Gen. Pharmac. Vol. 31, No. 1, pp. 47–50, 1998
Copyright 1998 Elsevier Science Inc.
Printed in the USA.
ISSN 0306-3623/98 $19.00 ? .00
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Analysis of PTZ-Induced Kindling in Mice
Lucimar F. da Silva,
Patrı ´cia Pereira and Elaine Elisabetsky*
Laborato ´rio de Etnofarmacologia, Depto. de Farmacologia,
and Curso de Po ´s Graduac ¸a ˜o em Cie ˆncias Biolo ´gicas—Bioquı ´mica,
Universidade Federal do Rio Grande do Sul, Caixa Postal 5072,
90041-970, Porto Alegre, RS, Brazil [E-mail: email@example.com]
ABSTRACT. 1. Glutamate seems to play a central role in epilepsy, and kindling is considered the
most useful experimental model in revealing plastic changes associated with epileptic features.
2. The aim of this study was to optimize pentylenetetrazol (PTZ)-kindling conditions in mice and
analyze glutamatergic changes associated with this phenomena.
3. A significant increase (85.7%) in seizuring animals was observed after four PTZ administra-
tions, with all subjects presenting full seizures after five administrations.
4. PTZ kindling, but not acute seizure, significantly increased (169.8%) the specific binding of
[3H]glutamate in the cerebral cortex.
5. The development of PTZ-induced kindling in mice was prevented by the coadministration of
phenobarbital or diazepam.
6. This study indicates that mice can be used in a reliable model of PTZ-induced kindling and that,
as in rats, the kindling increases the specific [3H]glutamate binding in the cerebral cortex, therefore
allowing for screening new drugs that can interfere in the plastic changes believed to underlie epileptic
gen pharmac 31;1:47–50, 1998.
1998 Elsevier Science Inc.
KEY WORDS. Binding, kindling, mice, pentylenetetrazol, glutamate, anticonvulsants
Concerning the transmitter systems in neuronal plasticity, much
attention has been focused on excitatory amino acids, especially on
glutamate. Under pathological conditions such as acute (hypoxia,
ischemia, hypoglycemia and seizures) and chronic (neurodegenera-
tive diseases) insults, an excessive release of glutamate results in neural
injury and cell death (Lipton and Rosemberg, 1994). Studies concern-
ing the neural excitotoxicity have indicated that glutamate plays cru-
cial roles in the initiation, spread and maintenance of epileptic activity
(Dingledine et al., 1990; Meldrum, 1994).
In this study, we determined conditions to optimize PTZ kindling
in mice. We evaluated the effects of age and those of diazepam and
phenobarbital on seizure threshold. In addition, the changes in glu-
tamate receptors in the kindling phenomenon was accessed by de-
termining glutamate-specific binding at cortex membranes of PTZ-
kindled mice in comparison with naive and acutely treated animals.
Kindling has been defined as the process whereby repeated applica-
tions of subconvulsive electrical or chemical stimuli to animals lead
to an increase in convulsive activity, resulting ingeneralized seizures
(Goddard, 1967; Goddard et al, 1969; Scho ¨der et al., 1993). The
kindling-induced enhanced seizure activity is understood to indicate
a long-lasting alteration of the neuronal excitability, which can be
regarded as suggestive of neuronal plasticity (Cain, 1989). As an ep-
ilepsy experimental model associating neuronal plasticity and sei-
zures, kindling is unique in providingopportunities to study the abil-
ity of drugs to modify these progressive changes. Such ability may
be due to the underlying mechanisms of drug action on seizure de-
velopment (Albertson et al., 1981).
Electrical kindling in rats has become a widely employed tech-
nique for studying seizure mechanisms and is considered to be a use-
ful experimental seizure model. An increased susceptibility of rats to
pentylenetetrazol (PTZ) seizures after repeated injections of this
drug was first observed in 1941 (Sacks and Glaser, 1941). PTZ kin-
dling therefore provides a means for studying a persistent decrease
in seizure thresholds, although this method has been much less uti-
lized than the electrical kindling. There are few studies using chemi-
cal kindling in mice (Karler et al., 1984, 1989; Piredda et al. 1986).
Earlier investigators questioned the soundness of PTZ-kindling in
mice, suggesting age as a major factor in decreasing seizure threshold
(Craig and Colasanti, 1989). Advantages in working with mice
rather than rats include the possibility of testing compounds available
in limited amounts, such as in the study of natural products or large
screening programs or both (Kupferberg, 1989; Swinyard et al. 1986).
Male SR-1 mice, 25–30 g, were used throughout the experiments.
They were housed at 20?2?C, maintained on a 12-hr light–dark cy-
cle, with food and water ad libitum. All procedures were carried out
according to institutional policies on experimental animal handling.
PTZ KINDLING. Animals (8 weeks old at the start of the experiment)
were divided into three groups. Each treatment consisted of two
drug administrations. All groups received saline intraperitoneally
and, 30 min later, one of the following subcutaneous injections:
(group I) saline, (group II) 40 mg/kg PTZ and (group III) 60 mg/kg
*To whom correspondence should be addressed.
Received 24 March 1997; accepted 3 October 1997.
L. F. da Silva et al.
Binding of [3H]glutamate
For measurement of the specific binding of [3H]glutamate, mem-
branes (100 ?g of protein) were incubated at 30?C for 15 min in 50
mM Tris/KCl buffer, pH 7.4, and 40 nM of [3H]glutamate, in a final
volume of 0.5 ml. The reaction was interrupted by cooling the tubes
and a further centrifugation for 20 min at 27,000g. The supernatant
was discarded. The walls of the tubes and the surface of the pellets
were quickly and carefully rinsed with cold distilled water. The pel-
lets were processed for radioactivity measurement in cintilador
Beckman. To determine the specific binding of [3H]glutamate, each
experiment was processed with parallel control tubes containing
[3H]glutamate, in the presence of 1,000 times of the amount of non-
labeled glutamate. Specific binding was defined as the difference in
[3H]glutamate binding between tubes without (total binding) and
with (nonspecific binding) unlabeled glutamate in excess concen-
To evaluate changes in glutamate binding associated with PTZ-
induced kindling, we studied acute and chronically PTZ treated ani-
mals.To comparechronicallyapplied PTZ(kindled mice) withacutely
induced convulsions, mice were given a single subconvulsive dose (60
mg/kg of PTZ, SC) or a single convulsive dose (88 mg/kg PTZ, SC).
Only mice presenting score ? 3 convulsions were selected for binding
assays, always carried out 24 hr after the last PTZ administration.
FIGURE 1. Percentage of mice presenting seizures with score
?3 after repeated PTZ (60 mg/kg, SC) administration, n?14.
*?P?0.05 and **?P?0.01, Fisher.
PTZ. These treatments were repeated once every third day, in a to-
tal of six treatments. Injections (0.1 ml/10 g) were given between
7:00 and 11:00 a.m.
After each PTZ injection, animals were placed in acrylic observa-
tion chambers for 30 min, and behavioral seizure activity was rated
according to the following scale [adapted from Ito et al. (1977)]: 0,
no convulsive behavior; 1, playing piano; 2, clonic forelimb convul-
sions lasting less than 3 sec; 3, clonic forelimb convulsions lasting
more than 3 sec; 4, generalized convulsions with tonic extension ep-
isodes and full status epilepticus; 5, death. Kindling was considered
to be established if the animal presented seizures of rate ?3.
The behavioral seizure activity as established by PTZ-induced kin-
dling in mice can be seen in Figure 1. With a single PTZ (60 mg/
kg) injection 21.4% of the animals presented seizures with score ?
3. A significant increase in the percentage of seizuring animals oc-
curred after the third PTZ injection. Repeated administration of sa-
line alone or PTZ 40 mg/kg failed to induce convulsions with scores
?3 at any point of the study (data not shown).
EFFECTS OF ANTICONVULSANTS ON PTZ KINDLING. Animals (8
weeks old at the start of the experiment) were divided into four
groups (n?10–14). As in the first experiment, each treatment con-
sisted of two drug administrations, repeated once every third day, in
a total of six treatments. The first IP injection was as follows: Group
I, 10 mg/kg phenobarbital; Group II, saline (phenobarbital vehicle);
Group III, 0.5 mg/kg diazepam; Group IV, propylenoglycol (PPG,
diazepam vehicle). Thirty minutes after the IP treatment, all groups
received 60 mg/kg of PTZ subcutaneously. Epileptic behavior was
evaluated as above.
Effects of anticonvulsants on PTZ kindling
The ability of anticonvulsants to counteract PTZ kindling is shown at
Figure 2. The development of PTZ-induced kindling was prevented by
EFFECT OF AGE ON SEIZURE THRESHOLD TO PTZ. The effect of age
on the seizure threshold was evaluated by using animals 3, 8, 11, 18
and 28 weeks of age. Animals received saline intraperitoneally and,
30 min later, a single dose of PTZ (60 mg/kg SC). Seizures were
evaluated as in the kindling experiment.
Effect of age on seizure threshold to PTZ
Figure 3 shows the effect of age on PTZ seizure threshold in mice.
Figure 3a shows that a significant increase in the percentage of seiz-
uring animals with acute adminsitration occurs only after 28 weeks.
MEMBRANE PREPARATION. Twenty-four hours after the last injec-
tion (PTZ or saline), animals were decapitated, and the brains were
rapidly removed. The cerebral cortex was dissected and used for the
biochemical studies. Membranes were prepared as described else-
where (Elisabetsky et al., 1995). The cerebral cortices were homoge-
nized (20:1 v:w) in 0.32 M sucrose containing 10 mM Tris/KCl
buffer, pH 7.4, and 1 mM MgCl2. All steps were carried out at 4?C.
The homogenate was centrifuged twice at 1,000g for 15 min and the
final pellet discarded. Both supernatants were pooled and centri-
fuged at 27,000g for 15 min. The supernatant was discarded and the
pellet was lysed (20:1 v:w) for 30 min in 10 mM Tris/HCl buffer,
pH 7.4. The lysed pellet was washed three times with lysing buffer
(20:1 v:w) by centrifuging at 27,000g for 15 min. Supernatants were
discarded and the final pellet was used for the experiments.
FIGURE 2. Protective effects of phenobarbital [?](diluted in sa-
line), saline alone [?] and diazepam [?](diluted in propylenegly-
col) propyleneglycol alone [?] on PTZ kindling in mice. n?10–
14. **?P?0.01, Fisher compared with respective control.
basis and neurochemical correlates of the kindling phenomena still
lack complete elucidation. PTZ-induced kindling has been reported
to be associated with a reduction in ?-aminobutyric acid-mediated
neurotransmission in the central nervous system (Corda et al., 1990;
Giorgi et al., 1991). Numerous studies have shown that N-methyl-
d-aspartate subtype of glutamate receptors plays an important role
in experimental epilepsy and kindling (McNamara et al., 1988;
Morimoto, 1989; Vezzani et al., 1988).
Here we reported that repeated administration of 60 mg/kg of
PTZ effectively induces kindling in mice (Fig. 1). Like that reported
by Craig and Colasanti (1989), our study detected an age-dependent
decrease in PTZ threshold. Nevertheless, the effect of age was not
detected in mice younger than 18 weeks, and it became significant
only after 28 weeks of age. Because significant expression of kindled is
seen already with 11-week old animals (Fig. 3a and b), this study con-
tradicts previous considerations in regard to the use of mice as reliable
subjects for PTZ kindling (Craig and Colasanti, 1989). In conclusion,
our data show a correlation between the behavioral expression of kin-
dling and an increse in [3H]glutamate binding in mice cortex mem-
branes, as previously reported for rats (Scho ¨der et al., 1993).
Kindling in mice can play an important role in the context of
screening new drugs that cold interfere with the plastic changes be-
lieved to underlie epileptic phenomena.
FIGURE 3. Effect of age on PTZ-induced convulsions. (a) Per-
centage of seizuring animals acutely treated with PTZ (60 mg/kg,
SC). *?P?0.05, Fisher. (b) Percentage of 11-week-old mice pre-
senting convulsions after acute or chronic (kindled) PTZ (60 mg/
kg, SC) administration. **?P?0.01, Fisher.
This work was supported by grants from CNPq.
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Figure 3b presents the percentage of seizuring mice at 11 weeks of
age, acutely or chronically (kindled) treated with PTZ.
Table 1 presents l-[3H]glutamate-specific binding (pmol/mg pro-
tein) in the cortex membrane of PTZ acutely treated or kindled
mice. At a ligand concentration of 40 nM, the specific l-[3H]gluta-
mate binding to the cerebral cortex membrane of kindled mice was
significantly increased compared with groups treated with saline and
with the group of convulsing animals acutely treated with PTZ.
PTZ kindling is a procedure of chemical kindling in which the re-
peated administration of this compound causes a progressive in-
crease in the excitability of the central nervous system so that the
threshold for generalized convulsions decreases over time (Corda et
al., 1990). Therefore, repeated administrations of otherwise subcon-
vulsant doses lead to generalized seizures. The neurophysiological
TABLE 1. Effect of PTZ kindling, acute convulsive PTZ (88.0
mg/kg, SC) and acute subconvulsive PTZ (60 mg/kg, SC) on
L-[3H]glutamate-specific binding in cortex membrane of mice
PTZ acute 60 mg/kg
PTZ acute 88 mg/kg
1.16 ? 0.04
3.13 ? 0.62**
1.45 ? 0.04
1.59 ? 0.23
Binding assays performed 20 h after the last drug administation. Data ex-
pressed as mean ? SE. **P ? 0.01, analysis of variance.
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L. F. da Silva et al.
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