Figure 1.
NMJs in young adult and old extensor digitorium longus and extraocular muscles.
Muscles from transgenic mice that expressed YFP in axons (green) were stained with BTX to label AChRs in the postsynaptic membrane (red). A) Young adult extensor digitorum longus (EDL). B) Two year-old EDL. C) Young adult extraocular muscle (EOM). D) Old EOM. Age-related alterations are striking in EDL but subtle in EOM. Scale Bar: 10 µm. Eight previously documented age-related alterations [19]were quantified from images such as those shown in Figure 1. E) Fragmentation of the postsynaptic membrane. F) Decreased AChR density. G) Partial denervation. H) Complete denervation. J) Nerve terminal sprouting. I) Preterminal axonal distension. K) Axonal dystrophy. L) Multiple innervation of a single postsynaptic site. Each bar represents mean ± SEM from at least 3 animals, with at least 100 NMJs counted per animal. *p<0.01 by t-test. Scale bar = 10 µm.
Figure 2.
Age-related alterations in neuromuscular junction of 2 year-old mice.
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 Fig. 1 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.
Table 1.
Fraction of NMJs exhibiting one or more age-related alterations.
Figure 3.
Structural alterations in NMJs during the third year.
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 t-test. Scale bar = 10 µm.
Figure 4.
Relationship of rostrocaudal position and segmental innervation to the incidence of age-related alterations in NMJs.
Muscles listed in Table 1 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 Table 1.
Figure 5.
Fiber type composition of muscles with NMJs that vary in severity of age-related changes.
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.
Figure 6.
NMJ morphology in Type I and Type II muscle fibers of old mice.
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 Fig. 5). 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.
Figure 7.
Motor unit size in muscles with NMJs that vary in severity of age-related changes.
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).
Figure 8.
Age-related changes in motor unit size.
Tracings from muscles such as the one shown in Figure 7A. 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.
Figure 9.
Branch order analysis of motor axons in young adult and aged animals.
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).
Figure 10.
Neuromuscular junctions in a mouse model of ALS.
A, B) Longitudinal sections from EDL (A) and EOM (B) of SOD-G93A mice transgenic mice. Axons and nerve terminals were stained with antibodies against neurofilament and synaptotagmin-2, (green) and postsynaptic sites were stained with BTX (red). Compare with young adult controls in Figure 1 and Figure S1. Alterations are striking in EDL but subtle in EOM. C) Alter ations quantified from images such as those shown in A–B. Each bar represents mean ± SEM from at least 3 animals, with at least 100 NMJs counted per animal. *p<0.02 by t-test. Scale bar = 10 µm.
Figure 11.
Extent of denervation in muscles from SOD-G93A mice.
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 Fig. 9. Scale bar = 10 µm. Some NMJs remain partially innervated in soleus, but most NMJs are fully denervated with dispersed postsynaptic structures in tibialis anterior.
Figure 12.
Relationship between the incidence of NMJ alterations in old mice and a mouse model of ALS.
Values are from Table 1 and Figure 10. NMJs in all muscles but the triangularis are similarly afflicted or spared by aging and ALS.
Figure 13.
Regulation of CRMP4a in aging and ALS.
A–D) CRMP4a immunoreactivity of motor neurons in caudal spinal cord (SC) from young adult wild type (A), old wild-type (B) and symptomatic SOD-G93A (C) mice and brainstem (BS) of a symptomatic SOD-G93A mouse (D). Somata are labeled with Neurotrace (A, B, D) or YFP (C). E) Percent of motor neurons immunoreactive for CRMP4a in spinal cord and brainstem of young adult, old, and symptomatic SOD-G93A mice. Each bar represents mean ± SEM. *p<0.015 by t-test. Scale bar = 20 µm.
Figure 14.
Level and localization of TDP-43 in aging and ALS.
A–D) TDP-43 immunoreactivity of motor neurons in caudal spinal cord from young adult wild type (A), old wild-type (B) and symptomatic SOD-G93A (C) mice. Somata are labeled with Neurotrace (A, B) or YFP (C). D) Percent of motor neurons with strong cytoplasmic immunoreactivity, cytoplasmic aggregates, or barely detectable levels of TDP-43 in spinal cord of young adult, old, and symptomatic SOD-G93A mice. Each bar represents mean ± SEM. Scale bar = 20 µm.