PURPOSE

The cell and molecular biology of extraocular muscle (EOM) is distinct from other skeletal muscles. 1, 2 Novel aspects of the innervation pattern of EOM include: high motoneuron discharge rates, presence of multiply innervated muscle fiber types in the adult, retention of embryonic acetylcholine receptor isoforms at some neuromuscular junctions (NMJs), absence of significant postjunctional membrane foldings at most NMJs, and sensitivity of EOM to neuromuscular transmission disorders (myasthenia gravis) and distinct response to botulinum toxin. The formation and maturation of the NMJ require a series of inductive interactions between axon and muscle fiber. These interactions culminate in the juxtaposition of a highly specialized nerve terminal with an elaborate postsynaptic apparatus. 3, 4 The sarcolemmal DGC and its associated proteins are central in this process of synapse formation and stabilization. 5, 6 The central hypothesis of this work was that the unique phenotype, and functional properties, of EOM may require muscle group-specific adaptations to organize and maintain the NMJ. Therefore, the signaling and sarcolemmal organization at the NMJ may differ between EOM singly (SIF) and multiply innervated fiber types (MIF) and between EOM and other skeletal muscles.

METHODS

Here, we have examined cryostat sections of EOM of adult mice using immunohistochemistry for the neuregulin/erbB and agrin/MuSK pathway molecules and important components of the junctional DGC. The en-plaque (on SIFs) and the en-grappe junctions (on MIFs) were identified by their morphologic pattern using Texas-red conjugated bungarotoxin (Molecular Probes; 4 μg/ml), which labels the AChRs. The other proteins were detected using monoclonal and polyclonal antibodies specific for: utrophin (NCL-DRP2, Novocastra Labs), α1-syntrophin (258), β1-syntrophin (37) and β2-syntrophin (28), rapsyn (1234), α-dystrobrevins DB-1 (670) and DB-2 (DB2)(all from Stan Froehner, University of Washington), s-laminin (D7; Joshua Sanes, Washington University), neuregulin (NDF; Jeffrey A Loeb, Wayne State University), erbB2 (Neu: sc-284; Santa Cruz Biotechnology, Inc. Santa Cruz), erbB3 and erbB4 (05-390, 06-572; Upstate Biotechnology, NY), agrin (AGR-550; StressGen Biotechnology Corp., Canada), and MuSK (Markus Ruegg, University of Basel). Junctions were examined for the cellular localization and colocalization of these proteins with AChRs using a scanning laser confocal microscope.

RESULTS

The components of the junctional DGC and the agrin/MuSK and the neuregulin/erbB nerve-muscle signaling pathways that we examined, colocalized with NMJ acetylcholine receptor aggregates in both the MIFs and the SIFs, similar to the typical skeletal muscles. There were some differences in the extrasynaptic localization of some DGC proteins, namely, β 1-syntrophin and α-dystrobrevin1 on EOM MIFs. The results are summarized in Table 1.