Brain Research Bulletin 131 (2017) 25–38

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Brain Research Bulletin

journal homepage: www.elsevier.com/locate/brainresbull

Research report

Altered functional efficacy of hippocampal interneuron during

epileptogenesis following febrile seizures
Yeon Hee Yu, Kahyun Lee, Dal Sik Sin, Kyung-Ho Park, Dae-Kyoon Park ∗ , Duk-Soo Kim ∗
Department of Anatomy, College of Medicine, Soonchunhyang University, Cheonan-Si, Chungcheongnam-Do, 31151, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Febrile seizure (FS) is the most common seizure type in infants and young children. FS may induce
Received 7 October 2016 functional changes in the hippocampal circuitries. Abnormality of excitatory and inhibitory neurotrans-
Received in revised form 17 February 2017 missions was previously related to wide-spread seizure attack in the hippocampus following recurrent
Accepted 23 February 2017
seizure onset. To clarify the involvement of expressional changes and functional alterations of hippocam-
Available online 7 March 2017
pal interneurons with epileptogenesis following FS, we investigated long-term effects following recurrent
seizure in a hyperthermia-induced seizure animal model. At 12 weeks following FS, the recurrent seizure
Keywords:
time period, local field potentials (LFP) revealed high amplitude potential and a sharp wave characteris-
Febrile seizure
GABAA -␣1
tic of epilepsy. Mossy fiber reorganization in the hippocampus was also detected as abnormal synaptic
Calretinin connection at 8 weeks. Calretinin (CR) −positive interneurons were transiently enhanced during epilep-
Field excitatory postsynaptic potential togenic period at 7–9 weeks after FS in the CA1 and DG region and it is double labeled with VGLUT-1.
Epileptogenesis However, although GABAA -␣1 immunoreactivities were un-changed as similar to control hippocampus
Paired-pulse response at 7–9 weeks after seizure onset, its expression was significantly enhanced at 4 weeks and 12 weeks
and it is colocalized with GABA. Furthermore, the field excitatory postsynaptic potential (fEPSP) and
the paired-pulse responses including population spike (PS) latency, excitability ratio and PS2/PS1 ratio
were markedly altered in the CA1 and DG region at 12 weeks after FS. Therefore, our findings in present
study indicate that these time-dependent changes may be based on the persistent alterations of hip-
pocampal neuronal circuits in balance between excitatory and inhibitory responses, and may lead to the
epileptogenesis and spread of seizure activity following FS.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction may sustain enhanced hippocampal excitatory circuits and con-

Febrile seizure (FS) induced by fever is the most common seizure
type in infants and young children, which occurs in 3–5% of chil-
dren between 6 months and 5 years of age (Toth et al., 1998; Virta
et al., 2002; Bender et al., 2003). Animal models have indicated that
seizures of immature animals through neurogenesis in the dentate
gyrus (DG) at postnatal 1–2 weeks may more effectively induce
the innervations of newly generated granule cells (Parent et al.,
1997; Koyama et al., 2012; Scott and Holmes, 2012). Therefore, pro-
longed FS may induce the alteration in hippocampal circuitry (Dube
et al., 2000; Bender et al., 2003; Kwak et al., 2008). These changes
Abbreviations: CR, calretinin; DG, dentate gyrus; LFP, Local field potentials; FS,
Febrile seizure; fEPSP, field excitatory postsynaptic potential; GABA, ␥-aminobutyric
acid; LTP, Long-term potentiation; PBS, phosphate-buffered saline; PS, population
spike; SE, status epilepticus; sTPS, strong theta-patterned stimulation; TLE, temporal
lobe epilepsy.
∗ Corresponding authors.
E-mail addresses: mdeornfl@sch.ac.kr (D.-K. Park), dskim@sch.ac.kr (D.-S. Kim).
tribute toward the development of temporal lobe epilepsy (TLE)
by enhanced hyperexcitatory neuronal circuit to epileptogenesis
during developmental stage in infant (Dubé et al., 2006).
␥-Aminobutyric acid (GABA) is the major inhibitory neuro-
transmitter. It is affected by the intermediation of GABAA and
GABAB receptors in the brain (Brooks-Kayal et al., 1998; Matthews
et al., 1998). GABAA receptors are main inhibitory receptors that
mediate many pathophysiological aspects (Sieghart et al., 1999).
However, calretinin (CR)-positive interneurons innervate diverse
interneuronal subtypes in cortical areas and have an apprecia-
ble disinhibitory effect on pyramidal neurons in the hippocampus
(Barinka and Druga, 2010; Barinka et al., 2012). CR-positive
interneurons control the contact of other interneuronal dendritic
and exodendritic with each other and selectively innervate several
interneurons (Gulyás et al., 1999; Bausch, 2005; Barinka and Druga
et al., 2010).
GABAergic interneurons are decreased in the hippocampus of
various epileptic models; these declines may involve epileptogen-
esis (Sloviter et al., 1991; Fritschy et al., 1999; Bouilleret et al., 2000;

http://dx.doi.org/10.1016/j.brainresbull.2017.02.009
0361-9230/© 2017 Elsevier Inc. All rights reserved.
26 Y.H. Yu et al. / Brain Research Bulletin 131 (2017) 25–38
Bausch, 2005; Kwak et al., 2005; Sloviter et al., 2006; Kwak et al.,
2008). Moreover, interneuron CR and GABAergic cells have been
implicated in epileptic animal models and in humans (Maglóczky
et al., 2000; André et al., 2001; Slézia et al., 2004; Van Vliet
et al., 2004; Tóth et al., 2010). GABAB receptor and parvalbumin-
mediated inhibitory alterations in the hippocampus have been
documented in an animal model of FS (Kwak et al., 2008). How-
ever, the effects of other interneuronal alterations including GABAA
receptor subunits and CR-positive interneurons during epilepto-
genic periods following FS in the hippocampus remain unclear.
We investigated the temporal involvement of expressional patterns
and functional alterations of GABAA -␣1 and CR in the hippocam-
pal interneurons with epileptogenesis following recurrent seizure
onset.
2. Materials and methods
2.1. Experimental animals
All experiments utilized the progeny of Sprague-Dawley (SD)
rats obtained from Experimental Animal Center, Soonchunhyang
University (Cheonan, South Korea). All animals were provided with
a commercial diet and water ad libitum under controlled tempera-
ture, humidity and lighting conditions (light/dark cycle 12:12, and
22 ± 2 ◦ C, 55 ± ; 5%). All animal protocols were approved by the
Administrative Panel on Laboratory Animal Care of Soonchunhyang
University (permit No. SCH15-0002). All possible efforts were to
avoid suffering of the rats and to minimize the number used during
the experiments.
2.2. FS induction
The hyperthermic seizure rat model of FS has been described
(Kwak et al., 2008). Briefly, rat pups (postnatal 11 days) were used,
because hippocampal developmental at that age is generally equiv-
alent to human infants (Baram et al., 1997; Gottlieb et al., 1977;
Scantlebury et al., 2005). After punch-marking of their ears, pups
were warmed in a plastic chamber measuring 10 × 13 cm at the
base and 12 cm in height. The chamber floor was covered with
paper towels. A 175W mercury vapor lamp was held 3 cm above
the chamber. Core temperatures were measured before the induc-
tion of hyperthermic seizure and checked every 5 min using an ear
thermoprobe. All animals showed generalized seizures at 5–10 min
after hyperthermic seizure induction, when core temperature was
41–43 ◦ C. Hyperthermic seizures were maintained in 40 min after
generalized seizure onset because the duration for febrile seizures
are maintained minimum 15 min more than one seizure in a 24 h
period, result in transient neuronal injury and associate adjust-
ing epilepsy (Maytal et al., 2000; Dubé and Baram, 2006; Graves
et al., 2012). During maintenance of hyperthermic seizures, ani-
mals showed multiple generalized seizures (Racine, 1972; stage 4
and 5). After hyperthermic seizure, rats were moved to a cool sur-
face, and were then returned to their mothers (mortality, n = 9 out
of 315, 3% approximately). Siblings of the rats were used as controls,
and they were placed in a chamber at room temperature. In order
to completely identify FS inductions, we were observed behavior
at recurrent seizures stage through video monitoring for 24 h. As a
result, recurrent seizure was observed at interval of approximately
4 h each day in the vivarium for general behavior (Racin scale crite-
ria, 2.3 ± 0.07). In addition, we have double cross-checked under
EEG monitoring for observation of FS induction. Thus, we have
utilized only completely inducted animal model for FS induction
animal groups in this study. After recurrent seizure onset (10–12
weeks after hyperthermic seizure induction, n = 95 out of 134, ∼71%
approximately), behavioral seizures were scored based on Racine
scale criteria [30,stage 1, mouth and facial movements; stage 2,
head nodding; stage 3, forelimb clonus; stage 4, rearing; stage 5,
rearing and falling].
2.3. Local field potentials (LFP)
At the designated times (1–12 weeks following control and FS,
n = 20 respectively), each animal was anesthetized by intraperi-
toneal injection of urethane 1.5 g/kg and placed in a stereotaxic
frame. Holes were drilled through the skull for the introduction of
electrodes. In young animals (1–6 weeks after FS), LFP was recorded
using a tungsten parylene electrode (0.005-inch outer diameter;
A-M Systems, USA). The coordinates (in mm) referenced to the
bregma were as follows (to neocortex): 1.0 posterior to bregma,
1.0 lateral to midline, 0.7 depths. In older animals (7–12 weeks
after FS), glass microelectrodes (microfilament capillary 1.2 outer
diameter; 5–10 M) filled with artificial cerebrospinal fluid (ACSF,
in mM; NaCl 126, KCl 5, CaCl2 2, MgCl2 2, NaH2 PO4 1.25, NaHCO3
26, d-glucose 10, pH 7.2) were used. The coordinates (in mm) ref-
erenced to bregma were as follows (in mm to dentate gyrus): 3.8
posterior to bregma, 2.5 lateral to midline, 2.9 depths. Signals were
recorded with QP511 AC amplifier (0.1–3000 Hz bandpass, GRASS
Technologies, USA) and data were digitized (5 kHz) and recorded
to obtain the baseline value for 2 h. The single-channel acquisition
was performed using the Axoscope 10.2 (Axon Instruments, USA)
software. Analysis of the single-channel electrical traces was car-
ried out using the Clampfit 10.2 (Axon Instruments, USA) software.
To analyze changes in normalized power of LFP, the amplitude
spectrum analysis of normalized power was estimated by event
frequency, and the root mean square (RMS) values were used to
derive estimates of spectral power (mV2 ) in the 1 Hz frequency
bins for each electrode site. Spectral power values were averaged
across all epochs within a single base-line, the resulting power
was expressed as mV2 /Hz. For each subject, fast fourier transform
(FFT) of the epochs with a resolution of 0.61 Hz was computed
for all electrodes and then averaged. Non-overlapping hamming
windows controlled spectral leakage. Moreover, The FFT power
value measurements within each frequency between 1 and 50 Hz
were averaged to create 50 non-overlapping <1 Hz frequency bins,
because the frequency bands of interest were defined as: ␦ (1–4 Hz),
␥ (25.0–50 Hz) (Schutter et al., 2003; Lee et al., 2004; Scheffer-
Teixeira et al., 2013; Fuggetta et al., 2014). After LFP recording, some
animals were used for paired-pulse responses, immunoreactivity,
immunofluorescence and Timm’s staining.
2.4. Paired-pulse responses
The paired-pulse responses from hippocampus following hyper-
thermic seizure were recorded (10–12 weeks after hyperthermic
seizure and control, n = 10 respectively) using published protocols
(Kim et al., 2007, 2008), with some modifications. Briefly, animals
were anesthetized (urethane, 1.5 g/kg, i.p.) and placed in a stereo-
taxic frame. Holes were drilled through the skull for introducing
electrodes. The coordinates (in mm) referenced to bregma were
as follows. For the unipolar recording electrode (to the dentate
gyrus); 3.8 posterior to bregma, 2.0 lateral to midline, 3.5 depths.
For the bipolar stimulating electrode (to the angular bundle);
8.0 posterior to bregma, 4.4 laterals to midline, 4.0 depths were
placed. Electrode depths were finally determined by optimizing the
evoked response. Glass microelectrodes (microfilament capillary
1.2 outer diameters; 5–10 M◦ ) filled with artificial cerebrospinal
fluid (ACSF, in mM: NaCl 126, KCl 5, CaCl2 2, MgCl2 2, NaH2 PO4
1.25, NaHCO3 26, d-glucose 10, pH 7.2) and bipolar tungsten stimu-
lating electrodes were used. Body temperature was monitored and
maintained at 37 ± 0.3 ◦ C by thermostat during recording. Stimuli
were applied as DC square pulses at 0.5 Hz with pairs of 150 ␮s
 Y.H. Yu et al. / Brain Research Bulletin 131 (2017) 25–38
constant current stimuli at 30-ms inter-stimulus intervals (GABAA
receptor-mediated inhibition, stimulus intensity; 2 × thresholds)
using a Digital Stimulus Isolation unit (Getting Instruments, CA,
USA) depending on the threshold intensity evoking the popula-
tion spike, whereas 70-ms and 150-ms of inter-stimulus interval
revealed the measurements of facilitation and slow inhibition
(GABAB receptor-mediated inhibition, Kwak et al., 2008). Dur-
ing baseline recording, the population spike threshold of dentate
gyrus was determined and stimulus intensity was increased until
a population spike was detected consistently. One hour after base-
line recording, paired-pulse responses were recorded. To obtain
input/output curve, stimulus intensities were changed from 0.5 to
4 × thresholds. Then responses were recorded in each train with
30 min inter-train intervals. Signals were recorded with a P55 A.C.
pre-amplifier (3–1000 Hz bandpass, Astro-Med Inc., USA) and data
were digitized (20 kHz) and analyzed using WinLTP ver 2.01 soft-
ware (WinLTP Ltd., USA). To analyze changes in evoked responses,
population spike (PS) amplitude was normalized by the baseline
values (the average of the PS amplitude of the first response)
from the same animal and the same recording session. The ratio
of the second PS amplitude/the first PS amplitudes was deter-
mined to measure GABAA -mediated inhibition. In addition, the
latency and amplitude of the PS following first evoked response
were analyzed for measuring the extent of glutamatergic synap-
tic transmission in the dentate gyrus before and after recurrent
seizure onset (Feng and Durand, 2004; Matzen et al., 2008). PS
amplitude and PS latency in first evoked responses were normal-
ized by the baseline values from the same animal and the same
recording session. Furthermore, in order to investigate the effi-
ciency of glutamatergic synaptic transmission in the dentate gyrus
the excitability ratio was calculated as the PS amplitude versus
the field excitatory postsynaptic potential (fEPSP) slope in the first
evoked response (Kwak et al., 2008). Briefly, the averages of the
PS amplitude and the fEPSP slope of the first response at stim-
ulus intensity 2 × threshold were used to normalize all of the PS
amplitudes and the fEPSP slope measurements during the record-
ing session from the same animal and the same recording session.
Paired-pulse responses for age-matched control animals were also
measured using the same method. After recording, animals were
used for immunohistochemistry, immunofluorescence or Timm’s
staining.
2.5. In vivo field EPSP recordings
Field potentials expressed as fEPSPs were recorded from the
CA1 of the hippocampus as described (Wong et al., 2007; Ge et al.,
2010; Cho et al., 2013), with some modifications. At the recur-
rent seizure time period (>7 weeks after FS and control, n = 10
respectively), animals were intraperitoneally anesthetized using
urethane 1.5 g/kg and placed into a stereotaxic frame. Rectal tem-
perature was maintained at 37 ± 0.3 ◦ C during the course of the
surgery using a temperature controller (Harvard Instruments, USA).
The scalp was opened and separated. Holes were drilled through
the skull for introducing electrodes. The coordinates (in mm) ref-
erenced to bregma were as follows. For the recording electrode
(to the Schaffer collateral): 4.0 posterior to bregma, 3.0 lateral to
midline, 2.5 depths. For the stimulating electrode (to the stratum
radiatum of CA1): 3.5 posterior to bregma, 2.0 lateral to midline, 3.5
depths were positioned in the hippocampus. Electrode depths were
finally determined by optimizing the evoked response. fEPSPs were
adjusted to ∼60% of maximal response size for testing. Stimulation
was generated by a BNC-2110 apparatus (National Instruments,
USA) and a Digital Stimulus Isolation unit (Getting Instruments,
CA, USA). Pyramidal neuron responses to the Schaffer collateral
stimulation were recorded with a P55A.C. pre-amplifier (3–1000 Hz
bandpass, Astro-Med Inc.) and analyzed using WinLTP ver 2.01 soft-
27
ware (WinLTP Ltd.). Responses were evoked by single pulse stimuli
and were delivered at 20-s intervals. A stable baseline was recorded
for 30–60 min. Long-term potential (LTP) was induced by the strong
theta- patterned stimulus (sTPS, four trains of 10 bursts of 5 pulses
at 400 Hz with a 200-ms inter-burst interval, 15-s inter-train inter-
val), because sTPS (bursts of 400 Hz stimuli) are NMDA-receptor
dependent and robust LTP of CA1 and DG areas are evoked by
sTPS in vivo (Cho et al., 2013; Farmer et al., 2004). To analyze the
changes of fEPSP, slopes of fEPSP were averaged over 60-s intervals
and expressed as percentages of the mean fEPSP slope measured
during the 30-min baseline period, which was expressed as 100%.
The responses of fEPSP for age-matched control animal were also
measured using the same method.
2.6. Timm’s staining
For identifying mossy fiber reorganization, Timm’s staining was
performed. Briefly, one to 12 weeks of the FS and control animal
model (n = 5 respectively) were intraperitoneally anesthetized with
urethane 150 mg/kg and perfused through the heart by 0.9% saline
for 10 min, by 0.6% sodium sulfide and 0.6% sodium phosphate for
10 min, and finally by 4% paraformaldehyde for 10 min. The same
method was used to perfuse age-matched control animals. The
brains were removed and postfixed in the same fixative for 12 h,
and rinsed in phosphate buffer (pH 7.4, PB) containing 30% sucrose
at room temperature for 1 day. Thereafter, the tissues were frozen
and sectioned with a cryostat at 30 ␮m and consecutive sections
were collected in six-well plates containing phosphate buffered
saline (PBS). Some hippocampal tissues attached to gelatin-coated
slides were incubated in the dark in a solution consisting of one-
part solution A (1 M AgNO3 ), 20 parts solution B (2% hydroquinone
and 5% citric acid in water), and 100 parts solution C (20% gum
arabic in water). Development took about 3 h, was monitored by
periodic evaluations under low light at 36.5 ◦ C. The reaction was
terminated by washing in water.
2.7. Immunohistochemistry and immunofluorescence
Anesthesized animals (urethane 1.5 kg/kg) were perfused tran-
scardially with PBS followed by 4% paraformaldehyde in 0.1 M PB
(1–12 weeks after hyperthermic seizure and control, n = 15 respec-
tively). The brains were removed and postfixed in the same fixative
for 4 h, and rinsed in PB containing 30% sucrose at 4 ◦ C for 2 days.
Thereafter, the tissues were frozen and sectioned with a cryo-
stat at 30 ␮m thickness and consecutive sections were collected
in six-well plates containing PBS. For cell counts, every sixth sec-
tion in the series throughout the entire hippocampus was used
in the same animals. Sections were incubated with each primary
antibody in PBS containing 0.3% Triton X-100 overnight at room
temperature: mouse anti-CR IgG (Chemicon, USA; diluted 1:1000)
and rabbit anti-GABAA -␣1 IgG (Millipore, USA; diluted 1:400). The
sections were washed three times for 10 min with PBS, incubated
sequentially in biotinylated goat anti-mouse and goat anti-rabbit
IgG (Vector, USA) and ABC complex (Vector), and diluted 1:200 in
the same solution as the primary antiserum. Between the incuba-
tions, the tissues were washed with PBS three times for 10 min each.
The sections were visualized with 3,3 -diaminobenzidine (DAB)
in 0.1 M Tris buffer and mounted on gelatin-coated slides. The
immunoreactions were observed using DMRB microscope (Leica,
Germany) and images were captured using a model DP72 digital
camera and DP2-BSW microscope digital camera software (Olym-
pus, Japan). To establish the specificity of the immunostaining,
a negative control test was carried out with pre-immune serum
instead of primary antibody. The negative control resulted in the
absence of immunoreactivity in any structures. To identify the
morphological changes induced by hyperthermic seizure in the
28 Y.H. Yu et al. / Brain Research Bulletin 131 (2017) 25–38
same hippocampal tissue, double immunofluorescence staining for
CR/vesicular glutamate transporter-1 (VGLUT-1) (7–9 weeks after
hyperthermic seizure and control, n = 15 respectively) and GABAA -
␣1/GABA (10–12 weeks after hyperthermic seizure and control,
n = 15 respectively) was performed. Brain tissues were incubated
overnight at room temperature in mixture of rabbit anti-CR IgG
(Abcam, USA, diluted 1:500)/guinea pig anti-VGLUT-1 IgG (Chemi-
con, USA, diluted 1:2500) and rabbit anti-GABAA -␣1 IgG (Millipore,
USA; diluted 1:200)/mouse anti-GABA IgG (Abcam, UK; diluted
1:300). After washing three times for 10 min with PBS, sections
were also incubated in a mixture of Cy2- and Cy3-conjugated sec-
ondary antisera (1:200, Amersham, USA) for 1 h room temperature.
Sections were mounted in Vectashield mounting media with DAPI
(Vector, USA). All images were captured using a model Fluoview
FV10i and FV10i software (Olympus, Japan).
2.8. Western blot
Based on the immunohistochemical results, of the expression
of CR and GABAA -␣1 protein was quantified in the control and FS
hippocampus as previously described (Blancher and Jones, 2001;
Mahmood and Yang, 2012). At designated times (4, 8 and 12 weeks
after FS and control, n = 5 respectively), immunoblot analysis was
performed. After sacrifice and removal of the hippocampus, the
tissues were homogenized in 50 mMTris-HCl (pH 8.0) contain-
ing 150 mM NaCl, 50 mM NaF, 0.5% NP-40, 5 mM EDTA, 1 mM
phenyl methyl sulfonyl fluoride (PMSF), and 1 mMA protinin. After
centrifugation at 14000 rpm, the protein concentrations in the
supernatants were determined as previously described (Bradford,
1976). Proteins were separated by 12% SDS-PAGE. Equal amounts
of protein (30 ␮g in all assays) were loaded in each lane with
loading buffer containing 1 M Tris-HCl (pH 6.8), 20% glycerol, 10%
SDS, 25% ␤-mercaptoethanol and 0.002% bromphenol blue. Sam-
ples were heated at 100 ◦ C for 5 min before gel loading. After
electrophoresis, the gels were transferred to nitrocellulose trans-
fer membranes (Bio-Rad, USA). To reduce background staining,
the filters were incubated with 5% non-fat skim milk in PBS for
1 h and sequentially incubated with the primary antibody in PBS
overnight at 4 ◦ C. Antibody used was mouse anti-CR IgG (Chemicon,
USA; diluted 1:1000) or rabbit anti-GABAA -␣1 IgG (Millipore, USA;
diluted 1:200). After washing, the membranes were incubated with
peroxidase-conjugated goat anti-rabbit IgG secondary antibody
for 1 h (Jackson ImmunoResearch Inc., USA; diluted 1:10000) and
anti-mouse IgG (Santa Cruz Biotechnology, USA; diluted 1:2000).
Immunoblots were visualized using an ECL kit (Advansta Inc., USA),
and the protein density was quantified by densitometric analysis
using Image J software (NIH, USA).
2.9. Quantification of data and statistical analysis
An optical fractionation was used to estimate the cell num-
bers. The optical fractionators (combination of performing counting
with the optical dissector, with fractionators sampling) is a stereo-
logical method based on a properly designed systematic random
sampling method that by definition yields unbiased estimates
of population number. The sampling procedure is accomplished
by focusing through the depth of the tissue (the optical dissec-
tor height, h; of 15 ␮m in all cases for this study). The number
of each cell type (C) in each of the subregion is estimated as:
C = ˙Q− × t/h × 1/asf × 1/ssf, where Q− is the number of cells actu-
ally counted in the dissectors that fell within the sectional profiles
of the subregion seen on the sampled sections, and asf is the areal
sampling fraction calculated by the area of the counting frame of the
dissector, a(frame) (50 × 50 ␮m2 in this study) and the area asso-
ciated with each x, y movement grid (x, y step) (of 250 × 250 ␮m2
in this study) {asf = [a(frame)/a(x,y step)]}. ssf is the fraction of the
sections sampled or section sampling fraction [minimum six tis-
sues per each animal models (1–12 weeks FS and control groups,
n = 15 respectively) in this study, since we used all sections in
the entire hippocampus]. The immunoreactive cells were counted
with a 40 × objective lens. All immunoreactive cells were counted
regardless the intensity of labeling. Cell counts were performed
by two different investigators who were blind to the classifica-
tion of tissues. For quantification of immunodensity [4,8 and 12
weeks after FS and control groups for WB (n = 5 respectively); 1–12
weeks following FS and control groups for Timm’s staining (n = 5
respectively)], CA1 and DG were delineated with a 2.5 × objective
lens. Each image was normalized by adjusting the black and white
range of the image using Adobe PhotoShop v. 8.0. Thereafter, 10
areas per rat (250 ␮m2 for each area) were selected and intensity
measurements represented as the mean number of a 256 gray scale
(using NIH Image 1.59 software). Values of background staining
were obtained from the corpus callosum. Optical density values
were corrected by subtracting the average values of background
noise obtained from five image inputs. All data obtained from the
quantitative measurements were analyzed using one-way analysis
of variance (ANOVA) to determine statistical significance. Bonfer-
roni’s test was used for post-hoc comparisons. A p-value < 0.01
or <0.05 was considered statistically significant (Kim et al., 2007,
2008).
3. Results
3.1. Representative LFP profiles following FS
Distinguishing characteristic of electroencephalography in the
control and epileptic hippocampus following FS were showed sig-
nificant differences. Although control rats showed a normal LFP in
the 8 weeks (Fig. 1A1 and C1) and 12 weeks (Fig. 1A3 and C3),
LFP signals at 12 weeks following FS showed epileptiform dis-
charges, which was characterized intermittently high amplitude
field potentials and sharp waves in DG and CA1 as compared to
control (Fig. 1A4 and C4), whereas its signal at 8 weeks follow-
ing FS revealed as similar to control groups (Fig. 1A2 and C2). In
addition, the power spectral analysis from the LFP recording of
the hippocampus following FS showed strong power in a lower
frequency of the hippocampus than the control (Fig. 1B1 and
D1), and its normalized power in DG and CA1 was more pow-
erful in the FS hippocampus (FS 8 weeks, 0.414 ± 0.08 in CA1,
0.456 ± 0.02 in DG; FS 12 weeks, 0.99 ± 0.25 in CA1, 0.45 ± 0.02 in
DG) as compared with the control (control 8 weeks, 0.248 ± 0.01
in CA1, 0.198 ± 0.03 in DG; control 12 weeks, 0.02 ± 0.25 in CA1,
0.02 ± 0.08 in DG; P < 0.01, Fig. 1B2 and 1D2). Especially, the delta
rhythm synchronous neuronal activity of hippocampus, which as
a unique LFP signaling marker of epilepsy (Di Gennaro et al.,
2003; Gelisse et al., 2011), was significantly elevated in the hip-
pocampus of 12 weeks following FS than in the control (FS 12
weeks, 67.40 ± 1.21 in CA1, 57.89 ± 2.97 in DG; control 12 weeks,
60.27 ± 1.38 in CA1, 41.78 ± 2.03 in DG; P < 0.01, Fig. 1B3 and D3),
whereas its signal at 8 weeks after FS was similar to control
groups (FS 8 weeks, 41.18 ± 3.76 in DG, 60.75 ± 1.70 in CA1; con-
trol 8 weeks, 40.86 ± 2.80 in DG, 54.68 ± 3.84 in CA1). However,
the gamma rhythm associated with hyper brain activity, which is
linked to GABAergic interneurons (White et al., 2000; Wendling
et al., 2002; Hughes, 2008), had a significantly declined rate in
the hippocampus of 12 weeks following FS than in the control
(FS 12 weeks, 2.17 ± 0.44 in CA1, 2.20 ± 0.55 in DG; control 12
weeks, 3.47 ± 0.31 in CA1, 10.02 ± 2.16 in DG; P< 0.01, P< 0.05,
Fig. 1B4 and 1D4), although its signals at 8 weeks following FS
shown similar result with control groups (FS 8 weeks, 3.042 ± 0.47
 Y.H. Yu et al. / Brain Research Bulletin 131 (2017) 25–38 29

Fig. 1. Local field potential profiles in DG and CA1 between the control and the epileptic hippocampus following FS. Normal control rats show a general representative LFP
signals at 8 weeks (A1 and C1) and 12 weeks of control groups (A3 and C3). At the recurrent seizure time period (12 weeks following FS), the LFP signals show significant
epileptiform discharges, as large amplitude spikes of irregularly sharp wave and multi-spikes (A4 and C4), although its signal is similar to control group at 8 weeks after FS
(A2 and C2). Power spectral analysis after FS revealed more power in lower frequency than the control group (B1 and D1). Representative average normalized power of 12
weeks after FS shows strong power more than control (B2 and D2). At the same time period, although synchronous neuronal activity of delta wave is markedly elevated,
gamma wave declines compared with control (B3-B4 and D3-D4), whereas these signals at 8 weeks after FS show strong normalized power only in the DG of hippocampus
(B2-B4 and D2-D4). All data are presented as mean ± SEM. Significant differences from the control. *p < 0.05, **p < 0.01, vs. control.

in CA1, 10.70 ± 0.53 in DG; control 8 weeks, 5.880 ± 1.25 in CA1, than control levels as shown synaptic reorganizations (P < 0.01,
9.907 ± 2.95 in DG). Fig. 2C1–C2, D1–D2 and E).

3.3. Immunohistochemical characterizations of CR and

3.2. Mossy fiber reorganization following hyperthermic seizure GABAA -˛1 expression following FS

In order to investigate the anatomical synaptic alterations of
development and adult stage following FS, we identified the ter-
minals of mossy fiber using Timm’s staining. Although diverse
synaptic fibers of mossy fiber in the dentate gyrus were observed
in the development stage of control (control 2 weeks, Fig. 2A1 and
A2), these fibers were declined on the hilar region following hyper-
thermic seizure, especially in the granule cell layer as compared
to controls (44.15 ± 2.03% in FS 2 weeks; 100 ± 2.53% in control 2
weeks; P < 0.01, Fig. 2B1–B2 and E). At the adult stage (8 weeks), the
axonal processes of mossy fiber in the dentate gyrus following FS
were significantly enhanced in the inner molecular (272.05 ± 2.58%
in FS 8 weeks; 100 ± 2.55% in control 8 weeks) and granule cell layer
(210.31 ± 3.02% in FS 8 weeks; 100 ± 2.53% in control 8 weeks) more
To identify the functional alteration of interneurons in the
hippocampus following hyperthermic seizure, immunohistochem-
istry analyses for CR and GABAA -␣1 receptors were done. The
immunoreactivities of CR −positive interneurons in the control
were observed in the CA1 and DG of hippocampus in the develop-
ing (Fig. 3A1 and A2) and adult hippocampus (Fig. 3C1 and C2). At
4 weeks after FS, CR immunoreactivity is enhanced only in the DG
than control (14 ± 0.70 in FS 4 weeks; 8.5 ± 1.06 in control 4 weeks;
P < 0.05, Fig. 3A1-A2, B1-B2 and F). No differences of immunoblot
analysis were apparent at 4 weeks of development (99.56 ± 26.77%
in FS 4 weeks; 100 ± 56.87%, in control 4 weeks; Fig. 3G and H). At 8
weeks following FS, CR-positive interneurons were markedly ele-
vated in each region more than control level (FS 8 weeks, 14.5 ± 0.35
30 Y.H. Yu et al. / Brain Research Bulletin 131 (2017) 25–38

Fig. 2. Timm’s staining in the dentate gyrus of control and epileptic hippocampus following FS during developmental (A1-B2) and adult stages (C1-D2). The staining reveals
dark labeling mainly in the hilar region of control animals including some of the granule cell layer (arrow, A1 and A2), while its labeling is reduced in the DG (GCL, HL and
IML) at 2 weeks following hyperthermic seizure (B1 and B2). At 8 weeks after recurrent seizure onset, the dense mossy fiber sprouting in the DG, including GCL and IML,
is observed (arrow, D1 and D2) more than in the control (C1 and C2). Rectangles in panels A1, B1, C1 and D1 indicate the high-magnification of panels A2, B2, C2 and D2.
Bar = 200 ␮m (panels A1, B1, C1 and D1), 25 ␮m (panels A2, B2, C2 and D2). Densitometric analysis for these staining is obtained same results in the GCL and IML (E). All data
are presented as mean ± SEM. Significant differences from the control. **p < 0.01, vs. control. Abbreviations: GCL, granule cell layer; HL, hilar region; IML, inner molecular
layer.

in DG, 14 ± 0.70 in CA1; control 8 weeks, 8 ± 0.70 in DG, 11 ± 0.17
in CA1; P < 0.05, Fig. 3C1–C2, D1–D2 and F). WB analysis for CR
expression also revealed significantly enhancements at 8 weeks
following FS more than control groups (265.30 ± 44.62% in FS 8
weeks; 100 ± 48.30% in control 8 weeks; P < 0.01, Fig. 3G and H).
In addition, at this time period, CR-positive interneurons were
double labeled with VGLUT-1 immunoreactivities in the CA1 and
DG region following FS, and its number of colocalized interneu-
rons were significantly enhanced more than control 8 weeks (FS
8 weeks, 226.98 ± 53.99% in CA1, 244.44 ± 33.89% in DG; con-
trol 8 weeks, 100 ± 25.25% in CA1, 100 ± 22.22% in DG; P < 0.05,
P < 0.01, Fig. 4A1–D4, E and F). However, the immunoreactivity of
CR-positive interneurons at recurrent seizure period (10–12 weeks
following hyperthermic seizure) was significantly down-regulated
in the CA1 (9.5 ± 0.62 in FS 12 weeks; 8.5 ± 0.35 in control 12 weeks)
and DG regions (11.75 ± 0.23 in FS 12 weeks; 10 ± 0.70 in control
12 weeks), similar to control 8 and 12 weeks (Fig. 3E1–E2 and F).
Immunoblot analysis also revealed similar results with immuno-
histochemistry data at 12 weeks after FS (95.00 ± 30.82% in FS 12
weeks; 100 ± 48.30% in control 8 weeks; Fig. 3G and H).
On the other hand, the immunoreactivity of GABAA -␣1 positive
interneurons was distinguished from the CR expressions follow-
ing FS. Briefly, GABAA -␣1 immunoreactivities at 4 weeks were
detected in the diverse interneruons of CA1 and DG regions in
the control hippocampus (Fig. 5A1–A3), whereas its expression
was remarkably elevated age-matched hippocampus following FS
(FS 4 weeks, 24.5 ± 1.76 in CA1, 32 ± 0.70 in DG; control 4 weeks,
15.5 ± 0.35 in CA1, 25 ± 0.77 in DG; P < 0.05, Fig. 5B1-B3 and G).
Immunoblot analysis also revealed same results with immuno-
histochemical data at this time points (189.62 ± 19.66% in FS 4
weeks; 100 ± 14.54% in control 4 weeks; P < 0.01, Fig. 5I and J). How-
ever, GABAA -␣1 immuonreactive interneurons at 8 weeks after FS
were significantly down-regulated both regions as similar to con-
trol groups (FS 8 weeks, 20.75 ± 0.51 in CA1, 22.5 ± 0.77 in DG;
control 8 weeks, 23 ± 1.41 in CA1, 19.5 ± 0.35 in DG; Fig. 5C1–C3,
D1-D3 and G), and western blot experiments also shown very sim-
ilar results with immunohistochemistry data (98.74 ± 14.13% in FS
8 weeks; 100 ± 13.02% in control 8 weeks; Fig. 5I and J). At the
recurrent seizure time period (12 weeks following hyperthermic
seizure), the numbers of GABAA -␣1 positive interneurons were re-
enhanced in the CA1 and DG regions as compared to the control
group (FS 12 weeks, 32 ± 0.71 in CA1, 30.75 ± 0.65 in DG; control
12 weeks, 22.5 ± 0.35 in CA1, 22.5 ± 0.35 in DG; P < 0.01, Fig. 5E1-
E3, F1-F3 and G). At the same time periods, immunofluorescence
staining revealed that GABAA -␣1 positive interneurons were dou-
ble labeled with GABA immunereactivity in both regions, and
its numbers of colocalized interneuronal population at 12 weeks
following recurrent seizure onset were significantly enhanced
more than control groups (Fig. 5H1–H4). Immunoblot and den-
sitometric analysis of GABAA -␣1 protein also showed as similar
results with immunohistochemical data (214.55 ± 16.64% in FS
12 weeks; 100 ± 13.02% in control 8 weeks; P < 0.01, Fig. 5I and
J).
 Y.H. Yu et al. / Brain Research Bulletin 131 (2017) 25–38 31

Fig. 3. Calretinin (CR) immunoreactivities and quantitative analyses in the control and the epileptic hippocampus following FS (A1- F). At four weeks after FS, CR immunore-
activity is markedly enhanced in the DG than control (open arrow, A2 and B2), whereas its expression in CA1 similar to control groups (A1 and B1). CR-positive interneurons
at 8 weeks following hyperthermic seizure in hippocampus are enhanced in CA1 and DG (open arrow, D1 and D2) as compared to control levels (C1 and C2). At recurrent
seizure time period (12 weeks following FS), the expressions of CR-positive interneuron are significantly reduced as similar to controls groups (E1 and E2). Bar = 50 ␮m
(panels A1- E2). The results of average cell number for CR-positive interneurons are shows as similar with immunohistochemical data (F). The immunoblot (G) and optical
density analyses (H) are revealed although the CR protein unchanged at 4 weeks after FS as compared to control, its protein level markedly elevated in the hippocampus at
8 weeks following seizure onset. All data are presented as mean ± SEM. Significant differences from the control, *p < 0.05, **p < 0.01, vs. control.

3.4. Paired-pulse responses and fEPSP following FS
To identify the glutamatergic synaptic transmissions by NMDA
receptors in the hippocampal CA1 region following FS because of
that NMDA receptor is an important role in regulating the GABAer-
gic input by GABA release (Maccaferri and Dingledine, 2002), we
investigated the activity-dependent synaptic plasticity under the
induction of NMDA receptors-mediated LTP at the time of recur-
rent seizure onset. The fEPSPs evoked by electrical stimulation
of the Schaffer collateral pathway at 0.05 Hz were recorded from
the stratum radiatum of the CA1 region. After a stable baseline
recording was obtained, LTP was induced by sTPS (four trains of
10 bursts of 5 pulses at 400 Hz with a 200 ms inter-burst inter-
val, 15-s intertrain interval), since robust LTP of CA1 and DG
areas are evoked by sTPS in vivo (Cho et al., 2013; Farmer et al.,
2004). As shown in Fig. 6A, this sTPS stimulation reliably elicited
LTP of the slope of fEPSPs in the CA1. At recurrent seizure time
periods, the amplitudes and the slope of evoked fEPSP follow-
ing LTP were significantly reduced as compared to control groups
(126.9 ± 2.5% in FS 12 weeks; 169.7 ± 3.9% in control 12 weeks;
P < 0.01, Fig. 6A–C). In order to investigate the evoked poten-
tials by paired-pulse stimulation from the dentate gyrus following
hyperthermic seizure, paired-pulse responses were recorded using
the perforant path stimulation. At recurrent seizure time period
(12week after FS), the PS2/PS1 ratio (the paired-pulse ratio) at
the 30-ms inter-stimulus interval (GABAA receptor-mediated fast
inhibition) was significantly reduced (0.62 ± 0.03) as compared
to control (1.18 ± 0.06 in control; P < 0.01, Fig. 6D and E), simi-
lar to other studies (Tsai and Leung, 2006; Kwak et al., 2008),
whereas 70-ms and 150-ms of inter-stimulus interval indicate a
facilitation and an action of GABAB receptor-mediated slow inhi-
bition. In addition, the excitability ratio at this time period was
elevated to 191 ± 24.4% (100 ± 8.4% in control 12 weeks; P < 0.01,
Fig. 6E). Moreover, the PS amplitude and the PS latency in the
first response, as well as the ratio of the second with the first
PS amplitude (PS2/PS1), were calculated to evaluate the change
of the neuronal excitability, synaptic transmission, and GABAergic
inhibition from interneurons (Feng and Durand, 2004). PS ampli-
tude after tetanic stimulation was increased at 12 weeks following
FS to 149.1 ± 5.7% (137.8 ± 7.0% in control 12 weeks; Fig. 6E),
whereas the PS latency after tetanic stimulation was significantly
down-regulated as compared to the condition before tetanic stim-
ulation to 90.5 ± 0.5% (100 ± 1.5% in control 12 weeks; P < 0.01,
Fig. 6E).
4. Discussion
Prior studies used diverse FS animal models to identify epilepto-
genic factors, because FS is a potential etiologic factor in human TLE
(Shinnar and Glauser, 2002; Dubé et al., 2006). These animal models
have indicated that hippocampal cell death or damage could pre-
lude epileptic seizures and mesial temporal lobe epilepsy (Cendes
et al., 1993; Kuks et al., 1993; VanLandingham et al., 1998; Sarkisian
et al., 1999; Tanabe et al., 2001). However, the models were limited
by un-matched age with human infants of FS patients (Dobbing
and Sands, 1973), absence of neuronal death or morphological
changes, and lower induction rate of FS (Baram et al., 1997; Bender
et al., 2003; Scantlebury et al., 2005; Dubé et al., 2006). Kwak et al.
(2008) developed a rat model to study the long-term effects of FS
and epileptogenesis. Advantages of these models include the high
morbidity to recurrent seizure using single hyperthermia (∼68%
of animals), morphological changes, lower mortality (∼2.9% of ani-
mals), and the age matched with human infants. Therefore, we used
the rat hyperthermic seizure model to investigate the long-term
effect of FS and the functional and/or morphological alterations of
hippocampal interneurons involved epileptogenesis.
32 Y.H. Yu et al. / Brain Research Bulletin 131 (2017) 25–38

Fig. 4. Double staining for CR and VGLUT1 in the hippocampus at 8 weeks after FS (A1-D4). DAPI counterstaining (blue, A1-A4); CR (green, B1-B4); VGLUT-1 (red, C1-C4);
Merged-images (yellow, D1-D4). CR immunoreactivity is detected in some interneurons of hippocampus following FS. Both CR and VGLUT1 are colocalized in the same
interneurons of the CA1 and DG at 8 weeks after FS than control groups (arrow). Bar = 17 ␮m (panels A1-D4). The results of average cell number for CR and VGLUT double
labeled interneurons are shows as similar with immunofluorescence staining (E and F). All data are presented as mean ± SEM. Significant differences from the control,
*p < 0.05, **p < 0.01, vs. control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4.1. LFP alterations following FS
During recurrent seizure time period, LFP signals generally
showed an epileptic wave as a fast spike and high amplitude multi-
spikes in the hippocampus; these signals were more noticeable in
the CA1 than in the DG region. The LFP signal in CA1 region appears
as an output signal of the local circuit, whereas these signals are an
input signal in the DG region incoming to the hippocampus (Takano
and Coulter, 2012). Thus, the present results indicate that the CA1
region is more vulnerable area for the epileptogenesis and the
seizure activity following FS, because of declined control abilities
to hyperexcitatory signal in the hippocampus. However, additional
experiments are necessary to verify the relationship between the
influences of input and output signals for epileptogenesis in this
animal model. On the other hand, delta rhythm synchronous neu-
ronal activity in the hippocampus offers a unique LFP signal marker
in epilepsy (Di Gennaro et al., 2003; Gelisse et al., 2011). In addi-
tion, gamma rhythm associated with hyper brain activities have
been linked to the influences of GABAergic interneurons (White
et al., 2000; Wendling et al., 2002; Hughes, 2008). According to the
properties of these brain waves, the present findings demonstrate
that the delta rhythm during the recurrent seizure period was sig-
nificantly increased in the hippocampus, whereas gamma rhythm
was reduced in CA1 and DG region. Therefore, our findings suggest
that these changes of LFP signal result in the enhanced epileptiform
discharges accompanied by abnormalities between hyperexcita-
tory neuronal transmission and inhibitory influences of GABAergic
interneurons at recurrent seizure time period following FS.
4.2. Morphological changes of synaptic connection following FS
In order to investigate the abnormal synaptic connections after
axonal reorganization, Timm’s staining was used from immature
(developmental stage) to mature (adult stage) brain following
hyperthermic seizure. The staining method labels zinc-rich ter-
minals of mossy fiber and can identify axon reorganization
(Buckmaster et al., 2002). Activity-dependent anatomical reorgani-
zation of mossy fiber is an important mechanism of development
and adult neural plasticity for recurrent connection of dentate
gyrus, principle neurons and interneurons (Cline, 2001; Wong and
Ghosh, 2002; Ge et al., 2006). Moreover, axonal reorganization is
important in the excitatory neuronal circuit in epileptic hippocam-
pus; a novel recurrent excitatory network involving glutamatergic
neurotransmission has been demonstrated in patients and in ani-
mal models of TLE (Victor Nadler et al., 1980; Tauck and Nadler,
1985; Buckmaster et al., 2002). These facts may contribute to
epileptogenesis that occurs through excitation by abnormal synap-
tic connection in FS (Kwak et al., 2008). Similar to these previous
reports, the present data demonstrate axonal reorganizations in
the DG region during development (2 weeks after FS). In addition,
the dense mossy fiber sprouting in the granule cell layer including
inner molecular layer of DG was detected at the adult time period
 Y.H. Yu et al. / Brain Research Bulletin 131 (2017) 25–38 33

Fig. 5. GABAA -␣1 immunoreactivities and quantitative analyses in CA1 and DG of the control and the epileptic hippocampus following FS (A1-G). At 4 weeks after FS,
GABAA -␣1 immunoreactivity is markedly increased in the hippocampal interneurons in both regions more than control (open arrow A1-B3). At 8 weeks following recurrent
seizure onset, GABAA -␣1 immunoreactivity is reduced in the hippocampal interneurons as similar to control levels (C1-D3). However, GABAA -␣1 positive interneurons are
significantly re-enhanced in the CA1 and DG region at recurrent seizure time period (12 weeks after FS) as compared to controls (open arrow E1-F3). Rectangles in panels
A1, B1, C1, D1, E1 and F1 indicate the high-magnification of panels A2-A3, B2-B3, C2-C3, D2-D3, E2-E3 and F2-F3. Moreover, rectangles in panels E2 and F2 indicate the right
low high-magnification of panels E2 and F2. Bar = 200 ␮m (panels A1, B1, C1, D1, E1 and F1), 50 ␮m (panels A2-A3, B2-B3, C2-C3, D2-D3, E2-E3 and F2-F3) and 25 ␮m (high-
magnification in panels E2 and F2). Quantitative analysis for average numbers of GABAA -␣1 positive interneurons is shown as similar with imuunohistochemical data (G).
All data are presented as mean ± SEM. Significant differences from the control, *p < 0.05, **p < 0.01, vs. control. Double immunofluorescence staining for GABAA -␣1 receptor
and GABA in the hippocampus at 8 weeks after FS (H1-H4). GABAA -␣1 receptor (green); GABA (red); Merged-images (yellow, H1-H4). GABAA -␣1 receptor immunoreactivity
is detected in some hippocampal interneurons following FS. Both GABAA -␣1 receptor and GABA are double labeled in the same interneurons of the CA1 and DG at 12 weeks
after recurrent seizure onset than control groups (arrow H3 and H4). Bar = 17 ␮m (panels H1-H4). Western blot (I) and optical density analysis (J) of GABAA -␣1 antibody are
revealed its protein levels at developmental and adult stages. Interestingly, at 4 weeks after recurrent seizure onset, GABAA -␣1 are transiently increased as compared to
control groups, while its expression is observed as similar between each groups at 8 weeks following seizure onset (I and J). According time course following FS, GABAA -␣1
protein levels also markedly re-enhanced at recurrent seizure time period (12 weeks after FS) as compared to age-matched control hippocampus (I and J). All data are
presented as mean ± SEM. Significant differences from the control, **p < 0.01, vs. control. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

(8 weeks after FS) following hyperthermic seizure. Inhibitory hilar
interneurons in the DG during neuronal damage contribute to net-
work instability by enhancing the relative impact in controlling
granule cell excitability (Hunt et al., 2011) and alteration of mossy
fiber sprout is caused the malfunction of inhibitory interneurons
by mossy cell degeneration (Sloviter et al., 2006). The present
findings further suggest that reorganization of newly generated
granule cells may result in the disruption of the gateway at the den-
tate gyrus, which contributes to increased seizure susceptibility, in
agreement with a prior study (Kwak et al., 2008).
4.3. Effects of hyperthermic seizure in functional efficacies of
inhibitory interneuron
Previously, some investigators reported that the preservation
of CR-containing cells in epilepsy (Blumcke et al., 1996) and the
enhanced numbers of CR-positive Cajal-Retzius cells in FS (Blumcke
et al., 1996; Thom et al., 2002). However, other investigators
demonstrated the increased vulnerability or loss of CR-positive
interneurons in human temporal lobe epilepsy (TLE), including
the CR-positive Cajal-Retzius cells (Maglóczky et al., 2000; Tóth
et al., 2010). In order to verify this controversy, thus we primarily
identified the expressional patterns of CR-positive interneuronal
population in the hippocampus following hyperthermic seizure.
In present study, our results show that immunoreactivity of CR-
positive interneurons was transiently increased in the adult time
period (7–9 weeks following hyperthermic seizure) in the CA1
and DG regions, although these expressions were reduced similar
to control level during the recurrent seizure time period (10–12
weeks after FS). These findings are consistent with a previous
study that demonstrated the preservation of CR-containing cells in
epilepsy (Blumcke et al., 1996) and the enhanced numbers of CR-
positive Cajal-Retzius cells in FS (Blumcke et al., 1996; Thom et al.,
2002). Thus, these discrepancies may reflect the compensatory
mechanisms including increased synaptic frequency of CR-positive
interneuronal terminals, and the time dependent phenomenon
induced by the developmental state of brain and the transient
accelerations of CR-positive cell production. Since a previous study
34 Y.H. Yu et al. / Brain Research Bulletin 131 (2017) 25–38

Fig. 6. Changes of the field excitatory post-synaptic potential (fEPSP) for synaptic plasticity in CA1 of the control and epileptic hippocampus. Representative traces for fEPSP
in the CA1 region at before and after tetanus in control and FS (A). The amplitude of evoked fEPSP (B) and the slope changes (C) following LTP at recurrent seizure time period
(12 weeks after FS) was reduced as compared to control. Arrows in panel B indicate the time point of sTPS application for LTP. The changes of paired-pulse responses for field
potentials in the DG of the control and FS animal hippocampus (D and E). Although the PS amplitude and the excitability ratio are increased at recurrent seizure time period
as compared to control levels, the PS latency and PS2/PS1 ratio are significantly declined in the DG (E). All data are presented as mean ± SEM. Significant differences from the
control, **p < 0.01, vs. control.

demonstrated, even with sensitivity and loss of CR-containing
interneurons in the DG, an increased frequency of CR-positive
interneuronal terminals in epilepsy (Maglóczky et al., 2000; Tóth
et al., 2010). This might mean that sprouting of the remaining CR-
containing inhibitory cells occurs as a compensatory mechanism
to offset the enhanced excitatory input on granule cell dendrites
(Tóth and Maglóczky, 2014). In addition, CR-positive interneurons
were colocalized with VGLUT-1 immunoreactivity at the adult time
period (7–9 weeks following hyperthermic seizure) in the CA1 and
DG regions. Because CR-positive interneurons may control via the
synaptic connections with other interneuronal dendrites and the
exodendritic synapses with each other, and may selectively inner-
vate with several interneurons and calbindin-containing inhibitory
interneurons (Barinka et al., 2012; Tóth et al., 2010; Bausch, 2005;
Gulyás et al., 1999), it seems to be modulated the inhibitory
interneuronal activities for controlling output of principal neurons.
In addition, VGLUT-1 positive neurons revealed the distinct exci-
tatory neuronal population and fast-spiking interneurons in the
cortical regions (Fattorini et al., 2015; Wouterlood et al., 2007;
Moutsimilli et al., 2005; Berghuis et al., 2004), and fast-spiking
GABAergic interneuron is forming synapses to control the neuronal
excitability in the perisomatic inhibitions of principal cell (Berghuis
et al., 2004), which is synchronizing action potential firing to con-
trol output of principal cells (Freund 2003). Therefore, our findings
in this study suggest that temporary enhancements of excitatory
neurotransmission via up-regulated VGLUT-1 expression in the
CR-positive interneurons following hyperthermic seizure may be
result in compensatory responses as the modulation of inhibitory
interneuronal activities in the hippocampus. Moreover, granule
cells and mossy cells also contain CR in mice, especially young
mice (Blasco-Ibáñez and Freund, 1997; Brandt et al., 2003), and
the induction of status epilepticus may transiently accelerate
the production of newly formed neurons (Kralic et al., 2005),
which are mostly granule cells (Hester and Danzer, 2013). Thus,
more CR-positive cells might be seen in the epileptic mice after
pilocarpine-induced epilepsy, although their number is decreased
at the chronic phase with the decreasing tendency of neuron
production and weakening of the CR immunoreactivity of prin-
cipal cells with time (Brandt et al., 2003; Kralic et al., 2005).
Therefore, the present findings suggest that transient elevations
of the CR-positive interneuronal population may underlie one of
the compensatory mechanisms according to the enhanced synap-
tic efficacies of CR-positive interneurons and the time dependent
events during epileptogenic period following FS. Further studies
are needed to assess this hypothesis.
On the other hand, although the slow inhibition is governed by
GABAB receptors, the fast inhibitory synaptic transmission in the
brain is modulated by the actions of GABAA receptors. The major-
ity of GABAA receptors contain two ␣ subunits, two ␤ subunits,
and a ␥ or ␦ subunit. These receptors are primarily responsi-
ble for mediating phasic and tonic inhibitions (Baumann et al.,
2002; Hines et al., 2012). In the present study, immunoreactiv-
ity of GABAA -␣1 receptor was markedly enhanced in the CA1
and DG regions 4 weeks after hyperthermic seizure compared to
control. Although these expressional patterns were recovered at
adult stage (7–9 weeks after FS) as similar to the control level,
GABAA -␣1 positive interneuron was significantly re-increased at
the recurrent seizure time period (10–12 weeks following FS) in
both areas. GABAA -␣1 positive interneurons, at this time period,
were revealed the colocalization with GABA, although the recep-
tors containing GABAA -␣1 receptor subunit are located dendritic
areas (Schwarzer et al., 1997) and extrasynapse both of hip-
pocampal interneurones (Semyanov et al., 2004) and dentate gyrus
granule cells (Nusser et al., 1995). These findings are consistent
with our paired-pulse responses data and the previous studies
that demonstrated the enhancements of paired-pulse inhibition at
 Y.H. Yu et al. / Brain Research Bulletin 131 (2017) 25–38
30-ms inter-pulse interval as GABAA receptor-mediated fast inhi-
bition in the DG following hyperthermia-induced seizures (Tsai
and Leung, 2006), and the increased GABAA receptor-mediated
inhibition in various seizure models including hippocampal kin-
dling (Tuff et al., 1983; Leung et al., 1994) and status epilepticus
(Kloosterman et al., 2001; Velišek and Velišková, 2002; Gorter et al.,
2002; Leung and Wu, 2003). In fact, the mRNA and protein lev-
els of GABAA receptor subunits including ␣1- and ␣4-subunit in
granule cells of DG were enhanced after status epilepticus (SE) in
rats induced by kainate and electrical stimulation (Schwarzer et al.,
1997; Tsunashima et al., 1997; Nishimura et al., 2005), and GABAA
receptor ␣1 -subunit in the DG was also increased in the acute
and chronic epileptic hippocampus (Loup et al., 2000; Pirker et al.,
2003; Sperk, 2007). In addition, Macdonald and their colleague
(Macdonald and Rogawski, 2007) reported that the main therapeu-
tic action of diverse antiepileptic drugs, such as benzodiazepines
and barbiturates, is to enhance GABAA receptor currents. Although
hyperthermic seizure suppressed GABAergic transmission in hip-
pocampal neurons of immature rats for shifting the excitation and
inhibition balance, the remaining interneurons can become hyper-
active to compensate for the loss of GABAergic inhibition (Rowley
et al., 1995; Beau and Alger, 1998; Isokawa, 1998; Cossart et al.,
2001) and the newborn DG granule cells following FS are a cause of
long-lasting increased GABAA receptor expression (Swijsen et al.,
2012). Thus, least in part, it is likely that the increased immunore-
activity of GABAA -␣1 receptor may be a common phenomenon in
the recurrent seizure models, such as FS model. Furthermore, the
present study also revealed the enhancement of GABAA -␣1 pos-
itive interneurons in the hippocampus during recurrent seizure.
Therefore, because of the abnormal neuronal discharge by dys-
function of GABAB receptor after FS, the present findings suggest
that an increased long-term efficacy of GABAA -␣1 receptor after
hyperthermic seizure may be related to a consequence of com-
pensated functional responses of excessive increased inhibitory
circuits in the hippocampus. Further studies are needed to confirm
this hypothesis.
4.4. Alterations of activity-dependent synaptic plasticity and
paired-pulse responses following hyperthermic seizure
LTP is regulated dependent to activation of NMDR, metabotropic
glutamate receptor (mGluR), acetylcholine (ACh) and many other
factors (Cho et al., 2013; McCoy et al., 2008; Huang et al., 1997).
Moreover, the LTP at glutamatergic synapses in the hippocam-
pus may play significant in the encoding of memory that is
activity-dependent effect of synaptic plasticity (Omrani et al., 2003;
Collingridge et al., 2004), and the postsynaptic calcium influx
was triggered for inducing LTP by activation of NMDA recep-
tors or voltage-dependent Ca+ channels (Malenka, 1991; Huber
et al., 1995; Chen et al., 2002; Omrani et al., 2003). Presently, the
induction of NMDA receptor-mediated LTP demonstrated that glu-
tamatergic synaptic transmissions in the hippocampal CA1 region
following hyperthermic seizure were significantly reduced at dur-
ing recurrent seizures as compared to control. This suppressed LTP
induction agrees with previous studies demonstrating the declined
induction of LTP by tetanic stimulation as consequence of network
changes from epileptogenesis in DG and amygdala of TLE patients
and many epileptic animal models (Beck et al., 2000; Schubert
et al., 2005; Salmani et al., 2007; Schubert and Albrecht, 2008;
Salmani et al., 2011). Thus, it is likely that the reduced LTP induc-
tions may represent the maximal potential of excitatory synapse,
because suppression of LTP in tetanic stimulation indicates that
synapses are already in maximum state (Rioult-Pedotti et al., 2000;
Savic et al., 2003; Abegg et al., 2004). Interestingly, NMDAR is
an important role in regulating the GABAergic input by GABA
release (Maccaferri and Dingledine, 2002) and LTP may accompany
35
enhancing feed-forward inhibitory pathway (Kairiss et al., 1987).
In addition to the arising of LTP by sufficient depolarization of
the membrane, this effect may due to the depression of inhibitory
responses (Davies et al., 1990). A previous study also showed the
block of excitatory synaptic transmission eliminated the afferent
inputs to the interneurons from both feed-forward and recurrent
inhibition circuits, indicating that this block could cause a decrease
in the interneuronal firing and a decline of their inhibitory action
on the pyramidal neurons in generating of epileptiform activity
(Matthews et al., 1981; Alger and Nicoll, 1982; Arai et al., 1995;
Feng and Durand, 2004; Liu et al., 2014; Houser, 2014). Therefore,
lease in part, the present findings suggest that prolonged reduced
LTP induction after hyperthermic seizure may be accompanied by
declined functional efficacies of inhibitory interneuron due to the
continuous changes in synaptic function after FS. However, it seems
to be need future experiments to investigate whether this declined
LTP induction following FS is closely involved to functional abnor-
malities of inhibitory interneurons in synaptic transmission of the
pyramidal neurons result in generating of epileptiform activities.
On the other hand, our findings reveal increased PS amplitude
and the excitability ratio from DG region at the recurrent seizure
time period following hyperthermic seizure. These findings are
consistent with a previous study that demonstrated the enhanced
PS amplitude in DG region after status epilepticus (SE) in an animal
model (Matzen et al., 2008). In fact, the PS amplitude is propor-
tional to the number of granule cells discharging an action potential
(Andersen et al., 1971) and the fEPSP slope is believed to be related
to the total amount of excitatory transmitter release by the perfor-
mant path volley (Lomo, 1971). In addition, the excitability ratio
was revealed the efficiency of glutamatergic synaptic transmission
in the DG as the PS amplitude versus the fEPSP slope in the first
evoked response. Thus, the present results seem to be imply the
declined synaptic balances by the altered neuronal circuit and the
enhanced hyperexcitatory signal by the excessive increased gluta-
matergic synaptic transmission in the DG, because the PS amplitude
and the excitability ratio indicate abnormalities of the excitability
and GABAergic inhibition in the hippocampus (Feng and Durand,
2004). Furthermore, PS latency and the PS2/PS1 ratio in DG region
significantly down-regulated during the recurrent seizure time
period after FS. Although progressively enhanced paired-pulse
facilitation accompanied by impaired late paired-pulse inhibition
has been observed in the DG following FS (Kwak et al., 2008), fast
paired-pulse inhibition enhanced in the DG has been described
in diverse animal models of epilepsy (Doherty and Dingledine,
2001; Kobayashi and Buckmaster, 2003; Cohen et al., 2003; Leroy
et al., 2004). Similarly, our study demonstrated the enhancement
of GABAA receptor-mediated fast inhibition (30-ms inter-stimulus
interval) following FS. In previous studies, GABA-mediated inhi-
bition was measured by the ratio of the second population spike
amplitude/the first population spike amplitude (Andersen et al.,
1966; Tuff et al., 1983), and paired-pulse stimulation identifies the
influences of feed-forward and feed-back inhibition in the principal
cells. In addition, feed-forward inhibition is reflected by frequency-
dependent inhibition of a single spike and feed-back inhibition is
showed by first spike amplitude-dependent inhibition of the sec-
ond spike (Sloviter, 1991). Thus, our findings such as remarkable
up-regulated GABAA receptor-mediated fast inhibition in this study
may seem to be related to the abnormalities of GABAergic neu-
rotransmission result in the persistent functional imbalances of
hippocampal neuronal circuits. However, it is need to investigate
whether these altered efficacies of GABAA receptor-mediated fast
inhibition are involved to a cause of excitatory neuronal circuits to
recurrent seizure spreading or accompanied by a consequence of
enhanced neuronal hyperexcitability as a compensatory response
in the DG following FS. Indeed, PS latency considered to be an
assess parameters for the characterizing hyperexcitatory states in
36 Y.H. Yu et al. / Brain Research Bulletin 131 (2017) 25–38
DG region (Matzen et al., 2008), evaluating the increase of neu-
ral excitability and the suppression of synaptic transmission (Feng
and Durand, 2004). The reduced PS latency after SE has been related
to DG excitability changes following recurrent seizures in vivo by
a higher release probability for glutamate on a presynaptic level
(Scimemi et al., 2006) and by the enhanced expression and altered
function of glutamate receptor (Blumcke et al., 1996; Mathern et al.,
1998) or changes in sodium or calcium currents (Gorter et al., 2002;
Ellerkmann et al., 2003). The prior and present findings suggest that
a long-term reduced PS latency at recurrent seizure time period
may be a result of excessive up-regulated excitatory neurotrans-
mission in the DG following hyperthermic seizure, and thus may
form the basis of epileptogenic changes that facilitate the genera-
tion of spontaneous seizures in the FS hippocampus.
In conclusion, abnormalities between glutamate-mediated exci-
tation and GABA-mediated inhibition lead to hyperexcitability with
epileptogenesis and emergence of seizures, which can generate
a reduction in inhibition coincident with subsequent formation
of recurrent excitatory networks and in part axonal sprouting of
excitatory principal cells (Bausch, 2005). Therefore, the present
findings reveal that time-dependent altered GABAA -␣1 and CR
immunoreactivities, and chronological changes in paired-pulse
responses in the FS hippocampus may be involved to the per-
sistent functional alterations of hippocampal neuronal circuits as
an imbalance of GABAergic and glutamatergic neurotransmission
by declined response abilities of excitatory and inhibitory actions.
These changes may lead to the epileptogenesis and the spreading
of seizure activity following hyperthermic seizure.
Conflict of interest
The authors declare that they have no competing financial inter-
est.
Acknowledgement
This work was supported by the Soonchunhyang University
Research Fund (No. 20120683) and a Basic Science Research Pro-
gram (NRF-2013R1A1A1076058) through the National Research
Foundation of Korea funded by the Ministry of Science.
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