46 C.B. Mantilla and G.C. Sieck
and/or IIb fibers is greater than at type I and IIa fibers (see below). Mitochondrial
volume density is also greater at presynaptic terminals innervating type I and IIa
fibers compared to those innervating type IIx and/or IIb fibers, possibly reflect-
ing the metabolic requirements of increased activation of these presynaptic ter-
minals. Taken together with the greater number of cycling vesicles at type I and
IIa fibers, these ultrastructural differences may contribute to a greater ability of
presynaptic terminals at type I or IIa fibers to sustain neuromuscular transmis-
sion with repeated activation (Johnson and Sieck 1993; Ermilov et al. 2007;
Rowley et al. 2007).
3.3 Ultrastructural Properties of Postsynaptic Motor End-Plates
At type I and IIa fibers, motor end-plates display less complex postsynaptic
folding but increased gutter depth compared to type IIx and/or IIb fibers (Fahim
and Robbins 1982; Fahim et al. 1983; Rowley et al. 2007). In addition, there is
frequent interposition of cellular organelles (e.g., mitochondria, endoplasmic
reticulum or myonuclei) between the motor end-plate and underlying myofibrils
at type I and IIa fibers, but not at type IIx and/or IIb fibers. Indeed, these features
of motor end-plate ultrastructure can be used to grossly distinguish among
muscle fiber types. The density and location of cholinergic receptors at the crest
of postsynaptic folds does not appear to differ across motor end-plates at different
muscle fiber types (Oda 1984; Fertuck and Salpeter 1974). However, the given
the larger surface area of motor end-plates at type IIx and/or IIb fibers, the actual
number of cholinergic receptors is greater at these fibers.
Fig. 4 Ultrastructural elements of type-identified NMJs. Presynaptic terminals at type-identified
rat diaphragm muscle fibers (Mantilla et al. 2004, 2007; Rowley et al. 2007) are full of synaptic
vesicles (top), which cluster around active zones (arrowheads) opposing postsynaptic folds
( bottom). The number and distribution of synaptic vesicles docked at each active zone is consistent
across fiber types
47Age-Related Remodeling of Neuromuscular Junctions
3.4 Aging Effects on Neuromuscular Junction Structure
With aging, there is increased fragmentation of nerve terminals at all muscle fibers

resulting in increased number of nerve terminal branches and greater complexity
(Prakash and Sieck 1998; Courtney and Steinbach 1981; Andonian and Fahim 1989;
Fahim and Robbins 1982; Fahim et al. 1983). The increased number of terminal
branches may reflect sprouting as motor neurons degenerate with aging. In the rat
diaphragm muscle, the number of nerve terminal branches increased with aging at
all muscle fiber types, but this increase was more pronounced at type IIx and/or IIb
fibers (Prakash and Sieck 1998). Whereas individual branch length remained
relatively unchanged, total branch length increased as a result of the greater number
of branches at type IIx and/or IIb fibers. The increased branching complexity
associated with aging resulted in a significant increase in the planar surface area of
presynaptic terminals at type IIx and/or IIb fibers (Prakash and Sieck 1998). This
occurs despite an actual age-related reduction in the cross-sectional area of type IIx
and/or IIb diaphragm muscle fibers. Similar fiber type-dependent changes in nerve
terminal size and complexity also occur in muscles of different fiber type composi-
tion (e.g., soleus muscle - composed of primarily slow-twitch fibers, and extensor
digitorum longus muscle – composed of predominantly fast-twitch fibers) in both
rats and mice (Andonian and Fahim 1989; Fahim and Robbins 1982; Fahim et al.
1983). In mice hind limb muscles, there are age-related reductions in the number
and density of mitochondria and synaptic vesicles at presynaptic terminals (Fahim and
Robbins 1982). Indeed, aging nerve terminals appear to be increasingly occupied by
smooth endoplasmic reticulum, cisternae, microtubules, neurofilaments and coated
vesicles. Accordingly, with age, acetylcholine content decreases at presynaptic
terminals in the rat diaphragm muscle, most likely reflecting increased acetylcholine
leakage despite increased synthesis rate and choline availability (Smith and Weiler
1987). Increased acetylcholine leakage may result from the overall increase in pre-
synaptic terminal area, but is not associated with increased frequency of miniature
end-plate potentials (indicative of spontaneous synaptic vesicle release) or a change
in Ca
2+
sensitivity for synaptic vesicle release (Smith 1988).

On the postsynaptic side, aging is also associated with increased branching and
complexity of junctional folds (Wokke et al. 1990; Rosenheimer and Smith 1985;
Prakash and Sieck 1998). In the rat diaphragm muscle, there is a corresponding age-
related increase in motor end-plate surface area, predominantly at type IIx and/or IIb
fibers (Prakash and Sieck 1998; Arizono et al. 1984). In addition, there is an age-
related increase in subsarcolemmal vesicles and appearance of lipofuscin deposits
(Fahim and Robbins 1982). With aging, there is a gradual decrease in the number of
cholinergic receptors at the motor end-plate and appearance of extra-junctional
receptors (Courtney and Steinbach 1981; Smith et al. 1990). These changes may be
the result of motor neuron loss and consequent denervation of some muscle fibers.
The incidence of nerve terminals projecting beyond the motor end-plate markedly
increases with aging (Prakash and Sieck 1998). This may reflect some general stimulus
for terminal sprouting, consistent with age-related motor neuron loss and denervation
48 C.B. Mantilla and G.C. Sieck
and/or the reduced activity levels associated with aging. In agreement with some
impact of age-related inactivity, similar morphological changes occur at an earlier age
in the extensor digitorum longus muscle compared to the soleus and diaphragm mus-
cles of rats (Kelly and Robbins 1983; Rosenheimer and Smith 1985). In older animals,
the extent of overlap between nerve terminals and motor end-plates (expressed as a
percent of end-plate area) remains greatest at type I and IIa fibers. At type IIx and/or
IIb fibers, the extent of overlap between nerve terminals and motor end-plates increases
with age but remains below that of type I and IIa fibers (Prakash and Sieck 1998).
4 Functional Properties of Neuromuscular Junctions
In mammals, functional properties of a neuromuscular junction depend on both
presynaptic release of acetylcholine and cholinergic receptor-induced postsynaptic
responses. These functional properties of neuromuscular junctions are matched to
the demands of muscle fibers particularly as they relate to activation level and
susceptibility to neuromuscular transmission failure. For example, functional prop-
erties of neuromuscular junctions at type I or IIa fibers must meet the functional
demands of higher activity levels and subsequent metabolic demands. These motor

units are often recruited to accomplish motor behaviors where failure cannot be
tolerated and therefore fidelity of the postsynaptic contractile response must be
maintained. Functional properties of neuromuscular junctions can be assessed
using a variety of techniques, including assessment of electromyographic record-
ings of evoked compound muscle action potentials, assessment of force loss during
nerve vs. direct muscle stimulation, and microelectrode measurements of synaptic
potentials and/or currents. Important in all these measures are dependencies on
fiber type, muscle fiber size (cross-sectional area) and frequency of activation.
4.1 Fiber Type Differences in Neuromuscular Transmission
As mentioned above, there are significant structural differences in presynaptic terminals
across fiber types that will affect the release of acetylcholine. For example, the total
number of active zones at type IIx and/or IIb fibers is substantially greater than at
type I or IIa fibers. Thus, while quantal size as reflected by average miniature end-
plate potential (mEPP) amplitude normalized for membrane input resistance (or
capacitance) does not vary across fiber types, quantal content (defined as the ratio
of EPP to mEPP) is significantly greater at type IIx and/or IIb fibers in diaphragm
muscle (Rowley et al. 2007; Ermilov et al. 2007). Comparing across muscle pre-
dominantly composed of type I or IIa (rat soleus muscle) vs. type IIx and/or IIb
fibers (rat extensor digitorum muscle), several investigators have also confirmed
higher quantal content at type IIx and/or IIb fibers (Reid et al. 1999; Wood and
Slater 1997).
49Age-Related Remodeling of Neuromuscular Junctions
The safety factor for neuromuscular transmission is defined as the ratio of EPP
amplitude to action potential activation threshold in muscle fibers. The action
potential activation threshold is highly dependent on the density of Na
+
channels
near the motor end-plate. Comparing across muscles predominantly comprising a
single fiber type, several studies (Ruff 1996; Harrison et al. 1997) have reported
that Na

+
channel density is much higher at type IIx and/or IIb fibers (e.g., extensor
digitorum longus muscle) than at type I fibers (e.g. soleus muscle). The impact of
higher Na
+
channel density and Na
+
input current would be mitigated by the larger
membrane surface area of type IIx and/or IIb muscle fibers that results in increased
membrane capacitance. For example, type IIx and/or IIb fibers in the rat diaphragm
are ~2-fold larger than type I or IIa muscle fibers, and accordingly, their membrane
capacitance would be ~4-fold higher. To maintain the same threshold for action
potential generation, Na
+
channel density and Na
+
input currents must be propor-
tionally higher in type IIx and/or IIb fibers. Indeed, in the rat diaphragm, we found
that the action potential activation threshold was lower at type IIx and/or IIb fibers
compared to type I and IIa fibers (Ermilov et al. 2007). Thus, if anything, it appears
that higher Na
+
channel density at type IIx and/or IIb fibers in the rat diaphragm
muscle more than compensates for differences in fiber size.
Due to both differences in EPP amplitude and action potential activation thresh-
old, the safety factor for neuromuscular transmission is higher at type IIx and/or IIb
fibers compared to type I or IIa fibers (Ermilov et al. 2007). However, during repeti-
tive stimulation, EPP amplitude progressively declines across all muscle fiber
types, but this decline is much greater at type IIx and/or IIb fibers (Rowley et al.
2007). The decline in quantal content varies across stimulation frequencies at type

IIx and/or IIb fibers but not at type I or IIa fibers. With continuous stimulation, the
decline in quantal content is dynamic, being very steep during the initial few pulses
followed by a much slower decrement until a plateau is reached where there is no
difference between fiber types (Rowley et al. 2007). It appears that the initial rapid
decline in quantal content reflects both a depletion of the readily releasable pool of
synaptic vesicles and a decrease in the probability of synaptic vesicle release. The
slower decline in quantal content during repetitive stimulation likely reflects a
balance between synaptic vesicle depletion and repletion at the readily releasable
pool. Synaptic vesicles at the readily releasable pool are replenished either by
recruitment from the immediately adjacent pool of vesicles (reserve pool) or by
recycling of released vesicles. Since the density of vesicles in the immediately
available pool is greater at type I and IIa fibers compared to type IIx and/or IIb
fibers this could contribute to the maintenance of quantal content in these fibers
compared to type IIx and/or IIb fibers. Based on the presynaptic uptake of styryl
dyes (e.g., FM4-64 or FM1-43), the extent of synaptic vesicle recycling is greater
at type I and IIa fibers compared to type IIx and/or IIb fibers (Mantilla et al. 2004;
Rowley et al. 2007). Both mechanisms likely contribute to the replenishment of the
readily releasable pool at type I and IIa fibers during repetitive stimulation reducing
susceptibility to neuromuscular transmission failure.
It is unclear whether the threshold for action potential generation changes with
repetitive activation, although this seems unlikely given the duration of the action
50 C.B. Mantilla and G.C. Sieck
potential refractory period in muscle fibers. Thus, with repetitive stimulation, it is
clear that the safety factor declines and that type IIx and/or IIb muscle fibers
become more susceptible to neuromuscular transmission failure. This has been
confirmed in the diaphragm muscle using a glycogen-depletion technique where
neuromuscular transmission failure is reflected by the failure to activate muscle
fibers and thus deplete their glycogen stores (Johnson and Sieck 1993).
4.2 Effects of Aging on Neuromuscular Transmission
No study to date has directly examined age-related changes in safety factor across

different fiber types. Miniature end-plate potential amplitude appears to be
unaffected by aging (Banker et al. 1983; Kelly and Robbins 1983). In the mouse
extensor digitorum longus and soleus muscles, EPP amplitude was reported to
increase with age (Kelly and Robbins 1983). The age-related change in EPP
amplitude is not related to an increase in cholinergic receptor density at motor
end-plates (Courtney and Steinbach 1981; Smith et al. 1990). Thus, it is likely that
quantal content increases with age in these limb muscles. These investigators found
that EPP amplitude in soleus muscle fibers was greater than in extensor digitorum
longus muscle fibers. In the mouse, the extensor digitorum longus muscle comprises
predominantly type IIx and/or IIb fibers and the soleus comprises predominantly
type I and IIa fibers. Thus, these observations were in contrast to other reports
where in a mixed muscle EPP amplitude (and quantal content) of type I and IIa
fibers was lower than that of type IIx and/or IIb fibers (Ermilov et al. 2007; Rowley
et al. 2007). Minimal age-related changes in EPP amplitude and quantal content
were reported for the mouse diaphragm muscle (Banker et al. 1983; Kelly and
Robbins 1987). In this sense, it is possible that the age-related increase in EPP
amplitude and quantal content are limited to limb muscles and do not reflect a
general response across all muscles.
An age-related decrease in cross-sectional area, in particular at type IIx and/or
IIb fibers, would be associated with increased input resistance and, thus, a lower
action potential activation threshold. Furthermore, with aging, there is either no
change or an increase in the density of Na
+
channels at skeletal muscle fibers
(Desaphy et al. 1998), which together with the change in fiber cross-sectional area
would tend to lower action potential activation threshold. However, further work
needs to be performed to confirm that there are age-related changes in action poten-
tial threshold at these fibers. If there is and age-related reduction in the threshold
for action potential generation, it is unclear whether this would be sufficient to miti-
gate reductions in EPP amplitude. There is little age-related change in type I and

IIa fiber cross-sectional areas, but there may be changes in Na
+
conductance in
these fibers; thus, it is difficult to predict whether the action potential activation
threshold is affected. Without a change in action potential threshold, any change in
EPP amplitude would result in a decrease in safety factor at these fibers. Comparing
across muscles of a predominant fiber type composition, it was reported that
51Age-Related Remodeling of Neuromuscular Junctions
age-related changes at the neuromuscular junction are well compensated across fiber
types with minimal impact on safety factor for neuromuscular transmission (Banker
et al. 1983; Kelly and Robbins 1987). However, it is very important to compare
across muscle fibers in a single muscle since type I muscle fibers in the soleus
muscle are generally much larger than type I fibers found in mixed muscles. The
safety factor for neuromuscular transmission decreases in the rat diaphragm muscle
with increasing age (Kelly 1978), but fiber type differences were not examined.
5 Conclusions
At present, there is surprisingly little direct information about the effects of aging on
the long-term plasticity of NMJs at different fiber types, especially in muscle of
mixed fiber type composition. Yet aging is clearly associated with changes that
could affect remodeling of the NMJ rendering it less resilient to perturbations
induced by disease or injury. Indeed, reductions in physical activity and the resulting
unloading of limb muscles affect NMJ structure and function to a greater extent in
older animals. Aging-related loss of motor neurons results in functional denervation
of muscle fibers that are then re-innervated by axons sprouting from remaining
neighboring motor neurons. The combined reduction in motor neuron number and
enlargement of motor unit size leads to loss of motor unit diversity. Importantly,
aging results in a disproportionate loss of those motor units able to generate greater
forces, which are also those that display the greatest reduction in muscle fiber size.
With aging, there appear to be effective compensatory mechanisms that provide
preservation of motor units required for low force, sustained motor behaviors, which

may be advantageous for example in the maintenance of adequate ventilation or
posture. However, aging-related changes in neuromuscular plasticity may be at the
expense of maintaining structural and functional diversity in motor unit properties.
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55
G.S. Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness,
DOI 10.1007/978-90-481-9713-2_4, © Springer Science+Business Media B.V. 2011
Abstract Aging has profound effects on skeletal muscle structure and function
with significant consequences for both the individual and society. In this short
review aging-related changes in the structure and function of the final functional
unit in the motor system, i.e., the motor unit, are discussed. This review does not

aim to give an overview of all aspects associated with aging-related changes in the
motor unit, but will focus on specific changes in motor unit structure and function,
such as the spatial organization of the muscle fibers innervated by a single alpha
motoneuron, i.e., motor unit fiber, as well as aging-related motor unit transitions,
and changes in motor unit physiological and firing properties.
Keywords Myosin
•
Motoneuron
•
Glycogen-depletion
•
Firing pattern
1 Introduction
Aging has profound effects on the motor system resulting in impaired coordination,
balance, speed and force with significant negative consequences for morbidity and
mortality in elderly citizens. That is, falls are a major cause of morbidity and mortality
L. Larsson (*)
Department of Clinical Neurophysiology, Uppsala University Hospital, Entrance 85,
3rd Floor, 751 85, Uppsala, Sweden
e-mail:
and
Department of Neuroscience, Clinical Neurophysiology, Uppsala University, Sweden
and
Department of Biobehavioral Health, the Pennsylvania State University, PA, USA
A. Cristea
Department of Neuroscience, Clinical Neurophysiology, Uppsala University, Sweden
D.E. Vaillancourt
Department of Kinesiology and Nutrition, Departments of Bioengineering and Neurology,
University of Illinois at Chicago, Chicago, IL, USA
Aging-Related Changes Motor Unit Structure

and Function
Alexander Cristea, David E. Vaillancourt, and Lars Larsson