Brain Stimulation 16 (2023) 1476–1485

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Brain Stimulation
journal homepage: www.journals.elsevier.com/brain-stimulation

Frequency-specific modulation of oscillatory activity in the rat auditory

cortex by vagus nerve stimulation
Shinichi Kumagai a, b, Tomoyo Isoguchi Shiramatsu b, Akane Matsumura b, Yohei Ishishita a,
Kenji Ibayashi a, Yoshiyuki Onuki a, Kensuke Kawai a, Hirokazu Takahashi b, *
a
Department of Neurosurgery, Jichi Medical University, Tochigi, Japan
b
Department of Mechano-Informatics, Graduate School of Information Science and Technology, The University of Tokyo, Tokyo, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Background: We previously found that vagus nerve stimulation (VNS) strengthened stimulus-evoked activity in
Vagus nerve stimulation the superficial layer of the sensory cortex but not in the deep layer, suggesting that VNS altered the balance
Auditory cortex between the feedforward (FF) and feedback (FB) pathways. Band-specific oscillatory activities in the cortex could
Acetylcholine
serve as an index of the FF-FB balance, but whether VNS affects cortical oscillations along sensory pathways
Noradrenaline
through neuromodulators remains unclear.
Auditory-evoked potentials
Oscillation Hypothesis: VNS modulates the FF-FB balance through the cholinergic and noradrenergic systems, which
modulate stimulus gain in the cortex.
Methods: We investigated the effects of VNS using electrocorticography in the auditory cortex of 34 Wistar rats
under general anesthesia while presenting click stimuli. In the time-frequency analyses, the putative modulation
of the FF and FB pathways was estimated using high- and low-frequency power. We assessed, using analysis of
variance, how VNS modulates auditory-evoked activities and how the modulation changes with cholinergic and
noradrenergic antagonists.
Results: VNS increased auditory cortical evoked potentials, consistent with results of our previous work.
Furthermore, VNS increased auditory-evoked gamma and beta powers and decreased theta power. Local
administration of cholinergic antagonists in the auditory cortex selectively disrupted the VNS-induced increase in
gamma and beta power, while noradrenergic antagonists disrupted the decrease in theta power.
Conclusions: VNS might strengthen the FF pathway through the cholinergic system and attenuate the FB pathway
through the noradrenergic system in the auditory cortex. Cortical gain modulation through the VNS-induced
neuromodulatory system provides new mechanistic insights into the effect of VNS on auditory processing.

1. Introduction activation of multiple neuromodulatory systems, including the cholin­

Vagus nerve stimulation (VNS) is a widely accepted palliative
treatment for drug-resistant epilepsy in humans since 1988 [1,2] and,
more recently, an alternative treatment for refractory depression [3,4],
post-stroke rehabilitation [5], and pain management [6,7]. VNS paired
with specific experience can drive plasticity to reorganize behaviorally
relevant neural circuits in the sensory and motor networks [8–12]. These
various clinical effects are probably obtained through VNS-induced
ergic system in the basal forebrain [13–16], noradrenergic system in the
locus coeruleus [13,17,18], and serotonergic system in the raphe nu­
cleus [19,20] projected from the nucleus tractus solitarius [21]. How­
ever, how VNS affects responses along sensory pathways through these
neuromodulators remains unclear. Considering the multiple targets of
VNS, an improved understanding of the mechanisms underlying
VNS-mediated changes is important to advance clinical applications of
VNS.

Abbreviations: A1, primary auditory cortex; AAF, anterior auditory field; ACh, acetylcholine; AEP, auditory-evoked potential; ANOVA, analysis of variance; ECoG,
electrocorticography; FB, feedback; FF, feedforward; Mec, mecamylamine; NA, noradrenaline; Phe, phentolamine; pps, pulse per second; STFT, short-time Fourier
transform; VNS, vagus nerve stimulation.
* Corresponding author. Department of Mechano-Informatics, Graduate School of Information Science and Technology, The University of Tokyo, 7-3-1 Hongo,
Bunkyo, Tokyo, 113-8656, Japan.
E-mail address: takahashi@i.u-tokyo.ac.jp (H. Takahashi).

https://doi.org/10.1016/j.brs.2023.09.019
Received 12 April 2023; Received in revised form 21 September 2023; Accepted 21 September 2023
Available online 29 September 2023
1935-861X/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
S. Kumagai et al. Brain Stimulation 16 (2023) 1476–1485

We previously demonstrated that VNS enhanced stimulus-evoked
activity in the superficial layer of the primary auditory cortex (A1) but
not in the deep layer [22]. Given that superficial layers in the primary
sensory cortex serve as hubs in feedforward (FF) pathways and deep
layers as hubs in feedback (FB) pathways [23–26], our results indicate
that VNS preferentially activates the FF pathways rather than the FB
pathways in the sensory system, and that VNS-induced modulation of
FF-FB balance may contribute to various clinical effects. Consistent with
our hypothesis, information theoretical analyses demonstrated that VNS
enhances thalamocortical FF transmission of sensory signaling [27].
Because FF and FB activations are associated with high- and
low-frequency oscillations, respectively [26,28,29], our hypothesis is
consistent with existing electroencephalogram studies in a resting state,
where VNS strengthens the high-frequency oscillation such as the

Fig. 1. VNS-induced modulation of AEPs. (A) Experimental protocol. (B) Grand average of pre-VNS (0hr; blue trace) and post-VNS (2hr; orange trace) AEPs at the
recording site showing the largest P1 amplitude in the pre-VNS condition in a representative rat (top). AEPs in the control group at 0hr and 2hr since the beginning of
recording are also shown for reference (bottom). The gray rectangle indicates the P1 time window. The shaded area around each solid line indicates the standard
error of the mean values. (C) Spatial map of P1 in the VNS group. The maps were made from data pooled across animals of pre-VNS (0 h; top) and post-VNS (2hr;
bottom) (Supplementary Fig. S1). Asterisks indicate the reference point to align the maps from individual animals, which was defined as the centroid of the P1
amplitude map in the putative core area. The most anterior and posterior columns and the most ventral row were excluded because they did not meet the inclusion
criteria for the main analysis. (D) Spatial map of VNS-induced P1 modulation in the auditory cortex. The cluster-based permutation test revealed a significant
difference between the post-VNS and pre-VNS conditions (top). Statistically significant time-space clusters are indicated by black dots (n = 10 rats, p < 0.05). No
significant modulation was found in the control group (bottom). ACtx, auditory cortex; A1, primary auditory cortex; AAF, anterior auditory field; AEP, auditory-
evoked potential; VNS, vagus nerve stimulation; A, anterior; D, dorsal; Mec, mecamylamine; Phe, phentolamine; ECoG, electrocorticography; ISI, inter-stimulus
interval. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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S. Kumagai et al.
gamma band and weakens the low-frequency oscillation such as the
theta band [30,31].
Acetylcholine (ACh) reportedly modulates the FF pathway [32–35]
and noradrenaline (NA) the FB pathway [36,37]. Lesions in the
cholinergic nucleus basalis reduce stimulus-evoked activities [38] and
impair stimulus perception, which leads to appropriate behaviors [39].
Lesions with ascending projections from the noradrenergic locus
coeruleus disrupt attention and impair task-focused behavior [40].
These findings are also consistent with our hypothesis that ACh and NA
are involved in the FF and FB pathways, respectively. The cholinergic
system activates gamma band oscillation in the basal forebrain and
drives plasticity in the sensory cortex when paired with stimuli [41–45].
ACh increases multiplicative gain, i.e., amplifies the response in the
sensory cortex [46–48] through its targeted selective projection to the
cortex [49]. Hence, the cholinergic system will likely strengthen the FF
pathway and cortical representation of ongoing stimuli. The noradren­
ergic system in the locus coeruleus decreases the low-frequency power
in the cortex [45,50,51]. NA enhances response gain; it increases the
signal-to-noise ratio [46,52,53] through its diffuse projection [49],
suggesting that NA plays functional roles in FB pathways that affect
sensory processing in a top-down manner [53]. The FF-FB balance could
play a crucial role in predictive coding [54–56]. Strengthening FF and
weakening FB pathways, i.e., increasing the precision of sensory signals
and decreasing the precision of prediction, might promote updating of
internal models [57]. Thus, VNS-induced modulation of the FF-FB bal­
ance may underlie various clinical effects [1–12].
Given our hypothesis that VNS modulates the FF-FB balance in the
sensory system, we reasoned that VNS-induced modulation of oscilla­
tory activities is more evident in stimulus-evoked activities than in a
resting state, which was tested previously [30,31,58]. This study
examined whether VNS modulates auditory-evoked oscillatory activity
in A1 in our animal model. We then hypothesized that the FF-FB balance
in A1, the balance of high- and low-frequency oscillations, is modulated
through the cholinergic and noradrenergic systems. To test these hy­
potheses, we investigated how VNS modulates oscillatory activity in A1
of anesthetized rats and how pharmacological blockage of ACh and NA
affects the modulation of oscillatory activity.
2. Material and methods
2.1. Animals
All procedures were performed in full compliance with the “Guiding
Principles for the Care and Use of Animals in the Field of Physiological
Science” published by the Japanese Physiological Society. The Com­
mittee on the Ethics of Animal Experiments at the Graduate School of
Information Science and Technology, the University of Tokyo (JA21-1),
approved the experimental protocol. Thirty-four male Wistar rats with
body weights of 200–300 g at 9–12 postnatal weeks were used in the
experiments.
We investigated how VNS modulates auditory-evoked potentials
(AEPs) in the auditory cortex of anesthetized rats (Fig. 1A). We
implanted VNS systems (VNS Therapy System Model 103, Cyberonics,
Houston, TX, USA) in rats and compared AEPs in pre-VNS and post-VNS
conditions (Fig. 1B, top). We also examined VNS effects on AEPs under
administration of either the nicotinic cholinergic antagonist mecamyl­
amine (Mec; Sigma-Aldrich, MO, USA) or the alpha-adrenergic receptor
antagonist phentolamine (Phe; Abcam, Cambridge, UK).
A VNS system was implanted in 23 rats divided into three groups. In
the VNS group (n = 10), sound-evoked activities in pre-VNS and post-
VNS conditions were compared to characterize VNS effects on sound-
evoked activities. Similarly, in the VNS + Mec group (n = 6) and VNS
+ Phe group (n = 7), VNS effects on sound-evoked activities were
characterized under administration of Mec and Phe, respectively. The
control group did not have the VNS implant (n = 11).
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Brain Stimulation 16 (2023) 1476–1485
2.2. Surgical procedures
The surgical procedures and VNS protocol have been described
previously [22]. Anesthesia was induced with isoflurane (5% at induc­
tion and 1.5–2.5% for maintenance) for implantation of either the VNS
system or the main electrophysiological experiments. The VNS system
was implanted at least five days before the physiological experiments. A
pulse generator was implanted subcutaneously into the back, and a
spiral electrode was attached to the left vagus nerve.
In the main physiological experiments, a craniotomy was performed
over the right auditory cortex, the dura mater above the auditory cortex
was then removed, and cerebrospinal fluid was drained from the
cisterna magna to minimize cerebral edema. The right eardrum was
ruptured and waxed to ensure unilateral sound input from the ear,
contralateral to the exposed cortex. A heating blanket and halogen lamp
were used to maintain the body temperature, and the cortical surface
temperature was maintained at approximately 35–37 ◦ C. The cortex was
covered with artificial cerebrospinal fluid (ARTCEREB®, Otsuka Phar­
maceutical Co., Ltd., Tokyo, Japan) and a neurosurgical pad with
excellent moisture absorbency and retention (BEMSHEETS™, Kawa­
moto Corporation, Osaka, Japan) to prevent desiccation and maintain
the electrolyte in the cerebrospinal fluid. Respiration and heart rate
were monitored throughout the experiment to ensure stable anesthesia.
2.3. VNS protocol
The electrical pulses from VNS were biphasic with charge balance to
avoid damaging the nerve fibers and asymmetric to preferentially acti­
vate afferent fibers to the brain [59]. The current from the pulse
generator was set to 500 μA with an interval of 130 μs at a rate of 30 Hz.
VNS of 900 pulses (30 s) was applied at 5-min intervals. The experiment
consisted of two conditions: pre- and post-VNS. After recording the
pre-VNS condition for 10 min, the VNS system was set to deliver inter­
mittent stimulation for 2 h. In the post-VNS condition, AEPs were
characterized after 2 h for 10 min. Similar experiments were performed
without VNS as controls. In the pharmacological blockage experiment,
we also recorded the pre-VNS condition for 10 min after the local
application of an antagonist. The VNS system was then set to deliver
intermittent stimulations for 2 h.
2.4. Auditory stimulation protocols
A click, a monophasic positive sound wave with a duration of 20 ms,
was provided as the test stimulus every second. The sound stimulus was
delivered through a speaker (DLS-108X; Alpine Electronics Inc., Tokyo,
Japan) positioned 10 cm from the left ear, contralateral to the exposed
cortex.
2.5. Electrophysiological recordings
A surface microelectrode array (NeuroNexus, Ann Arbor, MI, USA)
with 64 platinum electrodes was used to record the neuronal activity
epipially over the auditory cortex. The electrode diameter was 100 μm,
and the center-to-center distance was 500 μm. The array covered an area
of 4.5 × 3.0 mm. The array substrate had 0.3-mm diameter holes be­
tween the electrodes through which antagonists could be administered
locally onto the cortex [60]. A reference electrode was placed in the dura
over the contralateral parietal lobe. AEPs were recorded using amplifiers
with a gain of 1000 at a digital filter bandpass of 0.3–500 Hz and a
sampling frequency of 1 kHz (Cerebus Data Acquisition System, Black­
rock Microsystems LLC, Salt Lake City, UT, USA). The location of the
auditory cortex was identified from the spatial distribution of AEPs [61].
Empirically, the core region in the auditory cortex, including the A1 and
anterior auditory field, is 4–6 mm2 [62,63] and exhibits larger AEPs
than other regions [64]. Therefore, the core area was putatively defined
as 25 recording sites, i.e., 4.8 mm2, which showed the top 25 largest
S. Kumagai et al.
AEPs [61]. This empirical delineation might include the non-core re­
gion, but was sufficient for characterization of the most distinct AEPs in
the auditory cortex.
Recordings lasted 2 h and 20 min in total (Fig. 1A). AEPs charac­
terized in the analyses had the total average durations of 10 min (600
trials).
2.6. Administration of antagonists to the cortex
Hundred micromolar of Mec, a nicotinic cholinergic antagonist, or 1
mM of Phe, an alpha-adrenergic receptor antagonist, was locally applied
to the auditory cortex of rats with artificial cerebrospinal fluid using
BEMSHEETS™ for 10 min [34,65,66]. After the removal of BEM­
SHEETS™, the recording was started. The antagonists (50–100 μL) were
subsequently applied every 30 min, with which the antagonists were
effective [66,67].
2.7. Data analysis
Analyses were performed using MATLAB version 2022a (Mathworks,
Natick, MA, USA). The P1 amplitude was characterized as having the
largest amplitude within 50 ms of the onset of the click stimulus [60].
The AEP amplitudes were normalized by dividing the largest P1 among
all sites in the first 10 min before VNS (0 h in Fig. 1A) for each rat. The
spatial distributions of the P1 amplitudes were obtained by super­
imposing P1 maps across rats with respect to the core area centroid and
averaging the AEPs in each channel [60,61]. To align AEP maps across
animals, the centroid of P1 spatial maps in the first 10 min (0 h in
Fig. 1A) for each rat was determined using the MATLAB function
“regionprops.m” with the property of WeightedCentroid, and the
reference point was defined as the electrode closest to the centroid. AEP
maps were then overlayed across rats with respect to the reference point
(Supplementary Fig. S1, inset), and the average of normalized AEPs was
derived at each electrode. Data at the fringe of the pooled map, where
the number of samples was not identical to the number of test animals,
were excluded in the subsequent analyses.
To investigate the effects of VNS on auditory-evoked power, the
time-frequency representation of electrocorticography (ECoG) power
was derived using a short-time Fourier transform (STFT) from a single
trial AEP for each channel in the core region. Each single trial AEP was
segmented into 1000-ms epochs (with 500 ms each before and after the
click onset). Trials were rejected if outliers (5 standard deviations) of
background signals were present. STFT (MATLAB function “spectro­
gram.m”) was applied with a 200-ms Blackman window with 95%
overlap to obtain spectral decomposition. Data were then averaged
across 600 trials (10 consecutive minutes) every 30 min and normalized
in decibels with respect to the baseline, defined as a signal level between
− 500 and − 200 ms before the click stimulus onset. Spectrograms were
displayed for 100 ms pre-stimulus clicks to 300 ms post-stimulus la­
tency. The VNS-induced power changes were calculated for each fre­
quency range: theta, 4–8 Hz; alpha, 8–14 Hz; beta, 14–30 Hz; and
gamma, 30–150 Hz. The recording sites for statistical tests were deter­
mined to be located within the empirically determined core region in
each rat (25 sites/subject) and to be putatively identical in space among
the subjects between test groups (typically around 20 sites).
In the pharmacological blockage experiment, data were averaged in
the pre- and post-VNS conditions 2 h after VNS was applied.
2.8. Statistical analysis
A non-parametric cluster-based permutation test, written in MAT­
LAB with functions from the Fieldtrip toolbox [68], assessed the spatial
extent of VNS-induced modulatory changes for the P1 amplitude by
grouping the results at nearby time points and electrodes into clusters
based on the feature that neighboring time points and electrodes are
highly correlated to control the family-wise error rate [69]. The
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Brain Stimulation 16 (2023) 1476–1485
permutation distribution was obtained by randomly shuffling the time
points. Spatial clusters were formed by two or more neighboring sites in
space by thresholding at a critical alpha level of 0.05.
For multiple group comparisons, analysis of variance (ANOVA) fol­
lowed by Tukey’s post-hoc tests was performed using SPSS Statistics 26
(IBM, NY, USA).
3. Results
After implantation of the VNS system, we recorded ECoG using a
surface microelectrode array to investigate the effects of VNS on neural
activity in the auditory cortex. We compared click-evoked neural ac­
tivities, or AEPs, between the pre-VNS and post-VNS conditions, i.e.,
after VNS for 2 h (Fig. 1A). As reported previously [61,70], the grand
average of the AEPs had the first distinct component, P1 (Fig. 1B, top),
which is considered an FF input from the thalamus to the auditory cortex
[60]. The activation foci of P1 were obtained in the primary and anterior
auditory fields, or the core region in the auditory cortex [61] (Fig. 1C),
and these activation patterns and AEP maps were consistent across rats
with the reference point, i.e., the centroid of P1, at the same recording
site (asterisk in Fig. 1C) in 14 out of 21 rats and at the neighboring sites
in the remaining rats (AEP maps pooled across rats in Supplementary
Fig. S1). We compared the amplitude for 10–15 ms post-click latency,
including the rising portion and the peak of P1 shown in the gray rect­
angle, to investigate the effect of VNS on thalamocortical FF input. The
cluster-based permutation test revealed significant effects of VNS on the
AEP amplitudes around P1 latency (Fig. 1D, top, p < 0.05). Significant
increases in the P1 amplitude were observed in the core region in the
VNS group but not in the control group (dots in Fig. 1D). For our control
experiments, click sounds were also presented to rats without VNS, and
AEPs were grand-averaged across the trials (Fig. 1B, bottom). No sig­
nificant effects were observed for AEP amplitudes around P1 latency
without VNS in the entire auditory cortex (Fig. 1D, bottom). Consistent
with our previous work [22], these results suggest that VNS enhances
the P1 amplitude in the core region. To investigate the onset timing of
the VNS effect, we averaged the AEPs for 30 s (i.e., 30 clicks) before,
during, and after the second delivery of VNS (before, 5–5.5 min; during,
5.5–6 min; and after, 6–6.5 min in the timeline in Fig. 1A) and found that
the P1 amplitude increase was not significant during VNS delivery,
suggesting that the VNS effect on P1 accumulates over time, rather than
appearing immediately during VNS delivery, and is maintained there­
after (Supplementary Fig. S2).
Time-frequency representations within 300 ms post-stimulus latency
were obtained in the core region (230 sites in 10 rats with VNS and 253
sites in 11 rats without VNS) and averaged across both trials and rats.
Fig. 2 shows the effects of VNS on click-evoked oscillatory activity every
30 min during the experiments. The powers of click-evoked oscillatory
activities changed between the pre-VNS and post-VNS conditions in a
frequency-dependent manner (Fig. 2A, top). In contrast, fewer changes
were observed in the controls, i.e., in rats without VNS (Fig. 2A, bottom).
VNS tended to gradually increase the high-frequency power and
decrease the low-frequency power of click-evoked activities. These
band-specific powers were averaged within 300 ms of click onset and
quantified in decibels (Fig. 2B). A three-way repeated-measures ANOVA
with VNS condition, recording site, and time revealed a significant main
effect of VNS in some frequency bands: VNS increased the high-
frequency power, i.e., the gamma band (F(1,437) = 48.039, p < 0.001,
partial η2 = 0.099) and beta band (F(1,437) = 68.649, p < 0.001, partial
η2 = 0.136) and decreased the low-frequency power, i.e., the theta band
(F(1,437) = 18.942, p < 0.001, partial η2 = 0.042), while no significant
change was observed in the alpha band (F(1,437) = 0.265, p = 0.607,
partial η2 = 0.001). Significant interactions between the VNS condition
and time were found in the gamma band (F(3,1311) = 56.339, p < 0.001,
partial η2 = 0.114), beta band (F(3,1311) = 16.999, p < 0.001, partial η2 =
0.037), and theta band (F(3,1311) = 12.395, p < 0.001, partial η2 =
0.028). No interaction between the intervention and recording sites was
S. Kumagai et al. Brain Stimulation 16 (2023) 1476–1485

Fig. 2. VNS-induced modulation of time-frequency representations. (A) Time course of the spectral decomposition of click-evoked responses in the VNS (top; n = 10)
and control (bottom; n = 11) groups averaged across the core region. Colors indicate changes in power (dB scale, color bar on the right). The onset of evoked
responses appeared earlier than the click onset because of an artifact caused by smoothing and averaging with a 200-ms time window and 95% window overlap. (B)
Time course of VNS-induced modulation of band-specific power. The averages within 300 ms in post-click latency are shown with standard errors computed across
subjects (mean ± SEM; three-way repeated-measures analysis of variance: n. s., not significant; ***, p < 0.001). Blue, VNS; gray, control. VNS, vagus nerve stim­
ulation; SEM, standard error of the mean. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

found in the frequency bands.
To test whether ACh and NA mediated the VNS-induced band-spe­
cific modulations of click-evoked oscillatory activities, we conducted
similar experiments in another group of rats with pharmacological
blockade of the cholinergic and noradrenergic receptors. The core re­
gion included 200 sites in 10 rats with VNS, 120 sites in six rats with
VNS + Mec, 140 sites in seven rats with VNS + Phe, and 220 sites in 11
rats without VNS (controls). We found that topical application of a
cholinergic antagonist (Mec) in the auditory cortex disrupted the VNS-
induced increase in high-frequency power (Fig. 3A, left). Conversely, a
noradrenergic antagonist (Phe) disrupted the VNS-induced decrease in
low-frequency power. We then averaged the power within 300 ms post-
stimulus latency in each frequency band and statistically tested how the
intervention (VNS and antagonists) changed the oscillatory activities
(Fig. 3B). A two-way ANOVA with intervention and recording sites
revealed a significant main effect of the intervention on time-frequency
power but no interaction between the intervention and recording sites in
any frequency band. Tukey’s post-hoc test showed that the VNS-induced
increase in gamma power was disrupted by Mec administration (VNS +
Mec vs. Control, p = 0.948). Conversely, the effect of VNS on gamma
power was also observed under Phe administration (VNS + Phe vs.
Control, p < 0.001) at a level comparable to that without antagonists
(VNS + Phe vs. VNS, p = 0.760). Likewise, the VNS-induced increase in
beta power was disrupted by Mec (VNS + Mec vs. Control, p = 0.542),
whereas it was observed after Phe administration (VNS + Phe vs. Con­
trol, p = 0.021) at a level comparable to that without antagonists (VNS
+ Phe vs. VNS, p = 0.455). VNS-induced suppression of theta power was
observed with Mec (VNS + Mec vs. Control, p < 0.001) but was dis­
rupted by Phe (VNS + Phe vs. Control, p = 0.315). These results suggest
that the cholinergic system mediates VNS-induced increases in gamma
and beta powers, and the noradrenergic system mediates theta power
suppression.
4. Discussion
We investigated the acute effects of VNS on auditory-evoked activity
in the auditory cortex using intracranial electrophysiology recordings in
rats. Our results suggest that VNS enhances gamma and beta power
through ACh and attenuates theta power through NA. This band-specific
modulation supports our hypothesis that VNS strengthens the FF path­
ways and attenuates the FB pathways. Because the effect sizes of the
antagonist were larger in the high-frequency band than in the low-
frequency band (gamma, partial η2 = 0.098; beta, partial η2 = 0.063;
theta, partial η2 = 0.040), VNS-induced FF strengthening was likely
more predominant than FB attenuation.
Cholinergic and noradrenergic systems mediate the flexibility of
network dynamics, which underlies complex adaptive behavior [46,49].
The VNS-induced modulations of oscillatory activity in this study were

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Fig. 3. Effects of cholinergic and noradrenergic antagonists on VNS-induced modulation of time-frequency representations. (A) Grand-averaged spectrogram of click-
evoked responses in the core region with VNS after administration of mecamylamine (Mec; acetylcholine antagonist) (left; n = 6) and phentolamine (Phe;
noradrenaline antagonist) (right; n = 7). (B) Band-specific change in power. The average within 300 ms in post-stimulus latency was obtained in each recording site
(n.s., not significant, *, p < 0.05, ***, p < 0.001, two-way analysis of variance followed by Tukey’s post-hoc test). The horizontal line shows the mean value, the white
circle indicates the median, the bar includes 25th to 75th percentiles, and whiskers show the range of lower and upper adjacent values in each violin plot. VNS, vagus
nerve stimulation; Mec, mecamylamine; Phe, phentolamine.

similar to those induced by the activation of the cholinergic and
noradrenergic systems [41–45,50,51]. Furthermore, each neuro­
modulatory system produces a differentiated state to regulate adaptive
behavior: cholinergic activation induces a shift to a tonic firing mode,
while noradrenergic activation suppresses the cortical firing rate [45].
These neural modulations precede pupil dilation, which serves as an
indicator of arousal and attention levels and is correlated with neural
responsiveness, or gain [71]: Cholinergic activity is associated with
long-lasting pupil dilation, whereas noradrenergic activity is associated
with rapid pupil dilation [72]. These studies suggest that each neuro­
modulatory system influences neural gain in a different manner: The
cholinergic system is involved in multiplicative gain control, which
amplifies or attenuates specific neural responses by multiplying a scaling
factor, whereas the noradrenergic system is involved in response gain
control, which modulates the overall magnitude of neural responses
[46].
In our experiments, VNS enhanced gamma and beta oscillations,
likely mediated by ACh. Activation of the cholinergic system projected
from the nucleus basalis and pedunculopontine nucleus is associated
with cortical gamma band oscillation [41,42,44,45], and deactivation of
ACh receptors suppresses these oscillations in the primary auditory
cortex [73]. The cholinergic system enhances glutamatergic
thalamocortical transmission [74–78] and drives the excitation of py­
ramidal cells in the superficial layers, which triggers gamma oscillations
in an FF manner [79]. Cholinergic modulation also induces oscillations
in the primary auditory cortex and gates bottom-up flow from the pri­
mary auditory to higher-order association cortices [73,80]. Thus, the
cholinergic system will likely underlie the VNS-induced modulation of
gamma and beta oscillations and the enhancement of FF pathways.
While VNS can act via both nicotinic and muscarinic ACh receptors
in the cortex [81–84], our results suggest that VNS enhances the
high-frequency power via nicotinic ACh receptors. The activation of
nicotinic ACh signaling enhances thalamocortical transmission, whereas
muscarinic signaling suppresses intra-cortical connections and
strengthens the afferent influence [74,85,86]. Nicotinic ACh receptors
are expressed presynaptically at thalamic synapses in the thalamor­
ecipient granular layer and GABAergic interneurons in the sensory
cortex, which suppresses cortical activities outside the thalamorecipient
layer [48], whereas muscarinic receptors are highly expressed in
supragranular and infragranular layers [87]. These findings are consis­
tent with our finding that VNS strengthens the FF pathways through
nicotinic ACh receptors.
VNS suppressed theta oscillations, possibly through NA. This result is
consistent with previous findings that activation of the noradrenergic

1481
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system suppresses low-frequency power [45,50,51]. Low-frequency os­
cillations, including the theta band, coordinate activities between
distant brain regions [88,89] and are associated with top-down FB
control of stimulus processing [90–92]. In the auditory cortex, decreased
low-frequency oscillations can reduce sensory response variability in
response to repeated stimuli [93]. NA generally inhibits the auditory
cortex with profound effects [66,94,95]. Furthermore, neuromodulation
by NA is layer-specific and dose-dependent in the somatosensory cortex,
which generally inhibits spontaneous and stimulus-evoked activities
across all cortical layers. In contrast, it excites 10–40% of neurons in
deep layers in response to peripheral stimuli, specifically at low NA
concentrations [95–100]. These observations of neuromodulation are
consistent with our hypothesis that VNS regulates the balance between
FF and FB and plays a functional role in behavioral adaptation to
changing environments.
NA-dependent modulation through VNS is likely mediated by alpha-
adrenergic receptors [101–104]. Epileptiform activities are suppressed
by the activation of alpha-adrenergic receptors and enhanced by
beta-adrenergic receptors [105]. Alpha-adrenergic receptors are there­
fore likely to underlie the seizure-suppressing effect of VNS [103].
Alpha-adrenergic receptors are enriched in the deep layers of the sen­
sory cortex [95] and regulate the sensitivity of top-down inputs in py­
ramidal neurons [106]. These findings are consistent with our finding
that VNS attenuates FB pathways via alpha-adrenergic receptors.
Our study had some limitations. First, anesthesia may disrupt FB
pathways [107,108]. Anesthesia disrupts signaling along the apical
dendrites of pyramidal neurons in the deep layer, thereby suppressing
FB pathways arriving at the distal dendrites [109]. Minor modulation by
VNS in the alpha band, also associated with the FB process [110], is
potentially due to anesthesia, and VNS-induced modulation of FB
pathways might be overlooked. Second, we delineated the core region in
the auditory cortex based on the empirical distribution of AEPs [61],
which might vary across subjects. Precise delineation of the core, belt
and non-auditory cortices should be based on single/multi-unit activ­
ities [62–64]. Further studies are required to scrutinize whether and
how VNS modulates cortical activities in a region-specific manner.
Third, serotonergic neuromodulation has yet to be discussed, although
the vagus nerve has an anatomical projection to the serotonergic system
in the raphe nucleus [19,20]. The serotonergic system is involved in the
gain modulation of behaviorally salient stimuli [111], which might
underlie the clinical effects of VNS. Fourth, we only characterized the
acute effects of VNS and considered chronic effects beyond the scope of
the present study. Clinically, the therapeutic effects of VNS on seizure
control develop over time [1,112], the mechanism of which might differ
from the neuromodulation discussed above. Furthermore, VNS-induced
plasticity in chronic experiments has been the central focus of estab­
lishing the clinical application of VNS [1–12]. VNS induces cortical
plasticity lasting for weeks via ACh [15,113] and NA [101,114]. Our
working hypothesis of VNS-induced modulation of the FF-FB balance
might underestimate the effects of VNS-induced plasticity due to the
pairing of VNS with click trains at 1 pulse per second (pps) during the
experiment of 2 h. Stimulus paired with VNS might induce
repetition-rate-dependent plasticity in chronic experiments lasting over
weeks, which increases neural activities with high repetition rates (tone
pip train at 15 pps) but, in turn, decreases activities with low repetition
rates (5-pps tone train) [9]. Such VNS-induced plasticity might be less
effective in the present study, which was much shorter, i.e., 2 h, than the
previous studies, i.e., weeks. Yet, further studies are still required to
understand how VNS-induced modulation of the FF-FB balance is related
to the profound effects of VNS-induced plasticity. The theory of pre­
dictive coding suggests that FF-FB balance not only affects perception, i.
e., inference based on the internal model, but also promotes cortical
plasticity, i.e., updating the internal model, in the brain [54–57].
VNS-induced modulation of the FF-FB balance might underlie various
clinical effects of VNS [1–12].
1482
Brain Stimulation 16 (2023) 1476–1485
5. Conclusion
VNS acutely modulates AEPs and oscillatory activity in the primary
auditory cortex through ACh and NA. VNS might enhance FF pathways
through ACh via NA-mediated attenuation of FB pathways, which in­
dicates that VNS-induced neuromodulation can alter the FF-FB balance,
i.e., the neural gain, in the auditory cortex. Neural gain serves to
adaptively change the neural response in an environmental context and
relates to cognitive performance [115,116]. Thus, VNS-induced gain
modulation may influence auditory perception and behavioral adapta­
tion by changing auditory input and underlie various clinical effects of
VNS [1–12]. Further studies are needed to investigate the relationship
between VNS-induced changes in auditory signal processing and
cognitive task performance under awake conditions and the relationship
between VNS-induced modulation of the FF-FB balance and
VNS-induced plasticity in chronic experiments.
Funding
This study was supported by JSPS KAKENHI (21H05807, 23H03023,
23H04336 and 23H03465): JST (JPMJMS2296 and JPMJPR22S8);
AMED (JP22dm0307009); NEDO (181018060–0); and the Asahi Glass
Foundation. The funding agencies were not involved in the study design,
in the collection, analysis, interpretation of data, in the wiring the
report, and in the decision to submit the article for publication.
Data statement
The datasets analyzed in this study are available from the corre­
sponding author upon reasonable request.
CRediT authorship contribution statement
Shinichi Kumagai: Investigation, Formal analysis, Visualization,
Writing – original draft. Tomoyo Isoguchi Shiramatsu: Methodology,
Investigation, and, Funding acquisition. Akane Matsumura: Investi­
gation. Yohei Ishishita: Formal analysis. Kenji Ibayashi: Formal
analysis. Yoshiyuki Onuki: Formal analysis. Kensuke Kawai: Super­
vision, and, Funding acquisition. Hirokazu Takahashi: Conceptuali­
zation, Project administration, Supervision, Writing – review & editing,
and, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors thank Dr. Kenichi Usami of the National Center for Child
Health and Development for his technical support with VNS
implantation.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.brs.2023.09.019.
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