Limbic seizure and brain damage produced by kainic acid: Mechanisms and relevance to human temporal lobe epilepsy

030&4522/85 S3.00 + 0.00 Pergamon Pres Ltd IBRO

h’euroscience Vol. 14, No. 2, pp. 375GtO3. 1985 Printed in Great Britain

COMMENTARY LIMBIC SEIZURE AND BRAIN DAMAGE PRODUCED BY KAINIC ACID: MECHANISMS AND RELEVANCE TO HUMAN TEMPORAL LOBE EPILEPSY Y. BEN-ARI Laboratoire de Physiologic Nerveuse, Departement de Neurophysiologie appliquee, 91190 Gif-sur-Yvette, France “Perhaps the word kainic comes from Cam” U.S. Von Euler at a meeting CONTENTS Introduction Specific binding sites for kainate Potent excitatory effects of kainate Preferential action of kainate on limbic structures Parenteral administration of kainate: a limbic status epilepticus Intra-amygdaloid injection of kainate: dissociation between local and “distant” seizure-related damage Kainate-like endotoxins Mechanisms of local damage Mechanisms of “distant” damage Relevance to human epilepsy Clinical considerations Relevance of animal models for human temporal lobe epilepsy Conclusions

Kainic acid (KA-in Japanese literally the ghost of the sea) was isolated three decades ago from the seaweed Digenea simplex’“’ which has been extensively used in post-war Japan to eradicate ascariasis. The active principle of other biological extracts used for the same purpose has also been identified (notably domoic and quisqualic acids, ihid.). The antihelminthic action is shared by other analogues of glutamate in particular those having carboxyl (and carboxymethyl) substituents in the 2 and 3 positions. The next important step was made by Shinozaki and Konishi’“3.‘bl who showed that when microiontophoretically ejected upon cortical neurons these agents have a potent excitatory action, consisting of prolonged spike discharge. Kainate was the most potent analogue. It had been known for some time that parenterally administered monosodium glutamate destroys retinal ganglion cells in developing mice.” Olney’-“,‘” showed that glutamate also destroys neurons of various brain regions lying outside the blood-brain barrier, notably the arcuate nucleus. The neurotoxicity of KA was first shown by Olney er u!.‘~* using the same experimental paradigm. Parenteral KA in immature mice caused rapid necrotic changes 2deoxyglucose; butyric acid; KA, kainic acid.

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in the somata and dendrites of arcuate neurons; this was associated with hyperplasia of glial elements and followed by phagocytosis which could be manifest as early as 3-4 h after administration. Afferent fibers and axons in the necrotic area appeared normal (also see Ref. 134). In subsequent reports, the relationship between the structure of a series of glutamate analogues, their excitatory potency (as suggested from microiontophoretic experiments’%) and their toxicity toward the arcuate was studied.39.‘34.‘5*Since every analogue of glutamate which produced neuronal excitation also caused dendrosomatotoxic activity, the term “excitotoxin” was proposed by Olney and coworkers’36.13nwhich implies that neuronal cell death is due to excessive depolarization produced by the excitatory substances. It was proposed that the sensitivity of a given neuron to KA is determined by the percentage of its dendritic surface occupied by glutamatergic receptors. The resistance of axons was interpreted as resulting from the lack of amino acid receptors on fiber tracts. Intracerebral iontophoretic ejection of glutamate produces a lesion in the rat cerebral cortex.‘90 The demonstration that direct intracerebral injection of KA also produces an “axon-sparing lesion”‘* prompted the use of KA as a tool in neurobiology.“* The rational for this use stems primarily from the 375

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possibility to destroy selectively the neurons of a given brain region and thus to circumvent the problem of the contribution offibres depassuge, which are also destroyed by conventional (electrolytic) lesions. Such studies have provided several experimental models of human diseases such a hemiballism74 or Huntington’s chorea (e.g. Coyle et al.,” McGeer et ~1.‘~’ and Refs therein). Other more recent studies indicate, however, that both the use of KA as an axon-sparing device and the postulated mechanisms to explain KA neurotoxicity must be reassessed. (a) The concept that KA acts through glutamate receptors has been eroded by extensive biochemical and pharmacological studies;6~37~y3~‘2y~‘30~‘56 KA does not act on most glutamatergic receptors but it may involve a special class of glutamatergic receptors with unique pharmacological profile and regional distribution in the brain.‘15 (b) There is a clear-cut gradient of vulnerability of neurons to kainate; thus the pyramidal neurons of the CA3 region of the hippocampus are the most vulnerable neurons in the brain KA’*’ whereas neurons of the diagonal band of Broca or the mesencephalic trigeminal nucleus are resistant to its toxic effects.34,88 Even within the same region striking differences are noted, viz. the CA3 region and the granular layer of the hippocampal formation.‘*‘.‘*’ Several obsernotably those made by Nadler and vations, coworkers”“~‘2Z suggest that this gradient is not due to a differential distribution or density of glutamatergic receptors (also see Refs 89 and 157). Other potent excitatory analogues do not reproduce this gradient but destroy the neurons as a function of their proximity to the site of injection.2.88.96.‘2’ (c) Other observations suggest a dissociation between the excitatory and toxic properties of KA, for instance lesion of the corticostriatal pathway confers a protection of striatal neurons from the toxic action of KAl8.102 without altering the excitatory effect of KA on striatal neurons.“’ ’ (d) The axon-sparing characteristic of KA injections has been disputed9*.“‘.‘*’ and, at least in some brain regions, there is little doubt that fibre tracts are destroyed rapidly by local injection of KA.“’ (e) Intracerebral injections of KA also produces motor recurrent epileptiform discharge,“.6’ convulsions”,6’.‘6’ and subsequently damage at the site of injection and damage in structures distant The distant damage from the injected site. 11.61.135.161.198 which is not caused by other major excitatory analogues (notably ibotenic acidsa,‘*‘) was initially attributed to diffusion of the toxin combined with a greater vulnerability of the distant neurons to KA;‘35,‘98 we have however discovered the role of the limbic status epilepticus and paroxysmal discharge generated by KA as a cause of remote brain damage.” Since the regional distribution of this damage is reminiscent of that associated with human temporal lobe a better understanding of epilepsy 33.54,64.65,9'.151,155.172,175

the epileptogenic properties of KA is of more than theoretical interest. The preferential effect of KA on limbic structures is the main subject of the present commentary. The reader is referred to other monographs for more general reviews of the action of KA and other excitotoxins,36".37,6*,10*

SPECIFIC

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High and low afinity sites Following the initial observations of Simon et al I66the presence in the brain of saturable, specific biiding sites for KA has been confirmed and extended by several groups. 6,93~‘56.‘92 Whereas a single site was described in the earlier report, two sites were found in later studies. In the comprehensive work of London and CoyleT’ the high and low affinity sites (&s of 4- 16 and 27-66 nM respectively) were distinguished on the basis of their different association and dissociation constants. Kainate dissociates within seconds from the low affinity site, in contrast the dissociation from high affinity sites occurs in 1 h or more (M. L. Berger, in preparation). The affinity of various analogues for the two sites differs as well as the regional distribution in the brain.93 The striatum, hippocampus and forebrain regions are particularly enriched with high affinity sites whereas more than 90% of the total specific binding in the cerebellum or pons-medulla are to low affinity sites. The distribution of high affinity binding sites shows some correlation with the regional vulnerability to KA (see below). Regional distribution To gain a better understanding of the possible physiological significance of KA binding sites, it is essential to map carefully their distribution in the brain. In spite of the low specific activity of currently available radioactive KA, the distribution of KA binding sites has been described recently with autoradiographic techniques. ‘5,“4~‘88The highest level of specific binding sites in the brain is located in the stratum lucidum of CA3 in the hippocampus (see Fig. 1C) i.e. the region innervated by mossy fibers originating from the granular layer of the fascia dentata. This region is also the most vulnerable to the excitatory and toxic properties of KA (see below). Quantitative densitometry shows that the density of silver grains (representing bound kainate) in the mossy fiber region is ten times higher than in adjacent layers.15 This labelling is due to binding to the high affinity (slowly displaceable) sites (ibid.), as confirmed by a more recent study (M. L. Berger, G. Charton and Y. Ben-Ari, in preparation) in which the CA3 and CA1 plus fascia dentata were microdissected separately; in contrast to CA3, the CA1 and fascia dentata contained lower affinity sites. The autoradiographs also revealed that other forebrain regions are enriched in KA binding notably the amyg-

Limbic seizure and brain damage produced by kainic acid

deep layers of piriform cortex, striatum, reticularis nucleus of the thalamus, whereas most thalamic nuclei, the septum or the pons-medulla are poor in silver grains.‘5.“4,‘88

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Another crucial question, namely the pre- or postsynaptic localization of KA binding sites is still controversial. Thus, injections of kainate in the striatum at doses which destroys most neurons produce a 50-60% drop in specific binding sites, whereas destruction of major inputs to the striatum produce a modest or negligible reduction in specific KA binding sites,6,'8.37.78.156 A more recent study (M. L. Berger, in preparation) suggests that decortication has no effects on the number of KA binding sites but leads to a slight reduction in their affinity for the ligand. All of these observations suggest, in keeping with eiectrophysioiogical studies7’ ‘05*‘06a postsynaptic localization of both binding sites and neuroexcitatory actions of KA. Other observations suggest a presynaptic interaction between KA and the endogenous transmitter causes the neuronal damage. Thus decortication protects vulnerable neurons from being kiIled,‘s~‘o’without altering the excitatory effects produced by microiontophoretic applications of KA.“’ Interestingly, as this protection appears only after several days, this might be due to a slow degeneration of boutons possessing some of the KA binding sites (also see below). In the hippocampus, KA binding sites are readily and rapidly reduced following a variety of procedures. Thus, both colchicine treatment” and intracerebroventricular kainate12’ at doses which destroy respectively the granular Layer of the fascia dentata (and their mossy fibers) and the pyramidal layer of CA3 eliminate the labelling as demonstrated by autoradiographs.“4 We have recently reexamined this by using injections of KA in the amygdala to avoid direct administration of toxins to the hippocampus; we also used 9-12 day survival periods to avoid problems related to the reorganization of synaptic circuitry following KA.“’ A 60-707; reduction in high affinity KA sites was found together with a destruction of CA3 pyramidal layer but with limited change in the mossy fibers as assessed with Timm’s stain (M. L. Berger, G. Charton and Y. Ben-Ari, in preparation). These findings, in keeping with maturation studies (see below), suggest that the KA sites are primarily located on the postsynaptic side in this region. Kainate-glutamate

interactions

Electrophysiological studies made primarily in the spinal cord’94 suggest that there are various classes of excitatory amino acids including: (a) a N-methylD-aspartate-preferring site also activated by ibotenate, p-glutamate and antagonized by low concentrations of divalent metal cations and a series of

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D-amino acids; (b) a second class comprising quisqualate and kainate subpopulations (also see Ref. 73). However, since these studies rely on extracellular comparison of spike-inducing activity of various compounds, it is difficult to meet strict pharmacological criteria for receptor classification (e.g. reviews and discussion in Refs 129 and 130). Using autoradiographic techniques, Monaghan et a1.“5 have recently shown that KA sites in stratum lucidum of CA3 also bind [3H]glutamate although with ionic dependence and antagonist sensitivity which differs from the major glutamate site observed in biochemical studies. These authors suggest the presence in the hippocampus of four pharmacologically and anatomically distinct glutamate binding sites. Because of the particular vulnerability of the CA3 region to KA and the fact that this region is particularly enriched in KA high affinity binding sites, the pharmacological profile of these sites has been extensively studied on membrane preparations. Transmitters such as acetylcholine or centrally active agents (diazepam, cardiazol or morphine,‘56 but also flurazepam or [Leulenkephalin) do not displace KA (M. L. Berger and Y. Ben-Ari, unpublished). The mossy fibers are also particularly enriched in dynorphiniu3 and ZnZ+ ‘v’ (e.g. Fredericksanb” and Refs therein) but neither the former nor the latter (in millimolar concentrations) influence the binding of KA (M. L. Berger and Y. Ben-Ari, unpublished); we have aiso found no displacement of KA by nerve growth factor even if there are suggestions that this substance may be released in this region.“’ Putative endogenous kainate-like substances, notably folic acid or quinolinic acid (see chapter below on Kainate-like endotoxins) also produced negligible displacement of KA from the CA3 sites (ibid.). These observations suggest that the specific high affinity KA sites in CA3 have rather restricted conformational requirements. It bears stressing that the transmitter released by the mossy fibers has not beeu fully characterized; the evidence in favor of glutamate is for instance less compelhng than it is for the Schaffer collaterals or the perforant path.ls”.18~ POTENT EXCITATORY EFFECTS OF KAINATE

The particularly powerful excitatory action of KA has been described in several brain areaS 32.5R.72.105.1U6,l48.194 This effect has a slow onset and prolonged duration; in several preparations recovery is not obtained. The principal actions of KA on invertebrate preparations is to potentiate glutamateinduced depolarization.35,42.‘b3 In vertebrate spinal cord, KA directly produces a large and often irreversible depolarization’7,3~,5~ which is associated with a reduction in input resistance4’ and depends on the presence of extraceliular sodium.‘o0 As stressed by Nistri,“’ it is however possible that the activity of KA is due to an increase in Ca2+ permeability. This would readily explain the strong conductance increase and

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Fig. 1. Preferential metabolic alteration of the CA3 region. Following parenteral administration of KA, a rise in 2-deoxyglucose autoradiographic staining is noticeable in the CA3 region in immature (A-12 days of age) or adult animals (arrow, D) (B). In immature animals, this is the only brain region in which we have detected a rise in 2-DC. In adult, at doses which produce hmbic motor seizures other structures of the hmbic system are labelled (D). Furthermore in both immature (not shown) and adult (C) specific high affinity KA+ binding sites are concentrated in the CA3 region. The 2-deoxyglucose pictures in (A) and (B) were superimposed in the stained sections from which the autoradiography had been obtained (e.g. Ben-Ari et al.,14 Berger et al., I6 Tremblay et al.‘86). Abbreviations: cing, cingulate gyrus; Fi, fimbria; g, granular layer; h, hilus; mol, molecular layer; p, pyramidal layer; SR, stratum radiatum. Fig. 2. Selective and non-selective seizure-related damage produced by kainate. In (A) and (C), damage restricted to parts of CA3 by intra-amygdaloid injection of KA. (A) Neuronal cell loss with Nissl stain after survival period of 5 days; (B) extensive necrosis caused by intracerebroventricular (or also parenteral) KA. (C) Argyrophylia seen also after intra-amygdaloid injection of KA with short survival periods with a Fink-Heimer stain. Nissl stain. Fig. 3. Long-term sequelae of parenteral KA in adult animals. Two months after administration of KA. the animal displayed spontaneous seizures (A) with paroxysmal discharge in both the hippocampus (H) and amygdala (A). These seizures were elicited often by handling. (B) and (C) depict in the same animal the extent of neuronal cell loss in the hilar region (C) and dark degenerating cells in (B); clear-cut evidence of phagocytosis was also often observed suggesting a continuous process of neuronal degeneration (e.g. Tremblay and Ben-Ari’84). (A) Scale in microvolts; LMS, limbic motor seizures. Fig. 5. Parallelism between the distribution of neurons vulnerable to KA in the hilar region and GABAergic neurons revealed by glutamate decarboxylase (GAD) immunocytochemistry. With the Fink-Heimer technique following parenteral KA and short survival periods argyrophylic cells are particularly frequent in stratum oriens and pyramidale of CA1 and in the hilar zone (medial part of CA3 C and polymorph zone B). In (C)-(F), GAD immunocytochemistry was performed in control animals following injections of colchicine in the cortex (L. Nitecka, M. Tappaz, E. Tremblay and Y. Ben-Ari, unpublished observations); (e.g. also Riback et al.‘46 and Somogyi et al.“‘). The boxed area in (D) is shown at a higher magnification in (F). Arrows in (E) and (F) point to GABAergic neurons. Abbreviations: a, alveus; f, fimbria; g, granular layer; m, molecular layer; o, stratum oriens; p, stratum pyramidale; r. stratum radiatum. Fig. 7. Repetitive electrical stimulation of the perforant path produces a reduction of the Timm stain in the mossy fibers. (A), (C), (E) Control; (B), (D), (F) after electrical stimulation. from Sloviter’6x. Fig. 9. Bleaching of Zn’+ stain produced a K + pulse. Push-pull cannulae were stereotaxically introduced in the mossy fiber zone of each hemisphere. A K+ pulse (IOmin, 30mM) was applied to the right hemisphere and the animal perfused immediately at the end of the pulse. Note the reduction of the intensity of Zn*+ stain as compared to the contralateral (control) side. Timm stain (e.g. Ref. 168) counterstained with cresyl violet. Abbreviations: g, granular layer; h, hilus; IPSI, ipsilateral; p, pyramidal layer.

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Limbic seizure and brain damage produced by kainic acid the powerful depolarization, since Ca*+ carries more current than Na+ and has a very positive reversal potential because of its very low intracellular free concentration (e.g. Puil’“). Because of the particularly potent seizure-inducing effect of KA in the hippocampus, a number of studies have examined its electrophysiological effects in this region. Microiontophoretic applications of KA to the hippocampus in situ produce a prolonged increase in firing rate of pyramidal cells. The effect is particularly prominent in CA3 where removal of the iontophoretic retaining current is often sufficient to produce an intense activation of neurons.” CA1 and CA3 pyramidal cells have a similar sensibility to glutamate or ibotenic acid, thereby further stressing the unique features of the actions of KA in CA3. De Montigny et ~1.~ have also suggested that the GABAergic inhibition is not involved in this neuronal activation since (a) GABA does not exert any selective effect on the action of KA vs that of glutamate; (b) benzodiazepines selectively block the actions of KA in CAl-but not in CA3; this antagonism is not associated with enhancement of the inhibition produced by microiontophoretic appiications of GABA (ibid.). Other studies have however suggested that the action of kainate is at least partly due to a reduction of the GABAergic inhibitory drive. Thus, following both parenteral administration of KA in the intact animal16’ or bath applications of KA into slice preparation,‘06 there is a reduction of the recurrent inhibition measured with the paired pulse inhibition technique. A recent intracellular study” has indeed shown that the principal action of KA on in vitro CA3 and CA1 pyramidal neurons appears to be a synaptic depression, which first influence the inhibitory postsynaptic potentials and (with higher doses) the excitatory postsynaptic potentials. This depression of inhibition is associated with minimal effects on membrane properties, action potentials, fiber conduction, etc. It is not associated with alterations in the response produced by pulses of GABA, and it is best explained by a reduction of presynaptic GABA release although a postsynaptic effect is not completely ruled out. The excitatory phenomena which occur following limited applications of KA were thus interpreted as being secondary to decreased inhibition, in keeping with the hypothesis that the removal of GABAergic inhibition plays an important role in epileptogenesis in this region.’ The potent excitatory action of other agentsnotably acetylcholine’0~‘89-also involves a removal of the inhibition. Interestingly, another study’48 has shown that there is a large difference between the concentration of KA required to induce a reversible acceleration of spontaneous discharge (10 nM) and the one required to produce depolarization with apparently irreversible spike blockade (10 PM). This difference must be kept in mind when the mechanisms underlying the local and distant actions of KA are examined (see below).

385

PREFERENTIAL ACTIONS OF KAINATE ON LIMBIC STRUCTURES

Parenteral administration of kainate: a limbic status epilepticus

In the adult rat parenteral administration of the toxin produces a limbic seizure and brain damage syndrome which has not been reproduced by other excitatory amino acids. Since this illustrates, in a remarkable manner, the central role of limbic circuitry in the actions of KA, it will be described with some detail. Clinical signs. The clinical signs produced by i.v.94 or i.p. I4 injections can be divided in several distinct phases. During the first 20-30 min, the animals have “staring” spells. This is followed by head nodding and numerous wet-dog shakes for approximately 30min. The next phase which starts 1 h after KA is characterized by the occurrence of individual recurrent limbic motor seizures. These are identical to the seizures produced by repeated daily electrical stimulation of the amygdala or other limbic structures69 and include masticatory and facial movements, forepaws tremor, rearing and loss of postural control. The seizures then become progressively more complex and prolonged, with a reduction in the interictal pause. In the following phase (l-2 h), the animal displays a full status epilepticus i.e. continuous convulsions and abnormal electro encephalogram. We have referred to this syndrome as a limbic motor syndrome;14 tonico-clonic generalized convulsions (i.e. of the bicuculline or picrotoxin type) are not produced by kainate in adult animals, except with massive doses about four or five times the lethal dose (E. Tremblay and Y. Ben-Ari, unpublished). Therefore the latter terminology which has often been used in the literature for KA-induced seizures is inappropriate and should be reserved to describe procedures causing tonico-clonic convulsions and electrographic seizures generalized to the entire cortex from their onset. Electric activity. Electrographic records from cortical and subcortical structures have confirmed that the three main phases of behavioral abnormalities (staring, individual limbic seizures, status epilepticus) correspond respectively to (a) localized paroxysmal discharges in the hippocampus, (b) involvement of other limbic structures-notably the amygdala-and then (c) generalization to a number of other (nonlimbic) structures. The hippocampus has a particularly low threshold to parenteral KA.‘4,94 This is in keeping with electrophysiological studies showing that in the sllice preparation, seizure activity is produced by particularly low concentrations of KA.‘48 The 2-deoxyglucose autoradiographic method. This method also illustrates the central role played by the hippocampus in the initial stage which follows parenteral administration of KA (Fig. IB). Administration of lower doses of KA,94 or repeated injections of diazepam at doses which block the seizures except

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at the most susceptible sites (Y. Ben-Ari and E. Tremblay, unpublished) suggest that the temporal pole of the hippocampal formation is preferentially involved. A developmental study has also shown that before the end of the third week of age (in rats), the Ammon’s horn is the only brain region in which a rise in 2-deoxyglucose (2DG) labelling is observed following parenteral KA (Fig. 1A and Tremblay et ~l.‘*~). The lateral septum-which receives a direct projection from the Ammon’s horn-is also involved at this early stage. During the second stage when overt limbic motor seizures are produced, the 2DG maps reveal an intense activation of the entire limbic system (Fig. 1D). With almost no exception, the structures in which there is a metabolic rise have massive direct axonal connections with one or more of the following areas: the hippocampal formation, the amygdaloid complex and the mediodorsal thalamic complex (e.g. Refs 14 and 94, and Refs therein). Thus a rise in glucose consumption is noted in the target structures of the fornix pre- and postcommissural efferent projections of the hippocampal formation (i.e. the accumbens, ventral pallidum and claustrum, anterior thalamic nuclei and infra-lilnbic cortex). The amygdaloid complex-which is also connected with the entorhinal cortex and other components of the hippocampal formation-is also highly labelled as well as structures to which the amygdala projects (notably the stria terminals and its bed nucleus. Fig. ID). Finally, the labelhng in the cortical mantle is restricted to the “limbic” cortex i.e. the infra- and pre-limbic cortices as well as the agranular insular, retrosplenial and perirhinal cortices, several of these areas are projected upon by the mediodorsal nucleus and the amygdaloid complex. Although the 2DG method does not allow conclusions to be reached concerning the labelling at the cellular level. there is little doubt that the rise in metabolism occurs in relation to the propagation of paroxysmal discharges to neuronal populations which are axonally interconnected (e.g. Refs 14 and 94). The amygdala, hippocampal formation and mediodorsal thalamus occupy a central position in the postulated wiring diagram (ibid.). Another interesting feature of the 2DG maps is the metabolic decrease noted in several non-limbic structures such as most of the neocortex, striatum, ventrobasal thalamus, etc. ~~st~put~~~~l~~~~. The early histopathological changes (I -2 h after parenteral KA) are characterized by shrinkage and pycnosis of neuronal perikarya and swelling of dendrites. 136~‘6’.‘74 Pronounced oedema and swelling of astrocytes are also apparent shortly after KA application, at least with relatively high doses. These changes are largely distributed to the hemisphere with minor alterations in cerebellum, pons and medulla oblongata, and prominent changes in hippocampal formation and piriform cortex. These acute effects are associated with a dilatation of organelles; the golgi apparatus, endoplasmic reticulum and adjacent astrocytes are swollen (ihid.). With

argyrophylic stains, darkly impregnated neurons are restricted to limbic and associated structures.‘” With longer survival (1 day to 1 week) the histological changes, notably the neuronai cell loss, are largely restricted to the regions in which there had been a conspicuous rise in metabolism as reflected from the 2DG studies, i.e. hippocampus, amygdala, piriform lobe, septum, medial thalamus, etc. (Fig. 2). As far as the late brain damage is concerned subtotal or total tissue necrosis is found in the temporobasal portion of the brain where the damage extends through all cell layers from the piriform lobe to the occipital cortex up to the rhinal fissure dorsally (Fig. 3B and Refs 12, 14, I61 and 174). Conversely there are also regions of restricted damage such as the claustrum and adjacent deep cortical layers (claustrum insulare) or the vulnerable part Ammon’s horn (CA3) in which the neuronal cell loss is not associated with a full destruction of the neuropit. In the regions of total or subtotal necrosis, in addition to the neuronal cell loss, there is a loss of oligodendrocytes, demyelination, formation of astroglial scars and perivenous hemorrhages as wet1 as vascular sprouting.‘7’ ~(~~~-r~~~~ .~e4uelfft,. The long-term sequelae (I -3 months) of parenteral KA further depict the involvement of limbic structures. Firstly, in keeping with observations made following intracerebral injections of KA,” and animals display “spontaneous seizures” notably when handled.‘XJ These seizures are of a fimbic type and are accompanied with paroxysmal seizure discharges in the amygdala and hippocampus (Fig. 3A). Furthermore, examination of the brain (Fig. 3B) reveals a satellitosis in the Ammon’s horn and phagocytosis of neurons in strata oriens, radiatum and pyramidale several weeks after the parenteral injection of KA i.e. at a time when the toxin has been removed from the brain.“’ This reflects the on-going nature of the seizure and brain damage.“’ In the pyramidal layer this late damage is more conspicuous in CA3 than in CA 1. BIood,fhs and oxygen cwmmption. In order to test directly the possible contribution to nerve tissue damage by a mismatch between increased local metabolic consumption and oxygen supply. we have measured blood flow, as well as partial pressure of oxygen and carbon dioxide in the vulnerable CA3 region of unanaesthetized freely breathing rats following parenteral administration of KA.‘4”.““’ It was found that seizure (as noticed by means of hippocampal records as well as the motor signs) are associated with a rise in the local blood flow which more than compensates the increased oxygen consumption (Fig. 4). Thus the PO, and PCO, remains within normal limits during the observation period (2 h), at a time when damage is caused. Severe seizures are actually associated with hyperoxia (and not hypoxia). This provides compelling evidence that at least in this region the damage caused by parenteral kainate is not due to anoxic ischemic episodes.‘““’

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Fig. 4. The damage in CA3 produced by kainate is not caused by a mismatch between local blood flow and metabolism. The local partial pressure in CO, (PCOJ, oxygen (PO?), the blood flow (C.B.F.) and hippocampal electroencephalogram (EA Hipp) were continuously recorded in a chronically implanted, unanaesthetized freely breathing rat after parenteral administration of kainate. The PO, and PCO, were measured by gas spectrometry, the gases are drawn via a special cannula into the analysis chamber by an ultra high vacuum; the local blood flow was measured by the thermal clearance method. The cannulae and electrodes (for the hippocampal electroencephalogram) were chronically implanted in the CA3 region 2 weeks before the experiment. At the day of the experiment the animal was placed in a hammock, left to habituate for I h and the variables measured continuously and quantitatively before and (up to 3 h) after the administration of KA. The rat was anaesthetized and perfused Immediately after the recording sessions. Note that the seizures are associated with a rise in a local blood flow which more than compensates the increased metabolism. Damage in CA3 was found even though there were no hypoxia and hypercapnia in the CA3 region during the entire recording session. See e.g. Pinard et ~1.‘~’ and Refs therein for techniques used.

Neurochemistry. Neurochemical changes after parenteral KA include a highly significant reduction in the activity of glutamate decarboxylase, a marker for GABAergic synapses. This reduction is found in the hippocampus and amygdala but not in striatum or frontal cortex; the effect starts after 24 h and reaches a maximum 2 days later. “J’~ This is in keeping with the electrophysiological experiments described earlier, and further reflects the preferential involvement of these structures in the KA syndrome. Since both in the hippocampus’ and amygdala” GABAergic neurons are intrinsic, these observations reflect a direct damaging effect of the procedure on these structures. Neurons that are GABAergic are also particularly vulnerable to KA in other structures (ibid. and Ref. 191) and in epileptogenic foci.‘45 Electrophysiological studies on hippocampal slices from animals which received KA 2 weeks earlier also indicate a consistent reduction in the inhibitory mechanisms.59.9’ In keeping with these observations, the distribution of hippocampal neurons vulnerable to parenteral KA (as revealed with argyrophylic stains) is similar to the distribution of GABAergic interneurons as revealed by glutamate decarboxylase immunocytochemistry (Fig. 5, see also Ribak et ~1.‘~~ and Somogyi et d.“‘). In contrast, the cholinergic septohippocampal enteral

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par-

in the proof).

Ontogenesis of the ef/cts of parenteral kainate. In 3 week old animals, parenteral KA induces tonic or tonico-clonic convulsions, but not limbic motor signs.29,‘86 Limbic motor seizures are in fact first observed starting from the end of the third week; the shift from one type of syndrome to the other is sudden with almost no intermediary stage. This prompted us to study its anatomical, metabolic and biochemical correlates.‘6.‘3’.‘86 In young animals the rise in 2DG labelling induced by KA is exclusively restricted to the hippocampus (more precisely the CA3 region and hilus) (e.g. Fig. 1A) and to the lateral septum, thus confirming the unique susceptibility of these regions to KA. The rise in 2DG in this region is already observed in 3 day-old animals which have immature mossy fibers when studied anatomically and electrophysiologically.3~‘78 Furthermore, in spite of the severe tonico-clonic type convulsions, there is no brain damage before the end of the third week of life. It is only after, that the toxin produces limbic motor seizures associated with metabolic rise in hippocampus, amygdala, limbic cortex, medial thalamus, and with typical pathological changes.13’ Therefore the occurrence of limbic seizures and the presence of a fully mature mossy fiber system is necessary to cause damage in the Ammon’s horn. In the light of these results, the developing limbic system seemed an interesting subject for testing the

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ontogeny and role of KA binding sites. Both Scatchard analysis and autoradiographs revealed high affinity sites in the CA3 region at an early age but only low affinity sites in the amygdala.‘” The high affinity sites appear in the amygdala only at the end of the third week of life, i.e. at a time when the KA convulsions have a limbic aetiology (also see Ref. 25). Suggested sequence of events ,following parenteral kainate. To evaluate fully the sequence of events produced by parenteral KA, it is essential first of all to determine the amount of toxin reaching the brain, in particular the vulnerable regions of the limbic system. Earlier authors have underestimated the problem of the blood-brain barrier to KA and have interpreted the effects of KA as due to a direct action. This barrier is, however, highly impermeable to KA as much as to other acidic amino acids. Using the method described by Oldendorf’32 we found a brain uptake index for KA in the hippocampus and other limbic regions of 2-4x, this is close to the values found with the impermeant tracer saccharose (M. L. Berger, le Fauconnier, E. Tremblay and Y. Ben-Ari, in preparation). After systemic injection of [‘H]KA, about 250 fmol KA/mg wet tissue remained in brain tissue unremovable by perfusion (ibid.). It is estimated that the actual brain concentration of KA is sufficient to produce paroxysmal discharge in CA3’4X but two orders of magnitude lower than the concentrations required to directly produce damage by in situ injections.6’ These observations cannot be reconciled with the suggestions that the damage in the hippocampus following parental KA is due to a direct interaction of KA with (glutamatergic) receptors.“’ The progressive sequence of activation of the limbic circuitry and the lack of damage (even with lethal doses) to structures such as the striatum (rich in KA receptors” and readily destroyed by direct injection of the toxin37.3X) argue against the hypothesis of a injected KA. direct “toxic” action of parenterally A more likely explanation is that seizures are hrst elicited directly by KA in CA3. The disinhibition produced by removal of the powerful GABAergic inhibition will contribute to enhance the synchronous paroxysmal discharge. If the discharge is sustained, it will propagate to the entorhinal cortex and the lateral septum. Subsequently, it will recruit a limbic circuitry in which the amygdala has a key role. I suggest that recurrent activation of the amygdalohippocampal axis leads to the epileptogenic damage. Other studies in which the toxin was directly injected in vulnerable regions have also provided compelling evidence for a seizure-related damage produced by KA. These will be described prior to a discussion on the mechanisms of the damage.

Intra-atnygdaloid injection of kainate: dissociation between local and “distant” seizure-related damage Intra-amygdaloid tially limbic motor

injection of KA produces iniseizures.“.‘3 Later, the symptom-

atology in the rat is more complex and includes barrel rotation, circling behavior etc., reflecting the secondary involvement of extra-limbic structures. This is a made1 of limbic seizures with a focal origin. In the cat’*’ and monkey “.“* the symptomatology is also of an amygdaloid type (chewing, salivation, ipsilateral cloniae of the face in the cat and oral automatism in the monkey). There is a secondary generalization to other structures particularly in the cat but not so in the monkey (ibid.). The epileptiform activity which is first apparent in the injected amygdala rapidly propagates to the ipsilateral Ammon’s horn-notably the CA3 region-and the contralateral amygdala.’ ’ 2-Deoxyglucose autoradiographs’x7 reveal an ipsilateral regional rise in labelling reminiscent of that obtained after i.p. injection of the toxin. At the site of injection-in keeping with the local effect of KA in a large number of structures”.‘7.38.“X--there is a rapid neuronal cell loss associated with profuse microglial proliferation clearly apparent 1-2 days after the injection. Damage is also seen in several “distant” structures. The earliest pathological alterations appear after 30-40 min from the local injection after the animal has displayed a few limbic motor seizures and are exquisitely restricted to the CA3a,b region of the ipsilateral Ammon’s horn”.‘X7 (also see Fig. 2A,C). Patches of pyramidal neurons in CA3a (or b) and their distant apical dendrites are densely argyrophylic. The stratum lucidum where the mossy fibers terminate is in contrast vacuolated. Other parts of the ipsilateral Ammon’s horn are progressively affected: the CA3 area and hilus, then the CA1 area whereas CA2 and the granular layer of the fascia dentata are spared. In addition to the hippocampus, other “distant” areas show degeneration after intraamygdaloid KA, these are part of the limbic system,ll.l”7

The simplest explanation for the distant lesions is that KA diffuses (either directly or through the ventricle) and that a sufficient concentration of the toxin reaches vulnerable neurons (such as the CA3 pyramidal neurons) to produce directly the damage. However we have shown that administration of diazepam in doses sufficient to antagonize the limbic seizures blocked the distant damage in the CA3 region without affecting the damage at the site of injection.” It was therefore suggested that the KA brain damage following intra-amygdaloid injections had a dual aetiology: (a) local damage due to the direct toxic action of KA and (b) “distant” action mediated by the paroxysmal discharge and its associate convulsions. Other lines of supporting evidence for a major role of the epileptiform discharge in mediating the “distant” damage in the hippocampus include (e.g. Refs 8 and 12): (a) Similar metabolic and hippocampal changes can be produced following microiontophoretic applications of KA in the amygdala at doses which are not compatible with a significant diffusion to the hippo-

Limbic seizure and brain damage produced by kainic acid campus. *J*’ Also 2-5 nmol KA in the amygdala are enough to produce destruction of the vulnerable CA3 region: this concentration is similar to that found neurotoxic when applied directly into the ventricle’*’ and only twice the one which must be applied directly in the hippocampus to produce a similar damage*.6’ (b) With a lateral or an oblique approach to inject the toxin in the amygdala (i.e. conditions in which diffusion to the ventricle is unlikely) there is a similar hippocampal damage whereas injections of KA in the vicinity of the ventricle (i.e. in the septum-preoptic area’* or in the nucleus of the diagonal band36) do not readily produce this damage, in keeping with the fact that the symptomatology of the seizures produced by injections in the septum is not of the limbic type, at least with brief survival periods.‘* (c) The distribution of [3H]kainate following direct intracerebral injections in the striatum is not consistent with the hypothesis that the necrotizing action of KA in distant regions is due to diffusion.‘24,‘54 (d) The temporal pole of the hippocampus adjacent to the injected amygdala is less readily lesioned than the (distant) septal pole.” (e) There is an excellent correlation between the severity of epileptiform activity recorded in situ and the damage in the CA3 region. In an earlier study,‘* every second of the hippocampal electrographic activity was classified in five categories (normal, postictal depression, paroxysmal discharge with or without concomitant motor events and record with regular or irregular spikes); the hippocampal damage correlated significantly with the total duration of post-ictal depressions and paroxysmal discharges associated with motor events but not with other abnormalities. (f) Damage in the medial thalamus is occasionally more severe contralaterally to the injected site.“.‘* (g) Transection of the perforant path (through which the paroxysmal discharge probably propagates to the Ammon’s horn, see below) reduces post-ictal depressions and the distant but not the local damage.‘* In contrast, this procedure does not protect the hippocampal damage following intracerebroventricular injection of KA.‘20.‘22 (h) The correlations between the electrographic records, the metabolic maps and the regional distribution of neuronal damage suggest that axonal connections with the amygdala rather than proximity to the injection site play an important role. Several

*The statement made by French et aL6’ that doses used in the hippocampus in their study were as much as 1000 times lower than these previously employed in the work of Ben-Ari et al. (e.g. p. 2525) is unconvincing, since French et al. compare the lowest doses injected in the hippocampus which produce seizures and no damage, with the highest doses which produce damage when injected in the amygdala.“~12~‘87 The minimal dose producing damage in the hippocampus following local injection was 1.25 nmol (in 0.2 ~1) in the study of French

et al.t’

389

examples of this important feature of KA action have been provided in the literature following application of the toxin at various sites.s~‘2~36~‘so (i) There are major differences between the characteristics of local and distant damage, the former and not the latter is associated with a massive, early microglial proliferation” perhaps of vascular oripin.‘18 In contrast, glial proliferation in the hippocampus after injection of kainate into the amygdala is manifested 4-6 days after the injection, i.e. subsequent to the onset of neuronal damage. The direct action produced by KA on glial cells is indeed one of the most impressive and consistent effects of local damage. (j) Repetitive electrical stimulation of the perforant path, which plays a central role in mediating the damage caused in the hippocampus by intraamygdaloid KA,‘* reproduces a similar pattern of damage. 16’ The possibility that the distant damage is due to orthograde or retrograde transport of the toxin can be also excluded since it is not compatible with the time course of events especially in the CA3 region (e.g. Ref. 8). All of these observations therefore strongly suggest that following intra-amygdaloid KA, the paroxysmal discharge is generated in the entorhinal cortex where the amygdala heavily projects.‘43 This discharge will exert a powerful excitatory action on CA3 pyramidal neurons primarily via the granule cells and their mossy fibers. KAINATE-LIKEENDOTOXINS The observations that KA does not act through the predominant type of glutamic acid receptors raise the possibility that these effects could be mediated by another endogenous substance. The regional distribution of KA receptors as revealed by autoradiography in the Ammon’s horn reinforces this hypothesis. At present, the following candidates have been proposed as endogenous toxins since they reproduce some of the features of KA. Folates Ruck et al.‘49 reported that a folate derivative bound strongly to [3H]KA binding sites on rat cerebellar membranes and suggested that folates could be KA-like substances. This idea was reinforced by studies by Olney and coworkers’37 who showed that intra-amygdaloid injections of folates reproduce the seizures and distant brain damage caused by KA but not the damage at the site of injection. Other observations, however, cannot be reconciled with this scheme. In fact, recent studies have found that folates bind very weakly to [-‘H]KA binding sites in cerebellum, striatum, frontoparietal cortex and also major limbic structures including the hippocampus and amygdala (IC,, of about 2 mM to displace 20 nM KA).j7.1g5 In addition, injections of folates into the

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Y. Ben-Ari

hippocampus produce little or no motor abnormalities or pathological sequelae in contrast to KA.57 A detailed comparison of the electrographic, clinical, metabolic and histopathological sequelae of intraamygdaloid injections of folic acid and KA reveals major differences between the two syndromes’*’ and cannot support the observations of Olney et ~1.‘~’ Thus, following KA injections, the 2DG maps reveal an enhanced metabolism in the injected amygdala and adjacent piriform lobe, but the most conspicuous rise in labelling was noted in the entire frontoparietal cortex up to the cingulate region, and in the ventral thalamic complex and globus pallidus, i.e. structures not labelled following KA treatment.‘85,‘87 In keeping with this, we found a complete necrosis of the piriform lobe and a massive anoxic-ischemic type of damage in the frontoparietal cortex including status spongiosus, ischemic cell changes with or without incrustations in the superficial layers of the cortex, etc. Interestingly, several features of the damage caused by KA-in particular in the boundary zones of the frontoparietal cortex-are reminiscent of the pathological alterations induced in primates and subprimates species by anoxiceischemic episodes, notably atmospheric decompression or intracarotid air embolism,2’~‘27 conditions which are associated with convulsions. We suggest that folic acid produces those toxic actions of KA which are related to anoxic-ischemic changes caused by the convulsion but not the neuronal cell loss which is selectively and directly produced by the paroxysmal discharge in vulnerable regions (such as CA3, see below). Cholinomimetics Olney and coworkers’35” also reported that cholinomimetics injected in the amygdala caused a seizure and brain damage which is unsyndrome distinguishable from KA except that, like folates, these agents do not cause damage at the site of injection but only the distant damage. From the data presently available it would seem that the distant damage is also of the non-selective seizure-related type (see below). Quinolinic acid Schwartz and coworkers’59 have recently reported that nanomolar amounts of the tryptophan metabolite quinolinic acid, injected intracerebrally, produce axon-sparing neuronal damage. Neurochemical, clinical and pathological observations show that quinolinic acid mimics the effects of excitotoxins. Thus, the agent shares with ibotenic acid (but not KA) the ability to produce circumscribed damage with few convulsions and no distant seizure-related damage. Other observations derived from tissue culture experiments indicate that, like for the case of KA, the actions of quinolinic acid depend on the presence of normal synaptic patterns and appropriate synaptogenesis in regions where it produces a toxic effect.‘95 Further studies must be performed with this

agent in order to characterize other exogenous excitotoxins. MECHANISMS

it in the context

OF LOCAL

of

DAMAGE

Local damage includes that one produced at the site of injection or structures poorly protected by the blood-brain barrier following intracerebral or parenteral KA respectively. According to the excitotoxic hypothesis of neuronal death, interaction of KA or other excitatory amino acids with appropriate receptors (which in the initial version of the concept were assumed to be glutamatergic and located postsynaptically) produces intense depolarization. It was postulated that this depolarization will cause “energy-dependent homeostatic mechanisms to draw heavily on the ceil’s energy stores in an effort to restore ionic balance, with cell death ensuing when energy stores are exhausted or lethal alterations in the composition of the cells internal milieu have occurred”.’ In addition, glia at the site of injection are grossly oedematous and swollen, presumably because of the large increase in extracellular K + associated with the seizure. This may impair glial uptake, produce a reduction in extracellular space and contribute to neuronal damage. In keeping with the suggested sequence of events, electrophysiological studies have indeed shown that in hippocampal slices. high concentrations of KA produce intense depolarization associated with an irreversible loss of spike discharge.14’ This effect appears to depend neither on synaptic transmission’“‘~‘“’ nor on the presence of presynaptic terminals.‘“5 Extensive structure activity studies made primarily in the striatum.“” retina”’ or the arcuate region”15 suggest a relation between neuroexcitation potency and neurotoxicity. although the former has been usually assessed from extracellular studies made in different brain sites. For instance the dimethyl ester derivative of kainate or its N-acetyl derivative, both of which are electrophysiologically inactive, are devoid of neurotoxic effects at doses 40P 100 fold higher than KA itself. Strong support for the excitotoxic hypothesis comes from studies showing that KA also does not kill neurons insensitive to its excitatory actions (e.g. neurons of the mesencephalic trigeminal nucleus34,45) whereas. neurons which are readily excited by KA such as CA3 pyramidal neurons are also very vulnerable to its toxic effects.‘” Other studies however cannot be reconciled with this scheme and suggest a more complex aetiology. Thus blockade of the excitation produced by KA in cerebellar cultures does not prevent the damage.“’ Other observations suggest that the damage caused locally by KA is not explicable by the seizure discharge generated by the toxin. Thus. administration of anticonvulsants at doses sufficient to prevent the development of paroxysmal discharges, fails to prevent the local neurotoxicity of KA. In contrast, anaesthesia with y-butyrylacetone or chloralhydrate-

Limbic seizure and brain damage produced by kainic acid protects hippocampal neurons, pentobarbital suggesting that the anaesthetic and not the anticonvulsant action of drugs account for local protectionsZW (also see Ref. 12). Furthermore, a comparison of seizure and damage produced in the hippocampus by a variety of excitatory amino acids shows that the neurotoxicity poorly correlates with the neuroexcitatory effects;2~‘2’~‘y9 for instance Nmethyl-D-aspartate produces continuous electroencephalogram seizures for 2 h with little subsequent damage (ibid., also see Ref. 61). Lesion experiments suggest a dissociation between the excitatory (postsynaptic) and toxic actions (involving the presynaptic elements) of KA,‘8.‘0’.‘05and raise the possibility of different receptors for both effects.‘06 The toxicity of intrahippocampal KA depends on the presence of the septohippocampal (cholinergic) and perforant (presumably glutamater~c) pathways. ‘20.122 Interestingly, transection of the perforant path protects the granules of the fascia dentata from kainate but not from ibotenate lesions.89,‘J7 Other observations also emphasize the unique features of KA neurotoxicity which are not shared by other potent excitatory analogues. The actions of KA but not that of other excitants depends on the presence of mature synaptic patterns; thus KA but not ibotenate fails to produce damage before the end of the third week of age in the rat.25.‘3’.‘47Also, neurons are differentially susceptible to KA but uniformly vulnerable to other excitants, the damage produced by the latter depending simply on their proximity to the injection site.z.88.96.‘2’ Several neuronal populations resistant to local KA-notably the cholinergic neurons of the medial septum and the diagonal band-are readily destroyed by local ibotenate.** This suggests that the two agents exert their toxic effects by different mechanisms. To account for these observations, a number of alternative changes to the initial excitotoxic concept have been proposed. These include (a) accumulation of toxic concentrations of glutamate through inhibition of glutamate uptake by KA’O’ (e.g. also Johnston ef aLXS),(b) cooperative interaction between KA and endogenous glutamate released from presynaptic terminalsI and (c) presynaptic localization of KA receptors which would release toxic doses of glutamate and/or aspartate.56 There is however no compelling evidence in support of these mechanisms which have been challenged by other expe~menta~ observations. For instance, (a) dihydrokainate which is a more potent inhibitor of glutamate uptake is devoid of neurotoxic action,36” (b) the toxicity of KA is unaltered by coadministration of glutamate in the cerebellum63 and (c) the concentrations of KA required to release glutamate from cerebellar parailel fibers are 100 fold higher than the concentrations sufficient to kill the cells innervated by these fibers.63 Also the hypothesis that excitotoxicity is due to a presynaptic release of toxic concentrations of glutamate (or aspartatej is also handicapped by the poor

391

efficacy of these amino acids in causing damage when directly administered intracerebrally. Thus, to cause necrotic changes in the striatum, glutamate must be chronically infused for a week or more at particularly high concentrations. s-99 These high doses are assumed to be necessary in order to overcome the combined capacity of high and low affinity uptake systems. It is however not clear in this perspective how the damage would be so rapidly caused (l-2 h) following electrical stimulation or kainate administration in vulnerable regions (uide supra). Summing up, the mechanisms of the “local” damage remains to be clarified. In the opinion of the present reviewer, the local toxic action of KA is caused by a multiple and complex series of effects which are not necessarily due to a single mechanism and cannot be categorized in a single framework. The mechanism of the toxicity would differ according to the locus of injection and epileptogenicity of the injected structure, the experimental conditions (anaesthesia, concentrations, the doses used etc.). A possible direct effect of KA on the vasculature of the injected locus also deserves investigation. MECHANISMS

OF “DISTANT’* DAMAGE

This includes the damage produced in structures distant from the injection site or in structures protected by the blood-brain barrier following intracerebral or parenteral KA respectively. Two types of distant damage should be discussed separately (e.g. Fig. 2). Selective seizure-related damage

This is causally related to the propagation of seizure discharge through a wel~~haracteriz~ synaptic connection and without the involvement of deleterious effects associated with the convulsions. The evidence in favor of this type of damage is compelling for the CA3 region. Indeed, as already indicated above, the damage produced by parenteral KA in this region is not explicable by hypoxia or a mismatch between oxygen demand and blood suppl~.‘~* However a number of other structures may share with CA3 this feature, notably the claustrum, deepest layers of insular cortex (presumably claustrum insulare), lateral part of the amygdaloid complex etc. (see below). The evidence that damage to CA3 produced by parenteral or intra-amygdaloid KA is caused by the seizure has been discussed above and does not need further elaboration here. There is also evidence suggesting that seizure activity contributes to the hippocampal damage by intracerebroventricular KA.‘20.‘2) Nadler and coworkers have stressed the crucial role of the mossy fibers for the damage produced in CA3; in fact destruction of the mossy fibres provides immediate protection of the CA3 region against intracerebroventricular KA but not against direct intrahippocampal KA.‘20.‘22

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Y. Ben-Ari

Fig. 6. Delayed changes in extracellular and intracellular free-Ca2+ concentration [Ca’+] in CA2-3 region following repetitive stimulation of the fimbria. Upper [Ca?+] trace: extracellular recording with Ca’+-sensitive microelectrode. Lower [Ca2+l trace: intracellular recording with same electrode: note large fall in [Cal when electrode penetrated cell. Oscilloscope traces (at bottom) were recorded at times indicated by broken arrows. Initially &he hmbrial stimulation evoked an antidromic spike and a partly reversed inhibitory postsynaptic potential. During the 10 Hz tetanus (between arrows), there were only small fluctuations in [Ca’+] (reference barrel contained 3 M KCI), but there followed a period of post-tetanic depression, characterized by an absence of responses (including inhibitory postsynaptic potentials) to the same fimbrial stimulation. About 30s after the end of the tetanus, paroxysmal responses suddenly appeared, in the form of bursts of population spikes. This was accompanied by a huge increase in [Cal (to a peak of about 700 JIM) that lasted about 1 min. The return of [Cal to the intracellular base line level (close to 1 PM) was followed by gradual return to the inhibitory postsynaptic potential (now fully reversed). The upper [Cal trace shows a comparably delayed large fall in extracellular [Cal evoked in a second run by &hesame stimulation shortly after the intracellular recording. Data were obtained in situ, from a rat under urethane anaesthesia (K. Krnjevic, M. E. Morris and N. Ropert, unpublished observations).

Two types of mechanisms underline

the selective

can be proposed to seizure-related damage. The

first one implies large increase in jntracelluIar Ca2+ associated with the seizures; this will exceed the cell capacity to buffer the Ca?+ thus leading to an increase in intracellular free Ca*+ which, by means of proteolysis or blockade of the Ca2+ proton exchange across the mitochondrial membrane may cause neuronal damage.‘52 In support of this view, histochemical data indicate that the CA3 pyramidal cells have a limited Ca*.+ binding capacity whereas the granules of the fascia dentata and their mossy fibers are rich in Ca2+ binding proteinss3 (also see Ben-Ari’). Direct evidence in favor of this hypothesis has been recently obtained by Krnjevic and coworkers~,“~ who have determined the changes in extracellular and intracellular Ca2+ in the hippocampus during seizures with ion-sensitive electrodes. It was found that a particularly important decrease in extracellular Ca2 + occurs in the pyramidal layer of CA3 during seizures, and that this is associated with an increase in intracellular Ca*+ (Fig. 6). Other experiments suggesting the involvement of Ca*+ in excitotoxic damage have been reported.“‘.‘“’ Other observations also indicate that factor(s) perhaps exclusively localized to the mossy fibers, may be

released and produce damage during the seizures. A number of substances found in the mossy fibers deserve some discussion. For instance, the mossy fibers are rich in dynorphinio3 which conceivably could play a role in this event. Perhaps more likely is the possibility of an endogenous kainate-like compound since this region is amongst the richest in the brain in high affinity KA receptors.‘5~“4~‘88Interestingly, KA binding in the stratum lucidum (in the mossy fiber zone) and not in other hippocampal regions is decreased by Ca’+;“’ this is consistent with the observation of Beaumont et ~1.~that Ca’+ is a potent inhibitor of KA binding. Since a significant reduction in extracellular Ca*+ is readily observed during seizures, 9o this will further contribute to the toxicity by increasing the effectiveness of KA on its receptor sites. Nevertheless, it is possible that the toxic factor is not the transmitter itself but another agent released with the transmitter during severe seizure discharge. Zn2’ is a Iikely agent. Hence, (a) Zn2+ is particuIarly concentrated in the mossy fibers76 where it is found in synaptic vesicles75 (the actual concentration of Zn2+ in this region is amongst the highest in the brain3’,@). (b) Zn2+ is accumulated by the mossy fibers following parenteral administration of the

Limbic

seizure and brain damage produced by kainic acid

0 rmm

t

Time

Artificlol

393

CSF

Spontaneous

release

K+ (30 n-M) 2 min

(min)

Fig. 8. In situ release of endogeneous zinc in the Amman’s horn. A push-pull cannula was stereotaxically implanted in the CA3 zone in urethane anaesthetized animals. Zinc was measured by atomic adsorption. A spontaneous release of zinc was observed approximately twice the concentration of zinc was found in the artificial cerebrospinal fluid (CSF). A pulse of K+ (arrow, 30 mM, 2 min) caused a five-fold increase in the amount of zinc released followed by a prolonged decrease of zinc with levels close to the blank; IS-20 min elapsed before the recovery (Ben-Ari et al. *’, G. Charton, C. Rovira, Y. Ben-Ari and V. Leviel, submitted for publication).

radioactive metal.‘93 (c) There is some evidence that the population spike produced in CA3 by the activation of the mossy fibers is reduced by chelating agents or diet conditions in which zinc levels are reduced.79,‘93(d) During repetitive electrical stimulation of the fascia dentata (when CA3 damage is produced) there is a loss of zinc stain in the mossy fibers (Fig. 7, and Sloviter’68); this might be due to release of Zn2+ during the paroxysmal discharge. In a recent study, we have measured by atomic adsorption the spontaneous and evoked release of endogenous Zn2+ in the Ammon’s horn of unanaesthetized rats using push-pull cannulae stereotaxically implanted.%” It was found that when the cannula is located in the vicinity of the mossy fibers there is a significant spontaneous release of Zn2+ (two to four times the blank levels). A brief pulse of K+ (I min) produces an evoked release of Zn2+ to (maximal) levels which are two orders of magnitude above the blank. When the cannula was located in the fimbria or other parts of the Ammon’s horn there was neither spontaneous nor evoked release (e.g. Fig. 8, and G. Charton, C. Rovira, Y. Ben-Ari and V. Leviel, submitted for publication). Also, a K+ pulse applied unilaterally just prior to perfusion-fixation produced a bleaching of histologically demonstrable Zn2+ (Fig. 9 and Ref. 8a). Release of Zn ‘+ has also been found in slice preparations3”). (e) Earlier studies have also shown that intracerebral application of Zn2+ also produce seizures.30,‘40 The possible mechanisms through which Zn2+ might contribute to the damage are not yet clear in view of the large spectrum of cellular activities in which Zn2+ is involved.“3 It is however

of particular interest to note that zinc blocks very efficiently sodium-potassium adenosine triphosphatase46 and glutamate decarboxylase.‘97 Summing up, several features of the mossy fibers-CA3 synapse might cause the selective seizurerelated damage of the CA3 pyramidal neurons. Perhaps a likely sequence of events includes orthodromic generation of paroxysmal discharge in the granular layer of the fascia dentata by intra-amygdaloid KA; this causes a removal of the GABAergic inhibition in the hilar zone and thus a disinhibition of the mossy fiber input to CA3. Since the granular cells are resistant to epileptogenic procedures, they will discharge for prolonged periods. The release of an endokain will excite the CA3 pyramidal neurons by means of the postsynaptic KA receptors which might be directly involved in the control of Ca2+ channels. This is associated with a decrease in the extracellular Ca*+-and presumably an increased effectiveness of the endokain-and an increase in intracellular Ca2+ directly producing the damage. In addition, large shifts in the concentrations of zinc will contribute to kill the CA3 pyramidal neurons by unclarified mechanisms. It is probably the convergence of all these factors which underlines the particular vulnerability of the CA3 pyramidal neurons which can be destroyed by rather brief seizure episodes without an involvement of vascular or other deleterious conditions. Non -selective seizure -related damage

This is also related to the seizures and, as such, blocked by administration of anticonvulsants. How-

Y. Ben-Ari

394

ever, in contrast to the selective seizure-related damage, this is not associated with a specific synaptic connection, releasing a toxic factor but rather with more general deleterious conditions caused by the convulsions in some brain structures. This is best illustrated by the necrosis seen in the piriform region (e.g. Fig. 2) after parentera]“,‘” I” “’ or intracerebroventricular”‘.“5,“’ KA, but also intraamygdaloid folic acid.“’ The necrosis involves the entire frontobasal and temporobasal portions of the brain separated from normal cortex by the fissura rhinalis dorsally, reaching rostrally the olfactory bulb and occipital cortex. With survival times of l-3 days, the damage includes in addition to neuronal cell loss. demyelination, astroglial scaring perivenous hemorrhages and extensive vascular sprouting.‘7J As stressed recently by Sperk et al.,“” this incomplete parenchymal necrosis is a lesion typical ol anoxic-ischemic damage, although the topography 01 the lesions differ from that produced by generalized hypoxia or ischemia. The authors therefore suggested that the overactivity caused by KA in this region leads to release of water and metabolites and subsequently oedema; this by compressing drainage vessels against the skull in the front0 basal region result in local disturbance of blood flow and anoxic&schemic changes. This non-selective seizure-related damage is probably also produced in a number of other brain regions including the lateral septum, the CA1 field of the Ammon’s horn and perhaps also medial thalamic nuclei. A severe parenchymal necrosis is also produced in these regions’74 notably in CA1 which is more vulnerable to anoxic-ischemic episodes than CA3.*” Interestingly, these regions have little or uone of the features associated with the selective seizurerelated damage in CA3; for instance the lateral septum and medial thalamus are virtually devoid of high affinity specific KA receptors’5.‘h.“4.‘XX and zinc.7h RELEVANCE

Clinical

TO

HUMAN

EPILEPSY

considerations

Psychomotor or temporal lobe epilepsy or complex partial seizures as defined in the international classification”4 is seen mainly in adults.‘.“.hh Although seizure disorders frequently have their onset early in life, the seizures are more frequently of the generalized type in children younger than 15 years whereas temporal lobe epilepsy is much less frequent (21”,, as compared to 56:/, in adults@j). Furthermore since temporal lobe epilepsy is amongst the most difficult type to treat medically, surgery has become a useful form of therapy. 5’.55The symptomatology of temporal lobe epilepsy, its electrographic, metabolic and histopathological sequelae have been reviewed in earlier monographs to which the reader is refcrrc ~,24.54.64.hfi.97.151.1S5.l72.l7S This disorder is complex, has a large number of different aetiologies, includes a symptomatology which is not readily defined in

experimental animals. and i\ often a life time process with a complex interaction occurring between an initial event (occurring at birth) creating a scar$ the latter favors the occurrence of seizures which may also contribute to further brain damage. Therefore even if species difl’erences are put aside, an animal model cannot be expected to reproduce all the features of such a complex disorder. The researcher is faced with lifetime neurological disorders such as temporal lobe epilepsy v,ith the obvious necessity of adopting a very pragmatic attitude and accepting the fact that the tnodel will at best reproduce circumscribed features of the disease. Hence. I shall briefly present the aspects of human temporal lobe epilepsy which deserve particular attention as they can be partly interpreted in the framework of experimental data. The hippocur?rpu/ formution cm

trul positior2 in tcwiporirl

und am~~gdulu

occup~~

N

lohc of’ c>pikpsJ. Initial11

described by Jackson,“’ temporal lobe epilepsy is associated with a symptomatology which includes: (I) sensory symptoms (visual. olfactory. gustatory, sensations. etc. .): (2) mental symptoms (clouded consciousness, “forced thinking”, &jti VU,hallucinations, etc.); (3) visceral symptoms (epigastric sensations. chewing with salivation. oral automatisms, etc.); (4) somatomotor symptom5 (tonico-clonic movements.h’.h’.hu There is a general consensus that the temporal lobe occupies a central position in this syndrome. hence the name. Thus, although initially controversial, electroencephalographic studies using superficial and deep electrodes have shown that the clinical symptomatology is associated with the presence of paroxysmal discharge in the temporal lobe region.” More recently. the involvement or these regions is also suggested by positron emission tomography.“” Although the paroxysmal discharge is often present in the hippocampal formation~~~in keeping with its particularly tow threshold to epileptogenic procedures” the amygdaloid complex plays a more direct role in the occurrence of several symptoms notably the oro-alimentary signs.‘~hX.“7 Thus, in the case report made by Wieser,“‘h the episode began with recurring visceral symptoms. These were associated with hippocampal right discharges usually starting in the amygdala: bilateral hippocampal discharges were associated with an impairment of consciousness. Direct electrical stimulation of the amygdalabut not of the hippocampus reproduces several of these signs which closely resemble the symptoms displayed by patients during “spontaneous” seizures. Furthermore, Buser et cd.” have shown that whereas all patients display hippocampal-evoked responses to stimulation of the amygdala. an amygdaloid response to stimulation of the hippocampus is only observed in temporal lobe epilepsy, raising the possibility that the facilitation of hippocampoamygdaloid connections is instrumental for the aetiology of this disorder. This IS partIcularI\ important in view of the

Limbic seizure and brain damage produced by kainic acid anatomical evidence for the involvement of the amygdala (and not the hippocampus) in several clinical signs associated with epilepsy of the temporal lobe. In fact, in both rats and primates, the central amygdala directly projects to motor structures coordinating facial and masticatory movements; the amygdah is also in a position to directly control vegetative and autonomic function through its massive projections to the hypothalamus and brain stem centerss0s’43 (e.g. Ben-Ari’ and Refs therein). Projections from the amygdala to the striatum8’ are probably involved in some of the stereotypies associated with temporal lobe epilepsy. These anatomical observations are particularly relevant since they constitute, in the opinion of the present reviewer, a common ground for discussion between the clinician and the researcher. The temporal lobe region harbors the main pathological substrate of psychomotor epilepsy. Five decades after the observations of Bouchet and Cazauviehl” made in 18 15, Sommer made a detailed description of the “Ammon’s horn sclerosis” which consisted in pyramidal cell loss with a typical gradient of vulnerability. “* The observation that a sclerosis of the hippocampus constitutes the most frequent damage in post-mortem examination of chronic epileptics has been repeatedly confirmed (it is on average found in 50-7O’A of the cases). In the work of Margerison and Corsellis9’ and Sano and Malamud”’ the patients were clinically and electroencephalographically studied and a significant correlation between the symptoms and the subsequent brain damage was noted. The brain damage involves in addition to the hippocampus-where the most frequent degeneration affects the Sommer sector, the endfohum and in severe cases the granules of the fascia dentata-the amygdala, adjacent cortical regions, cerebellum, and midline thalamic structures.33*4’,54.64 Several of these regions are vulnerable to a variety of deleterious conditions such as febrile convulsions but also anoxia and other conditions which are not associated with convulsions. Temporal lobe seizures are particulary resistant to drug treatment and therefore surgery has been used for treatment of intractable epilepsy. With the advent of these procedures, the nature and characteristics of the lesions have been examined in more detail. Studies such as those of the Scheibels’53 (also see Brown”) have revealed the progressive time course of pathological changes. Several reports have shown that when the pathological abnormalities are removed, the patients benefit substantially from the procedure, i.e. there is general consensus that the sclerotic area contributes to the temporal lobe sei-

zures In contrast, there is less consensus amongst the clinicians on the aetiopathology of the brain damage. 33,54.64Initially Sommer”* suggested that the sclerosis was the cause of epilepsy; the studies of German pathologists notably Spielmeyer’ reversed N.S.C‘4,Z-B

395

the hypothesis and interpreted the lesion as ictal or more precisely as resulting from image hypoxic-ischemic episodes associated with the seizures. However, since epilepsy is associated with vascular dilatation and not constriction e.g. Ref. 141, other suggestions have been made including excessive increase in metabolism (i.e. a mismatch between metabolic demand and blood supply), increased permeability of the blood-brain barrier, or also increased brain volume (cerebral oedema which would lead to compression of vessels with herniation at the supratentorial region and compression of the posterior cerebral arteries). Other theories include early damage to the hippocampus due to birth or early infancy head trauma or febriles convulsions.33,h4,‘4’,‘55 Since, there is no clinical consensus for aetiopathology of the damage, animal models are useful to clarify this issue. Relevance of animal models for human temporal lobe epilepsy To bear some relevance to human temporal lobe epilepsy, the animal model must fulfill at least the following criteria: (a) the hippocampus, amygdala and other limbic structures must play a central role in the symptomatology; (b) it must reproduce a pattern of brain damage which is reminiscent of Ammon’s horn sclerosis; (c) it must reproduce the spontaneous and repetitive seizures of the temporal lobe type that are the hallmarks of epilepsy; (d) it must be relatively resistant to anticonvulsivants in parallel to human temporal lobe epilepsy’4’” in order to constitute a useful test model for developing new drug treatment. Several animal models have been used to experimentally reproduce temporal lobe epilepsy. Animal models which produce generalized tonico-clonic generalized convulsions. Parenteral administration of a number of potent convulsants such as bicuculline, allylglycine or pentetrazole, produce generalized convulsions (e.g. Refs 19, 108, 1 I I and 176, and Refs therein). These animal models however do not fulfil the above mentioned criteria. (a) Electrographic and clinical data indicate that the hippocampus and other limbic structures are not preferentially involved in these convulsions as the seizures are generalized from the onset to the entire cortical mantle (ibid.). In immobilized artificially ventilated rats, Siesjii and Abdul-Rahman’hS have shown by means of 2DG autoradiography a metabolic rise in the hippocampus; however in freely moving animals there is no rise in metabolism in hippocampus. I4 A recent study, in which a quantitative determination of 2DG in several brain regions was made, has confirmed and extended this observatiom4’ the overall picture with bicuculline in freely moving animals is that of a marked regional metabolic depression, including in the hippocampus. This also reflects the difficulties in extrapolating from immobilized preparations-on which most of the

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studies using bicuculline have been made-to the freely moving conditions. (b) In a number of studies made in immobilized artificially ventilated animals, bicuculline and similar convulsants produce h~ppocampal damage; however the lesions are quite widespread and include several non-limbic structures (superficial layers of neocortex, basal ganglia, cerebellum, etc.).9~‘08~‘W~“’ Other studies have failed to demonstrate significant hippocampal damage,14.23.52.160 In one study made in freely moving animals, damage to the basket cells of the cerebellum was the most consistent observation.‘4 Soderfeldt and coworkers have furthermore shown that after brief periods of recovery the oedema caused by the convulsants has vanished and most neurons look normal 4.170.171 (c) To the best of the present reviewer’s knowledge, “spontaneous’~ seizures are not observed after such procedures (e.g. Meldrum’08). Unless such seizures are produced-and particularly seizures of the limbic type-the relevance of these procedures to study temporal lobe epilepsy can be questioned.

Using the bicuculline model, Meldrum and coworkers initially concluded that damage to the hipp~ampus is primarily due to anoxic-Gschemic episodes.‘~~“” The vacuJization noted in several brain regions was interpreted as mitochrondrial swelling due to the anoxic events in keeping with earlier studies in which similar alterations were reproduced by anoxic episodes.2’ This view however has not been confirmed by Siiderfeidt and coworkers who showed that the vacuolization seen by light microscopy reflected dilatation of the endoplasmic re&dum and cytoplasmic vacuoles, and not mitochondrial swelling.“~“““~ In subsequent studies, Meldrum et ~1.“’ noted that the cerebellar damage was more reduced than the hippocampal one by paralyzing the animal, suggesting that the former may be causally related to the motor convulsions. Since in a later study” hypoxia surprisingly reduced-rather than enhanced as expected-the hippo~mpal damage, the authors suggested that the epileptic seizures per se could play an important role in the aetiology of the lesion. Subsequent studies in which the blood flow was measured provided further evidence that the damage was not solely due to an imbalance between metabolic demand and s~pply.‘~* These experiments have not been confirmed however by Soderfeldt et ul. using a similar preparation, since it was shown (a) that hypoxia augments rather than reduces the severity of the damagei70,‘7’and (b) the measurement of the ratio between the local cerebral blood flow and glucose consumption

indicates that a mismatch in the hippocampus probably plays an important role in the damage seen with the bicuculline model.” To summarize, procedures which produce tonico-clonic generalized convulsions have provided controversial results concerning the nature and regional distribution of the damage and the aetiology of this damage. It is the present reviewer’s opinion that the pathological sequelae noted after severe tonico-clonic generalized convulsion are explicable by the general deleterious conditions associated with the convulsions i.e. these procedures do not produce the selective seizure-related damage. To obtain compelling evidence for a causal role of the paroxysmal discharge per se it is essential: (a) to directly measure the Iocal PO,, PC02 and blood flow in vulnerable

regions in order to exclude local hypoxia as an important factor in the pathology; and (b) to define in detail the anatomical substrate of the propagation seizure activity and the possible preferential participation of a given excitatory input to the hippocampus or other vulnerable regions. Animal models which produce focal seizures with a limbic symptomatology. To produce focal-secondarily

generalized seizures with a limbic symptomatology, several experimental approaches have been used, including local injection into the amygdala of aluminum cream6’ or cobalt t ‘1y~‘26~128 Zn2+ into the lateral ventricle30,46or of ouabain into the lateral ventricle or septum. ‘39Another method is based on continuous electrical stimulation of the amygdala in previously kindled rat.““ Some of these procedures fulfil a number of the aforementioned criteria including hippocampal damage and ‘~spontaneous” seizures. The kainate model-which has been more extensively studied than the aforementioned proceduresis particularly useful for the study of the evolution, propagation and pathological consequences of epileptic discharge in the limbic system. It clearly fultils the criteria mentioned above with regard to human temporal lobe epilepsy since: (a) the hippocampus. amygdala and other limbic structures play a central role in its symptomatology’4.94”2.‘6’~‘R2(e.g. Ben-Ari’ and Refs therein); (b) the pattern of brain damage is clearly reminiscent of Ammon’s horn sclerosis with a similar gradient of vulnerability. A number of differences have been noted, however, including the neuronal cell loss in the granular layer of the fascia dentata in humans4’ whereas this is resistant in the animal models. However, cell counts were not made in these studies and in a more recent work, patches of necrosis were noted in this region after survival periods of I-2 months (L. Nitecka, E. Tremblay and Y. Ben-Ari, in preparation); (c) spontaneous seizures with a limbic symptomatology are consistently noted following parenterallg4 (Fig. 3) or intracerebral KA.27~28In keeping with this, electrophysiological studies in hippo~ampal slice suggest that the kainatelesioned hippo~ampi become epileptogeni~;s9,9’ (d) available anticonvulsants are weak against the seizures generated by KA.3’.‘79 The kainate model can shed some light on temporal lobe epilepsy in humans. For instance, the observation that selective seizure-related damage in CA3 can be produced without hypoxia or h~er~apn~a’42 suggests that restricted paroxysmal discharge may produce a selective seizure-related localized damage in the human hippocampus. Conversely, the damage noted in the human temporal cortex might be due to general disturbances associated with the seizures. If the Amman’s horn region to which the mossy fibers project is enriched in high affinity kainate receptors (present in human brain@), this may indicate that the release of an endokain from mossy fibers plays a role in the aetiopathology of the sclerosis. Autoradiographic techniques and zinc stains on human

Limbic seizure and brain damage produced by kainic acid post-mortem material can be used to study the biochemical organization of this region in normal and epileptic brains. It will be of particular interest to see whether there is also an abnormal sprouting of mossy fibers in the affected Ammon’s horn. In fact, Nadler and coworkers’*’ have recently shown that following destruction of the CA3 pyramidal layer, the mossy fibers deprived of their postsynaptic targets, establish new connections, with the granules of the fascia dentata. This new circuit increases the excitability of dentate granule cells which now become epileptogenic. Further animal studies should examine whether surgical removal of the necrotic region eliminates spontaneous seizures in temporal lobe epilepsy; there is some indication in the literature that this may be indeed the case.26 CONCLUSIONS

Kainate

is perhaps

not as useful a tool as initially

397

thought to produce axon-sparing lesions. On the other hand, studies made with kainate in the last few years reflect the considerable usefulness of KA and other excitotoxins to reproduce experimentally a number of models of interest to human pathology. As the emphasis of the present reviewer’s own research

suggests, it is the field of temporal lobe epilepsy which will particularly benefit from studies of the mechanisms of action of kainate. The use of KA undoubtedly will provide us with a better understanding of the normal and pathological functioning of the hippocampus and other limbic regions.

Acknowledgements-1

am indebted to Drs A. Nistri, M. Berger and R. Naquet for their criticism and remarks. I also acknowledge the help of Mrs D. Giraud for the bibliography. Our studies on the kainate model were supported by the Fondation pour la Recherce Medicale and INSERM (CRE 1953). I am also indebted to Dr. M. C. Demantay for her support.

REFERENCES

1. Aicardi J. and Chevrie J. J. (1970) Convulsive status epilepticus in infants and children. A study of 239 cases. Epilepsia 11, 167-197. 2. Aldinio C., French E. D. and Schwartz R. (1983) The effects of intrahippocampal ibotenic acid and its blockade by 2-amino-7-phosphonoheptanoic acid: morphological and electroencephalographic analysis. Expl Bruin Res. 51,3644. 3. Amaral D. G. and Dent J. A. (1981) Development of the mossy fibers of the dentate gyrus: I. A light and electron microscopic study of the mossy fibers and their expansions. J. camp. Neurol. 195, 51-86. 3a. Assaf S. Y. and Chung S. (1984) Release of endogenous Zn 2+ from brain tissue during activitv. Nature 308. 734-736. of brain lesions caused by 4. Atillo A., Soderfelt B., Kalimo H., Ollson Y. and Siesjo B. K. (1985) Pathogenesis experimental epilepsy. Light and electron microscopic changes in the rat hippocampus following bicuculline-induced status epilepticus. Acta neuropath., Berl. In press. 5. Bancaud J., Talairach J., Morel P. et Bresson M. (1966) La come d’Ammon et le noyau amygdalien: effets cliniques et electriques de leur stimulation chez I’homme. Revue Neural. 115, 329-352. 6. Beaumont K., Maurin Y., Reisine T. D., Fields J. Z., Spokes E., Bird E. D. and Yamamura H. I. (1979) Huntington’s disease and its animal model: alterations in kainic acid binding. Life Sci. 24, 809-816. 7. Ben-Ari Y. (1981) (ed.) (1981) The Amygdaloid Complex. Elsevier/North-Holland, Amsterdam. p. 516. 8. Ben-An Y. (1981) Epilepsy: changes in local glucose consumption and brain pathology produced by kainic acid. Adv. Biochem. Psychopharmac. 27, 385-394. and evoked release of Zn*+ in the Ammon’s 8a. Ben-Ari Y., Charton G., Leviel V. and Rovira C. (1985) Snontaneous horn of the anaesthetized rat. J. Physiol., Lond. In press. . 9. Ben-Ari Y., KmieviC K. and Reinhardt W. (1979) Hinnocamnal __ _ seizures and failure of inhibition. Can. J. Phvsiol. Pharmac. 57, 1462-1466. 10. Ben-Ari Y., KmjeviC K., Reinhardt W. and Ropert N. (1981) Intracellular observations on disinhibitory action of acetylcholine in the hippocampus. Neuroscience 6, 2475-2484. Il. Ben-Ari Y., Tremblay E. and Ottersen 0. P. (1980) Injections of kainic acid into the amvgdaloid complex of the rat: an electrographic, clinical and histological study in relation to the pathology of epilepsy: Neuroscience 5, 515-528. 12. Ben-Ari Y., Tremblav E., Ottersen 0. P. and Meldrum B. S. (1980) The role of enilentic activitv , in hiDDOCamDa1 1r --r and ~~~ “remote” cerebral lesions induced by kainic acid. Brain Res.- 191,’ 79-97. * * 13. Ben-Ari Y., Tremblay E., Ottersen 0. P. and Naquet R. (1979) Evidence suggesting secondary epileptogenic lesions after kainic acid: pre-treatment with diazepam reduces distant but local damage. Bruin Res. 165, 362-365. 14. Ben-An Y., Tremblay E., Riche D., Ghilini G. and Naquet R. (1981) Electrographic, clinical and pathological

alterations following systemic administration of kainic acid, bicuculline and pentetrazole: metabolic mapping using the deoxyglucose method with special reference to the pathology of epilepsy. Neuroscience 6, 1361-1391. 15. Berger M. and Ben-Ari Y. (1983) Autoradiographic visualization of ‘H kainic acid receptor subtypes in the rat hiDDOCamDUS. Neurosci. Leti. 39. 237-242. 16. Bdr’ger M.1 Tremblay E., Nitecka’L.and Ben-Ari Y. (1984) Maturation of kainic acid seizure-brain damage syndrome in the rat. III. Postnatal development of kainic acid binding sites in the limbic system. Neuroscience 13, 000-000. 17. Biscoe T. J., Evans R. H., Headley P. M., Martin M. R. and Watkins J. C. (1976) Structure activity relations of ^ excitatory ammo acids on frog and rat spmal neurones. Br. J. Pharmac. 58, 373-383. 18. Biziere K. and Coyle J. T. (1979) Effects of cortical ablation on the neurotoxicity and receptors binding of kainic acid in the striatum. J. Neurosci. Res. 4, 383-398. 19. Blennow G., Brierley J. B., Meldrum B. S., Siesjo B. K. (1978) Epileptic brain damage; the role of systemic factors that modify cerebral energy metabolism. Bruin 101, 687-700. 20. Bouchet C. et Cazauviehl B. (1825) De l’epilepsie consideree dans ses rapports avec I’aliination mentale. Archs ghn. Acad. 9, 510-542.

398

Y. Ben-Ari

21. Brierley J. B. (1976) Cerebral hypoxia. In Greer?fi’eld’s Neuroparhology (eds Blackwood W. and Corsellis J. A. N.), pp. 43-86. Edward Arnold Press, London. 22. Brown W. J. (1973) Structural substrates of seizure foci in the human temporal lobe. In Epilepsy: its Phenomena in Man (ed. Brazier M. A. B.), pp. 339-374. Academic Press. New York. 23. Brown W. J., Mitchell A. G. Jr., Babb T. L. and Crandall P. H. (1980) Structural and physiological studies in experimentally induced epilepsy. Expl Neural. 69, 543-562. 24. Buser P., Bancaud J. and Talairach J. (1973) Depth recording in man in temporal lobe epilepsy. In Epi1ep.s.y: its Phenomena in Man (ed. Brazier M. A. B.), pp. 67-97. Academic Press, New York. P. and Coyle J. T. (1978) Ontogenetic development of kainate neurotoxicity: correlates with 25. Campochiaro glutamatergic innervation. Proc. n&n. Acad. Sci. U.S.A. 75, 2025-2029. 26. Cavalheiro E. A., Calderazzo Filho L., Riche D.. Feldblum S. and Le Gal La Salle G. (1983) Amygdaloid lesion increases the toxicity of intrahippocampal kainic acid injection and reduces the late occurrence of spontaneous recurrent seizures in rats. Brain Res. 262, 201-207. E., Riche D. and Le Gal La Salle G. (1983) Long-term effects of intrahippocampal kainic acid in rats: 21. Cavalheiro a method for inducing spontaneous recurrent seizures. Electroenceph. clin. Neurophysiol. 53, 581-582. 28. Cepeda C., Tenaka T., Riche D. and Naquet R. (1982) Limbic status epilepticus: behavior and sleep alterations after intra-amygdaloid kainic acid microinjections in Pupio pupio baboons. Elecfroencephul. clin. Neurophvsiol. 54, 603-613. 29. Cherubini E., de Feo M., Mecardlli 0. and Ricci G. F. (1983) Behavioral and electrographic patterns induced by systemic administration of kainic acid in developing rats. Deal Bruin Res. 9, 69-77. 30. Chung S. H. and Johnson M. S. (1983) Divalent transition-metal ions (Ca” and Zn’+ ) in the brains of epileptogenic and normal mice. Bruin Res. 280, 323-334. 31. Clifford D. B., Lothman E. W., Dodson W. E. and Ferendelli J. A. (1982) IXects of anticonvulsant drugs on kaimc acid-induced epileptiform activity. ISpI Neural. 76, 156&167. 32. Collingridge G. L., Kehl R.. Cook and McLennan H. (1983) Effects of kainic acid and other amino acids on synaptic excitation in rat hippocampal slices. 1. Extracellular analysis. Expl Brain Res. 52, 170-17X. 33. Colloque sur 1es problemes d’anatomie normale et pathologique posCs par les dtcharges kpileptiques (1965). Acru neural. psychiat. Beg. 56. 34. Colonnier M., StCriade M. and Landry P. (1979) Selective resistance of sensory cells of the mesencephalic trigeminal nucleus to kainic acid-induced lesions. Bruin Res. 172, 552-556. 35. Constanti A. and Nistri A. (1976) A comparative study of the effects of glutamate and kainate on the lobster muscle fibre and frog spinal cord. Br. J. Pharmac. 57, 259-268. 36. Contestabile A. and Villani L. (1983) The use of kainic acid for tracing neuroanatomical connections in the septohabenulo interpeduncular system of the rat. J. camp. Neurol. 214, 459-469. 36a. Coyle J. T. (1983) Neurotoxic action of kainic acid. J. Neurochem. 41, 1-l 1. 37. Coyle J. T., Bird S. J., Evans R. H.. Gulley R. L., Nicklas W. J. and Olney J. W. (1981) Excitatory amino-acid neurotoxins: selectivity, specificity and mechanisms of action. Neurosci. Res. Prog. Bull. 19, l-427. 38. Coyle J. T., Molliver M. R. and Kuhar M. J. (1978) In situ injection of kainic acid: a new method for selectively lesioning neuronal cell bodies while sparing axons of passage. J. camp. Neural. 180, 301~318. 39. Coyle J. T., Zaczek R., Slevin J. and Collins J. (1981) Neuronal receptor sites for kainic acid: correlations with neurotoxicity. In Glutumutr us u Neurolrunsmirrer (eds Di Chiari G. and Gessd G. L.). pp. 337-346. Raven Press. New York. 40. Cudennec A., Duverger D., Lloyd K. G., McCulloch J. and McKenzie E. T. (1985) In Epilep.psl.und GABA Recep!or Aeonisrs: Ba.~ic und Therapeutic Reseurch (eds Bartholini G.. Bessi L.. Lloyd K. G. and Morselli P.). Raven Press, Niw York. In press. ’ 41. Dam A. M. (1980) Epilepsy and neuron loss in the hippocampus. Epilepsirr 21, 617-~629. 42. Daoud A. and Usherwood P. N. R. (1975) Action of kainic acid on a glutamatergic synapse. Comp. Biochem. Physiol. 52c, 51-53. 43. Della Bernardina B., Colamaria V., Capovilla G. and Bondavalli S. (1983) Nosological classification of epilepsies in the first three years of life. In Epilepsy: un Update on Research and Therupy (eds Nistico G.. Di Perri R. and Meinard H.), pp. 165-183. Alan Liss, New York. 44. De Montigny C., De Bonnel G., and Tardiff D. (1983) Electrophysiological studies on kainic acid induced neuronal activation. In Excitotoxins (eds Fuxe K., Roberts P. and Schwartz R.), pp. 157-169. McMillan Press, London. 45. De Montigny C. and Lund J. P. (1980) A microiontophoretic study of the action of kainic acid and putative neurotransmitters in the rat mesencephalic trigeminal nucleus. Neuroscience 5, 1621~ 1628. 46. Donaldson J., St Pierre T.. Minnich J. and Barbeau A. (1971) Seizures in rats associated with divalent cation inhibition of Na+-K’ ATPase. Can. J. Biochem. 48, 1217-1224. 47. Duce T. R., Donaldson P. L. and Usherwood P. N. R. (1983) Investigation into the mechanisms of excitant amino-acid cytotoxicity using a well-characterized glutamatergic system. Brain Res. 263, 77-87. 48. Earle K., Baldwin M. and Penfield W. (1953) Incisural sclerosis and temporal lobe seizures produced by hippocampal herniation at birth. Archs Neural. Psychiut.. Chicago 69, 27-38. 49. Engberg I., Flatman J. A. and Lambert J. D. C. (1978) The action of N-methyl-D-aspartic and kainic acid on motone&ones with special emphasis on conductance changes. Br. J. Pharmac. 64, 384. 50. Eneel J. Jr.. Brown W. J., Kuhl D. E., Phelps M. E., Mazziotta J. C. and Crandall P. H. (1982) Pathological findings underlying focal temporal lobe hypometadolism in partial epilepsy. Ann. Neurol. 12, 51&51?. 51. Engel J. Jr., Driver M. V. and Falconer M. A. (1978) Electrophysiological correlates of pathology and surgical results in temporal lobe epilepsy. Bruin Res. 98, 129-156. 52. Escobar A. and Nieto D. (1980) Absence of lesions in Ammon’s horn neurons following metrazol induced seizures in the hyperthermic rat. In Advances in Epileptology (eds Wada J. A. and Penry J. K.). pp. 494-501. Raven Press, New York. 53. Evans R. H. (1980) Evidence supporting the indirect depolarization of primary afferent terminals in the frog by excitatory amino acids. J. Physiol., Lund. 298, 25-35. 54. Falconer M. A. (1970) The pathological substrate of tempora! lobe epilepsy. Gu>,‘.s Hosp. Rep. 119, 47-60.

Limbic seizure and brain damage produced by kainic acid

399

55. Falconer M. A. and Serafetinides E. A. (1963) A follow up study of surgery in temporal lobe epilepsy. J. Neural. Neurosurg. Psychiat. 26, 1544165. 56. Ferkany, J. W. and Coyle J. T. (1983) Kainic acid selectively stimulates the release of endogenous excitatory acidic amino acids, J. Pharmac. exp. Ther. 223, 399-406. 57. Ferkany J. W., Slevin J. T., Zaczek R. and Coyle J. T. (1982) Failure of folic acid derivatives to mimic the actions of kainic acid in brain in L&O and in vitro. Neurobeh~. Toxicol. Terutol. 4, 573-580. 58. Fisher R. S. and Alger B. E. (1985) Electrophysiolo~cal action of kainic acid (KA) induced epileptiform activity in the rat hippocampal slice. J. Neurosci. In press. 59. Franck J. E. and Schwartzkroin P. A. (1983) Kainate lesioned hippocampi become epileptogenic. Sot. Neurosci. Abslr. 9, 908. 60. Frederickson C. J. (ed.) (1985) Neurobiology Zinc. A. R. Liss, New York. In press.

of

61. French E. D., Aldinio C. and Schwartz R. (1982) Intrahippocampal kainic acid, seizures and local neuronal degeneration: relationships assessed in unanesthetized rats. Neuroscienre, 7, 2525-2536. 62. Fuxe K., Roberts P. J. and Schwartz R. (eds) (1984) The Exeifofox~~s. McMillan Press, London. 63. Garthwaite J. and Wilkins G. P. (1982) Kainic acid receptors and neurotoxicity in adult and immature rat cerebellar slices. Neuroscience, 7, 2499-2514. 64. Gastaut H. (1980) L’ipilepsie temporale. Une nouvelle etude critique apres un quart de siecle. Concours med., Suppl. 15, l-48. 65. Gastaut H. and Fischer-Williams M. (1957) The physiopathology of epileptic seizures. In Handbook of Physiology -Neurophysiology, pp. 329-363. 66. Gaustaut H., Gaustaut J. L., Goncalvez Silva G. E. and Fernandez Sanchez G. P. (1975) Relative frequency of different types of epilepsy: a study employing the classification of the inte~ational ligue against epilepsy. Epjlepsiu 16,457-461. 67. Gastaut H., Vigouroux P. et Naquet R. (1952) Lesions ipileptogenes amygdalo-hippocampiques provoquees chez le chat par injection de “crime d’alumine”. Revue Neurol. 87, 607-609. 68. Gloor P., Olivier A. and Quesnay L. F. (1981) The role of the amygdala in the expression of psychic phenomena in temporal lobe seizures. In The Amygdaloid Complex (ed. Ben-Ari Y.), pp. 485498. Elsevier/North-Holland,

Amsterdam. 69. Goddard G. V., McIntyre D. C. and Leech C. K. (1969) A permanent change in brain function resulting from daily electrical stimulation. Expr Neural. 25, 295-330. 70. Goldschmidt R. B. and Stewart 0. (I 982) Neurotoxic effects of colchicine: differential susceptibility of CNS neuronal populations. Neuroscience 7, 695-714. 71. Green J. D. (1964) The hippocampus. Physiol. Rev. 44, 561-608. 72. Hall J. G., Hicks T. P. and McLennan H. (1978) Kainic acid and the glutamate receptor. Neurosci. Lett. 8, 171-175. 73. Hall J. G., Hicks T. P., McLennan H., Richardson T. L. and Wheal H. B. (lY7Y) ‘i’he excitatton ot mammaIjan central neurons by amino acids. J. Physiol., Lond. 286, 29-39. 74. Hammond C., Feger J., Bioulac B. and Souteyrand J. P. (1979) Experimental hemiballism in the monkey produced by unilateral kainic acid lesion in corpus luysii. Brain Res. 171, 577-580. 75. Haug F. M. S. (1967) Electron microscopilcal localization of the zinc in hippocampal mossy fibre synapses by a modified sulphide silver procedure. Hisiochemie 8, 3SS-368. 76. Haug F. M. S. (1973) Heavy metals in the brain. A light microscopic study of the rat with Timm’s sulphide silver method. Methodological considerations and cytological and regional staining patterns. Adv. Anat. Embryol. CeN Biol. 47, l-71. 77. Heggli D. E. and Malthe-S0renss~ D. (1982) Systemic injection of kainic acid: effect on neurotransmitter markers

in piriform cortex, amygdaloid complex and hippocampus and protection by cortical lesioning and anticonvulsants. Neuroscience 7, 125771264. 78. Henke H. and Cuenod M. (1979) L-Glutamate specific 3H kainic acid binding in the rat neostriatum after degeneration of the cortico-striatal pathway. Neurosci. Left. 979, 341-345. 79. Hesse G. W. (1979) Chronic zinc deficiency alters neuronal function of hippocampal mossy fibers. Science, N. Y. 205, 1005-1008. 80. Hopkins D. A. and Holstege G. (1978) Amygdaloid projections to the m~encephalon, pons and medulla oblongata in the cat. Bxp/ Brain Res. 32, 529-547. 8 1. Ingvar M. (1982)Regional cerebral metabolism and blood flow during experimentally induced seizures. Ph.D. Thesis, Lund. 82. Jackson J. H. (1888) On a particular variety of epilepsy (“intellectual aura”): one case with symptoms of organic disease. Brain 11, 179. 83. Jande S. S., Maler L. and Lawson E. M. (1981) Immunohistochemical mapping of vitamin D dependent calcium binding protein in brain. Nature 294, 765-767. 84. Jasper H. and Kershmann J. (1941) Electroencephalographic classification of the epilepsies. Archs Neurof. Psychiut., Chicago 45, 907-943. 85. Johnston G. A. R., Kennedy S. M. E. and Twitchin B. (1979) Action of the neurotoxin kainic acid on high affinity uptake of L-glutamatergic acid in rat brain slices. J. Neurochem. 32, 121-127. 86. Karnushina I., Susuki R., Padgett W. and Daly J. W. (1983) Degeneration of CA1 neurons in hippocampus after ischemia in mongolian gerbils: cyclic AMP systems. Brain Res. 268, 87-94.

87. Kelley A. E., Domesick V. B. and Nauta W. J. H. (1982) The amygdalostriatal projection in the rat-an anatomical study by anterograde and retrograde tracing methods. Neuroscience 7, 615630. 88. KBhler C. and Schwartz R. (1983) Comparison of ibotenate and kainate neurotoxicity in rat brain: a histological study. Neuroscience 8, 819-835. 89. Kohler C., Schwartz R. and Fuxe K. (1979) Hippocampal lesions indicate differences between excitotoxic properties of acidic amino acids. Neurosci Left. 175, 366-371. 90. Krnjevic K., Morris M. E. and Reiffenstein R. J. (1980) Changes in extracellular Ca*+ and K+ activity accompanying hippocampal discharges. Can. J. Physiol. Pharmac. 58, 5799583.

400

Y. Ben-Ari

91. Lancaster B., Wheal H. V. and Ashwood T. J. (1983) Hippocampal electrophysiology after kainic acid treatment: a chronic model of focal epilepsy. Sec. Neurosci. Absfr. 9, 908. 92. Le Gal La Salle G., Paxinos G., Emson P. and Ben-Ari Y. (1978) Neurochemical mapping of GABAergic systems in the amygdaloid complex and bed nucleus of the stria terminalis. Brain Res. 155, 397403. 93. London E. D. and Coyle J. T. (1979). Specific binding of ‘H kainic acid to receptor sites in rat brain. Molec. Pharmac. 15, 492-505. 94. Lothman E. W. and Collins R. C. (1981) Kainic acid-induced limbic motor seizures: metabolic, behavioral, electroencephalographic and neuropathological correlates. Bruin Res. 218, 299-3 18 95. Lucas D. R. and Newhouse J. P. (1976) The toxic effects of sodium L-glutamate on the inner layers of the retina. Archs Opthal., N.Y. 58, 193-204. 96. Mangano R. M. and Schwartz R. (1983) Chronic infusion of endogenous excitatory amino acids into rat striatum and hippocampus. Brain Res. Bull. 10, 47-51. 97. Margerison J. H. and Corsellis Y. A. N. (1966) Epilepsy and the temporal lobes. A clinical electroencephalographic and neuropathological study of the brain in epilepsy, with particular reference to the temporal lobes. Brain 89, 499-530. 98. Mason S. T. and Fibiger H. C. (1979) On the specificity of kainic acid. Science, N. Y. 204, 1339-1341. 99. McBean G. J. and Roberts P. J. (1984) Chronic infusion of L-glutamate causes neurotoxicity in rat striatum. Bruin Res. 294, 372-375. 100. McDonald J. F. and Porietis A. V. (1982) Two conductances activated by excitatory amino acids. Neurosci. Absir. 12, 796. 101. McGeer E. G., McGeer P. L. and Singh K. (1978) Kainate-induced degeneration of neostriatal neurons: dependency upon corticostriatal tract. Bruin Res. 139, 381-383. 102. McGeer E. G., Olney J. W. and McGeer P. L. (eds) (1978) Kainic Acid us u Tool in Neurobiology. Raven Press, New York. 103. McGinty J., Henriksen S. J., Goldstein A., Terenius L. and Bloom F. E. (1983) Dynorphin is contained within hippocampal mossy fibers, immunochemical alterations after kainic acid administration and colchicine induced neurotoxicity. Proc. natn. Acad. Sci. U.S.A. 80, 5899593. 104. McIntyre D. C., Natharison D. and Edson N. (1982) A new model of partial status epilepticus based on kindling. Brain Res. 250, 53-63. 105. McLennan H. (1980) The effect of decortication on excitatory amino acid sensitivity of striatal neurons. Neurosci. Left. 18, 3133316. 106. McLennan H., Collinridge G. L. and Kehl S. J. (1984) Electrophysiological actions of kainate and other excitatory amino acids and the structure of their receptors. In Excitotoxins (eds Fuxe K., Roberts P. and Schwartz R.), pp. 19-32. McMillan Press, London. 107. Meibach R. C., Brown L. and Brooks F. H. (1978) Histofluorescence of kainic acid-induced striatal lesions. Brain Res. 148, 219-223. 108. Meldrum B. S. (1983) Metabolic factors during prolonged seizures and their relation to nerve cell death. In Status Epilepticus Mechanisms of’ Brain Damage and Treatment (eds Delgado-Escueta A. V., Wansterlain C. G.. Treiman D. M. and Porter R. J.). Advances in Neurology, Vol. 34, pp. 261-275. Raven Press, New York. 109. Meldrum B. S. and Brierley J. B. (1973) Prolonged epileptic seizures in primates: ischemic cell changes and its relation to ictal physiological events. Archs Neural. Psychiat., Chicago 28, 10-47. 110. Meldrum B. S. and Horton R. W. (1973) Physiology of status epilepticus in primates. Archs Neural. Psychiat., Chicago 28, 1-9. II I. Meldrum B. S., Vigouroux R. A. and Brierley J. B. (1973) Systemic factors and epileptic brain damage. Prolonged seizures in paralyzed artificially ventilated baboons. Archs Neural. Psychiat., Chicago 29, 82-87. 112. Minini Ch., Meldrum B. S., Riche D., Silva-Comte C. and Stutzmann J. M. (1980) Sustained limbic seizures induced by intra-amygdaloid kainic acid in the baboon: symptomatology and neuropathological consequences. Ann. Neural. 8, 501-509. 113. Milvan A. S. (1970) Metals in enzyme catalysis. In The Enzymes (3rd Edn). Vol. II, pp. 445-536. 114. Monaghan D. T. and Cotman C. W. (1982) The distribution of ‘H kainic acid binding sites in rat CNS as determined by a&radiography. Bruin Res. 252, 91-100. 115. Monaahan D. T.. Holets V. R.. Tov D. W. and Cotman C. W. (1983) Anatomical distribution of four pharm\cologically ‘distinct ‘H L-glutamate binding sites. Nature 306, 176- 179. pyramidal 116. Morris M. E., Krnjevic K. and Ropert N. (1983) Changes in free Ca++ recorded inside hippocampal neurons in response to fimbrial stimulation. Sot. Neurosci. Abstr. 9, 395. 117. Munari C.. Bancaud J., Bonis A., Buser P., Talairach J., Szikla G. et Philippe A. (1979) Role du noyau amygdalien dans la survenue de manifestations oro-alimentaires au tours des crises tpileptiques chez l’homme. Rev. Electroenceph. Neurophysiol. 9, 236-240. 118. Murabe Y., Ibata Y. and Sano Y. (1980) Morphological studies on neuroglia. III. Macrophage response and “neuro gliocytosis” in kainic acid-induced lesions. Cell Tissue Res. 218, 75-86. 119. Mutaris R. (1967) Cobalt experimental hippocampal epilepsy. Epilepsia 8, 228-240. 120. Nadler J. V. and Cuthbertson G. J. (1980) Kainic acid neurotoxicity toward hippocampal formation: dependence on specific excitatory pathways, Brain Res. 195, 47-56. 121. Nadler J. V., Evenson D. A. and Cuthbertson G. J. (1981) Comparative toxicity of kainic acid and other acidic amino acids toward rat hippocampal neurons. Neuroscience 6, 2505-2517. 122. Nadler J. V., Evenson D. A. and Smith E. M. (1981) Evidence from lesion studies for epileptogenic and non-epileptogenic neurotoxic interactions between kainic acid and excitatory innervation. Brain Res. 205, 405410. 123. Nadler J. V., Perry B. W., Gentry C. and Cotman C. W. (1980) Degeneration of hippocampal CA3 pyramidal cells induced by intraventricular kainic acid. J. camp. Neural. 192, 333-359. 124. Nadler J. V., Shelton D. L., Perry B. W. and Cotman C. W. (1980) Regional distribution of ‘H kainic acid after intraventricular injection. L(fe Sri. 26, 1333138. 125. Nadler J. V., Tanck D. L.. Everson D. E. and Davis J. N. (1983) Synaptic rearrangements in the kainic acid model

Limbic seizure and brain damage produced by kainic acid

401

of Ammon’s horn sclerosis. In Excitotoxins (eds Fuxe K., Roberts P. and Schwartz R.), pp. 256-270. McMillan Press, London. 126. Naquet R. (1953) Sur les fonctions du rhinencephale d’aprts les resultats de sa stimulation chez I’homme. These de Doctorat d’Etat, Marseille. 127. Naquet R., Arfel G., Choux M., Dubois D. and Riche D. (1966) Etude experimentale de l’ambolie gazeuse par voie carotidienne chez le chat. Electroencepi. clin. Neurophysiol. 20, 181-196. 128. Naquet R., Tiberin P. et Vigouroux R. (1962) Rhinencephale et epilepsie experimentale, role respectifs du siege de la lesion cicatricielle primitive et des lesions secondaires de l’hippocampe. In Physiologic de I’Hippocumpe (ed. Passouant P.), pp. 120-128. CNRS, Paris. 129. Nistri A. (1985) Glutamate. In Neurotrunsmitter Action in the Vertebrate Nervous System (eds Barker J. L. and Roganski M. A.). Plenum Press, New York. In press. 130. Nistri A. and Constanti A. (1979) Pharmacological characterization of different types of GABA and glutamate receptors in vertebrates and invertebrates. Prog. Neurobiol. 13, 117-235. 131. Nitecka L., Tremblay E., Charton G., Bouillot J. P., Berger M. L. and Ben-Ari Y. (1984) Maturation of kainic acid seizure-brain damage syndrome in the rat. II. Histopathological sequelae. Neuroscience 13, 1073-1094. 132. Oldendorf W. H. (1970) Measurement of brain uptake of radiolabelled substances using a tritiated water internal substance. Brain Res. 24, 372-376. 133. Olney J. W. (1969) Brain lesion, obesity and other disturbances in mice treated with monosodium glutamate. Science, N.Y. 164, 719-721. 134 Ghiey J. W. (1978) Neurotoxicity of excitatory amino acids. In Kuinic Acid us a Tool in Neurobiology (eds McGeer E. G., Olney J. W. and McGeer P. L.), pp. 95-121. Raven Press, New York. 135 Olney J. W. and De Gubareff T. (1978) Extreme sensitivity of olfactory cortical neurons to kainic acid toxicity. In Kainic Acid as a Tool in Neurobiology (eds McGeer E. G., Olney J. W. and McGeer P. L.), pp. 201-217. Raven Press, New York. 135a. Olney J. W., De Gubareff T. and Labruyere J. (1982) Seizure-related brain damage produced by cholinergic agents. Nature 301, 520-522. 136. Olney J. W., Fuller T. and De GubarefI T. (1979) Acute dendrotoxic changes in the hippocampus of kainate treated rats. Brain Res. 176, 91-100. 137. Olney J. W., Fuller T. A. and De Gubareff T. (1981) Folates have kainate like neurotoxicity. Nufure 292, 165-167. 138. Olney J. W., Rhee V. and Ho 0. L. (1974) Kainic acid: a powerful neurotoxic analogue of glutamate. Brain Res. 77, 507-512.

139. Pealey T. A., Zuckerman E. C. and Glaser G. H. (1969) Epileptogenic effects of localized ventricular perfusion of ouabain on dorsal hippocampus. Expl Neurol. 25, 210-219. 140. Pei Y., Zhao D., Huang J. and Lao L. (1983) Zinc-induced seizures: a new experimental model of epilepsy. Epilepsiu 24, 169-176.

141. Penfield W. (1956) Epileptogenic lesions. Acta neural. psychiut. belg. 56, 75-88. 141a. Penry J. K. (1975) Perspectives in complex partial seizures. In Advances in Neurology (eds Penry J. K. and Daly 0. D.). Vol. 11, pp. 1-14. Raven Press, New York. 142. Pinard E., Ben-Ari Y., Tremblay E. and Seylaz J. (1983) Is intratissular hypoxia responsible for lesions during seizures induced by kainic acid? In Cerebral Blood Flow, Metabolism and Epilepsy (ed. Baldy-Moulinier M.), pp. 252-257. J. Libbey, London. 142a. Pinard E., Tremblay E. Ben-Ari Y. and Seylaz J. (1984) Blood flow compensates oxygen demand in the vulnerable CA3 region of the hippocampus during kainate-induced seizures. Neuroscience 13, 1039-1049. 143. Price J. L. (1981) The efferent connections of the amygdaloid complex in the rat, cat and monkey. In The Amygdaloid Complex (ed. Ben-Ari Y.), pp. 125-132. Elsevier/North-Holland, Amsterdam. 144. Puil R. (1981) L-Glutamate: its interactions with spinal neurons. Brain Res. 3, 229-322. 145. Ribak C. E., Bradbume R. M. and Basil-Harris A. (1982) A preferential loss of GABAergic, symmetric synapses in epileptic foci: a quantitative structural analyses of monkey neocortex. J. Neurosci. 2, 1725-1737. 146. Ribak C. E., Vaughn J. E. and Sato K. (1978) Immunocytochemical localization of glutamic acid decarboxylase in neuronal somata following cholchicine inhibition of axonal transport. Brain Res. 146, 315-332. 147. Roberts P. J.. McBean G. J.. Steiner M. Y.. Kohler C. and Schwartz R. (1983) Lesionine effects of ibotenate in the immature rat’ brain and protection by 2-amino-7-phosphoheptanoic acid.\ Sot.’ Neurosci.-Abstr. 9, 261. 148. Robinson J. H. and Deadwyler S. A. (1981) Kainic acid produces depolarisation of CA3 pyramidal cells in the in vifro hippocampal slice. Bruin Res. 221, 117-127. 149. Ruck A., Kramer S., Metz J. and Brennan M. J. W. (1980) Methyltetrahydrofolate is a potent and selective agonist for kainic acid receptors. Nature 287, 352-803. 150. Ruth R. E. (1981) Kainic acid lesions of the hippocampus produced iontophoretically: the problem of distant damage. Expl Neurol. 76, 508-527.

151. Sano K. and Malamud N. (1953) Clinical significance of sclerosis of the cornu Ammonis. Archs Neurol. Psychiat., Chicago 78, 40-53.

152. Schanne F. A. X., Kane A. B., Young E. E. and Forber J. L. (1979) Calcium dependence of toxic cell death: a final common pathway. Science, N.Y. 206, 700-702. 153. Scheibel M. E. and Scheibel A. B. (1973) Hippocampal pathology in temporal lobe epilepsy. A golgi survey. In Epilepsy. Its Phenomena in Man (ed. Brazier M. A. B.), pp. 311-337. Academic Press, New York. 154. Scherer-Singler V. and McGeer E. G. (1979) Distribution and persistence of kainic acid in the brain. Life Sci. 24, 1015-1022. 155. Scholz W. (1959) The contribution of pathoanatomical research to the problem of epilepsy. Epilepsia 1, 36-55. 156. Schwartz R. and Fuxe K. (1979) 3H Kainic acid binding: relevance for evaluating the neurotoxicity of kainic acid. Life Sci. 24, 1471-1480. 157. Schwartz R. and Klihler C. (1980) Evidence against an exclusive role of glutamate in kainic acid neurotoxicity. Neurosci. Lett. 19, 243-249.

158. Schwartz R., Scholcz D. and Coyle J. T. (1978) Structure activity relations for the neurotoxicity of kainic acid derivatives and glutamate analogues. Neuropharmacology 17, 1455 15 1.

402

Y. Ben-Ari

159. Schwartz R., Whetsell W. D. Jr. and Mangano R. M. (1983) Quinolinic acid: an endogenous metabolite that produces axon-sparing lesion in rat brain. Science, N.Y. 219, 316-319. 160. Schwartz I. R., Broggi G. and Pappas G. D. (1970) Fine structure of cat hippocampus during sustained seizures. Brain Rex. 18, 166-170. 161. Schwab J. E., Fuller T.. Price J. L. and Olney J. W. (1980) Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: a histological study. Neuroscience 5, 991-1014. 162. Seil F. J., Blank N. K., Woodward W. R. and Lehman A. L. (1981) Kainate effects in cerebellar cultures. Ado. Biochem. Psychopharmac. 27, 347-354. In Kainic Acid as a Tool in 163. Shinozaki H. (1981) Discovery of novel actions of kainic acid and related compounds. Neurobiology (eds McGeer E., Olney J. W. and McGeer P.). pp. 17-36. Raven Press. New York. 164. Shinozaki H. and Konishi S. (1980) Actions of several antihelminthics and insecticides on rat cortical neurons. Bruin Res. 24, 368-371. 165. Siesjij B. K. and Abdul-Rahman A. (1979) A metabolic basis for the selective status epilepticus. AC/Uphysiol. sand. 106, 377-378. 166. Simon J. R., Contrera J. F. and Kuhar M. J. (1976) Binding of )H kainic acid, an analogue of L-glutamate, to brain membranes. J. Neurochem. 26, 141-147. 167. Sloviter R. S. (1983) Epileptic brain damage in rats induced by sustained electrical stimulation of the perforant path. I. Acute electrophysiological and light microscopic studies. Bruin Res. Bull. 10, 675-698. 168. Sloviter R. S. (1984) A selective loss of Timm staining in the hippocampal mossy fiber pathway accompanies electrically-induced hippocampal granule cell seizure activity in rats. In Neurobiology q/Zinc (ed. Frederickson C. J.). A. R. Liss, New York. In press. 169. Sloviter R. S. and Damiano B. P. (1981) On the relationship between kainic acid-induced epileptiform activity and hippocampal neuronal damage. Neuropharmacology 20, 1003-101 I, 170. Soderfeldt B. (1982) Epileptic brain damage. Histopathological studies on rats with experimental status epilepticus. Ph.D. Thesis, Lund. 171. Siiderfelt B., Kalimo H., Olsson Y. and Siesjij B. (1981) Phatogenesis of brain lesions caused by experimental epilepsy: light- and electron microscopic changes in the rat cerebral cortex following bicuculline induced status epilepticus. Acta neuroputh., Berl. 54, 2 19-23 1 172. Sommer W. (1880) Erkrankung des Ammonshorn als aetiologisches Moment der Epilepsie. Arch. Psychiat. NercKrunkh. 10, 631-678. decarboxylase 173. Somogyi P. A., Smith A. D., Nunzi G., Gorio A., Takagi H. and Wu J. Y. (1983) Glutamate immunoreactivity in the hippocampus of the cat: distribution of immunoreactive synaptic terminal with special reference to the axon initial segment of pyramidal neurons. J. Neurosci. 373, 1450-1468. 174. Sperk G., Lassmann H., Baran H., Kish S. J., Seitelberger F. and Hornykiewicz 0. (1983) Kainic acid induced seizures: neurochemical and histopathological changes. Neuroscience 10, 1301- 13 15. 175. Spielmeyer W. (1927) Die pathogenesis des epileptischen krampfer. Z. Neurol. Psychiut. 109, 501-520. 176. Starzl T. E.. Niemer W. T. and Dell M. (1963) Cortical and subcortical electrical activity in experimental seizures induced by metrazol. J. Neuroputh. e-up. Neurol. 12, 262-276. C. J., Howell G. A. and Gage F. H. (1984) Cholinergic denervation-induced decrease 177. Stewart G. R.. Frederickson of chelatable zinc in mossy fiber region of the hippocampal formation. Brain Res. 290, 43-5 1. 178. Stirling R. V. and Bliss T. V. P. (1978) Hippocampal mossy fiber development at the ultrastructural level. Prog. Brain Res. 48, 191-198. and glutamate antagonists on the convulsive actions 179. Stone W. E. and Javid M. J. (1980) Effects of anticonvulsants of kainic acid. Archs inr. Phurmacodyn. Thhr. 243, 56-65. J. (1977) Localization of transmitter candidates in brain: the hippocampal formation as a model. 180. Storm-Matthisen Prog. Neurobiol. 8, 119- 129. identification of naturally occurring excitatory amino acids. In Kuinic 181. Takemoto T. (1978) Isolation and structural Acid as a Tool in Neurobiology (eds McGeer E. G., Olney J. W. and McGeer P.), pp. I-15. Raven Press, New York. features of kainic acid 182. Tanaka T., Kaijima M., Daita G., Oghani J., Yonemasu S. and Riche D. (1982) Electroclinical induced status epilepticus in freely moving cats. Microinjection to the dorsal hippocampus. Electroenceph. clin. Neurophysiol. 54, 288-300. J. (1984) Uptake of u-aspartate and L-glutamate in excitatory axon terminals in 183 Taxt T. and Storm-Mathisen and other amino acids in normal hippocampus: autoradiographic and biochemical comparison with y-aminobutyrate rats and in rats with lesions. Neuroscience 11, 79-100. kainic acid as a model of temporal lobe epilepsy. Ret,. 184. Tremblay E. and Ben-Ari Y. (1985) Usefulness of parenteral Eleclroenceph. Neurophysiol. In press. 185. Tremblav E.. Berzer M.. Nitecka L.. Cavalheiro E. and Ben-Ari Y. (1984) A multidisciplinary study of folic acid neurotoxicity: in&actions with kainate binding sites and relevance to the aetiology of epilepsy. Neuroscience 12, 5699589. E., Nitecka L., Berger M. L. and Ben-Ari Y. (1984) Maturation of kainic acid seizure-brain damage 186. Tremblay syndrome in the rat. I. Clinical, electrographic and metabolic observations. Neuroscience 13, 1051-1072. injections of kainic acid: regional 187. Tremblay E., Ottersen 0. P., Rovira C. and Ben-Ari Y. (1983) Intra-amygdaloid metabolic changes and their relation to the pathological alterations, Neuroscience 8, 299-315. 188. Unnerstall J. R. and Wamsley J. K. (1983) Autoradiographic localisation of high-affinity ‘H kainic acid binding sites in the rat forebrain. Eur. J. Pharmuc. 86, 361-371. 189. Valentino R. and Dingledine R. (198 1) Presynaptic, inhibitory effect of acetylcholine in the hippocampus. J. Neurosci. 1, 784792. 190. Van Harreveld A. and Fifkova E. (1971) Light and electron microscopic changes in central nervous tissue after electrophoretic injection of glutamate. E.xpl Molec. Path. 15, 61-81. 191. Vilani L.. Migani P., Poli A., Niso R. and Contestabile A. (1982) Neurotoxic effect of kainic acid on ultrastructure and GABAergic parameters in the goldfish cerebellum. Neuroscience 7, 25 15-2524. of striatal neurons. Life Sci. 24, 265-270. 192. Vincent S. R. and McGeer E. (1979) Kainic acid binding to membrane

Limbic seizure and brain damage produced by kainic acid

403

193. Von Euler C. (1962) On the significance of the high zinc content in the hippocampal formation. In Physiologic de I’Hippocampe (ed. Passouant P.). CNRS, Paris. 194. Watkins J. C. and Evans R. H. (1981) Excitatory amino acid transmitters. A. Reo. Pharmac. Toxicol. 21, 165-204. 195. Whetsell W. 0. W. Jr. and Schwartz R. (1983) Mechanisms of excitotoxins examined in organotypic cultures of rat central nervous system. In Exeitotoxins (eds Fuxe K., Roberts P. and Schwartz R.), pp. 207-218. McMillan Press, London. 196. Wieser H. G. (1980) Temporal lobe or psychomotor status epiiepticus. A case report. E~eetroe~ceph. cl&. ~earap~ys~a~. 48, 558-572.

197. Wu J. Y. and Roberts E. (1974) Properties of brain L-glutamate decarboxylase: inhibition studies. J. Neurochem. 23, 759-767. 198. Wuerthele S. M., Love11K. L., Jones M. Z. and Moore K. E. (1978) A histological study of kainic acid-induced lesions in the rat brain. Brain Res. 149, 489-497. 199. Zaczek R. and Coyle J. T. (1982) Excitatory amino acid analogues: neurotoxicity and seizures. ~europharmuco~ogy 21, 15-26. 200. Zaczek R., Nelson M. F. and Coyle J. T. (1978) Effects of anaesthetics and anticonvulsants acid in the rat hippocampus. Eur. J. Pharmac. 52, 323-327.

(Accepted

on the action of kainic

19 Jury 1984)

Nare added in proof. In a recent study (L. Nitecka, D. Riche, G. Ghilini and Y. Ben-Ari, in preparation) using an antibody directed against GABA, we have observed a rapid destruction of GABA-containing neurons in the instar zone after parenteral administration of KA.