iron-induced experimental cortical seizures: electroencephalographic mapping of seizure spread in...

Iron-induced experimental cortical seizures:Electroencephalographic mapping of seizurespread in the subcortical brain areas

Varsha Sharma a,*, P. Prakash Babu b, Arun Singh a,Sangeeta Singh a,c, Rameshwar Singh a

Seizure (2007) 16, 680—690

www.elsevier.com/locate/yseiz

aNeurobiology Laboratory, School of Life Sciences, Jawaharlal Nehru University,New Delhi 110067, IndiabDepartment of Biotechnology, University of Hyderabad, Hyderabad 500046, Andhra Pradesh, IndiacDepartment of Zoology, Bareilly College, Bareilly 243001, India

Received 16 June 2005; received in revised form 6 May 2007; accepted 23 May 2007

KEYWORDSEpilepsy;Iron epilepsy;Electroencephalo-graphy;Multi-unit activity;Ethosuximide;Mapping ofintracerebral epilepticactivity

Summary The iron-induced model of post-traumatic chronic focal epilepsy in ratswas studied by depth-electrode mapping to investigate the spread of epileptiformactivity into subcortical brain structures after its onset in the cortical epileptic focus.Electrical seizure activity was recorded in the hippocampal CA1 and CA3 areas,amygdala and caudate-putamen, in rats with iron-induced chronic cortical focalepilepsy. These experiments showed that the epileptiform activity with its onset inthe cortical focus synchronously propagated into the studied subcortical brain areas.Seizure behaviours seemed to increase in correspondence with the spread of theepileptic electrographic activity in subcortical areas. Comparison of the cortical focuselectroencephalographic and associatedmultiple-unit action potential recordings withthose from the subcortical structures showed that the occurrence and evolution of theepileptiform activity in the subcortical structures were in parallel with that in thecortical focus.The intracerebral anatomicprogressionanddelineationof seizure spread(mapped by field potential (EEG) and multiple-unit action potentials (MUA) recordings)indicated participation of these regions in the generalization of seizure activity in thismodel of epilepsy. The seizure-induced activation of the hippocampus appeared toevolve into an epileptic focus independent of the cortical focus. The present studydemonstrates the propagation of epileptic activity from the cortical focus into thelimbic and basal ganglia regions. Treatment of iron-induced epileptic rats with etho-suximide, an anti-absence drug, resulted in suppression of the epileptiform activity inthe cortical focus as well as in the subcortical brain areas.# 2007 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

* Corresponding author. Present address: Neurobiology Laboratory, 118 and 303, School of Life Sciences, Jawaharlal Nehru University,New Delhi 110067, India. Tel.: +91 9899008982.

E-mail address: [email protected] (V. Sharma).

1059-1311/$ — see front matter # 2007 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.seizure.2007.05.012

mailto:[email protected]

http://dx.doi.org/10.1016/j.seizure.2007.05.012

Iron-induced experimental cortical seizures 681

Introduction

Iron, in the form of FeCl2 or FeCl3, injected focally inthe cerebral cortex of rats produces a spontaneouslydischarging chronic epileptogenic focus.1 Thisexperimental epileptogenic focus constitutes anappropriate model of human post-traumaticepilepsy (complex partial seizures resulting fromtraumatic brain injury),2 and has often been inves-tigated to understand the mechanism of clinicalpost-traumatic epilepsy.1,3,4—7 The epileptiformelectrographic activity in the iron-induced focus isthought to result from the reaction of cortical neu-rons to iron-induced oxidative stress, i.e. the neu-ronal membrane lipid-peroxidation caused byreactive oxygen species.3,8,9 In humans, traumaticbrain injury (resulting from closed head injury) isknown to be a risk factor for subsequent develop-ment of clinical post-traumatic epilepsy. In theinjured brain tissue, hemolysis of extravasatedblood cells results in the deposition of iron withinthe brain tissue. The iron is thought to induceoxidative stress that may be responsible for post-traumatic epileptogenesis.4 In an iron-induced epi-leptic focus, the astrocytal uptake of glutamic acidwas found to be disrupted. This disruption appearedto be due to an oxidative stress-induced decrease inglial glutamate transporter protein.2 Decreasedlevels of the transporter protein would lead toincreased levels of extracellular glutamate whichis likely to contribute to epileptogenesis. Increasedlevels of excitatory amino acids were found to beassociated with traumatic brain injury.10,11 Acti-vated astrocytes themselves were also found torelease glutamate in experimental seizures.12

In FeCl3-induced epilepsy, the focal epileptiformactivity spreads from its site of origin into the entirecerebral cortex of both the cerebral hemi-spheres.13,14 In the process of this generalizationof seizure activity, multiple subcortical areas arelikely to operate as a network in the elaboration andexacerbation of spike—wave seizures after theirinitiation in the cortical focus.15 Identification ofsubcortical brain regions involved is necessary forunderstanding the process of seizure generalizationin the iron-model of epilepsy.16—18 Stereo-encepha-lographic [depth electroencephalographic (EEG)]studies will be needed to determine the subcorti-cal brain circuits or structures likely to beinvolved.14,17,19 Our initial study of iron epilepsyhas shown that the cortical focal epileptic activityis propagated to the thalamus, locus coeruleus andsubstantia nigra.20,21 The purpose of the presentstudy was to determine whether the subcorticalspread of the epileptiform activity involves someother brain regions.

In this paper, with a view to map the seizureprogression to various subcortical brain areas and tofurther characterize the iron-model of focal epi-lepsy, we have examined the hippocampus (CA1and CA3 subfields), amygdala and striatum (cau-date-putamen) by simultaneously recording theelectroencephalographic epileptic activity andmultiple-unit action potentials (MUA) from thesestructures. We also tested the effect of an antie-pileptic drug ethosuximide on the epileptic elec-trographic activity in this model. Ethosuximide hasbeen reported22 to desynchronize the hypersyn-chronizing electrophysiological activity in the reti-culo-thalmocortical circuit. This action of the drugmay be particularly responsible for its anti-absenceeffect in humans.22 Since in the iron-induced cor-tical focal epilepsy, synchronization of cortical andthalamic activity is involved,20 it would be of inter-est to see if ethosuximide is effective against theiron-induced epileptiform electrophysiological sei-zure activity. Recently, the hypothesis of the sub-cortical origin of absence seizures has beenchallenged,23,24 and it appears that a focal seizureinitiation site for the absence seizures is present inthe cerebral cortex rather than in the thalamus.Furthermore, ethosuximide microinfused into theperioral region of the primary somatosensory cortexof rats with genetically determined absence sei-zures has been found to abolish absence seizures.23

Ethosuximide’s effect on iron-induced seizureswould also be of further interest because the ironinduction of epilepsy is mediated by oxidativestress-induced lipid-peroxidation, and the drugethosuximide being a calcium channel antagonistmay have an anti-lipidperoxidative effect.25

Animals and drugs

Male Wistar rats (60) weighing 250—300 g werehoused individually in plastic cages in an air-condi-tioned room in the University animal house andmaintained on a 12-h light:12-h dark cycle, withfood and water available ad libitum.

FeCl3, urethane, ethosuximide and all other che-micals needed were purchased from Sigma ChemicalCompany (USA).

Surgical procedures

All experimental protocols were approved by theJawaharlal Nehru University, Institutional AnimalEthics Committee (IAEC).

Rats were anaesthetized with ketamine (50 mg/kg) for the duration of the surgery and placed in a rat

682 V. Sharma et al.

stereotaxic apparatus (Narishige, Japan). The skullsurface was exposed by a longitudinal midline inci-sion in the scalp. The scalp was retracted to the leftand right. A burr hole was made over the somato-sensory cortex at co-ordinates: relative to bregma(1mm posterior and 2 mm lateral to bregma at adepth of about 1.0 mm ventral to dura according tothe methods described earlier.5 The epileptogenicfocus was produced by a unilateral intracorticalinjection of FeCl3 (5 ml 100 mM) through the burrhole using a microliter Hamilton syringe held inposition by the stereotaxic electrode carrier. In fiverats the epileptogenic focus was produced in theright and in the other five in the left cerebral cortex.In control rats the same volume of saline wasinjected in place of FeCl3

5 to produce a sham focus.For electrocorticography, experimental and controlrats were chronically implanted with four screw orwire electrodes (Plastics one USA) in the parietalbone bilaterally, two in each parietal bone at co-ordinates (at and 3 mm posterior to bregma, 2 mmlateral to midline and 1.5 mm ventral to dura). Oneelectrode was placed at midline in the frontal boneto be used as a reference electrode. For stereoen-cephalography (depth EEG), these rats were alsoimplanted with bipolar intracerebral electrodes(obtained from Plastics one USA) in CA1 and CA3subfields of the hippocampus, amygdala and cau-date-putamen (stereotaxic co-ordinates, respec-tively, were: (CA1) 5.3 mm posterior and 3 mmlateral from bregma and 3 mm ventral to dura;(CA3) 3.8 mm posterior and 4 mm lateral frombregma and 5.5 mm ventral to dura; (amygdala),�1.3 mm anterior and 3.75 mm lateral and 8.75 mmventral to dura, and (caudate-putamen) 0.2 mmposterior, 3 mm lateral and 5 mm ventral to dura).In five animals, electrodes were implanted in theright cerebral hemisphere and in the other five, inthe left cerebral hemisphere. In order to check thatthere were no animals in this litter of rats showingspontaneous epileptiform activity, cortical andintra-cerebral recordings were also obtained fromfive normal rats (neither made epileptic by FeCl3 norinjected with saline (uninjected controls). Animalswere allowed to recover from surgery for 3 days. Inaddition to field potential recordings (electroence-phalographically recorded potentials), multiple-unit potentials (MUA) were also simultaneouslyrecorded from the five sites. This was done to verifythat there was an epileptic activity-associatedincrease in the neuronal firing at the recording sites.The MUA recording also ensured that the recordedstereoencephalographic epileptic activity repre-sented real epileptogenic EEG signals and cellularactivity changes rather than the accidentallyrecorded field potentials that might have spread

(volume conducted) from the nearby structures.Multiple-unit activity was amplified, filtered (GrassModel P511J or 7P511L amplifier, 300 Hz to 10 kHz),and electronically discriminated using a windowdiscriminator (WPI) and displayed on a storageoscilloscope. The standard pulses from the windowdiscriminator together with EEG activity were simul-taneously recorded on the polygraph as reportedearlier.26

Electrobehavioural seizure assessment

Electrocorticographic (ECoG) events showing a sud-den conspicuous increase in voltage (amplitude) andstanding out distinct from the background activitywere considered epileptic. The behaviour of animalsconcurrent to the epileptiform ECoG events wasstudied, and temporal changes in the behaviouralseizure severity were carefully observed accordingto Racine’s scale.27 Although synchronized video-EEGmonitoring (recording) of seizure behaviour wasnot performed, while observing concurrent beha-viour of epileptiform EEG events a limited number ofvideo photographs of animals for potentially epilep-tiform EEG events were taken by means of a digitalPanasonic camera (B/W 1/3 in 8 mm lens) and infra-red illumination (Tristate IR2) appropriatelymounted in the recording cage. Electrophysiologicalrecordings were obtained from conscious, unrest-rained experimental and control rats using a GrassPolygraph/EEG. Animals were allowed to acclima-tize to the recording chamber for 30 min beforecollecting experimental recordings. Animal’s beha-viour was critically checked for movement artefactsin the recordings. Epileptiform activity wasrecorded both monopolarly and bipolarly from ipsi-lateral and contralateral sites daily (between 11:00and 15:00 h) from day 3—5 after intracortical injec-tion. Development and progression of the epilepticactivity was followed for 3 months after the corticalFeCl3 or saline injection. Each recording sessionlasted for 4 h without interruption. Occurrence ofthe epileptiform EEG activity during waking periodsof (passive or, quiet wakefulness) was assessed.26

Recordings were also obtained from the epilepticanimals anaesthetized with urethane to control forthe appearance of movement artefacts in EEGrecordings.17,28,29 Electrode placements were con-firmed histologically using standard procedures(Fig. 6).20

For determining the effect of the drug ethosux-imide on iron-induced electrical seizure activity, thedrug (dissolved in physiological saline) was adminis-tered intraperitoneally (50 mg/kg) to epileptic rats.The effect of a single dose of the drug on the


epileptic EEG activity was determined in epilepticanimals on day 3,10,15 and 20 post FeCl3 intracor-tical injection. At each of these time points, thedrug effect was assessed in five animals. Besidesthis, the drug was given in single daily doses forconsecutive 12 days to five animals at day 10 afterFeCl3 intracortical injection and to another fiveanimals at day 20 after FeCl3 intracortical injection.In each case an equal number of control animalsreceived saline injection.

Figure 1 A representative EEG recording from controlcortical focus (sham) and intracerebral sites ipsilateral toit at day 8 post saline injection in the cortical site. CorticalEEG recording was obtained with one electrode at theperilesion site and the other at the reference point.Recording from the intracerebral sites were obtained bythe bipolar electrodes at the given sites.

Results

The epileptic ECoG events determined by changes inwaveform morphology and amplitudes comprisedisolated spikes, polyspikes, spike wave complexesand sharp waves. Behavioural seizure (concomitantwith epileptic ECoG events) consisted of only pausesin ongoing behaviour (exploratory behaviour) with-out loss of posture or additionally showed facialmovements (twitching of vibrissae, jaw automa-tisms), mild head noddings, tonic flexion concur-rently with biting and chewing of hindlimbextremity. Control rats did not show any electro-graphic and behavioural seizure activity in their EEGthrough the course of these experiments. The spon-taneous EEG activity in the control cortical focus,and the subcortical areas ipsilateral to it: hippo-campal CA1 subfield, amygdala, and striatum con-sisted of a low voltage fast activity pattern, whereasin the hippocampal CA3 subfield the activity wascomparatively of a higher voltage faster frequencypattern (Fig. 1). All rats that received intracorticaliron injection became epileptic. Distinct epilepti-form activity was readily observed chronically in thecortical and depth recordings from animals duringtheir wake behaviour around day 8 post iron injec-tion and onwards. Between day 3 and 8 only veryminor epileptiform seizure activities seemed toappear in the EEG records (Fig. 2A).

At day 8 post iron injection, the cortical focusshowed frequent electrical paroxysms consisting ofspike—wave complexes (Fig. 2B). Similar paroxysmswere evident in the recordings from hippocampalCA1 and CA3 subfields, and caudate-putamen area.The seizure activity in the recording from the amyg-dala consisted of comparatively low amplitudespike—wave complexes. Comparison of the seizureactivity in various leads showed that the epilepticactivity from the cortical focus had spread simulta-neously to all the subcortical sites. At this time point(day 8), the ECoG burst seizure activity appeared tobe accompanied by pauses in ongoing exploratorybehaviour without loss of posture. Facial automa-tisms were very scarce. At later time points (e.g.

day 18, 28) electrical paroxysms of longer durationconsisting of spike—wave complexes and polyspikingwere seen in cortical as well as in the depth record-ings (Fig. 2C and D). Behavioural seizure activity atthese time points consisted of more facial automa-tisms (twitches), some jaw automatisms, head nodsfollowing pauses in behaviour without loss of pos-ture, and tonic flexion concurrently with biting andchewing of hindlimb extremity. Severe tonic clonicbehavioural seizures, however were not observed.Recordings from the contralateral sites showedthat epileptiform EEG activity also occurred in thecontralateral cortical focus as well as in the corre-sponding contralateral subcortical structures. Thetemporal progression of the epileptiform activity inthe cortical focus and subcortical sites in the con-tralateral hemisphere followed a pattern similar tothat in the iron-induced cortical focus and subcor-tical sites ipsilateral to it. From Fig. 2C (day 18) it isapparent that in the hippocampal CA3 subfield, theelectrical seizure appeared earlier than in the cor-tical focus. In the recordings from the other threesubcortical structures, however, the seizure activitywas simultaneous with that in the cortical focus.

Simultaneous recordings of MUA and EEGs in thecortical focus and subcortical sites (Fig. 3) showedthat the increases in the bursts of MUA were con-current with EEG seizure paroxysms. The fre-quency of MUA concurrent to an EEG paroxysm


Figure 2 Temporal progression of the epileptiform activity in the cortical focus and ipsilateral intracerebral sites (Cx:cortex, CA3 and CA1 subfields of the hippocampus, AMG: amygdala, CPU: caudate-putamen). (A) EEG activity at day 3(post iron injection). Note very minor seizure activity at this time point. Video cursor showing the rat engaged inexploratory behaviour. (B) Electrographic seizure activity at day 8. Conspicuous electrographic paroxysms detected bymultiple channels. Video cursor showing the rat sitting stationary. (C) Electrographic seizure activity at day 18. Longduration paroxysms detected by multiple channels. Note that epileptiform activity is first detected in the CA3 lead andthen simultaneously in other leads. Video cursor showing the rat sitting stationary with dorsally convexed back. (D)Electrographic seizure activity at days 28. Long duration paroxysms detected simultaneously by all the channels. Videocursor showing the rat sitting stationary with raised head and fore and hind limbs slightly extended with back somewhatconvexed.

appeared to increase several times than thatoccurring during the interval between EEG parox-ysms indicating an increase in the cellular firingassociated with the corresponding EEG epilepti-

form activity. Fig. 4 (bottom panel) presents thetime course of changes in the ipsilateral epilepticactivity (in terms of MUA counts across the studiedtime points) in different brain structures. The top


Figure 3 Simultaneous multiple-unit activity (MUA) and electroencephalographic (EEG) recording from cortical focusand subcortical areas. (A) Control, and (B) seizure activity at day 15 post iron injection.

panel (Fig. 4A) shows the data recorded from thecontralateral regions. It is apparent from thesedata that levels of MUA counts progressivelyincreased with time in various brain regions (bothipsilateral and contralateral) as compared torespective controls (Fig. 4B, middle panel). Forexample, the MUA counts in the hippocampalCA3 region increased 6-fold with time at day 15,and 10-fold at day 28. In controls there were noincreases in the MUA counts across the time pointsstudied (Fig. 4, middle panel). Statistical compar-isons (Student’s t test) between the MUA countsfrom experimental and those from controls in eachbrain region showed a high significance ( p < .01)both in the ipsilateral and contralateral regions.

Effect of ethosuximide

A single dose of ethosuximide, given at various timepoints post iron-injection suppressed the EEG epi-leptic activity in all the epileptic animals tested(Fig. 5A compared with Fig. 5B). The drug effectappeared immediately and lasted 15—20 min, afterwhich the electrical paroxysms in the EEG reap-peared within 60—90 min (Fig. 5C). Further obser-vations showed that the drug given chronically oncea day for 12 days completely suppressed the epi-leptiform activity in the cortical focus and intra-cerebral sites. This effect was seen in animals inwhich the drug administration was started at day 10post seizure induction as well as in those in which

drug treatment was started at day 20 post seizureinduction.

Discussion

Stereoencephalographic studies to determine therole and seizure-induced activation of various sub-cortical structures in the iron-model of post-trau-matic epilepsy have not been carried out.14,20 Sincethe propagation of seizure activity from the site ofits initiation in the cortex to various subcorticalstructures may differ markedly in different epilep-sies because different epileptogenic agents maydifferentially affect the initiating and recruitingstructures, it is of interest to investigate differentmodels of epilepsy stereoencephalographically.17

The seizure-induced activation of subcortical brainregions as mapped by field potential and multiple-unit action potential recordings reflects an ana-tomic delineation of the seizure spread, and alsoparticipation of the affected brain regions in thegeneralization of seizure activity.30,31 Our previousobservations have shown that the iron-induced cor-tical epileptic activity propagates from the corticalfocus to the thalamus, locus coeruleus and substan-tia nigra.20,21 The present study has furthermoredemonstrated that the cortical focal seizure activitypropagates to the hippocampus, amygdala and stria-tum. The contralateral (mirror) focus together withthe contralateral subcortical sites also become fully


Figure 4 Time course of significant incremental changes in the MUA counts across the studied time points in ipsilateraland contralateral epileptic sites (bottom and top graphs), respectively ( p < 0.01). Saline injected control (middle graph)does not show significant changes in MUA counts of respective structures overtime.

active. The seizure activity in the subcortical sitesappeared simultaneously with that in the corticalfocus, and also increased in parallel with that inthe cortical focus. The EEG seizure activity wasassociatedwith the corresponding increases inmulti-ple-unit actionpotentials. Itwould, thus, appear thatan extensive intracerebral anatomical circuit devel-ops to subserve the spontaneous generalized seizureactivity in the cerebrum. The present data are con-

sistentwith animal experimental and humanepilepsystudies in which MUA and EEGs were simultaneouslyrecorded in cortical and subcortical structures forinvestigating the spread of cortical epileptic activityto subcortical structures.19,32,33a—33c

In the present study, no delay was observedbetween the appearance of epileptic activity inthe cortical focus and its occurrence in the subcor-tical regions. In a rat model of genetic petit mal like


Figure 5 Effect of ethosuximide on the epileptiformactivity of the cortical focus and intracerebral sites(Day 10 post iron injection). (A) Epileptiform activity justbefore ethosuximide injection, (B) suppression of epilep-tiform activity, (C) reappearance of the epileptiformactivity 20 min after ethosuximide injection; (all record-ings were obtained from the same animal on the sameday).

Figure 6 Representative line drawings of coronal sec-tions of the brain showing the location of cortical andsubcortical brain areas, where the electrodes wereimplanted for recording the EEGs and MUA, and somephotmicrographs showing histological verification of thepositions of electrode tips. (A and B) caudate-putamen;(C) cortex and amygdala; (D) CA3 subfield of hippocam-pus; (E and F) CA1 subfield of hippocampus.

seizures, a simultaneous initiation and synchronousoccurrence of epileptic activity was noticed in thecortex and various subcortical structures.34 A smalltime difference between epileptic discharges invarious sites, however, was found in some experi-mental epilepsy studies by using fast Fourier trans-formation of multiple EEG signals, cross-spectralphase and co-relational and coherence analyses thatreveal temporal relationships between the EEG andcellular firing.19,33 In the present study of iron-epi-lepsy model also small time differences between theoccurrence of epileptic activity at various sites areprobable because the propagation of activity fromone site to the other may involve small time delays.However, our data are limited in this respect sinceobservations were made by the visual inspectionof EEG epileptiform activity as in several otherstudies.17,34

In the iron-induced focal epilepsy model, the EEGseizure activity from the start becomes generalized

in the cerebral cortex.13,14 The model thus differsfrom those focal epilepsy models in which focalepileptiform activity does not become generalized.For example, in the g-aminobutyric acid (GABA)-withdrawal focal epilepsy model35 the epilepto-genic process somehow remains localized andlimited to the original focus. Our results furthershowed that, unlike several other generalized cor-tical seizure models,36 in the iron-epilepsy modelthe spike—wave epileptiform activity of the corticalfocus is propagated in to the limbic structures. Thespread of focal spike-and-wave seizure activity tothe hippocampal CA1 and CA3 areas makes the iron-model different from the rat model of petit malepilepsy32,34 in which the seizure activity, althougharises in corticothalamic system was not found toinvade the CA1 and CA3 areas of the hippocampusand the other limbic structures.

Our observations are also consistent with a recentstudy37 in which the fluid percussion injury-induced


fronto-parietal cortical post-traumatic epilepsy inthe rat was found to progress to mesial temporallobe epilepsy, and it was further found that inde-pendent cortical and hippocampal epileptic focievolved differentially overtime post injury. Ourobservations that the seizure activity in the hippo-campal CA1 area begins to lead the seizure activityof the cortical focus would be consistent with theconcept that independent neocortical and hippo-campal epileptic foci may coexist in the traumaticinjury-induced epilepsy.37,38 The hippocampus isknown to be a key structure for seizure generation,and its CA3 area may act as a pacemaker of syn-chronous epileptic activity.39,40 It has a role inhuman post-traumatic and temporal lobe epilep-sies. In fact both the CA1 and CA3 areas of thehippocampus may contribute to the generalizationand expression of seizure activity.15

In clinical temporal lobe epilepsy, electrographicseizures recorded from the hippocampus are notnecessarily linked to, or induced by, an excessiveoscillation of thalamocortical neurons.32 From thepresent data on iron-model, it would appear thathippocampal discharges increase in parallel withthose of the thalamocortical system.20 Thus, inthe iron-induced epilepsy the thalamocortical syn-chronized activity appears to be associated with theinduction of epileptic activity in the hippocampus. Ithas also been reported that seizure activity reachingthe hippocampus can lower its threshold to seizuresby increasing the basal release of endogenousacetylcholine.39 This may have implications forthe human post-traumatic epilepsy because likethe temporal lobe epilepsy, in the post-traumaticepilepsy also the hippocampus is affected. The iron-model is also similar to picrotoxin-induced general-ized epilepsy model in which the epileptic activityfollowing its initiation in the cortex radiates to thehippocampus and amygdala besides thalamus.19 Inthe rat model of human febrile seizures also,17

seizure propagation in the hippocampus and amyg-dala has been demonstrated.

The seizure activity propagated into limbicregions can in turn contribute to amplificationand generalization of cortical seizure activity, how-ever, this is sometimes considered uncertain as thelimbic seizure activity may not readily propagate tothe cortex.34 Seizure activity originating in theamygdala in some studies, however, has been foundto spread to the hippocampus and cortex.36 So to alimited extent there is a possibility that the seizureactivity generated in the limbic regions in theiron-model may be additive to the cortical seizureactivity and may thus enhance generalized seizureactivity. Seizure-induced caudate-putamen activa-tion seen in the present study would appear to be

similar to that found in the limbic seizures.36

Furthermore, a convulsive status which includesfacial—oral automatisms, sniffs and head noddingswhich may be due to the spread of the seizureactivity in the region of caudate-putamen, amyg-dala and other related structures34,36 is associatedwith limbic seizures. In the iron-epilepsy modelthese convulsive phenomena do occur but no majortonic—clonic type of convulsions have beenfound.13,14

Our ethosuximide experiments showed that thedrug suppressed the epileptiform activity in theiron-induced epilepsy model. Ethosuximide is effec-tive against pentylenetetrazol-induced seizures,28

but it is ineffective against maximal electroshockseizures.40 Pentylenetetrazol induces spontaneousseizure activity in hippocampal circuits by activa-tion of the entorhinal cortex.28 Therefore, ethosux-imide’s effectiveness against iron-induced seizuresmay at least in part be mediated by its possibleaction on the entorhinal cortex. Of further interestis the recent observation that ethosuximide is ableto act focally at cortical sites to abolish absenceseizures in the genetically determined absence epi-lepsy animal models.23 Ethosuximide’s mechanismof action is also presumed to be related to its effecton the thalamic calcium currents41,42 and on thehypersynchronizing electrophysiological activity inthe reticular-thalamic-cortical net.22 In our pre-vious work, we have found that the thalamocorticalcircuit is activated in the iron-induced epilepsy.20

Therefore, ethosuximide’s effect may also bemediated by the drug’s action on the thalamocor-tical circuit. Furthermore, ethosuximide as acalcium channel blocker is likely to possess anti-lipidperoxidative and glutathione peroxidase stimu-lating action.25 Therefore, the drug’s antioxidativeaction may also mediate its antiepileptic effects inthis model.

Acknowledgements

The author (VS) is grateful to the Council of Scien-tific and Industrial Research, New Delhi for the grantof a Research Associateship, and to the Departmentof Science and technology (DST) Government ofIndia, for the grant of a research project (SR/WOS-A/LS 503/2003).

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