Innervation and acetylcholine sensitivity of skeletal muscle cells differentiated<i>in vitro</i>from chick embryo

Kano, Masaakira; Shimada, Yutaka

doi:10.1002/jcp.1040780210

Cited by 46 publications

(14 citation statements)

References 31 publications

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“…Both the spontaneous and evoked potentials were completely inhibited by addition of d-tubocurarine, but were reversed after washout of the drug ( Fig.6c and d). Thus, these spontaneously depolarizing potential changes are similar to the miniature end-plate potentials and the evoked postsynaptic potentials are equally similar to the end-plate potentials in muscle cells cultured with spinal neurons, as reported in previous investigations (3,16,17). These results indicate that the nerve cells prepared by our present procedure possess the ability to form functional cholinergic synapses with muscle cells.…”

Section: Resultssupporting

confidence: 89%

Isolation and culture of motoneurons from embryonic chicken spinal cords.

Masuko¹,

Kuromi²,

Shimada³

1979

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

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A method is described for isolating cholinergic a motoneurons from the spinal cord of chicken embryos at stage [17][18] (Hamburger and Hamilton numbering), at the time when it has been shown that motoneurons withdraw from the mitotic cycle but neurons of other types and glia are still mitotic. Fragments of the ventral half of the spinal cord are incubated for 24 hr in the presence of 10 ,uM 1-fl-D-arabinofuranosylcytosine in order to eliminate dividing cells and are subsequently dissociated into a suspension of single cells. The following evidence has been obtained and suggests that these cells are neuronal and appear to be a motoneurons: (i) they are resistant to the lethal effect of arabinofuranosylcytosine, and thus are postmitotic at stage 17-18; (ii) when grown in vitro, they exhibit morphological characteristics similar to those of ventral spinal neurons, which include the ability to be stained with silver, Nissl, methylene blue vital stain at pH 6.5-7.0, and choline acetyltransferase histochemistry; (iii) they have high choline acetyltransferase activity; (iv) they are capable of forming functional synapses with muscle.Embryonic chicken spinal cords have been a favored source of neural tissue for culture. Recently, particular interest in them has been stimulated by the evidence that neurons dissociated from this tissue will form synapses with skeletal muscle cells in vitro (1-4). Although this culture system possesses the advantages of accessibility for analysis by microelectrodes, scanning electron microscopy, and other methods related to cell surface studies, it is complicated in that the dissociated spinal cord contains, in addition to a motoneurons, a variety of nerve cell types that do not usually make synaptic contacts with muscle in vivo; such cells may make nonspecific contacts with muscle in vitro and thus interfere with analysis of the properties exhibited by motoneurons in associations with muscle. Further, this culture, over a longer period of time, is overwhelmed by proliferating fibroblastic cells, glial cells, or both.In the present study, an attempt was made to isolate and culture a homogeneous population of a motoneurons from embryonic spinal cords. The procedure is based on the earlier observation that many of the cells destined for the ventral horn withdraw from the mitotic cycle in the developing chicken embryo by stage 17-18 of Hamburger and Hamilton (5), whereas other types of neurons as well as glia display an upward trend of mitosis at this state (6-8). Taking advantages of this difference in the period of mitosis, we were able to prepare the cell suspensions enriched in the former cells. By incubating fragments of ventral spinal cords (VSCs) in a medium containing an inhibitor of DNA synthesis (1-3-D-arabinofuranosylcytosine, Ara-C), dividing cells (non-motoneurons and glia) are eliminated. They are subsequently dispersed into a suspension of single cells for culture. MATERIALS AND METHODS Preparation of Cultures. Spinal cords from chicken embryosat 60-66 hr of incubation ...

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Section: Resultssupporting

confidence: 89%

Isolation and culture of motoneurons from embryonic chicken spinal cords.

Masuko¹,

Kuromi²,

Shimada³

1979

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

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“…This result accords with the notion that nerves affect gene expression and induce the differentiation of muscle fibers to specific types (Toyota and Shimada 1983). In particular, the observation that the changes occurred concurrently with or after formation of large AChR clusters (after stage 38) indicates that the formation of functional neuromuscular junctions (Kano and Shimada 1971;Fambrough et al 1974;Cohen and Fischbach 1977) could inhibit synthesis of isoproteins other than the adult types. Fluorescence micrograph of transverse section of chicken cervical muscles at stage 45 stained with antibodies against slow C-protein (sCP).…”

Section: Discussionsupporting

confidence: 88%

Differentiation of muscle-specific proteins in chicken somites as studied by immunofluorescence microscopy

Begum

Komiyama

Toyota

et al. 1998

Cell and Tissue Research

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Fluorescence microscopy of chicken cervical somites revealed that muscle-specific proteins began to appear at stage 11 (Hamburger and Hamilton numbering), and the onset of the expression of all the proteins examined in the present study had occurred by stage 17. Muscle proteins were classified into six groups according to the stage of their appearance. Since all these proteins were expressed before emergence of nerve fibers in myotomes, switching-on of their synthesis does not seem to require neuronal influence. However, since isoproteins other than adult muscle types disappeared and diversification of muscle fiber types occurred coordinately with the clustering of acetylcholine receptors in cervical muscles, switching-off of the synthesis of the nonadult isoforms might have been accelerated by the formation of functional neuromuscular junctions. The absence of nebulin and C-protein in early stages seems to indicate that these proteins are not required for the initial assembly of myofilaments and/or myofibrils. Further, this absence might be considered to facilitate exchangeabilities of proteins in nascent myofibrils, thereby changing the isoforms to adult types.

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“…Neuromuscular junctions have been shown to form when neurons, derived from spinal cord, are cultured with muscle fragments (11) or myotubes, formed either from primary (12)(13)(14)(15) or cloned (16) myoblasts. Further, Crain and Peterson (17) have reported junction formation between explants of sympathetic ganglia and muscle fragments.…”

Section: And 10)mentioning

confidence: 99%

Formation of cholinergic synapses between dissociated sympathetic neurons and skeletal myotubes of the rat in cell culture.

Nurse¹,

O'Lague²

1975

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

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Sympathetic principal neurons, dissociated from superior cervical ganglia of newborn rats, were plated into cultures containing rat skeletal myotubes formed from previously plated primary myoblasts. Electrophysiological evidence is presented that the neurons developed cholinergic synapses with the myotubes. In addition, the neurons developed cholinergic synapses with each other as previously reported [O'Lague et al. (1974) Proc. Nat. Acad. Sci. USA 71, 3602-36061. The

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Innervation and acetylcholine sensitivity of skeletal muscle cells differentiatedin vitrofrom chick embryo

Cited by 46 publications

References 31 publications

Isolation and culture of motoneurons from embryonic chicken spinal cords.

Isolation and culture of motoneurons from embryonic chicken spinal cords.

Differentiation of muscle-specific proteins in chicken somites as studied by immunofluorescence microscopy

Formation of cholinergic synapses between dissociated sympathetic neurons and skeletal myotubes of the rat in cell culture.

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