The proportion of fast and slow neuromuscular junctions in young and old mice

Abstract

Human skeletal mass and strength increase from birth until ~35 years of age, thereafter a decline in mass and strength occurs in a process called sarcopenia. Beyond 60 years of age muscular weakness falls to a point where it imposes physical disability and individuals lose the ability to live independently. The loss of muscle mass and strength can be attributed, at least in part, to a fast to slow muscle fibre type transition (increased proportion of slow type muscle fibres. An age associated change in the innervation of skeletal muscle with ageing is a probable underlying cause of this fibre type transition. Muscle fibre types are determined by their innervating axon; fast muscle fibres are innervated by fast axons forming fast neuromuscular junctions (NMJs) and slow muscle fibres are innervated by slow axons forming slow NMJs. If a muscle fibre is innervated by a motor axon of the wrong type e.g. a fast muscle fibre is innervated by a slow axon, that axon converts the muscle fibre type to slow. The present study hypothesised that the reduction in the proportion of fast muscle fibres seen in ageing may be caused by a selective loss of innervation of fast muscle fibres with a subsequent reinnervation by slow axons resulting in the conversion of the fast muscle fibres to slow muscle fibres. This fast to slow muscle fibre type change would therefore be coupled with an increased proportion of slow NMJs with a reduction of fast NMJs (a fast to slow NMJ transition). To determine this, a comparison of the proportion of fast and slow NMJs present in the soleus (containing both fast and slow muscle fibres), and extensor digitorum longus, (containing almost entirely fast muscle fibres) of young (2-2.4 months old) and old (20-24 months old) C57BL/6 mice was made. Immunohistochemistry was used to selectively label slow NMJs by targeting SV2a, a synaptic vesicle protein present only in the nerve terminals of slow axons. Nicotinic acetylcholine receptors (nAChRs) and Synaptophysin, present in all nerve terminals, were used as a general marker for NMJs. The proportion of SV2a positive (slow) NMJs was compared to the proportion of SV2a negative (fast) NMJs in young and old mice to determine whether there was a difference in the proportion of slow and fast NMJs with ageing. Sectioned muscles were viewed by widefield fluorescence microscopy. An average of 180 NMJs were counted in each soleus muscle and 162 in each EDL. A reduction in the proportion of fast nerve terminals with an increase in slow was found in the soleus muscle, 40.41 ± 3.3% of young NMJs were fast and 59.59 ± 3.3% were slow, of old NMJs 10.80 ± 4.1% were fast and 90.20 ± 4.1% were slow. No change was found in the EDL, 89.66 ± 6.3% of young NMJs were fast and 10.44 ± 6.3% were slow. Of old NMJs 90.06 ± 2.5% were fast and 9.94 ± 2.5% were slow. There was an insufficient number of EDL muscles used (n=2) meaning no valid statistical analysis could be drawn from the EDL results. These observations suggest that in the soleus there may be a deinnervation of fast muscle fibres with a reinnervation by slow axons occurring which promotes an increase in the proportion of slow NMJs with ageing and reduces the proportion of fast NMJs. This deinnervation-reinnervation process may underlie the increase in slow muscle fibres with a reduction in fast muscle fibres seen in the ageing soleus.