Pattern of electroencephalographic activity during light induced seizures in genetic epileptic chicken and brain chimeras

Neuroseience Letters, 145 (1992) 55-58

55

© 1992ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$05.00 NSL 08977

Pattern of electroencephalographic activity during light induced seizures in genetic epileptic chicken and brain chimeras N. G u y "'b, M . A . Teillet b, B. Schuler b, G. Le G a l la Salle c, N. Le D o u a r i n b, R. N a q u e t c a n d C. Batini a ~Laboratoire de Physiologic de la MotricitO, CNRS-URA 385 et UniversitO Pierre et Marie Curie, Paris (France), bInstitut d'Embryologie Cellulaire et Moldeulaire, CNRS-UMR 0009 et Colldge de France, Nogent-sur-Marne (France) and Clnstitut Alfred-Fessard, CNRS-UPR 2212, Gif-sur- Yvette (France)

(Received 5 March 1992;Revised version received26 June 1992;Accepted 1 July 1992) Key words:

Aviangenetic epilepsy;Brain chimeras; EEG

Genetic epilepsywas studied in Fayoumi epileptic(F.Epi) chickensand in neural chimeras obtained by selectivesubstitution of embryonicbrain vesicles of F.Epi donors in normal recipient chickens. Typical motor seizures accompanied by convulsions were evoked by intermittent light stimulation in F.Epi and in chimerashaving embryonicsubstitution of the prosencephalonand the mesencephalon.The motor seizurewas less severe in chimeras receiving only the prosencephalon. In the F.Epi, as well as in all the chimeras, the EEG during seizures was characterized by a desynchronized(or a flattening)pattern of activity. F.Epi and chimerashad a lower threshold to Metrazol inducedseizuresthan control chickens.The experimental animals show that, in this model, large prosencephalic and mesencephalic areas are involved in the epileptic disease. The epileptic character of this genetic dysfunctionis discussed.

Epilepsy is generally defined by the association of recurrent seizures and synchronous paroxysmal discharges of a group of neurons. It recognizes several clinical manifestations and a large etiological variety, including genetic. A valuable model for the study of the 'generalized' type of epilepsy is provided by the Fayoumi epileptic (F.Epi) strain of chickens [1] carrying a recessive autosomal gene mutation which shows typical generalized epileptic seizures consistently induced (although not only) by intermittent light stimulation (ILS) [1-3]. We report here the electroencephalographic (EEG) activity recorded during seizures in 15 F.Epi and as controls in 8 non-epileptic heterozygous (F.htz) hatchmates and 6 normal chickens of the ISA JA 57 strain (JA). The animals were of both sexes and 4-10 weeks old. The study was also extended to chimeras having a transfer of the genetic epileptic phenotype obtained [14] by substituting in normal JA embryos the primordia of both prosencephalon and mesencephalon of the stage matched F.Epi embryos. A grafted prosencephalon alone could not transfer full epileptic seizures but reproduced interictal E E G activity. EEGs were recorded in various chimeras at rest and during seizures and comCorrespondence: C. Batini, Laboratoire de Physiologiede la Motricit6, CNRS-URA 385 et Universit6Pierre et Marie Curie, Paris, France.

pared to EEGs of controls. Seven neural chimeras were obtained using the method described elsewhere [14] of which 3 had only the prosencephalon grafted and 3 both the prosencephalon and the mesencephalon. Among the latter different proportions of the mesencephalic vesicles were included in the graft. The types of brain substitutions performed and the age at which the animals were sacrificed are illustrated in Table I. Before the acute experiments all the animals were tested free moving for seizure susceptibility with ILS at 14 Hz, a frequency described as the best epileptogenic stimulus for F.Epi [3]. The F.Epi were selected by the ILS induced seizures which are characterized [2] by 3 phases: extension of the neck (I), loss of proper standing (II) and violent convulsions (III). The chimeras also displayed motor seizures but of a different severity depending on the individual graft performed. We have classified in Table I phases I and II as +; a short phase III (less than 5 s) was classified as ++ and a long phase III (more than 5 s) as +÷+. For the acute recording sessions all the animals were locally anesthetized at the points of surgery with lidoca~ne (2%) which was periodically repeated during the experiment. The head was placed in a stereotaxic frame and the animals were kept paralyzed with Flaxedil (galamine triethiodide) and artificially ventilated until

56

A

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TABLE l

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EMBRYONIC SUBSTITUTION OF BRAIN VESICLES FROM F.Epi DONORS AND SEVERITY OF MOTOR SEIZURES IN 6 CHIMERAS The left column shows the F.Epi graft performed in each animal (black areas); lines indicate the posterior border of the embryonic vesicles, prosencephalic (pro), mesencephalic (mes) and metencephalic (met). The names of the animals and the age at the recording sessions are listed in the middle columns. On the right column is represented the severity of motor seizures, tested before the experiment, for each chimera (see text for explanations); note that P874 had an inconsistent phase I (±) of the motor seizures, possibly because this chimera was only tested during the period after hatching when the F.Epi are described as partially resistant to ILS induced seizures [3].

Chimera, Non Paralyzed

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Fig. 1. A: resting EEG recording in a paralyzed F.htz. B,C: EEG and EMG recordings before (1), during (2) and after (3) ILS induced seizures, D: EEG recordings in a F.Epi paralyzed with Flaxedil before (1) and during (2) 1LS.

the end of the experiment. The body temperature was maintained around 39°C. Conventional bipolar EEG activity was recorded with transcranially introduced screw electrodes. EEGs were also recorded prior to the immobilization from 5 F.Epi, 4 F.htz, 1 JA and 1 chimera. In this case, to control artefacts strong movements were prevented by bandages. Needle electrodes were introduced in the neck muscles to record the electromyogram (EMG). During the experiments the animals were exposed to prolonged ILS (30-90 s) at intervals longer than 10 min to avoid stimulation during the refractory period between seizures. About 30 min after the last ILS application, 18 animals were tested for sensitivity to Metrazol (Pentylenetetrazol), a convulsant drug, injected intravenously. At the end of the experiments the animals were perfused intracardially with 10% formaline, then the whole brain was serially cut frozen and Nissl stained. The resting EEG of JA and F.htz (Fig. 1A), before and after immobilization, were both devoid of abnormal ep-

~C I I ~

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ileptiform waves. On the contrary, the EEG activity of the F.Epi and of all the chimeras was invariably characterized by typical high amplitude continuous slow waves, slow spikes and spike and wave complexes (Fig. I B-D). Immobilization with Flaxedil did not change the EEG patterns of activity (Fig. 1D) Whether the animals were paralyzed or not, in the 15 F.Epi recorded prolonged ILS invariabily resulted in the following EEG changes (Fig. 1B,D): several seconds after the stimulation was applied the spikes and spikes and waves were suppressed and replaced by a transient 'desynchronization' (D) of the rhythms, often followed by a 'flattening' (F) of the EEG. The two phenomena are related to the intensity of the seizure (D being less intense than F, and may be difficult to differentiate; for this reason the DF abbreviation will be used in the text). The DF may outlast the end of short ILS by a few seconds, but the typical interictal EEG spike and spike and wave activity may also return before the end of prolonged 1LS. During DF low voltage repeti-

57

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50~V L__ 5sec Fig. 2. E E G threshold of Metrazol induced seizures in a J A (A), a F.hzg (B), a F.Epi (C) a n d a chimera (D). E E G s p o n t a n e o u s epileptic seizure in a chimera (E). Metrazol was intravenously administered d u r i n g the bars over the traces at the indicated final dose.

tive potentials at the ILS frequency were rarely observed in the F.Epi (2 out of 13, before or after Flaxedil injection). In a few other F.Epi (6 of 15) a burst of spikes marked the end of the DF in some trials (not illustrated). ILS induced motor seizures were consistently recorded in the EMG of 5 F.Epi and 1 chimera (having pros- and mesencephalon grafted) non-paralyzed (Fig. 1B,C): rhythmical contractions of the neck muscles started at a low amplitude at the beginning of D, then progressively increased in amplitude invading all the skeletal muscles, with the transformation of D in DF, finally ceasing with the end of the DF. The frequency of the neck muscle contractions always followed that of the light stimulus. It did not persist during prolonged ILS and was abruptly interrupted at the end of short ILS. In our experimental conditions of partial constraint, extension of the neck, loss of equilibrium, uncoordinated movements of the legs and wings and collapsing on the floor as previously described [3] could not be observed, but the pecking and vocalizations were present. It is likely that low amplitude

rhythmical contractions of the neck muscles correspond to phase I and the generalization of the clonus corresponds to the typical convulsions of phase III, phase II being a transition phase. All the chimeras, whatever the graft, displayed DF, typical of the F.Epi seizures, during prolonged ILS. No clear evidence for shorter DF periods was found in the chimeras with grafted prosencephalon. We therefore conclude that the DF EEG pattern is specific to the prosencephalon grafted chimeras even in the absence of phase III. In addition, two animals, one for each type of substitution (P827 and EPll02), showed the common pattern of DF during ILS induced seizures and occasional transient EEG seizures consisting of continuous paroxysmal spikes during about 20 s. None of the 6 chimeras evidenced brain malformations in the post mortem histological control. Metrazol injected intravenously in paralyzed chickens and chimeras evoked characteristic EEG seizures in each group of recorded animals (Fig. 2A-D). However, the threshold (taken as the lowest dose inducing EEG paroxysmal seizures) was 5 mg/kg in F.Epi (n -- 4), 12.5 mg/kg in F.htz and 20 mg/kg in the JA (n -- 5). The susceptibility to epileptogenic drugs appears to be very high in the F.Epi and chimeras and higher in the F.htz than in the normal JA chickens. The results presented here raise an important question: is the DF observed in the F.Epi and chimeras (paralyzed or not) the expression of epileptic seizures? A close correlation between paroxysmal EEG symptoms and clinical symptoms would be expected. Since birds do not have a clearly defined cortex, a possible explanation is that the prosencephalon cannot express epileptic seizures as recorded in mammals. This hypothesis can be discarded since the EEG of the chicken can generate spikes and spikes and waves discharge spontaneously or under Metrazol injection as do mammals [8, 13, 15]. In addition, the low threshold for Metrazol induced seizures observed in our experiments is also described in other genetically epileptic animals [5, 8] as well as in some generalized epilepsies in man [6]. A desynchronization, or flattening, of the EEG rhythms is also described in rodent audiogenic seizures [12] whose epileptic character was questioned. Some observations (see refs. 4, 9 and 11), however, suggest that it could be generated from an epileptic brainstem therefore explaining the absence of EEG paroxysmal discharges. A similar interpretation cannot completely be applied to the F.Epi syndrome since the EEG shows interictal paroxysmal discharges. From the electrographic point of view, all types of chimeras show essentially the same ictal and interictal EEG activity. This is not the case for the motor manifestations, phase III being absent in the prosencephalon

58

grafted chimeras. In a previous work [14] it was postulated that cooperation of the pros- and mesencephalon is necessary to transfer the full pattern of seizures. This hypothesis implies that both parts of the grafted brain carry the elementary cellular dysfunction, the mechanisms of which need to be characterized. The present results confirm this hypothesis. In fact, ILS triggers the beginning of motor manifestations in the prosencephaIon grafted chimeras, implying a predisposition of the prosencephalon to seizures, and induces the full pattern of motor seizures in the pros- and mesencephalon grafted chimeras, implying that the 'seizure generator' is mostly located in the mesencephalon. To find and localize the 'epileptic generator' in this model needs further experimentation, but the present study already confirms (i) that the epileptic seizure is not necessarily accompanied by EEG paroxysms during seizures and (ii) that the genetic dysfunction of the F.Epi is carried by large brain areas including pros- and mesencephalon (part of this work has been previously reported, see ref. 7).

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9

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12

We wish to thank Dr. R.T. Kado for English corrections of the manuscript. R. Naquet and G. Le Gal La Salle were partially supported by the Fondation de France and the Fondation pour la Recherche Medicale.

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14 1 Crawford, R.D., Epileptiform seizures in domestic fowl, J. Hered., 61 (1970) 185-188. 2 Crawford, R.D., Genetics and behavior of the epi mutant chicken. In T. Ookawa (Ed.), The Brain and Behavior of the Fowl, Japan Scientific Society Press, Tokyo, 1983, pp. 259 269. 3 Crichlow, E.C. and Crawford, R.D., Epileptiform seizures in domestic fowl. II. Intermittent light stimulation and the electroencephalogram, Can. J. Physiol. Pharmacol., 52 (1974) 424429. 4 Faingold, C.L. and Anderson, C.A.B., Loss of intensity-induced

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