Motor Endplate Region Research Articles

Reinnervation of motor endplate-containing and motor endplate-less muscle grafts

Damaged muscle fibers degenerate and are phagocytosed, but their connective tissue sheaths and myogenic cells survive. New myofibers regenerate within the connective tissue sheath and functional reinnervation is restored. The present study compared regenerating myofibers and their reinnervation in muscle grafts where the original motor endplates were removed and when they were present. In regenerating muscles, where the endplates were present, axons made neuromuscular connections with the myofibers. High levels of CAT activity, characteristic of cholinergic innervation, were reached and maintained in these muscles. Also, motor endplate-containing grafts developed characteristic muscle fiber types and nerve-evoked muscle contraction was observed. In preparations lacking the original endplate region, CAT activity initially rose to high levels, but with time diminished to 17% of control, and muscle fiber types did not develop. Muscle contraction after nerve stimulation was not observed in these grafts. It was concluded that, although a small number of motor endplates are formed in grafts devoid of original motor endplates, the presence of the motor endplate region in regenerating muscle is critical for development and maintenance of myofibers and the axons that contact them.

Acetylcholinesterase like that of skeletal muscle in smooth muscle reinnervated by a motor nerve.

SKELETAL muscles contain several molecular forms of acetylcholinesterase (AChE) characterised by different sedimentation coefficients1–4. In rat muscle1,2 (but not human3 and chicken5 muscle) the heavy form of AChE is exclusively present at the motor endplate regions. In contrast, the heavy form of AChE has not been detected in cat6 or rat2 smooth muscles. The endplate AChE of rat skeletal muscle is selectively detached by treatment with collagenase1,7, an enzyme which has been shown specifically to digest the collagen-like tail of the heavy form of AChE from several sources8,9. The presence of endplate AChE is neurally regulated10, and there is evidence that this effect is partially mediated by muscle activity11 and by a trophic factor conveyed by axoplasmic transport12,13. Previous results from our laboratory have shown that reinnervation of the cat nictitating membrane smooth muscle by a motor nerve results in an increase of AChE activity14. We report here that collagenase treatment detaches active AChE from cat smooth muscle reinnervated by a cholinergic motor nerve, and that a new AChE form with a high sedimentation coefficient is induced in these muscles. Our results suggest that the presence of the motor nerve determines, in reinnervated smooth muscle, the appearance of an AChE similar to that found in skeletal muscle.

Attachment of acetylcholinesterase to structures of the motor endplate

The kinetics of AChE solubilization from intact motor endplates of mouse diaphragm, by collagenase, papain and hyaluronidase, was studied in parallel with the ultrastructural localization of AChE in treated neuromuscular junctions. Hyaluronidase did not solubilize more AChE from isolated motor endplate regions than Ringer's solution itself. Residual AChE activity could be demonstrated histochemically in motor endplates even after the plateau of solubilization by collagenase or papain was reached. Less than 35% of junctional AChE is left after collagenase, and less than 20% after papain treatment, as estimated by the percentage of AChE activity left in the isolated endplate region of the diaphragm after protease treatment. Cytochemically, both proteases had a similar effect on postsynaptic AChE. Residual AChE activity was distributed randomly, adhering to the sarcolemma of junctional clefts. Presynaptic AChE localized in the gap between axon terminal and Schwann cell appears to be resistant to collagenase but not to papain treatment. The mode of AChE attachment or the composition of the intercellular material in this gap may differ from that of the primary and secondary clefts.

The morphology and innervation of subepithelial "striated" muscle cells in the male guinea-pig urethra.

Light and electron microscopic techniques have been used to determine the distribution, morphology and innervation of subepithelial striated muscle cells in the wall of the proximal urethra of the male guinea-pig. These cells form a continuous layer, immediately beneath the urethral epithelium extending from the bladder neck to the termination of the ejaculatory ducts into the proximal urethra. They differ from "typical" striated muscle fibres (as seen in the external urethral sphincter) by their small size, rich acetylcholinesterase content and the irregular arrangement of intracellular myofilaments and sarcoplasmic reticulum. In addition, motor end plate regions have not been observed on these striated cells when examined using a light microscopic histochemical technique. The cells are related to acetylcholinesterase positive nerves which run between them in a manner compatible with the occurrence of "en passant" synaptic interactions. Using electron microscopy, axonal varicosities containing small (50 nm diameter) agranular vesicles are encountered 50 nm from the striated cells; membrane specialisations characteristic om motor end plates have not been observed on the cells. The findings are discussed, particularly in relation to the distribution, unusual morphology and innervation of these subepithelial muscle cells.

Pathological mechanisms in experimental autoimmune myasthenia gravis. II. Passive transfer of experimental autoimmune myasthenia gravis in rats with anti-acetylcholine recepotr antibodies.

Passive transfer of experimental autoimmune myasthenia gravis (EAMG) was achieved using the gamma globulin fraction and purified IgG from sera of rats immunized with Electrophus electricus (eel) acetylcholine receptor (AChR). This demonstrates the critical role of anti-AChR antibodies in impairing neuromuscular transmission in EAMG. Passive transfer of anti-AChR antibodies from rats with chronic EAMG induced signs of the acute phase of EAMG in normal recipient rats, including invasion of the motor end-plate region by mononuclear inflammatory cells. Clinical, eletrophysiological, histological, and biochemical signs of acute EAMG were observed by 24 h after antibody transfer. Recipient rats developed profound weakness and fatigability, and the posture characteristic of EAMG. Striking weight loss was attributable to dehydration. Recipient rats showed large decreases in amplitude of muscle responses to motor nerve stimulation, and repetitive nerve stimulation induced characteristic decrementing responses. End-plate potentials were not detectable in many muscle fibers, and the amplitudes of miniature end-plate potentials were reduced in the others. Passively transferred EAMG more severely affected the forearm muscles than diaphragm muscles, though neuromuscular transmission was impaired and curare sensitivity was increased in both muscles. Some AChR extracted from the muscles of rats with passively transferred EAMG was found to be complexed with antibody, and the total yield of AChR per rat was decreased. The quantitative decrease in AChR approximately paralleled in time the course of clinical and electrophysiological signs. The amount of AChR increased to normal levels and beyond at the time neuromuscular transmission was improving. The excess of AChR extractable from muscle as the serum antibody level decreased probably represented extrajunctional receptors formed in response to functional denervation caused by phagocytosis of the postsynaptic membrane by macrophages. The amount of antibody required to passively transfer EAMG was less than required to bind all AChR molecules in a rat's musculature. The effectiveness of samll amounts of antibody was probably amplified by the activation of complement and by the destruction of large areas of postsynaptic membrane by phagocytic cells. A self-sustaining autoimmune response to AChR was not provoked in animals with passively transferred EAMG.

Myopathic changes at the end-plate region induced by neostigmine methylsulfate

Administration of large dose of neostigmine caused very quickly marked myopathic changes at the motor end-plate region. With continued injections, however, some recovery of the structural features did occur suggesting the reconstructive changes in the affected regions.

N,O-di and N,N,O-tri ( 3 H) acetyl -bungarotoxins as specific labelling agents of cholinergic receptors.

1. alpha-Bungarotoxin isolated from the venom of Bungarus multicinctus was acetylated with [(3)H] acetic anhydride and N-[(3)H] acetyl imidazole. Tri-N-acetyl and hexa-N-acetyl derivatives were obtained from the former, and N,O-di, N,N,O-tri and N,N,N,O-tetraacetyl derivatives from the latter reaction, respectively.2. There were parallel decreases in both neuromuscular blocking action in the phrenic nerve-diaphragm preparation of rats and depression of acetylcholine response of the rectus abdominis muscle of frogs with increased acetylation. Also, a parallel but greater decrease of toxicity in mice was found.3. N,O-Di and N,N,O-triacetyl toxins were localized mostly in the motor endplate region of the rat diaphragm, whereas a slight nonspecific binding along the whole muscle fibre in addition to the peak in the endplate region was observed with N,N,N,O-tetraacetyl and tri-N-acetyl toxins. In contrast, there was a marked nonspecific binding with hexa-N-acetyl toxin and no peak was observed at the endplate zone.4. The specific binding was saturable and irreversible. The number of toxin-receptive sites in one endplate was 1.9-2.2 x 10(7) for all of the labelled toxins irrespective of their potency.5. (+)-Tubocurarine protected effectively against the binding as well as the irreversible neuromuscular blocking effect of the toxins.6. Denervation of the rat diaphragm caused an increase of toxin-receptive sites beginning from the endplate zone at 1-2 days and then along the whole muscle fibre, reaching the maximum at about 18 days. The total receptive sites increased by about 30-fold.7. The significance of the findings is discussed and it is concluded that N,O-di and N,N,O-tri-[(3)H] acetyl alpha-bungarotoxins are specific and irreversible labelling agents for the cholinergic receptors of skeletal muscle.

The effect of some foreign anions on suxamethonium blockade of the isolated rat diaphragm preparation.

1. The pharmacology of suxamethonium blockade of the rat phrenic nerve-diaphragm preparation has been studied in the presence of some foreign anions.2. Blockade proceeded in two distinct phases in both normal and modified Krebs solution.3. In normal Krebs solution the characteristics bore no close resemblance to either depolarizing or competitive type blockade.4. In the presence of foreign anions the characteristics of the blockade more closely resembled those of depolarization.5. There was an increase in the sensitivity of the motor end-plate region of the muscle to the depolarizing action of acetylcholine, carbachol and suxamethonium in the presence of the anions.6. Although the anions enhanced the depolarizing activity of suxamethonium, the blocking potency of the drug was unaltered.7. It is suggested that end-plate depolarization plays little part in suxamethonium blockade of the isolated rat diaphragm and that desensitization is the primary cause of the blockade.

神経損傷による筋麻痺の基礎的知見

Skeletal muscle paralysis is a clinical symptom in many cases of nerve injury or brain hemorrhage and it leads to the atrophy of muscles. The structural, biochemical, physiological and pharmacological changes of the denervated muscle might be the important subject of investigation at present. In this article, the experimental results of my colleagues and other many investigators are summarized in special references to the fundamental finding on the denervated muscle.The most obvious changes in a denervated muscle are weight loss and decrease in fiber diameter of individual muscle fibers. Weight loss and decrease in fiber diameter initially proceed at a rather rapid rate. After 7 weeks' denervation there is a 20 to 30 per cent weight loss in rat anterior tibial muscle. The nuclei of denervated muscle fiber may alter in shape and staining reaction and appear to increase in number. In the muscles denervated for relatively long periods,35 to 200days, fine structural changes in cell membrane were observed. Lamellar arrays of membrane enclosed cristernae were found in fibers denervated for 11 weeks or longer. Lamellar structure may be derived from the element of sarcoplasmic reticulum. Focal dissociation of the basement membrane from the plasma membrane is observed in muscle fibers denervated 11 weeks or longer. The number of myofilament remarkably decreases and Z-zone of myofibril seems to be indistinct. The nuclear membrane is found to be somewhat irregular in contour. Relatively clear areas of sarcoplasm appear in the perinuclear zone and the enlarged sarcotubles. Clamps of mitochondria, lysosomes and vacuoles are seen in the perinuclear area as well as in the interior of the fibers. At a motor-endplate region, the presynaptic membrane and cytoplasma of nerve-ending show degenerative changes and the basement membrane structure in postsynaptic membrane is found to be swelling and clouding in contour.After nerve section, a reduction of myoglobin content, succinic dehydrogenase and cytochrome oxidase activity occurs in 2 weeks. Myosin-ATPase activity does not decrease in the muscle at a time when there has been a great decline in the succinic dehydrogenase activity of mitochondria of muscle. Sarcolemma-ATPase activity examined by electronmicroscopic method, is found to reduce to the level, leading to the changes of potassium concentration in denervated muscle.Acetylcholinesterase is localized in the postsynaptic space and also in the axon-Schwann interface. In denervated muscle, a reduced acetylcholinesterase activity may explain the phenomenon of denervation hypersensitivity. There is, however, no parallelism between the reduced acetylcholinesterase activity and the hypersensitivity of muscle to acetylcholine.