Motor unit adaptation to disuse: crossing the threshold from firing rate suppression to neuromuscular junction transmission

Abstract

Neural conditioning to scenarios of muscle disuse is undoubtedly a cause of functional decrements that typically exceed losses of muscle size. Yet establishing the relative contribution of neural adaptation and the specific location in the motor pathway remains technically challenging. Several studies of healthy humans have targeted this system and have established that motor unit firing rate is suppressed following disuse, with a number of critical caveats. It is suppressed in the immobilized limb only, at relative and absolute force levels, and preferentially targets lower‐threshold motor units. Concomitantly, electrophysiological investigation of neuromuscular junction transmission (NMJ) stability of lower‐threshold motor units reveals minimal change following disuse. These findings contrast with numerous other methods, which show clear involvement of the NMJ but are unable to characterize the motor unit to which they belong. It is physiologically plausible that decrements observed following disuse are a result of suppressed firing rate of lower‐threshold motor units and impairment of transmission of the NMJ of higher‐threshold motor units. As such, motor units within the pool should be viewed in light of their varying susceptibility to disuse. image

Full text

J Physiol 0.0 (2024) pp 1–9 1
The Journal of Physiology
TOPICAL REVIEW
Motor unit adaptation to disuse: crossing the threshold
from ring rate suppression to neuromuscular junction
transmission
Mathew Piasecki
Centre of Metabolism, Ageing & Physiology (CoMAP), Medical Research Council/Versus Arthritis UK Centre of Excellence for Musculoskeletal Ageing
Research (CMAR), NIHR Nottingham Biomedical Research Centre, University of Nottingham, Derby, UK
Handling Editors: Laura Bennet & Martino Franchi
The peer review history is available in the Supporting Information section of this article
(https://doi.org/10.1113/JP284159#support-information-section).
Abstract gure legend Neuromuscular disuse scenarios of limb immobilization, reduced activity and bed rest result
in impairments of muscle strength that exceed losses of muscle size. Neural adaptations are an assured consequence
of disuse. The available evidence garnered from human and animal models highlights suppressed motor unit ring
rate (MUFR) that is more evident in lower-threshold motor units, in which electrophysiological methods indicate no
discernible impairment of the neuromuscular junction (NMJ). Numerous other methods corroborate NMJ impairment
following disuse, and this may be applicable only to higher-threshold motor units. The dashed arrow indicates the
theoretical and simplied relationship between motor unit recruitment and muscle force generation.
Mathew Piase cki is an Associate Professor within the Centre of Metabolism, Ageing and Physiology (COMAP) at the University
of Nottingham, UK. His research interests are focused on the neural input to muscle in ageing and disease and how decrements
of this might be alleviated with intervention.
© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. DOI: 10.1113/JP284159
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution
and reproduction in any medium, provided the original work is properly cited.

2M.PiaseckiJ Physiol 0.0
Abstract Neural conditioning to scenarios of muscle disuse is undoubtedly a cause of functional
decrements that typically exceed losses of muscle size. Yet establishing the relative contribution of
neural adaptation and the specic location in the motor pathway remains technically challenging.
Several studies of healthy humans have targeted this system and have established that motor
unit ring rate is suppressed following disuse, with a number of critical caveats. It is suppressed
in the immobilized limb only, at relative and absolute force levels, and preferentially targets
lower-threshold motor units. Concomitantly, electrophysiological investigation of neuromuscular
junction transmission (NMJ) stability of lower-threshold motor units reveals minimal change
following disuse. These ndings contrast with numerous other methods, which show clear
involvementoftheNMJbutareunabletocharacterizethemotorunittowhichtheybelong.It
is physiologically plausible that decrements observed following disuse are a result of suppressed
ring rate of lower-threshold motor units and impairment of transmission of the NMJ of
higher-threshold motor units. As such, motor units within the pool should be viewed in light
of their varying susceptibility to disuse.
(Received 7 February 2024; accepted after revision 21 October 2024; rst published online 4 November 2024)
Corresponding author Mathew Piasecki: Centre of Metabolism, Ageing & Physiology (CoMAP), Medical
Research Council/Versus Arthritis UK Centre of Excellence for Musculoskeletal Ageing Research (CMAR),
NIHR Nottingham Biomedical Research Centre, University of Nottingham, Derby DE223DT, UK. Email:
mathew.piasecki@nottingham.ac.uk
Introduction
Experimental approaches to physiological disuse have
primarily explored underlying mechanisms of reduced
neuromuscular function and muscle mass. These studies
have translational implications for clinical settings,
spaceight and, in some instances, are used as a model
of accelerated ageing (Deane et al., 2024). Methods of
intervention dier in their approach and range from
full bed rest to explore eects of whole-body disuse to
individual limb immobilization or suspension (Atherton
et al., 2016). Studies of individual limb disuse are arguably
more tolerable for participants and ethically agreeable,
andalthoughthislimitstheeectsofmoresystemic
disuse, it does enable the exploration of muscle- and
limb-specic adaptation, with the benet of using
non-immobilized opposing limbs as a comparison (Pre-
obrazenski, Janssen et al., 2023). Crucially, immobilization
studies also dier in duration, and collective evidence
suggests a rapid decline in function, with a plateau during
prolonged disuse (Preobrazenski, Seigel et al., 2023).
Several outcomes are unquestionable; disuse results in
a marked reduction in muscle size, which is exceeded
by a reduction in muscle strength and power (Campbell
et al., 2019; Hardy et al., 2022; Marusic et al., 2021). This
latter factor highlights a potent inuence on the neural
input to muscles, an area that has garnered comparatively
less research attention than the molecular mechanisms of
muscle contraction.
Of the data available in humans and animals, a
reduction of motor unit ring rate (MUFR) and
disruption at the neuromuscular junction (NMJ) are
clear consequences of disuse, but the underpinning
mechanisms are less clear. To identify potential
impairments in the process, it is essential to under-
stand fully the complex factors governing motor unit
(MU) activation and ring, in addition to eective
communication from nerve to muscle.
Motor pathway
The MU is the nal element of the motor pathway,
consisting of an α-motoneuron and its axon, numerous
NMJsandalltheindividualmusclebrestheyinnervate.
The amount of force generated with a voluntary
contraction is contingent upon the recruitment of MUs
and the rate at which they discharge action potentials (i.e.
theMUFR).RecruitmentofanMUisdependentonits
activation threshold, which is typically proportional to its
size. Smaller motoneurons with higher input resistance
reach activation threshold with lower synaptic input
compared with larger motoneurons. Smaller MUs are
typically composed of fewer and smaller muscle bres
(type I) and generate lower levels of force than larger bres
(type II) (Enoka & Duchateau, 2017). Both recruitment
and MUFR are altered to suit the desired level of force for
all movements and interactions with the environment,
and this can be described with a series of steps, each of
which can be viewed as an independent research eld and
is described briey herein.
In response to numerous stimuli, such as sight, sound
and aerent feedback from proprioceptive impulses,
signals from the primary motor cortex (M1) and reticular
© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

J Physiol 0.0 Motor unit adaptation to disuse 3
formation of the brainstem propagate down descending
corticospinal and reticulospinal tracts, where they are,
in turn, transmitted to interneurons and motoneurons
(Glover & Baker, 2022; Kiehn, 2016). The M1 region
in humans and higher primates diers from other
animal models and has distinct evolutionary origins. Sub-
dividedintoarostralregion(oldM1)andacaudal
region (new M1), the old M1 might have fewer or
more slowly conducting cortico-motoneuronal cells and
communicates with lower motoneurons via spinal inter-
neurons (Witham et al., 2016).
Of those descending cells that do not synapse directly
onto motoneurons, groups of excitatory and inhibitory
interneurons regulate the net excitability of motoneurons
(Zholudeva et al., 2021). In bilateral movements, the
regulation of each limb is partly reliant on the pre-
vention of contralateral motoneuron excitation, referred
to as crossed inhibition. This can occur directly via
inhibitory commissural neurons (CNs) or indirectly via
excitatory CNs acting on premotor inhibitory neurons.
Cross-excitationcanalsooccurviadirectCNexcitation
of interneurons/motoneurons (Maxwell & Soteropoulos,
2020) and is a prominent consideration for studies
of unilateral disuse, where detrimental eects can be
expected in the non-immobilized limb.
The net excitation of the motoneuron can occur from
thousands of synaptic inputs along its dendrites, ranging
from descending drive to sensory aerents, all of which
must be integrated and regulated. This is achieved via two
fundamental mechanisms: ionotropic input and neuro-
modulation. Postsynaptic ionotropic receptors depolarize
or hyperpolarize the cell via regulation of ion exchange
across the membrane to generate an excitatory or
inhibitory response (Heckman et al., 2009). The mono-
amines serotonin (5-HT) and noradrenaline (NA) are
potent neuromodulators released from caudal raphe
neurons and the locus coeruleus, respectively. These
neurotransmitters stimulate persistent inward currents
(PICs) via G-protein-coupled second messengers, which
act to amplify and prolong synaptic input to the
motoneuron and enable an input–output gain control of
motoneurons that facilitates motor output in response to
demand (Johnson & Heckman, 2014). Direct estimation
of PIC amplitude in human spinal motoneurons is not
possible, but the well-established ࢞Ftechnique, in which
MUFR onset–oset hysteresis is calculated during ramped
voluntary contractions, is able to estimate the inuence of
neuromodulatory inputs on the amplication of synaptic
input (Gorassini et al., 1998; Mesquita et al., 2024).
Much of the net excitation is common across multiple
motoneurons (Deluca & Erim, 1994), and monoaminergic
drive is highly diuse (Johnson & Heckman, 2014),
meaning that the selective activation of individual
motoneurons is improbable (Rossato et al., 2024). Once
depolarized, successive action potentials propagate along
the motoneuron axon and axonal branches to terminate
at NMJs. The NMJ has a highly specic role in the trans-
mission of ACh to the postsynaptic motor endplate and
initiates a muscle bre action potential. Indeed, this 1:1
ratio of a motoneuron and muscle bre action potential
has enabled an active eld of research; with appropriately
placed recording electrodes, action potentials are readily
detectable from the muscle and reveal structural and
functional characteristics of the MU (Del Vecchio et al.,
2020; Jones et al., 2021). There are scant combined
histological and electrophysiological data on the NMJ
from humans, and the relationship between the structural
appearance of the NMJ, often termed stability, and
the function of the NMJ, its transmission (in)stability,
has not been associated reliably. Put simply, an NMJ
of non-conventional appearance might well be able to
continue to regulate ACh release eectively.
Suppression of motor unit ring rate
Following 15 days of leg immobilization in healthy young
humans, vastus lateralis muscle size reduced by ∼15%,
which was far exceed by a reduction in strength of
thekneeextensorsof∼31% (Inns et al., 2022). The
MUFR recorded at submaximal contractions (10% and
25% maximum) was also reduced after disuse by ∼10%.
Given the pivotal role of MUFR in force generation,
this suppression of MUFR after disuse might initially be
viewed as a consequence of sampling MUs at reduced force
levels rather than a cause of lower force generation. Yet
thesamepatternofsuppressedMUFRwasapparentwhen
contraction levels were normalized to baseline maximum
strength (prior to loss of force), favouring suppressed
MUFR as a direct consequence of disuse (Inns et al., 2022).
In a similar yet independent study, following 10 days
of lower limb suspension in healthy young males the
vastus lateralis size was reduced by ∼4.5% and, again,
was far exceed by a ∼30% reduction in strength
(Sarto et al., 2022; Valli et al., 2023). The MUFR
during sustained contractions also decreased at 10% and
25% maximum voluntary contraction (MVC) by 12%
and 10%, respectively. Importantly, this was threshold
level specic, with MUFR at 50% MVC showing a
∼6% increase (Valli et al., 2023). Preceding these
studies by decades and underpinning their ndings,
Duchateau and Hainaut (1990) immobilized the hand for
6–8weeksandreportedareducedmaximalMUFRinthe
adductor pollicis and rst dorsal interosseous muscles of
∼41%. This suppression also disproportionately aected
low-threshold MUs. Likewise, a decrease of ∼31% and
37% of MUFR was reported following 3 and 6 weeks,
respectively,ofimmobilizationoftherstdorsalinter-
osseous (Seki et al., 2001). Similar to the unquestionable
decline in strength, the accumulated evidence in humans
© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

4M.PiaseckiJ Physiol 0.0
strongly suggests that suppressed MUFR plays a causal
role in the loss of strength induced by disuse, and
this appears to be targeted preferentially towards earlier
recruited lower-threshold MUs. However, the specic
factor causing the suppression of MUFR remains less
evident. At the simplest level, suppression of MUFR in
these studies can be viewed as an alteration in the gain
control of the motoneuron and/or an altered balance of
excitation and inhibition of motoneurons, inclusive of
the intrinsic properties of motoneurons; either ionotropic,
neuromodulatory, or both (Johnson & Heckman, 2014).
Consider an opposing stimulus to disuse, that of
resistance and/or endurance exercise training. Here,
convincing evidence highlights central neural adaptation
as a cause of improved performance (Pearcey et al.,
2021), including within intracortical circuits (Glover &
Baker, 2020) and descending drive (Glover & Baker,
2022). However, the temptation to view these opposing
interventions as having directly opposing eects should
be resisted; the commonly reported cross-education
eect noted with unilateral exercise (Altheyab et al.,
2024; Calvert & Carson, 2022), whereby an opposing
non-exercised limb also improves, has no opposing
equivalent with disuse, because no decrements of strength
or MUFR were noted in control limbs (Duchateau &
Hainaut, 1990; Inns et al., 2022; Preobrazenski, Janssen
et al., 2023). This was apparent in control muscles of
the lower limb and hand, indicating no bilateral eect
(or ‘cross-uneducation’) to muscles served predominantly
via interneurons or directly via cortico-motoneuronal
connections. This is further supported by functional
MRI data showing decreased activity in regions contra-
lateral to the immobilized limb only, alongside reduced
corticospinal excitability, with no ipsilateral adaptation
following unilateral immobilization (Avanzino et al.,
2011; Garbarini et al., 2019). Collectively, it seems
that impaired cortical activity and descending drive are
probable contributors to disuse-induced neuromuscular
impairment, and this is specic to the immobilized limb.
Fromapositiveperspective,vastuslateralisMUFR
at normalized contraction levels returned to baseline
followingashortresistanceexercisetrainingrehabilitation
programme (Sarto et al., 2022; Valli et al., 2023), and in
rst dorsal interosseous, decits observed after 6 weeks
of recovery were completely restored with an additional
6 weeks of non-interventional recovery (Seki et al.,
2001). These combined ndings strongly indicate that
conventional intervention methods, such as resistance
exercise training or even normal daily activity, eectively
restore MUFR after a period of disuse, albeit with current
evidence restricted to healthy young people.
Recall that monoaminergic (e.g. 5-HT and NA) drive
to spinal motoneurons has potent eects on the intrinsic
excitability of the motoneuron via PIC activation, which
amplies synaptic input (Heckman et al., 2009). The
onset–oset hysteresis of motoneuron ring as assessed
by ࢞Fis highly adaptable, with increases in response
to increased activity (Orssatto, Blazevich et al., 2023;
Orssatto, Rodrigues et al., 2023), decreases in response to
induced acute inhibition (Mesquita et al., 2022; Orssatto
et al., 2022), and is markedly lower in young males
compared with females (Jenz et al., 2023) and in older
compared with young subjects (Guo, Jones, Škarabot et al.,
2024; Hassan et al., 2021; Orssatto et al., 2021). In extended
analysis of data from the aforementioned 10 day lower
limb suspension study (Sarto et al., 2022; Valli et al.,
2023), ࢞Ffrom trapezoidal ramped contractions reduced
immediately following immobilization and recovered
following resistance exercise training (Martino et al.,
2024). The degree of adaptation of ࢞Fwas correlated
with MVC uctuations over this period, suggesting an
inuence of disuse on PIC-mediated sustained MU ring
(Martino et al., 2024). However, ࢞Fscales non-linearly
with contraction intensity in the vastus lateralis (Škarabot
et al., 2023), and lower normalized values following
strength loss might be a consequence of the reduced force
level at which it was calculated.
If the PIC contributions to motoneuron ring are
altered following disuse, further questions remain
regarding the causative mechanism; is it reduced
availability of 5-HT and NA, and/or a reduction in mono-
amine receptors or their impairment, or an altered balance
of excitatory and inhibitory inputs? All have physiological
plausibility and, to some extent, supporting evidence
from animal models. In rats, results of 5-HT activity
following short periods of immobilization are mixed,
showing increases (Takahashi et al., 1998) and decreases
(Clement et al., 1998). But, used as a model of stress
inducement, these brief periods of inactivity are unlikely
to translate directly to humans and might not reect
true eects of immobilization. Furthermore, there is a
strong likelihood that 5-HT neuronal activity is positively
correlated with motor output, as shown in cats (Jacobs
et al., 2002). Considering that MUFR in humans were
suppressed at multiple force levels (normalized to relative
and absolute maximal) after disuse (Inns et al., 2022), it
is possible that descending drive did not increase when
greater relative eort was required or that motoneurons
were less responsive to it. Notwithstanding this reduced
MUFR,theseforcesafterdisusewereachievableand,
presumably, were facilitated via greater MU recruitment;
however, current methods limit the reporting of MU
behaviour to only those which can be sampled.
There is also limited evidence to directly indicate
reduced or impaired receptors on motoneuron dendrites
following disuse. Indeed, the opposite might be more
likely, because synaptic scaling (the upscaling of post-
synaptic strength in response to reduced synaptic input)
is applicable to motoneurons (Santin et al., 2017). This
might partly explain the rapid recovery of the vastus
© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

J Physiol 0.0 Motor unit adaptation to disuse 5
lateralis (Martino et al., 2024; Sarto et al., 2022; Valli et al.,
2023) and rst dorsal interosseous (Seki et al., 2001) when
activity resumed after disuse.
A critical point of the available human data is that
the reduction in MUFR is threshold specic and pre-
ferentially aects lower-threshold MUs (Duchateau
& Hainaut, 1990; Valli et al., 2023). In response to
pain, the ring rates of lower-threshold (20% MVC)
tibialis anterior MUs were suppressed, and ring rates
of higher-threshold (70% MVC) MUs were increased
(Martinez-Valdes et al., 2020), which suggests a stronger
inhibitory inuence on the lower-threshold MUs.
Supporting this increased inhibition hypothesis from
a mechanistic perspective, 2 weeks of cast immobilization
in rats induced hyperalgesia (Ohmichi et al., 2012);
prolonged xed joints, such as adopted with disuse,
could reasonably alter muscle spindle activity (Lan & He,
2012), and the lack of contraction-induced deformation
of aerents might render them hypersensitive with reuse.
Furthermore, disuse-associated inammation might
also have inhibitory eects via group III/IV inhibitory
aerents (Amann, 2012; Jones et al., 2023).
The collective evidence points to suppression of MUFR
with disuse in humans, with several caveats: (i) it is specic
to the limb immobilized and has no negative cross-over
eects (‘cross-uneducation’); (ii) it remains suppressed
in comparison to baseline when assessed at relative and
absolute contraction forces; and (iii) it is more apparent in
lower-threshold MUs.
Transmission at the NMJ
The NMJ is the nal point of the motoneuron, bridging
nerve–muscle synaptic communication to initiate muscle
bre action potentials and contraction. Much of what is
known about the NMJ stems from animal models, but
exploration in humans is possible with electrophysiology
(Piasecki et al., 2021) and immunohistochemistry
(Boehm et al., 2020) techniques. Intramuscular electro-
myography during voluntary contractions in humans
enables the quantication of consecutive motor unit
potentials (MUPs) and their near-bre potentials across
multiple depths of muscle (Jones et al., 2021; Piasecki
& Stashuk, 2023). Trains of MUPs are used to generate
a MUP template, features of which can be used to infer
anatomical features of the MU (Jones et al., 2022).
High-pass ltering of the MUP and corresponding train
generates near-bre MUPs (Stashuk, 1999), and the
variability across consecutive observations can be used to
infer the variability of NMJ transmission times (Piasecki
et al., 2021). Termed NMJ transmission instability, this
assessment relates to the dynamics of ACh release and
postsynaptic binding, which is greater than the variability
observed in action potential conduction velocity of
axonal branches and muscle bres, hence it has a greater
inuence on near-bre MUP shape (Katz & Miledi, 1965).
This method was used in two independent studies of
leg immobilization in healthy young males, and both
reported no change in NMJ transmission instability after
10 (Sarto et al., 2022) and 15 days of disuse (Inns
et al., 2022). It is important to note limitations here.
These NMJs were assessed at fairly low contraction levels
(∼25% of maximum) and reveal little of the NMJs
of higher-threshold MUs, where alternative imaging
methods might be more advantageous.
Histological imaging of the human NMJ, particularly
the presynaptic region, poses signicant technical
challenges. The most detailed data often come from
amputated limbs, where access to full longitudinal
sections of muscle is possible (Boehm et al., 2019; Jones
et al., 2017). However, targeted human biopsy techniques
do improve NMJ yield beyond standard techniques
(Aubertin-Leheudre et al., 2020). Direct histochemical
imaging of the NMJ structure in young maturing rodents
following 10 weeks of limb suspension showed a marked
decrease in the size of the postsynaptic motor endplate,
with no change to presynaptic regions (Deschenes et al.,
2006). Similar eects on the postsynaptic endplate were
observed after only 10 days of unloading (Deschenes et al.,
2005), but no eect was observed in fully mature rodents
(Deschenes & Wilson, 2003). This form of structural NMJ
adaptation with disuse is age specic and more evident
in those still undergoing development (Deschenes et al.,
2021). Nevertheless, the importance of the methods of
immobilization/disuse are underscored using the rodent
model, because pharmacological inhibition of the NMJ
had opposing eects to the prevention of motoneuron
activation via spinal hemi-section (Mantilla et al., 2007);
blocking of the NMJ resulted in a reduced size of the
synaptic vesicle pool, which was increased when the
motoneuron soma was blocked (Mantilla et al., 2007).
This work shows that motoneuron activation, which
isdiculttoquantifyinhumandisusestudies,clearly
inuences peripheral NMJ adaptation.
More indirect imaging methods point to
disuse-induced NMJ disruption in humans. Neural
cell adhesion molecule (NCAM) is a muscle bre cell
surfaceproteinusedasoneofseveralmolecularmarkers
to highlight innervation status, with NCAM+bres
assumed to be denervated (Soendenbroe et al., 2021). The
proportion of NCAM+bres in human vastus lateralis
increased after 3 (Demangel et al., 2017), 10 (Monti
et al., 2021) and 14 days of bed rest (Arentson-Lantz
et al., 2016). RNA-sequencing analysis also highlighted
NMJ-specic alterations with 10 days of limb suspension
(Sarto et al., 2022); single-bre NCAM expression was
increased following spaceight (Murgia et al., 2022), and
a number of potential circulating biomarkers specic to
the NMJ have been proposed (Sirago et al., 2023).
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6M.PiaseckiJ Physiol 0.0
Several lines of human evidence call attention to NMJ
disruption with neuromuscular disuse, but a functional
impairment of the NMJ was not detectable with electro-
physiological methods at low- to mid-level contra-
ctions.Apossiblenotablecaveatofthisprocessisthe
susceptibility of some NMJs of higher-threshold MUs
to undergo disruption while those of lower-threshold
remain unaltered. Although neuromuscular eects of
ageing are not directly comparable to short-term disuse,
some similarities exist. In older rats, faster muscle bres
(presumably belonging to higher-threshold MUs) are
more susceptible to NMJ disruption and denervation than
slow muscle bres (Kadhiresan et al., 1996). The reasons
for this susceptibility might be related to dierences in
NMJ structure; when compared with type II bres, NMJs
innervating type I bres are smaller, have greater over-
lap between pre- and postsynaptic regions, and are less
fragmented (Sieck & Prakash, 1997). Type II (IIx and IIb in
rodents) NMJs are also more susceptible to transmission
failure (Deschenes et al., 1994; Sieck & Prakash, 1997).
Conclusion and future directions
It is probable that the eects of disuse on MUs are not
equally applied to all MUs, with a suppression of MUFR
that aects lower-threshold MUs disproportionally, and
with NMJ disruption aecting higher-threshold MUs
disproportionately. The two processes are not at odds,
anditisfeasiblethattheyaremutuallyexplanatory;the
increase in MUFR of higher-threshold MUs used during
higher contractions (50% MVC; Valli et al., 2023) might
act to overcome impaired muscle contraction caused by
NMJ disruption, which does not occur in lower-threshold
MUs. This is somewhat of a simplication, but the forces
at which MUs are sampled should be a key consideration
for future mechanistic studies in this eld.
Molecular aspects of spinal motoneurons in
humans will probably continue to elude us owing the
huge methodological constraints that prohibit their
investigation in vivo. Nonetheless, recent advances in
both EMG hardware and decomposition techniques
are enabling a greater number of MU spike trains and,
more importantly, from a greater range of contraction
intensities, to be sampled simultaneously across the
volume of muscle (Avrillon et al., 2024; Chung et al.,
2023; Škarabot et al., 2023).
Of the four human cohorts covered herein in which
individual MUs were sampled, 32 of the 33 combined
participants were male. This limitation might be
problematic given the documented dierences in male
and female MU function (Guo et al., 2022; Guo, Jones,
Smart et al., 2024; Jenz & Pearcey, 2022; Lulic-Kuryllo &
Inglis, 2022), and any further physiological constraints of
including females appear minimal, given that similar
methods of interrupted disuse have been used in
female-only cohorts (MacLennan et al., 2021).
Finally, although useful from a mechanistic standpoint,
the range of laboratory-based assessments performed
to date is somewhat limited. Unilateral MU behaviour
during submaximal isometric contractions might reveal
little of motor pathway commands during more dynamic
functions or those applicable to activities of daily living,
such as bilateral MU function during normal gait. This
dynamic assessment is methodologically challenging but
may generate a research capability in which the MU pool
is viewed not as single entity, but as a range with diering
susceptibility to intervention.
References
Altheyab,A.,Alqurashi,H.,England,T.J.,Phillips,B.E.,
& Piasecki, M.(2024). Cross-education of lower limb
muscle strength following resistance exercise training in
males and females: A systematic review and meta-analysis.
Experimental Physiology. Advance online publication.
https://doi.org/10.1113/EP091881.
Amann, M.(2012). Signicance of Group III and IV muscle
aerents for the endurance exercising human. Clinical and
Experimental Pharmacology and Physiology,39(9), 831–835.
Arentson-Lantz, E. J., English, K. L., Paddon-Jones, D., & Fry,
C. S.(2016). Fourteen days of bed rest induces a decline in
satellite cell content and robust atrophy of skeletal muscle
bers in middle-aged adults. Journal of Applied Physiology,
120(8), 965–975.
Atherton, P. J., Greenha, P. L., Phillips, S. M., Bodine, S. C.,
Adams, C. M., & Lang, C. H.(2016). Control of skeletal
muscle atrophy in response to disuse: Clinical/preclinical
contentions and fallacies of evidence. American Journal
of Physiology-Endocrinology and Metabolism,311(3),
E594-E604.
Aubertin-Leheudre, M., Pion, C. H., Vallée, J., Marchand, S.,
Morais, J. A., Bélanger, M., & Robitaille, R.(2020). Improved
human muscle biopsy method to study neuromuscular
junction structure and functions with Aging ed. Couteur
DL. The Journals of Gerontology: Series A,75, 2098–2102.
Avanzino, L., Bassolino, M., Pozzo, T., & Bove, M.(2011).
Use-dependent hemispheric balance. Journal of Neuro-
science,31(9), 3423–3428.
Avrillon, S., Hug, F., Enoka, R., Caillet, A. H., & Farina,
D.(2024). The decoding of extensive samples of motor
units in human muscles reveals the rate coding of entire
motoneuron pools. eLife,13, RP97085.
Boehm,I.,Alhindi,A.,Leite,A.S.,Logie,C.,Gibbs,A.,
Murray, O., Farrukh, R., Pirie, R., Proudfoot, C., Clutton,
R., Wishart, T. M., Jones, R. A., & Gillingwater, T. H.(2020).
Comparative anatomy of the mammalian neuromuscular
junction. Journal of Anatomy,237(5), 827–836.
Boehm,I.,Miller,J.,Wishart,T.M.,Wigmore,S.J.,Skipworth,
R. J. E., Jones, R. A., & Gillingwater, T. H.(2019). Neuro-
muscular junctions are stable in patients with cancer
cachexia. Journal of Clinical Investigation,130(3),
1461–1465.
© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

J Physiol 0.0 Motor unit adaptation to disuse 7
Calvert, G. H. M., & Carson, R. G.(2022). Neural mechanisms
mediating cross education: With additional considerations
for the ageing brain. Neuroscience & Biobehavioral Reviews,
132, 260–288.
Campbell, M., Varley-Campbell, J., Fulford, J., Taylor,
B., Mileva, K. N., & Bowtell, J. L.(2019). Eect of
immobilisation on neuromuscular function in vivo in
humans: A systematic review. Sports Medicine,49, 931–950.
Chung,B.,Zia,M.,Thomas,K.A.,Michaels,J.A.,Jacob,
A.,Pack,A.,Williams,M.J.,Nagapudi,K.,Teng,L.
H., Arrambide, E., Ouellette, L., Oey, N., Gibbs, R.,
Anschutz, P., Lu, J., Wu, Y., Kashe, M., Oya, T., Kersten,
R., … Mosberger, A. C. (2023). Myomatrix arrays for
high-denition muscle recording. eLife,12, RP88551.
Clement, H.-W., Kirsch, M., Hasse, C., Opper, C., Gemsa, D.,
& Wesemann, W.(1998). Eect of repeated immobilization
on serotonin metabolism in dierent rat brain areas and
on serum corticosterone. Journal of Neural Transmission,
105(10–12), 1155–1170.
Deane, C. S., Piasecki, M., & Atherton, P. J.(2024). Skeletal
muscle immobilisation-induced atrophy: Mechanistic
insights from human studies. Clinical Science,138(12),
741–756.
Del Vecchio, A., Holobar, A., Falla, D., Felici, F., Enoka, R.
M., & Farina, D.(2020). Tutorial: Analysis of motor unit
discharge characteristics from high-density surface EMG
signals. Journal of Electromyography and Kinesiology,53,
102426–102426.
Deluca, C., & Erim, Z.(1994). Common drive of motor units
in regulation of muscle force. Trends in Neurosciences,17(7),
299–305.
Demangel, R., Treel, L., Py, G., Brioche, T., Pagano, A. F.,
Bareille, M., Beck, A., Pessemesse, L., Candau, R., Gharib,
C., Chopard, A., & Millet, C.(2017). Early structural and
functional signature of 3-day human skeletal muscle disuse
using the dry immersion model. The Journal of Physiology,
595(13), 4301–4315.
Deschenes,M.R.,Covault,J.,Kraemer,W.J.,&Maresh,C.
M.(1994). The neuromuscular junction: Muscle bre type
dierences, plasticity and adaptability to increased and
decreased activity. Sports Medicine,17(6), 358–372.
Deschenes,M.R.,Patek,L.G.,Trebelhorn,A.M.,High,M.C.,
& Flannery, R. E.(2021). Juvenile neuromuscular systems
show amplied disturbance to muscle unloading. Frontiers
in Physiology,12, 754052.
Deschenes, M. R., Tenny, K. A., & Wilson, M. H.(2006).
Increased and decreased activity elicits specic
morphological adaptations of the neuromuscular junction.
Neuroscience,137(4), 1277–1283.
Deschenes, M. R., & Wilson, M. H.(2003). Age-related
dierences in synaptic plasticity following muscle
unloading. Journal of Neurobiology,57(3), 246–256.
Deschenes,M.R.,Wilson,M.H.,&Kraemer,W.
J.(2005). Neuromuscular adaptations to spaceight are
specic to postural muscles. Muscle and Nerve,31(4),
468–474.
Duchateau, J., & Hainaut, K.(1990). Eects of immobilization
on contractile properties, recruitment and ring rates of
human motor units. The Journal of Physiology,422(1),
55–65.
Enoka, R. M., & Duchateau, J.(2017). Rate coding and the
control of muscle force. Cold Spring Harbor Perspectives in
Medicine,7(10), a029702.
Garbarini, F., Cecchetti, L., Bruno, V., Mastropasqua, A.,
Fossataro, C., Massazza, G., Sacco, K., Valentini, M. C.,
Ricciardi, E., & Berti, A.(2019). To move or not to move?
functional role of ventral premotor cortex in motor
monitoring during limb immobilization. Cerebral Cortex,
29(1), 273–282.
Glover, I. S., & Baker, S. N.(2020). Cortical, corticospinal, and
reticulospinal contributions to strength training. Journal of
Neuroscience,40(30), 5820–5832.
Glover, I. S., & Baker, S. N.(2022). Both corticospinal and
reticulospinal tracts control force of contraction. Journal of
Neuroscience,42(15), 3150–3164.
Gorassini, M. A., Bennett, D. J., & Yang, J. F.(1998).
Self-sustained ring of human motor units. Neuroscience
Letters,247(1), 13–16.
Guo,Y.,Jones,E.J.,Inns,T.B.,Ely,I.A.,Stashuk,D.W.,
Wilkinson, D. J., Smith, K., Piasecki, J., Phillips, B. E.,
Atherton, P. J., & Piasecki, M.(2022). Neuromuscular
recruitment strategies of the vastus lateralis according to
sex. Acta Physiologica,235(2), 13803.
Guo,Y.,Jones,E.J.,Škarabot,J.,Inns,T.B.,Phillips,B.E.,
Atherton, P. J., & Piasecki, M.(2024). Common synaptic
inputs and persistent inward currents of vastus lateralis
motor units are reduced in older male adults. GeroScience,
46(3),3249–3261.
Guo, Y., Jones, E. J., Smart, T. F., Altheyab, A., Gamage, N.,
Stashuk,D.W.,Piasecki,J.,Phillips,B.E.,Atherton,P.J.,&
Piasecki, M.(2024). Sex disparities of human neuromuscular
decline in older humans. The Journal of Physiology.Advance
online publication. https://doi.org/10.1113/JP285653.
Hardy,E.J.O.,Inns,T.B.,Hatt,J.,Doleman,B.,Bass,J.J.,
Atherton, P. J., Lund, J. N., & Phillips, B. E.(2022). The time
course of disuse muscle atrophy of the lower limb in health
and disease. Journal of Cachexia, Sarcopenia and Muscle,
13(6), 2616–2629.
Hassan, A. S., Fajardo, M. E., Cummings, M., McPherson, L.
M.,Negro,F.,Dewald,J.P.A.,Heckman,C.J.,&Pearcey,
G. E. P.(2021). Estimates of persistent inward currents are
reduced in upper limb motor units of older adults. The
Journal of Physiology,599(21), 4865–4882.
Heckman, C. J., Mottram, C., Quinlan, K., Theiss, R., &
Schuster, J.(2009). Motoneuron excitability: The importance
of neuromodulatory inputs. Clinical Neurophysiology,
120(12), 2040–2054.
Inns,T.B.,Bass,J.J.,Hardy,E.J.O.,Wilkinson,D.J.,Stashuk,
D. W., Atherton, P. J., Phillips, B. E., & Piasecki, M.(2022).
Motor unit dysregulation following 15 days of unilateral
lower limb immobilisation. The Journal of Physiology,
600(21), 4753–4769.
Jacobs, B. L., Martı´
n-Cora, F. J., & Fornal, C. A.(2002).
Activity of medullary serotonergic neurons in freely moving
animals. Brain Research Reviews,40(1–3), 45–52.
Jenz,S.T.,Beauchamp,J.A.,Gomes,M.M.,Negro,F.,
Heckman, C. J., & Pearcey, G. E. P.(2023). Estimates of
persistent inward currents in lower limb motoneurons are
larger in females than in males. Journal of Neurophysiology,
129(6), 1322–1333.
© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

8M.PiaseckiJ Physiol 0.0
Jenz, S. T., & Pearcey, G. E. P.(2022). Sex matters in neuro-
muscular control. Acta Physiologica,235(2), e13823.
Johnson, M. D., & Heckman, C. J.(2014). Gain control
mechanisms in spinal motoneurons. FrontNeuralCircuits,
8, 00081.
Jones,E.J.,Chiou,S.-Y.,Atherton,P.J.,Phillips,B.E.,&
Piasecki, M.(2022). Ageing and exercise-induced motor unit
remodelling. The Journal of Physiology,600, 1839–1849.
Jones, E. J., Guo, Y., Martinez-Valdes, E., Negro, F., Stashuk, D.
W., Atherton, P. J., Phillips, B. E., & Piasecki, M.(2023).
Acute adaptation of central and peripheral motor unit
features to exercise-induced fatigue diers with concentric
and eccentric loading. Experimental Physiology,108(8),
827–837.
Jones, E. J., Piasecki, J., Ireland, A., Stashuk, D. W., Atherton,
P. J., Phillips, B. E., McPhee, J. S., & Piasecki, M.(2021).
Lifelong exercise is associated with more homogeneous
motor unit potential features across deep and supercial
areas of vastus lateralis. GeroScience,43(4), 1555–1565.
Jones,R.A.,Harrison,C.,Eaton,S.L.,LlaveroHurtado,M.,
Graham, L. C., Alkhammash, L., Oladiran, O. A., Gale, A.,
Lamont,D.J.,Simpson,H.,Simmen,M.W.,Soeller,C.,
Wishart, T. M., & Gillingwater, T. H.(2017). Cellular and
molecular anatomy of the human neuromuscular junction.
Cell Reports,21(9), 2348–2356.
Kadhiresan, V. A., Hassett, C. A., & Faulkner, J. A.(1996).
Properties of single motor units in medial gastrocnemius
muscles of adult and old rats. The Journal of Physiology,
493(2), 543–552.
Katz, B., & Miledi, R.(1965). The measurement of synaptic
delay, and the time course of acetylcholine release at the
neuromuscular junction. Proceedings of the Royal Society of
London, Series B: Biological Sciences,161, 483–495.
Kiehn, O.(2016). Decoding the organization of spinal circuits
that control locomotion. Nature Reviews Neuroscience,
17(4), 224–238.
Lan, N., & He, X.(2012). Fusimotor control of spindle
sensitivity regulates central and peripheral coding of joint
angles. Frontiers in Computational Neuroscience,6, 00066.
Lulic-Kuryllo, T., & Inglis, J. G.(2022). Sex dierences
in motor unit behaviour: A review. Journal of Electro-
myography and Kinesiology,66, 102689.
MacLennan, R. J., Ogilvie, D., McDorman, J., Vargas, E.,
Grusky, A. R., Kim, Y., Garcia, J. M., & Stock, M. S.(2021).
The time course of neuromuscular impairment during
short-term disuse in young women. Physiological Reports,
9(1), 14677.
Mantilla, C. B., Rowley, K. L., Zhan, W.-Z., Fahim, M. A., &
Sieck, G. C.(2007). Synaptic vesicle pools at diaphragm
neuromuscular junctions vary with motoneuron soma, not
axon terminal, inactivity. Neuroscience,146(1), 178–189.
Martinez-Valdes, E., Negro, F., Farina, D., & Falla, D.(2020).
Divergent response of low- versus high-threshold motor
units to experimental muscle pain. The Journal of Physio-
logy,598(11), 2093–2108.
Martino,G.,Valli,G.,Sarto,F.,Franchi,M.V.,Narici,M.V.,
& De Vito, G.(2024). Neuromodulatory contribution to
muscle force production after short-term unloading and
active recovery. Medicine & Science in Sports & Exercise,
56(9), 1830–1839.
Marusic, U., Narici, M., Simunic, B., Pisot, R., & Ritzmann,
R.(2021). Nonuniform loss of muscle strength and atrophy
during bed rest: A systematic review. Journal of Applied
Physiology,131(1), 194–206.
Maxwell, D. J., & Soteropoulos, D. S.(2020). The mammalian
spinal commissural system: Properties and functions.
Journal of Neurophysiology,123(1), 4–21.
Mesquita,R.N.O.,Taylor,J.L.,Heckman,C.J.,Trajano,G.
S., & Blazevich, A. J.(2024). Persistent inward currents
in human motoneurons: Emerging evidence and future
directions. Journal of Neurophysiology,132(4), 1278–1301,
jn.00204.2024.
Mesquita,R.N.O.,Taylor,J.L.,Trajano,G.S.,Škarabot,
J.,Holobar,A.,Gonçalves,B.A.M.,&Blazevich,A.
J.(2022). Eects of reciprocal inhibition and whole-body
relaxation on persistent inward currents estimated by two
dierent methods. The Journal of Physiology,600(11), 2765–
2787.
Monti, E., Reggiani, C., Franchi, M. V., Toniolo, L., Sandri,
M.,Armani,A.,Zampieri,S.,Giacomello,E.,Sarto,F.,
Sirago, G., Murgia, M., Nogara, L., Marcucci, L., Ciciliot,
S., Šimunic, B., Pišot, R., & Narici, M. V.(2021). Neuro-
muscular junction instability and altered intracellular
calcium handling as early determinants of force loss during
unloading in humans. The Journal of Physiology,599(12),
3037–3061.
Murgia,M.,Ciciliot,S.,Nagaraj,N.,Reggiani,C.,Schiano,
S.,Franchi,M.V.,Pišot,R.,Šimuni
ˇ
c, B., Toniolo, L., Blaauw,
B., Sandri, M., Biolo, G., Flück, M., Narici, M. V., & Mann,
M.(2022). Signatures of muscle disuse in spaceight and
bedrestrevealedbysinglemuscleberproteomics.The
Proceedings of the National Academy of Sciences Nexus,1(3),
pgac086.
Ohmichi, Y., Sato, J., Ohmichi, M., Sakurai, H., Yoshimoto, T.,
Morimoto, A., Hashimoto, T., Eguchi, K., Nishihara, M.,
Arai, Y.-C. P., Ohishi, H., Asamoto, K., Ushida, T., Nakano,
T., & Kumazawa, T.(2012). Two-week cast immobilization
induced chronic widespread hyperalgesia in rats. European
Journal of Pain,16(3), 338–348.
Orssatto, L. B. R., Blazevich, A. J., & Trajano, G. S.(2023).
Ageing reduces persistent inward current contribution to
motor neurone ring: Potential mechanisms and the role of
exercise. The Journal of Physiology,601(17), 3705–3716.
Orssatto,L.B.R.,Borg,D.N.,Blazevich,A.J.,Sakugawa,
R. L., Shield, A. J., & Trajano, G. S.(2021). Intrinsic
motoneuron excitability is reduced in soleus and tibialis
anterior of older adults. GeroScience,43(6), 2719–2735.
Orssatto,L.B.R.,Fernandes,G.L.,Blazevich,A.J.,&Trajano,
G. S.(2022). Facilitation–inhibition control of motor neuro-
nal persistent inward currents in young and older adults.
The Journal of Physiology,600(23), 5101–5117.
Orssatto, L. B. R., Rodrigues, P., Mackay, K., Blazevich, A. J.,
Borg,D.N.,Souza,T.R.D.e,Sakugawa,R.L.,Shield,A.J.,
& Trajano, G. S.(2023). Intrinsic motor neuron excitability
is increased after resistance training in older adults. Journal
of Neurophysiology,129(3), 635–650.
Pearcey, G. E. P., Alizedah, S., Power, K. E., & Button, D.
C.(2021). Chronic resistance training: Is it time to rethink
the time course of neural contributions to strength gain?
European Journal of Applied Physiology 1–10.
© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

J Physiol 0.0 Motor unit adaptation to disuse 9
Piasecki, M., Garnés-Camarena, O., & Stashuk, D. W.(2021).
Near-ber electromyography. Clinical Neurophysiology,
132(5), 1089–1104.
Piasecki, M., & Stashuk, D. W.(2023). Neuromuscular
Function: Intramuscular Electromyography. In Neuro-
muscular Assessments of Form and Function,ed.Atherton
P. J. & Wilkinson D. J., Neuromethods, (vol. 204,pp.
125–135). Springer US, New York, NY.
Preobrazenski, N., Janssen, I., & McGlory, C.(2023). The
eects of single-leg disuse on skeletal muscle strength
and size in the nonimmobilized leg of uninjured adults:
A meta-analysis. Journal of Applied Physiology,134(6),
1359–1363.
Preobrazenski, N., Seigel, J., Halliday, S., Janssen, I., &
McGlory, C.(2023). Single-leg disuse decreases skeletal
muscle strength, size, and power in uninjured adults: A
systematic review and meta-analysis. Journal of Cachexia,
Sarcopenia and Muscle,14(2), 684–696.
Rossato, J., Avrillon, S., Tucker, K., Farina, D., & Hug,
F.(2024). The volitional control of individual motor units
is constrained within low-dimensional neural manifolds
by common inputs. Journal of Neuroscience,44(34),
e0702242024.
Santin, J. M., Vallejo, M., & Hartzler, L. K.(2017). Synaptic
up-scaling preserves motor circuit output after chronic,
natural inactivity. eLife,6, e30005.
Sarto,F.,Stashuk,D.W.,Franchi,M.V.,Monti,E.,Zampieri,
S.,Valli,G.,Sirago,G.,Candia,J.,Hartnell,L.M.,Paganini,
M., McPhee, J. S., De Vito, G., Ferrucci, L., Reggiani, C., &
Narici, M. V.(2022). Eects of short-term unloading and
active recovery on human motor unit properties, neuro-
muscular junction transmission and transcriptomic prole.
The Journal of Physiology,600(21), 4731–4751.
Seki, K., Taniguchi, Y., & Narusawa, M.(2001). Eects of joint
immobilization on ring rate modulation of human motor
units. The Journal of Physiology,530(3), 507–519.
Sieck, G. C., & Prakash, Y. S.(1997). Morphological
adaptations of neuromuscular junctions depend on ber
type. Canadian Journal of Applied Physiology,22(3),
197–230.
Sirago, G., Pellegrino, M. A., Bottinelli, R., Franchi, M. V.,
& Narici, M. V.(2023). Loss of neuromuscular junction
integrity and muscle atrophy in skeletal muscle disuse.
Ageing Research Reviews,83, 101810.
Škarabot, J., Beauchamp, J. A., & Pearcey, G. E.(2023). Human
motor unit discharge patterns reveal dierences in neuro-
modulatory and inhibitory drive to motoneurons across
contraction levels. Neuroscience.bioRxiv. http://biorxiv.org/
lookup/doi/10.1101/2023.10.16.562612
Soendenbroe, C., Andersen, J. L., & Mackey, A. L.(2021).
Muscle-nerve communication and the molecular
assessment of human skeletal muscle denervation with
aging. American Journal of Physiology-Cell Physiology,
321(2), C317–C329.
Stashuk, D. W.(1999). Detecting single ber contributions to
motor unit action potentials. Muscle & Nerve,22, 218–229.
Takahashi,H.,Takada,Y.,Nagai,N.,Urano,T.,&Takada,
A.(1998). Extracellular serotonin in the striatum increased
after immobilization stress only in the nighttime.
Behavioural Brain Research,91(1–2), 185–191.
Valli, G., Sarto, F., Casolo, A., Del Vecchio, A., Franchi, M. V.,
Narici, M. V., & De Vito, G.(2023). Lower limb suspension
induces threshold-specic alterations of motor units
properties that are reversed by active recovery. Journal of
Sport and Health Science,13(2), 264–276.
Witham,C.L.,Fisher,K.M.,Edgley,S.A.,&Baker,S.
N.(2016). Corticospinal inputs to primate motoneurons
innervating the forelimb from two divisions of primary
motor cortex and Area 3a. Journal of Neuroscience,36(9),
2605–2616.
Zholudeva, L. V., Abraira, V. E., Satkunendrarajah, K.,
McDevitt, T. C., Goulding, M. D., Magnuson, D. S. K., &
Lane, M. A.(2021). Spinal interneurons as gatekeepers to
neuroplasticity after injury or disease. Journal of Neuro-
science,41(5), 845–854.
Additional information
Competing interests
The author has no competing interests to declare.
Author contributions
Sole author.
Funding
None.
Acknowledgements
I am grateful to several colleagues for the many discussions
around neuromuscular function in health, disease and disuse.
Keywords
disuse,ringrate,motoneuron,neuromuscularjunction
Supporting information
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