Affiliation:
1. Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8108, St. Louis, MO 63110, USA.
Abstract
Throughout the developing nervous system, competition between axons causes the permanent removal of some synaptic connections. In mouse neuromuscular junctions at birth, terminal branches of different axons are intermingled. However, during the several weeks after birth, these branches progressively segregated into nonoverlapping compartments before the complete withdrawal of all but one axon. Segregation was caused by selective branch atrophy, detachment, and withdrawal; the axon branches that were nearest to the competitor's branches were removed before the more distant branches were removed. This progression suggests that the signals that mediate the competitive removal of synapses must decrease in potency over short distances.
Publisher
American Association for the Advancement of Science (AAAS)
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5. 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′-dioctadecyloxacarbocyanine 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 (DiI) (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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