Age- and region-specific effects of anticonvulsants and

bumetanide on 4-aminopyridine–induced seizure-like events in

immature rat hippocampal–entorhinal cortex slices*Abdul Wahab, *Klaus Albus, and *yUwe Heinemann

*Institute of Neurophysiology, Charite Universitatsmedizin Berlin, Berlin, Germany; and yNeuroCure Center of Excellence,

Charite Universitatsmedizin Berlin, Berlin, Germany

SUMMARY

Purpose: Seizure-like events (SLEs) induced by 4-amino-

pyridine in rat organotypic slices cultures, which are pre-

pared early after birth, are resistant to standard

antiepileptic drugs. In this study we tested the hypothesis

that pharmacoresistance may be an intrinsic property of

the immature brain.

Methods: Frequently recurring SLEs presumably repre-

senting status epilepticus were induced by 4-aminopyri-

dine in acute rat hippocampal–entorhinal cortex slices

obtained from postnatal day 3–19 (P3–P19), and the

effects of carbamazepine, phenytoin, valproic acid, and

phenobarbital were examined. In addition, bumetanide

was tested, which blocks the Na+-K+-2Cl) (NKCC1)

cotransporter, and also acetazolamide, which blocks the

carbonic anhydrase and thereby the accumulation of

bicarbonate inside neurons.

Results: The efficacy of all antiepileptic drugs in blocking

SLEs was dependent on postnatal age, with low efficacy in

P3–P5 slices. Antiepileptic drugs suppressed SLEs more

readily in the medial entorhinal cortex (ECm) than in the

CA3. In P3–P5 slices, valproic acid and phenobarbital

increased both tonic and clonic seizure-like activities in

the CA3, whereas phenytoin and carbamazepine blocked

tonic-like but prolonged clonic-like activity. In P3–P5

slices, bumetanide often blocked SLEs in the CA3, but was

not as effective in the ECm. Like with other antiepileptic

drugs, the seizure-suppressing effects of acetazolamide

increased with postnatal age.

Conclusion: We conclude that pharmacoresistance may

be inherent to very immature tissue and suggest that

expression of the NKCC1 cotransporter might contribute

to pharmacoresistance.

KEY WORDS: Epilepsy, Antiepileptic drugs, Seizure, CA3,

NKCC1, Pharmacoresistance.

Epilepsies in early childhood and particularly in prema-turely born babies are frequently difficult to treat (Painteret al., 1999; Jensen, 2009). This may depend on physiologicimmaturities in ion homeostasis and expressions of ionchannels and receptors but also on the severity of early onsetepilepsy or origin of the epilepsy in the antepartum period.

Developmental in vitro studies on the pharmacosensitivityof provoked seizures are rare. In slices obtained from8–23 days old rats, ictaform discharges evoked by 4-amino-pyridine (4-AP) were blocked by standard antiepilepticdrugs (AEDs) (Fueta & Avoli, 1992). In contrast, in theintact 1-week-old corticohippocampal formation in vitro,low Mg2+-induced seizure-like events (SLEs) were notblocked by AEDs (Quilichini et al., 2003; Dzhala et al.,2008) and in organotypic hippocampal slice cultures

prepared from 6–12 days old rats, pharmacoresistance wasalso observed (Albus et al., 2008). It is thought that, duringthis time period, the response of c-aminobutyric acid(GABA)A receptors to the intrinsic agonist undergoes matu-ration from depolarizing to hyperpolarizing. This functionalswitch seems to occur around postnatal day 10 in rodents(Tyzio et al., 2007; Zhu et al., 2008) and has been attributedto age-related differences in the expression of cationchloride cotransporters, such as the K+-Cl) cotransporter(KCC2) and Na+-K+-2Cl) cotransporter (NKCC1) (Riveraet al., 1999; H�bner et al., 2001; Romo-Parra et al., 2008).Furthermore, a steep developmental upregulation of car-bonic anhydrase isoform VII in the pyramidal cells of theCA1, around postnatal day 12 (P12), may act as a switchin the maturation of the hippocampal neuronal network(Ruusuvuori et al., 2004).

The role of NKCC1 in central neurons and accordinglythe role of GABA in seizure susceptibility are, however, stillcontroversial. It was shown that absence of NKCC1 activityin CA3 pyramidal neurons in postnatal 9–13 (P9–P13) miceresults in a significant increase in cell excitability

Accepted July 20, 2010; Early View publication September 24, 2010.Address correspondence to Prof. Uwe Heinemann, Inst. Neurophysiol-

ogy, Charit� Universit�tsmedizin Berlin, Oudenarderstr. 16, Haus 10,13347 Berlin, Germany. E-mail: uwe.heinemann@charite.de

Wiley Periodicals, Inc.ª 2010 International League Against Epilepsy

Epilepsia, 52(1):94–103, 2011doi: 10.1111/j.1528-1167.2010.02722.x

FULL-LENGTH ORIGINAL RESEARCH

94

(Zhu et al., 2008; Sipil� et al., 2009). On the other hand, bu-metanide blocked seizures in immature rat hippocampus(Nardou et al., 2009) and enhanced phenobarbital efficacyin a neonatal in vitro seizure model (Dzhala et al., 2008).Bumetanide also reduced seizure-like activity in vivo and invitro in the high K+- model (Dzhala et al., 2005), but not inother in vitro seizure models (Kilb et al., 2007).

As reported herein, we studied the effects of standardAEDs on provoked SLEs in combined hippocampal–entorhinal cortex slices of rats during the first threepostnatal weeks, with the first postnatal week representinga stage roughly corresponding to the last trimester inprimate pregnancy (Vannucci et al., 1999; Tucker et al.,2009). In addition, a possible contribution of depolarizing/excitatory GABAergic mechanisms to seizure-like activitywas investigated by applying the bumetanide, an inhibitorof NKCC1, and acetazolamide, an inhibitor of carbonicanhydrase. We have found that SLEs are frequently resis-tant to AEDs in the P3–P5 age group, whereas they weresensitive to bumetanide. Furthermore, AEDs were moreeffective in blocking SLEs in the medial entorhinal cortex(ECm) than in the CA3, whereas bumetanide was moreeffective in blocking SLEs in the CA3 than in the ECm.We suggest that overexpression of the NKCC1 cotrans-porter in early postnatal age may contribute to pharmaco-resistance.

Methods

The experiments were performed as described previously(Dreier & Heinemann, 1990) on horizontal hippocampus–entorhinal cortex slices (400 lm) prepared from 3- to 10-day-old and 14- to 19-day-old Wistar rats. Methodologicdetails are given in supporting material. Animal procedureswere conducted in accordance with the guidelines of theEuropean Communities Council and the Landesamt f�rGesundheit und Soziales (LaGeSo) Berlin, T0068/02.Recordings were carried out in interface type chamber.

Changes in extracellular potassium concentration ([K+]o)in the pyramidal cell layer of the CA3 and upper layers ofthe ECm were measured with double-barreled K+-selective/reference glass microelectrodes. The reference electrodesrecorded the direct current (DC) potentials and populationspikes in the respective layers in the DC mode. SLEs wereinduced by adding 100 lM 4-AP to artificial cerebro-spinal fluid (ACSF) (Fueta & Avoli, 1992; Br�ckner &Heinemann, 2000). After at least five SLEs (control) in bothstructures, an AED of interest was added to ACSF contain-ing 4-AP and applied for 60–80 min.

SLEs were considered as blocked if ongoing SLEs disap-peared for the last 20 min of drug application (lasting60–80 min) and reappeared during the wash-out period.Drug effects were presented as values during the drugtreatment normalized to control values (set to 100%).Statistical significance was determined by the Wilcoxon

matched-paired rank test. (Please see Supporting Informa-tion for further details on methods).

Results

In entorhinal cortex–hippocampal slices from younganimals, application of 4-AP reliably induced SLEs charac-terized by tonic-like and clonic-like activity in parahippo-campal regions such as the ECm and in the hippocampusproper (Fig. 1). At postnatal age of 16–19 days (P16–P19),4-AP induced recurrent short discharges in the CA3 in asubset of slices, which were not further analyzed. TheSLEs were characterized by a slow or steady negative shiftof the field potential with superimposed tonic-like and laterclonic-like activity accompanied by rises in extracellularpotassium concentration ([K+]o). In very young slices, thenegative shift was not as apparent, in line with previousstudies of the relationship between rises in [K+]o and gen-eration of slow negative field potentials (Nixdorf-Bergwe-iler et al., 1994). In such cases, SLEs were identified onthe basis of potassium accumulation by >2 mM and on fieldpotential transients. The data presented in this study arebased on 185 slices from 124 rats in which SLEs in theCA3 and ECm were provoked by application of 100 lM

4-AP. Of these 185 slices, 49 slices were from 3- to 5-day-old animals, 55 slices from 6- to 10-day-old animals, and81 slices from 14- to 19-day-old animals. In most slices,SLEs occurred independently from each other in the CA3and ECm. In a few slices where SLEs synchronizedbetween the CA3 and ECm, seizure onset was changingbetween both regions. We did not analyze effects of drugson interictal discharges, as clinicians point out that seizureshould be treated but not the electroencephalography(EEG).

SLEs could be induced by 4-AP even in the youngestpreparations investigated (postnatal day 3; P3), althoughinduction was not always possible in the entorhinal cortexbetween P3 and P5.

Seizure parameters change significantly with age in theentorhinal cortex but not in the CA3. The onset to 4-AP–induced SLEs was shortened in the entorhinal cortex from[values in mean € standard error of the mean (SEM)]35 € 5 min at P3–P5 to 20.5 € 3 min at P14–P19, whereaslatency to onset of SLEs in the CA3 remained at around25 min for all groups. In the ECm, but not in the CA3, theduration of SLEs increased from 39 € 3.3 to 48 € 3.8 s onaverage and extracellular potassium accumulationsincreased with age from peak levels of 8 € 0.4 to17 € 1.2 mM on average. In the CA3, average peak levels inextracellular potassium concentration were around 10 mM

for all age groups.If the drugs altered but did not completely block seizure

activities, it was sometimes difficult to determine the begin-ning and end of an SLE. In these cases we used the periodsduring which [K+]o was elevated by >1 mM above baseline

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to determine the beginning and end of an SLE during drugapplications.

We chose seemingly high concentrations of AEDs. Inpharmacoresistant patients, clinicians use the highest possi-ble tolerated doses, which vary strongly between patients.In status epilepticus, AEDs are often administered at evenhigher doses than those tolerated by patients during persis-tent therapy. In cortical tissue from pharmacoresistantpatients, drug levels were found that compared well to thosein the plasma. We, therefore, chose concentrations corre-sponding to plasma levels for our analysis (Rambeck et al.,2006). These high doses enable us to produce a significantcomparison between pharmacoresistant and pharmacosensi-tive age groups.

CarbamazepineThe effects of carbamazepine (n = 28) were dose, age,

and region dependent. Although effects were usually revers-ible, recovery of drug effects was slow and often incomplete(not shown). In Fig. 1A, at postnatal day 5 (P5), SLEs in theCA3 were resistant to 100 lM carbamazepine, whereas theywere suppressed in ECm. In another example shown inFig. 1B, carbamazepine 100 lM completely blocked SLEsin both regions of a P16 slice. The summary histogram(Fig. 1C) demonstrates that the proportion of P3–P10 slicesin which 50 lM carbamazepine completely blocked SLEswas 43% in the CA3 and 57% in the ECm (n = 7). The sameconcentration of carbamazepine completely blocked SLEs

in both the CA3 and ECm in all P14–P19 slices tested(n = 6) (Fig. 1C). When we increased the concentration ofcarbamazepine to 100 lM, we found variability in its effectin the P3–P10 group. We, therefore, subdivided this groupinto P3–P5 and P6–P10, and found that the proportions ofslices in which SLEs were completely blocked at 100 lM

carbamazepine in the CA3 increased from 20% at subgroupP3–P5 (n = 5) to 100% at subgroup P6–P10 (n = 5) andgroup P14–P19 (n = 5). In the ECm, SLEs were completelyblocked in all slices from all age groups (Fig 1D).

SLEs, not blocked by carbamazepine, were significantlymodified (supplemental Tables S1 and S2). In these cases,it prolonged SLEs in the CA3 despite an almost completesuppression of the tonic-like period (supplementalTable S1). In addition, the incidences of SLEs and the peak[K+]o associated with SLEs were decreased (supplementalTable S1). In the ECm, only with 50 lM carbamazepinewere SLEs not always blocked. In such cases, the durationand incidence of SLEs and [K+]o were reduced (supplemen-tal Table S2).

PhenytoinPhenytoin (n = 27) showed dose-, region-, and age-

dependent effects. Similar to the effects of carbamazepine,SLEs were more resistant to phenytoin in P3–P10 slices andin the CA3. In Fig. 2A at P5, SLEs in the CA3 were resistantto 100 lM phenytoin, whereas they were completely sup-pressed in the ECm. Fig. 2B shows a similar experiment

A B

DC

Figure 1.

Carbamazepine (CBZ) showed

age-dependent and area-dependent

effects. (A) and (B) samples of field

potentials (FP) recordings with

extracellular potassium

measurements ([K+]0). (A) CBZ

(100 lM) completely blocked SLEs

in the ECm but, in the CA3, only

blocked tonic-like events in slices

prepared from 5-day-old rats. (B)

CBZ (100 lM) blocked SLEs in both

the ECm and CA3 of slices

prepared from 16-day-old rats. (C)

and (D) Bar graph demonstrating

the proportion of slices in which 50

and 100 lM CBZ completely

blocked SLEs.

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performed in a P14 slice, and here, 100 lM phenytoin com-pletely blocked SLEs in both regions. Fig. 2C shows thatthe proportion of P3–P10 slices in which 40 lM phenytoinblocked SLEs was 0% in the CA3 and 28% in ECm (n = 7),whereas this proportion rose to 17% in the CA3 and 83% inthe ECm in the P14–P19 group (n = 6) (Fig. 2C). When theconcentration of phenytoin was increased to 100 lM, itcompletely blocked the SLEs in the CA3 in 13% of slices,and in the ECm, in 50% of slices in the P3–P10 group(n = 8), whereas in the P14–P19 group, the proportion ofslices in which phenytoin (100 lM) blocked SLEs rose to50% in the CA3 and to 100% in the ECm (n = 6) (Fig. 2D).

Although phenytoin failed to block seizure-like activityin a number of slices, especially at P3–P10, it significantlymodified SLEs (supplemental Tables S1 and S2). The mod-ifications in the CA3 and ECm were similar to those seenwith carbamazepine. In the CA3, phenytoin caused aprolongation in the duration of SLEs due to the increasedduration of clonic-like activity in P3–P10 slices. Like carba-mazepine, it also completely suppressed tonic-like activityin the CA3 (Fig. 2A, supplemental Table S1). Similarly tocarbamazepine, phenytoin reduced the duration and inci-dence of SLEs in the ECm, and decreased the rise in [K+]o

(supplemental Table S2).

Valproic acidThe effects of valproic acid (n = 33) were dependent on

drug concentration, postnatal age, and region in the tempo-ral cortex. As with carbamazepine and phenytoin, the SLEs

in P3–P10 slices were more resistant to valproic acid thanthe SLEs in P14–P19 slices, and SLEs in the CA3 weremore resistant than the SLEs in the ECm. In Fig. 3A, SLEsin the CA3 were resistant to 2 mM valproic acid at P8, butthey were completely suppressed in the ECm. When a simi-lar experiment was performed in a P17 slice, valproic acidcompletely blocked SLEs in both regions, as shown inFig. 3B. The summary histogram (Fig. 3C) demonstratesthat in P3–P10 slices, 1 mM valproic acid failed to blockSLEs in both the CA3 and ECm (n = 5). In P14–P19 slices,proportions remained 0% in the CA3 but increased to 50%in the ECm (n = 6) (Fig. 3C). When testing valproic acid at2 mM, we observed variability in its effect on SLEs in groupP3–P10 (supplemental Table S1). Therefore, this group wasfurther subdivided into P3–P5 and P6–P10. Valproic acid(2 mM) still failed to block SLEs in the CA3 in both sub-groups of P3–P10 slices (n = 11) but at P14–P19, it blockedSLEs in the CA3 in 45% of the slices (n = 11). In the ECm,the proportions of slices in which SLEs were blocked by2 mM valproic acid increased from 0% at P3–P5, to 40% atP6–P10, and to 82% at P14–P19 (Fig. 3D).

SLEs not blocked by valproic acid displayed significantmodifications. In contrast to carbamazepine and phenytoin,tonic-like activity was not completely suppressed by valproicacid. In P3–P10 slices, 1 mM valproic acid increased theduration of SLEs in the CA3 (supplemental Table S1) bypreferentially increasing the duration of the clonic-likeperiod. A similar effect was seen in the CA3 when 2 mM

valproic acid was applied to subgroup P3–P5 slices

A B

C D

Figure 2.

Phenytoin (PHT) showed age-

dependent and area-dependent

effects. (A) and (B) samples of field

potentials (FP) recordings with

extracellular potassium

measurements ([K+]0). (A) PHT

(100 lM) completely blocked SLEs

in the ECm but, in the CA3, blocked

only tonic-like events in slices

prepared from 5-day-old rats. (B)

PHT (100 lM) blocked SLEs in both

the ECm and CA3 of slices prepared

from 14-day-old rats. (C) and (D)

Bar graphs demonstrating the

proportion of slices in which 40 and

100 lM PHT completely blocked

SLEs.

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(supplemental Table S1). Valproic acid applied at 1 mM inP14–P19 slices and 2 mM in subgroup P6–P10 and P14–P19slices reduced seizure severity in the CA3 as indicated bydecreases in SLE incidence, SLE duration, and peaks of[K+]o (supplemental Table S1). In the ECm, SLEs were alsoreduced in severity (supplemental Table S2).

PhenobarbitalWe analyzed the effects of phenobarbital on 4-AP–

induced SLEs in 34 slices. Like other AEDs, SLEs in theCA3 were more resistant to phenobarbital than SLEs in theECm in the P3–P10 group. A typical experiment is depictedin Fig. 4A. Phenobarbital (200 lM) applied to a P4 sliceblocked SLEs in the ECm but failed to block SLEs in theCA3 (Fig. 4A). In another example shown in Fig. 4B, phe-nobarbital (200 lM) reversibly blocked SLEs in both areasin a P15 slice. The summary histogram (Fig. 4C) demon-strates that, at P3–P10, phenobarbital 100 lM failed to sup-press SLEs in both the CA3 and the ECm (n = 6), whereasat P14–P19 it blocked SLEs in the CA3 in 25% and in theECm in 100% of slices (n = 8). At a concentration of200 lM, the effects of phenobarbital were very variable inthe P3–P10 age group. We, therefore, subdivided this groupagain into P3–P5 and P6–P10 slices. The proportions ofslices with SLEs blocked by 200 lM phenobarbital in theCA3 were 0% at P3–P5 (n = 5), 38% at P6–P10 (n = 8),and 43% at P14–P19 (n = 7), whereas in the ECm, SLEswere blocked at all postnatal ages in all slices tested(Fig. 4D).

The effects of phenobarbital on the parameters of drugrefractory SLEs were similar to the effects of valproicacid. In the CA3 of subgroup P3–P5, 200 lM phenobarbi-tal significantly increased the SLE duration, with adecrease in SLE incidence (supplemental Table S1). Inthe remaining cases (100 lM phenobarbital at P14–P19,and 200 lM phenobarbital at subgroups P6–P10 and P14–P19) phenobarbital reduced seizure severity as indicatedby reduced duration and incidence of SLEs as well as bysmaller rises in [K+]o during SLEs in the CA3 (supple-mental Table S1).

BumetanideWe tested the effects of bumetanide, a loop diuretic that

blocks NKCC1, on 4-AP–induced SLEs in 29 slices. Theeffects were again region and age dependent. In Fig. 5A,10 lM bumetanide reversibly blocked SLEs in the CA3 ofa P4 slice, but failed to block SLEs in the ECm (Fig. 5A).In another example (Fig. 5B) bumetanide reversiblyblocked SLEs in the ECm but not the CA3 at P7. Thepostnatal age and region-dependent action of bumetanideis illustrated in the summary histogram (Fig. 6B). Tocompare the effects of bumetanide with AEDs, we dividedthe rats into several age groups. The seizure-suppressingeffects of bumetanide in the CA3 was restricted to P3–P5,at which time SLEs were completely suppressed in 91%of the slices tested (n = 11) (Fig. 6B). At later postnataltimes, SLEs were no longer suppressed in the CA3. In theECm, we did not find the strong suppressing effect of

A

CD

B

Figure 3.

Valproic acid (VPA) showed age-

dependent and area-dependent

effects. (A) and (B) samples of field

potentials (FP) recordings with

extracellular potassium

measurements ([K+]0). (A) VPA

(2 mM) completely blocked SLEs in

the ECm but not in the CA3 in

slices prepared from 8-day-old rats.

(B) VPA (2 mM) blocked SLEs in

both the ECm and CA3 of slices

prepared from 17-day-old rats. (C)

and (D) Bar graphs demonstrating

the proportion of slices in which

VPA 1 & 2 mM completely blocked

SLEs.

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bumetanide in very young age as compared to its effect inthe CA3. In the ECm the proportions of slices with SLEsuppressions were 9% at P3–P5 (n = 11), 25% at P6–P10(n = 8), and 10% at P14–P19 (n = 10) (0% in P14–P16and 14% in P17–P19) (Fig. 6B). The seizure-suppressingeffects of bumetanide were thus weaker in the ECm ascompared to the CA3. In slices in which bumetanidefailed to block SLEs, it increased the duration of SLE

with a decrease in SLE incidence in both the CA3 andECm (supplemental Tables S1 and S2).

Comparison between proportions of pharmacoresistantSLEs during the postnatal period (Fig. 6A) and the seizure-suppressing effects of bumetanide (Fig. 6B) revealed strongdiscrepancies. In the CA3 after postnatal day 5, bumetanidefailed to suppress SLEs, whereas the other AEDs gainedefficacy.

A B

CD

Figure 4.

Phenobarbital (PHB) showed age-

dependent and area-dependent

effects. (A) and (B) samples of field

potentials (FP) recordings with

extracellular potassium

measurements ([K+]0). (A) PHB

(200 lM) completely blocked SLEs

in the ECm but not in the CA3 in

slices prepared from 4-day-old rats.

(B) PHB (200 lM) blocked SLEs in

both the ECm and CA3 of slices

prepared from 15-day-old rats. (C)

and (D) Bar graphs demonstrating

the proportion of slices in which

PHB (100 & 200 lM) completely

blocked SLEs.

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Bumetanide (BUM) showed age-

dependent and area-dependent

effects. (A) and (B) samples of field

potentials (FP) recordings with

extracellular potassium

measurements ([K+]0). (A) BUM

(10 lM) blocked SLEs in the CA3 but

not in the ECm of slices prepared

from 4-day-old rats, (a) control with

4-AP (100 lM), (b) BUM (10 lM)

and 4-AP (100 lM), (c) wash out of

BUM. (B) BUM (10 lM) could not

block SLEs in the CA3 but blocked

SLEs in the ECm of slices prepared

from 7-day-old rats, (a) control with

4-AP (100 lM), (b) BUM (10 lM),

and 4-AP (100 lM) (c) wash out of

BUM.

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AcetazolamideA possible contribution of bicarbonate-mediated GABA-

dependent depolarization to 4-AP–induced SLEs wasinvestigated by applying acetazolamide (0.5 or 2 mM) to34 slices. Acetazolamide at 0.5 mM failed to suppress SLEsin the CA3 at all postnatal ages, whereas it blocked SLEsin the ECm in 20% and 30% of slices at P3–P10 (n = 5)and P14–P19 (n = 7), respectively. Analysis of data with2 mM acetazolamide forced us to break the P3–P10 groupinto two (P3–P5 and P6–P10) for this dose and we foundthat the proportions of slices in which SLEs were blockedin the CA3 were 33% at P3–P5 (n = 6), 100% at P6–P10(n = 6), and 80% at P14–P19 (n = 10). The respective pro-portions in the ECm were 83%, 100%, and 90%. The modi-fications of SLEs parameters in slices where acetazolamide

failed to block SLEs are shown in supplemental Tables S1and S2.

Discussion and Conclusions

Our present study shows that AEDs used in clinical prac-tice do not necessarily block SLEs in the first postnatalweek. The entorhinal cortex was generally more sensitive toAEDs than the hippocampus. Although carbamazepine andphenytoin blocked tonic-like activity, valproic acid and phe-nobarbital did not. We made an attempt to relate pharmaco-resistance to the depolarizing action of GABA and foundthat bumetanide had strong blocking effects in the CA3 until5 days postnatally. In the ECm the effects of bumetanidewere weaker than in the CA3, but spread over the whole per-iod studied.

Although SLEs induced in slices from very young ani-mals were often not blocked by AEDs, SLEs in entorhinal–hippocampal slices prepared from adult animals, whetherinduced by elevation of potassium (Leschinger et al., 1993),lowering of calcium (Heinemann et al., 1985), lowering ofmagnesium (Dreier et al., 1998), or by application of 4-AP(Br�ckner & Heinemann, 2000), usually responded to stan-dard AEDs, whereas interictal discharges and late recurrentshort discharges (RSDs) were pharmacoresistant, the latterevolving from SLEs. Our data are in agreement withfindings that, in the intact 1-week-old corticohippocampalformation in vitro, low Mg2+-induced SLEs in the CA1 andneocortex were not blocked by AEDs used at similarconcentrations (Quilichini et al., 2003; Dzhala et al., 2008).We here demonstrate that in acute entorhinal–hippocampalslices prepared from rats shortly after birth, 4-AP–inducedSLEs in the CA3 and the ECm showed comparatively moreresistance to AEDs when they were compared with the SLEsof slices from rats in the second and third postnatal weeks.

Pharmacoresistance might be due to severity of the dis-ease. Seizure severity might be indicated by the duration ofSLEs and the amplitude of rises in extracellular potassiumconcentration ([K+]o), as well as by the latency for inductionof SLEs. Latency to onset of SLEs was similar in the CA3for all ages, but decreased with age in the ECm, suggestingthat differences in seizure susceptibility do not account forthe appearance of pharmacoresistance. This is in contrast toobservations with low Mg2+- and low Ca2+-induced epilep-tiform activities, where seizure onset times were shorter inyoung slices than in adult slices (Albrecht & Heinemann,1989; Gloveli et al., 1995). The most significant measure ofseverity was the duration of seizure activity and the eleva-tion in [K+]o. Interestingly, K+ elevations were similar in theCA3, whereas they increased in the ECm with age in thesecond and third postnatal weeks, often beyond the ceilinglevel of 10–12 mM (Krnjevic & Morris, 1974; Heinemann& Lux, 1975, 1977). The findings in the CA3 region seem tobe at odds to a previous study from our lab, where abnor-mally large rises in [K+]o in response to stimulation were

A

B

Figure 6.

A comparison between the effects of antiepileptic drugs

(AEDs) and bumetanide on 4-AP–induced seizure-like activity

in the early postnatal temporal cortex. (A) Bars give the pro-

portions of slices in which the AEDs tested completely sup-

pressed SLEs in the CA3 and ECm. Only the results with the

higher concentrations of the 4 AEDs tested were considered in

this histogram. (B) Bars give the proportions of slices in which

10 lM bumetanide completely suppressed SLEs in the CA3 and

ECm.

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seen during the second and third postnatal weeks in the CA1(Nixdorf-Bergweiler et al., 1994). This would suggest thatSLEs in the CA3 were not too severe and that drug resis-tance is not related to severity of SLEs as indicated by potas-sium accumulation. In addition, the duration of SLEs wasnot altered over time in the CA3, whereas SLEs becamelonger in the ECm. The duration of SLEs is, therefore, notan indicator of pharmacoresistance.

Excitatory/depolarizing actions of GABA contributingto pharmacoresistance of 4-AP–induced SLEs

The gradients of anions that flow through open GABAA

receptors, namely Cl) and HCO3), determine whether neu-

rons depolarize or hyperpolarize. Chloride homeostasis iscontrolled mostly by cation chloride cotransporters such asthe Cl) extruding cotransporter KCC2 and the Cl) importerNKCC1. The depolarizing actions of GABA (Muelleret al., 1983; Ben Ari et al., 1989) in the first postnatalweek were ascribed to a strong expression of the cation-chloride cotransporter NKCC1, which causes accumulationof Cl) inside the cells setting the reversal potential forinhibitory postsynaptic potentials to depolarized levelswith respect to resting membrane potential. A steep upreg-ulation of the chloride-extruding potassium–chloridecotransporter KCC2 and the consequent increase in theefficacy of neuronal Cl) extrusion is thought to account forthe ‘‘developmental switch,’’ which converts depolarizingand excitatory GABA responses of immature neurons toclassical hyperpolarizing (Rivera et al., 2005). In theWistar rat CA3 pyramidal cells, the change in action ofGABA from excitatory to inhibitory occurs at around P8–10, via GABAA receptors (Tyzio et al., 2007; Romo-Parraet al., 2008).

We have found that in the CA3, the anticonvulsanteffects of NKCC1 blocker-bumetanide were obvious onlyin the first five postnatal days. In the ECm, the anticonvul-sive effects of bumetanide were, on one hand, weaker atthe chosen concentration, but persisted over a longer periodof postnatal life. Therefore, our data indicate the contribu-tion of depolarizing/excitatory GABA to the pharmacore-sistance of seizure-like activity with some regionalvariation. Indeed, the lack of effect of phenobarbital andvalproic acid in early age might be related to depolarizingeffects of GABA. These agents augment GABAergic syn-aptic transmission, although by different means, and thedepolarizing effects of GABA might be involved in theirrelative inefficiency. Differential maturation of Cl) trans-port has been observed in cortical versus subcorticalregions (Glykys et al., 2009) and it is, therefore, possiblethat the different effects of bumetanide in the CA3 andECm may also be related to differential expression of thistransporter in these structures.

Previous reports have indicated that NKCC1 expressionmay be upregulated in chronic animal models of epilepsyand in human epileptic tissue. Selective upregulation of

mRNA for NKCC1 with a resultant reduction in GABAergicinhibition was observed in an amygdala kindling model(Okabe et al., 2002). Analyses of tissue from patients withdrug-resistant epilepsy revealed upregulation of NKCC1(Palma et al., 2006; Aronica et al., 2007) and depolarizingGABA responses (Cohen et al., 2002; Wozny et al., 2003).Anticonvulsant activities of NKCC1 blocker bumetanidehave been observed in vivo and in vitro in the highK+- model (Dzhala et al., 2005; Kilb et al., 2007), and in akindling model (Mazarati et al., 2009). Another inhibitor ofNKCC1, furosemide, has also been reported to have anti-convulsant effects using in vivo and in vitro seizure models(Hochman et al., 1995, 1999; Schwartzkroin et al., 1998;Gutschmidt et al., 1999; Hesdorffer et al., 2001); however,at concentrations where likely other effects were involvedin the anticonvulsant effects.

Carbonic anhydrases II, IV, V, and VII (CA VII), whichoccur in neurons of the hippocampus (Ghandour et al.,2000; Wang et al., 2002), also affect actions of GABA.Expression of the cytosolic carbonic anhydrase (CA) may,therefore, influence the efficacy of GABAergic drugs.Indeed, depolarizing responses to GABA were seen duringhigh frequency stimulation of hippocampal interneurons,during application of high concentrations of neurosteroids(Burg et al., 1998) or during epileptiform activity (Kaila &Voipio, 1987). Membrane permeable CA inhibitors suchas sulthiame and acetazolamide (Leniger et al., 2002;Ruusuvuori et al., 2004) prevent GABAergic depolarizationby decreasing the intracellular bicarbonate concentrationand probably, in addition, by inducing a modest intracellularacidosis. Similarly to other AEDs, acetazolamide showedlow efficacy in P3–P5 slices and it blocked SLEs moreefficiently in the ECm than in the CA3.

In conclusion, our experiments indicate that the pharma-coresistance of seizure-like activity in rat temporal cortexregions is temporally correlated with both the immaturity ofthe tissue and with particular properties of the chloridehomeostasis. It should be noted that gestational age is ratherdifferent between rodents and man. Rodents are prema-turely born and P3–P5 in rats corresponds to 23–30 weeksof gestation in humans and P12–P13 in rats is roughlyequivalent to the time of birth in humans (Romijn et al.,1991; Vannucci et al., 1999; Hagberg et al., 2002; Stadlinet al., 2003; Tucker et al., 2009). It has been reported thatshort gestational age is associated with an increased risk ofseizures in humans (Vestergaard et al., 2002; Sun et al.,2008). Our data might thus explain the severe pharmacore-sistance noted frequently in babies, particularly when theyare born preterm and indicate that NKCC1 inhibitors maybe used as an adjuvant for treatment.

Acknowledgments

This research was supported by grants from the Hertie Foundation, theSFB TR3, EU IP 037315 ‘‘Epicure’’ and the Sander Foundation. We would

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like to thank Dr. S. Gabriel for helpful discussions and Dr. Kate ElizabethGilling for improving the English of manuscript.

Disclosure

We confirm that we have read the Journal’s position on issues involvedin ethical publication and affirm that this report is consistent with thoseguidelines. None of the authors has any conflict of interest to disclose.

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Supporting Information

Additional Supporting Information may be found in theonline version of this article:

Data S1. Supporting information for method section.Table S1. Effects of drugs on the properties of tonic-

clonic SLEs in area CA3 of each age group. Changes arepresented in percentages as the values present during treat-ment with the drug normalized to the control value.

Table S2. Effects of drugs on the properties of tonic-clonic SLEs in medial entorhinal cortex of each age group.Changes are presented in percentages as the values presentduring treatment with the drug normalized to the controlvalue.

Please note: Wiley-Blackwell is not responsible for thecontent or functionality of any supporting informationsupplied by the authors. Any queries (other than missingmaterial) should be directed to the corresponding author forthe article.

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