of metrazol during ontogenesis in rats. Activ. Nerv. Super. 22: 264-268. MARES, P., AND L. VEL~SEK. 1982. Electrocorticographic effectsof metrazol and ethosuximide during ontogenesis in rats. Physiol. Bohemoslov. 31: 455. MOSH& S. L. 1981. The effect of age on the kindling phenomenon. Dev. Psychobiol. 14: 75-81. MOSHB, S. L. 198 1. The kindling phenomenon and its possible relevance to febrile seizures. Pages 59-63 in K. H. NELSON AND J. H. ELLENBERG, Eds., Febrile Seizures. Raven Press, New York. O’DONOHOE, N. V. 1981. Epilepsies of Childhood, pp. 25-28. Butterworths, London. SCHICKEROV& R., P. MARES, AND S. TROJAN. 1982. Correlations entre le comportement et EEG pendant les crises epileptiques induites par metrazol au cows de l’ontogen& chez le rat. .I. Physiol. (Paris) 78: 46A. SWINYARD,E. A. 1973. Assay of antiepileptic drug activity in experimental animals. Stand& tests.Pages47-65 in J. MERCIER, Ed., Anticonvulsant Drugs, Int. Encycl. Pharma&Ther., Vol. 1, Sect. 19. Pergamon Press, Oxford. VERNADAKIS,A., AND D. M. WOODBURY. 1969. The developing animal as a model. Epilepsia lo: 163-178. VIDA, J. A. 1977. Anticonvulsants. Medicinal Chemistry: A Series ofMonographs, Vol. 15. Academic Press, New York. ZOUHAR, A., P. MARES, AND G. BROZEK. 1980. Electrocorticographic activity elicited by metrazol during ontogenesis in rats. Arch. Int. Pharmacodyn. Ther. 248: 280-288.