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Epilepsia, 46(4):499–508, 2005
Blackwell Publishing, Inc.
C
2005 International League Against Epilepsy
A 5-Month Period of Epilepsy Impairs Spatial Memory, Decreases
Anxiety, but Spares Object Recognition in the Lithium-pilocarpine
Model in Adult Rats
∗Julien Detour, †Henri Schroeder, †Didier Desor, and ∗Astrid Nehlig
∗INSERM U398, Faculty of Medicine, Strasbourg; and †Laboratoire des Sciences Animales, INRA UC 12340, INPL-UHP,
Nancy, France
Summary: Purpose: In temporal lobe epilepsy (TLE), interictal
behavioral disorders affect patients’ quality of life. Therefore
we studied long-term behavioral impairments in the lithium-
pilocarpine (li-pilo) model of TLE.
Methods: Eleven li-pilo adult rats exhibiting spontaneous re-
current seizures (SRSs) during 5 months were compared with
11 li-saline rats. Spatial working memory was tested in a radial
arm maze (RAM), anxiety in an elevated plus-maze (EPM), and
nonspatial working memory in an object-recognition paradigm.
Neuronal loss was assessed on thionine brain sections after be-
havioral testing.
Results:Inthe RAM, the time to complete each session and
the number of errors per session decreased over a 5-day period
in li-saline rats but remained constant and significantly higher in
li-pilo rats. In the EPM, the number of entries in and time spent
on open arms were significantly higher in li-pilo than li-saline
rats. In the object-recognition task, the two groups exhibited
a comparable novelty preference for the new object. Neuronal
loss reached 47–90% in hilus, CA1, amygdala, and piriform and
entorhinal cortex.
Conclusions:Inli-pilo rats having experienced SRS for
5 months, performance in the object-recognition task is spared,
which suggests that object discrimination remains relatively in-
tact despite extensive damage. Neuronal loss in regions mediat-
ing memory and anxiety, such as hippocampus, entorhinal cor-
tex, and amygdala, may relate to impaired spatial orientation
and decreased anxiety. KeyWords: Temporal lobe epilepsy—
Lithium-pilocarpine—Spatial memory—Object recognition—
Anxiety—Neuronal damage.
It is generally agreed that patients with temporal lobe
epilepsy (TLE) are more prone to behavioral disorders
and cognitive impairments than is the general population
(1). These interictal impairments usually disrupt the pa-
tient’s everyday life, often more than the seizures them-
selves (2,3). Furthermore, depression or anxiety often de-
velops several years after epilepsy onset (2,4), and the
control of the seizures with pharmacologic treatment does
not always suppress these disorders (5). Among cognitive
impairments, memory problems are frequently observed
in patients with TLE. The most reliable observations are
deficits in declarative memory (ability to acquire facts and
events related to one’s personal past, 6) and in the perfor-
mance of visuospatial tasks (7–9). Moreover, long dura-
tion of refractory TLE seems to be associated with cogni-
tive deterioration (10–12). Thus understanding the neural
mechanism underlying these disturbances is an important
Accepted November 28, 2004.
Address correspondence and reprint requests to Dr. J. Detour at
INSERM U666, Clinique Psychiatrique, Hˆopitaux Universitaires, 1
place de l’hˆopital, BP426, 67091 Strasbourg Cedex, France. E-mail:
Julien.Detour@chru-strasbourg.fr
issue in the management of TLE. In animal models of
TLE, the molecular, lesional, and metabolic characteris-
tics have been quite extensively studied. Conversely, the
cognitive or behavioral validity of the models was less
studied, especially after a long-lasting period of sponta-
neous seizures.
The model of epilepsy induced in rats by pilocarpine
alone or associated with lithium reproduces most clin-
ical and neuropathologic features of human TLE (13–
17). In adult rats, the injection of lithium and pilocarpine
(li-pilo) leads to status epilepticus (SE) followed by a la-
tent seizure-free period of a mean duration of ∼3 weeks,
after which all animals exhibit spontaneous recurrent
seizures (SRSs) that last for their whole life (chronic pe-
riod). During the latent period, neuronal loss, mossy fiber
sprouting, gliosis, and synaptic reorganization participate
in the constitution of a hyperexcitable circuit that underlies
the occurrence of SRSs. Neuronal loss is located mainly in
hippocampus, parahippocampal cortices, amygdala, and
thalamus. Most of these structures are involved in be-
haviors like memory and anxiety (18,19). The behavioral
consequences of TLE cannot be solved by the traditional
499
500 J. DETOUR ET AL.
approach that usually disrupts one or two regions of in-
terest at the most. However, the lesions induced by li-pilo
SE that represent multifocal brain damage resembling the
neuropathology of human TLE can be used as an ade-
quate tool for the understanding of interictal behavioral
disorders in patients with TLE (17).
Previous studies on the behavioral consequences of
SRSs consecutive to pilocarpine- or kainate-induced SE
have concentrated mostly on memory. Thus working
memory appears impaired in the Morris water maze dur-
ing the latent period (20), and spatial working memory as-
sessed in a radial arm maze is impaired during the chronic
period (17,21–24). In the present work, we evaluated the
long-term behavioral consequences of li-pilo SE induced
in adult rats. The main difference with previous studies
is that behavioral performance was assessed after a quite
long period of epilepsy (i.e., 5 months of SRSs). To evalu-
ate spatial working memory, we used the eight-arm maze
(25). Deleterious effects of hippocampal lesions in learn-
ing and spatial memory have been reported in this task
(18,26,27). We also used two other tasks that involve be-
haviors mediated by structures quite vulnerable to li-pilo
SE, like the parahippocampal cortices or amygdala: the
elevated-plus maze and an object-recognition task. The
elevated-plus maze is designed to assess the level of anxi-
ety in rodents (28), which depends partly on the amygdala
(19). The last test was a nonspatial object-recognition task.
Recognition memory is generally regarded as the ability
to discriminate the familiarity of things previously en-
countered (29), which is critical for everyday life. This
behavior, which seems to depend mostly on structures out-
side of the hippocampus, mainly perirhinal and entorhinal
cortices (for review, see refs. 29 and 30), has never been
explored in the li-pilo model. In addition, the extent of
neuronal loss was assessed in the regions of interest of the
same rats.
MATERIALS AND METHODS
Animals
In total, 58 adult Sprague–Dawley rats (Janvier Breed-
ing Center, Le Genest-St-Isle, France) weighing 300–
350 g were used for the study. All rats where housed in
quiet, uncrowded facilities in a room maintained at 21–
22◦Cona12/12-h light/dark cycle (lights on at 7.00 a.m.)
with food and water available ad libitum. Rats were main-
tained in groups of four animals before the pilocarpine
protocol and were then housed individually for the re-
mainder of the experiment. Males only were used for the
experiments to eliminate confounding effects of variable
estrogen levels on neuronal excitability. All animal exper-
imentation was performed in accordance with the rules
of EC Council Directive (86/69/EEC) of November 24,
1986, and the French Department of Agriculture (license
no. 67–97). All efforts were made to minimize animal
suffering.
Pilocarpine protocol
Animals were randomly divided into an experimental
(li-pilo group, n =41) and a control group (li-saline group,
n=17). All rats received lithium chloride (3 mEq/kg;
Sigma, St. Louis, MO, U.S.A.) intraperitoneally. Then,
20 h later, the li-pilo group received 1 mg/kg methylscopo-
lamine bromide (s.c., Sigma) to limit the peripheral ef-
fects of the convulsant. SE was induced 30 min later by
the subcutaneous injection of pilocarpine hydrochloride
(25 mg/kg, Sigma). Diazepam (2.5 mg/kg; Valium, Roche,
Meylan, France) was injected i.m.2hafter SE induc-
tion and4hlater to improve survival. The li-saline group
received methylscopolamine and saline instead of pilo-
carpine. All animals were aged 90 days at the time of SE
induction.
The occurrence of SRSs was observed in the li-pilo
group starting from day 14 after the pilocarpine injec-
tion. The observation period was conducted daily from
9.00 a.m. to 6.00 p.m. All rats were observed until the
occurrence of at least one seizure.
Behavioral testing
Among the 21 epileptic rats that survived SE, the 11
li-pilo rats that exhibited the most frequent SRSs were
selected for behavioral studies and compared with 11 li-
saline rats randomly selected within the 17 available. Be-
havioral performance and neuronal loss were assessed at
∼5 months after SE. To test anxiety, spatial working mem-
ory, and novelty discrimination, all rats were tested in the
elevated-plus maze, the eight-arm maze, and an object-
recognition task, respectively, over a period of ∼1 month.
Three weeks before the first behavioral testing, rats were
housed under an inverse 12/12h dark/light cycle (lights off
at 7.00 a.m.). All behavioral testing was performed ≥3h
after the last motor seizure, and no spontaneous seizure
occurred in any rat during behavioral testing.
The elevated-plus maze
The elevated-plus maze is a validated test that evaluates
anxiety in rodents (28). The apparatus was totally made of
transparent Plexiglas. It comprises two open arms (50 ×
10 cm), two enclosed arms (10 ×40 ×50 cm), and a
central platform (10 ×10 cm). The configuration has the
shape of a plus sign, and the apparatus is elevated 50 cm
above the floor level. Grip on the open arms is facilitated
by inclusion of a small edge (0.5 cm high) around their
perimeter. Fortesting, rats were brought to the room2hbe-
fore the test and were tested individually. Before each trial,
the maze was cleaned thoroughly with a 30% ethanol so-
lution. At the beginning of the test, rats were placed on the
central platform always facing the same open arm. The test
lasted 5 min in standard laboratory conditions under red
light (2 ×60 W). The testing device was video-recorded,
and the experimenter supervised the test in an adjacent
room. Videotapes were scored by a na¨ıve trained observer
using a software developed in our laboratory. Behaviors
Epilepsia, Vol. 46, No. 4, 2005
LONG-LASTING EPILEPSY AND BEHAVIOR IN ADULT RATS 501
were encoded afterward directly on a PC keyboard. Data
were then transferred for statistical analysis.
Activity and anxiety-related behaviors were assessed.
Standard measures comprised: the total number of arm
entries (arm entry defined as all four paws entering an
arm), the number of open- and closed-arm entries, and
the time spent in different sections of the maze (open and
closed arms and central platform). In addition to conven-
tional measures, three specific behavioral measures were
recorded: rearing frequency and duration, head-dipping
frequency (exploratory movement of head/shoulders over
the sides of the maze), grooming frequency and duration
(typical sequences beginning with snout, progressing to
ears, and ending with whole-body groom).
The eight-arm maze
Rats were tested in the eight-arm maze according to the
procedure originally proposed by Olton and Samuelson
(25) and described elsewhere (31). The animals were food
deprived to 85% of their body weight before testing and
maintained at that weight during the 5 days of testing. The
apparatus was a wooden gray, enclosed eight-arm radial
maze with walls and entirely covered with a transparent
top. Each of the arms (60 ×12 ×17 cm) projected from
one side of an octagonal center measuring 50 cm in diam-
eter. One food pellet (45 mg) was positioned at the far end
of each arm. Before each trial, every arm of the maze was
baited with a food pellet. Reinforcement was not replaced
during the test. The whole apparatus was video-recorded
from above under red light (2 ×60 W). At the beginning
of the test, a white plastic cylinder (45 cm in diameter) was
used to place the rats in the central platform. The cylinder
was taken back, and animals were left in the maze until
they had either entered all eight arms or until 15 min had
elapsed, whichever occurred first. Placing all four paws
inside an arm was recorded as an arm entrance. Times of
arm entrances, the identity of each arm entered, and the
serial order were recorded. All rats were tested for 5 days
(one test per day).
The object-recognition task
Apparatus. The object-recognition task is a nonre-
warded paradigm based on the spontaneous exploratory
behavior of rats, which implies working memory. The test
apparatus consisted of a fenced cage (50 ×40 ×30 cm)
with a front door. Three objects were fixed on one side
(25 cm above the floor) and could not be displaced by the
rats. Distance between objects and walls was equal. Two
of the three objects were made up of wood and were circu-
lar (diameter, 40 mm). The third one was made of plastic
and had roughly the same aspect but was denticulate and
smaller (diameter, 25 mm). The two types of objects had
the same color. The objects had no genuine significance
for rats and had never been associated with reinforcement.
A video camera was mounted opposite to the cage and was
used to record performance.
Procedure. The object-recognition task was performed
as follows. On the first day, rats were placed in the cage
and were allowed to move freely for 5 min; the three ob-
jects were identical. Rats were then put back to their home
cage. The same procedure was repeated on 3 consecutive
days (one session per day). On the fourth day, the familiar
object on the right was removed and replaced by the new
denticulate object. This paradigm was chosen because of
expected working-memory impairment in the li-pilo group
(17) and to allow a habituation period (the environment of
the fenced cage) before the recognition-testing day. Thus
the intertrial interval was 24 h. To avoid the presence of
olfactory trails, the apparatus and the objects were thor-
oughly cleaned with a 30% ethanol solution.
Video tapes were scored afterward by a na¨ıve trained
observer. Behaviors were encoded directly on a PC key-
board. The screen was divided into three zones, each zone
being centered on one object. The following behaviors
were then scored during every session: the time spent in
each zone and the number of transitions between each
zone. The median number and median duration of each
period of object sniffing in each zone (sniffing an object
was defined as follows: directing the nose to the object at a
distance ≤2cmand/or touching it with the nose) also were
rated. In addition, the cumulative median time spent sniff-
ing each object and the total median time spent sniffing all
objects was calculated. The median number and duration
of rearing and grooming in each zone were recorded.
Neuropathologic studies
Long-term pathologic changes were assessed in all the
li-saline (n =11) and li-pilo (n =11) rats subjected to
behavioral testing. Brains were removed 5 days after the
last behavioral testing. They were rapidly removed and
frozen in isopentane chilled to –25◦C and cut into 20-µm
coronal sections. Quantification of cell density was per-
formed with a 10 ×10 box, 1-cm2microscopic grid on
coronal sections stained with cresyl violet. The grid of
counting was placed on a well-defined area of the cere-
bral structure of interest, and counting was carried out
with a microscopic enlargement of 200- or 400-fold, de-
fined for each single cerebral structure. Cell counts were
performed bilaterally in three adjacent sections for each re-
gion by a single observer unaware of the animal treatment.
The number of cells obtained in the six counted fields in
each cerebral structure was averaged. This procedure was
used to minimize the potential errors that could result from
double counting, leading to overestimation of cell num-
bers. Neurons touching the inferior and right edges of the
grid were not counted. Counts involved only neurons with
cell bodies larger than 10 µm. Cells with small cell bod-
ies were considered glial cells and were not counted. This
method has been described elsewhere (32).
As we performed in all our previous studies concern-
ing neuronal damage induced by li-pilo SE (32,33), brain
Epilepsia, Vol. 46, No. 4, 2005
502 J. DETOUR ET AL.
sections were selected at three different levels [i.e., (a) the
lateral thalamus and amygdala, (b) dorsal hippocampus
and piriform cortex, and (c) ventral hippocampus and en-
torhinal cortex]. The anteroposterior level of the sections
was selected according to stereotaxic coordinates of the rat
brain atlas of Paxinos and Watson (34) and were –3.30 mm
from bregma for the mediodorsal and lateral thalamus and
medial and basolateral amygdala, –4.30 mm from bregma
for the dorsal hippocampus (CA1, CA3 subfields, and the
hilus of the dentate gyrus and piriform cortex), and –5.20
mm from bregma for ventral hippocampus and entorhinal
cortex).
Statistical analysis
Most behavioral data did not follow gaussian distribu-
tion, and variances were not equal. Therefore we used
medians and inferior and superior quartiles as statistical
variables of central tendency and dispersion and nonpara-
metric statistical analysis that is more powerful than para-
metric statistics in that case (35). Data from the li-pilo
group were compared with those from the li-saline group
by using a Mann–Whitney Utest; a Friedman test for
within-groups analyses, followed by a post hoc modified
Mann–Whitney Utest also was applied when adapted.
Neuronal damage was expressed as the percentage of re-
maining neurons compared with control levels ±SEM.
The significance of the differences observed between the
li-pilo and li-saline groups was evaluated by using an anal-
ysis of variance followed by a Scheff´e’s ttest for multiple
comparisons.
RESULTS
Characteristics of lithium-pilocarpine–induced status
epilepticus in adult rats
Control animals receiving lithium and saline did not ex-
hibit any behavioral alteration. Rats subjected to lithium
and pilocarpine developed the characteristic features of
SE, which started ∼50 min after pilocarpine injection, as
previously described (33,36,37). Among the 41 rats sub-
jected to li-pilo, 38 (93%) rats developed SE; 18 rats died
within the first 48 h after SE (13 between 2 and 6 h after
onset of SE, five during the following night), and two rats
died a few days after SE. Thus 21 (55%) of 38 rats survived
SE. Within the 38 rats that developed SE, the mean latency
to the first 4/5 stage seizure was 26.3 ±6.8 min (mean
±SD), the latency to the onset of SE reached a mean
value of 54.7 ±8.1 min, and the mean duration between
the first 4/5 stage seizure and SE was 27.7 ±10.1 min.
Among the 21 rats that survived li-pilo SE, 16 developed
SRS, whose severity matched stages 4/5 of kindling, with
a mean latency of 25 ±7 days.
Behavioral studies
Elevated-plus maze
Three li-pilo–treated rats jumped from the apparatus
after 20, 57, and 81 s, respectively, and were not included
in the final analysis, which comprised eight li-pilo and 11
li-saline rats.
The total number of arm entries was significantly higher
in the li-pilo group than in the li-saline group (U =10.5, p
=0.004; Fig. 1A). This high locomotor activity was rather
oriented toward open arms because the number of closed-
arms entries was not different between the two groups
(U =32.5, p =0.351; Fig. 1A), whereas the number of
entries was significantly increased for open arms in the li-
pilo group compared with the li-saline animals (U =4.5,
p<0.001; Fig. 1A). The time spent on open and closed
arms was significantly higher and lower, respectively, in
li-pilo compared with li-saline rats (U =8, p =0.002 and
U=2, p <0.001 for open and closed arms, respectively;
Fig. 1B). The number of rearings was higher in the li-
saline group (U =9, p =0.003; Table 1), but their median
duration was similar in the two groups (U =24.5, p =
0.109; Table 1). The li-pilo–treated rats made far more
head-dips than li-saline rats (U =1.5, p <0.001; Table 1).
A - Number of arm entries
0
10
20
30
40
50
60
70
80
Li-saline Li-pilo Li-saline Li-pilo Li-saline Li-pilo
TOTAL ENTRIES OPEN ENTRIES CLOSED ENTRIES
Number of entries
**
***
B - Time spent on arms
0
50
100
150
200
250
300
350
Li-saline Li-pilo Li-saline Li-pilo
OPEN ARMS CLOSED ARMS
Time (s)
**
***
FIG. 1. Effect of li-pilo SE induced in adult rats on open and
closed arm entries (A) and time spent on arms (B) in the elevated
plus maze. Values represent medians, first and third quartiles,
first and ninth deciles (bars), and extreme values (points)ofeach
measure of 11 control rats and eight epileptic rats. ∗∗p<0.01;
∗∗∗p<0.001; statistically significant differences between li-saline
and li-pilo rats.
Epilepsia, Vol. 46, No. 4, 2005
LONG-LASTING EPILEPSY AND BEHAVIOR IN ADULT RATS 503
TABLE 1. Effect of li-pilo SE induced in adult rats on specific
behaviors in the elevated plus-maze
Li-saline Li-pilo
Variable group (n =11) group (n =8)
Number of rearings 18 (14.5–20.5) 7 (5–9.25)a
Number of head-dips 8 (3–9) 24 (20.75–25.75)b
Number of groomings 2 (1.5–3.5) 0 (0–0.25)b
Rearing duration 1.6 (1.32–2.15) 1.1 (0.82–1.68)
Grooming duration 4 (3.17–6.14) 0 (0–0.58)b
Values represent median number or median duration (s), with quartiles
in parentheses.
ap<0.01, bp<0.001: statistically significant differences between
li-pilo and li-saline rats.
The li-pilo group did not show any grooming behavior
during the test, conversely to li-saline rats, which led to a
highly significant difference between the two groups (U
test, number and duration, U =9.5, p =0.003 and U =7,
p=0.001, respectively; Table 1).
The eight-arm maze
In the li-saline group, the total time necessary to enter
all eight arms of the maze decreased over the 5 days of
testing from 192 to 73 s (χ2,F(4,11) =25.81, p <0.001;
Fig. 2A). As expected, this group learned the task since
the number of errors and the number of arms visited per
session decreased (χ2,F(4,11) =18.50, p =0.001; Fig. 2B
and C), and the number of arms visited per min increased
over the 5 days (χ2F(4,11) =24.51, p <0.001; Fig. 2D).
A - Total time per session
0
50
100
150
200
250
12345
Session
Time (s)
Li-salin e
Li-pilo
*** ** *** ***
B -Total number of errors per
session
0
2
4
6
8
10
12345
Session
Number of arms
Li-saline
Li-pilo
*
**
*****
C - Total number of arms visited per
session
0
5
10
15
20
12345
Session
Number of arms
Li-saline
Li-pilo
**
*****
*
D -Total number of arms visited
per min
0
2
4
6
8
10
12345
Session
Number of arms
Li-saline
Li-pilo
*** **
FIG. 2. Effects of li-pilo SE induced in
adults rats on the behavior in the eight-arm
maze. Data represent the medians of 11
control rats and 11 epileptic rats. ∗p<0.05;
∗∗p<0.01; ∗∗∗ p<0.001; statistically sig-
nificant differences between li-saline and
li-pilo rats.
Conversely, the li-pilo group took approximately the same
time to complete the task during the five sessions (171–
230 s; χ2,F(4,11) =2.50, p =0.644; Fig. 2A). The total
number of errors per session, the total number of arms
visited per session, and the total number of arms visited
per minute did not significantly differ between sessions
(χ2,F(4,11) =3.00, p =0.557, χ2,F(4,11) =3.00, p =
0.557; and χ2,F(4,11) =5.75, p =0.219, respectively;
Figs. 2B-D).
Except for the first day, the total time per session was
significantly higher in li-pilo compared with li-saline rats
(7.10−5<p<0.008; Fig. 2A). The total number of errors
per session and the total number of arms visited per session
were significantly higher in the li-pilo than in the li-saline
group for each session (5.10−4<p<0.044; Figs. 2B and
C). The median number of arms entered per minute was 5.8
for the li-pilo group and 3.9 for the li-saline group during
the first session; this number was significantly higher in
li-pilo than in li-saline rats during sessions 2, 4, and 5
(0.001 <p<0.016; Fig. 2D).
The object-recognition task
The total time spent sniffing all three objects was con-
stant during the four sessions in the two groups (χ2F(3,11)
=0.59, p =0.901 and χ2,F(3,11) =1.80, p =0.615 in
li-saline and li-pilo rats, respectively). It was lower for
the central object compared with the lateral ones. The
time spent sniffing all objects and the number of sniff-
ing sequences of all objects (data not shown) were never
Epilepsia, Vol. 46, No. 4, 2005
504 J. DETOUR ET AL.
significantly different between the two groups in any of
the four sessions. When taking each object individually,
no difference in the time spent sniffing the central ob-
ject was observed between the two groups (Fig. 3B). The
same was observed for the left object except for session
3 (Fig. 3A), during which li-pilo rats spent significantly
more time than li-saline rats sniffing this left object (U =
29.5; p =0.040). The total time spent sniffing the right
object during the first three sessions ranged from 11.0 to
11.8 s for li-saline and from 10.3 to 15.4 s for li-pilo rats
(Fig. 3C). On day 4, when the novel (right) object was
introduced, the two groups exhibited a longer time sniff-
ing this object (18.9 and 24.6 s, respectively, for li-saline
and li-pilo rats). The time spent sniffing this new object
showed a statistically significant variation over the four
sessions in the li-pilo group (χ2,F(3,11) =9.44, p =0.024
and χ2,F(3,11) =5.86, p =0.119, respectively, for li-pilo
and li-saline groups). In this group, session 4 was signif-
icantly different from sessions 1–3 (0.004 <p<0.047).
However, in the two groups, a comparable novelty prefer-
ence could be observed, reflected by the median duration
of each sniffing sequence for the right object, which was
higher during session 4 compared with sessions 1–3 (0.001
<p<0.023 and 0.001 <p<0.016 for li-saline and li-pilo
groups, respectively; data not shown). The total time spent
sniffing the new object during session four (Fig. 3C) as the
total number of sniffing behaviors (data not shown) during
this session were not significantly different between the
li-pilo and li-saline group (U =49, p =0.478 and U =
39, p =0.171, respectively).
Concerning locomotor activity, the number of tran-
sitions between the three delimited zones of the cage
(Fig. 4A) remained unchanged during the four sessions in
both groups (χ2,F(3,11) =1.51, p =0.679 for the li-pilo
group and χ2,F(3,11) =5.68, p =0.127 for the li-saline
group). However, the level of transitions was lower in the
li-saline than in the li-pilo group. This difference was sta-
tistically significant for all sessions (U =12, p <0.01
for the first session; U <5, p <0.001 for sessions 2–4).
The total time spent rearing (Fig. 4B) and the total number
of rearings (data not shown) were similar in both groups
and remained constant within each group over the four
sessions. Finally, grooming activity was totally absent in
epileptic rats and 9–24 times lower than in li-saline rats.
The difference between the two groups was statistically
significant in each session (2.10−6<p<0.005, Fig. 4C).
Histologic observations and neuronal loss
In the present study performed on cresyl-violet stained
sections taken from rats having experienced 5 months of
epilepsy (Fig. 5), two structures showed intense or to-
tal neuronal loss either with no tissue replacement (hole)
or with swollen tissue replacement consisting mainly of
filaments and glial cells bodies; these were the piriform
cortex (70% and 90% cell loss in layers II and III–IV, re-
A - Time spent sniffing the left
object
0
5
10
15
20
25
30
1234
Session
Time (s)
Li-saline
Li-pilo
*
B - Time spent sniffing the central
object
0
5
10
15
20
25
30
1234
Session
Time (s)
Li-saline
Li-pilo
C - Time spent sniffing the right
object
0
5
10
15
20
25
30
1234
Session
Time (s)
Li-saline
Li-pilo
FIG. 3. Effects of li-pilo SE induced in adults rats on sniffing be-
haviors in an object-recognition task. Values represent the median
time of sniffing the left (A), central (B), and right (C) object of 11
control rats and 11 epileptic rats. ∗p<0.05; statistically significant
differences between li-saline and li-pilo rats.
Epilepsia, Vol. 46, No. 4, 2005
LONG-LASTING EPILEPSY AND BEHAVIOR IN ADULT RATS 505
A - Number of transitions
0
20
40
60
80
1234
Session
Li-saline
Li-pilo
** *** *** ***
B - Total time of rearing
0
10
20
30
40
1234
Session
Time (s)
Li-saline
Li-pilo
C - Total time of grooming
0
10
20
30
1234
Session
Time (s)
Li-saline
Li-pilo
**
*** **
**
FIG. 4. Effects of li-pilo SE induced in adults rats on three be-
havioral measurements in an object-recognition task. Values rep-
resent medians of the number of transitions between each zone
of the maze (A), total time of rearing (B), and total time spent
grooming (C) of 11 control rats and 11 epileptic rats. ∗∗p<0.01;
∗∗∗p<0.001; statistically significant differences between li-saline
and li-pilo rats.
spectively) and the entorhinal cortex (∼50% loss in layers
II and III–IV). The amygdala underwent a marked loss
of neurons (70% in the basolateral nucleus, 47% in the
medial nucleus). Neuronal loss occurred also in the hip-
pocampus, 53% in the hilus, ∼30% in pyramidal cell layer
CA3 and 67% in CA1. Neuronal dropout reached 58% in
the lateral thalamus with more shrunken neurons (Fig. 5).
Neuronal loss was statistically significant in every struc-
ture analyzed (at least p <0.01).
DISCUSSION
The data of the present study represent the first report of
the behavioral consequences of SRSs lasting for a period
as long as 5 months. In accordance with previous stud-
ies, spatial learning was severely impaired: animals were
unable to learn the eight-arm maze. Avoidance for open
arms in the elevated-plus maze was abolished. Nonethe-
less, li-pilo–treated rats performed well during the object-
recognition task. The most damaged areas were the hip-
pocampal CA1 area, the hilus, piriform and entorhinal
cortices, and the amygdala.
Spatial memory
As reported in previous studies performed a few weeks
after the occurrence of SRSs induced by pilo or li-pilo SE
(17,22,24), the present data confirm that rats subjected to
li-pilo SE and experiencing a 5-month duration of epilepsy
are unable to learn the task in the radial-arm maze. The
same types of results were reported in rats rendered epilep-
tic by KA-induced SE (21,23,38). In the present study,
no improvement of the performance of epileptic rats was
noted along the 5 days of testing, neither for total time
nor for total number of errors per session. The absence of
learning and goal-oriented behavior also was reflected by
a higher number of arms visited per session in the li-pilo
than in the li-saline group. This difference is indicative of
lower performance and poor strategy, and directly in line
with the longer time needed by li-pilo compared with li-
saline rats to achieve the task. In this maze, li-pilo rats did
not appear hyperactive. These data indicate that spatial
working memory was adversely affected, as previously
reported (17).
The hippocampus has been reported to be prominently
involved in spatial working memory (18,26,27,39). CA1
and CA3 pyramidal neurons as well as intact connections
between these two regions appear critical in learning and
retrieval of spatial memory (40,41). Indeed, neurons from
layers II and III of the entorhinal cortex provide the en-
torhinal input to the hippocampus (42), and numerous
studies report a prominent role of the entorhinal cortex in
memory (for review, see ref. 43). The combination of le-
sions of the hippocampus and entorhinal cortex, observed
in li-pilo rats, causes impairment of learning and retention
(44), which may support the poor performance of these rats
in the radial-arm maze in the present study.
Compared with previous neuropathologic data from our
laboratory collected after a 2-month period of SRSs (33), a
3 months longer duration of epilepsy significantly worsens
neuronal loss in the CA1 subfield of the hippocampus and
layer II of the entorhinal cortex. Hence, it appears possi-
ble that not only SE but also SRSs, which both differently
contribute to neuronal loss, might contribute to behavioral
Epilepsia, Vol. 46, No. 4, 2005
506 J. DETOUR ET AL.
Neuronal damage in li-pilo rats 5 months after SE
0
20
40
60
80
100
HILUS CA1 CA3 BLA
AMY
Me AMY LAT
THAL
MD
THAL
PIR II PIR III-IV ENT II ENT III-IV
% of control
***
*** *** ***
***
***
***
**
** **
*
FIG. 5. Number of neurons remaining in
selected areas of adult rats that under-
went li-pilo SE as adults and studied 5
months after SE (n =11). Values, ex-
pressed as percentage of control levels (n
=11), represent mean ±SEM. ∗p<0.05;
∗∗p<0.01; ∗∗∗ p<0.001; statistically sig-
nificant differences between the li-pilo and
control group. HILUS, hilus of the dentate
gyrus; CA1, CA3 pyramidal cell layers of
the hippocampus; BLA AMY, basolateral
amygdala; Me AMY, medial amygdala; LAT
AMY: lateral amygdala; LAT THAL: lateral
thalamus; MD THAL: mediodorsal thala-
mus; PIR II, layer II of the piriform cortex;
PIR III–IV, layers III–IV of the piriform cor-
tex; ENT II, layer II of the entorhinal cortex;
ENT III–IV, layers III–IV of the entorhinal
cortex.
impairments in the li-pilo model of TLE. In the Morris
Water Maze task, impaired learning appears as early as 10
days after KA-induced SE, which suggests that the long-
term deficits of KA seizures are due to SE rather than to
SRSs (45). Conversely, in the li-pilo model, the latency
to reach the platform in the Morris Water Maze increases
with the occurrence of SRSs (20). Thus impaired spatial
learning may be the result of both SE-induced damage
and SRSs. It seems that in both humans (46) and animals
(47), the deterioration of cognitive functions could relate
to the duration of the epilepsy. In humans, the association
between the severity of structural damage and the duration
of epilepsy, and lifetime number of seizures, was reported
in both hippocampus (48) and entorhinal cortex (49). Fur-
ther studies testing the performance of rats along the time
course of the chronic period would be necessary to clarify
this issue.
Anxiety
The present data represent the first report on the con-
sequences of li-pilo–induced SE and epilepsy on the per-
formance of rats in the elevated-plus maze. In a recent
study, Kubova et al. (50) reported that all rats that they
subjected to li-pilo SE as adults jumped off the maze and
did not perform the task. In the present study, only three
of 11 rats jumped off the maze, whereas the other eight
completed the task. This difference could be linked to the
time at which performance was assessed, 3 months af-
ter SE in the study by Kubova et al. (50) and 5 months
in the present study. In the elevated-plus maze, li-pilo–
treated rats showed increased overall activity reflected by
a higher number of entries in open arms, whereas entries
in closed arms remained unchanged. The open-arm ac-
tivity is usually taken as the measure of the anxiety level
(28). Moreover, the median number of head-dippings and
rearings was higher in the li-pilo–treated group, which
rather reflects disinhibited hyperactive behavior (51). Our
results are in line with data obtained after a ventral le-
sion of the hippocampus, which potentially mediates the
unconditioned fear response resulting from the exposure
to a threatening situation, such as the elevated-plus maze
(52). In the present model of TLE, lesions are as extensive
in the ventral as in the dorsal hippocampus (53,54), and
large lesions also are observed in numerous amygdala nu-
clei, mainly the basolateral, and in the entorhinal cortex
(32,33,36,54, the present study). The ventral part of the
hippocampus and the entorhinal cortex have bidirectional
connections with the amygdala (55), which suggests that
these structures may organize fear expression as a single
integrated system (52). The disruption of these networks
in li-pilo–treated rats might give rise to a misevaluation of
threatening situations, which could in turn reduce anxiety
and/or enhance impulsive inadapted behavior.
Object recognition
To our knowledge, no published data concern lesional
animal models of TLE and the object-recognition task.
In this task, li-pilo rats recognized the novel object as
well as li-saline rats did, as reflected by similar total time
spent sniffing the novel object. As in the elevated-plus
maze, increased locomotor activity was observed in li-pilo
rats, as shown by a higher number of transitions between
zones. Grooming was almost abolished in li-pilo rats, as
previously reported (50), but all other behavioral variables
were identical in both groups. These data suggest a similar
habituation in the two groups.
Our data are in accordance with previous articles that
support spared object-discrimination capacities after hip-
pocampal damage (29,30,56). Moreover, when taking into
account the extent of the lesions in the entorhinal and pir-
iform cortices, our study supports the view that simple
object discrimination might be independent of those struc-
tures. In rodents, object discrimination has been shown to
rely on visual and olfactory cues (57). In this task, even if
objects were thoroughly cleaned after each trial, the new
object was made up of a different matter (plastic instead
Epilepsia, Vol. 46, No. 4, 2005
LONG-LASTING EPILEPSY AND BEHAVIOR IN ADULT RATS 507
of wood), which might provide an olfactory cue. The ol-
factory bulb presents reciprocal connections with the en-
torhinal (58) and the piriform cortex (59). In the li-pilo
and pilo models of TLE, the whole olfactory system is
severely damaged, at the level of the piriform cortex and
the anterior olfactory nuclei (14,36), which might disrupt
olfactory skills. Alternately, there could be visual compen-
sation via the spared perirhinal cortex (60) that has been
proposed to be critically involved in visual-recognition
memory (61,62) and may be sufficient to solve this sim-
ple task.
Finally, our task design might not rely entirely on sim-
ple object discrimination and pure working memory. Ac-
cording to reported impaired spatial learning abilities in
li-pilo treated rats, we chose to change the right object
after the third session of habituation. This could lead to
overlearning of environmental cues and contamination of
the task by reference memory. To try to prevent this phe-
nomenon, we chose a 24-h intertrial delay because it was
previously shown that this duration made discrimination
more difficult for control rats (63).
In conclusion, the present study shows behavioral
deficits occurring after 5 months of epilepsy in the li-pilo
model of TLE. The new finding of this study concerns
the normal performance of epileptic rats in object dis-
crimination, which suggests that such abilities do not en-
tirely relate on hippocampal integrity. Neuronal loss was
prominent in regions such as the hippocampus, entorhi-
nal cortex, and amygdala, which is in accordance with
severe learning impairment in the eight-arm maze and
disinhibited behavior in the elevated-plus maze. In ad-
dition, rats were quite hyperactive, which reflects a lack
of goal-oriented activity. The present data confirm that the
li-pilo model of TLE represents a useful tool to explore be-
havioral disorders associated with the disease as well as
potential pharmacologic treatments to prevent cognitive
decline.
Acknowledgment: This work was supported by the Institut
National de la Sant´eetdelaRecherche M´edicale (U 398) and
the Fondation pour la Recherche M´edicale. We thank E. Koning
and A. Ferrandon for friendly, professional, and helpful technical
assistance.
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