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. 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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: lars.larsson@neurofys.uu.se 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 . 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. 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. Metabolic and phenotypic adaptations of diaphragm muscle fibers with inactivation. Journal of Applied Physiology, 82, 1145–1153. 55 G.S. Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness,