Muscle-derived agrin in cultured myotubes: expression in the basal lamina and at induced acetylcholine receptor clusters.

The synaptic basal lamina (SBL) directs key aspects of the differentiation of regenerating neuromuscular junctions. A range of experiments indicate that agrin or a closely related molecule is stably associated with the SBL and participates in inducing the formation of the postsynaptic apparatus after damage to adult muscle. The selective concentration of agrin-related molecules in the SBL suggests that agrin is secreted locally by cellular components of the nerve-muscle synapse. In vivo studies on aneural embryonic muscle indicate that the muscle cell is one source of the agrin-like molecules in the SBL. Here we have used cultured chick muscle cells to study the expression of agrin-related molecules in the absence of innervation. Immunofluorescence and immunoelectron microscopy show that myogenic cells in culture express agrin-related molecules on their surfaces, and that at least a subset of these molecules are associated with the basal lamina. Moreover, in short term cultures agrin-like molecules accumulate on the surfaces of myogenic cells grown in unsupplemented basal media. We quantified the expression of agrin-like molecules on the cell surface using a solid-phase radioimmune assay. The expression of these molecules is relatively low during the first 6 days of culture and increases fourfold during the second week. The stimulation of the expression of agrin-related molecules in these long-term cultures requires the presence of chick embryo extract or fetal calf serum. We also characterized the expression of muscle-derived agrin-like molecules at clusters of AChR. These agrin-related molecules are not consistently colocalized at spontaneous AChR aggregates; however, they are selectively concentrated at greater than or equal to 90% of the AChR clusters that are induced by Torpedo agrin. These data, together with previous results from in vivo developmental experiments indicate that the agrin-like molecules in the synaptic basal lamina are derived at least in part from the muscle cell. In addition, the expression of agrin-like molecules can be regulated by soluble factors present in CEE and FBS. Finally, the selective localization of these molecules at induced AChR clusters, taken together with their localization in the basal lamina, suggests that agrin-like molecules secreted by the muscle cell play an important role in the formation and/or the stabilization of the postsynaptic apparatus.

The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering.

Agrin derived from Torpedo electric organ induces the clustering of acetylcholine receptors (AChRs) on cultured myotubes. As a first step toward characterizing the plasma membrane receptor for agrin, we have examined agrin binding to cultured myotubes. Agrin binding is saturable as measured by radioimmunoassay and, like agrin-induced AChR clustering, requires extracellular calcium. Immunofluorescence shows that on myotubes incubated with agrin at 4 degrees C, agrin binds in a uniform, finely punctate pattern that correlates poorly with the distribution of AChRs. Myotubes stimulated with agrin at 37 degrees C for greater than or equal to 2 hr show a coclustering of agrin binding sites and AChRs. By contrast, if anti-AChR antibodies are used either to cluster or to internalize AChRs, the distribution and number of agrin binding sites remain unchanged. The aggregation and calcium dependence of the putative agrin receptor may represent important control points in postsynaptic differentiation.

Aging rat neuromuscular junctions: a morphometric study of cholinesterase-stained whole mounts and ultrastructure.

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 evidence of continued reorganization at the aging (11-26 months) rat soleus neuromuscular junction.

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.

Ultrastructural evidence indicating reorganization at the neuromuscular junction in the normal rat soleus muscle.

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.

Ultrastructural localization of calcium in unfertilized sea-urchin eggs.

The pyroantimonate technique was employed to identify the binding sites for calcium in unfertilized Arbacia punctulata and Strongylocentrotus purpuratus eggs. Since antimony is non-specific and binds with a variety of cations, the indentification of calcium was established by specific chelation with ethyleneglycol tetra-acetic acid (EGTA) and X-ray microprobe analysis. Antimony deposits were observed on the egg's membranes, i.e. plasma, cortical (secretory) granule, pigment granule, smooth-surfaced vesicle, and yolk platelet. Deposits were also observed in the mitochondria, rod-containing vesicles, and the vitelline layer. Two types of yolk platelets were observed: a more numerous electron-opaque platelet which had precipitate along its limiting membrane as well as within the stored-matrix substance, and a less-frequently seen platelet with lower electron opacity which contained precipitate only along its limiting membrane. Deposits were reduced at all sites following exposure of eggs to EGTA either prior to or after osmium-antimonate fixation. Initial fixation in glutaraldehyde followed by postfixation in osmium-antimonate solutions provided better preservation of structure but less precipitation than direct fixation in osmium-antimonate. The organelle sites of calcium binding identified within unfertilized sea-urchin eggs may participate in stimulus-secretion coupling (exocytosis of the cortical granules) and the activation of embryogenesis at fertilization.

Role of estrogen receptor binding and transcriptional activity in the stimulation of hyperestrogenism and nuclear bodies.

The effects of estradiol and nafoxidine on nuclear estrogen receptor binding, RNA polymerase activities, and uterine ultrastructure were studied. Animals were either injected with estradiol, implanted with estradiol/paraffin pellets, or injected with nafoxidine. Animals treated with nafoxidine or estradiol implants showed sustained long-term nuclear retention of estrogen receptor and increased nuclear RNA polymerase activities for up to 72 hr. A single injection of estradiol caused initial increases in these variables which returned to control levels by 24 hr after hormone treatment. Uterine tissue was examined by light and electron microscopy 72 hr after hormone treatments. Uteri from eith estradiol-implanted or nafoxidine-treated animals showed markedly increased hypertrophy of the luminal epithelial cells. Nuclei in sections of the uteri of these hyperestrogenized animals displayed a large number and wide array of nuclear bodies composed of a filamentous capsule and granular cores. We conclude that hyperestrogenization, a condition that eventually results in abnormal cell growth, is correlated with increased and sustained nuclear binding of the estrogen receptor, increased and sustained RNA polymerase activity, and the appearance of nuclear bodies.

A method for the chemical isolation of individual muscle fibers and its application to a study of the effect of denervation on the number of nuclei per muscle fiber.

A method is described for isolating intact, individual skeletal muscle fibers from glutaraldehyde fixed muscle. This method was conceived to climinate the many limitations of determining muscle nuclear numbers in histological cross section. Glutaraldehyde fixed fibers are isolated by dissection while in a solution of low concentration guanidine in a borate buffer at high pH. Electron miscroscopy demonstrates that single fibers, isolated in this manner, are free of their microvasculature and connective tissue. Their basal laminas and the structures within them, including their satellite cells, are preserved. This method is employed to determine whether changes in nuclear numbers occur within the basal lamina of individual muscle cells from 1 to 28 days following denervation of mouse gastrocnemius muscle. The total number of nuclei located within the basal lamina of individual muscle fibers (i.e. muscle and satellite cell nuclei) does not change after denervation. This rules out the possibility that additional nuclei are arising from an influx of cells outside the basal lamina or by mitotic division of nuclei within the basal lamina. However, the possibility of a change in the ratio of satellite cell nuclei, to muscle cell nuclei, is not eliminated. Other possible applications of this isolation method are discussed.

An analysis of nuclear numbers in individual muscle fibers during differentiation and growth: a satellite cell-muscle fiber growth unit.

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.