a novel type of multiterminal motor endplate in human

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Liu, J-X., Pedrosa-Domellöf, F. (2018)A novel type of multiterminal motor endplate in human extraocular musclesInvestigative Ophthalmology and Visual Science, 59(1): 539-548https://doi.org/10.1167/iovs.17-22554

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Eye Movements, Strabismus, Amblyopia and Neuro-Ophthalmology

A Novel Type of Multiterminal Motor Endplate in HumanExtraocular Muscles

Jing-Xia Liu1 and Fatima Pedrosa Domellof1,2

1Department of Integrative Medical Biology, Section for Anatomy, Umea University, Umea, Sweden2Department of Clinical Science, Ophthalmology, Umea University, Umea, Sweden

Correspondence: Fatima PedrosaDomellof, Department of ClinicalScience, Ophthalmology, Umea Uni-versity, 901 85 Umea, Sweden;[email protected].

Submitted: July 5, 2017Accepted: December 19, 2017

Citation: Liu J-X, Pedrosa Domellof F. Anovel type of multiterminal motorendplate in human extraocular mus-cles. Invest Ophthalmol Vis Sci.2018;59:539–548. https://doi.org/10.1167/iovs.17-22554

PURPOSE. To investigate the relation between type of motor endplate, acetylcholine receptor(AChR) subunit composition, and fiber types in human extraocular muscles (EOMs).

METHODS. EOM samples collected from subjects aged 34 to 82 years were serially sectionedand processed for immunohistochemistry, with specific antibodies against different myosinheavy chain (MyHC) isoforms, neurofilament, synaptophysin, and adult epsilon (e) and fetalgamma (c) AChR subunits as well as a-bungarotoxin.

RESULTS. A novel type of motor endplate consisting of large, multiterminal en plaque endingswas found in human EOMs, in addition to the previously well-described single en plaque andmultiple en grappe endplates. Such novel endplates were abundant but exclusively observedin myofibers lacking MyHC slow and fast IIa but containing MyHC extraocular (MyHCeom),isoforms. Multiple en grappe endings were found only in myofibers containing MyHC slow-tonic isoform and contained fetal c AChR subunit. Adult e and fetal c AChR subunits, alone orcombined, were found in the multiterminal endplates. Distinct AChR subunits were presentin adjacent motor endplates of a given myofiber containing MyHCeom.

CONCLUSIONS. Human EOMs have a more complex innervation pattern than previouslydescribed, comprising also a novel type of multiterminal motor endplate present in myofiberscontaining MyHCeom. The heterogeneity in AChR subunit composition in a given myofibersuggests the possible presence of polyneuronal innervation in human EOMs.

Keywords: extraocular muscles, motor endplate, multiterminal motor endplate, en grappe, enplaque, polyneuronal innervation, myosin heavy chain, acetylcholine receptor subunit

Human extraocular muscles (EOMs) have a very complexarchitecture, muscle fiber composition, and innervation

patterns that are thought to reflect their highly specializedfunction in the execution of the different types of eyemovements. The fibers of EOMs are organized into twoseparate layers: a thin orbital layer facing the orbital wall anda global layer comprising the remaining central part of themuscle.1–3 Among the special features of EOMs is the presenceof multiply innervated fibers (MIFs), which are not normallyfound in adult mammalian skeletal muscles, in addition to singlyinnervated fibers (SIFs).4 MIFs display multiple small en grappemotor endplates generally closely located in a row, in contrastto the typical, large, single en plaque endings found on theSIFs.5 En grappe motor endplates exist only in muscle fiberscontaining myosin heavy chain slow-tonic (MyHCsto) isoformand exhibit tonic mode of contraction.6,7 MIFs in the orbitallayer generally have several en grappe motor endplates alongthe fiber length, and a single en plaque motor endplate in themiddle portion.1,8 Approximately 15% to 20% of fibers are MIFswhereas the remaining are SIFs, innervated by a single large enplaque motor endplate in the middle portion of each musclefiber.2,3,7 SIFs typically contain MyHC fast IIa (MyHCIIa) and areregarded as fast myofibers or lack both MyHCIIa and MyHCslow (MyHCI) but contain MyHC extraocular (MyHCeom), anisoform almost exclusive to the EOMs, and are thereforeclassified as MyHCeom fibers.7,9

Another striking feature of the EOMs is that their myofibersshow unusual content of acetylcholine receptor (AChR)subunits. AChRs are highly concentrated in the postsynapticregion of neuromuscular junctions (NMJs), and are pentamericproteins existing in two isoforms in mammalian NMJs: the fetalisoform a2dcb and the adult isoform a2deb.10 In limb muscle,the fetal c subunit is expressed in AChR only duringdevelopmental stages or in denervated skeletal muscles.11,12

In contrast, the EOMs are the only adult muscles that normallyexpress both the fetal (a2dcb) and the adult (a2deb) subunits ofAChR.12–14 RT-PCR14 and immunohistochemical12 data indicatethat en plaque endings of SIFs express the adult e but not thefetal c AChR subunit whereas en grappe endings of MIFs fibersexpress the fetal c but not the adult e AChR subunit in theEOMs of rats and mice, respectively. However, more complexresults have also been reported in another immunohistochem-ical study of the rat EOMs,13 and it remains to be determinedwhether such direct correlation between innervation pattern,AChR subunits, and fiber types is true for the human EOMs.

The present study aimed to systematically investigate thetype of motor endplate of the different fiber types and toexamine the distribution of adult e and fetal c subunits of AChRin human adult EOMS. In addition to the previously describedtypes of motor endplates, we report the wide occurrence of anovel type of multiterminal innervation in MyHCeom fibers anda very complex AChR subunit composition in the human EOMs.

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MATERIALS AND METHODS

Human Muscle Samples

A total of 23 EOM samples (8 superior rectus, 7 medial rectus,4 lateral rectus, 3 inferior obliques, and 1 unspecified EOM)were collected at autopsy from six adult (34–59 years old,males, 14 specimens) and five elderly (72–82 years old, 3 malesand 2 females) subjects. None of the subjects was known tosuffer from neuromuscular disease. In addition, a vastuslateralis muscle sample from a 23-year-old male and the tibialisanterior muscle from a fetus collected at 20 weeks of gestationwere also used to test the specificities of the AChR antibodies.The muscle samples were collected with the approval of theRegional Ethical Review Board in Umea, Sweden, in adherenceto the recommendations of the Declaration of Helsinki.

The muscle samples were mounted on cardboard withoptimal cutting temperature (OCT) Cryomount (HistolabProducts AB, Vastra Frolunda, Sweden), rapidly frozen inpropane chilled with liquid nitrogen, and stored at�808C untilsectioned. Serial sections, 5-lm thickness, comprising thewhole muscle cross section or serial longitudinal sectionscomprising the whole muscle thickness and part of or thewhole muscle length, were obtained at�238C using a ReichertJung cryostat (Leica, Heidelberg, Germany).

Antibodies and Immunofluorescence

Motor endplates were detected either by rhodamine-conjugat-ed a-bungarotoxin (a-BTx) labeling (Molecular Probes, Inc.,Eugene, OR, USA) or by a mixture of monoclonal antibodies(mAbs) against neurofilament protein (clone NR4; 70kD; Dako,Glostrup, Denmark) and against synaptophysin (SY38; Boeh-ringer Mannheim Biochemica, Indianapolis, IN, USA). The useof a mixture of antibodies against neurofilament protein andsynaptophysin allows the simultaneous visualization of theaxon and the motor endplates using a single fluorochrome. Arat mAb against the e AChR subunit15,16 (mAb168; purchasedfrom Socrates J. Tzartos, University of Patras, Rio, Greece) anda mouse mAb against the c AChR subunit16 (GTX74890;Gentex, Landskorna, Sweden) were used to detect the adult eAChR and the fetal c AChR subunits, respectively. In addition,rabbit polyclonal antibody MYH14/7b against MyHCsto iso-form17 (gift from Stefano Schiaffino, CNR Institute of Neuro-science, Padova, Italy), mAbs A4,951 and BA-D5 against MyHCIisoform,18 A4.74 against MyHCIIa isoform,18 and N2.261against MyHCI, MyHCIIa, and MyHCeom18,19 were used todistinguish different types of muscle fibers (DevelopmentalStudies Hybridoma Bank, Department of Biological Sciences,University of Iowa, Iowa City, IA, USA). Because each myofiberand its motor endplates can be reliably studied only on a singlelongitudinal section, given the very small diameter of themyofibers in the EOMs, double or triple immunofluorescentlabeling was carried out: AChR subunits (e or c) þ MyHCisoforms (MyHCI, MyCIIa, or MyHCeom) þ a-BTx; AChRsubunits (e or c) þ neurofilament mixed with synaptophysinþMyHC isoform; adult e AChR subunitþ fetal c AChR subunitþMyHC isoform.

Immunohistochemistry was performed on air-dried tissuesections, as previously described.9 In brief, the tissue sectionswere air-dried, rehydrated in 0.01 M PBS, and then blockedwith 5% donkey serum for 15 minutes. Sections were thenincubated with the first primary antibody (AChR subunit) atþ48C overnight. All antibodies were diluted in 0.01 M PBScontaining 0.1% bovine serum albumin and used at theiroptimal dilutions. The next day, after washing in PBS and anadditional blocking with 5% donkey serum for 15 minutes,sections were incubated for 30 minutes at 378C with

appropriate secondary antibody. Thereafter, the second prima-ry antibody (the other AChR subunit or neurofilament þsynaptophysin) was applied (378C for 60 minutes), followed byincubation with appropriate secondary antibodies for 30minutes at 378C. Subsequently, immunolabeling for the thirdprimary antibody against MyHCs was performed, followed byincubation with the appropriate secondary antibodies for 30minutes at 378C. Detailed information on the secondaryantibodies used to detect each primary antibody is providedin Supplementary Table S1. Control sections were treated asabove, except that the primary antibodies were excluded. Nostaining was observed in control sections.

Microscopy and Motor Endplate Quantification

The sections were examined and photographed with a Spotcamera (RT KE slider; Diagnostic Instruments, Inc., SterlingHeights, MI, USA) connected to a Nikon microscope (Eclipse,E800; Tokyo, Japan). The images were processed using theAdobe Photoshop software (Adobe Systems, Inc., MountainView, CA, USA).

The samples were evaluated with respect to motorendplates, fiber types, and AChR subunits. For quantificationof labeled motor endplates, double-labeled sections combininga-BTx or neurofilament and synaptophysin (NFþSyn) with oneof the antibodies against AChR subunits (a-BTxþc, a-BTxþe,NFþSynþc, NFþSynþe) were evaluated. In addition, triple-labeled sections combining antibodies against fetal c AChRsubunit, adult eAChR subunit, and one of the MyHC isoformswere also evaluated. The total area of each muscle section wasexamined, and every motor endplate identified with a-BTx orNFþSyn was counted and evaluated as either positive ornegative for the antibodies used, carefully taking into accountthe background staining level and excluding lipofuscin dotspresent under both green and red filters. The same criteriawere used for the antibodies against MyHC isoforms and AChRsubunits. In addition, care was taken to properly identifymuscle spindles in cross sections and palisade endings as wellas myomyous junctions in longitudinal sections, to avoid anypossible misinterpretation of the results. A total of 768myofibers, in both cross and longitudinal sections, of all EOMshaving motor endplate(s) were typed and evaluated for AChRsubunit composition.

When necessary, following microscopic evaluation andphotographing, a sequential immunolabeling procedure withan additional MyHC antibody was performed in order to allowtyping of all myofibers. For example, a muscle section that hadbeen immunolabeled with antibodies against adult e AChRsubunit (fluorescein isothiocyanate [FITC], green), against fetalc AChR subunit (Rhodamine Red-X, IgG, red), and againstMyHCIIa (Alexa Fluor 647) was, after evaluation and photo-graphing, additionally incubated with the antibody againstMyHCI (Alexa Fluor 594, IgG2b, red). Thereby we couldexamine the section and photograph it again and classify themyofibers into those containing MyHCI or MyHCII or, in casethey were unlabeled by both antibodies, myofibers containingMyHCeom.

RESULTS

Selective Antibody Specificity Against Fetal c andAdult e AChR Subunit Epitopes

The specificity of the two antibodies against fetal c and adult eAChR subunits in human tissue was tested on a tibialis anteriormuscle from a fetus at 20 weeks of gestation and a sample fromadult vastus lateralis muscle (Fig. 1). MAb GTX74890, raised

A Novel Type of Motor Endplate in Extraocular Muscles IOVS j January 2018 j Vol. 59 j No. 1 j 540


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against AChR c subunit, immunolabeled all the NMJs detectedwith a-BTx in fetal limb muscle (Figs. 1A–C) whereasimmunoreactivity with this antibody was not found in NMJsof human adult vastus muscle (Figs. 1G–I), indicating thatGTX74890 specifically recognizes the fetal c AChR subunit alsoin human muscle tissue. In contrast, antibody mAb 168, raisedagainst e AChR subunit, labeled only a few NMJs of limbmuscles at 20 weeks gestation very weakly (Figs. 1D–F) butstrongly labeled all the NMJs of adult limb muscles (Figs. 1J–L),confirming its specificity to the adult e AChR subunit in humanmuscle tissue.

Muscle Fiber Types in Human EOMs

Three groups of muscle fibers exhibiting different stainingpatterns with the antibodies against MyHC isoforms wereidentified with immunohistochemistry in the human EOMs, aspreviously described7,9: (1) muscle fibers strongly labeled withthe antibody against MyHCsto and in the vast majority, alsostrongly labeled with the antibody against MyHCI and thereforeclassified as muscle fibers containing MyHCsto/I, as previous-ly9; (2) muscle fibers strongly or moderately labeled with theantibody against MyHCIIa and hereinafter referred to as musclefibers containing MyHCIIa; (3) muscle fibers unlabeled with allof the antibodies above but moderately labeled with antibodyN2.26, which recognizes MyHCIþMyHCIIaþMyHCeom,7,19

therefore referred to as muscle fibers containing MyHCeom.9

Three Major Types of Motor Endplates in theHuman EOMs

The well-known single large en plaque and small multiple engrappe motor endplates were readily identified in the musclefibers of the human EOMs. In addition, a novel type of motorendplate was detected, mostly in the myofibers of the global

layer. We list all three major types of motor endplates below,together with a schematic illustration (Fig. 2) and relation toAChR subunits and muscle fiber types (Figs. 3–6; Table).

Single En Plaque Motor Endplates. Single large enplaque motor endplates, the typical endplates of SIFs, werereadily detected with a-BTx or antibodies against neurofila-ment and synaptophysin in all the above-mentioned threegroups of muscle fibers of the human EOMs (myofiberscontaining MyHCsto/I, myofibers containing MyHCIIa, andmyofibers containing MyHCeom), in both orbital and globallayers (Fig. 2A).

The single en plaque motor endplates in muscle fiberscontaining MyHCIIa were always labeled with the mAb againstadult e AChR subunit but were generally unlabeled with themAb against fetal c AChR subunit (Figs. 3A–D). The number ofmyofibers containing MyHCIIa varied widely between speci-mens, but generally, NMJs were less frequently found on thisfiber type. In muscle fibers containing MyHCI and/orMyHCsto/I in the orbital layer, en plaque motor endplateswere in most cases labeled with antibodies against fetal c AChRsubunit (Figs. 3I–L) but motor endplates labeled with both fetalc and adult e AChR subunits were also encountered (Figs. 3E–H; Table). The AChR composition of myofibers containingMyHCsto/I varied between EOMs from the same subject andbetween subjects, but we could not find any clear relationbetween fiber location and AChR subunit composition.

Typical En Grappe Motor Endplates. Typical en grappemotor endplates were readily identified in longitudinal sectionsof the human EOMs (Figs. 2B, 2C, 4). These multiple small engrappe endplates were exclusively seen in myofibers contain-ing MyHCsto/I, in both the orbital and global layers (Fig. 4). Engrappe motor endplates were typically present along a line,only on one side of the longitudinally cut muscle fibers (Figs.2B, 2C, 4A–C). When it was possible to track nerve terminals,it was clear that these multiple small en grappe endings

FIGURE 1. Immunoreactivity of antibodies against AChR subunits in cross sections of human fetal tibialis anterior muscle (A–F) and adult vastuslateralis muscle (G–L). Labeling with the antibody against fetal c AChR subunit (A, C, Alexa Fluor 488, green) was strong on motor endplatesidentified with rhodamine-conjugated a-BTx (B, C, red) in fetal tibialis anterior muscle but absent in adult vastus lateralis muscle (G, I). Labelingwith antibody against adult e AChR subunit (D, F, FITC, green) was absent on motor endplates identified with rhodamine-conjugated a-BTx (E, F,red) in fetal tibialis anterior muscle but strong (J, L, FITC, green) on motor endplates identified with rhodamine-conjugated a-BTx (K, L, red) in adultvastus lateralis muscle. The right column (C, F, I, L) shows the merged images for each antibody and a-BTx. The arrows denote motor endplateslabeled with a-BTx but unlabeled with the antibodies used.



originated from one common axon (Fig. 2C). Thus, weconfirmed that these were MIFs.

The multiple en grappe motor endplates were labeled withmAb against fetal c AChR subunit (Figs. 4A, 4C, 4F, 4G) butwere not labeled with mAb against adult e AChR subunit (Figs.4E, 4G).

In the most distal part of the EOMs, palisade endings werefound in the global layer in the vicinity of myotendinousjunctions and only in muscle fibers containing MyHCsto. Theaxons supplying the palisade endings ran closely along eitherside of the myofibers or intertwined with the myofibers. In asubset of MyHCsto fibers receiving palisade endings, at leasttwo or more small motor endplates labeled with the antibodyagainst c AChR subunit were found (not shown).

Novel Multiterminal En Plaque Motor Endplates. Inlongitudinal sections, a novel type of motor endplatesconsisting of several large en plaque endplates was observedin the myofibers containing MyHCeom isoform in both globaland orbital layers, but mostly in the global layer, irrespective ofthe age of the subject or EOM examined (Figs. 2D, 5). Theselarge multiterminal en plaque motor endplates were readilyfound along myofiber segments that were up to ~520 lm long

(Figs. 2D, 5B, 5L, 6B). Approximately 77 6 11% of theendplates were rather shallow, in contrast to the deepjunctional folds seen in typical single en plaque endplates.The length of each motor endplate was approximately 15 to 40lm and the distance between adjacent endplates variedbetween 25 and 240 lm. These multiterminal en plaquemotor endplates were clearly distinct from the multiple engrappe endplates typically seen on muscle fibers containingMyHCsto/I. In contrast to the multiple en grappe endingswhich were mostly aligned on the same side of the musclefiber, some of these multiterminal large en plaque motorendplates were present on opposite sides of the longitudinallycut muscle fibers (Figs. 2D, 5B). In addition, the diameter ofMyHCeom fibers possessing the multiterminal en plaqueendings was always larger than that of MyHCsto/I onespossessing the multiple en grappe endings, in agreement withour previous findings that the size of MyHCeom fibers waslargest whereas that of MyHCsto/I fibers was smallest (590 6

210 vs. 320 6 190 lm2, respectively).7

In the present study, approximately 63 6 10% of themyofibers containing MyHCeom and displaying motor end-plates on the sections analyzed had multiterminal en plaque

FIGURE 2. Light microscopy images and schematic illustrations of the three major types of motor endplates in the human EOMs. (A) Typical singleen plaque motor endplate. Immunostaining with rhodamine-conjugated a-BTx (red) revealed a large en plaque motor endplate (long arrow) in themidportion of a longitudinally cut myofiber. (B, C) Typical multiple small en grappe motor endplates. Immunostaining with antibodies againstneurofilament and synaptophysin (NFþSyn; Rhodamine Red-X, red) revealed multiple small motor endplates (short arrows) lying on one side of themyofiber, in longitudinal sections. Note the two motor endplates (short arrows in [C]) arising from the same nerve axon (NA). (D) Novelmultiterminal large en plaque motor endplates. Six large en plaque endplates (arrowheads) labeled with rhodamine-conjugated a-BTx (red) werelocalized on both sides of a long segment of a longitudinally cut myofiber. The background staining level has been intensified to clearly show thecontinuity of the underlying myofiber. In all micrographs, red lipofuscin granules are also seen, but under the microscope these can easily bedistinguished from the specific immunolabeling.



FIGURE 3. AChR subunit composition in single en plaque motor endplate of myofibers containing MyHCIIa (A–D) and MyHCI (E–L) in cross-sectioned (A–H) or longitudinally sectioned (I–L) samples. Adult e AChR subunit was present (A, C, FITC, green) whereas fetal c AChR subunit wasabsent (B, C, Rhodamine Red-X, red) in myofibers containing MyHCIIa (D, Alexa Fluor 647, gray). Copresence of adult e (E, G, arrow; FITC, green)and fetal c (F, G, arrow; Rhodamine Red-X, red) AChR subunits was found in motor endplates of myofibers containing MyHCsto/I (H, Alexa Fluor594, red). Presence of fetal c AChR subunit (J, arrow; Rhodamine Red-X, red) but absence of adult e AChR subunit (I, arrow; FITC, green) was alsofound on motor endplates of MyHCsto/I myofiber (L, Alexa Fluor 647, gray).

FIGURE 4. AChR subunit composition in multiple en grappe motor endplates of longitudinally cut myofibers containing MyHCsto. Fetal c AChRsubunit (A, C, F, G, arrows; Alexa Fluor 488, green) was present on all en grappe motor endplates labeled with neurofilament and synaptophysin(NFþSyn; B, C, Rhodamine Red-X, red) in myofibers containing MyHCsto (D, H, Alexa Fluor 594, red). In contrast, the adult e AChR subunit (E, G,FITC, green) was absent.



FIGURE 5. AChR subunit composition in multiple large en plaque motor endplate of myofibers containing MyHCeom in longitudinally (A–C, K–M) ortransversely (D–J) cut EOMs. Note the three combinations of AChR subunit composition of motor endplates. (A–C) Adult e AChR subunit (A, arrows;FITC, green) was absent but fetal c AChR (B, arrows; Rhodamine Red-X, red) was present on the multiterminal en plaque endings. (D–J) Copresenceof adult e (D, E, arrowheads; FITC, green; a-BTx, red) and fetal c AChR subunits (F, G, arrowheads; Alexa Fluor 488, green; a-BTx, red) on motorendings of myofibers lacking MyHCI (H, Alexa Fluor 488, green) and MyHCIIa (I, Alexa Fluor 488, green) but labeled with the antibody againstMyHCIþIIaþeom (J, Alexa Fluor 488, green), that is, a myofiber containing MyHCeom. (K–M) Two adjacent en plaque motor endplates in a givenmyofiber showed distinct AChR subunits. The left endplate contained solely adult e AChR subunit (K, M, FITC, green; marked with single asterisk)whereas the endplate to the right contained solely fetal c AChR subunit (L, M, Rhodamine Red-X, red; marked with two asterisks).



motor endings, whereas the remaining MyHCeom fibers hadsingle en plaque endings. It should be noted that thispercentage was evaluated on longitudinally sectioned speci-mens where longer and shorter myofiber segments werepresent, but an analysis of the full length of each myofiber isnever possible. In other words, we could not determinewhether those MyHCeom myofibers displaying a singleendplate in the segment analyzed had additional motorendplates along the rest of their length, and therefore thepercentage above may be underestimated. Furthermore, thepercentage of MyHCeom myofibers varied within differentportions of EOMs and between EOMs from different subjects,but we could not detect any clear-cut differences betweendifferent EOMs in terms of incidence and distribution of thisnovel multiterminal en plaque motor endplate.

Three different patterns regarding AChR subunit composi-tion were noted for the multiterminal large en plaque motor

endplates (Fig. 5; Table): (1) fetal c AChR subunit alone (Figs.5A–C); (2) coexpression of adult e and fetal c AChR subunits(Figs. 5D–G); and (3) less frequently, adult e AChR subunitalone (Figs. 5K–M). In a number of myofibers containingMyHCeom, the AChR subunit composition varied from oneendplate to another, along the length of a single myofiber; thatis, one en plaque endplate contained fetal c AChR subunitsolely whereas another contained adult e AChR subunit only(Figs. 5K–M) or contained both adult e and fetal c AChRsubunits.

In addition, sporadic MyHCeom myofibers displaying acombination of multiterminal large en plaque and multiplesmall en grappe motor endplates were also observed,displaying at least two small en grappe endplates along thefiber segments examined (Fig. 6). The multiterminal en plaqueendplates were labeled with mAb against fetal c AChR subunit(Figs. 6A–C) and in some cases there was coexistence of bothadult e and fetal c AChR subunits in the same endplate. Thesmall en grappe endplates were generally labeled with mAbagainst fetal c AChR subunit (Figs. 6B, 6E) and either labeled(Figs. 6A, 6D) or unlabeled with mAb against adult e AChRsubunit.

Finally, sporadic myofibers containing MyHCsto/I or MyH-Ceom displayed several small ‘‘grape-like’’ endplates on eitherside of the muscle fiber (Fig. 7), as previously described in theanterior part of the inferior oblique muscle of childrenundergoing strabismus surgery and two young adults.20 Theseclustered small grape-like endplates were labeled with the mAbagainst adult e AChR subunit (Fig. 7), and we could notdetermine whether they contained fetal c AChR subunit, asonly very sporadic myofibers with this type of motor endingswere found.

DISCUSSION

The present study reports a novel major type of motorendplate in human EOMs, in addition to the previously

FIGURE 6. Combination of multiterminal en plaque motor endplates (arrowheads) and multiple en grappe motor endplates (arrows, shown athigher magnification in the right column) in sporadic MyHCeom myofibers. Note the absence of adult e AChR subunit (A, C) but the presence offetal c AChR subunit (B, C, Rhodamine Red-X, red) on multiterminal en plaque motor endplates (arrowheads) and the copresence of both adult e(A, C, D, F, arrows; FITC, green) and fetal c (B, C, E, F, arrows; Rhodamine Red-X, red) AChR subunits in the two en grappe motor endplates.

TABLE. Relation Between Myofiber Type, AChR Subunit Composition,and Motor Endplate Types in Human EOMs

Fiber Type SIF/MIF AChR Subunit

Type of Motor

Endplate

MyHCIIa SIF e þ, c � Large en plaque

MyHCsto/I * e �, c þ Large en plaque

e þ, c þMyHCsto/I MIF e �, c þ Multiple small en

grappe

MyHCeom MIF e �, c þ Multiterminal large

en plaquee þ, c þe þ, c �

Distinct subunits in

adjacent endplates

* We were unable to determine whether this type of motor endingwas present in the myofibers containing MyHCsto/I and having engrappe endings (MIFs) or whether these could be true SIFs.



described en plaque and en grappe endings. Furthermore, thisis, to the best of our knowledge, the first study clearlycorrelating fiber types, motor endings, and AChR subunitcomposition in human EOMs (Table). The major findings canbe summarized as follows. (1) A novel type of motor endplateconsisting of multiterminal en plaque endings was found alongthe length of myofibers containing MyHCeom, indicating thatthis is a new category of multiply or polyneuronally innervatedmyofibers; (2) adult e and fetal c AChR subunits were presentsimultaneously in a subgroup of en plaque endings ofmyofibers containing either MyHCeom or MyHCsto/I; (3)distinct AChR subunits were present in neighboring en plaquemotor endplates of a given myofiber containing MyHCeom; (4)we confirm that typical en grappe motor endings are presentonly in myofibers containing MyHCsto/I and contain the fetal cAChR subunit.

Here we report a novel type of multiterminal motorendplate that differed from traditional multiple en grappeendplates in several aspects. First, this novel type ofmultiterminal motor endplate consisted of several large enplaque endings, with longer length and greater distancebetween two adjacent endplates than the conventional smallmultiple en grappe motor endplates. Second, they wereexclusively observed in myofibers containing MyHCeomwhereas conventional en grappe endplates were found onlyin myofibers containing MyHCsto/I. Third, unlike en grappemotor endplates, which were always lined up in a row on thesame side of the longitudinally cut myofibers, several en plaquemotor endplates appeared on both sides of a given myofiber.Finally, care was taken to ensure that these novel en plaqueendplates were not incorrectly identified palisade endings ormuscle spindles and that no myomyous junctions were presentin these muscle fiber segments. The present findings clearlyindicate a new group of myofibers with multiterminalinnervation and containing MyHCeom isoform in humanEOMs. Although the material of the present study did notallow a clear evaluation of whether these multiterminal enplaque motor endplates are supplied by single or multipleaxons, and although there is uncertainty regarding the totalpercentage of myofibers with such multiterminal endings, it is

clear that a higher percentage of myofibers in human EOMsthan previously recognized do not have single en plaque motorendings. The myofibers containing MyHCeom have previouslybeen reported to account for approximately 25% of the fibersin the global layer,7 and in the midbelly of the EOMs they arethe absolutely most abundant fiber type. In the present study,we estimated that approximately 63% of these myofibers hadmultiterminal en plaque motor endplates. In addition, approx-imately 14% to 16% of the fibers in human EOMs havepreviously been recognized as MIFs,7 meaning that, altogether,at least one-third of the myofibers in human EOMs are multiplyor polyneuronally innervated.

Kjellevold and Bruenech21 reported the presence ofmultiple en plaque motor endplates in serial transversesections of human EOMs and concluded that these wererelated to aging.21 In addition, they did not classify the fibertype possessing these multiple en plaque motor endplates. Inthe present study investigating a very large number oflongitudinally cut myofibers, we found multiterminal en plaquemotor endplates in the EOMs from both younger and elderlysubjects, indicating that aging is not a confounder for thepresence of multiterminal en plaque motor endplates butrather that this is a true major type of endplate in myofiberscontaining MyHCeom. Oda20 reported two types of multiplemotor endplates, Type B and Type C, in the most anteriorportion of the inferior oblique muscles from childrenundergoing strabismus surgery (4–11 years old) and two youngadults (17 and 19 years old).20 Type C is identical to the well-known multiple small en grappe motor endplates, whereasType B is formed by endplates consisting of several smallparticles stained by AChE and regularly spaced on musclefibers.20 The latter were called ‘‘grape-like’’ endplates and werepresent in approximately 5% of the myofibers and only inmyofibers of intermediate diameter. Altogether, the morpho-logic appearance of such multiple grape-like endplates andtheir myofibers was completely different from the novelmultiterminal en plaque endplates in the large MyHCeomfibers of the present study. Furthermore, grape-like motorendplates were sporadically found in the present study, in bothMyHCsto/I and MyHCeom fibers. The fact that we did notobserve such motor endings as often as reported by Oda20 mayreflect differences between EOMs or may be related tostrabismus, given that the study by Oda was performed inthe most anterior portion of the inferior oblique muscles ofpatients undergoing strabismus surgery.

In the present study, single en plaque endplates inmyofibers containing MyHCIIa generally contained adult eAChR subunit whereas multiple en grappe endplates inmyofibers containing MyHCsto/I contained only fetal c AChRsubunit, as reported in other species.12,14 In contrast, single enplaque endplates seen in myofibers containing MyHCsto/I andmultiterminal en plaque endplates in myofibers containingMyHCeom showed a complex AChR subunit composition,including fetal c or adult e AChR subunit alone, and thecoexpression of both subunits. The current results are partiallydivergent from previous findings on adult EOMs of rats andmice, where the en plaque endplates always have adult e AChRsubunit only.12,14 Furthermore, the coexpression of adult e andfetal c AChR subunits in small en grappe endplates in ratEOMs13 was never observed in human EOMs in the currentstudy, suggesting that differences between species or antibod-ies used may exist.

The distinct AChR subunit composition is likely related tothe fiber type, its physiological properties, and motor axonsupply. AChRs containing fetal c subunit differ from thosecontaining adult e subunit in their ion conductance and ionchannel open speed.22 The opening time of ion channel isreduced from 11 to 6 ms during the switch from fetal c to adult

FIGURE 7. Several small grape-like endplates (arrows) identified withneurofilament and synaptophysin (NFþSyn, Rhodamine Red-X, red) ona longitudinally cut MyHCeom myofiber showing strong labeling withmAb against adult e AChR subunit (FITC, green).



e AChR subunit during limb muscle development.22 Thus, thepresent findings that single en plaque endplates of myofiberscontaining MyHCIIa exclusively contained adult e AChRsubunits whereas multiple en grappe endplates of myofiberscontaining MyHCsto/I exclusively expressed fetal c AChRsubunits fit the difference in contraction speed between thesetwo fiber types. Abnormalities in NMJ size, number, and AChRsubunit composition have been previously reported in theEOMs of subjects with infantile nystagmus syndrome.23 Itwould be important to study the muscles of such patients inlongitudinally cut sections and taking into consideration thenew knowledge of an additional type of motor endings and thecomplex AChR subunit composition of the human EOMs.

Unfortunately, with the freshly frozen muscle samplesavailable, we did not succeed in determining whether themultiterminal en plaque endings were supplied by a single orby several axons, that is, whether these myofibers weremultiply or polyneuronally innervated. In order to assesswhether the multiterminal en plaque motor endings arepolyneurally or multiply innervated, further studies using, forexample, fixed samples and/or silver impregnation of wholeEOMs are needed. Such an approach was not possible with thematerial available, given the way it had been collected andprocessed. However, the finding of neighboring en plaqueendings with distinct AChR subunit composition on a givenmyofiber favors the possibility of polyneuronal innervation.The presence of polyneuronal innervation has been recognizedelectrophysiologically in the EOMs of mammals, includingcat,24–28 monkey,26,29 and rat.30 Electrophysiological dataelegantly show that the sum of twitch and tetanic tensions inresponse to individual nerve branch stimulation is greater thanthat when the whole EOM nerve is simultaneously stimulated,indicating the presence of polyneuronal innervation.27,28 Inaddition, by using silver teasing and/or acetylcholinesterasestaining method, Dietert5 and Oda20 demonstrate multitermi-nal endplates that are innervated by several different motoraxons, indicating the presence of polyneuronal innervationalso in human EOMs.

The multiterminal and possible polyneuronal innervation ofEOM myofibers could play a role in protecting the EOMs andmaintaining normal eye motility in neuromuscular diseaseinvolving loss of motor neurons, for example, amyotrophiclateral sclerosis (ALS). We have previously shown that EOMsare remarkably well preserved in comparison to the severelyaffected limb muscles from the same ALS patients, althoughsome morphologic alterations were observed.31–34 However,we very recently reported significant loss of myofiberscontaining MyHCsto/I in both layers of the medial rectus interminal ALS patients, suggesting that the EOMs as a whole arerelatively spared but that the MyHCsto/I fibers, which aremultiply innervated from endings arising from the same motoraxon, are vulnerable.35

In summary, human EOMs have a more complex innerva-tion pattern with respect to motor endplate number and AChRcomposition than previously recognized. Further studies areneeded to determine the extent of multiple and polyneuronalinnervation and its relation to fiber types, motor endplatemorphology, and AChR subunit composition.

Acknowledgments

The authors thank Anna-Karin Olofsson for excellent technicalassistance, Mona Lindstrom for valuable advice, Stefano Schiaffinofor the gift of antibody MYH14/7b, and Gustav Andersson for theschematic illustration.

Supported by grants from the Swedish Research Council (2015-02438; Stockholm, Sweden), County Council of Vasterbotten(Umea, Sweden), Ogonfonden (Umea, Sweden), Kronprinsessan

Margaretas Arbetsnamnd for Synskadade (Valdemarsvik, Sweden),and the Medical Faculty, Umea University (Umea, Sweden).

Disclosure: J.-X. Liu, None; F. Pedrosa Domellof, None

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a novel type of multiterminal motor endplate in human

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