Epilepsy Research 46 (2001) 191– 203

www.elsevier.com/locate/epilepsyres

Behavioral and metabolic features of repetitive seizures in

immature and mature rats
Patricia Szot a,b,*, Sylvia S. White a,b, Elizabeth B. McCarthy c,
Andrew Turella c, Starr X. Rejniak c, Philip A. Schwartzkroin c,d
a
Geriatric Research, Education and Clinical Center (GRECC) (182B), VA Puget Sound Health Care System,
1660 South Columbian Way, Seattle, WA 98108, USA
b
Department of Psychiatry and Beha6ioral Science, Uni6ersity of Washington, Seattle, WA 98195, USA
c
Department of Neurological Surgery, Uni6ersity of Washington, Seattle, WA 98195, USA
d
Department of Physiology/Biophysics, Uni6ersity of Washington, Seattle, WA 98195, USA
Received 28 February 2001; received in revised form 14 May 2001; accepted 16 May 2001

Abstract

Seizure incidence varies significantly with age, with seizure susceptibility particularly high during the first few years
of life. Of significant concern is what effects do brief, repetitive seizures have on the developing brain. We approached
this issue by examining the change in seizure threshold, and related markers of neuronal activity and metabolic
activity (c-fos mRNA and 2-deoxyglucose [2DG]), as a function of repetitive seizure episodes in immature and mature
rats. Starting on postnatal day 15 (P15) (immature) or P60 (adult) rats were given two flurothyl seizures a day for 5
days (nine or ten seizures). The seizure latency profile, our measure of threshold, in immature versus adult rats across
the 5-day testing period was different. In immature rats, threshold for the second seizure on each day was significantly
lower than for the first seizure, suggesting that there was little refractoriness after the first seizure of the day. In
contrast, the mature animal had a significantly longer threshold latency to the second seizure for the first 3 days of
testing. The immature animal was also more likely than the adult to exhibit tonic extension as a feature of the first
seizure of the day. Following repetitive seizures, more regions of the CNS showed c-fos mRNA expression in the
immature animal than adults, suggesting that repetitive seizures in the immature animal activated a greater percentage
of the brain. Compared with the effects of a single seizure, repetitive seizures resulted in less 2DG labeling in most
regions of the brain (except the hippocampus); in the immature brain this difference was more distinct than in adults.
The consequences of repetitive seizures in the immature animal results in distinctly different seizure behavior and
neuronal activity pattern (c-fos expression) than that observed in the mature animal. © 2001 Published by Elsevier
Science B.V.

Keywords: Repetitive seizure; Immature brain; Flurothyl; c-fos; 2-Deoxyglucose; Hippocampus; Ventromedial hypothalamus;
Pediatric epilepsy

* Corresponding author. Tel.: + 1-206-7642-308; fax: + 1-206-7642-569.

E-mail address: szot@u.washington.edu (P. Szot).

0920-1211/01/$ - see front matter © 2001 Published by Elsevier Science B.V.

PII: S 0 9 2 0 - 1 2 1 1 ( 0 1 ) 0 0 2 8 5 - 6
192 P. Szot et al. / Epilepsy Research 46 (2001) 191–203
1. Introduction
The occurrence of seizures is significantly higher
in children than in young-to-mid-life adults
(Hauser and Kurland, 1975; Ellenberg et al., 1984).
Seizures at this time in development may have
devastating effects on developmental outcome, de-
pending on the number and severity of the seizure
(Holdsworth and Whitmore, 1974; Fowler et al.,
1985; Seidenberg et al., 1986; Austin et al., 1998).
Animal research has focused on the conse-
quences of status epilepticus (prolonged seizures)
or of a single severe seizure on the developing
animal (de Casrilevitz et al., 1971; Wasterlain and
Plum, 1973; Moshe et al., 1983; de Feo et al., 1985;
Cavalheiro et al., 1987; Sperber and Moshe, 1988;
Zouhar et al., 1989; Hirsch et al., 1992; Stafstrom
et al., 1992; Velisek et al., 1992; Weller and Mostof-
sky, 1995). However, short repetitive seizures are
far more common in the affected childhood popu-
lation. Recent studies have begun to determine the
effects of brief, repetitive seizures on the brains of
immature animals. Seizures during development,
whether repetitive or prolonged, lead to an in-
creased sensitivity of the adult animal to a variety
of stimuli (Moshe and Albala, 1982, 1983; Holmes,
1983; Holmes et al., 1984, 1998, 1999; Okada et al.,
1984; Ferland and Applegate, 1998b; Huang et al.,
1999; Liu et al., 1999), suggesting that the early
seizure event triggers a long-lasting alteration in
critical neuronal networks. Also, seizures during
development, whether repetitive or prolonged, are
correlated with impaired learning and memory of
the animal in adulthood (Wasterlain and Plum,
1973; Wasterlain, 1976; Holmes et al., 1984; de Feo
et al., 1986; Holmes et al., 1998, 1999; Huang et al.,
1999; Liu et al., 1999). Investigators have suggested
that such changes in learning/memory and seizure
susceptibility may be associated with cellular dam-
age (i.e. cell loss) and reorganization (mossy fiber
sprouting) induced by the repetitive seizures (Neill
et al., 1996; Holmes et al., 1998, 1999; Huang et al.,
1999; Liu et al., 1999). However, single seizures (or
even status epilepticus) results in less damage in the
immature animal than observed in the adult animal
(Albala et al., 1984; Nitecka et al., 1984; Okada et
al., 1984; Cavalheiro et al., 1987; Hirsch et al., 1992;
da Silva Fernandes et al., 1999). Therefore, it is
unclear if a correlation exists between hippocampal
damage associated with seizures in the immature
animal and later to changes in learning/memory
and/or seizure susceptibility.
The main objective of this study was to determine
the effects of brief, repetitive seizures on the devel-
oping brain. Toward this end, we have examined
changes in seizure threshold in immature and
mature rats following ten flurothyl (FL)-induced
seizures, delivered over 5 days. We then assessed
markers of neuronal activity (c-fos mRNA expres-
sion) and metabolic activity (2-deoxyglucose
(2DG) labeling) in these animals. These measures
provide an initial step toward gaining insight into
the consequences of repetitive seizures, and toward
understanding the differences in outcome between
the immature and mature rat.
2. Materials and methods
2.1. Immature animals
Litters of Sprague– Dawley rat pups (with dams)
were purchased from Bantam and Kingman (Seat-
tle, WA) on postnatal day 10 (P10), and housed as
litters in standard cages in a controlled environ-
ment with 12 h light/dark cycle. Food and water
were provided to dams ad libitum, and nursing
times of pups were monitored on days of seizure
threshold testing to ensure that experimental ani-
mals had sufficient opportunity to nurse. On P15,
animals were weight-matched into pairs, and re-
spective members of each pair were randomly
assigned to experimental or control groups.
Weights were monitored from P15 to P19, the days
of seizure threshold testing. All animal use proce-
dures were in accordance with University of Wash-
ington and NIH guidelines.
2.2. Mature animals
Adult male Sprague–Dawley rats were pur-
chased from Bantam and Kingman on P58 and
housed in standard cages, as above. On P60,
animals were weight-matched into pairs, and re-
spective members of each pair were assigned ran-
domly into experimental or control groups. The
 P. Szot et al. / Epilepsy Research 46 (2001) 191–203
animals were weighed daily during 5 days of seizure
threshold testing. All animal use procedures were
in accordance with University of Washington and
NIH guidelines.
2.3. Seizure induction
Flurothyl (FL, bis-2,2,2-trifluoroethyl ether,
Aldrich) was used to induce brief, repetitive
seizures in immature and mature rats. Each animal
was placed in an air-tight Plexiglas chamber, and
the volatile convulsant was infused (20 ul/min) onto
filter paper from which it vaporized (Prichard et al.,
1969).
2.3.1. Seizure threshold testing
The latencies (s) to the first myoclonic jerk (MJ,
focal seizure) and to generalized (clonic tonic (CT))
seizure were recorded for each animal for each
seizure; these latencies constitute the seizure
‘threshold’ measurements. Two seizures were ad-
ministered on each of 5 consecutive days with 2-h
interval between seizures on a given day. Seizures
were administered at the same time each day to
minimize circadian effects on seizure vulnerability.
Drug exposure was terminated by opening the
chamber and removing the animal at the first sign
of generalized seizure activity; however, some ani-
mals progressed rapidly to tonic extension. The
number of animals, which exhibited tonic exten-
sion, was recorded for each group, for each seizure
on each day. Age- and weight-matched control
animals were treated equivalently to the experimen-
tal animals (experiencing repetitive seizures), but
were exposed to a benign odorant instead of FL.
2.3.2. Test groups
Exposure time of the control animals was yoked
to the FL exposure times of the experimental
animals. The control animals consisted of two
groups, (a) those that experienced no seizure (‘con-
trol’) (n=7 immature; n =7 mature); and (b) those
that received one FL seizure corresponding in time
to the last seizure of the experimental group (‘sin-
gle’ seizure) (n=10 immature; n = 6 mature). The
‘single’ seizure group allowed us to assess the
difference in response to one seizure versus repeti-
tive seizures. Experimental animals were also di-
193
vided into two groups, (a) those that received nine
seizures (only one seizure on the fifth day) (n= 7
immature; n= 6 mature) and (b) those that received
ten seizures (two on the fifth day) (n= 9 immature;
n= 7 mature). The nine seizure group was added
to our analysis since preliminary results revealed a
significant seizure latency difference between the
first and second seizure of a given day. The nine
seizure group allowed us to dissect within day
differences from differences associated with repeti-
tive seizure occurrence (over 5 days). Statistical
comparisons of latencies were made using Student’s
paired t-test with Bonferroni correction for laten-
cies and  2 or Fisher’s exact test for behavioral
variables.
14
2.4. C-2 deoxyglucose (2 -DG) labeling
[14C]-2DG (New England Nuclear, Boston, MA)
(0.05 uCi/g for mature [approximately 90 ml] and
0.08 uCi/g for immature animal [approximately 30
ml]) was administered to the animal through the
lateral tail vein. On the fifth day of threshold
testing, each animal was given a bolus injection of
[14C]-2DG, then placed into the FL chamber for
seizure induction. Seizure threshold testing pro-
ceeded as usual, with animals removed from the FL
chamber when generalized seizure activity was
observed. Forty-five minutes after the injection of
[14C]-2DG, the animal was sacrificed and the brain
was removed and frozen on dry ice. The brains were
sectioned at 20 mm on a cryostat and mounted onto
Fisher Superfrost slides (Fisher Scientific, Houston,
TX). Two sets of slides with consecutive sections
were collected from each brain. Slides were stored
at −70 °C until assayed. One set was dried for 10
min on a slide warmer and opposed to Hyperfilm
b-max (Amersham, Arlington Heights, IL)— for 1
week for immature animals and 2 weeks for mature
animals— in order to produce autoradiographic
images of the [14C]-2DG labeling. Exposure times
were different between the immature and mature
animals to adjust for the difference in radioactiv-
ity that was administered. A [14C] standard was also
placed onto each sheet of film in order to allow
quantification of the images using the Mi-
croComputer Imaging Device System (MCID,
Imaging Research, Ont., Canada). Separate opti-
194 P. Szot et al. / Epilepsy Research 46 (2001) 191–203
cal densities were made of the left and right
hemispheres over three consecutive sections, which
were anatomically matched across animals accord-
ing to the atlas of Paxinos and Watson (1986).
Thus, mean9S.E.M. reported here is the average
of six density readings for each animal. Glucose
utilization was determined in the following regions,
i.e. cortex, striatum, thalamus, hippocampus, sub-
stantia nigra and locus coeruleus (LC). Statistical
comparisons were made by analysis of variance
(ANOVA) with post-hoc Scheffe test; statistical
differences were significant at P B0.05. Statistical
comparison was made only between each treatment
group (control vs. single vs. nine vs. ten seizures)
at a given age. Comparison of optical density values
between immature and mature animals was not
attempted; however, the relative pattern of change
in 2DG (and c-fos, see below) between immature
and mature animals was noted.
2.5. c-fos mRNA in situ hybridization
The second set of slides were used to determine
the expression of c-fos mRNA. Tissue preparation
and labeling of the c-fos oligonucleotide was per-
formed as described previously (Szot et al., 1997).
The c-fos oligonucleotide probe was a 51-base
probe complementary to nucleotides 270– 319 of
the c-fos mRNA (Curran et al., 1987). The oligonu-
cleotide probe was 3%-end-labeled with [33P]-dATP
(New England Nuclear) using terminal deoxyri-
bonucleotidyl transferase (Life Technologies,
Gaithersburg, MD) and then purified on NEN-
Sorb columns (New England Nuclear). The c-fos
hybridization buffer for the immature animals
contained 0.30× 106 cpm/50 ul, and for mature
animals contained 0.37×106 cpm/50 ul. Hyperfilm
b-max (Amersham) was exposed to slides contain-
ing tissue hybridized with c-fos [33P] oligonucle-
otide —72 h for immature and 48 h for mature
animals. The difference in exposure time between
the immature and mature animals was to allow
adequate hybridization signal for data analysis.
c-fos mRNA analysis utilized the alternate set of
slides that were used to measure 2DG labeling;
therefore, measures had to be taken to prevent the
[14C]-2DG labeling from interfering with [33P]-
dATP labeled oligonucleotide on the film. Toward
this end, a layer of plastic wrap was placed over the
slides following [33P]-dATP labeling, but before the
Hyperfilm was opposed to the slides. To quantitate
c-fos mRNA expression in the specific regions of
the CNS, all sections were processed, hybridized,
and washed in the same session; each sheet of
Hyperfilm contained both control and experimen-
tal tissue sections. Tissue from immature animals
was run as one assay, and tissue from mature
animals was run as a second assay. Optical densities
were measured from films using MCID (Imaging
Research). Separate optical density measurements
were made of the left and right hemispheres over
three successive sections, which were anatomically
matched across animals according to the atlas of
Paxinos and Watson (1986). Background optical
density was subtracted from each image. c-fos
mRNA expression was determined in the following
regions, i.e. cingulate cortex, neocortex, septum,
striatum, ventromedial hypothalamus (VMH),
hippocampal CA1, CA3, DG, and LC. Each
mean9 S.E.M. reported here is the average of six
optical density readings (after background subtrac-
tion) for each animal. Statistical comparisons were
made using ANOVA followed by post-hoc Scheffe
test; statistical significance was considered as PB
0.05. As indicated for 2DG labeling, statistical
comparison was made only between each treatment
group (control vs. single vs. nine vs. ten seizures)
within immature and mature age groups. Compari-
son of optical density values across age groups was
not quantified; however, the relative pattern of
change in c-fos expression was noted for immature
versus mature animals.
3. Results
As reported by others (Holmes et al., 1998, 1999;
Huang et al., 1999; Liu et al., 1999; Schmid et al.,
1999), the immature animal tolerated repetitive FL
induced seizures with about 5% mortality. Mortal-
ity in the immature animal always occurred with
the very first seizure; once past this initial seizure,
all immature animals survived the repetitive seizure
paradigm. Mature animals also had about 5%
mortality; unlike the immature animals, mature
animals died after the ninth or tenth seizure.
 P. Szot et al. / Epilepsy Research 46 (2001) 191–203 195

3.1. Seizure threshold
The seizure latency profile of the immature and
mature animals was different across 5 days of
threshold testing (Fig. 1). In immature animals,
the latency to the first seizure of the day (MJ and
CT) was the same across 5 days. On the fifth day,
latencies (MJ and CT) to the first seizure on that
day (the ninth seizure) were identical to latencies
in animals that experienced only a single seizure
(P19). However, for all days latencies for the
second seizure of the day were significantly lower
(MJ and CT) than for the first seizure. Also in
immature animals, there was a gradual increase in
the number of animals, which exhibited tonic
extension in response to the first seizure of every

Fig. 1. Flurothyl seizure thresholds in immature and mature rats following ten consecutive generalized seizures. Histograms on the
left side represent the immature animal; while the histograms on the right side represent the mature animal. Latency to MJ for each
of the seizures across 5 test days are observed in the top two histograms. Latency to generalized CT seizures for each seizure across
5 days are demonstrated in the middle two histograms. Percentage of animals exhibiting tonic extension for each seizure across 5
test days are demonstrated in the bottom two histograms. On P60 for mature animals, none of the animals exhibited tonic extension
with either the first or second seizure. * Represents statistical (P B0.05) difference to the first seizure on that day. c Represents
statistical (PB 0.05) difference to first/second seizure on the first day (P15 or P60).
196 P. Szot et al. / Epilepsy Research 46 (2001) 191–203
day. At the ninth seizure, 100% of the immature
animals exhibited tonic extension. In contrast, the
second seizure on each day was less likely to induce
tonic extension. The second seizure on each day
started to demonstrate an increase in tonic exten-
sion on the fourth day, reaching a maximum of 40%
of the animals on the fifth day with the tenth
seizure.
In adult animals, the latency to the first MJ of
the day, over the course of 5 days, declined grad-
ually, with the shortest latency observed on the fifth
day. However, the latency to the first CT seizure of
the day, across 5 days, did not change. For the first
3 days of testing, seizure threshold latency (both
MJ and CT) was significantly longer for the second
seizure of the day compared with the first seizure
latency. However, after the third day, the latencies
to MJ and CT were similar for the first and second
seizures of the day, suggesting that a refractoriness
induced by the first seizure of the day was lost.
Tonic extension in adults occurred in an increasing
percentage of animals over days 2– 4; a maximum
60% of animals exhibited tonic extension with the
eighth seizure.
3.2. c-Fos mRNA expression
3.2.1. Immature animals
The c-fos mRNA expression profile in the imma-
ture and mature animal, following either single or
repetitive seizures, varies across different brain
regions (Fig. 2). In the immature animal a single FL
seizure resulted in increased c-fos mRNA expres-
sion in the cingulate cortex, neocortex, septum and
striatum. In the hippocampus, the response to a
single FL was variable. Of the ten immature
animals that received a single FL seizure, six had
c-fos mRNA expression comparable to no seizure
controls, but the other four animals showed ele-
vated c-fos mRNA expression in the CA3 and
dentate gyrus (DG) (Fig. 3a, no seizure; compared
with b, single seizure).
Repetitive FL seizures in immature animals sig-
nificantly elevated c-fos mRNA expression in the
cingulate cortex, neocortex, septum, LC, VMH and
hippocampal CA3 and DG (Fig. 3a, no seizure; b,
single seizure; c, nine seizures; and d, ten seizures
in immature animal). The hippocampal CA1 region
did not show increased c-fos mRNA expression
(compared with control) for any seizure paradigm.
The expression of c-fos mRNA in the striatum, LC
and VMH was significantly different for repetitive
FL seizures versus single FL seizures. In the stria-
tum, c-fos mRNA expression was significantly
elevated with the single FL seizure, and similar to
control with repetitive FL seizures; thus there
appears to be a loss of c-fos mRNA expression (and
possibly activation) in the striatum with repetitive
FL seizures. In the LC, there was a correlation
between the number of seizures an animal experi-
enced and the level of c-fos mRNA expression;
c-fos mRNA expression was greatest with ten FL
seizures, lower with nine seizures and even less with
a single seizure. LC was the only region that showed
such a correlation of c-fos expression with the
number of seizures. In the VMH, both nine and ten
repetitive FL seizures increased c-fos mRNA ex-
pression versus control and single seizure; however,
the nine FL seizure group showed the greatest
increase in c-fos mRNA expression (Fig. 3a– d,
autoradiograms).
3.2.2. Mature animals
In the mature animals, a single FL seizure
increased c-fos mRNA expression in the cingulate
cortex, neocortex and striatum (Fig. 2). Unlike the
immature animals, none of the seven mature ani-
mals which had a single FL seizure showed expres-
sion of c-fos mRNA in any of the hippocampal
regions.
Repetitive FL seizures in the mature animals
increased c-fos mRNA expression in the cingulate
cortex, septum and the hippocampal CA1, CA3
and DG (Figs. 2 and 3e, no seizure; f, single seizure;
g, nine seizures; and h, ten seizures in mature
animals). The DG had the greatest change in c-fos
mRNA expression of any of the regions studied
(Fig. 3e–h, autoradiograms). The VMH and LC,
which in the immature animals had very large
changes in c-fos mRNA expression with repetitive
FL seizures, showed c-fos mRNA expression com-
parable to control levels in adult brains (Fig. 3e, h
for VMH).
3.2.3. VMH and c-fos mRNA
Expression of Fos protein in the VMH has been
been hypothesized to correlate to the expression
 P. Szot et al. / Epilepsy Research 46 (2001) 191–203 197

Fig. 2. c-Fos mRNA expression in immature and mature rats following no seizures (control), single seizure, nine seizures and ten
seizures. c-Fos mRNA expression was measured in the cingulate cortex (Cing. ctx), neocortex (Ctx), septum, striatum, VMH, LC
and hippocampal CA1, CA3 and DG. * Represents statistical (PB 0.05) difference compared with control. c Represents statistical
(PB 0.05) difference compared with a single seizure. a Represents statistical (P B0.05) difference between nine and ten repetitive
seizures.

of hindlimb seizures (i.e. tonic extension) (Samor-
iski et al., 1997a,b; Ferland and Applegate, 1998a).
Fig. 4 shows the level of c-fos mRNA expression
in animals exhibiting tonic extension with nine or
ten FL seizures. Immature animals sacrificed after
nine FL seizures all exhibited tonic extension, and
all expressed significant levels of c-fos mRNA in the
VMH. Immature animals sacrificed after ten FL
seizures also showed a significant increase in c-fos
mRNA expression in the VMH, but not to the
degree that was observed in the nine seizure group;
this increase was similar whether or not tonic
extension was observed. In adult animals, there was
no correlation between c-fos mRNA expression in
the VMH and tonic extension during seizures.
There was elevated c-fos mRNA expression in both
nine seizure and ten seizure groups with or without
tonic extension.
198 P. Szot et al. / Epilepsy Research 46 (2001) 191–203

3.3. 2 -Deoxyglucose (2DG) labeling
2DG labeling was determined in sections adja-
cent to those in which c-fos mRNA expression was
measured. Due to the brevity of the FL seizure,
2DG labeling in these animals reflects not only
changes in glucose use during the seizure, but also
(and perhaps primarily) during the postictal period.
Since the majority of time between the 2DG bolus
injection and animal sacrifice consisted of postictal
activity, we also examined a group of animals
(adults) that we sacrificed directly after their FL
generalized seizures (the 2DG bolus given 45 min
before or immediately before seizure).
Using the standard injection of 2DG-seizure
induction and then sacrificing 45 min later proto-
col, we found significant decreases in 2DG labeling
in immature animals following repetitive (nine or
ten) FL seizures compared with control and single
seizure animals; this change was found in most
regions of the brain (except the hippocampus) (Fig.
5, immature). A single seizure, however, had little
effect on glucose utilization. Adult animals showed
rather variable changes in 2DG labeling following
single or repetitive seizures. A significant decrease
in 2DG labeling was observed in the cortex and
thalamus (Fig. 5, mature). In animals where a
prolonged status epilepticus was generated (pilo-
carpine), there was a significant increase in 2DG
labeling in many brain regions.
4. Discussion
This study has focused on the consequences of
repetitive seizure activity in rat, and particularly
on the differences between immature and mature
animals. Our results clearly do not yield informa-
tion about any causal relationship between
seizures and c-fos mRNA expression (and/or glu-

Fig. 3. Autoradiograms of c-fos mRNA expression at the level of VMH and hippocampal CA1, CA3 and dentate gyrus (DG) in
immature (a – d) and mature (e –h) animals following no seizure (control) (a and e), single seizure (b and f), nine seizures (c and g)
and ten seizures (d and h).
 P. Szot et al. / Epilepsy Research 46 (2001) 191–203 199

1992; Stafstrom et al., 1992; Velisek et al., 1992;

Fig. 4. Correlation of c-fos mRNA expression in the VMH of
immature and mature rats to tonic extension following nine
FL seizures and ten FL seizures. With nine FL seizures:
+tonic, tonic extension occurring after the ninth seizure; and
−tonic, tonic extension did not occur after the ninth seizure.
With ten FL seizure: − /− tonic, tonic extension did not
occur following either the ninth or tenth seizure; + /− tonic,
tonic extension occurred following the ninth seizure, but not
following the tenth seizure and + /+ tonic, tonic extension
occurred following both the ninth and tenth seizure.
Weller and Mostofsky, 1995). These studies have
generally focused on sensitivity to a single, acutely-
induced seizure. We have, in contrast, examined
changes in responsiveness to a series of seizure-in-
ducing stimuli. Overall, both age groups tolerated
the repetitive seizure paradigm very well; however,
mortality in the immature animal occurred after the
first seizure, while mortality in the adults occurred
after several seizures (first death after the eighth
seizure) had been administered.
Enhanced seizure susceptibility of the immature
animal was apparent in our paradigm primarily in
the lower threshold latency of the second of two
daily seizure trials. That is, once the immature 12
animal experienced a seizure on a given day, it
was more vulnerable to the second stimulus.
Adult animals, in contrast, were more resistant to
the second daily seizure provoking stimulus (at
least for the first 3 of 5 days of testing). This

cose utilization). However, the results provide some

important insight into the consequences of repeti-
tive seizures in the immature animal. Using seizure
latency as a measure of threshold, and c-fos activa-
tion as a measure of neuronal activity, we found
rather different patterns of response in immature
versus mature brains. However, seizure-related glu-
cose utilization—2DG distribution — did not show
an increase in either the immature and mature
animal. 2DG distribution may not be a sensitive
enough technique to measure changes in the
metabolic demands of these brief FL seizure. Until
more adequate measures can be utilized, it is
unclear what differences in the metabolic demands
of repetitive seizures exist between the immature
and mature animals.

4.1. Seizure threshold and se6erity

Many studies have shown that immature animals
are extremely sensitive to many perturbations that
result in seizures (de Casrilevitz et al., 1971; Waster-
lain and Plum, 1973; Moshe et al., 1983; de Feo et
al., 1985; Cavalheiro et al., 1987; Sperber and
Moshe, 1988; Zouhar et al., 1989; Hirsch et al.,
Fig. 5. [14C]-2DG labeling in immature and mature animals
following no seizures (control), single seizure, nine seizures and
ten seizures. 2DG labeling was measured in the cingulate
cortex (Cing. ctx), neocortex (Ctx), striatum (STR), thalamus,
LC and hippocampus. * Represents statistical (PB 0.05) dif-
ference to control. c Represents statistical (PB0.05) differ-
ence to a single seizure.
200 P. Szot et al. / Epilepsy Research 46 (2001) 191–203
result suggests that the immature animals lack
refractoriness following a seizure—a feature also
observed by Holmes and Thompson (1987) with
electrical kindling. It should be noted that an
increase in seizure response with repetitive seizures
has not, however, always been observed in the
immature animal. Liu et al. (1999) noted an in-
crease in FL seizure latency in immature rats with
repeated (25 and 50) seizures; and Sarkisian et al.
(1997) saw an increase in kainate seizure latency in
immature animals, but no change in adult rats.
Interestingly, in our study the adult animals ap-
peared to lose refractoriness after the sixth seizure
(although they never showed an increased respon-
siveness to seizures as observed with the immature
animals).
Another indication of the greater susceptibility
of immature animals compared with adults with FL
stimuli, is their more intense seizure severity (i.e.
expression of tonic extension). All the immature
animals in our study showed tonic extension when
FL seizures were induced repetitively. This en-
hanced seizure response was observed mainly with
the first seizure of every day. Interestingly, there
was a lower incidence of tonic extension with the
second seizure, suggesting the influence of some
form of refractoriness in these immature animals,
perhaps localized to hindbrain regions where the
origins of tonic extension are thought to be local-
ized (Browning et al., 1993; Samoriski et al.,
1997a,b; Ferland and Applegate, 1998a). Adult
animals also exhibited tonic extension; however,
only a percentage of the animals, 60% maximum,
showed such a response on any given day. Samor-
iski and Applegate (1997) noted that with mice, as
the number of seizures the animal experienced was
increased, the percentage of animals which exhib-
ited tonic extension also increased. It appears then
that brief, recurrent FL seizures in both mice and
rats will result in increased seizure severity.
4.2. c-Fos mRNA expression and 2DG labeling
Behavioral data indicate that immature animals,
in contrast to adults, become more sensitive to
repeated convulsant stimuli. We, therefore, asked
whether there were changes in the distribution of
seizure activity in the brains of these rats. That is,
‘do repetitive seizures involve more/different brain
regions?’ and ‘is the seizure representation different
in immature versus mature rats?’ Expression of the
immediate early gene, c-fos, was used to determine
which regions in the brain were active during a
single seizure, or following repetitive seizures with
a 45-min post-seizure time point. Interestingly,
even following a single FL seizure, the immature
brain showed more regions expressing c-fos mRNA
than the mature brain; the ‘additional’ regions in
the immature animal included primarily the septum
and occasionally, the hippocampus. In the imma-
ture animal, a single FL seizure resulted in in-
creased c-fos mRNA expression in the
hippocampus (DG and CA3) in a percentage of the
animals, while a single FL seizure in adult animals
did not alter c-fos expression in any region of the
hippocampus in any animal. Repetitive seizures in
the immature animal recruited more regions than
seen after a single FL seizure; in particular the
VMH, LC, and hippocampal CA3 and dentate
gyrus region were activated in all animals with
repetitive seizures but not with the single seizure.
In addition, repetitive FL seizures in immature
animals resulted in more regions with elevated c-fos
mRNA expression than seen in the mature brains;
c-fos expression in the VMH and LC was not
evident in adult animals but clearly seen in imma-
ture animals. These data indicate that in the imma-
ture animal, in comparison to adult animals under
similar seizure conditions, more regions in the brain
are activated when single or repetitive FL seizures
are induced.
In addition to the wider distribution of c-fos
mRNA expression in the immature animal, the
intensity of labeling per region also appeared
greater in the immature animal than in the adult
(based on differences between control and seizure
groups for immature and mature animals). In the
immature rat, the regional change in c-fos mRNA
expression in the septum, striatum, VMH, LC and
hippocampal CA3 was greater than the changes
observed in the adult animal for comparable re-
gions. Indeed, the DG was the only region in the
adult animal that showed a large increase in c-fos
mRNA expression when compared with control
animals. Similar observations have been made in
studies of repetitive seizures in immature rats and
adult mice using a non-quantitative immunohisto-
 P. Szot et al. / Epilepsy Research 46 (2001) 191–203
chemical determination of Fos protein (Samoriski
et al., 1997a,b; Liu et al., 1999).
During repeated seizures there is a change in
seizure phenotype from clonic/tonic (‘forebrain’)
seizure to tonic extension (‘hindbrain’) seizures.
This behavioral pattern has been correlated by
other investigators to an increase in the expression
of the Fos protein in the VMH (Samoriski et al.,
1997a,b; Ferland and Applegate, 1998a). In our
study, both the immature and mature animals
showed a progression of seizure phenotype from
forebrain to hindbrain seizure. However, this pro-
gression of seizure phenotype was not always
associated with the induction of c-fos mRNA in the
VMH. For example, in response to the tenth FL
stimulus, some immature animals had tonic exten-
sion seizures and some did not; however, all the
immature animals showed c-fos mRNA expression
in the VMH. In adult animals, expression of c-fos
mRNA expression in the VMH did not correspond
with tonic extension at either the ninth or tenth
seizure.
In addition to observing cellular activity via c-fos
mRNA expression, we also measured 2DG labeling
as a reflection of the metabolic demands on neurons
in different brain regions. Previous studies have
shown that status epilepticus results in dramatically
increased 2DG labeling (Ben-Ari et al., 1981; White
and Prince, 1993; Handford and Ackermann,
1995). However, this measurement generally
reflects the metabolic demand of the brain during
very intense, prolonged seizure activity. In contrast,
FL seizures are very brief in duration. Given the
methodology for obtaining 2DG labeling, it is
reasonable to assume that 2DG pattern seen after
a FL seizure reflects primarily the metabolic de-
mands following the seizure, during the postictal
period. This factor probably explains the absence
of 2DG increases following a single seizure in both
mature and immature animals. Interestingly, fol-
lowing repeated FL seizures in both immature and
mature animals, a decrease in 2DG labeling was
observed. This decrease may, in fact, reflect a
significantly lower level of activity in many brain
regions during the period following multiple
seizures (posticital depression).
This study was designed to determine the effects
of repetitive seizures on behavior, neuronal activity
201
and metabolism in developing animals, and to
compare these changes to those observed in the
adult. Both seizure behavior and neuronal activity
pattern (c-fos expression) were different in imma-
ture compared with mature animals. 2DG method-
ology to measure metabolic activity was not
sufficiently sensitive to reveal distinct FL seizure-
related changes.
Acknowledgements
This work was supported by the University of
Washington Pediatric Epilepsy Research Center,
Department of Veterans Affairs (P.S.), and NIH
grant NS-18895 (P.A.S.).
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