Relationship of rostrocaudal position and segmental innervation to the incidence of age-related alterations in NMJs.

<p>Muscles listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034640#pone-0034640-t001" target="_blank">Table 1</a> are arranged by the rostrocaudal position of the motor pool that innervates them. The ordinate is the percentage of NMJs with age-associated defects from column 6 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034640#pone-0034640-t001" target="_blank">Table 1</a>.</p

Structural alterations in NMJs during the third year.

<p>NMJs from EDL (A), frontalis (B) and EOM (C) of 3 year-old mice. D) Incidence of age-related alterations in NMJs of 3 month-, 2 year-, and 3 year-old mice. Each bar represents mean ± SEM from at least 3 animals, with at least 100 NMJs counted per animal. *p<0.025 by <i>t</i>-test. Scale bar = 10 µm.</p

Extent of denervation in muscles from SOD-G93A mice.

<p>A, B) NMJs in muscles from symptomatic (A) or end-stage (B) SOD-G93A mice listed were scored as fully innervated, partially denervated or fully denervated. Values are shown for 14–16 weeks old symptomatic (A) and 19 weeks old end-stage mice (B). C) NMJs from soleus and tibialis anterior muscles of end stage mice, stained as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034640#pone-0034640-g009" target="_blank">Fig. 9</a>. Scale bar = 10 µm. Some NMJs remain partially innervated in soleus, but most NMJs are fully denervated with dispersed postsynaptic structures in tibialis anterior.</p

Fraction of NMJs exhibiting one or more age-related alterations.

<p>Columns 2 and 3 show the percentage of NMJs in young adult and old muscles that exhibit one or more of five structural features that characterize old NMJs in limb muscles: decreased AChR density, partial denervation, complete denervation, nerve terminal sprouting, fragmentation of the postsynaptic membrane. Column 4 and 5 show values for only the first four of these features, taking account of the fact that the postsynaptic membrane is fragmented in young adult NMJs in some muscles. Column 6 shows difference between values in columns 4 and 5. Values represent average (and SD) from at least 3 animals, with at least 100 NMJs counted per animal.</p

Fiber type composition of muscles with NMJs that vary in severity of age-related changes.

<p>A) Cross-sections of soleus, EDL, and interscutularis muscles from young adult mice stained with antibodies specific for myosin Type I, IIA or IIB. Scale Bar: 20 µm. B) Fiber type composition of muscles, determined from micrographs such as those in A. NMJs in both soleus and EDL suffer severe age-related changes but differ in fiber type composition. In contrast, NMJs in frontalis, levator auris longus and intercutularis are largely spared from age-related changes, even though their fiber type composition of these muscles is similar to that of the EDL.</p

Age-related alterations in neuromuscular junction of 2 year-old mice.

<p>Incidence of age-related alterations shown in NMJs from sternomastoid, Gracilis, Soleus Diaphragm, Gastrocnemis, Anal Sphincter, EDL and EOM. A) Fragmentation of the postsynaptic membrane. B) Decreased AChR density. C) Partial denervation. D) Complete denervation. E) Nerve terminal sprouting. F) Preterminal axonal distension. G) Axonal dystrophy. H) Multiple innervation of a single postsynaptic site. Values from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034640#pone-0034640-g001" target="_blank">Fig. 1</a> are replotted for comparison. Each point represents mean ±SEM from at least 3 animals, with at least 100 NMJs counted per animal. Dashed lines are drawn to emphasize that values for external anal sphincter, frontalis and EOM are generally lower than those from other muscles.</p

Branch order analysis of motor axons in young adult and aged animals.

<p>A, B) Branching trees from motor units in young adult (A) and old (B) omohyoid muscles. Color of circles represents the degree of AChR occupancy by nerve terminals (color code, bottom right corner). C) No correlation between AChR occupancy and distance of the nerve terminal from the first branch point of the axonal arbor in either the young adult (r = 0.09) or the aged animal (r = 0.05).</p

NMJ morphology in Type I and Type II muscle fibers of old mice.

<p>A, B) EDL and Soleus muscles from 2 year-old mice were stained for Type 1 muscle fibers. Type 1 muscle fibers are found in old (A) but not young EDL muscles (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034640#pone-0034640-g005" target="_blank">Fig. 5</a>). C, D) NMJs in Type I and Type II fibers exhibit age-related changes in EDL (C) and soleus (D). Each bar represents mean ± SEM. Scale bar = 20 µm.</p

Age-related changes in motor unit size.

<p>Tracings from muscles such as the one shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034640#pone-0034640-g007" target="_blank">Figure 7A</a>. A–D) Motor units from the omohyoid (A, B) and extraocular muscles (C, D) of young adult (A, C) and old (B, D) mice. E, F) Motor unit size in young adult and old omohyoid (E) and extraocular (F) muscles. Each bar represents mean ± SEM from at least 4 motor units per muscle and age. Scale bar = 50 µm.</p

Motor unit size in muscles with NMJs that vary in severity of age-related changes.

<p>A) Frontalis muscle of a young adult mouse in a simple motor axon was YFP-positive (green). The muscle was also labeled with BTX (red) to mark all postsynaptic sites. Scale bar: 50 µm. B) Motor unit size determined from muscles such as that in A. Bars indicate mean (±SD) of number of motor units in parentheses. Data on neck muscles (sternomastoid, clavotrapezius, and cleidomastoid) are replotted from Schaefer et al., (2005).</p