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LOW-FREQUENCY ELECTRICAL STIMULATION ENHANCES
THE EFFECTIVENESS OF PHENOBARBITAL ON GABAergic
CURRENTS IN HIPPOCAMPAL SLICES OF KINDLED RATS
AZAM ASGARI,
a
SAEED SEMNANIAN,
a
NAFISEH ATAPOUR,
b
AMIR SHOJAEI,
a
HOMEIRA MORADI-CHAMEH,
a
SAMIREH GHAFOURI,
a
VAHID SHEIBANI
b
AND JAVAD MIRNAJAFI-ZADEH
a
*
a
Department of Physiology, Faculty of Medical Sciences,
Tarbiat Modares University, Tehran, Iran
b
Neuroscience Research Center, Institute of
Neuropharmacology, Kerman University of Medical Sciences,
Kerman, Iran
Abstract—Low frequency stimulation (LFS) has been pro-
posed as a new approach in the treatment of epilepsy. The
anticonvulsant mechanism of LFS may be through its effect
on GABA
A
receptors, which are the main target of
phenobarbital anticonvulsant action. We supposed that
co-application of LFS and phenobarbital may increase the
efficacy of phenobarbital. Therefore, the interaction of LFS
and phenobarbital on GABAergic inhibitory post-synaptic
currents (IPSCs) in kindled and control rats was investi-
gated. Animals were kindled by electrical stimulation of
basolateral amygdala in a semi rapid manner (12 stimula-
tions/day). The effect of phenobarbital, LFS and phenobarbi-
tal + LFS was investigated on GABA
A
-mediated evoked and
miniature IPSCs in the hippocampal brain slices in control
and fully kindled animals. Phenobarbital and LFS had
positive interaction on GABAergic currents. In vitro
co-application of an ineffective pattern of LFS (100 pulses
at afterdischarge threshold intensity) and a sub-threshold
dose of phenobarbital (100 lM) which had no significant
effect on GABAergic currents alone, increased the ampli-
tude and area under curve of GABAergic currents in CA1
pyramidal neurons of hippocampal slices significantly.
Interestingly, the sub-threshold dose of phenobarbital
potentiated the GABAergic currents when applied on the
hippocampal slices of kindled animals which received LFS
in vivo. Post-synaptic mechanisms may be involved in
observed interactions. Obtained results implied a positive
interaction between LFS and phenobarbital through GABA
A
currents. It may be suggested that a combined therapy of phe-
nobarbital and LFS may be a useful manner for reinforcing the
anticonvulsant action of phenobarbital. Ó2016 IBRO.
Published by Elsevier Ltd. All rights reserved.
Key words: amygdala, GABAergic currents, hippocampus,
low frequency stimulation, phenobarbital, seizure.
INTRODUCTION
One of the most common types of epilepsy is temporal
lobe epilepsy in which different areas of the limbic
system including amygdala and hippocampus are
involved. Anticonvulsant medication is yet the most
common method for treating patients with epilepsy. It is
effective in controlling the seizures in up to 70% of
patients. However, the rest continue to suffer from
seizures, illness and risk of mortality (Sander, 2004).
Phenobarbital is using as one of the main and the
oldest antiepileptic drugs in many countries
(Radhakrishnan, 2009; Satishchandra and Nagappa,
2012). Despite development of successive generations
of antiepileptic drugs, it has maintained an unrivaled role
in the therapeutic facilities and is recommended as first
line for partial and tonic–clonic seizures in developing
countries in all age groups (Anderson, 2002; Abou
Khaled and Hirsch, 2008). At ‘therapeutic’ dose, pheno-
barbital produces moderate changes in membrane con-
ductance, but displays its anticonvulsant effect mainly
by increasing c-amino butyric acid (GABA)-ergic-
mediated postsynaptic inhibition (Anderson, 2002;
Schmidt and Loscher, 2009). Phenobarbital interacts with
the GABA
A
receptor, which contains binding site for barbi-
turates and benzodiazepines and acts as a Cl
ion-
selective channel (Olsen, 2002). Phenobarbital increases
the mean channel open duration without affecting channel
conductance or opening frequency (Anderson, 2002;
Schmidt and Loscher, 2009).
As the main inhibitory neurotransmitter, GABA can
effectively control the excitability of neurons through
activation of its receptors in the central nervous system.
A-type GABA receptors are the major Cl
permeable
ion channels activated by GABA and the most
abundant fast inhibitory neurotransmitter receptors in
the mammalian brain. The GABA
A
receptors control
basal information processing in the central nervous
system, therefore allowing it to affect a wide variety
of physiological and pathophysiological processes
http://dx.doi.org/10.1016/j.neuroscience.2016.05.038
0306-4522/Ó2016 IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Address: Department of Physiology, Faculty
of Medical Sciences, Tarbiat Modares University, PO Box: 14115-
331, Tehran, Iran. Tel: +98-21-82883865; fax: +98-21-82884555.
E-mail address: mirnajaf@modares.ac.ir (J. Mirnajafi-Zadeh).
Abbreviations: ACSF, artificial cerebrospinal fluid; AD, afterdischarge;
ADT, afterdischarge threshold; EGTA, ethylene glycol tetraacetic acid;
eIPSC, evoked inhibitory post-synaptic current; HEPES, 4-(2-hydro
xyethyl)-1-piperazineethanesulfonic acid; IPSCs, inhibitory post-
synaptic currents; LFS, low-frequency stimulation; mIPSC, miniature
inhibitory post-synaptic current; TTX, tetrodotoxin.
Neuroscience 330 (2016) 26–38
26
(Twyman et al., 1989; DeLorey and Olsen, 1992; Brickley
and Mody, 2012; Rudolph and Mohler, 2014).
Low-frequency stimulation (LFS) has been considered
as a potential way for treatment of epilepsy. A lot of
evidences indicate the anticonvulsant action of LFS in
epileptic patients (Chkhenkeli and Chkhenkeli, 1997;
Yamamoto et al., 2002; Koubeissi et al., 2013) and exper-
imental models of epilepsies including kindling (Goodman
et al., 2005; Schiller and Bankirer, 2007; Rashid et al.,
2012). Application of LFS in amygdala inhibits epileptoge-
nesis by modulating the network of the limbic system
(Wang et al., 2014). LFS can be considered as a potential
therapeutic manner for pharmacoresistant patients. A sig-
nificant number of epileptic patients, especially those with
temporal lobe epilepsy, are pharmacoresistant (Engel,
1996; Regesta and Tanganelli, 1999; Bialer and White,
2010). Surgical incision may be considered as a treatment
choice in a small percent of these patients and it is asso-
ciated with serious side effects (Picot et al., 2008). There-
fore, many studies are performed to find new therapeutic
ways for treatment of these drug resistant patients with
epilepsy.
The mechanisms underlying antiepileptic effect of
LFS have still remained unclear. These mechanisms
may involve the activation of GABAergic terminals
and subsequent GABA release (Windels et al., 2000;
Mantovani et al., 2006). Consistent with this idea,
Cuellar-Herrera et al. have suggested that electrical stim-
ulation of para-hippocampal cortex is effective in patients
with less severe epilepsy, an effect which was associated
with a high GABA tissue content and a low rate of cell loss
(Cuellar-Herrera et al., 2004). Autoradiography experi-
ments also revealed an increase in benzodiazepine
receptor binding in basolateral amygdala following appli-
cation of LFS in amygdala-kindled rats; suggesting that
the antiepileptic effects of LFS may involve activation of
GABA-benzodiazepine system (Lopez-Meraz et al.,
2004). This anticonvulsant effect was related to enhance-
ment of the expression level of a5 subunits of extrasynap-
tic GABA
A
receptor (Shen et al., 2013). In addition, it has
been shown that the application of LFS may protect
against seizures by modulating the expressions of a
1
and b
2
subunits of GABA
A
receptor (Yang et al., 2014).
Our previous study implied an interaction between
LFS and phenobarbital in reducing seizure parameters
and suggested that a combined therapy of phenobarbital
and LFS may be a useful manner for drug resistant
epilepsies (Asgari et al., 2014). We supposed that if a
positive interaction exists between LFS and phenobarbi-
tal, then a combined therapy of animals with sub-
effective dose of phenobarbital and ineffective pattern of
LFS can significantly exert anticonvulsant effect (i.e.
greater than the sum of their individual effect). However,
the precise mechanism of this interaction remains to be
determined. Considering the fact that application of LFS
prevents the kindling-induced hyperexcitability in hip-
pocampal CA1 area (Ghotbedin et al., 2013), in present
study we tried investigate the possible interaction
between LFS and phenobarbital on GABA
A
receptor-
mediated currents in hippocampal CA1 pyramidal cell in
amygdala-kindling rat. Therefore, in continue to our
previous study, we applied an ineffective pattern of LFS
combined to a sub-effective dose of phenobarbital to
investigate whether a positive interaction exists between
the anticonvulsant effects of LFS and phenobarbital on
GABAergic currents.
EXPERIMENTAL PROCEDURES
Animals
Male Wistar rats (4 weeks at the time of surgery) were
housed with an ambient temperature of 22–25 °C and a
12-h light/dark cycle under water and food ad libitum. All
experimental and animal care procedures were
performed according to international guidelines on the
use of laboratory animals and approved by Tarbiat
Modares University Ethical Committee for Animal
Research, which is in line with the ‘‘NIH Guide for The
Care and Use of Laboratory Animals’’. Efforts were
made to minimize both the number of animals used and
their suffering.
Surgery
The rats were deeply anesthetized by a ketamine/
xylazine mixture (100/10 mg/kg, i.p.), fixed in a
stereotaxic frame (Paxinos and Watson, 1986). A bipolar
stimulating and a monopolar recording electrode were
twisted together and chronically implanted in the basolat-
eral amygdala of the right hemisphere, using the following
coordinates in mm: anteroposterior 2.5 and lateral 4.8
from bregma, depth 7.5 from skull surface. These
Teflon-coated, stainless steel electrodes were insulated
except at their tips (A-M Systems, Inc., WA, USA).
Another electrode connected to stainless steel screw
positioned in the skull as reference and ground elec-
trodes. All electrodes were connected to pins of a multi-
channel socket as a head stage and fixed to the skull with
dental acrylic.
The location of implanted electrodes was histologically
confirmed in animals at the end of experiments. Rats
were deeply anesthetized and perfused with 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.4)
prior to staining to verify electrode placements.
Kindling procedure
Following a post-surgical recovery period (7–10 days),
the afterdischarge (AD) threshold was determined by a
train of stimuli (1-ms monophasic square pulses, 50 Hz
for 3 s) as previously described (Asgari et al., 2014).
The stimulating current was initially delivered at 30 lA,
and then its intensity was increased in increments of
10 lA at 5-min intervals until ADs of at least 8 s were
recorded. Rats were electrically stimulated at the AD
threshold 12 times a day with an interval of 5 min. Epilep-
tiform ADs were continuously recorded following kindling
stimulations using a PC-based data acquisition system
(D3107; Science Beam Co., Tehran, Iran). The behavioral
seizure stages (1–5) were rated according to Racine’s
scale (Racine et al., 1977). After the appearance of the
stage 5 seizure, the animals were subjected to one
A. Asgari et al. / Neuroscience 330 (2016) 26–38 27
stimulation/ day in a chronic kindling manner. The animals
were considered as fully kindled when they exhibited
three consecutive stage 5 seizures.
Whole-cell patch clamp recording
Whole-cell patch clamp recording was performed in
hippocampal slices. Young adult rats were
anaesthetized with ether, euthanized by decapitation,
and the brains were quickly removed. The right
hippocampus (ipsilateral to the kindling site) was
dissected out, and transverse slices (400 lm) were cut
using a vibroslicer (Vibratome 1000 plus) in ice-cold
cutting solution containing the following component (in
mM): 238 sucrose, 2.5 KCl, 0.5 CaCl
2
, 2 MgSO
4
,1
NaH
2
PO
4
, 26.2 NaHCO
3
, and 11
D
-glucose and
equilibrated to a pH of 7.2–7.4 by 1 M KOH when it was
contentiously bubbled with carbogen (95% O
2
and 5%
CO
2
). The osmolarity was adjusted to 290–300 mOsm.
Thereafter, the slices were incubated in a holding
chamber containing carboxygenated artificial
cerebrospinal fluid (ACSF) composed of (in mM): 125
NaCl, 3 KCl, 2 CaCl
2
, 2 MgSO
4
, 1.25 NaH
2
PO
4
,25
NaHCO
3
, and 10
D
-glucose at pH of 7.2–7.4 and
osmolarity of 290–300 mOsm was kept at 32–35 °C for
60 min. Then, the slices were maintained at room
temperature (23–25 °C) before being transferred to a
recording chamber for at least 30 min. Individual slices
were transferred to a submerged recording chamber on
the stage of an upright microscope (Axioskop 2 FS
MOT; Carl Zeiss, Germany), which was continuously
superfused with the same ACSF used during incubation
at a rate of 2 ml/min. Whole-cell recordings under
voltage clamp condition were obtained from CA1
pyramidal neurons visually identified by an infrared CCD
camera (IR-1000, MTI, USA) with a 40- water
immersion objective. All recordings were made at room
temperature (23–25 °C) using filamented borosilicate
capillary glass (1.5 mm O.D. and 0.86 mm I.D., Sutter,
USA) filled with intracellular pipette solution containing
(in mM): 140 CsCl, 1CaCl
2
, 5 lidocaine N-ethyl bromide
(QX-314), 10 HEPES, 2 MgCl
2
,2Mg
2
ATP and 2
Na
2
GTP, 10 EGTA. The pH of the internal solution was
set to 7.2–7.4 by 1 M CsOH and the osmolarity was
adjusted to 290–300 mOsm. Electrode tip resistance in
the bath was typically 4–6 MO, and series resistance
ranged from 12 to 25 MO. Cells were rejected if series
resistance changes were more than 20% during
experiment. Capacitance compensation and bridge
balance were carried out. Data were low-pass filtered at
3 kHz and acquired at 10 kHz with a Multiclamp 700B
amplifier equipped with Digidata 1440 A/D converter
(Molecular Devices, CA, USA). The signal was recorded
on a PC for offline analysis using the Axon pClamp 10
and minianalysis software. After giga seal (more than
2GO) establishment, the whole-cell configuration was
attained simply by applying a brief suction.
The changes in GABAergic transmission were
evaluated by recording the evoked and miniature
GABA
A
receptor-mediated inhibitory postsynaptic
currents (IPSCs) in voltage clamp mode at a holding
potential of -60 mV in the continuous presence of
3-[[(3,4-Dichlorophenyl)methyl]amino]propyl]diethoxymethyl)
phosphinic acid (CGP; 5 lM) as GABA
B
receptor antagonist,
2-amino-5-phosphopentanoic acid (AP5; 50 lM) as NMDA
receptor antagonist and 6-cyano-7-nitroquinoxaline-2,3-
dione (CNQX; 20 lM) as AMPA receptor antagonist. To
record the evoked IPSCs (eIPSCs) in pyramidal cells, a
concentric bipolar stimulation electrode was placed close to
the stratum pyramidal layer approximately 150–200 lm
away from the recorded pyramidal neurons. The
stimulation strength was set at 1.5 times above eIPSC
threshold. The monophasic stimuli were delivered at
0.05 Hz with a pulse duration of 0.1 ms. In some
experiments, paired pulse ratio was also calculated by
stimulating the input with two pulses separated by 50 or
100-ms inter-pulse interval. Miniature IPSCs (mIPSCs)
were recorded at holding potential of -60 mV for 10 min.
mIPSCs were recorded in the presence of tetrodotoxin
(TTX; 1 lM) to block the action potential-dependent
responses. At the end of each experiment, the recorded
GABA
A
currents were confirmed through their blockade by
bicuculline (20 lM). The parameters of eIPSCs were
calculated by averaging of 6–8 consecutive records.
Occurrence of mIPSCs and their parameters were
analyzed off-line by minianalysis software (6). The
amplitude and area under curve of eIPSCs, amplitude and
inter-event intervals of mIPSCs and paired-pulse indices
were calculated.
Drugs
Phenobarbital was dissolved in 1 M NaOH and then was
diluted to desired concentration by ACSF. CGP, TTX
and CNQX were dissolved in ACSF. AP5 and bicucullin
were dissolved in dimethyl sulfoxide (DMSO) and then
were diluted to desired concentration by ACSF. All
chemicals were purchases from Sigma except CGP
which purchased from Tocris.
Experimental design
In experiment 1, animals were divided into kindled, sham
(underwent surgery but did not receive stimulation) and
control groups. Hippocampal slices were prepared at
24 h following the last kindling stimulation in kindled
group and at a similar time scale in sham and control
groups. Slices of both groups were assigned into three
subgroups which treated by phenobarbital, LFS or a
combination of phenobarbital and LFS. In LFS
subgroup, following the baseline recording (10 min)
monophasic square wave pulses of 0.1 ms duration
were applied at 1 Hz in the stratum radiatum. LFS was
applied at different number of pulses (20, 100 and 200
pulses) and different intensities (equal to AD threshold
(ADT) and 1.5 ADT of each animal). In other subgroup,
phenobarbital was superfused in the recording chamber
solution at concentrations of 50,100, 200 and 500 lM
following 10-min baseline recording. According to
obtained results of these two subgroups, slices of third
subgroup received a sub-threshold dose of phenobartital
(100 lM) along with the ineffective pattern of LFS (100
pulses at the intensities of equal to AD threshold) to
28 A. Asgari et al. / Neuroscience 330 (2016) 26–38
investigate the interaction between them. In this
subgroup, following 10 min baseline recording,
phenobarbital was added to the ASCF and 20 min later
LFS was applied in the presence of phenobarbital. Four
to five fully kindled animals were used in each
experimental group.
In experiment 2, animals were divided into sham
(control) and kindled groups. In kindled group, fully
kindled animals received the ineffective pattern of LFS
(1 Hz, 100 pulses at the intensity equal to AD threshold)
just before slice preparation. Animals of the control
group were also received LFS at the same time scale.
Phenobarbital (100 lM) was superfused in recording
chamber solution following an initial baseline recording
(10 min). In all experiments the number of animals in
different groups (control, sham and kindled) was 4–5.
Statistical analysis
Data were averaged and expressed as mean ± SEM.
Statistical analysis was performed using GraphPad
Prism version 6.01 for Windows (GraphPad Software,
Ca, USA). To compare the data of different groups
following application of different doses of phenobarbital
and/or different patterns of LFS during the recording
times one-way, two-way or three-way analysis of
variance (ANOVA) was used as appropriate, followed by
Tukey post hoc test. Student’s paired or unpaired t-test
was used when two groups of data were compared. To
construct cumulative probability plots, 400 random
events of mIPSCs were selected from each neuron and
their distributions were analyzed by Kolmogorov–
Smirnov test using Minianalysis software. Cumulative
plots were generated from amplitude and inter-event
intervals collected from individual cells under similar
experimental conditions. Recordings were made from
only one cell from each slice. Therefore, the reported n
refers to the number of cells or slices. A Pvalue of less
than 0.05 was considered statistically significant.
RESULTS
Effect of LFS on eIPSCs in CA1 pyramidal neurons in
control and kindled animals
In experiment 1, we studied the effect of different LFS
patterns on GABAergic currents. Application of 20 and
100 pulses of LFS at the intensity of ADT had no
significant effect on eIPSC parameters in control and
kindled groups (Fig. 1). 200 pulses of LFS at the
intensity of ADT increased the amplitude and area
under curve of eIPSCs in control and kindled groups
significantly (Fig. 1). As the Fig. 1 shows, the effect of
200 pulses of LFS on eIPSC parameters was long term
and sustained for 30 min. Similar results were obtained
when LFS was administered at the intensity of 1.5 ADT
(data not shown). Therefore, the effect of LFS on
evoked GABAergic currents was related to the number
of pulses. According to these results 100 pulses of LFS
at the intensity of ADT was considered as an ineffective
pattern of LFS to be used in the next experiments.
Effect of phenobarbital on eIPSCs in CA1 pyramidal
neurons in control and kindled animals
Phenobarbital was added to the aCSF following 10 min
baseline recording and then eIPSCs recording was
extended for 40 min. This chemical significantly
potentiated eIPSCs at the doses of 200 and 500 lMin
slices of both control and kindled animals (Fig. 2).
Although administration of phenobarbital at the dose of
200 lM had no significant effect on eIPSCs amplitude
and area-under curve in the control group (Fig. 2A),
however, when we measured the decay time constant
and slope of eIPSCs, a significant increase was
observed in these parameters following administration of
phenobarbital at the dose of 200 lM in the control group
(data not shown). Administration of phenobarbital at the
doses of 50 (not shown) and 100 lM(Fig. 2) had no
significant effect on eIPSC parameters. The effect of
phenobarbital on eIPSCs was dose dependent.
According to these results, 100 lM was selected as the
sub-threshold dose of phenobarbital to be used in the
next experiments.
Effect of co-administration of phenobarbital and LFS
on eIPSCs in CA1 pyramidal neurons in control and
kindled animals
In this experiment, following 10 min baseline recording,
phenobarbital (100 lM) was added to the aCSF and
LFS (100 pulses at ADT intensity) was applied 20 min
later. Similar to previous experiment, phenobarbital did
not significantly affect the recorded eIPCSs during the
first 20 min post its administration. However,
application of LFS (at its ineffective pattern) at the
presence of phenobarbital changed eIPSC parameters
significantly. In slices of kindled animals, amplitude
and area under curve of eIPSCs were increased
significantly when LFS was applied in the presence of
phenobarbital (Fig. 3B). In slices of the control group,
only the area under curve increased significantly
(Fig. 3A). The observed increase in the mentioned
parameters was lasting for 20 min following LFS
application. The bar diagrams in the right side of
Fig. 3 show the mean values of each parameter. Here
the data of LFS group were obtained from the first
experiment (i.e. Fig. 1). These data clearly show that a
combination of sub-threshold dose of phenobarbital
and ineffective pattern of LFS effectively changed the
eIPSC parameters. These changes were higher than a
simple additive effect.
Paired pulse ratio was also calculated by applying
two stimuli with 50- or 100-ms interval and measuring
the amplitude ratio of second to first eIPSC during
baseline and following application of LFS or
phenobarbital or a combination of both. There was no
change in paired-pulse ratio compared to baseline
following co-administration of LFS and phenobarbital at
inter-pule interval of 50 ms (data not shown)
and 100 ms (Fig. 4) which suggests a role for
postsynaptic mechanisms in mediating the effects this
co-administration.
A. Asgari et al. / Neuroscience 330 (2016) 26–38 29
200 pulses
100 pulses
20 pulses
20pu
lses
100 pulses
200 pulses
eIPSC Amplitude
(% of Baseline)
4 8 12 16 20 24 28 32 36 40
50
100
150
200
eIPSC Area Under
Curve (% of Baseline)
4 8 12 16 20 24 28 32 36 40
50
100
150
200
eIPSC Amplitude
(% of Baseline)
4 8 12 16 20 24 28 32 36 40
50
100
150
200
Time (min)
Time (min)
200 pulses
100 pulses
20 pulses
LFS 20 Pulses LFS 100 Pulses LFS 200 Pulses
B
50 pA
100 ms
ALFS 20 Pulses LFS 100 Pulses LFS 200 Pulses
50 pA
100 ms
50
100
150
200
***
20pu
lses
100 pulses
200 pulses
50
100
150
200
***
eIPSC Area Unde r
Curve (% of Baseline)
4 8 12 16 20 24 28 32 36 40
50
100
150
200
50
100
150
200
*
20pu
lses
100pulse
s
200 pulses
50
100
150
200
*
Before After
Before After
Fig. 1. Effect of LFS on eIPSCs in control (A) and kindled (B) groups. In A and B upper traces show sample records of eIPSCs before (pale line) and
after (dark line) application of different LFS patterns. The intensity of LFS in each animal was equal to its afterdischarge threshold. The time-course
diagrams on the left show the amplitude and area under curve of eIPSCs as percentage of baseline. Each graph shows the response before (as 10 min
baseline recording) and after application of LFS (black triangle). The bar graphs on the right show the mean value of data during baseline (Before) and
30 min following LFS (After) for three patterns of LFS. LFS had a significant increasing effect on amplitude and area under curve in control and kindled
groups when applied at 200 pulses. Values are mean ± SEM.
*
p< 0.05 and
***
p< 0.001 (n= 6–7 in control and n= 5–7 in kindled group).
30 A. Asgari et al. / Neuroscience 330 (2016) 26–38
Effect of co-administration of phenobarbital and LFS
on mIPSCs in CA1 pyramidal neurons in control and
kindled animals
At first, we determined the effect of phenobarbital or LFS
alone on GABA
A
-mediated mIPSC in kindled and control
hippocampal slices. The amplitude and inter-event
interval were calculated for all events. Similar to
previous experiment, application of phenobarbital
(100 lM) or LFS (100 pulses at ADT intensity) alone
had no significant effect on mIPSCs parameters in
control and kindled groups. However, co-application of
both phenobarbital and LFS significantly changed the
parameters of mIPSCs and potentiated the miniature
GABAergic currents. As Fig. 5 shows, co-administration
of phenobarbital and LFS significantly increased the
amplitude of mIPSCs in control and kindled groups as
denoted by a rightward shift of the cumulative probability
distributions; while there was no shift in the graph after
application of phenobarbital or LFS alone in both control
and kindled groups (Fig. 5). In addition, there was no
significant change in the inter-event intervals of mIPSCs
and no shift was observed in cumulative probability plots
of this parameter following co-administration of LFS and
phenobarbital (Fig. 5).
Effect of in vivo LFS on responses of hippocampal
IPSCs to in vitro phenobarbital in control and kindled
animals
After observing a positive interaction between LFS and
phenobarbital on the hippocampal slices of both control
Phenobarbital concentration (µM)
50 pA
100 ms
B
A
Before Pheno After Pheno
Phenobarbital concentration (µM)
Phenobarbital concentration (µM) Phenobarbital concentration (µM)
Pheno 100
µ
M Pheno 200
µ
M Pheno 500
µ
M
50 pA
100 ms
Pheno 100
µ
M Pheno 200
µ
M Pheno 500
µ
M
Before Pheno After Pheno
eIPSC Amplitude
(% of Baseline)
100 200 500
50
75
100
125
150
***
eIPSc Area Under
Curve (% of Baseline)
100 200 500
50
100
150
200
*
eIPSC Amplitude
(% of Baseline)
100 200 500
50
75
100
125
150
***
***
eIPSC Area Under
Curve (% of Baseline)
100 200 500
50
100
150
200
*
*
Fig. 2. The effect of phenobarbital on eIPSCs in control (A) and kindled (B) groups. In A and B upper traces show sample records of eIPSCs before
(pale line) and after (dark line) application of different concentrations of phenobarbital. Lower bar graphs show the changes in amplitude and area
under curve. The mean response of cells before application of phenobarbital (Before Pheno) was considered as baseline and the effect of
phenobarbital (After Pheno) was calculated as % of baseline. Phenobarbital had significant increasing effect in control group (A) when applied at the
concentration of 500 lM and in kindled group (B) when applied at the concentrations of 200 and 500 lM. Values are mean ± SEM.
*
p< 0.05 and
***
p< 0.001 (n= 6–7 in control and kindled groups).
A. Asgari et al. / Neuroscience 330 (2016) 26–38 31
and kindled animals in vitro, the question was raised
whether in vivo application of LFS at the seizure focus,
i.e. amygdala in our experiments, can also change the
responsiveness of hippocampal slices to phenobarbital.
Therefore, in this experiment the ineffective pattern of
LFS (100 pulses at 1 Hz) was applied in the amygdala
and immediately after application of LFS, brain slices
were prepared from control and kindled animals.
Similar to previous experiments, 10 min baseline
recording was done at first, then the sub-threshold dose
2 6 10 14 18 22 26 30 34 38 42 46 50
50
100
150
200
250
eIPSC Amplitude
(% of Baseline)
2 6 10 14 18 22 26 30 34 38 42 46 50
50
100
150
200
Time (min)
eIPSC Area Under
Curve (% of Baseline)
50
100
150
200
250
eIPSC Amplitude
(% of Baseline)
2 6 10 14 18 22 26 30 34 38 42 46 50
50
100
150
200
250
eIPSC Amplitude
(% of Baseline)
Time (min)
eIPSC Area Under
Curve (% of Baseline)
2 6 10 14 18 22 26 30 34 38 42 46 50
50
100
150
200
***
50
100
150
200
250
eIPSC Amplitude
(% of Baseline)
***
eIPScAreaUnder
Curve (% 0f Baseline)
LFS
Pheno
LFS+Pheno
50
100
150
200
***
***
B
A
eIPSC Area Under
Curve (% of Baseline)
LFS
Pheno
FS+Pheno
50
100
150
200
***
*
L
Fig. 3. The interaction of phenobarbital (100 lM) and LFS (100 pulses, 1 Hz) on eIPSCs in control (A) and kindled (B) groups. Graphs in the left are
the time-course diagrams showing the amplitude and area under curve of eIPSCs as percentage of baseline. Each graph shows the changes in
IPSC parameters before (as 10 min baseline recording) and after application of phenobarbital. The thick line above each graph shows the duration
of phenobarbital application. LFS (black triangle) was applied 20 min following phenobarbital. The bar graphs on the right show the mean % of
baseline values of data following application of phenobarbital alone (Pheno) and after application of LFS in the presence of phenobarbital (Pheno
+ LFS). The first bar, named LFS, has been drawn according to the data of Fig. 1 when LFS was applied alone. Although application of LFS and
phenobarbital alone had no significant effect on eIPSC parameters, co-administration of both of them increased amplitude (in control and kindled
groups) and area under curve (in kindled group). Values are mean ± SEM.
*
p< 0.05 and
***
p< 0.001 (n= 7–10 in control and n= 6 in kindled
group).
32 A. Asgari et al. / Neuroscience 330 (2016) 26–38
of phenobarbital (100 lM) was added to the aCSF and
eIPSCs or mIPSCs were recorded in different slices.
Phenobarbital had no significant effect on different
parameters of eIPSCs in the control group. However,
there was a significant increase in area under curve of
eIPSCs recorded in kindled animals which received
in vivo LFS (Fig. 6A2). No changes were observed in
the amplitude of eIPSCs in control and kindled groups
(Fig. 6A). In addition, application of phenobarbital
(100 lM) in animals which received in vivo LFS,
increased area under curve, but not the amplitude of
mIPSC in control and kindled animals (Fig. 6B). Paired-
pulse ratio was also measured during baseline and after
phenobarbital application. There was no significant
change in this ration for eIPSC amplitude at inter-
stimulus interval of 50 ms (data not shown) and 100 ms
(Fig. 6C).
DISCUSSION
Obtained results indicated that pretreatment of either the
seizure focus or hippocampal slices by an ineffective
pattern of LFS can increase the responsiveness of
GABAergic currents to phenobarbital. On the other
hand, co-application of a sub-threshold dose of
phenobarbital and an ineffective pattern of LFS can
significantly affect the evoked and miniature IPSCs in
hippocampal pyramidal neurons. These data are in
parallel to our previous experiment which showed a
positive interaction between the anticonvulsant effects of
LFS and phenobarbital (Asgari et al., 2014). In addition,
the present results suggest that potentiation of the
GABAergic currents can be considered as a probable
mechanism for the positive interaction between LFS and
phenobarbital.
Amygdala kindling is accompanied with neuronal
hyperexcitability in seizure focus and other areas which
have a role in seizure generation and propagation, such
as hippocampus (Mirnajafi-Zadeh et al., 2002;
Ghotbedin et al., 2013; Shojaei et al., 2014; Moradi
Chameh et al., 2015). Our previous experiments showed
that application of LFS prevented the amygdala kindling-
induced hyperexcitability of hippocampal CA1region by
measuring both passive and active membrane properties
(Ghotbedin et al., 2013). Therefore, the observed effects
of LFS in the present study are in line with our previous
experiments. Obtained data clearly showed that LFS
can potentiate the GABAergic currents in hippocampal
slices in a long-term manner so that it was stable for at
least 30 min. This action of LFS was dependent on its
parameters so that increasing the number of pulses and
intensity of LFS resulted in the increment of its effect.
Our previous experiments also showed that the anticon-
vulsant effect of LFS is dependent on these parameters
in a similar manner (Ghorbani et al., 2007; Shahpari
et al., 2012; Asgari et al., 2014). It has to be noted that
control brain sections do not present the same effects
as those obtained by kindled sections. In fact, the effect
of LFS is dependent on the baseline synaptic activity
(Faingold, 2004; Liu et al., 2013). Therefore, in the pre-
sent study the different effects of LFS on GABAergic cur-
rents in control and kindled animals may be related to the
different level of neuronal activity of these two groups.
To explore the positive interaction between LFS and
phenobarbital on GABA
A
currents, we selected an
ineffective pattern of LFS (100 pulses) and a
sub-threshold dose of phenobarbital (100 lM). We
applied LFS after treating the slices by phenobarbital to
mimic our previous in vivo experiments in which the
phenobarbital was injected intraperitoneally 30 min
before LFS administration (Cuellar-Herrera et al., 2010).
Using this protocol, we observed a significant effect of
phenobarbital and LFS on IPSCs parameters, while they
had no effect on measured currents when applied alone.
It this way, we could confirm the interaction of LFS with
the phenobarbital-mediated enhancement of GABA
A
cur-
rents in both control and kindled groups.
Several mechanisms have been suggested for LFS
anticonvulsant effects including those involved in LTD or
depotentiation (Velisek et al., 2002; Ozen et al., 2008;
Wu et al., 2008) such as activation of opioid (Lopez-
Meraz et al., 2004), galanin (Sadegh et al., 2007) and
adenosine (Mohammad-Zadeh et al., 2009) receptors.
A1
LFS Pheno LFS+Pheno
A2
B
50 pA
100 ms
pp Index
LFS Pheno Pheno+LFS
50
75
100
125
150
pp Index
LFS Pheno Pheno+LFS
50
75
100
125
150
Before After
Before After
Fig. 4. Effect of LFS, phenobarbital and their co-administration on
paired pulse indices of eIPSCs in control (A1-2) and kindled (B)
groups. Sample records show the eIPSCs in response to paired
pulses with inter-pulse interval of 100 ms before (pale) and after
(dark) application of LFS (left), phenobarbital (middle) and pheno-
barbital + LFS (right) in control group. In both control (A2) and
kindled (B) groups there was no significant different in paired pulse
indices before and after application of LFS, phenobarbital and
phenobarbital + LFS. Values are mean ± SEM (n= 7–10 in control
and n= 6 in kindled group).
A. Asgari et al. / Neuroscience 330 (2016) 26–38 33
A1
LFS
Before
A2
B
Inter-event Interval (m s)
Inter-event Interval (m s)
Cumulative frequency
Amplitude (pA) Amplitude (pA)
Amplitude (pA)
Inter-event Interval (m s)
Cumulative frequency
Inter-event Interval (m s) Inter-event Interval (m s)
Cumulative frequency
Amplitude (pA) Amplitude (pA)
Amplitude (pA)
P
LFS
Pheno
heno+LFS
40 pA
1 s
Before After
***
mIPSC Inter-event
Interval (% of Baseline)
50
75
100
125
150
mIPSC Amplitude
(% of Baseline)
LFS
Pheno
Pheno+LFS
50
75
100
125
150
**
mIPSC Inter-event
Interval (% of Baseline)
50
75
100
125
150
mIPSC Amplitude
(% of Baseline)
50
75
100
125
150
Inter-event Interval (m s)
Cumulative frequency
0 300060009000
0.0
0.2
0.4
0.6
0.8
1.0
0 3000 6000 9000
0.0
0.2
0.4
0.6
0.8
1.0
0300060009000
0.0
0.2
0.4
0.6
0.8
1.0
20 40 60 80
0.0
0.2
0.4
0.6
0.8
1.0
20 40 60 80
0.0
0.2
0.4
0.6
0.8
1.0
20 40 60 80
0.0
0.2
0.4
0.6
0.8
1.0
0 3000 6000 9000
0.0
0.2
0.4
0.6
0.8
1.0
0 3000 6000 9000
0.0
0.2
0.4
0.6
0.8
1.0
0 3000 6000 9000
0.0
0.2
0.4
0.6
0.8
1.0
20 40 60 80
0.0
0.2
0.4
0.6
0.8
1.0
20 40 60 80
0.0
0.2
0.4
0.6
0.8
1.0
20 40 60 80
0.0
0.2
0.4
0.6
0.8
1.0
Before After
Phenobarbital LFS+Phenobarbital
After
Fig. 5. The interaction of phenobarbital (100 lM) and LFS (100 pulses, 1 Hz) on mIPSCs in control (A1-2) and kindled (B) groups. Sample records
show the mIPSCs before (pale) and after (dark) application of LFS (left), phenobarbital (middle) and phenobarbital + LFS (left) in control group.
While application of LFS and phenobarbital (Pheno) alone had no significant effect on mIPSCs parameters, their co-administration (Pheno + LFS)
significantly increased mIPSC amplitude, but not mIPSC inter-event intervals in both control (A2) and kindled (B) groups. Similarly, the cumulative
frequency curves obtained after application of LFS or phenobarbital (dark lines) perfectly had overlap with curves obtained before their application
(dark lines). However, the cumulative frequency curve for mIPSC amplitude had shift to right after application of phenobarbital + LFS (dark)
compared to before (pale) their application. Values are mean ± SEM.
**
p< 0.01 and
***
p< 0.001 (n= 11–12 in control and n= 6–8 in kindled
group).
34 A. Asgari et al. / Neuroscience 330 (2016) 26–38
Activation of GABAergic terminals and subsequent GABA
release may also be considered as a mechanism of LFS
action (Yamamoto et al., 2002; Koubeissi et al., 2013).
Consistent with this idea, Cuellar-Herrera et al. (2004)
have suggested that the anticonvulsant action of electrical
stimulation of parahippocampal cortex in epileptic patients
is associated with a high GABA tissue content. In addi-
tion, Dostrovsky and Lozano (2002) hypothesize that
the short duration of inhibition produced by electrical stim-
ulation of basal ganglia is due to activation of the
GABAergic terminals of neurons, leading to release of
GABA and inhibition of the postsynaptic neuron.
Added to above evidences, previous studies have
suggested that a1 and a5 GABA
A
receptors are the
major isoforms underlying GABAergic inhibition in
hippocampal CA1 pyramidal cells (McKernan and
Whiting, 1996; Pirker et al., 2000; Caraiscos et al.,
2004; Serwanski et al., 2006; Glykys et al., 2008). It has
been reported that LFS application increases the expres-
sion of GABA
A
receptor a5 subunit (Shen et al., 2013). A
recent study has also shown that application of LFS in the
hippocampus increases the content of GABA in the brain
through expression of SV2A (a synaptic vesicle protein)
(Wang et al., 2014). Meanwhile, it is suggested that the
beneficial effect of the electrical stimulation on focal epi-
lepsy may be due to its desynchronizing and GABA-
induced hyperpolarizing effects (Durand and Warman,
1994). Therefore, some of LFS anticonvulsant actions
might be mediated through the activation of GABA-
benzodiazepine receptors. Our results provided the first
report which shows the direct effect of LFS on GABAergic
currents and confirms the above mentioned reports. In
addition to GABAergic synapses, the occurrence of some
changes in glutamatergic neurotransmission can be also
postulated. For example, SV2A is expressed in gluta-
matergic neurons of the hippocampus (Bajjalieh et al.,
1994; Acsady et al., 1998) and therefore LFS-induced
changes in SV2 expression can affect not only GABAer-
gic, but also glutamatergic transmission. In the present
study the latency between LFS application and changes
in IPSCs parameters was very short (about 2–3 min).
Therefore, obtained results less likely could be related
to gene or protein expression.
It has to be noted that according to the results of the
present study, similar to phenobarbital action, LFS
*
50
100
150
200
50
100
150
200
*
mIPSC Area Under
Curve (% of Baseline)
50
100
150
200
*
mIPSC Area Under
Curve (% of Baseline)
50
100
150
200
pp Index
Control
Before
Kindled
80
100
120
140
2 6 10 14 18 22 26 30 34 38
50
100
150
200
eIPSC Area Under
Curve (% of Baseline)
After
Before
After
2 6 10 14 18 22 26 30 34 38
50
100
150
200
eIPSC Area Under
Curve (% of Baseline)
A2
A1
B2
B1
C
2 6 10 14 18 22 26 30 34 38
50
75
100
125
150
Time (min)
eIPSC Amplitude
(% of Baseline)
Baseline
LFS+Pheno
50
75
100
125
150
2 6 10 14 18 22 26 30 34 38
50
75
100
125
150
Time (min)
eIPSC Amplitude
(% of Baseline)
Baseline
LFS+Pheno
50
75
100
125
150
mIPSC Amplitude
(% of Baseline)
Baseline
LFS+Pheno
50
75
100
125
150
mIPSC Amplitude
(% of Baseline)
Baseline
LFS+Pheno
50
75
100
125
150
3
Fig. 6. The interaction of in vivo application of LFS (100 pulses, 1 Hz)
and in vitro phenobarbital (100 lM) on eIPSCs and mIPSCs in control
(A1 and B1) and kindled (A2 and B2) groups. Animals received LFS
before slice preparation. In A1 and B1, graphs in the left are the time-
course diagrams showing the amplitude and area under curve of
eIPSCs as percentage of baseline. Each graph shows the changes in
amplitude or area under curve of eIPSCs before (as 10 min baseline
recording) and after application of phenobarbital. The thick line above
each graph shows the duration of phenobarbital application. The bar
graphs on the right show the mean value of data during baseline and
30 min following phenobarbital (LFS + Pheno). The sub-threshold
dose of phenobarbital (100 lM) increased the area under curve of
eIPSC in kindled group, significantly. Similarly, a significant increase
in area under curve (but not amplitude) of mIPSCs in response to
sub-threshold dose of phenobarbital (100 lM) in control (B1) and
kindled (B2) animals. However, no significant changes were
observed in paired-pulse indices following sub-threshold dose of
phenobarbital in control and kindled groups (C). Values are mean
± SEM.
*
p< 0.05 (n= 6–10 in control and n= 6 in kindled group).
A. Asgari et al. / Neuroscience 330 (2016) 26–38 35
increases GABAergic neurotransmission. It is well known
that combined therapy using different targets results in
better effects instead of using the same target.
Therefore, in the future studies it is necessary to
combine LFS with non-GABAergic antiepileptic drugs.
There are few reports about the combined therapy of
electrical stimulation and anticonvulsant drugs. For
example, application of high-frequency stimulation to the
ventral hippocampus combined with drug therapy would
get a better control of seizures in rats (Cuellar-Herrera
et al., 2010). Deep stimulation of both the amygdala and
hippocampus can also decrease the required dosage of
anticonvulsant drugs in some patients (Vonck et al.,
2002; Kerrigan et al., 2004). Similarly, the results
obtained in the present study and our previous report
showed the first approach to evaluate the effects of LFS
combined with an antiepileptic drug on the intensity and
incidence of seizure activity and GABAergic currents
(Asgari et al., 2014). However, while we used LFS, other
mentioned studies used high frequency stimulation which
may exert its effects through different mechanisms com-
pared to LFS. In addition, the results of the present study
have to be validated also in different animal model of epi-
lepsy and with other anticonvulsant drugs.
Our results indicate that application of phenobarbital
and LFS enhances postsynaptic GABA
A
receptor
functions without altering the GABAergic transmission
onto pyramidal neurons through increasing presynaptic
GABA release because co-application of phenobarbital
and LFS increased only the amplitude (confirmed by the
rightward shift in the cumulative frequency curves of
mIPSC amplitude) without influencing the inter-event
interval of mIPSCs. This was ensured further by
observing no change in paired-pulse index of GABA
A
receptor-mediated eIPSCs because changes in the
action of postsynaptic receptors are usually relates to
no changes in paired-pulse index. Therefore, all of the
changes in evoked and miniature IPSCs can be related
to the changes in GABA
A
receptor function not to
alteration in GABA release. However, additional
experiments need to perform to find the exact
mechanism(s) involved in this phenomenon.
Interestingly, when LFS was applied in vivo in
seizure focus (i.e. amygdala in this study) it could
affect the responsiveness of hippocampal neurons to
phenobarbital. In this situation, LFS was applied at first
and phenobarbital was administered several hours after
on. It means that, the positive interaction between LFS
and phenobarbital can be observed following different
manners of their application and is not limited to in vitro
administration of LFS and phenobarbital. On the other
hand, it seems that the efficacy of LFS to interact with
another anticonvulsant agent is not limited to a specific
situation. In addition to kindled group, application of LFS
in control animals changes the response of brain slices
to the sub-effective dose of phenobarbital. However, this
action of LFS was lower than that of kindled group and
only the area under curve of mIPSCs was changes
significantly. As discussed previously, this difference
may be related to the different level of neuronal activity
of these two groups. It must be noted that in experiment
2 the interval between LFS application (in vivo) and
phenobarbital administration (in vitro) was at least
90 min (the time of brain slice preparation and slice
incubation). Therefore, the magnitude of interaction
between LFS and phenobarbital was lower than what
observed in experiment 1. Of course, because there
was an increase in the lonely eIPSCs or mIPSCs area
under curve but not in the amplitude of IPSCs, we can
only suggest a possible effect of phenobarbital on
GABA
A
receptor kinetic not a strong effect.
CONCLUSION
Our findings showed that application of an ineffective
pattern of LFS, with no significant effect on GABAergic
currents, can potentiate these currents when applied
following a sub-threshold dose of Phenobarbital. These
results confirm a positive interaction between LFS and
phenobarbital and suggest a potential combined therapy
in drug resistant epileptic patients to achieve a
satisfactory clinical effect.
CONFLICT OF INTEREST
The authors claim that there is no financial or other
conflicts of interest that might be construed as
influencing the results or interpretation of this study.
Acknowledgment—This study was supported by a grant from
Tarbiat Modares University and Kerman Neuroscience Research
Center (No 93-16/A).
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(Accepted 16 May 2016)
(Available online 26 May 2016)
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