Regeneration and innervation in cultures of adult mammalian skeletal muscle coupled with fetal rodent spinal cord

EXPERIMENTAL

36,

NECROLOGY

Regeneration

136-159

and

Mammalian

Innervation Skeletal

Fetal EDITH

(1972)

R.

Saul R. Corey Department Albert Eiwtei,r. Collcgc

Muscle

Coupled

Spinal

Cord

Rodent

PETERSON

in Cultures

AND

STANLEY

M.

CRAIN

of

Adult

with

1

of Neurology and fhe Departlrwut of Physiology. of Medic&-, Yeshiva Ullh,cvsifAl, NCW York 10461 Receizvd

March

8. 1972

In a collagen-substrate culture, long strips of teased adult skeletal muscle fibers oriented toward ventral-root nerve fibers of fetal rodent spinal cord explant will regenerate rapidly as the neural outgrowth makes contact with the muscle. Trophic enhancement of the early regenerative capacity of muscle is relatively nonspecific and can also be produced by contacts with a variety of nonneural fetal cells. Myotubes of neurally deprived muscle can regenerate, show fibrillatory contractions, and develop transient cross striations at their peak of development iti vitro, but soon afterwards, as bc zizro. they atrophy. Further differentiation, maturation, and long-term maintenance require innervation, i+t vitro as well as irt zizfo. In culture this is dependent upon the organotypic development of both the central and peripheral nerve network of the spinal cord complex. Although electrophysiologic studies show that neuromuscular transmission can occur early in the second week after coupling, the junctional structures are primitive and definitive loci of cholinesterase activity are not discernable (at the light microscopic level) until about a week later. During the following weeks in culture gradual maturation of the motor endplate structure occurs, including increased complexity of nerve terminals and postsynaptic specializations, as illustrated by silver impregnation, cholinesterase staining, and other histologic techniques. The ordered progression of skeletal muscle regeneration and maturation through spinal cord innervation ilt zltro is quite comparable to basic aspects of regeneration in viva. Organotypic cultures provide, therefore, a reliable model system for studies of some of the mechanisms underlying muscle regeneration and the restoration of functional neuromuscular relations which may be difficult to analyze in situ. 1 This study was supported by grants NS-06735, NS-06545 and NS-08770 from the National Institute of Neurological Diseases and Stroke, and the Nancy Louise Tryner Memorial Grant (No. 433) from the National Multiple Sclerosis Society. Dr. Crain is a Kennedy Scholar at the Rose F. Kennedy Center for Research in Mental Retardation and Human Development (Albert Einstein College of Medicine). Preliminary reports of this work have been published (31, 32). The authors would like to thank Dr. Murray B. Bornstein for his interest, advice and support during the course of these studies, and Dr. Edmund B. Masurovsky for invaluable constructive suggestions and critical review. 136 Copyright All rights

0 1972 by Academic Press, of reproduction in any form

Inc. reserved.

MUSCLE

CYLTURES

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Introduction

Recent morphological and physiological studies have demonstrated that functional neuromuscu!ar junctions can form ix zlitro between spatially separated esplants of fetal mammalian spinal cord and fetal skeletal muscle 17, 30; see also review of similar studies with chick embryo cultures in Refs. (17, 39)]. Th e 1x-esent report focuses on cultures of adult skeletal muscle coupled in zbitr-owith fetal spinal cord (or other fetal tissues), in which dramatic evidence of growth-stimulating and trophic influences on muscle regeneration has been obtained. Mature adult muscle fibers cultured in isolation on a collagen substrate produce a very limited outgrowth of cells from which no muscle fibers develop. Muscle regeneration can, however. be triggered by neuritic or other cellular contacts from fetal tissues grown with the adult muscle. This is in direct contrast to the intrinsic capacity of fetal muscle to develop independently. Even in late periods of gestation fetal rodent muscle contains a large complement of proliferating myoblasts-the undifferentiated cells of Kelly and Zacks (20). This embryonic component, together with newly formed primitive muscle cells, contribute largely to the adaptability of fetal tissue to muscle development in culture. In the adult animal there are indeed residual undifferentiated cells. the satellite cells (23 j. sparsely distributed within the basement membrane of mature muscle fibers (see Discussion). These are considered to be stem-cell myoblasts which can be triggered by injury to replicate again (4, 5. 26, 28, 38). \Vhether these cells are solely responsiMe for muscle regeneration. or whether myonuclei can also contribute to this process (15. 16. 36, -la), is a question not yet fully resolved. Injury alone is sufficient only for an abortive muscle development in collagen-substrate cultures of isolated adult muscle, in contrast to the rapid sequenceof regenerative events evoked by neuritic contacts. Full maturation, however, both for muscle developing from embryonic tissue as well as muscle regenerating from adult fibers is dependent on innervation. Long-term cultures of these dc WOZ’O innervated muscle fibers of adult origin show that motor endplate structures develop with a high degree of organization, as demonstrated by the light microscopic evidence described helow and by correlative electron microscopy (29). Characteristic neuromuscular transmission has been demonstrated with electrophysiologic techniques after regeneration and innervation of these muscle fibers in zpitro (11.). Materials

and

Methods

Spinal cord with meningeal covering and attached dorsal root ganglia were dissected from fetal rats or mice at 14-15 days in ~rtruo. Whole cross sections the width of a ganglion (appros. 0.5%mm) were explanted singly

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GRAIN

FIG. 1. Photomicrographs of living cultures of adult rodent skeletal muscle strips (m, and m2) positioned at various distances from fetal rodent spinal cord-ganglion complex explanted 4 days earlier. The muscle strips are oriented towards the ventral (A) or lateral (B) surface of the spinal cord (SC). Note attached dorsal root ganglia (G). (-4) : Two days after cord-muscle coupling, outgrowth from cord-

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on collagen-coated coverslips (l), provided with a drop of nutrient fluid, and sealed into Maximow slide assemblies (33). The nutrient fluid contained 33% human placental serum 53% Eagle’s minimum essential medium (with glutamine), 10% rat or chick embr\-o estract, 600 mg% gluclose, and 1.2 h~g/ml nchromycin with ascorbic acid. Cultures were incubated at 33-33 C in lying-drop position. Four days later, 3-6 mm lengths of teased skeletal muscle fibers, varying from two to 12 in nunber, were positioned with the aid of a dissecting microscope, around the ventrolateral surface of previously explanted spinal cord cross sections, close to the zone of neuritic outgrowth (Fig. 1 ). The muscle was obtained from the thigh of adult rodents or, in a few cases, from human biopsies (Part B-4 in Results j. Twice a week. the cultures were washed in Simms’ balanced salt solution and fed a drop of nutrient fluid. The nutrient fluid and substrate in all cultures used in this study were selected in order to favor the maturation of organized spinal cord tissue (30). The coupled cord-muscle cultures were maintained for up to 30 weeks in ztitr-o, and were observed daily at a magnification of 600X. Cultures were selected at representative stages for bioelectric and histologic studies (‘7, 8, 11 for details of the electrophysiologic techniques). Nerve terminals were demonstrated by a modified Holmes silver impregnation (43 ) and by methylene-blue vital staining (6 j. Koelle-Friedenwald (121 ) and Karuovsky (1s. 19) methods were used to demonstrate cholinesternse activity at the newly formed as well as the original endplates. The cholinesterase preparations were embedded in .iquamount in order to preserve the myelin sheaths for subsequent study of the innervated cross-striated muscle fibers with polarized light. Results

Explants of isolated adult muscle fibers, comparable in size to those used for neural coupling. show few or no signs of muscle regeneration during maintenance for several months in this culture medium. In all newly explanted adult muscle details of the fine cross-striated pattern are rapidly lost, but a remarkable degree of orgasized coarse striations are retained in these isolated fibers, lying dormant .-1. Isolated

A4d~~lt Muscle

Explant.

ganglion complex has reached the proximal muscle explant (m, ). Muscle regeneration is at early stage (see high-power view in Fig. 3-q; also, similarly oriented culture after 1 month irr ztifro in Fig. 4.4 ). (B) : Ten days after coupling, muscle regeneration in the proximal muscle explant (m, ) is extensive (see high-power views in Fig. 3B, C), while still at beginning stages in the distal explant (m,). Scale: 1 mm. ( Notr : Calibration bar in lower right-hand corner applies to all photomicrographs in each montage except those specified by additional bar directly on photomicrograph.)

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features of isolated adult rodent skeletal muscle fibers in culture. FIG. 2. Cytologic (A) : 2 days in z~itro (in living state). Note typical coarse cross striations (see also Fig. 3B, C : “P”), and cluster of nuclei at a motor endplate (arro\nr ). ( B) : 35 days irr vitro (d.i.v.) (stained for cholinesterase by Koelle-Friedenwa!d rr.ethod). Motor endplate still shows postsynaptic grooves (arrow) ; coarse cross-striated appearance has not changed. Scale : 50 p.

during months in culture (Fig. 2). Since the nutrient fluid in the Masimow slide chambers becomes extremely alkaline in cultures composed entirely of muscle, this variable is controlled by adding small explants of fetal lung or liver along with the muscle explants. The small fragments of rapidly growing tissue, explanted some distance away from the muscle, maintain the nutrient fluid within the normal pH range but do not alter the negative regenerative response of the adult muscle fibers (see, however, Part C). Even the inclusion of cord-ganglion explants at distances from the muscle well beyond th? range of the outgrowing nerve fibers does not lead to significant mu:c!e regeneration. although fibroblasts and endothelial cells occajional!y migrate out of the muscle explant. Isolated muscle maintained in this essentially inert state in slitro still shows marked cholinesterase activity at the old motor endplates. even after 2-3 months (Fig. 2B). Furthermore, the enzyme-active borders of the “pretzel”shaped subneural apparatus show only partial fusion and little or no fragmentation after this long dormant period. Recently HEPES buffer (‘lo-‘M ) has been incorporated into the nutrient fluid for more efficient pH stabilization ( 12 ) in the Maximow slide chamber where rigorous control of the gas phase is difficult to maintain’. This can be critical in the early stages of culturing slow-growing or very small fragments, since their metabolic activity mav be insufficient to pre2 Based on earlier unpublished observations tures in Maximow chambers ( E. B. Masurovsky

of HEPES-buffered and R. P. Bunge).

neural

tissue

cul-

MUSCLE

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141

vent an excessive alkaline shift of the nutrient fluid. 14-hen isolated muscle Strips are grown in the HEPES-buffered medium, about 40% of the explants produce a modest fibroblastic outgrowth, as well as periodic nucleated “budlike” structures and slender spindle cells on or along the muscle surface (Fig. 3-1: Part B-2). In only a few instances did even binucleated forms develop. Regeneration does not progress beyond these early stages, nor are there overt signs of degeneration during 6 weeks ilz vitra. Occasional myol)lasts migrate away hut most retain a close relationship to the parent fibers. The latter show no further alteration, and retain the coarse cross-striated appearance (zlidc s~pva) until fisation 5 weeks later (Fig. 2B ) . B. Paired E.t-plal1t.s of Adult IIKW~C anti Frtal Spiml Cord. Although cell contact with outgrowth from a varietv of non-neural fetal tissues, as well as peripheral ganglia, can trigger the early stages of regeneration of adult muscle in our cultures (Part C), the ordered sequence of events evoked by arrival of spinal cord outgrowth will he described first since this orgnnotypic muscle-regeneration process provides a firm baseline for evalu. sting the more limited de\-elopments which occur after noncord contacts. 1. Cord ad mm-c/r r.rplant away. In cord-ganglion cultures an earl: period of rapid neuritic growth lasts for about 7-10 days after explantation. Muscle was added at 4-S days, since by this time organization of the peripheral nerve comples is already apparent and more meaningful placement of muscle can be made. Strips of teased adult muscle fibers were placed around the ventrolateral region of the cord-ganglion explant, within 23-hr reach of neuritic outgrowth. In some cultures 1 or 2 additional mu.c+ cle esplants were placed l-2 nini beyond the first explant. progressively more distal from the spinal cord (Fig. 1). Some muscle fibers were slightly traumatized prior to placement. but this did not appear to de crease, or enhance, their capacity for regeneration. Severely traumatized muscle loses much of its capacity to regenerate. Its behavior in culture differs from that of undamaged or slightly traumatized muscle fibers. Freshly tensed muscle fibers often appear to he in a hypersensitive state and tend to contract and coil when grasped with forceps. Manipulations associated with arranging the fibers on the culture coverslip mav easily result in breakage, with the formation of numerous small retracted coarsely cross-striated compartments, separated from each other by amorphous granular segments, hut all held together 1)~ apparently intact sarcolemnia. In direct contrast to the stal)i!itv of undaniaged fibers, the small compartments of muscle tissue are very labile and the fibrillar organization lx-e&s down quite rapidly. forniin g swirls and then losing all organized fibrillar pattern. 1Vhen muscle regeneration occurs in the mechanicallv disrupted fillers it appears to involve myoMastic lmdding from the

FIG. 3. Early phases of adult skeletal muscle regeneration (triggerable by nonspecific cell contacts : see text), in this case initiated by outgrowth from fetal spinal cord complex (photomicrographs of living cultures). (li) : 2 d.i.v. Neuritic growth has just reached muscle fiber (arrow). Fibroblasts (F) have begun to migrate away from muscle surface. Myonucleus (Mn) is visible in the muscle fiber. ( B) : 10 c!.i.v. Distal muscle fiber (Fig. 1B) shows earlier phases of muscle regeneration. Note mononucleated budlike formations (black arrows) and multinucleated fusions (white arrows) in close association with the intact parent muscle fiber ( P). (C) : Along the entire length of a more proximal muscle fiber (Fig. 1B). long myotubes (1 and 2) have formed, only portions of which are illustrated here. The less mature myotube

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fibrillar compartments into the amorphous segments, or projection of lateral spurs from weakened areas of the sarcolenmal membrane. This regeneration is rarely of the orderly type described below following neurotrophic stimulation of undisrupted muscle fibers. Moreover. escessive phagocytic round-cell activity often eliminates a large proportion of the potentially viable muscle tissue, so that this type of regenerated muscle is less abundant and consists of small-caliber fibers. ,7. Early ~n~~sclc rrgcm~~atio~~. Within 1-3 days after cord neurites reach the muscle fibers and spread over the muscle surfaces, early signs of organized muscle regeneration can be detected. Small nucleated swellings or buddings appear to erupt along the entire length of muscle fiber at irregular intervals (Fig. 3A j . These buds gradually increase in number, and fuse to forin large broad nmltinucleated swellings on the parent muscle surface, without appearing to detach from the original fibers. This is in contrast to the near-absence of fusion in isolated muscle cultures (Fig. 2 : Part =\ 1). Adjacent swellings gradually fuse into myotubes (Fig. 3B ). Concomitantly the volume of cytoplasm increases and partial cross striations may form. The newly formed myotubes may cross-fuse to form one broad crossstriated fiber or they develop as two or more separate narrower fibers. 14s the new muscle forms, the central core of old muscle shrinks in diameter, hut continues to retain its characteristic coarsely striated appearance (Fig. 3B, Cj. The latter may constrict periodically and form long avoids surrounded by a continuum of regenerated muscle. Granular round cells now become very active, clingin g tenaciously to the parent muscle surface, and phagocytizing rapidly the deteriorating old muscle (Fig. 3C ). As they penetrate deeper into the “ragged” remnants of old muscle. these engorged cells may double or even triple their size. They may become niultinucleated, although this is usually obscured by the dense yellow granularity of their cytoplasm. Following completion of phagocytosis the round cells migrate away from the new muscle. I’igorous phagocytic activity is also often observed as an early process at the cut ends of muscle fibers which are the areas of greatest trauma. Spontaneous contractions of the growing muscle regions begin to occur quite early, even before fusion of adjacent swellings is completed. They are often seen as local quiverings occurring asynchronously in different regions

(1) above the parent muscle fiber ( P) shows fihrillar cytoplasm and a cluhtcr of myonuclei (arrows). In the more differentiated myotuhe (2), the nuclei are distributed in linear array (arrows) and dc IIUTYJ cross striations have formed. The coarse cross-striated fihrillar structure of the parent fiber is interrupted by phagocytic granular cells (g), which appear to be associated with rapid disappearance of the parent sarcoplasm concomitant tvith maturation of the adjacent myntubes. Scale : 50 8.

141

PETERSON

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CRAIK

of the muscle, and continue at high frequencies for long periods of time. In the Leginning, the old muscle seems to move with the contractions as though it is an integral part of the regenerating complex, but this appearance could be due to passive pulling of the closely associated old muscle mass. In preliminary electrophysiologic studies during this early stage of regeneration, not only was there no evidence of cord-evoked muscle contractions. but even application of electric stimuli (with microelectrodes placed directly on the muscle fibers) was ineffective, Several days later, however, muscle contractions can be readily elicited by these direct electric stimuli, and neuromuscular transmission has become demonstrable in some cultures by the begimiing of the second week of coupling (Crain and Peterson, in preparation). When several strips of muscle were explanted at various distances from the cord, the proximal explant regenerates before the distal (Fig. 1B). Kegeneration within each explant does not begin until the outgrowth from the cord complex arrives at the muscle fibers. In a lo-day culture, for example, muscle regeneration may still be at a very early phase in the distal explant, while regeneration may be complete-and neuromuscular transmission already futlctioning-in the proximal explant, where all vestiges of parent muscle have already disappeared. More distant muscle explants IOcated beyond the range of cord outgrowth have remained for weeks in a state of “suspended animation” comparable to the isolated muscle cultures. An attempt has been made to clarify the nature of this dormant or inert state of isolated adult muscle fibers in culture, by explanting a cord-ganglion fragment in close proximity to such one-month-old muscle fibers. Contacts of cord outgrowth now induce only fibroblastic, endothelial and phagocytic cell migration. but they no longer evoke even abortive muscle regeneration. Instead. granular round cells rapidly phagocytize the old muscle fibers within a week’s time. Further study is required to determine the length of the critical period during which isolated adult muscle can retain the capability to undergo active regeneration when triggered by appropriate cellular contacts. 3T. Muscle diflevrntiution. The newly regenerated muscle is usually restricted to the coniines of the original muscle explant. One to four new muscle fibers may form in place of each old muscle fiber. Some of these may run the entire length of the original fiber (Figs. lB, 3C), but more are of shorter lengths. Where the explant consists of a few (two to four) long muscle fibers which happen to be long enough to completely encircle the cord explant, only a portion of the muscle regenerates and differentiates. When the original explant consists of many (12 or more) shorter fibers there is a greater tendency to form muscle spurs, extending beyond the boundary of, or at angles to, the original explant. Although there are

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further increases in volume after cross striations have formed, the mature muscle fiber in culture rarely exceeds one-half to two-thirds of the cliameter of the fiber of origin. By 2 weeks iw vitro the regenerated muscle nxty appear well differenaitated. with prominent cross striations and peripherally positioned nuclei. In addition to asynchronous fibrillations of individual muscle fibers, mauy of the fibers now show widespread coordinated contractions in response to selective stimulation of innervating cord neurons. Siniultaneous microelectrode recordings of cord and muscle responses to local cord or ventral root stimuli (Fig. 3 ) demonstrate characteristic neuromuscular transmission of impulse;. There is also normal phamacologic sensitivity to selective blockatle by d-tuhocurarine ( l-10 pg/ml ) and enhancement by eserine ( 11 ). Xeuronluscular transmission has, indeed. been demonstrated as early as S days after addition of a muscle explant to a cord culture, although no signs of cholinesterase activity could be detected in these muscle fibers with standard staining procedures (‘see Methods ). Only after 20 days in zfitro has it been possible to demonstrate loci of cholinesterase activity iii the form of lightly stained hut distinctive :~ccumulations of granules on the muscle fibers. More differentiated endplate structures are observed in 4 (Fig. S ) to 12-wee!< old (Fig. 6) cultures. Related nerve fibers and terminal Sclinnnn cdl (30) nxlei can he identified in some of the chclinesterase-stained endplates (Figs. 5. 6). Endplates are far more difficult to find in the living unstained cultures, hut in some cases they have heen itlentifietl 1)~ systeand sulxequent confirimtion hy matic study during develolxnent in 7ifro cholinesteras= staining. Sonle of these structures resemble siqle pretzelshaped forms conq~aral~le to those observed on young adult musc!e fibers in sift ( Fig. 6a). In the neural component of the cultures the spinal cord differentiates fairly rapidly so that II\- the end of the first week complex synaptic networks can he demonstrated hioelectrically ( 11) and hy electron microscopy (29). Myelination 0: central nerve fibers in the cord is usually lvell developed hv the end of the second week. The peripheral nerve coq!es en:erging from the spinal cord differentiates more gradually. During the earl) perio,l of rapid cord outgrowth (al:cmt 10 days ) the neuritic pol~ulation consist5 essentially of smxll caliber fibers. In n:ouse tissue, which telltl5 to he far less organized than that of rat, the initial neuritic outgroc-th is nearly radial and lx-ol~alJy arises from neurons which would not normally contribute to the peripheral nervous system. 1\Zany of these fillers reach into the muxle, and their peripheral regions appear to be bare, although closer to the cord they may he acconpnied 1,~ neuroglia. By about 2 weeks. howeve*-. the fibers either retract or degenerate. Inhereas the

146

PETERSOK

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: Mouse spinal cord-ganglion explant (SC : G) coupled with regenFIG. 4. (A) erated muscle (m) derived from adult mouse skeletal muscle (Holmes’ silver impregnation; 1 month ill rjitro). Ventral root fibers (arrows) proceed into and terminate on the regenerated muscle. Scale: 1 mm. (B) : Large neurons in another spinal cord culture (Holmes’ silver impregnation). In this particularly thin-spread explant (5 months in vifvo), the motor neuron perikarya could be visualized in the living state and directly stimulated with microelectrodes to trigger muscle contractions [Refs. 7, Ill. Scale: SO ,u.

hi USCLE

CULTURES

l-+7

Schwann cell-enveloped fascicles, both sensory and motor, continue to develop normally. Rarely do these “internuncial” neurites grow out from rat cord ganglion explants since they are efficiently retained by meningeal boundaries. The ensheathed fibers gradually increase in diameter and begin to become myelinated, usually between the second and third week. Myelination of peripheral nerve is more gradual than central myelination and continues actively for the nest 2-3 weeks ( 11, 3.3). By 5-6 weeks the peripheral nerve complex is usually well differentiated. and broad axons are often present among the muscle fibers (Figs. 5. 7, S ). Ker\-e terminals become increasingly complex in older cultures (S-12 weeks ipz z~ifro), show-

FIG. 5. Regenerated rodent skeletal muscle coupled \vith cord-ganglion complex, stained for cholinesterase (Karnovsky) after 1 month ill 7tfr.o. ( .4 ) : I)P now motor endplate structures show some infoldings (arrow), but are still immature (cf. Figs. 2B and 6). A broad myelinated axon “a” runs parallel to the muscle. (R ) : Terminal Schwann cell can be seen in relation to another stained endplate. (C) : Same region as in (B) viewed \vith polarized light to demonstrate integrity of cross striations (as in A). Scale : 50 +

FIG. 6. More mature motor endplate stx:turcs in cultures simi!ar to Fig. 5, stained for cholinesterase (Karnovsky) after 2-4 months in s~ifro. Note well-developed cross striations of muscle fibers associated with the endplates. (A) : Two pretzel-shaped structures (1 and 2) show compact pattern with neural grooves comparable to adult of the muscle fiber associated with endplates, although smaller in size. The width the line of positive staining extending beyond endplate “1” is indicated by arrows;

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CVLTVRES

ing terminal arborizations with terminal swellings (Fig. S), in contrast to the simple terminals seen in younger cultures (Fig. 7L4, B) . 1. Cross-sp~ies coupling. Functional neuromuscular junctions have iormed not only in cross couplings of mouse muscle with rat spinal cord, but also by coupling human muscle with mouse cord. Correlative electrophysiologic tests were carried OLIN in one of the latter groups. Since the muscle regeneration of this specimen was particularly favorable, some background data are included. Muscle from the left quadriceps was obtained from an adult male in whom a myoglobinuria had been demonstrated 4 months prior to the biopsy. 3 Although this muscle was relatively fragile, it was possible to dissect out fibers which were only slightly traumatized. These were explanted into 5-day old mouse cord cultures. The human muscle fibers retained a coarse cross-striated appearance similar to that observed in isolated rodent muscle. About 3 days after neuritic overgrowth from the cord, nuclei of newly regenerating muscle were observed at the periphery of the old fibers. Portions of old muscle gradually retracted into more granular large ovoids, appearing somewhat like large granular inclusions in broad new fibrillar muscle fibers. In some cases, however, linear arrays of nuclei could be seen near the surface of the parent fibers, suggesting that direct reorganization of sarcoplasm may also occur (Fig. 9A). Fiber diameters of the regenerated muscle fibers, in this case, were comparable to the old fibers. By 16 days in z&o, several fibers showed contractions and de novo cross striations. By 21 days, all of the regenerated muscle was well cross striated (Fig. 91 ) and synchronous contractions of many fibers were observed. After 1 month ill vifro selective stimulation of the mouse spinal cord esplant or ventral root neurites evoked coordinated contractions of large groups of human muscle fibers, demonstrating functional neuromuscular transmission quite similar to that generally observed in intraspecies COLIplings (11). Muscle action potentials occurred with latencies of several milliseconds following ventral root stimuli, and they were evoked at a critical stimulus threshold. The muscle potentials were preceded by smaller 3 The Liveson

biopsy tissue was (The Saul R. Corey

obtained through the Dept. of Neuro!ogy).

courtesy

of

Drs.

A.

Spiro

and

J.

this endplate is tlot the muscle boundary. Such linear stainings are observed from time to time, but their significance is not clear. (B) : Endplate structure with welldefined terminal Schwann cell (ts). ( C) : Endplate structure with well-developed infoldings and terminal Schwann cell (ts). Secondary foci of the ChE staining (arrows) may be found. (D) : Branch of a nerve fiber (arroivs) terminating at ChE-stained endplate structure. Scale : SO p.

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FIG. 7. Axon terminal arborizations on regenerated rodent skeletal muscle after about 1 month in culture. (-4-C) : Holmes’ silver impregnation (low-power view in Fig. 4A). (A) : Long terminal bifurcation (white arrow) on a young regenerated cross-striated muscle fiber. Note periodic thickenings along the neurites (black arrows). Terminals of this type may not be permanent or may undergo further modification in time. (B) : Another immature arborizing neurite terminates on crossstriated muscle fibers (arrows). (C) : T erminal arborization with greater subneural specialization. Note myonuclei of a soleplate (mn) and a faintly visible terminal Schwann cell (ts). (D, E) : Methylene blue vital stain. (D) : Terminal arborization with bouton endings (arrow) on a muscle fiber. (E) : Plate-like neural ending with tiny brush processes (arrow). Scale : 50 p.

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FIG. S. More mature neuromuscular junctions after 5-9 weeks in culture (Holmes silver impregnation ). (A) : Fine terminal arborization (arrows) of delicate branch of a study axon on mature muscle fiber. Kate terminal Schwann cell (ts) and subneural specialization of sole plate myonuclei (mn). (B) : Terminal branch with delicate arhorization and bouton structures (arrows) on well-differentiated muscle fiber. Note terminal Schwann cell (ts) and sole plate myonuclei 1mn). (C) : Complex arborization of nerve terminal in a well-differentiated endplate structure. Note myonuclei (Inn) and terminal Schwann cell (ts). Scale : 50 U.

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spikes of shorter duration which appeared to be generated in presynaptic nerve terminals. Neurally evoked muscle action potentials and contractions were blocked after introduction of d-tubocurarine (1 pg/ml), while presynaptic neural spikes could still be elicited, followed by potentials resembling muscle endplate potentials. and contractions were still triggered by direct electric stimuli to the muscle. Eserine (1 pg/ml) accelerated recovery of some of the curarized muscle fibers and progressive stages during restoration of normal transmission could be demonstrated with brief tetanic stimuli. The time required to produce neuromuscular block with d-tubocurarine and the recovery period after drug removal were well within the range of variation observed in couplings with rodent muscle [further details in Ref. ( 11) ] . C. Paired Explants of Adult M~~scle and Misceilawoas Fetal Tissues (Lung, Liver, Meninges and Peripheral Ganglia). Since muscle regeneration appeared to respond to contacts of migrating neuritic elements of the spinal cord complex, it seemedappropriate to test the specificity of this response by coupling muscle with dorsal root ganglia and sympathetic gan-

FIG. 9. Adult human skeletal muscle Z-3 weeks after coupling with fetal mouse spinal cord complex (living culture). (A) : Two weeks irz r4tl.o. Early phase of regeneration. Note linear arrays of myonuclei (arrows), fibrillar pattern of the cytoplasm and absence of cross striations. (B) : Three weeks ill rlifvo. Cross striations are now prominent throughout the regenerated muscle fibers. Scale: 50 IL

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glia. as well as with nonneuronal tissues, such as lung, liver. or meninges. Of the tissues selected, dorsal root ganglia and meninges were generally present as components of the spinal cord complex noted above. Each of these fetal tissues were explanted in close proximity to the adult muscle fibers, so that coutact with cellular outgrowth could be made within 2-L-@ hr. The cultures were nlaintained for 5 weeks. AU of the tissues did, indeed. trigger the cnrljl stages of muscle regeneration as observed following coupling with the spinal cord conlples. i.e.. budding along the muscle fiber surface and fusion into myotubes. The muscle reaction was especially rapid with liver and lung tissue, but generally less ordered. Often spurs formed and projected at angles off the original muscle fiber array, and terminal budding frequently occurred, such as described by earlier investigators (13, 1-F. 27, 33) using plasma clot substrate. Remnants of parent fibers were very rapidly phagocytized. The nmscle regenerates remained irregular in form and often showed fusiform swellings and central nuclei quite reminiscent of the atrophic appearance of fetal muscle cultured in isolation for manv weeks ( 30 1. Transient cross striations were observed from time to time. With spinal ganglia, sympathetic ganglia and meninges the initial budding response was similar, but regeneration did not appear to advance as rapidly, nor were the parent fibers phagocytized as quickly. Often reninnnts: of parent fibers remained for several weeks. The muscle regenerates were usually composed of long thin fibers. Again, transient cross striations were observed, persisting perhaps a little longer in one of the couplings with sympathetic ganglia. In all cases the degree of muscle differentiation was stunted drastically in comparison to that observed with spinal cord couplings. Furthermore, in spite of the profuse arborization of sympathetic ganglion neurites in these regenerating skeletal muscle explants, no electrophysiologic evidence of neuromuscular transmission could be detected. even though many of the fibers contracted in response to direct electric stimuli. These observations add further support to the organotypic specificity of the cord-innervation phenomenon in rodent tissues in culture. In this regard, it is of interest to note that cholinergic parasympathetic and possibly sympathetic nerves can, under some conditions. actually make functional synaptic connections with skeletal muscle in the frog in sitar (22‘). Discussion

The sequence of events in muscle regeneration observed during in vitro coupling of adult skeletal muscle with the spinal cord complex presents two distinct response phenomena which closely mimic the in BGUOresponse of muscle to injury. First, adult muscle retains an intrinsic capacity to undergo early stages of regeneration (e.g.. 41). In this state the muscle is responsive to extrinsic influences which may be of a trophic nature, enhanc-

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ing the intrinsic regenerative capacity. Secondly, however, full muscle differentiation, growth, and maintenance are dependent on specific neurotrophic influences and stimulation. Evidence of the innate capacity of adult mammalian skeletal muscle to regenerate has been demonstrated in cultures using a plasma clot substrate (13, 14, 27, 34). In contrast to the collagen substrate used in the present study, which provides only for surface growth, the plasma clot also permits growth within the fibrin meshwork. Cells and budding projections of the muscle tissue embedded in this substrate are provided a matrix through which, or upon which, they can migrate. In addition to facilitating proliferation from the muscle, the plasma clot enveloping the muscle (and associated endomysium and capillaries), may also serve as a diffusion barrier to retain a concentration of metabolites liberated by cells which could trophically enhance muscle development. The investigations of Pogogeff and Murray (34) and of Godman ( 13, 14) were concerned less with the explant and more with the outgrowth from the uniformly cut slabs of muscle tissue undergoing gradual necrosis. After a lag period of l-3 weeks, muscle outgrowth was ohserved which Murray (27) described as highly variable morphological forms, suggestive of modulations in phases of muscle regression and redifferentiation. Goclman ( 14) noted that the most significant mode of “muscle regeneration” in similar types of cultures was through terminal budding of fiber stumps, although isolated mononucleated and binucleated, fusiform cells and multinucleated giant cells also appeared to he of muscle origin. Differentiation progressed to myotube formation, contractility, and formation of cross striations of a transient nature. On the collagen-film substrate used in this study, the regenerative response of isolated teased muscle fibers was minimal; rounded budlike cells and myoblasts formed on the surface of some muscle fibers but progressed no farther. Enhancement of the regenerative capacity of these muscle fibers is dramatically displayed, however, when a variety of fetal tissues are explanted in close proximity to the muscle. The burst of regenerative growth appears to be triggered by contacts of cells migrating to the muscle fibers. contacts which may be providing trophic influences. In these neurally deprived modes of muscle regeneration, long broad myotubes may form hoth in the more organized array parallel to the parent fiber and in angled less organized patterns, away from the parent fibers which are now rapidly phagocytized. Although portions of these regenerated fibers may become cross striated, they inevitably atrophy. The organized. early regenerative response of the muscle, developing in arrays parallel to the parent fibers, is comparable to that of the neurally enriched muscle cultures. Differentiation is limited, however, to a peak of partial or transient cross striation, comparable to muscle grown in plasma clot cultures. The suhsecluent irreversible

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atrophy is comparable more to neurally deprived muscle regeneration ia zliz~o (-I1 ). The plasma clot substrate, in contrast to the gradually aging collagen film, can he intermittently renewed. Possibly this is a sufficient stimulus to permit the waves of rejuvenation esperienced in the prolonged maintenance of muscle in long-term plasma clot cultures (3-F ) . The CJi-@iliZed rapid muscle regeneration triggered by the cord-ganglion comples requires that the esplxnted muscle fibers be well-preserved, relnsetl, and stretched flat on the collagen surface, so that these intact fibers can provide guidance to the regenerating fibers. In this setting. organotypic regeneration can be observed sequentially from inception through phases of development, leading to full differentiation of mature imiervated muscle fibers. The sequence of changes observed in early stages of regeneration preceding myotube formation suggests that our cultures may provide a valuable experimental system to clarify current ambiguities regarding the nature of the myoblasts and satellite cells associated with these complex developmental processes. The generally accepted concept of muscle regeneration subscribes to a formation of myoblasts, their proliferation, and subsequent fusion into multinucleated myotubes. Considerable controversy centers on the origin of these myoblasts. Mauro (23 ) first described a mononucleated cell enclosed within the striated muscle fiber which he termed the satellite cell. Identification of this cell, which lies between the basement membrane and the plasmalemma, requires electron microscopy. Many investigators ( e.g., 3, 5, 26, 2s. 35) consider the satellite cell to be a residual stem-cell myolhst solely responsible for the formation of proliferating myoblasts in regeneration. Others (15. 16, 36, 32) suggest that satellite cells are also formed after injury to muscle, by myonuclear budding (16’) or by separation of the myonucleus with a thin rim of cytoplasm and subsequent formation of new plasma membrane (36). Autoradiographic evidence of satellite cell involvement in muscle growth (25) is most impressive. On the other hand, the possibility of myonuclear activation and transformation into satellite cells during regeneration is also supported by autoradiography ( 36‘) and warrants further consideration. Recognizing the limitations of the light microscope for identifying the muscle nuclei. satellite cells and n~yolhsts. and the intimacy of their relationship to the parent muscle fiber, correlative electron microscopy of these structures during development in r&o is now in progress. The dynamic sequence of events leading to differentiation of the new muscle fibers is undoubtedly influenced by concomitant development of the peripheral nerve complex emerging from the cord explant. Studitsky (40, 41.) wrote of the plastic state of injured muscle when its regenerative response appears to be more sensitive to extrinsic factors inducing growth, differentiation and maturing processes. He also provided evidence for a pe-

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riod of neurotrophic regulation of muscle growth and differentiation prior to functional maturation. Seven days after implantation of minced muscle, nerve fibers began to grow into the primitive muscle regenerate. Following neuritic invasion the muscle tissue differentiated progressively [also Ref. (3)], although functional maturity was not reached until the twenty-first day. Similarly, in our cultures where neuritic invasion of the muscle tissue may begin l-2 days after esplantation, rapid differentiation of the regenerated muscle

fibers occurs

during

the following

week,

well

before

functional

transmission has been detected. In both systems, however, there may be earlier primitive modes of functional interaction which are not expressed as overt musclecontractions in responseto neural stimulation (37). Sustained maintenance of differentiated cross-striated muscle fibers after regeneration in culture is dependent upon innervation by organotypic spinal cord tissue. Thus, improperly oriented muscle explants, not in the path of ventral root fibers, will generally atrophy. Sensory (dorsal root) and sympathetic ganglia do indeed provide a stimulus to muscle regeneration, but not to sustained maintenance of the differentiated state. Selective degeneration of the spinal cord portion of the explant results in gradual muscle atrophy, even though healthy dorsal root ganglia retain their intimate relationship with the muscle. It will be of interest to determine whether introduction of sympathetic nerve fibers after development of the organotypic cord-muscle system in culture can sustain the differentiated muscle state following denervation in zritro, as has been observed in the adult cat in viz~ (24). The latter experiments in the cat are particularly interesting since they demonstrate clearcut neurotrophic effects on skeletal muscle by adrenergic nerve fibers which have not apparently made functional comlections with the muscle. This neurotrophic effect appears, therefore, to be present in both cholinergic and adrenergic nerve fibers and may not require preferential transfer via neuromuscular junctions. Correlative electron microscopic data is neededto clarify this problem. Electrophysiological studies have demonstrated the onset of neuromuscular function during the second weeek after coupling. Preliminary esperiments suggest a possible immaturity of the early junction, as indicated by repetitive muscle responsesto a single neural impulse, instead of the usual simple rapidly quenched twitch response, This may be related to the presence of multiple transient synapseson the young muscle fibers (35, also Part B-2). Myelination of peripheral nerves is not yet complete at this stage in vitro, and the relationship of the terminal Schwann cell to the junctional apparatus may still be immature. Furthermore, since characteristic endplate structures have not been detected by cholinesterase staining (at the light microscope level) until about 2 weeks later, repetitive muscle responsesmay be due to prolonged ACh action at these AChE-deficient

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junctions. Electron lnicroscopic studies show that even after 5 weeks of coupling the postsynaptic endplate structure is not extensively infolded, although a normal terminal Schwann cell relationship to the presynaptic ter:ninal is present (29). Further differentiation of the postsynaptic membrane develops gradually, and at 10 weeks in zlifro comples infoldings characteristic of niature motor endplates are present. The ordered progression of skeletal muscle regeneration and differentiation through innervation by spinal cord in vitro closely resetnhles regeneration iuz zG7v and suggests that these cultures provide a reliable system for studying sotne of the nlore elusive controversial features of nmscle regeneration which have been difficult to clarify ill sift. The unique advantage of this experimental method is that all regions of the living culture from the nerve centers to the niuscle can be monitored with high-power light niicroscopy. Questions such as the satellite cell relationship to early muscle regeneration, trophic neuronluscnlar interactions, and the role of synaptic transniitter agents in maturation are now under investigation. utilizing coordinated cytologic, electron microscopic, and electrophysiologic techniques carried out on cultures maintained in a variety of experimental niedia for various periods of development in vitro ( 10, 29). References 1. BORNSTEIX, hl. B. 1955. Reconstituted rat-tail collagen used as substrate for tissue culture on coverslips in Maximolv slides and roller tubes. Lab. Thirst. 7: 134-140. 2. BVNGE, M. B., R. I’. BUNGE, and E. R. PETERSON. 1967. The onset of synapse formation in spinal cord culture as studied by electron microscopy. DGI Rrs. 6 : 728-749. 3. CARLWK. B. Al. 1968. Regeneration of the completely excised gastrocnemius muscle in the frog and rat from minced muscle fragments. J. Xorplzol. 125 : 447-471. 4.

CHL-R( H. J. C. T. 1970. Cell quantitation in regenerating bat web muscle, pp. 101-117. IJC “Regeneration of Striated Muscle and blyogenesis.” A. Maw-o, S. -4. Shafiq, and A\. T. Milhorat [Eds.]. Excerpta Medica, Amsterdam.

5. CHVRCII,

6. 7. 8.

J. C. T. 1970. r\ model for myogenesis using the concept of the satellite cell segment, pp. 115-121. Ijt “Regeneration of Striated Muscle and h#Iyogenesis.” A. hlauro, S. A. Shafiq, and -4. T. Milhorat [Eds.]. Excerpta Rledica, .Amsterdam. COEKS, C., and A4. L. WOOLF. 1959. “The Innervation of Muscle.” C. C. Thomas, Springfield, IL. CI
S. AI. 1970. Bioelectric and skeletal muscle after

interactions imlervation

between

cultured

fetal

in vitro. J. Exp. Zo~~l.

rodent 173:

spinal 353-370.

GRAIN. S. hl. 1972. Microelectrode recording in brain tissue cultures. IJI “Methods in Physiological Psychology : 1. Recording of Bioelectric A4ctivity.” R. F. Thompson and ?vl. hi. Patterson [Eds.]. ;\cademic Press, New York ( in press )

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9. GAIN, S. M., and E. R. PETERSON. 1957. Onset and development of functional interneuronal connections in explants of rat spinal cord-ganglia during maturation in culture. Brnill Rrs. 6 : 75S762. 10. CRAIN, S. M., and E. R. PETERSON. 1971. Development of paired explants of fetal spinal cord and adult skeletal muscle during chronic exposure to curare and hemicholinium. 111 L’itro 6 : 373. 11. CRAIN, S. M., L. ALFEI, and E. R. PETERSON. 1970. Neuromuscular transmission in cultures of adult human and rodent skeletal muscle after innervation irl elitvo by fetal rodent spinal cord. J. Nclrrobiol. 1 : 471-488. 12. EAGLE, H. 1971. Buffer combination for mammalian cell culture. Science 174: 50&503. 13. GODMAN, G. C. 1957. On the regeneration and redifferentiation of mammalian striated muscle. J. Movp/zol. 100 : 27-81. 14. GODIWAN, G. C. 1958. Cell transformation and differentiation in regenerating striated muscle, pp. 381-416. III “Frontiers in Cytology.” S. L. Palay [Ed.]. Yale University Press, New Haven, CT. 15. HAY, E. D. 1970. Regeneration of muscle in the amputated amphibian limb, pp. 3-24. 11t “Regeneration of Striated Muscle and Myogenesis.” A. Mauro, S. A. Shafiq, and A. T. Milhorat [Eds.]. Excerpta Medica, Amsterdam. 16.

HESS, A., and S. ROSNER. 1970. The satellite cell bud and myoblast in denervated mammalian muscle fibers. A11lev. J. .4pmt. 129 : 21-40. 17. KANO, M., and Y. SHIMAIIA. 1971. Innervation and acetylcholine sensitivity of skeletal muscle cells differentiated ilo zfitro from chick embryo. J. Cc/l. I’hgsiol. 76 : 233-242. 18. KARNOVSKY, M. J. 1963. The localization of cholinesterase activity in rat cardiac muscle by electron microscopy. J. Cell Biol. 23 : 217-232. 19. KARNOVSECY, M. J. 1964. “Direct coloring” thiocholine method for cholinesterases. J. Hisfoclzorz. Cytochcw. 12 : 21’s221.

20. 21. 22. 23. 24. 25.

KELLY, A. M., and S. I. ZACKS. 1969. The histogenesis of rat intercostal muscle. J. Cdl Biol. 42 : 135-153. KOELLE, G. B., and J. S. FRIEDENIZ’ALD. 1949. A histochemical method for localizing cholinesterase activity. Pvoc. Sot. Exp. Biol. Med. 70: 617-622. LANDMESSER, L. 1971. Contractile and electrical responses of vagus-innervated frog sartorius muscles. J. Pliysiol. 213 : 707-725. MAURO. A. 1961. Satellite cell of skeletal muscle fibers. J. Bioph.~s. Biorkcrrz. C‘yfol. 9 : 493-495. MENDER, J., C. ARANDA, and J. V. Luco. 1970. Antifibrillary effect of adrenergic fibers on denervated striated muscles. J. Ncurophysiol. 33 : 882-890. Moss, F. P.. and C. P. LEBLOXD. 1971. Satellite cells as the source of nuclei in muscles of growing rats. ill~af. Rrc. 170 : 421-435.

26.

MUIR, .4. R. 1970. The structure ‘and distribution of satellite cells, pp. 91-100. III. “Regeneration of Striated Muscle and Myogenesis.” A. Mauro, S. A. Shafiq, and A. T. Milhorat [Eds.]. Excerpta Medica. Amsterdam.

27.

MURRAY, M. in Culture,”

28.

NAMEROFF, M., and H. HOLTZER. 1970. Interference with myogenesis, pp. 251266. II, “Regeneration of Striated Muscle and Myogenesis.” A4. Mauro. S. A. Shafiq. and -4. T. Milhorat [Eds.]. Excerpta Medica, Amsterdam.

R. 1965. Muscle tissues Vol. 2. E. N. Willmer

ill ~itvo. pp. 311-372. 1~1 “Cells [Ed.]. Academic Press, New

and Tissues York.

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PAPPAS, G. D.. E. R. PETEKSOK, E. B. MASURWSK~. and S. M. CRAIS. 1971. The fine structure of developing neuromuscular synapses ijz Gtvo. .Irzrr. 1V.Y. .dcad. sci. 183 : 33-45. 30. PETERSON, E. R., and S. M. CKAIX. 1970. Innervation in cultures of fetal rodent skeletal muscle by organotypic explants of spinal cord from different animals. %. Zcllfwsch. 108 : 1-21. 31. PETEKSON, E. R.. L. .\LPEI. and S. M. CRAIN. 1969. Innervation ilt aifro of adult human and rodent skeletal muscle by fetal neurons from rodent spinal cord. J. Crll l3iol. 43 : 104a. 32. PETEI~SON, E. R., L. I-\LFEI, and S. M. CRAIN. 1970. Regeneration and innervaJ. .Ycrrropath. h-p. .‘Fr~rvol. 29: tion, tlr //ozw. of adult skeletal muscle ill Gtro. 134. 33. PETEIISON, E. R.. S. 31. GRAIN, and M. R. MuI