Effects of a Snake α-Neurotoxin on the Development of Innervated Skeletal Muscles in Chick Embryo

Abstract

The evolution of the cholinergic (nicotinic) receptor in chick muscles is monitored during embryonic development with a tritiated a-neurotoxin from Naja nigricollis and compared with the appearance of acetylcholinesterase. The specific activity of these two proteins reaches a maximum around the 12th day of incubation. By contrast, choline acetyltransferase reaches an early maximum of specific activity around the 7th day of development, and later continuously increases until hatching. Injection of a-toxin in the yolk sac at early stages of development causes an atrophy of skeletal and extrinsic ocular-muscles and of their innervation. In 16-day embryos treated by the a-toxin, the endplates revealed by the Koelle reaction are almost completely absent. The total content and specific activities of acetylcholinesterase and choline acetyltransferase in atrophic muscles are markedly reduced.Snake venom a-neurotoxins bind with a high affinity and a low reversibility to the cholinergic (nicotinic) receptor site (1-3).They have therefore been widely used to assay, characterize, and identify the protein that carries this site (see ref. 4).These neurotoxins can also be used to create a chronic block of neuromuscular transmission at the postsynaptic level and to analyze the consequences of this block in the differentiation, morphogenesis, and stability of a neuromuscular synapse.In this paper, we report on the cholinergic receptor sites in chick-embryonic muscles and on their evolution during development, in comparison to the appearance of choline acetyltransferase and acetylcholinesterase. Injection of the atoxin from Naja nigricollis at early stages of development causes a marked atrophy of skeletal muscles and of their innervation, accompanied by characteristic changes in their content of choline acetyltransferase and acqtylcholinesterase. MATERIALS AND METHODS[3H]a-Toxin. The al-isotoxin was purified from the venom of Naja nigricollis by the method of Boquet et al. (5) The stock solution contained 24 MM a-toxin (10.2 Ci/mmol); 72% of the molecules of a-toxin were active.Embryos. Chick embryos were obtained from fertilized eggs of a local strain and incubated at 380 at a relative humidity of 70-80%; 3-, 4-, 5-, 6-, 8-, 11-, 12-, 14-, 16-, 18-, and 21-dayold embryos were used. Muscles were dissected under a stereomicroscope and quickly frozen; most samples were lyophilized and stored at -45°. With 3-to 6-day-old embryos, assays were made on posterior limb buds; from the 8th day of incubation and after, the posterior muscles of the leg were used.The embryos were treated with a-toxin by three successive injections (the 3rd, 8th, and 12th day of incubation) in the yolk sac with a Hamilton syringe, through a small window in the

Differentiation of Cell Membranes in Cultures of Embryonic Chick Breast Muscle

“…1B and 4). This value closely agrees with levels of receptor reported for embryonic chick muscle in culture (3) and in ovo (17) and is comparable to the amounts of receptor per mg of protein in electrolplax of electric eel, estimated by affinity labeling (19).…”

“…Cholinesterase activity was demonstrated in avian muscle by Nachmansohn (16), and this activity has been recently reported to increase in parallel with receptor in embryonic chick muscle during development in ovo (17). The rapid kinetics shown by the chick muscle cultures with respect to cell fusion 3208…”

“…Cholinesterase activity was demonstrated in avian muscle by Nachmansohn (16), and this activity has been recently reported to increase in parallel with receptor in embryonic chick muscle during development in ovo (17). The and receptor elaboration allow a precise comparison of changes in activity patterns of acetylcholine receptor and acetylcholinesterase with differentiation.…”

“…The acetylcholine receptor appears 5-10 hr after the main period of fusion in control cultures, and the time and extent of receptor elaboration are unaffected when fusion is blocked by lowering the Ca++ concentration in the medium (Fig. 1).Cholinesterase activity was demonstrated in avian muscle by Nachmansohn (16), and this activity has been recently reported to increase in parallel with receptor in embryonic chick muscle during development in ovo (17). The rapid kinetics shown by the chick muscle cultures with respect to cell fusion 3208…”

Human muscle cultured in monolayer and cocultured with fetal rat spinal cord: importance of dorsal root ganglia for achieving successful functional innervation

“…It has been suggested that, while clustering of AChRs at the nervemuscle contact does not depend on muscle activity, the loss of multi-endplate status and accumulation of AChE may depend on muscle activity (Giacobini et al, 1973;Lomo and Slater, 1978;Rubin et al, 1980;and review in Bennett, 1983). Development of organized neuromuscular junctions in the rat has also been considered to be linked to muscle activity (Lomo et al, 1984).…”