Ben-Ari00 Kainic Acid Review

R E V I E W

580 TINS Vol. 23, No. 11, 2000 0166-2236/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S0166-2236(00)01659-3

ANIMAL MODELS of epilepsy do not provide all thecomplex etiologies and variety of syndromes thathave been identified in humans. Nevertheless, becauseof similar basic features, experimental models haveallowed the determination of the basic molecular andcellular mechanisms of epileptogenesis and its relationto brain damage. This is particularly exemplified in thefield of temporal lobe epilepsy (TLE). Indeed, studiesusing the kainate model of epilepsy have considerablyimproved our understanding of important issues inthe field of the epilepsies: are seizures the cause or theconsequence of brain damage? Does hyperactivity perse lead to cell loss? And, if so, how? Where are seizuresgenerated and why? Since the publication of an earliercomprehensive review1, the combined use of molecu-lar biology, patch clamp and imaging techniques aswell as novel in vitro preparations have significantlyimproved our understanding of the mechanisms ofaction of kainate. Here we compare recent and earliermodels proposed to explain how kainate generatesseizures in the hippocampus. In particular, this reviewconcentrates on the effects of kainate on the multiplefacets of GABA-mediated inhibition and their contri-bution to epileptogenesis. We propose a model inwhich kainate excites all its targets in the hippo-campus, i.e. pyramidal neurons and interneurons viadifferent kainate receptor subtypes. The excitation ofinterneurons leads to a massive increase of tonicGABA-mediated inhibition in principal cells. This,however, fails to prevent epileptiform activitiesbecause of the strong excitation of CA3 pyramidalneurons, first by the selective activation of kainatereceptors at mossy fiber synapses and second by gluta-matergic recurrent collateral synapses that have a lowthreshold for the generation of synchronized activ-ities. Thus, the generation of seizures by kainate is notcaused by a collapse of inhibition, a conclusion thathas been reinforced by observations made in chronicmodels of epilepsy in which there is no general failureof action-potential driven inhibition.

The kainate experimental animal model of TLE

Studies performed two decades ago have shown thatsystemic or intracerebral injections of kainate cause

epileptiform seizures in the CA3 region of the hippo-campus. These seizures propagate to other limbic struc-tures and are followed by a pattern of cell loss that issimilar to that seen in patients suffering from TLE1,2.

CA3 pyramidal neurons are indeed amongst themost responsive neurons to kainate in the brain,because they readily degenerate following local or dis-tal injections of kainate. However, studies using thekainate model have also shown that CA3 pyramidalneurons are highly vulnerable to network hyperactiv-ity per se and readily degenerate following recurrentseizures probably because of a sustained release of glu-tamate leading to an activation of kainate receptors.Thus, injections of kainate in structures that are distalfrom the hippocampus, at concentrations that do notdiffuse to the hippocampus, are sufficient to generatea seizure and brain damage syndrome that includesCA3 damage1. In addition, the neuronal damage inCA3 following distal injections is prevented by block-ade of seizures using diazepam injections suggestingthat this damage is caused by the repetitive activationof afferent pathways during seizures. This is furthersupported by the direct relationship between epilep-tiform activities and the extent of damage in CA3 andby the fact that lesion of hippocampal afferent path-ways abolishes most of the damage in the hippo-campus. The conclusion that damage in that region isselectively caused by seizure per se is confirmed by thedirect measure of local blood flow and oxygen con-sumption in situ, which shows that there is no imbal-ance between oxygen supply and neuronal activity(reviewed in Ref. 1). Furthermore, repetitive high-fre-quency stimulation of CA3 pyramidal neuronsinduces a selective cell loss in this region also suggest-ing that excessive activation of synaptic inputs to CA3pyramidal neurons is toxic3. Therefore, CA3 pyramidalneurons are susceptible to repetitive synchronizedactivities that leads to cell loss probably as a result ofthe sustained release of glutamate.

The CA3 region is also the hippocampal pacemakerfor the generation of synchronized activities. This islargely the consequence of the dense network of recur-rent collateral glutamatergic axons (associated to AMPA receptor-mediated synapses) that interconnect

Kainate, a double agent that generatesseizures: two decades of progressYehezkel Ben-Ari and Rosa Cossart

Studies using kainate, an excitatory amino acid extracted from a seaweed, have provided majorcontributions to the understanding of epileptogenesis.Here we review pioneering and more recentstudies aimed at determining how kainate generates seizures and, in particular, how inhibition isaltered during seizures.We focus on target and subunit-specific effects of kainate on hippocampalpyramidal neurons and interneurons that lead to an excitation of both types of neurons and thusto the parallel increase of glutamatergic and GABAergic spontaneous currents.We propose thatkainate excites all its targets,the net consequence depending on the level of activity of the network.Trends Neurosci. (2000) 23, 580587

Yehezkel Ben-Ariand Rosa Cossart

are at the INMED,INSERM U29, Parc

scientifique deLuminy, BP 13,

13273 Marseille,France.

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pyramidal neurons. More recently, extensive physiologi-cal and modeling studies have shown that activation ofeven a small percentage of recurrent excitatory collateralsynapses that interconnect pyramidal neurons is suffi-cient to generate synchronized activities4. Because ofthis feature, which is unique in the hippocampus, awide range of convulsive agents with different modes ofaction, such as bicuculline, kainate, carbachol or 4-AP,generate seizures in CA3 but not in the isolated CA1.Therefore, the CA3 region is the pacemaker for the gen-eration of synchronized activities that subsequentlypropagate to CA1 and other brain regions.

Several observations suggest that the epileptogeniceffects of kainate in CA3 are largely caused by the acti-vation of high-affinity kainate receptors that are pref-erentially expressed in the mossy fiber synaptic region.This was first suggested by the earlier observation thatin both humans and various animal species, the mossyfiber synaptic region (stratum lucidum) is enrichedwith high-affinity kainate receptors (Kd within therange of 520 nM)5,6 that can be activated even by thesmall concentrations of kainate crossing thebloodbrain barrier during seizures induced by sys-temic injections of the toxin7. Furthermore, selectivelesion by neonatal irradiation of the granule cells andtheir mossy fibers eliminates the epileptogenic effectsof kainate but not that of high K1 concentrations8

(Fig. 1b). Parallel developmental studies show that theneuronal damage induced by kainate is only observedonce granule cells and mossy fiber synapses are opera-tional, at approximately the third postnatal week1. Inaddition, in vivo9 and slice recordings1012 indicate thatlow concentrations of kainate (50250 nM) that selec-tively activate kainate and not AMPA receptors13 gen-erate seizures in CA3 pyramidal neurons that propa-gate to CA1 and to other limbic structures10,12. Evenhigh concentrations of kainate (greater than micro-molar) do not generate seizures in the disconnectedCA1 area suggesting that the epileptiform activitiesobserved in CA1 following local infusions of kainate14

are in fact generated in CA3 following diffusion of thetoxin. This has also been directly implemented in theintact hippocampus preparation in vitro15.

These studies suggest that local or systemic injec-tions of kainate first activate CA3 pyramidal neuronsvia high-affinity receptors present in mossy fibersynapses. The activation of CA3 recurrent collateralsynapses generates synchronized network-driven glutamatergic currents that propagate to other hippo-campal regions. It is important to stress that the cru-cial role of the mossy fiber synapses is also confirmedby the observations that episodes of status epilepticus,such as those generated by kainate, also lead to theformation of novel aberrant mossy fiber synapses onCA3 pyramidal cells and on granule cell neurons1618,to an increased density of kainate receptors and to areduction of seizure threshold (Fig. 1a). Therefore,seizures beget seizures because although seizuresinduce damage through neuronal hyperactivity, theneuronal hyperactivity will facilitate seizure genera-tion via the formation of novel mossy fiber synapses.

However, to unravel the precise mechanisms of theaction of kainate, it is essential to analyze its action onionotropic receptor-mediated currents and on voltage-gated currents. Studies performed primarily in CA1pyramidal neurons have reported a plethora of effectsof kainate including: (1) a reduction of the amplitude

of evoked GABAergic IPSCs and of the frequency ofminiature IPSCs (but see below) effects that arethought to facilitate seizure generation; (2) an increaseof tonic inhibition that might have opposite conse-quences; (3) a blockade of two currents that are impor-tant in regulating cell excitability: IAHP and IH (Ref. 19)and (4) a reduction of voltage-gated Ca21 currents20 andof glutamate release21 (but see below). However, the rel-evance of these effects to the epileptogenic action ofkainate is not clear as a wide range of concentrations ofkainate were used (50 nM to 27 mM) and the effects arenot consistently directly associated to kainate receptor-mediated synaptic currents. The following discussionfirst concentrates on the effects induced by low con-centrations (submicromolar) of kainate that generate

Y. Ben-Ari and R. Cossart Kainate and seizures RE V I E W

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Fig. 1. Seizures are generated in CA3 by activation of kainate receptors located at mossyfiber synapses. Seizures induce collateral sprouting of mossy fibers in the CA3 region45,(a) shows photomicrographs depicting the sprouting of mossy fibers (Timm-stained) in the CA3region of a control (left) versus an epileptic rat (right). Note the aberrant infrapyramidal bandof mossy fibres (arrows). (b) Photomicrographs of Timm-stained mossy fibers from control (left)and irradiated (right) hippocampi of an adult rat (P40). The traces below show the field-poten-tial recordings. In the non-irradiated CA3 region (left trace), bath application of kainate (KA)(300 nM) induced spontaneous and evoked epileptiform discharges. In the right trace from theirradiated CA3 region, KA (500 nM) decreased the amplitude of the field potentials and did notinduce bursts. Neonatal irradiation reduces the density of Timm-stained mossy fibers and pre-vents the epileptic action of kainate8. (c) Photomicrographs from receptor autoradiographyusing 3[H]kainate on coronal sections from wild-type (left) and GluR6 mutant mice (right).GluR6 deficient mice are less susceptible to systemic administration of kainate (20 mg kg1)than control mice as revealed by the number of mice showing seizures25, 6 out of 6 for controland 1 out of 17 for GluR6 knockout mice. (a) Adapted, with permission, from Ref. 45, (b)adapted, with permission, from Ref. 8 and (c) adapted, with permission, from Ref. 25.

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paroxysmal discharges in the hippocampus. Theseeffects are associated to synaptic currents and are, atleast in part, target and subunit selective.

Target and subunit-selective effects of kainateassociated to synaptic currents

Activation of GluR6-containing kainate receptors at mossy fibersynapses located on CA3 pyramidal neurons

Repetitive electrical stimulation of the mossy fiberpathway generates slow EPSCs in CA3 pyramidal neur-ons that are mediated by kainate receptors and notAMPA receptors because they are resistant to the selec-tive AMPA receptor antagonist GYKI53655 (Refs 22,23).

The mossy fiber synapses are located close to the somaof pyramidal neurons and should therefore generateEPSCs that will efficiently propagate to the cell bodyand its intracellular machinery. These EPSCs are notgenerated in CA1 pyramidal neurons, confirming thespecific involvement of high-affinity kainate receptorsfor synaptic transmission in CA3 pyramidal cells24.

Cloning of several kainate receptor subunits andgeneration of knockout mice have made it possible toidentify the subunit involved in kainate receptor-medi-ated synaptic currents. Thus, granule cells and CA3pyramidal cells are enriched with GluR6-containingkainate receptors and the synaptic currents generatedby the stimulation of mossy fibers, after blockade ofAMPA receptors, are eliminated in GluR6 knockouts25

(Fig. 1c). In GluR6 knockouts higher concentrations ofkainate are also required to generate seizures. Theseobservations provide direct evidence that GluR6 sub-units mediate the epileptogenic actions of kainate inCA3. Collingridge and colleagues suggested that mossyfiber synapses also include pre- and postsynaptic GluR5containing kainate receptors26. However, the selectiveGluR5 ag-onist ATPA does not generate a postsynapticcurrent in CA3 pyramidal cells26 and the mRNAsencoding GluR5 are weakly expressed in granule cellsor CA3 pyramidal cells27. In addition, these data arebased on the inhibition by a GluR5 antagonist of EPSCsthat are considered to be mediated by mossy fibersynapses. However, stimulating the fascia dentata notonly activates the mossy fibers but also activates theextensively arborized glutamatergic recurrent collater-als of the CA3 pyramidal cell axons. Because mossyfiber EPSCs are larger than recurrent collateral ones28, itwill be interesting to determine the effects of GluR5antagonists on identified mossy fiber-mediated EPSCs.

These observations and the dramatic effects of gran-ule cell and mossy fiber lesion by neonatal irradiation(see above) suggest that the epileptogenic actions ofkainate are mediated at least in part by GluR6-contain-ing kainate receptors present on mossy fiber synapses.The secondary activation of the CA1 region, the majoroutput gate from the hippocampus, leads to the propa-gation of seizures to other limbic structures, notably tothe entorhinal cortex and other cortical structures andthus to the generation of a limbic partial type ofseizure. Therefore, postsynaptic kainate receptors con-taining GluR6 subunits and located on CA3 mossyfiber synapses are key players in the generation ofseizures by kainate. In spite of this, evidence also existsfor the presence of presynaptic kainate receptors inmossy fiber synapses, but their role in the epilepto-genic effects of kainate is presently unclear (see below).Activation of GluR5 containing receptors located oninterneurons

Two recent studies have shown that applications oflow concentrations (submicromolar) of kainate in thepresence of selective NMDA and AMPA receptorsantagonists produce a massive long-lasting depolar-ization of CA1 interneurons and a powerful and sus-tained barrage of action potentials13,24. As expected,the consequence of this strong excitation of interneu-rons is an increase of the spontaneous inhibitionrecorded in CA1 pyramidal neurons (Fig. 2); indeed,an eightfold increase of the frequency of tonic IPSCswas attained using 250 nM kainate. This effect is selec-tive for interneurons because similar or higher con-centrations of kainate do not significantly depolarize

Y. Ben-Ari and R. Cossart Kainate and seizuresRE V I E W


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Fig. 2. Activation of GluR5-containing kainate receptors located on CA1 interneuronsincreases tonic GABA-mediated inhibition on pyramidal cells13. (a) Diagram showing CA1interneuron as the blue cell and the CA1 pyramidal neuron as the red cell. (b) and (c) Lefttraces shown kainate (KA) depolarization in CA1. Current clamp recordings (I holding: 0 pA)were measured in the presence of GYKI53655 (30 mM) and D-APV ( 50 mM). KA (250 nM) orATPA (1 mM), the selective agonist for GluR5-containing kainate receptors, caused a reversibledepolarization of the membrane potential and repetitive action potential firing. Right tracesshow that kainate increases tonic inhibition in CA1 pyramidal cells. Voltage-clamp recordingsof spontaneous IPSCs at the reversal potential for glutamatergic currents (Vhold: 110 mV). Inthe presence of GYKI53655 (30 mM) and D-APV (50 mM), KA (250 nM) or ATPA (1 mM)reversibly increases the spontaneous IPSCs frequency. (d) Left trace shows that the kainate-receptor-mediated EPSP evoked after stimulation (shown by arrow) in stratum radiatum in thepresence of GYKI53655 (30 mM), D-APV (50 mM) and bicuculline (20 mM) triggers a burst ofaction-potentials. Right trace shows that the EPSP evoked in CA1 pyramidal cells by stimula-tion (arrow) in s. radiatum is completely blocked by GYKI53655 (30 mM) and D-APV (50 mM).Adapted, with permission, from Ref. 13.

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CA1 pyramidal neurons. Morphological identificationof the recorded interneurons indicated that a widerange of GABAergic neurons in strata oriens, radiatumor lacunosum moleculare that project to the cell bodyor to the apical dendrites of pyramidal neurons areactivated by kainate (Fig. 3). This suggests that this isa widespread property of various interneurons types.Preliminary observations suggest that CA3 interneur-ons are also depolarized massively by kainate. Mostimportantly, kainate receptor-mediated synaptic cur-rents could be generated by electrical stimulation ofstratum radiatum in CA1 inter-neurons but not inpyramidal neurons. Kainate receptor-mediated EPSCshave slower kinetics than AMPA receptor-mediatedEPSCs in the same neurons (rise time, 6 ms versus 2ms; decay time, 30 ms versus 13 ms, respectively),which appears to be a general property of kainatereceptor-mediated synaptic currents. The mechanismsunderlying this difference are unknown, but it is pos-sible that it results from a distal distribution of kainatereceptors at the edge of the postsynaptic density.Interestingly, at low concentrations, kainate did notalter other parameters of inhibition in pyramidalneurons, including miniature and evoked inhibition(see below). Therefore, there is a network of postsy-naptic kainate receptors present in interneurons butnot in pyramidal cells. Activation of this network pro-duces a paradoxical overinhibition of the target neur-ons that might reduce seizure generation.

Cossart et al.13 also reported that the effects ofkainate are mediated in part by GluR5-containingreceptors because they could be mimicked by theselective GluR5 subunit agonist ATPA and blocked bythe relatively selective antagonist, LY293558. Mostinterneurons in stratum oriens responded to ATPA(Fig. 3). By contrast, only 20% of stratum radiatuminterneurons were affected by the GluR5 agonist,although most of them were depolarized using kainate(250 nM). Therefore, kainate excites most interneurontypes, some via GluR5 subunit-containing receptors,others via different receptor subtypes. Indeed, a recentstudy suggested that kainate was still able to depolar-ize stratum radiatum interneurons in mice that lackedthe GluR5 subunit29. Therefore, it is probable that inaddition to the GluR5-mediated network of interneu-rons, other interneurons overinhibit principal neur-ons via activation of different conformations ofkainate receptors. Nevertheless, activation of GluR5subunit-containing receptors appears to be restrictedto interneurons in CA1 (and also in other hippo-campal regions) and thus might act to reduce thepropagation of seizures and the generation of syn-chronized activities. The heterogeneity of interneurontypes results in a wide-range of selective modulationby kainate of GABA-mediated inhibition and of net-work excitability. Future studies are required to deter-mine whether the selective activation of the inhibitorynetwork is sufficient to prevent epileptogenesis.

Other effects of kainate

Presynaptic effects of kainate on the release of GABAThe observation that kainate enhances spontaneous

GABAA-mediated inhibition was unexpected becauseintuitively epileptogenesis should be associated with areduction of GABA-mediated inhibition. In fact, a col-lapse of inhibition has been repeatedly suggested tounderlie the epileptogenic effects of kainate11,14,30. Two

parameters have been examined in detail: evokedGABA-mediated IPSCs and miniature TTX-insensitiveIPSCs. However, in contrast to the clear cut effects ofkainate on tonic inhibition, these effects are contro-versial and require high concentrations of agonist(Box 1) even if they can be mimicked by glutamatereleased during repetitive high-frequency stimula-tion31. The decrease of evoked GABA-mediated inhibi-tion has been proposed to be mediated by a presynap-tic subtype of kainate receptor involving ametabotropic function32. This interpretation hasrecently been challenged in a study showing that theeffects of kainate on evoked IPSCs could be explainedby indirect mechanisms resulting from the highamount of GABA released by interneurons duringkainate application33 [(Box 1) but see also Ref. 32].

Another important point to stress is that there areseveral parameters to consider in order to evaluatethe strength of inhibition and the IPSC evoked byelectrical stimulation is not necessarily the most rel-evant. Also, the hypothesis that epileptogenesis isassociated to a general fall of the GABA-mediatedinhibitory drive has not been confirmed in acuteand chronic models of epilepsy (Box 2). Indeed, thenotion of kainate-induced decrease of inhibitiondepends upon the parameter used to assess the levelof inhibition. For example, in the most recent stud-ies examining the fate of GABA-mediated inhibitionin the kainate model of TLE, the modifications ofinhibition are specific for each inhibitory pathwayand locus-dependent within each specific pathway.Thus, measuring only one parameter might pointto a deficit of inhibition even though, globally, inhibition is enhanced. This stresses the necessity tocheck all the parameters characterizing inhibitionand not one in particular (Box 2).Presynaptic effects of kainate on the release of glutamate

Early morphological and biochemical studies sug-gest that kainate receptors are located presynapticallyon mossy fiber terminals8,17,34. Thus, the selective



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Fig. 3. Different types of interneurons tested for their sensitivity to kainate . Reconstructionsusing the Neurolucida system of four types of interneurons depolarized by kainate (cell bodyand dendrites are shown in red and axons are shown in black). (a) 100% of stratum oriensinterneurons are depolarized by kainate (250 mM) or by ATPA (the GluR5 agonist). (b) 85%of stratum radiatum interneurons are depolarized by kainate (250 nM) and 20% of stratumradiatum interneurons are depolarized by APTA (Ref. 13). Abbreviations: SO, stratum oriens;SP, stratum pyramidale; SR, stratum radiatum; SLM, stratum lacunesum moleculare.

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lesion of dentate gyrus neurons and of mossy fibersmarkedly reduced the high-affinity binding sites instratum lucidum (Fig. 1) and immunolabeling ofGluR6/7 subunits was observed in unmyelinatedaxons of the CA3 region34. Furthermore, it wasrecently shown that low concentrations of kainateaugment the presynaptic afferent volley recorded inCA3 following stimulation of mossy fibers, an effectsuggested to result from depolarization of mossy fiberaxons35. By contrast, kainate application does notaffect the frequency of miniature EPSCs recorded in

CA3 pyramidal neurons (see below and Ref. 24) evenif a selective modulation by kainate of mossy fiberminiature EPSCs, which are large amplitude eventsoccurring at a low frequency28, cannot be excluded.

Several observations suggest that different concen-trations of kainate can have opposite effects. Thus, inthe elegant study of Kamiya and Ozawa35, low concen-trations of kainate (200 nM) that generate seizures inCA3 (Ref. 36), increase the mossy fiber afferent volleybut reduce the mossy fiber EPSP. Higher concentra-tions of kainate (3 mM) reduce both the afferent volley


Kainate has repeatedly been shown to depress GABA-mediated inhi-bition in CA1 pyramidal cells, but both the mechanisms and thephysiological relevance of this effect are unclear.

(1) The disinhibitory action of kainate hypothesis relies mainlyon the decrease of the evoked IPSC amplitude by kainate. However,evoked responses cannot conclusively distinguish either a mecha-nism (pre- or postsynaptic, or direct or indirect) or a general level ofinhibition (see Box 2). First, a decrease of the amplitude of theevoked IPSCs can occur as a result of a pure postsynaptic phenom-enon [i.e. a change in postsynaptic membrane resistance (seeFig. I)], or to an indirect presynaptic phenomena such as exhaustionof the terminal, a change in the probability of transmission failurein the axon or activation of presynaptic GABAB receptors [see points(3) and (4) and Fig. I]. Thus, the strongest evidence for a presynap-tic population of kainate receptors on GABAergic terminals shouldbe based on the study of miniature IPSCs or on the study of theeffects of kainate on evoked IPSCs obtained with paired recordingsfrom connected neurons. The latter experiment has not been per-formed and the former has generated contradictory resultsac.Furthermore, the decrease in the evoked IPSC amplitude observedonly with high concentrations of kainate does not imply that thereis a reduction of GABA quantal release. Marty et al.d have shown

that a reduction of evoked IPSC can be associated with an increaseof the miniature IPSCs frequency.

(2) Reduction of evoked IPSCs by kainate requires micromolarconcentrations (Fig. I). Thus, in the study of Rodriguez-Moreno etal.c, the depressant effect of kainate on evoked IPSCs was shown tofollow a bell-shaped curve, with optimal concentrations of1030 mM. This bell-shaped curve effect was suggested to occur as aresult of the fact that low and high concentrations of kainate hin-der steady-state receptor activity: low concentrations are unable toactivate receptors and high concentrations rapidly desensitizes thereceptors. However, in the same study, kainate was bath applied for30 min, which is long enough for the receptors to be largely desen-sitized. In the study of Cossart et al.a, the reduction of evoked inhi-bition by kainate is also observed at high (greater than micromolar)but not at low (submicromolar) concentrations (Fig. I). By contrast,the target and subunit-specific effects of kainate have been reportedat low concentrations.

(3) Nicoll and co-workerse suggested that the reduction of theevoked IPSCs by kainate could be explained by indirect mechanisms:kainate increased firing in interneurons leading to an enhanced releaseof GABA. The resulting massive activation of postsynaptic GABAAreceptors augments passive shunting of the postsynaptic membrane,and the increase of GABA release activates presynaptic GABAB receptorsthat in turn depress GABA releasee. The involvement of GABAB recep-tors in the depressant action of kainate could account for themetabotropic action of kainate proposed by Lerma and co-workersf,g.

(4) An additional problem is caused by the fact that in mostexperiments the connections between CA3 and CA1 were not sur-gically removed, thus enabling the propagation of seizures fromCA3 to CA1. For example, in the pioneering study of Alger andFisherh, the reduction of the evoked IPSP occurs concomitantly withpropagated epileptiform activities.

(5) Finally, we have recently shown that activation of presynap-tic kainate receptors, selectively located at inhibitory synapses onCA1 interneurons, does not decrease but instead increases GABAquantal release (R. Cossart et al., unpublished observations).

Referencesa Cossart, R. et al. (1998) GluR5 kainate receptor activation in interneurons

increases tonic inhibition of pyramidal cells. Nat. Neurosci. 1, 470478b Frerking, M. et al. (1998) Synaptic activation of kainate receptors on

hippocampal interneurons. Nat. Neurosci. 1, 479486c Rodriguez-Moreno, A. et al. (1997) Kainate receptors presynaptically

downregulate GABAergic inhibition in the rat hippocampus. Neuron 19,893901

d Glitsch, M. and Marty, A. (1999) Presynaptic effects of NMDA in cerebellarPurkinje cells and interneurons. J. Neurosci. 19, 511519

e Frerking, M. et al. (1999) Mechanisms underlying kainate receptor-mediated disinhibition in the hippocampus. Proc. Natl. Acad. Sci. U. S. A.96, 1291712922

f Rodriguez-Moreno, A. and Lerma, J. (1998) Kainate receptor modulationof GABA release involves a metabotropic function. Neuron 20, 12111218

g Rodriguez-Moreno, A. et al. (2000) Two populations of kainate receptorswith separate signaling mechanisms in hippocampal interneurons. Proc.Natl. Acad. Sci. U. S. A. 97, 12931298

h Fisher, R.S. and Alger, B.E. (1984) Electrophysiological mechanisms ofkainic acid-induced epileptiform activity in the rat hippocampal slice.J. Neurosci. 4, 13121323

Box I. Does kainate presynaptically reduce GABA-mediated inhibition?


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Fig. I. Does kainate presynaptically reduce GABA-mediated inhibition?Kainate-induced disinhibition of CA1 pyramidal neurons requires high con-centrations of agonist13. (a) Kainate (KA) (250 nM) has no effect on the ampli-tude of the evoked IPSC in CA1 pyramidal cells or on the frequency of miniatureIPSCs (shown in cumulative probability plot, right) recorded in the presence ofGYKI53655 (30 mM) and D-APV (50 mM) (Vhold: 110 mV). (b) Increasing theconcentration of kainate to 10 mM depresses the evoked IPSC amplitude andreduces the frequency of miniature IPSCs from 60% of the CA1 pyramidal cellsrecorded. n= x cells/y.

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Box 2.The fate of inhibition in temporal lobe epilepsy

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Fig. I. The fate of inhibition in temporal lobe epilepsy. (a) Interneurons are alsorecruited during kainate-induced epileptiform discharges 17. Bath application of kainate(KA) (250 nM) in the neonatal intact hippocampal formation in vitro induces ictal activ-ity in a CA3 stratum oriens interneuron shown by current clamp recording. Differentphases of the ictal episode are marked by arrows (1,2) and shown on the traces belowon an expanded time scale. 1-interictal phase, 2-tonic oscillations. (b) The fate ofinterneurons in animal models of temporal lobe epilepsy (TLE). (i) Neurolucida recon-struction of a biocytin-labeled interneuron from a KA-treated rat. (ii) Voltage clamprecordings (cell attached configuration) of evoked and spontaneous epileptiform dis-charges in interneurons from slices of KA-treated ratsd showing that interneurons arehyperexcitable in TLE. (iii) Distribution of spontaneous firing frequencies from controland epileptic interneurons recorded in the cell attached mode showing that interneu-rons are hyperactive in TLE. Traces show typical cell attached recordings from controland TLE interneurons (right). Part (i), adapted, with permission from Ref. d.

GABAA receptor-mediated inhibition is a concept that encompassesa constellation of variables, including the tonic activity ofGABAergic interneurons, the properties of the presynapticGABAergic terminals impinging upon their target and the propertiesof postsynaptic receptors. The multiplicity of inhibitory interneu-rons types, each defining morphologically, physiologically and func-tionally distinct classesa,b adds to the difficulty in measuring inhibi-tion. In the kainic acid model of temporal lobe epilepsy (TLE),several parameters characterizing inhibition have been investigatedin two morphologically and functionally different inhibitory path-ways in the CA1 region of the hippocampus: the pathway compris-ing the class of interneurons that specifically project to the periso-matic region of CA1 pyramidal cells and which tightly control theiroutput and the pathway comprising the class that project to the den-drites and which control excitatory inputs and dendritic firingc.According to the parameter being measured, inhibition can appearunchangedd, decreasedeg or increasedgi in TLE. The following alter-ations have been reported in brain slices from epileptic animals:

(1) A reduction of synaptic and extrasynaptic GABAA receptor-mediated currents following a probable modification of the com-position of GABAA receptors subunits

d,j.(2) A deficit of GABA quantal release at perisomatic synapses and

a depletion of the reserve pool of GABA-containing vesicles consis-tent with the suggestion of an impairment of vesicular release,although the number of perisomatic GABAergic terminals on pyra-midal neurons is not modified in TLE (Ref. f).

(3) A reduction of the frequency of spontaneous IPSCs in dendriticbut not somatic recordings that is probably caused by the loss of den-dritic projecting interneuronsk.

(4) An increase of the excitability of various populations ofinterneurons, i.e. both the number and the firing frequency of spon-taneously firing interneurons are increased by 50% in TLE (Ref. l).

These observations clearly show a multiplicity of modifications,which can go in opposite directions even within a giveninhibitory pathway. However, it is possible to get an overall viewof inhibition in TLE by looking at the spontaneous GABAergiccurrents received by the soma and dendrites of pyramidal cellsduring steady state. This measurement reveals that the net flux of

Cl2 through GABAA receptors is increased by 50% in somata inTLE, i.e. the hyperactivity of perisomatic projecting interneuronsmore than compensates for the pre- and postsynaptic deficits. Bycontrast, the inhibitory drive is decreased in the dendrites, i.e. thehyperactivity of the surviving dendritic projecting interneuronsdoes not compensate totally for the loss of other populations ofdendritic projecting interneurons and for the postsynaptic deficit.This example demonstrates that the assessment of the fate of inhi-bition necessitates the evaluation of each of the parameters thatdefine inhibition and for each subspecific pathway.

Referencesa Freund, T.F. and Buzsaki, G. (1996) Interneurons of the hippocampus.

Hippocampus. 6, 347470b Parra, P. et al. (1998) How many subtypes of inhibitory cells in the

hippocampus? Neuron 20, 983993c Miles, R. et al. (1996) Differences between somatic and dendritic inhibition

in the hippocampus. Neuron 16, 815823d Esclapez, M. et al. (1997) Operative GABAergic inhibition in hippocampal

CA1 pyramidal neurons in experimental epilepsy. Proc. Natl. Acad. Sci.U. S. A. 94, 1215112156

e Buhl, E.H. et al. (1996) Zinc-induced collapse of augmented inhibition byGABA in a temporal lobe epilepsy model. Science 271, 369373

f Hirsch, J.C. et al. (1999) Deficit of quantal release of GABA inexperimental models of temporal lobe epilepsy. Nat. Neurosci. 2, 499500

g Gibbs, J.W. et al. (1997) Differential epilepsy-associated alterations inpostsynaptic GABA(A) receptor function in dentate granule and CA1neurons. J. Neurophysiol. 77, 19241938

h Nusser, Z. et al. (1998) Increased number of synaptic GABA(A) receptorsunderlies potentiation at hippocampal inhibitory synapses. Nature395, 172177

i Brooks-Kayal, A.R. (1998) Selective changes in single cell GABA(A)receptor subunit expression and function in temporal lobe epilepsy. Nat.Med. 4, 11661172

j Gibb, J.W. et al. (1996) Characterization of GABAA receptor function inhuman temporal cortical neurons. J. Neurophysiol. 75, 14581471

k Bernard, C. et al. (1997) Selective loss of GABAergic inhibition in theapical dendrites of CA1 pyramidal neurons in temporal lobe epilepsy. Soc.Neurosci. Abstr. 838.10

l Bernard, C. et al. (1999) Increased inhibitory GABAergic drive in the somaof CA1 pyramidal cells in experimental epilepsy. Soc. Neurosci. Abstr.340.10

586 TINS Vol. 23, No. 11, 2000

and the EPSP probably because of a conduction blockas a result of axonal membrane depolarization. Thisstudy not only reflects the importance of the dose ofkainate used but also the lack of direct relationshipbetween epileptogenesis and reduction of the evokedEPSP. In physiological conditions, i.e. no glutamateantagonists and intact preparations15, low concentra-tions of kainate are sufficient to generate seizures,whereas larger concentrations (micromolar doses) usu-ally produce an irreversible loss of synaptic activity,presumably as a result of cell swelling and cell death.

Kainate has also been suggested to modulate gluta-mate release in the CA1 region. Indeed, kainatereduces the release of [3H]L-glutamate from synapto-somes and the amplitude of evoked NMDA receptor-mediated EPSCs (Ref. 21). However, because this effectrequires particularly large concentrations (1100 mM)and prolonged applications (1030 min), its physio-logical relevance remains to be established.

Therefore, a presynaptic action of kainate that canreduce glutamate release from mossy fibers probablyplays an important role in mediating some effects ofkainate, but it is presently unclear how this partici-pates in the epileptogenic actions of the toxin.Other non-direct effects of kainate

Kainate has additional effects that might enhanceor reduce the excitability of pyramidal neurons.Although these early studies were carried out beforethe availability of selective AMPA receptor antagon-ists, the effects observed with low concentrations ofkainate were mediated by kainate and not AMPAreceptors because only kainate receptors are activatedwith submicromolar concentrations13. The followingeffects deserve emphasis.

(1) Low concentrations of kainate (100200 nM)facilitate the repetitive firing of CA1 pyramidal neur-ons37. This effect is mediated by the attenuation of theCa21-dependent K1 current (IAHP), which is responsiblefor the afterhyperpolarization following Na1 spikesand by the reduction of the inward rectifier K1 current(IQ) (Ref. 19).

(2) In the elegant study of Nistri and Cherubini20,kainate (50400 nM) depressed the L-type Ca21 cur-rent. This effect was prevented by intracellular dialysiswith BAPTA, suggesting that it is mediated by anincrease in the inactivation of Ca21 currents via a risein free intracellular Ca21. It is possible that such a riseof intracellular Ca21 mediates other effects of kainate.

(3) Kainate reduces postsynaptic GABAB receptor-activated K1 currents38, further stressing possible second-messenger cascades-mediated effects.

Concluding remarks

Kainate acts as a double-agent controlling thehippocampal network activity via the activation of anheterogeneous network of kainate receptors differen-tially distributed among inhibitory interneurons andexcitatory pyramidal cells. Hence, kainate in thenanomolar range generates seizures in CA3 at least inpart through the activation of GluR6-containing recep-tors localized postsynaptically at mossy fiber synapseson pyramidal cells. Kainate, at similar low concentra-tions, massively increases tonic inhibition via the acti-vation of GluR5-containing receptors localized at glu-tamatergic synapses on GABAergic inter-neurons. Thisdifferential expression of kainate receptors betweenneuronal subtypes is reminiscent of other pathways,

for example of glutamate acting on metabotropicreceptors39 or ACh acting on nicotinic receptors40.

The multiple facets of inhibition (i.e. evoked, spon-taneous or miniature) and the extremely diversifiednetwork of interneurons provide a rich repertoire ofexcitability modulations that cannot be classified sim-ply as an increase or decrease of inhibition. Thus,there is now direct evidence for two distinct forms ofinhibition on pyramidal cells41 originating from twobroad classes of interneurons: those that innervate thedendrites, control the input of the hippocampal net-work and the propagation to the soma of large calciumcurrents, and those that innervate the soma, controlthe generation of Na1 action potential and hence theoutput of the hippocampal network. Furthermore, apopulation of interneurons is specialized to innervateother interneurons42, thereby enabling a fine controlof the excitability of these cells. As the distribution ofkainate receptor subtypes appears to be age-, subunit-and target-selective, the net consequence of the acti-vation of kainate receptors will vary: an increase of theinterneuronal activity by kainate might even result ina paradoxical reduction of its epileptogenic effects.

However, because only evoked kainate responseshave been observed, the physiological conditionsunder which kainate receptors are activated are notpresently clear. Interestingly, it has been recentlyreported43 that pure kainate receptor-mediated sponta-neous PSCs (not associated to AMPA receptor-mediatedPSCs) are observed at early stages of maturation. Thissuggests a differential activation of AMPA and kainatereceptors that can be regulated by the neuronal activ-ity level or conditioned by a concomitant activation ofother transmitters or synaptic pathways. All these pos-sibilities point to a wider repertoire of modulation ofionotropic glutamatergic synapses than previouslyenvisaged. Interestingly, activation of kainate receptor-mediated PSCs in CA3 pyramidal neurons requiresbrief tetani, whereas in interneurons a single stimulusis sufficient suggesting that under physiological condi-tions postsynaptic kainate receptors on interneuronsmight be more frequently recruited than those onpyramidal cells at mossy fiber synapses. Furthermore,there are some examples where the efficacy of EPSP-spike coupling is particularly strong and precise ininterneurons resulting in an efficient feed-forwardrecruitment of interneur-ons44. If this is the case,kainate receptor-mediated EPSPs might play an impor-tant role in resetting endogenous rhythmic activitiesthat are controlled by interneurons during the genera-tion of seizures in addition to normal physiologicalconditions. It remains to determine the mechanismsresponsible for the shift of the inhibitory/excitatorybalance in the somato-dendritic compartments thatwill ultimately lead to epileptogenesis.

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2 Nadler, J.V. (1981) Minireview. Kainic acid as a tool for the studyof temporal lobe epilepsy. Life Sci. 29, 20312042

3 Sloviter, R.S. (1996) Hippocampal pathology and pathophysiologyin temporal lobe epilepsy. Neurologia 11, 2932

4 Miles, R. and Wong, R.K (1983) Single neurones can initiatesynchronized population discharge in the hippocampus. Nature306, 371373

5 Monaghan, D.T. and Cotman, C.W. (1982) The distribution of[3H]kainic acid binding sites in rat CNS as determined byautoradiography. Brain Res. 252, 91100


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6 Tremblay, E. et al. (1985) Autoradiographic localization of kainic acidbinding sites in the human hippocampus. Brain Res. 343, 378382

7 Berger, M.L. et al. (1986) Limbic seizures induced by systemicallyapplied kainic acid: how much kainic acid reaches the brain? Adv.Exp. Med. Biol. 203, 199209

8 Gaiarsa, J.L. et al. (1994) Neonatal irradiation prevents theformation of hippocampal mossy fibers and the epileptic action ofkainate on rat CA3 pyramidal neurons. J. Neurophysiol. 71, 204215

9 Debonnel, G. et al. (1990) Neurotoxic effect of domoic acid:mediation by kainate receptor electrophysiological studies in therat. Can. Dis. Wkly Rep. 16, 5968

10 Ben-Ari, Y. and Gho, M. (1988) Long-lasting modification of thesynaptic properties of rat CA3 hippocampal neurones induced bykainic acid. J. Physiol. 404, 365384

11 Fisher, R.S. and Alger, B.E. (1984) Electrophysiologicalmechanisms of kainic acid-induced epileptiform activity in therat hippocampal slice. J. Neurosci. 4, 13121323

12 Robinson, J.H. and Deadwyler, S.A. (1981) Kainic acid producesdepolarization of CA3 pyramidal cells in the vitro hippocampalslice. Brain Res. 221, 117127

13 Cossart, R. et al. (1998) GluR5 kainate receptor activation ininterneurons increases tonic inhibition of pyramidal cells. Nat.Neurosci. 1, 470478

14 Rodriguez-Moreno, A. et al. (1997) Kainate receptorspresynaptically downregulate GABAergic inhibition in the rathippocampus. Neuron 19, 893901

15 Khalilov, I. et al. (1999) Maturation of kainate-inducedepileptiform activities in interconnected intact neonatal limbicstructures in vitro. Eur. J. Neurosci. 11, 34683480

16 Cronin, J. and Dudek, F.E. (1988) Chronic seizures and collateralsprouting of dentate mossy fibers after kainic acid treatment inrats. Brain Res. 474, 181184

17 Represa, A. et al. (1987) Kainate binding sites in the hippo-campal mossy fibers: localization and plasticity. Neuroscience20, 739748

18 Nadler, J.V. et al. (1980) Loss and reacquisition of hippocampalsynapses after selective destruction of CA3CA4 afferents withkainic acid. Brain Res. 191, 387403

19 Gho, M. et al. (1986) Kainate reduces two voltage-dependentpotassium conductances in rat hippocampal neurons in vitro.Brain Res. 385, 411414

20 Nistri, A. and Cherubini, E. (1991) Depression of a sustainedcalcium current by kainate in rat hippocampal neurones in vitro.J. Physiol. 435, 465481

21 Chittajallu, R. et al. (1996) Regulation of glutamate release bypresynaptic kainate receptors in the hippocampus. Nature379, 7881

22 Castillo, P.E. et al. (1997) Kainate receptors mediate a slow post-synaptic current in hippocampal CA3 neurons. Nature 388, 182186

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27 Bahn, S. et al. (1998) Kainate receptor gene expression in thedeveloping rat brain. J. Neurosci. 14, 55255545

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29 Bureau, I. and Mulle, C. (1998) Potentiation of GABAergicsynaptic transmission by AMPA receptors in mouse cerebellarstellate cells: changes during development. J. Physiol.509, 817831

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AcknowledgementsThe authors areindebted toC. Bernard,M. Esclapez andJ.C. Hirsch for theirmajor contributionto most of theresults reviewed inthis paper and fortheir helpful comments on themanuscript.

B O O K R E V I E W S

Most theoretical articles and books onpain begin nowadays with the official IASPdefinition of pain: an unpleasant sensoryand emotional experience associated withactual or potential tissue damage, ordescribed in terms of such damage1. Theythen go on to agree with the definition andproceed with their discussion. DonaldPrice in his book Psychological Mechanismsof Pain and Analgesia follows a differenttack. He disagrees with the definition,arguing that it does not emphasize the

experiential nature of pain. He proposes anew definition: pain is a somatic perceptioncontaining (1) a bodily sensation with qualitieslike those reported during tissue-damagingstimulation, (2) an experienced threat associ-ated with this sensation, (3) a feeling ofunpleasantness or other negative emotionbased on this experienced threat.

Notice how much Price packs into hisdefinition of pain. Each unit of pain con-tains the sensation of pain itself, the feelingthat one is somehow being threatened and

a negative affective reaction to the sen-sation and feeling. However, this is toomuch, for two reasons. First, we can dis-sociate the negative affective reactionsfrom pain sensations, either pharmacologi-cally using, for example, fentanyl, or bio-logically using, for example, a frontal lobot-omy. Understanding and treating theconcomitant emotional reactions to painsensations are important and Price is cor-rect in stating that scientists and cliniciansdo not pay enough attention to this aspectof pain patients. However, acknowledgingthese facts does not make reactions topain part of pain itself.

Psychological Mechanisms of Pain and Analgesiaby Donald D. Price, IASP Press, 1999. $69.00 (xiii 1 248 pages) ISBN 0 931092 29 9

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Ben-Ari00 Kainic Acid Review