Transcranial Focal Electrical Stimulation Reduces Seizure Activity

and Hippocampal Glutamate Release During Status Epilepticus*

Csar E. Santana-Gmez, David Alcntara-Gonzlez, Hiram Luna-Munguia, Ivette Bauelos-Cabrera,
Vctor Magdaleno-Madrigal, Michael Tamayo, Luisa L. Rocha, and Walter G. Besio, Senior Member, IEEE

Abstract

Previously we demonstrated that noninvasive

transcranial focal electrical stimulation (TFS) with sub-effective
doses of diazepam reduces status epilepticus (SE)-induced
neuronal damage. However, it was unclear if this
neuroprotective effect is a consequence of the decrease in the
glutamate release. The aim of the present study was to evaluate
the effects of TFS on -Aminobutyric acid (GABA) and
glutamate release in the hippocampus during pilocarpineinduced SE. After pilocarpine administration, the rats showed
progressive behavioral changes that culminated in SE with a
significant increase of GABA and glutamate (95 and 128%
respectively), even more evident at the end of the experiment
(120 and 182% respectively), 5 hours after pilocarpine injection
and was associated with the prevalence of high-voltage rhythmic
spikes and increased spectral power in the 4-90 Hz bands. The
TFS application during the SE decreased the convulsive
expression, the prevalence of high-voltage rhythmic spikes and
spectral power in 4-8 Hz and 30-90 Hz bands. These effects were
associated with lower release of GABA and glutamate in the
hippocampus. These results support the anticonvulsive and
neuroprotective effects induced by TFS.

I. INTRODUCTION
Status epilepticus (SE) is a neurologic emergency that
requires immediate management to avoid brain injury [1-4].
At present, pharmacological strategies to control SE and its
consequences in patients are inadequate. The efficacy of
diazepam (DZP) and similar first-line abortive SE treatments
are ineffective and SE often continues [5, 6]. Some SE drugs
induce secondary effects such as respiratory depression,
sedation, hypotension and cardiac dysrhythmias [7].
Electrical modulation of the brain such as deep brain
stimulation (DBS) and vagal nerve stimulation are alternative
treatments to control pharmacoresistant epilepsy [8].
However, few neuromodulation strategies have been
considered as treatment options for SE. Low frequency
repetitive transcranial magnetic stimulation applied for one
hour sessions for 8 days decreased the expression of SE [9].
Other studies indicate that DBS of brain areas such as the
thalamus, may increase the latency to SE under specific
experimental conditions [10]. We previously described that
high-frequency DBS applied in the hippocampus combined
with subeffective doses of diazepam or phenobarbital reduce
the expression of lithium pilocarpine induced SE [11].
However, experimental evidence indicates that the presence
of intracranial electrodes may facilitate seizure activity [12].
*This

research was supported in part by the National Institute of

Neurological Disorders and Stroke (R21NS061335) and by the Fogarty
International Center of the National Institutes of Health (R21TW009384).
C. E. Santana-Gmez, D. Alcntara-Gonzlez, H. Luna-Munguia; I.
Bauelos-Cabrera, and L. L. Rocha are with the Dept. of Pharmacobiology,
Center for Research and Advanced Studies, Mexico City, Mexico.

978-1-4244-9270-1/15/$31.00 2015 IEEE

In previous studies we reported that noninvasive

transcranial focal electrical stimulation (TFS) applied via
tripolar concentric ring electrodes (TCREs) during
pilocarpine-induce SE in rats decreased the seizure expression
[13]. We also demonstrated that TFS combined with subeffective doses of diazepam applied before the induction of
pilocarpine-induce SE, reduces the incidence of mild and
severe generalized seizures, which significantly reduced
neuronal damage [14]. These results lead to suggest that TFS
represents a noninvasive therapy for SE. However, the
mechanism is unclear by which TFS is able to reduce the SE
seizure activity and the associated cell loss.
Neuronal damage induced by SE has been associated with
glutamatergic excitotoxicity mediated by N-Methyl-Daspartate (NMDA) receptors [15-17]. SE also decreases the
inhibitory neurotransmission mediated by -aminobutyric
acid (GABA) [18-20] and the neuroprotective effects of
benzodiazepines [21]. Accordingly, we cannot discard that a
decrease in the glutamatergic or/and an increase in the
GABAergic neurotransmission play an important role in the
protective effect of TFS on SE induced neuronal damage.
Identification of new non-invasive therapeutic strategies to
arrest SE, and its consequences, represents an important
achievement. The present study investigated if TFS applied
during SE is able to reduce seizure activity, avoid the increase
in the glutamate release, and/or augment the GABA release in
the hippocampus of rats when applied immediately after the
onset of the SE.
II. METHODS
A. Animals
Adult male Wistar rats initially weighing 250300 g were
the subjects of the present study. All procedures involving
animals described in this paper were conducted in agreement
with the Mexican Official Standard (NOM-062-ZOO-1999)
and approved by the Ethical Committee of the Center for
Research and Advanced.
B. Surgery
Rats were anesthetized with a mixture of ketamine (80 mg/kg,
i.p.) and xylazine (20 mg/kg, i.m.). Then, a guide cannula
attached to a bipolar electrode, consisting of two twisted
strands of stainless steel wire and insulated except at the
cross-section of their tips, was stereotactically implanted into
V. Magdaleno-Madrigal, is with the Division for Neuroscience Research,
National Institute of Psychiatry. Mexico City, Mexico.
M. Tamayo and W. G. Besio are with the Electrical, Computer, and
Biomedical Engineering Department, University of Rhode Island, Kingston,
RI 02881 USA (e-mail: besio@uri.edu).

6586

the right hippocampus [22]. The tip of the cannula was located
-4.5 mm below the skull surface. A 6.0 mm dia. TCRE was
placed centered on the cranium, as close to 5 mm behind the
Bregma as possible. Stainless steel screws were threaded into
the cranium over the frontal and cerebellar cortices to fix the
electrode assembly to the skull with dental acrylic. Animals
were allowed to recover for 7 days.
C. Transcranial focal electrical stimulation
The TFS consisted of 200 s symmetrical biphasic square
charge-balanced constant current pulses at a rate of 300 Hz
and at an intensity of 100 to 5000 A, depending on the
experimental protocol (see below). TFS was applied through
the outer ring (external diameter of 6.0 mm) and disc of a
TCRE. For this purpose, we used two Grass Technologies S48
square pulse stimulators each with a SIU-C constant current
stimulus isolation unit (Grass Technologies, West Warwick,
RI). One S48 provided the positive pulses and the other
provided the negative pulses.
D. Afterdischarge threshold and amino acid release
We first carried out an experiment to determine the current
intensity necessary to induce changes in amino acid release in
the hippocampus of control animals. Rats (n=8) received daily
administration of saline solution for 5 days. Twenty four
hours after the last saline injection, a perfused dialysis probe
constructed by ourselves as previously described [23],
designed to protrude 3 mm beyond the cannula tip in order to
stay in the hippocampus, was inserted into the guide cannula
and fixed with dental acrylic. The active part of the dialysis
probe (3 mm) was a polyacrylonitrile membrane.
The dialysis probe inlet was connected to a syringe and
microperfusion pump (model 1001; BAS). The dialysis
system was continuously perfused during the entire
microdialysis experiment at a rate of 2 l/min with freshfiltered sterilized artificial cerebrospinal fluid. Following a 2h recovery period under basal conditions, the afterdischarge
threshold (ADT) was determined to estimate the hippocampal
excitability. The ADT was established by administering, in
the right ventral hippocampus, a series of stimulations (1 ms
rectangular pulses, 60 Hz for 1 s) at 1-min intervals beginning
at 10 A and incrementally increased in steps of about 20%
of the previous current. The threshold was defined as the
lowest intensity producing a behavioral change or an
afterdischarge with duration of at least 3 s. The afterdischarge
duration was recorded from the bipolar electrode implanted in
the right hippocampus using a P511 amplifier (0.1 to 100 Hz)
and digitized (PVA-16, 16-bit, 1000 samples/s) data
acquisition card controlled by Polyview on the computer (all
from Grass Technologies West Warwick, RI.).
Two minutes after the determination of the ADT, TFS was
applied constantly during 30 min with an intensity from 100
to 5000 A, the TFS ADT. The ADT was determined for a
second time 30 min after the end of the TFS. The dialysates
were continuously collected at 10 min intervals during the
TFS application and 30 min after. Then, they were collected
at 30 min intervals during 3 additional hours. Immediately
after recovery high performance liquid chromatography
(HPLC) was performed on the dialysates to determine the

concentrations of GABA and glutamate. A control group

(n=5) was manipulated as described above, except that
animals did not receive TFS.
E. Effects of TFS on the amino acid released during the SE
a. SE-TFS group (n=6). Rats were manipulated as described
above (D). Following the 2-h recovery period under basal
conditions, animals were administered methylscopolamine (1
mg/kg, i.p.), and then pilocarpine (300 mg/kg, i.p.) was
applied 30 min later. Then, continuous behavioral monitoring
was carried out to identify the establishment of SE, i.e., when
animals presented seizures continuously for longer than 2 min
without recovery between them. Five
minutes after the establishment of the SE, TFS was applied
constantly during 2 h at a rate of 300 Hz and at the lowest
intensity to induce changes in the extracellular levels of
GABA and glutamate according the previous experiment (D,
100 A). The dialysates were sampled at 30 min intervals
under basal conditions (2 h), beginning of the SE (first
recovery after SE establishment), during the TFS application
(2 h) and after it (2 additional h). Behavioral and
electrographic recordings were obtained during the
experiment.
b. SE group (n=6). Rats were manipulated as indicated
previously for SE-TFS group without receiving TFS.
F. High performance liquid chromatography (HPLC)
An internal standards solution was combined with
perfusate-HClO4, mixed with ophthalaldehyde acid, and
injected into the solvent stream of an HPLC system 2 min
later. The quantification of amino acids was carried out by
peak height measurements against standard solutions [24].
G. Evaluation of the electrographic activity
The hippocampal electrical activity was recorded as in (D).
Off-line spectral analysis using fast Fourier transform (FFT)
methods during periods of one minute at each stage of interest
(baseline, 5, 60 and 150 min after the beginning of SE) were
performed [25,26]. The power corresponding to the different
bands were normalized against the maximum power of each
band analyzed (0.14, 4-8, 8-13, 13-30 and 30-90 Hz). Spike
counting was performed to quantify the difference in SE
electrographic seizure activity between control and TFStreated rats. Briefly, the mean of the absolute values of the
data file was calculated and a threshold of one standard
deviation from the mean was set. A count of the absolute
spikes above the threshold was then calculated for each min.
E. Statistical analysis
Values were expressed as mean S.E.M. An unpaired t-test
was applied to examine ADT values and the spike counts. The
extracellular levels of GABA and glutamate in the course of
the microdialysis experiments were analyzed by one-way
ANOVA followed by the post hoc Fisher's PLSD test.
Pearsons correlation coefficients were estimated to establish
the potential impact of TFS on the ADT and release of GABA
and glutamate. The power spectra were evaluated using a oneway ANOVA test followed by a post-hoc Bonferrionis
multiple comparison test. In all statistical comparisons,
p<0.05 was used as criterion for significance.

6587

III. RESULTS
Histological analysis proved the electrode tips were located
within the ventral hippocampus in all experimental groups.
A. Amino acid release and electrographic activity under
control and basal situations
Under control (control group) and basal condition (SE and
SE-TFS groups), the hippocampal electrographic activity
demonstrated prevalence of 0.1-4 and 30-90 Hz bands. The
extracellular levels of GABA and glutamate were maintained
stable over the experimental period (GABA, 0.36 0.02 M;
glutamate, 0.76 0.08 M) (data not shown).
B. Effects of TFS on the afterdischarge threshold and amino
acid release
Under basal conditions, rats showed a homogeneous
distribution of their pre-TFS ADT values (173 26 A
ranging from 100 to 300 A). Once the animals received the
TFS, post-TFS ADT values exhibited a non-significant
increase (189 25 A; 9.7%, p<0.64), when compared with
their pre-TFS values. No significant correlation was detected
between the percentage of change on ADT values and the
current intensity values for the TFS (p = 0.1129). Statistical
analysis revealed significant correlations between the current
intensity of TFS and the changes in GABA and glutamate
release. At current intensities of 2800 A or lower of TFS,
decreased extracellular levels of glutamate and increased
GABA were detected, effects more evident at the lower
intensities (glutamate, 76% decrease at 400 A; GABA, 90%
increase at 620 A). No significant changes were detected at
higher intensities (>4800 A). Fig. 1 shows the averages of
all rats for GABA and glutamate.

When compared with the SE group, the rats receiving TFS

(SE-TFS group) demonstrated lower intensity of the
behavioral seizure activity. During the SE, the analysis of the
electrographic activity demonstrated lower voltage and less
rhythmic spiking during and after TFS application. In the
spectral analysis, the activity showed a lower power of the 48 and 30-90 Hz bands, and an increase in other bands analyzed
(0.1-4, 8-13 and 13-30 Hz) (p<0.001). The spike count did not
show a significant difference however visually there appears
to be obvious difference (Fig.2).

Fig. 2. Panel 1 (TFS-treated) A. spectrogram, B. spike count,

C. absolute hippocampal EEG. Panel 2 is for a control rat not
receiving TFS. Black vertical dashed line is SE onset. The
vertical dashed green lines are the start and end of the TFS.
The red horizontal line is the threshold.

Concerning the amino acid release, the SE-TFS group had

a non-significant increase of glutamate (44%, p=0.182) and
decrease in GABA (24%, p=0.154) at the onset of the SE.
Thereafter, no significant changes were detected during
(glutamate, 5%, p=0.911; GABA, 21%, p=0.171) and 1 h after
the end of the TFS (glutamate, 24%, p=0.700; 15%, p=0.716).
IV. DISCUSSION/CONCLUSION

Fig. 1. Grand averages of GABA and glutamate in the TFStreated control rats (not having SE) over time. There is a clear
increase of GABA and decrease of glutamate.

Accordingly we decided to apply TFS at 100 A during the

SE experiments.
C. Amino acid release and electrographic activity during SE
Administration of pilocarpine caused progressive behavioral
changes that culminated in SE in 100% of the animals. The
analysis of the electrographic activity of the SE group
revealed faster, high-voltage rhythmic spikes and increased
spectral power in 4-90 Hz bands (p<0.001).
After the initiation of the SE, animals from the SE group
demonstrated a significant progressive increase in GABA and
glutamate release and reached 120% (p<0.001) and 182%
(p<0.001), respectively, by the end of the microdialysis
experiment (240 min after the beginning of the SE).
D. Effects of TFS on the amino acid released and
electrographic activity during the SE

We conclude that TFS applied during SE, in rats, reduces: (a)

seizure activity, (b) power of the electrographic activity, and
(c) the release of extracellular GABA and glutamate. There
was a decrease of power in 4-8 Hz (theta) and 30-90 Hz
(gamma) bands in the hippocampus. These results correlated
with a significant decrease of SE-evoked increases in GABA
and glutamate release.
In the present study, we found that TFS avoided increases
in release of glutamate during the SE, an effect that can
explain the reduced seizure activity of animals receiving TFS
after the initiation of the ictal event induced by pilocarpine,
pentylenetetrazol and penicillin G [13,27-30]. The control
experiments revealed that TFS applied at low current
intensities results in enhanced GABA and decreased
glutamate extracellular levels in the hippocampus. The SETFS experiments support the notion that the high voltage fast
activity in the hippocampus during SE is characterized by the
prevalence of delta, theta and gamma waves [31-34]. We also
found that TFS applied during the SE significantly reduced
the prevalence of theta and gamma waves and avoids the
increased release of glutamate. The rhythmically discharging
GABAergic interneurons play an important role in the
modulation of the theta and gamma oscillations in

6588

hippocampus. It is possible that the lessened glutamate release

in the hippocampus is associated with the decreased power of
theta and gamma waves and the anticonvulsant effects
induced by TFS when applied during the seizure activity
[28,29,34].

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