Yonago Acta medica 2000;43:95–101

Persistence of Acquired Epileptogenesis in Amygdaloid-Kindled Rats:
Relationship between the Initial Kindling Stages and Seizure Development in Rekindling
Shigeki Takeo, Shin-ichi Yoshioka, Kazuhisa Maeda, Junji Takasu, Toshiyuki
Ueda and Ryuzo Kawahara
Department of Neuro-Psychiatry, Faculty of Medicine, Tottori University, Yonago 683-0826,
Japan
In order to gain insight into mechanisms underlying the persistence of the partial
kindling effect, we suspended amygdaloid kindling at different seizure stages in rats,
and investigated the effects on subsequent rekindling after a rest period of 2 months.
Ten-week-old rats, implanted with bipolar electrodes, were separated by various initialkindling stages into 5 groups of rats, partially kindled to stages 1 (n = 7) and 2 (n = 10),fully kindled to stages 4 (n = 11) and 5 (n = 11), and control rats (n = 12) which were
implanted but not stimulated initially. The number of stimulations required to elicit
the first stage 5 seizure during rekindling was significantly lower in the fully kindledgroups (P < 0.01 in the stage 4 group and P < 0.001 in the stage 5 group) than thecontrol group. The cumulative afterdischarge duration during rekindling was significantly shorter in the fully kindled groups (P < 0.01 in both groups) than the control
group. The latency of the first stage 5 seizure during rekindling was significantly shorterin the partially and fully kindled groups than the control group. These results suggestthat epileptogenesis acquired at the partially kindled stage is different than that acquiredat the fully kindled stage. However, the effects of the initial kindling on the latency to
produce the stage 5 seizure during rekindling persisted both in the partially and fully
kindled rats.
Key words: amygdaloid kindling; epileptogenesis; partial seizure; rat; rekindling

One potential reason for the intractability of
epileptic seizures has been reported as symptomatic localized epilepsy with complex partialand secondarily generalized seizures (Okuma
and Kumashiro, 1981) as well as a high incidence of seizures before the start of treatment(Reynolds, 1990). In addition, the possible roleof the long-term persistence of epileptogenesis
continues to be debated.
Limbic kindling is a well-known model of
temporal lobe epilepsy (Sato et al., 1990), witha long-term persistence of acquired susceptibility (Goddard et al., 1969; Wada et al., 1974;
Wada, 1980; Sato et al., 1990). As for the degreeof kindling relationship between the seizure
stage before the completion of kindling and thepersistence of its effect, Moshé and Albala(1982) reported that the kindling effectpersisted into adulthood when the previously
fully and partially kindled pups were rekindledat maturity. Similarly, Dennison et al. (1995)
reported that a partial kindling effect was
retained during a 12-week period in adult rats.
However, it is not clear at what stage of partial
kindling the effect becomes persistent. In thepresent amygdaloid kindling study, therefore,
we investigated the effects of various stages of
initial kindling on the pattern of subsequent
rekindling following a rest period of 2 months.

Abbreviation: AD, afterdischarge

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clonus; stage 4, rearing; stage 5, rearing andfalling. Stages 1 and 2 are thought to correspond to the partial seizure stages, and stages 4
and 5, to the secondarily generalized seizure
stages.

Materials and Methods

Animals
Fifty-one 10-week-old male Wistar rats wereused. The rats were randomly divided into an
experimental group (n = 39) and control group(n = 12), maintained on lab chow and water ad
libitum in an environmentally controlled room
(12/12 h light/dark cycle, lights on at 0700,
temperature 20–22˚C, humidity 30–40%) in the
Laboratory Animal Research Center, Faculty of
Medicine, Tottori University. All experiments
conformed to the Guidelines for Animal
Experimentation at the Faculty of Medicine,
Tottori University.

Initial kindling and rekindling
The control rats (n = 12) were implanted withelectrodes but not stimulated for 2 months until
the time of rekindling. The experimental group(n = 39) was initially kindled according to theabove-described procedures, and divided into
the following 4 subgroups: Stage-1 (n = 7),
Stage-2 (n = 10), Stage-4 (n = 11) and Stage-5(n = 11). Stimulation was suspended after astage 1 seizure developed in the Stage-1 group,and similarly after a seizure of stages 2, 4 and 5
developed in the Stage-2, -4 and -5 groups,
respectively. The rats of the Stage-1 and Stage2 groups were arbitrarily categorized as
partially kindled, and those of the Stage-4 andStage-5 groups, as fully kindled.
Two months later, the animals were rekindled at the AD threshold which was determined with the same method as in the initialkindling. The rats received electrical stimulation at the maximum AD threshold for eachgroup once a day till a stage 5 seizure was induced for 5 consecutive days. Behavioral
seizure stages during rekindling and the duration of ADs after each rekindling stimulation
were recorded and evaluated with the same
method as in the initial kindling.

Surgery
Under pentobarbital anesthesia (intraperitoneal
administration of 40-mg/kg Nembutal; Abott,
Chicago, IL), stimulating and recording
stainless-steel bipolar electrodes 200 µm in
diameter were stereotaxically (König andKlippel, 1970) implanted into the right amygdala (P = 3.0 mm, L = 4.0 mm and H = 8.8 mm
relative to the bregma) and a reference electrode was placed on the skull over the frontalbone. After a 1-week postoperative recovery
period, the kindling study was started.

Kindling procedures
Kindling was begun with 60-Hz sinusoidal
electrical stimulations applied to the right
amygdala at the afterdischarge (AD) thresholdfor 1 s once every day. The AD threshold wasdetermined by increasing the stimulation intensity by 50 µA every 15 min from the initial intensity of 100 µA until an AD was induced.
Electrographic recording was made throughout
to measure the duration of ADs, which werecorrelated with behavioral seizure stages. The
behavioral seizure stages of amygdaloid kindling were assessed with the classification
proposed by Racine (1972): stage 0, AD but noconvulsions; stage 1, mouth and facial movement; stage 2, head nodding; stage 3, forelimb

Data analysis
The number of electrical stimulations thatelicited the first seizure in stages 1 to 4 in theinitial kindling was recorded in each rat fromthe 4 experimental groups. The number ofelectrical stimulations used in one of the seizure
stages to elicit the first stage 5 seizure during
rekindling was recorded in each rat of thecontrol and 4 experimental groups.
Furthermore, we totaled up the duration of
ADs at the manifestation of respective seizure
stages during rekindling in the control and 4
experimental groups. Peterson et al. (1981)96

Persistence of kindling epileptogenesis

significantly lower than that in the control
group; in fact, the number of stimulations fromstages 1 to 4 was zero in the Stage-5 group
because this group directly progressed to stage
5 in rekindling. In the Stage-4 group, the number of stimulations needed to develop the firststage 5 seizure in rekindling was also significantly lower than that in the control group.
The number of stimulations needed in stages 0
and 3 in rekindling was also significantly lower
than that in the control group. In the Stage-1
and Stage-2 groups in which the initial kindlingwas suspended at the partial seizure stage, thenumber of stimulations that elicited the firststage 5 seizure in rekindling was not significantly different from that in the control group inrekindling. On the other hand in the Stage-2
group, the number of stimulations needed instages 0 and 3 in rekindling was significantly
lower than that in the control group. No significant differences were seen between the Stage-1
and control groups in the number of stimulations needed in each seizure stage in rekindling.

reported that the cumulative AD duration was amore reliable indicator in the development ofkindling than the actual number of stimulations
required to elicit ADs. So, we calculated the
AD duration cumulated in one seizure stage andthe cumulative AD duration totaled up from thefirst stimulation until the completion of kindling, and compared the obtained results among
the control and experimental groups.
The latency of the onset of generalized
seizures, i.e., the time needed to produce a stage
5 seizure after the start of stimulation, was recorded for 5 consecutive days during rekindlingin the control and 4 experimental groups. Data
were expressed as means ± SD. Statisticalanalysis was carried out with Mann-Whitney’s
U-test or Student's t-test.

Results

Number of stimulations in initial kindling and rekindling
Table 1 shows the number of stimulations
needed to elicit the first seizure of each stage in
the initial kindling and used in each seizure
stage during rekindling. In the Stage-5 group,
the number of stimulations used to develop thefirst stage 5 seizure in rekindling was significantly lower than that in the control group. In
addition, the number of stimulations totaled up
from stages 0 to 4 during rekindling was also

Cumulative AD duration in rekindling
The means of AD duration cumulated in each
seizure stage during rekindling classified bygroups are shown in Table 2. In both the Stage4 and Stage-5 groups, the cumulative AD
duration totaled up from stages 0 to 4 was significantly shorter than that in the control group.

Table 1. The effects of initial kindling stages on seizure manifestations during rekindlingGroup
Initialkindling†

Stage 0

Number of stimulations
Rekindling‡
Stage 1 Stage 2
Stage 3

Stage 4

Stage 5Control [12]—2.9 ± 2.0
1.4 ± 1.8 0.5 ± 0.5 0.5 ± 0.5
0.7 ± 0.8
7.0 ± 3.9
Stage-1 [ 7]4.1 ± 1.9 3.3 ± 4.5
2.3 ± 3.0 1.0 ± 1.4 0.1 ± 0.4
1.3 ± 1.4
9.0 ± 6.5
Stage-2 [10]9.4 ± 3.5 1.2 ± 1.6*
0.4 ± 0.7 0.9 ± 0.7 0.1 ± 0.3* 0.7 ± 0.8
4.3 ± 1.5
Stage-4 [11] 10.6 ± 3.7 0.3 ± 0.7*** 0.3 ± 0.7 0.4 ± 0.5 0.1 ± 0.3* 0.5 ± 0.7
2.5 ± 1.0**
Stage-5 [11]9.6 ± 4.8 0.2 ± 0.6***
0*
0**
0**
0**
1.2 ± 0.6***
Mean ± SD.
[ ], number of animals used.
† Number of stimulations that elicited the first seizures of stage 0 to stage 5 in the initial kindling.
‡ Number of stimulations used in one of the seizure stages or required to elicit the first stage 5 seizure during
rekindling.
* P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group (Mann-Whitney’s U-test).

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Table 2. The effect of initial kindling stages on the cumulative AD duration needed to elicit
the first stage 5 seizure during rekindlingGroup
Stage 0Stage 1

Cumulative AD duration (s)Stage 2
Stage 3

Stage 4Control
186.2 ± 217.4
107.1 ± 156.1 44.3 ± 54.5 50.5 ± 59.6
48.8 ± 65.1
Stage-1
191.0 ± 387.5
149.0 ± 247.4 50.1 ± 68.5 15.6 ± 41.2
93.1 ± 96.0
Stage-2
33.9 ± 52.8*
20.6 ± 46.5
65.5 ± 66.9
2.2 ± 7.0* 64.7 ± 86.9
Stage-4
3.6 ± 9.2*
15.1 ± 33.7
11.6 ± 18.9
3.6 ± 11.8* 43.0 ± 81.1
Stage-5
3.5 ± 11.5*
0*
0*
0*
0*
AD, afterdischarge.
* P < 0.05, **P < 0.01 compared with the control group (unpaired Student's t-test).

Total
436.8 ± 403.2
498.9 ± 554.7
186.9 ± 114.1
76.8 ± 72.7**
3.5 ± 11.5**

Discussion

However, in the Stage-5 group, the cumulative
AD duration in stages 1 to 4 was zero due to thedirect progression to stage 5 in rekindling. No
significant differences were seen between theStage-1 and control groups in the cumulative
AD duration totaled up in all seizure stages
during rekindling.

In the present study, the previously partially andfully kindled rats were rekindled 2 months later,
and the effects of differences in seizure stages
during the initial kindling in seizure manifestations during rekindling were investigated.
The fully kindled groups showed significantly
lower levels in the number of stimulations
required to elicit the first stage 5 seizure during
rekindling than the control group did. The difference was not significant between the partially kindled groups and the control group. In
other words, susceptibility acquired at thepartial seizure stage was different from that

Latency of the onset of generalized seizures in rekindling

Latency of stage 5 seizures

Figure 1 shows the mean values of latency of
the first stage 5 seizure during rekindlingclassified by groups. The mean latencies weresignificantly shorter in all 4 experimentalgroups than in the control
group. In the Stage-5(s)group, the mean latencies
80
were significantly shortControl
70
er than those in the conStage-1
Stage-2trol group, even when the
60
Stage-4stage 5 seizures occurred
50
Stage-5on days 2 and 3, but not
40
significant in the 3 other*30
experimental groups**
other than in the control
20**
group. On days 4 and 5*
10
also, the differences in0mean latency of the first
0
1
2
3
4
5
stage 5 seizures duringDayrekindling were not sigFig. 1. The latencies needed to elicit a stage 5 seizure for 5 consecutive
nificant between the condays during rekindling. Symbols are means of the latencies. *P < 0.05
trol group and any of thecompared with the control group (Student’s t-test). On day 1, the latencies
4 experimental groups.
in the Stage-1, -2, -4 and -5 groups are significantly shorter than in thecontrol group. On days 2 and 3, only the Stage-5 group shows asignificantly shorter mean than the control group.

98Persistence of kindling epileptogenesis

acquired at the secondarily generalized seizure
stage, and only the latter persisted to make an
impact on rekindling carried out 2 months later.
This finding is consistent with the differential
effects observed in cumulative AD duration(Table 2).
On the other hand, the latency of the firststage 5 seizure during rekindling was significantly shorter in both the partially and fully
kindled groups than in the control group. Thisfinding conflicted with the results that the difference in number of stimulations and cumulative AD duration during rekindling was not
significant between the partially kindled andcontrol rats. The reason why the latency of thefirst stage 5 seizure during rekindling was
shortened even in the partially kindled rats isdifficult to explain from the results of thepresent experiments only. However, our results
clearly showed that the latencies on days 2 and3 were significantly shorter only in the Stage-5
group than in the control group. The kindlingeffects in the Stage-5 group were stronger andmore persistent than in the other experimentalgroups.
Dennison et al. (1995) carried out kindlingexperiments in rats which reached stages 1, 3
and 5, and rekindled 12 weeks after kindlingsuspension. They compared the number ofstimulations that elicited generalized seizures inthe 3 experimental and control groups, andfound that the number of stimulations that elicited generalized seizures was significantly lower in both the Stage-3 and Stage-5 groups thanin the control group. The difference was not
significant between the Stage-1 group and thecontrol group. This Stage-1 group had onsets of
seizures even after the first stimulation during
rekindling. Hence, they concluded that the effects of kindling were persistent without further
stimulation, and were also retained to somedegree even in the group partially kindled tostage 1. Moshé and Albala (1982) performed
amygdaloid kindling in developing rats: in bothfully and partially kindled rats, the kindling rateduring rekindling in the same, adult rats was
significantly lower than in the control rats.
Thus, kindling-induced neurobiological changes
in developing rats persisted into adulthood evenin the partially kindled. However, the AD
duration of the first seizure and the AD durationof the first generalized seizure during
rekindling were only significantly longer in the
fully kindled group than in the control group.
Therefore, they concluded that the degree ofretention of the kindling effects was stronger inthe fully kindled group than in the partiallykindled group. On the other hand, since both
the AD duration and seizure stage were retained
during rekindling in rats that had been kindled
to stages 1 to 4 and then left unstimulated for 45days, regression of the kindling effect is notdependent on the seizure stage prior to interruption of stimulation (Homan and Goodman,1988).
In the present experiments, both the number ofstimulations that elicited the first stage 5 seizure
and the AD duration during rekindling in thepartially kindled group, unlike in the fully
kindled group, were not significantly different
from those in the control group. However, theinitial kindling exerted an effect on the latencyof the first stage 5 seizure during rekindling,even in the partially kindled group. Hence, afurther detailed investigation is needed toclarify whether a long-term persistence of epileptogenesis is acquired in partial kindling ornot.
As for the anatomic relationship between
amygdaloid kindling and the seizure development stage following kindling stimulation, it isthought that a functional connection is formed
between the amygdala and the cerebral cortexof the motor area on the stimulation side in thecase of progression from partial seizure stages
to stage 3 (Wada, 1982). In addition, a functional connection is thought to be formed
among the cortical and subcortical nuclei inboth hemispheres in monkeys, especially in thebilateral motor areas, when stage 3 progresses
to stage 4 and the seizures become secondarily
generalized (Baba et al., 1986). Burchfiel andApplegate (1989) hypothesized, on the basis of
their results using a kindling antagonism model,that seizure development in kindling is not acontinuous process but one that follows a stepwise progression. In their view, stages 1 and 2
are the stages of transient neural reorganizationin which the forebrain gate is opened, and stage
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S. Takeo et al.

3 involves permanent neural reorganization inwhich the brain stem gate is opened, leading to
the generalized seizures of stages 4 and 5.
Therefore, the differences in the degree ofretention of kindling effects between partiallyand fully kindled groups in the present study
may represent differences in the degree of functional connection between the forebrain andthe brain stem.
It is thought that amygdaloid kindling induces morphometric changes in the nervous
system, such as mossy fiber sprouting (Cavazos
and Sutula, 1990) and neurogenesis (Scott et al.,1998) in the dentate gyrus of the hippocampus,
leading to synaptic reorganization and thedevelopment of epileptogenesis. However,Osawa et al. (1995) reported that the degrees ofneural sprouting in the mossy fiber of the dentate gyrus of the hippocampus of amygdalakindled rats which had reached stage 5 andthose that had only reached stage 3 were notsignificantly different as compared with thedegree in the control rats. Thus, they concluded
that their neuropathological findings were notsufficient to explain kindled seizure susceptibility by neural sprouting in the dentate gyrus of
the hippocampus. Uemura et al. (1996) performed rekindling following a rest period of 2
weeks after the initial kindling in perforantpath-kindled rats, but they found no correlations between the degree of mossy fiber sprouting and the AD duration or seizure stage during
rekindling. Therefore, they concluded thatmossy fiber sprouting has no relationship tokindling development. Recently, Scott et al.
(1998) reported that granular cells were newly
formed in the dentate gyrus of the hippocampusof rats which had reached stage 5 by amygdaloid kindling. However, they found that there
was no new growth of neural cells in rats whichshowed only localized seizure manifestations
after several kindling stimulations. Hence, they
concluded that the stage of seizure elicited bykindling and the degree of new neural cell
growth are closely related. Causes of the differential effects of partial and full kindling remain
to be elucidated. Evaluation of a possible relationship between morphometric changes mentioned above and the differential effects ofkindling described in the present study may
shed some light on this question.

Acknowledgments: The authors thank Prof. Juhn A.
Wada (Health Sciences Center Hospital, Universityof British Columbia) for his helpful comments on the
manuscript.
This study was supported in part by a Grant-inAid for Scientific Research (C) (No. 10670899)
from the Japanese Ministry of Education, Science,Sports and Culture.

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Received June 20, 2000; accepted August 10, 2000
Corresponding author: Dr. Shin-ichi Yoshioka