Development of the neuromuscular junction in extraocular muscles of white Leghorn chicks

Abstract

Relatively little is known about the development of the neuromuscular junction of extraocular muscles (EOMs). In recent years, chicks have been increasingly used as a developmental model in ophthalmological research. To utilize this model system for understanding the development and plasticity of the extraocular motor system, we investigated the structural changes that occur at the developing neuromuscular junction in the chick between embryonic day 14 (E14) and posthatch day 2 (P2). Axons and nerve terminals were visualized with fluorescent neurofilament antibodies and motor endplates with rhodamine-conjugated alpha-bungarotoxin. Nerve fibers and endplates were colabeled within the same tissue samples. Motor endplates (density, length, width, and area) were measured and numbers of axons per neuromuscular junction were counted using confocal and conventional microscopy. In P2 chicks, densities of motor endplates were significantly greater in the superior oblique muscle when compared with the superior rectus and lateral gastrocnemius muscle. EOMs showed a two- to threefold larger area of motor endplate size as compared to gastrocnemius muscle. Motor endplate size also differed among EOMs with the superior oblique muscle having endplates with a larger area than those of the superior rectus muscle. The period of synapse elimination was similar between EOM and gastrocnemius muscle. Synapse elimination began at about E18 and was completed by P2. By describing the normal morphological changes in developing EOMs, this study provides a baseline for future work to elucidate underlying molecular mechanisms that regulate EOM innervation and strength.