Cytological Localization of Cholinesterase in Developing Chick Embryo Skeletal Muscle

Mumenthaler, Marco; Engel, Werner

doi:10.1159/000141814

Cited by 123 publications

(32 citation statements)

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“…The localized activity of AChE in the embryonic muscle of the vertebrate has been found to be concomitant with the formation of the discrete region of nerve termination (Mumenthaler & Engel, 1961;Teraviinen, 1968;Atsumi, 1971) and no definite experiments have been reported which demonstrate the preliminary presence of the esterase far ahead of the synapse formation. The activity of the enzyme is initially very low around the newly formed synapses in co-cultured myotubes and nerve explants (Robbins & Yonezawa, 1971;Koenig, 1973).…”

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confidence: 99%

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Development of neuromuscular transmission in a larval tunicate

Ohmori

Sasaki

1977

The Journal of Physiology

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confidence: 99%

“…There is no evidence of delayed release at new synapses. AChE activity in myoneural junctions progressively increases during development (Mumenthaler & Engel, 1961;Teraviinen, 1968;Atsumi, 1971). Thus the slow time course of the falling phase may be partly due to low activity of AChE.…”

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confidence: 99%

Development of neuromuscular transmission in a larval tunicate

Ohmori

Sasaki

1977

The Journal of Physiology

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“…Later, it is found within and at the surface of the differentiating muscle fibers and at the embryonic motor endplates. After birth, most of the AChE activity becomes restricted to the adult motor endplates (3,4). In the chicken, several molecular forms of AChE occur in embryo twitch muscles that are virtually-undetectable in the same muscles after hatching, and large amounts of AChE are released by cultured muscle fibers and appear in plasma from intact embryos (2).…”

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Regulation of Newly Synthesized Acetylcholinesterase in Muscle Cultures Treated with Diisopropylfluorophosphate

Wilson

Walker

1974

Proc. Natl. Acad. Sci. U.S.A.

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Brief treatment with 0.1 mM diisopropylfluorophosphate inhibited an average of 89% of the acetylcholinesterase (EC 3.1.1.7; acetylcholine hydrolase) activity of cultures of chick embryo muscle. As long as protein synthesis occurred, an average of 78% of the activity returned within 4 hr. Newly synthesized acetylcholinesterase did not stain cytochemically, was rapidly and extensively degraded or released in the presence of 10 1M cycloheximide, and consisted mainly of low-molecularweight forms. Acetylcholinesterase activity first appeared around the nucleus, about 4 hr after treatment with diisopropylfluorophosphate, and then spread to the rest of the cell about the time release of acetylcholinesterase was detected in the medium. With time, more and more of the enzyme was retained in the cells after treatment with cycloheximide, and the proportions of low-molecularweight forms decreased and high-molecular-weight forms increased. The results suggest that newly synthesized acetylcholinesterase undergoes an orderly process of binding, movement, and assembly in diisopropylfluorophosphate treated, and probably also in untreated, embryo muscle fibers.Localization of acetyleholinesterase (EC 3.1.1.7; acetylcholine hydrolase; AChE) at the neuromuscular junction is the culmination of a complicated path of development. AChE first appears in mononucleated myoblasts before they fuse to form multinucleated myotubes (1, 2). Later, it is found within and at the surface of the differentiating muscle fibers and at the embryonic motor endplates. After birth, most of the AChE activity becomes restricted to the adult motor endplates (3, 4). In the chicken, several molecular forms of AChE occur in embryo twitch muscles that are virtually-undetectable in the same muscles after hatching, and large amounts of AChE are released by cultured muscle fibers and appear in plasma from intact embryos (2).The characteristics of AChE in embryo muscle suggest that newly synthesized enzyme undergoes an orderly process of movement and assembly. To examine the process, chick embryo muscle fibers were grown in culture and treated with diisopropylfluorophosphate (DFP), an irreversible inhibitor of AChE; recovery of enzyme activity was studied.Pectoral muscles from 11-day-old chick embryos were dissociated with trypsin, and mononueleated cells were plated onto 35-mm plastic petri dishes coated with collagen. The medium contained 10% (v/v) horse serum, screened for cytotoxicity, 2% embryo extract, and 88% Eagle's minimal essential medium with Earle's salts (5). Temperature was 380, pH was 7.2-7.5, and the atmosphere was a humidified mixture of CO2 and air. Antibiotics were not used. Cholinesterase activities were determined with acetyl-and butyrylthiocholine esters and with the specific inhibitors 10 uM 284C51 (for AChE) and 0.1 mM iso-OMPA (for nonspecific cholinesterases (6). Previous experiments showed that amounts of nonspecific cholinesterases were low in the cultured cells

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“…Motor end-plates were stained for cholinesterase according to the modification of the thioacetic acid technique described by Mumenthaler & Engel (1961).…”

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confidence: 99%

The developing motor end‐plate: pharmacological studies in the chick embryo

Drachman¹

1963

The Journal of Physiology

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Spontaneous contractions of skeletal muscle can be observed as early as the 4th day of incubation in the embryo of the domestic fowl. It is not yet known whether they arise as the result of impulses initiated in the muscle membrane or transmitted from motor nerves. Nevertheless, from their inception such movements may be blocked by curare (Kuo, 1939) or curare-like agents. These drugs are thought to act at the level of the receptor substance of the neuromuscular junction; it is therefore of interest to examine their pharmacological effects during a period featured by significant changes in the morphology of the junction. In the present study the minimum paralytic dosages (MPDs) of curare, succinylcholine and decamethonium were determined in chick embryos from 7 to 15 days of incubation age. Histochemical staining demonstrated the profound structural changes in the neuromuscular junction which take place within the same period. METHODSEach egg was 'candled' in order to determine the position of the embryo and of a large overlying chorio-allantoic vein. The air sac was punctured, and a relatively large window was made in the shell and shell membrane so as to afford an optimum view of the embryo. The egg was placed in a warmed chamber under a dissecting microscope, brightly illuminated by an adjustable lamp. The chorio-allantoic membrane was kept moist with unbuffered isotonic salt solution. A polyethylene microcatheter was inserted into a large chorio-allantoic vein, according to the method previously described by Drachman & Coulombre (1962). Drug voluimes of 2-5-10 ,lI. were delivered by a micrometer-driven Becton-Dickinson 0-25 ml. syringe, sealed with high-vacuum grease. This technique of intravenous injection has the advantages of permitting accurate dosage directly into the extra-embryonic circulation, and at the same time of presenting a clear view of the undisturbed embryo. The drugs used in these experiments included tubocurarine chloride (Squibb), succinylcholine chloride, and decamethoniulm bromide (Burroughs Weilcome), diluted with isotonic saline solution.After the injection was complete a 5 mini period was allowed to elapse for the drug to take full effect. The embryo was then observed directly through the dissecting microscope for a 5 min test period, during which all discrete muscular movements seen were recorded on a 45Physiol. 169

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Cytological Localization of Cholinesterase in Developing Chick Embryo Skeletal Muscle

Cited by 123 publications

References 2 publications

Development of neuromuscular transmission in a larval tunicate

Development of neuromuscular transmission in a larval tunicate

Regulation of Newly Synthesized Acetylcholinesterase in Muscle Cultures Treated with Diisopropylfluorophosphate

The developing motor end‐plate: pharmacological studies in the chick embryo

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