Neuroscience 347 (2017) 48–56
AUDITORY PROCESSING ASSESSMENT SUGGESTS THAT WISTAR
AUDIOGENIC RAT NEURAL NETWORKS ARE PRONE TO ENTRAINMENT
HYORRANA PRISCILA PEREIRA PINTO, a
VINÍCIUS REZENDE CARVALHO, a,b
DANIEL DE CASTRO MEDEIROS, a,b,c
ANA FLÁVIA SANTOS ALMEIDA, a
EDUARDO MAZONI ANDRADE MARÇAL MENDES a,b,c
AND MÁRCIO FLÁVIO DUTRA MORAES a,b,c*
a
Núcleo de Neurociências (NNC), Departamento de Fisiologia e
Biofı´sica, Instituto de Ciências Biológicas – Universidade Federal de
Minas Gerais, Belo Horizonte, Minas Gerais CEP 31270-901, Brazil
b
Programa de Pós-Graduação em Engenharia Elétrica – Escola
de Engenharia – Universidade Federal de Minas Gerais,
Belo Horizonte, Minas Gerais CEP 31270-901, Brazil
c
Centro de Tecnologia e Pesquisa em Magneto Ressonância
– CTPMAG – Universidade Federal de Minas Gerais, Av.
Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil
Abstract—Epilepsy is a neurological disease related to the
occurrence of pathological oscillatory activity, but the basic
physiological mechanisms of seizure remain to be under-
stood. Our working hypothesis is that specific sensory pro-
cessing circuits may present abnormally enhanced
predisposition for coordinated firing in the dysfunctional
brain. Such facilitated entrainment could share a similar
mechanistic process as those expediting the propagation
of epileptiform activity throughout the brain. To test this
hypothesis, we employed the Wistar audiogenic rat (WAR)
reflex animal model, which is characterized by having sei-
zures triggered reliably by sound. Sound stimulation was
modulated in amplitude to produce an auditory steady-
state-evoked response (ASSR;
53.71 Hz) that covers
bottom-up and top-down processing in a time scale compat-
ible with the dynamics of the epileptic condition. Data from
inferior colliculus (IC) c-Fos immunohistochemistry and
electrographic recordings were gathered for both the con-
trol Wistar group and WARs. Under 85-dB SLP auditory
stimulation, compared to controls, the WARs presented
higher number of Fos-positive cells (at IC and auditory tem-
poral lobe) and a significant increase in ASSR-normalized
energy. Similarly, the 110-dB SLP sound stimulation also
statistically increased ASSR-normalized energy during ictal
and post-ictal periods. However, at the transition from the
physiological to pathological state (pre-ictal period), the
WAR ASSR analysis demonstrated a decline in normalized
energy and a significant increase in circular variance values
compared to that of controls. These results indicate an
*Correspondence to: M.F.D. Moraes, Núcleo de Neurociências,
Departamento de Fisiologia e Biofı́sica, Universidade Federal de
Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG 31270-
901, Brazil.
E-mail address: mfdm@ufmg.br (M. F. D. Moraes).
Abbreviations: AS, audiogenic seizure; ASSR, auditory steady-state-
evoked response; IC, inferior colliculus; ROIs, regions of interest;
WAR, Wistar audiogenic rat.
http://dx.doi.org/10.1016/j.neuroscience.2017.01.043
0306-4522/Ó 2017 IBRO. Published by Elsevier Ltd. All rights reserved.
48
enhanced coordinated firing state for WARs, except immedi-
ately before seizure onset (suggesting pre-ictal neuronal
desynchronization with external sensory drive). These
results suggest a competing myriad of interferences among
different networks that after seizure onset converge to a
massive oscillatory circuit. Ó 2017 IBRO. Published by Else-
vier Ltd. All rights reserved.
Key words: auditory steady state response, hyperexcitability,
desynchronization, audiogenic seizures.
INTRODUCTION
Information coding at the neuronal level is very much
dependent on the time-changing interactions between
neurons, which is paramount in determining how
sensory-motor integration is able to elicit appropriate
behavioral responses (Engel et al., 2001). Accordingly,
the phase-locked/synchronized firing among large popu-
lations of neurons has been proposed as a key mecha-
nism for rapidly combining the computational power of
different neural substrates, forming both local and large-
scale network discharge patterns (Fries, 2005; Buzsáki,
2006). Inhibitory feedback loops that exist throughout
the brain have been shown to produce rhythmic neural
activity that in turn generates temporal pockets with
increased probability of discharge, thus providing the
ideal architectural framework for coordinated firing
(Womelsdorf et al., 2007; Cardin et al., 2009).
Beenhakker and Huguenard (2009) proposed that patho-
logical states, e.g., epileptic seizures, could generate ‘‘at-
tractors” that would hijack otherwise functional brain
oscillators into abnormally synchronized large neural
ensembles. Using the same argument, even at a lesser
degree of abnormal coupling, there could still be a signif-
icant compromise of proper brain function (Lopes da Silva
et al., 2003a,b), thus leading to the characteristic signs
and symptoms found in epilepsy (Fisher et al., 2005)
and its myriad of associated comorbidities. Therefore,
the transition from physiological brain activation to a state
of epileptic seizure could be viewed as shades of gray
embraced by the same principal process.
The concept of seizure initiation and propagation from
focal microcircuits to more distal networks has been
suggested to bear a straight relation to the above-
described hypothesis of abnormal coupling among
oscillators (Jiruska et al., 2013). This may be the case
even for the so named ‘‘primarily generalized epilepsies,”
 H. P. P. Pinto et al. / Neuroscience 347 (2017) 48–56
for which it has been proposed that initial epileptogenic
microdomains rapidly recruit large portions of the brain
(Jiruska et al., 2013). Altogether, the mechanisms
involved in the ictogenic process may use the same frame-
work that is necessary for physiological neural processing
that, although sharing common substrates, must differ at
some level between the normal and epileptic brain. Our
working hypothesis is that the dysfunctional brain compro-
mises physiological neural dynamics in specific processes
by abnormally enhancing coordinated firing, thus facilitat-
ing the overall entrainment of sensory processing net-
works, which is closely associated to the etiology of the
epileptic condition. Reflex seizures are ideal models to test
our hypothesis because both physiological and pathologi-
cal states may be triggered by the same sensory input,
with outcome dependent on stimulus intensity. Suitably,
the Wistar audiogenic rat (WAR) is a genetically selected
strain that is susceptible to generalized tonic–clonic sei-
zures induced by high-intensity acoustic stimulation
(110 dB) (Garcia-Cairasco et al., 1992), with known brain-
stem dependency (Dutra Moraes et al., 2000). The inferior
colliculus (IC) is a brainstem structure essential for trigger-
ing the WAR audiogenic seizure (AS); in fact, bilateral IC
lesions completely block the acoustically induced tonic–
clonic seizures (Garcia-Cairasco and Sabbatini, 1989). It
is important to highlight that the audiogenic reflex seizure
is initiated only if acoustic stimulation surpasses 110-dB
SPL; otherwise no paroxysmal neural discharges are vis-
ible. Therefore, the WAR model of reflex seizure allows
the study of seizure-prone circuits during acoustic sensory
processing under physiological and pathological states.
Adversely, auditory event-related potential (P300)
recordings did not show any indications of abnormality in
patients with medically intractable temporal lobe epilepsy
unless recorded during the immediate post-ictal period
(Abubakr and Wambacq, 2003). At a glance, these data
suggest that auditory circuitry operates normally in an
epileptic brain; however, transient evoked potentials have
the disadvantage of not properly evaluating both bottom-
up and top-down neural modulations on oscillatory activity
(Regan, 1989). The auditory steady-state-evoked response
(ASSR) is an electrophysiological recording methodology
that allows the real-time monitoring of continuous sensory
processing by modulating the sound carrier frequency in
amplitude (Picton et al., 2003). Thus, the modulation fre-
quency would work as an external oscillator entraining pri-
mary auditory neural network activity, recruited by the
carrier frequency stimulation, to discharge at a specific
phase lag (Picton et al., 2003). The ASSR approach allows
the evaluation of mechanistic processes that not only differ
from those elicited by transient stimulation but also more
closely reflect the hypothesis of abnormal coupling
expected in the epileptic brain. It is important to highlight
that, to the best of our knowledge, this is the first time ASSR
has been used to evaluate epileptic susceptibility, auditory
processing during AS onset, and network propensity to
abnormal phase-locking to stimuli in any animal model of
epilepsy, or even clinically. Therefore, the main objective
of this work was to analyze the ASSR response of deep
electrodes placed in the IC at different moments of the
AS: pre-, during, and post-paroxysmal discharges.
49
EXPERIMENTAL PROCEDURES
Animals
All experiments were performed in accordance with the
Ethics Committee for Animal Experimentation (Comitê
de Ética em Experimentação Animal – CETEA) of the
Federal University of Minas Gerais, and procedures of
animal care were previously approved by this
organization under the Protocol 24/2013. Eighteen adult
Wistar rats (control group) and 19 adult WARs
(experimental group) from our own breed were used in
this study (weighing 270–300 g). Animals were screened
by three acoustic stimuli, with a 48-h interval between
successive stimuli. All rats from the control group
presented no behavioral sign of seizure (0.0 severity
index), whereas rats from the WAR group had tonic–
clonic seizure (0.85 severity index) (Garcia-Cairasco
et al., 1992).
Experimental groups
Rats were divided into two experimental procedures: EEG
recordings (Wistar and WAR groups: total 14 animals)
and c-Fos immunohistochemistry (Wistar and WAR
groups further divided into sound and no sound
subgroups: 21 total animals).
Surgery
All animals were anesthetized with ketamine (30 mg/kg)
and xylazine (5 mg/kg) and positioned in a stereotaxic
frame (David Koft Model 960) for electrode implants
(0.00500 , TeflonTM coated to a final thickness of 0.00700 ,
#7915, A-M Systems) in IC (
9.0 mm from bregma,

1.8 mm lateral, and
3.2 mm ventral) (Paxinos and
Watson, 1997). The EEG activity was recorded by a refer-
ential (monopolar) montage using a stainless-steel screw
in the nasal bone, anterior to the olfactory bulb, as ground
reference (0 V). Once the electrodes were implanted, they
were affixed to the skull with dental cement. The proximal
ends of the electrodes were then soldered to stripped cop-
per wires that were crimp connected to a four-conductor
standard phone connector (RJ-11), which in turn was
fixed to the rat’s skull by an acrylic helmet. Following sur-
gery, animals were allowed to recover for at least 7 days
before any recording procedure was tested. Animals were
sacrificed with an anesthetic overdose of urethane
(140 mg/ml) after EEG recording, and their brain was col-
lected for histological confirmation of the recorded
anatomical sites. A small lesion current (0.1 mA) was
applied for 2 s to produce a mark that could be easily visu-
alized in histology slices (50–100 mm), which were stained
with neutral red (2%) for the histological identification of
electrode position.
Acoustic stimulation
All animals were acoustically stimulated with a sinusoidal
tone (carrier frequency of 10 kHz) modulated in amplitude
(53.71 Hz). The sound intensity was set for either 85 or
110-dB sound pressure level (SPL) to analyze sound
processing in either a physiological or pathological
50 H. P. P. Pinto et al. / Neuroscience 347 (2017) 48–56
paradigm, respectively. EEG protocol 1 consisted of a
sound stimulation with three trials (30 s each; 30 s of
silent period before, between, and after the trials), with
intensity set at 85-dB SPL. EEG protocol 2 consisted of
a single 60-s sound stimulation trial (with 30 s of silent
period before and after the trial), with intensity set at
110 dB. The c-Fos protocol consisted of a single 60-s
sound stimulation, with intensity set at 85-dB SPL,
whereas the control group was not exposed to sound.
Recording procedure
The EEG signal was amplified (10,000 gain/85-dB SPL;
2000 gain/110 dB) and filtered (from 1 to 2-kHz
bandpass filter) by a signal conditioner (Cyberamp 380,
Axon Instruments). Data were sampled at 20 kHz for all
channels using an A/D converter (National Instruments
Inc., mod NIDAQ 6023E). Simultaneous with the
recording, live image of the animal was captured
through a video camera (TVnPC P6). To conduct
recording sessions, animals were placed one at a time
into a stimulation chamber. This soundproof Faraday
cage had a piezoelectric tweeter (Le Son; 80 W)
mounted at the top that is capable of delivering up to
110-dB SPL of prerecorded auditory stimuli. All EEG
signal processing was done in Matlab (R2011a); the
codes can be made available to the scientific community
upon request.
c-Fos immunohistochemistry brain tissue
preparation
Animals were anesthetized (urethane 14% w/v; 10 mL/kg)
and submitted to a transcardiac perfusion with cold (4 °C)
phosphate-buffered saline (PBS) followed by
paraformaldehyde (PFA) in PBS (4% w/v). The brains
were removed, postfixated in PFA, and maintained at
4 °C. After 24 h, the brains were transferred to a
sucrose PBS solution (30% w/v) and maintained at 4 °C
for the next 3 days.
All the 21 brains from the c-Fos subgroup of animals
were frozen in 99% isopentane at
45 °C and
cryosectioned (Cryostat 300 e ANCAP Ltd) at 40-mm
thickness. The IC sagittal cut was standardized because
it is a more stable slice preparation (minimizing risks of
detachment from the brainstem) and has clearer
neuroanatomical markers for more precise target
localization. To obtain both sagittal and coronal slices
from the same brain, a first coronal cut was performed
slightly anterior to the superior colliculus, preserving the
anterior portion of the brain for auditory temporal lobe
(ATL) coronal slices. The remainder posterior portion of
the brain was then cut in the sagittal plane, allowing
sagittal cryosectioning of IC slices. The IC slices were
quantified in a sagittal section at a fixed mediolateral
coordinate of
1.9 mm (referenced to medial suture).
The ATL slices were quantified in a coronal section at a
fixed anteroposterior coordinate of
3.5 mm (referenced
to bregma suture). Slices were stored at
20 °C in
PBSAF (PBS with 20% sucrose, 15% ethylene glycol,
and 0.05% NaN3). The sections were washed thrice for
6 min (3  6 min) in 0.01 M Trizma base-buffered saline
(TBS) before being placed in hydrogen peroxide (3%
H2O2 in TBS) for 10 min. Next, the sections were
washed 3  6 min in TBS followed by 2 h of incubation
in a blocking solution (3% normal goat serum in
TBS/0.3% Triton X-100). The primary antibody against
c-Fos (Santa Cruz, sc-52), diluted 1:1000, was added
and incubated overnight at room temperature. Next day,
the sections were washed 3  6 min in TBS with 0.3%
Triton X-100 and incubated with the secondary antibody
(1:1000, biotinylated anti-IgG antibody goat antirabbit;
Vector Laboratories, Burlingame, CA, USA) for 2 h at
room temperature. Following this, the sections were
washed 3  6 min in TBS with 0.3% Triton X-100 and
incubated with avidin–biotin horseradish peroxidase
complex (1:500 in TBS with 0.3% Triton X-100; Vector
Laboratories, Burlingame, CA, USA) for 1 h at room
temperature. Subsequently, the sections were washed
3  6 min in TBS and 3  6 min in 174 mM acetate
buffer. The sections were developed with a solution
containing 0.2 mg/ml diaminobenzidine, 25 mg/ml nickel
sulfate, and 0.0025% H2O2 in acetate buffer for 15 min.
Finally, the sections were washed 3  6 min in 174 mM
acetate buffer and mounted on gelatin-coated slides, air
dried, dehydrated in xylene, and embedded in Entellan
(Fonseca et al., 2013).
Data analysis
EEG recordings. To extract spectral magnitude and
phase synchrony information, the local field potentials
recordings were analyzed according to the short-time
Fourier transform (STFT), a time–frequency
decomposition method. The energies of different
frequency bands (corresponding to the ASSR and EEG
rhythms) were calculated along each trial with windows
composed of 65,536 data points and normalized by the
respective baseline values (30 s before sound onset).
For quantifying the amount of phase synchronization
of each sample, the resulting phase values of the ASSR
were subtracted from the ones from the sound channel.
To assess the angular dispersion of these values along
the time axis, circular variance was applied, which was
given by Mardia and Jupp (1999):

CV ¼ 1
R
where CV is circular variance, and R is the mean resultant
length:
 
 X N 
 ¼  1
R

eið/ASSR ðtj Þ
/sound ðtj ÞÞ 
N j¼1 
where N is the number of time-axis samples of each signal
and /_ASSR and /_sound are the STFT phase values for
the ASSR and sound signals, respectively, on the
frequency bin of interest. In this case, windows with
4096 data samples were used.
c-Fos expression. Photomicrographs of stained brain
sections were obtained using a digital camera (AxioCam
MRm, Zeiss) through the software Carl Zeiss Axiovision
4.8 (1388  1040 pixels image; 0.5123 mm  0.5123 mm
 H. P. P. Pinto et al. / Neuroscience 347 (2017) 48–56
pixel size). The ZVI-format micrographs were analyzed
using ImageJ software (http://rsbweb.nih.gov/ij/). The
ImageJ threshold tool was used to construct a mask
separating stained cells from the background (same
pixel threshold level, 60% darker than background,
was held for all images analyzed). An identified object
was only counted as a cell if the size (lm2) was 25–100
with circularity of 0.00–0.80. To increase the spatial
resolution of the acquired images, each slice with the
specific target structure was assembled from a mosaic
of 50–60 individual snapshots of sequential 40%
overlapping images from the microscope (Zeiss system;
Axio Imager.M2; 1388  1040 pixels each) using a 5
objective. Once the WAR had an AS triggered by a
high-intensity sound stimulation, we selected two
auditory pathway areas to evaluate the neuronal
activation, called here as regions of interest (ROIs). The
ROIs were selected as encompassing the whole
targeted structure at a particular anteroposterior coronal
slice for the ATL (
3.5-mm bregma) and at a particular
mediolateral coordinate for the sagittal IC slices
(
1.9-mm medial suture). The specific slice coordinates
were chosen, instead of a rostrocaudal (coronal
sections) or mediolateral (sagittal sections) sweep, to
evaluate all animals at the same optimal cross section
of the targeted areas. Thus, only one slice of each
structure per animal was quantified. Total counts were
made within the ROIs, depicted in the schematic
diagram of the rat brain atlas shown in Fig. 1: right IC
and right auditory temporal lobe. To provide proper
inter-ROI comparison, the total number of cell counts
was normalized by the area of each ROI in mm2. In
addition, no significant difference was observed for the
total area analyzed for correlated slices between animal
groups and/or condition (data not shown); in other
words, the size of same-type ROIs did not differ among
animals within a group or within different groups.
Statistical analysis
All data are presented as means ± SEM. The
approximation to the normal distribution was confirmed
by the Kolmogorov–Smirnov normality test. Statistical
comparisons were made using a two-way repeated-
measures analysis of variance, when appropriate
followed by Bonferroni’s post hoc test in accordance
with the coefficient of variation, except in Fig. 4 in which
we used Student’s t-test. Values of p < 0.05 were
considered statistically significant. Data were analyzed
using GraphPad Prism 6.0 Software and MATLAB, The
Mathworks, Natick, USA.
RESULTS
The results demonstrated distinct immunohistochemistry
and electroencephalographic patterns for the WAR and
Wistar groups, suggesting hyperexcitability of the
assessed neural network. The c-Fos immunostaining
showed a statistical increase in positively labeled cells
of WARs in the IC (Sound: Wistar n = 5, WAR n = 5;
No sound: Wistar n = 5, WAR n = 6; F1,17 = 3.09;
p = 0.021) and in the auditory temporal lobe (Sound:
51
Wistar n = 4, WAR n = 4; No sound: Wistar n = 4,
WAR n = 6; F1,14 = 4.033; p = 0.020) during 85-dB
SPL sound presentation (Fig. 1). Intragroup analyses
showed a significant increase in c-Fos labeling in the IC
and the ATL for the WAR group.
The electroencephalographic results, similar to the
immunohistochemistry data, indicated a hyper-activation
of the recorded neuronal population in the WAR’s IC.
Fig. 2 shows that WARs have significantly higher
ASSR-normalized energy that is steady throughout the
entire seizure subthreshold acoustic stimulation
(30 s–85-dB SPL) recorded at 53.71 Hz (Wistar: n = 07;
WAR: n = 07, subdivided in 11 epochs; F10,120 = 18.19;
p < 0.05). As expected, because low sound intensity
levels fail to induce a reflex seizure, there were no
observed statistically significant energy increase in the
EEG frequency bands (delta: F10,120 = 1.073, p > 0.05;
theta: F10,120 = 0.7000, p > 0.05; alpha: F10,120 =
0.6012, p > 0.05; beta: F10,120 = 0.7242, p > 0.05;
gamma: F10,120 = 1.067, p > 0.05) or a significant
change in the circular variance in the IC-evoked
responses for both groups (F10,120 = 0.9335; p > 0.05).
In contrast, when the sound stimulation was provided
at 110-dB SPL (EEG Protocol 2; Wistar: n = 07; WAR:
n = 07, subdivided in 18 epochs), a reflex seizure was
evoked only in the WARs, reaching at least 95% of the
severity index score (Table 1, Video 1). During the
suprathreshold sound stimulation, there was a clear
change in the EEG behavioral dynamics for the WAR
group (Fig. 3, Table 1). For the WARs’ IC LFP
recordings, during the transition from the physiological
to pathological state (pre-ictal period: time epochs 0–1),
there was a decrease in ASSR 53.71-Hz normalized
energy (p = 0.0070), which was accompanied by a
significant increase in circular variance (p = 0.0468). No
statistical difference was found in the frequency bands
at this specific seizure time-point, except for the delta
band, showing a significant normalized energy increase
(delta: F17,204 = 3.690, p < 0.05; theta: F17,204 = 4.708,
p < 0.05; alpha: F17,204 = 9.453, p < 0.05; beta:
F17,204 = 12.43, p < 0.05; gamma: F17,204 = 33.66,
p < 0.05). During the ictal period (time epochs 4–8), the
ASSR 53.71-Hz normalized energy statistically
increased concomitantly with certain frequency bands of
the spectral EEG (alpha, beta, and gamma bands).
However, no statistical difference was found for circular
variance during the ictal period. During the post-ictal
period (epochs 9–17) the before-mentioned increase in
ASSR-normalized energy remained until the end of
sound stimulation. In addition, there was a decrease in
energy for all frequency bands (except delta). There
was no statistically significant change in the circular
variance. It is important to highlight that no behavioral
(Video 2) or electrographic changes regarding seizure-
like activity were observed in the control Wistar rats.
Although the 53.71-Hz normalized energy was
maintained during the entire sound stimulation in the
WAR group, its levels were significantly reduced in the
pre-ictal period (p = 0.0070), linked with a statistical
increase in the circular variance (p = 0.0468) during the
same time period (Fig. 4). It is important to highlight that
52 H. P. P. Pinto et al. / Neuroscience 347 (2017) 48–56

et al. (2005) (Mesquita et al., 2005) and

reduced GABAergic modulation recorded
from hippocampal slices (Drumond et al.,
2011). These data are suggestive of com-
promised inhibitory modulation, which
often relates to increased excitatory tonus
and facilitated neural synchronization.
The circular variance in ASSR, a measure
of angular dispersion along time, may be
interpreted as an estimate of facilitated
network phase synchronization capabili-
ties. Although the WARs’ circular variance
showed a consistent tendency for being
lower than that of controls (Fig. 2H), there
were no statistically significant differences
between the groups.
Interestingly, if suprathreshold sound
stimulation (110-dB SPL) levels are
used, the hyper-responsive ASSR
potentials are no longer evident and
circular variance rises sixfold when
compared to that of controls (Fig. 3B, H).
If the temporal dynamics of seizure
onset are analyzed in a little more detail,
some potentially interesting data become
evident. Fig. 4 depicts the immediate first
and second epochs (epoch 0 and 1,
respectively, after sound presentation)
for both 85-dB SPL stimulation and 110-
dB SPL. During subthreshold sound
stimulation, the ASSR energy and
Fig. 1. The c-Fos expression in all the ROIs analyzed. (A, B) Illustrative figures of ROIs: circular variance seem fairly constant
IC – inferior colliculus, ATL – auditory temporal lobe. The images were captured using a 5 throughout the sound presentation
magnifying lens. Scale bar 1 mm. (C, D) Illustrative figures of the different analyzed groups (Fig. 4A, C). Conversely, at 110 dB SPL,
with and without sound stimulation at 85-dB SPL. The images were captured using a 20
there is a significant reduction in the
magnifying lens. Scale bar 200 mm. (E, F) Number of c-Fos-labeled cells per unit area (mm ).
2

Data are represented as mean ± S.E.M. # denotes p < 0.05 Wistar sound  WAR sound;
** denotes p < 0.01 WAR sound  WAR no sound. Group comparisons performed by a
two-way ANOVA with Bonferroni’s post hoc.
this particular neuronal dynamic was found only for
suprathreshold (i.e., for seizure induction) external
stimulation in the pre-ictal phase. Accordingly, no such
difference was observed during the 85-dB SPL
stimulation.
DISCUSSION
The results confirm our hypothesis that the WARs’
epileptic brain is prone to entrainment even when
operating within sound stimulation intensities that are
subthreshold to ASs (85-dB SPL), as suggested by the
hyper-responsive ASSR potentials at the modulatory
frequency (53.71 Hz, Fig. 2B). Unexpectedly, we found
no significant changes in ‘‘self-generated” EEG energy
bands during the before-mentioned ASSR stimulation
(Fig. 2C–G). These results were also corroborated by
increased c-Fos labeling in the WARs when compared
to the controls (Fig. 1), depicting abnormal network
recruitment of the primary auditory pathway during
subthreshold sound stimulation. Accordingly, the WARs
showed reduced hyperpolarizing GABAergic currents as
seen in whole-cell patch-clamp studies by Mesquita
ASSR potentials and an increase in the
circular variance (Fig. 4B, D), which
indicate a decrease in synchronization
between the IC and the external
oscillator (sound stimulus) at the pre-ictal
phase. The desynchronization at early phases of
seizure onset has been demonstrated from cellular
(Bower et al., 2012; Kramer et al., 2012) to network levels
(Le Van Quyen et al., 2001; Mormann et al., 2003;
Wendling et al., 2003; Schiff et al., 2005) and may indicate
a neural processing ‘‘interference” of regions outside the
seizure focus by the coalescence of multiple and dis-
tributed micro-oscillators (Jiruska et al., 2013) that would
lead to full-scale seizure (Medeiros et al., 2014). How-
ever, after such ictogenic ‘‘interfering” circuits are no
longer apparently active, i.e., during the post-ictal phase,
the IC auditory circuits behave as if they were not compro-
mised by the seizure (Fig.3C–H). This is demonstrated by
the ASSR circular variance reaching close to perfect
entrainment at some time windows and by the returning
to 85-dB SPL levels of the ASSR-normalized energy.
The normalized energy of all EEG spectral bands ana-
lyzed (except for delta, naturally) are also significantly
lower after the seizure, as expected, because of the very
well-characterized post-ictal electrodecremental
response.
 H. P. P. Pinto et al. / Neuroscience 347 (2017) 48–56 53

Fig. 2. Physiological ASSR processing by IC. The graphs demonstrate the IC LFP analysis during the subthreshold sound stimulation of 85-dB SPL
(sound stimulation signalized area). (A) EEG illustrative figures during the subthreshold sound stimulation. (B) ASSR-normalized energy. (C–G)
LFP-normalized energy at specific band frequencies. (H) ASSR circular variance. (I–J) The polar plot illustrates the ASSR amplitude and phase
dispersion during the sound stimulation in WAR 01 (black dots) and Wistar 01 (gray dots). The arrows show the respective mean values. Window
size, 4096 samples. Data are represented as mean ± S.E.M.; * denotes p < 0.05, ****p < 0.0001. Group comparisons performed by a two-way
ANOVA with Bonferroni’s post hoc.

Table 1. Duration in seconds of the sequential events during an audiogenic seizure. Pre-ictal: period before the first behavioral audiogenic seizure
expression and after stimuli onset. Wild running: running pattern preceding the tonic-clonic seizures. Ictal: Generalized tonic-clonic convulsions. Post-
ictal: Immobility behavioral expressed after the end of convulsion up to a total observation window of 60 s. Severity index (SI): Behavioral categories
assessment of the audiogenic seizure, ranging from 0 to 1. IS of 0.95: tonic–clonic convulsion with fore limb extension. IS of 1.00: tonic-clonic
convulsion with fore and hind limb extension

Animal Pre-ictal Wild running Ictal Post-ictal Total SI

WAR 01 4 5 18 33 60 1.00
WAR 02 6 5 18 31 60 0.95
WAR 03 6 7 16 31 60 0.95
WAR 04 7 8 15 30 60 0.95
WAR 05 6 7 17 30 60 0.95
WAR 06 5 4 19 32 60 0.95
WAR 07 5 5 21 29 60 1.00
Mean 5.5 5.9 17.1 30.8 – –
Std. Deviation ±0.9 ±1 ±2 ±1 – –
Std. Error ±0.4 ±0.5 ±0.7 ±0.5 – –

It is important to emphasize that the acoustic startle acoustic network until the lower motor neuron, having a
was present in all animals with, however, different latency of 8 ms. However, the magnitude of the acoustic
degrees of intensity. The primary acoustic startle circuit startle reflex can vary once this network is modulated by
in the rat consists of a few structures coming from the the cingulated, amydaloid, and hippocampal circuits,
54 H. P. P. Pinto et al. / Neuroscience 347 (2017) 48–56

Fig. 3. Ictal dynamics alter the ASSR processing by IC. The graphs demonstrate the IC LFP analysis during the AS using a suprathreshold sound
stimulation of 110-dB SPL (sound stimulation signalized area). (A) EEG illustrative figures during the suprathreshold sound stimulation. In the WAR
recording (black recording), we can note distinct LFP patterns during the sound stimulation due to the AS: basal (epoch-1), pre-ictal (epochs 0–1),
wild running (not shown in the illustration – epochs 2–3), ictal (red dots – epochs 4–8), and post-ictal (epochs 9–17). The number represented above
the EEG indicates (1) pre-ictal, (2) wild running, (3) ictal, and (4) post-ictal periods. (B) ASSR-normalized energy. (C to G) LFP-normalized energy at
specific band frequencies. (H) ASSR circular variance. (I–K) The polar plot illustrates the ASSR amplitude and phase dispersion during the sound
stimulation in WAR 01 during the suprathreshold stimulation of 110-dB SPL. (L–N) The polar plot illustrates the ASSR amplitude and phase
dispersion during the sound stimulation in Wistar 01 during the suprathreshold stimulation of 110-dB SPL. The arrows show the respective mean
values. Data are represented as mean ± S.E.M.; * denotes p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Group comparisons performed by
a two-way ANOVA with Bonferroni’s post hoc.

which can modify its final expression (Lee et al., 1996;
Caine et al., 1992; Angrilli et al., 1996; Pissiota et al.,
2003).
Altogether, our results corroborate the conventional
rationale in epileptology that if a dominant oscillatory
frequency is imposed upon an epilepsy-prone network,
abnormal coupling would promote an expansion of
recruited neural populations, implicating an increasingly
distributed synchronized activity (Spencer, 2002; Nair
and Thomas 2004; Kramer et al., 2012; Korzeniewska
et al., 2014; Khambhati et al., 2015). Khambhati et al.
(2016), using mathematical models to predict the out-
come from virtual dissections, argued that a push–pull
interaction between synchronizing and desynchronizing
circuits would create a scenario similar to a ‘‘tug of war”
between seizure onset zones and surrounding tissue.
The outcome of an unbalanced contest would result in
an abnormally recruited neural population being entrained
by a central dominant oscillatory frequency. Our result
most clearly fit this framework on which endogenous
epileptogenic generators desynchronize with the external
sensory drive at different periods between pre- and post-
ictal periods.
CONCLUSIONS
In summary, our results suggest the existence of a neural
facilitation mediated by the primary auditory pathway in
WARs. The hyperexcitability, demonstrated by
immunohistochemistry and electrophysiology, may
compromise physiological neural dynamics during the
 H. P. P. Pinto et al. / Neuroscience 347 (2017) 48–56
Fig. 4. Audiogenic seizure onset disrupts IC ASSR possessing. The graphs depict
the IC ASSR analysis of initial epochs (epochs 0 and 1) for both sound intensities:
85- (A and C) and 110-dB SLP (B and D). The data are presented by the normalized
value of the epochs (epoch 1/epoch 0) for ASSR-normalized energy (A and B) and
ASSR circular variance (C and D). Data represented as mean ± S.E.M.; * denotes
p < 0.05 and **p < 0.01 by unpaired Student’s t-test.
sound processing in the pre-ictal moment (Fig. 4)
because of epileptic microdomain desynchronization
(Jiruska et al., 2013). After seizure initiation, there is an
increasing synchronization as the seizure progresses
(Fig. 3B, H), leading to an overall entrainment of the
related neural networks. If these results are proven to
be a generalized property of epileptogenic neural net-
works for different expressions of epilepsy, the imposition
of external oscillatory activity to access how prone net-
works are to entrainment might be an useful diagnostic
tool or may even be predictive of brain state shifts that
lead to epileptic seizures.
DECLARATION OF INTEREST
The authors declare no competing of interests, including
any financial, personal, or other relationships with other
people or organizations.
Acknowledgments—This work was supported by FAPEMIG
(CBB-APQ-02290-13), CNPq (306767/2013-9; 454458/2014-2),
and CAPES (PROCAD 88881.068460/2014-01; BEX 5826/15-
2). A special thank you to Dr. Sydney Cash (MGH/Harvard) for
his willingness to review and discuss the manuscript.
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APPENDIX A. SUPPLEMENTARY DATA
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.neuroscience.2017.01.043.