DEVELOPMENTAL

BIOLOGY

145,356-366

(19%)

Neural Influence on the Expression of Acetylcholinesterase Molecular Forms in Fast and Slow Rabbit Skeletal Muscles FRANCIS BACOU~ AND PIERRE VIGNERON Unitk

Di&!renciation

cellulaire

et Croissance,

INRA-ENSA, Accepted

I Place March

Pierre

Viala,

34060 Montpellier

Cedez

I, France

4, 1991

With the aim of investigating the roles of motor innervation and activity on muscle characteristics, we studied the molecular forms of acetylcholinesterase (AChE) in fast-twitch (semimembranosus accessorius; SMa) and slow-twitch (semimembranosus proprius: SMp) muscles of the rabbit. We have shown that SMa and SMp express different patterns and tissue distribution of AChE forms and that the effect of long denervation varies with age. Three principal findings concerning expression of AChE molecular forms emerge from these studies. (1) The activity of AChE and the pattern of its molecular forms are particularly altered in adult denervated SMa and SMp muscles. AChE activity increases by lo-fold in both muscles, but asymmetric forms disappear in SMa and increase by 20-fold in SMp muscles. A similar alteration of AChE is found after tenotomy of these muscles, showing that the effect of denervation may be partly due to suppression of muscle activity. (2) The different changes occurring in the composition of AChE molecular forms in adult denervated SMa and SMp muscles are consistent with fluorescent staining with anti-AChE monoclonal antibodies and with DBA or VVA lectins, which bind to AChE asymmetric, collagen-tailed forms. These lectins poorly stain denervated SMa muscle surfaces but intensely stain neuromuscular junctions and extrasynaptic areas in denervated SMp muscle. (3) In contrast with the adult, denervation of l-day-old muscles does not markedly modify the total amount of AChE or the proportions of its molecular forms, despite dramatic effects on muscle structure. These results are supported by studies of labeling with fluorescent DBA: the lectin only slightly stains the muscle fiber surface of denervated 15-dayold SMp muscle. Taken together, these data show that denervated muscles escape physiological regulation, producing increased levels of AChE with highly variable cellular distribution and patterns of molecular forms, depending on the age of operation and on the type of muscle. z 1991 Academic Press. Inc.

which enables them to bind detergent micelles and may anchor them in membranes. AChE asymmetric forms consist of one (AJ, two (A,), or three (A12) tetramers, covalently linked by disulfide bonds to a collagen-like tail (Abramson et al., 1989). They do not interact with detergents but are solubilized at high salt concentration. Asymmetric forms are attached to extracellular matrix by strong interactions with other molecules. Using histochemical or immunocytochemical methods, AChE has been localized in muscle to the region of nerve-muscle contacts. Because of its physiological role and its localization at the neuromuscular junction, AChE is an important marker of synaptogenesis and subsequent nerve-muscle interactions. Therefore, the appearance and regulation of AChE forms, particularly asymmetric ones, have generated considerable interest (Fernandez-Valle and Rotundo, 1989; Rotundo et al,, 1989). Ifl, viva, the formation of myoneural contact sites and the establishment of evoked muscle contraction correlate with the appearance of asymmetric AChE forms in innervated regions (Hall, 1973; Koenig and Vigny, 1978; Lsmo and Slater, 1980). However, the reappearance of asymmetric AChE forms at ectopic sites after reinnervation (Weinberg and Hall, 1979) and their induction by electrical stimulation in denervated muscle

INTRODUCTION

In vertebrates, two different enzymes hydrolyze acetylcholine. Acetylcholinesterase (EC 3.1.1.7; AChE)2 is largely responsible for terminating the action of the neurotransmitter acetylcholine at the neuromuscular junction. The other enzyme, butyrylcholinesterase (EC 3.1.1.8), hydrolyzes acetylcholine as well as many other esters, but has no established physiological function (Berman et ab, 1987; Chatonnet and Lockridge, 1989). Cholinesterases constitute not one but a family of structurally related polymers of catalytic subunits (MassouliQ and Bon, 1982). AChE globular forms G,, G,, and G, contain one, two, and four subunits. Globular forms can be subdivided into hydrophilic and amphiphilie forms (Toutant and Massoulie, 1987; Massoulii! and Toutant, 1988). The latter contain a hydrophobic domain * To whom correspondence should be addressed. ’ Abbreviations used: SMm, semimembranosus muscles; SMa, fasttwitch semimembranosus accessorius; SMp, slow-twitch semimembranosus proprius; AChE, acetylcholinesterase; AChR, acetylcholine receptor; HST, high salt Triton buffer; EGTA, ethyleneglycol-bis-(@aminoethyl ether) N,N,N’,N’-tetracetic acid; Iso-OMPA, tetraisopropyl pyrophosphoramide; DBA, Dolichos OiJorus agglutinin; VVA, Vicicr villosa-B, isolectin; GalNAc, N-acetylgalactosamine. 0012-1606/91 Copyright All rights

$3.00

IS’ 1991 by Academic Press, Inc. of reproduction in any form reserved.

356

BACOUANDVIGNERON

AChE

Expvxssicm

TABLE

irr Rabbit

357

Muscles

1

EVOLUTION OF AChE SPECIFICACTIVITY (mu. min-‘mg . prot-r) AND AChE MOLECULAR FORMS IN FAST (SMa) AND SLOW (SMp) RABBIT MUSCLES AFTER DENERVATION AND TENOTOMY Control muscle Fast-twitch SMa AChE (mu. min-‘mg. G, G, G, -4, A,,

prott’)

(3 S) (5 S) (10.5 S) (13 S) (16.2 S)

Slow-twitch SMp AChE (mu. min~rmy . prot -‘) G1 (3 S) G, (5 S)

G, (10.5 S) A, (13 S) A,, (16.2 S)

2.3 1.0 (46%) 0.1 (4%)

0.1 (4%) 0.2 (10% )

l-Month denervated muscle

29.3 24.2 (83%)

-

0.1 (3%) 0.2 (6%) 0.4 (11% ) 0.4 (11%)

l-Month tenotomized muscle

62.1 48.4 (789 ) 3.6 (6% ) 7.6 (12%)

1.8 (6%) 3.2 (11% )

0.8 (36% ) 4.3 3.0 (69%)

Z-Month denervated muscle

23 17.5 1.4 3.2 0.2 0.7

0.7 (1%)

( 1 (13%) (24% )

(76% ) (6% ) (14% )

(1%‘) (3%’ )

(48% )

(10% ) (9% ) (13% ) (20% )

IVote. The proportions of the various molecular forms were estimated from the sedimentation profiles (Fig. 1, Bacou et oL. 1982), and the specific activity of each form was then obtained from the total AChE activity. Although the minor asymmetric A, form represents a significant component in some muscles (4% in control SMp), its contribution is generally negligible and is not listed.

(Lomo et al., 1985) indicate that muscle activity rather than the presence of a motor nerve is essential for the synthesis of asymmetric molecular forms. However, it is well known that motor innervation exerts a profound influence on protein synthesis in muscle. Following denervation, skeletal muscles undergo a series of structural and functional changes which eventually lead to their complete degeneration. Denervation produces either a rapid and large decrease in muscle AChE activity in rat (Vigny et al., 1976a), mouse (Goudou et al., 1985), and human (Carson et al., 1979) or a large increase in muscle AChE activity in chicken (Vigny et al., 1976b), guinea pig (Lai et al., 1986), and rabbit (Tennyson et ah, 1977; Bacou et al., 1982). These modifications are accompanied by alterations in the polymorphism of AChE, particularly of asymmetric forms. Rabbit muscles represent a good model for studying the influence of innervation on the synthesis of AChE molecular forms. In a previous paper (Bacou et crl., 1982), we have shown that the molecular forms of AChE in the slow-twitch part and in the fast-twitch part of the semimembranosus muscle (SMm) change in different ways following denervation. In the semimembranosus accessorius muscle (the fast-twitch SMm part; SMa), the smaller globular forms increase after denervation, while the collagen-tailed forms decrease gradually and disappear 1 month after the operation. The semimembranosus proprius muscle (the slow-twitch SMm part; SMp) follows a very different evolution: there is a marked increase of collagen-tailed forms, both in proportion and in absolute level, which is maintained for at

least 2 months. In order to assessthe nerve-muscle interactions governing the synthesis of AChE in fast and slow muscles of the rabbit, we have examined the biosynthesis of AChE molecular forms under different experimental conditions. In the adult, we compared the alteration of AChE synthesis in SMm muscle after sciatic denervation or deprivation of activity after tenotomy. In adult innervated or denervated muscles, we studied the distribution of AChE in the extracellular matrix of skeletal muscle fibers by staining with monoclonal antibodies raised against rabbit brain AChE globular forms. We also took advantage of the specific binding of the lectins Dolichos ti&n+us agglutinin (DBA) and Vicia villosa-B, isolectin (VVA) with asymmetric AChE forms (Scott et al., 1988) to detect their distribution on the synaptic and extrasynaptic basal lamina of muscle fibers. These data were compared with those obtained on immature SMa and SMp muscles of rabbit denervated at birth, during postnatal muscle differentiation. MATERIALS

AND

METHODS

Surgical Procedures This study was carried out with New Zealand white rabbits from our own breeding. All surgical experiments were performed under aseptic conditions. Adult rabbits (2.5 kg) were anesthetized with sodium pentobarbitone (Nembutal, 30 mg/kg iv), and l-day-old rabbits (65 g) were anesthetized with ketamine (Ketalar 50, 100 ~1/100 g ip). Tenotomy of SMa and SMp muscles was performed on adult rabbits. Muscle tendons were cut at

358

DEVELOPMENTAL BIOLOGY

VOLUME 145,1991

nervation. The several branches innervating the SMa and SMp muscles were sectioned and ligated twice, and their proximal parts were reflected carefully backward to prevent reinnervation. Immunojuorescent

e

10

20

30

40

10

20

Fractions

FIG. 1. Sedimentation profiles of AChE in adult rabbit semimembranosus muscles. Control SMa (a) and SMp (b); 2-month denervated SMa (c) and SMp (d); l-month tenotomized SMa (e) and SMp (f) muscles. The proportions and specific activities of the different forms of AChE are indicated in Table 2.

Monoclonal antibodies against rabbit brain AChE globular forms (F, 61, F, 22, F, 43, F, 61, Fd 69; Mintz and Brimijoin, 1985) were a gift of Dr. S. Brimijoin (Mayo Clinic, Rochester, NY). DBA and VVA were biotinylated using N-biotinyl-o-aminocaproic acid N-hydroxysuccinimide ester according to Scott et al. (1988). Rhodamine-cu-bungarotoxin was prepared as described by Ravdin and Axelrod (1977). Unfixed muscles were mounted on metal chucks with gum tragacanth and frozen in liquid N,-cooled isopentane. Cross sections 4-6 pm thick were cut in a cryostat. The sections were incubated at room temperature for 30 min with biotinylated lectin or antibody and then incubated for 30 min with an appropriate fluorescein-conjugated reagent: avidin (Cappel Laboratories) for biotinylated lectins and goat anti-mouse IgG (Cappel Laboratories) for monoclonal antibodies. Fluorescein-avidin was dissolved in carbonate-bicarbonate buffer, pH 9.5, to reduce nonspecific binding of avidin; other incubations and all washes were in PBS. To identify neuromuscular junctions, rhodamine-a-bungarotoxin, which binds to acetylcholine receptors in the postsynaptic membrane, was included in either the first or the second incubation, Stained sections were mounted in glycerol containing 1 mg/ml paraphenylenediamine and viewed with fluorescein and rhodamine optics with a Zeiss Axiophot microscope. Extract&m

the level of their insertion on the knee joint and reflected to prevent adhesion. Particular precautions were taken to prevent vascular sections which might induce additional effects. Adult rabbit SMa and SMp muscles were denervated unilaterally by transection of the sciatic nerve near its roots and removal of a l-cm nerve stump. SMa and SMp muscles from l-day-old rabbits were specifically denervated under binocular lens according to the surgical procedure previously described (Bacou et ab, 1985). Briefly, a longitudinal section of the gracilis muscle provided access to the SMm and its in-

Staining

and Assay of AChE

To extract total AChE, whole SMa or SMp muscles were homogenized in a glass-glass Potter homogenizer in 10 vol of a “HST” detergent-saline buffer (0.01 M Tris-HCl, pH 7.2; 1 M NaCl; 1 mlM EGTA; 1% Triton X-100; 5 TIU/I aprotinin; 1 mM benzamidin) and centrifuged at 25,OOOg, 4°C for 30 min. The supernatant was used for assays of AChE activity and distribution of AChE molecular forms. AChE molecular forms were analyzed by velocity sedimentation in 5-20s sucrose gradients in extraction buffer and generally run at 4°C 40,000 rev/min for 18 hr

FIG. 2. Cross sections of rabbit semimembranosus muscles doubly stained with anti-AChE monoclonal antibodies, followed by fluoreseeinsecond antibody plus rhodamine-a-bungarotoxin. Fluorescein optics (left panels) shows antibody staining, while illumination of the same fields with rhodamine optics (right panels) reveals a-bungarotoxin-stained neuromuscular junctions. Anti-AChE monoclonal antibodies stain only end plates in control SMa (a, b) and SMp (c, d) muscles; denervated SMa (e, f) and SMp (g, h) muscles show AChE staining sometimes devoid of rhodamine-a-bungarotoxin. Bar in h, 50 pm.

BACOUANDVIGNERON

AChE

Expression

in Rabbit Muscles

359

DEVELOPMENTALBIOLOGY

360

V0~~~~145,1991

AChE binding to lectins was determined with isolated globular G, or asymmetric A,, forms from l-month denervated adult rabbit SMp muscles. The molecular forms were separated in sucrose gradients as described above, and the G, and A,, fractions were dialyzed against extraction buffer for 3 hr at 4°C. DBA or VVA lectins (0.5 mg) prepared in 50 ~1 of HST buffer without EGTA were preincubated with 600 ~1 of G, or A,, AChE forms for 1 hr at 37°C. When necessary, AChE binding to lectins was blocked by a 15-min, room temperature preincubation of DBA or VVA lectins with N-acetylgalactosamine (GalNAc), its carbohydrate potential inhibitor. Six hundred microliters of each of the lectin-AChE preparations was layered on top of 5-20% sucrose gradients and analyzed by velocity sedimentation as described above.

,

RESULTS

Efect of Denervaticrn and Tenotomy on AChE in Adult

0.

10

20

30 Fractions

FIG. 3. Binding of asymmetric and globular AChE to VVA isolectin and DBA (the results are illustrated for VVA but were identical for DBA). The G, and A,, forms were isolated from l-month denervated adult rabbit SMp muscles and layered on top of sucrose gradients, as described under Materials and Methods. Sedimentation analysis indicates that the globular forms do not bind VVA (A), while asymmetric forms are shifted in sedimentation (B). Preincubation of VVA with GalNAc does not modify AChE activity, but inhibits A,,-lectin binding (B). The G, form was allowed to sediment for 19 hr and the A,, form for 16 hr (incubations of each fraction in Ellman’s reagent: 1 hr for gradients A and 2 hr for gradients B). (D) control AChE sedimentation profiles; (A) AChE + VVA; (0) AChE + VVA + GalNAc.

unless specified otherwise, in a SW-41 Beckman or a TST-41 Kontron rotor. Horse liver alcohol dehydrogenase (4.8 S) and Escherichia coli P-D-galactosidase (16 S) were used as internal sedimentation standards. Each of the fractions was assayed for AChE activity by a slightly modified version of the calorimetric method of Ellman et al. (1961) in the presence of 0.1 mM tetraisopropyl pyrophosphoramide (Iso-OMPA).

Denervation and tenotomy alter the fiber type pattern in different ways. Either by specific SMm denervation or by sciatic section, both muscles undergo alterations characterized by an important fatty degeneration in the fast-twitch SMa and a general atrophy of the slow-twitch SMp muscles (Bacou et al., 1982,1985). After tenotomy, the ATPase properties and area of the fasttwitch fibers constituting the SMa muscle are maintained. On the contrary, the slow-twitch fibers of the SMp muscle are considerably atrophied, with an important fatty degeneration (not shown). Despite these morphological differences, AChE is expressed similarly after denervation and tenotomy of SMa and SMp muscles. In both cases, the AChE specific activity increased markedly, reaching about 10 times the original level in both SMa and SMp 1 month after the operations (Table 1). Figure 1 illustrates the changes occurring in the composition of AChE molecular forms. In SMa, the smaller globular forms increase after sciatic denervation or tenotomy, while the collagen-tailed forms decrease gradually, disappearing 1 month after denervation (Bacou et ab, 1982). They reappear by 2 months in significant proportion, with the same general pattern as that observed in l-month tenotomized muscles (Fig. 1 and Table 1). As previously observed in denervated SMp (Bacou et al., 1982), tenotomy of this muscle induces a

FIG. 4. Cross sections of adult rabbit semimembranosus muscles doubly stained with biotinylated DBA lectin, followed by fluorescein-avidin plus rhodamine-a-bungarotoxin. Fluorescein optics (left panels) shows lectin staining, while illumination of the same fields with rhodamine optics (right panels) reveals a-bungarotoxin-stained neuromuscular junctions. The lectin stained only end plates in control SMa (a, b) and SMp (c, d) muscles; (e, f) l-month denervated SMa muscle sections contain less DBA staining than Lu-bungarotoxin staining; (g, h) in l-month denervated SMp muscle sections, DBA stains both synaptic and extrasynaptic portions of the muscle fiber surface (g, h). Bar in h, 50 pm.

BACOUANDVIGNERON

AC//E

E.rprwsim

in Rabbit

iV~csclcs

361

362

DEVELOPMENTAL

ALTERATION

OF AChE

MOLECULAR

FORMS

DURING

POSTNATAL

Birth

Note.

Estimation

. prott’)

7.4 2.8 0.9 1.0 0.4 2.4

. prot-‘)

of specific

16.6 5.1 2.0 1.7 2.5 5.4 activity

TABLE 2 GROWTH

Cont.

of AChE in Adult

DENERVATED

14.6 7.8 2.6 1.1 0.7 2.6

(54%) (18%) (8%) (5%) (18%)

(31%) (12%) (10%) (15%) (33%)

20.9 10.5 3.0 1.1 2.0 4.8

(50%) (14%) (5%) (9%) (23%)

17.2 7.8 2.1 1.4 2.4 3.8

(45% ) (12%) (8%) (14%) (22%)

of each form

Muscle Sections

Muscle Sections by DBA

and VVA

To determine whether DBA or WA bound to specific AChE molecular forms, isolated G, and A,, forms from l-month denervated adult rabbit SMp muscles were incubated with the lectins and sedimented in sucrose gradients. Figure 3 shows that the lectins affected neither the activity nor the sedimentation coefficient of the G, form. On the contrary, the sedimentation of the A,, form was shifted by these lectins, showing that DBA and WA bind to GalNAc residues borne by asymmetric forms. Preincubation of the lectins with GalNAc inhibited binding to A,, AChE. These results are in good

9.3 5.5 1.9 0.3 0.2 1.5

(59% ) (21%) (4%) (2%) (16% 1

18.1 7.0 1.8 1.4 3.4 4.4

(39% (10% : (8%) (19%) (25%)

as in Table

Den.

21.7 12.6 4.5 1.8 0.5 2.0

(58%) (21%) (9%) (2%) (9%)

23.7

1. Cont.,

MUSCLES 1 Month

Cont.

(55% ) (13%) (7%) (4%) (20%)

min-‘mgeprott’)

SEMIMEMBRANOSUS

15 Days

22.5 12.4 2.9 1.6 0.8 4.6

As shown in Fig. 2, anti-AChE monoclonal antibodies stain only neuromuscular junctions on both SMm control muscle sections (Figs. 2a-2d). One-month denervated SMa muscle sections are characterized by the small size of their end plate and by numerous rhodamine-a-bungarotoxin patches devoid of AChE staining (Figs. 2e, 2f). These data are in apparent contradiction with the increase in AChE expression as measured biochemically (Table 1). Unlike SMa muscle, denervated SMp muscle sections show abundant AChE end platelike patches, some of them lacking rhodamine-a-bungarotoxin staining (Figs. 2g, 2h). These results are in agreement with those obtained after histochemical staining of AChE (not shown). Staining of Adult Lectins

OF ~-DAY-OLD

(38%) (12%) (13%) (5%) (32% )

(mu.

145,199l

Den.

marked increase of the collagen-tailed forms, both in proportion and in absolute level (Fig. 1 and Table 1). Dis&mtion

VOLUME

5 Days

Cont. Fast-twitch SMa AChE (mu. min’mg G, (3 S) G, (5 3 G, (10.5 S) A, (13 S) A,, (16.2 S) Slow-twitch SMp AChE (mu. min’mg G, (3 S) Gz (5 S) G, (10.5 S) A, (13 S) A,, (16.2 S)

BIOLOGY

12.2 2.9 2.4 2.0 4.3 control;

Cont.

5.3 2.5 1.3 0.2 0.1 1.1

(47%) (25%) (5%) (2%) (21%)

12.1 (12%) (52%) (10%) (9%) (18%)

4.7 1.1 1.1 2.6 2.5

Den.

12.8 7.3 3.2 1.0 0.4 1.3

(57%) (25%) (8%) (3%) (10%)

18.2 (9%) (39%) (9%) (22%) (21%)

2.0 8.5 2.0 1.7 4.0

(11%) (47%) (11%) (9%) (22% )

Den., denervated.

agreement with those previously obtained with a different approach by Scott et aZ. (1988). Both lectins stain only neuromuscular junctions in control muscles (Figs. 4a-4d) in agreement with the results obtained with anti-AChE monoclonal antibodies. After denervation of SMa muscle, lectin staining was faint and numerous sites were stained by rhodamine-abungarotoxin but not by lectins (Figs. 4e, 4f). On the contrary, the sites of denervated SMp junctions, labeled by rhodamine-a-bungarotoxin, were stained by the two lectins. In fact, denervation of SMp muscle induced a large increase in lectin staining which spread around muscle fibers, presumably on their basal lamina (Figs. 4g, 4h). These results are in good agreement with the increase in A,, activity observed in denervated SMp muscles (Table 1). AChE Expression Rabbit

in Denervated

Muscles

of New Born

AChE specific activity reaches a maximum around Day 5 after birth in both muscles (Table 2). Denervation affects AChE specific activity much less when performed at birth than in the adult. AChE activity increases slightly, reaching about 2 times and 1.5 times the control levels in l-month SMa and SMp muscles, respectively (Table 2). Despite the striking muscle fiber alteration after denervation of newborn rabbit muscles, changes in the AChE molecular form pattern are not as dramatic as those in adult denervated muscles. During postnatal growth, l-day denervated SMa muscles are characterized by a decrease in the percentage of asymmetric forms even on l-month operated muscles. However, there is no significant change in the pattern of

ilUtE

BACOUANDVIGNER~N

AChE molecular forms born SMp muscle.

following

denervation

Staining of Developing Muscle Sections Anti-AChE Monoclonal Antibodies

of new-

by Lectins

and

Anti-AChE monoclonal antibodies as well as DBA and VVA lectins intensely stain only neuromuscular junctions in 15-day-old SMa or SMp muscles (Figs. 5a5d). In denervated 15-day-old SMm muscle sections, the anti-AChE antibody stains end plate-like structures, some of which are not stained by rhodamine-abungarotoxin. In denervated SMa muscle, the lectins stain neuromuscular junctions only (not shown). On the contrary, DBA and VVA also slightly stain the extrasynaptic region of the denervated SMp muscle surface (Figs. 5e, 5f). These results are in good agreement with those observed for denervated adult SMp muscle, with either anti-AChE antibody or lectin staining. DISCUSSION

Expression of AChE in Adult Tenotomixed Muscles

Denervated

w

Muscle structure is considerably affected by both operations. Denervation induces a fatty degeneration of the fast-twitch SMa muscle and a general atrophy of the slow-twitch SMp muscle which, however, maintains its ATPase properties (Bacou et al., 1982). Tenotomized muscles follow a different behavior. The SMa muscle retains its histological characteristics, but the SMp muscle is dramatically affected by an extensive infiltration of fatty inclusions. These differences may be related to the anatomy of the SMa and SMp muscles. The former is a large muscle inserted on a large area of the knee joint by muscle fibers themselves, without visible tendon. Thus, tenotomy of SMa muscle in fact resembles a muscle section rather than a true tenotomy. However, the sectioned muscle stump is considerably atrophied. On the contrary the SMp muscle is much smaller and is inserted on the knee joint by a long and very distinct tendon, which makes its section easier. Despite postoperation histological differences, denervation and tenotomy affect AChE in much the same way. It is well known that activity plays an important role in the development and maintenance of muscle properties (Salmons and Sreter, 1976; Lsmo, 1989). Moreover, the biosynthesis of AChE has been shown to be highly sensitive to electrical stimulation in denervated muscle (Lomo and Slater, 1980; Lomo et al., 1985). Our results confirm that the activity of AChE and the patterns of its molecular forms vary strongly with muscle activity. Both denervated or tenotomized SMa and SMp muscles are characterized by a large increase in

E.cprcwior~

ire Rubbit

Muxl~s

363

AChE specific activity and by a similar alteration of AChE polymorphism pattern. The effects of denervation on AChE thus appear to be due, at least in part, to the loss of activity. Expression of AChE Molecular Forms in Adult Muscles as Observed lay Antibody or Lectin Staining Anti-AChE monoclonal antibodies and lectins stain only neuromuscular junctions in control SMm muscle sections. In denervated SMa muscles, rhodamine-cubungarotoxin labels smaller patches and some of them appear devoid of AChE or lectin staining (Figs. 2 and 4). An obvious possibility is that rhodamine-Lu-bungarotoxin stains new patches of acetylcholine receptors (AChR). It is well known that denervation induces a considerable increase in the biosynthesis of AChR, the density of which increases from about 10 to more than 600 AChR molecules/~m2 at the extrasynaptic membrane (Fambrough, 1979). Moreover, in vitro studies have shown that the accumulation of AChR and of AChE during the formation of neuromuscular junctions is apparently not regulated in the same way. Particularly, muscle activity seems to be the crucial factor for accumulation of AChE, which does not occur in the absence of synaptic transmission (Rubin et al, 1980). Thus, either the formation of ectopic AChR patches or a distinct regulation of AChR and AChE may explain the discrepancy between AChE and AChR stainings of inactive SMa denervated muscles. However, although the lack of some lectin staining might be related to the disappearance of asymmetric forms, the low level of AChE staining on neuromuscular junctions is particularly striking with respect to the high level of AChE specific activity in denervated SMa muscle (Table 1). This apparent discrepancy might be related to the monoclonal antibodies which were raised against rabbit brain AChE globular forms, most of them being intracellular. Moreover, the increase in the activity of AChE in SMa muscle results from the very large increase of globular forms, particularly the soluble G, form which might escape staining under our conditions of immunocytochemistry. In contrast, AChE and lectin staining are particularly intense in denervated SMp muscles. Moreover, the number of AChR patches, as stained by rhodamine-a-bungarotoxin, is lower than anti-AChE monoclonal antibody staining, which is highly concentrated on end plate-like structures. The fact that AChE is not confined to original synaptic sites is consistent with the histochemical distribution of AChE activity, which is detected all around the muscle fiber surface (not shown). These data are in agreement with a previous study of Tennyson et al. (1977), who showed that after denervation of rabbit gastrocnemius muscles, the AChE

364

DEVELOPMENTALBIOLOGY

V0~~~~145.1991

FIG. 5. Cross sections of 15-day-old rabbit semimembranosus muscles doubly or anti-AChE antibodies, followed by fluorescein-avidin or fluorescein-second (left panels) shows lectin or antibody staining, while illumination of the same toxin-stained neuromuscular junctions, AChE antibody (a, b) and DBA (c, d) muscle sections. The same results are obtained with SMa muscles (not shown). stains the extrasynaptic surface on newborn denervated 15-day-old SMp muscle

activity increased mostly in the extrajunctional part of muscle fibers, and to a lesser extent at intracellular sites. Using lectins which bind to asymmetric but not to globular AChE forms, we have shown that in contrast to control SMm and to denervated SMa muscle sections,

stained with biotinylated DBA lectin (same results with VVA) antibody plus rhodamine-u-bungarotoxin. Fluorescein optics fields with rhodamine optics (right panels) reveals a-bungarostain only neuromuscular junctions of 15-day-old rabbit SMp However, DBA stains neuromuscular junctions and slightly surface (e, f). Bar in f, 50 Fm.

the lectins intensely stain both neuromuscular junctions and extrasynaptic areas in denervated SMp muscle (Fig. 4). A similar observation has been reported recently by Scott et al. (1990) on denervated rat muscle sections

stained with VVA lectin. Although the GalNAc containing molecules concentrated at the neuromuscular junction include both glycolipids and the collagen-tailed AChE (Kaupmann and Jockusch, 1988; Scott et a,l., 1988), the levels and the distribution of lectin staining on SMa and SMp muscles might be related to asymmetric AChE in our observations. Indeed, biochemical analysis shows that, in contrast to SMa muscle, denervation of SMp muscles induced a marked increase of the collagentailed forms, both in proportion and in absolute level (Fig. 1 and Table l), which is consistent with DBA staining in both muscles. It is thus very likely that AChE collagen-tailed forms disappear from the end plate on denervated SMa muscle, but remain concentrated at the neuromuscular junction and also spread largely at the extrasynaptic surface of denervated SMp muscle fibers.

In a previous study (Bacou and Vigneron, 1988) we have shown that both SMa and SMp muscles are heterogeneous at birth with respect to their fiber types, with an equivalent percentage of type I and II fibers in the SMa and SMp muscles. During postnatal development, these muscles progressively acquire their specific properties and become homogeneous by 2 months. A similar postnat,al evolution has been observed in other mammalian species (Karpati and Engel, 1968; Okada et d., 1984) and it is well known that motor innervation is essential for muscle differentiation after birth (Hanzlikova and Schiaffino, 1973; Betz et ul., 1980; Okada et a.l., 1984). Further studies on denervated muscles also showed that type II fibers are more affected than slow-twitch fibers. (Curless, 1977; Lowrie and Vrbova, 1984). We have shown (Bacou and Vigneron, 1988) that after denervation both muscles undergo a rapid but different alteration. The fast-twitch SMa muscle quickly undergoes fatty degeneration. The remaining type II fibers are considerably atrophied and muscle degeneration is almost complete by 2 months. The slow-twitch SMp muscle is much less affected by denervation. Type I fibers remain similar to control with respect to their ATPase properties but show a slight hypertrophg. Type II fibers are dramatically atrophied, then gradually disappear as observed in the SMa muscle. By 1 month, the denervated SMp muscle contains almost only type I fibers (Bacou and Vigneron, 1988). Despite these dramatic morphological changes, the AChE content of the muscles was much less affected by denervation than in adult muscles. We observed a slight increase in AChE specific activity and minor changes in the pattern of its molecular forms. However, anti-AChE antibodies and DBA or VVA lectins intensely stain the

neuromuscular junction in normal developing muscles, but denervation induced changes which slightly differed from those observed in the adult, particularly in the case of the SMa muscle. This muscle presented numerous AChR patches after denervation and in addition AChE patches which did not correspond to rhodamin-nbungarotoxin sites. On the contrary, the staining of denervated newborn SMp with anti-AChE antibodies or lectins resembled that of the adult, although the extrasynaptic area was less intensely stained with DBA. In summary, our results further illustrate the role of muscle activity in the control of AChE polymorphism in the adult and provide evidence for the localization of a fraction of AChE in the extracellular matrix. Moreover, these data invite speculation on nerve-muscle interactions in the adult and during development. In the adult, neuromuscular junctions are stabilized and incorporate elements from both motor nerve and muscle (Bacou et ul., 1985). We can speculate that during development, a reciprocal balance is progressively established between nerve and muscle, until all components of the neuromuscular junction are expressed as in the adult. This evolution includes the loss of polyneuronal innervation which is almost complete by 15 days (Bixby and Van Essen, 19’79;Van Essen et nl., 1990) and the synaptic reorganization occurring in juvenile (3-5 weeks) rabbit muscles (Gordon and Van Essen, 1985; Soha et a,l., 198’7). We thank MarylPne Rostagno and Louis Marger for excellent tcchnical assist,ance. This work was supported by AIP “Cholinesterase” INRA grant and an “Association Franqaise contre les Mgopathies” contract. REFERENCES ABRAMSON, S. N., ELLISMAN, M. H., DEERINCK, T. J., MAULET, Y., GENTRY, M. K., DOCTOR, B. P., and TAYLOR, P. (19X9). Differences in structure and distribution of the molecular forms of aret>rlcholinesterase. J. Cell Binl. 108, 2301-2311. BACOU. F., and VIGNERON, P. (19%). Polymorphisme de 1’acCtylcholinestkrase et de la myosine au tours du developpement drs muscles rapide et lent denerves chez le lapin nouveau-n&. RF pud

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