Alterations in neuromuscular junction morphology during fast-to-slow transformation of rabbit skeletal muscles

Chronic low frequency stimulation of motor nerves results in transformation of muscle fibre phenotype from fast- to slow-twitch. We examined the light and electron microscopic structure of neuromuscular junctions in normally fast twitch muscles, tibialis anterior and extensor digitorum longus of rabbit after 3 weeks of stimulation to determine whether synaptic structure is also modified during fibre type transformation. Neuromuscular junctions of stimulated and unstimulated (control) tibialis anterior and extensor digitorum longus muscles and unstimulated slow twitch soleus muscle were visualized with rhodamine-conjugated alpha-bungarotoxin. Video Light microscopic images of neuromuscular junctions were digitized to allow quantification of their surface areas, perimeters, lengths and widths. Three weeks of stimulation resulted in a decrease in the maximal velocity of muscle fibre shortening and augmentation of mitochondrial volume in fast muscles, demonstrating the efficacy of the stimulation protocol employed in altering muscle fibre phenotype. Neuromuscular junctions of control tibialis anterior and extensor digitorum longus are thin, compact, and continuous, with complex branching patterns. In contrast, those of slow-twitch soleus are thicker and discontinuous. Neuromuscular junctions in control tibialis anterior and extensor digitorum longus are larger than those in soleus. Three weeks of stimulation causes a marked decrease in the size of neuromuscular junctions in tibialis anterior and extensor digitorum longus, as reflected in the significant reduction in neuromuscular junction surface area, length and width. Electron microscopy of these junctions suggests that secondary postsynaptic folds in stimulated muscles are more closely spaced. Also, axon terminals of stimulated muscles appear to contain more densely packed synaptic vesicles and mitochondria than controls. Decreases in neuromuscular junction dimensions can be partly explained by muscle fibre atrophy. However, the decrease in neuromuscular junction size is proportionately greater than that of muscle fibre diameter in both muscles, indicating that factors other than fibre atrophy may contribute to the reduced neuromuscular junction size in stimulated muscles. Neuromuscular junctions of stimulated tibialis anterior and extensor digitorum longus muscles exhibit some features characteristic of normal soleus neuromuscular junctions, indicating structural adaptations consistent with the altered muscle fibre phenotype. On the other hand, neuromuscular junctions of 3 week stimulated tibialis anterior and extensor digitorum longus and their synaptic branches remain as thin and continuous as those of unstimulated controls, suggesting that the transformation of neuromuscular junctions towards a morphology characteristic of slow muscle, is only partial. These results demonstrate that an altered pattern of impulse activity causes significant synaptic remodelling in adult rabbit skeletal muscles.