Figure 1.
Life history and survival curves for the female C57BL/6J mice used in this study.
(A) Between 2- and 8-months of age all the mice were engaged in stock breeding. From 8-months mice were group-housed under our care (see Methods). Arrows indicate the ages at which mice were killed for analysis. Some boxes of mice received a running wheel from late middle-age until sacrificed (horizontal grey bars). (B) Survival curves. Filled circles reflect the percentage of mice in our care that died of natural causes or were euthanized due to disease (starting number 79). Filled squares show results for mice provided access to a running wheel from late middle age (starting number 31). The horizontal grey lines in this panel indicate the age ranges recommended for life-stage comparisons while the dashed line shows comparative survival data for female C57BL/6J mice, both from Jackson Laboratories [25].
Figure 2.
Typical NMJs from the TA muscle of young and old mice viewed en face.
Confocal maximum projection images of individual NMJs from the TA muscle were stained for postsynaptic AChR and nerve terminal synaptophysin. (A–C) NMJ from a 2-month old mouse showing AChRs (A), synaptophysin (B) and a merge of the pre- and post-synaptic staining (C). (D–F) NMJ from a 25-month old mouse at which the AChR-stained primary synaptic gutters were more fragmented but remained largely covered by synaptophysin staining. (G–I) NMJ from a 25-month month old mouse at which the AChR-rich area of the endplate was only partially covered by synaptophysin staining. Scale bar in panel A is 10 µm.
Figure 3.
Changes with age in the area of nerve terminal synaptophysin and postsynaptic AChRs.
(A) Average area of nerve terminal synaptophysin staining (Nerve; green squares) and postsynaptic AChRs (red diamonds) with age. The area of overlap between synaptophysin and AChR staining (synaptic overlap) is indicated by amber triangles (* P<0.05, ** P<0.01, *** P<0.001 relative to 14-month mice). (B) Changes in the area of synaptic overlap, expressed as a percentage of the endplate AChRs area (filled circles; # P<0.05 relative to 19-month old mice). Filled symbols in A and B show the mean ± SEM for n = 3mice (19-months) or n = 4 mice (2-, 14-, 22-, 25- and 28-months). The full list of significant differences by one-way ANOVA with Bonferroni's multiple comparison post-test were as follows. AChR area: 2- vs. 14- and 22-months (P<0.05); 2- vs. 28-months, 14- vs. 19- and 22-months (P<0.1); 14- vs. 28-months (P<0.001). Nerve area: 2- vs. 25-months and 19- vs. 28-months (P<0.05); 2- vs. 28-months, 14- vs. 25- and 28-months (P<0.01). Overlap area: 2- vs. 25-months, 14- vs. 25-months (P<0.05); 2- vs. 28-months, 14- vs. 28-months (P<0.01). Percentage overlap: 19- vs. 25- and 28-months (P<0.05). The decline in average synaptic overlap could be explained, in part, by an increase in the percentage of endplates with ≤ 10% synaptic overlap (from pooled data).
Figure 4.
A subset of endplates lose nearly all their synaptophysin in old age.
(A) Frequency distributions show the synaptophysin-positive nerve terminal area of endplates at 2, 14, 19, 22, 25 and 28 months of age. Note the leftward shift toward endplates with ≤50 µm2 synaptophysin area from 22-months onwards. (B) Frequency distributions for the AChR-rich area of the endplate. Data represent 60 - 80 NMJs pooled from 3–4 mice at each age. Arrows indicate a statistically-significant shift compared to the immediately-preceding age (**P<0.01; Kruskal-Wallis test followed by Dunn's multiple comparisons post-test). The full set of significant differences were as follows. Synaptophysin area: 2- vs. 25-months (P<0.05); 14- vs 22- and 19- vs. 22- (P<0.01); 2- vs. 28-, 14- vs. 25-, 14- vs. 28-, 19- vs. 25-, 19- vs. 28- (P<0.001). AChR area: 2- vs. 22-months and 14- vs. 19- (P<0.01); 2- vs. 28-, 14- vs. 22-, 14- vs. 28- (P<0.001).
Figure 5.
Age-related nerve terminal shrinkage correlates poorly with AChR changes.
(A) The percentage of endplates composed of five or more discrete AChR clusters (fragmented endplates) increased with the onset of old age (mean ± SEM for n = 4 mice; *P<0.05, **P<0.01 by one-way ANOVA followed by Bonferroni's multiple comparisons post-test). (B) The percentage AChR covered by nerve (synaptophysin) was similar for fragmented endplates (open bars) and non-fragmented endplates (1–4 AChR clusters; filled bars). The only significant difference was at 19-months (*P<0.05, unpaired Student t-test). (C) Scatter plot comparing the percentage of the AChRs covered by synaptophysin staining to the total area of AChRs at an endplate. Each symbol represents an individual endplate from a 25-month old mouse. No correlation was found between the postsynapic area occupied by AChRs and the extent to which the AChRs remained covered by synaptophysin staining.
Figure 6.
Voluntary wheel running by aging mice.
(A) One week of typical circadian running activity by 12-month old mice (arrowheads indicate midnight). Distances run are expressed as the average per mouse. (B) Circadian running activity by 27-month old mice. (C) Decline with age in the average daily distance run per mouse. Blue diamonds show results for mice that received a wheel at 18-months of age. Red squares show results for mice that received a wheel at 10-months of age (the latter mice were not included in the NMJ analysis). Data represents the mean ± SEM for 3–5 boxes of mice at each time-point.
Figure 7.
Effect of voluntary wheel running upon the aging NMJ.
(A & B) Sample endplate from a 25-month old sedentary mouse (A) and from a 25-month old mouse provided with a running wheel from 21-months of age onward (B). Red fluorescence shows AChRs, green fluorescence shows nerve staining and white pixels indicate the area of overlap. Scale bar = 10 µm. (C) The effect of wheel running upon the area of nerve terminal synaptophysin for mice analyzed at 25- and 28months of age (Nerve area). (D) The effect of wheel running upon the area of overlap between AChR and synaptophysin staining. (E) The effect of wheel running upon the area of endplate AChRs. (F) Wheel running did not significantly alter the percentage of endplates composed of five or more AChR clusters (fragmented endplates). Data represent the mean ± SEM (n = 4 control mice, n = 5 wheel mice; P<0.05, unpaired 2-sided Student t-test). The horizontal dashed line in panels C–F shows the mean value for 19-month old mice that did not have access to a wheel (data replotted from Figs. 2 and 5A).