Neuroscience 348 (2017) 3340
MOTOR TRAINING AND THE COMBINATION OF ACTION
OBSERVATION AND PERIPHERAL NERVE STIMULATION
RECIPROCALLY INTERFERE WITH THE PLASTIC CHANGES INDUCED
IN PRIMARY MOTOR CORTEX EXCITABILITY
AMBRA BISIO, LAURA AVANZINO, MONICA BIGGIO,
PIERO RUGGERI AND MARCO BOVE *
Department of Experimental Medicine, Section of Human
Physiology, University of Genoa, 16132 Genoa, Italy
AbstractAO-PNS is a stimulation protocol combining
action observation (AO) and peripheral nerve stimulation
(PNS) to induce plasticity in the primary motor cortex (M1)
(increased excitability). Another method to increase M1
excitability is motor training. The combination of two proto-
cols, which individually induce long-term potentiation
(LTP)-like plasticity in overlapping neural circuits, results
in a transitory occlusion or reverse of this phenomenon.
This study aimed to understand the neurophysiological
mechanisms underlying AO-PNS by testing whether AO-
PNS and motor training induced LTP-like plasticity in, at
least partially, overlapping neural networks. One group of
participants practiced a motor training (nger opposition
movements) followed by AO-PNS, whereas another group
performed the two protocols in reverse order. Motor perfor-
mance was evaluated by means of a sensor-engineered
glove and transcranial magnetic stimulation was used to
assess M1 excitability before and after each conditioning
protocol. Motor training increased movement frequency,
suggesting the occurrence of motor learning in both
groups. When applied on rst, both motor training and
AO-PNS signicantly increased the motor-evoked potential
(MEP), but occluded the increase of cortical excitability
expected after the following protocol, leading to a signi-
cant decrease of MEP amplitude. These results suggest that
motor training and AO-PNS act on partially overlapping neu-
ronal networks, which include M1, and that AO-PNS might
be able to induce LTP-like plasticity in a similar way to overt
movement execution. This candidates AO-PNS as methodol-
ogy potentially useful when planning rehabilitative interven-
tions on patients who cannot voluntarily move. 2017
IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Address: Department of Experimental Med-
icine, Section of Human Physiology, Viale Benedetto XV 3, 16132
Genoa, Italy. Fax: +39-0103538194.
E-mail address: marco.bove@unige.it (M. Bove).
Abbreviations: AO, action observation; PNS, peripheral nerve
stimulation; M1, primary motor cortex; LTP, long-term potentiation;
MEP, motor-evoked potential; APB, abductor pollicis brevis muscle;
TMS, transcranial magnetic stimulation; S1mV, motor-evoked potential
acquired at a TMS intensity able to evoke 1 mV MEP amplitude in
resting muscles; NIBS, non-invasive brain stimulation.
http://dx.doi.org/10.1016/j.neuroscience.2017.02.018
0306-4522/ 2017 IBRO. Published by Elsevier Ltd. All rights reserved.
33
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Key words: associative plasticity, long-term potentiation,
motor training, action observation, peripheral nerve electrical
stimulation, transcranial magnetic stimulation.
INTRODUCTION
Formation of motor memories is required for learning new
motor skills. This phenomenon is accompanied by
neurophysiological changes resulting in the output
reorganization of the motor cortex in humans (Pascual-
Leone et al., 1995; Classen et al., 1998; Liepert et al.,
1999; Muellbacher et al., 2001, 2002), non-human pri-
mates (Nudo et al., 1996), and rodents (Kleim et al.,
1998). The neurophysiological mechanism underlying
these events is that synaptic strength of neuronal connec-
tions at the level of motor areas is modied through long-
term potentiation (LTP) (Sanes and Donoghue, 2000). In
humans, a long-term increase of the excitability of the pri-
mary motor cortex (M1), which can be induced by motor
training and by non-invasive brain stimulation techniques,
has been labeled LTP-like plasticity eect (Rioult-Pedotti
et al., 1998; Stefan et al., 2000; Nitsche et al., 2008).
Furthermore, experimental evidence suggests that
motor areas are recruited also when actions are
mentally simulated (for review see Runo, 2017) or sim-
ply observed (for review see Rizzolatti and Craighero,
2004). Specically concerning action observation (AO),
this eect may vanish if movement execution is not con-
current or immediately follows its observation (Bove
et al., 2009; Zhang et al., 2011), indicating that the tempo-
ral distance between these two events plays a crucial role
in consolidating the AO eects. In a previous study we
showed that a stimulation paradigm, the AO-PNS, where
AO was delivered in conjunction with a peripheral nerve
stimulation (PNS), induced an increase of the M1
excitability in the area of the stimulated muscle which out-
lasted the stimulation period and was present up to
45 min after the stimulus administration. These results
suggested that AO-PNS can induce long-term plastic
changes in M1 (Bisio et al., 2015a). For this reason we
speculated that M1 might be one of the locui where the
information coming from the mirror neuron system acti-
vated by AO and from the electrical stimulation of the
peripheral nerve converged causing the increase of its
excitability. A possible way to test whether the changes
induced by AO-PNS in M1 might be ascribed to LTP-like
 34 A. Bisio et al. / Neuroscience 348 (2017) 3340
eects is to evaluate how AO-PNS interacts with another
protocol known to evoke LTP-like plasticity in M1, as
motor training.
Several studies showed that the combination of two
protocols, which individually induce LTP-like plasticity in
M1, evoke an occlusion or a reverse of this eect, a
phenomenon that has been suggested to be related to
the history of the synapses involved in the interaction
(Ziemann, 2004; Stefan et al., 2006; Jung and Ziemann,
2009). This was the case of motor learning when interact-
ing with non-invasive brain stimulation protocols able to
evoke long-lasting changes in M1 excitability, as for
instance paired associative stimulation (Ziemann et al.,
2004; Stefan et al., 2006; Rosenkranz et al., 2007; Jung
and Ziemann, 2009) and anodal transcranial direct cur-
rent stimulation (Cantarero et al., 2013a,b).
Therefore, the aim of the present study was to
investigate whether the changes in cortical excitability
evoked by a motor training paradigm, resulting in a
motor learning through LTP-like plasticity, interfered with
the increase of M1 excitability induced by AO-PNS, and
what happened when the two protocols were
administered in reverse order.
One group of participants (GROUP TRAINING-rst)
practiced a motor training, which consisted in a
sequence of nger opposition movements performed as
fast and accurate as possible. Afterward, subjects were
exposed to AO-PNS. The second group received AO-
PNS and then performed the motor training task
(GROUP AO-PNS-rst). Motor-evoked potential (MEP)
were acquired before and after each protocol to evaluate
changes in M1 excitability. Finger opposition movement
rate was monitored before and after the motor training to
evaluate the occurrence of motor learning.
EXPERIMENTAL PROCEDURES
Participants
Thirty-seven participants (20 females and 17 males,
mean age std = 26.3 5.4), naive to the purpose of
the experiment, were recruited for this study. Twenty of
them were assigned to GROUP TRAINING-rst and
seventeen to GROUP AO-PNS-rst. They reported no
previous history of neurological disorders or orthopedic
problems for the right-dominant hand, as determined by
the Edinburgh Handedness Inventory (Oldeld, 1971).
Subjects had no contraindication to transcranial magnetic
stimulation (TMS), and they participated in this study after
giving an informed written consent. The study was
approved by the local ethics committee and conducted
in accordance with the Declaration of Helsinki.
Study design
The experiment consisted of two sessions. The rst
session (SESSION 1) was designed to test the eect of
AO-PNS protocol on motor cortical excitability. The
amplitude of the MEP in the abductor pollicis brevis
(APB) muscle was evaluated before, immediately after,
and 30 and 45 min after AO-PNS. In GROUP
TRAINING-rst of 20 subjects, 15 of them were enrolled
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for the second session, while in GROUP AO-PNS-rst
of 17 subjects, 15 of them continued the experiment.
Indeed, only subjects showing after AO-PNS a long-
lasting increase in MEP amplitudes were admitted to the
following experimental session (SESSION 2), which
occurred at least one week after SESSION 1.
In SESSION 2 the AO-PNS and the motor training
protocols were combined. Participants received a
priming protocol and afterward a test protocol. In the
GROUP TRAINING-rst the priming protocol was motor
training and the test protocol was AO-PNS, while in the
GROUP AO-PNS-rst the order of administration of the
two protocols was reversed. To evaluate the motor
performance and the occurrence of motor learning, a
sensor-engineered glove worn by the participants
measured the nger movement rate (Hz) before and
after the motor training: namely, in GROUP TRAINING-
rst the behavioral evaluation was performed at baseline
and at POST-priming epochs, while in the GROUP AO-
PNS-rst it was performed in POST-priming and POST-
test epochs. Participants were required to perform as
fast and as accurate as possible one block of 5
sequences of nger opposition movements (for details
see Evaluation: Movement rate). MEP amplitudes were
measured before and after each conditioning protocol,
always before the execution of the motor task, when
present. The experimental paradigm is shown in Fig. 1.
Intervention: AO PNS protocol
Participants were requested to relax and look at a
computer screen where a video showing a right hand
performing repetitive nger opposition movements at
natural frequency (2 Hz) (McAuley et al., 2006; Bove
et al., 2007) was displayed. This movie clip was obtained
by lming on a black background the right hand of a
human demonstrator who performed a nger opposition
movement sequence (thumb toward index, middle, ring,
and little ngers) paced with a metronome at 2 Hz for
4 s. While observing the visual stimulus, a total of 200
electrical stimuli were delivered on the median nerve of
the right wrist, for a total duration of 840 s (i.e., 14 min).
The frequency of the electrical stimulation was set in
order to administer to the subject an electrical stimulus
every 8 ngers opposition movements (in correspon-
dence to the thumb-index closing phase). Electrical stim-
uli were applied through a bipolar electrode (cathode
proximal) connected to a Digitimer constant current stim-
ulator (DS7AH HV, Digitimer Ltd, UK), using a square
wave pulses (duration 0.2 ms) at an intensity of three
times the perceptual threshold, able to evoke a small
twitch in the APB muscle. All subjects tolerated this inten-
sity of stimulation. Electrical stimuli were delivered during
the ngers closing phase by means of custom-made
MatLab software that managed the synchronization
between the video presentation and the electrical stimula-
tion. The video was continuously repeated until the end of
the peripheral electrical nerve stimulation. No audio
accompanied the video presentation. Additionally, to keep
participants attentive on the visual stimulus, a dot was
superimposed for 1 s over the video. A total of 18 dots
appeared on the screen during the video administration
 A. Bisio et al. / Neuroscience 348 (2017) 3340
Fig. 1. Experimental protocol (SESSION 2). The experimental
protocol consisted in evaluation and intervention phases. The
evaluation phases consisted in the acquisition of motor-evoked
potentials (MEPs) recorded at a TMS intensity able to evoke
1 mV MEP amplitude at rest in the abductor pollicis brevis (APB).
This was followed by the execution of a nger-opposition movement
sequence (5 repetitions) at maximal velocity in order to evaluate
participants movement rate. The evaluation phases were performed
before (Baseline) and after (POST-priming) the priming protocol, and
after the test protocol (POST-test). In the GROUP TRAINING-rst the
priming protocol was a motor training during which participants
repeated 15 times a sequence of nger opposition movements at
maximal rate, while the test protocol was the Action Observation-
Peripheral Nerve Stimulation (AOPNS), during which electrical
stimulations of the median nerve of the right wrist were administered
while the participant was observing a video showing the same nger
opposition movement sequence. In the GROUP AO-PNS-rst the
interventions were administered in reverse order.
in a random position with respect to the depicted hand.
Participants were asked to count the total number of dots
appearing during video observation and the experimenter
questioned them during the experiment.
Intervention: Motor training protocol
Participants were instructed to execute as fast and
accurate as possible the following sequence of nger
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35
opposition movements: thumb to index, medium, ring
and little ngers. An eyes-closed paradigm was chosen
to avoid possible confounding eects. All the
participants had a short familiarization session during
which they had to perform few trials of the task at a
natural velocity. After 35 sequences, all participants
reported being comfortable with the task. The motor
training consisted in performing 15 blocks of 5
sequences with 10-s rest between the blocks (300 nger
movements in total which consisted of 4 ngers
opposition movements  5 seq  15 blocks). This
training paradigm was adopted because in our previous
study (Avanzino et al., 2015) it was demonstrated to be
ecient to induce a plastic increase of M1 excitability.
Evaluation: Movement rate
Participants were seated in a comfortable chair in a quiet
room and wore a sensor-engineered glove (Glove
Analyzer System (GAS), ETT S.p.A., Italy) on their right
hand. Before and after the motor training (baseline and
POST-priming in GROUP TRAINING-rst; POST-
priming and POST-test in GROUP AO-PNS-rst)
participants kinematics was acquired by means of GAS
during the execution of ve nger-opposition movement
sequences at maximal velocity. Data from glove were
processed with customized software and the nger
movement rate was extracted and expressed in Hz.
Evaluation: MEP
TMS was used to evaluate changes in the left M1
excitability induced by the conditioning protocols.
Intensities were expressed as a percentage of the
maximum output of the stimulator. TMS was performed
with a single Magstim 2002 magnetic stimulator
(Magstim Co Ltd, UK) connected with a gure-of-eight
coil with wing diameters of 70 mm. The coil was placed
tangentially to the scalp with the handle pointing
backward and laterally at a 45 angle to the sagittal
plane inducing a postero-anterior current in the brain.
This orientation was chosen based on the ndings that
the lowest motor threshold is achieved when the
induced electrical current ows approximately
perpendicular to the line of the central sulcus (Werhahn
et al., 1994). The optimal position for activation of the
abductor pollicis brevis (APB) muscle was determined
by moving the coil in 0.5-cm steps around the presumed
motor hand area. Prior to the experimental procedure,
the intensity of stimulation was individually assessed to
reliably elicit peak-to-peak MEP amplitude of approxi-
mately 1 mV in the APB muscle at rest (S1 mV). MEP val-
ues were acquired before and after each stimulation
protocols. Twenty trials were performed at each testing
epoch, and the average MEP amplitude was taken as
MEP size. A minimum time of 4 s was kept as TMS
inter-stimulus interval.
EMG recording
MEPs were recorded from the right APB muscle using
silver disk surface electrodes taped to the belly and
 36 A. Bisio et al. / Neuroscience 348 (2017) 3340

tendon of the muscles. The ground electrode was placed RESULTS

at the elbow. Electromyographic signals (EMG) were
digitalized, amplied and ltered (20 Hz to 1 kHz) with a
1902 isolated pre-amplier controlled by the Power 1401
acquisition interface (Cambridge Electronic Design
Limited, Cambridge, UK), and stored on a personal
computer for display and later oine data analysis.
Each recording epoch lasted 400 ms, of which 100 ms
preceded the TMS. Participants were constantly
reminded to always keep their hand relaxed during the
whole experiment. EMG signal was monitored visually
by the experimenter and trials with background EMG
activity were excluded from analysis.
Fifteen out of 20 participants of GROUP TRAINING-rst,
and 15 out of 17 of GROUP AO-PNS-rst were
responsive to AO-PNS in SESSION 1. The results of
these subjects are considered in the statistical analyses.
Session 1
The AO-PNS protocol produced the expected eects of
MEP facilitation immediately and up to 45 min after its
administration in both groups (Fig. 2) (Bisio et al.,
2015a). ANOVA showed a signicant eect of TIME (F
(3,84) = 11.01, 2 = 0.28, p  0.0001), while no dier-

Statistical analysis
We checked that variables were normally distributed
(ShapiroWilk W test) and that sphericity was respected
(Mauchly tests). In SESSION 1, to analyze the eect of
AO-PNS protocol on the left M1 excitability, a repeated-
measures ANOVA with GROUP (2 levels: GROUP
TRAINING-rst, GROUP AO-PNS-rst), as between-
subject factor, and TIME (4 levels), as within-subject
factor, was applied to compare mean MEP amplitudes
before (PRE), immediately, 30 min and 45 min after the
stimulation (POST 0, POST 30, and POST 45,
respectively).
In SESSION 2, in order to test whether AO-PNS when
primed by the motor training paradigm was able to induce
plastic changes in M1 excitability, S1mV MEP values
have been subjected to a mixed ANOVA with GROUP
(2 levels: GROUP TRAINING-rst, GROUP AO-PNS-
rst) as between-subject factor, and TIME (2 levels:
POST-priming and POST-test in GROUP TRAINING-
rst; baseline and POST-priming in GROUP AO-PNS-
rst) as within-subject factors.
Conversely, in order to test whether AO-PNS had a
priming eect on plastic changes in M1 excitability
induced by the motor training, a mixed design ANOVA
with GROUP (2 levels) as between-subject factor, and
TIME (2 levels: baseline and POST-priming in GROUP
TRAINING-rst; POST-priming and POST-test in
GROUP AO-PNS-rst) was performed on mean MEP
values. Furthermore, aiming to evaluate whether the
ecacy of motor training was inuenced by the priming-
induced eect of AO-PNS, a mixed design ANOVA with
GROUP (2 levels) as between-subject factor, and TIME
(2 levels: baseline and POST-priming in GROUP
TRAINING-rst; POST-priming and POST-test in
GROUP AO-PNS-rst) was performed on ngers
opposition movement rate.
At last, to evaluate how motor training and AO-PNS
interacted when the rst primed the second and vice
versa, a mixed ANOVA with TIME (3 levels: baseline,
POST-priming, POST-test) as within-subject factor, and Fig. 2. First experimental session (SESSION 1): Amplitude of the
GROUP (2 levels) as between-subject factor, was motor-evoked potentials recorded before (PRE), immediately after
applied on S1mV values recorded in SESSION 2. (POST 0), and 30 and 45 min (POST 30, and POST 45, respectively)
NewmannKeuls post hoc tests were applied to analyze after the AO-PNS protocol in the two groups (A. GROUP TRAINING-
rst, B. GROUP AO-PNS-rst) at a TMS intensity able to evoke peak-
signicant interactions. The probability level taken to to-peak MEP amplitude of approximately 1 mV at rest (S1 mV) in the
indicate the signicance was p < 0.05. Data are abductor pollicis brevis (APB) muscle. Values are mean standard
expressed as mean MEP amplitude SE. error.

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 A. Bisio et al. / Neuroscience 348 (2017) 3340
ences appeared between the two groups (p  0.05). Post
hoc analysis revealed that immediately after the AO-PNS
protocol MEP amplitude signicantly increased (POST 0:
1.22 0.05) with respect to PRE (0.89 0.03) (PRE vs.
POST 0 p  0.0001) and this eect was maintained up to
45 min after (POST 30: 1.25 0.08; POST 45: 1.16
0.07, PRE vs. POST 30 p  0.0001; PRE vs. POST
45 p  0.001).
Session 2: Eect on plastic changes in M1 excitability
induced by AO-PNS when applied as priming vs. test
protocol (Fig. 3)
The result of the mixed ANOVA on MEP amplitude
acquired before and after AO-PNS in the two groups
showed a signicant GROUP * TIME interaction (F
Fig. 3. Second experimental session (SESSION 2): Amplitude of the
motor-evoked potential (MEPs). MEPs were recorded at a TMS
intensity able to evoke a response of approximately 1 mV at rest in
the abductor pollicis brevis muscle in GROUP TRAINING-rst (A) and
in GROUP AO-PNS-rst (B) at the dierent time epochs: at the
beginning of the experimental session (baseline), after the priming
protocol (POST-priming) and after the test protocol (POST-test).
Values are mean standard error.
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37
(1,28) = 21.48, 2 = 0.43, p  0.0001). The Newman
Keuls post hoc comparisons showed that, when
AO-PNS was applied as priming protocol (GROUP AO-
PNS-rst, Fig. 3B), mean MEP value acquired after AO-
PNS administration (POST-priming: 1.32 0.09)
signicantly increased with respect to baseline (0.92
0.13) (p  0.01), as shown also in SESSION 1.
Dierently, MEP amplitudes of the GROUP TRAINING-
rst measured after AO-PNS protocol (POST-test: 1.04
0.12) signicantly decreased with respect to those
acquired before its administration (POST-priming: 1.35
0.13) (p  0.05) (Fig. 3A).
Session 2: Eects on plastic changes in M1
excitability induced by motor training when applied
as priming vs. test protocol
ANOVA applied on MEP amplitudes acquired before and
after motor training showed a signicant GROUP * TIME
interaction (F(1,28) = 27.52, 2 = 0.49, p  0.0001).
Post hoc analysis showed a signicant increase of
mean MEP amplitude after motor training in the GROUP
TRAINING-rst (baseline: 0.92 0.09; POST-priming:
1.35 0.13; p  0.001) (Fig. 3A), while a signicant
decrease of M1 excitability was observed after motor
training when it was performed as test protocol in
GROUP AO-PNS-rst (POST-priming: 1.32 0.09;
POST-test: 1.05 0.14; p  0.01) (Fig. 3B).
Session 2: Interaction between AO-PNS and motor
learning-induced plasticity
When comparing the results of the two sequential
treatments in SESSION 2 a signicant eect of TIME
was found (F(2,56) = 17.74, 2 = 0.39, p  0.001) and
the post hoc showed that MEP values in POST-priming
epoch signicantly increased with respect to the
baseline (p  0.001), while they decreased and reached
the baseline value after the administration of the test
protocol (baseline vs. POST-test, p  0.05; POST-
priming vs. POST-test, p  0.001). No dierence
appeared between the two groups as well as no
signicant interaction, suggesting that the priming eect
exerted by the two protocols was similar.
Session 2: Eects on motor learning induced by
motor training when applied as priming vs. test
protocol
The t-test comparing nger movement rate at baseline in
the two groups revealed no signicant dierences
(baseline GROUP TRAINING-rst: 2.83 0.15;
baseline GROUP AO-PNS-rst: 3.12 0.15; p  0.05).
The ANOVA evaluating the results of the motor training
in the two groups showed a signicant increase of nger
movement rate after the training (POST-priming
GROUP TRAINING-rst: 4.16 0.22; POST-test
GROUP AO-PNS-rst: 3.98 0.22; TIME: F(1,28)
= 75.64, 2 = 0.73, p  0.0001), meaning that
irrespective the motor training was primed or not with
AO-PNS, the motor performance signicantly improved
in term of increased movement rate (Fig. 4).
 38 A. Bisio et al. / Neuroscience 348 (2017) 3340

from the frontal part of the mirror neu-

Fig. 4. Second experimental session (SESSION 2): nger opposition movement rate (Hz). The
columns refer to the performance of participants of (A) the GROUP TRAINING-rst before
(baseline) and after (POST-priming) motor training, and (B) the GROUP AO-PNS-rst before
motor training (i.e., after AO-PNS, POST-priming) and after it (POST-test). The gray symbols
indicate nger opposition movement rate at each repetition of the training paradigm (T1. . . T15). changes in M1 excitability following
Values are mean standard error.
DISCUSSION
The aim of the present study was to explore whether a
motor training paradigm, resulting in a motor learning
through LTP-like plasticity, interfered with the increase
of M1 excitability induced by a protocol that combines
AO and peripheral nerve stimulation (AO-PNS). In a rst
experimental session, we conrmed the increase in
cortical excitability following the administration of AO-
PNS in the two groups that took part to the study. In the
GROUP TRAINING-rst during the second session the
administration of the AO-PNS protocol was preceded by
a motor training, which consisted in the repetition of a
sequence of nger opposition movements as fast and
accurate as possible. The results showed that motor
training interfered with the eects of AO-PNS occluding
the increase of M1 excitability induced by the AO-PNS
administration. In the GROUP AO-PNS-rst the AO-
PNS administration preceded the motor training
paradigm, and this combination resulted in the occlusion
of the increase of M1 excitability expected after
repeated movement execution.
In the AO-PNS paradigm (Bisio et al., 2015a) a 14-min
video showing thumb-index tapping movement at 2 Hz
was combined with the stimulation of the median nerve
that innervated the muscle that was active in the observed
movement. This paradigm has been proven to lead to an
increase in M1 excitability that persisted up to 45 min after
the stimulus administration. Further, in another work
(Bisio et al., 2015b), we found that the observation of
rhythmical actions at a frequency higher than the sponta-
neous movement tempo (SMT) combined with PNS
evoked lasting changes in SMT even in absence of imme-
diate movement execution. In view of these ndings, we
proposed that AO-PNS might be able to induce the forma-
tion of a new motor memory where movement patterns
are those acquired via AO and consolidated via PNS.
These changes might involve a neuronal network which
includes M1. Indeed, it is well known that AO activates
M1 through cortico-cortical connections, likely starting
ron system (Fadiga et al., 1995). In
turn, the aerent information gener-
ated by PNS reaches M1 during the
AO-induced activation and are likely
responsible of the neurophysiological
and behavioral outcomes described
above (Bisio et al., 2015a).
In the present study the AO-PNS
protocol induced an increase in M1
excitability that was maintained up to
45 min after its administration in
agreement with our previous study
(Bisio et al., 2015a). However, the
results of the present research take
a step further, by showing that in the
GROUP TRAINING-rst the ability of
the motor cortex to develop plastic
AO-PNS protocol was abolished
when AO-PNS was primed by motor
training.
During the motor training participants were requested
to repeat a sequence of nger opposition movements as
fast and accurate as possible. As proved by the
behavioral data, the task was eective in inducing motor
learning, resulting in an increase in nger opposition
movement rate. Motor learning is associated with
functional changes in a distributed network that includes
the primary motor, premotor, supplementary motor,
dorsolateral prefrontal, parietal cortices, the cerebellum,
and sub-cortical regions, such as basal ganglia and
thalamus. The present experiments were focused on M1
because our previous study showed that M1 is involved
in AO-PNS protocol, too. Further, a large body of
evidence showed that motor learning is accompanied by
noticeable changes in M1 functioning, including the
modication of the cortical representation of the trained
muscle (Pascual-Leone et al., 1995; Classen et al.,
1998) and the increase in the cortical excitability
(Muellbacher et al., 2001, 2002), all consistent with the
idea that motor learning is associated with LTP-like plas-
ticity in M1. Moreover, similarly to animal studies and in
agreement with the theory of homeostatic plasticity
(Abraham and Bear, 1996; Turrigiano and Nelson,
2004), researches in humans have shown that when
LTP-like plasticity is induced in M1, this potentiation pre-
vents the occurrence of further LTP-like plasticity in the
same cortical area (Ziemann et al., 2004; Stefan et al.,
2006; Rosenkranz et al., 2007). In particular, it has been
shown that motor learning may alter the response of M1
to other plasticity protocols applied immediately after
(Ziemann et al., 2004; Stefan et al., 2006; Rosenkranz
et al., 2007; Avanzino et al., 2015) and, conversely, a
paradigm known to induce LTP-like plasticity in M1 may
interfere with the subsequent motor learning (Jung and
Ziemann, 2009). On the basis of these studies, the pre-
sent ndings suggest that motor learning and AO-PNS
may involve partially overlapped neural substrates,
including M1. The neurophysiological results here
described in the GROUP AO-PNS-rst support this con-

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 A. Bisio et al. / Neuroscience 348 (2017) 3340
clusion. Indeed, when the AO-PNS administration primed
the motor training the increase of M1 excitability expected
after a repeated movement execution was prevented, and
M1 excitability decreased towards the baseline value.
The interaction between motor learning and AO-PNS
might be explained considering the dependence of
motor cortical plasticity on the activation history of M1
(Siebner et al., 2004; Ziemann et al., 2004). Metaplastic-
ity, i.e. the plasticity of the synaptic plasticity (Abraham
and Bear, 1996), is a term that is commonly referred to
the Bienenstock, Cooper and Munro (BMC) theory
(Bienenstock et al., 1982), which states that recent high
synaptic activity makes LTP-like plasticity harder to
induce. If we assume that both motor learning and AO-
PNS lead to LTP-like plasticity in M1, then the BMC the-
ory would predict that both will result in an increase of
the threshold for subsequent induction of LTP, which will
cause a reduction of the probability to induce further
LTP, having a homeostatic impact on plasticity
(Karabanov et al., 2015). Another possible explanation
of the present ndings involves the theory of the satura-
tion of LTP-like plasticity (Monls and Teskey, 2004;
Hodgson et al., 2005). It has been proposed that when
learning occurs after motor skill training, M1 synaptic con-
nections strengthen up into the range of saturation, lead-
ing to an occlusion of further LTP induction. The decrease
of the M1 excitability here observed when motor training
preceded and followed the AO-PNS administration is thus
compatible with both theories.
Interestingly, the occlusion of cortical plasticity when
AO-PNS primed motor training was not sucient to
prevent motor learning, and the motor performance
improved signicantly after motor training. Therefore,
AO-PNS was not able to completely saturate LTP in the
motor learning circuitry. In literature most of the studies
involving the interaction between motor learning and
non-invasive brain stimulation (NIBS) techniques
demonstrated that learning may have a homeostatic
impact on plasticity induced by subsequent NIBS
protocols, while the evidence for a reverse interaction is
less consistent. As also suggested in a previous study
where a paired associative stimulation protocol primed a
motor learning paradigm (Jung and Ziemann, 2009), a
possible explanation for these ndings is that motor learn-
ing involves more extensive neuronal circuits than NIBS
protocols (here represented by AO-PNS), which may be
able to guarantee the possibility to improve the motor per-
formance regardless M1 occlusion.
CONCLUSION
The present study is an attempt to elucidate the
neurophysiological mechanisms underlying the
combination of AO and peripheral electrical nerve
stimulation. First, the interaction between motor learning
and AO-PNS suggests that these two paradigms act, at
least in part, on overlapping neuronal networks,
including M1. Furthermore, the occlusion of plastic
changes in M1 excitability after AO-PNS administration
when primed by motor learning, and after motor learning
when preceded by AO-PNS is most likely attributable to
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39
the ability of AO-PNS to induce LTP-like plasticity.
However, the improvement of the motor performance
observed when the motor training was primed by the
AO-PNS suggests that this protocol acts only on a part
of the motor learning circuitry, which likely includes M1,
i.e. the site of the plastic modications induced by AO-
PNS administration.
This study supports the eectiveness of AO-PNS in
evoking plastic increase in the excitability of motor
cortical areas in a way that is similar to overt movement
execution and proposes AO-PNS as methodology
potentially useful when planning rehabilitative
interventions on patients who cannot voluntarily move.
Indeed, the possibility to train the motor system without
moving represents an appealing way to restore the
functioning of the motor cortical circuits.
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(Received 10 January 2017, Accepted 8 February 2017)

(Available online 15 February 2017)

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