A numerical analysis of changes in the populations of nuclei in individual, intact muscle fibers was made to study how multinucleation arises during normal differentiation and growth. Gastrocnemius muscle fibers from pre- and post-natal mice were isolated with guanidine (Cardasis and Cooper, '75) and examined. Satellite cells associated with muscle fibers were first observed at 19 days of gestation. The number of nuclei per muscle fiber (muscle + satellite cell nuclei) averages 83 at this age, 157 at birth and continues to increase to 354 by 63 days of age. However, the rate of increase during growth is not constant. Estimates of satellite cell and muscle nuclei in histological cross sections indicate that there is a decrease in the percentage of satellite cells from 32% at birth to 6% in the adult. However, the numbers of satellite cells associated with individual muscle fibers, calculated from these percentages and the nuclear counts on whole fibers, decreases only between 2 and 4 weeks of age. Cytosine arabinoside was injected subcutaneously during the first two weeks of age. Pairs of satellite cells, abnormal nuclei and elevated percentages of satellite cells were observed. This evidence as well as the numerical analysis of nuclear populations in whole fibers lends further support to the hypothesis that satellite cells account for the increase in muscle nuclei from birth to maturity.

The ultrastructural organization of 40 soleus neuromuscular junctions from ten normal young adult male and female Sprague-Dawley (SD)-derived rats (Charles River Breeders, CD-Crl:COBS (SD)BR) has been studied. A smaller sample of motor endplates from the gastrocnemius, diaphragm, and extensor digitorum longus muscles of these rats as well as from the soleus muscles of two adult Wistar (W) rats (Crl:COBS(WI)BR) was included. Widespread ultrastructural reorganization was evident at the soleus neuromuscular junction during the growth period from three to five months of age. A major characteristic of reorganization is the presence of junctional folds not associated with axonal terminals; such sites occur within a single endplate adjacent to areas with typical intact synaptic associations. Additional features possibly related to remodelling are: 1) spatial separation of axonal terminals from the myofiber, 2) intervention of Schwann cell cytoplasm between an axon terminal and myofiber, 3) aggregates of satellite cells, and 4) folded or multilayered basal lamina. These features are most pronounced in the soleus muscle but occur to varying degrees in the neuromuscular junctions of other muscles of SD-derived rats. Distinctive characteristics of the rat soleus postjunctional sarcoplasm include the widespread occurrence of myofibrillar components, abundant free and membrane-associated polysomes, and triads oriented in various planes. Away from such discrete sites, myofibers possess the usual highly oriented organization of myofibrils, T tubules, sarcoplasmic reticulum, and mitochondria. The soleus muscle is a postural muscle that responds directly to rising workload imposed by continuous body growth during young adulthood by steady myofiber hypertrophy and conversion of motor units (Kugelberg, '76). This changing structural-functional relationship may be reflected also by ultrastructural remodelling of the neuromuscular junctions reported here.

No abstract

Previous morphological studies of the adult rat soleus neuromuscular junction (NMJ) indicate that its ultrastructure changes with age, a consequence of a dynamic, ongoing remodeling of pre- and postsynaptic elements. With aging, the balance between axonal sprouting and withdrawal appears to shift in favor of denervation. Here we have examined the extent and direction of the remodeling process during the adult life of the rat (5-111 weeks) by quantification and morphometry of NMJ structure as seen in electron microscopic composites and in cholinesterase (ChE) stained whole mounts. We have found that the net effect of axonal sprouting and withdrawal on the entire NMJ region varies with age: at 5-12 weeks there is rapid growth and maturation; at 12-48 weeks slower growth occurs, and at 48-82 weeks there is a gradual shift in direction to result in progressive loss of synaptic contact in individual NMJs; finally, at 82-111 weeks, there is loss of entire NMJs with some continued reinnervation at others. The diaphragm NMJ also exhibits continuous structural remodeling; however, between 82-111 weeks many changes in the ChE staining pattern appear abruptly.

Ultrastructural remodeling, with evidence of focal deafferentation and reinnervation, occurs within normal young adult rat soleus neuromuscular junctions (Cardasis and Padykula, 1981). This may be related to normal variations in function. Recognition of this plasticity provides a basis for analysis of aging changes in junctional ultrastructure. Thirty soleus junctions were studied between 11 and 26 months of life. In these junctions, compared to younger ones (3-5 months) synaptic sites with the conventional ultrastructure become increasingly sparse. There is an increase in extent and frequency of exposed junctional folds, of intervention of Schwann cell cytoplasm between axon and junctional folds, and of numbers of lysosomes in all cytoplasmic profiles. Often primary clefts are shallow or missing, and secondary folds are widened and contain collagen. Features limited largely to these older junctions include highly pleomorphic myonuclei, deeply invaginated by myofibrils, and an increase in cellular profiles between basal lamina and sarcolemma. The identity of these profiles is unknown. At other locations within many of the same endplates, small intact terminals are associated with larger expanses of junctional folds, and several small terminals occur within the same primary cleft. Such terminals frequently contain dense-cored vesicles. These observations suggest continuation of some terminal axonal regeneration. Thus, the ultrastructure of these aging neuromuscular junctions reveals the same degenerative and regenerative events suggested by the ultrastructure of younger junctions, but suggests a shift in the balance between them.