Identification of acetylcholine receptor subunits differentially expressed in singly and multiply innervated fibers of extraocular muscles

PURPOSE. To identify the acetylcholine receptor ( AChR) isoforms among the neuromuscular junctions ( NMJs) of singly and multiply innervated fibers ( SIFs and MIFs) of rat extraocular muscles ( EOMs). METHODS. EOMs were dissected from adult rats and serially sectioned. Sections were simultaneously stained for acetylcholinesterase and with an antibody to the slow myosin heavy chain to identify NMJ topography and fiber types in the same section. Synapses and subsynaptic regions of SIFs and MIFs were isolated by laser capture microdissection and the AChR subunits identified by RT-PCR. RESULTS. The en plaque endings of SIFs expressed only the adult epsilon subunit, not the fetal gamma subunit, of the AChR, whereas the en grappe endings of the MIFs expressed only the gamma subunit, and not the epsilon subunit. Although the expression of the epsilon subunit was confined to the NMJ region of the SIFs, the gamma subunit was expressed both synaptically and extrasynaptically within the MIFs. The gamma subunit in MIFs correlated with the expression of the myogenic regulatory factor myogenin. Moreover, an unusual neuronal AChR subunit, alpha 9, was found in the EOMs, but not in the limb muscles. CONCLUSIONS. The adult epsilon and fetal gamma subunits of the AChRs are segregated into distinct synapses on distinct fiber types. The maintenance of the fetal subunit in a population of fibers is probably linked to the expression of myogenin and is a unique attribute of the EOM allotype.