Progression of spontaneous seizures after status epilepticus is associated with mossy fibre...

Progression of spontaneous seizures after statusepilepticus is associated with mossy ®bre sprouting andextensive bilateral loss of hilar parvalbumin andsomatostatin-immunoreactive neurons

J. A. Gorter,1,2 E. A. van Vliet,1 E. Aronica3 and F. H. Lopes da Silva1,2

1Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, Amsterdam, The Netherlands2Stichting Epilepsie Instellingen Nederland, Heemstede, The Netherlands3Department of Neuropathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Keywords: hilus, mossy ®bre sprouting, parvalbumin, progression, rat, seizures, somatostatin, temporal lobe epilepsy

Abstract

The development of spontaneous limbic seizures was investigated in a rat model in which electrical tetanic stimulation of the

angular bundle was applied for up to 90 min. This stimulation produced behavioural and electrographic seizures that led to a

status epilepticus (SE) in most rats (71%). Long-term EEG monitoring showed that the majority of the rats (67%) that underwentSE, displayed a progressive increase of seizure activity once the ®rst seizure was recorded after a latent period of about 1 week.

The other SE rats (33%) did not show this progression of seizure activity. We investigated whether these different patterns of

evolution of spontaneous seizures could be related to differences in cellular or structural changes in the hippocampus. This wasthe case regarding the following changes. (i) Cell loss in the hilar region: in progressive SE rats this was extensive and bilateral

whereas in nonprogressive SE rats it was mainly unilateral. (ii) Parvalbumin and somatostatin-immunoreactive neurons: in the

hilar region these were almost completely eliminated in progressive SE rats but were still largely present unilaterally innonprogressive SE rats. (iii) Mossy ®bre sprouting: in progressive SE rats, extensive mossy ®bre sprouting was prominent in the

inner molecular layer. In nonprogressive SE rats, mossy ®bre sprouting was also present but less prominent than in progressive

SE rats. Although mossy ®bre sprouting has been proposed to be a prerequisite for chronic seizure activity in experimental

temporal lobe epilepsy, the extent of hilar cell death also appears to be an important factor that differentiates between whether ornot seizure progression will occur.

Introduction

Animals in which a status epilepticus (SE) is induced via injection of

drugs (pilocarpine or kainate) or via certain forms of electrical brain

stimulation are considered to be valid experimental models for the

study of human mesial temporal lobe epilepsy (MTLE) (Ben-Ari,

1985; Lothman et al., 1990; Cavalheiro et al., 1991). These models

display characteristic neuropathological aspects of the syndrome viz.

hippocampal sclerosis (neuron loss and gliosis), synaptic reorganiza-

tion, such as mossy ®bre sprouting, and the recurrence of seizures.

The recurrence and subsequent development of spontaneous electro-

graphic seizures have been described in the kainate model (Stafstrom

et al., 1992; Hellier et al., 1998; Bouilleret et al., 1999), the

pilocarpine model (Cavalheiro et al., 1991) and the electrogenic

model (Bertram & Cornett, 1994; Nissinen et al., 2000). The status

epilepticus induces a large individual variation of later chronic

seizure activity and it is not clear which neuropathological changes

are responsible for this differential evolution of seizure frequency and

severity.

A hallmark of MTLE is the extensive neuronal hilar cell loss

without a similarly severe cell loss of granule cells in the dentate

gyrus, also known as `endfolium sclerosis' (Margerison & Corsellis,

1966; de Lanerolle et al., 1989; Sloviter, 1991). Because of extensive

hilar cell loss and synaptic reorganization, it has been proposed that

the dentate gyrus, the main gateway to the hippocampus, is a major

source for epileptogenicity (Heinemann et al., 1992; Lothman et al.,

1992; Wasterlain et al., 1996). Another feature of the syndrome is

mossy ®bre sprouting (MFS), which has been suggested to be a

candidate in seizure progression (Sutula et al., 1989; Mathern et al.,

1997; Patrylo & Dudek, 1998). Indeed there is evidence that sprouted

mossy ®bres may form recurrent excitatory collaterals on the granule

cell dendrites in the inner molecular layer (Golarai & Sutula, 1996;

Wuarin & Dudek, 1996; Lynch & Sutula, 2000). Nevertheless, it has

also been proposed that MFS is just an epiphenomenon that does not

play a crucial role in epileptogenicity (Longo & Mello, 1997).

Although MTLE is generally considered to be a progressive

neurological syndrome (Engel, 1996), the factors contributing to this

progressive character are still poorly understood. It is important to

unravel the underlying mechanisms, in order to be able to achieve

better control or even to prevent seizures. The present study was

carried out with the main objective to discover factors that may

account for the fact that after SE some rats may develop progressive

Correspondence: Dr Jan A. Gorter, SILS, PO Box 94084, 1090 GBAmsterdam, The Netherlands.E-mail: [email protected]

Received 24 July 2000, revised 25 October 2000, accepted 29 November 2000

European Journal of Neuroscience, Vol. 13, pp. 657±669, 2001 ã Federation of European Neuroscience Societies

seizures with a high frequency of recurrence, whereas others do not.

Seizure activity was measured and quanti®ed during 3±6 months

using continuous hippocampal EEG monitoring after electrically

induced SE (according to a similar protocol as described by Lothman

et al. (1989). We determined whether seizure evolution was related to

mossy ®bre sprouting and/or cell loss. Moreover, since a large

population of GABAergic interneurons in the dentate gyrus consists

of parvalbumin (PV) and somatostatin (SOM) -immunoreactive (IR)

interneurons, we also investigated whether changes in these neuronal

populations were associated with the character of the seizure

evolution. The results suggest that the presence of PV- and SOM-

IR hilar interneurons and the extent of mossy ®bre sprouting are both

critical in determining whether seizure progression occurs or not.

Materials and methods

Experimental animals

Adult, male Sprague±Dawley rats (Harlan CPB laboratories, Zeist,

The Netherlands) weighing 350±550 g at the time of the experiment

were used in this study. The rats were housed in individual cages

under a controlled environment (21 6 1 °C; humidity 60%; lights on

08.00±20.00 h). Food and water were available ad libitum. One

group of rats (n = 35) was used for continuous monitoring of EEG

and seizure activity, and for subsequent histological analysis,

including 14 sham-operated controls.

Electrode implantation

Rats were anaesthetized with an intramuscular injection of ketamine

(57 mg/kg; Alfasan, Cuyk, The Netherlands) and xylazine (9 mg/kg;

Bayer AG, Germany) and placed in a stereotactic apparatus. In order

to record hippocampal EEG, a pair of insulated stainless steel

electrodes (70 mm, tips were 0.8 mm apart) were implanted into the

left dentate gyrus of the hippocampus under electrophysiological

control using the following co-ordinates: 3.9 mm anterior-posterior

(AP), 1.7 mm mediolateral, nosebar ±3.9 mm. For stimulation of the

angular bundle, an insulated stainless steel electrode was implanted

(7.2 mm anterior-posterior, 4.5 mm mediolateral). The location of

the electrodes was veri®ed during the operation by recording ®eld

potentials evoked in the granule cell layer by stimulation of the

angular bundle. Electrode pins were placed in a small six-pin

connector and the assembly was attached to the skull with small

stainless steel screws and dental acrylic. The experimental protocols

followed the European Communities Council directive 86/609/EEC

and the Dutch Experiments on Animal Act (1997), and were

approved by the Dutch animal welfare committee (DEC).

Seizure induction and EEG monitoring

Two weeks after recovery from the operation, each rat was

transferred to a special cage (40 3 40 3 80 cm) and connected to

a recording and stimulation system with a shielded, multistrand cable

and electrical swivel (Air Precision, Le Plessis Robinson, France). A

week after habituation to the new condition, the rats underwent a

series of tetanic stimulations (50 Hz) of the hippocampus in the form

of a succession of trains of pulses every 13 s. Each train had a

duration of 10 s and consisted of biphasic pulses (pulse duration

0.5 ms, maximal intensity 500 mA), similar to the protocol of

Lothman et al. (1989). Stimulation was stopped when the rats

displayed sustained forelimb clonus and strong salivation for minutes.

This behavioural condition was usually reached within 1 h; if not,

stimulation was continued, but it never lasted longer than 90 min.

Usually, immediately after the termination of the stimulation,

periodic epileptiform discharges (PEDs) at a frequency of 1±2 Hz

were evident in the hippocampal EEG. In this way a state

characterized by limbic electrographic seizure activity that we call

here status epilepticus was induced. In the present context, we make

the following operational de®nition of this status: the occurrence of

SE was scored when PEDs continued to be displayed for at least 4 h

after the series of tetanic stimuli. Note that we did not include in this

de®nition a criterion regarding the occurrence of behavioural

seizures, since the latter could have a variety of forms of appearance

while the EEG criterion was more reliable. Rats were intra-

peritoneally injected with pentobarbital (Nembutal, Sano® SanteÂ

B.V, France; 60 mg/kg) 4 h after termination of the tetanic stimuli to

avoid a severe and prolonged SE possibly leading to death. Twenty-

one rats were submitted to this form of tetanic stimulation. These rats

were placed under continuous EEG monitoring for 24 h/day for as

long as 3 to 6 months. Sham-operated control rats (n = 14) had

electrodes implanted, were handled and recorded in the same way,

but did not receive tetanic stimulation.

EEG signals were ampli®ed (103) via a FET transistor that

connected the headset to a differential ampli®er (503; CyberAmp,

Axon Instruments, Burlingame, CA, USA), ®ltered (1±60 Hz) and

digitized by a PC (Pentium 133 MHz). A seizure detection program

(Harmonie, Stellate Systems, Montreal, Canada) sampled the incom-

ing signal at a frequency of 200 Hz per channel, but since seizures

were sometimes missed by automatic detection software, we recorded

the EEG continuously. Every 24 h, recordings were interrupted

brie¯y to transfer previous day's recordings to a central review

computer. EEG recordings were reviewed afterwards. Seizures were

quanti®ed by the automatic system and further controlled by visual

inspection of the EEG records and in some cases of video recordings.

All EEG records were stored on compact discs; this procedure was

repeated daily, and was the guarantee not to miss any electrographic

limbic seizures that the rats experienced since the SE until the time

the rats were killed for histological and immunocytochemical

evaluation.

Tissue preparation and histology

Rats were disconnected from the EEG recording set-up and deeply

anaesthetized with pentobarbital (Nembutal, 60 mg/kg). The animals

were perfused through the ascending aorta with 300 mL of 0.37%

Na2S solution, 300 mL 4% paraformaldehyde and 0.2% glutaralde-

hyde in 0.1 M phosphate buffer, pH 7.4. The brains were post®xed

overnight at 4 °C. After post®xation, the brains were dissected and

the ipsilateral and contralateral parts were separated. Both parts were

cryoprotected in 30% phosphate-buffered sucrose solution, pH 7.4.

After an overnight incubation at 4 °C, the brain parts were frozen in

isopentane (±25 °C) and stored at ±80 °C until cutting was

performed. Brains were cut on a sliding microtome and alternate

horizontal sections (40 mm) were mounted on gelatine-coated slides

and either stained with 0.5% cresyl violet (Nissl staining) in 0.3%

acetic acid or processed by a modi®ed Timm method (for details see

Sloviter, [1982]) or processed for immunocytochemistry. Sections of

the different groups were processed for Timm stain at the same time

so that histological development time (55 min) was the same, to

enable comparison between groups.

Parvalbumin and somatostatin immunostaining

Horizontal sections at the midlevel of the hippocampus were washed

in 0.05 M phosphate-buffered saline (PBS), pH 7.4 and incubated for

30 min in 0.3% hydrogen peroxidase in PBS to inactivate

endogenous peroxidase. Sections were then washed (2 3 10 min)

in 0.05 M PBS, followed by washing (1 3 60 min) in PBS + 0.5%

658 J. A. Gorter et al.

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 13, 657±669

Triton X-100 + 0.4% bovine serum albumin (BSA). The sections

were incubated with monoclonal mouse antiparvalbumin (1 : 500;

clone PA-235; Sigma-Aldrich, Germany) or polyclonal rabbit

antisomatostatin serum (1 : 2500; no. 8001; Peninsula Laboratories,

Belmont, CA, USA) in PBS + 0.1% Triton X-100 + 0.4% BSA at

4 °C. After 24 h, the sections were washed in PBS (3 3 10 min) and

then incubated for 1.5 h in biotinylated sheep antimouse or antirabbit

IgG (Amersham, Pharmacia Biotech, Germany), diluted 1 : 200 in

PBS + 0.1% Triton X-100 + 0.4% BSA. Sections were washed in

PBS (3 3 10 min) and incubated for 1.5 h in streptavidin-horserad-

ish peroxidase (Zymed Laboratories, San Francisco, CA, USA),

diluted 1 : 200 in PBS + 0.1% Triton X-100 + 0.4% BSA. After

washing in 0.05 M Tris-HCl, pH 7.9, the sections were stained with

3,3¢-diaminobenzidine tetrahydrochloride (30 mg DAB, Sigma-

Aldrich) and 750 mL 1% hydrogen peroxide in a 100-mL solution

of Tris-HCl. The staining reaction was stopped by washing sections

in Tris-HCl. After mounting on gelatine-coated slides, the sections

were air dried, dehydrated in alcohol and xylene and coverslipped

with Entellan (Merck, Darmstadt, Germany). A subset of sections of

control and SE rats were incubated with a neuronal marker, a

monoclonal mouse antibody against Neu-N (1 : 1000, Chemicon,

Temecula, CA USA) or polyclonal rabbit antisomatostatin serum

(1 : 2500; no. 8001; Peninsula Laboratories). After 24 h, the sections

were washed in PBS (3 3 10 min) and then incubated for 2 h with a

1 : 200 dilution of ALEXA 488 goat antimouse (or antirabbit) IgG

antisera (Molecular probes, Eugene, OR, USA). Following three

additional washes in PBS, sections were mounted using Fluoromount

G (Southern Biotechnic, Birmingham, AL, USA) and used for

¯uorescent microscopy.

Cell counts and data analysis

The number of neurons was counted at the midlevel of the

hippocampus (5400±5700 mm below cortex surface). Neuronal

Nissl cell counts in CA3 (at the level of the ®mbria) and the

proximal portion of the CA1 area were performed at 4003

magni®cation over a microscope grid by an investigator unaware of

the treatment of the animals. All hilar Nissl-stained neurons (diameter

>10 mm) of a particular section were counted at 2003 magni®cation,

as described previously (Okazaki et al., 1999). The hilus was de®ned

by its border with the granule cell layer and by straight lines drawn

from the ends of the granule cell layer to the proximal end of the CA3

pyramidal cell layer. Per section, the cell density was given as the

number of cells divided by the hilar surface area of that particular

section. This was calculated using Scion Image (Scion Corporation,

release beta 3B, Frederick, MD, USA). Thus, cell density was

initially expressed as cell numbers per unit area (mm2) and then

normalized to control sections (100%). Statistical comparisons

between each experimental and control group were performed on

the cell density countings using a Student's t-test. The extent of

synaptic reorganization of the mossy ®bres was evaluated by two

observers according to a standardized 0±5 scale according to the

extent and density of zinc-stained granules (Cavazos et al., 1992):

0 No zinc staining between the tips and crest of the dentate gyrus

(DG).

1 Sparse zinc staining in the supragranular region in a patchy

distribution between the tips and crest of the DG.

2 More abundant staining in the supragranular region with patchy

distribution between tips and crest of the DG, and a continuous

pattern near the tips.

3 Prominent staining in the supragranular region in a continuous

pattern between the tips and crest of the DG.

4 Prominent staining in the supragranular region that forms a

con¯uent dense laminar band in the inner molecular layer (IML)

but does not completely ®ll it.

5 Con¯uent dense laminar band of staining in the supragranular

region that completely occupies the IML.

Intermediary values were scored in case the suprapyramidal blade

had a different score than the infrapyramidal blade.

Quanti®cation immunostaining

The number of PV- or SOM-IR neurons was counted per subarea (e.g.

hilar region) and expressed as cell densities/mm2 and then normalized

to control sections (100%). Statistical comparisons between experi-

mental and control groups were performed on the PV- and SOM-IR

cell density countings, respectively, using a Student's t-test.

Results

EEG during and shortly after stimulation

Twenty-one rats were stimulated and electrographically monitored

for 3 to 6 months. All rats showed limbic behavioural and

electrographic seizures during the stimulation. The electrical stimu-

lation produced an electrographic status epilepticus in 15/21 (= 71%)

of the rats (SE rats) that continued for 4 h after which the SE was

stopped with an intraperitoneal injection of pentobarbital (Nembutal;

60 mg/kg). PEDs often reappeared after the rat recovered from the

pentobarbital anaesthesia and could continue for several more hours.

In the other rats (non-SE rats; n = 6; 29%) PEDs did not appear, or if

they occurred, disappeared quickly (< 1 h) after the tetanic stimula-

tion. These rats were often stimulated for longer than 1 h, but never

longer than 90 min in an attempt to induce SE. In both SE and non-

SE rats, several spontaneous electrographic seizures could still be

detected within the ®rst 24 h after the stimulation (= the acute seizure

period). After this initial acute seizure period, that lasted no longer

than 48 h, the rats entered a latent period without electroclinical

seizures. As shown in Table 1, the latent period was considerably

shorter in the SE rats (1 week) than in the non-SE rats (34 days). In

the latent period, EEG epileptiform spike activity was frequently

recorded in SE rats, as well as occasional short bursts of EEG spikes

TABLE 1. Behavioural and electrophysiological characteristics of the three groups of epileptic rats

n

Number of stageIV±V seizuresduring stimulation

PEDduration(h)

Latencyto ®rstseizure (days)

Seizureindex

Progressive SE 10 8.7 6 0.6 12.2 6 0.8 7.2 6 1.5 4.10 6 0.89Nonprogressive SE 5 8.5 6 1.3 9.7 6 1.7 8.6 6 3.4 0.24 6 0.06*Non-SE 6 4.1 6 1.0* 0.61 6 0.20* 34.2 6 6.7* 0.14 6 0.03*

Seizure index is de®ned as cumulative number of seizures after stimulation/number of poststimulation days. t-test comparison with progressive SE group.*P < 0.001.

Progression of seizure activity after status epilepticus 659


(< 3 s) accompanied with jaw movements and blinking. Spikes were

less frequent in non-SE rats.

Electrographic seizure development after the ®rst spontaneousseizure

Both SE and non-SE rats displayed spontaneous seizures. Figure 1A

shows a record of a spontaneous seizure taken 2 months after

stimulation. After the latent period, seizures were frequently recorded

in most SE rats, but with a remarkable variability in the frequency of

seizure occurrence at the stage where a steady state was reached. If

we compare the seizure frequency at the start (individual average of

the ®rst 2 weeks of recording, starting from the day the ®rst seizure

was encountered) with the seizure frequency at the end of the

observation period (average of last 2 weeks of recording) 2±3 months

later we can clearly distinguish two groups (Fig. 1B). One group

(n = 10) has a clear increase in number of seizures experienced per

day (circles above the line in Fig. 1B). Since there is a steady

progression of the number of seizures experienced per day, these rats

will be further referred as `progressive SE rats'. These 10 out of 15

rats (67%) showed an increase in seizure frequency during the ®rst

2 months, after which seizure activity appeared to reach a steady state

at about 8±12 seizures a day (Fig. 1C). The other ®ve SE rats (all

around or below the line in Fig. 1B) showed occasional seizures

without an increase in seizure frequency until at least 12 weeks after

the ®rst registered spontaneous seizure (Fig. 1C). Of course we

cannot exclude that in this group, progression of seizures could have

FIG. 1. (A) Example of electrographic seizure recorded in the dentate gyrus at 2 months after the induction of status epilepticus (SE). (B) Individual averagenumber of seizures per day during the last 2 weeks of recording vs. the average number of seizures per day during the ®rst 2 weeks, counted from the ®rstrecorded spontaneous seizure. Ten rats (indicated by closed circles) show a much higher score during the last weeks than during the ®rst weeks and therebyshow seizure progression. The other 11 chronic epileptic rats, six of which were non-SE rats, showed no progression of seizures. (C) Average number ofseizures for each 1-week block in progressive SE rats (n = 10), nonprogressive SE rats (n = 5) and rats (n = 6) that did not display an SE after electricalstimulation (non-SE rats). A progressive increase in seizure frequency is observed only in progressive SE rats during the ®rst 9 weeks after the ®rst registeredspontaneous seizure after which seizure frequency stabilizes. (D) Number of seizures during the light period (white bars) and the dark period of theprogressive SE rats (black bars) during each consecutive week.



occurred at a later time-point than 3 months. Nevertheless, since

there was no increase in seizure frequency during the recording

period of 3 months, these rats will be referred as `nonprogressive

rats'. In the chronic epileptic phase, the duration of seizures did not

differ between these two groups and lasted on average about 1 min.

Figure 1D shows that seizures were more frequent during the day

(sleep period) than during the night (active period), as has been

reported before (Bertram & Cornett, 1994). We constructed a `seizure

index' (Table 1), equal to the cumulative number of seizures divided

by the total number of days poststimulation. This resulted in a seizure

index of 4.1 (seizures/day) for the progressive SE rats and 0.24

(seizures/day) for the nonprogressive SE rats. The duration of the

initial stimulation and the duration of the subsequent PED period

tended to be shorter in nonprogressive vs. progressive SE rats. The

occurrence of the ®rst spontaneous seizure was after about 7±9 days

in both groups (Table 1). EEG seizure activity, in the initial period of

about 5 weeks, was characterized by high voltage amplitude oscil-

lations. Later on, the EEG seizure pattern often changed. In many

cases the same rat could display different types of EEG seizure

activity; some started with high-frequency, low-voltage oscillations

that increased in amplitude and lasted over 1±2 min; these seizures

were often followed by a ¯attening of the EEG; others started with

high-frequency, low-voltage oscillations that remained of lower

voltage amplitude and had a shorter duration.

Non-SE rats also develop spontaneous seizures

In non-SE rats the latent period was on average four to ®ve times

longer than in both groups of SE rats (Table 1). As shown in Table 1,

the average duration of PEDs was just over half an hour. As can be

seen in Fig. 1B, the evolution of seizures was strikingly different

between most SE and non-SE rats. In the ®rst week after the end of

the latent period, non-SE rats experienced one seizure every 2±

3 days. Non-SE rats showed no further increase of seizure activity; on

the contrary in most rats we observed a decrease of seizure frequency

over time within the 3 months of continuous monitoring reaching

about one seizure every 10 days at 13 weeks. Non-SE rats experi-

enced an average of 0.14 seizures/day.

Behavioural seizures

During stimulation we observed the rats' behaviour and the number

of stage IV±V seizures (according to Racine's scale [Racine, 1972])

were counted. Both SE and non-SE rats displayed behavioural stage

IV±V seizures during stimulation. However, SE rats experienced

more than twice the number of stage IV±V seizures (expressed per

hour) than non-SE rats (Table 1). After stimulation, SE rats had a

variable behavioural pattern ranging from increased exploratory

behaviour with occasional tonic±clonic convulsions to frequent and

sustained tonic±clonic convulsions. Non-SE rats often showed

increased exploratory behaviour with an occasional stage IV seizure

during the ®rst hours after stimulation, but never sustained

behavioural seizures. Although we did not perform a continuous

video monitoring, we made some simultaneous video and electro-

graphic recordings of individual SE rats in the chronic stage.

Spontaneous EEG seizures were initially accompanied by jaw

movements, rearing and blinking of the eyes. Seizures often became

generalized (stage IV±V); this behaviour changed after about

2 months when generalized seizures were interchanged by seizures

consisting of only forelimb clonus and jaw movements (Stage II±III).

Sometimes, only muscle jerks of the body were visible associated

with high-voltage EEG amplitude oscillations. Interictal behaviour

was characterized by extreme sensitivity to auditory and tactile

stimuli in progressive SE rats. Non-SE rats did not seem to behave

differently from control rats.

Neuron loss after status epilepticus

Nissl-stained sections taken from the horizontal midlevel of the

hippocampus were examined to determine whether neuronal loss had

occurred and whether the differences in seizure evolution could be

associated with a differential cell loss. In progressive SE rats,

bilateral neuronal loss was evident in hippocampal areas CA1±CA3

and the hilar region of the DG. This was also demonstrated using the

speci®c neuronal marker Neu-N (Fig. 2). Also, outside the hippo-

campus (piriform cortex, entorhinal cortex [mainly layer III], septal

area, amygdala and midline thalamic nuclei) damage was often

extensive and bilateral, as has been described before (Du et al., 1995;

Bertram, 1997; GoncËalves Pereira et al., 2000). Figure 3A±D shows a

typical example of a Nissl-stained section of the hippocampus

(contralateral side) in a control rat, a non-SE rat, a nonprogressive SE

rat and a progressive SE rat. In Table 2 we show the average

percentage of hilar and CA cell densities in horizontal midsections of

the hippocampus. Non-SE rats are similar to controls except in the

ipsilateral CA1 where more cell loss was observed. In all progressive

SE rats > 70% of the hilar cells (> 10 mm) had disappeared in both

hippocampi. Hilar cell loss was more extensive at the infrapyramidal

than at the suprapyramidal side. In nonprogressive SE rats, hilar

neuron loss was mainly unilateral. Hilar interneuron number was only

slightly reduced in the contralateral hippocampus. Interestingly, CA3

appeared to be affected equally in the nonprogressive SE and in the

progressive SE rats, and the extensive cell loss (> 35%) that was

observed in this area was associated with a shrunken hippocampus

and enlarged ventricles. Cell loss in CA1 region was also consider-

able (> 35%), though less cell loss was found in contralateral CA1 of

progressive SE rats, compared with the ipsilateral side.

FIG. 2. Images (inverted ¯uorescence) of hippocampal sections of the CA1±CA3 region of a control (A) and post-status epilepticus (SE) rat (B).Immunostaining with the neuronal marker Neu-N shows cell loss in the CAregion indicated by the arrows (B). Dentate gyrus sections of a control rat(C); section of post-SE rat shows extensive neuronal loss in the hilus (D).Calibration bar, 200 mm.



Parvalbumin-immunoreactive neurons

Parvalbumin immunoreactivity in control rats was similar to what has

been described before (Kosaka et al., 1987; Sloviter, 1989). A dense

PV-IR ®bre plexus surrounds the CA pyramidal neurons and the

granule cells of the DG. Most PV-IR cell bodies are present in the

stratum pyramidal and in stratum oriens of the CA regions. In the

apical dendritic layer of the CA region, PV-IR neurons are rare. In the

DG, PV is present in a subset of interneurons in the hilus and in the

granule cell layer. A few cell bodies can be detected in stratum

moleculare. The dendrites of PV-IR cells run parallel to the principal

neuron dendrites. Most of these cells have bitufted dendrites with

frequent branching close to the soma.

Table 3 shows the average percentage of PV-IR neuron densities in

the hilus and granule cell layer at horizontal DG midsections. PV-IR

densities in sections of non-SE rats were similar to controls. The most

conspicuous difference between the two SE groups was the dramatic

reduction of the ®bre plexus around the granule cell layer in

progressive, but not in nonprogressive SE rats (compare Fig. 4C with

Fig. 4D). This was correlated with extensive and bilateral PV-IR

neuron loss in the hilus. Adjacent Nissl sections demonstrated that the

loss of immunoreactivity was associated with a general cell loss. In

progressive SE rats, the number of hilar PV-IR neurons was reduced

to 8±16% of the control densities. There was also a signi®cant

reduction of PV-IR neurons in the granule cell layer of progressive

SE rats to 44±53% of control densities. The number of PV-IR neurons

in the molecular layer, however, was not signi®cantly changed in any

of the epileptic groups compared with control (range 1±5 neurons per

section; percentage see Table 3). In nonprogressive SE rats, the

reduction of PV-IR neurons was unilateral and similar to what was

FIG. 3. Horizontal midsection of the contralateral hippocampus stained with cresyl violet in a control (A), non-status epilepticus (SE) (B), nonprogressive SE(C) and progressive SE rat (D). Arrows indicate extensive cell loss, e.g. in CA3 and hilar region of the progressive SE rats. Calibration bar, 500 mm.

TABLE 2. Cell densities in the hilar region and Timm scores in the inner

molecular layer in the mid-hippocampus rats

Mid level Control Non-SENonprogressiveSE

ProgressiveSE

Cell densities (% 6 SEM)Hilar cells ipsilateral 100 6 7 88 6 11 33 6 4* 34 6 5*Hilar cells contralateral 100 6 6 102 6 10 71 6 9*² 26 6 4*CA3 ipsilateral 100 6 5 117 6 4 60 6 10* 57 6 4*CA3 contralateral 100 6 4 101 6 3 65 6 3* 64 6 6*CA1 ipsilateral 100 6 8 84 6 3* 64 6 4* 61 6 3*CA1 contralateral 100 6 6 92 6 6 63 6 5* 86 6 4*²

Timm score ipsilateral 0.6 6 0.1 1.3 6 0.3* 3.5 6 0.3* 4.5 6 0.2*Timm score contralateral 0.6 6 0.1 0.7 6 0.2 2.7 6 0.3*² 4.5 6 0.2*

The cell densities of all control animals were averaged; the average value wasassigned 100%. The de®nition of the Timm scores (0±5) is given in text.*P < 0.01, compared with control; ²P < 0.01, contralateral compared withipsilateral side.



observed in progressive SE rats. In the contralateral hilus, however,

the number of immunoreactive neurons was slightly but not

signi®cantly reduced to 85% of control densities (Table 3).

Somatostatin-immunoreactive neurons

The distribution of SOM-IR neurons in control rats was as described

before (Sloviter & Nilaver, 1987; Kosaka et al., 1988; Vezzani et al.,

1996; Buckmaster & Dudek, 1997). These neurons were mainly

present in the hilar region of the dentate gyrus and in stratum oriens

of CA1 region and stratum oriens and radiatum of CA3 region with a

few cells in the pyramidal cell layer. A ®bre plexus was present in the

stratum lacunosum moleculare and in the outer two thirds of the

molecular layer (ML) of the dentate gyrus (Fig. 5A arrow). Table 3

shows the quanti®cation of the SOM-IR neurons in the hilar region.

SOM-IR densities in sections of non-SE rats were similar to controls.

SOM-IR neuron densities in the hilus were reduced to 27% in

progressive SE rats, while there was a reduction to 68% of control

densities in nonprogressive SE rats in the contralateral side. The

ipsilateral side was equally affected in both SE groups (> 70% loss).

The ®bre plexus in the outer ML was less intense in progressive SE

TABLE 3. PV-IR and SOM-IR cell densities in hilus and the granule cell

layer of the hippocampus

Mid levelControl(n = 10)

Non-SE(n = 5)

NonprogressiveSE (n = 4)

ProgressiveSE (n = 6)

PV-IR cell densities (% 6 SEM)PV hilus ipsilateral 100 6 20 96 6 10 12 6 4* 8 6 4*PV hilus contralateral 100 6 11 91 6 8 85 6 21² 16 6 6*PV GCL ipsilateral 100 6 23 78 6 22 48 6 7* 44 6 6*PV GCL contralateral 100 6 12 90 6 13 71 6 11*² 43 6 11*PV IML ipsilateral 100 6 25 100 6 25 125 6 33 125 6 43PV IML contralateral 100 6 12 109 6 20 106 6 31 113 6 28

SOM-IR cell densities (% 6 SEM)SOM hilus ipsilateral 100 6 12 94 6 10 29 6 10* 28 6 7*SOM hilus contralateral 100 6 9 104 6 14 68 6 16*² 27 6 7*

Average values of controls are assigned as 100%. *P < 0.01, compared withcontrol; ²P < 0.01, contralateral compared with ipsilateral side. GCL, granulecell layer; IML, inner molecular layer.

FIG. 4. Horizontal midsection of the contralateral dentate gyrus showing parvalbumin immunoreactivity in a control (A), non-status epilepticus (SE) (B),nonprogressive SE (C) and progressive SE rat (D). Note that the parvalbumin-immunoreactive (PV-IR) ®bre plexus has almost disappeared in progressive SErats, while PV-IR densities in the section of a nonprogressive SE rat is similar to densities of control and non-SE rats. Note also the survival of PV-IRneurons in the molecular layer of SE rats. Calibration bar, 250 mm.



rats (Fig. 5D) while it was clearly visible in nonprogressive SE rats,

at the contralateral side, and in non-SE rats, at both sides (Fig. 5B and

C). In hippocampal sections of progressive SE rats there was a

remarkable band of increased SOM immunoreactivity at the border of

the inner and middle ML (small arrows in Fig. 5D; see also Fig. 5F).

The remaining hilar neurons in both SE groups were rather large and

had a darker appearance than SOM-IR neurons in non-SE or control

rats, as described previously (Buckmaster & Dudek, 1997) (Fig. 5C

and D; at higher magni®cation in 5E).

Mossy ®bre sprouting after status epilepticus

In order to investigate the relation of mossy ®bre sprouting (MFS) to

the progression of seizure activity, we performed a Timm staining.

Typical examples of horizontal hippocampal midlevel sections of rats

of the three different epileptic groups and the control group are shown

in Fig. 6(A±D). Control rats had a very low Timm score with a score

that was considerably higher in the temporal pole than in the mid and

septal regions, as has been described previously (data not shown; see

FIG. 5. Horizontal midsection of the contralateral hippocampus showing somatostatin immunoreactivity in a control (A), non-status epilepticus (SE) (B),nonprogressive SE (C) and progressive SE rat (D). Note that only a few large somatostatin-immunoreactive (SOM-IR) cells are left in the suprapyramidal partof the hilar region of progressive SE rats. Calibration bar, 400 mm. (E) Higher magni®cation of clustered large SOM-IR neurons of a progressive SE ratperfused 3 months after SE. (F) Higher magni®cation of the DG molecular layer shows SOM-IR band in the IML at a distance of the GCL as detected with¯uorescent immunolabelling (inverted ¯uorescence image). Calibration bar (E and F), 100 mm. Arrows indicate band of SOM immunoreactivity in themolecular layer that is present in hippocampal sections of progressive SE rats. IML, inner molecular layer, OML, outer molecular layer, GCL, granule celllayer.



Cavazos et al., [1992]). At the time the animals were killed

(> 13 weeks after stimulation), all progressive SE rats had very

high Timm scores (4±5) along the entire septal-temporal axis (only

the data at midlevel are shown). There was a signi®cant negative

correlation (Spearman rank correlation Rs = 0.81; P < 0.01) between

Timm score and hilar Nissl-positive cell densities (Fig. 7A).

A high degree of progression of seizure activity (expressed as the

individual difference (D) between the number of seizures/day during

the last 2 weeks of recording and the number of seizures/day during

the ®rst 2 weeks after the ®rst registered seizure) was associated with

a higher Timm score (Fig. 7B; Spearman rank correlation coef®cient

Rs = 0.88; P < 0.001; n = 21). Nevertheless, if we consider both

groups of epileptic rats that have no seizure progression (nonpro-

gressive and non-SE rats) there is no signi®cant association with

sprouting (Rs = ±0.21; P < 0.53; n = 11) indicating that an increased

MFS does not necessarily lead to an increase in seizure frequency.

Nonprogressive SE rats had experienced a similar number of seizures

as non-SE rats but nevertheless had signi®cantly more abundant

Timm staining than non-SE rats.

Discussion

The main ®ndings of the present study can be summarized as follows.

(i) Progression of spontaneous seizures is observed in most but not all

rats that experienced an (SE). (ii) Seizure progression is associated

with bilateral hilar cell loss and extensive mossy ®bre sprouting in the

dentate region. (iii) Seizure progression is not observed in rats in

which PV and SOM interneurons are still abundantly present

unilaterally or bilaterally in the hilar region. (iv) SE rats that have

no seizure progression can have abundant MFS, though this is always

less prominent than progressive SE rats. (v) Rats that have not

experienced an SE (non-SE) after stimulation have no or only a slight

increase in MFS when compared with control rats; these rats

experience spontaneous seizure activity which, however, does not

progress in time.

This study con®rms the observation that a status epilepticus

induced by electrical stimulation can lead to a plastic process that

often culminates in an increase and intensi®cation of electrographic

seizures and worsening of the epileptic condition (Bertram & Cornett,

1994). However, our ®ndings also indicate that a status epilepticus

does not always lead to a progressive evolution of electrographic

seizure frequency. The factors that condition whether this will or not

occur are probably complex. However, we were able to reveal a

possible factor of importance in this aspect. We found that rats that

had extensive MFS and that had lost PV-IR and SOM-IR hilar cells in

both hippocampi experienced an increasingly progressive number of

seizures per day. Accordingly, preservation of these hilar interneur-

ons, even when this was limited to one hippocampus, was associated

FIG. 6. Horizontal midsection of the contralateral hippocampus stained by the Timm method in control (A), non-status epilepticus (SE) rat (B) thatexperienced a total of 24 seizures during 167 days poststimulation, nonprogressive rat that experienced 23 seizures during 106 days post-SE (C), progressiveSE rats that experienced 676 seizures during 106 days post-SE (D). Calibration bar, 500 mm.



with the lack of seizure progression, despite the fact that MFS on the

`preserved' DG was often rather robust (scores between 2 and 4).

These ®ndings may imply that the two hippocampal formations

should be envisaged as one network system in the sense that the

stability of the whole system depends, among other factors, on the

number of surviving inhibitory interneurons, such as PV- and SOM-

IR hilar neurons. If this number decreases below a given level, the

whole network will tend to display seizures more easily than if this

number were larger than that level. This may be a simpli®ed picture

of reality since here we only assessed a selected population of

neurons. Nevertheless the latter was chosen since it is likely that

(inhibitory) interneurons play a crucial role in the stability conditions

of the dynamic neuronal networks of the hippocampal system. The

fact that cell loss in CA1±CA3 and other limbic regions of

nonprogressive rats was bilateral supports this idea (this study and

P.M. GoncËalves-Pereira, P.J. Lucassen, E.A. van Vliet, F.H. Lopes da

Silva and J.A. Gorter, unpublished observations).

Although MFS was associated with chronic seizure activity, the

fact that MFS in nonprogressive SE rats is abundant while non-SE

rats with similar seizure scores show only minimal MFS, indicates

that MFS is not linearly related with seizure frequency. This con®rms

and extends recent ®ndings of PitkaÈnen et al. (2000). We cannot

exclude the fact that seizures become progressive only when

sprouting is prominent (Timm score > 4). However, this is not very

likely. Taking into consideration both the mossy ®bre sprouting and

the hilar cell loss ®ndings in our model, we propose that a critical

relation between hilar cell loss and the extent of mossy ®bre sprouting

is the determining factor regarding whether seizure progression will

occur or not: if cell loss in the hilus is extensive, bilateral and above a

certain threshold, and MFS is substantial, progression of seizures will

occur. If cell loss is less extensive, MFS can occur but this does not

necessarily lead to seizure progression.

Loss of parvalbumin and somatostatin immunoreactivity in thehilus

We observed a bilateral extensive loss of PV and SOM immuno-

reactivity in the hilar region of progressive SE rats. In nonprogressive

SE rats this loss was unilateral only. The loss of hilar PV and SOM

immunoreactivity in our SE rats is associated with general cell loss,

since Nissl staining from adjacent sections demonstrated the disap-

pearance of most hilar neurons. Dentate hilar cell loss is one of the

most conspicuous changes in other models of MTLE (Ben-Ari, 1985;

Mello et al., 1993; Buckmaster & Dudek, 1997), although to variable

extent and less extensive, as described for our post-SE model. The

fact that there is such an extensive hilar interneuron loss makes it less

likely that a substantial number of `dormant' interneurons would

remain in progressive SE rats, at least not in the dentate hilar region.

The `dormant' basket cell theory postulates that interneurons become

inactive after SE because of lack of excitatory drive by mossy cells

(Sloviter, 1991). It is possible that interneurons in the granule cell

layer and inner molecular layer of the dentate gyrus may become

innervated again by reorganized mossy ®bres, especially in

nonprogressive SE rats where interneuron cell loss is less extensive

(discussed later). Molecular layer PV-IR neurons in the dentate

FIG. 7. (A) Relation between Timm score and hilar cell densities in contralateral hippocampus. Higher Timm scores are related to lower hilar cell densities(r = 0.81). (B) Individual Timm score vs. the difference (D) between the individual average number of seizures/day during the last 2 weeks of the recordingand the number of seizures/day during the ®rst 2 weeks, after the ®rst recorded spontaneous seizure. Nonprogressive status epilepticus (SE) rats had Timmscores in between non-SE and progressive SE rats; progressive SE rats had high Timm scores without exception.



region did not appear affected after SE. Similar observations were

reported in the stimulated hippocampus of the perforant path

stimulation model (Sloviter, 1991).

The extent of PV-IR hilar cell loss that was observed in progressive

SE rats is similar to that seen in MTLE patients with endfolium

sclerosis pathology. In these patients, no PV-IR hilar neurons are

observed and only a few PV-IR neurons are present in the granule cell

layer (Sloviter et al., 1991; Zhu et al., 1997).

When we compare our results with those reported in investigations

of other experimental MTLE models, it is obvious that hilar cells are

amongst the most vulnerable ones in all these models. The cell loss in

the post-SE model is more extensive than has been described for the

pilocarpine or kainate model. Buckmaster and coworkers

(Buckmaster & Dudek, 1997; Buckmaster & Jongen-Relo, 1999)

showed considerable bilateral loss of hilar PV- and SOM-IR neurons

in chronic epileptic rats after kainate injection. The extent of hilar cell

loss in kainate-injected rats was considerably less than the extent of

cell loss in our progressive SE rats (42% in kainate vs. an average of

66±74% in our progressive SE rats). No correlation was found

between PV or SOM-IR cell loss and the epileptic state of the

kainate-injected rats. However, no electrographic seizure activity was

monitored in these studies, and no detailed account was made on

seizure progression or frequency of spontaneous seizures. In a recent

study in which we determined the temporal evolution of cell death

after SE, we found similar percentages of cell loss scores in the septal

and temporal poles of the hilar region as at the mid-level Ð unlike

what has been found in many other TLE models Ð so that it is

unlikely that the differences can be ascribed to differences in location

within the septal-temporal axis of the hippocampus (GoncËalves

Pereira et al., 2000). Unilateral cell loss is observed in the perforant

path stimulation model (Sloviter, 1991), but to lesser extent as

observed in our nonprogressive SE rats. Whether rats that are

stimulated according to Sloviter's protocol experience nonprogres-

sive spontaneous seizures has not been reported.

PV neurons are a subset of GABAergic interneurons, which play a

major role in the inhibition of pyramidal and granule cell activity

(Celio, 1986; Kosaka et al., 1987). Other important populations of

GABAergic hilar interneurons consist of calretinin and neuropeptide

(e.g. SOM) -containing neurons (Sloviter & Nilaver, 1987; Kosaka

et al., 1988; Miettinen et al., 1992; Freund & Buzsaki, 1996). Since

all SOM-IR hilar neurons are colocalized with GAD and send their

axons to the outer two-thirds of the molecular layer, they are

supposed to control inhibition at the level of the perforant path

(Freund & Buzsaki, 1996). This staining is preserved in nonpro-

gressive SE rats but practically absent in progressive SE rats,

suggesting that progression of seizure activity is less likely to occur

when inhibitory neurons in the dentate region are preserved. In

addition to the larger morphology of the SOM-IR neurons that were

often present after SE (in progressive as well as nonprogressive SE

rats), an additional dark band of SOM immunoreactivity could be

observed at a distance of the granule cells at the external border of the

IML in progressive SE rats. Whether this re¯ects aberrant SOM-IR

commissural ®bres, as has been suggested after perforant path lesions

(Deller et al., 1995), needs further investigation.

The role of mossy ®bre sprouting in epileptogenesis

Synaptic reorganization of the mossy ®bres in the dentate gyrus is a

typical hallmark of MTLE (Sutula et al., 1989; Represa et al., 1990;

Babb et al., 1992; Dudek et al., 1994). We show that abundant MFS

is not necessarily related to progression of spontaneous seizures later

in life. In nonprogressive SE rats, MFS was rather abundant and the

corresponding Timm scores were similar to those observed in rats

that showed seizure progression at 4±5 weeks after SE (most of

which experience 20±25 seizures/week at this time-point; J.A. Gorter

unpublished observations). Even at 3 months after SE, nonprogres-

sive SE rats still experienced only 1±3 seizures per week. Thus, the

extent of MFS does not appear to be correlated simply to the

progression of seizures. On the other hand, all progressive SE rats

show extensive MFS at the time of sacri®ce, suggesting that

progression of seizures is more likely to occur when both extensive

bilateral cell loss and MFS are present. What is the contribution of

MFS to seizure activity? Support for a crucial role for MFS in

epileptogenesis comes from various studies in which an increase of

excitatory postsynaptic currents is observed in slices of kainate-

injected rats (Molnar & Nadler, 1999; Wuarin & Dudek, 1996). On

the other hand, it has also been suggested that in addition to

excitatory connections, new inhibitory connections may be formed

after kainate-induced SE: (1) an increase in inhibition is observed

using paired pulse-evoked ®eld potential protocols (Sloviter, 1992;

Buckmaster & Dudek, 1997). (2) Electron microscopic analysis

revealed that sprouted mossy ®bre terminals can make synapses with

inhibitory interneurons in the granule cell layer (Kotti et al., 1997). It

has been reported that mossy ®bres in control rats do not make

random synaptic contacts but prefer to make selective synaptic

contacts with inhibitory neurons (Acsady et al., 1998). The fact that

in nonprogressive SE rats considerably more interneurons are

preserved in the granule cell layer than in progressive SE rats, may

explain why the balance, though still abnormal, could be restored

towards inhibition. Sprouting of mossy ®bres to the remaining

interneurons could then contribute to recover, at least partially, the

initially lost inhibition. In this scenario, basket cells would become

activated again by reorganized mossy ®bres. The data suggest that the

extent of inhibitory control in the dentate region could be an

important factor in determining whether seizures progress or not. In

this context, it is also appropriate to note that in the hippocampal-

kindling model of epileptogenesis, the dentate inhibition is enhanced

(King et al., 1985; Kamphuis et al., 1992; Zhao & Leung, 1992). In

this model, spontaneous seizures are rarely observed which reinforces

the idea that an important inhibitory control in the dentate gyrus is not

compatible with a progressive evolution of spontaneous seizures.

Conclusion

In most patients that suffer from MTLE, the epileptic condition

worsens with time and seizures can be progressive (Engel, 1996). In

this study, we have shown an association between seizure progression

and the extent of loss of inhibitory control in the hilar region. SE

leads to extensive cell death in speci®c brain areas and to synaptic

reorganization, typical hallmarks of MTLE. However, a severe SE

does not necessarily lead to progressive evolution of seizures. Future

research needs to be performed in order to determine whether

selective loss of hilar inhibitory PV- and SOM-IR neurons, or

additional cellular changes can be a crucial factor in determining the

progressive character of the seizure condition.

Acknowledgements

This work was supported by the `Nationaal Epilepsie Fonds' (NEF; J.A.Gorter and F.H. Lopes da Silva) and the `Teding van Berkhout' fellowshipof the `Christelijke Vereniging voor de Verpleging van Lijders aan Epilepsie'(E. Aronica). We thank W.P. Meun and S. van Mechelen for expertphotography.



Abbreviations

BSA, bovine serum albumin; DG, dentate gyrus; GCL, granule cell layer;IML, inner molecular layer; IR, immunoreactive; MFS, mossy ®bre sprouting;ML, molecular layer; MTLE, mesial temporal lobe epilepsy; OML, outermolecular layer; PBS, phosphate-buffered saline; PED, periodic epileptiformdischarge; PV, parvalbumin; SE, status epilepticus; SOM, somatostatin.

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Progression of spontaneous seizures after status epilepticus is associated with mossy fibre...

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