Mode-dependent effect of high-frequency electrical stimulation of the anterior thalamic nucleus on amygdala-kindled seizures in rats.

Abstract

Deep brain stimulation (DBS) is an emerging treatment of epilepsy. Anterior nucleus of the thalamus (ANT) is considered to be an attractive target due to its close connection to the limbic structures and wide regions of neocortex. The present study aimed to investigate the effects of high frequency stimulation (HFS) targeting the ANT on amygdala-kindled seizures in Wistar rats in two different stimulation modes i.e. pre-treatment and post-treatment stimulations, mimicking the scheduled and responsive stimulations in clinical use respectively. When fully-kindled seizures were achieved by daily amygdala kindling (1 s train of 1 ms pulses at 60 Hz), HFS (15 min train of 100 μs pulses at 150 Hz and 450-800 μA) was applied in two modes for 10 days. Bilateral post-treatment with HFS reduced the incidence of generalized seizures and the mean behavioral seizure stage and shortened average afterdischarge duration (ADD) and generalized seizure duration (GSD), while bilateral pre-treatment with HFS resulted in a similar but much weaker inhibition of seizures. On the other hand, we also found the two stimulation modes both increased the afterdischarge threshold (ADT) and the differences of current intensity between ADT and generalized seizure threshold (GST) i.e. Δ(GST-ADT). However, Δ(GST-ADT) increased by at least 20 μA in bilateral post-treatment group, while less in bilateral pre-treatment group. Additionally, unilateral post-treatment with HFS failed to inhibit seizures. Our data show that anti-epileptic effect of bilateral post-treatment with HFS of ANT is much stronger than that of bilateral pre-treatment HFS, indicating bilateral responsive stimulation might be more appropriate for clinical anti-epileptic treatment of ANT HFS.

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MODE-DEPENDENT EFFECT OF HIGH-FREQUENCY ELECTRICAL
STIMULATION OF THE ANTERIOR THALAMIC NUCLEUS
ON AMYGDALA-KINDLED SEIZURES IN RATSI
Q. ZHANG, Z. C. WU, J.-T. YU,* N. N. YU X. L. ZHONG AND
L. TAN*
Department of Neurology, Qingdao Municipal Hospital, School
of Medicine, Qingdao University, Qingdao 266071, PR China
Abstract—Deep brain stimulation (DBS) is an emerging
treatment of epilepsy. Anterior nucleus of the thalamus
(ANT) is considered to be an attractive target due to its close
connection to the limbic structures and wide regions of neo-
cortex. The present study aimed to investigate the effects of
high frequency stimulation (HFS) targeting the ANT on
amygdala-kindled seizures in Wistar rats in two different
stimulation modes i.e. pre-treatment and post-treatment
stimulations, mimicking the scheduled and responsive
stimulations in clinical use respectively. When fully-kindled
seizures were achieved by daily amygdala kindling (1 s train
of 1 ms pulses at 60 Hz), HFS (15 min train of 100 ls pulses
at 150 Hz and 450–800 lA) was applied in two modes for
10 days. Bilateral post-treatment with HFS reduced the
incidence of generalized seizures and the mean behavioral
seizure stage and shortened average afterdischarge dura-
tion (ADD) and generalized seizure duration (GSD), while
bilateral pre-treatment with HFS resulted in a similar but
much weaker inhibition of seizures. On the other hand, we
also found the two stimulation modes both increased the
afterdischarge threshold (ADT) and the differences of cur-
rent intensity between ADT and generalized seizure thresh-
old (GST) i.e. D(GST ? ADT). However, D(GST ? ADT)
increased by at least 20 lA in bilateral post-treatment group,
while less in bilateral pre-treatment group. Additionally, uni-
lateral post-treatment with HFS failed to inhibit seizures. Our
data show that anti-epileptic effect of bilateral post-treat-
ment with HFS of ANT is much stronger than that of bilateral
pre-treatment HFS, indicating bilateral responsive stimula-
tion might be more appropriate for clinical anti-epileptic
treatment of ANT HFS. ? 2012 IBRO. Published by Elsevier
Ltd. All rights reserved.
Key words: epilepsy, anterior nucleus of the thalamus (ANT),
highfrequencystimulation,
dependent.
amygdala-kindled,mode-
INTRODUCTION
Epilepsy is a highly prevalent brain disorder affecting
approximately 1% of the population worldwide, and
around one-third of epileptic patients do not respond ade-
quately to medical treatment (Kwan and Brodie, 2000;
Fisher et al., 2010). Moreover, for these intractable cases,
some patients are not recommended for resective surgery
and vagus nerve stimulation cannot control seizures opti-
mally (Laga et al., 2010).
The recent success of deep brain stimulation (DBS)
for the treatment of movement disorders and pain, com-
bined with the advantages of reversibility, adjustability
and remarkable safety, have promoted the application of
DBS for patients with drug refractory epilepsy (DRE)
(Hamani et al., 2005; Lim et al., 2008). To date, there
have been various brain regions targeted for DBS both
experimentally and clinically, e.g. the hippocampus, the
cerebellum,the caudate
nucleus (STN), the centromedian thalamic nucleus
(CM), and the anterior thalamic nucleus (ATN) (Laga
et al., 2010; Jobst, 2010a). Despite promising results,
the optimal targets for stimulation and stimulus parame-
ters for preventing or disrupting seizure remain unknown.
Anterior nucleus of the thalamus (ANT) is emerging as
an attractive target for DBS due to its close connection to
the limbic structures and wide regions of neocortex
(Takebayashi et al., 2007). Positive results in animal
models and preliminary human studies demonstrate that
scheduled brain electric stimulation of the ANT is an
effective treatment of epileptic seizure (Mirski et al.,
1997; Hodaie et al., 2002; Hamani et al., 2004; Kerrigan
et al., 2004; Osorio et al., 2005, 2007; Lee et al., 2006;
Lim et al., 2007; Takebayashi et al., 2007; Zhong et al.,
2011a; Zhang et al., 2012). Recently, the ANT was
chosen as the target for the first large-scale multicenter,
double-blind, randomized trial of bilateral ANT high
frequency stimulation (HFS) with a significant reduction
in the seizure frequency (Fisher et al., 2010).
nucleus,thesubthalamic
0306-4522/12 $36.00 ? 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neuroscience.2012.05.009
qThis work was supported by Grants from the National Natural
Science Foundation of China (81000544, 81171209), the Shandong
Provincial Natural Science Foundation, China (ZR2010HQ004,
ZR2011HZ001), the Project supported by the Qingdao Bureau of
Science and Technology (10-3-3-4-19-nsh, 11-2-3-2-(1)-nsh) and the
Shandong Provincial Outstanding Medical Academic Professional
Program.
*Corresponding authors. Address: Department of Neurology, Qing-
dao Municipal Hospital, School of Medicine, Qingdao University, No.5
Donghai Middle Road, Qingdao 266071, PR China.
E-mail addresses: yu-jintai@163.com (J.-T. Yu), dr. tanlan@163.com
(L. Tan).
Abbreviations: AD, anterodorsal nucleus; ADD, afterdischarge dura-
tion; ADT, afterdischarge threshold; ANOVA, analysis of variance;
ANT, anterior nucleus of the thalamus; AV, anteroventral nucleus; CM,
centromedian thalamic nucleus; DBS, deep brain stimulation; DRE,
drug refractory epilepsy; EEGs, electroencephalograms; GS, general-
ized seizures; GSD, generalized seizure duration; GST, generalized
seizure threshold; HFS, high frequency stimulation; SM, stria medul-
laris of the thalamus; STN, subthalamic nucleus.
Neuroscience 217 (2012) 113–122
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Responsive neurostimulation, the other mode of DBS,
is another novel therapy for DRE aiming to suppress
epileptiform activity by delivering stimulation directly in
response to the electrographic activity (Sun et al.,
2008). Recently, a multicenter, double-blind, randomized-
controlled trial suggests that responsive cortical stimula-
tion that targeted the seizure focus is an effective,
safe and well tolerant treatment for the adults with
DRE (Morrell, 2011). On the other hand, a study in
epilepsy patients indicates that high-frequency respon-
sive stimulation is also effective in patients who received
stimulation to the ANT (Osorio et al., 2005). However, it
is still unknown whether it is better to give the HFS to
the ANT before or after the epileptiform activity.
Most previous animal studies investigated the role of
ANT stimulation in chemical-induced animal models of
epilepsy (Jobst et al., 2010b). The amygdala-kindling
model of epilepsy, as a model of complex partial epilepsy
with secondary generalization, is widely used to study
alternative therapeutic approaches to intractable epilepsy
(Barcia and Gallego, 2009). In the present study, we used
pre- and post-treatment HFS to mimic the scheduled and
responsive stimulations respectively. Accordingly, we ex-
plored the effects of bilateral HFS of ANT applied in the
two stimulation modes (pre- and post-treatment stimula-
tions) and compared the effects of unilateral and bilateral
post-treatmentHFSofANT
seizures in rats.
on amygdala-kindled
EXPERIMENTAL PROCEDURES
Animals
Adult male Wistar rats weighing (260–300 g, provided by the
Experimental Animal Center, Qingdao University, China) were
housed individually in cages with a ambient temperature of
23–25 ?C and a 12-h light/12-h dark cycle (lights on from 7:00
to 19:00). All animals were provided with water and food ad libi-
tum. They were acclimated for at least 1 week before surgery.
The experimental protocol was approved by the Animal Care
and Management Committee of the Qingdao University and
was in complete compliance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals (NIH Publica-
tion No.80-23) revised in 1996. Efforts were made to minimize the
number of animals used in the study and their suffering.
Surgery procedures
Rats were fixed in a stereotactic apparatus under intraperitoneal
chloral hydrateanesthesia (400 mg/kg).
implanted into the right basolateral amygdala (AP: ?3.0 mm, L:
?4.8 mm, V: ?8.8 mm ventral to the dura mater) and the right
or bilateral ANT (AP: ?1.8 cm, L: ±1.8 mm, V: ?5.8 mm ventral
to the dura mater). All coordinates were measured in mm from
bregma according to the atlas of Paxinos and Watson (1998).
The electrodes were made of two twisted Teflon-coated stainless
steel wires (0.16 mm in diameter, A.M. Systems, USA) insulated
except for 50 lm at the tip. The tip separation was 0.5–0.3 mm.
The electrodes were connected to a miniature receptacle, which
was fixed to the skull with three screws and dental acrylic
cement. After electrode implantation, the animals were treated
with penicillin for 5 days to prevent infection and allowed to
recover for 10 days.
Electrodeswere
Fully kindled acquisition
Kindling stimulation of the right amygdala was applied by a con-
stant current stimulator (AD Instruments, Australia), and electro-
encephalograms (EEGs) were recorded with the PowerLab
system (AD Instruments, NSW, Australia) through the same elec-
trodes. The afterdischarge threshold (ADT) was defined for each
animal by using a 1 s, 60-Hz sine-wave stimulus of 1-ms per pulse
on the first day of the amygdala-kindling period. The initial stimu-
lus intensity was 50 lA, and was increased in 20 lA steps every
30 min until at least 5 s of afterdischarge (AD) was induced. The
current intensity was defined as the ADT and used to one daily
kindling stimulation at the right amygdala. Seizure severity was
classified according to a modification of Racine (Racine, 1972):
(1) facial movement; (2) head nodding; (3) unilateral forelimb clo-
nus; (4)bilateral forelimbclonusand rearing; and(5) bilateralfore-
limb clonus and rearing and falling. An animal was regarded as
fully kindled until at least three consecutive stage 5 seizures were
elicited. Stages 1–3 were considered focal seizures, while stages
4 and 5 were considered generalized seizures (GS).
HFS in fully kindled animals
The day that the rats were fully kindled was defined as day 0. The
fully kindled animals were randomly divided into one of four
groups: the control group (n = 10), the bilateral HFS + K group
(n = 10), the bilateral K + HFS group (n = 10) and the unilateral
K + HFS group (n = 10). ADT and generalized seizure thresh-
old (GST) was determined on day 0 and retested on days 5
and 10 by the same procedure used for kindling ADT determina-
tion. The method for ADT measurement was as same as that for
initial ADT. Then the current intensity was increased in incre-
ments of 20 lA until a GS was evoked and this current intensity
was defined as GST. Consecutive trials were separated by at
least 30 min. After the ADT was retested, animals were subjected
to the HFS and kindling stimulation.
In experiment 1, three groups of fully kindled rats were des-
ignated to evaluate whether it is better to give the HFS before
or after the epileptiform activity. From the day 1, the bilateral
HFS + K group received HFS of bilateral ANT immediately be-
fore the kindling stimulation (bilateral pre-treatment). The bilateral
K + HFS group received the HFS of bilateral ANT immediately
after the kindling stimulation (bilateral post-treatment). The con-
trol rats were also connected to the HFS-frequency stimulator
for 15 min, but no current was delivered.
In experiment 2, another group of fully kindled animals (the
unilateral K + HFS group) was compared with the control and
bilateral K + HFS group in experiment 1 to test the effect of uni-
lateral post-treatment HFS of ANT. The unilateral K + HFS
group received the HFS of the right ANT immediately after the
kindling stimulation.
All animals received daily HFS and kindling stimulation for
10 days. The intensity of kindling stimulation was the same as
the value of pre-kindling ADT. HFS (each side, 150 Hz, a squared
biphasic pulse of 100 ls, 1 V) lasted 15 min. The current intensity
of HFS was gradually increased from 100 lA until a marked in-
crease in motor and vigilant behavior was observed in each
experimental animal. Then it was decreased to 70% of this point
within the range of 450–800 lA (Takebayashi et al., 2007).
K means kindling stimulation. Furthermore, the severity of the
kindled seizures was accessed by the following parameters: sei-
zure stage, GS incidence, afterdischarge duration (ADD) and
generalized seizure duration (GSD). The GSD was defined as
the duration of bilateral forelimb clonus.
Histology
Electrode location was histologically verified at the end of the
experiments (Fig. 1A). Animals were sacrificed at the end of
the experiments by deep anesthesia with chloral hydrate
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(400 mg/kg, i.p.) and transcardiac perfusion with 0.9% saline
followed by a 10% formaldehyde fixative solution. They were then
frozen at ?20 ?C, sliced into 12 lm sections and stained with
Toluidine Blue O. The stained slices were qualitatively analyzed
for electrode position using a light microscope. Only animals with
electrodes lying within both the basolateral amygdala and the
ANT were included in the statistical analysis. In our experiments,
37 of 40 rats fulfilled this criterion, i.e. the electrodes of the 37 rats
were within the ANT (including AV (anteroventral nucleus), SM
(stria medullaris of the thalamus), and AD (anterodorsal nucleus))
(Fig. 1B). In addition, in the slides of the animals with electrodes
correctly located in the ANT and amygdala, there was no appar-
ent morphological abnormality in the neurons of these areas.
Statistical analysis
During HFS in fully kindled animals, values for ADD and behav-
ioral seizure score were averaged after each stimulation. The
data used in this analysis were generated from only those stimu-
lations by electrodes that were situated within both the basolat-
eral amygdala and the ANT. All data were presented as
mean ± S.E.M. Statistical analysis was carried out by SPSS11.5
for Windows. Analysis of the mean behavioral seizure stage and
ADD during HFS was performed by two-way analysis of variance
(ANOVA) for repeated measures with Huynh–Feldt correction.
Bonferroni post hoc tests were used to determine the significance
of differences between groups. A Chi-square test was used to
compare the incidence of GS. One-way ANOVA followed by
Dunnett’s test was used in the other comparisons. For all analy-
ses, the tests were two-sided and a p < 0.05 was considered
significant.
RESULTS
During 15 min of HFS delivery, no noticeable abnormal
behavior was observed in these animals. We also com-
pared the average ADD and GSD on day 0 among groups
after all rats were fully kindled. No significant difference
was found in the two parameters (p < 0.05, Fig. 2).
Experiment 1: effect of HFS administration in bilateral
pre- and post-treatment modes on amygdala-kindled
seizures
The present study shows that there were significant differ-
ences in average incidence of GS (F = 25.4), seizure
stage (F = 26.2), mean ADD (F = 28.5) and GSD
(F = 24) among the control, bilateral pre- and post-
treatment groups (all p’s < 0.001, Fig. 3). The evoked
seizures were significantly decreased in the bilateral
HFS + K group, which received daily HFS immediately
before the kindling stimulation (bilateral pre-treatment).
The average incidence of GS was decreased to 60%
comparedwith thatofthe
p < 0.01; Fig. 3A). Additionally, bilateral pre-treatment
HFS of the ANT also significantly suppressed the seizure
stage, shortened average ADD and GSD compared with
those of the control group (3.3 ± 0.4 vs. 5.0; 49.2 ± 5
vs. 71.5 ± 0.5; 21 ± 3.2 vs. 33.4 ± 0.3, all p’s < 0.01;
Fig. 3). Interestingly, in the bilateral K + HFS group which
controlgroup(100%,
Fig. 1. (A) Photomicrograph of a rat that underwent anterior thalamic nucleus stimulation showing the electrode track (arrow heads) and the
location of the tip of the electrode. AD, anterodorsal nucleus; AV, anteroventral nucleus; SM, stria medullaris of the thalamus. (B) Schematic
representation of coronal rat brain sections from an atlas showing the region in which the tips of the electrodes were identified. For clarity, we did not
plot the tips of each electrode but rather indicated the boundaries of the regions where they were identified (circles). Numbers on the left denote the
anteroposterior position from the bregma.
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received daily HFS immediately after the kindling stimula-
tion (bilateral post-treatment), evoked seizures were
reduced more markedly. Average incidence of GS, sei-
zure stage, mean ADD and GSD were significantly de-
creasedto 27 ± 8.8%, 1.7 ± 0.4,
8.9 ± 3.0, respectively when compared with the control
group (all p’s < 0.001; Fig. 3). It is worth mentioning that
the inhibition of GS incidence, seizure stage, average
ADD and GSD induced by bilateral post-treatment HFS
was stronger than that by bilateral pre-treatment HFS
(p < 0.05; Fig. 3).
Fig. 4 gave the mean (±S.E.M) values of behavioral
seizure stage and ADD for each group at each time-point.
There were significant overall effects of HFS, time effects
andstimulation–timeinteractiononbothbehavioralseizure
stage (F2,25= 17.6, p < 0.001; F2,57= 45.6, p < 0.001;
F5,57= 13.8,
p < 0.001,
(F2,25= 2734.2, p < 0.001; F6,138= 1704.7, p < 0.001;
F11,138= 717.7, p < 0.001). The average behavioral sei-
zure stage (Bonferroni post hoc tests, pre-treatment,
p < 0.01; post-treatment stimulation, p < 0.001, respec-
tively) and ADD (Bonferroni post hoc tests, pre-treatment,
p < 0.001; post-treatment stimulation, p < 0.001, respec-
tively) at the time between days 4 and 10 of HFS were sig-
nificantly suppressed by the pre- and post-treatment
stimulations of the bilateral ANT compared with the control
group. In addition, the effects of bilateral post-treatment
HFS were significantly stronger than those of bilateral
pre-treatment HFS (Bonferroni post hoc tests, behavioral
seizure stage, p < 0.05; ADD, p < 0.001).
As shown in Table 1, there were no differences in ADT
and GST among groups on the day 0. On the 5th day, the
ADT of the control group changed from 128 ± 7.8 lA to
116 ± 6.0 lA (90.6% of ADT on day 0, p > 0.05), and
the value decreased to 92 ± 4.0 lA (71.9% of pre-HFS
ADT, p < 0.001) on the 10th day. However, bilateral
pre-treatment with HFS (bilateral HFS + K) elevated
ADT from 134 ± 6.5 lA to 214 ± 9.2 lA (159.7% of
30.9 ± 4.7 and
respectively)andADD
ADT in day 0, p < 0.001) on the 5th day, and to
247 ± 8.4 lA (184.3% of ADT in day 0, p < 0.001) on
the 10th day. For the bilateral post-treatment HFS group
(bilateral K + HFS), HFS markedly increased ADT from
128 ± 7.6 lA to 234 ± 8.4 lA (182.8% of ADT in day
0, p < 0.001) on the 5th day, and to 274 ± 6.5 lA
(214% of ADT in day 0, p < 0.001) on the 10th day. In
addition, ADT in the bilateral pre- and post-treatment
HFS groups was significantly higher than that of the con-
trol group on the 5th and 10th day (p < 0.001). ADT of
bilateral post-treatment HFS group was slightly higher
than that of the bilateral pre-treatment HFS group
(p > 0.05).
On the other hand, the differences of current intensity
between ADT and GST (D(GST ? ADT)) in rats on day 0
were not statistically different among the three groups.
However, therewere significant
D(GST ? ADT) on the 5th and 10th day during the HFS
stimulation(F = 10.5and
p < 0.001).When post-treatment with HFS was applied
to the bilateral ANT, the value was significantly increased
from 10 ± 3.3 to 38 ± 3.6 on the 5th day (p < 0.001),
and to 62 ± 3.6 on the 10th day (p < 0.001). The bilateral
pre-treatment HFS increased the value from 11 ± 4.8 to
22 ± 5.2 slightly on the 5th day (p > 0.05) and to
42 ± 4.0 significantly on the 10th day (p < 0.001)
(Table 1). Furthermore, the elevation of D(GST ? ADT)
induced by bilateral post-treatment HFS was much
stronger than that by bilateral pre-treatment HFS on
10th day (62 ± 3.6 vs. 42 ± 4.0, F = p < 0.001).
differences in
59.2, respectively,
Experiment 2: effect of unilateral post-treatment HFS
of ANT and comparison between unilateral and
bilateral post-treatment stimulation
As shown in Fig. 3, there were significant differences in
averageincidenceofGS
(F = 59.1), mean ADD (F = 67.7) and GSD (F = 60.4)
(F = 58),seizurestage
Fig. 2. The (A) average afterdischarge (AD) duration and (B) average generalized seizures (GS) duration of the rats in different groups on day 0.
Data are shown as mean ± S.E.M. One-way ANOVA was used for statistical analysis, followed by the Dunnett’s t-test.
116 Q. Zhang et al./Neuroscience 217 (2012) 113–122

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among the control, bilateral and unilateral post-treatment
group (all p’s < 0.001). When we compared the unilateral
post-treatment stimulation group with the control group,
there was no significance in GS incidence (94 ± 2.7%),
average seizurestage(4.8 ± 0.06),
(69.5 ± 0.8) and GSD (31.5 ± 0.7) (p > 0.05). It is worth
mentioning that there were significantly less generalized
seizure incidence, shorter average ADD, seizure stage,
and average GSD in the bilateral K + HFS group com-
pared to the unilateral K + HFS group (p < 0.001;
Fig. 3).
As shown in Fig. 4, there were significant overall ef-
fects of HFS, time effects and stimulation–time interaction
onbothbehavioral seizure
p < 0.001; F3,65= 45.6, p < 0.001; F5,65= 28.2, p<
0.001, respectively) and ADD (F2,25= 4981.2, p <
0.001;
F6,157= 807.4,
p < 0.001;
p < 0.001). However, unilateral post-treatment ANT
HFS had no significant effect on average seizure stage
(Bonferroni post hoc tests, p = 1) or ADD (p = 0.06). In
addition, the effects of post-treatment HFS of the bilateral
ANT were significantly stronger than those of the unilate-
ral ANT (p < 0.001). Furthermore, there were significant
differences in ADT and D(GST ? ADT) among the con-
trol, bilateral and unilateral post-treatment group on the
5th and 10th day (ADT, F = 91.6 and 261 respectively;
D(GST ? ADT),
averageADD
stage(F2,25= 37.5,
F13,157= 1133.8,
F = 13and89.1,respectively;all
p’s < 0.001, Table 1). However, both ADT and GST were
not changed by unilateral post-treatment HFS once daily
for 5 days (p > 0.05, Table 1). Interestingly, statistical
significance of ADT was observed between the control
and unilateralK + HFS
(p < 0.01, Table 1).
Representative EEGs recorded from the right amyg-
dala during the two experimental sessions are shown in
Fig 5. When the 10-day HFS stimulation treatment was
finished, all rats were continuously kindled without HFS
for 3 weeks. After HFS was stopped for 1 week, four rats
in bilateral pre-treatment group still failed to exhibit GS,
whereas all rats in the other three groups exhibited GS
(p < 0.05). After HFS was stopped for 2 weeks, eight rats
in bilateral pre-treatment group and all rats in post-treat-
ment groups and control group exhibited GS (p > 0.05).
After HFS was stopped for 3 weeks, all rats exhibited gen-
eralized seizure (Fig. 6).
groupon the 10thday
DISCUSSION
DBS is an emerging treatment of epilepsy. Stimulation
frequency seems to be a pivotal parameter for stimula-
tion-induced activation of seizure-gating networks, which
can impede seizure propagation resulting in seizure sus-
ceptibility change (Gale, 1992; Lado et al., 2003; Good-
man et al., 2005). Several studies have demonstrated
Fig. 3. Effects of pre- and post-treatment with high frequency stimulation (HFS) targeting the anterior nucleus of the thalamus (ANT) on (A)
incidence of generalized seizures (GS), (B) seizure stage, (C) average afterdischarge (AD) duration, (D) average GS duration from days 1 to 10 in
fully kindled rats. Data are shown as mean ± S.E.M.⁄p < 0.01,⁄⁄p < 0.001 represent statistically significant differences as compared with the
control group.#p < 0.05 represents statistically significant difference as compared with the bilateral pre-treatment HFS group.?p < 0.01 represent
statistically significant differences as compared with the group receiving bilateral post-treatment HFS.
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that low frequency stimulation synchronized the EEG
activity making the cortex more susceptible to seizures,
while HFS lead to EEG desynchronization resulting in less
susceptibility to seizures (Lim et al., 2008). In the past few
Fig. 4. Effects of high frequency stimulation (HFS) of the anterior nucleus of the thalamus (ANT) on (A) behavioral stage of seizures and (B)
afterdischarge duration during 10 days of HFS treatment.⁄p < 0.01,⁄⁄p < 0.001 represent statistically significant differences as compared with the
control group#p < 0.05,##p < 0.001 represents statistically significant difference as compared with the bilateral pre-treatment HFS group.?
p < 0.001 represent statistically significant differences as compared with the group receiving bilateral post-treatment HFS.
Table 1. The ANT and GST changes during 10 days’ HFS treatment in fully amygdala-kindled rats
Groups
n
Pre-kindling
ADT (lA)
Day 0Day 5 Day 10
ADT (lA)
D(GST ? ADT)
(lA)
ADT (lA)
D(GST ? ADT)
(lA)
ADT (lA)
D(GST ? ADT)
(lA)
Control
Bilateral HFS + K
Bilateral K + HFS
Unilateral K + HFS
9
9
10
9
235 ± 24.6
230 ± 20.5
235 ± 18.7
225 ± 25.3
128 ± 7.8
134 ± 6.5
128 ± 7.6
132 ± 9.7
13 ± 4.7
11 ± 4.8
10 ± 3.3
11 ± 3.5
116 ± 6.0
214 ± 9.2**,?
234 ± 8.4**,?
130 ± 6.7a
8.9 ± 4.8
22 ± 5.2
38 ± 3.6*,?
15 ± 4.4a
92 ± 4.0?
247 ± 8.4**,?
274 ± 6.5**,?
127 ± 7.0*,a
6.7 ± 3.3
42 ± 4.0**,?
62 ± 3.6**,#,?
6.7 ± 3.3a
Data are shown as means ± S.E.M.
*p < 0.01 represent statistically significant differences as compared with controls.
**p < 0.001 represent statistically significant differences as compared with controls.
#p < 0.001 represent statistically significant differences as compared with the bilateral HFS + K group.
?p < 0.001 represents statistically significant differences as compared with the value of day 0.
ap < 0.01 represent statistically significant differences as compared with the bilateral K + HFS group.
118Q. Zhang et al./Neuroscience 217 (2012) 113–122

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decades, HFS was applied widely and has met with great
success on seizure control (Zhong et al., 2011a,b). How-
ever, the principal questions of where to stimulate and
what type of stimulation will be most effective remain
unclear.
To date, two modes of brain stimulation, scheduled
and responsive stimulations are used clinically for the
treatment of intractable epilepsy. The ANT is one of the
most explored targets for scheduled DBS, which is pro-
gramed to provide intermittent or continuous stimulation
irrespective of the brain’s physiological electrical activity.
Numerous research findings have suggested that bilateral
scheduled HFS of ANT was able to control seizures effec-
tively in both animal investigation and limited human trials
(Mirski et al., 1997; Hodaie et al., 2002; Hamani et al.,
2004; Kerrigan et al., 2004; Lee et al., 2006; Lim et al.,
2007; Osorio et al., 2007; Takebayashi et al., 2007; Fisher
et al., 2010). Alternatively, the responsive stimulation
device (the RNS System) is capable of performing real-
time evolution of abnormal activity and delivering respon-
sive electrical stimulation to abort the seizure propagation
before clinical onset (Gigante and Goodman, 2011).
Animal and human data support the concept that respon-
sive stimulation is a safe and effective treatment option for
epilepsy, with improved quality of life (Sun et al., 2008;
Morrell, 2011). Among these studies, a small-case human
study indicates that high-frequency responsive stimula-
tion (100–200 Hz) of ANT is also effective in patients with
DRE(Morrell,2011).In
conducted by Hamani et al. suggests that HFS (130 Hz)
contrast,a recentstudy
Fig. 5. Electroencephalographic examples of afterdischarge (AD) during 10-days high frequency stimulation (HFS) treatment in fully amygdala-
kindled rats. Examples were fully kindled rats that received no HFS treatment, bilateral pre-treatment, bilateral and unilateral post-treatment HFS
targeting the ANT. Records are shown after the 5th (A) and the 10th (B) HFS. The EEGs were recorded from the right amygdala using a PowerLab
system. Linear bars indicate artifacts induced by HFS. ES represents electrical kindling stimulation.
Q. Zhang et al./Neuroscience 217 (2012) 113–122
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of ANT is not able to arrest effectively the ongoing pilocar-
pine-induced seizures in rats (Hamani et al., 2008).
In present study, we used pre-treatment and post-
treatment HFS to mimic the scheduled and responsive
stimulationsin clinical use
explored the effects of bilateral HFS of ANT applied in
the two stimulation modes on amygdala-kindled seizures
in rats. The results provided evidence that both pre- and
post-treatments of bilateral ANT with HFS (150 Hz) can
effectively reduce the severity of amygdala-kindled sei-
zures. The mean incidence of rats exhibiting GS in bilat-
eral K + HFS group was 27% compared with an
incidence of 100% of the control group. This finding, con-
sistent with the results of Morrell, 2011, is contradictory to
the results reported by Hamani et al. (2008). The discrep-
ancy of these results might be due to the different stimu-
lation parameters (i.e. frequency, intensity), time delay of
stimulation (immediately vs. 5–15 min after seizure onset)
and model of epilepsy (amygdala-kindled vs. polocarpine-
induced). Additionally, we also found that the mean
values of seizure stage, ADD and GSD were markedly
suppressed by post-treatment with HFS of bilateral
ANT. Most importantly, bilateral post-treatment with
HFS produced a much stronger inhibitory effect on
evoked seizures than bilateral pre-treatment with HFS.
So it is likely that post-treatment with HFS is more
preferable for clinical anti-epileptic treatment.
ADT and GST represent the susceptibility of the
animal to generation and propagation of the seizure activ-
ity. The current difference between ADT and GST, i.e.,
D(GST ? ADT) represents the likelihood of epileptic
discharges spreading from the kindled site, and finally
eliciting a GS (LO¨scher et al., 2000). In this study, we
found that both bilateral post- and pre-treatments with
HFS significantly elevated the ADT, but there is no signif-
respectively andfirstly
icance between the two groups. We also found that
D(GST ? ADT) significantly increased by at least 20 lA
in bilateral post-treatment group, while less in bilateral
pre-treatment group. Thus, our findings at least indicate
that bilateral pre-treatment HFS of ANT inhibits kindled
seizures mainly by preventing AD initiation and bilateral
post-treatment HFS of ANT aborts evoked seizures
mainly by suppressing AD propagation. Accordingly, it
was not surprising that when HFS was stopped for
1 week, four rats in bilateral pre-treatment group still failed
to exhibit GS, whereas all rats in the other three groups
exhibited GS (p < 0.05).
The ANT appears to closely interact with the circuit of
Papez, the circuit of thalamic reticular nucleus (RTN),
superior frontal and temporal lobe structures which are
commonly involved in seizures. Because various path-
ways involved in generalization of seizures and part of
the ANT efferents have bilateral projections (Hamani
et al., 2004), it is not surprising that in our study only bilat-
eral ANT stimulation is effective in inhibiting seizures, and
unilateral post-treatment with HFS is completely ineffec-
tive. Additionally, unilateral post-treatment HFS signifi-
cantly increases the ADT compared with that of the
control group, but with a less significance than the bilat-
eral post-treatment HFS.
The exact mechanisms of action of ANT electrical
stimulation in reducing seizures are not fully understood.
Lozano and Eltahawy (2004) found that DBS invokes a
mixture of excitatory and inhibitory effects, ultimately
resulting in disruption of neural networks. In hippocampal
slice model systems, HFS causes negative slow potential
shifts and increased extracellular potassium accumula-
tion, inhibiting synchronized neural excitability (Durand,
1986; Gluckman et al., 1996). Additionally, seizure
aborting effect of DBS may be due to synaptic release
Fig. 6. Persistent effect of pre-HFS of the bilateral ANT on the seizure development induced by amygdala kindling. No further HFS was
administrated during the 3-week period following the 10 days of kindling. Four rats in bilateral pre-treatment group still failed to exhibit generalized
seizures after 1 week. After 3 weeks, all rats reached the fully kindled state at the initial stimulation parameters.
120 Q. Zhang et al./Neuroscience 217 (2012) 113–122

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of inhibitory neurotransmitters (Guo et al., 2010; Jiruska
et al., 2010). Furthermore, HFS resulted in the depression
of synaptic transmission manifesting as a reduction in
excitatory postsynaptic potential amplitudes (Schiller
and Bankirer, 2007). In addition to the local inhibition, it
has been considered that the effect on projections leaving
from the stimulation site to other key nervous structures
may be the most likely mechanism of anticonvulsant
effect of ANT stimulation (Boon et al., 2009; Zhong
et al., 2011b).
CONCLUSION
Our study demonstrated that bilateral post-treatment with
HFS of ANT can markedly inhibit amygdala-kindled sei-
zures and its anti-epileptic effect is much stronger than
that of bilateral pre-treatment HFS. This indicated that
responsive stimulation might be more appropriate for clin-
ical anti-epileptic treatment of ANT HFS. Further studies
are needed to make comparison of the two stimulation
modes in other seizure models and human cases and to
provide evidence for clinical use.
Acknowledgements—This work was supported by Grants from
the National Natural Science Foundation of China (30870884)
and the Shandong Provincial Outstanding Medical Academic
Professional Program.
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