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Kainic acid seizure syndrome and binding sites in developing rats YEHEZKEL BEN-ARI, EVELYNE TREMBLAY, MICHAEL BERGER and LILIANA NITECKA* Laboratoire de Physiologie Nerveuse, CNRS, 91190 Gif-sur-Yvette (France)

(Accepted February 22nd, 1984) Key words: kainic acid - - maturation - - seizures - - brain damage - - limbic system - - binding sites - - autoradiography

In rats up to 16 days after birth, parenteral kainic acid (KA) produced tonico-clonic convulsions, metabolic activation limited to the hippocampus, and no brain damage. Starting with the 19th day after birth, KA produced limbic seizures associated with metabolic activation and subsequent damage in the hippocampus, the amygdala, and other limbic structures. Membranes prepared from hippocampi 10 days after birth bound [3H]KA with a high affinity component, which was localized in the mossy fiber region by slice autoradiography. In contrast, on amygdaloid membranes this component appeared only 17-19 days after birth. Our results further stress the crucial role of the amygdala in the KA seizure syndrome. Limbic structures, notably the hippocampus and the amygdaloid complex, occupy a central position in the etiology of human temporal lobe epilepsy 8. The hippocampus represents the most susceptible brain structure to epileptogenic procedures 9, whereas the amygdala plays a crucial role in several features of its clinical symptomatology 16, Systemic or intracerebral injections of the potent neuroexcitatory agent kainic acid 13 (KA) in rats reproduce this brain disorder with a striking broad scale of electrographic, clinical, metabolic, and histopathological correlates2,3,19. The molecular events underlying the various actions of the neurotoxin are poorly understood. A central question concerns the relevance of specific K A binding sites 20 to the etiology of the syndrome. We have therefore studied the ontogenesis of both the K A seizure-brain damage syndrome and the K A binding sites in the rat hippocampus and amygdala. Parts of this study have been published as abstracts 5,22. Male Wistar rats were used at 5 developmental stages from 3 up to 35 days after birth. In a pre-experimental series (n = 67), the effects of various doses of i.p. K A (from ineffective to lethal ones) were tested and the lowest amounts of K A to consistently induce convulsions were found to rise with age (also see Cherubini et al.7), probably due to a progressive

maturation of the b l o o d - b r a i n barrier. Thus, for the following series (see below), the doses which had been chosen for 3, 9-12, 14-16 and 19-35 day old rats were 1, 2, 4 and 9 mg/kg, respectively. Electrographic records before and after K A were obtained from two 10 day old rats with twisted wires which were implanted under stereotaxic guidance in the hippocampus. To evaluate metabolic alterations a pulse of [lac]2-deoxyglucose (2-DG, 100/~Ci/kg, 52 Ci/mol, C . E . A . Saclay) was administered i.p. to 3 day (n = 7), 9 - 1 0 day (n = 7) and 2 1 - 2 4 day (n = 4) old ratsll. 14 which had received KA, and to controls from the same litter (n = 3, 4 and 2, respec-tively). Animals were sacrificed 45 min later and their brains processed for autoradiography as described elsewhere 3. Developed films were subjected to quantitative densitometry. Histopathological sequelae were studied in 33 rats of all ages with various survival times (24 h up to 14 days), using argyrophilic methods and Nissl stains (paraffin embedded sections). For [3H]KA binding studies, 11 rats from the same litter were sacrificed at various ages; dissected regions were stored at - - 2 0 °C and processed together for membrane preparation. The same results have been obtained in a second series with 26 animals of various ages taken from different litters (not illus-

* Present address: University of Gdansk, Department of Anatomy, Academy of Medicine, 80210 Gdansk, Poland. Correspondence: Y. Ben-Ari, Laboratoire de Physiologie Nerveuse, CNRS, 91190 Gif-sur-Yvette, France. 0165-3806/84/$03.00 ~) 1984 Elsevier Science Publishers B.V.

285 trated). Tissues were h o m o g e n i z e d in a glass-teflon potter; 50 m M Tris-acetate ( p H 7.0) was used t h r o u g h o u t all steps; washed m e m b r a n e s were preinc u b a t e d for 20 rain at 37 °C and centrifuged once more; incubation time 90 rain, 20 n M [3H]KA (2

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Fig. 1. KA-induced metabolic alterations and high affinity KA binding sites in developing rat brain. Pattern of glucose utilization after injection of saline (controls, A and D) or KA (2 and 9 mg/kg in 10 and 24 day old rats, respectively, B and E). Note that KA seizures accumulated radioactivity in the amygdala 24 days (E) but not 10 days after birth (B); seizures were of limbic type only in the 24 days old rats. C and F: [3H]KA autoradiographs. Frontal brain sections were prepared from 10 day old (n = 2) and 23 day old rats (n = 2) and incubated in 20 nM [3H]KA (2 Ci/mmol, Amersham) as described elsewhereS; exposure time on the film 195 days. Note that the hippocampal CA3 region is strongly labeled already 10 days after birth (C), whereas deep layers of the cerebral cortex are preferentially labeled in more mature tissue (F). Developmental changes in the amygdala are not evident from this approach and only revealed by detailed Scatchard analysis of membrane suspension experiments, tool, molecular layer; TV, ventrobasal complex of the thalamus; AB, AC, AM, basal, central, medial nucleus of the amygdala; rf, rhinal fissure; A, amygdala; d, days.

286 activity measured by liquid scintillation counting. For detailed Scatchard analysis of slowly dissociating [3H]KA, hippocampi and amygdalae + piriform cortices were dissected 10 days after birth from 22 rats, and 19 days after birth from 5 rats. [3H]KA concentrations ranged from 0.5 to 133 nM. Saturation curves were subjected to non-linear Gauss-Newton computer regression analysis. The autoradiographic visualization of slow dissociation rate [3H]KA binding sites has been accomplished as described elsewhere 4. KA injected i.p. induced severe convulsions at all ages tested. To the end of the third week after birth, however, the type of expressed seizure activity changed radically. Whereas rats up to 16 days after

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birth manifested clonic or tonico-clonic convulsions, they started with the 19th day after birth to develop the whole repertoire of limbic motor signs, i.e. the characteristic reaction of adult rats to parenteral KA application. This alteration in the etiology of the convulsion is not due to differences in the dosage of KA used (see above) since, in agreement with others 7, limbic seizures were not produced before P19 and tonico-clonic seizures after that age whatever the dosage used (from ineffective to lethal doses). The relative steep time course of this change, allowing for almost no intermediary transition stage, prompted us to search for its neuroanatomical substrate. Thus, we visualized the KA-induced metabolic changes in rat brains before and after the critical age by means of the 2-DG method. KA induced an accumulation of radioactivity in the hippocampus both 10 days (Fig. 1A, B) and 24 days after birth (Fig. 1D, E), in keeping with electrographic data showing KA-induced paroxysmal discharge in this structure already 10 days after birth (not illustrated). However, only at the older age, did KA induce metabolic activation in a series of other limbic structures, notably the amygdaloid complex and piriform lobe region (Fig. 1E) but also the hypothalamus and mediodorsal and adjacent medial thalamic nuclei (ibid). This was followed by first signs of irreversible brain damage in these same regions, notably in the hippocampal formation (Fig. 2). In rats younger than 18 days, KA did not induce the slightest neuronal damage, in agreement with the resistance of immature striatal6 and hippocampal neurons (C. K6hler, personal communication) to locally applied KA. It is interesting to note

that in KA-treated rats the amygdala was metabolically activated only after the critical age (Fig. 1E), not 10 days after birth (Fig. 1B). This observation further stresses the particular importance of the amygdala for the expression of limbic signs as known from animal models 3 as well as from clinical studies on humans16; the anatomical basis for this particular role has been identified l~.~s. In light of these results, the developing limbic system presented itself as a promising subject to put on test the relevance of specific KA membrane binding sites. Thus, we established the ontogenesis of specific [3H]KA binding sites with slow and fast dissociation kinetics 12, respectively, in the hippocampal formation and the amygdaloid region. With amygdaloid membranes, we observed a rise in 'slow' binding of 47% between the 17th and the 22nd day after birth (P < 0.02), i.e. when rats change in their reaction to KA from tonico-clonic to limbic motor seizures (Fig. 3); neither before nor after this period did we find significant changes in 'slow' binding. Scatchard analysis revealed that this rise was due to the appearance of a high affinity component which was absent from amygdaloid membranes 10 days after birth (see upper insert in Fig. 3). In hippocampal membranes, 'slow' binding appeared earlier. Between the 12th and the 17th day after birth, binding sites more than doubled. Scatchard analysis, however, indicated only quantitative and no qualitative changes, since already 10 days after birth high affinity components were found in hippocampal membrane suspensions (see lower insert in Fig. 3: it should be noted that for

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tissues taken from the same litter, see above). This is in keeping with results from slice autoradiography, 10 days after birth, the visualization of specifically bound slowly dissociating [3H]KA in the hippocampus is already reminiscent of the adult distribution4, ]5 (Fig. 1C), particularly in the mossy fiber region. The ontogenetic study of the KA-induced limbic

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with the postnatal development of specific membrane binding sites for the neurotoxin in the limbic system on the other, may allow for some suggestions concerning the possible relevance of KA binding sites. Thus, high affinity receptors in the CA3 region may be responsible for KA-induced paroxysmal discharge and metabolic activation of the hippocampus as soon as 10 days after birth; receptors with similar kinetic properties residing in the amygdala + piriform lobe seem to play a crucial role in KA's symptomatology of the limbic type, leading to the recruitment of the whole limbic circuitry into prolonged seizure activity. Interestingly, seizures will only result in pared

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Fig. 3. Age-dependent binding of slowly dissociating [3H]KA to membranes prepared from the amygdala + piftform lobe and the hippocampus. Radioligand concentration 20 nM; S.E.M. values indicated by bars; triplicates. Upper trace: seizure type induced by KA in rats of various age groups. Note the rise in amygdaloid KA binding sites 17-22 days after birth, when seizures change from tonico-clonicto limbic. The inserts show Scatchard analyses 10 and 19 days after birth. In most cases, non-linear regression resulted in a better fit than linear regression. Underlying a model of two different binding sites, the followingK d (nM) and Bmax values (fmol/mgwet tissue, in parentheses) were obtained: hippocampus (hpc) 2.4 (2.0) and 42 (13); hpc 19 days, 2.6 (2.6) and 40 (14); amygdala complex (am-cx) 10 days, 23 (10.5) (only 1 site, linear regression); amcx 19 clays 4.8 (3.1) and 54 (19). B, pH]KA bound (fmol/mg wet tissue); B/F, bound/free (10-6); TC, tonico-clonicconvulsions; LMS, limbic motor seizures; SE, status epilepticus; d, days. these analyses membranes have been prepared separately from different litters; quantitative changes are reflected more reliably by the main frame figure, since membranes have been prepared together from 1 Amaral, D. G. and Dent, J. A., Development of the mossy fibers of the dentate gyrus: I. A light and electron m i c r o scopic study of the mossy fibers and their expansions, J. comp. Neurol., 195 (1981) 51-86. 2 Ben-Aft, Y., Tremblay, E., Ottersen, O. P. and Meldrum, B. S., The role of epileptic activity in hippocampal and 'remote' cerebral lesions induced by kainic acid, Brain Res., 191 (1980)79-97. 3 Ben-Aft, Y., Tremblay, E., Riche, D., Ghilini, G. and Naquet, R., Electrographic, clinical and pathological alterations followingsystemic administration of kainic acid, bicu-

brain damage if they are of the limbic type. Thus, mature limbic connections seem to be more crucial for the generation of brain damage than the direct interaction of KA with membrane binding sites. The resistance of the immature CA3 region to KA correlates with the delayed maturation of the granule cells in the dentate gyrus and their mossy fibers1; this, in keeping with other observationstT,21, further indicates the involvement of this fiber system in KA-induced damage to CA3 pyramidal cells. We thank G. Ghilini, J.-P. and Bouillot and G. Charton for technical assistance. Supported by 'Foundation pour la Recherche M6dicale', Minist6re de la Recherche et de l'Industrie, INSERM (CRE. 1093), and by the Austrian Science Research Fonds (for M.B., Project E0002). culline and pentetrazoh metabolic mapping using the deoxyglucose method with special reference to the pathology of epilepsy, Neuroscience, 6 (1981)1361-1391. 4 Berger, M. and Ben-Ari, Y., Autoradiographic visualization of [3H]kainic acid receptor subtypes in the rat hippocampus, Neurosci. Lett., 39 (1983) 237-242. 5 Berger, M., Tremblay, E., Nitecka, L. and Ben-Aft, Y., Ontogenesis of kainic acid induced seizures and brain damage: the crucial role of high affinity [3H]kainic acid receptors in the amygdala, Soc. Neurosci. Abstr., 9 (1983) 77.9. 6 Campochiaro, P. and Coyle, J. T., Ontogenetic devel-

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[3H]kainic acid binding sites in rat CNS as determined b~ autoradiography, Brain Res., 252 (1982) 91- l llii 16 Munari, C.. Bancaud, J., Bonis, A.. Buser. P.. t'alairaeh, J., Szikla, G. et Philippe, A., R61e du noyau amygdalien dans la survenue de manifestations oro-alimcntaires all cours des crises 6pileptiques chez l'homme, Rev. FE(; Neu~ rophysiol., 9 (1979) 236-240. 17 Nadler, S. V. and Cuthbertson, G. J., Kanic acid neurotoxicity toward hippocampal formation: dependence on specific excitatory pathways, Brain Res., 195 (1980)47-56. 18 Price, J. L., The efferent projections of the amygdaloid complex in the rat, cat and monkey. In Y. Ben-Ari (Ed.), The Amygdaloid Complex, Elsevier, Amsterdam. 1981, pp. 121-132. 19 Schwob, J. E., Fuller, T., Price, J. L. and OIncy, J. W., Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: a histological study, Neuroscience, 5 (1980) 991-1014. 20 Simon, J. R., Contrera, J. F. and Kuhar. M. J., Binding of [3H]kainic acid, an analogue of L-glutamate, to brain membranes, J. Neurochem., 26 (1976) 141-147. 21 Sloviter, R. S., Epileptic brain damage in rats induced by sustained electrical stimulation of the perforant path. I. Acute electrophysiological and light microscopic studies, Brain Res. Bull., 10(1983) 675-698. 22 Tremblay, E. and Ben-Ari, Y., Clinical and metabolic aiterations induced by systemic injection of kainic acid in iramature rats, Soc. Neurosci. Abstr., 8 (1982) 287.