No abstract

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 cause significant synaptic remodelling in adult rabbit skeletal muscles.

Nerve fibers extend from spinal cord explants of larval frog in an enhanced and directed manner when cocultured with limb mesenchyme target tissue. In order to gain a better understanding of the events involved in target directed neurite extension, a detailed examination of the nerve growth cone was undertaken. The growth cones of spinal neurites that had elongated in the presence or absence of target tissue were examined by light and electron microscopy. Scanning electron microscopy revealed that growth cones of cord+limb cultures were elaborate in form with numerous and long filopodia, while those cultured in the absence of the target tissue appeared relatively simple with few, short filopodia. A morphological parallel existed between those growth cones that were cultured without the target and those in cord+limb cultures but which grew from the side of the cord explant away from the mesenchyme tissue. When examined with the transmission electron microscope, growth cones under target influence were organelle-rich in contrast to target-deprived growth cones, which lacked the extensive array of vesicles, endoplasmic reticulum, and filaments. When the attachment substratum of polylysine was substituted by collagen, the dramatic differences in growth cones were not realized, although enhanced, oriented growth still occurred in the presence of limb target tissue. It appears that growth cone morphology is a dynamic reflection of the growth effects elicited by a target tissue factor that in turn may be mediated by the nature of the extracellular environment.

The extracellular matrix has been shown to play an important role in the differentiation of neuromuscular junctions during reinnervation in frogs. Peanut agglutinin, a lectin, is known to specifically bind to some glycoconjugates in the extracellular matrix at the frog neuromuscular junction and myotendinous junction. In order to determine if innervation has any role in regulating the specific binding of peanut agglutinin at neuromuscular junctions and myotendinous junctions, the distribution of peanut agglutinin binding was examined in muscles chronically denervated for various periods. Short-term denervated muscles (less than or equal to 2 months) showed no changes in peanut binding agglutinin binding at neuromuscular junctions and no extrajunctional binding. In contrast, long-term denervation (greater than 2 months - 7.5 months) resulted in altered peanut agglutinin distribution and a substantial reduction or a total loss in its binding at denervated neuromuscular junctions; binding at myotendinous junctions was not affected. Results of electron microscopic studies suggest that the presence of Schwann cells at denervated endplates delays the loss of peanut agglutinin binding. Reinnervation restores normal peanut agglutinin binding at neuromuscular junctions following long-term denervation. This report demonstrates that although the distribution of peanut agglutinin binding molecules is unchanged short-term denervation, intact innervation is necessary for the long-term maintenance of these molecules at neuromuscular junctions.

No abstract