Proc. Nati. Acad. Scd. USAVol. 74, No. 7, pp. 3073-77, July 1977Neurobiology

Reinnervation of original synaptic sites on muscle fiber basementmembrane after disruption of the muscle cells

(neuromuscular junction/topographic specificity/regeneration/denervation/synaptogenesis)

LAWRENCE M. MARSHALL, JOSHUA R. SANES, AND U. J. MCMAHANDepartment of Neurobiology, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115

Communicated by Stephen W. Kuffler, April 18, 1977

ABSTRACT Regenerating axons form new synapses pre-cisely at sites of original syapses in denervated skeletal muscle.To determine what role the muscle cell plays in this phenome-non, we studied reinnervation of frog muscle at intervals aftercrushing the nerve and damaging the muscle fibers. Damagedmuscle fibers degenerate and are phagocytized, but theirbasement membrane persists and acts as a scaffold for regen-erating muscle cells. Specializations of the basement membraneserve to mark original synaptic sites after nerve and muscle havedegenerated. Regenerating axons enter the region of damageand form functional synapses with regenerating myofibers. Thenew nerve terminals are found almost exclusively at the originalsynaptic sites, demonstrating that the integrity of the originalpostsynaptic cell is not necessary for topographically precisereinnervation of denervated muscle.

Connectivity in the nervous system is ordered and specific:neurons form synapses with only certain classes of potentialtargets (cellular specificity) and, in some cases, contact onlycertain portions of their target cell's surface (topographicspecificity). A particularly striking example of topographicspecificity, and one that may be accessible to detailed study,is observed in mature skeletal muscles. After a motor nerve iscut or crushed, the damaged axons regenerate and establish newsynapses on the denervated muscle fibers. The regeneratingaxons usually return precisely to the sites of the original neu-romuscular junctions (nmjs) (e.g., refs. 1-5), even though thesesites may cover only 0.1I% of each muscle fiber's surface.The experiment presented here was undertaken to determine

if the muscle cell governs this topographic specificity. A sheathof basement membrane covers the surface of each skeletalmuscle fiber and extends through the synaptic cleft at the nmj.If the muscle is damaged, the myofibers degenerate and arephagocytized, but the sheaths of basement membrane remainintact and eventually new myofibers regenerate within them(6-8). We deliberately damaged muscles at the same time thatwe denervated them, causing the postsynaptic (i.e., myofibers)as well as the presynaptic (i.e., nerve terminals) components ofthe nmj to degenerate. Within 2 weeks, myofibers regeneratedwithin the sheaths of basement membrane, nerves grew backto their surface, and functional nmjs were established. Thesheaths had features that enabled us to recognize where theoriginal nmjs had been and thus to determine if the new nmjsformed preferentially at old sites. We found regenerated nerveterminals situated almost exclusively at original sites andtherefore conclude that the topographic specificity of synapseformation at the denervated nmj does not require the integrityof the original postsynaptic cell for its expression. Instead, theregenerating axons must use cues that are produced and/ormaintained outside of the muscle cell.

METHODSExperiments were performed on the cutaneous pectoris musclesof 5-cm-long male frogs (Rana pipiens). Preparations werefixed, stained, and embedded whole in thin wafers of plastic,so that the same structures could be studied by light and electronmicroscopy (5, 9). To enhance the contrast of basement mem-branes for electron microscopy, we stained muscles with ru-thenium red (10).

RESULTSDegeneration and regeneration of nerve and muscleThe paired cutaneous pectoris muscles lie just beneath the skinof the thorax. A nerve enters each thin muscle from the lateraledge and courses across it (Fig. la); one or two blood vesselsenter and run with the nerve. Axons leave the nerve to formsingle nmjs on each of the approximately 500 fibers in themuscle. Most of the nmjs are near the nerve trunk and its largebranches, in a band across the central region of the muscle (seerefs. 5 and 9 for a detailed description).To damage muscle fibers while preserving the integrity of

the main nerve trunk and blood vessels and as many synapticsites as possible, we performed the following operation. The skinof an anesthetized (5) frog was cut open to expose both cuta-neous pectoris muscles. A rectangular slab was cut from themuscles on each side of the nerve trunk, leaving behind a bridge(3-4 mm long; 1-1.5 mm wide) of muscle fiber segments ex-tending between strips of undamaged fibers at the muscle'smedial and lateral edges (Fig. la). The nerve was then crushedwith fine forceps 2-3 mm from the muscle's lateral border.Finally, the wound was closed with sutures.The damaged myofibers in the bridge degenerated and were

phagocytized, leaving behind their sheaths of basementmembrane. By 4 days after the operation, phagocytosis wasextensive and the collapsed profiles of the basement membranesheath (Fig. lb) enclosed numerous small cells, probablymacrophages and myoblasts (8). Although scattered fragmentsof plasma membrane remained attached to the inner surfaceof the basement membrane (Fig. ic) and myofibrillar debriswas occasionally still present within the sheaths, undisruptedextrafusal muscle fibers were never found in the region ofdamage. Nerve terminals also degenerated within 4 days of theoperation and were phagocytized by Schwann cells, as occursin denervated but undamaged muscle (5, 11, 12).By 2 weeks postoperatively, myofibers had formed within

many of the basement membrane sheaths and nerves hadgrown back to the muscle. Large portions of each bridge, as wellas the undamaged medial fibers (see Fig. la), twitched whenthe nerve was stimulated, indicating that functional nmjs had

3073

Abbreviations: nmj, neuromuscular junction; ChE, cholinesterase.

3074 Neurobiology: Marshall et al.

FIG. 1. (a) Paired cutaneous pectoris muscles. Muscle and nerve(arrow) on left are intact. Muscle on right has been damaged as de-scribed in text, leaving behind a bridge of muscle fiber segments(arrowhead). (b) At 4 days after muscle was damaged and denervated,muscle cell has been extensively phagocytized, but its sheath ofbasement membrane survives. The sheath stains darkly with ru-thenium red and surrounds small cells and debris. Region at largearrow (lower left) is magnified in c. (c) Debris (arrows) adheres to theinside of the basement membrane. Cross-cut collagen fibers lie outsidethe basement membrane. (Bar in b indicates 8mm for a, 5 ,um for b,and 0.5 ,m for c.)

been established across the muscle. Electron microscopy andintracellular recording showed that the new synapses were

structurally and physiologically similar to [although oftenstructurally less mature than (see ref. 5)] normal nmjs (Fig.2).Synaptic sites on basement membrane sheaths afterdegeneration of nerve and muscleEven after both the muscle and nerve were extensivelyphagocytized, the sites of the original nmjs could be identifiedin two ways.

Cholinesterase. In normal muscles stained for cholinesterase(ChE), reaction product fills the synaptic cleft of the nmj (Fig.Sa). After degeneration of the nerve and muscle, discretepatches of basement membrane stained for ChE (Fig. Sb); thisobservation is consistent with the suggestion (13) that synapticChE is contained in or connected to the basement membrane.The patches usually lay beneath processes of Schwann cells,which are known to remain at denervated end plates after theyphagocytize the nerve terminals (5, 11, 12).Basement Membrane of Junctional Folds. In normal

muscle, periodically arranged junctional folds indent thepostsynaptic membrane, and projections of basement mem-

brane extend into the folds (Fig. 4a). Projections of basementmembrane with similar dimensions and periodicity were seenbeneath Schwann cells after nerve and muscle had degenerated(Fig. 4b). Although abnormal in appearance, these projections

FIG. 2. Neuromuscular junctions from normal muscle (a) andfrom reinnervated, regenerating muscle that had been damaged anddenervated 14 days previously (b). (Bar is 1 Mm.) (Inset in b) End-plate potential recorded intracellularly from a fiber in this muscle;muscle was curarized (3 X 10-6 g/ml) to prevent twitching duringrecording. Amplitude of end-plate potential is 1.0 mV and trace spans75 msec.

were easily distinguishable from the "wrinkles" seen elsewherein the basement membrane (Fig. lb), and they thus served tomark old synaptic sites by a method independent of ChEstaining.

Regenerating axons return to original sites onbasement membraneWe used both ChE stain and the basement membrane ofjunctional folds as markers to learn whether the original syn-aptic sites were reinnervated when axons returned to thebasement membrane sheaths and regenerating myofibers ofthe bridge.ChE as a Marker. Two weeks postoperatively, when re-

generation of nerve and muscle was underway, nerve terminalswere almost exclusively adjacent to ChE-stained patches (Fig.Sc). We found terminals by systematically following the pe-rimeters of cross-sectioned basement membrane sheaths in theelectron microscope at a magnification of X10,000; our surveyincluded profiles with ChE-stained patches and profiles thatdid not have stained patches. Overall, we counted 523 ChE-stained patches and 229 nerve terminals in sections from 12bridges. About 40% (226/523) of the patches were approachedby nerve terminals, and 99% (226/229) of the terminals werewithin 1 Asm of a patch.

Several observations suggested that these ChE-stainedpatches were components of old synaptic sites and had not beensynthesized de novo during regeneration. In undamagedmuscle (Fig. 5a), the terminal arborizations outlined by ChEare formed by the branches of a single axon that runs along the

Proc. Natl. Acad. Sci. USA 74 (1977)

Proc. Natl. Acad. Sci. USA 74 (1977) 3075

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FIG. 3. ChE-stained synaptic sites in normal muscle (a) and inmuscles damaged and denervated 7 (b) or 14 (c) days previously. Stainfills synaptic cleft at normal nmj (a). After degeneration of nerve andmuscle (b), ChE remains on basement membrane beneath Schwanncell process (S). When nerve and muscle regenerate (c), new terminalsare apposed to stained site. (Bar in c is 1 ,m for a, b, and c.)

muscle fiber's surface, roughly in parallel to the fiber's long axis(5, 9). Similar bands of ChE stain were present on the bridgesthat we used to study reinnervation (Fig. 5b). Although fainterand somewhat wrinkled, these bands were generally similar innumber, size, and arrangement to those on normal muscle(compare Fig. 5 a and b) and far more complex than thoseformed when nerves are forced to form completely new

synapses on previously uninnervated regions of muscle (K.Buckley and U. J. McMahan, unpublished data). Moreover,similar patterns of ChE staining were seen on bridges that werekept denervated for 23 days by recrushing the nerve at frequentintervals (Fig. 5c); thus, old sites were doubtless present on the14-day-old bridges that we used to study reinnervation. Finally,some of the ChE-stained bands in reinnervated bridges werecovered by a nerve terminal along only part of their length (Fig.6), suggesting that a regenerating axon had grown back to butonly partially filled a preexisting site.To determine whether all of the ChE-stained bands repre-

sented old synaptic sites or whether any had been formed

d~~~~ ,i4s-vtAP -FIG. 4. (a) Junctional folds indent the postsynaptic membrane

of normal muscle, and basement membrane extends into the folds.(b and c) Projections of basement membrane persist in muscle dam-aged and denervated 5 (b) or 14 (c) days previously. New terminalslie above projections after nerve and muscle regenerate (c). (d)Drawing from c to emphasize basement membrane. In all parts, ar-rowheads point to intersections between basement membrane ofjunctional folds and that of the synaptic cleft. M, muscle; N, nerveterminal; S, Schwann cell processes. (Bar in d indicates 1 Am for a andb, 0.5 um for c and d.)

during the first 2 weeks of regeneration (e.g., by the new nerveterminals), we treated muscles in situ with an irreversible in-hibitor of ChE at the time that we damaged and denervatedthem, to eliminate the activity at old sites (c.f. ref. 14). Twoweeks later, we stained the bridges for ChE, reasoning that anybands of reaction product that appeared would represent "new"ChE, synthesized subsequent to operation. The inhibitor, di-isopropylfluorophosphate dissolved in saline to a concentrationof 10 mM immediately before use, was applied topically byplacing a piece of gauze soaked in it directly on the bridgefor 30 min before rinsing the muscle with fresh saline andclosing the wound. Electron microscopy revealed that bothnerve and muscle degenerated and regenerated in DFP-treatedbridges as they did in unpoisoned preparations, and nmjs weremicroscopically and electrophysiologically demonstrable 14days postoperatively. In a light microscopic survey of severalwhole bridges fixed 14 days after operation and poisoning, wecould find no ChE-stained bands that resembled those onsimilar but unpoisoned preparations (compare Fig. 5 b and d).Thus, most if not all of the ChE-stained bands in reinnervatedbridges mark old synaptic sites.The possibility remained that some terminals were apposed

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3076 Neurobiology: Marshall et al.

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FIG. 5. ChE-stained muscles: light microscopy of whole mounts. (a) Normal muscle. (b) Reinnervated bridge, 14 days after denervation andmuscle damage. (c) Bridge kept denervated for 23 days by recrushing the nerve at 4- or 5-day intervals. (d) Bridge, 14 days after denervation,damage, and application of diisopropylfluorophosphate. Reaction product outlines synaptic sites near nerve branch (N) in a, b, and c. Synapticsites are also present in d but, because ChE has been extensively inhibited, reaction product is very sparse and visible only at high power withoil-immersion optics (not shown). Muscles in a, b, and c were incubated in ChE reaction mixture (5,9) for 10 min, d for 30 min. (Bar in d is 100gm for a, b, c, and d.)

to newly synthesized patches of ChE that were too faint to berevealed by low-power light microscopy. We excluded thispossibility by correlating light and electron microscopic ob-servations on unpoisoned, reinnervated bridges. The positionof over 95% of the innervated, ChE-stained patches seen in theelectron microscope could easily be recognized in the light

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FIG. 6. (a) ChE-stained band on regenerating muscle fiber seg-ment in bridge, 14 days after denervation and damage; light micro-graph. A cellular process covers only part of the band; arrow markstip of process. Electron micrographs of cross sections through thisband at regions marked by arrowheads show that a nerve terminal isapposed to part of the band (b), while an adjacent region is uncovered(c). (Bar in a is 20 Am; bar in c is 1 ,um for b and c.)

microscope, and the patches were seen to be portions of bandsthat extended longitudinally on muscle fibers.Basement Membrane of Junctional Folds as a Marker. In

reinnervated bridges 2 weeks postoperatively, basementmembrane projections of original junctional folds sometimeslay beneath the new nerve terminals (Fig. 4c). Because theseprojections were not visible in every section, we examined shortruns of serial sections through 15 terminals. In 14 of the 15 se-ries, clearly recognizable projections of basement membranewere situated beneath the terminal in at least one section, in-dicating that these terminals had returned to old synapticsites.

DISCUSSIONAfter the nerve to a frog's muscle is crushed, the damaged axonsregenerate to cover nearly all of the original postsynapticmembrane, and they make few if any synaptic contacts else-where on the muscle fiber's surface (5). Original synaptic sitesare also reinnervated in denervated mammalian and avianmuscle (1-4), although new sites are clearly formed in somecases (e.g., refs. 15 and 16). The experiment described here usestwo independent histological criteria to demonstrate that nerveterminals can return to original synaptic sites on the basementmembrane sheath of muscle fibers after the muscle cellsthemselves have been disrupted and phagocytized. Muscle cellswell may play a role in the transformation of growing motoraxons into nerve terminals; our study does not deal with the issueof neuronal differentiation. However, it is clear from our resultsthat neither the integrity of the original muscle fiber nor anyappreciable portion of its contents is necessary for the expressionof topographic specificity in denervated skeletal muscle.When axons regenerate after a simple nerve crush, as in the

present study, they often grow through the cellular tubes that

Proc. Nati. Acad. Sci. USA 74 (1977)

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Proc. Natl. Acad. Scd. USA 74 (1977) 3077

originally surrounded the parent axons (1-5). This observationshows that these tubes can provide a pathway for the regen'er-ating axons to reach the original synaptic sites. However, re-generating axons precisely cover long stretches of synapticmembrane after they leave the perineurial tubes (5). Moreover,axons growing outside or beyond the original pathways can passover long extrasynaptic stretches of muscle fibers to form sy-napses at original sites (1-3, 5). Thus, there must be componentsof the original site itself that account at least in part for itsprecise reinnervation. Our demonstration that a principal el-ement of the original synaptic site-the original muscle cellitself-is not required permits one to focus on the remainingelements: the Schwann cell, the basement membranes of themuscle and the Schwann cell, and components of the musclefiber's surface that adhere tightly to the basement membrane.Any or all of these might provide chemical or mechanical cuesto the regenerating axon.

We are grateful to Mary Hogan, Shirley Wilson, and Linda Yu forassistance. This work was done during the tenure of a Research Fel-lowship of the Muscular Dystrophy Association (L.M.M.) and wassupported by U.S. Public Health Service Grants NS 70606, NS 12922,NS 02253, and NS 05731.The costs of publication of this article were defrayed in part by the

payment of page charges from funds made available to support theresearch which is the subject of the article. This article must thereforebe hereby marked "advertisement" in accordance with 18 U. S. C.§1734 solely to indicate this fact.

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