Density and distribution of α-bungarotoxin-binding sites in postsynaptic structures of regenerated rat skeletal muscle

Abstract

Acetylcholine receptors (AChR) are organized in a discrete and predictable fashion in the postsynaptic regions of vertebrate skeletal muscle . When muscle is damaged, nerves and myofibers including muscular elements of the endplate degenerate, but the connective tissue elements survive. Muscle fibers regenerate within the basal lamina of the original myofiber . Postsynaptic differentiation in regenerated mammalian skeletal muscle can occur in different ways : (a) at the site of the original endplate in the presence or absence of the nerve, or (b) at ectopic regions of the regenerated myofiber in the presence of the nerve when the original endplate is not present. The present study used '251-a-bungarotoxin (1251 -a-BuTX) and EM autoradiography to examine the density and distribution of AChR in postsynaptic structures regenerated at the site of the original endplate in the absence of the nerve and at ectopic sites of the myofiber in the presence of the nerve when the original endplate was removed . In regenerated myofibers, the density of a-BuTX-binding sites fell within the range of densities observed in uninjured muscle whether postsynaptic differentiation occurred at the site of the original endplate in the absence of the nerve or at an originally ectopic position of the regenerated myofiber . In addition, the distribution of a-BuTX-binding sites within the regenerated postsynaptic regions closely resembled the distribution of a-BuTX-binding sites in uninjured muscle . Morphometric analysis was performed on postsynaptic structures formed at the site of the original endplate in the absence of the nerve or at an ectopic position of the regenerated myofiber by interaction of the nerve and muscle . Although variation in the depth of the primary cleft occurred, there was little difference between the overall structure of regenerated postsynaptic structures and that of endplates of uninjured muscles.The density of acetylcholine receptors (AChR) at innervated motor endplates of skeletal muscle has been determined by both physiological (23, 25) and morphological (16,28,30,(33)(34)(35) means. Various studies (for review see reference 12) have shown that the number of AChR at endplates in mice (2), rats (14, 31), frogs (31), and humans (13) is quite similar. Electron microscope autoradiographic studies have demonstrated a relatively constant density (16,28,30,(33)(34)(35) and distribution (16,(33)(34)(35) of AChR at endplates of vertebrate skeletal muscle .When skeletal muscle grafts are made, the neuromuscular junctions are disrupted by the degeneration of the myofibers and the nerves that supply them (7,10,29,37, and footnote 1).' Carlson, B. M., P. Hnik, S. Tucek, R. Vejsada, and D. Bader. 338It has been established that synapses are formed in the regenerated muscle of amphibians (29, 37) and mammals (1,3,10). Marshall et al . (29) have shown that, in regenerating frog muscle, neuromuscular connections are almost always at the site of the original endplate . Burden et al . (7) have demonstrated that postsyna...

Denervation increases the degradation rate of acetylcholine receptors at end‐plates in vivo and in vitro.

“…It appears, therefore, that even at established adult end-plates receptor density and receptor metabolism are not tightly coupled. This is consistent with previous observations that during development receptor aggregation, receptor function and receptor metabolism appear to be independently controlled (Steinbach et al 1979;Fischbach & Schuetze, 1980;Reiness & Weinberg, 1981;Michler & Sakmann, 1980;Steinbach, 1981a (Burden, Sargent & McMahan, 1979;Bader, 1981). It is possible that molecules in the basal lamina, which are removed or modified by collagenase treatment, also play a role in determining the metabolic stability of junctional ACh receptors; these molecules may be sensitive to enzymes released at end-plates after denervation.…”

Laminin, fibronectin, and collagen in synaptic and extrasynaptic portions of muscle fiber basement membrane.

“…Despite these uncertainties, however, the firm conclusion from our experiments is that all five proteins studied are components of muscle fiber basement membrane and are closely associated with the basal lamina . Our interest in comparing synaptic and extrasynaptic basement membrane stems from the fording that the synaptic region is functionally specialized : When new neuromuscular junctions form following injury, regenerating axons preferentially contact original synaptic sites on the basement membrane and differentiate into nerve terminals at these sites in the absence ofthe muscle fiber (36); acetylcholine receptors on regenerating myotubes cluster at original synaptic sites on the basement membrane in the absence of the nerve (1,5). In addition, acetylcholinesterase, which terminates neurotransmitter action at the neuromuscular junction, is connected to or contained in synaptic basement membrane (19,28), and adhesion of nerve to muscle at the neuromuscular junction may be mediated by components of synaptic basement membrane (2,36).…”

“…A small fraction of the basement membrane, -0.1% of the total, occupies the synaptic cleft between nerve and muscle at the neuromuscular junction. Recent experiments have demonstrated that this synaptic portion of the basement membrane is specialized and plays important roles in neuromuscular function and regeneration (1,2,5,19,28,36) . These results motivate molecular analysis of muscle-fiber basement membrane and comparison of its synaptic and extrasynaptic portions.…”

Distribution and turnover rate of acetylcholine receptors throughout the junction folds at a vertebrate neuromuscular junction.

“…The top ~2,000 A of postjunctional membrane (pjm) has a distinct electron density, a cytoplasmic filamentous substructure (4) and is rich in intramembrane particles (13,26,28). This thickened region has a high density of receptors (~10,000-20,000 sites/#m 2) (2,15,16, 19,23,27). At the bottom of the folds, the membrane is morphologically indistinguishable from that at extrajunctional regions, and the receptor site density is considerably reduced (~ 1,000 sites//.tm 2, or ~5-10% the site density at the top of the folds).…”