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Volumn 282, Issue 5393, 1998, Pages 1508-1511

Synaptic segregation at the developing neuromuscular junction

Author keywords

[No Author keywords available]

Indexed keywords

ANIMAL TISSUE; ARTICLE; MOUSE; NERVE ENDING; NERVE FIBER GROWTH; NERVOUS SYSTEM DEVELOPMENT; NEUROMUSCULAR SYNAPSE; NEWBORN; NONHUMAN; POSTSYNAPTIC MEMBRANE; PRIORITY JOURNAL; SENSORY NERVE; SIGNAL TRANSDUCTION; TARGET CELL;

EID: 0032553603     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5393.1508     Document Type: Article
Times cited : (89)

References (34)
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    • Neonatal mice (between P0 and P17) were anesthetized with 0.1 ml of sodium pentobarbital. Sternomastoid muscles were then dissected and placed in petri dishes in physiological saline. In some cases, junctional AChRs were labeled for 10 min with tetramethyl rhodamine-conjugated α-bungarotoxin (5 μg/ml) (Molecular Probes, Eugene, OR). Sharp electrodes (5 to 10 megohms, as measured with 3 M KCl) were backfilled with a 1% solution of 3,3′-dioctadecytoxacarbocyanine perchlorate (DiO) (Molecular Probes) in a 100% solution of methylene chloride (Sigma) and positioned on a superficial neuromuscular junction. Depolarizing current (200 ms, 1 to 10 nA, and 1 Hz) was applied for a few seconds until a dye crystal was deposited at the junction. The muscle was then fixed in a 4% solution of paraformaldehyde for 12 hours, over which time the fluorescent DiO lipid labeled many terminals of one motor unit that were distributed over several hundred micrometers. For the labeling of two competing axons at the same neuromuscular junction, two electrodes [each containing 1% solutions of either 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) (Molecular Probes) or DiO in a 100% solution of methylene chloride] were used to deposit dye. In this way, two different subsets of axon terminals, which by chance occasionally converged at the same junction, were labeled. All labeled junctions were imaged with confocal microscopy (Noran Odyssey, Olympus Fluoview, and Bio-Rad MRC1024) with 1.4-numerical aperture objectives, and three-dimensional (3D) reconstructions were generated with the Bio-Rad MRC1024 software.
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    • note
    • We thank all the members of our lab for helpful discussion of this work; J. R. Sanes, R. O. Wong, and M. L. Nonet for comments on the manuscript; and S. G. Turney for technical help. This work was supported by grants from NIH and the Muscular Dystrophy Association. W.-B.G. was supported by a National Research Service Award from NIH.


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