Yonago Acta medica 2004;47:45–51

Positive Transfer Effect of Amygdaloid Kindling in Developing
Rats
Kazuyoshi Matsuda, Shin-ichi Yoshioka, Junji Takasu, Hideaki Mitani and Ryuzo
Kawahara
Division of Neuropsychiatry, Department of Multidisciplinary Internal Medicine, School of
Medicine, Tottori University Faculty of Medicine, Yonago 683-8504 JapanTo study the hypothesis that seizure susceptibility in the young rat brain is higher than thatin the adult brain, positive transfer effect (PTE) in amygdaloid kindling in rats was investigated at varing ages: 15 days, 18 days, 28 days, 40 days and 70 days. Although PTE wasobserved regardless of age, it was more pronounced in weaning rats than in adult rats.
Key words: amygdaloid kindling; development; rat; seizure susceptibility; transfer effect

Kindling is the progression of behavioral andelectrographical seizure induced by repeated activation of neural pathways with initial subclinicalstimulations (Goddard et al., 1969). Once kindlingis established, the kindling effect is sustained fora long period without electrical stimulations. In
addition, after the establishment of kindling at onesite in the brain (primary site, PS), a significantly
lower number of stimulations can induce kindlingat another site (secondary site, SS), called the “positive transfer effect” (PTE) (Goddard et al., 1969;
Racine, 1972; Cain, 1985). These data suggest thatthe separate secondary epileptic foci can be createdwith minor stimuli in comparison to the original
stimuli required for the induction of the 1st focus.
We previously reported that young rats developed amygdaloid kindling with a smaller numberof stimulations and had a higher susceptibility thanadult rats (Matsuda et al., 1993, 1995). Differenceof the PTE between young and adult brains may add
the deeper understanding on the age dependency ofkindling induction in rat brains, but only reports on
16-day-old rats (Kawawaki et al., 1987) and 20-day-

old rats (Chiba et al., 1994) are available to date.
No reports are available on the PTE in the younger
rats. To shed a light on a working hypothesis thatthe young rat brain is more susceptible to kindling,
we investigated the PTE in rats at various ages,
including those before weaning.

Materials and Methods
Animals
Male Wistar rats aged 70- [n = 6] postnatal days
(referred to as adult rats) and rats of either sex
aged 15- [n = 5], 18- [n = 6], 28- [n = 5] and 40[n = 5] postnatal days (referred to as young rats)
were used in these studies. The animals were kept
in an environmentally controlled room (12/12 hlight/dark cycle, lights on at 0700, temperature
20–22˚C, humidity 30–40%) in the Animal Care
Center at Tottori University, with food and water
available ad libitum except during the experimental
procedure. All animal experiments were performed

Abbreviations: AD, after-discharge; ADD, AD duration; ADT, AD threshold; PS, primary site; PTE, positive transfer
effect; SS, secondary site

45

K. Matsuda et al.

in accordance with the Guidelines set out by the
Tottori University Committee for Animal Experimentation.

Stage-5 seizures were regarded as a completion ofkindling. In the present study, the inter-stimulationintervals and durations of stimulation were different among the rats at respective ages because
the PS (right) kindling and SS (left) kindling must
be completed within a single day. After one day
following completion of the PS kindling in 15-,18-, 28-, 40- and 70-day-old groups, SS kindlingwas performed until Stage-5 seizures appeared.
It is known that the behavioral manifestationsof amygdaloid kindling seizures in young rats´
were different from those in adult rats (Moshe,1981). Therefore, we chose 2 different classification scales of kindling seizures for young andadult rats. The behavioral seizure stages wereassessed with the classification proposed by Moshé(1981) for 15-, 18- and 28-day old groups: Stage0, no manifestations; Stage 1, facial movements;
Stage 2, head turning to stimulation site or headnodding; Stage 3, alternating independent forelimb
clonus or unilateral forelimb clonus; Stage 4, bilateral clonus or rotatory movements of tonicallyextended forelimbs and Stage 5, loss of balance;and by Racine (1972) for 40- and 70-day-old
groups: Stage 0, no manifestations; Stage 1, mouthand facial movements; Stage 2, head nodding;Stage 3, contralateral forelimb clonus; Stage 4,bilateral forelimb clonus with rearing and Stage5, rearing and falling. Electrographic recording
in the amygdala was also performed before andduring kindling stimulation, and the AD durations(ADDs) elicited by each stimuli were measured.
ADDs elicited by the 1st ADT stimulation at PS
kindling and SS kindling (1st response ADDs) andat the 1st Stage-5 seizure (1st Stage-5 ADDs) were
measured. Locations of the electrodes tips wereconfirmed histologically.

Kindling preparation
Bipolar stainless-steel electrodes were implanted
3 days prior to the kindling stimulations in the15-, 18- and 28-day-old groups, and 7 days priorin the 40- and 70-day-old groups. The rats wereanesthetized with pentobarbital (Nembutal, Abbot,
Chicago, IL; 25 mg/kg intraperitoneally for young
rats and 50 mg/kg intraperitoneally for adult rats)
and then placed in a streotaxic apparatus with theskull set level. Bipolar stainless-steel electrodes(0.1 mm in diameter for young rats and 0.2 mm indiameter for adult rats) were implanted bilaterally
into the amygdala according to the streotaxic atlasof Sherwood and Timiras (1970) for young rats andKönig and Klippel (1979) for adult rats: the site of
implantation in the both amygdala to the bregma
was 1.8 mm posterior, 3.5 mm lateral and 7.2 mm
inferior in 15- and 18-day-old groups; 2.0 mm
posterior, 3.8 mm lateral and 7.4 mm inferior in the
28-day-old group; 2.5 mm posterior, 3.8 mm lateraland 7.8 mm inferior in the 40-day-old group; and 3.0mm posterior, 4.8 mm lateral and 8.8 mm inferior
in the 70-day-old group. A reference electrode wasplaced in the frontal sinus in the young rats, andin the frontal bone in the adult rats. All electrodes
were secured to the skull with dental cement.
Kindling procedure
Kindling procedure followed according to the method described by Yoshioka et al. (2000). Briefly,
the after-discharge (AD) threshold (ADT) was
determined by 60 Hz electrical stimulation for 1 s
beginning at 50 µA and increasing its intensity by
50 µA every 10 min until AD was elicited. After
determining the ADT, the rats were subjected tostimulation at an ADT for 1 s every 30 min in the
15- and 18-day-old groups; for 3 s every 30 min
in the 28-day-old group; and for 3 s every 60 min
in the 40- and 70-day-old groups until Stage-5
seizures appeared 5 times. The 5 appearances ofData analysis
All data were expressed as mean ± SEM. Statistical analysis of the data was performed using
Wilcoxon’s rank sign sum test, Student’s paired
t-test, Kruskall-Wallis’ test and one-way analysis ofvariance followed by post hoc analysis of Fisher’s
protected least significant difference.

46������� �����

PTE of kindling in developing rats

��
� �
� �
�
�

� �
�
�
�
�
�
�
�
�

�

�

�
�
�

�

��

��

��

�

��

������� �����

������ �� ������������

�
�
�

�
�
�

�

�

��

�
�
��

��

��

�

�

�

�

�

�

��

��

�

�

��

��

��

��

�

��

������ �� �������������� ���������� ��������

�

��

�

�

�

�

�

�

�

�

������ �� ������������ ������ �� ������������

�

�

� �
�
�
�
�

�

�

��

��

��

��

������ �� ������������

Fig. 1. Amygdaloid kindling development stimulated at the primary site (PS) and the secondary site (SS) in young
and adult rats aged 15 (A), 18 (B), 28 (C), 40 (D) and 70 days (E). The group mean seizure stages are plotted
over the number of stimulations in rats. Significance of differences in the seizure stages at equivalent number ofstimulations compared with PS of each group are indicated by asterisks (*P < 0.05) as obtained from Wilcoxon’s
rank sum singed test.
�� ��������

������ �� �������������� ��������
�����Results

���

��

���

Figure 1 shows kindling development stimulated at
the PS and SS in 5 groups of 15-, 18-, 28-, 40- and70-day-old rats. Kindling progressed more rapidly
at the SS than at the PS in all groups.
The number of stimulations required to reachthe 1st Stage-5 seizure (kindling rate) was comparedin PS and SS kindling at each age: the rate wassignificantly lower in SS kindling (P < 0.05) thanin PS kindling in all groups (Fig. 2). The kindlingrate during PS kindling of the 5 groups in the
age-increasing order was 13.4 ± 1.1, 23.5 ± 1.5,
21.2 ± 3.0, 18.2 ± 2.7 and 7.3 ± 1.4, respectively.
The 70-day-old group was significantly faster inkindling than 15- (P < 0.05), 18- (P < 0.0001), 28(P < 0.0001) and 40-day old groups (P < 0.001).
Among younger groups, the 15-day-old group was
significantly faster in kindling than 18- (P < 0.01)
and 28-day-old groups (P < 0.05).

��
��

���

�

��

�

���

�

�

���

���

���

���

�

�

�

�
���

�

�

�

�

��� �����
Fig. 2. Comparison of the number of stimulations
required to the 1st Stage-5 seizure between the primarysite (PS) and the secondary site (SS) kindling in rats
at different ages of 15 (A), 18 (B), 28 (C), 40 (D) and70-days (E). At every age, the number of stimulations
required to the 1st Stage-5 seizure is significantly
smaller at SS than PS. Values are mean ± SEM.
Brackets indicate the number of animals. Significanceof differences in the number of stimulations until the
1st Stage-5 appearance compared with PS kindling areindicated by asterisks (*P < 0.05) as obtained from
Wilcoxon’s rank sum singed test.

47

K. Matsuda et al.

Table 1. After-discharge durations (ADDs) at 1st after-discharge threshold stimulation and 1st
Stage-5 appearance at primary site (PS) and secondary site (SS) kindling in rats at different ages
Rats

Kindling

First response ADDs (s)First Stage-5 ADDs (s)15-day-old [5]

PS
SS
PS
SS
PS
SS
PS
SS
PS
SS

16.4 ± 1.4
39.0 ± 5.8
12.3 ± 1.1
23.0 ± 14.1
12.2 ± 1.5
35.6 ± 9.1
15.0 ± 3.9
20.0 ± 5.3
21.8 ± 5.0
41.5 ± 18.3

82.4 ± 10.3
118.0 ± 27.6
74.2 ± 12.5
74.8 ± 12.5
60.2 ± 13.2
70.4 ± 14.3
74.8 ± 15.1
65.2 ± 16.4
106.0 ± 17.8
91.5 ± 19.9

18-day-old [6]28-day-old [5]40-day-old [5]70-day-old [6]**Values are mean ± SEM.
[ ], the number of animals.
* P < 0.05 by Studen’s paired t-test.

The ratio of the kindling rate in SS kindlingto that in PS kindling (SS/PS) was analyzed: the 5groups showed, in the age-increasing order, 0.30 ±0.10, 0.17 ± 0.03, 0.35 ± 0.12, 0.36 ± 0.09 and 0.51
± 0.03, respectively. Analysis of variance indicatedsignificant differences in SS/PS ratio between
young and adult groups [F(4, 22) = 3.033; P < 0.05].
The SS/PS level of the 18-day-old group was
significantly lower (P < 0.01) by post hoc analysis
than that of the 70-day-old group.
Table 1 compares ADDs at the time of 1st
stimuli and 1st Stage-5 development between PS
and SS kindling: differences in 1st-response ADDs
between PS and SS kindling were significant in the15- (P < 0.05) and 28-day-old groups (P < 0.05),
but not in the other groups. In addition, ADDs atthe 1st Stage-5 appearance did not differ between
PS and SS kindling among all groups.

in PS kindling. These results indicate that PTE in
amygdaloid kindling occurred in developing rats at
various ages as well as adult rats.
Moshé et al. (1995) reported that postnatal
age-related changes in seizure susceptibility were
not linear. The seizure susceptibility to amygdaloid
kindling in rats was decreased in the 1st postnatal
week, but increased in the 2nd and 3rd weeks. In
addition, 30- to 35-day-old rats were resistant to
the development of amygdaloid kindling (Moshé,
1981; Moshé et al., 1981) and kainic acid-induced
seizures (Albala et al., 1984). In the present
study, the 70-day-old group reached Stage 5 more
rapidly as compared with other groups. Further,
the 15-day-old group also reached more rapidly
as compared with 18- and 40-day-old groups.
Hence, it seems that adult rats have an increased
seizure susceptibility as compared with young
rats. However, Chiba et al. (1992) reported nodifferences in kindling rate among 20-, 30- and60-day-old rats when hourly electrical stimulation
at a same suprathreshold current level for 1 s wasapplied in all groups 10 times daily. Moshé (1981)
also reported that when the rats received the same
electrical stimulation at a 400-µA current for 1 s
hourly, the kinding rates were similar among 15-,17-, 18-, 48- and 72-day-old rats, whereas morestimulations were necessary for the 34-day-old rats.
The reasons for the different rates of progression

Discussion
Most studies on PTE have been of adult animals.
In the present study, we investigated whether
PTE may occur age-dependently in developing
rats. The kindling rate in 70-day-old adult rats
was significantly faster in SS kindling than in PS
kindling. Furthermore, the rate in younger ratgroups was significantly faster in SS kindling than48

PTE of kindling in developing rats

between the literature (Moshé, 1981; Chiba et al.,1992) and our results can be attributed not only tocharacteristics related to differences in age but also
to differences in experimental conditions, such as
inter-stimulation intervals, durations of stimulation,
strength of stimuli or implantation sites of electrodes.
In order to examine the seizure susceptibility
at a precise age, we performed kindling with thestimulation conditions set to the extent possible
so that the kindling stimulations at PS and SS
kindling would be completed within 1 day for eachof the age. However, the experimental conditions
could not be made entirely consistent. Firstly,
the inter-stimulation intervals were different
among groups. Moshé et al. (1983) reported that
stimulations at 60-min intervals induced kindlingof the amygdala in both adult and sucking rats.
However, stimulations at 15-min intervals disrupted
kindling in adult rats, but induced kindling insuckling pup rats. These results suggest that thepup rats appear to have relatively short seizure
refractory periods. Therefore, the 40- and 70-dayold rats received electrical stimulation of longer
inter-stimulus intervals as compared with the 15-,
18- and 28-day-old rats. Secondly, the durationsof stimulation were also different among groups.
Holmes and Thompson (1987) have found that in
rapid kindling model, 29-day-old rats kindled when
the duration of stimulation was 10 s. However, theanimals stimulated with 1-s stimulation did not.
They suggested that the key factor in rapid kindlingwas the duration of stimulation. Thus, we chosethe longer duration of stimulation for kindlingprocedure for the 28-, 40- and 70-day-old groups
than for the 15- and 18-day-old groups. Thirdly,
Moshé et al. (1981) also reported that the ADTs
varied with age, being highest in the suckling rats,
lowest in the 35-day-old rats and intermediate in
the 60- and 85-day-old rats. However, previous
studies (Goddard et al., 1969; Racine, 1972) havedemonstrated that the kindling development did not
significantly differ at the current intensities used
for their stimulus between the ADT level and thesuprathreshold level. In the present experiments,
we chose the ADT levels as the strength of stimuli

in all the groups. However, the possibility thatthe differences in the durations of stimulation andstrength of stimuli among the groups influence
the kindling development needs to be taken into
account. Finally, it is well known that the rate
at which the animals developed kindled seizures
showed some differences according to the regional
distribution of the electrode site (Goddard et al.,1969). In the present experiments, the electrodetips were located in the amygdala in all groups.
The coordinates of implantation of electrodes to thebregma were somewhat different among all groups;
therefore, the possibility cannot be excluded thatthe present age-related changes in the kindlingrats were due to the differences in the sites of
implantation.
Increased seizure susceptibility in the young
brain has been obtained in experiments including
studies on amygdala kindling (Moshé, 1981;
Moshe´ et al., 1983), hippocampal kindling (Haaset al., 1990), or kainic acid- (Albara et al., 1984) or
pentylentetrazole- (Weller and Mostofsky, 1995)
induced seizure. In addition, Haas et al. (1990)
have found that young rats receiving alternating
stimulations in the amygdala and hippocampus
develop progressively more severe seizures thanadult rats, indicating that the young brain might
be less able to suppress the generalization of
seizures than the adult brain. Our observations on
PTE revealed that SS/PS ratios were significantly
smaller in the 18-day-old rats than in the adult rats
when the kindling rates in PS and SS kindling werecompared for calculating SS/PS ratios. However,there were no significant differences in SS/PS ratios
between the 15-, 28- or 40-day-old rats and 70-dayold ones. These results indicate that the savingsin the number of stimulations required to produce
the 1st Stage-5 seizure during the SS kindlingwere larger only in weaning rats than in adultrats. Chiba et al. (1994) analyzed SS/PS ratios
until the appearance of generalized convulsions
and the prolongation of ADDs at each stage in
SS kindling: based on the findings, they reported
that PTE was more strongly manifested in 20-dayold rats than in mature rats (Chiba et al., 1994):
among our data, those of the 18-day-old group49

K. Matsuda et al.

alone were consistent with theirs. Le Gal La Salle(1981) analyzed the relationship between the initial
ADDs and the kindling rate, and observed thatrats which initially responded to the amygdaloid
stimulation by long AD tended to kindle faster thanthose which responded by short AD. In the present
study, the kindling rates in SS kindling wereshorter than in PS kindling, but ADDs at 1st ADT
stimulation in SS kindling were not always longer
than in PS kindling. In addition, comparisons of
ADDs showed no difference in excitability. These
results were in disagreement with those reported by
Chiba et al. (1994). Absence of increased ADDs atthe 1st Stage-5 seizure during SS kindling suggeststhat increased ADDs are not a necessary correlateof enhanced kindling susceptibility.
What are the factors that may be responsible
for the enhanced seizure susceptibility of theyoung brain? In young rats, the level of brain
norepinephrine is lower than in mature rats
(Milby et al., 1979; Morris et al., 1980), andthe γ -aminobutyric acid inhibition system inthe substantia nigra in the former is less welldeveloped (Wurpel et al., 1988). Recently, we havefound that α-2 agonist clonidine facilitates kindlingdevelopment in 14-day-old rats, whereas it retards
in adult rats (Yoshioka et al., 2000). The increased
susceptibility to kindling development in young
rats might be due to these ontogenetic changes in
the neurochemical system.
Some reports have suggested involvement of
the brain stem in generalized convulsions and PTE
in mature rat amygdaloid kindling (McIntyre, 1975;
Cain, 1985; Chiba and Wada, 1997). Chiba andWada (1997) reported that electrolytic lesioning of
the midbrain prior to amygdala kindling resulted
in not only retardation at PS kindling but alsoabolishment of PTE. These results suggest that thebrain stem participates not only in the developmentof PS kindling but also in the mechanism oftranshemispheric PTE. In the present study, it
was found that the PTE was observed in rats
regardless of ages. These findings suggest that theepileptiform activity at the PS developed into thecontralateral hemisphere via the brain stem in rats
at various ages. Thus, a similar study of the brain-stem lesioning reported by Chiba and Wada (1997)
may give some answers on the role of the brain
stem responsible for the PTE formation in youngrats.
Acknowledgments: We are grateful to Professor Juhn A.
Wada (Health Sciences Center Hospital, University of
British Columbia) for his valuable discussion and help
with the manuscript.
This study was supported in part by a Grant-in Aid
for Scientific Research (C) from the Japanese Ministryof Education, Science, Sports and Culture (10670899)
and by Japan Epilepsy Research Foundation.

References
1 Albala BJ, Moshé SL, Okada R. Kainic-acid-induced
seizures: a developmental study. Dev Brain Res
1984;37:43–58.
2 Cain DP. Transfer of kindling: clinical relevance and a hypothesis of its mechanism. Prog
Neuropsychopharmacol Biol Psychiat 1985;9:467–
472.
3 Chiba S, Muneoka Y, Kamata S, Inaba H, Miyagishi
T. Positive transfer effect of the amygdala kindlingin developing rats. Tenkan O Megutte1994;14:54–
62 (in Japanese with English abstract).
4 Chiba S, Muneoka Y, Kamata S, Inaba H, Sato Y,
Miyagishi T. The amygdaloid kindling in developing,
adult and aged rats. Jpn J Psychiat Neurol 1992;46:516–517.
5 Chiba S, Wada JA. The effect of electrolytic lesioning of the midbrain prior to amygdala kindling inrats. Neurosci Lett 1997;227:83–86.
6 Goddard GV, McIntyre DC, Leech CK. A permanent change in brain function resulting from daily
electrical stimulation. Exp Neurol 1969;25:295–330.
7 Haas KZ, Sperber EF, Moshé SL. Kindliing in developing animals: expression of severe seizures andenhanced development of bilateral foci. Dev Brain
Res 1990;56:275–280.
8 Holmes GL, Thompson JL. Rapid kindling in the
prepubescent rat. Dev Brain Res 1987;36:281–284.
9 Kawawaki H, Lee S-S, Nakajima S, Hattori H,
Matsuoka O, Murata R. Transfer phenomenon in
amygdaloid kindling in sucking rats. Jpn J Psychiat
Neurol 1987;41:537–538.
10 König JFR, Klippel RA. The rat brain: a stereotaxic
atlas of the forebrain and lower parts of the brain
stem. New York: Krieger; 1970.

50

PTE of kindling in developing rats

11 Le Gal La Salle G. Amygdaloid kindling in therats: regional differences and general properties. In:
Wada JA, ed. Kindling 2. New York: Raven Press;
1981. p. 31–47.
12 Matsuda K, Kawahara R, Takeshita H, Komatsu
K, Ito K, Hazama H. Postmaturational changes inseizure susceptibility acquired by amygdaloid kindling in developing rats. Neurosciences 1993;19:31–
36.
13 Matsuda K, Kawahara R, Yoshioka S, Katayama S,
Sunami M. Amygdaloid kindling in variously aged
infant rats and rekindling after maturation. Seishin
Shinkeigaku Zasshi 1995;97:199–216 (in Japanesewith English abstract).
14 McIntyre DC. Split-brain rat: transfer and interference of kindled amygdala convulsions. Can J
Neurol Sci 1975;2:429–437.
15 Milby KH, Mefford IN, Keller RW, Adams RN. Developmental changes in rat brain epinephrine levels.
Brain Res 1979;169:398–400.
16 Morris MJ, Dausse J-P, Devynck M-A, Meyer P.
Ontogeny of α1 and α2-adrenoceptors in rat brain.
Brain Res 1980;190:268–271.
17 Moshé SL. The effects of age on the kindlingphenomenon. Dev Psychobiol 1981;14:75–81.
18 Moshé SL, Albala BJ, Ackermann RF, Engel J Jr.
Increased seizure susceptibility of the immature
brain. Dev Brain Res 1983;7:81–85.
19 Moshé SL, Sharpless NS, Kaplan J. Kindlingin developing rats: variability of afterdischarge
thresholds with ages. Brain Res 1981;211:190–195.
20 Moshé SL, Shinnar S, Swann JW. Partial (focal)
seizures in developing brain. In: Schwartzkroin
PA, Moshé SL, Noebels JL, Swann JW, eds. Braindevelopment and epilepsy. New York: Oxford
University Press; 1995. p. 34–65.
21 Racine RJ. Modification of seizure activity byelectrical stimulation: II. Motor seizure. Electroencephalogr Clin Neurophysiol 1972;32:281–294.
22 Sherwood NM, Timiras PS. A stereotaxic atlas ofthe developing rat brain. London: University ofCalifornia Press; 1970.
23 Weller A, Mostofsky DI. Ontogenetic development
and pentylenetetrazol seizure thresholds in rats.
Physiol Behav 1995;57:629–631.
24 Wurpel JND, Tempel A, Sperber EF, Moshé SL.
Age-related changes of muscimol binding in thesubstantia nigra. Dev Brain Res 1988;43:305–308.
25 Yoshioka S, Mitani H, Maeda K, Takeo S, Matsuda
K, Mizukawa R, et al. Age-specific effects of noradrenergic alpha-2 agonist clonidine on the development of amygdaloid kindling in developing rats.
Dev Brain Res 2000;119:283–288.
Received June 23, 2004; accepted August 25, 2004
Corresponding author: Shin-ichi Yoshioka, MD

51