Brain Research, 523 (1990) 87-91 Elsevier
87
BRES 15706
Mapping of spontaneous spike and wave discharges in Wistar rats with genetic generalized non-convulsive epilepsy Marguerite Vergnes 1, Christian Marescaux 2 and Antoine Depaulis 1 t D.N.B.C., Centre de Neurochimie du C.N.R.S. Strasbourg (France) and 2Clinique Neurologique, C.H.U., Strasbourg (France) (Accepted 23 January 1990)
Key words: Generalized non-convulsive epilepsy; Electroencephalography; Spike and waves; Localization; Rat
Electrical activity was recorded in different parts of the brain in Wistar rats from a strain with genetic generalized non-convulsive epilepsy (GNCE or absence epilepsy). Movable bipolar electrodes were lowered stereotaxically by 1 mm steps into the brain in immobilized animals. Spontaneous spike and wave discharges (SWD) of the largest amplitude were recorded in the cortex and in lateral nuclei of the thalamus where they appeared occasionally to precede. Smaller amplitude SWD were recorded in the striatum, hypothalamus, tegmentum and substantia nigra. No SWD were recorded in limbic structures. Partial limbic seizures induced by the introduction of the electrode did not interfere with occurrence of cortical SWD. These results confirm the primacy of thalamocortical involvement in SWD of GNCE. The absence of spread to limbic structures and the implication of a precisely limited substrate in GNCE accounts for the clinical and pharmacological specificity of this particular kind of epilepsy. INTRODUCTION A strain of Wistar rats affected with spontaneous generalized non-convulsive epilepsy ( G N C E ) (absence epilepsy) was selected in our laboratory. All the animals have s p o n t a n e o u s seizures, recorded on the E E G as rhythmic (6-10 c/s) spike and wave discharges (SWD) which occur in awake, quiet states, at a m e a n frequency of l/min, and last from 1 to more than 100 s. They are recorded on cortical E E G , where they are bilateral and synchronous and p r e d o m i n a t e in the frontoparietal cortex 16"2°. In a previous study, we found that the lateral parts of the thalamus, namely the relay nuclei, participate actively in the SWD: in this part of the brain, SWD have a high amplitude and occur simultaneously with and sometimes before the S W D recorded in the cortex. Therefore a leading role has been proposed for the thalamus in the elaboration of SWD in the rat 21. No structural lesions of any kind which could account for the occurrence of absence epilepsy have ever been described. In contrast with partial seizures, generalized absence seizures are not generated in a localized focus, but are sustained by a b n o r m a l activity in an extensive network. To determine the brain structures which participate in the SWD of rats with G N C E , movable bipolar depth electrodes were lowered through the brain and the local E E G was recorded. Special attention was paid to limbic structures which have a low threshold to epilep-
togenic stimuli and are involved in partial non-convulsive seizures of temporal lobe epilepsy. A m a p p i n g of the SWD according to their amplitude and regularity was obtained. The present results were obtained in animals immobilized with curare, allowing a large n u m b e r of data to be collected in each animal. MATERIALS AND METHODS Fourteen adult male rats from our selected strain with GNCE were used. Several days before the experiment, the animals were prepared under pentobarbital (40 mg/kg i.p.) anesthesia. Two stainless steel screw electrodes were fixed unilaterally over the frontoparietai cortex and connected to a microconnector embedded in acrylic cement fixed to the skull. On the other side, small holes were drilled in the skull at various stereotaxic coordinates to allow penetration of depth electrodes. The skin was sutured and the animals were allowed to recover for at least 3 days. A control EEG showed the occurrence of SWD in the cortex. For depth recording at several levels, movable bipolar electrodes were used. They were made of twisted enameled stainless steel wires, with 1 mm vertical distance between the tips. Under ether anesthesia and local xylocalne, the rat was tracheotomized and a canula was introduced into its trachea. After injection of Dtubocurarine (2 mg/kg i.p.) the rat was connected to a respirator for rats and placed in a stereotaxic frame. Electrocardiogram and ECoG were continuously monitored. Animals' body temperature was maintained above 37 °C. Xylocaine was injected repeatedly into the wounds and pressure points in the ears. A small dose of a morphinomimetic, fentanyl (5/~g/kg), which was shown not to alter the EEG and occurrence of SWD was injected i.p. in order to protect animals from stress after recovery from anesthesia. Through the permanently implanted electrodes the cortical EEG was constantly recorded. After 1-2 h recovery the EEG was character-
Correspondence: M. Vergnes, D.N.B.C., Centre de Neurochimie du CNRS, 5, rue Blaise Pascal, 1:67084 Strasbourg Cedex, France. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
88 istic of wakefulness and SWD occurred spontaneously. When a sleep pattern appeared on the control EEG, gentle tactile stimulations were applied to keep the animal awake. Then a bipolar electrode was lowered stereotaxically into the brain. A first record was made at a depth of 2 mm from the surface of the skull. Then EEG was recorded after lowering the electrode by 1 mm steps. Each position was kept for at least 10 min or until several SWD had occurred on the control EEG. After reaching a depth of 8 or 9 mm the electrode was withdrawn and lowered into other sites. Several tracks could be used successively in most animals. EEG was recorded from 459 locations along 61 electrode tracks. After completion of the experiment (4-6 h) the animal was killed with an overdose of pentobarbital. The brain was removed for processing of histological slides. The electrode tracks were located on brain sections and recording locations were reconstructed. The SWD were scored as large or small according to their voltage compared to the baseline activity in the same structure: large SWD exceeded at least 5 times the mean amplitude of the baseline EEG.
RESULTS The cortical E E G recorded on the p e r m a n e n t l y implanted hemisphere was used as a control for the occurrence of SWD and the state of the animal. The cortical E E G was usually characteristic of a waking state. Sleep occurred and was allowed for short periods when no recording in depth electrodes was performed, as very few S W D occur in asleep animals. Heart rate was stable. The first location recorded with the movable bipolar
electrode was so that the lower tip of the electrode was at 2 m m from the surface of the skull, and was located in the cortex (Fig. 1). In most of the cortical areas SWD were large and regular. Their voltage varied from 500 to 2000/~V, representing a r o u n d 10-fold the m e a n voltage of baseline activity. Cortical SWD recorded through bipolar electrodes were more sustained and always exceeded the voltage of SWD recorded between two single contact surface electrodes. The SWD were reduced or even absent near the midline. In the lateral nuclei of the thalamus (ventrolateral, reticular, posterior nuclei) SWD were always present, with an amplitude of 250-450/~V exceeding 8-10 times the baseline activity. They occasionally preceded the occurrence of the cortical control SWD. The delay between appearance of the SWD in the thalamus and in the cortex varied from zero to several seconds and was different from one SWD to another in the same animal. In contrast, the cortical SWD never preceded the thalamic ones. No S W D or only very small ones were recorded from medial thalamic nuclei• In the striatum, the SWD appeared usually with a delay after the cortical SWD. Their amplitude was unstable, oscillating between 100 and 300 ~V, exceeding 2-5 times the baseline. Small SWD were recorded in the lateral hypothalamus and the area of the medial forebrain
......
..1 •
".-1
~1---~,~----,,,~
,-,-rF~lll.Wrl~l,~l~l~',,-].,l|-~ll,l.-,
Fig. 1. Stereo-EEG at dorsoventral coordinates 2-9 mm from the surface of the skull. The movable electrode was lowered by 1 mm steps• Each trace was successively recorded during a concomitant cortical SWD (not shown). The horizontal bars at 4 and 9 mm represent the simultaneous cortical SWD; calibration: 200/~V, 1 s. A schematic representation of a brain section (modified from ref. 17) shows the localization of the movable electrode with the sites where the EEG was recorded. Am, amygdala; CM, central medial thalamic nucleus; DH, dorsal hippocampus; DM, dorsomedial hypothalamic nucleus; LH, lateral hypothalamic area; MD, mediodorsal thalamic nucleus; Po, posterior thalamic nucleus; VM, ventromedial hypothalamic nucleus; VP, ventroposterior thalamic nucleus.
89
A
I)
C
Am
G
14
Fig. 2. Schematic mapping of recorded sites on coronal sections at 1 mm intervals of a rat brain (AP extending from 10.2 (A) to 3.2 (H) mm anterior to the interaural line according to Paxinos~7). Filled symbols, large and sustained SWD; Striped symbols, small SWD; Open symbols, no SWD recorded. Thalamic nuclei: AV, anteroventral; CM, central medial; LD, laterodorsal; LG, lateral geniculate; LP, lateral posterior; MD, mediodorsal; MG, medial geniculate; Po, posterior; Re, reuniens; Rt, reticular; VL, ventrolateral; VM, ventromedial; VP, ventroposterior. Ac, accumbens nucleus; Am, amygdala; CC, corpus callosum; CG, central gray; CPu, caudate putamen; CxFP, frontoparietal cortex; Cxo, occipital cortex; DB, diagonal band; DM, dorsomedial hypothalamic nucleus; f, fornix; GP, globus pallidus; H, hypothalamus; Hb, habenula; Hi, hippocampus; ic, internal capsule; LH, lateral hypothalamus; M, mammillary body; ml, medial lemniscus; Pa, paraventricular hypothalamic nucleus; PO, preoptic area; R, red nucleus; S, septum; SC, superior colliculus; SN, substantia nigra; Tu, olfactive tubercle; VTA, ventral tegmental area; ZI, zona incerta.
bundle. In the mesencephalic t e g m e n t u m and especially in the substantia nigra, small and fluctuating S W D were present. The dorsal h i p p o c a m p u s was examined carefully be-
cause of its proximity with the cortex and the thalamus. W h e n both tips of the e l e c t r o d e were located within the hippocampus, no S W D was ever r e c o r d e d from this structure. No discharges were r e c o r d e d in the septum,
90 Cx
Hi
Cx
.+ ..JJ,,~LI
i L.I ,IIL~. ,la+,..,
. ~.Ll,lh,
,
,.,~lJ~,l~.Ju
Hi
Cx
I Fig. 3. Simultaneous recording of the cortical (Cx) reference EEG, and of the hippocampus (Hi). When the bipolar electrode is lowered into the hippocampus an artefact is seen (upper Hi trace) followed by a hippocampal seizure. Concomitant spontaneous SWD occur independently in the cortex. Ten seconds separate each pair of traces. Calibration, 2 s, 500 ~V.
amygdala and piriform cortex (Fig. 2). In structures where no SWD were recorded, no modification of baseline E E G was noted in relationship with occurrence of cortical SWD. Penetration of the electrode into the cortex frequently elicited a flattening of the cortical activity most probably as a consequence of a spreading depression. No SWD occurred in any lead as long as the cortex had not fully recovered its normal activity. Penetration into the hippocampus or the amygdala elicited local high voltage seizures which were independent of cortical SWD and were followed by reduced baseline activity in these structures. However, limbic local seizures did not prevent SWD occurring, and the two types of seizures were sometimes recorded simultaneously, i.e. SWD on the cortex and a partial limbic seizure in the hippocampus or the amygdala (Fig. 3). DISCUSSION The present data show the primary involvement of the thalamocortical circuits in rats' GNCE. The predominance of the SWD in the neocortex and lateral thalamus recorded with movable bipolar electrodes in the present experiment confirms previous recordings made with chronically implanted rats from our strain with genetic GNCE 2~. As previously observed, thalamic discharges
occasionally preceded the cortical ones. The involvement of the cortex and thalamus in SWD was also observed in other strains of rats with spontaneous discharges 2'7,8']3']9, although the interpretation of the significance of the SWD differed among the authors (for review see ref. 22). Our data are also in agreement with the thalamo-cortical distribution of penicillin-induced SWD in cats 3"4. In the feline penicillin model of petit mal, the spike and waves were shown to result from an abnormal oscillatory pattern involving a thalamocortical loop. Although the cortex appears to play a leading role in this model, neither the cortex nor the thalamus can sustain the spike-and-wave pattern on its own H,]5. In men, E E G recorded simultaneously in cortex and thalamus during absence seizures showed that synchronous SWD occur in both structures and sometimes are initiated in the thalamus 23. The primary involvement of a corticothalamic system in animal as well as in human absences is now commonly admitted 1"6. The involvement of the striatum in the SWD is likely to result from its strong connections with the cortex and the thalamus. The delayed appearance and the small and fluctuating amplitude of the striatal SWD are not in favor of a leading role for this structure. Similarly, SWD spread to the tegmental structures, which in turn are involved in the control of SWD. Indeed the substantia nigra appears to participate in the control of SWD: lesions of the dopaminergic neurons in the substantia nigra increase occurrence of SWD (unpublished data), and microinjections of GABA agonists into the substantia nigra suppress SWD 9. Similarly, the mesencephalic reticular formation participates in the control of SWD. Electrical stimulations of this structure suppress SWD in rats 12. Administration of the anticonvulsant drugs, carbamazepine or phenytoin, which depress excitatory pathways from the reticular formation ~° aggravate GNCE 16, whereas drugs effective against absence seizures depress inhibitory pathways from the reticular formation ~°. Finally, SWD occur mainly during quiet wakefulness and are suppressed by arousing stimulations 14. A major point is that the SWD of the generalized non-convulsive seizures do not spread to the limbic structures in spite of their high excitability. Conversely, limbic seizures do not readily spread to the cortex. In models of limbic epilepsy (kindling or kainic acid-induced seizures) the paroxystic discharges reach the cortex only after repeated stimulations or a prolonged status 5"18. In our experiments occurrence of a local seizure in the hippocampus or the amygdala does not interfere with SWD of GNCE recorded in the cortex, demonstrating that the two kinds of seizures function independently with distinct substrates. The separation of the anatomic substrates of generalized non-convulsive (absence) sei-
91 zures and partial limbic seizures is in a g r e e m e n t with their clinical and p h a r m a c o l o g i c a l specificities 1. In spite of their classification as generalized seizures, bilateral and synchronous S W D in absence seizures are restricted to the cortex and lateral nuclei of the thalamus and directly connected structures with the exclusion of the limbic system. This precisely limited substrate for the
S W D in absence seizures contributes to the specificity of this particular type of epilepsy.
Acknowledgements. Special thanks are given to Any Boehrer for histological processing of brains and help in selection of rat strains and Gaby Rudolf for preparation of the figures. This work was supported by a grant, Contrat de Recherche Externe 866017, from I.N.S.E.R.M. and by la Fondation pour la Recherche M6dicale.
REFERENCES 1 Aird, R.B., Masland, R.L. and Woodbury, D.M., Hypothesis: the classification of epileptic seizures according to systems of the CNS, Epilepsy Res., 3 (1989) 77-81. 2 Albe-Fessard, D. and Lombard, M.C., Use of an animal model to evaluate the origin of and protection against deafferentation pain. In J.J. Bonica (Ed.), Advances in Pain Research and Therapy, Raven, New York, 1983, pp. 691-700. 3 Avoli, M. and Gloor, P., Interaction of cortex and thalamus in spike and wave discharges of feline generalized penicillin epilepsy, Exp. Neurol., 76 (1982) 196-217. 4 Avoli, M., Gloor, P., Kostopoulous, G. and Gotman, J., An analysis of penicillin induced generalized spike and wave discharges using simultaneous recordings of cortical and thalamic single neurons, J. Neurophysiol., 50 (1983) 819-837. 5 Ben-Aft, Y., Tremblay, E., Riche, D., Ghilini, G. and Naquet, R., Electrographic, clinical and pathological alterations following systemic administration of kainic acid, bicuculline or pentetrazole: metabolic mapping using the deoxyglucose method with special reference to the pathology of epilepsy, Neuroscience, 6 (1981) 1361-1391. 6 Berkovic, S.E, Andermann, E, Andermann, E. and Gloor, P., Concepts of absence epilepsies: discrete syndromes or biological continuum?, Neurology, 37 (1987) 993-1000. 7 Buzsaki, G., Bickford, R.G., Ponomareff, G., Thai, L.J., Mandel, R.J. and Gage, EH., Nucleus basalis and thalamic control of neocortical activity in the freely moving rat, J. Neurosci., 8 (1988) 4007-4026. 8 Chocholova, L., Incidence and development of rhythmic episodic activity in the electroencephalogram of a large rat population under chronic conditions, Physiol. Bohemoslov., 32 (1983) 10-18. 9 Depaulis, A., Vergues, M., Marescaux, C., Lannes, B. and Warter, J.M., Evidence that activation of GABA receptors in the substantia nigra suppresses spontaneous spike-and-wave discharges in the rat, Brain Research, 448 (1988) 20-29. 10 Fromm, G.H. and Terrence, C.F., Effect of antiepileptic drugs on the brain stem. In G.H. Fromm, C.L. Faingold, R.A. Browning and W.M. Burnham (Eds.), Epilepsy and the Reticular Formation Neurology and Neurobiology, Vol. 27, Liss, New York, 1987, pp. 119-136. 11 Gloor, P. and Fariello, R.G., Generalized epilepsy: some of its cellular mechanisms differ from those of focal epilepsy, Trends Neurosci., 11 (1988) 63-68. 12 Golden, G.T. and Fariello, R.G., The epileptogenic action of
13 14
15
16
17 18 19 20
21
22
23
some direct GABA agonists: effects of manipulation of the GABA and glutamate systems. In R.G. Fariello, P.L. Morselli, K.G. Lloyd, L.F. Quesney and J. Engel Jr. (Eds.), Neurotransmitters, Seizures and Epilepsy II, Raven, New York, 1984, pp. 237-244. Klingberg, E and Pickenhain, L., Das Auftreten yon 'Spindelentladungen' bei der Ratte in Beziehung zum Verhalten, Acta Biol. Med. Germ., 20 (1968) 45-54. Lannes, B., Micheletti, G., Vergnes, M., Marescaux, Ch., Depaulis, A. and Warter, J.M., Relationship between spikewave discharges and vigilance levels in rats with spontaneous petit mal-like epilepsy, Neurosci. Lett., 94 (1988) 187-191. McLachlan, R.S., Avoli, M. and Gloor, P., Transition from spindles to generalized spike and wave discharges in the cat. Simultaneous single-cell recordings in cortex and thalamus, Exp. Neurol., 85 (1984) 413-425. Marescaux, C., Micheletti, G., Vergnes, M., Depaulis, A., Rumbach, L. and Warter, J.M., A model of chronic spontaneous petit mal-like seizures in the rat: comparison with pentylenetetrazol-induced seizures, Epilepsia, 25 (1984) 326-331. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, Sydney, 1982. Racine, R.J., Burnham, W.M., Gilbert, M. and Kairiss, E.W., Kindling mechanisms: I. Electrophysiological studies. In J.A. Wada (Ed.), Kindling, Vol. 3, Raven, New York, 1986. Semba, K. and Komisaruk, B.R., Neural substrates of two different rhythmical vibrissal movements in the rat, Neuroscience, 12 (1984) 761-774. Vergnes, M., Marescaux, Ch., Micheletti, G., Reis, J., Depaulis, A., Rumbach, L. and Warter, J.M., Spontaneous paroxysmal electroclinical patterns in rat: a model of generalized non-convulsive epilepsy, Neurosci. Lett., 33 (1982) 97-101. Vergnes, M., Marescaux, Ch., Depaulis, A., Micheletti, G. and Warter, J.M., Spontaneous spike and wave discharges in thalamus and cortex in a rat model of genetic petit mal-like seizures, Exp. Neurol., 96 (1987) 127-136. Vergnes, M., Marescaux, Ch., Depaulis, A., Micheletti, G. and Warter, J.M., The spontaneous spike and wave discharges in Wistar rats: a model of genetic generalized non convulsive epilepsy. In M. Avoli, P. Gloor, G. Kostopoulos and R. Naquet (Eds.), Generalized Epilepsy: Cellular, Molecular and Pharmacological Approaches, Birkh~iuser Boston Inc., 1990, pp. 238253. Williams, D., A study of thalamic and cortical rhythms in petit mal, Brain, 76 (1953) 50-69.
87
BRES 15706
Mapping of spontaneous spike and wave discharges in Wistar rats with genetic generalized non-convulsive epilepsy Marguerite Vergnes 1, Christian Marescaux 2 and Antoine Depaulis 1 t D.N.B.C., Centre de Neurochimie du C.N.R.S. Strasbourg (France) and 2Clinique Neurologique, C.H.U., Strasbourg (France) (Accepted 23 January 1990)
Key words: Generalized non-convulsive epilepsy; Electroencephalography; Spike and waves; Localization; Rat
Electrical activity was recorded in different parts of the brain in Wistar rats from a strain with genetic generalized non-convulsive epilepsy (GNCE or absence epilepsy). Movable bipolar electrodes were lowered stereotaxically by 1 mm steps into the brain in immobilized animals. Spontaneous spike and wave discharges (SWD) of the largest amplitude were recorded in the cortex and in lateral nuclei of the thalamus where they appeared occasionally to precede. Smaller amplitude SWD were recorded in the striatum, hypothalamus, tegmentum and substantia nigra. No SWD were recorded in limbic structures. Partial limbic seizures induced by the introduction of the electrode did not interfere with occurrence of cortical SWD. These results confirm the primacy of thalamocortical involvement in SWD of GNCE. The absence of spread to limbic structures and the implication of a precisely limited substrate in GNCE accounts for the clinical and pharmacological specificity of this particular kind of epilepsy. INTRODUCTION A strain of Wistar rats affected with spontaneous generalized non-convulsive epilepsy ( G N C E ) (absence epilepsy) was selected in our laboratory. All the animals have s p o n t a n e o u s seizures, recorded on the E E G as rhythmic (6-10 c/s) spike and wave discharges (SWD) which occur in awake, quiet states, at a m e a n frequency of l/min, and last from 1 to more than 100 s. They are recorded on cortical E E G , where they are bilateral and synchronous and p r e d o m i n a t e in the frontoparietal cortex 16"2°. In a previous study, we found that the lateral parts of the thalamus, namely the relay nuclei, participate actively in the SWD: in this part of the brain, SWD have a high amplitude and occur simultaneously with and sometimes before the S W D recorded in the cortex. Therefore a leading role has been proposed for the thalamus in the elaboration of SWD in the rat 21. No structural lesions of any kind which could account for the occurrence of absence epilepsy have ever been described. In contrast with partial seizures, generalized absence seizures are not generated in a localized focus, but are sustained by a b n o r m a l activity in an extensive network. To determine the brain structures which participate in the SWD of rats with G N C E , movable bipolar depth electrodes were lowered through the brain and the local E E G was recorded. Special attention was paid to limbic structures which have a low threshold to epilep-
togenic stimuli and are involved in partial non-convulsive seizures of temporal lobe epilepsy. A m a p p i n g of the SWD according to their amplitude and regularity was obtained. The present results were obtained in animals immobilized with curare, allowing a large n u m b e r of data to be collected in each animal. MATERIALS AND METHODS Fourteen adult male rats from our selected strain with GNCE were used. Several days before the experiment, the animals were prepared under pentobarbital (40 mg/kg i.p.) anesthesia. Two stainless steel screw electrodes were fixed unilaterally over the frontoparietai cortex and connected to a microconnector embedded in acrylic cement fixed to the skull. On the other side, small holes were drilled in the skull at various stereotaxic coordinates to allow penetration of depth electrodes. The skin was sutured and the animals were allowed to recover for at least 3 days. A control EEG showed the occurrence of SWD in the cortex. For depth recording at several levels, movable bipolar electrodes were used. They were made of twisted enameled stainless steel wires, with 1 mm vertical distance between the tips. Under ether anesthesia and local xylocalne, the rat was tracheotomized and a canula was introduced into its trachea. After injection of Dtubocurarine (2 mg/kg i.p.) the rat was connected to a respirator for rats and placed in a stereotaxic frame. Electrocardiogram and ECoG were continuously monitored. Animals' body temperature was maintained above 37 °C. Xylocaine was injected repeatedly into the wounds and pressure points in the ears. A small dose of a morphinomimetic, fentanyl (5/~g/kg), which was shown not to alter the EEG and occurrence of SWD was injected i.p. in order to protect animals from stress after recovery from anesthesia. Through the permanently implanted electrodes the cortical EEG was constantly recorded. After 1-2 h recovery the EEG was character-
Correspondence: M. Vergnes, D.N.B.C., Centre de Neurochimie du CNRS, 5, rue Blaise Pascal, 1:67084 Strasbourg Cedex, France. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
88 istic of wakefulness and SWD occurred spontaneously. When a sleep pattern appeared on the control EEG, gentle tactile stimulations were applied to keep the animal awake. Then a bipolar electrode was lowered stereotaxically into the brain. A first record was made at a depth of 2 mm from the surface of the skull. Then EEG was recorded after lowering the electrode by 1 mm steps. Each position was kept for at least 10 min or until several SWD had occurred on the control EEG. After reaching a depth of 8 or 9 mm the electrode was withdrawn and lowered into other sites. Several tracks could be used successively in most animals. EEG was recorded from 459 locations along 61 electrode tracks. After completion of the experiment (4-6 h) the animal was killed with an overdose of pentobarbital. The brain was removed for processing of histological slides. The electrode tracks were located on brain sections and recording locations were reconstructed. The SWD were scored as large or small according to their voltage compared to the baseline activity in the same structure: large SWD exceeded at least 5 times the mean amplitude of the baseline EEG.
RESULTS The cortical E E G recorded on the p e r m a n e n t l y implanted hemisphere was used as a control for the occurrence of SWD and the state of the animal. The cortical E E G was usually characteristic of a waking state. Sleep occurred and was allowed for short periods when no recording in depth electrodes was performed, as very few S W D occur in asleep animals. Heart rate was stable. The first location recorded with the movable bipolar
electrode was so that the lower tip of the electrode was at 2 m m from the surface of the skull, and was located in the cortex (Fig. 1). In most of the cortical areas SWD were large and regular. Their voltage varied from 500 to 2000/~V, representing a r o u n d 10-fold the m e a n voltage of baseline activity. Cortical SWD recorded through bipolar electrodes were more sustained and always exceeded the voltage of SWD recorded between two single contact surface electrodes. The SWD were reduced or even absent near the midline. In the lateral nuclei of the thalamus (ventrolateral, reticular, posterior nuclei) SWD were always present, with an amplitude of 250-450/~V exceeding 8-10 times the baseline activity. They occasionally preceded the occurrence of the cortical control SWD. The delay between appearance of the SWD in the thalamus and in the cortex varied from zero to several seconds and was different from one SWD to another in the same animal. In contrast, the cortical SWD never preceded the thalamic ones. No S W D or only very small ones were recorded from medial thalamic nuclei• In the striatum, the SWD appeared usually with a delay after the cortical SWD. Their amplitude was unstable, oscillating between 100 and 300 ~V, exceeding 2-5 times the baseline. Small SWD were recorded in the lateral hypothalamus and the area of the medial forebrain
......
..1 •
".-1
~1---~,~----,,,~
,-,-rF~lll.Wrl~l,~l~l~',,-].,l|-~ll,l.-,
Fig. 1. Stereo-EEG at dorsoventral coordinates 2-9 mm from the surface of the skull. The movable electrode was lowered by 1 mm steps• Each trace was successively recorded during a concomitant cortical SWD (not shown). The horizontal bars at 4 and 9 mm represent the simultaneous cortical SWD; calibration: 200/~V, 1 s. A schematic representation of a brain section (modified from ref. 17) shows the localization of the movable electrode with the sites where the EEG was recorded. Am, amygdala; CM, central medial thalamic nucleus; DH, dorsal hippocampus; DM, dorsomedial hypothalamic nucleus; LH, lateral hypothalamic area; MD, mediodorsal thalamic nucleus; Po, posterior thalamic nucleus; VM, ventromedial hypothalamic nucleus; VP, ventroposterior thalamic nucleus.
89
A
I)
C
Am
G
14
Fig. 2. Schematic mapping of recorded sites on coronal sections at 1 mm intervals of a rat brain (AP extending from 10.2 (A) to 3.2 (H) mm anterior to the interaural line according to Paxinos~7). Filled symbols, large and sustained SWD; Striped symbols, small SWD; Open symbols, no SWD recorded. Thalamic nuclei: AV, anteroventral; CM, central medial; LD, laterodorsal; LG, lateral geniculate; LP, lateral posterior; MD, mediodorsal; MG, medial geniculate; Po, posterior; Re, reuniens; Rt, reticular; VL, ventrolateral; VM, ventromedial; VP, ventroposterior. Ac, accumbens nucleus; Am, amygdala; CC, corpus callosum; CG, central gray; CPu, caudate putamen; CxFP, frontoparietal cortex; Cxo, occipital cortex; DB, diagonal band; DM, dorsomedial hypothalamic nucleus; f, fornix; GP, globus pallidus; H, hypothalamus; Hb, habenula; Hi, hippocampus; ic, internal capsule; LH, lateral hypothalamus; M, mammillary body; ml, medial lemniscus; Pa, paraventricular hypothalamic nucleus; PO, preoptic area; R, red nucleus; S, septum; SC, superior colliculus; SN, substantia nigra; Tu, olfactive tubercle; VTA, ventral tegmental area; ZI, zona incerta.
bundle. In the mesencephalic t e g m e n t u m and especially in the substantia nigra, small and fluctuating S W D were present. The dorsal h i p p o c a m p u s was examined carefully be-
cause of its proximity with the cortex and the thalamus. W h e n both tips of the e l e c t r o d e were located within the hippocampus, no S W D was ever r e c o r d e d from this structure. No discharges were r e c o r d e d in the septum,
90 Cx
Hi
Cx
.+ ..JJ,,~LI
i L.I ,IIL~. ,la+,..,
. ~.Ll,lh,
,
,.,~lJ~,l~.Ju
Hi
Cx
I Fig. 3. Simultaneous recording of the cortical (Cx) reference EEG, and of the hippocampus (Hi). When the bipolar electrode is lowered into the hippocampus an artefact is seen (upper Hi trace) followed by a hippocampal seizure. Concomitant spontaneous SWD occur independently in the cortex. Ten seconds separate each pair of traces. Calibration, 2 s, 500 ~V.
amygdala and piriform cortex (Fig. 2). In structures where no SWD were recorded, no modification of baseline E E G was noted in relationship with occurrence of cortical SWD. Penetration of the electrode into the cortex frequently elicited a flattening of the cortical activity most probably as a consequence of a spreading depression. No SWD occurred in any lead as long as the cortex had not fully recovered its normal activity. Penetration into the hippocampus or the amygdala elicited local high voltage seizures which were independent of cortical SWD and were followed by reduced baseline activity in these structures. However, limbic local seizures did not prevent SWD occurring, and the two types of seizures were sometimes recorded simultaneously, i.e. SWD on the cortex and a partial limbic seizure in the hippocampus or the amygdala (Fig. 3). DISCUSSION The present data show the primary involvement of the thalamocortical circuits in rats' GNCE. The predominance of the SWD in the neocortex and lateral thalamus recorded with movable bipolar electrodes in the present experiment confirms previous recordings made with chronically implanted rats from our strain with genetic GNCE 2~. As previously observed, thalamic discharges
occasionally preceded the cortical ones. The involvement of the cortex and thalamus in SWD was also observed in other strains of rats with spontaneous discharges 2'7,8']3']9, although the interpretation of the significance of the SWD differed among the authors (for review see ref. 22). Our data are also in agreement with the thalamo-cortical distribution of penicillin-induced SWD in cats 3"4. In the feline penicillin model of petit mal, the spike and waves were shown to result from an abnormal oscillatory pattern involving a thalamocortical loop. Although the cortex appears to play a leading role in this model, neither the cortex nor the thalamus can sustain the spike-and-wave pattern on its own H,]5. In men, E E G recorded simultaneously in cortex and thalamus during absence seizures showed that synchronous SWD occur in both structures and sometimes are initiated in the thalamus 23. The primary involvement of a corticothalamic system in animal as well as in human absences is now commonly admitted 1"6. The involvement of the striatum in the SWD is likely to result from its strong connections with the cortex and the thalamus. The delayed appearance and the small and fluctuating amplitude of the striatal SWD are not in favor of a leading role for this structure. Similarly, SWD spread to the tegmental structures, which in turn are involved in the control of SWD. Indeed the substantia nigra appears to participate in the control of SWD: lesions of the dopaminergic neurons in the substantia nigra increase occurrence of SWD (unpublished data), and microinjections of GABA agonists into the substantia nigra suppress SWD 9. Similarly, the mesencephalic reticular formation participates in the control of SWD. Electrical stimulations of this structure suppress SWD in rats 12. Administration of the anticonvulsant drugs, carbamazepine or phenytoin, which depress excitatory pathways from the reticular formation ~° aggravate GNCE 16, whereas drugs effective against absence seizures depress inhibitory pathways from the reticular formation ~°. Finally, SWD occur mainly during quiet wakefulness and are suppressed by arousing stimulations 14. A major point is that the SWD of the generalized non-convulsive seizures do not spread to the limbic structures in spite of their high excitability. Conversely, limbic seizures do not readily spread to the cortex. In models of limbic epilepsy (kindling or kainic acid-induced seizures) the paroxystic discharges reach the cortex only after repeated stimulations or a prolonged status 5"18. In our experiments occurrence of a local seizure in the hippocampus or the amygdala does not interfere with SWD of GNCE recorded in the cortex, demonstrating that the two kinds of seizures function independently with distinct substrates. The separation of the anatomic substrates of generalized non-convulsive (absence) sei-
91 zures and partial limbic seizures is in a g r e e m e n t with their clinical and p h a r m a c o l o g i c a l specificities 1. In spite of their classification as generalized seizures, bilateral and synchronous S W D in absence seizures are restricted to the cortex and lateral nuclei of the thalamus and directly connected structures with the exclusion of the limbic system. This precisely limited substrate for the
S W D in absence seizures contributes to the specificity of this particular type of epilepsy.
Acknowledgements. Special thanks are given to Any Boehrer for histological processing of brains and help in selection of rat strains and Gaby Rudolf for preparation of the figures. This work was supported by a grant, Contrat de Recherche Externe 866017, from I.N.S.E.R.M. and by la Fondation pour la Recherche M6dicale.
REFERENCES 1 Aird, R.B., Masland, R.L. and Woodbury, D.M., Hypothesis: the classification of epileptic seizures according to systems of the CNS, Epilepsy Res., 3 (1989) 77-81. 2 Albe-Fessard, D. and Lombard, M.C., Use of an animal model to evaluate the origin of and protection against deafferentation pain. In J.J. Bonica (Ed.), Advances in Pain Research and Therapy, Raven, New York, 1983, pp. 691-700. 3 Avoli, M. and Gloor, P., Interaction of cortex and thalamus in spike and wave discharges of feline generalized penicillin epilepsy, Exp. Neurol., 76 (1982) 196-217. 4 Avoli, M., Gloor, P., Kostopoulous, G. and Gotman, J., An analysis of penicillin induced generalized spike and wave discharges using simultaneous recordings of cortical and thalamic single neurons, J. Neurophysiol., 50 (1983) 819-837. 5 Ben-Aft, Y., Tremblay, E., Riche, D., Ghilini, G. and Naquet, R., Electrographic, clinical and pathological alterations following systemic administration of kainic acid, bicuculline or pentetrazole: metabolic mapping using the deoxyglucose method with special reference to the pathology of epilepsy, Neuroscience, 6 (1981) 1361-1391. 6 Berkovic, S.E, Andermann, E, Andermann, E. and Gloor, P., Concepts of absence epilepsies: discrete syndromes or biological continuum?, Neurology, 37 (1987) 993-1000. 7 Buzsaki, G., Bickford, R.G., Ponomareff, G., Thai, L.J., Mandel, R.J. and Gage, EH., Nucleus basalis and thalamic control of neocortical activity in the freely moving rat, J. Neurosci., 8 (1988) 4007-4026. 8 Chocholova, L., Incidence and development of rhythmic episodic activity in the electroencephalogram of a large rat population under chronic conditions, Physiol. Bohemoslov., 32 (1983) 10-18. 9 Depaulis, A., Vergues, M., Marescaux, C., Lannes, B. and Warter, J.M., Evidence that activation of GABA receptors in the substantia nigra suppresses spontaneous spike-and-wave discharges in the rat, Brain Research, 448 (1988) 20-29. 10 Fromm, G.H. and Terrence, C.F., Effect of antiepileptic drugs on the brain stem. In G.H. Fromm, C.L. Faingold, R.A. Browning and W.M. Burnham (Eds.), Epilepsy and the Reticular Formation Neurology and Neurobiology, Vol. 27, Liss, New York, 1987, pp. 119-136. 11 Gloor, P. and Fariello, R.G., Generalized epilepsy: some of its cellular mechanisms differ from those of focal epilepsy, Trends Neurosci., 11 (1988) 63-68. 12 Golden, G.T. and Fariello, R.G., The epileptogenic action of
13 14
15
16
17 18 19 20
21
22
23
some direct GABA agonists: effects of manipulation of the GABA and glutamate systems. In R.G. Fariello, P.L. Morselli, K.G. Lloyd, L.F. Quesney and J. Engel Jr. (Eds.), Neurotransmitters, Seizures and Epilepsy II, Raven, New York, 1984, pp. 237-244. Klingberg, E and Pickenhain, L., Das Auftreten yon 'Spindelentladungen' bei der Ratte in Beziehung zum Verhalten, Acta Biol. Med. Germ., 20 (1968) 45-54. Lannes, B., Micheletti, G., Vergnes, M., Marescaux, Ch., Depaulis, A. and Warter, J.M., Relationship between spikewave discharges and vigilance levels in rats with spontaneous petit mal-like epilepsy, Neurosci. Lett., 94 (1988) 187-191. McLachlan, R.S., Avoli, M. and Gloor, P., Transition from spindles to generalized spike and wave discharges in the cat. Simultaneous single-cell recordings in cortex and thalamus, Exp. Neurol., 85 (1984) 413-425. Marescaux, C., Micheletti, G., Vergnes, M., Depaulis, A., Rumbach, L. and Warter, J.M., A model of chronic spontaneous petit mal-like seizures in the rat: comparison with pentylenetetrazol-induced seizures, Epilepsia, 25 (1984) 326-331. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, Sydney, 1982. Racine, R.J., Burnham, W.M., Gilbert, M. and Kairiss, E.W., Kindling mechanisms: I. Electrophysiological studies. In J.A. Wada (Ed.), Kindling, Vol. 3, Raven, New York, 1986. Semba, K. and Komisaruk, B.R., Neural substrates of two different rhythmical vibrissal movements in the rat, Neuroscience, 12 (1984) 761-774. Vergnes, M., Marescaux, Ch., Micheletti, G., Reis, J., Depaulis, A., Rumbach, L. and Warter, J.M., Spontaneous paroxysmal electroclinical patterns in rat: a model of generalized non-convulsive epilepsy, Neurosci. Lett., 33 (1982) 97-101. Vergnes, M., Marescaux, Ch., Depaulis, A., Micheletti, G. and Warter, J.M., Spontaneous spike and wave discharges in thalamus and cortex in a rat model of genetic petit mal-like seizures, Exp. Neurol., 96 (1987) 127-136. Vergnes, M., Marescaux, Ch., Depaulis, A., Micheletti, G. and Warter, J.M., The spontaneous spike and wave discharges in Wistar rats: a model of genetic generalized non convulsive epilepsy. In M. Avoli, P. Gloor, G. Kostopoulos and R. Naquet (Eds.), Generalized Epilepsy: Cellular, Molecular and Pharmacological Approaches, Birkh~iuser Boston Inc., 1990, pp. 238253. Williams, D., A study of thalamic and cortical rhythms in petit mal, Brain, 76 (1953) 50-69.
