New insights from the use of pilocarpine and kainate models

J.P. Leite a,*, N. Garcia-Cairasco b, E.A. Cavalheiro c

a Department of Neurology, University of Sao Paulo School of Medicine at Ribeirao Preto, Campus Universitario, CEP 14049-900,

Ribeirao Preto, Brazilb Department of Physiology, University of Sao Paulo School of Medicine at Ribeirao Preto, Ribeirao Preto, Brazil

c Division of Experimental Neurology, Federal University of Sao Paulo, Sao Paulo, Brazil

Abstract

Local or systemic administration of pilocarpine and kainate in rodents leads to a pattern of repetitive limbic seizures

and status epilepticus, which can last for several hours. A latent period follows status epilepticus and precedes a chronic

phase, which is characterized by the occurrence of spontaneous limbic seizures. These distinct features, in a single

animal preparation, of an acute damage induced by status epilepticus, a silent interval between injury and the onset of

spontaneous seizures, and a chronic epileptic state have allowed antiepileptic drug (AED) studies with different

purposes, (a) in the acute phase, identification of compounds with efficacy against refractory status epilepticus and/or

neuroprotection against damage induced by sustained seizures; (b) in the latent period, identification of agents with a

potential for preventing epileptogenesis and/or against seizure-induced long-term behavioral deficits and (c) in the

chronic phase, testing drugs effective against partial and secondarily generalized seizures. Studies on pilocarpine and

kainate models have pointed out that some AEDs or other compounds exert an antiepileptogenic effect. The analogy of

the latent phase of pilocarpine and kainate models with the acquisition of amygdala kindling should encourage testing

of drugs that have proved to suppress the evolution of amygdala kindling. Drug testing in the chronic phase should not

address only the suppression of secondarily generalized motor seizures. Most of current tools used to quantify

spontaneous seizure events need to be coupled to electrophysiology and more sophisticated systems for recording and

analyzing behavior. # 2002 Elsevier Science B.V. All rights reserved.

Keywords: Antiepileptic drugs; Animal models; Prevention; Epilepsy

1. Introduction

Pilocarpine and kainate models replicate several

phenomenological features of human temporal

lobe epilepsy and can be used as animal prepara-

tions to understand the basic mechanisms of

epileptogenesis (Ben-Ari, 1985; Turski et al.,

1989; Buckmaster and Dudek, 1997). Local or

systemic administration of pilocarpine and kainate

in rodents leads to a pattern of repetitive limbic

seizures and status epilepticus, which can last for

several hours (Cavalheiro et al., 1982; Turski et al.,

1983; Leite et al., 1990; Furtado et al., 2002). A

somewhat variable latent period follows status

epilepticus and precedes the chronic phase, which

is characterized by the occurrence of spontaneous

limbic seizures. The brain damage induced by

* Corresponding author. Tel.: �/55-16-602-2556; fax: �/55-

16-6330760

E-mail address: jpleite@fmrp.usp.br (L. J.P.).

Epilepsy Research 50 (2002) 93�/103

www.elsevier.com/locate/epilepsyres

0920-1211/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 9 2 0 - 1 2 1 1 ( 0 2 ) 0 0 0 7 2 - 4

status epilepticus in such preparations may beconsidered an equivalent of the initial precipitating

injury event, usually a prolonged febrile convul-

sion, which is commonly found in patients with

mesial temporal lobe epilepsy (Mathern et al.,

1996).

Neuropathological changes such as neuron loss

in several hippocampal subfields and reorganiza-

tion of mossy fibers into the molecular layer of thefascia dentata are observed in both models and are

similar to hippocampi from patients with hippo-

campal sclerosis (Tauck and Nadler, 1985; Sutula

et al., 1989; Babb et al., 1991; Mathern et al., 1993;

Mello et al., 1993; Mathern et al., 1995). This

abnormal synaptic reorganization has been

suggested to be an anatomical substrate for

epileptogenesis (Tauck and Nadler, 1985; Babbet al., 1991; Buckmaster and Dudek, 1997).

The distinct features, in a single animal pre-

paration, of an acute damage induced by status

epilepticus, a silent interval between injury and the

onset of spontaneous seizures, and a chronic

epileptic state have allowed antiepileptic drug

(AED) studies with different purposes, (a) in the

acute phase, identification of compounds withefficacy against refractory status epilepticus and/

or neuroprotection against damage induced by

sustained seizures; (b) in the latent period, identi-

fication of agents with a potential for preventing

epileptogenesis and/or against seizure-induced

long-term behavioral deficits and (c) in the chronic

phase, testing drugs effective against spontaneous

recurrent partial and secondarily generalized sei-zures.

2. Methods of induction

2.1. Pilocarpine

The status epilepticus induced by pilocarpine

can be achieved by high doses of intraperitonealinjections, usually over 300 mg/kg (Turski et al.,

1983; Leite et al., 1990; Cavalheiro et al., 1991).

Pre-treatment with lithium chloride 24 h prior to

pilocarpine injection, at a dose of 3 mEq/kg, i.p.,

potentiates the epileptogenic action of pilocarpine

and the amount of the drug can be reduced by ten

times (Honchar et al., 1983; Jope et al., 1986;Clifford et al., 1987). Acute behavioral manifesta-

tions and the pattern of seizure-induced brain

damage after high doses of pilocarpine and

lithium�/pilocarpine models are very similar; how-

ever, there is evidence that some antiepileptic

compounds respond differentially to status epilep-

ticus, suggesting that distinct biochemical mechan-

isms control seizures in these two preparations(Ormandy et al., 1989; Sofia et al., 1993). Chaudh-

ary et al. (1999) have shown that lithium chloride

pretreatment schedule can be adopted anytime

between 2 and 24 h with results similar to the 24 h

interval. It seems that the lithium�/pilocarpine

protocol reduces mortality, and avoids many of

the peripheral cholinomimetic side effects of high

doses of pilocarpine (Chaudhary et al., 1999).More recently it has been shown that lithium�/

pretreatment, followed by several low doses of

pilocarpine, efficiently produces status epilepticus

and chronic epilepsy in rats with much lower

mortality rates than a single dose of pilocarpine

(Glien et al., 2001). The reason for the reduced

mortality is unknown, but it seems plausible that

repeated low-doses intervals allow individualdosing with respect to inter-rat differences in

sensitivity to the convulsant action of pilocarpine,

which is not possible with the single-dose admin-

istration (Glien et al., 2001).

Local administration of pilocarpine delivered

either intracerebroventricularly or directly into the

hippocampus has been used in studies assessing

the seizure-induced changes in amino acid levelsand the effectiveness of some anti-epileptic agents

(Croiset and De Wied, 1992; Millan et al., 1993;

Croiset and De Wied, 1997; Smolders et al., 1997;

Lindekens et al., 2000). In none of these studies a

detailed behavioral, electrophysiological and mor-

phological analysis of the model was made. More

recently, however, Furtado et al. (2002) have

shown that intrahippocampal-pilocarpine injec-tion (2.4 mg/ml; injected volume 1.0 ml) induces

status epilepticus with near zero mortality. Timm

positive-mossy fiber sprouting and spontaneous

recurrent seizures are also observed in intrahippo-

campal-pilocarpine injected rats, with similar sei-

zure frequency than that observed in systemically

injected animals (Furtado et al., 2002).

J.P. Leite et al. / Epilepsy Research 50 (2002) 93�/10394

2.2. Kainic acid

Limbic status epilepticus in rats can be induced

by kainic acid with either local administration

(intracerebroventricular or intrahippocampal, at

doses of 0.1�/3.0 mg per hemisphere) or injected

systemically (usually at doses of 15�/30 mg/kg).

Such treatment protocols have often been asso-

ciated with a relatively high mortality rate and alow percentage of rats becoming epileptics (Nadler

et al., 1980; Cavalheiro et al., 1982; Cronin and

Dudek, 1988; Cronin et al., 1992; Mathern et al.,

1993; Mikati et al., 1994). Hellier et al. (1998)

proposed a modified treatment protocol using

multiple low doses (5 mg/kg, i.p.) of kainate.

This protocol had a relatively low mortality rate

(around 15%) and nearly all kainate-treated rats(97%) had two or more spontaneous motor

seizures months after treatment (Hellier et al.,

1998).

3. Acute phase

Several drugs have proved to efficiently abort or

attenuate status epilepticus when injected eitherbefore or right after pilocarpine or kainate admin-

istration. In fact, benzodiazepines and barbiturates

have been broadly used to prevent the relatively

high mortality associated with prolonged seizures

in both models.

In the pilocarpine model, interruption of status

epilepticus by pentobarbital and diazepam after

different duration times indicates that early statusepilepticus suppression can prevent spontaneous

recurrent seizures. In addition, severe cell loss and

synaptic reorganization are prevented if treatment

is established with 30 min of status epilepticus

(Lemos and Cavalheiro, 1995). In the intrahippo-

campal pilocarpine preparation, diazepam (4 mg/

kg; i.p.) injected 90 min after sustained seizures

can suppress status epilepticus and reduce acutemortality to nearly zero even though this protocol

does not hamper the expression of spontaneous

recurrent seizures and the presence of Neo-Timm

positive mossy fiber sprouting (Furtado et al.,

2002). Clonazepam, ED50 0.35 mg/kg (0.25�/0.49),

phenobarbital, 23.4 mg/kg (18.5�/29.6), and val-

proic acid, 286 mg/kg (202�/405), when adminis-tered before systemic pilocarpine, prevented the

buildup of limbic seizures and protected against

seizure-related brain damage. Pretreatment with

trimethadione, 179 mg/kg (116�/277) resulted in a

moderate protection against pilocarpine-induced

seizures, whereas diphenylhydantoin, 10�/200 mg/

kg and carbamazepine, 10�/50 mg/kg, had no effect

and did not prevent pilocarpine-induced braindamage (Turski et al., 1987). In the lithium�/

pilocarpine preparation, however, carbamazepine

displays a protective effect while felbamate is far

more effective than in animals treated with high

doses of pilocarpine alone (Sofia et al., 1993). Both

preparations seem to respond differentially to N -

methyl-D-aspartate (NMDA) receptor antago-

nists. Pretreatment with MK-801 (dizocilpine), anoncompetitive NMDA receptor agonist, pro-

duces an effective dose-dependent anticonvulsant

action in the lithium�/pilocarpine model but not in

rats treated with pilocarpine alone (Ormandy et

al., 1989). In spite of the lack of effect in blocking

the onset of status epilepticus in the pilocarpine

model, MK-801 (4 mg/kg), given 20 min prior to

pilocarpine, prevents later spontaneous recurrentseizures and protects against CA1 pyramidal

damage (Rice and DeLorenzo, 1998).

In addition, MK-801 seems to have a synergistic

effect with diazepam against refractory status

epilepticus. Early pilocarpine-induced status epi-

lepticus responds rapidly to diazepam treatment,

whereas status epilepticus of longer duration turns

increasingly less responsive to treatment. MK-801pretreated rats respond rapidly to diazepam treat-

ment, even after 60 min of status epilepticus,

indicating that NMDA receptor activation plays

a role in the seizure-induced refractoriness to

benzodiazepines in status epilepticus (Rice and

DeLorenzo, 1999). Ketamine, another noncompe-

titive NMDA receptor antagonist, at a dose of 100

mg/kg, i.p., exerts a time-dependent protection ofhippocampal cells and prevents late spontaneous

recurrent seizures and spatial memory deficits in

the Morris water maze (Hort et al., 1999).

Studies on the kainate model have indicated that

compounds with an antagonistic effect on gluta-

mate receptors, both on NMDA and non-NMDA

types, can have a protective effect on neurotoxic-

J.P. Leite et al. / Epilepsy Research 50 (2002) 93�/103 95

induced damage and reduce late spontaneousseizures and/or behavioral deficits, when given

either before or right after kainate treatment.

Stafstrom et al. (1993) have shown that MK-801

pretreatment prior to kainate-induced status epi-

lepticus, while not substantially altering the acute

epileptic behavior, reduces spontaneous recurrent

seizure frequency and flurothyl seizure

susceptibility. NBQX, an AMPA receptor antago-nist, when given at different times after kainate

treatment, at a dose of 30 mg/kg per dose, reduced

the severity of status epilepticus. Later, animals

receiving kainate and treated with NBQX did not

show memory impairment or histologic damage in

the CA1 and CA3 subfields, however, spontaneous

recurrent seizure frequency was not different from

non-treated animals (Mikati et al., 1999).D-cycloserine, a partial agonist for the NMDA

receptor-associated glycine-binding site, exerts a

potent, dose-dependent and long lasting anti-

convulsant effect against kainate-induced seizures.

Contrary to MK-801 and diazepam, D-cycloserine

did not significantly alter the number of auto-

matisms (wet dog shakes) induced by kainate

(Baran et al., 1994). MacGregor and coworkershave shown that when the GABA-A agonist

muscimol, dizocilpine (MK-801) and the adeno-

sine A1 receptor agonist R-N6-phenylisopropyl-

adenosine are administered together with chlor-

methiazole, at their respective ED25 doses, a

potentiation was apparent in the degree of neuro-

protection. These findings suggest that the combi-

nation of neuroprotective agents with differentmechanisms of action can lead to a synergistic

protection against excitotoxicity (MacGregor et

al., 1997).

Studies on the intrahippocampal kainate model

have shown that treatment with phenobarbital (60

mg/kg, s.c., once daily) after kainate, for 5 days,

suppresses seizure activity, protects against hippo-

campal excitotoxic damage, reduces mossy fibersprouting and completely abolishes the increased

susceptibility to kindling associated with kainic

acid (Sutula et al., 1992). Vigabatrin (gamma-vinyl

GABA, 1000 mg/kg, i.p.), given 24 h before

kainate treatment, decreased neuronal damage in

the CA3a and CA1 hippocampal subfields, atte-

nuated the severity of seizures but had no effect on

the development or generalization of convulsions(Halonen et al., 1995). Using higher doses (1500

mg/kg) Mecarelli et al. (1997) have shown that

vigabatrim reduces the incidence of epileptic

manifestations and the subsequent mortality.

Felbamate also seems to have some long-term

neuroprotective effects after kainate-induced sta-

tus epilepticus. Thirty-day-old rats treated with

felbamate (300 mg/kg) 1 h after kainate and tested50 days later have longer latencies to flurothyl-

induced seizures and performed better on water

maze, open field and handling tests (Chronopou-

los et al., 1993).

4. Latent period

The importance of drug testing in the latentperiod has been stressed by many groups because

there is a great deal of evidence that in this phase

several molecular changes occur and may contri-

bute to the process of epileptogenesis (Loscher,

1998). A putative compound with the property of

inhibiting the process underlying the development

of the epileptic condition may be considered a true

antiepileptic or antiepileptogenic drug. In thissense, the latent period of pilocarpine and kainic

acid models can be considered analogous of the

acquisition or development phase of amygdala

kindling in rats (Cavalheiro et al., 1991; Silver et

al., 1991). Several drug studies in pilocarpine and

kainate models initiate after the period of status

epilepticus and last from few days to several

weeks. Hence, such drug studies carried out inthe latent period may overlap with the period of

spontaneous recurrent seizures (chronic period).

Bolanos et al. (1998) compared in 35-day-old

rats (P35) the long-term effects of valproate and

phenobarbital after kainate. Phenobarbital, given

from P36�/75, did not have a protective effect on

the prevention of learning impairment in the water

maze, recurrent seizures or histologic lesions in theCA3, CA1 and dentate hilus. Valproate-treated

rats, however, had no spontaneous seizures, no

deficits in visuospatial learning and had fewer

histologic lesions than animals receiving kainate

alone (Bolanos et al., 1998). This antiepileptogenic

effect of valproate confirms a previous study in the

J.P. Leite et al. / Epilepsy Research 50 (2002) 93�/10396

amygdala-kindling model (Silver et al., 1991). Incontrast, Pitkanen et al. (1999) have shown that

vigabatrin, 1 or 2 h or 7 days after the beginning of

kainic acid-induced status epilepticus did not

prevent mossy fiber sprouting regardless of when

treatment was started. Cilio et al. (2001) have

shown that rats treated with gabapentin had a

reduced incidence of spontaneous recurrent sei-

zures, less intense pathological lesions and lessaggressiveness when compared with saline treated

controls.

There is a growing body of experimental data

which suggests that there are differences between

the mechanisms underlying epileptogenesis and the

mechanisms involved in maintaining the expres-

sion of seizures once an epileptogenic process has

been established. Therefore, it is reasonable toassume that drugs which prevent epileptogenesis

may be quite different from drugs that prevent

seizures (Silver et al., 1991; Shinnar and Berg,

1996; Loscher, 1998). Hence, the use of com-

pounds that interfere on trophic factors which

have impact on neuroprotection or damage-in-

duced plastic changes, may also have an antiepi-

leptogenic effect. Supporting this concept, Liu andHolmes (1997) have shown that administration of

basic fibroblast growth factor for 7 days, starting 2

days before kainate, has neuroprotective effect,

reduces spontaneous recurrent seizures, and pre-

vents memory deficits on kainate-treated rats. In

the pilocarpine model, local i.c.v. injections of

antibodies against nerve growth factor suppress

the cholinergic sprouting in the hippocampus butdo not interfere with mossy fiber sprouting. The

impact of this selective change on seizure fre-

quency is unknown since a concomitant behavioral

study is lacking (Holtzman and Lowenstein, 1995).

On the other hand, studies from Longo and

Mello have indicated in pilocarpine and kainate

models that cycloheximide, a potent protein syn-

thesis inhibitor, was able to completely blockmossy fiber sprouting without changing the fre-

quency of spontaneous seizures (Longo and Mello,

1997, 1998). This finding would argue against a

contributory role of mossy fiber sprouting on

epileptogenesis. However, this finding has not

been confirmed by Williams et al. (2000) in the

pilocarpine model. The reasons for these incon-

sistencies are still unknown, but differences in ratstrains might be a determinant (Longo and Mello,

1997, 1998).

5. Chronic period

It is surprising that there are not many studies of

AEDs during the chronic phase of pilocarpine andkainate models. Most of the studies overlap with

the silent period and were already mentioned in

the previous section. In the pilocarpine model,

AEDs that are efficient to suppress status epilepti-

cus are not necessarily the same that are effective

on controlling spontaneous recurrent seizures.

Diazepam, phenobarbital, valproic acid and tri-

methadione protect against pilocarpine-inducedstatus epilepticus while phenytoin and carbamaze-

pine are ineffective. In the chronic phase, carba-

mazepine and phenytoin were effective against

spontaneous seizures. Valproic acid was also

effective against spontaneous seizures at the dose

of 600 mg/kg and ethosuximide was ineffective

against these seizures (Turski et al., 1987; Leite

and Cavalheiro, 1995).In the kainate model, ketogenic diet seems to be

effective on reducing spontaneous recurrent sei-

zures. Ketogenic diet-fed rats had significantly

fewer and briefer spontaneous seizures and less

supragranular mossy fiber sprouting, although the

degree of hippocampal pyramidal cell damage was

similar in both groups. (Muller-Schwarze et al.,

1999).

6. Coupling neuroethology and EEG to evaluate

behavioral seizure sequences

Intractable complex partial seizures rarely gen-

eralize even when drug doses are tapered during

video-EEG monitoring. Nevertheless these oligo-

symptomatic seizures are often associated withlack of responsivity and amnesia to the seizure

event and consequently have a considerable mor-

bidity (Kotagal, 1991). Although in experimental

epilepsy models it is possible to see analogous

behavioral alterations, most of them are, because

of their subtlety, frequently overlooked.

J.P. Leite et al. / Epilepsy Research 50 (2002) 93�/103 97

Fig. 1

J.P. Leite et al. / Epilepsy Research 50 (2002) 93�/10398

One of the reasons is that secondary generalized

motor seizures, rather than partial ones, are easily

detected by video monitoring setups. Although it

is possible to couple motion detectors to video

monitoring, class 4�/5 limbic seizures are preferen-

tially used as markers of epileptogenicity, in

contrast with, for example, classes 1, 2 or 3

(Racine, 1972). Thus, it will be of fundamental

importance for AED studies to precisely detect

subtle behavioral entities that present strong

correlation with, for example, hippocampal and

amygdala EEG seizure activity. For instance, a

drug that suppresses a secondarily generalized

seizure does not necessarily prevent episodes that

would be an equivalent to a complex partial

seizure in humans.For this purpose, not only a high-resolution

video capturing system is needed, but also a

reliable system of behavioral sequence reconstruc-

tion. The method we will present here is based on

quantitative ethology, first applied to epilepsy

studies by Garcia-Cairasco and coworkers study-

ing acute and kindled audiogenic seizures (Garcia-

Cairasco et al., 1992, 1993, 1996). When behavior,

captured in videos is analyzed, this approach

detects statistically significant interactions (P B/

0.05; X2-test) of behavioral pairs (dyads) that are

displayed in flowcharts (see Fig. 1). Then, depend-

ing of the type of behavioral sequence that is

detected, some rules for the involved circuitry can

be suggested.

The initial recording of the behavior, using a

regular video camera and a video capturing board

in order to digitalize the images, gives us time-

behavior pairs such as those presented in Fig. 1A.

The consequent behavioral sequence can be de-

picted as the flowchart shown in Fig. 1B, where

rectangles represent specific behaviors and arrows

represent links between behaviors. In Fig. 1A and

B, the behavioral sequence of a spontaneous

seizure induced by systemic application of pilo-

carpine is represented. Observe in this case a

complex pattern of oro-facial automatisms, fore-

limb myoclonus, rearing and falling. Furthermore,

in order to get a dynamic picture of what happens

with several animals, it is possible to display the

average of a number of observation windows

where spontaneous seizures are captured. In the

current case, for each time window we used as

behavioral trigger the presence of class six limbic

seizures (Pinel and Rovner, 1978). In Fig. 1C is

illustrated the resultant flowchart of nine sponta-

neous seizures observed after status epilepticus

induced by systemic pilocarpine (380 mg/kg). In

Fig. 1D, the calibration values of frequency and

duration of behaviors are illustrated, as well as the

statistical values, that link each behavioral pair in

the flowcharts. In Fig. 1E and F are shown typical

video-EEG screens of a partial seizure with

recordings in the hippocampus and amygdala

similar to Furtado et al. (2002).

Even secondarily generalized motor seizures can

be easily detected with this ethologically based

method and compared with the sequences of

partial seizures shown in Fig. 1. Therefore, a

comprehensive and coherent picture of both par-

tial and generalized seizures can be evaluated,

when specific observation times are chosen, for

example, after drug treatments or when looking

for age related effects. The observation time

windows will depend on the experimental proto-

col.

The rational implicit in the current ethological

evaluation is that the expression of brain activity,

seen in linked motor acts, is highly correlated with

behavioral sequences (Fentress and Stilwell, 1973;

Norton, 1977; Garcia-Cairasco et al., 1992, 1996).

Thus, behavioral events are recorded by conven-

Fig. 1. Neuroethological evolution of spontaneous seizures in the pilocarpine model. (A) Time and behavioral sequence, as they are

decoded from the digitally-captured video of a rat having a spontaneous recurrent seizure (7 s duration) induced by systemic injection

of pilocarpine. (B) Flowchart of behaviors for the seizure recorded in (A). (C) Flowchart of the mean of nine spontaneous seizures (60 s

observation windows each) after status epilepticus induced by systemic pilocarpine (320 mg/kg). Animals reaching class 6 (several

limbic class 5: falling) according to Pinel and Rovner (1978). (D) Flowcharts calibration and statistical associations (scales) are

depicted as, (1) behavior frequency, rectangle height; (2) behavior duration, rectangle base; (3) statistical significance, X2 log. (E)

Typical video-EEG split screen in a setup (MP 100 and Acknowledge 3.0, Biopac; Cyberamp, Axon Instruments) adapted according to

Moraes et al. (2000). (F) Hippocampal and amygdala epileptiform activity from (E) associated to orofacial automatisms (Class 1;

Racine, 1972) after status epilepticus induced by intrahippocampal pilocarpine injection (2.4 mg/0.2 ml).

J.P. Leite et al. / Epilepsy Research 50 (2002) 93�/103 99

tional video cameras and digitized by means ofvideo capturing boards. After reviewing the se-

quences, times and behaviors are collected and

processed in specifically developed software (Etho-

matic ) that allows the reconstruction of, not only

the sequence, but the statistical strength of beha-

vioral interactions (Garcia-Cairasco et al., 1992).

The flowcharts, which are the graphic expression

of these sequences, are calibrated for behaviorfrequency, duration and statistical values. When

evaluating either GABA and glutamate related

drugs in experimental tonic-clonic seizures, neu-

roethological tools have proven to be more

appropriate than seizure conventional scales, be-

cause flowchart analysis reveals behavioral asso-

ciations that seizure severity scores usually do not

detect (Terra and Garcia-Cairasco, 1992, 1994).Thus, for AED studies in pilocarpine and kainate

models, sequential analysis will enable to build

precise and reliable correlations between pharma-

cological effects on seizure behavior and involved

brain substrates.

Analogous behavioral approaches to those de-

scribed here are ethological studies after kainic

acid evoked seizures in animals and studies basedon cluster and flowcharts analysis in temporal and

frontal lobe epileptic patients (Engel et al., 1991;

Wieser 1991; Kotagal et al., 1995; Manford et al.,

1996).

7. Conclusions

We conclude that pilocarpine and kainic acidare models that are useful for drug testing. In the

status epilepticus phase, several drugs have proved

to be effective. There is general agreement in

clinical practice that prolonged seizures require

aggressive therapy and that current AEDs are

effective. Prolonged seizures can be terminated in

most cases with intravenous administration of

benzodiazepines, phenytoin, barbiturates andother compounds (Shinnar and Berg, 1996).

Therefore, the need for new drugs does not seem

to be of high priority. Studies on the latent phase

seem to be more critical since there is a good

chance that drugs or molecules that can block

damage-induced plastic changes may also suppress

the development of epilepsy. Studies from pilocar-pine and kainate models have pointed out that

AEDs such as valproate or other non-convulsant

compounds such as basic fibroblast growth factor

exert an antiepileptogenic effect (Liu and Holmes,

1997). The analogy of this latent phase with the

acquisition of amygdala kindling should encou-

rage testing of drugs that have proved to suppress

the evolution of amygdala kindling (Cavalheiro etal., 1991; Silver et al., 1991). Levetiracetam and

other effective compounds might be strong candi-

dates (Loscher et al., 1998). Drug testing in the

chronic phase should not address only the

suppression of secondarily generalized motor

seizures. Most of current tools used to quantify

spontaneous seizure events need to be coupled to

electrophysiology and more sophisticated systemsfor recording and analyzing behavior, such as the

neuroethological approach discussed here.

Acknowledgements

To Brazilian Foundations FAPESP (Grants 99/

06586-8 and 99/11729-2), CNPq and PRONEX for

financial support. JPL, NG-C and EAC are

recipients of CNPq-Brazil Research Fellowships.

To M.A. Furtado a student from NGC Lab, whokindly provided and mounted, from his Ph.D.

research project, data presented in Fig. 1.

References

Babb, T.L., Kupfer, W.R., Pretorius, J.K., Crandall, P.H.,

Levesque, M.F., 1991. Synaptic reorganization by mossy

fibers in human epileptic fascia dentata. Neuroscience 42,

351�/363.

Baran, H., Loscher, W., Mevissen, M., 1994. The glycine/

NMDA receptor partial agonist D-cycloserine blocks kai-

nate-induced seizures in rats. Comparison with MK-801 and

diazepam. Brain Res. 652, 195�/200.

Ben-Ari, Y., 1985. Limbic seizure and brain damage produced

by kainic acid: mechanisms and relevance to human

temporal lobe epilepsy. Neuroscience 14, 375�/403.

Bolanos, A.R., Sarkisian, M., Yang, Y., Hori, A., Helmers,

S.L., Mikati, M., Tandon, P., Stafstrom, C.E., Holmes,

G.L., 1998. Comparison of valproate and phenobarbital

treatment after status epilepticus in rats. Neurology 51, 41�/

48.

J.P. Leite et al. / Epilepsy Research 50 (2002) 93�/103100

Buckmaster, P.S., Dudek, F.E., 1997. Neuron loss, granule cell

axon reorganization, and functional changes in the dentate

gyrus of epileptic kainate-treated rats. J. Comput. Neurol.

385, 385�/404.

Cavalheiro, E.A., Riche, D.A., Le Gal La Salle, G., 1982.

Long-term effects of intrahippocampal kainic acid injection

in rats: a method for inducing spontaneous recurrent

seizures. Electroencephalogr. Clin. Neurophysiol. 53, 581�/

589.

Cavalheiro, E.A., Leite, J.P., Bortolotto, Z.A., Turski, W.A.,

Ikonomidou, C., Turski, L., 1991. Long-term effects of

pilocarpine in rats: structural damage of the brain triggers

kindling and spontaneous recurrent seizures. Epilepsia 32,

778�/782.

Chaudhary, G., Malhotra, J., Chaudhari, J.D., Gopinath, G.,

Gupta, Y.K., 1999. Effect of different lithium priming

schedule on pilocarpine-induced status epilepticus in rats.

Methods Find Exp. Clin. Pharmacol. 21, 21�/24.

Chronopoulos, A., Stafstrom, C., Thurber, S., Hyde, P.,

Mikati, M., Holmes, G.L., 1993. Neuroprotective effect of

felbamate after kainic acid-induced status epilepticus.

Epilepsia 34, 359�/366.

Cilio, M.R., Bolanos, A.R., Liu, Z., Schmid, R., Yang, Y.,

Stafstrom, C.E., Mikati, M.A., Holmes, G.L., 2001. Antic-

onvulsant action and long-term effects of gabapentin in the

immature brain. Neuropharmacology 40, 139�/147.

Clifford, D.B., Olney, J.W., Maniotis, A., Collins, R.C.,

Zorumski, C.F., 1987. The functional anatomy and pathol-

ogy of lithium�/pilocarpine and high-dose pilocarpine

seizures. Neuroscience 23, 953�/968.

Croiset, G., De Wied, D., 1992. ACTH: a structure-activity

study on pilocarpine-induced epilepsy. Eur. J. Pharmacol.

229, 211�/216.

Croiset, G., De Wied, D., 1997. Proconvulsive effect of

vasopressin; mediation by a putative V2 receptor subtype

in the central nervous system. Brain Res. 759, 18�/23.

Cronin, J., Dudek, F.E., 1988. Chronic seizures and collateral

sprouting of dentate mossy fibers after kainic acid treatment

in rats. Brain Res. 474, 181�/184.

Cronin, J., Obenaus, A., Houser, C.R., Dudek, F.E., 1992.

Electrophysiology of dentate granule cells after kainate-

induced synaptic reorganization of the mossy fibers. Brain

Res. 573, 305�/310.

Engel, J.J., Bandler, R., Griffith, C., Caldecott-Hazard, S.,

1991. Neurobiological evidence for epilepsy-induced inter-

ictal disturbances. Adv. Neurol. 55, 97�/111.

Fentress, J.C., Stilwell, F.P., 1973. Grammar of a movement

sequence in inbred mice. Nature 244, 52�/53.

Furtado, M.A., Braga, G.K., Oliveira, J.A., Del Vecchio, F.,

Garcia-Cairasco, N., 2002. Behavioral, morphological and

electroencephalographic evaluation of seizures induced by

intrahippocampal microinjection of pilocarpine. Epilepsia,

in press.

Garcia-Cairasco, N., Doretto, M.C., Prado, R.P., Jorge, B.P.,

Terra, V.C., Oliveira, J.A., 1992. New insights into beha-

vioral evaluation of audiogenic seizures. a comparison of

two ethological methods. Behav. Brain Res. 48, 49�/56.

Garcia-Cairasco, N., Terra, V.C., Doretto, M.C., 1993. Mid-

brain substrates of audiogenic seizures in rats. Behav. Brain

Res. 58, 57�/67.

Garcia-Cairasco, N., Wakamatsu, H., Oliveira, J.A., Gomes,

E.L., Del Bel, E.A., Mello, L.E., 1996. Neuroethological

and morphological (Neo-Timm staining) correlates of

limbic recruitment during the development of audiogenic

kindling in seizure susceptible Wistar rats. Epilepsy Res. 26,

177�/192.

Glien, M., Brandt, C., Potschka, H., Voigt, H., Ebert, U.,

Loscher, W., 2001. Repeated low-dose treatment of rats

with pilocarpine: low mortality but high proportion of rats

developing epilepsy. Epilepsy Res. 46, 111�/119.

Halonen, T., Miettinen, R., Toppinen, A., Tuunanen, J., Kotti,

T., Riekkinen, P.J., Sr, 1995. Vigabatrin protects against

kainic acid-induced neuronal damage in the rat hippocam-

pus. Neurosci. Lett. 195, 13�/16.

Hellier, J.L., Patrylo, P.R., Buckmaster, P.S., Dudek, F.E.,

1998. Recurrent spontaneous motor seizures after repeated

low-dose systemic treatment with kainate: assessment of a

rat model of temporal lobe epilepsy. Epilepsy Res. 31, 73�/

84.

Holtzman, D.M., Lowenstein, D.H., 1995. Selective inhibition

of axon outgrowth by antibodies to NGF in a model of

temporal lobe epilepsy. J. Neurosci. 15, 7062�/7070.

Honchar, M.P., Olney, J.W., Sherman, W.R., 1983. Systemic

cholinergic agents induce seizures and brain damage in

lithium-treated rats. Science 220, 323�/325.

Hort, J., Brozek, G., Mares, P., Langmeier, M., Komarek, V.,

1999. Cognitive functions after pilocarpine-induced

status epilepticus: changes during silent period precede

appearance of spontaneous recurrent seizures. Epilepsia

40, 1177�/1183.

Jope, R.S., Morrisett, R.A., Snead, O.C., 1986. Characteriza-

tion of lithium potentiation of pilocarpine-induced status

epilepticus in rats. Exp. Neurol. 91, 471�/480.

Kotagal, P., 1991. Seizure symptomatology of temporal lobe

epilepsy. In: Luders, H. (Ed.), Epilepsy Surgery. Raven

Press, New York, pp. 143�/156.

Kotagal, P., Luders, H.O., Williams, G., Nichols, T.R.,

McPherson, J., 1995. Psychomotor seizures of temporal

lobe onset: analysis of symptom clusters and sequences.

Epilepsy Res. 20, 49�/67.

Leite, J.P., Bortolotto, Z.A., Cavalheiro, E.A., 1990. Sponta-

neous recurrent seizures in rats: an experimental model of

partial epilepsy. Neurosci. Biobehav. Rev. 14, 511�/517.

Leite, J.P., Cavalheiro, E.A., 1995. Effects of conventional

antiepileptic drugs in a model of spontaneous recurrent

seizures in rats. Epilepsy Res. 20, 93�/104.

Lemos, T., Cavalheiro, E.A., 1995. Suppression of pilocarpine-

induced status epilepticus and the late development of

epilepsy in rats. Exp. Brain Res. 102, 423�/428.

Lindekens, H., Smolders, I., Khan, G.M., Bialer, M., Ebinger,

G., Michotte, Y., 2000. In vivo study of the effect of

valpromide and valnoctamide in the pilocarpine rat model

of focal epilepsy. Pharm. Res. 17, 1408�/1413.

J.P. Leite et al. / Epilepsy Research 50 (2002) 93�/103 101

Liu, Z., Holmes, G.L., 1997. Basic fibroblast growth factor is

highly neuroprotective against seizure-induced long-term

behavioural deficits. Neuroscience 76, 1129�/1138.

Longo, B.M., Mello, L.E., 1997. Blockade of pilocarpine- or

kainate-induced mossy fiber sprouting by cycloheximide

does not prevent subsequent epileptogenesis in rats. Neu-

rosci. Lett. 226, 163�/166.

Longo, B.M., Mello, L.E., 1998. Supragranular mossy fiber

sprouting is not necessary for spontaneous seizures in the

intrahippocampal kainate model of epilepsy in the rat.

Epilepsy Res. 32, 172�/182.

Loscher, W., 1998. New visions in the pharmacology of

anticonvulsion. Eur. J. Pharmacol. 342, 1�/13.

Loscher, W., Honack, D., Rundfeldt, C., 1998. Antiepilepto-

genic effects of the novel anticonvulsant levetiracetam (ucb

L059) in the kindling model of temporal lobe epilepsy. J.

Pharmacol. Exp. Ther. 284, 474�/479.

MacGregor, D.G., Graham, D.I., Stone, T.W., 1997. The

attenuation of kainate-induced neurotoxicity by chlor-

methiazole and its enhancement by dizocilpine, muscimol,

and adenosine receptor agonists. Exp. Neurol. 148, 110�/

123.

Manford, M., Fish, D.R., Shorvon, S.D., 1996. An analysis of

clinical seizure patterns and their localizing value in frontal

and temporal lobe epilepsies. Brain 119, 17�/40.

Mathern, G.W., Cifuentes, F., Leite, J.P., Pretorius, J.K.,

Babb, T.L., 1993. Hippocampal EEG excitability and

chronic spontaneous seizures are associated with aberrant

synaptic reorganization in the rat intrahippocampal kainate

model. Electroencephalogr. Clin. Neurophysiol. 87, 326�/

339.

Mathern, G.W., Babb, T.L., Pretorius, J.K., Leite, J.P., 1995.

Reactive synaptogenesis and neuron densities for neuropep-

tide Y, somatostatin, and glutamate decarboxylase immu-

noreactivity in the epileptogenic human fascia dentata. J.

Neurosci. 15, 3990�/4004.

Mathern, G.W., Babb, T.L., Leite, J.P., Pretorius, K., Yeoman,

K.M., Kuhlman, P.A., 1996. The pathogenic and progres-

sive features of chronic human hippocampal epilepsy.

Epilepsy Res. 26, 151�/161.

Mecarelli, O., De Feo, M.R., Del Priore, D., 1997. Vigabatrin

versus carbamazepine and phenytoin in kainic acid-treated

pubescent rats. Pharmacol. Res. 36, 87�/93.

Mello, L.E., Cavalheiro, E.A., Tan, A.M., Kupfer, W.R.,

Pretorius, J.K., Babb, T.L., Finch, D.M., 1993. Circuit

mechanisms of seizures in the pilocarpine model of chronic

epilepsy: cell loss and mossy fiber sprouting. Epilepsia 34,

985�/995.

Mikati, M.A., Holmes, G.L., Chronopoulos, A., Hyde, P.,

Thurber, S., Gatt, A., Liu, Z., Werner, S., Stafstrom, C.E.,

1994. Phenobarbital modifies seizure-related brain injury in

the developing brain. Ann. Neurol. 36, 425�/433.

Mikati, M.A., Werner, S., Gatt, A., Liu, Z., Rahmeh, A.A.,

Rachid, R.A., Stafstrom, C.E., Holmes, G.L., 1999. Con-

sequences of a-amino-3-hydroxy-5-methyl-4-isoxazolepro-

pionic acid receptor blockade during status epilepticus in

the developing brain. Brain Res. Dev. Brain Res. 113, 139�/

142.

Millan, M.H., Chapman, A.G., Meldrum, B.S., 1993. Extra-

cellular amino acid levels in hippocampus during pilocar-

pine- induced seizures. Epilepsy Res. 14, 139�/148.

Moraes, M.F.D., Galvis-Alonso, O.Y., Garcia-Cairasco, N.,

2000. Audiogenic kindling in the Wistar rat: a potential

model for recruitment of limbic structures. Epilepsy Res. 39,

251�/259.

Muller-Schwarze, A.B., Tandon, P., Liu, Z., Yang, Y., Holmes,

G.L., Stafstrom, C.E., 1999. Ketogenic diet reduces sponta-

neous seizures and mossy fiber sprouting in the kainic acid

model. Neuroreport 10, 1517�/1522.

Nadler, J.V., Perry, B.W., Gentry, C., Cotman, C.W., 1980.

Degeneration of hippocampal CA3 pyramidal cells induced

by intraventricular kainic acid. J. Comput. Neurol. 192,

333�/359.

Norton, S., 1977. The study of sequences of motor behavior. In:

Iversen, L.C., Iversen, S.D., Snyder, S.H. (Eds.), Principles

of Behavioral Pharmacology. Plenum Press, New York, pp.

82�/105.

Ormandy, G.C., Jope, R.S., Snead, O.C., III, 1989. Antic-

onvulsant actions of MK-801 on the lithium�/pilocarpine

model of status epilepticus in rats. Exp. Neurol. 106, 172�/

180.

Pinel, J.P., Rovner, L.I., 1978. Experimental epileptogenesis:

kindling-induced epilepsy in rats. Exp. Neurol. 58, 190�/202.

Pitkanen, A., Nissinen, J., Jolkkonen, E., Tuunanen, J.,

Halonen, T., 1999. Effects of vigabatrin treatment on status

epilepticus-induced neuronal damage and mossy fiber

sprouting in the rat hippocampus. Epilepsy Res. 33, 67�/85.

Racine, R.J., 1972. Modification of seizure activity by electrical

stimulation. II. Motor seizure. Electroencephalogr. Clin.

Neurophysiol. 32, 281�/294.

Rice, A.C., DeLorenzo, R.J., 1998. NMDA receptor activation

during status epilepticus is required for the development of

epilepsy. Brain Res. 782, 240�/247.

Rice, A.C., DeLorenzo, R.J., 1999. N -methyl-D-aspartate

receptor activation regulates refractoriness of status epilep-

ticus to diazepam. Neuroscience 93, 117�/123.

Shinnar, S., Berg, A.T., 1996. Does antiepileptic drug therapy

prevent the development of ‘chronic’ epilepsy. Epilepsia 37,

701�/708.

Silver, J.M., Shin, C., McNamara, J.O., 1991. Antiepileptogenic

effects of conventional anticonvulsants in the kindling

model of epilespy. Ann. Neurol. 29, 356�/363.

Smolders, I., Khan, G.M., Lindekens, H., Prikken, S., Marvin,

C.A., Manil, J., Ebinger, G., Michotte, Y., 1997. Effective-

ness of vigabatrin against focally evoked pilocarpine-

induced seizures and concomitant changes in extracellular

hippocampal and cerebellar glutamate, g-aminobutyric acid

and dopamine levels, a microdialysis-electrocorticography

study in freely moving rats. J. Pharmacol. Exp. Ther. 283,

1239�/1248.

Sofia, R.D., Gordon, R., Gels, M., Diamantis, W., 1993.

Effects of felbamate and other anticonvulsant drugs in

J.P. Leite et al. / Epilepsy Research 50 (2002) 93�/103102

two models of status epilepticus in the rat. Res. Commun.

Chem. Pathol. Pharmacol. 79, 335�/341.

Stafstrom, C.E., Holmes, G.L., Thompson, J.L., 1993. MK801

pretreatment reduces kainic acid-induced spontaneous sei-

zures in prepubescent rats. Epilepsy Res. 14, 41�/48.

Sutula, T., Cascino, G., Cavazos, J., Parada, I., Ramirez, L.,

1989. Mossy fiber synaptic reorganization in the epileptic

human temporal lobe. Ann. Neurol. 26, 321�/330.

Sutula, T., Cavazos, J., Golarai, G., 1992. Alteration of long-

lasting structural and functional effects of kainic acid in the

hippocampus by brief treatment with phenobarbital. J.

Neurosci. 12, 4173�/4187.

Tauck, D.L., Nadler, J.V., 1985. Evidence of functional mossy

fiber sprouting in hippocampal formation of kainic acid-

treated rats. J. Neurosci. 5, 1016�/1022.

Terra, V.C., Garcia-Cairasco, N., 1992. Neuroethological

evaluation of audiogenic seizures and audiogenic-like sei-

zures induced by microinjection of bicuculline into the

inferior colliculus. II. Effects of nigral clobazam microinjec-

tions. Behav. Brain Res. 52, 19�/28.

Terra, V.C., Garcia-Cairasco, N., 1994. NMDA-dependent

audiogenic seizures are differentially regulated by inferior

colliculus subnuclei. Behav. Brain Res. 62, 29�/39.

Turski, W.A., Cavalheiro, E.A., Schwarz, M., Czuczwar, S.J.,

Kleinrok, Z., Turski, L., 1983. Limbic seizures produced by

pilocarpine in rats: behavioural, electroencephalographic

and neuropathological study. Behav. Brain Res. 9, 315�/335.

Turski, W.A., Cavalheiro, E.A., Coimbra, C., da Penha

Berzaghi, M., Ikonomidou-Turski, C., Turski, L., 1987.

Only certain antiepileptic drugs prevent seizures induced by

pilocarpine. Brain Res. 434, 281�/305.

Turski, L., Ikonomidou, C., Turski, W.A., Bortolotto, Z.A.,

Cavalheiro, E.A., 1989. Review: cholinergic mechanisms

and epileptogenesis. The seizures induced by pilocarpine: a

novel experimental model of intractable epilepsy. Synapse 3,

154�/171.

Wieser, H.G., 1991. Ictal manifestations of temporal lobe

seizures. Adv. Neurol. 55, 301�/315.

Williams, P.A., Wuarin, J.P., Dou, P., Ferraro, D.J., Dudek,

F.E., 2000. Reassessment of the effect of cyclohexamide in

the pilocarpine model of temporal lobe epilepsy. Epilepsia

41, 76.

J.P. Leite et al. / Epilepsy Research 50 (2002) 93�/103 103