Sequence of Age-Associated Changes to the Mouse Neuromuscular Junction and the Protective Effects of Voluntary Exercise

Abstract

Loss of connections between motor neurons and skeletal muscle fibers contribute to motor impairment in old age, but the sequence of age-associated changes that precede loss of the neuromuscular synapse remains uncertain. Here we determine changes in the size of neuromuscular synapses within the tibialis anterior muscle across the life span of C57BL/6J mice. Immunofluorescence, confocal microscopy and morphometry were used to measure the area occupied by nerve terminal synaptophysin staining and postsynaptic acetylcholine receptors at motor endplates of 2, 14, 19, 22, 25 and 28month old mice. The key findings were: 1) At middle age (14-months) endplate acetylcholine receptors occupied 238±11 µm2 and nerve terminal synaptophysin 168±14 µm2 (mean ± SEM). 2) Between 14-months and 19-months (onset of old age) the area occupied by postsynaptic acetylcholine receptors declined 30%. At many endplates the large acetylcholine receptor plaque became fragmented into multiple smaller acetylcholine receptor clusters. 3) Between 19- and 25-months, the fraction of endplate acetylcholine receptors covered by synaptophysin fell 21%. By 28-months, half of the endplates imaged retained ≤50 µm2 area of synaptophysin staining. 4) Within aged muscles, the degree to which an endplate remained covered by synaptophysin did not depend upon the total area of acetylcholine receptors, nor upon the number of discrete receptor clusters. 5) Voluntary wheel-running exercise, beginning late in middle-age, prevented much of the age-associated loss of nerve terminal synaptophysin. In summary, a decline in the area of endplate acetylcholine receptor clusters at the onset of old age was followed by loss of nerve terminal synaptophysin from the endplate. Voluntary running exercise, begun late in middle age, substantially inhibited the loss of nerve terminal from aging motor endplates.

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

“…The protocols described here have revealed an age-associated decline in the area of nerve terminal synaptophysin at motor endplates as mice progress into old age. The same methods revealed that voluntary exercise could largely prevent the reduction in presynaptic nerve terminal area 22 , consistent with previous work by other groups 4 . Loss of neuromuscular connections also occurs in the SOD1G93A mouse model of amyotrophic lateral sclerosis 1.…”

“…Thus, it was possible to demonstrate significant 30-40% reductions in synaptic area in mice injected with IgG from anti-MuSK-positive myasthenia gravis patients, compared to controls with a sample size of three mice 17,20,32 . Elderly mice displayed greater animal-to-animal variation in endplate parameters than young mice 22 . Consequently experiments involving aged mice might require larger sample sizes.…”

“…Table I lists areas of pre-and post-synaptic specializations for NMJs for healthy 2 month old (young adult) female C57Bl/6J mice from previous studies. The areas of both pre-and post-synaptic specializations declined with sedentary aging 22 . AChR area was also markedly reduced in mice injected with IgG from anti-MuSK myasthenia gravis patients 17,21 .…”

“…13. Prepare a spreadsheet of data sample means, calculate and plot standard deviations and standard errors as histograms or scatterplots 20,22 . Note that the value of n generally represents the number of mice per sample group for statistical purposes.…”

“…For such images a minimum threshold (just above extrasynaptic background fluorescence) will readily isolate the AChRrich or synaptophysin-rich area of the endplate (horizontal dashed lines in (Figure 5A' and B')). In aged mice and in some disease states endplate staining for AChR may be less intense, AChR cluster edges may appear blurred and there may be higher levels of extrasynaptic autofluorescence ( Figure 5D; 17,22 ). Fluorescence staining with indistinct boundaries can introduce error into estimates of synaptic area.…”