Epilepsy&

Epilepsy & Behavior 5 (2004) 180–191

Behavior

www.elsevier.com/locate/yebeh

Long-term behavioral and morphological consequences ofnonconvulsive status epilepticus in rats

Pavel Kr�sek, Anna Mikuleck�a, Rastislav Druga, Hana Kubov�a, Zden�ek Hli�n�ak,Lucie Suchomelov�a, and Pavel Mare�s*

Institute of Physiology, Academy of Sciences of the Czech Republic, V�ıde�nsk�a 1083, CZ 142 20 Prague 4, Czech Republic

Received 23 July 2003; revised 24 November 2003; accepted 25 November 2003

Abstract

The aims of the present study were to ascertain whether nonconvulsive status epilepticus (NCSE) could give rise to long-term

behavioral deficits and permanent brain damage. Two months after NCSE was elicited with pilocarpine (15mg/kg ip) in LiCl-pre-

treated adult male rats, animals were assigned to either behavioral (spontaneous behavior, social interaction, elevated plus-maze,

rotorod, and bar-holding tests) or EEG studies. Another group of animals was sacrificed and their brains were processed for Nissl and

Timm staining as well as for parvalbumin and calbindin immunohistochemistry. Behavioral analysis revealed motor deficits (shorter

latencies to fall from rotorod as well as from bar) and disturbances in the social behavior of experimental animals (decreased interest

in juvenile conspecific). EEGs showed no apparent abnormalities. Quantification of immunohistochemically stained sections revealed

decreased amounts of parvalbumin- and calbindin-immunoreactive neurons in the motor cortex and of parvalbumin-positive neurons

in the dentate gyrus. Despite relatively inconspicuous manifestations, NCSE may represent a risk for long-term deficits.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Lithium–pilocarpine status epilepticus; Epileptic brain damage; Motor performance; Behavior; Calcium-binding proteins

1. Introduction

It has been proved that convulsive status epilepticuscauses serious brain damage via excitotoxic mechanisms

[e.g., 1]. A complex neurotoxic cascade consisting of

multiple serial and parallel processes leading to both

necrotic and apoptotic cell death has been described

[2,3]. It is now evident that ‘‘epileptic‘‘ brain damage

results from excessive neuronal activity during status

epilepticus (SE), with complicating systemic factors

playing an additional role. Consequently, there is also aconceptual framework for brain damage resulting from

nonconvulsive status epilepticus (NCSE). However, al-

though a number of clinical reports support the view

that convulsive SE represents a serious risk for the hu-

man brain, possible harmful effects of NCSE are still a

source of controversy.

* Corresponding author. Fax: +420-2-41062488.

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

1525-5050/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.yebeh.2003.11.032

It has repeatedly been demonstrated that complex

partial status epilepticus, one of the two main types of

NCSE in humans, can be accompanied by considerableneurological morbidity, as well as mortality [4–8]. These

clinical observations are supported by MRI findings [9]

as well as by studies on serum neuron-specific enolase,

an accepted marker of acute brain injury [10,11]. How-

ever, most deficits are completely reversible [12,13]. It is

also stressed that the poor outcome of patients may be

related to the course of a systemic disease and not to

complex partial SE itself [14,15]. It is therefore doubtfulwhether clinical studies can definitively answer questions

concerning harmful effects of complex partial SE on the

brain.

Some electrically and pharmacologically induced

animal models of NCSE have been introduced. Hosford

recently asserted that the following models satisfy the

criteria for an animal model for complex partial SE:

kainic acid model, pilocarpine model, prolonged kind-ling, and Lothman�s self-sustaining limbic SE [16]. All

these models cause extensive brain damage. However, a

P. Krsek et al. / Epilepsy & Behavior 5 (2004) 180–191 181

marked discrepancy between the dire consequences ofartificially induced SE in animals and the relatively good

outcome of patients after complex partial SE has been

reported [15]. We suggest that one possible explanation

of this disagreement is that these experimental models

represent much more severe insults compared with hu-

man complex partial SE. Similarly, Drislane recently

suggested that EEG discharges recorded in the majority

of animal models are better suited to convulsive SE thanto complex partial SE, with its usually less dramatic

electroencephalographic patterns [17].

Short-term and self-limited NCSE induced by the

administration of low doses of pilocarpine in lithium

chloride-pretreated rats was developed in our laboratory

[18]. NCSE consisted of approximately 90 minutes of

abnormal behavior characterized by the occurrence of

various epileptic automatisms, e.g., chewing, nodding,face washing, and exploratory behavior as if in a new

environment. Both cortical and hippocampal spikes,

isolated or in bursts, as well as other types of epileptic

EEG activity, accompanied these behavioral features.

Profound impairment of responsiveness to exteroceptive

stimuli correlating with the occurrence of epileptic EEG

activity was observed [19]. One and two weeks after

NCSE, seizure-related brain damage consisting of darkand shrunken neurons in Nissl-stained brain sections

was observed mainly in the motor neocortical fields

[18,19].

On the basis of these observations, we were able to

define a new pharmacologically induced model of hu-

man complex partial SE. However, it remained to be

ascertained whether this relatively brief NCSE could

cause long-term functional and morphological conse-quences. This presumption was supported by the study

of Cook and Persinger, who described a deficit in long-

term memory using the radial maze paradigm in rats

injected with a subconvulsive dose of pilocarpine 5

months before testing [20].

To determine the possible delayed consequences of

NCSE, video EEG monitoring, behavioral testing, and

morphological examination were conducted 2 monthslater. Histological examination of brain sections was

performed to clarify whether morphological damage

observed 1 and 2 weeks after NCSE was permanent.

Moreover, brain slices were immunostained using anti-

bodies to the calcium-binding proteins calbindin D-28k

and parvalbumin to specifically evaluate affected cell

populations in both the neocortex and limbic structures.

2. Methods

2.1. Animals

Adult (90-day-old) male albino Wistar rats (N ¼ 114)

weighing 250–350 g were used. They were housed in

standard plastic cages in a temperature-controlled room(22� 1 �C) with a 12-hour light/dark cycle (lights on at

6:00 AMAM). Food and water were available ad libitum in

the home cage. In addition, 32 juvenile rats 21 days old

were used in the social interaction test. All experiments

were conducted under approval of the Animal Care and

Use Committee of the Institute of Physiology of the

Academy of Sciences in agreement with the Animal

Protection Law of the Czech Republic (fully compatiblewith the guidelines of European Community Council

Directives 88/609/EEC).

2.2. Experimental procedure

NCSE was elicited with pilocarpine (15mg/kg ip) 24

hours after lithium chloride pretreatment (3mEq/kg ip).

The rats were placed in separate plastic boxes and theirbehavior was observed for 120 minutes. EEG recording

was not performed in this study because of data from

the previous study [18]. Experimental groups were

formed only by animals exhibiting clear-cut features of

NCSE (N ¼ 63). Control rats (N ¼ 51) received an

equal volume of saline instead of pilocarpine; other

conditions were the same as for the pilocarpine-treated

animals. These controls were put together from all on-going studies on nonconvulsive status. Two rats in-

cluded in the control group received pilocarpine also but

they did not develop any signs of NCSE. Animals ex-

hibiting short-lasting behavioral signs of nonconvulsive

seizures (N ¼ 3) were not included in the present study;

it would be necessary to have more rats to make an

additional group. Animals exhibiting motor seizures

were included in other studies focused on convulsivestatus epilepticus.

All experiments were performed 2 months after

NCSE. Three groups of animals were established. First,

video EEG monitoring was conducted in 6 pilocarpine-

treated and 2 control animals. The second group, con-

sisting of 46 pilocarpine-treated and 44 control rats, was

started on behavioral testing. Rats in the third group (11

pilocarpine-treated rats and 5 controls) were sacrificedand their brains processed for morphological tech-

niques. Animals from the first and the third groups were

not tested behaviorally to ensure that possible changes

are due to their previous nonconvulsive status.

2.3. Video EEG monitoring

Animals were anesthetized with ketamine (100mg/kgip) and xylazine (20mg/kg im). Two flat silver electrodes

were placed over the sensorimotor areas of both hemi-

spheres (AP¼ 0, L ¼ 2mm), two Teflon-coated stain-

less-steel electrodes were inserted into the dorsal

hippocampus (AP¼ 3.5, L ¼ 2, H ¼ 3:8mm), and an

indifferent electrode was inserted into the nasal bone.

The electrodes were fixed to the skull with dental acrylic.

182 P. Krsek et al. / Epilepsy & Behavior 5 (2004) 180–191

One week after surgery, animals were individuallyplaced in plastic boxes (30� 40� 45 cm) and attached to

the EEG apparatus (BrainScope) with flexible wires and

swivels so that their movement was not restricted. The

EEG was continuously recorded for 5 hours for six ex-

perimental and two control rats. Behavior was simul-

taneously videotaped.

Localization of hippocampal electrodes was checked

histologically after the end of the experiment. Again, allanimals included in EEG study had tips of electrodes

placed in the CA3 field of the dorsal hippocampus.

2.4. Behavioral procedure

Animals (N ¼ 90) were tested between 9:00 AMAM and

3:00 PMPM. All the experimental devices used were thor-

oughly cleaned before a new animal was tested. In boththe spontaneous behavior test and the social interaction

test, rat behavior was monitored using a video camera

located 100 cm away from the arena. The videotape was

scored by an experienced observer (intrarater reliabil-

ity> 0.9) using the Observer program (Noldus Infor-

mation Technology). Rats were never used for two

behavioral tests; only some animals were exposed to one

behavioral and one motor (rotorod or bar holding) test.

2.5. Spontaneous behavior test

Animals (14 pilocarpine-treated rats and 14 controls)

were placed individually in the square arena (plexiglas

cage, 45� 45� 30 cm) and their behavior was monitored

for 5 minutes. The following categories of spontaneous

behavior were distinguished: walking (movementaround the arena); rearing (upright posture both against

and away from the wall); sniffing (investigation of the

floor, walls, and space); face washing (brief period of

vibrating movements of the forepaws in front of the

snout followed by nose or head washing and paw lick-

ing); grooming (face washing with a subsequent cepha-

locaudal progression); and immobility (lying or sitting).

The total number, total duration (in seconds), and meanduration (a derived measure: total duration divided by

total number) of individual categories were evaluated.

2.6. Social interaction test

On the day of the experiment, both pilocarpine-

treated (N ¼ 16) and control (N ¼ 16) adults and juve-

niles (21 days old, N ¼ 32) were housed individually.After 1 hour of adaptation to the experimental room, an

adult animal was placed in the experimental arena

(identical to that described above) to explore the new

environment. Two minutes later, a juvenile conspecific

was placed in the arena. Each interaction lasted 5 min-

utes. Reexposure to the same juvenile occurred 30

minutes after the first interaction. Between successive

exposures, both adults and juveniles were maintainedindividually. The total number and total duration of

social investigations directed toward the juvenile (ap-

proaching, nosing, and body sniffing) were calculated.

2.7. Elevated plus-maze test

Pilocarpine-treated (N ¼ 16) and control (N ¼ 14)

animals were placed individually on the open arm of theplus-maze, and the transfer latency (time required for an

animal to move from the open arm to the enclosed arm)

was recorded. The second session was carried out 24

hours later. If the rat did not enter the enclosed arm

within 120 seconds, it was excluded from the evaluation

(2 of the original group of 30 rats).

2.8. Motor performance

2.8.1. Rotorod test

The rotorod apparatus was set to rotate at a constant

5 rpm. The rats (25 pilocarpine-treated and 22 control

rats; these groups were formed by animals exposed to

the open field and social interaction test) were placed on

the rod with the body axis perpendicular to the rod�slong axis and with the head directed against the direc-tion of rotation. The maximal score in maintaining

equilibrium was arbitrarily fixed at 60 seconds. To ob-

serve the behavioral strategy used by an animal to

maintain equilibrium, rats were placed on the rod indi-

vidually. Three strategies were observed: grasping (mo-

tionless grasping of the rod), walking (walking on the

rod), and orientation (described as scanning relative to

the rod and attempting to stand upright).

2.8.2. Bar-holding test

Rats (14 pilocarpine-treated and 14 control rats, the

same animals as in the spontaneous behavior test) were

hung by both forepaws on a wooden rod (25 cm long,

1 cm in diameter) located 1m above a landing platform

covered with a thick sheet of soft plastic to cushion falls.

The duration of grasping was evaluated.

2.9. Morphology

2.9.1. Fixation

Two months after treatment, an overdose of urethane

(2 g/kg ip) was administered and animals were perfused

with 0.37% sulfide solution (1mL/g) for 10 minutes

followed by 4% paraformaldehyde in 0.1M phosphatebuffer, pH 7.4 (1mL/g), for 10 minutes. The brains were

removed from the skull and postfixed in buffered 4%

paraformaldehyde for 3 hours and then cryoprotected in

a solution containing 20% glycerol in 0.02M potassium-

buffered saline for 24 hours. The brains were frozen in

dry ice and stored at )70 �C until cut. Brains were sec-

tioned in the coronal plane (50 lm, 1-in-5 series) with a

P. Krsek et al. / Epilepsy & Behavior 5 (2004) 180–191 183

sliding microtome and sections were stored in a cryo-protectant tissue-collecting solution (30% ethylene gly-

col, 25% glycerol in 0.05M sodium phosphate buffer) at

)20 �C until processed. Adjacent series of sections were

used for Nissl and Timm staining.

2.9.2. Nissl and Timm staining

The first series of 1-in-5 sections were mounted onto

gelatin-coated slides and stained with cresyl violet. Forthe Timm sulfide/silver method, all sections (1-in-5 se-

ries) in which hippocampus was present were mounted

on gelatin-coated slides, air-dried, and stained in the

dark according to the following protocol: A working

solution containing arabic gum (300 g/L), sodium citrate

buffer (25.5 g/L citric acid monohydrate and 23.4 g/L

sodium citrate), hydroquinone (16.9 g/L), and silver ni-

trate (84.5mg/kg) was poured into the staining dish,which contained the slides. The sections were developed

until an appropriate staining intensity was attained

(approximately 60 minutes). Then the slides were rinsed

with tap water for 30 minutes and placed in 5% sodium

thiosulfate for 12 minutes. Finally, sections were dehy-

drated through an ascending series of ethanol, cleared in

xylene, and coverslipped. Nissl-stained slices were ex-

amined to identify the cytoarchitectonic boundaries aswell as the possible distribution of neuronal damage and

to localize hippocampal electrodes in animals tested

electrophysiologically.

2.9.3. Immunohistochemistry

Sections were sequentially incubated in 0.15% hy-

drogen peroxide in PBS (0.01M, pH 7.4) for 10 minutes,

rinsed with PBS four times, and then incubated withblocking solution containing 2% normal horse serum

(Vector Laboratories, Burlinghame, CA, USA) and

0.1% Triton X-100 in PBS at room temperature. The

sections were incubated with the primary antibody (anti-

parvalbumin and/or anti-calbindin monoclonal anti-

body, Sigma, dilution; 1:5000 in PBS containing 1.5%

normal horse serum and 0.1% Triton X-100) for 48

hours at 4 �C and then rinsed four times in PBS. Sectionswere incubated for 1 hour at room temperature with

secondary antibody, biotinylated anti-mouse antibody

made in horse serum (Vector); dilution 1:50 in PBS

containing 1.5% normal horse serum and 0.1% Triton

X-100. After this step, the tissue was rinsed with PBS

four times and covered with the ABC reagent (Vecta-

stain Kit, Vector) for 1 hour at room temperature. After

being rinsed, sections were incubated for 5–7 minuteswith a mixture of 0.02% diaminobenzidine and 0.05%

hydrogen peroxide in PBS. Control sections were

prepared with the same procedures, but omitting the

primary antibody. All sections were mounted onto gel-

atin-coated slides, coverslipped, and examined by light

microscopy (Olympus AX 70 microscope with bright-

field optics).

2.9.4. Analysis of the sections

Synaptic reorganization (mossy fiber sprouting) was

analyzed in sections stained the using the Timm method.

Sprouting was analyzed along the septotemporal axis of

the hippocampus; method was described in detail by

Nissinen et al. [21]. The septal end included the coronal

sections between AP levels 2.3 and 6.0mm posterior

from bregma [22]. The dorsal and ventral midportions

of the dentate gyrus included dorsal and ventral parts ofhippocampus where the granule cell layer of the septal

and temporal ends becomes fused and forms a stan-

dardized ‘‘oval-shaped’’ layer (AP level 6.1–6.7mm

posterior to bregma). Mossy fiber sprouting was scored

according to Cavazos et al. [23]. The distribution of

Timm granules in the supragranular layer was rated

from 0 to 5. While the lowest levels (0 and 1) indicate

negative finding or sparse granules, respectively, morenumerous granules are scored as 2, occasional patches

of granules as 3, presence of a dense laminar band of

granules as 4, and extension into the inner molecular

layer as 5.

Parvalbumin-immunoreactive (PV-IR) neurons were

counted throughout layers II–VI of motor neocortical

fields Fr 1, Fr 2, and Fr 3 (where the most prominent

effect was observed 1 and 2 weeks after NCSE in theprevious studies [18,19]). For each animal 12 sections in

the range of stereotaxic planes AP 2.2 and AP )0.3 were

counted [22]. Calbindin-immunoreactive (CB-IR) neu-

rons were counted in the same anteroposterior range of

motor fields using 0.3� 0.3-mm squares localized ran-

domly in neocortical layers V and VI. Sixteen squares

per each of 12 sections from each rat were evaluated.

Classification of the neocortical areas was based on thecytoarchitectonic criteria of Zilles [24].

PV-IR neurons in the dentate gyrus were counted in

the whole mediolateral extent of both blades in 12 sec-

tions per each animal in the range of stereotaxic planes

AP )2.8 and AP 4.3. PV-IR neurons in CA1 and CA3

hippocampal fields were counted in 0.3� 0.3-mm

squares (12 squares per each animal). PV-IR neurons in

the CA1 field were restricted to the segment betweensagittal planes 1 and 2 or 1 and 3 in more caudal sec-

tions, respectively. Terminology used in description of

the limbic structures is according to Paxinos and Wat-

son [22]. All analyses were conducted blind to treatment

group.

2.10. Statistics

Behavioral data were analyzed nonparametrically. To

compare the differences between independent groups

(pilocarpine-treated animals vs controls) within a given

session, the Mann–Whitney U test was used. To com-

pare the differences within matched pairs (subsequent

sessions for both pilocarpine-treated and control rats) in

the social interaction and elevated plus-maze tests, the

184 P. Krsek et al. / Epilepsy & Behavior 5 (2004) 180–191

Wilcoxon matched-pairs signed-ranks test was used.Morphological data (numbers of PV-IR and CB-IR

neurons) were analyzed parametrically with the t test.Statistical significance was accepted at P < 0:05 (two-

tailed).

Fig. 1. Relative representation of the occurrence (left) and duration

(right) of individual behavioral patterns. C, control group (n ¼ 14); P,

pilocarpine group (n ¼ 14). Statistical significance, P < 0:05 (two-

tailed): �Pilocarpine versus control group (only duration of face

washing exhibited a significant difference).

3. Results

NCSE was elicited in all 63 experimental animals in-

cluded in this study. It was characterized by hyper-

salivation, piloerection, licking, swallowing, and chewing

starting immediately after pilocarpine administration.

These phenomena could not be taken as epileptic; rather,

they represented the peripheral action of pilocarpine.

Then automatisms like face washing, head nodding, and

creep walking (ataxia-like movements) were observed.Fully developed ictal behavior was characterized by

immobility (with chewing and head nodding) interrupted

by periods of creep walking. Gradual disappearance of

the above-mentioned abnormal behavior was accompa-

nied by repeated periods of exploration/searching activ-

ity (in a well-known cage) and self-grooming. These

activities also subsided so that there were no apparent

behavioral changes in the experimental animals 2 hoursafter pilocarpine treatment. Rats were returned to the

animal facility and examined 2 months later.

3.1. Video EEG monitoring

Evaluation of video EEG recordings revealed no

apparent EEG as well as behavioral abnormalities in

experimental as well as control rats.

3.2. Behavioral data

3.2.1. Spontaneous behavior test

There was no difference in the total frequency of all

recorded behavioral patterns between pilocarpine-trea-

ted animals (48.1� 5.6, mean� SEM) and controls

(42.5� 5.7). Analysis of particular patterns revealed nochange except for face washing (Fig. 1). The total time

spent and the mean duration of face washing were sig-

nificantly increased in pilocarpine-treated animals.

Fig. 2. Total time spent by adult males in investigation of juveniles in

the social interaction test (means+SEM). The second exposure to the

same juveniles was performed 30 minutes after the first one. Open bars:

control group (n ¼ 16); crosshatched bars: pilocarpine group (n ¼ 16).

Statistical significance, P < 0:05 (two tailed): �Pilocarpine versus

control group, dsecond session versus first session.

3.2.2. Social interaction test

When compared with the controls (Fig. 2), a signifi-

cant reduction in the total duration of social investiga-

tion was observed for pilocarpine-treated animalsduring the initial exposure. No difference in social in-

vestigation was found between pilocarpine-treated and

control rats during the second exposure. Intersession

comparisons of social investigation revealed signifi-

cantly lower levels during the second exposure for both

experimental and control rats.

3.2.3. Elevated plus-maze test

No statistical difference in transfer latency between

pilocarpine-treated and control animals was noted dur-

ing both the initial session and the second session.

As compared with the initial session, a significant

P. Krsek et al. / Epilepsy & Behavior 5 (2004) 180–191 185

shortening of the transfer latency during reexposure wasnoted in both groups of animals.

3.3. Motor performance: rotorod and bar-holding tests

Maintenance of equilibrium on the rotorod without

falling (Fig. 3) was significantly decreased in pilocar-

pine-treated animals. Moreover, the strategy of the pi-

locarpine-treated animals was different from that of thecontrols. While control rats walked on the rod and

maintained equilibrium, pilocarpine-treated rats at-

tempted to rear, scan, and turn in the direction of the

rotation and to sit; 16 of 25 pilocarpine-treated animals

fell.

In the bar-holding test, the duration of grasping was

significantly decreased in the pilocarpine-treated ani-

mals. Similarly to controls, pilocarpine-treated rats at-tempted to climb the bar; however, they were less skillful

(Fig. 3).

3.4. Morphology

3.4.1. Nissl and Timm staining

Whole brain was always checked in Nissl-stained

sections. There were dark and shrunken neurons inlayers V and VI of the motor neocortical areas (fields Fr

1–3) in pilocarpine-treated rats (Figs. 4A,B). Small

numbers of dark neurons were inconsistently found in

the corpus striatum and CA3 hippocampal field. Exact

quantification of the changes was precluded by the

thickness of the slices; however, the number of affected

neurons appeared to be smaller in comparison with the

numbers 1 and 2 weeks after NCSE [18,19]. No obviouschanges were observed in any other brain structure.

Fig. 3. Motor performance in the rotorod test (left, expressed as the time sp

expressed as the time of grasping). Data are expressed as means+SEM. Ope

pilocarpine group (n ¼ 25 and 14, respectively). Statistical significance, P <

Evaluation of Timm-stained sections showed nosprouting of mossy fibers in the hippocampus of pilo-

carpine-treated animals, i.e., level 0 according to Cava-

zos et al. [23].

3.4.2. Immunohistochemistry

In control animals, PV-IR cells were found in all

cortical layers except layer I, and exhibited a variety of

somatic and dendritic morphologies (Fig. 4C). Multi-polar cell bodies predominated in the upper layers while

small pyramidal and bipolar neurons were less frequent.

In layers V and VI, some bipolar and triangular (pyra-

midal-like) neurons were distinguished in addition to the

prevailing multipolar neurons. Most PV-IR cells were

moderately to heavily labeled. The highest density of

PV-IR neurons was located in the supragranular layers

of the motor area.Two populations of CB-IR cells could be distin-

guished. First, weakly to moderately positive neurons

predominated in layers II and III and could also be

found in layer V. Many weakly and moderately CB-IR

cells were obscured by dense diffuse staining of the

neuropil in layers I–III. Second, heavily labeled CB-IR

neurons were found scattered in layers II–VI but most of

them were localized in two belts corresponding to layersII–III and V–VI. Both bipolar and multipolar perikarya

were present in the supragranular layers, while the ma-

jority of heavily labeled cells in layer V–VI were multi-

polar (Fig. 4E).

In pilocarpine-treated animals, a significant reduc-

tion in both PV-IR and CB-IR neurons in the motor

neocortical area was evident in comparison with con-

trols (Figs. 4D,F). The number of PV-IR neuronsthroughout layers II–VI was 47.7� 2.88 (mean� SEM)

ent on the rod without falling down) and the bar-holding test (right,

n bars: control group (n ¼ 22 and 14, respectively); crosshatched bars:

0:05 (two-tailed): �Pilocarpine versus control group.

Fig. 4. Photomicrographs demonstrating parvalbumin-containing cells in the sensorimotor cortex (A,B), calbindin-containing cells in the motor

cortex (C,D), and parvalbumin-containing cells in the dentate gyrus (E,F). Control rats: A, C, E; animals after nonconvulsive status: B, D, F.

Bars¼ 500lm. Roman numerals denote cortical layers. WM, white matter. CA3, hippocampal field CA3; sm, stratum moleculare; sg, stratum

granulare. Arrowheads in (E) and (F) denote parvalbumin-positive cells on the borderline between the granular layer and hilus of the dentate gyrus.

186 P. Krsek et al. / Epilepsy & Behavior 5 (2004) 180–191

in pilocarpine-treated and 94.3� 4.72 in control ani-

mals. A decline in PV-IR cells was particularly prom-

inent in layers II and III. Some of the persisting

neurons exhibited weaker immunopositivity of peri-

karya and dendrites when compared with controls. The

average number of CB-IR neurons in a 0.3� 0.3-mmarea localized randomly in layers V and VI was

4.57� 0.13 in pilocarpine-treated rats and 6.33� 0.26

in the control group (Fig. 5).

In control animals, PV-IR neurons predominated in

the stratum pyramidale and oriens of the hippocampus

and in the stratum granulosum and hilus of the dentate

gyrus (Fig. 4G). A decline in the number of PV-IR

neurons was demonstrated in both blades of the dentategyrus in pilocarpine-treated rats (Fig. 4H). The average

number of PV-IR cells in the dentate gyrus was

6.75� 0.49 in pilocarpine-treated and 10.27� 0.82 in

control animals. On the other hand, no significant dif-

ferences in the numbers of PV-IR neurons were found in

the CA1 (17.6� 1.78 pilocarpine-treated vs 20.7� 1.0

controls) and CA3 (12.2� 0.72 pilocarpine-treated vs12.4� 0.89 controls) hippocampal fields (Fig. 6).

4. Discussion

Classic pilocarpine-induced SE represents a model of

secondary generalized convulsive status epilepticus

[25,26]. Studies dealing with subconvulsive doses ofpilocarpine are rare. Two original studies described

Fig. 5. Average number of calbindin-positive neurons counted in the square of 0.3� 0.3mm localized randomly in layers V and VI of the motor

neocortex (left); average number of parvalbumin-positive neurons counted in the whole range of layers V and VI of the motor neocortical fields Fr 1–

3 of both hemispheres (right). Open bars: control group (n ¼ 5); crosshatched bars: pilocarpine group (n ¼ 11). Data are expressed as means+SEM.

Statistical significance, P < 0:05 (two-tailed): �pilocarpine versus control group.

Fig. 6. Average number of parvalbumin-positive neurons in the den-

tate gyrus (counted in the whole extent of both blades) and in the CA1

and CA3 hippocampal fields (counted in a square of 0.3� 0.3mm).

Open bars: control group (n ¼ 5); crosshatched bars: pilocarpine

group (n ¼ 11). Data are expressed as mean+SEM. Statistical signif-

icance, P < 0:05 (two-tailed): �Pilocarpine group versus control group.

P. Krsek et al. / Epilepsy & Behavior 5 (2004) 180–191 187

effects of nonconvulsant pilocarpine doses in rats notpretreated with lithium chloride [27,28]. Sixty to one

hundred twenty minutes of abnormal behavior concur-

rently with EEG changes (theta rhythm and spiking in

the hippocampus and low-voltage fast activity in thecortex) were observed. However, we found only one

subsequent study that focused on these phenomena

and reported expression of the c-fos protein in certain

limbic areas induced by a subconvulsant dose of

pilocarpine [29].

Our previous results demonstrated that the model of

NCSE induced by a subconvulsant dose of pilocarpine in

lithium chloride-pretreated rats, characterized by be-havioral automatisms and spikes (isolated as well as in

series) in hippocampal and cortical EEGs, is equivalent

to human CPSE [18,19]. The striking result of the anal-

ysis of this model was the finding of morphological

damage in the motor neocortex 1 and 2 weeks after

NCSE. As one of the most critical questions regarding

NCSE in humans concerns the risk of long-term harmful

sequelae to the brain, we turned our attention to ana-lyzing both behavioral and morphological consequences

of nonconvulsive seizures in our model. The possibility of

behavioral sequelae was suggested by the study of Cook

and Persinger [20], a pioneering morphological study.

Video EEG monitoring did not reveal any epileptic

activity in our animals. This contrasts sharply with data

for convulsive status epilepticus (lithium–pilocarpine

model with pilocarpine doses of about 40mg/kg and/orhigh-dose pilocarpine model) where life-long spontane-

ous seizures were recorded under conditions similar to

those for our monitoring [30].

Behavioral tests revealed some deficits 2 months after

NCSE. Conversely to the prominent disturbances ob-

served in rats during pilocarpine-induced NCSE [18,19],

discernible long-term behavioral consequences were

only subtle. No long-term structural disintegration

188 P. Krsek et al. / Epilepsy & Behavior 5 (2004) 180–191

of spontaneous behavior was observed. However, theanimals experiencing NCSE displayed more face wash-

ing. In intact animals, this behavioral component

occupies the second position in the sequential organi-

zation of grooming behavior [31,32]. An increased

display of face washing in animals experiencing NCSE

probably reflects different levels of responsiveness

following exposure to a stressor when placed into a new

environment. It has been proved that the groomingresponse is related to age [33,34] and, among others, also

to neurohormonal modulation [35,36].

The social interaction test revealed decreased inves-

tigation by both pilocarpine-treated and control animals

reexposed to the same juvenile 30 minutes after the

initial interaction, suggesting that the animals are able

to recognize juvenile conspecifics [37–40]. However,

duration of investigation during the initial exposure wasmarkedly decreased in animals after NCSE. One possi-

ble explanation is that the decreased interest in juveniles

by pilocarpine-treated animals might reflect deficits in

sensitivity as well as in responsiveness of animals ex-

posed for the first time to social novelty. On the other

hand, pilocarpine-treated rats remembered their juvenile

conspecifics; there was a significant reduction in dura-

tion of investigation during the second exposure.The reduction in the transfer latency in the elevated

plus-maze test during the second session showed that

spatial orientation and/or short-term memory of pilo-

carpine-treated animals were not impaired. This, to a

certain extent, contrasts with the finding of Cook and

Persinger [20], who reported long-term memory distur-

bance in animals 5 months after treatment with a sub-

convulsive dose of pilocarpine. However, it is necessaryto note that animals tested on the elevated plus-maze

were apparently submitted to a less difficult memory

task in comparison with the eight-arm radial maze used

by Cook and Persinger.

Long-term motor deficits were observed in animals

that experienced NCSE. These may be caused by either a

muscular tone deficiency or impaired skillfulness of the

pilocarpine-treated animals. The static component ofthe equilibrium, in particular, seemed to be impaired.

Moreover, pilocarpine-treated animals attempted to turn

the body in the direction of the rotation on the rotorod

and therefore fell. Equilibrium behavior depends on a

number of sensory cues and on training [41,42]. The

different behavioral strategy of the pilocarpine-treated

animals could be related to deficits in perception, for

example, of visual, proprioceptive, vestibular, and tactilestimuli. It has been demonstrated that the olivocerebellar

pathway is involved in the temporal organization of

motor learning and skills [43,44]. However, no discern-

ible damage was observed in the brainstem (including

oliva inferior) in our experimental rats. Therefore, a

more plausible explanation is that the motor deficits

reflect damage to the motor neocortical areas.

Both histological and immunohistochemical tech-niques demonstrated damage in different brain struc-

tures 2 months after NCSE, strongly supporting the

view that short-term NCSE is able to damage the brain

permanently. Damage to motor neocortical fields cor-

responding to motor deficits was the most conspicuous

morphological finding. This is in accordance with our

former observations [18]. It was suggested that the mo-

tor neocortex might be damaged by projection of theepileptic activity from the limbic structures via the

amygdala [19].

Calcium-binding proteins like parvalbumin and cal-

bindin play an important role as calcium transporters, a

buffering system for intracellular calcium ions, and

represent one of the most important calcium compart-

ments in the brain [45]. The decrease in immunoreac-

tivity (and probably in the concentrations of PV andCB) may thus increase neuronal susceptibility to calcium

fluctuations, leading to structural and functional

impairment.

Analysis of tissue obtained from patients undergoing

epilepsy surgery indicated reduced numbers of PV-IR

and CB-IR neurons in human neocortical epileptic foci

[46]. Loss of PV-IR neurons was also detected in the

neocortex of the temporal pole in patients with intrac-table temporal epilepsy [47]. A decrease in glutamate

decarboxylase and PV immunostaining was observed in

the high-dose pilocarpine model in rats surviving 60

days after the onset of chronic seizures [48]. These

findings support an involvement of neocortex in tem-

poral lobe epilepsy with secondary generalization.

The distribution of PV-IR and CB-IR neurons in the

motor cortical areas (fields Fr1, Fr2, and Fr3) of controlanimals was similar to that reported in the studies of

Celio [49] and van Brederode et al. [50]. In our pilocar-

pine-treated rats, a decline in both PV-IR and CB-IR

neurons was demonstrated in layers II–VI and V–VI of

the motor neocortical fields, respectively. This finding

indicates that NCSE influenced neuronal expression of

the calcium-binding proteins in the same direction as

convulsive SE. PV-IR and CB-IR cells represent a sub-population of cortical GABAergic inhibitory neurons

[49]. PV is localized in fast-spiking, metabolically active

large basket cells and chandelier cells in the neocortex.

On the contrary, CB-IR cells are typically regular-spik-

ing double-bouquet cells (some are fast-spiking ones)

[51–53]. A reduction in neocortical PV and CB immu-

noreactivity thus suggests an impairment of GABAergic

inhibition as a consequence of nonconvulsive SE.Our prior work failed to demonstrate an apparent

effect on limbic structures, where the most prominent

epileptic brain damage has repeatedly been described in

humans [1,54], as well as in the majority of animal

models of SE [2,16]. For that reason, discovery of the

dentate gyrus lesion demonstrable by PV immunohis-

tochemistry could be regarded as a crucial result of the

P. Krsek et al. / Epilepsy & Behavior 5 (2004) 180–191 189

present work. Although dark neurons were found insome pilocarpine-treated animals, examination of Nissl-

stained brain sections failed to reveal discernible damage

to limbic structures 2 months after NCSE. However, it

was suggested that conventional cresyl violet staining of

the hippocampus might not be able to demonstrate cell

loss because of a lack of sensitivity [55].

The pattern of PV-IR cells in the hippocampus in

control rats was similar to that known from previousstudies [56,57]. PV is present exclusively in interneurons

of all hippocampal subregions [54]. These neurons can

be classified as basket or chandelier cells [57]. Similarly

to the neocortex, these cells are regarded as GABAergic

interneurons [58] and constitute 25–31% of the GABA-

ergic cell population in the normal hippocampus [56].

These cells innervate the somata, proximal dendrites,

and axon initial segments of principal neurons. Theymay thus control the pattern and timing of output of

principal cells and synchronize their action potential

discharges [57,59]. Concerning the fascia dentata, the

existence of two almost separate populations of GABA-

ergic nongranular cells—somatostatin- and PV-contain-

ing neurons—has been reported. It was demonstrated

that PV-IR neurons are less sensitive to seizure-induced

calcium overload than somatostatin-containing cells[60]. Granular cell pathology was observed only in ani-

mals that exhibited a loss of adjacent hilar neurons [61].

A severe decrease in PV-IR cells was reported in the

hippocampal formation of rats after convulsive pilo-

carpine-induced SE [62]. Partial loss of PV-IR neurons

and markedly reduced pericellular innervation of den-

tate granular cells by PV-positive axons was recently

described in the lithium–pilocarpine model as well asafter intrahippocampal injection of kainic acid [63,64].

Similarly, there was a significant reduction in PV-IR and

somatostatin-IR neurons in the hilar region accompa-

nied by extensive mossy fiber sprouting after tetanic

stimulation of the angular bundle [65]. A decrease in

PV-IR neurons was detected in the dentate gyrus not

only after stimulation of the angular bundle but also in

systemic kainic acid-induced SE [66]. In contrast to theabove-mentioned findings, a loss of PV-IR neurons oc-

curred in the kainic acid model in areas CA1–CA3 of the

ipsilateral hippocampus following a unilateral lesion,

but not in the dentate gyrus [67,68]. A decrease in PV

immunoreactivity does not necessarily mean cell death;

it might be due to decreased PV expression. Anyway, it

was suggested that a decrease in PV CB immunorectivity

is associated with decreased phosphorylation and sub-sequent degradation of neurofilaments, leading eventu-

ally to neuronal degeneration [69]. On the other hand, a

decrease in the number of PV-IR cells in the stratum

oriens of region CA1 in pilocarpine-induced chronic

seizures was ascribed to the death of these neurons [70].

It was, however, proven that PV- and CB-IR neurons

are not spared in pilocarpine-induced brain damage.

In contrast to a severe decrease in PV-IR cells reported inthe hippocampal formation of rats experiencing convul-

sive pilocarpine-induced SE [62], the changes caused by

NCSE were understandably less pronounced. Neverthe-

less, the loss of the specific and important population of

nonpyramidal interneurons in the dentate gyrus might be

responsible for a chronic alteration of inhibition.

Our results support the view that no direct evidence

had been obtained linking these calcium-binding pro-teins to specific protection against calcium-mediated cell

injury [49]. It should be emphasized again that PV and

CB immunonegativity can mean either that the cells

have died or that neurons may be spared but do not

express PV and CB [67]. No matter which of these is

true, because calcium-binding proteins are present

mainly in GABAergic interneurons, their disturbance

could thus refer to an alteration of inhibitory mecha-nisms as the consequence of NCSE. Such an alteration

was demonstrated in the dentate gyrus of rats exposed

previously to convulsive pilocarpine-induced SE. Den-

tate granule cells exhibited prolonged EPSPs and dis-

charged more action potentials in comparison with

controls. In addition, IPSP conductances as well as

frequency of GABA-A spontaneous and miniature

IPSCs were decreased, thus confirming a loss of inhibi-tion of granule cells [71]. This alteration might be re-

sponsible for the long-term deficits in equilibrium

behavior and grasping observed in the rotorod and bar-

holding tests.

It is not easy to answer the question whether this

effect on the dentate gyrus is directly responsible for

the behavioral changes revealed by the social interac-

tion test. As part of the hippocampal formation, thedentate gyrus participates in the processes of learning,

memory, motivation, integration of cognitive func-

tions, alerting responses, and awareness as well as in

cardiovascular, endocrine, and reproductive functions

[72]. Via the entorhinal cortex, the dentate gyrus in-

tegrates inputs from a variety of cortical regions [73].

The loss of inhibition in the dentate gyrus may cause

hyperexcitability of the CA3 and CA1 subregions,and this changed excitability state in the whole hip-

pocampal formation might cause diverse behavioral

abnormalities.

In conclusion, pilocarpine-induced NCSE represents

a suitable model for studying possible consequences of

complex partial SE (without secondary generalization)

in the brain. It is necessary to study other well-defined

neuronal populations (e.g., somatostatin-positive neu-rons), changes in excitability of at least neocortex and

hippocampal formation, and memory functions in our

model. The present finding of seizure-related brain

damage associated with long-term behavioral deficits

following this relatively subtle epileptic condition warns

against underestimating the harmful effects of complex

partial SE in patients.

190 P. Krsek et al. / Epilepsy & Behavior 5 (2004) 180–191

Acknowledgments

This study was supported by Grants 309/00/1643 and

309/03/0770 of the Grant Agency of the Czech Republic

and by Research Project AVOZ 5011922.

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