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N.A. Rubinstein, T.S. Khurana, S. Fraterman; Laser Capture Microscopy–based Molecular Analysis of Acetylcholine Receptors From Singly and Multiply Innervated Fibers of Extraocular Muscles . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5724.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Unlike most mammalian skeletal muscles, extraocular muscles (EOMs) contain both singly and multiply innervated fibers (SIFs & MIFs, respectively). Prior studies analyzing acetylcholine receptor (AChR) subunit expression in SIFs and MIFs have yielded contradictory results. We used laser capture microscopy (LCM) to unambiguously define subcellular expression of AChR subunits at sub–synaptic nuclei of MIFs and SIFs Methods: LCM coupled with cytochemical and immunohistochemical markers for synapses and fiber types was used to clearly identify and isolate mRNA from subsynaptic nuclei of neuromuscular junctions (NMJs) of MIFs and SIFs of rat EOM. RT–PCR based cloning and sequencing was used to determine AChR subunit mRNAs at these nuclei. Results:In adult EOMs, there is a strict segregation of the adult ε and fetal γ subunit mRNAs between SIF and MIF neuromuscular junctions. The subsynaptic region of all SIFs expressed only the ε subunit mRNA, not the γ subunit mRNA; beyond the SIF NMJ zone, no AChR subunit mRNAs were detected . This is identical to the situation in other skeletal muscles. In stark contrast, all MIF subsynaptic regions express the γ, and not the ε, subunit mRNA. Moreover, unlike the situation in SIFs, the γ subunit mRNA was found in inter–NMJ regions. Since the muscle regulatory factor, myogenin, has been implicated in developmental control of the γ subunit, we investigated whether myogenin is present in MIFs. Myogenin mRNA was found exclusively at MIF NMJs and not in SIFs or in inter–NMJ regions. Conclusions: Regulation of AChR subunit expression may differ between SIFs and MIFs; and myogenic regulatory factors may play a role in maintaining synthesis of the fetal AChR subunit in MIFs.
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