Molecular Organization of the Extraocular Muscle Neuromuscular Junction: Partial Conservation of and Divergence from the Skeletal Muscle Prototype

Abstract

purpose. The phenotypically novel extraocular muscles (EOMs) exhibit fundamental differences in innervation and neuromuscular junction (NMJ) morphology from other skeletal muscles. In the current study, the morphology and molecular organization of NMJs of EOM singly innervated (SIF) and multiply innervated (MIF) fiber types were evaluated and the distribution of molecules involved in formation and maintenance of NMJs were specifically characterized.

methods. Adult mouse EOM NMJ organization was examined by immunofluorescence and confocal microscopy. Differential cellular localization of components of two established synaptic signaling pathways, (1) neuregulin and erbB receptors 2, 3, and 4 and (2) agrin, MuSK, and rapsyn and select NMJ-associated structural proteins were studied for EOM SIF and MIF populations. Endplate topography and structure were also studied, using both confocal microscopy and transmission electron microscopy, with NMJ morphologic organization correlated with specific EOM fiber types.

results. Confocal fluorescence microscopy demonstrated that, for NMJs of both EOM SIFs and MIFs, components of neuregulin and agrin pathways and the major components of the junctional dystrophin-glycoprotein complex (DGC) colocalized with acetylcholine receptor (AChR) aggregates. However, EOM exhibited novel fiber-type–specific extrasynaptic localization of two key DGC signaling-related molecules: α-dystrobrevin 1 (global MIFs) and syntrophin β1 (global MIFs and orbital MIFs and SIFs).

conclusions. The data establish that the molecular organization of EOM SIF and MIF NMJs includes the same signaling and structural molecules previously characterized for other skeletal muscles. By contrast, divergence in other aspects of the synaptic and nonsynaptic sarcolemmal organization of EOM fiber types may underlie the unique responses of these muscles in a variety of neuromuscular disorders.

Extraocular muscle (EOM) represents a distinctive class among mammalian skeletal muscles, with a novel phenotype, molecular signature, and disease responsiveness. ^{1 2 3 4} EOM may be the only skeletal muscle that does not fit the widely accepted myofiber type classification schemes. There is compelling evidence that the unique fiber type composition of EOM may be related to demands imposed by the diversity of eye movement control systems and the discharge patterns of oculomotor motoneurons. ^{1 5 6} Alternatively, such differences between EOM and the other skeletal muscles may, at least in part, relate to their embryonic origin from isolated mesenchymal condensations that are of neither somitic nor branchial arch origin that typifies other muscles. ^{7 8} In all likelihood, both epigenetic and cell lineage factors collaborate in development and maintenance of the novel EOM fiber types. 

Activity and non–activity-based mechanisms at the neuromuscular junction (NMJ) are key trophic determinants of skeletal muscle fiber properties. The nature of nerve-muscle interactions in EOM are likely to be more complex than in other muscles, because EOM contains singly innervated twitch fibers (SIFs), the standard for mammalian skeletal muscle, as well as atypical multiply innervated nontwitch fiber types (MIFs), that are rare in mammals but common in birds and amphibians. There are both general and fiber-type–specific differences in NMJs in EOM compared with other skeletal muscles, including the conspicuous sparseness of subjunctional folds, coexpression of adult and fetal acetylcholine receptor (AChR) isoforms, and increased susceptibility to the neurotransmission disorder, myasthenia gravis. ⁹ Moreover, the nature of ocular motoneuron–EOM communication has system-specific properties, including the requirement for innervation by appropriate motoneuron pools to mediate EOM primordia survival in an organotypic coculture model. ¹⁰ In this context, the structural and functional properties of EOM NMJs are likely to exhibit both conserved and divergent features in comparison to those of more typical skeletal muscle. 

Because of its accessibility, the NMJ represents the best-studied synapse (for review, see Sanes and Lichtman ¹¹ ). Presynaptic motoneuron axons enter a muscle, branch to innervate multiple muscle fibers, lose the myelin sheath immediately adjacent to each fiber, and then form a mitochondria and synaptic vesicle–filled expansion, or bouton, that closely contacts individual myofibers at one site only. The presynaptic boutons lie in deep synaptic gutters formed by invagination of the myofiber sarcolemma, thereby minimizing neurotransmitter diffusion distance and isolating the cell–cell communication events from much of the extracellular milieu. Additional sarcolemmal specializations, the postjunctional folds, increase the surface area for synaptic interaction and segregate molecular events at the synapse. In contrast to this idealized synaptic profile, there is considerable variability at the NMJs. The presence of MIFs in EOM represents an extreme in variability of neuromuscular structural organization. 

A generalized view of the molecular events controlling synapse induction, maturation, and maintenance has been constructed using data from a rather restricted subset of skeletal muscles (e.g., diaphragm and sternocleidomastoid; reviewed in Sanes and Lichtman ¹² ). Two principal motoneuron-derived signaling pathways mediate NMJ molecular organization: agrin–MuSK and neuregulin–erbB pathways. ^{13 14 15 16 17} These are not independent, but rather are interlinked in regulating synaptogenesis. Nerve-derived agrin acts through a myotube sarcolemmal MuSK receptor complex to induce synthesis and aggregation of both MuSK and erbB receptors. MuSK activation by agrin, in turn, initiates focal clustering of AChRs and other synaptic proteins (e.g., utrophin and rapsyn) and, later, induces structural specializations of the postsynaptic apparatus. Rapsyn functions downstream of the agrin-MuSK pathway as a scaffold protein to cocluster AChRs, dystroglycan, utrophin, and erbB3 at the postsynaptic membrane. Neuregulin acts through agrin-recruited erbB receptors to accelerate AChR transcription by subsynaptic myonuclei. Synapse-mediated electrical activity is required to maintain the neuregulin- and agrin-dependent complex at the NMJ. 

Cytoskeletal and basal lamina components, particularly the dystrophin-glycoprotein complex (DGC), s-laminin (β2 laminin), spectrin, and ankyrin_G, also are critical for the stabilization and maintenance of the AChR clusters and maturation of postsynaptic structure, including the formation of postsynaptic folds. ^{18 19 20 21 22 23} Except for retention of developmental AChR isoforms ^{24 25 26} and the localization of some components of the dystrophin-glycoprotein complex (dystrophin, utrophin, the dystroglycans, and the sarcoglycans), ²⁷ nothing is known of the molecular organization of EOM NMJs. 

On the basis of established morphologic divergences between EOM and typical skeletal muscles, and novel differences between SIF and MIF NMJs, we hypothesized that EOM may use distinctive synaptic signaling and structural proteins. Such differences are important, in that they may account for the differential susceptibility of EOMs to neuromuscular disorders, such as myasthenia gravis and muscular dystrophy. 

In the current study, we examined the cellular localization of established neuromuscular synaptic cell signaling and structural proteins at EOM NMJs, differentiating the synaptic organization of the SIF and MIF types. We first correlated endplate topography and structure with the distinctive EOM fiber types. Next, we established that the EOMs share the basic postsynaptic molecular architecture described for the other skeletal muscles. Finally, we identified EOM-specific features of the DGC. 

Methods

Muscle Tissue

Adult C57Bl/6 (Jackson Laboratories, Bar Harbor, ME) mice were asphyxiated with carbon dioxide. Animal use procedures received prior approval from the Institutional Animal Care and Use Committee at Case Western Reserve University and were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Globes, with intact EOMs and associated connective tissue, were obtained by orbital dissection. Enucleated specimens were freeze protected with 30% sucrose, embedded in optimal cutting temperature (OCT) compound, snap frozen with isopentane cooled in liquid nitrogen, and stored at −80°C. For muscle cross-sectional studies, 8-μm-thick cryostat sections transverse to the globe axis were collected and mounted on coated microscope slides (Vectabond-coated Superfrost/Plus; Fischer Scientific, Pittsburgh, PA). For whole muscle mounts, the EOMs of five mice were isolated after asphyxiation. Individual rectus muscles were carefully dissected, from origin to insertion. The muscle sheath was stripped, and muscles were fixed in 2% paraformaldehyde for 30 minutes. After they were washed with PBS, muscles were incubated with Texas red–conjugated α-bungarotoxin (TR-αBtx 1 μg/mL; Molecular Probes, Eugene, OR) overnight at 4°C. Muscles then were washed with PBS and mounted in antifade reagent in glycerol (SlowFade; Molecular Probes), coverslipped, and sealed with nail enamel for observation under a confocal fluorescence microscope. 

Immunohistochemistry

Frozen sections were air dried, hydrated for 30 minutes with PBS, and then blocked with 10% goat serum in 1% bovine serum albumin (BSA) for 30 minutes. Primary antibodies were diluted in 1% BSA/PBS and applied to the sections overnight at 4°C. The sections were washed three times for 5 minutes each with PBS. The appropriate secondary antibody was then applied, along with 5 μg/mL TR-αBtx, for 1.5 hours at room temperature. Sections were washed and coverslipped with antifade reagent in glycerol (SlowFade; Molecular Probes) and sealed with nail enamel. 

Antibodies

NMJ proteins were detected with primary monoclonal and polyclonal antibodies specific for ankyrin_G (RDI-ANKYRGabmX; Research Diagnostics, Inc., Flanders, NJ); utrophin (NCL-DRP2; Novocastra/Vector Laboratories, Burlingame, CA); α1-syntrophin (SYN258), β1-syntrophin (SYN37), β2-syntrophin (SYN28), rapsyn (1234), α-dystrobrevins DB-1 (670), and DB-2 (DB2) (all gifts from Marvin Adams and Stanley Froehner, University of Washington, Seattle, WA); s-laminin (D7; Joshua Sanes, Washington University, St. Louis, MO); nNOS (DiaSorin, Stillwater, MN); neuregulin (NDF; Jeffrey A. Loeb, Wayne State University, Detroit, MI); erbB2 (Neu:sc-284; Santa Cruz Biotechnologies, Santa Cruz, CA); erbB3 and erbB4 (05-390 and 06-572; Upstate Biotechnology, Lake Placid, NY); agrin (anti-agrin 4, 8 (606051); Regeneron Pharmaceuticals, Tarrytown, NY); and MuSK (Markus Ruegg, University of Basel, Switzerland). The antibody to slow myosin heavy chain (NCL-MHC slow) was obtained from Novocastra/Vector Laboratories. The secondary antibodies, anti-mouse and anti-rabbit IgGs, were obtained from Molecular Probes, Inc. 

Confocal Microscopy

Confocal microscopy was performed with a laser scanning microscope (model LSM 410; Carl Zeiss, Göttingen, Germany). For cross-sectional studies, images were acquired with the 40× objective (1.3 numeric aperture; Plan-NeoFluar; Carl Zeiss), with a constant pinhole setting used to preserve the thickness of the confocal plane. For dual color imaging, TR-αBtx and green-fluorescence–conjugated secondary antibody labels were excited sequentially using the 568-nm and the 488-nm excitation lines of the krypton/argon laser. The laser intensity and gain settings were adjusted on a per-image basis. 

For endplate topography, whole muscle mounts were labeled for AChR with TR-αBtx and imaged using the 568-nm excitation line and a 100× objective (1.3 numeric aperture; Plan-NeoFluar; Carl Zeiss), with a zoom setting of 2×. For endplates that were in the plane of observation, z-sectioning was performed to view the entire thickness of the junction. For each NMJ, the final image was obtained by combining 6 to 16 images taken every 0.6 μm along the z-axis into a single projected view, using the software provided by the manufacturer. Approximately 100 NMJs were examined by confocal microscopy. All digital images were then processed (Photoshop; Adobe, Mountain View, CA). 

Electron Microscopy

For ultrastructural analyses, mice were perfused with physiological saline followed by 1% paraformaldehyde/2% glutaraldehyde fixative solution in 0.1 M phosphate buffer. EOMs were removed, postfixed in 4% glutaraldehyde fixative solution, followed by 1% osmium tetroxide in 0.1 M phosphate buffer, and processed into plastic resin, according to standard procedures. ²⁸ Sections were photographed by electron microscopes (Zeiss; or JEOL, Tokyo, Japan). Delineation of fiber-type–specific NMJ properties relied on recognition of fiber types by the criteria of Spencer and Porter, ⁹ which include distinctive differences in myofibril size and in the number, size, and distribution of mitochondria. 

Results

Mouse extraocular rectus muscles, when labeled with TR-αBtx, showed a single distinct innervation band, similar to those found in other skeletal muscles. Singly innervated fiber types (SIF; Fig. 1A ) had single en plaque junctions (Fig. 1B) , and all were localized in the common endplate zone in the muscle midbelly (Fig. 1D) . In contrast to other skeletal muscles, EOMs also exhibited small-diameter, MIFs (Fig. 1A) with multiple en grappe endplates (Fig. 1C) scattered along the length of the fibers. MIF NMJs were found both proximal and distal to the central endplate zone. Cross-sections through the midbelly of rectus muscles labeled with TR-αBtx showed numerous endplates (Fig. 1D) . The morphology of most of the NMJs was typical of en plaque endplates. The slow fibers (identified by immunoreactivity to slow myosin heavy chain antibody ⁵ ) showed much smaller punctate labeling with TR-αBtx, representative of the en grappe morphology. En grappe endplates were concentrated in sections from the proximal and distal ends of the EOM (Fig. 1E) . 

Confocal and Ultrastructural Characterization of Endplate Morphology for SIF and MIF Types

To better characterize the morphology of EOM endplates, approximately 100 NMJs from EOMs of five mice were visualized en face in unsectioned whole-muscle mounts, after labeling with TR-αBtx. We identified four distinctive NMJ types in EOM (Figs. 2A 2B 2C 2D) . The synaptic gutters of all en plaque endings exhibited the continuous, branched morphology typical of skeletal muscles, with uniform labeling of AChRs within the gutters. Most of the en plaque endplates were large and oval, approximately 20 μm in width, with length varying from 35 to 45 μm (Fig. 2C) . Less typical junctions associated with SIFs were smaller and more rounded (Fig. 2B) . Some SIFs had unusually long (∼65 μm) junctions (Fig. 2D) . These usually curved around the circumference of the fiber (these typically were associated with small myofibers approximately 25 μm in diameter). This last type was not seen in the accessory EOMs (retractor bulbi and levator palpebrae superioris). All rectus muscle en plaque endplates were more intricately branched than those of levator (Fig. 2E) and retractor bulbi muscles (Figs. 2F 2G) . Rectus muscle en grappe junctions were small, approximately 10 μm, and their substructure was not complex, as viewed by confocal microscopy (Fig. 2A) . This endplate type was not found in the accessory EOMs. 

By electron microscopy, distinct neuromuscular junction types were associated with the identified EOM MIF and SIF types (Fig. 3) . NMJs of orbital layer SIFs exhibited traits consistent with the long, myofiber-encircling junctions on confocal microscopy (compare Figs. 3A 3B with Fig. 2D ). These NMJs had elongated synaptic contact areas embedded in deep depressions in the myofiber surface, and postjunctional folds were sparse. By contrast, NMJs of both orbital and global MIFs were characterized by small, punctate axon terminals that were not embedded in sarcolemmal depressions, and did not exhibit postjunctional folds (Figs. 3C 3D 3E) . This morphology is consistent with the small, noncomplex (en grappe) nerve terminals seen by confocal microscopy (Fig. 2A) . The global SIFs were identified on the basis of mitochondrial features (see the Methods section). Their NMJs showed a range in cytoarchitecture, varying in size and degree of postjuctional folding (Figs. 3F 3G) . Typically, postjunctional folding was modest, although a high density of folds was observed in some global SIFs (usually global pale SIFs; Fig. 3G ). 

Collectively, these observations demonstrate that EOM SIFs have a range of endplate morphologies, including some endplates that are relatively large in relationship to the diameter of the fiber itself. These data are consistent with prior observations ²⁹ that the ratio of endplate size to fiber diameter is markedly higher in EOM than in limb muscle. 

Cellular Localization of Synaptic Signaling Proteins at SIF and MIF Endplates

The neuregulin/erbB receptor and agrin/MuSK pathways are critical for the establishment and maintenance of the communication between nerve, muscle, and perisynaptic Schwann cell that lies behind the precise topological arrangement at typical skeletal NMJs (for review see Sanes and Lichtman ¹² ). ErbB2, erbB3, and erbB4, as well as neuregulin, agrin, and MuSK and rapsyn, are concentrated at the limb, diaphragm, and sternocleidomastoid NMJs. To determine whether these same molecules may be involved in the maintenance of the synapse in the phenotypically unique EOMs, with attention to potential differences in the NMJs of the MIFs and SIFs, we examined the localization of synaptic proteins in EOM. 

As a positive control, we first examined sections of adult rat gastrocnemius-soleus muscle to confirm that the antibodies used revealed the expected enrichment of neuregulin, the erbBs, agrin, rapsyn, and MuSK at the NMJ. NMJs were identified by colabeling with TR-αBtx. Consistent with previous results, these proteins were enriched at the NMJ (data not shown). There was no evidence for extrajunctional sarcolemmal localization of any of the synaptic molecules in the gastrocnemius and soleus. 

Cross-sections of adult mouse EOM were evaluated for components of the neuregulin/erbB and agrin/MuSK signaling pathways. NMJs were identified by AChR labeling with TR-αBtx. For the EOM SIF en plaque endplates, clear colocalization of agrin/MuSK and NDF/erbB pathway proteins with AChR receptors, was noted (Figs. 4 and 5) . MIF en grappe endplates were identified in cross-section by their small size compared with the en plaque endplates and their localization on the slow fibers, often distal and proximal to the endplate zone (Fig. 1E) . Observations confirmed the presence of all components of agrin/MuSK and NDF/erbB pathway proteins at en grappe endplates (Figs. 4 5) . Thus, the major synaptic signaling pathway molecules previously identified in other skeletal muscles were conserved for EOM SIF and MIF types. 

Expression of the Cytoskeletal Proteins at SIF and MIF Endplates

The multimolecular DGC is found at synaptic and extrasynaptic regions of the skeletal myofibers and is important for postsynaptic development and muscle membrane stability and signaling. ³⁰ The DGC includes dystrophin or its homologue utrophin, three groups of transmembrane–intramembrane proteins (dystroglycans, sarcoglycans, and sarcospan) and two groups of soluble membrane-associated proteins, the dystrobrevins and syntrophins. The DGC at the postsynaptic membrane is highly specialized. Whereas dystrophin, the dystroglycans, the sarcoglycans, syntrophins α and β1, and α-dystrobrevin 2 are present throughout the sarcolemma, with enrichment at the synapse, utrophin, syntrophin β2, and α-dystrobrevin-1 are largely restricted to synaptic sites. Deletions of the various junctional DGC components alter skeletal muscle postsynaptic specializations, including postsynaptic folds and AChR distribution. ^{18 19 20 21}  

Here, we studied the localization of select DGC components at EOM endplates by immunohistochemistry. As shown in Figures 6 and 7 , EOM SIF and MIF NMJs showed the presence of important elements of the DGC—namely, utrophin, α-dystrobrevin-1 and -2, α-syntrophin, β1- and β2-syntrophin, and a signaling molecule related to α-syntrophin, neuronal nitric oxide synthase (nNOS). This localization pattern is identical with that in other skeletal muscles. We also evaluated the distribution of the basal lamina protein s-laminin (β2-laminin) and the cytoskeletal molecule ankyrin_G. Laminin β2 is involved in the differentiation and maturation of the presynaptic apparatus. ³¹ We noted laminin localization at both the MIF and SIF junctions. Ankyrin_G is a cytoskeletal protein that binds voltage-gated sodium channels and is important in organizing the secondary folds at the junction. ²³ EOM SIFs and MIFs showed localization of ankyrin_G along the entire sarcolemma with enrichment at the synapse, as is typical of other skeletal muscles (data not shown). 

In the course of the synaptic localization studies, we consistently observed the localization of syntrophin β1 at the extrasynaptic sarcolemma of three of the six distinct EOM fiber types: the global MIF (Fig. 8) and the orbital MIF and SIF types. By contrast, syntrophin β1 was restricted to NMJs of global SIFs. These data represent a striking divergence of EOM from other skeletal muscles, because prior studies have established that syntrophin β1 is preferentially enriched at the sarcolemma of fast-twitch glycolytic (IIB) muscle fibers and is absent or at low levels in skeletal slow-twitch muscle fiber types. ³² Global pale SIFs are the only EOM fibers with IIB myosin and none of the global SIFs showed extrasynaptic immunostaining. 

The distribution of dystrobrevins in limb and sternocleidomastoid muscle includes DB-1 and -2 enrichment at synaptic sites, but both isoforms also are found at the sarcolemma, where DB-2 is more abundant than DB-1. ³⁰ Sarcolemmal distribution of DB-1 on the muscle fibers has been reported by some investigators ³³ and not by others, depending on the antibody used for detection. A previous study on the sternocleidomastoid and limb muscle using the same antibody (Ab 670) showed only synaptic distribution of the DB-1 isoform. ³⁴ Although EOM conserves the skeletal muscle pattern of DB-2 and -1 expression at both SIF and MIF NMJs, the DB-1 extrasynaptic immunostaining pattern was distinctive. Although light, inconsistent extrasynaptic immunostaining was seen on most fiber types, the global MIFs showed consistent bright staining of the sarcolemma. This fiber-type–specific pattern of extrasynaptic immunostaining was confirmed by labeling adjacent section with slow myosin (marker for MIFs; Fig. 8 ). Thus, although we noted conservation of the synaptic DGC, we found divergence in the molecular specialization of the extrasynaptic DGC in EOM. 

Discussion

EOM fiber types, and their innervation patterns, are fundamentally distinct from other skeletal muscles. ^{1 9} Although four of the six fiber types in adult EOM exhibit the skeletal muscle prototypical pattern of single innervation and twitch contractions (SIFs), the remainder of the fibers (MIFs) show an unusual innervation pattern with multiple NMJs distributed along the length of the single fibers and a tonic contraction mode. We extended these studies by delineating the morphologic endplate types and the synaptic signaling and structural molecules that are associated with the distinctive EOM fiber types. Our data are the first to show that, despite the unique innervation patterns of the MIF and SIF types, the molecular organization of skeletal muscle NMJs is conserved in all EOM fiber types. In the course of these studies, we also identified an important EOM fiber-type–specific divergence in the properties of the extrasynaptic DGC. 

NMJ Morphology

Acetylcholinesterase histochemistry has been used in conjunction with light and electron microscopy to identify two principal morphologic motor endplates types in EOM fibers ^{35 36} : single and multiple. Moreover, additional junctional adaptations are correlated with the various EOM fiber types. ^{9 37 38 39 40} Our data closely paralleled these prior studies in demonstrating the endplate structural variability that is associated with EOM. AChR visualization and confocal optical sectioning of entire endplates allowed reconstruction of projected surface views that were comparable with prior scanning electron microscopic studies. By this approach, we identified one small en grappe type and three principal en plaque types, differing mainly in size, shape, and branching pattern of the synaptic gutter. Correlation of confocal-generated projected images with ultrastructure enabled us to associate NMJ morphology with identified EOM fiber types. 

The orbital SIFs exhibited highly branched endplates, leading to a distinctive appearance with several synaptic profiles distributed around the perimeter of individual myofibers in transverse sections (Fig. 3B) . This morphology was consistent with the elongated, encircling NMJ profiles seen with confocal microscopy (Fig. 2D) . Our data also confirmed that EOM endplates show a notable absence or paucity of postjunctional folds in all fiber types. Only the global pale SIFs exhibited NMJs with significant postjunctional folding (Fig. 3G) . Postjunctional folds are thought to increase synaptic area and segregate postjunctional structural and signaling proteins. The absence of postjunctional folds at most EOM NMJs, however, alters synaptic organization by eliminating the spatial segregation of molecules normally found in the depths of the folds (e.g., ankyrin_G, dystrophin, DB-2, and syntrophin α1 and β1) from those that usually occupy the fold crests (e.g., AChR; α- and β-dystroglycan; α-, β-, γ-, and δ-sarcoglycan; MuSK; and erbB). 

Despite the paucity of postjunctional folds, EOM en plaque endplates are relatively large in relationship to myofiber diameter, when compared with those of the accessory EOMs and other nonocular skeletal muscles. The long axon terminal–myofiber contact zones may offset the need for augmentation of synaptic area with postjunctional folds. Endplate morphology and branching pattern have been correlated with fiber types of typical skeletal muscle (diaphragm). NMJ structural complexity typically increases from type I to type IIB fibers. ⁴¹ NMJ cytoarchitecture also has been linked to motor unit properties and to selective recruitment patterns that are responsible for various motor behavior. Thus, the fiber type differences in EOM NMJ morphology may relate to the unique properties of the fibers and their innervating motoneurons. 

Agrin- and Neuregulin-Based Signaling Molecules in EOM

Based on the novel physiology, fiber types, gene expression profile, innervation pattern, and endplate morphology of EOM (present data), ^{1 2 3 4} we hypothesized that the synapse signaling molecules that play essential roles at skeletal muscle NMJs, agrin/MuSK and neuregulin/erbB, may not be fully shared by the oculomotor motoneuron/EOM system. Recent findings ⁴² support this notion by showing that NMJ synaptogenesis may have muscle-group–specific adaptations not suggested by current models. In particular, the unusual EOM MIFs may require novel molecular architecture at the synapse. Yet, there have been no studies to establish the presence of the NMJ agrin- and neuregulin-based signal transduction molecules at any mammalian, avian, or amphibian multiply innervated, muscle fiber type. 

Because EOM possesses a unique combination of SIF/twitch and MIF/tonic muscle fiber types and distinct oculomotor motoneuron classes innervate SIFs and MIFs, ⁴³ differences in synaptic signaling molecules were anticipated for these two major muscle fiber classes. By contrast, our data establish that the molecular framework for the principal NMJ signal transduction pathways, agrin/MuSK and neuregulin/erbB, is conserved in EOM. We did not detect differences in signaling pathway components between either EOM SIFs and MIFs or between EOM SIFs and published skeletal muscle data. These findings establish that the fundamental skeletal muscle synaptic organization molecules are essential, even in the highly divergent EOM fiber types. 

Our data have bearing on the nature of the signals necessary to initiate and maintain the neuregulin-dependent signaling complex at the NMJ. EOM MIFs present an interesting dilemma for models of synaptogenesis. In allowing the formation of distributed synaptic sites along the entire length of individual myotubes, MIFs ignore skeletal muscle mechanisms that normally exclude NMJs from other sarcolemmal sites once the initial synapse is formed. Recent evidence in skeletal muscle suggests uninnervated myotubes are not passive in synaptogenesis, but rather that synaptic sites may be preordained by myotube MuSK expression patterns. ^{44 45 46} The formation and maintenance of the NMJ requires both myofiber MuSK expression and the secretion of agrin from motor nerve terminals. Lack of action potential propagation in MIFs may be mechanistic in allowing the formation of multiple synaptic sites. Thus, EOM MIFs represent an excellent model to test NMJ synaptogenesis concepts. During synaptogenesis, both the motoneuron and the myotube exhibit considerable molecular heterogeneity, ^{47 48} that could determine whether a particular myotube is destined as an MIF or SIF, and bidirectional cues at the developing NMJ may favor contacts between appropriate partners. 

Divergence of the EOM DGC

The DGC plays essential roles in the structural and functional organization of the sarcolemma and its NMJ postjunctional specializations. ³⁰ Although we have shown that many DGC components are conserved in EOM, ²⁷ our present study is the first to describe a muscle-group–specific pattern in the extrasynaptic DGC. In most skeletal muscles, syntrophin β1 is distributed along the entire sarcolemma at birth, but rapidly becomes concentrated at the NMJs in all fiber types and at the sarcolemma in type IIB myofibers. ^{32 49} In the current study, we identified adult preservation of the embryonic expression pattern of sarcolemmal syntrophin β1 for both orbital fiber types and the global MIF (notably, these do not expresses IIB myosin ⁵ ). Prior studies suggest that syntrophin may not function in the mechanical role of the DGC, but rather may serve as a scaffold to recruit signaling complexes to the sarcolemma. Because of the relatively low homology among syntrophin isoforms and variations in isoform binding partner preferences, ³² EOM fiber type differences in syntrophin expression probably reflect the recruitment of distinct subsets of signal transduction molecules. Consistent with this view, studies in two muscular dystrophy models, dystrophin-deficient mdx and dystrophin/utrophin–deficient double-knockout mice, ⁵⁰ suggest that the three EOM fiber types identified herein with atypical syntrophin β1 distribution may have substantial plasticity in DGC expression. 

In addition, we showed in the current study that EOM extrasynaptic DB-1 does not fit the established skeletal muscle pattern. Instead of localization to the sarcolemma of all fiber types, as in other skeletal muscles, ^{33 51} DB-1 consistently localized to the sarcolemma of EOM global MIFs only. Dystrobrevin complexes contain dystrophin and syntrophin β1, ³² a finding that may explain the colocalization of syntrophin β1 and the syntrophin-binding protein DB-1, at the MIF sarcolemma. Knockout mouse studies suggest that dystrobrevin is likely to play signaling, rather than structural, roles at the sarcolemma. ²⁰ Together with present data, this suggests that dystrobrevin-mediated signaling mechanisms are tailored to the requirements of the various EOM fiber types. The absence of both dystrophin and utrophin, which displaces all DGC components from the membrane, leads to a maturational arrest of select EOM myofibers and their NMJs at an early myotube stage ⁵⁰ that does not occur in other EOM fiber types and in other skeletal muscles. ^{52 53} These findings suggest that DGC functional roles are, at least in part, different for EOM and may relate to the complete sparing of the EOMs in the DGC-based muscular dystrophies. ^{50 54 55 56 57 58}  

Finally, the DGC is known to play a key role in maturation of the skeletal muscle NMJ. Deletion of any one of several DGC components alters synaptic properties including formation of postjunctional folds. ^{18 19 20 59 60} Because EOM NMJs generally lack postjunctional folds, particularly those three fiber types with sarcolemmal distribution of syntrophin β1, the DGC organizational differences between EOM and other skeletal muscles identified here may contribute toward the distinctive morphology and function of NMJs. 

Developmental Traits in EOM

A prevalent theme in EOM biology is adult retention of traits generally considered to be embryonic or neonatal in most other skeletal muscles (e.g., embryonic and perinatal myosin heavy chain isoforms). ^{5 61 62} The fiber-type–specific sarcolemmal localization of syntrophin β1 shown herein contributes to this pattern. One interpretation of adult muscle retention of developmental traits is the partial developmental arrest of some EOM myofiber types. An alternative explanation is that EOM requires the full range of plasticity available to skeletal muscle to execute its complex and diverse range of tasks. ⁶³ Known NMJ properties further reflect the utilization of ontogenic or phylogenetically primitive traits in adult EOM. 

The embryonic pattern of sarcolemmal syntrophin β1 was noted in EOM orbital layer fiber types that also retain embryonic myosin heavy chain, express both embryonic (α₂β_γδ) and adult (α₂β_δε) AChRs, and retain the embryonic neural cell adhesion molecule (NCAM) distribution pattern at the sarcolemma. ^{24 25 26 64} Global layer MIFs share some of these same traits and, in our data, were the only fiber type exhibiting sarcolemmal DB-1. The global layer MIFs exhibit simple NMJs, with small synaptic boutons, shallow to absent synaptic gutters, absent postjunctional folds, and AChRs that are exclusively of the embryonic type. ²⁵ That these same developmentally primitive traits are found at NMJs of other skeletal muscles in AChR ε-subunit–knockout mice ⁶⁵ supports the operation of either common or coordinated mechanisms in determining NMJ properties. These data further suggest that absence of the requisite signal for the γ-to-ε AChR subunit switch, possibly an as yet uncharacterized neurotrophic factor, ⁶⁶ underlies the novel properties of the MIF NMJ. This signaling difference may not reside solely in the agrin or neuregulin axis, because we have shown in this study that MIFs use both of these pathways. Collectively, our data advance the concept that select EOM fiber types have a very different developmental program than either other EOM fiber types or traditional skeletal muscle types. 

Summary

The present findings established that major components of the two key synapse-signaling mechanisms are conserved in EOM. Although we have shown that the essential elements of both the agrin- and neuregulin-based signaling pathways were present in both EOM MIFs and SIFs, there are important fiber-type–specific specializations in the nonsynaptic sarcolemma. Data also extend the concept that EOM maintains protein distribution patterns that are considered as developmental transients in other skeletal muscles. Many of these probably have their origin in oculomotor motoneuron- and EOM-specific adaptations to the complex and diverse functional roles served by this muscle group. Finally, divergence in the synaptic and nonsynaptic sarcolemmal organization of EOM versus other skeletal muscles: (1) suggests that DGC complexes organized by some EOM fiber types may be functionally distinct from those in other skeletal muscles and (2) may underlie the paradox of the greater sensitivity of EOM to neurotransmission disorders such as myasthenia gravis, but total sparing in the muscular dystrophies. ^{50 54 55 56 57 58}  

 Supported by grants from the Knights-Templar Eye Research Foundation, Chicago, Illinois; and Grants R01 EY09834 and R01 EY12779 (JDP), R01 EY013238 (HJK), and P30 EY11370 from the National Eye Institute, Bethesda, Maryland; the Muscular Dystrophy Association (JDP); and a Departmental Grant and a Senior Scientific Investigator Award (JDP) from Research to Prevent Blindness. JDP is supported by the Carl F. Asseff, MD, Professorship in Ophthalmology.

 Submitted for publication August 30, 2002; revised October 16 and November 11, 2002; accepted November 26, 2002.

 Disclosure: S. Khanna, None; C.R. Richmonds, None; H.J. Kaminski, None; J.D. Porter, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Confocal photomicrographs of rectus EOM NMJ endplates labeled with TR-αBtx. (A) The SIF has one en plaque endplate (arrowhead), whereas the MIF has multiple small en grappe junctions (arrowheads) along its length. En face views of the en plaque (B) and en grappe (C) endplates as seen in confocal imaging of whole muscle mounts. Merged confocal images of 8-μm-thick muscle cross-section showing the endplate zone (D) and distal muscle (E). En grappe junctions were identified in cross-section by small, punctate TR-αBtx staining (arrowheads) on the slow fibers (labeled green with fluorescence–conjugated-slow myosin heavy chain antibody). Scale bar in (C) applies to (B).

Figure 2.

 

Confocal immunofluorescence-projected views of the morphologic types of the junctions imaged en face on whole-muscle mounts of EOM. The rectus muscle en grappe endplate (A) is small without much resolvable substructure. The en plaque endplates on rectus SIFs (B–D) show a range of variability in size and shape and are more intricate in branching pattern in the NMJs of levator palpebrae superioris (E) and retractor bulbi muscles (F, G).

Figure 3.

 

Electron photomicrographs of EOM NMJ types. (A, B) Orbital SIF NMJs encircled individual myofibers, with long terminals (t) embedded in deep depressions of the myofiber surface. Terminals were capped by Schwann cells (S), postjunctional folding was either absent or sparse (arrows), and postjunctional accumulations of myonuclei (mn) and mitochondria (m) were evident. MIF NMJs were small and did not lie in sarcolemmal depressions for both orbital (C, D) and global (E) fiber types. The range of variability in global SIF NMJs included most junctions with few or no folds (F), but occasional junctions with a high density of postjunctional folds (G) were found, primarily on the global pale SIF type.

Figure 4.

 

Confocal immunofluorescence images of cross-sections of EOM endplates double labeled with antibody to synaptic proteins (agrin, rapsyn, and MuSK; green) and TR-αBtx (red). The merged images show colocalization (yellow) of the proteins with AChR at both en plaque and en grappe (arrowheads) endplates. The scale bar in each row applies to all panels in the row.

Figure 5.

 

Expression of neuregulin/erbB pathway proteins at EOM NMJs. Confocal immunofluorescence images of cross-sections of EOM endplates double-labeled with antibody to synaptic protein (neuregulin [ndf] and erbB receptors) and TR-αBtx. The merged images show colocalization of the proteins with AChR at both en plaque and en grappe (arrowheads) endplates. Scales bars: 25 μm.

Figure 6.

 

Localization of DGC proteins at EOM NMJs. Confocal immunofluorescence images of cross-sections of EOM showing en plaque and en grappe endplates double labeled with antibody to DGC protein (utrophin, dystrobrevin 1 and 2) and TR-αBtx. The merged images show colocalization of the DGC proteins with AChRs at both the en plaque and en grappe (arrowheads) endplates. DB-2 was present all along the sarcolemma of all the fiber types. Scale bar at bottom left applies to all panels, unless otherwise indicated.

Figure 7.

 

Localization of DGC proteins at EOM NMJs. Confocal immunofluorescence images of cross-sections of EOM showing en plaque and en grappe endplates double labeled with antibody to DGC proteins (α-syntrophin, β1 and β2 syntrophins, and nNOS) and TR-αBtx. The merged images show colocalization of the DGC proteins with AChRs at both the en plaque and en grappe (arrowheads) endplates. α-Syntrophin and nNOS are present along the entire sarcolemma of all the fiber types, with enrichment at the synapse. Scale bar applies to all.

Figure 8.

 

Extrasynaptic expression of syntrophin β1 and α-dystrobrevin-1 on MIFs. Photomicrographs of adjacent serial sections of EOM showing fibers labeled with antibody to the DGC protein and antibody to slow myosin heavy chain. The fibers that showed ringing of the sarcolemma with these two DGC protein antibodies were the same that labeled positively with slow myosin heavy chain in the adjacent section, thereby confirming the slow nature of these fibers (see Brueckner et al. ⁵ ).

The authors thank Denise Hatala, MaryAnn Pendergast, Midori Hitomi, Mark Harrod, and Anita Merriam for technical assistance and Marvin Adams for helpful discussions.