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pilepsy Research (2008) 78, 178—185

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hanges of cortical epileptic afterdischarges aftertatus epilepticus in immature rats

rygoriy Tsenov, Hana Kubova, Pavel Mares ∗

nstitute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, CZ-14220 Prague 4, Czech Republic

eceived 7 August 2007; received in revised form 20 November 2007; accepted 22 November 2007

KEYWORDSPilocarpine;Status epilepticus;Corticalafterdischarges;Ontogeny;Rat

Summary Status epilepticus (SE) in developing rats leads to neuronal degeneration in manybrain structures including neocortex but the functional consequences of cortical damage werestudied only exceptionally.

Lithium—pilocarpine SE was elicited in 12- (P12) and 25-day-old (P25) rats, convulsions wereinterrupted after 2 h by paraldehyde. Cortical electrodes were implanted 3, 6, 9, 13 and/or26 days after SE. Low-frequency stimulation of sensorimotor cortex was repeated with atleast 10-min intervals with a stepwise increasing intensity (0.2—14 mA). Thresholds for move-ments elicited by stimulation, spike-and-wave afterdischarges (ADs), clonic seizures, mixedADs (transition into a limbic type of ADs) and recurrent ADs as well as duration of ADs wereevaluated.

The first three phenomena were not influenced by SE with the exception of lower thresholdsfor movements during stimulation. Transition into limbic seizures and recurrent seizures weredelayed in both age groups and threshold intensities for limbic ADs were at some intervals

higher in SE than in control animals. Duration of ADs was changed only at short intervals afterSE; it was shortened at 3 and 6 days in P25 and 3 days in P12 rats, respectively. P12 group thenexhibited a transient increase in duration of ADs 6 days after SE.

Our results did not prove a higher cortical excitability after SE in either age group. On thecontrary, there were some signs of a decreased excitability.

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© 2007 Elsevier B.V. All righ

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ithium—pilocarpine-induced status epilepticus (SE) in ratsepresents a model of brain damage inducing chronic

∗ Corresponding author. Tel.: +420 24106 2495;ax: +420 24106 2488.

E-mail address: maresp@biomed.cas.cz (P. Mares).

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920-1211/$ — see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.eplepsyres.2007.11.008

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pileptogenesis. It was demonstrated that it leads to degen-ration of neurons in many brain structures in adult rodentsTurski et al., 1983; Olney et al., 1986). It is clear thatithium—pilocarpine SE induced at early stages of develop-ent may also result in neuronal degeneration but these

hanges are age-dependent (Sankar et al., 1998, 2000;ubova et al., 2001, 2002, 2004). Other models of SEay lead to different patterns as well as age profile

f damage in the developing brain (kainic acid-inducedE—–Sperber et al., 1991; Leite et al., 1996; electrical

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stimulation-induced SE—–Sankar et al., 2000), i.e. neuronaldegeneration is also model-dependent (Sankar et al., 1998,2000). There are descriptions of damage in limbic struc-tures after lithium—pilocarpine SE (Cavalheiro et al., 1987;Sankar et al., 1997; Nairismagi et al., 2006), age-dependentchanges in subunit composition of GABA-A (decrease of �1subunit after SE induced during adulthood in contrast to anincrease if SE was elicited at postnatal day 10 or 20—–Zhanget al., 2004; Brooks-Kayal, 2005; Raol et al., 2006) and exci-tatory amino acid receptors (Porter et al., 2006) in dentategyrus. Functional studies were also focused on hippocam-pal formation. Among behavioral studies Morris water maze(test of spatial memory dependent on hippocampus—–Morris,1984) is most common (Mares, 2007), studies of excitabilityare also focused on limbic structures in a pilocarpine modelof SE (amygdala kindling—–Cilio et al., 2003) as well as inother developmental models (Jensen et al., 1992, 1998; Leeet al., 1995; Chen et al., 1999; Dube et al., 2000; Jensenand Baram, 2000; Cilio et al., 2003; Zhang et al., 2004).Recently morphological changes of thalamus were describedin detail (Kubova et al., 2001, Druga et al., 2005) but neo-cortical damage was sometimes shortly mentioned (Turskiet al., 1983; Cavalheiro et al., 1987) but not studied indetail in spite of the dominant role of cerebral cortex inchildhood epilepsies (Aicardi, 1998). Irreversibly damagedneurons were demonstrated after lithium—pilocarpine SE incortex of 3- and 4-week-old rats (Sankar et al., 1997). Activ-ity of cortical neurons analyzed in Cavalheiro’s (Sanabria etal., 2002) and as well as in our laboratory (Doczi et al., 2003)revealed changes in the firing pattern of nerve cells afterSE induced at early developmental stages. Benzodiazepinereceptors in the cerebral cortex also exhibit age-dependentchanges: an increase 1 week after SE elicited at P12 and adecrease 3 months after SE induced in adult rats (Rocha etal., 2007). In connection with these findings we started adetailed study of morphological and functional changes ofsensorimotor cortical area. Our first study described oppo-site changes of transcallosal (interhemispheric) responsesin the sensorimotor cortex of rats exposed to SE at the ageof 12 and/or 25 days (Mares et al., 2005). There were nosignificant differences 3, 6 and 9 days after SE in compar-ison with control rats. Changes were observed only 2 andmore weeks after SE, amplitude of responses was lower inanimals undergoing SE at P12 than in corresponding con-trols whereas input—output curve was steeper in rats withSE at P25 than in their control group. Cortical excitabilitywas also studied by means of rhythmic stimulation: series offive stimuli can induce frequency potentiation and/or fre-quency depression. Our study of these phenomena did notreveal any marked changes of cortical excitability after SEelicited at the age of 12 and/or 25 days (Tsenov and Mares,2007).

Cortical epileptic afterdischarges represent another pos-sibility how to study cortical excitability (Mares and Kubova,2006). This model elicited by low-frequency stimulationof sensorimotor cortical area is frequently used in ourlaboratory in adult as well as immature rats (Lojkova

et al., 2006; Mares and Slamberova, 2006). It allows tostudy four different phenomena, three of them epilepticin nature: movements directly elicited by stimulation, EEGafterdischarges of the spike-and-wave type, clonic seizuresaccompanying this type of afterdischarges, and with high

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timulation intensities a transition from spike-and-wave intonother type of afterdischarges identical with that inducedy hippocampal stimulation. Ontogeny of these phenomenaas described in detail (Mares et al., 2002) so that longitu-inal study after SE could demonstrate not only a markedathology but also a modification of brain development.

ethods

nimals

ale albino rats of Wistar strain (breeding of the Institute of Phys-ology, Academy of Sciences of the Czech Republic) were used.nimals were kept under standard conditions (12/12 h light/darkycle, temperature 22 ± 1 ◦C, humidity 50—60%) with a free accesso food and water. The experimental protocol was approved by Ani-al Care and Use Committee of the Institute of Physiology to be in

greement with Animal Protection Law of the Czech Republic (whichs fully compatible with European Community Council directives6/609/EEC as well as with NIH Guidelines).

tatus epilepticus

tatus epilepticus (SE) was elicited in 12- and 25-day-old rats byilocarpine (40 mg/kg, i.p.; # P6503, Sigma Chemical Co., St. Louis,O) 20—24 h after LiCl (3 mequiv./kg i.p.; # L-0505, Sigma Chemicalo., St. Louis, MO) pretreatment. Paraldehyde (0.3 ml/kg, i.p.; #6260, Fluka Chemie AG, Buchs, Switzerland) was injected afterh of continuous convulsions to prevent mortality. Younger animalsere returned to their mothers, older ones needed an intensive care

or at least 2 days after SE. Control animals received saline insteadf pilocarpine, other treatments were the same as in SE group.

urgery

ortical stimulation and registration electrodes were implantednder ether anesthesia 3, 6, 9, 13 and/or 26 days after SE. Silverlectrodes were implanted epidurally—–two over the right senso-imotor cortical area (AP + 1 and −1; L = 2 mm) for stimulation;egistration electrodes over left sensorimotor cortical area (AP = 0;= 2 mm), over left association parietal area (AP = 3; L = 3 mm), andver left and right visual cortical regions (AP = 6; L = 4 mm). Theoordinates in parentheses are for adult rats; they were recalcu-ated for immature animals on the basis of bregma-lambda distance.n indifferent electrode was put into nasal bone, ground electrodever cerebellum. All silver electrodes were fixed to the skull byast curing dental acrylic (Duracrol® Dental, Prague). Implanta-ion of electrodes lasted less 8—10 min, then ether anesthesia wasnterrupted. The experiment started after 1-h rest, rat pups withmmature thermoregulation (up to the end of the third postnataleek) were on pad heated to 34 ◦C during this period as well asuring the experiment.

timulation procedure

nimals were placed individually into plastic boxes and connectedo the amplifier and stimulator. Every rat had an easy access to foodellets and water.

Cortical epileptic afterdischarges were elicited by a constanturrent stimulator (15-s series of 1-ms biphasic rectangular pulsesith 8-Hz frequency). Intensity was increased stepwise from 0.2 to

5 mA; at least 10-min interval was preserved between stimulationsith individual intensities (Fig. 1). EEG was digitalized at a rate of00 Hz and saved on the hard-disc (Kaminskij Biomedical Researchnstruments, Prague). It was recorded before, during and 2 min (asinimum) after stimulation. The behavior of animals was coded into

180 G. Tsenov et al.

Figure 1 Original recordings of cortical afterdischarges fromthe left sensorimotor area of 18-day-old (12 + 6) control rat pup.Individual traces represent increasing stimulation intensities(indicated by black numbers). Clear spike-and-wave ADs wereelicited by intensities from 0.6 to 5.0 mA, mixed type of ADs wasinduced by 6.0-mA stimulation. Individual recordings from left:let

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Figure 2 Thresholds for cortically induced phenomena(mean + S.E.M.). From top to bottom: movements during stimu-lation; spike-and-wave afterdischarges; clonic seizures; mixedafterdischarges (transition into the limbic type); recurrentafterdischarges. Left column: P12; right column: P25 group.Abscissae: intervals after SE in days: 3, 6, 9, 13 and 26; ordi-ncd

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ast 2 s of 15-s stimulation (limited by a line) and 40 s after thend of stimulation. Time mark 2 s, amplitude calibration equalso 1 mV.

he EEG recording. The type, duration, and thresholds of ADs andotor phenomena were evaluated. As duration of ADs is concerned,

hree calculations were done: total duration without relation to theype of ADs; duration of spike-and-wave ADs (i.e. from mixed ADsnly a part formed by SW activity was taken); duration of mixed ADs.odified Racine’s five-point scale (0: no activity; 1: motor activitiesot synchronous with stimuli and/or EEG spikes; 2: head jerks; 3:lonic forelimb movements; 4: clonic forelimb movements + rearing;: clonic forelimb movements + rearing + falling) and symbols weresed for quantification of behavior (Racine, 1972; Mares et al.,002). Rats could be used only once, 7—10 animals formed eachroup.

tatistics

ata for thresholds and duration of afterdischarges and intensityf motor phenomena were statistically evaluated by means of Rankum Mann—Whitney test. Incidence of mixed and recurrent ADs wasvaluated by means of Fisher Exact Test (SigmaStat® SYSTAT). Theevel of statistical significance was put on 5%.

esults

hresholds

12 groupevelopmental changes of thresholds for the first three phe-omena (movements during stimulations, spike-and-wavefterdischarges and clonic seizures) in control rats exhib-

ted an U-shape pattern with the lowest value at the agef 21 days. This developmental curve was distorted in SEnimals—–there was an increase of threshold values at the-day interval after SE (i.e. at the age of 21 days). BothE and control rats exhibited the highest thresholds at the

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ates: threshold current intensity in mA. Control group: whiteolumns and SE: dashed columns. Asterisks at pairs of columnsenote a significant difference between SE and control animals.

ongest interval, i.e. at the age of 38 days. Significantlyower threshold for movements directly elicited by stimu-ation was found at 3-day interval after SE in comparisonith the age-matched control group. The same change was

ound at 13-day interval (Fig. 2). Threshold current inten-ities for elicitation of spike-and-wave afterdischarges andlonic seizures tended to be lower 3, 6 and 13 days afterE than in corresponding control groups but the level ofignificance was not reached.

The incidence of mixed and recurrent ADs was negligiblet short intervals after SE and increased gradually with ageTable 1). This behavior was especially expressed at 3-daynterval, when mixed type of afterdischarges was recordedn 4 out of 10 control animals but in none of 7 rats after SE;nd in 7 out of 9 control animals compared with 4 out of 9ats 6 days after SE. Recurrent ADs were recorded since thege of 18 days only in control rats (4 out of 9); 3 days lateri.e. 9 days after SE) recurrent AD appeared in 1 out of 8 SE

ats compared to 5 out of 7 animals in the control group.inor changes were observed at longer intervals.

Threshold for mixed type of ADs was higher in SE than inontrol groups after 9 and 26 days, no significant differences

Changes of cortical epileptic ADs after SE in rats 181

Table 1 Incidence of mixed seizures and recurrent after discharges

P12 P25

3 days 6 days 9 days 13 days 26 days 3 days 6 days 9 days 13 days 26 days

Mixed ADsControl 4/10 7/9 6/7 5/7 9/10 7/7 8/8 10/10 10/10 10/10SE 0/7 4/9 5/8 8/8 10/10 4/9 6/8 10/10 10/10 8/8

Recurrent ADsControl 0/10 4/9 5/7 6/7 10/10 7/7 7/8 9/10 10/10 10/10

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were found at other intervals. Significantly higher thresh-olds were found for recurrent afterdischarges only at 26-dayinterval after SE when compared with controls.

P25 groupNo significant changes were found in P25 group as the thresh-olds of the first three phenomena (movements bound tostimulation, spike-and-wave ADs, and clonic seizures accom-panying these ADs) are concerned (Fig. 2). Developmentalchanges were only marginal in both SE and control groups.

Incidence of mixed type and recurrent ADs showed thatspread of epileptic activity into limbic system was not con-siderably influenced (Table 1). Four out of 9 rats 3 days afterSE generated mixed type of afterdischarges and only oneexhibited recurrent ADs in comparison with control groupwhere all animals had both phenomena. This was the onlydifference found; SE and control animals at other intervalsyielded similar results. Significant differences of thresholdswere found for mixed type of ADs: animals 9 days after SEneeded higher current intensities than control rats. Simi-lar difference was found for threshold of recurrent ADs inanimals 6 days after SE.

Duration

P12 groupTotal duration of ADs 3 days after SE was shorter than incontrols if intensities from 3 to 14 mA were used. Durationof spike-and-wave activity was shorter, too, significant dif-ferences were the same as for total duration of ADs (Fig. 3).Opposite changes (longer ADs in SE rats) were registered 3days later (i.e. 6 days after SE); the level of statistical sig-nificance was reached with low stimulation intensities (from1 to 4 mA) for both total duration of ADs and spike-and-waveADs. Mixed ADs which appeared in SE group regularly for thefirst time at the 9-day interval (i.e. at the age of 21 days) didnot exhibit any significant changes (data not shown). Dura-tion of either type of ADs was not systematically changedat longer intervals after SE, only isolated significant differ-ences were found.

P25 groupRats in SE P25 group exhibited shorter total as well asspike-and-wave ADs than control rats not only 3 but also6 days after SE (Fig. 3). This difference was observed at

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mber animals in group in both rats with SE at P12 (left part) and

any stimulation intensities, especially at the interval ofdays (from 1.4 to 8 mA, differences in values for 10- and

2-mA stimulation intensities did not reach the level of sta-istical significance). Significant differences in duration ofpike-and-wave ADs were again the same as those in totaluration. Mixed type of ADs participated in changes in totaluration especially if higher stimulation intensities weresed (data not shown but see the difference between totaluration and duration of spike-and-wave ADs at higher stim-lation intensities in Fig. 3) but the statistical results wereot changed. Again, there were no systematic differencest longer intervals after SE.

otor phenomena

ntensity of movements directly elicited by stimulation asell as of clonic seizures accompanying spike-and-wave typef ADs were not significantly changed at any interval afterE in both P12 and P25 groups (data not shown).

iscussion

ndividual phenomena evaluated in our experimentalaradigm are generated by different mechanisms. Move-ents directly elicited by stimulation represent an

ctivation of the motor system—–sensorimotor cortex is stim-lated and movements started always at the opposite sidef the body. Spike-and-wave EEG activity is generated byhalamo-cortical mechanisms; it was demonstrated for spon-aneous spike-and-wave episodes in rats (Avanzini et al.,992) and it is highly probable that this activity char-cteristic for the first type of ADs has the same origin.lonic seizures accompanying spike-and-wave ADs are againreflection of activation of the motor system—–individual

erks are synchronous with sharp elements in the EEG.he second type of ADs, elicited only by high intensitiesf stimulation current (Mares et al., 2002) is due to apread of epileptic activity into limbic system (EEG as wells behavioral correlates are identical with those inducedy hippocampal stimulation—–(Swartzwelder et al., 1979;

ragin et al., 1997)). This transition is realized by meansf polysynaptic connections between thalamus and limbictructures and mediodorsal thalamic nucleus could be a pri-ary structure in this transition (Bertram et al., 2001) but

ther possibilities have to be taken into account.

182 G. Tsenov et al.

Figure 3 Duration of afterdischarges (mean + S.E.M.) elicited by individual current intensities. Upper box: P12 + 3 and P12 + 6—–P12g ay ini on ofO

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roups; lower box: P25 + 3 and P25 + 6—–P25 groups at 3- and 6-dn mA; ordinates: upper part of each graph denotes total duratither details as in Fig. 2.

The above-mentioned phenomena are differently influ-nced by SE. Thresholds for movements bound to stimulationere significantly decreased only in P12 group at the short-st interval and again 13 days after SE. The acute change is ingreement with our finding on the response to a series of fivetimuli (frequency potentiation—–Tsenov and Mares, 2007)ut a decreased threshold at the longer interval is in contra-iction with changes in single interhemispheric responses,hey are lower in SE rats than in controls at that inter-al (Mares et al., 2005). Thresholds for spike-and-wave ADsnd clonic seizures were not changed in either group atny interval after SE. Again, it is in contrast to a steeper

nput—output curve of single responses at long intervalsfter SE in P25 group (Mares et al., 2005). These discrep-ncies between changes in evoked potentials and epilepticfterdischarges suggest that their mechanisms of genera-ion are different—–pure cortical in single evoked responses

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terval, respectively. Abscissae: stimulation current intensitiesADs, bottom part: duration of spike-and-wave ADs in seconds.

or at least of their initial components—–Grafstein, 1959)nd an involvement of reverberatory thalamo-cortical cir-uits in origin of spike-and-wave ADs (Avanzini et al., 1992;nead, 1992; Seidenbecher et al., 1998). Unfortunately,eurotransmitter mechanisms of cortical epileptic ADs areot known. Our pharmacological data with NMDA receptorntagonists indicate an important role of these receptors indult (Lojkova et al., 2006) as well as immature rats (Maresnd Slamberova, 2006; data on file). Analysis of a partici-ation of these as well as other receptors in generation andaintenance of cortical epileptic afterdischarges represents

urther direction of our research.

Changes in transition into the limbic type of ADs and

ppearance of recurrent ADs were completely different.hese phenomena were suppressed at short intervals andignificant increase in threshold current intensities wereound at different intervals in both age groups. Their

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delayed appearance in P12 group (transition into the lim-bic type of ADs appeared in more than half of experimentalanimals 9 days after SE and recurrent AD even 4 days laterwhereas control rats exhibited these phenomena at earlierage, Table 1) can be interpreted as a developmental delaydue to an insult—–SE. This insult takes place at the momentof brain growth spurt (Dobbing and Sands, 1971) thereforeextreme activity during SE might interfere with processesnecessary for normal development. Developmental delayafter SE in this age group was found also in body weight as asimple measure of development (Kubova et al., 2004) as wellas in a complex behavioral process—–acute habituation in theopen field (Kubova et al., in preparation). Another factor tobe taken into account is the damage of the most probablepathway through mediodorsal thalamic nucleus (Bertram etal., 2001); marked degeneration of neurons was describedin this nucleus even in rats undergoing SE at P12 (Kubovaet al., 2001, 2002; Druga et al., 2005). The probability ofparticipation of these two processes is supported by lessexpressed changes in P25 group where the connections (notonly through mediodorsal nucleus) are mature and nearly allcontrol rats exhibit this transition (present results, Mares etal., 2002).

Age-related changes in duration of afterdischarges werefound in our experiments. Duration of ADs was significantlydecreased 3 days after SE in both age groups. This decreasewas observed even 6 days after SE in P25 rats, P12 animalsexhibited a significant prolongation of ADs at this inter-val. This increase in duration is in controversion with anincrease of benzodiazepine receptors in the cerebral cortex(Rocha et al., 2007) as well as of �1 subunit of GABA-A receptors in granule cells of the dentate gyrus 1 weekafter SE in immature rats. This increase is taken for anadaptive anticonvulsant mechanism (Raol et al., 2006). Onthe other hand, an impairment of inhibition mediated byGABA-B receptors was found in limbic structures (Manganand Lothman, 1996); the GABA-B system plays an importantrole in arrest of cortical epileptic afterdischarges (Mares,2006; Mares, submitted). The reversal of changes in P12rats between the third and sixth day after SE with sub-sequent normalization suggests a possibility of a transientimpairment of GABA-B or other inhibitory system. Thesepossibilities have to be further analyzed.

Changes in cortical excitability after SE are much lessexpressed than changes in hippocampal excitability afterdifferent models of SE in immature rats. In contrast topapers studying hippocampal excitability (Jensen et al.,1992, 1998; Lee et al., 1995; Chen et al., 1999; Dube etal., 2000; Jensen and Baram, 2000; Cilio et al., 2003; Zhanget al., 2004) data on neocortical excitability are sparse.They originate either from our (Doczi et al., 2003; Mareset al., 2005; Tsenov and Mares, 2007) or from Cavalheiro’s(Sanabria et al., 2002; Silva et al., 2005a,b) laboratoryand they all demonstrate rather subtle changes includingchanges in inhibitory and excitatory receptors (Silva et al.,2005a,b; Rocha et al., 2007). The same is true for mor-phological damage—–marked damages in limbic structures

and only moderate changes in neocortex (Cavalheiro et al.,1991; Leite et al., 1996; Mares et al., 2005; Nairismagiet al., 2006). Degenerating neurons in cortical motor andsomatosensory areas (which are rather sparse) have a shapeof interneurons meanwhile pyramidal cells remain intact

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Mares et al., 2005). In spite of the fact that morphologicalhanges in P12 are not so extensive as in P25 or adult group,arked degeneration of pyramidal neurons was observed in

ippocampus (especially in CA1 field) in rats undergoing SEt the age of 12 days whereas degenerating interneurons areot numerous (Druga et al., in preparation). Thus the mor-hological damage in neocortex and hippocampus is not onlyuantitatively but also qualitatively different. All these find-ngs are in agreement with our data on spontaneous seizuress a late consequence of SE—–all seizures in rats with SE atD12 are nonconvulsive and epileptic activity is restricted toippocampus (Kubova et al., 2004). Failure of generalizationor at least of spread into cerebral cortex) of seizure activ-ty in rats with SE at PD12 is probably not due to changesn neocortex—–there is only a moderate difference from ani-als with SE at PD25 (Mares et al., 2005) where convulsive

eizures with corresponding electrocorticographic patternere recorded (Kubova et al., 2004). It might indicate dif-

erences in structures mediating connections between oldnd new cortical structures. Further analysis will be nec-ssary to understand differences in consequences of SE ineocortex and limbic system. Participation of serious wors-ning of the conditions after SE (acute changes in circuitryesponsible for shorter afterdischarges 3 days after SE),ompromising of inhibitory systems and a possible develop-ental delay remains to be elucidated. Changes of cortical

pileptic afterdischarges found in the present study caneflect rather a developmental delay than a shift to an easypileptogenesis. They might be interpreted that neocorti-al damage is not sufficient to form a basis for generation ofpileptic seizures in this brain structure in either age grouptudied as well as for an easy spread of epileptic activityriginating in limbic structures into the cerebral cortex inats with SE at PD12. The impact of level of brain matura-ion at the moment of SE induction is reflected also in theact that SE induced in adult rats resulted in appearance ofeizures in nearly all animals (Cavalheiro et al., 1991) mean-hile SE at PD25 led to approximately 75% and SE at PD12

o 25% of epileptic rats, respectively (Kubova et al., 2004).nimals in the present study were not videoEEG monitoredut the relatively long latency of spontaneous seizures afterE in immature rats (Raol et al., 2006) means that all ourecordings were made during the latent period. Longer inter-als after SE and a combination of videoEEG monitoring withtimulation have to be used for future analysis.

cknowledgements

his study was supported by grant nos. 309/03/0770 and04/05/2582 of the Grant Agency of the Czech Republic andy a research project AV0Z 50110509. The authors would likeo express their thanks to Ms. Irina Necheva for excellentechnical assistance.

eferences

icardi, J., 1998. Syndromes of infancy and early childhood. In:Engel Jr., J., Pedley, T.A. (Eds.), Epilepsy: A ComprehensiveTextbook. Lippincott, Raven, Philadelphia, pp. 2263—2265.

vanzini, G., de Curtis, M., Marescaux, C., Panzica, F., Spreafico,R., Vergnes, M., 1992. Role of the thalamic reticular nucleus in

1

B

B

B

C

C

C

C

D

D

D

D

G

J

J

J

K

K

K

L

L

L

M

M

M

M

M

M

M

M

N

O

P

R

R

R

S

S

84

the generation of rhythmic thalamo-cortical activities subserv-ing spike and waves. J. Neural Transm. Suppl. 35, 85—95.

ertram, E.H., Mangan, P.S., Zhang, D., Scott, C.A., Williamson,J.M., 2001. The midline thalamus: alterations and a potentialrole in limbic epilepsy. Epilepsia 42, 967—978.

ragin, A., Penttonen, M., Buzsaki, G., 1997. Termination ofepileptic afterdischarge in the hippocampus. J. Neurosci. 17,2567—2579.

rooks-Kayal, A.R., 2005. Rearranging receptors. Epilepsia 46(Suppl. 7), 29—38.

avalheiro, E.A., Silva, D.F., Turski, W.A., Calderazzo-Filho, L.S.,Bortolotto, Z.A., Turski, L., 1987. The susceptibility of rats topilocarpine-induced seizures is age-dependent. Dev. Brain Res.37, 43—58.

avalheiro, E.A., Leite, J.P., Bortolotto, Z.A., Turski, W.A., Ikonomi-dou, C., Turski, L., 1991. Long-term effects of pilocarpine in rats:structural damage of the brain triggers kindling and spontaneousrecurrent seizures. Epilepsia 32, 778—782.

hen, K., Baram, T.Z., Soltesz, I., 1999. Febrile seizures in thedeveloping brain result in persistent modification of neuronalexcitability in limbic circuits. Nat. Med. 5, 888—894.

ilio, M.R., Sogawa, Y., Cha, B.H., Liu, X., Huang, L.T., Holmes,G.L., 2003. Long-term effects of status epilepticus in the imma-ture brain are specific for age and model. Epilepsia 44, 518—528.

obbing, J., Sands, J., 1971. Vulnerability of developing brain. IX.The effect of nutritional growth retardation on the timing of thebrain growth-spurt. Biol. Neonate 19, 363—378.

oczi, J., Bernaskova, K., Kubova, H., Detari, L., Vilagi, I., Druga,R., Mares, P., 2003. Long-term changes of activity of corticalneurons after status epilepticus induced at early developmentalstages. Neurosci. Lett. 352, 125—128.

ruga, R., Kubova, H., Mares, P., 2005. Degenerative neuronalchanges in the rat thalamus induced by status epilepticus atdifferent developmental stages. Epilepsy Res. 63, 43—65.

ube, C., Chen, K., Eghbal-Ahmadi, M., et al., 2000. Prolongedfebrile seizures in the immature rat model enhance hippocampalexcitability long term. Ann. Neurol. 47, 336—344.

rafstein, B., 1959. Organization of callosal connections in supra-sylvian gyrus of cat. J. Neurophysiol. 22, 504—515.

ensen, F.E., Baram, T.Z., 2000. Developmental seizures inducedby common early-life insults: short- and long-term effects onseizure susceptibility. Ment. Retard. Dev. Disabil. Res. Rev. 6,253—257.

ensen, F.E., Holmes, G.L., Lombroso, C.T., Blume, H.K., Firkusny,I.R., 1992. Age-dependent changes in long-term seizure suscep-tibility and behavior after hypoxia in rats. Epilepsia 33, 971—980.

ensen, F.E., Wang, C., Stafstrom, C.E., Liu, Z., Geary, C., Stevens,M.C., 1998. Acute and chronic increases in excitability in rat hip-pocampal slices after perinatal hypoxia in vivo. J. Neurophysiol.79, 73—81.

ubova, H., Druga, R., Lukasiuk, K., Suchomelova, L., Haugvicova,R., Jirmanova, I., Pitkanen, A., 2001. Status epileptus causesnecrotic damage in the mediodorsal nucleus of the thalamus inimmature rats. J. Neurosci. 21, 3593—3599.

ubova, H., Druga, R., Haugvicova, R., Suchomelova, L., Pitkanen,A., 2002. Dynamic changes of status epilepticus-induced neu-ronal degeneration in the mediodorsal nucleus of the thalamusduring postnatal development of the rat. Epilepsia 43 (Suppl. 5),54—60.

ubova, H., Mares, P., Suchomelova, L., Brozek, G., Druga, R.,Pitkanen, A., 2004. Status epilepticus in immature rats leads tobehavioural and cognitive impairment and epileptogenesis. Eur.

J. Neurosci. 19, 3255—3265.

ee, C.L., Hrachovy, R.A., Smith, K.L., Frost Jr., J.D., Swann, J.W.,1995. Tetanus toxin-induced seizures in infant rats and theireffects on hippocampal excitability in adulthood. Brain Res. 677,97—109.

S

G. Tsenov et al.

eite, J.P., Babb, T.L., Pretorius, J.K., Kuhlman, P.A., Yeoman, K.M.,Mathern, G.W., 1996. Neuron loss, mossy fiber sprouting, andinterictal spikes after intrahippocampal kainate in developingrats. Epilepsy Res. 26, 219—231.

ojkova, D., Zivanovic, D., Mares, P., 2006. Different effects of non-NMDA and NMDA receptor antagonists (NBQX and dizocilpine)on cortical epileptic afterdischarges in rats. Brain Res. 1124,167—175.

angan, P.S., Lothman, E.W., 1996. Profound disturbances of pre-and postsynaptic GABA-B receptor-mediated processes in regionCA1 in a chronic model of temporal lobe epilepsy. J. Neurophys-iol. 76, 1282—1296.

ares, P., 2006. GABA-B system plays a role in arresting corticalseizures in immature rats. In: Abstracts of the Fifth Forum ofEuropean Neuroscience, Vienna, p. 340.

ares, P., 2007. Cognitive and affective effects of seizures:immature/developing animals. In: Schachter, S.C., Holmes,G.L., Kasteleijn-Nolst Trenite, D. (Eds.), Behavioral Aspects ofEpilepsy: Principles and Practice. Demos, New york, pp. 29—33.

ares, P., Kubova, H., 2006. Electrical stimulation-induced mod-els of seizures. In: Pitkanen, A., Schwartzkroin, A., Moshe, S.L.(Eds.), Models of Seizures and Epilepsy. Elsevier, Amsterdam, pp.153—159.

ares, P., Slamberova, R., 2006. Opposite effects of a GABA-B antag-onist in two models of epileptic seizures in developing rats. BrainRes. Bull. 71, 160—166.

ares, P., Haugvicova, R., Kubova, H., 2002. Unequal developmentof thresholds for various phenomena induced by cortical stimu-lation in rats. Epilepsy Res. 49, 35—43.

ares, P., Tsenov, G., Aleksakhina, K., Druga, R., Kubova, H.,2005. Changes of cortical interhemispheric responses after sta-tus epilepticus in immature rats. Epilepsia 46 (Suppl. 5), 31—37.

orris, R., 1984. Developments of a water—maze procedure forstudying spatial learning in the rat. J. Neurosci. Methods 11,47—60.

airismagi, J., Pitkanen, A., Kettunen, M.I., Kauppinen, R.A.,Kubova, H., 2006. Status epilepticus in 12-day-old ratsleads to temporal lobe neurodegeneration and volumereduction: a histologic and MRI study. Epilepsia 47, 479—488.

lney, J.W., Collins, R.C., Sloviter, R.S., 1986. Excitotoxic mecha-nisms of epileptic brain damage. Adv. Neurol. 44, 857—877.

orter, B.E., Cui, X.-N., Brooks-Kayal, A.R., 2006. Status epilepticusdifferentially alters AMPA and kainate receptor subunit expres-sion in mature and immature dentate granule neurons. Eur. J.Neurosci. 23, 2857—2863.

acine, R.J., 1972. Modification of seizure activity by electricalstimulation. II. Motor seizures. Electroenceph. Clin. Neurophys-iol. 32, 281—294.

aol, Y., Zhang, G., Lund, I.V., Porter, B.E., Maronski, M.A.,Brooks-Kayal, A.R., 2006. Increased GABA-A receptor �1 subunitexpression in hippocampal dentate gyrus after early-life statusepilepticus. Epilepsia 47, 1665—1673.

ocha, L., Suchomelova, L., Mares, P., Kubova, H., 2007. Effectsof LiCl/pilocarpine-induced status epilepticus on rat brain muand benzodiazepine receptor binding: regional and ontogeneticstudies. Brain Res. 1181, 104—112.

anabria Garrido, E.R., Silva, A.V., Spreafico, R., Cavalheiro, E.A.,2002. Damage, reorganization and abnormal cortical hyperex-citability in the pilocarpine model of temporal lobe epilepsy.Epilepsia 43 (Suppl. 5), 96—106.

ankar, R., Shin, D.H., Wasterlain, C.G., 1997. Serum neuron-specific enolase is a marker for neuronal damage following status

epilepticus in the rat. Epilepsy Res. 28, 129—136.

ankar, R., Shin, D.H., Liu, H., Mazarati, A., Pereira de Vasconcelos,A., Wasterlain, C.G., 1998. Patterns of status epilepticus-induced neuronal injury during development and long-termconsequences. J. Neurosci. 18, 8382—8393.

S

S

T

T

Changes of cortical epileptic ADs after SE in rats

Sankar, R., Shin, D.H., Mazarati, A., Liu, H., Katsumori, H., Lezama,R., Wasterlain, C.G., 2000. Epileptogenesis after status epilep-ticus reflects age- and model-dependent plasticity. Ann. Neurol.48, 580—589.

Seidenbecher, T., Staak, R., Pape, H.C., 1998. Relations betweencortical and thalamic cellular activities during absence seizuresin rats. Eur. J. Neurosci. 10, 1103—1112.

Silva, A.V., Regondi, M.C., Cavalheiro, E.A., Spreafico, R., 2005a.Disruption of cortical development as a consequence of repeti-tive pilocarpine-induced status epilepticus in rats. Epilepsia 46(Suppl. 5), 22—30.

Silva, A.V., Regondi, M.C., Cipelletti, B., Frassoni, C., Cavalheiro,E.A., Spreafico, R., 2005b. Neocortical and hippocampal changes

after multiple pilocarpine-induced status epilepticus in rats.Epilepsia 46, 636—642.

Snead III, O.C., 1992. Pharmacological models of generalizedabsence seizures in rodents. J. Neural Transm. Suppl. 35,7—19.

Z

185

perber, E.F., Haas, K.Z., Stanton, P.K., Moshe, S.L., 1991. Resis-tance of the immature hippocampus to seizure-induced synapticreorganization. Dev. Brain Res. 60, 88—93.

wartzwelder, H.S., Johnson, C.G., Cooley, B.C., Howell, W.E.,Dyer, R.S., 1979. Alcohol-induced alterations in hippocampalafterdischarge thresholds: dose—response studies. Neurobehav.Toxicol. 1, 253—258.

senov, G., Mares, P., 2007. Depression and/or potentiation of corti-cal responses after status epilepticus in immature rats. Physiol.Res. 56, 485—491.

urski, W.A., Cavalheiro, E.A., Schwarz, M., Czuczwar, S.J., Klein-rok, Z., Turski, L., 1983. Limbic seizures produced by pilocarpinein rats: behavioural, electroencephalographic and neuropatho-

logical study. Behav. Brain Res. 9, 315—336.

hang, G., Raol, Y.S.H., Hsu, F.-C., Brooks-Kayal, A.R., 2004.Long-term alterations in glutamate receptor and transporterexpression following early-life seizures are associated withincreased seizure susceptibility. J. Neurochem. 88, 91—101.